Sphingosine, W-7, and Trifluoperazine Inhibit the Elevation in Cytosolic Calcium Induced by High K+Depolarization in Synaptosomes

Abstract: A possible role for protein kinases in the regulation of free cytosolic Ca²⁺ levels in nerve endings was investigated by testing the effect of several kinase inhibitors on the increase in cytosolic Ca²⁺ (monitored with the Ca²⁺-sensitive dye fura-2) induced by depolarization with 15 or 30 mM K⁺. The ability of various drugs to inhibit the cytosolic Ca²⁺ response appeared to correlate with their reported mechanism of action in inhibiting protein kinases. W-7 and trifluoperazine, drugs reported to inhibit calmodulin-dependent events, were effective inhibitors of the increase in cytosolic Ca²⁺ induced by high K⁺ depolarization, as was sphingosine, a drug that inhibits protein kinase C by binding to the regulatory site, but which also inhibits calcium/calmodulin kinase. On the other hand, drugs that inhibit protein kinases by binding to the catalytic site, such as H-7 (1 m/W ), staurosporine (1μ M ), and K252_a(1μ M ), were ineffective. Activation of protein kinase C, which is blocked by each of these drugs, does not appear to be essential to the maintenance of elevated cytosolic Ca²⁺ in depolarized synaptosomes. All of the drugs, including sphingosine, that functionally inhibit the depolarization-induced elevation in cytosolic Ca²⁺ have in common the ability to bind to calmodulin. Because the drugs that inhibit protein kinases by competing with ATP binding at the active catalytic site did not block the response in this system, we suggest that a calmodulin or a calmodulin-like binding site participates in the regulation of Ca²⁺ increases after depolarization.     

Involvement of Calcium Channels in Depolarization-Evoked Release of Adenosine from Spinal Cord Synaptosomes

Abstract: The potential involvement of L- and N-type voltage-sensitive calcium (Ca²⁺) channels and a voltage-independent receptor-operated Ca²⁺ channel in the release of adenosine from dorsal spinal cord synaptosomes induced by depolarization with K⁺ and capsaicin was examined. Bay K 8644 (10 n M ) augmented release of adenosine in the presence of a partial depolarization with K⁺ (addition of 6 m M ) but not capsaicin (1 and 10 μ M ). This augmentation was dose dependent from 1 to 10 n M and was followed by inhibition of release from 30 to 100 n M . Nifedipine and nitrendipine inhibited the augmenting effect of Bay K 8644 in a dose-dependent manner, but neither antagonist had any effect on release of adenosine produced by K⁺ (24 m M ) or capsaicin (1 and 10 μ M ) ω-Conotoxin inhibited K⁺-evoked release of adenosine in a dose-dependent manner but had no effect on capsaicin-evoked release. Ruthenium red blocked capsaicin-induced release of adenosine but had no effect on K⁺-evoked release. Although L-type voltage-sensitive Ca²⁺ channels can modulate release of adenosine when synaptosomes are partially depolarized with K⁺, N-type voltage-sensitive Ca²⁺ channels are primarily involved in K⁺-evoked release of adenosine. Capsaicin-evoked release of adenosine does not involve either L- or N-type Ca²⁺ channels, but is dependent on a mechanism that is sensitive to ruthenium red.     

Tachykinins Potentiate N-Methyl-D-Aspartate Responses in Acutely Isolated Neurons from the Dorsal Horn

Abstract: Substance P and neurokinin A both potentiated N -methyl- d -aspartate (NMDA)-induced currents recorded in acutely isolated neurons from the dorsal horn of the rat. To elucidate the mechanism underlying this phenomenon, we measured the effects of tachykinins and glutamate receptor agonists on [Ca²⁺]_i in these cells. Substance P, but not neurokinin A, increased [Ca²⁺]_i in a subpopulation of neurons. The increase in [Ca²⁺]_i was found to be due to Ca²⁺ influx through voltage-sensitive Ca²⁺ channels. Substance P and neurokinin A also potentiated the increase in [Ca²⁺]_i produced by NMDA, but not by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, or 50 m M K⁺. Phorbol esters enhanced the effects of NMDA and staurosporine inhibited the potentiation of NMDA effects by tachykinins. It is concluded that activation of protein kinase C may mediate the enhancement of NMDA effects by tachykinins in these cells. However, the effects of tachykinins on [Ca²⁺]_i can be dissociated from their effects on NMDA receptors.     

Analysis of γ-Aminobutyric AcidA Receptor Subunits in the Mouse Cochlea by Means of the Polymerase Chain Reaction

Abstract: Unlike 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces consistent decreases in levels of striatal dopamine (DA) with considerably smaller and more variable effects on mouse brain levels of serotonin (5-HT) and norepinephrine (NE), a novel amine-substituted MPTP analogue, 1-methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH₂-MPTP), administered in a standard mouse dosing paradigm for MPTP (20 mg/kg X 4) did not affect striatal DA but led to marked reductions (60–70%) in levels of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and NE measured in frontal cortex and hippocampus 1 week after treatment. Another 2'-substituted MPTP analogue, 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine, affected cortical and hippocampal 5-HT, 5-HIAA, and NE only minimally, while markedly reducing the DA content in striatum (90%), thus indicating that the substituent (-NH₂ versus -CH₃) at the 2'position is important for the differential effects of these MPTP analogues. In a replication study with a 3-week end point, hippocampal and cortical 5-HT, 5-HIAA, and NE levels remained depressed with no indication of recovery. These results suggest that 2'-NH₂-MPTP may be a novel, regionally selective neurotoxin for serotonergic and norad-renergic nerve terminals.     

INTRACELLULAR LOCALIZATION OF THE TASTE/ODOR METABOLITE 2-METHYLISOBORNEOL IN OSCILLATORIA LIMOSA (CYANOPHYTA)1

The monoterpene derivative, 2-methylisoborneol (MIB), is produced by many blue-green algae and often is responsible for the “musty” taste/odor in aquaculturally raised finfish and potable water supplies. Although previous researchers have suggested that taste/odor metabolites are partitioned among cell constituents and that coregulation with pigment biosynthesis occurs, no structural evidence for these hypotheses exists. MIB was localized in cells of Oscillatoria limosa (Roth) Agardh at the ultrastructural level using standard gold-labeled antibody techniques. There was no apparent relationship between age of the cells and MIB synthesis; cells that were and were not undergoing active division had similar amounts of MIB label. There was no consistent partitioning of MIB label within cells. However, occasionally, specific label was observed along photosynthetic lamellae, suggesting a potential linkage of MIB synthesis and/or binding to the pigment systems.     

ANTIVIRAL ACTIVITY OF CULTURED BLUE-GREEN ALGAE (CYANOPHYTA)1

Lipophilic and hydrophilic extracts from approximately 600 strains of cultured cyanophytes, representing some 300 species, were examined for antiviral activity against three pathogenic viruses. Approximately 10% of the cultures produced substances that caused significant reduction in cytopathic effect normally associated with viral infection. The screening program identified the order Chroococcales as commonly producing antiviral agents.     

Cationic and Anionic Requirements for the Binding of 2β-Carbomethoxy-3β-(4-Fluorophenyl)[3H]tropane to the Dopamine Uptake Carrier

Abstract: The present study reports the ion dependency of 2β-carbomethoxy-3β-(4-fluorophenyl)[³H]tropane ([³H]- CFT) binding to the dopamine transporter in the rat striaturn. The results indicate that [³H]CFT binding to synaptosomal P₂ membranes requires low concentrations of Na⁺ (peak binding between 20 and 50 m M Na⁺), is stimulated by phosphate anion or l^-, but is unaffected or only slightly affected by F^-, Cl^-, Br^-, NO₃^-, or SO₄^2-, Concentrations of Na⁺ of >50 m M become inhibitory except in the presence of l^-, which shifts peak binding levels toward higher Na⁺ concentrations and also elevates the peak binding level. K⁺ strongly decreased [³H]CFT binding with a shallow inhibition curve, and Na⁺ could not overcome this effect. Saturation analysis of [³H]CFT binding revealed a single binding site changing its affinity for CFT depending on the concentration of sodium phosphate buffer (6, 10, 30, 50, 130, or 200 m M ; 1 mM plus 49 mM NaCIversus 10 m M plus 40 m M NaCI; or 1 mM plus 129 m M Nal versus 10 m M plus 120 m M Nal). No differences were observed in the density of CFT binding sites between any of the conditions examined.     

Optimization of alkyl beta-D-galactopyranoside synthesis from lactose using commercially available beta-galactosidases

Commercially available lactase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) enzymes produced from Kluyveromyces fragilis and Kluyveromyces lactis were accessed as catalysts for use in the production of beta-galactopyranosides of various alcohols using lactose as galactosyl donor. The yield of galactoside was enhanced by using the highest practical concentrations of both lactose and alcohol acceptor. The concentrations and thus yield, were limited by the solubility of the substrates. The increase in galactoside yield with increasing lactose concentration appeared to be specific to the lactose substrate and not due to water activity alterations, because addition of maltose to a fixed concentration of lactose had no effect. During the course of the reaction, the yield of galactoside peaked after around 70% to 80% of the lactose was consumed, due to hydrolysis of the product by the enzyme. A wide variety of compounds with primary or secondary hydroxyl groups could act as acceptors, the essential requirement being at least some water solubility. Addition of organic cosolvents had little effect on galactoside yield except when it increased the water solubility of sparingly soluble alcohols. Some galactosides were synthesized on a gram scale to determine practical product recoveries and improve purification methods for large-scale synthesis. Initial purification by hydrophobic chromatography (for galactosides of hydrophobic alcohols) or strong anion-exchange chromatography (for galactosides of hydrophilic alcohols) separated galactosides, galactobiosides, and higher oligomers from reducing sugars. A facile separation of the galactoside and galactobioside could then be effected by flash chromatography on silica gel. (c) 1993 John Wiley & Sons, Inc.     

Factors affecting bio-oxidation of sulfide minerals at high concentrations of solids: a review

Bio-oxidation has proved to be a viable process for the oxidative pretreatment of refractory gold-bearing sulfides. Generally, the oxidation rate is maximal at 20% solids for high sulfide content materials [ca. 30% sulfur]. Low grade ores [1% sulfur] have been successfully oxidized at 55% solids, indicating a link between the sulfide grade of the material and the optimal solids concentration for operation. Concentrations of high solids have been reported to lower oxidation rates, increase lag times, and decrease the ultimate extent of oxidation. This review discusses the various factors that have been proposed as causes of these phenomena. The factors include oxygen and carbon dioxide availability, low bacteria-solids ratio; mechanical damage or inhibition of the bacteria, inhibition of bacterial attachment, and the buildup of toxic leach products or other detrimental substances such as some flotation reagents. (c) 1993 John Wiley & Sons, Inc.