Biomedical subjects

Tiffany Huang

Publications and source records attributed to Tiffany Huang.

7 recordsLinked to original sources

PubMed2005

Commensal microbiota alter the abundance and TCR responsiveness of splenic naïve CD4+ T lymphocytes.

The epidemiologic risk of certain systemic immunologic diseases is affected by commensal or environmental microbiota, but the cellular basis of the "hygiene hypothesis" is poorly understood. In this study, we demonstrate that composition of the commensal microbiota affects the functional state of the peripheral naïve (CD62L(hi)CD44(lo)) T lymphocyte populations. Restricted flora (RF) mice (stably colonized with excess nonpathogenic Clostridium sp., and changes in other bacterial and fungal taxa) were distinguished after the neonatal period by a progressive deficiency in absolute numbers of naïve CD4+ and CD8+ T lymphocytes. SPF and RF mice had comparable levels of memory CD4+ and CD8+ T cells. This phenotype was attributable to the altered levels of certain commensals and their products, since germ-free mice had normal absolute numbers of splenic CD4+ and CD8+ T cells and their respective naïve and memory subsets. The naïve CD4+ T cell subset was functionally distinguished in RF mice versus SPF mice by TCR hyperresponsiveness, pro-inflammatory cytokine production, and increased activation-induced cell death. Biochemically, these traits were associated with higher basal phosphorylation of the TCR signaling proteins ZAP-70, Lck, and LAT. These findings indicate that enteric microbial products, through unknown cellular circuitry, influence steps in CD4 T cell differentiation moderating basal TCR signaling and immune responsiveness.

Animals↗

PubMed2004

Developing a genetic system in Deinococcus radiodurans for analyzing mutations.

We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the beta-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif(r) system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif(r) in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

Amino Acid Sequence↗

PubMed2003

Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome.

Mutations in the rpoB gene of Escherichia coli result in resistance to the antibiotic rifampicin (Rif(r)) by altering the beta subunit of RNA polymerase. Previous studies have identified 39 single base substitutions in the rpoB gene that lead to Rif(r) at 37 degrees C and an additional two mutations that result in temperature sensitive cells. We have extended this work and identified an additional 30 single base substitutions that result in the Rif(r) phenotype. With these mutations the rpoB/Rif(r) system now allows the monitoring of 69 base substitutions at 37 degrees at 37 sites (base pairs) distributed among 24 coding positions. Each of the six possible base substitutions is represented by 8-17 mutations. More than 90% of the mutations are within a small enough region of the rpoB gene to allow PCR amplification with a single pair of oligonucleotide primers, followed by sequencing with a single primer, leading to rapid analysis of numerous mutations. The remaining mutations can be monitored using an additional primer pair. To calibrate this system we sequenced over 500 mutations in rpoB occurring spontaneously or generated by different mutagens and mutators with known specificity. These results show that rpoB/Rif(r) is an accurate and easy to employ detection system, and offers the advantage of allowing analysis of mutations occurring on the chromosome rather than on an extrachromosomal element. The mutS, mutT, mutY, M mutators, as well as the mutagenic agents ethyl methanesulfonate (EMS), ultraviolet (UV) irradiation, 2-aminopurine (2AP), 5-azacytidine (5AZ), and cisplatin (CPT) gave results predicted by their characterized specificities. The number of different sequence contexts is sufficient to reveal significant hotspots among the spontaneous mutS, 2-aminopurine, ultraviolet light, 5-azacytidine, and cisplatin mutational spectra. The cisplatin distribution is particularly striking, with 68% of the mutations resulting from an A:T-->T:A transversion at a single site. Because of the conservation of key regions of RNA polymerase among many microorganisms, using the Rif(r)/rpoB system may be a general method for studying mutational processes in microorganisms without well developed genetic systems.

2-Aminopurine↗

PubMed2003

Competition between MutY and mismatch repair at A x C mispairs In vivo.

We show that the MutY protein competes with the MutS-dependent mismatch repair system to process at least some A. C mispairs in vivo, converting them to G. C pairs. In the presence of an increased dCTP pool resulting from the loss of nucleotide diphosphate kinase, the frequency of A. T-->G. C transitions at a hot spot in the rpoB gene is 30-fold lower in a MutY-deficient derivative than in the wild type.

Adenosine Triphosphatases↗

PubMed2002

Escherichia coli strains (ndk) lacking nucleoside diphosphate kinase are powerful mutators for base substitutions and frameshifts in mismatch-repair-deficient strains.

Nucleoside diphosphate (NDP) kinase is one of the enzymes that maintains triphosphate pools. Escherichia coli strains (ndk) lacking this enzyme have been shown to be modest base substitution mutators, and two members of the human family of NDP kinases act as tumor suppressors. We show here that in E. coli strains lacking NDP kinase high levels of mispairs are generated, but most of these are corrected by the mismatch-repair system. Double mutants that are ndk mutS, lacking both the NDP kinase and mismatch repair, have levels of base substitutions 15-fold higher and levels of certain frameshifts up to 10-fold higher than those of the respective mutations in mutS strains that are NDP kinase proficient. A sequence analysis of the specificity of base substitution mutations generated in ndk and ndk mutS backgrounds as well as other experiments suggests that NDP kinase deficiency stimulates polymerase errors that lead to A:T --> G:C transitions and that the editing capacity of cells may be affected, leading to additional uncorrected mispairs and to A:T --> T:A transversions.

Alleles↗

PubMed2002

Pseudomonas fluorescens encodes the Crohn's disease-associated I2 sequence and T-cell superantigen.

Commensal bacteria have emerged as an important disease factor in human Crohn's disease (CD) and murine inflammatory bowel disease (IBD) models. We recently isolated I2, a novel gene segment of microbial origin that is associated with human CD and that encodes a T-cell superantigen. To identify the I2 microorganism, BLAST analysis was used to identify a microbial homologue, PA2885, a novel open reading frame (ORF) in the Pseudomonas aeruginosa genome. PCR and Southern analysis identified Pseudomonas fluorescens as the originating species of I2, with homologues detectable in 3 of 13 other Pseudomonas species. Genomic cloning disclosed a locus containing the full-length I2 gene (pfiT) and three other orthologous genes, including a homologue of the pbrA/pvdS iron response gene. CD4(+) T-cell responses to recombinant proteins were potent for I2 and pfiT, but modest for PA2885. pfiT has several features of a virulence factor: association with an iron-response locus, restricted species distribution, and T-cell superantigen bioactivity. These findings suggest roles for pfiT and P. fluorescens in the pathogenesis of Crohn's disease.

Amino Acid Sequence↗

PubMedDate not supplied

Phytoestrogens as cardioprotective agents.

Epidemiologic studies have shown that women with a higher dietary intake of phytoestrogens, plant-derived compounds with partial estrogen agonist properties, have a lower incidence of cardiovascular disease and breast and uterine cancer than women with a lower dietary intake of these substances. The most-studied category of phytoestrogens is the isoflavones found in soybeans, chick peas, and other legumes. Genistein and daidzein are the 2 major forms of isoflavones and have demonstrated lipid-lowering properties, especially in hypercholesterolemic subjects. The mechanism for the hypocholesterolemic effects is unclear, but may involve altered hepatic metabolism, antioxidant properties, tyrosine kinase inhibition, and improved vascular reactivity. Other phytoestrogens, including coumestans and lignans, have shown unfavorable results with regard to their role in the prevention of coronary artery disease. Food and food supplements containing phytoestrogens are often advocated as an alternative to hormone replacement therapy (HRT) in women with contraindications to the use of conventional estrogen replacement or those wanting a natural alternative. In light of the recent trial results with HRT (estrogen plus progesterone), it would be prudent not to recommend phytoestrogens as cardioprotective substances until adequate safety and efficacy studies are completed.

Cardiotonic Agents↗