Purification of a new extracellular 90-kDa serine proteinase with isoelectric point of 3.9 from Bacillus subtilis (natto) and elucidation of its distinct mode of action.

A new extracellular 90-kDa serine proteinase with an isoelectric point (pI) of 3.9 was purified from Bicillus subtilis (natto). Microheterogeneity was detected in the 50-kDa protease of bacillopeptidase F with pI 4.4 reported previously by Wu et al. and the sequence for the first 25 amino acids in the internal region of the enzyme was analyzed: ATDGVEWNVDQIDAPKAWALGYDGA. The cleavage sites in the oxidized B-chain of insulin by the proteinase were CySO3H7-Gly8, Val12-Glu13, Try16-Leu17, and Phe25-Tyr26. The activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, while the activity was not inhibited by proteinaceous Streptomyces subtilisin inhibitor (SSI) or alpha 2-macroglubulin.     

Antitumor effect of RBS (rice bran saccharide) on ENNG-induced carcinogenesis

We examined whether orally administered RBS (rice bran saccharide), prepared from rice bran by hot water extraction, increases immunocompetence, inhibits gastrointestinal carcinogenesis with N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) or shows an antitumor effect. After the administration of RBS, phytohemagglutinin (PHA)- and pokeweed mitogen (PWM)-stimulated blastogenesis of lymphocytes derived from the mesenteric lymph nodes and peripheral blood was enhanced, and the helper/ suppressor T-cell ratio was elevated, and migration activity of peritoneal macrophages was also increased in rats treated continuously with ENNG. ENNG-induced gastrointestinal carcinomas were observed in 43% of those administered RBS (ENNG-RBS) as compared with 88% in the control (ENNG) and 94% in the prednisolone (PRD) group (ENNG-PRD). The 12-month survival rate of rats bearing gastrointestinal cancer was 58% in the ENNG-RBS group as compared with 25% in the ENNG group and 15% in the ENNG-PRD group. RBS prevented the reduction in immunocompetence in the course of carcinogenesis, suppressed carcinogenesis, and prolonged the survival of rats with gastrointestinal cancer. Antitumor activities of RBS are thought to be a kind of host mediated action. The growth inhibition ratio of transplantable ENNG-induced cancer in Wistar rats was 42.1% in the RBS and 51.8% in the 5-FU group. Since little is known about the potent antitumor activity of -glucan, it would be interesting to consider the relationship between the structure and the biological activities of polysaccharides.     

Formation of Nitrosylleghemoglobin in Nodules of Nitrate-Treated Cowpea and Pea Plants

The formation of nitrosylleghemoglobin (LbNO) was examined incowpea and pea nodules in relation to the inhibition of nitrogenfixation by nitrate. Leghemoglobin was of the ferrous type andwas mainly converted to LbNO in cowpea nodules when the acetylene-reducingactivity decreased to 45% of control values as a result of thesupply of nitrate. In nodules of nitrate-treated pea plants,leghemoglobin was also of the ferrous type and LbNO was a minorcomponent of leghemoglobin. The levels of LbNO isolated fromnodules corresponded to the levels of LbNO calculated from equilibriumconstants for LbNO and the concentration of nitrite in nodules.The dissociation rate constants for LbNO from both cowpea andpea were much smaller than those for LbO₂ or LbCO, as is alsothe case in soybean. These results indicate that the inhibition of the functionsof leghemoglobin, due to the accumulation of LbNO, induces adecrease in nitrogen fixation in cowpea nodules, and that theinhibition of nitrogen fixation in pea nodules is not relatedto the formation of LbNO. (Received July 2, 1990; Accepted October 9, 1990)     

Heterochromatic differentiation in barley chromosomes revealed by C- and N-banding techniques

Summary Heterochromatin distribution in barley chromosomes was investigated by analyzing the C- and N-banding patterns of four cultivars. Enzymatic maceration and air drying were employed for the preparation of the chromosome slides. Although the two banding patterns were generally similar to each other, a clear difference was observed between them at the centromeric sites on all chromosomes. Every centromeric site consisted of N-banding positive and C-banding negative (N⁺ C^–) heterochromatin in every cultivar examined. An intervarietal polymorphism of heterochromatin distribution was confirmed in each of the banding techniques. The appearance frequencies of some bands were different between the two banding techniques and among the cultivars. The heterochromatic differentiation observed is discussed with respect to cause.     

Requirement of c-kit for development of intestinal pacemaker system.

A discovery that the protooncogene encoding the receptor tyrosine kinase, c-kit, is allelic with the Dominant white spotting (W) locus establishes that c-kit plays a functional role in the development of three cell lineages, melanocyte, germ cell, and hematopoietic cell which are defective in W mutant mice. Recent analyses of c-kit expression in various tissues of mouse, however, have demonstrated that c-kit is expressed in more diverse tissues which are phenotypically normal in W mutant mice. Thus, whether or not c-kit expressed outside the three known cell lineages plays a functional role is one of the important questions needing answering in order to fully elucidate the role of c-kit in the development of the mouse. Here, we report that some of the cells in smooth muscle layers of developing intestine express c-kit. Blockade of its function for a few days postnatally by an antagonistic anti-c-kit monoclonal antibody (mAb) results in a severe anomaly of gut movement, which in BALB/c mice produces a lethal paralytic ileus. Physiological analysis indicates that the mechanisms required for the autonomic pacing of contraction in an isolated gut segment are defective in the anti-c-kit mAb-treated mice, W/Wv mice and even W/+ mice. These findings suggest that c-kit plays a crucial role in the development of a component of the pacemaker system that is required for the generation of autonomic gut motility.     

Cloning and sequence analyses of cDNAs encoding vasotocin and isotocin precursors of chum salmon,Oncorhynchus keta: evolutionary relationships of neurohypophysial hormone precursors

Summary The nucleotide sequences of cloned cDNAs were used to determine the primary structures of the precursors of vasotocin (sVT) and isotocin (sIT) from the hypothalamus of the chum salmon,Oncorhynchus keta. Two different cDNAs were obtained for each of sVT and sIT precursors (sVT-I and sVT-II; sIT-I and sIT-II). Both sVT and sIT precursors were found to contain a signal peptide and hormone that is connected to a neurophysin by a Gly-Lys-Arg sequence. Northern and Southern blot analyses showed that the sVT and sIT genes are expressed by the same chum salmon hypothalamus, but not by the liver and kidney. Microheterogeneity was found in the nucleotide and amino acid sequences of sVT precursors between our results and the previously reported data (Heierhorst et al. 1990). The conspicuous difference is the occurrence of a stop codon in the middle of sVT-II cDNA. The carboxyl termini of both sVT and sIT neurophysins are about 30 amino acids longer than neurophysins of toad and mammalian neurohypophysial hormone precursors. Although these extended regions do not contain a glycosylation site, they show striking similarity with the glycopeptide moiety (copeptin) of toad vasotocin and mammalian vasopressin precursors. The central portion of the neurophysins shows highest homology among corresponding regions of sVT and sIT precursors. Moreover, calculation of nucleotide substitution rates suggests that a recent gene conversion may have occurred which encompasses the exon that encodes the central segment of the sVT and sIT precursors. A possible pathway for the evolution of precursor molecules of neurohypophysial hormones is discussed.Abbreviations AVP vasopressin - C carboxyl - h human - IT isotocin - MT mesotocin - N amino - OXT oxytocin - S chum salmon - SDS sodium dodecyl sulfate - t toad - VT vasotocin     

Gelatinases of metastatic cell lines of murine colonic carcinoma as detected by substrate-gel electrophoresis

Gelatinases were detected in the conditioned medium of murine colonic carcinoma cells by SDS-polyacrylamide gel electrophoresis using gels copolymerized with gelatin. Several gelatinase activities differing in molecular weight were detected but the major activities migrated with molecular weights of 60,000 and 95,000. The enzymes did not hydrolyze bovine serum albumin or casein, and required calcium for activity. All of the gelatinase activities were inhibited by EDTA, 1,10-phenanthroline and dithiothreitol but not by N-ethylmaleimide and phenylmethylsulfonyl fluoride. The 95,000 dalton gelatinase was separated from the 60,000 dalton gelatinase by affinity chromatography on Ricinus communis agglutinin-agarose, and the former activity was markedly increased in highly metastatic cell lines as compared with its activity in poorly metastatic cell lines.     

The Site of Synthesis and Accumulation of Rice Storage Proteins

Electron microscopy showed that the two types of protein bodies(PB) in starchy endosperms of rice were formed differently duringthe period of storage protein accumulation. Two routes for thetransport of storage protein from the site of synthesis at therough endoplasmic reticulum (RER) to the site of accumulationwere also proposed. PB-I, bound by a single membrane to whichribosomes were attached, was thought to develop inside the cisternaeof RER, while the PB-II membrane was thought to originate fromthe vacuole. In the wheat germ cell-free translation system, storage protein-relatedpolypeptides of developing rice endosperms, including a precursorof glutelin and putative precursors of prolamin, were directedby membrane-bound polysomes but not by free-polysomes. Immunoassayof the total translation products directed by a PB fractionshowed that 46% were storage protein-related polypeptides. Rice storage proteins (prolamin) that accumulate in PB-I appearto be synthesized by membrane-bound polysomes attached to PB-Ior RER and to pass through the membrane into the lumen wherethey aggregate and are deposited. The proteins (glutelin andglobulin) that accumulate in PB-II, however, seem to be synthesizedby membrane-bound polysomes as a large precursor and to becomesequestered into the cisternal space of RER, from where theyare transferred to the vacuolar precursor of PB-II. (Received August 6, 1985; Accepted November 6, 1985)     

Characteristics of a Urate-Degrading Diamine Oxidase-Peroxidase Enzyme System in Soybean Radicles

The cadaverine content of soybean radicles showed a maximumpeak 3–4 days after planting. The variation coincidedwith radicle uricase activity during seed germination. The uricase activity could not be fractionate when the bufferpH for the extraction was at 6.0. The addition of 1 M KCl orNaCl to the buffer allowed the extraction of the uricase activity,but an addition of 1 M MgCl₂ or BaCl₂ inhibited this enzyme'sactivity. The urate-degrading enzyme system was purified 248-fold permilligram of protein from soybean radicles. The respective K_mvalues of the diamine oxidase activity for cadaverine and ofthe urate-degrading activity for hydrogen peroxide and uratewere 1.25, 2.93 and 50.3 µM. Analysis by gel electrophoresisof the partially purified enzyme fraction revealed that theurate-degrading enzyme system consisted of a peroxidase thatdegrades urate with hydrogen peroxide and a diamine oxidasethat releases hydrogen peroxide. These data are evidence that a urate-degrading diamine oxidaseand peroxidase system exists in soybean radicles and that thereaction rate of urate-degradation is controlled by the concentrationof cadaverine. (Received November 28, 1984; Accepted April 8, 1985)     

Efficient synthesis and secretion of a thermophilic alpha-amylase by protein-producing Bacillus brevis 47 carrying the Bacillus stearothermophilus amylase gene.

Bacillus subtilis and Bacillus brevis 47-5, carrying the Bacillus stearothermophilus alpha-amylase gene on pUB110 (pBAM101), synthesized the same alpha-amylase as the donor strain as determined by the enzyme's thermal stability and NH2-terminal amino acid sequence. Regardless of the host, the 34-amino acid signal peptide of the enzyme was processed at exactly the same site between two alanine residues. B. brevis 47-5(pBAM101) secreted the enzyme most efficiently of the hosts examined, 100, 15, and 5 times more than B. stearothermophilus, Escherichia coli HB101(pH1301), and B. subtilis 1A289(pBAM101), respectively. The efficient secretion of the enzyme in B. brevis 47-5(pBAM101) was suggested to be due to the unique properties of the cell wall of this organism.