NH2-terminal processing of Bacillus subtilis alpha-amylase

Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by removal of the NH2-terminal 41-amino acid sequence. To study the mechanism of this processing, the extracellular forms of alpha-amylase were analyzed for B. subtilis N7 alpha-amylase cloned and expressed in B. subtilis. The major form (form N34) isolated from log phase cultures in L-broth had an NH2 terminus corresponding to position 34 from the initiator Met but appeared to be microheterogeneous, as judged by native gel electrophoresis. The major forms from stationary phase cultures had NH2 termini at positions 40 (form N40) or 42 (form N42) and were homogeneous. The conversion of the larger to smaller forms could be achieved in culture supernatants or partially purified samples. The process N34----N40 was inhibited by EDTA; N40----N42 was facilitated by Ca2+. Phenylmethylsulfonyl fluoride inhibited the former but not the latter process. These results suggest that the signal peptidase cleavage site 30 decreases 35 is -Ala-Ala-Ala-Ser-Ala-Glu-Thr- (arrow or further upstream) and that proteolytic maturation occurs after secretion, which involves at least two different processing enzymes.     

Estrogen participation in induction of cervicovaginal adenosis-like lesions in immature mice exposed prenatally to diethylstilbestrol

The structure of human zygapophyseal joint synovial folds as seen by high-power light microscopy and transmission electron microscopy is described. Small myelinated nerves are demonstrated in association with some capillaries in the synovial folds. This may have clinical significance in the field of spinal pain.     

Peptidoglycan synthetic activities in membranes of Escherichia coli caused by overproduction of penicillin-binding protein 2 and rodA protein

Penicillin-binding protein (PBP)-2 and the RodA protein are known to function in determining the rod shape of Escherichia coli cells. Peptidoglycan biosynthetic reactions that required these two proteins were demonstrated in the membrane fraction prepared from an E. coli strain that overproduced both of these two proteins and which lacked PBP-1B activity (the major peptidoglycan synthetase activity in the normal E. coli membranes). The cross-linked peptidoglycan was synthesized from UDP-N-acetylmuramylpentapeptide and UDP-N-acetylglucosamine in the presence of a high concentration of cefmetazole that inhibited all of PBPs except PBP-2. The peptidoglycan was synthesized via a lipid intermediate and showed up to 30% cross-linking. The cross-linking reaction was strongly inhibited by the amidinopenicillin, mecillinam, and by other beta-lactam antibiotics that have a high affinity for PBP-2, but not by beta-lactams that had very low affinity for PBP-2. The formation of peptidoglycan required the presence of high levels of both PBP-2 and the RodA protein in the membranes, but it is unclear which of the two proteins was primarily responsible for the extension of the glycan chains (transglycosylation). However, the sensitivity of the cross-linking reaction to specific beta-lactam antibiotics strongly suggested that it was catalyzed by PBP-2. The transglycosylase activity of the membranes was sensitive to enramycin and vancomycin and was unusual in being stimulated greatly by a high concentration of a chelating agent.     

The effect of concentration on the antioxidant effectiveness of alpha-tocopherol in lipid peroxidation induced by superoxide free radicals

Egg yolk phosphatidylcholine liposomes were rapidly oxidized in the presence of chelated iron and a superoxide-generating system. alpha-Tocopherol incorporated in the bilayer was oxidized at the same time. No lipid or alpha-tocopherol oxidation occurred in liposomes composed of dimyristoyl phosphatidylcholine. The antioxidant did not inhibit lipid peroxidation until its concentration reached a critical level, which depended on the effectiveness of the oxidative stress. Beyond this level, peroxidation was inhibited completely and, simultaneously, the rate of oxidation of tocopherol was lowered. The results suggest that the antioxidant efficiency of alpha-tocopherol depends on its ability to react mainly with the chain-initiating or chain-propagating lipid radicals. This, in turn, is closely tied to the tocopherol content of the membrane. Ascorbate inhibited the consumption of alpha-tocopherol, possibly by regenerating its reduced form.     

MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C.

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.     

In vivo and in vitro characterization of Ser477X mutations in polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. 61-3: effects of beneficial mutations on enzymatic activity, substrate specificity, and molecular weight of PHA

Evolutionary engineered polyhydroxyalkanoate (PHA) synthases from Pseudomonas sp. 61-3 enhance PHA accumulation and enable the monomer composition of PHAs to be regulated. We characterized a newly screened Ser477Arg (S477R) mutant of PHA synthase by in vivo analyses of P(3-hydroxybutyrate) [P(3HB)] homopolymer and P(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] copolymer productions in the recombinants of Escherichia coli. The results indicated that the S477R mutation contributed to a shift in substrate specificity to smaller monomers containing a 3HB unit rather than to an enhancement in catalytic activity. Multiple mutations of S477R with other beneficial mutations, for example, Ser325Cys, exhibited synergistic effects on both an increase in PHA production (from 9 wt % to 21 wt %) and an alteration of substrate specificity. Furthermore, the effects of complete amino acid substitutions at position 477 were characterized in terms of in vivo PHA production and in vitro enzymatic activity. The five mutations, S477Ala(A)/Phe(F)/His(H)/Arg(R)/Tyr(Y), resulted in a shift in substrate specificity to smaller monomer units. The S477Gly(G) mutant greatly enhanced activity toward all different sizes of substrates with carbon numbers ranging from 4 to 12. These results indicated that the residue 477 contributes to both the catalytic activity and substrate specificity of PHA synthase. In recombinant E. coli, the S477A/F/G/H/R/Y mutations consistently led to increases (up to 6 times that of wild-type enzyme) in weight average molecular weights of P(3HB) homopolymers. On the basis of our studies, we created a structural feasibility accounting for the mutational effects on enzymatic activity and substrate specificity of PHA synthase.     

The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.     

Characterization of d-Alanyl-(d)-meso-2,6-diaminopimelic Acid Endopeptidase from Streptomyces globisporus 1829

d-Alanyl-(d)-meso-2,6-diaminopimelic acid endopeptidase was purified 47.4-fold with a yield of 40.5% from mutanolysin, which was partially purified from the cultural supernatant of Streptomyces globisporus 1829, by using ion exchange column chromatographies and a molecular sieve column. The purified enzyme was electrophoretically homogeneous. This enzyme had a molecular weight of 13,500 and an isoelectric point of pI 9.0. This enzyme was most active at pH 8.5 and stable between pHs 8.0 and 9.0. The hydrolyzing activity of this enzyme was enhanced by Co^{+ +} and Ca^{+ +} but inhibited appreciably by Zn^{+ +}, Cu^{+ +} and EDTA. The enzyme activity was not affected by β-lactam antibiotics and vancomycin. The Km values for bisdisaccharide heptapeptide and its derivative modified chemically by BOC-S were calculated to be 5.7 × 10^-4 and 4.0 × 10^-4 m, respectively.     

Compilation of Japanese fisheries statistics for the Japanese eel, <Emphasis Type="Italic">Anguilla japonica</Emphasis>, since 1894: a historical dataset for stock assessment

Fishery sustainability and the extinction risk of the Japanese eel, Anguilla japonica, are of global concern. The landings of the Japanese eel in Japan comprise a large part of the landings in East Asia. This study provides a compiled dataset of the annual fisheries statistics of the Japanese eel in Japan for stock assessment. The Japanese government has been recording Japanese eel statistics annually since 1984 in five series of annual reports by conducting systematic questionnaire surveys of fisheries managers and associations; however, most of these data are stored in analog format. The key variables in the dataset include the harvest weight of eels, the harvest weight and number of seeds for aquaculture, the number of eels stocked, and the number of management entities engaged in the eel fishery. The levels of spatial aggregation of the variables include the site (river and lake), prefecture, inland and coastal waters, and total in Japan. We also incorporated location data (latitude and longitude) of the site and prefecture into the dataset. Eel harvest includes primarily yellow eels (late juvenile stage) and silver eels (mature stage). Seed harvest in inland waters includes glass eels (intermediate stage between leptocephalus and elver) and elvers (early juvenile stage). Seed harvest from coastal waters comprises glass eels. This dataset provides information to assess long-term trends in the Japanese eel population.