Isolation of mitochondrial and plastid ftsZ genes and analysis of the organelle targeting sequence in the diatom Chaetoceros neogracile (Diatoms,Bacillariophyceae)

Mitochondria and plastids multiply by division in eukaryotic cells. Recently, the eukaryotic homolog of the bacterial cell division protein FtsZ was identified and shown to play an important role in the organelle division process inside the inner membrane. To explore the evolution of FtsZ proteins, and to accumulate data on the protein import system in mitochondria and plastids of the red algal lineage, one mitochondrial and three plastid ftsZ genes were isolated from the diatom Chaetoceros neogracile, whose plastids were acquired by secondary endosymbiotic uptake of a red alga. Protein import into organelles depends on the N‐terminal organelle targeting sequences. N‐terminal bipartite presequences consisting of an endoplasmic reticulum signal peptide and a plastid transit peptide are required for protein import into diatom plastids. To characterize the organelle targeting peptides of C. neogracile, we observed the localization of each green fluorescent protein‐tagged predicted organelle targeting peptide in cultured tobacco cells and diatom cells. Our data suggested that each targeting sequences functioned both in tobacco cultured cells and diatom cells.     

The initial step of the thermal unfolding of 3-isopropylmalate dehydrogenase detected by the temperature-jump Laue method

A temperature-jump (T-jump) time-resolved X-ray crystallographic technique using the Laue method was developed to detect small, localized structural changes of proteins in crystals exposed to a temperature increase induced by laser irradiation. In a chimeric protein between thermophilic and mesophilic 3-isopropylmalate dehydrogenases (2T2M6T), the initial structural change upon T-jump to a denaturing temperature (approximately 90 degrees C) was found to be localized at a region which includes a beta-turn and a loop located between the two domains of the enzyme. A mutant, 2T2M6T-E110P/S111G/S113E, having amino acid replacements in this beta-turn region with the corresponding residues of the thermophilic enzyme, showed greater stability than the original chimera (increase of T:(m) by approximately 10 degrees C) and no T-jump-induced structural change in this region was detected by our method. These results indicate that thermal unfolding of the original chimeric enzyme, 2T2M6T, is triggered in this beta-turn region.     

Chlorella virus-encoded deoxyuridine triphosphatases exhibit different temperature optima

A putative deoxyuridine triphosphatase (dUTPase) gene from chlorella virus PBCV-1 was cloned, and the recombinant protein was expressed in Escherichia coli. The recombinant protein has dUTPase activity and requires Mg(2+) for optimal activity, while it retains some activity in the presence of other divalent cations. Kinetic studies of the enzyme revealed a K(m) of 11.7 microM, a turnover k(cat) of 6.8 s(-1), and a catalytic efficiency of k(cat)/K(m) = 5.8 x 10(5) M(-1) s(-1). dUTPase genes were cloned and expressed from two other chlorella viruses IL-3A and SH-6A. The two dUTPases have similar properties to PBCV-1 dUTPase except that IL-3A dUTPase has a lower temperature optimum (37 degrees C) than PBCV-1 dUTPase (50 degrees C). The IL-3A dUTPase differs from the PBCV-1 enzyme by nine amino acids, including two amino acid substitutions, Glu81-->Ser81 and Thr84-->Arg84, in the highly conserved motif III of the proteins. To investigate the difference in temperature optima between the two enzymes, homology modeling and docking simulations were conducted. The results of the simulation and comparisons of amino acid sequence suggest that adjacent amino acids are important in the temperature optima. To confirm this suggestion, three site-directed amino acid substitutions were made in the IL-3A enzyme: Thr84-->Arg84, Glu81-->Ser81, and Glu81-->Ser81 plus Thr84-->Arg84. The single substitutions affected the optimal temperature for enzyme activity. The temperature optimum increased from 37 to 55 degrees C for the enzyme containing the two amino acid substitutions. We postulate that the change in temperature optimum is due to reduction in charge and balkiness in the active cavity that allows more movement of the ligand and protein before the enzyme and substrate complex is formed.     

One-step purification of Escherichia coli H(+)-ATPase (F0F1) and its reconstitution into liposomes with neurotransmitter transporters.

About 30% of the protein in the inner membrane of Escherichia coli strain DK8/pBWU13 is H(+)-ATPase (F0F1), and practically homogeneous F0F1 could be obtained by gradient centrifugation after solubilization of these membranes. The recombinant plasmid pBWU13 carries the unc operon for F0F1. When reconstituted into liposomes, F0F1 formed an ATP-dependent proton gradient and membrane potential. Proteoliposomes reconstituted with F0F1 and solubilized transporters from chromaffin granules or synaptic vesicle membranes could transport serotonin, dopamine, and norepinephrine dependent on ATP hydrolysis. F0F1 can be obtained rapidly from DK8/pBWU13, and its reconstitution into liposomes with transporters may be useful for monitoring these transporters during their purification.     

Effects of intraplantar injections of nociceptin and its N-terminal fragments on nociceptive and desensitized responses induced by capsaicin in mice

Injection of capsaicin into the hindpaw has been employed as a model of chemogenic nociception in mice. Intraplantar injection of nociceptin (30–240 pmol) produced a significant and dose-dependent antinociceptive activity in the capsaicin test. The nociceptin N-terminal fragments, (1–11) and (1–13), were also active with a potency higher than nociceptin and comparable to nociceptin, respectively. Intraplantar injection of the nociceptin (1–7) fragment had no effect on capsaicin-induced nociception. Antinociception induced by nociceptin or nociceptin (1–13) was reversed significantly by intraplantar co-injection of [Nphe¹]nociceptin (1–13)NH₂, an orphan opioid receptor-like 1 (ORL1) receptor antagonist, whereas local injection of the antagonist did not interfere with the action of nociceptin (1–11). Nociceptin (1–11) was approximately 2.0-fold more potent than naturally occurring peptide nociceptin, and 10-fold more active than intraplantar morphine. Nociceptive licking/biting response to intraplantar injection of capsaicin was desensitized by repeated injections of capsaicin at the interval of 15 min. Desensitization induced by capsaicin was attenuated significantly by co-injection of nociceptin at much lower doses than antinociceptive ED₅₀ for nociceptin. Capsaicin desensitization was also decreased by co-injection of nociceptin (1–11) and (1–13) to a similar extent. The present results indicate that not only nociceptin but also the N-terminal fragment (1–13) possesses a local peripheral antinociceptive action, which may be mediated by peripheral ORL1 receptors. In addition, the difference of the effective doses suggests that the antinociceptive action and inhibition of capsaicin-induced desenitization by nociceptin, nociceptin (1–11) and (1–13), may involve distinct mechanisms at the level of the peripheral nerve terminal.     

Prolactin-releasing peptide is essential to maintain the prolactin level and osmotic balance in freshwater teleost fish

We administered prolactin-releasing peptide (PrRP) or anti-PrRP antiserum to goldfish in fresh water and analyzed their effects on prolactin and osmoregulatory mechanisms. The pituitary mRNA level of prolactin increased by PrRP but decreased by anti-PrRP. The rate of water inflow in the gills decreased by PrRP and increased by anti-PrRP, showing that PrRP restricts branchial water permeability, as also restricted by prolactin. PrRP also expanded the mucous cell layers on the scales, which may restrict efficiently water inflow by the mucous system. Eventually, the plasma osmotic pressure decreased by anti-PrRP. We conclude that PrRP is essential to maintain prolactin levels and osmotic balance in fresh water.     

Norepinephrine triggers Ca2+-dependent exocytosis of 5-hydroxytryptamine from rat pinealocytes in culture

5-hydroxytryptamine (5-HT) is a precursor and a putative modulator for melatonin synthesis in mammalian pinealocytes. 5-HT is present in organelles distinct from l-glutamate-containing synaptic-like microvesicles as well as in the cytoplasm of pinealocytes, and is secreted upon stimulation by norepinephrine (NE) to enhance serotonin N-acetyltransferase activity via the 5-HT2 receptor. However, the mechanism underlying the secretion of 5-HT from pinealocytes is unknown. In this study, we show that NE-evoked release of 5-HT is largely dependent on Ca2+ in rat pinealocytes in culture. Omission of Ca2+ from the medium and incubation of pineal cells with EGTA-tetraacetoxymethyl-ester inhibited by 59 and 97% the NE-evoked 5-HT release, respectively. Phenylephrine also triggered the Ca2+-dependent release of 5-HT, which was blocked by phentolamine, an alpha antagonist, but not by propranolol, a beta antagonist. Botulinum neurotoxin type E cleaved 25 kDa synaptosomal-associated protein and inhibited by 50% of the NE-evoked 5-HT release. Bafilomycin A1, an inhibitor of vacuolar H+-ATPase, and reserpine and tetrabenazine, inhibitors of vesicular monoamine transporter, all decreased the storage of vesicular 5-HT followed by inhibition of the NE-evoked 5-HT release. Agents that trigger L-glutamte exocytosis such as acetylcholine did not trigger any Ca2+-dependent 5-HT release. Vice versa neither NE nor phenylephrine caused synaptic-like microvesicle-mediated l-glutamate exocytosis. These results indicated that upon stimulation of a adrenoceptors pinealocytes secrete 5-HT through a Ca2+-dependent exocytotic mechanism, which is distinct from the exocytosis of synaptic-like microvesicles.     

Novel application of Shochu distillery by-products to prophylaxis against mammary carcinogenesis induced by 7,12-dimethylbenz[a]anthracene in rats

The effect of the heat-dried product of Shochu distillery by-products (HSDB) derived from sweet potato on mammary carcinogenesis in rats was investigated. HSDB was fed at 2.5% or 5% of the total feed weight. Dietary HSDB at the 5% level suppressed the incidence and number of tumors, and delayed the latency of mammary tumor development relative to the control diet. Experiments were conducted to determine the relative polarity of the anticarcinogenic constituent(s). The number of tumors per tumor-bearing rat was lower in the diet group fed with an ethyl acetate extract of HSDB than in the control group. The tumor incidence evaluated at both palpation and autopsy was slightly lower in the group fed with a methanol extract than in the control group. These results suggest that HSDB contained at least two constituents of differing polarity that counteracted mammary carcinogenesis.     

Negative regulation of the E3 ubiquitin ligase itch via Fyn-mediated tyrosine phosphorylation

Conjugation of ubiquitin (Ub) to a protein substrate targets the substrate for degradation or functional modification, which is tightly controlled by diverse mechanisms including phosphorylation of the substrate. An emerging mechanism involves regulation of the E3 Ub ligase, for example, the JNK-dependent phosphorylation and activation of Itch E3 ligase, which controls the turnover of Jun proteins and T cell differentiation. Here we show that Itch is also modulated by an Src kinase Fyn via tyrosine phosphorylation at the Tyr371 residue. Fyn associates with Itch, and loss of Fyn results in reduced Itch phosphorylation. Importantly, tyrosine phosphorylation of Itch appears to reduce its interaction with its substrate JunB. The turnover of JunB is accelerated in Fyn-deficient T cells, which is further reconstituted by Itch Tyr371 mutation. Thus, in contrast to the activation pathway mediated by serine/threonine phosphorylation, tyrosine phosphorylation of Itch plays a negative role in modulating Itch-promoted ubiquitination.