SK&F 96365, a novel inhibitor of receptor-mediated calcium entry.

A novel inhibitor of receptor-mediated calcium entry (RMCE) is described. SK&F 96365 (1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H- imidazole hydrochloride) is structurally distinct from the known 'calcium antagonists' and shows selectivity in blocking RMCE compared with receptor-mediated internal Ca2+ release. Human platelets, neutrophils and endothelial cells were loaded with the fluorescent Ca2(+)-indicator dyes quin2 or fura-2, in order to measure Ca2+ or Mn2+ entry through RMCE as well as Ca2+ release from internal stores. The IC50 (concn. producing 50% inhibition) for inhibition of RMCE by SK&F 96365 in platelets stimulated with ADP or thrombin was 8.5 microM or 11.7 microM respectively; these concentrations of SK&F 96365 did not affect internal Ca2+ release. Similar effects of SK&F 96365 were observed in suspensions of neutrophils and in single endothelial cells. SK&F 96365 also inhibited agonist-stimulated Mn2+ entry in platelets and neutrophils. The effects of SK&F 96365 were independent of cell type and of agonist, as would be expected for a compound that modulates post-receptor events. Voltage-gated Ca2+ entry in fura-2-loaded GH3 (pituitary) cells and rabbit ear-artery smooth-muscle cells held under voltage-clamp was also inhibited by SK&F 96365; however, the ATP-gated Ca2(+)-permeable channel of rabbit ear-artery smooth-muscle cells was unaffected by SK&F 96365. Thus SK&F 96365 (unlike the 'organic Ca2+ antagonists') shows no selectivity between voltage-gated Ca2+ entry and RMCE, although the lack of effect on ATP-gated channels indicates that it discriminates between different types of RMCE. The effects of SK&F 96365 on functional responses of cells thought to be dependent on Ca2+ entry via RMCE were also studied. Under conditions where platelet aggregation is dependent on stimulated Ca2+ entry via RMCE, the response was blocked by SK&F 96365 with an IC50 of 15.9 microM, which is similar to the IC50 of 8-12 microM observed for inhibition of RMCE. Adhesion and chemotaxis of neutrophils were also inhibited by SK&F 96365. SK&F 96365 is a useful tool to distinguish RMCE from internal Ca2+ release, and to probe the role of RMCE in mediating functional responses of cells. However, SK&F 96365 is not as potent (IC50 around 10 microM) or selective (also inhibits voltage-gated Ca2+ entry) as would be desirable, so caution must be exercised when using this compound.     

Effects of gonadotropins and granulosa cell secretions on the maturation and fertilization of rat oocytes in vitro

Fully grown germinal vesicle-stage oocytes are induced to resume meiosis and acquire the capacity to undergo fertilization in response to a surge of gonadotropins. The present study examined possible direct and indirect roles of gonadotropins in the maturation and fertilization of rat oocytes by determining 1) the effect of exogenous administration of gonadotropins (priming) to immature rats prior to oocyte collection on the capacity of oocytes to undergo maturation and fertilization in vitro, 2) the effect of follicle-stimulating hormone (FSH) in the maturation media on the resumption of meiosis and subsequent capacity of oocytes to undergo fertilization, and 3) the capacity of oocytes to undergo maturation and fertilization following culture in preovulatory follicular fluid or in conditioned media obtained from gonadotropin-stimulated granulosa cell (GC) cultures. In the first experiment, oocytes from unprimed rats underwent spontaneous meiotic maturation in vitro and 17% underwent subsequent fertilization. Priming increased the proportion of oocytes undergoing fertilization. Maturation of oocytes in media supplemented with various concentrations of FSH or for various lengths of time (6-16 h) in medium with 500 ng FSH/ml indicated that FSH slowed the rate of meiotic maturation, but had no effect on the capacity of the oocytes to be fertilized. Oocytes obtained from primed animals and cultured in the presence of preovulatory follicular fluid were fertilized in proportions similar to those cultured in serum-containing medium. In the third experiment, medium conditioned by FSH-stimulated GC for 40 h slowed the rate of meiotic maturation; the addition of luteinizing hormone (LH) to the FSH-stimulated cells produced a medium in which the rate of oocyte maturation was not different from that of control oocytes (in medium from unstimulated cells). Medium conditioned by FSH- or LH-stimulated GC, but not fibroblasts, increased the proportions of oocytes undergoing fertilization following maturation in those media. FSH + LH stimulation of GC increased the fertilization of oocytes to proportions significantly higher than with either gonadotropin alone. These data suggest that GC respond to gonadotropin stimulation by providing a factor(s) that regulates the rate of oocyte maturation and promotes the capacity of oocytes to undergo fertilization.     

Evaluation of free D-glutamate in processed foods

Monosodium glutamate (MSG) is added to many processed foods at significant levels for flavor enhancement. It is also naturally occurring at high levels in some foods. The enantiomeric composition of free glutamate in foods was examined and all foods analyzed were found to contain D -glutamate. The relative percent of D -glutamate in the food products studied depended on the origin of the glutamate. Foods to which MSG was added by the manufacturer had a high total level of MSG but a lower relative percentage of the D -enantiomer (usually less than 0.8%). In comparison, fermented foods tend to have high relative levels of D -glutamate but a lower total amount of the amino acid. The relative percent of D -glutamate in nonfermented foods containing no added MSG was also found to be low compared to fermented products. In some cases the percent D -glutamate could be related to the relative amounts of other food ingredients such as cheese. © 1994 Wiley-Liss, Inc.     

Effects of decreased atmospheric deposition on the sulfur budgets of two Dutch moorland pools

The chemical composition of surface waters of two Dutch moorland pools and of incident precipitation, was monitored from 1982 to 1990. For this period, sulfur and water budgets were calculated using a hydrochemical model developed for well-mixed non-stratifying lakes. Total atmospheric deposition of S decreased significantly after 1986 at both locations. A model describing the sulfur budget in terms of input, output and reduction/oxidation processes predicted a fast decrease of pool water SO₄ ²⁻ concentrations after a decrease of atmospheric input. However, SO₄ ²⁻ concentrations in the surface water was lowered only slightly or remained constant. Apparently a source within the lake caused the unexpectedly high SO₄ ²⁻ concentrations. The possible supply of SO₄ ²⁻ from the sediment through regulation by (K-)Al-SO₄ containing minerals or desorption of SO₄ ²⁻ from positively charged surfaces in the sediment was evaluated. Solubility calculations of pore water with respect to alunite, basaluminite and jurbanite indicated that SO₄ ²⁻ concentration was not regulated by these minerals. It is suggested here (1) that desorption of SO₄ ²⁻ from peaty sediments may account for the estimated SO₄ ²⁻ supply provided that the adsorption complex is periodically recharged by partial oxidation of the upper bottom sediments and (2) that because of exposure of a part of the pool bottom to the atmosphere during dry summers and subsequent oxidation of reduced S, the amount of SO₄ ²⁻ may be provided which complements the decreasing depositional SO₄ ²⁻ input. In future research these two mechanisms need to be investigated.     

Transient expression of GUS and anthocyanin constructs in intact maize immature embryos following electroporation

Electroporation was used for the delivery and subsequent expression of GUS and anthocyanin reporter genes into intact maize immature embryos. The optimal conditions consisted of culturing immature embryos for 4 days on N6 1-100-25-Ag medium prior to electroporation (375 V/cm; 960 µF capacitance) in EPR buffer containing DNA and 0.07 M sodium glutamate at room temperature (22°C) after a 10 min heat shock at 37°C. Under these conditions, over 40 spots of GUS transient activity were observed per immature embryo. Transient gene expression after electroporation was further demonstrated using an anthocyanin construct, which is specific for expression in plant cells.     

HLA polymorphism in the Havasupai: evidence for balancing selection.

The characterization and analysis of genetic variation at the HLA loci provides important insight for population geneticists trying to understand the evolutionary forces that have shaped human populations. This study describes the HLA-A and HLA-B loci serotyping and statistical analysis on an isolated Native American population, the Havasupai of Arizona. Four alleles at the HLA-A locus were identified, while eight alleles were found at the HLA-B locus. These variants were present as 20 of 32 potential two-locus haplotypes, with five of the six most common haplotypes exhibiting high positive linkage disequilibrium. Significant homozygote deficiency (heterozygosity excess) was detected both at HLA-A and at HLA-B. This deviation from Hardy-Weinberg proportions was not attributable to nonselective causes such as different allele frequencies in males and females or avoidance of consanguineous matings. In addition, the distribution of alleles at both HLA-A and HLA-B was more even than expected from neutrality theory; that is, the observed Hardy-Weinberg homozygosity was only 62.4% of that expected under neutrality. These observations suggest that balancing selection is of major importance in maintaining genetic variation at HLA-A and HLA-B.     

K+ channels of stomatal guard cells: Abscisic-acid-evoked control of the outward rectifier mediated by cytoplasmic pH

The activation by abscisic acid (ABA) of current through outward-rectifying K⁺ channels and its dependence on cytoplasmic pH (pH_i) was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with multibarrelled and H⁺-selective microelectrodes to record membrane potentials and pH_i during exposures to ABA and the weak acid butyrate. Potassium channel currents were monitored under voltage clamp and, in some experiments, guard cells were loaded with pH buffers by iontophoresis to suppress changes in pH_i. Following impalements, stable pH_i values ranged between 7.53 and 7.81 (7.67±0.04, n = 17). On adding 20 M ABA, pH_i rose over periods of 5–8 min to values 0.27±0.03 pH units above the pH_i before ABA addition, and declined slowly thereafter. Concurrent voltage-clamp measurements showed a parallel rise in the outward-rectifying K⁺ channel current (I_{K, out}) and, once evoked, both pH_i and I_{K, out} responses were unaffected by ABA washout. Acid loads, imposed with external butyrate, abolished the ABA-evoked rise in I_{K, out}. Butyrate concentrations of 10 and 30 mM (pH₀ 6.1) caused pH_i to fall to values near 7.0 and below, both before and after adding ABA, consistent with a cytoplasmic buffer capacity of 128±12 mM per pH unit (n = 10) near neutrality. Butyrate washout was characterised by an appreciable alkaline overshoot in pH_i and concomitant swell in the steady-state conductance of I_{K, out}. The rise in pH_i and i_{K, out} in ABA were also virtually eliminated when guard cells were first loaded with pH buffers to raise the cytoplasmic buffer capacity four- to sixfold; however, buffer loading was without appreciable effect on the ABA-evoked inactivation of a second, inward-rectifying class of K⁺ channels (I_{K, in}). The pH_i dependence of I_{K, out} was consistent with a cooperative binding of at least 2H⁺ (apparent pK_a = 8.3) to achieve a voltage-independent block of the channel. These results establish a causal link previously implicated between cytoplasmic alkalinisation and the activation of I_{K, out} in ABA and, thus, affirm a role for H⁺ in signalling and transport control in plants distinct from its function as a substrate in H⁺-coupled transport. Additional evidence implicates a coordinate control of I_{K, in} by cytoplasmic-free [Ca²⁺] and pH_i.Abbreviations ABA abscisic acid - [Ca²⁺]_i cytoplasmic free [Ca²⁺]_i - E_K K⁺ equilibrium potential - I_{K, out}, I_{K, in} outward-, inward-rectifying K⁺ channel (current) - I-V current-voltage (relation) - Mes 2-(N-morpholino)ethanesulfonic acid - pH_i cytoplasmic pH - Tes 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-amino}ethanesulfonic acid - V_m membrane potential We are grateful to G. Thiel (Pflanzenphysiologisches Institut, Universität Göttingen, Germany) for helpful discussions. This work was possible with equipment grants-in-aid from the Gatsby Charitable Foundation, the Royal Society and the University of London Central Research Fund. F.A. holds a Sainsbury Studentship.     

Cytological and molecular characterization of a chromosome interchange and addition lines in Cadet involving chromosome 5B of wheat and 6Ag of Lophopyrum ponticum

Efforts to transfer wheat curl mite (Eriophyes tulipae Keifer) resistance from Lophopyrum ponticum 10X (Podb.) Love to bread wheat (Triticum aestivum L.) have resulted in the production of a number of cytogenetic stocks, including an addition line of 6Ag, a ditelo addition line, and a wheat-Lophopyrum translocation line. Characterization of these lines with C-banding, in situ hybridization with a Lophopyrum species-specific repetitive DNA probe (pLeUCD2), and Southern blotting with pLeUCD2 and a 5S ribosomal DNA probe (pScT7) confirmed that the distal portion of the short arm of 6Ag was translocated onto the distal portion of 5BS (5BL. 5BS-6AgS). It was also determined that the ditelo addition was an acrocentric chromosome of 6AgS.     

Detoxification mechanisms for 1,2,4-benzenetriol employed by a Rhodococcus sp. BPG-8

A Rhodococcus sp. BPG-8 produces 1,2,4-benzenetriol during the transformation of resorcinol by phloroglucinol induced cell-free extract. The oxidation of 1,2,4-benzenetriol to 2-hydroxy-1,4-benzoquinone produces superoxide radicals that may have potential deleterious effects on cellular integrity. It has been shown that both superoxide dismutase (SOD) and catalase retard the autoxidation of 1,2,4-benzenetriol to 2-hydroxy-1,4-benzoquinone. Termination of the free radical chain reaction between superoxide radical and 1,2,4-benzenetriol seems to prevent this autoxidation. A NAD(P)H-dependent reductase appears to convert the 2-hydroxy-1,4-benzoquinone back to 1,2,4-benzenetriol. Both of these mechanisms appear to stabilize 1,2,4-benzenetriol so that it may be cleaved by meta cleavage enzymes. The enzymes responsible for the stabilization of 1,2,4-benzenetriol appear not to be inducible.