The effect of alpha2-macroglobulin from bovine serum on bovine alpha-chymotrypsin.

Bovine alpha2-globulin contains a protein which increases the activity of bovine alpha-chymotrypsin against synthetic substrates. The active protein fraction migrates slowly on polyacrylamide gel electrophoresis, so it was named slow alpha2-globulin (Salpha2). The fraction was isolated from bovine serum and purified. Its sedimentation constant S20 was 18.5 S. It was thus identified with the alpha2-macroglobulin (alpha2M). By kinetic studies, the dissociation constant of the alpha-chymotrypsin-alpha2 M complex was calculated to be of the order of 10(-7) l/mol. The purified alpha2 M was shown to bind alpha-chymotrypsin at a definite rate. If the binding ratio was assumed to be 1:2, the molecular weight was calculated to be about 8 X 10(5).     

Alpha reduced nicotinamide adenine dinucleotide-dependent reductase reactions of rat liver microsomes.

The kinetics of alpha-NADH-dichlorophenolindophenol (DCPIP) and alpha-NADH-cytochrome c reductase reactions of rat liver microsomes showed that the reactio ns proceeded by a ping-pong mechanism, and that the oxidation of alpha-NADH was the rate-determining reaction. The DCPIP-reducing activity with alpha-NADH in the presence of ADP was about 1% of that with beta-NADH. ADP inhibited the alpha-NADH-DCPIP reductase reaction in a competitive manner with respect to alpha-NADH and a value of 1.2 mM for the inhibition constant was obtained. ADP also inhibited cytochrome b5 reduction with alpha-NADH. More than 90% of cytochrome b5 was reduced under conditions where 90% of the alpha-NADH-DCPIP reductase activity was suppressed with ADP. The reduction of DCPIP with alpha-NADH preceded that of cytochrome b5, but the reductions partly overlapped. From these results, a diversed electron flow from alpha-NADH to cytochrome b5 and electron sharing between cytochrome b5 and DCPIP were indicated. alpha-NAD+ also inhibited the alpha-NADH-DCPIP reductase reaction. Analyses of the inhibition indicated that two types of alpha-NADH-DCPIP reductase reaction existed, one of which was resistant to alpha-NAD+ inhibition. In contrast to the reoxidation of beta-NADH-reduced cytochrome b5, the process was largely monophasic when cytochrome b5 was reduced with alpha-NADH.     

Molecular cloning and expression analysis of novel wheat cysteine protease

A cDNA clone encoding a novel papain-like cysteine protease was isolated from wheat germ (Triticum aestivum). This cDNA encoded a 371-residue protein, designated WCP2, composed of signal peptide followed by a propeptide and a mature protease containing active site residues that are highly conserved among the papain family. The mature WCP2 protein (26 kDa) was detected in the quiescent embryo and its level of expression in the germinating embryo was greatly increased.     

Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor.

We have previously shown that Madin-Darby canine kidney (MDCK) epithelial cells grown in collagen gels in the presence of fibroblasts or fibroblast-conditioned medium (CM) form branching tubules, instead of the spherical cysts that develop under control conditions. We now report that the fibroblast-derived molecule responsible for epithelial tubulogenesis is hepatocyte growth factor (HGF). First, addition of exogenous HGF to cultures of MDCK cells induces formation of epithelial tubules. Second, the tubulogenic activity of fibroblast CM is completely abrogated by antibodies to HGF. These results demonstrate that HGF, a polypeptide that was identified as a mitogen for cultured hepatocytes, has the properties of a paracrine mediator of epithelial morphogenesis, and suggest that it may play important roles in the formation of parenchymal organs during embryonic development.     

Colcemid treatment of myeloma prior to cell fusion increases the yield of hybridomas between myeloma and splenocyte

Effect of Colcemid treatment of myeloma (X63-Ag8-6.5.3.) prior to fusion with mouse spleen cell was studied in terms of hybridoma formation. Spleen cells from BALB/c mice immunized with various soluble antigens were fused with the myeloma cells by using polyethylene glycol solution. Colcemid treatment of myeloma cells prior to fusion increased the average number of hybridoma colonies per well by 26-570%. The yield of hybridomas producing antigen-specific antibodies was also higher with the Colcemid treatment. The results suggest that most of the proliferative hybridomas are formed by fusion of cells in the M-phase of the cell cycle.     

Aromatic amino acid transaminase in rat intestine

The transamination of aromatic l-amino acids (5-hydroxytryptophan, tryptophan, tyrosine, phenylalanine and kynurenine) was shown to be catalysed by enzyme preparations from rat small intestine. On the basis of the partial purification and characterization of these aromatic amino acid transaminases, it is suggested that rat small intestine contains several kinds of aromatic amino acid transaminases.     

Molecular cloning of three homoeologous cDNAs encoding orthologs of the maize KNOTTED1 homeobox protein from young spikes of hexaploid wheat

The plant knotted1 (kn1)-like homeobox genes are known to play important roles in the maintenance of shoot apical meristem (SAM), determination of cell fate and differentiation of vegetative tissues. To study structural diversity of the three homologous loci encoding a KN1-like homeobox protein in the hexaploid wheat genome, we isolated clones from a cDNA library of young spikes of Japanese common wheat cultivar 'Norin 26'. Three different but highly homologous cDNAs were isolated and their sequences were determined. The mean homology of the deduced amino acid sequences was 96% as compared to the barley ortholog KNOX3. The wheat kn1-like homeobox proteins named WKNOX1 are encoded by a single set of homologous genes on the homologous group 4 chromosomes in the three component genomes of common wheat, i.e. 4A, 4B and 4D. The nucleotide sequence data and the Southern blot pattern suggested that the three homologous loci of wknox1 genes are highly conserved through polyploid evolution of wheat. They were expressed in SAM-containing shoots and young spikes but not in developed leaves, glumes and lemmas and callus tissues. The ectopic expression of the wknox1 was observed in lemma of wheat Hooded (Hd) mutants. The result suggested that the Hd gene is a dominant allele of the wknox1 locus on chromosome 4A.     

Human aquaporin adipose (AQPap) gene. Genomic structure, promoter analysis and functional mutation.

Aquaporin adipose (AQPap), which we identified from human adipose tissue, is a glycerol channel in adipocyte [Kishida et al. (2000) J. Biol. Chem. 275, 20896-20902]. In the current study, we determined the genomic structure of the human AQPap gene, and identified three AQPap-like genes that resembled (approximately 95%) AQPap, with little expression in human tissues. The AQPap promoter contained a putative peroxisome proliferator response element (PPRE) at -46 to -62, and a putative insulin response element (IRE) at -542/-536. Deletion of the PPRE abolished the pioglitazone-mediated induction of AQPap promoter activity in 3T3-L1 adipocytes. Deletion and single base pair substitution analysis of the IRE abolished the insulin-mediated suppression of the human AQPap gene. Analysis of AQPap sequence in human subjects revealed three missense mutations (R12C, V59L and G264V), and two silent mutations (A103A and G250G). The cRNA injection of the missense mutants into Xenopus oocytes revealed the absence of the activity to transport glycerol and water in the AQPap-G264V protein. In the subject homozygous for AQPap-G264V, exercise-induced increase in plasma glycerol was not observed in spite of the increased plasma noradrenaline. We suggest that AQPap is responsible for the increase of plasma glycerol during exercise in humans.     

cis‐Regulatory analysis for later phase of anterior neuroectoderm‐specific foxQ2 expression in sea urchin embryos

The specification of anterior neuroectoderm is controlled by a highly conserved molecular mechanism in bilaterians. A forkhead family gene, foxQ2, is known to be one of the pivotal regulators, which is zygotically expressed in this region during embryogenesis of a broad range of bilaterians. However, what controls the expression of this essential factor has remained unclear to date. To reveal the regulatory mechanism of foxQ2, we performed cis‐regulatory analysis of two foxQ2 genes, foxQ2a and foxQ2b, in a sea urchin Hemicentrotus pulcherrimus. In sea urchin embryos, foxQ2 is initially expressed in the entire animal hemisphere and subsequently shows narrower expression restricted to the anterior pole region. In this study, as a first step to understand the foxQ2 regulation, we focused on the later restricted expression and analyzed the upstream cis‐regulatory sequences of foxQ2a and foxQ2b by using the constructs fused to short half‐life green fluorescent protein. Based on deletion and mutation analyses of both foxQ2, we identified each of the five regulatory sequences, which were 4–9 bp long. Neither of the regulatory sequences contains any motifs for ectopic activation or spatial repression, suggesting that later mRNA localization is regulated in situ. We also suggest that the three amino acid loop extension‐class homeobox gene Meis is involved in the maintenance of foxQ2b, the expression of which is dominant during embryogenesis.