Signalling by protein kinase C isoforms in the heart

Understanding transmembrane signalling process is one of the major challenge of the decade. In most tissues, since Fisher and Krebs's discovery in the 1950's, protein phosphorylation has been widely recognized as a key event of this cellular function. Indeed, binding of hormones or neurotransmitters to specific membrane receptors leads to the generation of cytosoluble second messengers which in turn activate a specific protein kinase. Numerous protein kinases have been so far identified and roughly classified into two groups, namely serine/threonine and tyrosine kinases on the basis of the target amino acid although some more recently discovered kinases like MEK (or MAP kinase kinase) phosphorylate both serine and tyrosine residues.Protein kinase C is a serine/threonine kinase that was first described by Takai et al. [1] as a Ca- and phospholipid-dependent protein kinase. Later on, Kuo et al. [2] found that PKC was expressed in most tissues including the heart. The field of investigation became more complicated when it was found that the kinase is not a single molecular entity and that several isoforms exist. At present, 12 PKC isoforms and other PKC-related kinases [3] were identified in mammalian tissues. These are classified into three groups. (1) the Ca-activated -, -,and -PKCs which display a Ca-binding site (C2); (2) the Ca-insensitive -, -, -, -, and -PKCs. The kinases that belong to both of these groups display two cystein-rich domains (C1) which bind phorbol esters (for recent review on PKC structure, see [4]). (3) The third group was named atypical PKCs and include , , and -PKCs that lack both the C2 and one cystein-rich domain. Consequently, these isoforms are Ca-insensitive and cannot be activated by phorbol esters [5]. In the heart. evidence that multiple PKC isoforms exist was first provided by Kosaka et al. [6] who identified by chromatography at least two PKC-related isoenzymes. Numerous studies were thus devoted to the biochemical characterization of these isoenzymes (see [7] for review on cardiac PKCs) as well as to the identification of their substrates.This overview aims at updating the present knowledge on the expression, activation and functions of PKC isoforms in cardiac cells. (Mol Cell Biochem 157: 65–72, 1996)     

Characterization of an abscisic acid carrier in suspension-cultured barley cells

The uptake of [³H]-abscisic acid in barley (Hordeum vulgareL. cv. Heartland) cell cultures was found to be mediated throughboth non-saturable and saturable components. The kinetic parametersof the saturable component, determined at pH 4.5 and 21 °C,showed a K_m for natural or (+ )-ABA of 1.3±0.7µMand an apparent V_mex of 7.0 ± 2.8 nmol g^–1 cellsh^–1. The carrier showed a strong preference for the naturalenantiomer of ABA as compared to the unnatural one. Other substancestested, e.g. amino acids, organic acids, and other growth regulators,did not appear to interfere with the carrier-mediated uptakeof ABA. At low external concentrations of ABA (below 2.0 µM),the saturable component was greater than the diffusion component.Similarly, between pH 4.0 and 6.0, the saturable uptake wasresponsible for more than 50% of the total uptake. The carriermay be important in vivo for mediating uptake when endogenouslevels of ABA are low (c. 1 µM). The carrier specificity was evident in inhibition experimentsdone with ABA analogues. Our data showed that the carrier couldaccommodate small modifications in the ABA structure. Four analogueswere able to compete efficiently with ( + )-ABA for the bindingsite of the carrier. Three of these competitors were of the(+)-series. Only one ( –)-analogue, (–)-ABA, wasable to inhibit markedly the saturable uptake of ( + )-ABA.The induction of the ABA-respons-ive gene WCS120 (Houde et al.,1992) presented stricter requirements for the ABA molecule thanthe carrier, although with a similar preference for the ( +)-analogues. Besides ( + )-ABA itself, only two of the analoguestested, both ( + )-series, were able to induce the WCS120 geneafter a 24 h incubation period. The absence of correlation betweenthe activity of the analogues as ABA inhibitors in the carriersystem, and their capacity to induce the WCS120 gene tend tosuggest that the carrier is not directly involved in the signaltransduction pathway leading to the induction of this specificgene. Key words: Abscisic acid, barley, gene induction, Hordeum vulgare, uptake carrier     

Structure and function of the photosynthetic reaction center fromRhodobacter sphaeroides

The three-dimensional structure of the photosynthetic reaction center fromRhodobacter sphaeroides is described. The reaction center is a transmembrane protein that converts light into chemical energy. The protein has three subunits: L, M, and H. The mostly helical L and M subunits provide the scaffolding and the finely tuned environment in which the chromophores carry out electron transfer. The details of the protein-chromophore interactions are from studies of a trigonal crystal form that diffracted to 2.65-Å resolution. Functional studies of the multi-subunit complex by site-specific replacement of key amino acid residues are summarized in the context of the molecular structure.This work was supported in part by the U.S. Department of Energy, Office of Health and Environmental Research, under Contract No. W-31-109-ENG-38 and by Public Health Service Grant GM36598.     

Cloning and expression of distinct nitrite reductases in tobacco leaves and roots

Summary Three tobacco nitrite reductase (NiR) cDNA clones were isolated using spinach NiR cDNA as a probe. Sequence analysis and Southern blot hybridization revealed four genes in tobacco. Two of these genes presumably derived from the ancestral species Nicotiana tomentosiformis, the other two from the ancestor N. sylvestris. Northern blot analysis showed that one gene from each ancestral genome was expressed predominantly in leaves, whilst RNA from the other was detected mostly in roots. The accumulation of both leaf and root NiR mRNAs was induced by nitrate and repressed by nitrate- or ammonium-derived metabolites. In addition, the expression of the root NiR gene was detectable in leaves of a tobacco nitrate reductase (NR)-deficient mutant. Thus, the regulation of expression of tobacco NiR genes is comparable to the regulation of expression of barley NR genes.     

Micropropagation of Cowania subintegra and Cowania stansburiana

Both Cowania subintegra Kearney and C. stansburiana Torr. were successfully propagated in vitro. Shoot proliferation occurred from shoot tips of green-house grown C. subintegra using a modified Murashige and Skoog medium supplemented with 4.4 M 6-benzyladenine and 0.5 M indole butyric acid. Excised microshoots (1.5–3.0 cm long) of both species were rooted using a two-step process in which they were cultured for 3 days in a root initiation medium with 2.7 M naphthaleneacetic acid and then transferred to a low nitrogen root elongation medium without auxin. Plantlets were successfully transferred to soilless potting mix.     

Rank relations among sisters in semi-free-ranging Barbary macaques (Macaca sylvanus)

Although semi-free-ranging Barbary macaque females are able to outrank older females from lower-ranking matrilines (matrilineal rank acquisition), they do not systematically outrank their older sisters, as is known to be the case for semi-free-ranging rhesus monkeys (Macaca mulatta) and Japanese macaques (Macaca fuscata). We test the hypothesis that differences in the support received by younger sisters against their older sisters and against older lower-ranking females might account for this interspecific difference. Thirty-one sister dyads, members of a group of 109 Barbary macaques living at La Montagne des Singes, France, were observed during 16 months. The results indicate that (1) all females were dominant to their younger sisters, and the latter were never observed to challenge their older sisters; (2) younger sisters received as much kin support against their older sisters as against older lower-ranking females; (3) only very young females received support from their kin against their older sisters; (4) younger sisters received much more support from nonkin females against lower-ranking females than against their older sisters; and (5) Barbary macaque females appear to be supported against their older sisters less frequently than rhesus macaque females are. We conclude that the lack of nonkin support is the main factor accounting for the failure of younger sisters to outrank their older sisters in Barbary macaques. Initially this might result from kin support not being sufficient to induce younger sisters to challenge and to solicit support against their older sisters.     

Development and validation of a high-performance liquid chromatographic method for the determination of methocarbamol in human plasma

An isocratic HPLC method was developed and validated for the quantitation of methocarbamol in human plasma. Methocarbamol and internal standard in 200 μl of human plasma were extracted with ethyl acetate, evaporated to dryness and reconstituted in water. Separation was achieved on a reversed-phase C₁₈ column with a mobile phase of methanol—0.1 M potassium phosphate monobasic—water (35:10:55, v/v/v). The detection was by ultraviolet at 272 nm. Linearity was established at 1–100 μg/ml (r > 0.999). The limit of quantitation was designed as 1 μg/ml to suit pharmacokinetic studies. Inter-day precision and accuracy of the calibration standards were 1.0 to 3.6% coefficients of variance (C.V.) and −2.0 to +1.6% relative error (R.E.). Quality controls of 3, 20 and 70 μg/ml showed inter-day precision and accuracy of 2.5 to 3.6% C.V. and −0.9 to −0.4% R.E. Recovery of methocarbamol was 91.4–100.3% in five different lots of plasma. The method was shown to be applicable on different brands of C₁₈ columns.