Bone morphogenetic protein (BMP)-4 and BMP-7 suppress granulosa cell apoptosis via different pathways: BMP-4 via PI3K/PDK-1/Akt and BMP-7 via PI3K/PDK-1/PKC

BMP-4 and BMP-7 are associated with the suppression of granulosa cell apoptosis. LY294002 (PI3K inhibitor) or UCN-01 (PDK-1 inhibitor) increased the percentage of apoptotic cells in the granulosa cells treated with BMP-4 or BMP-7. The inhibitors of ERK and p38 (SB203580) did not increase the percentage of apoptotic cells in the granulosa cells treated with BMP-4 or BMP-7. Akt inhibitor did not induce apoptosis in the BMP-4-treated granulosa cells, whereas it did induce apoptosis of the BMP-7-treated granulosa cells. In the granulosa cells treated with BMP-4, the PKC inhibitor increased the percentage of apoptotic cells. Our data show that BMP-4 and BMP-7 are associated with granulosa cell survival via several non-Smad specific pathways: BMP-4 via the PI3K/PDK-1/PKC and BMP-7 via the PI3K/PDK-1/Akt.     

recA genotyping of Salmonella enteritidis phage type 4 isolates by restriction fragment length polymorphism analysis

AIMS: To subtype Salmonella enteritidis phage type 4 isolates by using recA genotyping. METHODS AND RESULTS: Random amplified polymorphic DNA analysis using a primer ERIC2 of 76 isolates of Salmonella enteritidis phage type 4 obtained in Northern Ireland in 1998 and in 1999 demonstrated the presence of five genotypes. Restriction fragment length polymorphism analysis, using a degenerate primer pair designed to amplify a segment (about 640 bp in length) of the recA gene from several members of the Enterobacteriaceae with restriction enzymes, HhaI and Sau3AI, showed that the resulting fragments could differentiate the isolates into three groups, respectively. CONCLUSION: recA gene amplification and HhaI and Sau3AI restriction digestion was demonstrated to increase the differentiating power between isolates of Salmonella enteritidis phage type 4 by combining the patterns of the random amplified polymorphic DNA analysis procedure using a primer ERIC2. Significance and Impact of the Study: A novel restriction fragment length polymorphism assay for isolates of Salmonella enteritidis phage type 4, based on the amplification of the recA gene was attained and its comparison and its combination with random amplified polymorphic DNA analysis was provided.     

Structural stability of covalently linked GroES heptamer: advantages in the formation of oligomeric structure

In order to understand how inter-subunit association stabilizes oligomeric proteins, a single polypeptide chain variant of heptameric co-chaperonin GroES (tandem GroES) was constructed from Escherichia coli heptameric GroES by linking consecutively the C-terminal of one subunit to the N-terminal of the adjacent subunit with a small linker peptide. The tandem GroES (ESC7) showed properties similar to wild-type GroES in structural aspects and co-chaperonin activity. In unfolding and refolding equilibrium experiments using guanidine hydrochloride (Gdn-HCl) as a denaturant at a low protein concentration (50 microg ml(-1)), ESC7 showed a two-state transition with a greater resistance toward Gdn-HCl denaturation (Cm=1.95 M) compared to wild-type GroES (Cm=1.1 M). ESC7 was found to be about 10 kcal mol(-1) more stable than the wild-type GroES heptamer at 50 microg ml(-1). Kinetic unfolding and refolding experiments of ESC7 revealed that the increased stability was mainly attributed to a slower unfolding rate. Also a transient intermediate was detected in the refolding reaction. Interestingly, at the physiological GroES concentration (>1 mg ml(-1)), the free energy of unfolding for GroES heptamer exceeded that for ESC7. These results showed that at low protein concentrations (<1 mg ml(-1)), the covalent linking of subunits contributes to the stability but also complicates the refolding kinetics. At physiological concentrations of GroES, however, the oligomeric state is energetically preferred and the advantages of covalent linkage are lost. This finding highlights a possible advantage in transitioning from multi-domain proteins to oligomeric proteins with small subunits in order to improve structural and kinetic stabilities.     

Macrophage inflammatory protein-1α (MIP-1α) enhances a receptor activator of nuclear factor κB ligand (RANKL) expression in mouse bone marrow stromal cells and osteoblasts through MAPK and PI3K/Akt pathways

Osteolytic lesions are rapidly progressive during the terminal stages of myeloma, and the bone pain or bone fracture that occurs at these lesions decreases the patients’ quality of life to a notable degree. In relation to the etiology of this bone destruction, it has been reported recently that MIP-1α, produced in large amounts in myeloma patients, acts indirectly on osteoclastic precursor cells, and activates osteoclasts by way of bone-marrow stromal cells or osteoblasts, although the details of this process remain obscure. In the present study, our group investigated the mechanism by which RANKL expression is induced by MIP-1α and the effects of MIP-1α on the activation of osteoclasts. RANKL mRNA and RANKL protein expressions increased in both ST2 cells and MC3T3–E1 cells in a MIP-1α concentration-dependent manner. RANKL mRNA expression began to increase at 1 h after the addition of MIP-1α; the increase became remarkable at 2 h, and continuous expression was observed subsequently. Both ST2 and MC3T3-E1 cells showed similar levels of increased RANKL protein expression at 1, 2, and 3 days after the addition of MIP-1α. After the addition of MIP-1α, the amount of phosphorylated ERK1/2 and Akt protein expressions showed an increase, as compared to the corresponding amount in the control group. On the other hand, the amount of phosphorylated p38MAPK protein expression showed a decrease from the amount in the control group after the addition of MIP-1α. U0126 (a MEK1/2 inhibitor) or LY294002 (a PI3K inhibitor) was added to ST2 and MC3T3-E1 cells, and was found to inhibit RANKL mRNA and RANKL protein expression in these cells. When SB203580, a p38MAPK inhibitor, was added, RANKL mRNA and RANKL protein expression were increased in these cells. MIP-1α was found to promote osteoclastic differentiation of C7 cells, an osteoclastic precursor cell line, in a MIP-1α concentration-dependent manner. MIP-1α promoted differentiation into osteoclasts more extensively in C7 cells incubated together with ST2 and MC3T3-E1 cells than in C7 cells incubated alone. These results suggested that MIP-1α directly acts on the osteoclastic precursor cells and induces osteoclastic differentiation. This substance also indirectly induces osteoclastic differentiation through the promotion of RANKL expression in bone-marrow stromal cells and osteoblasts. The findings of this investigation suggested that activation of the MEK/ERK and the PI3K/Akt pathways and inhibition of p38MAPK pathway were involved in RANKL expression induced by MIP-1α in bone-marrow stromal cells and osteoblasts. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.     

Feasibility study of a pilot-scale sewage treatment system combining an up-flow anaerobic sludge blanket (UASB) and an aerated fixed bed (AFB) reactor at ambient temperature

A feasibility test of a 17 m3-pilot-scale sewage treatment system was carried out by continuous feeding of raw municipal sewage under ambient temperature conditions. The system consisted of a UASB and an aerated fixed bed reactor. Some of the effluent from the fixed bed reactor was returned to the UASB influent in order to provide a sulfate source. The total BOD of 148-162 mg l(-1) in the influent was reduced to a more desirable 11-25 mg l(-1) in the final effluent. The levels of methane-producing activity from acetate and H2/CO2 gas at 10 degrees C were only 2% and 0% of those at 35 degrees C, respectively. On the other hand, the sulfate-reducing activity levels of the UASB sludge were relatively high at 10 degrees C, for example, 18% for acetate and 9% for H2/CO2 gas, compared to the activity levels at 35 degrees C. Therefore, BOD oxidization by sulfate reduction in the UASB was greater than that by methane production under low temperature conditions. This sulfate-reducing activity tended to be proportional to the copy number of adenosine-5'-phosphosulfate (APS) reductase genes in DNA extracted from the sludge.     

Characterization of a proteolytic enzyme derived from a Bacillus strain that effectively degrades prion protein

AIMS: The purpose of this paper was to screen candidate bacterial strains for the production of proteases suitable for application to the degradation of pathogenic forms of prion protein (PrP(Sc)). This paper describes the biochemical characteristics and proteolytic activity of the isolated protease. METHODS AND RESULTS: After screening more than 200 bacterial proteases for keratinolytic activity, we identified a Bacillus stain that produced a protease exhibiting high-degradation activity against a scrapie PrP(Sc). Sequence analysis indicated that this serine-protease belonged to the Subtilisin family and had optimum pH and temperature ranges of 9-10 and 60-70 degrees C. Western blotting analysis revealed that the protease was also capable of decomposing bovine spongiform encephalopathy-infected brain homogenate. In addition, the protease was demonstrated to degrade dried PrP(Sc) that had become firmly attached to a plastic surface considerably more effectively than proteinase K or PWD-1, a previously reported keratinase. CONCLUSIONS: These results indicate that the isolated protease exhibited higher activity for PrP(Sc) degradation compared with other proteases examined. SIGNIFICANCE AND IMPACT OF THE STUDY: This protease could be used under moderate conditions for the decontamination of precision instruments that are susceptible to PrP(Sc) contamination.