Construction of a new shuttle expression vector for Bacillus subtilis and Escherichia coli by using a polycistronic system.

A shuttle vector has been constructed by fusing the Bacillus subtilis trimethoprim-resistance-carrying (TpR) plasmid pNC601 with the Escherichia coli plasmid pBR322. The resultant plasmid pNBL1 can replicate in both B. subtilis and E. coli, conferring Tp resistance on both cells and ampicillin resistance (ApR) on E. coli. The B. subtilis dihydrofolate reductase operon (dfr) on pNC601 and therefore on pNBL1 consists of the thymidylate synthase B gene (thyB) and the TpR-dihydrofolate reductase gene lacking the C-terminal seven codons (designated as drfA' as compared with the complete dfrA gene). A direct-expression vector pNBL3 has been constructed by inserting synthetic oligodeoxynucleotides containing a Bacillus ribosome-binding site (RBS) and the ATG codon downstream from dfrA' on pNBL1. When the E. coli lacZ gene was placed downstream from the dfrA' gene in pNBL3, efficient synthesis of beta-galactosidase was observed in both cells, showing that the polycistronic expression system is suitable for directing expression of heterologous genes. Translational efficiency of the lacZ gene on pNBL3 was further examined in B. subtilis by changing the sequence upstream from lacZ. Unlike the results previously reported [Sprengel et al., Nucleic Acids Res. 13 (1985) 893-909], when RBS was present, the high level of lacZ expression was preserved irrespective of spacing between the stop codon of the upstream dfrA' gene and the start codon of the downstream lacZ gene. However, in the absence of RBS, the spacing between both genes affected lacZ expression. That is, translational coupling of dfrA'-lacZ was observed, although the translational efficiency was very low.

A possible role of the pineal gland in acute immobilization-related suppression of naloxone-induced LH release in ovariectomized estrogen-primed rats.

It has been recently reported that acute immobilization stress almost completely suppresses the luteinizing hormone (LH) release induced by naloxone, a mu-opioid antagonist, in ovariectomized estrogen-primed rats. The present study examined the possible involvement of the pineal gland in the acute immobilization-related suppression of the naloxone-induced LH release. An intraventricular (ICV) injection of 15 microg naloxone produced an abrupt increase in circulating LH concentrations in non-stressed rats. The naloxone-induced LH release was completely eliminated when tested 60 min after the end of a 30 min session of acute immobilization. The same stress conditions did not affect LH-releasing hormone (LHRH)-induced LH release, suggesting that the stress-related suppression of the naloxone-induced LH release was a suprapituitary event. In chronically-pinealectomized rats, but not in sham-pinealectomized rats, naloxone injected 60 min after the end of the stress session evoked a significant increase in serum LH concentrations. However, naloxone injected ICV during the acute immobilization did not elicit LH release in either pinealectomized or sham-operated rats. Under non-stressed conditions, the LH secretory response to naloxone was similar in pinealectomized and sham-operated animals. The same stress (30 min immobilization) significantly increased pineal melatonin content as well as plasma melatonin concentrations in rats bearing intact pineal glands, indicating that stress actually affected the pineal function. These results provide evidence for a role of the pineal in the suppression of the LH response to naloxone after stress, but not during stress.

Melatonin inhibits naloxone-induced luteinizing hormone release in ovariectomized estrogen-primed rats.

The present study aimed to examine the effect of melatonin on naloxone-induced luteinizing hormone (LH) secretion in ovariectomized estrogen-primed rats. A single intracerebroventricular (i.c.v.) injection of naloxone (mu opioid receptor blocker, 15 micrograms) or an intravenous (i.v.) injection of LH-releasing hormone (LHRH, 50 ng/kg) elicited a transient and significant increase in the serum LH concentration within 10 min. While an i.c.v. injection of 100 ng melatonin by itself did not change the basal LH release, it almost completely inhibited the naloxone-induced LH release. Melatonin (10 ng) also significantly reduced the effect of naloxone. However, an i.c.v. injection of 100 ng melatonin did not affect the LHRH-induced LH release. In separate experiments, the effect of melatonin on naloxone-induced pulsatile LH secretion was studied in estrogen-treated rats. A continuous i.v. infusion of naloxone (20 mg/kg/h) induced LH pulses in rats treated i.c.v. with saline. An i.c.v. administration of 100 ng melatonin, which by itself did not affect basal LH secretion, significantly reduced the frequency, but not the amplitude, of LH pulses induced by the naloxone infusion. These results show that melatonin has a suprapituitary site of action to inhibit naloxone-induced LH release, and suggest that melatonin has an effect in inhibiting the activity of the hypothalamic LHRH pulse generator, either directly or indirectly, in female rats.

Molecular cloning and nucleotide sequence of a DNA fragment from Bacillus natto that enhances production of extracellular proteases and levansucrase in Bacillus subtilis.

A DNA fragment from Bacillus natto IFO3936 has been cloned which enhances the production of both extracellular alkaline and neutral proteases in Bacillus subtilis. The DNA sequence analysis around the gene responsible for the hyperproduction, prtR, revealed one open reading frame (comprising 60 amino acid residues) which was bounded by potential transcriptional and translational regulatory signals in its preceding and following regions. This open reading frame was not homologous to the published sequences of the structural genes of the two proteases. The calculated molecular weight (7,109) of the polypeptide predicted from the DNA sequence is much smaller than those of the two proteases, indicating that the gene product is distinct from those enzymes. In-frame fusion between the N-terminal region of the coding sequence and the lacZ gene of Escherichia coli demonstrated that the coding region was indeed translated in vivo. By deletion analysis it was suggested that prtR was the structural gene for the 60-amino-acid polypeptide. Cells carrying a prtR plasmid secreted both proteases 40 to 400 times more than the cells carrying the vector alone. Furthermore, it was found that prtR also enhanced the production of levansucrase by 1 or 2 orders of magnitude. There was no difference, however, in the amount of the other extracellular enzymes such as alpha-amylase, RNase, and alkaline phosphatase. These results indicate that prtR is specific for the hyperproduction of the proteases and levansucrase.

Enhanced secretion of Escherichia coli beta-lactamase by a spontaneous erythromycin-resistant mutant of Bacillus subtilis.

An extracellular-protease-deficient mutant, ME142, was isolated from Bacillus subtilis as a spontaneous erythromycin-resistant (Eryr) clone. This mutant showed conditional sporulation and only sporulated normally in the absence of erythromycin. In the presence of the antibiotic, sporulation was greatly reduced. Production of extracellular proteases by ME142 also exhibited conditional deficiency, possibly due to pleiotropic effects of the sporulation deficiency. The production of protease was 2-10% that of the wild-type level in the presence of erythromycin. ME142 showed poor competence for transformation even in the absence of erythromycin; however, derivatives of ME142 were isolated which had the same Eryr phenotype but which exhibited normal competence. One such mutant, ME162, was used as a host for the secretion of Escherichia coli beta-lactamase. The amount of beta-lactamase in the culture supernatants of ME162 increased significantly when the cells were cultured with erythromycin, suggesting that proteolysis of the beta-lactamase in the supernatants of ME162 was greatly reduced as compared to that in the supernatants of the wild-type strain.

prtR enhances the mRNA level of the Bacillus subtilis extracellular proteases.

Studies were performed on the prtR gene which enhances the production of the Bacillus subtilis extracellular proteases and levansucrase, but not the alpha-amylase, RNase, and alkaline phosphatase. To investigate the mode of action of prtR, the Escherichia coli bla gene was placed under the control of two promoters. One was the promoter of the alkaline protease gene (aprE), and the other was the promoter of B. subtilis dihydrofolate reductase gene (dfrA). Expression of the bla gene was enhanced by prtR only when the apr promoter was used. From these results, it was concluded that the apr promoter or its vicinity was the target of prtR and that prtR does not affect the process after transcription. The mRNA levels of aprE and nprE (the neutral protease gene) were significantly increased by prtR, but the half-life of the aprE mRNA was not affected. These results show that the prtR gene product enhances protease production by increasing the rate of transcription initiation.

Effects of elevated temperature on growth, respiratory-deficient mutation, respiratory activity, and ethanol production in yeast.

Some mesophilic yeasts and a thermotolerant strain of Saccharomyces cerevisiae were found to grow at 40 degrees C in complex media containing 1% yeast extract when an inoculum of 10(6) or more cells.mL-1 was used. Yeast extract (6%) permitted Saccharomyces cerevisiae to grow at 40 degrees C even with a smaller inoculum size (10(5) cells.mL-1). The fraction of respiratory-deficient (petite) mutants in 40 degrees C grown culture was less than 10% except for the thermotolerant strain, which showed greatly increased levels depending on culture conditions. Seven of eight yeast strains exhibited extremely reduced cytochrome oxidase activity when grown at 40 degrees C irrespective of the frequency of the petite mutation. In contrast, the accumulation of ethanol in the medium and the ethanol-producing activity of the cells were not affected by growth at 40 degrees C.

20 KDa homologous restriction factor of complement resembles T cell activating protein.

We previously identified a 20KDa membrane glycoprotein 1F5 antigen which inhibits the assembly of homologous complement membrane attack complexes and we designate it as HRF20 standing for 20KDa homologous restriction factor. The amino acid sequence deduced from its coding base sequence resembles that of T cell activating protein, most conspicuously in cysteine residues, 10 out of 11 of which occupy identical positions in an overall sequence homology of 24.8%. Furthermore, proliferation of human T cells was stimulated by monoclonal antibody to HRF20.

Differential inhibition of NMDA- and naloxone-induced LH release by NMDA receptor antagonist and CRH in ovariectomized estrogen-primed rats.

A possible functional relationship between endogenous opioid peptides (EOPs), corticotropin-releasing hormone (CRH) and excitatory amino acids (EAAs) in the control of LH secretion was investigated in ovariectomized estrogen-primed rats. An intraventricular (icv) injection of an EAA agonist, N-methyl-D-aspartate (NMDA), or an EOP antagonist, naloxone, produced an abrupt increase in the serum LH level. While icv pretreatment of the animals with 2-amino-5-phosphonovaleric acid, a specific NMDA receptor antagonist, did not affect by itself basal LH levels, it significantly suppressed the NMDA-induced and also the naloxone-induced LH release. An icv injection of CRH also interfered with the naloxone-induced LH release. However, the NMDA-induced LH release was not affected by an icv injection of CRH or of beta-endorphin. These results suggest that the sites of EOP and CRH inhibition may be located upstream of the site of NMDA stimulation on the GnRH neuronal pathway, and that CRH can inhibit LH secretion without mediation by EOP neurons.

The N-terminal region of NS3 serine proteinase of hepatitis C virus is important to maintain its enzymatic integrity.

Hepatitis C virus codes a serine proteinase in nonstructural protein 3 (NS3) to produce viral replicative machinery. Recently, we reported that the activity of NS3 proteinase (region 1050-1214) was efficiently inhibited by some chelators. Kinetic analysis revealed that its K(m) value was 3.9 mM. In contrast, an enzyme covering region 1027-1214 (including N-terminal region of NS3) was found to show an improved K(m) of 0.3 mM and a remarkably reduced susceptibility to EDTA. These results suggest that the N-terminal region of NS3 is not essential for the proteinase activity but indispensable to maintain its structural integrity.