E-2(S)-amino-3-methyl-3-pentenoic acid from coniogramme intermedia

A new amino acid, E-2(S)-amino-3-methyl-3-pentenoic acid was isolated from Coniogramme intermedia. The structure was elucidated by elementary analysis, optical rotation, catalytic hydrogenation, ¹H and ¹³C NMR spectra.     

Identification of the Carboxyl-Terminal Processing Protease for the D1 Precursor Protein of the Photosystem II Reaction Center of Spinach

The enzyme involved in the carboxyl-terminal processing of theD1 precursor protein (pD1) of the photosystem II reaction centerwas purified from extracts of sonicated spinach thylakoids bya method that included chromatography on quaternary aminoethylanion-exchange, hydroxylapatite, copper-chelating affinity andgel-filtration columns. The enzyme was identified, from itschromatographic behavior, to be a monomeric protein of about45 kDa. The sequence of the amino-terminal 27 amino acids ofthis protein was determined directly, which exhibited low butappreciable (37%) homology to that deduced from a gene (ctpA)in Synechocystis sp. PCC 6803 that was proposed recently toencode the processing protease from results of genetic complementationanalysis. ³Present address: Asahi Kasei Chem. Co.     

Reversible Inhibition of Gap Junctional Intercellular Communication, Synchronous Contraction, and Synchronism of Intracellular Ca Fluctuation in Cultured Neonatal Rat Cardiac Myocytes by Heptanol

We analyzed by Fotonic Sensor, a fiber-optic displacement measurement instrument, the effects of heptanol on synchronized contraction of primary neonatal rat cardiac myocytes cultured at confluent density. We also examined the effect of heptanol on the changes in gap junctional intercellular communication by using the microinjection dye transfer method, and on intercellular Ca²⁺ fluctuation by confocal laser scanning microscopy of myocytes loaded with the fluorescent Ca²⁺ indicator fluo 3. In addition, we studied expression, phosphorylation, and localization of the major cardiac gap junction protein connexin 43 (Cx43) using immunofluorescence and Western blotting. At Day 6 of culture, numerous myocytes exhibited spontaneous, synchronous contractions, excellent dye coupling, and synchronized intracellular Ca²⁺ fluctuations. We treated the cells with 1.5, 2.0, 2.5, and 3.0 mmol/liter heptanol. With 1.5 mmol/liter heptanol, we could not observe significant effects on spontaneous contraction of myocytes. At 3.0 mmol/liter, the highest concentration used in the current experiment, heptanol inhibited synchronous contractions and even after washing out of heptanol, synchronous contraction was not rapidly recovered. On the other hand, at the intermediate concentrations of 2.0 and 2.5 mmol/liter, heptanol reversely inhibited synchronized contraction, gap junctional intercellular communication, and synchronization of intracellular Ca²⁺ fluctuations in the myocytes without preventing contraction and changes of intracellular Ca²⁺ in individual cells. Brief exposure (5-20 min) to heptanol (2.0 mmol/liter) did not cause detectable changes in the expression, phosphorylation, or localization of Cx43, despite strong inhibition of gap junctional intercellular communication. These results suggest that gap junctional intercellular communication plays an important role in synchronous intracellular Ca²⁺ fluctuations, which facilitate synchronized contraction of cardiac myocytes.     

Inhibition of Ischemia-Induced Fodrin Breakdown by a Novel Phenylpyrimidine Derivative NS-7: An Implication for Its Neuroprotective Action in Rats with Middle Cerebral Artery Occlusion

Abstract: The effect of a novel neuroprotective compound, NS-7[4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride], on ischemia-induced fodrin breakdown was examined both in vitro and in vivo. The fodrin breakdown was measured by western blot followed by a densitometric analysis. In slices of the rat cerebral cortex, a pronounced fodrin breakdown was observed under hypoxic and hypoglycemic conditions. The enhancement of fodrin breakdown was completely blocked by omission of extracellular Ca²⁺ and significantly inhibited by calpain inhibitors such as E-64 and calpain inhibitor-I, thereby suggesting that the fodrin breakdown induced by hypoxia/hypoglycemia is due to the activation of Ca²⁺-stimulated neutral protease calpain. NS-7 (1–30 µ M ) produced a concentration-dependent inhibition of hypoxia/hypoglycemia-induced fodrin breakdown. In rats with unilateral middle cerebral artery occlusion (MCAO), a pronounced fodrin breakdown was observed in the cerebral cortex and striatum, although the time course for the development of the fodrin breakdown was much slower in the cerebral cortex than in the striatum. NS-7 (0.5 mg/kg i.v.), when injected immediately after MCAO, suppressed not only the fodrin breakdown but also the infarction in the cerebral cortex. From these results it is suggested that inhibition of calpain activation is implicated in the neuroprotective action of NS-7.     

A mechanism of mitochondrial damage induced by tert-butyl hydroperoxide and microsomes in vitro

Isolated potato ( Solanum tuberosum L. cv. Dansyaku) tuber mitochondria showed a significant loss in respiratory activity when treated with tert -butyl hydroperoxide (BHP), especially in the presence of microsomes. The following alterations appeared in parallel with the gradual decrease in the respiratory activity: The outer membrane became leaky, probably due to peroxidation of phospholipids. The level of sulfhydryl (SH) groups in mitochondrial proteins decreased in contrast to non-protein SH groups. A considerable amount of phospholipids was degraded and lost. A mechanism of the mitochondrial damage induced by BHP and microsomes is discussed with respect to a significant role of free radicals which may be formed at the onset of senescence or physiological disorders.     

Prediction of the coding sequences of mouse homologues of KIAA gene: IV. The complete nucleotide sequences of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse homologues of human KIAA and FLJ genes since 2001. As an extension of these projects, we herein present the entire sequences of 500 mKIAA cDNA clones and 4 novel cDNA clones that were incidentally identified during this project. We have isolated cDNA clones from the size-fractionated mouse cDNA libraries derived from 7 tissues and 3 types of cultured cells. The average size of the 504 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 807 amino acid residues. We assigned the integrity of CDSs from the comparison with the corresponding human KIAA cDNA sequences. The comparison of mouse and human sequences revealed that two different human KIAA cDNAs are derived from single genes. Furthermore, 3 out of 4 proteins encoded in the novel cDNA clones showed moderate sequence similarity with human KIAA proteins, thus we could obtain new members of KIAA protein families through our mouse cDNA projects.     

Autonomous migration of human fetal skin fibroblasts into a denuded area in a cell monolayer is mediated by basic fibroblast growth factor and collagen

Summary Human fetal skin fibroblasts (TIG-3S) were found to migrate into a denuded area in a cell monolayer when cultured in both serum-depleted and serum-supplemented media, unlike adult-donor skin fibroblasts which migrated well only when cultured in serum-supplemented medium. Therefore, a series of experiments was carried out to determine whether autocrine factors are involved in their migration. The migration of TIG-3S cells in serum-depleted medium was suppressed by the addition of suramin, a factor with growth factor antagonist properties, which suggests that growth factors are important for cell migration. The suramin-induced inhibition was reversed completely by adding excess basic fibroblast growth factor (bFGF) to the culture medium and partially by platelet-derived growth factor (PDGF). Treatment with neutralizing anti-PDGF antibody did not suppress TIG-3S cell migration, whereas neutralizing anti-bFGF antibody did, which indicates that bFGF is an autocrine and PDGF a paracrine factor involved in cell migration. Next, an experiment was performed to ascertain whether the extracellular matrix is involved in TIG-3S cell migration. Monensin, an inhibitor of extracellular matrix secretion, inhibited cell migration, which was reversed by adding excess type I collagen, but not excess plasma fibronectin. In addition, further evidence for the involvement of collagen was provided by the observation that ethyl-3,4-dihydroxybenzoate, a specific inhibitor of collagen synthesis, suppressed cell migration. These results suggest that the autonomous migration of TIG-3S human fetal skin fibroblasts is mediated by bFGF and type I collagen, which they produce and secrete.     

IDENTIFICATION OF WHALE SPECIES BY LIQUID CHROMATOGRAPHIC ANALYSIS OF SARCOPLASMIC PROTEINS

Liquid chromatography (LC) was applied to identify whale species by analyzing water-soluble sarcoplasmic proteins in skeletal muscles. Twenty-five samples from four baleen whale species (fin whale, sei whale, Bryde's whale, and minke whale) and eight toothed whale species (sperm whale, Baird's beaked whale, short-finned pilot whale, Dall's porpoise, northern right whale dolphin, Pacific white-sided dolphin, common dolphin, and striped dolphin) were analyzed. Water-soluble sarcoplasmic proteins were extracted from each sample and analyzed using a UV-VIS spectrophotometric detector at 280 nm and a pho-todiode array detector. The chromatographic profiles of each sample showed distinctive qualitative and quantitative characteristics for each whale species, making species identification possible. A photodiode array detector was useful for further accurate identification of whale species by obtaining the absorption spectra of separated protein peaks. These results suggest that the LC method is readily applicable to rapid, simple, and reliable identification of whale species.     

Stereoselective and manganese-dependent sulfation and urinary excretion of -form and -form meta-tyrosine O-sulfate by Sprague–Dawley rats

In our previous studies, we have demonstrated the stereoselective and manganese-dependent sulfation of tyrosine and Dopa isomers by human monoamine (M)-form phenol sulfotransferase (PST). In the present study, we investigated the occurrence of these phenomena in vivo using Sprague–Dawley rats as an experimental model. Three groups of six male rats were orally introduced with 1 ml of, respectively, 3 mM, 10 mM and 30 mM MnCl₂ with a constant level of 0.2 mM -m-tyrosine per day for 7 days. Their urine was collected and analyzed for the presence of sulfated -m-tyrosine ( -m-TyrS) by ion-pair HPLC using a C18 reversed-phase column. The level of urinary -m-TyrS, which was detected in the urine of the MnCl₂-treated rats but not control rats, appeared to increase proportionally to the amount of MnCl₂ administered. Chiral HPLC was employed to differentiate the -form and -form m-TyrS present in the urine sample of MnCl₂-treated rats. Both -m-TyrS and -m-TyrS were detected, with the -form being present at significantly higher level than the -form.