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Biomedical subjects

R L Sinsheimer

Publications and source records attributed to R L Sinsheimer.

At least 19 recordsLinked to original sources
PubMed2001

Salmonella DNA adenine methylase mutants confer cross-protective immunity.

Salmonella isolates that lack or overproduce DNA adenine methylase (Dam) elicited a cross-protective immune response to different Salmonella serovars. The protection afforded by the Salmonella enterica serovar Typhimurium Dam vaccine was greater than that elicited in mice that survived a virulent infection. S. enterica serovar Typhimurium Dam mutant strains exhibited enhanced sensitivity to mediators of innate immunity such as antimicrobial peptides, bile salts, and hydrogen peroxide. Also, S. enterica serovar Typhimurium Dam(-) vaccines were not immunosuppressive; unlike wild-type vaccines, they failed to induce increased nitric oxide levels and permitted a subsequent robust humoral response to diptheria toxoid antigen in infected mice. Dam mutant strains exhibited a low-grade persistence which, coupled with the nonimmunosuppression and the ectopic protein expression caused by altered levels of Dam, may provide an expanded source of potential antigens in vaccinated hosts.

Animals↗
PubMed2001

DNA adenine methylase is essential for viability and plays a role in the pathogenesis of Yersinia pseudotuberculosis and Vibrio cholerae.

Salmonella strains that lack or overproduce DNA adenine methylase (Dam) elicit a protective immune response to different Salmonella species. To generate vaccines against other bacterial pathogens, the dam genes of Yersinia pseudotuberculosis and Vibrio cholerae were disrupted but found to be essential for viability. Overproduction of Dam significantly attenuated the virulence of these two pathogens, leading to, in Yersinia, the ectopic secretion of virulence proteins (Yersinia outer proteins) and a fully protective immune response in vaccinated hosts. Dysregulation of Dam activity may provide a means for the development of vaccines against varied bacterial pathogens.

Animals↗
PubMed2000

In vivo gene expression and the adaptive response: from pathogenesis to vaccines and antimicrobials.

Microbial pathogens possess a repertoire of virulence determinants that each make unique contributions to fitness during infection. Analysis of these in vivo-expressed functions reveals the biology of the infection process, encompassing the bacterial infection strategies and the host ecological and environmental retaliatory strategies designed to combat them (e.g. thermal, osmotic, oxygen, nutrient and acid stress). Many of the bacterial virulence functions that contribute to a successful infection are normally only expressed during infection. A genetic approach was used to isolate mutants that ectopically expressed many of these functions in a laboratory setting. Lack of DNA adenine methylase (Dam) in Salmonella typhimurium abolishes the preferential expression of many bacterial virulence genes in host tissues. Dam- Salmonella were proficient in colonization of mucosal sites but were defective in colonization of deeper tissue sites. Additionally, Dam- mutants were totally avirulent and effective as live vaccines against murine typhoid fever. Since dam is highly conserved in many pathogenic bacteria that cause significant morbidity and mortality worldwide, Dams are potentially excellent targets for both vaccines and antimicrobials.

Adaptation, Physiological↗
PubMed2000

Assessment of bacterial pathogenesis by analysis of gene expression in the host.

A number of techniques have been developed to assess the expression of microbial virulence genes within the host (in vivo). These studies have shown that bacteria employ a wide variety of mechanisms to coordinately regulate the expression of these genes during infection. Two tenets have emerged from these studies: bacterial adaptation responses are critical to growth within the host, and interactions between microorganisms and the microenvironments of their hosts cannot be revealed from in vitro studies alone. Results that support these tenets include (i) the prevalent class of in vivo expressed genes are involved in adaptation to environmental stresses, (ii) pathogens recovered from host tissues (versus laboratory growth) are often more resistant to host killing mechanisms, and (iii) virulence gene expression can differ in the animal compared to laboratory media. Thus, pathogenicity comprises the unique ability to adapt to the varied host milieus encountered as the infection proceeds.

Bacteria↗
PubMed1999

An essential role for DNA adenine methylation in bacterial virulence.

Salmonella typhimurium lacking DNA adenine methylase (Dam) were fully proficient in colonization of mucosal sites but showed severe defects in colonization of deeper tissue sites. These Dam- mutants were totally avirulent and were effective as live vaccines against murine typhoid fever. Dam regulated the expression of at least 20 genes known to be induced during infection; a subset of these genes are among those activated by the PhoP global virulence regulator. PhoP, in turn, affected Dam methylation at specific genomic sites, as evidenced by alterations in DNA methylation patterns. Dam inhibitors are likely to have broad antimicrobial action, and Dam- derivatives of these pathogens may serve as live attenuated vaccines.

Adenine↗
PubMed1998

Differential patterns of acquired virulence genes distinguish Salmonella strains.

Analysis of several Salmonella typhimurium in vivo-induced genes located in regions of atypical base composition has uncovered acquired genetic elements that cumulatively engender pathogenicity. Many of these regions are associated with mobile elements, encode predicted adhesin and invasin-like functions, and are required for full virulence. Some of these regions distinguish broad host range from host-adapted Salmonella serovars and may contribute to inherent differences in host specificity, tissue tropism, and disease manifestation. Maintenance of this archipelago of acquired sequence by selection in specific hosts reveals a fossil record of the evolution of pathogenic species.

Animals↗
PubMed1995

Applications for atomic force microscopy of DNA.

Tapping mode atomic force microscopy (AFM) of DNA in propanol, dry helium, and aqueous buffer each have specific applications. Resolution is best in propanol, which precipitates and immobilizes the DNA and provides a fluid imaging environment where adhesive forces are minimized. Resolution on exceptional images of DNA appears to be approximately 2 nm, sufficient to see helix turns in detail, but the smallest substructures typically seen on DNA in propanol are approximately 6-10 nm in size. Tapping AFM in dry helium provides a convenient way of imaging such things as conformations of DNA molecules and positions of proteins on DNA. Images of single-stranded DNA and RecA-DNA complexes are presented. In aqueous buffer DNA molecules as small as 300 bp have been imaged even when in motion. Images are presented of the changes in shape and position of circular plasmid DNA molecules.

Bacteriophage lambda↗
PubMed1993

Atomic force microscopy of DNA in aqueous solutions.

DNA on mica can be imaged in the atomic force microscope (AFM) in water or in some buffers if the sample has first been dehydrated thoroughly with propanol or by baking in vacuum and if the sample is imaged with a tip that has been deposited in the scanning electron microscope (SEM). Without adequate dehydration or with an unmodified tip, the DNA is scraped off the substrate by AFM-imaging in aqueous solutions. The measured heights and widths of DNA are larger in aqueous solutions than in propanol. The measured lengths of DNA molecules are the same in propanol and in aqueous solutions and correspond to the base spacing for B-DNA, the hydrated form of DNA; when the DNA is again imaged in propanol after buffer, however, it shortens to the length expected for dehydrated A-DNA. Other results include the imaging of E. coli RNA polymerase bound to DNA in a propanol-water mixture and the observation that washing samples in the AFM is an effective way of disaggregating salt-DNA complexes. The ability to image DNA in aqueous solutions has potential applications for observing processes involving DNA in the AFM.

1-Propanol↗
PubMed1992

Atomic force microscopy of single- and double-stranded DNA.

A method has been developed for imaging single-stranded DNA with the atomic force microscope (AFM). phi X174 single-stranded DNA in formaldehyde on mica can be imaged in the AFM under propanol or butanol or in air. Measured lengths of most molecules are on the order of 1 mu, although occasionally more extended molecules with lengths of 1.7 to 1.9 mu are seen. Single-stranded DNA in the AFM generally appears lumpier than double-stranded DNA, even when extended. Images of double-stranded lambda DNA in the AFM show more sharp kinks and bends than are typically observed in the electron microscope. Dense, aggregated fields of double-stranded plasmids can be converted by gentle rinsing with hot water to well spread fields.

Bacteriophage lambda↗
PubMed1990

Imaging single-stranded DNA, antigen-antibody reaction and polymerized Langmuir-Blodgett films with an atomic force microscope.

The combination of an (AFM) atomic force microscope together with microfabricated cantilevers that have integrated tips opens many possibilities for imaging systems of great importance in biology. We have imaged single-stranded 25mer DNA that was adsorbed on treated mica or that was covalently bound with a crosslinker to a polymerized Langmuir-Blodgett (LB) film, the top monolayer of a bilayer system. At low magnification the AFM shows cracks between solid domains, like in an image taken with a fluorescence microscope. At higher magnification, however, the AFM reveals much finer cracks and at still higher magnification it reveals rows of individual molecules in the polymerized LB film with a spacing of 0.45 nm. We have also imaged a LB film consisting of lipids in which 4% of the lipids had hapten molecules chemically bound to the lipid headgroups. Specific antibodies can then bind to these hapten molecules and be imaged with the AFM. This points to the possibility of using the AFM to monitor selective antibody binding.

Antibodies↗
PubMed1977

Separation of the complementary strands of DNA fragments on polyacrylamide gels.

32P-labeled (in vivo) phiX174 RFI DNA was restricted by Hinc II. Three aliquots of the same digest: a) nondenatured, b) heat denatured, and c) denatured by 5 mM Me-HgOH were analyzed on 3-15% acrylamide gel gradients or on 3% gels with reduced N,N'-methylene-bis-acrylamide. The autoradiography of the gels showed that the nondenatured sample migrates two times faster than the denatured samples. After denaturation each original fragment appeared as a doublet. Using in vitro synthesized RFI DNA labeled only in negative strand with 32P we could identify the position of the negative strand in each denatured doublet. The single strand DNA fragments could be recovered from the gel slices on a semi-preparative scale by electrophoresis into dialysis tubing.

Coliphages↗