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No Sex For 40 Million Years? No Problem

Posted by tumicrobiology on March 20, 2007

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A group of organisms that has never had sex in over 40 million years of existence has nevertheless managed to evolve into distinct species, says new research published today. The study challenges the assumption that sex is necessary for organisms to diversify and provides scientists with new insight into why species evolve in the first place 

The research, published in PLoS Biology, focuses on the study of bdelloid rotifers, microscopic aquatic animals that live in watery or occasionally wet habitats including ponds, rivers, soils, and on mosses and lichens. These tiny asexual creatures multiply by producing eggs that are genetic clones of the mother — there are no males. Fossil records and molecular data show that bdelloid rotifers have been around for over 40 million years without sexually reproducing, and yet this new study has shown that they have evolved into distinct species.

Using a combination of DNA sequencing and jaw measurements taken using a scanning electron microscope, the research team examined bdelloid rotifers living in different aquatic environments across the UK, Italy and other parts of the world. They found genetic and jaw-shape evidence that the rotifers had evolved into distinct species by adapting to differences in their environment.

Dr Tim Barraclough from Imperial College London’s Division of Biology explained: “We found evidence that different populations of these creatures have diverged into distinct species, not just because they become isolated in different places, but because of the differing selection pressures in different environments.

“One remarkable example is of two species living in close proximity on the body of another animal, a water louse. One lives around its legs, the other on its chest, yet they have diverged in body size and jaw shape to occupy these distinct ecological niches. Our results show that, over millions of years, natural selection has caused divergence into distinct entities equivalent to the species found in sexual organisms.”

Previously, many scientists had thought that sexual reproduction was necessary for speciation because of the importance of interbreeding in explaining speciation in sexual organisms. Asexual creatures like the bdelloid rotifers were known not to be all identical, but it had been argued that the differences might arise solely through the chance build-up of random mutations that occur in the ‘cloning’ process when a new rotifer is born. The new study proves that these differences are not random and are the result of so-called ‘divergent selection’, a process well known to cause the origin of species in sexual organisms.

Dr Barraclough adds: “These really are amazing creatures, whose very existence calls into question scientific understanding, because it is generally thought that asexual creatures die out quickly, but these have been around for millions of years.

“Our proof that natural selection has driven their divergence into distinct species is another example of these miniscule creatures surprising scientists — and their ability to survive and adapt to change certainly raises interesting questions about our understanding of evolutionary processes.”

Note: This story has been adapted from a news release issued by Imperial College London.

Science Daily

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Study: Antiviral protein may fight Ebola

Posted by tumicrobiology on March 20, 2007

German scientists have found an antiviral protein shown to inhibit other viruses might protect against Ebola and Marburg virus infections.

The Ebola and Marburg viruses belong to the Filoviridae family and cause severe hemorrhagic fever in humans and non-human primates. Filovirus infections are characterized by high fever, hemorrhages and shock and are responsible for mortality rates up to 90 percent. Currently, there is no vaccine or therapy available for treating infected patients.

In a previous study researchers found the zinc finger antiviral protein, or ZAP, capable of inhibiting Moloney murine leukemia virus and Sindbis virus replication.

In the new study, ZAP was tested for its antiviral activity in cells infected with Ebola and Marburg. Results showed up to 95 percent inhibition of Ebola, while Marburg was less significantly inhibited suggesting the antiviral effectiveness of ZAP may depend on the filovirus species.

The study conducted at the Bernhard-Nocht Institute for Tropical Medicine in Hamburg is reported in the Journal of Virology.

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Bacterial Virus Gene Confers Disease Resistance In Tall Fescue Grass

Posted by tumicrobiology on March 20, 2007

Researchers at North Carolina State University have discovered that inserting a specific gene from a bacterial virus into tall fescue grass makes the grass resistant to two of its biggest enemies
The NC State researchers showed that the inserted gene – the T4 lysozyme gene, a gene found in bacteriophages, or bacterial viruses – conferred high resistance to gray leaf spot disease in six of 13 experimental grasses. Three of the six resistant grasses also showed high resistance to brown patch disease. These two diseases are arguably the most important – and severe – fungal diseases affecting tall fescue grass.

The finding has the potential to have wide applications in engineering resistance to a variety of fungal diseases in not only tall fescue grass – the most widely planted turfgrass in North Carolina and a commonly utilized grass in the southeastern United States – but various other crops.

The collaborative research involves four faculty members: Dr. Ron Qu in the Department of Crop Science, Drs. H. David Shew and Lane Tredway from the Department of Plant Pathology, and Dr. Eric Miller, in the Department of Microbiology. The research was mainly performed by Dr. Shujie Dong, a post-doctoral researcher who was a graduate student of Qu’s, with assistance from two other scientists in Qu’s lab – Drs. Jianli Lu and Elumalai Sivamani.

About half of the turfgrass planted in North Carolina – one million acres – is tall fescue grass, a cool-season grass that has a high tolerance for the heat and drought of North Carolina summers, Tredway says. It is ubiquitous in the Southeast, found on lawns, golf courses and commercial acreages.

Gray leaf spot disease is caused by the Magnaporthe grisea fungus, the pathogen that also causes rice blast – the major disease of rice plants. Gray leaf spot causes round or oval tan spots that turn gray when there’s high humidity. It infects blades to make the grasses die rapidly.

Brown patch disease, caused by the soil-dwelling fungus Rhizoctonia solani, a major pest to various plant species, brings about circular, brown lesions on grass. Lawns with brown patch disease appear wilted, even if watered sufficiently, the researchers say.

Miller, the microbiologist, says that the bacterial viruses exist widely in different environments, and produce an array of products that are harmful to bacteria; as viruses attempt to spread, which they need to do in order to survive and thrive, the T4 lysozyme gene produces the enzymes that chew through the bacterial cell walls.

Miller says that the lysozyme now made by the grass does essentially the same thing to a fungus when it tries to infect, thereby providing anti-fungal properties in tall fescue and allowing the grass to withstand fungal disease.

Tredway says the benefits of potential applications may be felt economically and environmentally.

“A lot of money is spent on fungicides, which also have an impact on the environment,” he said. “Disease-resistant plants have the potential to reduce those economic and environmental impacts for many years.”

Qu says that future research will replicate this experiment in the field, rather than just in the lab, and that other disease resistance genes show anti-fungal properties in tall fescue. He also hopes to study how the group’s genetically altered plants interact with other important fungal diseases to further test their anti-fungal mettle.

Much of the work was funded by NC State’s Center for Turfgrass Environmental Research and Education and the Turfgrass Council of North Carolina.

Reference: “Expression of the Bacteriophage T4 Lysozyme Gene in Tall Fescue Confers Resistance to Gray Leaf Spot and Brown Patch Diseases”

Authors: Shujie Dong, H. David Shew, Lane P. Tredway, Jianli Lu, Elumalai Sivamani, Eric S. Miller and Rongda Qu, North Carolina State University

Published: February 2007 in Transgenic Research

Note: This story has been adapted from a news release issued by North Carolina State University.

Source: North Carolina State University
(Sciencedaily)

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Protein Found In Chickens May Help Protect Against Food-Borne Pathogens

Posted by tumicrobiology on March 20, 2007

Researchers from The Netherlands have identified a protein in the digestive tract of chickens that may serve as an antimicrobial agent against food-borne pathogens. They report their findings in the March 2007 issue of the journal Antimicrobial Agents and Chemotherapy

Food-borne pathogens, responsible for most cases of food poisoning in developed countries, are commonly affiliated with poultry products including chicken. Therapeutic doses of antibiotics in chicken feed have been administered since the 1950s, but are now discouraged due to increasing rates of antibiotic resistance.

In the study researchers tested for B-defensin gallinacin-6 (Gal-6) protein expression in chickens and explored antimicrobial activity against gram-positive and gram-negative bacteria as well as yeast. Researchers observed high expression of Gal-6 in the esophagus and crop and moderate expression in the glandular stomach. Colony-counting tests showed strong bactericidal activity against Campylobacter jejuni, Salmonella enterica serovar Typhimurium, Clostridium perfringens, and Escherichia coli, all major food-borne pathogens. Fungicidal activity was also noted. In a kill-curve study results showed treatment with Gal-6 reduced C. perfringens survival within sixty minutes.

“In conclusion, to our knowledge, this is the first report of a chicken B-defensin highly expressed in the digestive tract and displaying strong bactericidal activity against food-borne pathogens.” say the researchers.

(A. van Dijk, E.J.A. Veldhuizen, S.I.C. Kalkhove, J.L.M. Tjeerdsma-van Bokhoven, R.A. Romijn, H.P. Haagsman. 2006. The B-defensin gallinacin-6 is expressed in the chicken digestive tract and has antimicrobial activity against food-borne pathogens. Antimicrobial Agents and Chemotherapy, 51. 3: 912-922.)

Note: This story has been adapted from a news release issued by American Society for Microbiology. (Science Daily — )

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Method for detection and identification of multiple chromosomal integration sites in transgenic animals created with lentivirus

Posted by tumicrobiology on December 4, 2006

Elizabeth C. Bryda, Michael Pearson, Yuksel Agca, and Beth A. Bauer

BioTechniques® December 2006
Volume 41, Number 6: pp 715-719

Abstract
Transgene delivery systems, particularly those involving retroviruses, often result in the integration of multiple copies of the transgene throughout the host genome. Since site-specific silencing of trangenes can occur, it becomes important to identify the number and chromosomal location of the multiple copies of the transgenes in order to correlate inheritance of the transgene at a particular chromosomal site with a specific and robust phenotype. Using a technique that combines restriction endonuclease digest and several rounds of PCR amplification followed by nucleotide sequencing, it is possible to identify multiple chromosomal integration sites in transgenic founder animals. By designing genotyping assays to detect each individual integration site in the offspring of these founders, the inheritance of transgenes integrated at specific chromosomal locations can be followed efficiently as the transgenes randomly segregate in subsequent generations. Phenotypic characteristics can then be correlated with inheritance of a transgene integrated at a particular chromosomal location to allow rational selection of breeding animals in order to establish the transgenic line.

INTRODUCTION

Production of genetically engineered animals by pronuclear injection or retroviral delivery systems has been a successful strategy for generating animal models to better understand the functionality of genes. In transgenic animals created by embryo microinjection, the site of integration of the transgene within the genome is a random event. Thus, when multiple embryos have been injected or infected with the same DNA, the integration site will be different in each founder animal. When the integration events have occurred at the one-cell stage, they should exhibit germline transmission with the potential to be inherited by the founder’s offspring. If the integration occurs at a later stage, the resulting mosaic founder may or may not exhibit germline transmission of the transgene. In the case of pronuclear injection, there is typically one insertion site, although multiple transgene copies are often found in a tandem array at that integration site (1).

Lentivirus transgenesis is becoming an increasingly attractive alternative to pronuclear injection because it is more efficient in terms of successful transgene incorporation into the host genome, less invasive to the embryo, and technically less demanding to perform (2). Lentiviral delivery systems have been used successfully to generate transgenic mice, rats, pigs, and cattle (2–7). The disadvantage of lentivirus is that there are often multiple integration events with random transgene insertions on several chromosomes.

Independent of the method of transgene delivery, the insertion site can have profound effects on transgene expression. This can lead to phenotypic effects in the transgenic animal that are not due to the transgene per se, but are a consequence of the integration site, a phenomenon referred to as position effect (8). It is critical to correlate phenotype with genotype, particularly in animals created via lentivirus transgenesis, since not all copies of the transgene may be contributing equally to the phenotype.

Determining transgene integration sites is challenging. A number of PCR-based methods, often referred to as chromosome walking techniques, have been developed to isolate DNA fragments adjacent to known sequences, including inverse PCR (9), ligation-mediated PCR (LMPCR) (10), randomly primed PCR (RP-PCR) (11,12), and T-linker PCR (13). The method described in this paper incorporates several elements of these techniques in a unique way that allows the capture of DNA fragments containing the chromosomal region flanking the transgene. Our method enables quick and inexpensive determination of multiple independent transgene integration sites in founder animals and their offspring. Here, we demonstrate that this method is useful for identifying and monitoring multiple transgene integration sites in transgenic animals created using lentivirus.

MATERIALS AND METHODS

Animals

Lewis rat lines carrying an enhanced green fluorescent protein (EGFP) transgene were created by using the EGFP DNA construct and experimental protocol described by Lois et al. (2). Transgene positive founder animals were identified using an EGFP PCR assay (14). To assess GFP expression, tail biopsies were examined for fluorescence under a Nikon SMZ1500 UV dissecting scope (Nikon Instruments, Melville, NY, USA). Founders (F0) were bred to wild-type Lewis rats obtained from Harlan Sprague Dawley (Indianapolis, IN, USA) to generate the N1. N1 animals were genotyped using integration site-specific PCR assays. Selected N1 animals were mated to wild-type Lewis rats to generate the N2. N2 animals were genotyped using integration site-specific genotyping assays, and GFP expression was confirmed. Several of these lines (F455.5, F456.9, F458.7, F463.1, and F463.5) demonstrated stable transmission of the integration site-specific transgene coupled with robust GFP expression and were donated to the Rat Research and Resource Center (RRRC) at the University of Missouri. All other rat lines and mouse strains used are available either through the RRRC (Web site (external)) for rats or the University of Missouri/Harlan Mutant Mouse Regional Resource Center (MMMRC; Web site (external)) for mice.

Preparation of DNA

DNA was isolated from tail biopsies using the DNeasy® Tissue kit (Qiagen, Valencia, CA, USA). Restriction endonuclease digestion was performed with PstI and HhaI (Figure 1, step 1). These enzymes were chosen because they created 3′ overhangs and their recognition sites were not present within the transgene sequence. Three micrograms genomic DNA were digested with 20 U enzyme in a total reaction volume of 30 μL as recommended by the manufacturer. Reactions were incubated for 2 h for partial digestion.

PCR Amplification and Linker Ligation

Three transgene-specific nested primers were designed to both the 5′ and 3′ regions of the transgene using PrimerQuest available from Integrated DNA Technologies (IDT; Coralville, IA, USA). Primer 1 was farthest from the junction between the known transgene sequence and the unknown integration site, whereas primer 3 was closest (Figure 1). All gene-specific primers were designed to have an optimum melting temperature (Tm) of 60°C, optimum primer length of 24 bp, and an optimum % GC content of 50. Y-linker and primer sequences for the Y-linker and their relationships are provided in Figure 2. Y-linker A (Figure 2) contains a terminal inverted T at the 3′ end to inhibit extension by DNA polymerases. The Y-linker was prepared by combining equal volumes of 8 μM Y-linker A and 8 μM Y-linker E, incubating at 95°C for 5 min, and then letting the reaction cool on the benchtop. The sequences of the genespecific primers used for the lentivirus-generated rat lines are available upon request or are listed under the genotyping assay for the specific lines at Web site (external). Primers and linkers were synthesized by IDT.

All PCRs were performed in a 25-μL volume containing 2.5 μL 10× FastStart Taq with 20 mM MgCl2 (Roche Diagnostics, Indianapolis, IN, USA), 0.2 mM each dNTP, 1.6 μM each primer, and 1.25 U FastStart Taq buffer. In the first PCR, 1 μL restriction digest from above and transgenespecific primer 1 were used, and a single round of PCR was performed with the following thermal cycling conditions: 94°C for 10 min, 60°C for 1 min, and 72°C for 10 min (Figure 1, step 2). Following amplification, a 10-μL reaction containing 7 μL this PCR, 1 μM Y-linker, 400 U T4 DNA Ligase (New England BioLabs, Ipswich, MA, USA), and 1 μL 10× T4 DNA Ligase buffer supplied with the enzyme was incubated at 16°C for 16 h (Figure 1, step 3). Transgene-specific primer 2, Y-primer D, and 1 μL ligase reaction were used in the second PCR with the following cycling conditions: 94°C for 5 min, 20 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 1.5 min, and one cycle of 72°C for 10 min (Figure 1, step 4). The same cycling conditions were used in the third PCR. This final reaction utilized transgene-specific primer 3, Y-primer G, and 1 μL from the second PCR (Figure 1, step 5).

PCR Product Purification and Analysis

Amplification products from the third PCR were separated by gel electrophoresis on 1%-3% agarose gels, and individual products were gel-purified using QIAquick® Gel Extraction kit (Qiagen). When multiple bands were detected, all bands were isolated. The nucleotide sequence of each amplification product was determined using the transgene-specific primer 3 as a sequencing primer (Figure 1, step 6). Nucleotide sequencing was performed either at our DNA Core (University of Missouri-Columbia) or by SeqWright (Houston, TX, USA). The nucleotide sequences were aligned and examined to confirm the presence of the expected known transgene sequence and determine the flanking sequence representing the insertion site. The insertion site sequence was analyzed using Basic Local Alignment Search Tool (BLAST) (15) to determine the chromosomal location of the transgene.

RESULTS AND DISCUSSION

Lentivirus was used as a delivery system to create Lewis rat lines carrying an EGFP transgene (2). A total of nine transgene positive founder (F0) animals were recovered. While all nine founders were positive for the presence of the EGFP transgene, eight founders expressed EGFP based on epifluorescent microscopy of tail biopsies, while one founder (456) had no detectable fluorescence (Table 1). Our method for determining chromosomal integration sites was used to identify the chromosomal location(s) of the transgene in each of the nine founders. PCR products ranged in size from 100–1000 bp. Longer PCR fragments were generally necessary for determining the insertion site when the region contained repetitive elements. Several sites were successfully identified with recovered products as small as 110 bp, which included as little as 20 bp genomic sequence. For seven of the founders, we identified one to four integration sites depending on the founder. We failed to identify the integration site in two founders using PstI and HhaI digestion, and we did not pursue these further. It is possible that by using other restriction enzymes with 4–6 bp recognition sites or increasing PCR extension times to generate larger products, we would have successfully identified the integration sites in these founder lines. For seven lines, site-specific genotyping assays were developed for every integration site identified in the founder. In one case (463), we recovered genomic sequence that matched a sequence found on many chromosomes, so we could not assign a chromosomal location to this integration site. Nonetheless, the genotyping assay based on this sequence allowed the transgene integration site to be followed not only in the founder but in his offspring.

To follow segregation of the integration sites, the offspring from matings between each of the seven founders and wild-type Lewis rats were monitored for presence and expression of the transgene at each integration site using the site-specific genotyping assays and microscopic examination for GFP fluorescence. One founder (452) was infertile. For the remaining six founder lines, offspring were obtained, and both the insertion sites and the GFP expression were determined.

Founder 455 had two independent transgene insertion sites: one on chromosome 1, and a second on chromosome 5. When this founder was mated to a wild-type Lewis animal, two N1 offspring were recovered. One N1 carried the chromosome 1 integration site, but did not have detectable GFP expression. Lack of expression may have been due to positional effects. The other N1 carried the chromosome 5 integration site and did have GFP expression. The chromosome 5 transgene positive N2 offspring (n = 5) continued to have high fluorescence, demonstrating that GFP expression in this line was associated with the chromosome 5 transgene, and could be stably transmitted from generation to generation. This illustrates the importance of determining which transgene insertion site was correlated with GFP expression in order to successfully maintain a GFP-expressing line.

Line 456, which carried two transgene insertion sites, was unusual in that the founder did not display GFP fluorescence. Of the offspring recovered from this founder, three carried the chromosome 17 transgene insertion only and one carried the chromosome 9 transgene insertion only. While animals carrying the chromosome 17 insertion did not have detectable fluorescence, robust GFP fluorescence was seen in the animal that inherited the chromosome 9 transgene insertion. The chromosome 9 transgene positive N2 animals continued to have high fluorescence, demonstrating that GFP expression in this line was associated with the chromosome 9 transgene. We speculate that GFP expression was suppressed at the chromosome 17 insertion due to a position effect and that the founder was mosaic for the chromosome 9 insertion, resulting in undetectable GFP expression. In subsequent generations, inheritance of the chromosome 9 insertion was germline, and expression occurred in all cells leading to detectable fluorescence.

For line 458, three insertion sites were detected. By correlating GFP expression with inheritance of the various transgene insertion sites, it was possible to show that the chromosome 7 integration gave good GFP expression. This was confirmed in N2 animals (n = 10) carrying only the chromosome 7 integration. It should be noted that while none of the N1 animals carried the chromosome 10 integration site, this site was confirmed in the original founder by integration site-specific genotyping. It is possible that the founder was mosaic for the chromosome 10 integration site.

For line 459, which had a single detected transgene insertion on chromosome 2, two offspring were recovered that inherited the chromosome 2 transgene insertion site. However, neither of these animals exhibited GFP fluorescence. This could be due to a positional effect associated with the particular insertion site on chromosome 2 coupled with inheritance through the male lineage, or alternatively, we may have missed an insertion site in founder 459 that was associated with the fluorescence seen in the founder, which was not inherited by these two offspring.

Founder 463 had the greatest number of insertion sites; by continuing to correlate GFP expression with inheritance of specific transgene insertion sites, it was possible by the N2 generation to identify animals with single transgene insertion sites that maintained high GFP expression.

In addition to the lentivirus experiment described above, we have successfully used this technique to determine the lentivirus integration sites and to generate rat lines with single transgene integration sites for a Sprague-Dawley GFP transgenic (RRRC: 0065), derived from the line created by Lois et al. (2), and a rat model containing the human presenilin-1 gene (RRRC: 0061). To test whether our method for identifying insertion sites had broader application beyond just determining chromosomal locations of lentivirus transgene integration, we attempted to identify the transgene integration site of three additional rodent strains: (i) a mouse transgenic strain (MMRRC: 000366-MU/H) carrying the EGFP gene on the FVB inbred genetic background (16); (ii) a knockout mouse line (MMRRC: 000352-MU/H) involving the acetylcoenzyme A dehydrogenase long chain gene (Acadl), which carries a duplication of exons 3 and 4 with insertion of the neocassette into the approximately 11 kb intron 4 (17); and (iii) a transgenic rat line (RRRC: 0043), which contains a mutated version of the human HLA-B2705 gene on a Lewis genetic background (18). In the case of the FVB-EGFP strain and the Acadl knockout strain, we were able to determine precisely the insertion site on chromosome 3 and within the large Acadl intron 4, respectively. Our method failed for the HLA-B2705 transgenic rat line, which has a high transgene copy number (24 copies) in homozygous animals (18). The insertions are integrated at a single locus (18), and the multiple copies have probably integrated as concatamers.

A major advantage of determining the precise chromosomal integration site in animals created via pronuclear injection is that genotyping assays can be developed that allow animals heterozygous for a transgene to be easily distinguished from homozygous animals. Without this type of information, PCR-based genotyping assays can distinguish only whether animals carry the transgene. While it is possible to use Southern blot analysis and densitometry to measure transgene copy number as an alternative method to distinguish homozygotes from heterozygotes, it is laborious, timeconsuming, and not practical for many labs.

In summary, we have described a quick and straightforward method for determining the location of multiple chromosomal integration sites in animals created by lentivirus transgenesis. Our studies underline the need to carefully correlate a particular integration site with gene expression over multiple generations in order to create stable models. We have also found that this method has applicability for detecting transgene integration sites in other cases of random integration, but is probably limited to situations where very few tandem copies of the transgene have been integrated at the insertion site.

ACKNOWLEDGMENTS

E.C.B. and M.P contributed equally to this work. This work was supported in part by grants from the National Institutes of Health (NIH; U42 RR014821 and P40 RR16939). We thank James Sparks for technical assistance and Howard Wilson for assistance with graphics.

COMPETING INTERESTS STATEMENT

The authors declare no competing interests.

REFERENCES

1. Lo, C.W., M. Coulling, and C. Kirby. 1987. Tracking of mouse cell lineage using microinjected DNA sequences: analyses using genomic Southern blotting and tissue-section in situ hybridizations. Differentiation 35:37-44.

2. Lois, C., E.J. Hong, S. Pease, E.J. Brown, and D. Baltimore. 2002. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295:868-872.

3. Hofmann, A., B. Kessler, S. Ewerling, M. Weppert, B. Vogg, H. Ludwig, M. Stojkovic, M. Boelhauve, et al. 2003. Efficient transgenesis in farm animals by lentiviral vectors. EMBO Rep. 4:1054-1060.

4. Whitelaw, C.B., P.A. Radcliffe, W.A. Ritchie, A. Carlisle, F.M. Ellard, R.N. Pena, J. Rowe, A.J. Clark, et al. 2004. Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector. FEBS Lett. 571:233-236.

5. Pfeifer, A., M. Ikawa, Y. Dayn, and I.M. Verma. 2002. Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc. Natl. Acad. Sci. USA 99:2140-2145.

6. Hofmann, A., V. Zakhartchenko, M. Weppert, H. Sebald, H. Wenigerkind, G. Brem, E. Wolf, and A. Pfeifer. 2004. Generation of transgenic cattle by lentiviral gene transfer into oocytes. Biol. Reprod. 71:405-409.

7. van den Brandt, J., D. Wang, S.H. Kwon, M. Heinkelein, and H.M. Reichardt. 2004. Lentivirally generated eGFP-transgenic rats allow efficient cell tracking in vivo. Genesis 39:94-99.

8. Clark, A.J., P. Bissinger, D.W. Bullock, S. Damak, R. Wallace, C.B. Whitelaw, and F. Yull. 1994. Chromosomal position effects and the modulation of transgene expression. Reprod. Fertil. Dev. 6:589-598.

9. Rosenthal, A. 1992. PCR amplification techniques for chromosome walking. Trends Biotechnol. 10:44-48.

10. Rosenthal, A. and D.S. Jones. 1990. Genomic walking and sequencing by oligocassette mediated polymerase chain reaction. Nucleic Acids Res. 18:3095-3096.

11. Shyamala, V. and G.F. Ames. 1989. Genome walking by single-specific-primer polymerase chain reaction: SSP-PCR. Gene 84:1-8.

12. Parker, J.D., P.S. Rabinovitch, and G.C. Burmer. 1991. Targeted gene walking polymerase chain reaction. Nucleic Acids Res. 19:3055-3060.

13. Yuanxin, Y., A. Chengcai, L. Li, G. Jiayu, T. Guihong, and C. Zhangliang. 2003. Tlinker-specific ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends. Nucleic Acids Res. 31:e68.

14. Lai, L., K.W. Park, H.T. Cheong, B. Kuhholzer, M. Samuel, A. Bonk, G.S. Im, A. Rieke, et al. 2002. Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells. Mol. Reprod. Dev. 62:300-306.

15. Altschul, S.F., W. Gish, W. Miller, E.W. Myers, and D.J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.

16. Kisseberth, W.C., N.T. Brettingen, J.K. Lohse, and E.P. Sandgren. 1999. Ubiquitous expression of marker transgenes in mice and rats. Dev. Biol. 214:128-138.

17. Kurtz, D.M., P. Rinaldo, W.J. Rhead, L. Tian, D.S. Millington, J. Vockley, D.A. Hamm, A.E. Brix, et al. 1998. Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation. Proc. Natl. Acad. Sci. USA 95:15592-15597.

18. Taurog, J.D., S.D. Maika, N. Satumtira, M.L. Dorris, I.L. McLean, H. Yanagisawa, A. Sayad, A.J. Stagg, et al. 1999. Inflammatory disease in HLA-B27 transgenic rats. Immunol. Rev. 169:209-223.

Received 6 July 2006; accepted 22 August 2006.

Address correspondence to Beth A. Bauer or Yuksel Agca, University of Missouri-Columbia, Department of Veterinary Pathobiology, 1600 E. Rollins St., Columbia, MO 65211, USA. e-mail: bauerbe@missouri.edu; e-mail: agcay@missouri.edu.

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Painkillers May Threaten Power Of Vaccines

Posted by tumicrobiology on November 29, 2006

With flu-shot season in full swing and widespread anticipation of the HPV vaccine to prevent cervical cancer, a new University of Rochester study suggests that using common painkillers around the time of vaccination might not be a good idea
Researchers showed that certain nonsteroidal anti-inflammatory drugs (NSAIDs), also known as cyclooxygenase inhibitors, react with the immune system in such a way that might reduce the effectiveness of vaccines.

The research has widespread implications: study authors report that an estimated 50 to 70 percent of Americans use NSAIDs for relief from pain and inflammation, even though NSAIDs blunt the body’s natural response to infection and may prolong it.

“For years we have known that elderly people are poor responders to the influenza vaccine and vaccines in general,” said principal investigator Richard P. Phipps, Ph.D., a professor of Environmental Medicine, and of Microbiology and Immunology, Oncology and Pediatrics. “And we also know that elderly people tend to be heavy users of inhibitors of cyclooxygenase such as Advil, aspirin, or Celebrex. This study could help explain the immune response problem.”

The study is available online in the Dec. 1, 2006, Journal of Immunology, and was funded in part by the National Institutes of Health.

When a person is vaccinated, the goal is to produce as many antibodies as possible to effectively neutralize the infection. To do this, white blood cells called B-lymphocytes, or B cells, spring into action to produce those antibodies. B cells also serve as the immune system’s memory for future protection against the illness.

But Phipps and colleagues discovered that human B cells also highly express the cyclooxygenase-2 (cox-2) enzyme, which is not intrinsically bad unless it is overproduced, causing pain and fever. So, when a person takes a drug to block the cox-2 enzyme — and thereby reduce pain and fever — the drug also reduces the ability of B cells to make antibodies.

“The next step is to figure out the worst time to take drugs that inhibit cox-2 in the context of getting vaccinated. Is it the day before, the day of, or the day after” The timing is likely to be very important,” Phipps said. “But meanwhile, we believe that when you reach for the medicine cabinet to reduce pain at the injection site, that is probably the wrong thing to do.”

The findings are based on laboratory studies of blood samples from people who participated in early clinical trials for the HPV vaccine, and on studies of mice.

For the animal portion of the study, researchers vaccinated normal mice and mice engineered to be cox-2 deficient with a component form of the HPV vaccine. They analyzed the amount of antibodies the animals produced, focusing on the critical virus-neutralizing antibodies. The cox-2 deficient mice made 50 to 70 percent less of these key antibodies.

The same experiment was done on preserved blood samples from people who had been vaccinated against HPV-16, the strain linked to cervical cancer. Scientists reactivated the B cells in the blood samples and watched them churn out antibodies, as expected. But when researchers treated the B cells with a cox-2 inhibiting drug, the cells significantly diminished their production of antibodies — showing that cox-2 is essential for an optimal immune response against HPV 16.

This study is not questioning the effectiveness of the newly marketed HPV vaccine, the Rochester scientists said. They pointed out that in many clinical trials involving thousands of women, the vaccine offered complete protection against the development of cervical cancer. And presumably some of these women were taking NSAIDs at the time.

“There’s no doubt the HPV vaccine showed 100 percent efficacy. Still, our data does suggest that it might be wise to limit the use of NSAIDs when you receive any vaccine,” said co-author Robert Rose, Ph.D., associate professor of Medicine and Microbiology and Immunology at the University of Rochester, and one of the virologists whose work led to the development of the new cancer vaccine.

Scientists do not completely understand the mechanism by which cox-2 influences the immune response in humans. They do believe the response may depend upon the dose and frequency of NSAID use.

The negative effects of blocking cox-2 could be more pronounced in people with compromised immune systems, such as AIDS or cancer patients, the study noted. Moreover, if a vaccine is in short supply and needs to be given in lower-than-optimal doses, taking an NSAID could hamper the immune response even more.

In addition to Phipps and Rose, graduate student Elizabeth Ryan was a co-author on the study, with assistance from students Matt Bernard and Christine Malboef.

Source: University of Rochester Medical Center

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Causes Of Global Death And Disease In The Next 25 Years

Posted by tumicrobiology on November 29, 2006

In 1993, the World Bank sponsored the 1990 Global Burden of Disease study carried out by researchers at Harvard University and the World Health Organization (WHO). This study provided the first comprehensive global estimates of death and illness by age, sex, and region. It also provided projections of the global burden of disease and mortality up to 2020. The study and its projections have been crucial in national and international health policy planning. Colin Mathers and Dejan Locar (from the World Health Organization, Geneva) have now updated the projections based on 2002 data on mortality and burden of disease and published their results in the international open-access journal PLoS Medicine.

As for the earlier report, the researchers used projections of socio-economic development to model future patterns of mortality and illness for three different scenarios: a baseline scenario, a pessimistic scenario that assumes a slower rate of socio-economic development, and an optimistic scenario that assumes a faster rate of growth.

They predict that between 2002 and 2030 under all three scenarios life expectancy will increase around the world, fewer children under the age of 5 years will die, and the proportion of people dying from non-communicable diseases such as heart disease and cancer will increase. Although deaths from infectious diseases will decrease overall, HIV/AIDS deaths will continue to increase. Despite this increase, 50% more people are predicted to die of tobacco-related disease than of HIV/AIDS in 2015. By 2030, the three leading causes of illness will be HIV/AIDS, depression, and ischemic heart disease in the baseline and pessimistic scenarios. In the optimistic scenario, road-traffic accidents (which increase with socioeconomic development) will replace heart disease as the number 3 killer.

In an accompanying editorial, the PLoS Medicine editors ask whether they are publishing “the right stuff”, i.e. research and commentary whose goal it is to reduce mortality and suffering from the most relevant conditions–and whether research funding and health expenditure are consistent with these results.

Citation: Mathers CD, Loncar D (2006) Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med 3(11): e442. (http://dx.doi.org/10.1371/journal.pmed.0030442)

Source: Public Library of Science

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Mass Extinction 250 Million Years Ago Sparked Dramatic Shift To Complex Marine Ecosystems

Posted by tumicrobiology on November 29, 2006

The earth experienced its biggest mass extinction about 250 million years ago, an event that wiped out an estimated 95% of marine species and 70% of land species. New research shows that this mass extinction did more than eliminate species: it fundamentally changed the basic ecology of the world’s oceans.

Ecologically simple marine communities were largely displaced by complex communities. Furthermore, this apparently abrupt shift set a new pattern that has continued ever since. It reflects the current dominance of higher-metabolism, mobile organisms (such as snails, clams and crabs) that actually go out and find their own food and the decreased diversity of older groups of low-metabolism, stationary organisms (such as lamp shells and sea lilies) that filter nutrients from the water.

So says research published in Science on November 24, 2006. An accompanying article suggests that this striking change escaped detection until now because previous research relied on single numbers–such as the number of species alive at one particular time or the distribution of species in a local community–to track the diversity of marine life. In the new research, however, scientists examined the relative abundance of marine life forms in communities over the past 540 million years.

One reason they were able to do this is because they tapped the new Paleobiology Database (http://www.pbdb.org), a huge repository of fossil occurrence data. The result is the first broad objective measurement of changes in the complexity of marine ecology over the Phanerozoic.

“We were able to combine a huge data set with new quantitative analyses,” says Peter J. Wagner, Associate Curator of Fossil Invertebrates at The Field Museum and lead author of the study. “We think these are the first analyses of this type at this large scale. They show that the end-Permian mass extinction permanently altered not just taxonomic diversity but also the prevailing marine ecosystem structure.”

Specifically, the data and analyses concern models of relative abundance found in fossil communities throughout the Phanerozoic. The ecological implications are striking. Simple marine ecosystems suggest that bottom-dwelling organisms partitioned their resources similarly. Complex marine ecosystems suggest that interactions among different species, as well as a greater variety of ways of life, affected abundance distributions. Prior to the end-Permian mass extinction, both types of marine ecosystems (complex and simple) were equally common. After the mass extinction, however, the complex communities outnumbered the simple communities nearly 3:1.

The other authors are Scott Lidgard, Associate Curator of Fossil Invertebrates at The Field Museum, and Matthew A. Kosnik, from the School of Marine and Tropical Biology at the James Cook University in Townsville, Queensland, Australia.

“Tracing how marine communities became more complex over hundreds of millions of years is important because it shows us that there was not an inexorable trend towards modern ecosystems,” Wagner said. “If not for this one enormous extinction event at the end of the Permian, then marine ecosystems today might still be like they were 250 million years ago.”

These results also might provide a wake-up call, Wagner added: “Studies by modern marine ecologists suggest that humans are reducing certain marine ecosystems to something reminiscent of 550 million years ago, prior to the explosion of animal diversity. The asteroid that wiped out the dinosaurs couldn’t manage that.”

Lidgard added, “When Pete walked into my office with his preliminary results, I simply couldn’t believe them. Paleontologists had long recognized that ecosystems had become more complex, from the origin of single-celled bacteria to the present day. But we had little idea of just how profoundly this one mass extinction–but not the others like it–changed the marine world.”

Source: Field Museum

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Evolution Of Typhoid Bacteria: Researchers Warn Of Increased Spread Of Resistant Strains

Posted by tumicrobiology on November 29, 2006

In a study published in the latest issue of Science (24 November, 2006), an international consortium from the Max-Planck Society, Wellcome Trust Institutes in Britain and Vietnam, and the Institut Pasteur in France have elucidated the evolutionary history of Salmonella Typhi. Typhi is the cause of typhoid fever, a disease that sickens 21 million people and kills 200,000 worldwide every year. The results indicate that asymptomatic carriers played an essential role in the evolution and global transmission of Typhi. The rediscovered importance of the carrier state predicts that treatment of acute disease, including vaccination, will not suffice to eradicate this malady. The results also illuminate patterns leading to antibiotic resistance after the indiscriminate use of antibiotics. Fluoroquinolone treatment in southern Asia over two decades has resulted in the emergence of multiple, independent nalidixic acid-resistant mutants, of which one group, H58, has multiplied dramatically and spread globally. The prevalence of these bacteria hampers medical cure of clinical disease via antibiotics.

Typhoid fever remains a major health problem in the developing world and continues to cause disease in Europe and on the american continent. The evolutionary history and population structure of Typhi were poorly understood, partly because these bacteria show little genetic diversity. Now a team led by Mark Achtman and Philippe Roumagnac from the Max Planck Institute for Infection Biology, Berlin, has applied population genetic experience from prior work with Yersinia pestis, Escherichia coli, Helicobacter pylori and Neisseria meningitidis to provide novel insights into the evolution of this pathogen. The team combined its resources to assemble for the first time a globally representative collection of 105 strains of Typhi and investigated the sequence diversity within 90,000 base pairs per strain. Eighty-eight informative sequence differences were detected, showing that the population structure has evolved over the last 10,000 to 43,000 years. Amazingly, the ancestral strain continues to exist today, as do many of its direct descendents, indicating a neutral population structure, whereas normally selective forces lead to extinction of intermediate genotypes. Furthermore, these bacteria are distributed globally, demonstrating that Typhi has spread inter-continentally on multiple occasions.

The authors propose that the unusual population structure of Typhi reflects long-term carriage by asymptomatic carriers, who reached public notoriety at the beginning of the 20th century with “Mr. N the milker” in England and Typhoid Mary (Mary Mallon) in the U.S.A. These individuals infected 100s of people over the decades while they worked in the food production industry. Healthy carriers may have allowed Typhi to survive in hunter-gatherer populations prior to the Neolithic expansion of city states and facilitated its intercontinental spread. Healthy carriers are also consistent with the observation that individual genotypes of Typhi persist for many decades within each country.

Increasing resistance to antibiotics in recent decades has hampered efforts of clinicians to cure typhoid fever. The indiscriminate use of fluoroquinolones, which is a cost-effective, standard treatment for typhoid fever, has been accompanied by a frightening increase in the numbers of resistant Typhi. Investigations of a large strain collection from southern Asia revealed that many different genotypes independently acquired resistance to nalidixic acid, a quinolone. One of these genotypes, H58, has become predominant throughout southern Asia and has even spread to Africa. In Vietnam, up to 95% of Typhi are now resistant to nalidixic acid and many other antibiotics. Although these cases can still be treated with newer antibiotics, those antibiotics are much more expensive than standard fluoroquinolones, which raises the cost of medical treatment. Furthermore, it is likely that Typhi will develop resistance to these antibiotics as well.

The combination of these investigations raises problems for public health measures. Indiscriminate antibiotic usage results in real-time evolution of bacteria that resist treatment. Furthermore, the healthy carrier state provides a safe reservoir for these bacteria which allows them to evade short-term antibiotic treatment and vaccination, indicating that typhoid fever will remain a major health problem for the foreseeable future.

The research was carried out collaboratively by the Max Planck Institute for Infection Biology in Berlin, the Wellcome Trust Sanger Institute, Hinxton Hall, the Institut Pasteur, Paris, and the Oxford University Clinical Research Unit, Ho Chi Minh City, with assistance from hospitals in Ho Chi Minh City and Hanoi and the International Vaccine Institute in Seoul. Financial support was by the Wellcome Trust, UK.

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Researchers find smallest cellular genome

Posted by tumicrobiology on October 29, 2006

Researchers find smallest cellular genome
The smallest collection of genes ever found for a cellular organism comes from tiny symbiotic bacteria that live inside special cells inside a small insect.

The bacteria Carsonella ruddii has the fewest genes of any cell. The bacteria’s newly sequenced genome, the complete set of DNA for the organism, is only one-third the size of the previously reported “smallest” cellular genome.

“It’s the smallest genome — not by a bit but by a long way,” said co-author Nancy A. Moran, UA Regents’ Professor of ecology and evolutionary biology and a member of the National Academy of Sciences. “It’s very surprising. It’s unbelievable, really. We would not have predicted such a small size. It’s believed that more genes are required for a cell to work.”

Carsonella ruddii has only 159,662 base-pairs of DNA, which translates to only 182 protein-coding genes, reports a team of scientists from The University of Arizona in Tucson and from Japan.

The finding provides new insights into bacterial evolution, the scientists write in the Oct. 13 issue of the journal Science.

Atsushi Nakabachi, a postdoctoral research associate in UA’s department of ecology and evolutionary biology and a visiting scientist at RIKEN in Wako, Japan, is the first author on the research report, “The 160-kilobase genome of the bacterial endosymbiont Carsonella.” The research was conducted in senior author Masahira Hattori’s laboratory in Japan and in Moran’s lab at the UA.

Hackberry petiole gall psyllid just after emerging from the gall on a hackberry. The leaf is yellow because it is autumn. The insect is only 3 to 4 millimeters long….

Many insects feed on plant sap, a nutrient-poor diet. To get a balanced diet, some sap-feeders rely on resident bacteria. The bacteria manufacture essential nutrients, particularly amino acids, and share the goodies with their hosts.

In many such associations, the bacteria live within the insect’s cells and cannot survive on their own. Often the insect host cannot survive without its bacteria, known as endosymbionts.

The relationship between some insects and their endosymbionts is so close and so ancient that the insects house their resident bacteria in special cells called bacteriocytes within specialized structures called bacteriomes.

Studying the genomes of such endosymbionts can provide clues to how microorganisms’ metabolic capabilities contribute to both their hosts and to biological communities.

An organism’s genome, its complete complement of DNA, provides the operating instructions for everything the organism needs to do to survive and reproduce.

Endosymbiotic bacteria live in an extremely sheltered world and have a pared-down lifestyle, so they need a simpler set of instructions. Many of the metabolic pathways that free-living bacteria maintain have been lost after so many generations of living within insects.

Nakabachi and Hattori were interested in sequencing the genome of the bacteria Carsonella.

Moran had done some previous work on the Carsonella genome and found its DNA composition and evolution to be unusual. She suggested the team pursue the Carsonella that lived inside an Arizona psyllid insect called Pachypsylla venusta. The insect has only one species of endosymbiotic bacteria, which would simplify the genomic analysis.

The researchers collected Pachypsylla venusta psyllids from hackberry trees (Celtis reticulata) on the UA campus and around Tucson. The team extracted the Carsonella DNA and sequenced it.

Even though endosymbionts need fewer operating instructions to survive, the bacteria’s itsy bitsy genome was a surprise.

“It lost genes that are considered absolutely necessary. Trying to explain it will probably help reveal how cells can work,” said Moran, who is a member of UA’s BIO5 Institute.

The scientists speculate that in the bacteria’s evolutionary past, some of its genes were transferred into the insect’s genome, allowing the insect to make some of the metabolites the bacteria needed. Once the insect shouldered those responsibilities and provided the bacteria with those metabolites, the bacteria lost those genes.

Animal and plant cells have specialized structures inside them called organelles that are derived from symbiotic bacteria that became incorporated into the cell over the course of evolution.

Carsonella’s stripped-down genome may indicate that it is on its way to becoming an organelle, the researchers write in their article.

Source: University of Arizona

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Biomineralization of Gold: Biofilms on Bacterioform Gold

Posted by tumicrobiology on July 16, 2006

Frank Reith,1,2* Stephen L. Rogers,1,4 D. C. McPhail,1,2 Daryl Webb3

Bacterial biofilms are associated with secondary gold grains from two sites in Australia. 16S ribosomal DNA clones of the genus Ralstonia that bear 99% similarity to the bacterium Ralstonia metallidurans—shown to precipitate gold from aqueous gold(III) tetrachloride—were present on all DNA-positive gold grains but were not detected in the surrounding soils. These results provide evidence for the bacterial contribution to the authigenic formation of secondary bacterioform gold grains and nuggets.

Source: Science 14 July 2006:
Vol. 313. no. 5784, pp. 233 – 236
DOI: 10.1126/science.1125878

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Researchers Discover How Bacteria Sense Their Environments

Posted by tumicrobiology on June 3, 2006

When humans taste or smell, receptors unique to each nerve cell detect the chemical and send signals to the brain, where many cells process the message to understand what we are smelling or tasting. But a bacterium is just a single cell, and it must use many different receptors to sense and interpret everything around it.

Bacteria can sense in their environments changes in molecular concentrations as small as 0.1 percent, the equivalent of one drop diluted in a pool of a 1,000 drops. How do they do it?

New Cornell research, highlighted on the cover of the May issue of Nature Structural and Molecular Biology, reveals that receptors assemble into a kind of cooperative lattice on a bacterium’s surface to amplify infinitesimal changes in the environment and kick off processes that lead to specific responses within the cell.

“Bacteria sense a lot of different things. But assume it’s a sugar molecule that a bacterium needs as a nutrient — even a 0.1 percent change in concentration can be detected, and this sensitivity is maintained over five orders of magnitude in nutrient concentration,” said Brian Crane, assistant professor of chemistry and chemical biology and corresponding author of the paper. “Biologically, I know of no other system that is so sensitive over such a large range.”

Crane believes the kind of cooperative lattice found in bacterial receptors may in fact point to a general mechanism for cellular signaling and serve as inspiration for developing molecular devices. Such devices could be used to sense a wide range of chemicals, light, ionic strength (salt), pH and heavy metals with great sensitivity, gain and dynamic range. Scientists are interested in developing synthetic systems with such sensing properties as well as engineering bacteria that respond to stimuli such as pollutants or explosives.

Using a combination of X-ray crystallography to determine the structure of receptors and enzymes and a novel spectroscopic technique for measuring interactions between them, Crane’s group was able to develop a model for how the complex of receptors is organized. Jack Freed, Cornell professor of chemistry and chemical biology and director of the National Biomedical Center for Advanced ESR Studies at Cornell, developed the spectroscopic technique, called pulsed electron spin resonance dipolar spectroscopy.

The researchers suggest that when one receptor detects, for example, a sugar in its environment, communication of some sort triggers an array of linked receptors to rearrange itself, much like when water freezes, all the water molecules assort themselves into a new structure. Through this reorganization, the bacterium’s receptor array amplifies the signal that a specific molecule has been sensed outside the cell. This structural shift then activates kinases, or enzymes, within the cell, starting a chain reaction that leads to a response, such as changing how the flagella (or tails) spin. This allows the bacterium to move toward or away from what it has sensed.

Such a mechanism of amplification allows signals from the receptors to travel hundreds of angstroms, a distance used in atomic physics that is a virtual marathon in the world of intracellular communication. Ten angstroms equal a nanometer, which is one-billionth of a meter.
Source: Cornell University

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New Potential Drug Target In Tuberculosis

Posted by tumicrobiology on May 30, 2006

Tuberculosis remains one of the deadliest threats to public health. Every year two million people die of the disease, which is caused by the microorganism Mycobacterium tuberculosis. Roughly one third of the world’s population is infected and more and more bacterial strains have developed resistance to drugs. Researchers from the Hamburg Outstation of the European Molecular Biology Laboratory (EMBL) and the Max Planck Institute for Infection Biology (MPIIB) in Berlin have now obtained a structural image of a protein that the bacterium needs for survival in human cells. This image reveals features of the molecule that could be targeted by new antibiotic drugs. The results appear in this week’s online issue of the Proceedings of the National Academy of Sciences (PNAS).
Tuberculosis is dangerous because it hides and persists in the immune cells of our bodies. “It can only persist there because of the activity of key molecules,” says Matthias Wilmanns, Head of EMBL Hamburg. “We are investigating the functions of tuberculosis proteins and determining their atomic structures, in hopes of finding weak points and new inhibitors.”

A protein called LipB is essential for the organism because it activates cellular machines that drive the bacterium’s metabolism. Stefan Kaufmann’s department at the MPIIB has specialised in the biology of M. tuberculosis infection and its ability to survive in immune cells. They discovered that LipB is highly active in acutely infected cells, particularly in patients infected by multidrug-resistant forms of M. tuberculosis.

“In these cells we see a 70-fold increase in the production of LipB when compared to other cells,” says Stefan Kaufmann, Director at the MPIIB. “This strongly indicates an involvement in pathogenesis and makes it a particularly interesting target where traditional drugs have lost their efficacy.”

A structural picture of the protein – a kind of technical diagram of its building plan – has yielded important clues about its activity. Qingjun Ma from Wilmanns’ group purified LipB and obtained crystals of the molecule. Using the high-energy synchrotron radiation beamlines at EMBL Hamburg, on the campus of the German Electron Synchrotron Radiation Facility (DESY), he created an atom-by-atom map of the protein’s structure. A high-resolution picture of the active site of LipB bound to a lipid inhibitor helped to determine the function of the enzyme. In collaboration with EMBL’s Proteomics Core Facility in Heidelberg and researchers from the University of Illinois (USA), the Hamburg group discovered how LipB attaches specific fatty acids onto other proteins.

“LipB is a very promising drug target,” Wilmanns says, “because it belongs to a vital pathway. Unlike other organisms M. tuberculosis has no backup mechanism that could take over LipB’s role. This means that an inhibitor blocking its active site would shut down key processes the bacterium needs to survive and replicate. This would be a very effective strategy for a drug.”

The scientists will now search for compounds that can do so. At the same time, they are continuing to look for other proteins as drug targets. Wilmanns and his colleagues from various other institutes are now focusing on structures of molecules that help M. tuberculosis to persist in its dormant state and could become drug targets.

Over the past three years, EMBL has coordinated an “M. tuberculosis structural proteomics” consortium, supported by the German Ministry of Education and Research (BMBF), and produced high resolution images of more than 30 proteins. “Structure-based drug discovery has been a big success in the battle against many other diseases. We are now applying these tools to tuberculosis, one of the most devastating infectious diseases of mankind,” Wilmanns concludes.

Source: European Molecular Biology Laboratory

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New Treatment For Food Poisoning

Posted by tumicrobiology on May 12, 2006

A team of researchers working at the University of Bristol has found a potential new treatment for listeriosis, a deadly form of food poisoning. Their work is reported in Nature Medicine.

The group, led by Professor Jose Vazquez-Boland, has shown that one particular antibiotic — fosfomycin — can treat Listeria in the body, despite it being ineffective in laboratory conditions.

Because it was not effective in the laboratory, this drug has never been considered for the treatment of listeriosis, in spite of it reaching the infection sites more effectively than other antibiotics.

Professor Vazquez-Boland said: “Our results illustrate that antibiotic resistance in the laboratory does not always mean that the drug will not work in the infected patient. This work brings some optimism to the highly worrying problem of the increasing resistance to antibiotics.”

The Listeria bacteria causes the food-borne disease, listeriosis. It often triggers a brain infection and kills up to 30% of those affected.

To test whether antibiotics are effective, bacteria are taken from patients and tested in the laboratory. These tests measure whether antibiotics can halt the growth of Listeria in laboratory conditions. Such tests are usually a measure of how effective the drug will be in the body.

When tested this way, Listeria had been shown to be resistant to the antibiotic, fosfomycin. As a consequence, this drug has never been considered for the treatment of listeriosis.

Dr Mariela Scortti, lead author on the paper, added: “Our findings warn about the need to revise laboratory methods currently in use to determine the susceptibility or resistance of bacteria to such drugs, so that the tests reflect better what actually happens in the body.”
Source: University of Bristol

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How Bad Is Malaria Anemia? It May Depend On Your Genes

Posted by tumicrobiology on May 12, 2006

Cell and animal studies conducted jointly by scientists at Johns Hopkins, Yale and other institutions have uncovered at least one important contributor to the severe anemia that kills almost half of the 2 million people worldwide who die each year of malaria. The culprit is a protein cells make in response to inflammation called MIF, which appears to suppress red blood cell production in people whose red blood cells already are infected by malaria parasites.
The parasite that causes malaria – known as plasmodium – is carried through blood by mosquito bites, and in parts of the world where mosquitoes thrive, millions are infected, most of them by early childhood. Once in the bloodstream, plasmodium invades liver and red blood cells and makes more copies of itself. Eventually, as red cells break and free plasmodium to infect other cells, and as the body’s immune system works to kill infected cells, the total number of red blood cells drops, causing anemia.

But not everyone infected with malaria develops severe, lethal anemia. And there are cases where patients who have been cured of infection still develop severe anemia.

This report provides the rationale for a simple, genetic test to sort out which children may be most susceptible to this lethal complication of malarial infection and to identify treatments targeted to them especially, the study’s authors suggest.

“This is important because in places where malaria is endemic, drug treatment resources are scarce,” says the study’s primary author, Michael A. McDevitt, M.D., Ph.D., an assistant professor of medicine and hematology at the Johns Hopkins School of Medicine.

“There are many difficulties with blood transfusion safety and access in Africa, especially in rural areas where most of the malaria-related deaths occur,” says McDevitt. “That led us to search for a better way to identify those most at risk and a better way to treat the disease,” he says.

The study, published online April 24 in the Journal of Experimental Medicine, adds to a growing amount of evidence that an individual’s unique genetic makeup can affect the prevalence and outcome of diseases, in this case the individual risk of malarial anemia.

A number of human proteins, including MIF (which stands for migration inhibitory factor), were long suspected to cause malarial anemia because they are known to reduce red blood cell counts as part of the body’s normal response to such inflammatory conditions as rheumatoid arthritis or some cancers.

Using immature blood cell precursors grown in a dish, the research team showed that adding MIF to the cells decreases both the final number and maturity of red blood cells. The researchers believe this effect can lead to anemia.

When infected with plasmodium, mice genetically engineered to lack MIF experience less severe anemia and are more likely to survive. Without MIF around to prevent blood cells from maturing, the mice appear better able to maintain their oxygen carrying capacity and don’t lose as much hemoglobin, the protein found in red blood cells responsible for binding to oxygen molecules.

“Demonstrating that MIF clearly contributes to severe anemia suggests new ideas for therapies that can block MIF in malaria patients,” says the study’s senior author, Richard Bucala, M.D., Ph.D., a professor of medicine at Yale University School of Medicine.

The research team also found different versions of “promoter” DNA sequences next to the MIF gene that control how much MIF protein a cell makes in response to infection. One version of the MIF promoter leads to less MIF protein made, while cells containing another version of the MIF promoter make much more MIF protein. Differences in the MIF promoter also have been linked to the severity of other inflammatory diseases.

The researchers continue to collaborate in an effort to develop drugs that might block MIF and treat severe anemia in malaria patients.

The researchers were funded by the National Institutes of Health, the Office of Research on Minority Health, a Howard University General Clinical Research Center grant, and the Department of Medicine at Johns Hopkins.

Authors on the paper are McDevitt, Ganapathy Shanmugasundaram and Jeffrey Keefer of Johns Hopkins; Jianlin Xie and Christine Metz of the Feinstein Institute for Medical Research; Jason Griffith, Aihua Liu, Courtney McDonald, Lin Leng and Bucala of Yale; Philip Thuma of the Macha Malaria Research Institute in Choma, Zambia, a field unit of the Johns Hopkins Bloomberg School of Public Health; Victor Gordeuk of Howard University; Robert Mitchell of the James Graham Brown Cancer Center; and John David of Harvard School of Public Health.

Source: Johns Hopkins Medical Institutions

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Mars Meteorite Similar To Bacteria-etched Earth Rocks

Posted by tumicrobiology on May 12, 2006

A new study of a meteorite that originated from Mars has revealed a series of microscopic tunnels that are similar in size, shape and distribution to tracks left on Earth rocks by feeding bacteria.

And though researchers were unable to extract DNA from the Martian rocks, the finding nonetheless adds intrigue to the search for life beyond Earth.

Results of the study were published in the latest edition of the journal Astrobiology.

Martin Fisk, a professor of marine geology in the College of Oceanic and Atmospheric Sciences at Oregon State University and lead author of the study, said the discovery of the tiny burrows do not confirm that there is life on Mars, nor does the lack of DNA from the meteorite discount the possibility.

“Virtually all of the tunnel marks on Earth rocks that we have examined were the result of bacterial invasion,” Fisk said. “In every instance, we’ve been able to extract DNA from these Earth rocks, but we have not yet been able to do that with the Martian samples.

“There are two possible explanations,” he added. “One is that there is an abiotic way to create those tunnels in rock on Earth, and we just haven’t found it yet. The second possibility is that the tunnels on Martian rocks are indeed biological in nature, but the conditions are such on Mars that the DNA was not preserved.”

More than 30 meteorites that originated on Mars have been identified. These rocks from Mars have a unique chemical signature based on the gases trapped within. These rocks were “blasted off” the planet when Mars was struck by asteroids or comets and eventually these Martian meteorites crossed Earth’s orbit and plummeted to the ground.

One of these is Nakhla, which landed in Egypt in 1911, and provided the source material for Fisk’s study. Scientists have dated the igneous rock fragment from Nakhla – which weighs about 20 pounds – at 1.3 billion years in age. They believe that the rock was exposed to water about 600 million years ago, based on the age of clay found inside the rocks.

“It is commonly believed that water is a necessary ingredient for life,” Fisk said, “so if bacteria laid down the tunnels in the rock when the rock was wet, they may have died 600 million years ago. That may explain why we can’t find DNA – it is an organic compound that can break down.”

Other authors on the paper include Olivia Mason, an OSU graduate student; Radu Popa, of Portland State University; Michael Storrie-Lombardi, of the Kinohi Institute in Pasadena, Calif.; and Edward Vicenci, from the Smithsonian Institution.

Fisk and his colleagues have spent much of the past 15 years studying microbes that can break down igneous rock and live in the obsidian-like volcanic glass. They first identified the bacteria through their signature tunnels then were able to extract DNA from the rock samples – which have been found in such diverse environments on Earth as below the ocean floor, in deserts and on dry mountaintops.

They even found bacteria 4,000 feet below the surface in Hawaii that they reached by drilling through solid rock.

In all of these Earth rock samples that contain tunnels, the biological activity began at a fracture in the rock or the edge of a mineral where the water was present. Igneous rocks are initially sterile because they erupt at temperatures exceeding 1,000 degrees C. – and life cannot establish itself until the rocks cool. Bacteria may be introduced into the rock via dust or water, Fisk pointed out.

“Several types of bacteria are capable of using the chemical energy of rocks as a food source,” he said. “One group of bacteria in particular is capable of getting all of its energy from chemicals alone, and one of the elements they use is iron – which typically comprises 5 to 10 percent of volcanic rock.”

Another group of OSU researchers, led by microbiologist Stephen Giovannoni, has collected rocks from the deep ocean and begun developing cultures to see if they can replicate the rock-eating bacteria. Similar environments usually produce similar strains of bacteria, Fisk said, with variable factors including temperature, pH levels, salt levels, and the presence of oxygen.

The igneous rocks from Mars are similar to many of those found on Earth, and virtually identical to those found in a handful of environments, including a volcanic field found in Canada.

One question the OSU researchers hope to answer is whether the bacteria begin devouring the rock as soon as they are introduced. Such a discovery would help them estimate when water – and possibly life – may have been introduced on Mars.
Source: Oregon State University

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‘Coffee Ring’ Formations Found In Drying Drops Of DNA

Posted by tumicrobiology on May 12, 2006

Coffee drinkers are familiar with the ring-shaped stains that result from spilled drops that have dried, in which the brown stain is not evenly distributed, but instead concentrated at the edge. Now, a team led by Gerard Wong, a professor of materials science and engineering, physics, and bioengineering at the University of Illinois at Urbana-Champaign has found the same “coffee-ring” formation in drying drops of DNA.
To gain insights into the physics behind the ring phenomenon, Wong’s team experimentally studied the dynamics of drying DNA droplets on glass surfaces. They report their findings in a paper accepted for publication in the journal Physical Review Letters, and posted on its Web site.

“As the droplet evaporated, DNA chains were transported outward by water flow to the drop’s perimeter,” Wong said. “At the droplet edge, the DNA became increasingly concentrated and formed a liquid crystal with concentric chain orientations. (Liquid crystals are materials that flow like a liquid, but can align in a preferred direction like a crystalline solid.) During the final stages of drying, stresses propagated from the rim inward through the liquid crystal, creating cracks that formed a periodic zigzag pattern.”

To examine the structure and behavior of the DNA liquid crystal, the researchers used a relatively new imaging technique developed at Kent State University. Called fluorescence confocal polarizing microscopy, the technique imaged the DNA in the drying droplet in three dimensions.

“The DNA alignment parallel to the droplet’s edge was counterintuitive,” Wong said. “We had expected the DNA to extend along the direction of flow, which was mainly in the radial direction. But, instead of resembling the spokes of a bicycle wheel, the transported DNA resembled the rim of a bicycle wheel.”

Since nearly all the DNA is concentrated in a narrow ring with almost no DNA in the rest of the stain, these effects should be accounted for in the design of arrays in which DNA droplets are sequentially deposited onto a glass surface for hybridization studies, the researchers report.

“Without optimization of the wetting conditions, it is possible to miss all the DNA in the ring stain of a dried droplet, resulting in false negatives,” Wong said. “We need to think of strategies to minimize this effect.”

The co-authors of the paper are postdoctoral research associate Ivan Smalyukh, graduate students Olena Zribi and John Butler, and professor Oleg D. Lavrentovich, director of the Liquid Crystal Institute at Kent State.
Source: University of Bristol

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Follow The Nitrogen To Extraterrestrial Life; Narrow Search For Water May Miss Important Clues, Say USC Geobiologists

Posted by tumicrobiology on May 6, 2006

The great search for extraterrestrial life has focused on water at the expense of a crucial element, say geobiologists at the University of Southern

Writing in the Perspectives section of the May 5 issue of Science, four USC researchers propose searching for organic nitrogen as a direct indicator of the presence of life. Nitrogen is essential to the chemistry of living organisms.

Even if NASA were to find water on Mars, its presence only would indicate the possibility of life, said Kenneth Nealson, Wrigley Professor of earth sciences in the USC College of Letters, Arts and Sciences.

“It’s hard to imagine life without water, but it’s easy to imagine water without life,” Nealson said.

The discovery of nitrogen on the Red Planet would be a different story.

“If you found nitrogen in abundance on Mars, you would get extremely excited because it shouldn’t be there,” Nealson said.

The reason has to do with the difference between nitrogen and carbon, the other indispensable organic element.

Unlike carbon, nitrogen is not a major component of rocks and minerals. This means that any substantial organic nitrogen deposits found in the soil of Mars, or of another planet, likely would have resulted from biological activity.

Dimming the hopes of life-on-Mars believers is the makeup of the planet’s atmosphere. The abundant nitrogen in Earth’s atmosphere is constantly replenished through biological activity. Without the ongoing contribution of living systems, the atmosphere slowly would lose its nitrogen.

The extremely low nitrogen content in the Martian atmosphere suggests that biological nitrogen production is close to zero.

However, the authors write, it is possible that life existed on Mars at some hypothetical time when nitrogen filled the atmosphere.

Co-author Douglas Capone, Wrigley Professor of environmental biology in USC College, said NASA should establish a nitrogen detection program alongside its water- seeking effort. He noted that next-generation spacecraft will have advanced sampling capabilities.

“What we’re suggesting here is basically drilling down into geological strata, which they’re going to be doing for water anyway,” Capone said.

“The real smoking gun would be organic nitrogen.”

Said Nealson: “If your goal is to search for life, it would be wise to include nitrogen.”

In their acknowledgments, the authors thanked the students of the Spring 2004 Geobiology & Astrobiology course at USC, with whom Nealson and Capone began their discussion on how to search for life outside earth.

“That’s really what stimulated this [paper],” Nealson said.

The authors also thanked NASA, the Department of Energy and the National Science Foundation for their financial support.

Along with Nealson and Capone, USC graduate student Beverly Flood and former USC Research Professor Radu Popa (now a professor of biology at Portland State University) contributed to the Perspectives paper.
Source: University of Southern California

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The Next Flu Pandemic: When It Happens, Restricting Air Travel Won’t Help

Posted by tumicrobiology on May 6, 2006

Restricting air travel from countries where there is a serious influenza outbreak will do little to hold back the spread of the infection, according to the findings of a study conducted at the UK Health Protection Agency and published in the journal PLoS Medicine.

Sometimes a new type of influeza virus appears that causes an illness that is more serious than is usually the case for flu. This happened, for example, in 1918, when a flu pandemic killed between 20 million and 100 million people. Recently, there have been concerns about the new type of bird (avian) flu. At present the virus responsible does not pass easily from birds to humans, and it does not seem to pass from one human to another. However, the fear is that the virus might change and that human-to-human infection could then be possible. Should all this happen, the changed virus would be a major threat to human health.

With current technology, it would take several months to produce enough vaccine against such a new virus for even a small proportion of the world’s population. By that time, it would probably be too late; the virus would already have spread to most parts of the world.

Health authorities must therefore consider all the methods that might control the spread of the virus. With the increase in international travel that has taken place, the virus could spread more quickly than in previous pandemics. Restrictions on international travel might be considered necessary, particularly travel by air. However, it is important to estimate how useful restrictions on air travel might be in controlling the spread of a flu virus. Travel restrictions are usually unpopular and could themselves be harmful. If they are not effective, resources could be wasted on enforcing them.

Researchers of the Centre for Infections, Health Protection Agency, UK used the techniques of mathematical modelling. In other words, complex calculations were done using information that is already available about how flu viruses spread, particularly information recorded during a worldwide flu outbreak in 1968-1969. Using this information, virtual experiments were carried out by simulating worldwide outbreaks on a computer. The researchers looked at how the virus might spread from one city to another and how travel restrictions might reduce the rate of spread. Their calculations allowed for such factors as the time of the year, the number of air passengers who might travel between the cities, and the fact that some people are more resistant to infection than others.

From the use of their mathematical model, the researchers concluded that restrictions on air travel would achieve very little. This is probably because, compared with some other viruses, the flu virus is transmitted from one person to another very quickly and affects many people. Once a major outbreak was under way, banning flights from affected cities would be effective at significantly delaying worldwide spread only if almost all travel between cities could be stopped almost as soon as an outbreak was detected in each city. It would be more effective to take other measures that would control the spread of the virus locally. These measures could include use of vaccines and antiviral drugs if they were available and effective against the virus.

{Citation: Cooper BS, Pitman RJ, Edmunds WJ, Gay NJ (2006) Delaying the international spread of pandemic influenza. PLoS Med 3(6): e212. (http://dx.doi.org/10.1371/journal.pmed.0030212)}

Source: Public Library of Science

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Stomach Receptor For H. Pylori Discovered

Posted by tumicrobiology on May 6, 2006

Scientists have determined that decay-accelerating factor (DAF), a protein found in epithelial cells in the stomach, acts as a receptor for the bacteria Helicobacter pylori. Blocking this interaction could lead to new drugs that reduce the risk of peptic ulcer disease or gastric cancer. The research appears as the “Paper of the Week” in the May 12 issue of the Journal of Biological Chemistry, an American Society for Biochemistry and Molecular Biology journal.

Helicobacter pylori are spiral shaped bacteria that live in the thick layer of mucus that covers the stomach lining. The bacteria are found everywhere in the world, but are especially prevalent in developing countries, where up to 80% of children and 90% of adults can have laboratory evidence of an H. pylori infection–usually without having any symptoms.

The vast majority of H. pylori in colonized hosts are free-living, but approximately 20% bind to gastric epithelial cells. This binding induces an immune response and alters the morphology and behavior of the epithelial cells due to injection of bacterial proteins into the cells. This interaction can lead to peptic ulcer disease, gastric adenocarcinoma, and non-Hodgkins lymphoma of the stomach.

“Ulcers are a breach in the gastric or duodenal mucosa and gastric adenocarcinoma is one type of cancer of the stomach,” explains senior author Dr. Richard M. Peek, Jr. of the Vanderbilt University School of Medicine. “H. pylori alters acid production and can lead to increased acid outputs which causes peptic ulcer disease. The means through which H. pylori causes gastric cancer are more complex but likely involve alterations in gastric epithelial cell responses that are perturbed within the context of a chronic gastric inflammatory infiltrate, which can persist for decades.”

A membrane-imbedded protein found in the stomach called decay-accelerating factor (DAF) has been shown to function as a receptor for several microbial pathogens. Peek and his colleagues were curious as to whether DAF was also involved in H. pylori adherence. To do this, the researchers measured the number of H. pylori that bound to cells that either expressed or did not express DAF. They found that the bacteria do indeed adhere to cells with DAF. They also discovered that H. pylori induce DAF expression in cultured gastric epithelial cells and that mice lacking DAF develop attenuated stomach inflammation.

“Our results indicate that H. pylori can co-opt a host protein as a receptor and that it can increase expression of this receptor in gastric epithelial cells,” says Peek. “Further, absence of this receptor abolishes the inflammatory response that H. pylori induces in infected mice, suggesting that this receptor mediates H. pylori-induced injury in the stomach.”

These findings suggest that drugs that interfere with DAF binding could be used to prevent or treat peptic ulcer disease or distal gastric adenocarcinoma. These new drugs will be a welcome alternative to the current treatment for H. pylori infections which typically involves taking 3 to 4 medications over a 10 to 14 day period.
Source: American Society for Biochemistry and Molecular Biology

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New Treatment Against Persistent Ulcer-inducing Bacteria Successful

Posted by tumicrobiology on May 6, 2006

For those who suffer from stomach ulcers, the daily routine of breakfast, lunch and dinner can be painful. A common cause of these ulcers, as well as other gastric malignancies, is a bacterium called Helicobacter pylori. For some, this infection can be persistent and difficult to treat.
Many approaches have been taken in an attempt to clear such infections, but with limited or unsuccessful outcomes. In a recent meta-analysis of therapies published in the April issue of The American Journal of Gastroenterology, Levofloxacin-based triple therapy was found to be better tolerated and more effective than bismuth-based quadruple therapy for patients with persistent H. pylori despite previous treatment attempts. Levofloxacin is commonly prescribed to treat such infections as pneumonia, bronchitis and urinary tract infections.

According to author William D. Chey, “Helicobacter pylori is a highly prevalent chronic infection with a worldwide prevalence of nearly 50% and U.S. prevalence of 20-40%.” This bacterial infection is particularly difficult to treat because of its ability to adapt to the harsh environment in the stomach. The bacterium guards itself in the lining of the stomach, which prevents the body’s natural defenses (Killer T Cells) from attacking it.

Levofloxacin-based triple therapy may offer an effective and safe treatment option for patients with persistent H. pylori infection, according to researchers. With so many people living with this infection, it has become increasingly important to achieve effective methods of treatment. Levofloxacin-based therapy may prove to be this method.

This study is published in The American Journal of Gastroenterology.

William D. Chey, MD is Associate Professor, Department of Internal Medicine and Director, Gastrointestinal Physiology Laboratory at the University of Michigan. Dr. Chey is conducting research and has extensively published in the areas of GERD and Helicobacter pylori. Dr Chey is conducting research and has published extensively in the areas of functional bowel disease, GERD, and Helicobacter pylori.
Source: Blackwell Publishing Ltd.

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New Study Reveals Structure Of E. Coli Multidrug Transporter Protein; Could Aid In Fight Against Drug-resistant Bacteria

Posted by tumicrobiology on May 6, 2006

Scientists at The Scripps Research Institute have determined the x-ray structure of EmrD, a multidrug transporter protein from Escherichia coli (E. coli), a common bacteria known to cause several food-borne illnesses. Proteins like EmrD that expel drugs from cells contribute significantly to the continued rise in multidrug resistant bacteria, and the re-emergence of drug-resistant strains of diseases such as tuberculosis that were once thought to have been eradicated.

This new study could potentially help researchers find new ways to avoid the problem of multidrug resistance and enhance the potency of existing drug compounds.

The study is being published in ScienceXpress, an advance online edition of the journal Science, on May 5.

“The development of antibiotics to treat infectious disease is being seriously undermined by the emergence of drug-resistant bacteria,” says Geoffrey A. Chang, Ph.D., a Scripps Research associate professor and a member of the Skaggs Institute for Chemical Biology, who led the study. “Multidrug resistance develops in part through the expulsion of drugs by integral membrane transporters like EmrD. Determining the structure of this transporter will add significantly to our general understanding of the mechanism of drug transport through the cell membrane and provide the structural basis for how these proteins go about selecting specific drugs to expel.”

Multidrug resistant bacterial infections raise the cost of medical treatment and are far more expensive than treating normal infections. Treating drug-resistant tuberculosis, for example, requires so-called second-line drugs if standard treatment fails. According to the Centers for Disease Control, second-line drugs can cost as much as “$33,000 per patient in industrialized countries compared to $84 for first-line drugs.” In addition, the centers noted, second-line drugs need to be taken for longer periods of time-from 18 to 36 months-and may require substantial patient monitoring, making these treatments difficult if not impossible to “be available in many of the resource-poor nations where drug-resistant tuberculosis is emerging.”

EmrD belongs to the Major Facilitator Superfamily, a group of transporters among the most prevalent in microbial genomes. These transporters are distinctive in their ability to recognize and expel a highly diverse range of amphipathic compounds. Amphipathic molecules contain both hydrophobic and hydrophilic groups-molecules that repel or are attracted to water, respectively.

The x-ray structure of the EmrD transporter-determined with data collected at the Stanford University Synchrotron Radiation Laboratory and the Advanced Light Source at the University of California, Berkeley-revealed an interior composed primarily of hydrophobic residues. This finding is consistent with its role of transporting hydrophobic or lipophilic molecules-and similar to the interior of another multidrug transporter, EmrE, which Chang and his colleagues uncovered in a study that was published last year in the journal Science.

This internal cavity is the “most notable difference” between EmrD and most non-Major Facilitator Superfamily multidrug transporters that, the new study noted, typically transport “a relatively narrow range of structurally related” compounds. The hydrophobic residues in the EmrD internal cavity are likely to contribute to the general mechanism transporting various compounds through the cell membrane, and may play “an important role in dictating a level of drug specificity” through a number of molecular interactions.

The study also suggests that EmrD intercepts and binds cyanide m-chlorophenyl hydrazone, a known efflux pump inhibitor, before it reaches the cell cytoplasm. This binding is likely facilitated by hydrophobic interactions within the internal cavity of EmrD. The researchers speculate that cyanide m-chlorophenyl hydrazone is either expelled from the bacterial cell or into the periplasmic space-the space between the outer membrane and the plasma membrane in gram-negative bacteria like E. coli.

“While EmrD and EmrE are completely different proteins from different molecular families,” Chang said, “both are multidrug transporters that help bacteria develop multidrug resistance. Together with MsbA, another MDR structure that our laboratory is studying, this new x-ray structure adds another important view of some general structural features across multi-drug resistant transporter families.”

Other authors of the study include Yong Yin, Xiao He, Paul Szewczyk, and That Nguyen of The Scripps Research Institute.

The study was supported by the National Institutes of Health and the Skaggs Institute for Chemical Biology.
Source: Scripps Research Institute

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Virus reproduction and spread are studied

Posted by tumicrobiology on April 7, 2006

Wake Forest University scientists have made a surprising discovery about a powerful virus — a discovery that may lead to better vaccines and medications.

The biochemists have identified a protein that plays an important role in the ability of the vesicular stomatitis virus to invade healthy cells and reproduce.

Although VSV infects animals, it is not a human pathogen. Nevertheless, scientists study it because of its similarity to the Ebola, rabies and Marburg hemorrhagic fever viruses.

“VSV is a good model of a variety of other viruses,” said John Connor, an assistant professor of biochemistry. “Our research has given us a better understanding of how viruses like these are able to do the nasty things they do.”

Normally, VSV is extremely powerful, with the ability to shut down a cell’s system for making proteins. VSV then controls the cell’s protein-making machinery, making its own proteins so it can replicate and spread. The scientists were able to weaken that power by altering the matrix protein, so VSV cannot make as much protein and does not reproduce.

The study is reported in the April issue of the Journal of Virology.

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DNA links abductees from Japan, S.Korea

Posted by tumicrobiology on April 7, 2006

TOKYO, April 7 (UPI) — A DNA test has shown the husband of a Japanese woman kidnapped by North Korea in the 1970s is a South Korean who also was abducted by the North.

North Korea had insisted that Megumi Yokota, a Japanese national kidnapped by North Korean agents in 1977 at age 13, married North Korean agent Kim Chol Jun in 1986 but committed suicide in 1994.

However, Japan has concluded that Kim is not North Korean, as claimed by Pyongyang, but an abducted South Korean, the JoongAng Ilbo reported Friday.

"After conducting DNA tests on family members of five abducted South Koreans, one matched the DNA of the daughter of Mr. Kim," Japanese officials said Thursday.

The Japanese government obtained DNA samples from Kim Hye Gyong, the daughter of Kim and Yokota, in 2002 during an interview in Pyongyang. Japan also collected DNA samples from the relatives of five South Korean men allegedly abducted by North Korea to see if any of them matched the DNA of Yokota's daughter.

The Japanese government reportedly is discussing with South Korea when they will make a formal announcement of their findings.

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Saliva might become common diagnostic tool

Posted by tumicrobiology on April 7, 2006

A University of Kentucky research study suggests the use of saliva might help change the diagnosis and treatment methods for periodontal disease.

Scientists wanted to determine if saliva could be tested for signs of periodontal disease, a chronic bacterial infection affecting millions of Americans. The study's results showed it could.

"Our research team has been working on methods and point-of-care devices that could allow saliva to be used as a diagnostic fluid," said Craig Miller, primary investigator and professor of oral medicine. "This could impact the practice of dentistry and medicine in the very near future, as healthcare practitioners use saliva, possibly instead of blood, to diagnose and monitor oral and systemic health."

He said eventually portable devices might be created to diagnose a wide variety of disease conditions using saliva.

The research appears in the March issue of the Journal of American Dental Association.

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