A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus-and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10 6 to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10 6 cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.

For the detection and identification of predominant bacteria in human feces, 16S rRNA-gene-targeted groupspecific primers for the Bacteroides fragilis group, Bifidobacterium, the Clostridium coccoides group, and Prevotella were designed and evaluated. The specificity of these primers was confirmed by using DNA extracted from 90 species that are commonly found in the human intestinal microflora. The group-specific primers were then used for identification of 300 isolates from feces of six healthy volunteers. The isolates were clearly identified as 117 isolates of the B. fragilis group, 22 isolates of Bifidobacterium, 65 isolates of the C. coccoides group, and 17 isolates of Prevotella, indicating that 74% of the isolates were identified with the four pairs of primers. The remaining 79 isolates were identified by 16S ribosomal DNA sequence analysis and consisted of 40 isolates of Collinsella, 24 isolates of the Clostridium leptum subgroup, and 15 isolates of disparate clusters. In addition, qualitative detection of these bacterial groups was accomplished without cultivation by using DNA extracted from the fecal samples. The goal for this specific PCR technique is to develop a procedure for quantitative detection of these bacterial groups, and a real-time quantitative PCR for detection of Bifidobacterium is now being investigated

An analytical system based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) was established for the precise evaluation of human intestinal microbiota. Group-and species-specific primer sets for Clostridium perfringens, Lactobacillus spp. (six subgroups and three species), Enterococcus spp., and Staphylococcus spp. targeting 16S rRNA gene sequences were newly developed for the quantitative analysis of such subdominant populations in human intestines. They were used together with previously reported group-specific primer sets for Enterobacteriaceae, Pseudomonas spp., and six predominant bacterial groups (the Clostridium coccoides group, the Clostridium leptum subgroup, the Bacteroides fragilis group, Bifidobacterium spp., the Atopobium cluster, and Prevotella spp.) for the examination of fecal samples from 40 healthy adults by RT-qPCR with lower detection limits of 10 2 to 10 4 cells per g of feces. The RT-qPCR method gave data equivalent to those yielded by qPCR for predominant populations of more than 10 8 cells per g of feces and could quantify bacterial populations that were not detectable (Staphylococcus and Pseudomonas) or those only detected at lower incidences (Prevotella, C. perfringens, Lactobacillus, and Enterococcus) by qPCR or the culture method. The RTqPCR analysis of Lactobacillus spp. at the subgroup level revealed that a subject has a mean of 4.6 subgroups, with an average count of log 10 (6.3 ؎ 1.5) cells per g of feces. These results suggest that RT-qPCR is effective for the accurate enumeration of human intestinal microbiota, especially the entire analysis of both predominant and subdominant populations.The human intestinal tract harbors a large, active, and complex microbial community, which consists of several hundred bacterial species (8) with concentrations of 10 11 viable microorganisms per g of content (47). Human intestinal microbiota have been reported to have numerous important functions, including the digestion of food, the metabolism of endogenous and exogenous compounds, immunopotentiation, protection against pathogens, and the regulation of host fat storage (22,26,51). For almost a century, human intestinal microbiota have been investigated in great detail using anaerobic culture techniques (35). Conventional culture techniques for the enumeration of different populations involve the use of selective microbiological media, followed by the isolation of pure cultures and the application of confirmatory biochemical tests. Despite the effectiveness and advantage of sensitivity, these procedures are extremely labor-intensive and time-consuming and therefore might be undesirable. Moreover, classification and identification based on phenotypic traits do not always provide clear-cut results and sometimes are unreliable.Recently, in place of cultivation-based techniques, a number of molecular methods based on the use of rRNA gene sequence information have been applied to the analyses of intestinal microbiota (46,59,60). Techniques such as the clone library method (8,11,26,4...

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Activity-dependent changes in neuropsin gene expression in the hippocampus implies an involvement of neuropsin in neural plasticity. Since the deduced amino acid sequence of the gene contained the complete triplet (His-Asp-Ser) of the serine protease domain, the protein was postulated to have proteolytic activity. Recombinant full-length neuropsin produced in the baculovirus/ insect cell system was enzymatically inactive but was readily converted to active enzyme by endoprotease processing. The activational processing of prototype neuropsin involved the specific cleavage of the Lys 32 -Ile 33 bond near its N terminus. Native neuropsin that was purified with a purity of 1,100-fold from mouse brain had enzymatic characteristics identical to those of active-type recombinant neuropsin. Both brain and recombinant neuropsin had amidolytic activities cleaving Arg-X and Lys-X bonds in the synthetic chromogenic substrates, and the highest specific activity was found against Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. The active-type recombinant neuropsin effectively cleaved fibronectin, an extracellular matrix protein. Taken together, these results indicate that this protease, which is enzymatically novel, has significant limbic effects by changing the extracellular matrix environment.Some proteases have been suggested to be related to neural cell dynamics in such processes as cell death, migration, cellto-cell adhesion and de-adhesion, process elongation, pathfinding, and axonal rearrangement (1-5). These phenomena have been investigated by supplying known proteases involved in blood coagulation, fibrinolysis, or digestion to neural cell cultures. However, the observations that the proteases are mainly localized in and released from non-neural cells do not support all of such neural effects (5-7). Thus, we postulated that neurons themselves may produce and release their own proteases to participate in the neural cell dynamics described above. Neuropsin (NP)1 was cloned from the mouse brain and was shown to be localized in mouse hippocampal pyramidal neurons (8). These results and the observation that its mRNA showed marked activity-dependent changes caused by plasticity-inducible stimuli are suggestive of some neural effects in limbic plasticity (8, 9). However, it is still not known whether NP protein has enzyme activity as suggested by the deduced amino acid sequence (8). We postulated that the enzyme activity might be a molecular basis for the physiological responses induced by various stimuli. Therefore, in the present study, we examined whether recombinant NP (r-NP) and brain NP had proteolytic activity against synthetic and natural substrates. EXPERIMENTAL PROCEDURESMaterials-Mono S, Sepharose 2B, CNBr-activated Sepharose 4B and CL-6B, Superdex-75HR, Superose 12, Resource S, and Protein G-Sepharose were from Amersham Pharmacia Biotech. Silver staining kits were from Bio-Rad. Diisopropyl fluorophosphate (DFP), benzamidine, bestatin, soybean trypsin inhibitor, human plasma thrombin (EC 3.4.4.13), and TNM-FH insect cel...

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We found no difference in histopathologic features and outcomes between men and women treated with RNU for UTUC. Nevertheless, epidemiologic and mechanistic molecular studies should be encouraged to design, analyze, and report gender-specific associations to aid in our understanding of gender impact on UTUC incidence, progression, and metastasis.