Hydrolysis rate constants at 10-25 °C can be more than doubled by a short anaerobic pre-hydrolysis at 35 °C.

Hydrolysis is the first step of the anaerobic digestion of complex wastewater and considered as the rate limiting step especially at low temperature. Low temperature (10-25 °C) hydrolysis was investigated with and without application of a short pre-hydrolysis at 35 °C. Batch experiments were executed using cellulose and tributyrin as model substrates for carbohydrates and lipids. The results showed that the low temperature anaerobic hydrolysis rate constants increased by a factor of 1.5-10, when the short anaerobic pre-hydrolysis at 35 °C was applied. After the pre-hydrolysis phase at 35 °C and decreasing the temperature, no lag phase was observed in any case. Without the pre-hydrolysis, the lag phase for cellulose hydrolysis at 35-10 °C was 4-30 days. Tributyrin hydrolysis showed no lag phase at any temperature. The hydrolysis efficiency of cellulose increased from 40 to 62%, and from 9.6 to 40% after 9.1 days at 15 and 10 °C, respectively, when the pre-hydrolysis at 35 °C was applied. The hydrolysis efficiency of tributyrin at low temperatures with the pre-hydrolysis at 35 °C was similar to those without the pre-hydrolysis. The hydrolytic activity of the supernatant collected from the digestate after batch digestion of cellulose and tributyrin at 35 °C was higher than that of the supernatants collected from the low temperature (≤25 °C) digestates.

Proteomic profiling of k-11706 responsive proteins.

Erythropoietin promotes the production of red blood cells. Recombinant human erythropoietin is illicitly used to improve performance in endurance sports. Expression of the ERYTHROPOIETIN gene is negatively controlled by the transcription factor GATA-binding protein (GATA). Specific GATA inhibitors have recently been developed as novel drugs for the management of anemia. These drugs could, therefore, be illicitly used like recombinant human erythropoietin to improve performance in sports. To examine alterations in levels of plasma protein after administration of GATA inhibitors, proteomic analyses were conducted on mouse plasma samples treated with the potent GATA inhibitor K-11706. The analysis based on gel electrophoresis identified 41 protein spots differentially expressed when compared with normal plasma. Each spot was identified with liquid chromatography coupled to tandem mass spectrometry and 2 of them, fetuin-B and prothrombin, were verified by Western blotting. The results showed that the expression of fetuin-B in mice plasma was increased by K-11706, but not by recombinant human erythropoietin or hypoxia. These results suggest the potential of proteomic-based approaches as tools to identify biomarkers for the illegal use of novel drugs (e.g., GATA inhibitors). Also, fetuin-B could be a sensitive marker for the detection of abuse of GATA inhibitors.

Caffeine and taurine enhance endurance performance.

Caffeine enhances endurance performance; however, its effect on accumulated lactate remains unclear. Conversely, taurine, which also enhances endurance performance, decreases accumulated lactate. In this study, the effect of combination of caffeine and taurine on endurance performance was assessed. Mice ran on a treadmill, and the accumulated lactate was measured. In addition, muscle fibers from the gastrocnemius muscle of the mice were stained with ATPase and analyzed. The use of caffeine and taurine over a 2 week period enhanced endurance performance. Moreover, taurine significantly decreased the accumulated concentration of lactate over long running distances. However, the diameter of the cross-sections and ratios of Types I, IIA, and IIB muscle fibers were not affected.

Nicking sites in a subunit of cholera toxin and Escherichia coli heat-labile enterotoxin for Vibrio cholerae hemagglutinin/protease.

We analyzed the nicking site of the A subunit of Escherichia coli heat-labile enterotoxin for hemagglutinin/protease produced by Vibrio cholerae non-O1 (NAG-HA/P). The determined nicking site was the Thr193-Ile194 junction, which was distinct from that for a protease of V. cholerae (Ichinose et al., European Journal of Epidemiology 8, 743-747, 1992). We further analyzed proteolytic cleavage by NAG-HA/P of a synthetic peptide corresponding to the nicking region of cholera toxin A subunit and determined the cleavage site to be preferentially between Ser194 and Met195, and in addition between Ser193 and Ser194.

[Inflammatory pseudotumor of the ureter: a case report].

A 57-year-old male was admitted because of the right flank pain. The image examinations, retrogradeurography, abdominal CT and MRI, showed a mass located at the upper right ureter. Although the tumor was not typical as ureteral cancer, we could not make a diagnosis of a benign tumor by image examinations. Therefore nephroureterectomy that was surgical method for ureteral cancer was performed. The tumor was diagnosed as inflammatory pseudotumor of the ureter by histological findings. Inflammatory pseudotumor is extremely rare for ulogeital organs. And this lesion is difficult to distinguish from malignancy only by image examinations. Therefore, the surgical resection and pathological studies are necessary.

[Pitfall in 201Tl-99mTc subtraction scintigraphy: a case of 99mTc-pertechnetate uptake in a parathyroid hyperplasia].

201Tl-99mTc subtraction scintigraphy has been recognized as a useful procedure in the preoperative localization of hyperfunctioning parathyroid glands. We experienced a case which showed 99mTc-pertechnetate uptake in a parathyroid hyperplasia. This case warned us to focus a lot of attention on the detection for preoperative localization. There has been no such case reported in the previous Japanese literatures. Hypervascularity and thick fibrous capsule presumed explanation for a rare case of marked pertechnetate uptake into a parathyroid hyperplasia.

In vitro proteolytic processing and activation of the recombinant precursor of El Tor cytolysin/hemolysin (pro-HlyA) of Vibrio cholerae by soluble hemagglutinin/protease of V. cholerae, trypsin, and other proteases.

Vibrio cholerae produces a cytolytic toxin named El Tor cytolysin/hemolysin which is encoded by the hlyA gene. This cytolysin is produced as a 79-kDa precursor form (pro-HlyA) into the culture supernatant after cleavage of the signal peptide of the hlyA product (prepro-HlyA). The pro-HlyA is then processed to a 65-kDa mature cytolysin (mature HlyA) after cleavage of the 15-kDa N-terminal peptide (pro region) of the 79-kDa precursor, usually at the bond between Ala-157 and Asn-158. We investigated whether proteases could process the recombinant 79-kDa pro-HlyA to the 65-kDa mature HlyA. We observed that the soluble hemagglutinin/ protease (HA/protease; a major protease of V. cholerae), trypsin, alpha-chymotrypsin, subtilisin BPN', papain, and thermolysin all processed the pro-HlyA to the 65-kDa mature form of the protein. Along with this, the protease-processed HlyA showed drastically increased hemolytic activity. The N-terminal amino acid of the mature form of cytolysin generated by HA/protease was Phe-151, and that due to trypsin was Ser-149. Other proteases also cleaved the pro-HlyA at a nearby site, between Leu-146 and Ser-153, and all the processed cytolysins showed increased hemolytic activity. These data suggest that the active El Tor cytolysin of V. cholerae could be derived from the C-terminal region of a pro-HlyA following proteolytic cleavage of the bonds in the vicinity of Leu-146 to Asn-158 by any of a wide variety of proteases.

Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1.

Haemaglutinin/protease (HA/P) is one of the virulence factors of Vibrio cholerae O1 and pathogenic strains of V. cholerae non-O1. In this study, we examined protease activity of a new serogroup of Vibrio cholerae recently designated as O139 synonym Bengal. The protease activity was produced by all eight isolates of V. cholerae O139 from Bangladeshi patients. Purification and partial characterization of the protease from V. cholerae O139 demonstrated the purified protease (O139-P) was indistinguishable from that previously reported for HA/P of V. cholerae non-O1 (NAG-HA/P) and V. cholerae O1 (Vc-HA/P). These results prove that V. cholerae O139 produces a protease belonging to HA/P, and suggest that the protease is another virulence factor found in newly emerged V. cholerae O139, as in V. cholerae O1.

Characterization of two forms of hemagglutinin/protease produced by Vibrio cholerae non-O1.

Two forms (34 kDa and 32 kDa) of hemagglutinin/protease produced by Vibrio cholerae non-O1 were characterized. The hemagglutinin/protease purified by immunoaffinity column chromatography using a monoclonal antibody was essentially a 34-kDa form. By incubation of the purified 34-kDa form at 37 degrees C, it was processed (autodigested) to the 32-kDa form. The N-terminal 20 amino acid sequences of both the 34- and 32-kDa forms were identical, suggesting that proteolytic processing at the C-terminal region of the 34-kDa hemagglutinin/protease resulted in the 32-kDa form. With this shift, protease activity increased, but hemagglutinating activity decreased, suggesting that the C-terminal region of the hemagglutinin/protease is related to hemagglutinating activity.

A simple purification method of Vibrio cholerae non-O1 hemagglutinin/protease by immunoaffinity column chromatography using a monoclonal antibody.

A new simple purification method (I) for Vibrio cholerae non-O1 hemagglutinin/protease (NAG-HA/P) was developed. The method (I) requires only an immunoaffinity column chromatography using a monoclonal antibody against NAG-HA/P. The method (I) is much simpler than previously reported purification method (II) (Honda, T. et al, Infection and Immunity 57: 2799-2803, 1989) which required four or more complicated chromatographic procedures. Method (I) also gave an improved recovery rate (about 27%) compared with (II). The molecular weight of NAG-HA/P purified by method (I) was mainly 34 kilodaltons (kDa) with a little of 32 kDa, whereas that of NAG-HA/P purified by (II) was usually 32 kDa. Immunological analysis by the Ouchterlony double gel diffusion test and Western blotting test using polyclonal antibody against 32 kDa protein revealed that the 34 and 32 kDa proteins are immunologically indistinguishable and thus it is supposed that 34 K protein is an isoform or a preform of the 32 K protein.

Production of monoclonal antibodies against a hemagglutinin/protease of Vibrio cholerae non-01.

Two hybridoma cell lines producing monoclonal antibodies (MAbs) against a hemagglutinin/protease (HA/P) from Vibrio cholerae non-01 were produced and characterized. The two MAbs contained the kappa light chain and were IgG1 type. They similarly neutralized HA/P protease activity derived from both V. cholerae non-01 and V. cholerae 01, whereas they were unable to neutralize the hemagglutinating activity of HA/P, suggesting that the epitopes for protease and hemagglutination activities are different. Western blotting analysis and the cross-neutralization test with the two MAbs confirmed the identity of HA/P produced by V. cholerae non-01 and 01. This study also suggests that HA/P of V. cholerae and a protease of V. parahaemolyticus are immunologically unrelated.

[Effects of phorbol ester and forskolin on steroidogenesis and protein phosphorylation in cultured rat granulosa cells].

Granulosa cells obtained from immature estradiol-treated SD rats (10 rats/experiment) were employed in elucidating the control mechanism of steroid secretion. Phorbol 12-myristate 13-acetate (PMA) inhibited estradiol production by cultured rat granulosa cells with IC50 less than 1 nM. PMA, however, stimulated small but significant increases in progesterone production in a dose-dependent manner with ED50 of 14 nM to 3.5-fold above the basal control level. These effects could not be induced by calcium ionophore A23187. Forskolin-stimulated progesterone production was inhibited by the concomitant addition of PMA with IC50 less than 1 nM. The phosphorylation of proteins by [32P] orthophosphate-labelled cells was examined by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Treatment of cells with forskolin altered the intensity of 40 kDa acidic phosphoprotein compared to that of the control. On the other hand, treatment of cells with PMA altered the intensity of 78 and 32 kDa acidic phosphoproteins. These results suggest, therefore, that PMA can modulate steroidogenesis in rat granulosa cells, presumably through activation of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C).

Inflammatory fibroid polyp of the stomach.

Fourteen cases of inflammatory fibroid polyp of the stomach were studied in terms of immunohistochemistry and ultrastructure. They occurred as polypoid lesions in the antrum, except for two found in the body of the stomach. Out of the 14 cases, two were found to be multiple and the remainder solitary. In all but one lesion, the mucosal layer was involved and six lesions were entirely localized within the mucosal layer, suggesting that inflammatory fibroid polyps of the stomach originate in the mucosal layer. Neither S100 protein, factor VIII-related antigen, alpha 1-antitrypsin nor lysozymes were found in the cytoplasm of the proliferating cells. The ultrastructures of the proliferating cells were fibroblastic rather than neurogenic, angiogenic, or myofibroblastic. These findings suggest that the cells are fibroblasts, though the possibility that they are facultative fibroblasts remains. An interesting observation made with electron microscope was the infection of micro-organisms similar to mycoplasma. This seems to deserve further investigation as a possible etiologic factor of the disease.

[Clinicopathological study on 100 Japanese patients with peritoneal mesothelioma in Japan].

We report a clinicopathological study on 100 japanese patients with peritoneal mesothelioma encountered between 1911 and 1982. Fifty-six were males and 44 were females, they ranged in age from 10 months to 83 years. As the inducement materials, asbestos was reported in 5 and thorium in 2 patients. Clinical symptoms were abdominal distension in 56 and abdominal pain in 50 patients. Thrombocytosis and hypoglycemia were observed in 7 and 3 patients, respectively. Macroscopically, the peritoneal mesothelioma was localized in 7, diffuse in 86 and not described in 7 patients. The pathological classification was benign in 5 and malignant in 77 cases. According to Stout's classification, the peritoneal mesothelioma was tubular in 43 mixed in 25 and fibrous type in 11 cases. Based on our findings we suggest that peritoneal mesotheliomas are on the increase in Japan.

Culture of isolated glomeruli from normal and nephritic rabbits. I. Characterization of outgrowing cells.

The existence of four types of cells in the glomerular outgrowths from both normal and nephritic rabbits was confirmed, and their characteristics by phase contrast, scanning electron, and transmission electron microscopy are described. Type I (arborized), Type II (fusiform), Type III (round), and Type IV (paving stone-like) cells presumably originate from visceral epithelial cells, mesangial cells, monocytes, and parietal epithelial cells of Bowman's capsule, respectively, though conclusive evidence is still lacking. The Type III cells, unlike the other types of cells, have a remarkable phagocytic capacity as well as Fc and C3 receptors.