Pivotal Roles for pH, Lactate, and Lactate-Utilizing Bacteria in the Stability of a Human Colonic Microbial Ecosystem.

Lactate can be produced by many gut bacteria, but in adults its accumulation in the colon is often an indicator of microbiota perturbation. Using continuous culture anaerobic fermentor systems, we found that lactate concentrations remained low in communities of human colonic bacteria maintained at pH 6.5, even when dl-lactate was infused at 10 or 20 mM. In contrast, lower pH (5.5) led to periodic lactate accumulation following lactate infusion in three fecal microbial communities examined. Lactate accumulation was concomitant with greatly reduced butyrate and propionate production and major shifts in microbiota composition, with Bacteroidetes and anaerobic Firmicutes being replaced by Actinobacteria, lactobacilli, and Proteobacteria Pure-culture experiments confirmed that Bacteroides and Firmicutes isolates were susceptible to growth inhibition by relevant concentrations of lactate and acetate, whereas the lactate-producer Bifidobacterium adolescentis was resistant. To investigate system behavior further, we used a mathematical model (microPop) based on 10 microbial functional groups. By incorporating differential growth inhibition, our model reproduced the chaotic behavior of the system, including the potential for lactate infusion both to promote and to rescue the perturbed system. The modeling revealed that system behavior is critically dependent on the proportion of the community able to convert lactate into butyrate or propionate. Communities with low numbers of lactate-utilizing bacteria are inherently less stable and more prone to lactate-induced perturbations. These findings can help us to understand the consequences of interindividual microbiota variation for dietary responses and microbiota changes associated with disease states.IMPORTANCE Lactate is formed by many species of colonic bacteria, and can accumulate to high levels in the colons of inflammatory bowel disease subjects. Conversely, in healthy colons lactate is metabolized by lactate-utilizing species to the short-chain fatty acids butyrate and propionate, which are beneficial for the host. Here, we investigated the impact of continuous lactate infusions (up to 20 mM) at two pH values (6.5 and 5.5) on human colonic microbiota responsiveness and metabolic outputs. At pH 5.5 in particular, lactate tended to accumulate in tandem with decreases in butyrate and propionate and with corresponding changes in microbial composition. Moreover, microbial communities with low numbers of lactate-utilizing bacteria were inherently less stable and therefore more prone to lactate-induced perturbations. These investigations provide clear evidence of the important role these lactate utilizers may play in health maintenance. These should therefore be considered as potential new therapeutic probiotics to combat microbiota perturbations.

Effect of traditional Chinese medicine compounds on rumen fermentation, methanogenesis and microbial flora in vitro.

This study was conducted to investigate the effects of traditional Chinese medicine compounds (TCMC) on rumen fermentation, methane emission and populations of ruminal microbes using an in vitro gas production technique. Cablin patchouli herb (CPH), Atractylodes rhizome (AR), Amur Cork-tree (AC) and Cypsum were mixed with the weight ratios of 1:1:1:0.5 and 1:1:1:1 to make up TCMC1 and TCMC2, respectively. Both TCMC were added at level of 25 g/kg of substrate dry matter. In vitro gas production was recorded and methane concentration was determined at 12 and 24 h of incubation. After 24 h, the incubation was terminated and the inoculants were measured for pH, ammonia nitrogen, volatile fatty acids (VFA). Total deoxyribonucleic acid of ruminal microbes was extracted from the inocula, and populations were determined by a real-time quantitative polymerase chain reaction. Populations of total rumen methanogens, protozoa, total fungi, Ruminococcus albus, Fibrobacter succinogenes and Ruminococcus flavefaciens were expressed as a proportion of total rumen bacterial 16S ribosomal deoxyribonucleic acid. Compared with the control, the 2 TCMC decreased (P ≤ 0.05) total VFA concentration, acetate molar proportion, acetate to propionate ratio, gas and methane productions at 12 and 24 h, hydrogen (H) produced and consumed, and methanogens and total fungi populations, while the 2 TCMC increased (P ≤ 0.05) propionate molar proportion. Traditional Chinese medicine compound 1 also decreased (P ≤ 0.05) R. flavefaciens population. From the present study, it is inferred that there is an effect of the TCMC in suppressing methanogenesis, probably mediated via indirect mode by channeling H2 utilized for methanogenesis to synthesis of propionate and direct action against the rumen microbes involved in methane formation. In addition, the relative methane reduction potential (RMRP) of TCMC2 was superior to that of TCMC1.

Vascular endothelial growth factor expression and signaling in the lens.

PURPOSE: Previous studies have identified sequences encoding vascular endothelial growth factor (VEGF)-A and one of the VEGF receptors (VEGFR2, Flk-1, KDR) in lens fiber cells. The current study was undertaken to determine the distribution of VEGF-A protein in the lens, whether signaling through VEGF receptors occurs in lens cells, the pattern of VEGF-A expression during lens development, and the effect of hypoxia on VEGF-A expression. METHODS: VEGF-A and VEGFR2 were localized using immunocytochemistry. VEGF-A and VEGFR2 protein were identified and quantified by Western blot analysis. Activated (tyrosine phosphorylated) VEGFR2 was detected by immunoprecipitation with an anti-phosphotyrosine antibody followed by Western blot analysis with antibody to VEGFR2. Levels of VEGF-A mRNA were measured by quantitative PCR. Suturing the lids of adult mouse or rabbit eyes for 3 days was used to induce lens hypoxia. RESULTS: VEGFR2 sequences were present in adult human lens epithelial cells, and VEGF-A transcripts were detected in chicken embryo, adult human, and mouse lens epithelial cells. VEGF-A protein localized to the ends of mouse embryo lens fiber cells at developmental stages when the fetal vasculature was forming. At later stages, VEGF-A was distributed uniformly throughout the cytoplasm of cortical fiber cells. VEGFR2 was present in mouse lens epithelial and fiber cells and was tyrosine phosphorylated at all stages examined. VEGF-A protein was barely detectable in lens epithelial cells during the first postnatal week, but increased as the capillaries of the anterior pupillary membrane regressed. VEGF-A levels were highest in adult lenses. Suturing the eyelid caused an increase in VEGF-A mRNA and protein in lens epithelial and fiber cells. CONCLUSIONS: VEGF-A secreted by lens cells may stimulate the formation of the fetal vasculature, but regression of these vessels is not likely to be caused by a reduction in VEGF-A production by the lens. An active VEGF-A signaling system of unknown function appears to be active in the lens. It is likely that VEGF-A expression is regulated by tissue hypoxia at all stages of lens development.

Regulation of enhanced vacuolar H+-ATPase expression in macrophages.

The proton-translocating vacuolar ATPase (V-ATPase) acidifies the endocytic network of eukaryotic cells. Although all eukaryotic cell types require low to moderate levels of V-ATPase, some proton-secreting cells express amplified levels for use in specialized membrane domains. To characterize genetic elements required for this heightened expression, we studied transcription and stability of mRNA encoding the V-ATPase c subunit in a low expressing fibroblast cell line (NIH 3T3) and a high expressing macrophage cell line (RAW 264.7). Isolation of the promoter and mapping of the transcriptional start site indicated that the c subunit promoter is TATA-less and initiates transcription at a single site. Promoter activity was regulated through the same transcription factor binding sites in both cell types, which showed no discernible difference in rates of c subunit transcription. In contrast, c subunit transcripts showed markedly greater stability in RAW cells than in 3T3 cells, as did other constitutively expressed V-ATPase subunit transcripts. Only the B and 'a' subunits, which are expressed in multiple isoforms, were not regulated solely by mRNA stability. These results suggest that overall expression levels of the V-ATPase are set primarily by regulation of mRNA stability and that transcriptional mechanisms determine subunit composition in varying cell types.