Rubber-like elasticity and volume changes in the isolated spasmoneme of giant Zoothamnium sp. under Ca2+-induced contraction.

Using glycerinated spasmoneme of giant Zoothamnium sp., the physical properties of spasmoneme before and after Ca2+-induced contraction (pCa 4.5) were investigated. The volume change of spasmoneme contraction was measured under zero tension. The length and diameter decreased by about 50% of their initial value as a result of contraction, which means that contraction is nearly isotropic. Thus the volume of spasmoneme decreased drastically by 86% of its original value. The swollen ratio of extended and contracted spasmoneme were 0.07 and 0.37, respectively. Tension-extension relationships of extended and contracted spasmonemes were measured. By applying the theory of rubber elasticity, the number of segments of a chain in originally extended spasmoneme was only 3.3, i.e., the chain was almost a straight one. On the other hand, the number of segments of a chain in contracted spasmoneme was more than 100, i.e., the chain was essentially a random one. Furthermore, the total number of chains in single spasmoneme was the same in extended and contracted spasmoneme. This means that the interchain cross-links of chains were not influenced by addition or removal of Ca2+. Moreover, the molecular weight of a chain is estimated to be at most about 50 kd. By considering all these results, it is concluded that the contractile mechanism of spasmoneme originates in the intramolecular folding and unfolding induced by Ca2+ binding and detaching.     

Role of N-glycosylation in cathepsin E. A comparative study of cathepsin E with distinct N-linked oligosaccharides and its nonglycosylated mutant.

Cathepsin E (CE), a nonlysosomal, intracellular aspartic proteinase, exists in several molecular forms that are N-glycosylated with high-mannose and/or complex-type oligosaccharides. To investigate the role of N-glycosylation on the catalytic properties and molecular stability of CE, both natural and recombinant enzymes with distinct oligosaccharides were purified from different sources. An N-glycosylation minus mutant, that was constructed by site-directed mutagenesis (by changing asparagine residues to glutamine and aspartic acid residues at positions 73 and 305 in potential N-glycosylation sites of rat CE) and expressed in normal rat kidney cells, was also purified to homogeneity from the cell extracts. The kinetic parameters of the nonglycosylated mutant were found to be essentially equivalent to those of natural enzymes N-glycosylated with either high-mannose or complex-type oligosaccharides. In contrast, the nonglycosylated mutant showed lower pH and thermal stabilities than the glycosylated enzymes. The nonglycosylated mutant exhibited particular sensitivity to conversion to a monomeric form by 2-mercaptoethanol, as compared with those of the glycosylated enzymes. Further, the high-mannose-type enzymes were more sensitive to this agent than the complex-type proteins. A striking difference was found between the high-mannose and complex-type enzymes in terms of activation by ATP at a weakly acidic pH. At pH 5.5, the complex-type enzymes were stabilized by ATP to be restored to the virtual activity, whereas the high-mannose-type enzymes as well as the nonglycosylated mutant were not affected by ATP. These results suggest that N-glycosylation in CE is important for the maintenance of its proper folding upon changes in temperature, pH and redox state, and that the complex-type oligosaccharides contribute to the completion of the tertiary structure to maintain its active conformation in the weakly acidic pH environments.     

Fertilizing ability in vitro of golden hamster spermatozoa after acute testicular X-irradiation]

The present study is concerned with the effect of radiation to the testis on fertilizing ability in vitro using golden hamster spermatozoa. Male hamsters at 6 and 8 weeks of age were given acute testicular X-irradiation (200 kVp, 20 mA, 0.47-0.48 Gy/min). Spermatozoa were collected from the cauda epididymides at different times after irradiation and then they were suspended in fertilization medium. After preincubation for 4-5 hr, the spermatozoa were cultured with the eggs collected from mature hamsters treated with PMSG-hCG. Fertilized eggs were examined for incidence of sperm penetration and formation of pronuclei at 4-5 hr after insemination. The fertilization rate (47.7%) at the 6th week after irradiation with a dose of 2 Gy was much lower in comparison with the control value (92.6%). However, the fertilization rates at the 3rd and 9th weeks after irradiation were 97.7 and 90.6%, respectively. In these period, no difference was found between the irradiated groups and the control groups. From the changes in sperm concentration after irradiation with a dose of 2 Gy, it was found that the fertilization rate was the lowest at the 6th week. The sensitive stage to radiation during spermatogenesis with reference to the reduction of fertilizing ability after irradiation coincides with that of decrease in the sperm concentration and sperm motility. The results of fertilization rate at the 6th week after different doses of X-irradiation (0.25-6 Gy) indicated that the reduction of fertilization rate is nearly expressed as a dose-response relationship.(ABSTRACT TRUNCATED AT 250 WORDS)     

Whole-Genome Sequencing and Comparative Genome Analysis of Bacillus subtilis Strains Isolated from Non-Salted Fermented Soybean Foods

Bacillus subtilis is the main component in the fermentation of soybeans. To investigate the genetics of the soybean-fermenting B. subtilis strains and its relationship with the productivity of extracellular poly-γ-glutamic acid (γPGA), we sequenced the whole genome of eight B. subtilis stains isolated from non-salted fermented soybean foods in Southeast Asia. Assembled nucleotide sequences were compared with those of a natto (fermented soybean food) starter strain B. subtilis BEST195 and the laboratory standard strain B. subtilis 168 that is incapable of γPGA production. Detected variants were investigated in terms of insertion sequences, biotin synthesis, production of subtilisin NAT, and regulatory genes for γPGA synthesis, which were related to fermentation process. Comparing genome sequences, we found that the strains that produce γPGA have a deletion in a protein that constitutes the flagellar basal body, and this deletion was not found in the non-producing strains. We further identified diversity in variants of the bio operon, which is responsible for the biotin auxotrophism of the natto starter strains. Phylogenetic analysis using multilocus sequencing typing revealed that the B. subtilis strains isolated from the non-salted fermented soybeans were not clustered together, while the natto-fermenting strains were tightly clustered; this analysis also suggested that the strain isolated from “Tua Nao” of Thailand traces a different evolutionary process from other strains.     

Taxol derivatives are selective inhibitors of DNA polymerase alpha

During screening for mammalian DNA polymerase inhibitors, we found and succeeded in isolating a potent inhibitor from a higher plant, Taxus cuspidate. The compound was unexpectedly determined to be taxinine, an intermediate of paclitaxel (taxol) metabolism. Taxinine was found to selectively inhibit DNA polymerase alpha (pol.alpha) and beta (pol.beta). We therefore, tested taxol and other derivatives and found that taxol itself had no such inhibitory effect, and only taxinine could inhibit both pol.alpha and beta. The other compounds used, one derivative, cephalomannine, and five intermediates synthesized chemically inhibited only the pol.alpha activity in vitro. None of the compounds, including taxinine, influenced the activities of the other DNA polymerases, which are reportedly targeted by many pol.beta inhibitors. With both pol.alpha and beta, all of the compounds tested noncompetitively inhibited with respect to both the DNA template-primer and the dNTP substrate.     

Precise structural elucidation of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase alpha

The X-ray crystal structure of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase alpha, has previously been reported. We show that dehydroaltenusin exists in an equilibrium mixture of two tautomers possessing gamma-lactone or delta-lactone in polar solvents by NMR experiments. Acetylation of dehydroaltenusin afforded two types of diacetates and two types of monoacetate, possessing gamma-lactone or delta-lactone, respectively. The inhibitory activities of these acetate derivatives against DNA polymerase alpha were all much weaker than that of dehydroaltenusin.     

Nonspecific immune response of rainbow trout (Oncorhynchus mykiss Walbaum) in relation to different status of vitamin E and highly unsaturated fatty acids

This study was designed to examine the effects of dietary vitamin E (VE) on modulation of immune responses when supplied with two levels of n-3 highly unsaturated fatty acids (n-3 HUFA) in rainbow trout, Oncorhynchus mykiss. Six semipurified diets were prepared containing three levels of dietary VE (0, 100 or 1000 mg alpha-tocopheryl acetate kg(-1) diet) and n-3 HUFA either at 20 or 48% of dietary lipid provided from fish oil or docosahexaenoic acid (DHA) concentrated fish oil respectively. The diets were fed to rainbow trout (100 g initial mean weight) for 15 weeks. The VE, vitamin C (VC) content in plasma and tissues and the nonspecific immune responses, both humoral (alternative complement activity, total immunoglobulin) and cellular (phagocytosis, nonspecific cytotoxicity) were examined. VE contents in the kidney reflected the dietary input but were lower in fish fed 48% n-3 HUFA diets, and could have impaired some of immune responses compared to fish fed 20% n-3 HUFA. VC contents in kidney followed the same pattern as VE. Both humoral and cellular immune functions deteriorated in fish fed VE deficient diets whereas improvement in most of the parameters corresponded to its supplementation. However, the higher dose of dietary VE did not substantially enhance the responses assayed compared to the 100 mg dose. Besides clearly indicating the role of VE in maintaining the immune functions in fish in relation to dietary n-3 HUFA, this study has revealed that optimum health benefits could be achieved when VE is maintained slightly above the levels generally recommended for normal growth.     

Activin-like signaling activates Notch signaling during mesodermal induction

Both activin-like signaling and Notch signaling play fundamental roles during early development. Activin-like signaling is involved in mesodermal induction and can induce a broad range of mesodermal genes and tissues from prospective ectodermal cells (animal caps). On the other hand, Notch signaling plays important roles when multipotent precursor cells achieve a specific cell fate. However, the relationship between these two signal pathways is not well understood. Here, we show that activin A induces Delta-1, Delta-2 and Notch expression and then activates Notch signaling in animal caps. Also, in vivo, ectopic activin-like signaling induced the ectopic expression of Delta-1 and Delta-2, whereas inhibition of activin-like signaling abolished the expression of Delta-1 and Delta-2. Furthermore, we show that MyoD, which is myogenic gene induced by activin A, can induce Delta-1 expression. However, MyoD had no effect on Notch expression, and inhibited Delta-2 expression. These results indicated that activin A induces Delta-1, Delta-2 and Notch by different cascades. We conclude that Notch signaling is activated when activin-like signaling induces various tissues from homogenous undifferentiated cells.