Quantitative PCR with 16S rRNA-gene-targeted species-specific primers for analysis of human intestinal bifidobacteria

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.     

A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-kappaB activation

Nod1 and Nod2 are intracellular proteins that are involved in host recognition of specific bacterial molecules and are genetically associated with several inflammatory diseases. Nod1 and Nod2 stimulation activates NF-kappaB through RICK, a caspase-recruitment domain-containing kinase. However, the mechanism by which RICK activates NF-kappaB in response to Nod1 and Nod2 stimulation is unknown. Here we show that RICK is conjugated with lysine-63-linked polyubiquitin chains at lysine 209 (K209) located in its kinase domain upon Nod1 or Nod2 stimulation and by induced oligomerization of RICK. Polyubiquitination of RICK at K209 was essential for RICK-mediated IKK activation and cytokine/chemokine secretion. However, RICK polyubiquitination did not require the kinase activity of RICK or alter the interaction of RICK with NEMO, a regulatory subunit of IkappaB kinase (IKK). Instead, polyubiquitination of RICK was found to mediate the recruitment of TAK1, a kinase that was found to be essential for Nod1-induced signaling. Thus, RICK polyubiquitination links TAK1 to IKK complexes, a critical step in Nod1/Nod2-mediated NF-kappaB activation.     

Release of erythrocytes from the spleen during exercise and splenic constriction by adrenaline infusion in the rainbow trout

Erythrocyte-supplying function of the spleen was examined in the rainbow troutSalmo gairdneri under exercise. The spleen showed remarkable reduction, about 70% in weight and about 85% in hemoglobin content, after forced exercise of 15 min. The amount of erythrocytes released from the spleen was 2.33 ml/kg body, and this amount corresponds to about 20% of the total volume of circulating erythrocytes in resting condition. No damage was observed at the spleen, splenic artery and splenic vein after the exercise. Examination of the vascular system by a corrosion casting method showed that no place other than the venous circulation exists for the erythrocytes released from the contracted spleen. The spleen was strongly constricted by infusion of adrenaline into the organ. These facts imply that the fish spleen supplies stored hemoglobin into the circulating blood in response to an increased demand of oxygen during exercise, under the control of the sympathetic nervous system.     

Interaction between a negative regulator (Msa2/Nrd1) and a positive regulator (Cpc2) of sexual differentiation in Schizosaccharomyces pombe

The sexual differentiation of Schizosaccharomyces pombe is controlled by many cellular components which have not been fully characterized. We isolated a gene called msa2 as a multi-copy suppressor of a sporulation abnormal mutant (sam1). Msa2p is identical with Nrd1p which has been characterized as a factor that blocks the onset of sexual differentiation. The yeast two-hybrid system was used to identify Cpc2p, a fission yeast homolog of the RACK1 protein, that interacted with Msa2p/Nrd1p. We confirmed that Msa2p/Nrd1p interacted with Cpc2p in S. pombe cells. An epistatic analysis of msa2/nrd1 and cpc2 suggests that Msa2p/Nrd1p was an upstream regulator for Cpc2p. A localization analysis of Cpc2p and Msa2p/Nrd1p indicates that both proteins were predominantly localized in the cytoplasm. The interaction of negative regulator Msa2p/Nrd1p with positive regulator Cpc2p suggests a new regulatory circuit in the sexual differentiation of S. pombe.     

Recovery of phosphorous from swine wastewater through crystallization

All the phosphate rock Japan needs must be presently imported from abroad because the country has no subterranean phosphorous resources. Therefore, there is a need to accelerate the development of and establish the technologies for phosphorous recovery from waste and wastewater. Swine wastewater has a high potential for phosphorous recovery in Japan. A reactor for removing and recovering phosphorous from swine wastewater was designed with dual functions, crystallization through aeration and separation of formed struvite by settling. However, a dehydration, composting and characterization process was first needed before using sediment sludge, including struvite, on farmland, since the struvite will settle along with huge amounts of other suspended solids (organic matter). For the recovery of pure struvite, an accumulation device was designed and its efficiency examined. The device has a struvite-accumulation face made of stainless steel wire mesh (1 mm in diameter, 1 cm(2) square) to reduce its total weight. During submergence in the aeration column of the demonstration reactor, struvite cross-bridged and accumulated on the face of the device. The struvite could be scraped off easily with only a light brushing, and was found to be approximately 95% pure. Because this device is a very simple structure, it is thought to be acceptable to swine farmers.     

Production of trehalose synthase from a basidiomycete, Grifola frondosa, in Escherichia coli

The genomic DNA and cDNA for a gene encoding a novel trehalose synthase (TSase) catalyzing trehalose synthesis from α-d-glucose 1-phosphate and d-glucose were cloned from a basidiomycete, Grifola frondosa. Nucleotide sequencing showed that the 732-amino-acid TSase-encoding region was separated by eight introns. Consistent with the novelty of TSase, there were no homologous proteins registered in the databases. Recombinant TSase with a histidine tag at the NH₂-terminal end, produced in Escherichia coli, showed enzyme activity similar to that purified from the original G. frondosa strain. Incubation of α-d-glucose 1-phosphate and d-glucose in the presence of recombinant TSase generated trehalose, in agreement with the enzymatic property of TSase that the equilibrium lay far in the direction of trehalose synthesis. Received: 12 January 1998 / Received revision: 20 February 1998 / Accepted: 20 March 1998