Conjugal Transfer of Genetic Information in Group N Streptococci

Streptococcus lactis strains ML3 and C₂O and S. lactis subsp. diacetylactis strains DRC3, 11007, and WM₄ were found to transfer lactose-fermenting ability to LM0230, an S. lactis C2 lactose-negative (Lac⁻) derivative which is devoid of plasmid deoxyribonucleic acid (DNA). Lactose-positive streptomycin-resistant (Lac⁺ Str^r) recombinants were found when the Lac⁺ Str^s donor was mixed with Lac⁻ Str^r LM0230 in solid-surface matings. Transduction and transformation were ruled out as the mechanism of genetic exchange in strains ML3, DRC3, 11007, and WM₄, nor was reversion responsible for the high number of Lac⁺ Str^r recombinants. Furthermore, chloroform treatment of the donor prevented the appearance of recombinants, indicating that transfer of lactose-fermenting ability required viable cell-to-cell contact. Strain C₂O demonstrated transduction as well as conjugation. Transfer of plasmid DNA during conjugation for all strains was confirmed by demonstrating the presence of plasmid DNA in the transconjugants by using agarose gel electrophoresis. In some instances, a cryptic plasmid was transferred in conjunction with the lactose plasmid by using strains DRC3, 11007, and WM₄. In S. lactis C2 × LM0230 matings, the Str^r marker was transferred from LM0230 to C2, suggesting conjugal transfer of chromosomal DNA. The results confirm conjugation as another mechanism of genetic exchange occurring in dairy starter cultures.     

Trifluoroethylamines as amide isosteres in inhibitors of cathepsin K

The P2-P3 amide of dipeptide cathepsin K inhibitors can be replaced by the metabolically stable trifluoroethylamine group. The non-basic nature of the nitrogen allows the important hydrogen bond to Gly66 to be made. The resulting compounds are 10- to 20-fold more potent than the corresponding amide derivatives. Compound 8 is a 5 pM inhibitor of human cathepsin K with >10,000-fold selectivity over other cathepsins.     

Novel effects of the prototype translocating Escherichia coli, strain C25 on intestinal epithelial structure and barrier function

Intestinal bacteria play an etiologic role in triggering and perpetuating chronic inflammatory bowel disorders. However, the precise mechanisms whereby the gut microflora influences intestinal cell function remain undefined. Therefore, the effects of the non-pathogenic prototype translocating Escherichia coli, strain C25 on the barrier properties of human T84 and Madine-Darby canine kidney type 1 epithelial cells were examined. T-84 cells were also infected with commensal E. coil, strains F18 and HB101, and enterohaemorrhagic E. coli, serotype O157:H7. Strains F18 and HB101 had no effect on transepithelial electrical resistance (TER) of T84 monolayers. By contrast, epithelial cells infected with strain C25 displayed a time-dependent decrease in TER, preceded by an altered distribution of the cytoskeletal protein alpha-actinin, comparable to infection with E. coli O157:H7. E. coli C25 infection also led to activation of nuclear factor kappaB (NF-kappaB), interleukin-8 secretion and alterations in localization of claudin-1, but not zona occludens-1 or claudin-4, in T84 cells. There were adherent C25 bacteria on the intact apical surface of infected T84 cells, while mitochondria appeared swollen and vacuolated. These novel findings demonstrate the ability of a translocating commensal bacterium to adhere to and modulate intestinal epithelial barrier function and to induce morphological changes in a manner distinct from the known enteric pathogen, E. coli O157:H7.     

Heterologous overexpression and purification of cytochrome c' from Rhodobacter capsulatus and a mutant (K42E) in the dimerization region. Mutation does not alter oligomerization but impacts the heme iron spin state and nitric oxide binding properties

Rhodobacter capsulatus cytochrome c' (RCCP) has been overexpressed in Escherichia coli, and its spectroscopic and ligand-binding properties have been investigated. It is concluded that the heterologously expressed protein is assembled correctly, as judged by UV-vis absorption, EPR, and resonance Raman (RR) spectroscopy of the unligated protein as well as forms in which the heme is ligated by CO or NO. To probe the oligomerization state of RCCP and its potential influence on heme reactivity, we have compared the properties of wild-type RCCP with a mutant (K42E) that lacks a salt bridge at the subunit interface. Analytical ultracentrifugation indicates that wild-type and K42E proteins are both monomeric in solution, contrary to the homodimeric structure of the crystalline state. Surprisingly, the K42E mutation produces a number of changes at the heme center (nearly 20 A distant), including perturbation of the ferric spin-state equilibrium and a change in the ferrous heme-nitrosyl complex from a six-coordinate/five-coordinate mixture to a predominantly five-coordinate heme-NO species. RR spectra indicate that ferrous K42E and wild-type RCCP both have relatively high Fe-His stretching frequencies, suggesting that the more favored five-coordinate heme-nitrosyl formation in K42E is not caused by a weaker Fe2+-His bond. Nevertheless, the altered reactivity of ferrous K42E with NO, together with its modified ferric spin state, shows that structural changes originating at the dimer interface can affect the properties of the heme center, raising the exciting possibility that intermolecular encounters at the protein surface might modulate the reactivity of cytochrome c' in vivo.     

Polymorphism analysis of six selenoprotein genes: support for a selective sweep at the glutathione peroxidase 1 locus (3p21) in Asian populations

Background

There are at least 25 human selenoproteins, each characterized by the incorporation of selenium into the primary sequence as the amino acid selenocysteine. Since many selenoproteins have antioxidant properties, it is plausible that inter-individual differences in selenoprotein expression or activity could influence risk for a range of complex diseases, such as cancer, infectious diseases as well as deleterious responses to oxidative stressors like cigarette smoke. To capture the common genetic variants for 6 important selenoprotein genes (GPX1, GPX2, GPX3, GPX4, TXNRD1, and SEPP1) known to contribute to antioxidant host defenses, a re-sequence analysis was conducted across these genes with particular interest directed at the coding regions, intron-exon borders and flanking untranslated regions (UTR) for each gene in an 102 individual population representative of 4 major ethnic groups found within the United States.

Results

For 5 of the genes there was no strong evidence for selection according to the expectations of the neutral equilibrium model of evolution; however, at the GPX1 locus (3p21) there was evidence for positive selection. Strong confirmatory evidence for recent positive selection at the genomic region 3p21 in Asian populations is provided by data from the International HapMap project.

Conclusion

The SNPs and fine haplotype maps described in this report will be valuable resources for future functional studies, for population specific genetic studies designed to comprehensively explore the role of selenoprotein genetic variants in the etiology of various human diseases, and to define the forces responsible for a recent selective sweep in the vicinity of the GPX1 locus.