Yasuo Aizono

TGTCACA Motif Is a Novel cis-Regulatory Enhancer Element Involved in Fruit-specific Expression of the cucumisin Gene*

Cucumisin, a subtilisin-like serine protease, is expressed at high levels in the fruit of melon (Cucumis melo L.) and accumulates in the juice. We investigated roles of the promoter regions and DNA-protein interactions in fruit-specific expression of the cucumisin gene. In transient expression analysis, a chimeric gene construct containing a 1.2-kbcucumisin promoter fused to a β-glucuronidase (GUS) reporter gene was expressed in fruit tissues at high levels, but the promoter activities in leaves and stems were very low. Deletion analysis indicated that a positive regulatory region is located between nucleotides −234 and −214 relative to the transcriptional initiation site. Gain-of-function experiments revealed that this 20-bp sequence conferred fruit specificity and contained a regulatory enhancer. Gel mobility shift experiments demonstrated the presence of fruit nuclear factors that interact with thecucumisin promoter. A typical G-box (GACACGTGTC) present in the 20-bp sequence did not bind fruit protein, but two possiblecis-elements, an I-box-like sequence (AGATATGATAAAA) and an odd base palindromic TGTCACA motif, were identified in the promoter region between positions −254 and −215. The I-box-like sequence bound more tightly to fruit nuclear protein than the TGTCACA motif. The I-box-like sequence functions as a negative regulatory element, and the TGTCACA motif is a novel enhancer element necessary for fruit-specific expression of the cucumisin gene. Specific nucleotides responsible for the binding of fruit nuclear protein in these two elements were also determined.

Molecular cloning and characterization of a cDNA and a gene for subtilisin-like serine proteases from rice (Oryza sativa L.) and Arabidopsis thaliana.

The complete nucleotide sequences of a cDNA (RSP1) that encodes a subtilisin-like serine protease (subtilase) of rice (Oryza sativa L.) and a gene (ASP48) for Arabidopsis subtilase were analyzed. The RSP1 cDNA and ASP48 DNA encoded 736- and 757-residue pre-pro-polypeptides including a signal peptide with molecular masses of 78,668 Da and 79,414 Da, respectively. RSP1 is the first known serine protease in rice, and ASP48 is a gene for ara12 cDNA. Sequence comparison and phylogenetic analysis showed that RSP1 is distantly related to all other plant subtilases and ASP48 is closely related to a tomato subtilase, SBT1. The ASP48 gene was found to lack introns. The Arabidopsis subtilase gene appears to consist of a small gene family. The RSP1 was found to be expressed in seed and shoots of seedlings while ASP48 transcripts was found to be accumulated in immature silique and flowers, indicating that both RSP1 and ASP48 are organ-specific and may be involved in the specific proteolytic events that occur during organ development.

The carbachol-induced release of prothoracicotropic hormone from brain-corpus cardiacum-corpus allatum complex of the silkworm, Bombyx mori

The in vitro release of prothoracicotropic hormone (PTTH) from brain-corpus cardiacum-corpus allatum complex (brain-CC-CA complex) of Bombyx mori was stimulated significantly with carbachol (acetylcholine agonist) in the range of 0.01–10 mM. One mM musucarine also induced the release of PTTH within 5 min, while 1 mM nicotine exerted no effect. In addition, 5 mM atropine (muscarinic antagonist) inhibited the PTTH release induced with 0.1 mM carbachol. From these results, it became apparent that the muscarinic acetylcholine receptor was associated with the release of PTTH. To explore the signal transduction mediated by the muscarinic acetylcholine receptor in the PTTH release, the effects of phospholipase C (PLC)-inhibitor, calmodulin antagonist and protein kinase C (PKC)-inhibitor on the PTTH release induced with 0.1 mM carbachol were examined. 2-nitro-4-carboxyphenyl-N,N-diphenylcalbamate (NCDC: PLC-inhibitor), N-(6-aminohexyl)-5-chloro-1-1-naphthalenesulfonamide (W-7: calmodulin antagonist) and calphostin C (PKC-inhibitor) caused dose-dependent suppression of the carbachol-induced release of PTTH, and 10 μM NCDC, 500 μM W-7 and 10 μM calphostin C suppressed the release of PTTH completely. These results suggested that activation of phospholipase C, and subsequent protein phosphorylations by PKC and calcium/calmodulin dependent protein kinase (CaMK) were involved in the carbachol-induced signal transduction in the PTTH release.

Prothoracicotropic hormone-producing neurosecretory cells in the silkworm, Bombyx mori, express a muscarinic acetylcholine receptor.

Using an anti-muscarinic acetylcholine receptor (mAChR) antibody and an anti-prothoracicotropic hormone (PTTH) antibody, double immunofluorescence staining was performed on brain sections of the silkworm, Bombyx mori. Four pairs of dorsolateral neurosecretory cells, along with some intercerebral neurosecretory cells, were immunoreactive to anti-mAChR antibody. Among these immunoreactive cells, two pairs of dorsolateral neurosecretory cells were identified to be PTTH-producing neurosecretory cells. Nerve fibers in the median and paramedian protocerebral areas, and nerve terminals in the corpus allatum also showed immunoreactivity to the anti-mAChR antibody. Some of these nerve terminals expressing mAChRs were overlapped by immunostaining with the anti-PTTH antibody. These results indicated that PTTH-producing neurosecretory cells of Bombyx mori expressed an mAChR, and that muscarinic, cholinergic transmission might directly regulate PTTH release from neurosecretory cells.

Characteristics of Psychrophilic Alkaline Phosphatase

The phosphatase of a psychrophile (Shewanella sp.) was purified by ammonium sulfate fractionation, followed by sequential column chromatographies. The purified enzyme was electrophoretically homogeneous on native- and SDS-PAGE. Its molecular weight was 41,826 by its amino acid composition. The enzyme had its optimal pH for the activity at 9.8, and a broad substrate specificity to dephosphorylate ATP, pyrophosphate, glycerophosphate, and so on. Its activity was increased by metal ions including Mg(2+), Mn(2+), and Co(2+). The maximal activity was observed at 40°C, and the enzyme at 0°C showed 39% of activity at 40°C. The enzyme, however, tended to lose its activity at 20°C and pH 9.8. These results indicated that purified enzyme was an alkaline phosphatase with characteristics; high catalytic efficiency at low temperature and gradual inactivation at an intermediate temperature.

Cloning of Cold-active Alkaline Phosphatase Gene of a Psychrophile, Shewanella sp., and Expression of the Recombinant Enzyme

A psychrophilic alkaline phosphatase (EC 3.1.3.1) from Shewanella sp. is a cold-active enzyme that has high catalytic activity at low temperature [Ishida et al. (1998) Biosci. Biotechnol. Biochem., 62, 2246–2250]. Here, we identified the nucleotide sequence of a gene encoding the enzyme after cloning with the polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence of the enzyme contained conserved amino acids found among mesophilic alkaline phosphatases and showed some structural characteristics including a high content of hydrophobic amino acid residues and the lack of single α-helix compared with the alkaline phosphatase of Escherichia coli, which were possibly efficient for catalytic reaction at low temperatures. The recombinant enzyme expressed in E. coli was purified to homogeneity with the molecular mass of 41 kDa. The recombinant enzyme had a specific activity of 1,500 units/mg and had high catalytic activity at low temperatures.

Light activates a 46-kDa MAP kinase-like protein kinase in soybean cell culture

Light induced rapid and transient activation of a 46‐kDa protein kinase in soybean photomixotrophic cell culture. This kinase was designated as LAP kinase (light signal‐activated protein kinase). Activation of LAP kinase in response to light was associated with tyrosine phosphorylation of the kinase, and treatment of the kinase with protein tyrosine phosphatase abolished its activity. The LAP kinase efficiently phosphorylated myelin basic protein and histone, but did not phosphorylate casein. Phospho‐amino acid analysis indicated that the LAP kinase was a serine/threonine protein kinase. These results indicated that the LAP kinase is related to the MAP kinase family of protein kinases.

Crystal Structure of Cold-Active Alkaline Phosphatase from the Psychrophile Shewanella sp.

The crystal structure of a cold-active alkaline phosphatase from a psychrophile, Shewanella sp. (SCAP), was solved at 2.2 Å. A refined model showed a homodimer with six metal-ligand sites. The arrangement of the catalytic residues resembled those of alkaline phosphatases (APs), suggesting that the reaction mechanism of SCAP was fundamentally identical to those of other APs. SCAP had two distinct structural features: (i) a loop with Arg122 that bound to the phosphate moiety of the substrate suffered no constraints from the linkage to other secondary structures, and (ii) Mg3-ligand His109 was considered to undergo repulsive effect with neighboring Trp228. The local flexibility led by these features might be an important factor in the high catalytic efficiency of SCAP at low temperatures.

Small GTP-binding proteins in the brain-corpus cardiacum-corpus allatum complex of the silkworm, Bombyx mori: Involvement in the secretion of prothoracicotropic hormone

At least three GTP-binding proteins (G-proteins), 28, 25, and 21 kDa, were found in the brain-corpus cardiacum-corpus allatum complex (BR-CC-CA) of the silkworm, Bombyx mori. They bound to GTP and GDP specifically among nucleotides tested, indicating that these proteins are small G-proteins. The 25 kDa G-protein showed a cross-reactivity to anti-rab3A antibody, while it did not cross-react with anti-rhoA, rab3B, and anti-ras antibodies. On the other hand, the 28 and 21 kDa G-proteins showed no cross-reactivity to any of those antibodies tested. Immunoblot analysis using the anti-rab3A antibody demonstrated that the 25 kDa G-protein was detected preferentially in the BR-CC-CA, and to some extent in the suboesophageal ganglion, but not in the salivary gland, fat body, prothoracic gland, and oesophagus. These results suggested that the 25 kDa G-protein was a member of the rab family of G-proteins. Furthermore, 1 mM GTP gamma S capable of activating G-proteins induced BR-CC-CA to release PTTH under the conditions that stimulation of the PTTH release with hetero-trimeric G-protein was suppressed. These results indicated that the small G-proteins may possibly contribute to PTTH release in Bombyx mori.

The in vitro release of prothoracicotropic hormone (PTTH) from the brain—corpus cardiacum—corpus allatum complex of silkworm, Bombyx mori

Abstract An in vitro system for evaluation of the exact PTTH release from the brain-CC-CA complex of Bombyx mori was developed. The PTTH release induced with a high potassium ion concentration reached the maximal level in 30 min and was dependent on the concentration of the potassium ion. The PTTH release was also dependent on the calcium ion and was induced with a calcium ionophore, indicating that increasing intracellular calcium ion might be essential for its release. Carbachol (acetylcholine agonist) induced the PTTH release fastest, followed by dopamine and serotonin in that order, while norepinephrine exerted no effect. Carbachol might act in the brain of brain-CC-CA complex.