Cytosolic free Ca2+ concentration and intracellular calcium distribution of Ca2+-tolerant isolated heart cells

The cytosolic free Ca2+ concentration of calcium-tolerant rat myocytes has been measured by the null point titration technique using arsenazo III as a Ca2+ indicator and digitonin to permeabilize the plasma membrane. The mean value obtained for 8 separate preparations was 270 +/- 35 nM. The distribution of releasable calcium between the mitochondrial and sarcoplasmic reticular compartments was measured by the successive additions of uncoupler and A23187 to cells pretreated with ruthenium red. The relative distribution of calcium in each pool was independent of the cell calcium content up to the maximum value of releasable calcium investigated (4.5 nmol/mg of cell dry weight) and was distributed in the approximate ratio of 2:1 in favor of the sarcoplasmic reticulum. The cells contained 1 nmol of calcium/mg of cell dry weight in a form nonreleasable by A23187, which was independent of the total cell calcium content as measured by atomic absorption spectroscopy. It is calculated that the calcium content of mitochondria in heart under physiological conditions is about 5 nmol/mg of mitochondrial protein. At this level, the mitochondria are likely to provide effective buffering of the cytosolic free Ca2+ concentration of quiescent heart cells. The corresponding intramitochondrial free Ca2+ is in a range above values needed to regulate the activity of Ca2+-dependent enzymes of the citric acid cycle in heart. The physiological calcium content of the sarcoplasmic reticulum in heart cells is estimated to be about 2.5 nmol/mg of cell dry weight, which is at least 5-fold greater than the amount of calcium release calculated to cause maximum tension development of cardiac muscle.     

A heat-shock promoter fusion to the Ac transposase gene drives inducible transposition of a Ds element during Arabidopsis embryo development

A heat-shock promoter fusion to the Ac transposase gene (hs::TPase) was constructed and introduced into Arabidopsis. In five transformants containing the fusion the abundance of transposase mRNA increased approximately 120-fold on exposure to high temperatures. Hybrid plants containing hs::TPase and a Ds element inserted in a streptomycin resistance gene (Ds::SPT) were made and these plants were self-fertilized either after heat shocking at different stages in development or without exposure to high temperature. The progeny of these plants were sown on streptomycin-containing medium and the frequency with which variegated or streptomycin-resistant (strep^R) seedlings occurred was used as an indication of the frequency of Ds excision. Very few of the progeny of plants not exposed to heat shock or of those heat shocked only during vegetative development were variegated or strep^R. However, plants that were heat shocked after the appearance of flower buds and during seed development produced high frequencies (approaching 100%) of variegated, but very few strep^R, progeny. Furthermore, when variegated seedlings were grown to maturity and self-fertilized without further exposure to heat shock then strep^R seedlings often occurred at high frequency among their progeny. Southern analysis indicated that the majority of these strep^R plants contained a transposed Ds at a new location. These data indicate that in response to heat shock Ds excision frequently occurs in embryonic cells which ultimately give rise to the gametes, as well as in cells of the developing cotyledons. The importance of an inducible transposon system for transposon tagging is discussed.     

Intraspecific Variation in Chemical Attraction of Rice to Insect Predators

The olfactory response of predators of the brown planthopper,Nilaparvata lugensStål, to different genotypes of rice (14 cultivars and breeding lines ofOryza sativaL. and 1 wild species,Oryza nivaraSharma et Shastry) was measured in an airflow olfactometer. Odor from rice plants attracted more females of the mirid predatorCyrtorhinus lividipennisReuter than plain air (control) on only 6 of the 15 rice genotypes. Orientation ofC. lividipennistoward volatiles of certain rice genotypes was apparent even when the plants were free of the brown planthopper. However, the predator distinguished between prey-infested and uninfested plants and preferred plants with eggs over plants with nymphs. The predator did not distinguish different stages of plant growth (vegetative, booting, or flowering). Plants artificially injured to simulate brown planthopper oviposition wounds were not as attractive to the predator as plants on which the planthopper had oviposited. The preassay preconditioning on the cultivar TN1 did not produce a predator bias for this genotype. This suggests that rearing effects or chemically mediated associative learning reported for some natural enemies did not influenceC. lividipennis'host response. Results with another predator, the coccinellidMicraspis hirashimaiSasaji, produced less consistent behavior. Planthopper-infested plants attracted more females ofM. hirashimaithan unifested plants in only 1 of the 12 rice genotypes evaluated. Implications for augmenting predators by rice cultivar selection and modification are discussed.     

TCEN-49, a monoclonal antibody that identifies a central body antigen in the planarian Dugesia (Girardia) tigrina. Implications for pattern formation and positional signalling mechanisms

We have produced monoclonal antibodies (mAbs) against antigens of the freshwater planarian Dugesia (G.) tigrina (Girard) using standard protocols. One of these mAbs, TCEN-49, detects an antigen (TCEN-49Ag) present in most cells of the central area of the body, including the pharynx. Labelled cells seem more related by position than by lineage, suggesting that TCEN-49Ag is involved somehow in the expression of central body positional identity. The spatial and temporal changes in TCEN-49Ag expression during growth/degrowth and regeneration have been monitored and the implications of these results are discussed.     

Regulation of L-Type Calcium Channels in GH4 Cells via A1 Adenosine Receptors

Abstract: Identification of A₁ adenosine receptors (A₁Rs) in a tumor cell line derived from rat pituitary (GH₄ cells) was performed by ligand binding and immunological experiments. Subsequently, the involvement of A₁Rs in the regulation of calcium conductance was studied in these cells. The agonist N ⁶-( R )-(2-phenylisopropyl)adenosine ( R -PIA) did not modify the intracellular calcium basal levels, whereas it inhibited the increase produced by 15 m M KCl depolarization. The antagonist 1,3-dipropyl-8-cyclopentylxanthine led to the opening of voltage-dependent cell surface calcium channels in the absence of exogenous KCl. The channels were of the L type because the effect was abolished by calciseptine and by verapamil. These results suggest that endogenous adenosine exerts a tonic inhibitory effect on calcium transport. This was confirmed by the high adenosine concentration found in cell supernatants (up to 1 µ M ) and by the calcium mobilization produced by exogenously added adenosine deaminase. In depolarizing conditions, the calcium peak in the presence of adenosine deaminase was reduced when cells were preincubated with R -PIA, thus suggesting that A₁R activation regulates the intensity of depolarization. These results demonstrate that adenosine is an important regulator of the physiological state of pituitary tumor cells by modulating, in an autocrine manner, the activity of L-type voltage-dependent calcium channels.     

Number and sex of offspring of ΔF508 carriers outside cystic fibrosis families

Number and sex of offspring were determined in a group of 7,841 randomly selected blood donors who were screened for the F508 mutation. We did not find any evidence for differences in number or sex ratio of offspring between F508 carriers and non-carriers.     

Effects of branched chain alpha-ketoacids on the metabolism of isolated rat liver cells. I. Regulation of branched chain alpha-ketoacid metabolism.

alpha-Ketoisocaproate (ketoleucine) is shown to be metabolized to ketone bodies rapidly by isolated rat liver cells. Acetoacetate is the major end product and maximum rates were observed with 2 mM substrate. Studies with 2-tetradecylglycidic acid (an inhibitor of long chain fatty acid oxidation) showed that ketogenesis from alpha-ketoisocaproate and from endogenous fatty acids were additive. With alpha-ketoisocaproate present as soole substrate at 2 mM, leucine production was less than 10% of alpha-ketoisocaproate uptake and only 30% of the acetyl coenzyme A generated was oxidized in the citric acid cycle. Metabolism of alpha-ketoisocaproate was inhibited by fatty acids, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and pyruvate. Oxidation of acetyl-CoA generated from alpha-ketoisocaproate was suppressed by oleate and by pyruvate, but was enhanced by lactate. Metabolism between the different branched chain alpha-ketoacids was mutually competitive. When alpha-ketoisocaproate (2 mM) was added in the presence of high pyruvate concentrations (4.4 mM), flux through pyruvate dehydrogenase was decreased, and the proportion of total pyruvate dehydrogenase in the active form (PDHa) also fell. With lactate as substrate, PDHa was only 25% of total activity and was little affected by addition of alpha-ketoisocaproate. These data suggest that enhanced oxidation of acetyl-CoA from alpha-ketoisocaproate by lactate addition is caused by a low activity of pyruvate dehydrogenase combined with increased flux through the citric acid cycle in response to the energy requirements for gluconeogenesis. However, acetyl-CoA generation from pyruvate is apparently insufficiently inhibited by alpha-ketoisocaproate to cause a diversion of acetyl-CoA formed during alpha-ketoisocaproate metabolism from ketone body formation to oxidation in the citric acid cycle. Measurements of the cell contents of CoASH, acetyl-CoA, acid-soluble acyl-CoA, and acid-insoluble fatty acyl-CoA indicated that when the branched chain alpha-ketoacids were added as sole substrate, their oxidation was limited at a step distal to the branched chain alpha-ketoacid dehydrogenase. Acid-soluble acyl-CoA derivatives were depleted after oleate addition in the presence of alpha-ketoisocaproate, suggesting an inhibition of the branched chain alpha-ketoacid dehydrogenase by the elevation of the mitochondrial NADH/NAD+ ratio observed during fatty acid oxidation. This effect was not observed in the presence of oleate and 2-tetradecylglycidic acid.     

Relationship between hydroxypyruvate and the production of oxalate in vitro

Chicken liver lactate dehydrogenase (l-lactate: NAD⁺ oxidoreductase, EC 1.1.1.27) catalyses the reversible reduction reaction of hydroxypyruvate to l-glycerate. It also catalyses the oxidation reaction of the hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form to glycolate. At pH 8, these latter two reactions are coupled. The coupled system equilibrium is attained when the NAD⁺/NADH ratio is greater than unity.Hydroxypyruvate binds to the enzyme at the same site as the pyruvate. When there are substances with greater affinity to this site in the reaction medium and their concentration is very high, hydroxypyruvate binds to the enzyme at the l-lactate site. In vitro and with purified preparation of lactate dehydrogenase, hydroxypyruvate stimulates the production of oxalate from glyoxylate-hydrated form and from NAD; the effect is due to the fact that hydroxypyruvate prevents the binding of non-hydrated form of glyoxylate to the lactate dehydrogenase in the pyruvate binding site. At pH 8, the l-glycerate stimulates the production of glycolate from glyoxylate-non-hydrated form and NADH since hydroxypyruvate prevents the binding of glyoxylate-hydrated form to the enzyme.     

The binding of [3H]R-PIA to A1 adenosine receptors produces a conversion of the high- to the low-affinity state

Kinetic evidence for negative cooperativity on the binding of [3H]R-PIA to A1 adenosine receptors was obtained from dissociation experiments at different ligand concentrations and from the equilibrium isotherm. The dissociation curves indicate that there is an apparent ligand-induced transformation of high- to low-affinity states of the receptor. At concentrations of 18.2 nM R-PIA or higher there was only found the low-affinity state of the receptor. In view of these results equilibrium binding data were analyzed by the usual two-state model (assuming that there is an interconversion between them) and by the negative cooperativity model employing the Hill equation.     

Cloning and nucleotide sequence of celA1, and endo-beta-1,4-glucanase-encoding gene from Streptomyces halstedii JM8.

The celA1 gene encoding an endo-beta-1,4-glucanase from a mesophilic actinomycete, strain JM8, identified as Streptomyces halstedii, was cloned and expressed in S. lividans JI66. From the nucleotide sequence of a 1.7-kb DNA fragment we identified an open reading frame of 963 nucleotides encoding a protein of 321 amino acids, starting at TTG (instead of ATG). The Cel1 mature enzyme is a protein of 294 amino acids (after signal peptide cleavage) and can be included in the beta-glycanase family B (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991). The Cel1 enzyme lacks a cellulose-binding domain as predicted by computer analysis of the sequence and confirmed by Avicel binding experiments. The promoter region of celA1 was identified by S1 mapping; the -35 region closely resembles those of housekeeping Streptomyces promoters. Three imperfectly repeated sequences of 15, 15, and 14 nucleotides were found upstream from celA1 [ATTGGGACCGCTTCC-(N85)-ATTGGGACCGCTTCC-(N2)-TGGGAGC GCTCCCA]; The 14-nucleotide sequence has a perfect palindrome identical to that found in several cellulase-encoding genes from Thermomonospora fusca, an alkalophilic Streptomyces strain, and Streptomyces lividans. This sequence has been implicated in the mechanism of induction exerted by cellobiose. Using an internal celA1 probe, we detected similar genes in several other Streptomyces species, most of them cellulase producers.