Evidence for an intermediate conformational state of LacY

LacY mutant Cys154 → Gly exhibits a periplasmic-closed crystal structure identical to the WT, but is periplasmic-open in the membrane. The mutant hardly catalyzes transport, but binds galactosides from either side of the membrane with the same affinity and is resistant to site-directed proteolysis relative to the pseudo-WT. Site-directed alkylation was also applied to 11 single-Cys mutants in Cys154 → Gly LacY in right-side-out membrane vesicles or after solubilization and purification in dodecyl-β-D-maltopyranoside (DDM). Unlike the pseudo-WT, Cys replacements on the periplasmic side of the Cys154 → Gly mutant label rapidly in the membrane without sugar, but labeling decreases markedly after the mutant proteins are purified. Thus, Cys154 → Gly LacY likely favors a higher-energy intermediate periplasmic-open conformation in situ, but collapses to a lower-energy periplasmic-closed conformation in DDM after purification. Notably, branched-chain or neopentyl glycol maltoside detergents stabilize Cys154 → Gly LacY in the membrane-embedded form.     

Association of TOMM40 Polymorphisms with Late-Onset Alzheimer’s Disease in a Northern Han Chinese Population

Mitochondrial dysfunction is an early defect in the pathogenesis of late-onset Alzheimer’s disease (LOAD). One interesting candidate gene for mitochondrial dysfunction in LOAD is the translocase of outer mitochondrial membrane 40 homolog (TOMM40) gene. Several single nucleotide polymorphisms (SNPs) within TOMM40 have been shown to affect susceptibility to LOAD in Caucasians, while there are no studies on the association of the polymorphisms with LOAD risk in Han Chinese. Here, the association of TOMM40 polymorphisms in LOAD was investigated in a large Northern Han Chinese cohort consisting of 1,578 individuals. Both allelic and genotypic associations of three SNPs (rs157580, rs2075650, and rs11556505) with LOAD risk were observed in the total sample as well as in the non- APOE ε4 carriers. For rs1160985, the allele and genotype frequencies differed significantly only in APOE ε4 carriers. After adjustment for age, gender, and APOE ε4 status, the association remained statistically significant only for the rs157580 but not for rs2075650 and rs11556505. In contrast, the rs1160985 exhibited significant risk effect after adjustment. In addition, haplotype analysis confirmed that the haplotypes derived from SNPs in rs2075650, rs11556505, and rs1160985 were associated with either risk or protective effects. In summary, our findings suggest that the TOMM40 polymorphisms may play a role in the pathogenesis of LOAD in Han Chinese.     

Butyrate induces reactive oxygen species production and affects cell cycle progression in human gingival fibroblasts

Background and Objective: Short‐chain fatty acids, such as butyric acid and propionic acid, are metabolic by‐products generated by periodontal microflora such as Porphyromonas gingivalis, and contribute to the pathogenesis of periodontitis. However, the effects of butyrate on the biological activities of gingival fibroblasts (GFs) are not well elucidated. Material and Methods: Human GFs were exposed to various concentrations of butyrate (0.5–16 mm ) for 24 h. Viable cells that excluded trypan blue were counted. Cell cycle distribution of GFs was analyzed by propidium iodide‐staining flow cytometry. Cellular reactive oxygen species (ROS) production was measured by flow cytometry using 2’,7’‐dichlorofluorescein (DCF). Total RNA and protein lysates were isolated and subjected to RT‐PCR using specific primers or to western blotting using specific antibodies, respectively. Results: Butyrate inhibited the growth of GFs, as indicated by a decrease in the number of viable cells. This event was associated with an induction of G0/G1 and G2/M cell cycle arrest by butyrate (4–16 mm ) in GFs. However, no marked apoptosis of GFs was noted in this experimental condition. Butyrate (> 2 mm ) inhibited the expression of cdc2, cdc25C and cyclinB1 mRNAs and reduced the levels of Cdc2, Cdc25C and cyclinB1 proteins in GFs, as determined using RT‐PCR and western blotting, respectively. This toxic effect of butyrate was associated with the production of ROS. Conclusion: These results suggest that butyrate generated by periodontal pathogens may be involved in the pathogenesis of periodontal diseases via the induction of ROS production and the impairment of cell growth, cell cycle progression and expression of cell cycle‐related genes in GFs. These events are important in the initiation and prolongation of inflammatory processes in periodontal diseases.