Copper elicits an increase in cytosolic free calcium in cultured tobacco cells

At concentrations greater than 0.1 mM, CuSO(4) provoked a rapid and sustained increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), in tobacco suspension culture cells expressing apoaequorin, a Ca(2+)-sensitive photoprotein. The increase was suppressed by treatment with LaCl(3), indicating that the increase is due to an influx of Ca(2+) from the apoplast through plasma membrane Ca(2+) channels. Although stimulation of H(2)O(2) production upon the CuSO(4) treatment (0.1 mM) was observed, treatment with catalase did not inhibit the increase in [Ca(2+)](cyt), and treatment with H(2)O(2) dose-dependently suppressed or delayed the increase. These results suggested that active oxygen species generated through copper-mediated reactions, or copper-mediated oxidative damages to plasma membrane, are not responsible for the increase. Treatment with sulfhydryl reagents, which alkylate or oxidize thiol groups, or acidification of the culture medium suppressed the increase in [Ca(2+)](cyt). These results demonstrated that copper causes an influx of Ca(2+) through plasma membrane Ca(2+) channels, and that plasma membrane thiol groups play an important role in activating the Ca(2+) channels.     

Magnesium gating of cardiac gap junction channels

We aimed to study kinetics of modulation by intracellular Mg²⁺ of cardiac gap junction (Mg²⁺ gate). Paired myocytes of guinea-pig ventricle were superfused with solutions containing various concentrations of Mg²⁺. In order to rapidly apply Mg²⁺ to one aspect of the gap junction, the non-junctional membrane of one of the pair was perforated at nearly the connecting site by pulses of nitrogen laser beam. The gap junction conductance (G_j) was measured by clamping the membrane potential of the other cell using two-electrode voltage clamp method. The laser perforation immediately increased G_j, followed by slow G_j change with time constant of 3.5 s at 10 mM Mg²⁺. Mg²⁺ more than 1.0 mM attenuated dose-dependently the gap junction conductance and lower Mg²⁺ (0.6 mM) increased G_j with a Hill coefficient of 3.4 and a half-maximum effective concentration of 0.6 mM. The time course of G_j changes was fitted by single exponential function, and the relationship between the reciprocal of time constant and Mg²⁺ concentration was almost linear. Based on the experimental data, a mathematical model of Mg²⁺ gate with one open state and three closed states well reproduced experimental results. One-dimensional cable model of thirty ventricular myocytes connected to the Mg²⁺ gate model suggested a pivotal role of the Mg²⁺ gate of gap junction under pathological conditions.     

Distribution of cytoplasmic determinants in unfertilized eggs of the ascidian Halocynthia roretzi

 Cytoplasmic determinants that specify the fate of endoderm, muscle and epidermis cells are known to be localized in specific areas of fertilized eggs of ascidians. The presence of such cytoplasmic determinants in unfertilized eggs was demonstrated in previous studies, but no information has yet been proved about their distribution. To investigate the distribution of cytoplasmic determinants in unfertilized eggs, we devised a method for distinguishing the polarity of unfertilized eggs using vital staining and we performed cytoplasmic-transfer experiments by fusing blastomeres and cytoplasmic fragments from various identified regions of unfertilized eggs. Cytoplasmic fragments, that contained cortical and subcortical material, from five different positions along the animal-vegetal axis were prepared, and they were fused with a4.2 (presumptive-epidermis) or A4.1 (non-epidermis) blastomeres. The ectopic development of endoderm, muscle and epidermis cells that was promoted by the transplanted cytoplasm was assessed by examining the expression of alkaline phosphatase (ALP), myosin and epidermis-specific antigen, respectively. Differentiation of endoderm and muscle was observed at higher frequencies as cytoplasmic fragments closer to the vegetal pole were transplanted. Conversely, formation of epidermis was observed at higher frequencies as cytoplasmic fragments closer to the animal pole were transplanted. The results suggest that, in cortical and subcortical regions of unfertilized ascidian eggs, endoderm and muscle determinants are widely distributed along a gradient, with maximum activity at the vegetal pole, whilst epidermis determinants are also distributed along a gradient but with maximum activity at the animal pole. Recieved: 10 June 1996 / Accepted: 12 September 1996     

Linear elements are stable structures along the chromosome axis in fission yeast meiosis

Chromosoma - The structure of chromosomes dramatically changes upon entering meiosis to ensure the successful progression of meiosis-specific events. During this process, a multilayer proteinaceous...     

Cumulative Effect of Amino Acid Replacements Results in Enhanced Thermostability of Potato Type L α-Glucan Phosphorylase

The thermostability of potato type L α-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations—Phe39→Leu (F39L), Asn135→Ser (N135S), and Thr706→Ile (T706I)—by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60°C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65°C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.     

Structure-activity relationship studies of S1P agonists with a dihydronaphthalene scaffold

Structure-activity relationship (SAR) of sphingosine-1-phosphate receptor agonists with a dihydronaphthalene scaffold was investigated. Compound 1 was modified to improve S1P(1) agonistic activity and in vivo peripheral lymphocyte lowering (PLL) activity without impairing selectivity over S1P(3) agonistic activity. A detailed SAR study of the terminal lipophilic part revealed that the introduction of substituents on the propylene linker and the terminal benzene ring influences in vitro and PLL activities. Compound 6n bearing a (S)-methyl group at the 2-position on the propylene linker and chlorine at the para-position on the terminal benzene ring showed potent hS1P(1) agonistic activity with excellent selectivity over hS1P(3) and in vivo PLL activity in mice.     

Molecular Cloning and Expression of a Novel Cholinephosphotransferase Involved in Glycoglycerophospholipid Biosynthesis of <Emphasis Type="Italic">Mycoplasma fermentans</Emphasis>

A gene, mf1, encoding a novel cholinephosphotransferase in glycoglycerophospholipid (GGPL) biosynthesis of Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, and expressed in Escherichia coli. The mf1 gene comprises an open reading frame of 777 bp encoding 258 amino acids. The mf1 gene product, Mf1, has 23% amino acid homology with LicD of Haemophilus influenzae but no homology with genes of other Mycoplasma species in the GenBank database. The reaction product of Mf1 using α-glucopyranosyl-1,2-dipalmitoilglycerol and cytidine 5′-diphosphocholine (CDP-choline) as substrates showed the specific protonated molecule at m/z 896, which corresponded to GGPL-I as determined by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem mass spectrometry (MS) analysis of protonated molecules at m/z 896. These results identified mf1 as a novel cholinephosphotransferase and showed that the phosphocholine transfer step is involved in the GGPL biosynthesis pathway of M. fermentans. This is the first report of a GGPL biosynthesis enzyme.     

A human gene encoding a protein homologous to ribosomal protein L39 is normally expressed in the testis and derepressed in multiple cancer cells

We identified and characterized a gene encoding a protein that was 92% identical to human ribosomal protein L39. This gene was located on the long arm of chromosome 3, and was composed of three exons and two long introns. Analysis of mRNA expression in 16 types of normal human tissues showed that this gene was expressed specifically in the testis, in sharp contrast to the ubiquitous expression of the ribosomal protein L39 gene. Surprisingly, the new gene was expressed in 19 out of 24 human cancer samples of various tissue origins. When the new gene was expressed in the cell, a translated product was observed by immunofluorescence microscopy in the nucleus, especially strongly in the nucleolus, and in the cytoplasm. Association of this protein with the large subunit of cytoplasmic ribosomes was detected by polyacrylamide-agarose composite gel electrophoresis followed by immunodetection. These immunochemical data suggest a relationship between the new gene and the ribosome.     

Localization of group IB phospholipase A(2) isoform in the gills of the red sea bream,Pagrus (Chrysophrys) major

We previously reported that PLA(2) activity in the gills is higher than that in other tissues in red sea bream and purified PLA(2) from the gills belongs to the group IB PLA(2) as well as other red sea bream PLA(2)s. In this study, we reconfirmed that the level of PLA(2) activity is extremely high in the gills compared with other tissues, and gill PLA(2) was detected only in the gills by immunoblotting and inhibition test using anti-gill PLA(2) monoclonal antibody. The level of PLA(2) activity and protein expression in the gills are well correlated. Fish can be roughly divided into high and low groups based on the level of PLA(2) activity. Gill PLA(2) was detected in the gills of the high group, but not the low group by immunoblotting. In the gills of the high group, gill PLA(2) was detected in the mucous cells and pavement cells located on the surface of gill epithelia by immunohistochemistry. On the other hand, positive signals were observed only in the mucous cells by in situ hybridization. We also isolated inactive proPLA(2), having AR propeptide, preceding the mature enzyme from the gill extract. These results suggest that gill PLA(2) is synthesized as an inactive proPLA(2) in the mucous cells and is secreted to the surface of gill epithelia.