Low intensity pulsed ultrasound exposure increases prostaglandin E2 production via the induction of cyclooxygenase-2 mRNA in mouse osteoblasts

Both prostaglandin E2 (PGE2) and low intensity pulsed ultrasound (U/S) exposure have been reported to accelerate fracture repair. We hypothesized that these two pathways are interactive. To verify this hypothesis, we examined the regulation of PGE2 production by U/S exposure (sine wave of 1.5MHz repeating at 1kHz, 30mW/cm2, 20 minutes) in mouse osteoblastic cell line, MC3T3-E1. The production of PGE2 in osteoblasts was augmented by U/S, which was threefold at 60 min. in comparison with unexposed samples. Then we evaluated the expression of cyclooxygenase-2 (COX-2) mRNA, which is a critical enzyme for PGE2 production. U/S rapidly up-regulated the expression of COX-2 mRNA in a time dependent manner. In addition, PGE2 production by U/S was drastically suppressed by a selective inhibitor of COX-2. These results provide strong evidence that PGE2 production in osteoblasts is dependent upon the induction of COX-2 mRNA expression by U/S and offer a mechanistic insight into how U/S accelerates fracture repair.     

Bovine milk lactoferrin modulates the proliferative lymphocyte response to phytohemagglutinin

Bovine milk lactoferrin (2 to 20 g ml^–1) changed enhancement of [³H]thymidine incorporation by phytohemagglutinin-stimulated rat spleen lymphocytes into suppression as their lactoferrin-withdrawal incorporation increased to greater than 10000 cpm culture^–1 under the present isotope-labeling conditions. The enhancement disappeared by 15-min delayed addition of lactoferrin after addition of lectin. There was no lactoferrin effect when the cells were stimulated with 12-O-tetradecanoylphorbol-13-acetate plus ionomycin. Thus, lactoferrin has a certain extracellular effect on lymphocyte proliferation in response to the lectin.     

Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer

Fetal liver, the major site of hematopoiesis during embryonic development, acquires additional various metabolic functions near birth. Although liver development has been characterized biologically as consisting of several distinct steps, the molecular events accompanying this process are just beginning to be characterized. In this study, we have established a novel culture system of fetal murine hepatocytes and investigated factors required for development of hepatocytes. We found that oncostatin M (OSM), an interleukin-6 family cytokine, in combination with glucocorticoid, induced maturation of hepatocytes as evidenced by morphological changes that closely resemble more differentiated hepatocytes, expression of hepatic differentiation markers and intracellular glycogen accumulation. Consistent with these in vitro observations, livers from mice deficient for gp130, an OSM receptor subunit, display defects in maturation of hepatocytes. Interestingly, OSM is expressed in CD45(+) hematopoietic cells in the developing liver, whereas the OSM receptor is expressed predominantly in hepatocytes. These results suggest a paracrine mechanism of hepatogenesis; blood cells, transiently expanding in the fetal liver, produce OSM to promote development of hepatocytes in vivo.     

The pro-peptide of Streptomyces mobaraensis transglutaminase functions in cis and in trans to mediate efficient secretion of active enzyme from methylotrophic yeasts

Transglutaminase (TGase) from the actinomycete Streptomyces mobaraensis is a useful enzyme in the food industry, and development of an efficient production system for it would be desirable. Herein we report secretion of TGase in an enzymatically active form by methylotrophic yeasts as expression hosts. Secretory production of active TGase required a pro-peptide from TGase. When an artificial Kex2-endopeptidase recognition site was placed between the pro-peptide and mature TGase, secretion and in vitro maturation of TGase depended on Kex2-dependent cleavage. Unexpectedly, coexpression of unlinked pro-peptide with mature TGase yielded efficient secretion of the active enzyme. These results indicate that the pro-peptide from TGase functions not only in an intramolecular but also in an intermolecular manner. Site-directed mutagenesis of putative N-glycosylation sites increased the productivity of the active TGase further. A recombinant Candida boidinii strain was found to secrete active TGase up to 1.83 U/ml (about 90 mg/l) after 119 h of cultivation.     

An Aneurinibacillus sp. strain AM-1 produces a proline-specific aminopeptidase useful for collagen degradation

AIMS: We have been for a species of thermophilic bacteria that can effectively decompose collagen and collagen peptides that tend to be hard-to-degrade proteins because of their high content of proline residues. This study focused upon the enzymatic degradation of prolyl peptides by thermophilic bacteria. METHODS AND RESULTS: A strain, AM-1, producing a proline-specific aminopeptidase was isolated using a medium containing gelatin that was taken from soil samples collected at Arima Hot Spring located near Kobe, Japan. The strain showed the strongest level of hydrolysing activity toward prolyl-p-nitroanilide, and the activity proved to be thermostable. Phylogenetic analysis based on 16S rDNA sequences revealed that the isolated strain AM-1 was closest to Aneurinibacillus thermoaerophilus DSM10154T in its characteristics. Analysis of the purified proline-specific aminopeptidase suggested that the enzyme is an aminopeptidase containing metal that includes important disulphide bond(s). The strain AM-1 aminopeptidase has more similarities with leucyl aminopeptidases, but its activity level differs greatly with prolyl peptides. CONCLUSIONS: The proline-specific aminopeptidase from strain AM-1 is the first from the genus Aneurinibacillus and may be a new type of aminopeptidase for hydrolysing prolyl peptide. This enzyme also contributed to the degradation of collagen when used in combination with another collagenolytic protease. SIGNIFICANCE AND IMPACT OF THE STUDY: The proline-specific aminopeptidase obtained from strain AM-1 may be used in the treatment of wastewater containing collagen that is encountered in the meat industries, and for decreasing bitter peptides in milk products.     

The budget of dissolved trace metals in Lake Biwa,Japan

A comprehensive study on the dynamics of dissolved elements (Mg, Al, Si, P, Ca, V, Cr, Mn, Fe, Ni, Zn, As, Sr, Y, W, and U) in Lake Biwa was carried out using a clean technique. Lake water samples (n = 523) were collected from six stations in the North Basin and three stations in the South Basin. River water samples (n = 178) were collected from 14 major rivers flowing into the North Basin. Rainwater samples (n = 89) were collected at Otsu. The river water was enriched with Mn, Al, Fe, P, and Zn and the rainwater was enriched with Zn, Al, Fe, and Mn compared to North Basin water during winter mixing. The residence times of dissolved species were estimated on the basis of input through the rivers and rain. The residence times for Ca, Mg, and Sr were about 8 years, the same as that for water. Mn, Al, Fe, and Zn showed the shortest residence times (0.05–0.19 year). A budget calculation suggested that more than 60% of the input of dissolved Si, P, V, Cr, Mn, Fe, Ni, and Zn was scavenged and retained in the lake sediments and/or discharged as suspended particles.     

Mechanism of action of low recurrence of gastritis caused by Helicobacter pylori with the type II urease B gene

Background. Low recurrence of gastritis is seen in patients infected with Helicobacter pylori carrying the type II urease B gene, compared with H. pylori carrying types I and III. The underlying mechanism has been studied in terms of the urease activity and interleukin (IL)‐8 production capacity of different strains of H. pylori. Materials and Methods. Forty‐five patients infected with different strains of H. pylori (type I; 15, type II; 15 and type III; 15) were enrolled in the study. H. pylori was isolated from gastric mucosa and cultured in the presence of urea at pH 5.5 to evaluate urease activity. The capacity of different strains of H. pylori to induce IL‐8 mRNA and IL‐8 from a human gastric cancer cell line and human peripheral blood mononuclear cells was evaluated. Results. The urease activity of type II H. pylori[523 ± 228 µg of ammonia/dl/10⁸ colony‐forming units (CFU)/ml] was significantly lower than that of type I (1355 ± 1369 µg of ammonia/dl/10⁸ CFU/ml) and type III (1442 ± 2229 µg of ammonia/dl/10⁸ CFU/ml) (p < .05). Gastric cancer cells cocultured with type II H. pylori produced lower levels of IL‐8 mRNA compared with type I and type III H. pylori. The levels of IL‐8 were also significantly lower in cultures induced by type II H. pylori compared with those induced by type I and type III H. pylori. Peripheral blood mononuclear cells also produced lower levels of IL‐8 when cocultured with type II compared with type I H. pylori. Conclusions. These results indicate that both the lower level of urease activity and the low IL‐8‐inducing capacity of type II H. pylori might underlie the lower recurrence rate of gastritis caused by type II H. pylori.     

A novel in vitro infection model of Helicobacter pylori using mucin-producing murine gastric surface mucous cells

BACKGROUND: Helicobacter pylori is found within the gastric surface mucous gel layer and in the epithelial surface. Gastric cancer cells have been used in experimental H. pylori infection in vitro, although cancer cells have some abnormalities in cellular properties. The aim of this study was to develop an in vitro H. pylori infection model using normal gastric surface cells that produce gastric mucin. MATERIALS AND METHODS: Normal murine gastric surface mucous cells (GSM06) were cultured by the liquid interface method using a serum-free medium and a collagen gel containing a fibroblast cell line (L929) and infected with H. pylori. Infection by H. pylori was assessed by enumerating the colony-forming units (CFU) of H. pylori adhered to GSM06 cells and by transmission electron microscopy. The production of mucin was determined by a lectin binding assay, sugar analysis, and MUC5AC gene expression. RESULTS: GSM06 cells cultured under these conditions produced mucin containing N-acetylgalactosamine and MUC5AC as the core protein. Significantly higher numbers of H. pylori adhered to GSM06 cells under mucin-producing conditions than under nonproducing conditions. Microscopic observation showed a filamentous structure resembling a type IV secretion system apparatus formed between the surface of GSM06 cells and H. pylori. CONCLUSIONS: This study demonstrates a novel in vitro H. pylori infection model using mucin-producing murine GSM06 cells for early stages of infection.