Inhibition of Excitatory Amino Acid-Induced Phosphoinositide Hydrolysis as a Possible Mechanism of Nitroprusside Neurotoxicity

Abstract: Inclusion of sodium nitroprusside {Na₂[Fe²⁺-(CN)₅NO]} into the culture medium is toxic to cultured rat cerebellar granule neurons. A possible underlying mechanism may be the inhibition of phosphoinositide (PI) response to excitatory amino acids (EAAs) because activation of glutamate receptors can be neuroprotective and neurotrophic in differentiating neurons. Sodium nitroprusside selectively inhibited the PI response to EAAs (NMDA > glutamate = quisqualate > kainate) without affecting that to carbachol or KCI. In contrast, S-nitroso-N-acetylpenicillamine (SNAP), another nitric oxide (NO) donor, potentiated NMDA-induced PI hydrolysis. Hemoglobin reversed the effects of nitroprusside and SNAP. However, NO may not be involved because NO solution was without effect and N-acetylpenicillamine, a SNAP analogue that does not contain a NO moiety, also potentiated NMDA-induced PI hydrolysis in a hemoglobin-sensitive manner. Furthermore, the metabolites of NO (nitrate and nitrite), l -arginine, reduced glutathione, 8-bromo-cyclic guanosine 3′:5′-cyclic monophosphate (8-Br-cGMP), and atrial natriuretic peptide, which accelerates the production of cGMP independent of NO, were ineffective as modulators. However, potassium ferrocyanide {K₄[Fe²⁺(CN)₆]}, but not potassium ferricyanide {K₃[Fe³⁺(CN)₆]}, inhibited NMDA-induced PI hydrolysis as effectively as nitroprusside, but this inhibition was not reversed by hemoglobin. Cyanide, a product from the disintegration of nitroprusside, potentiated rather than inhibited NMDA-induced PI hydrolysis. Taken together, these results suggest that the parent molecule itself, nitroprusside, contributes primarily in inhibiting EAA-induced PI hydrolysis. Inhibition of EAA-induced PI hydrolysis may in part mediate the mechanisms of nitroprusside toxicity in primary cultures of differentiating cerebellar granule neurons.     

Desensitization of the Neurokinin 1 Receptor Is Mediated by the Receptor Carboxy-Terminal Region, but Is Not Caused by Receptor Internalization

Abstract: The carboxy-terminal cytoplasmic regions of the rat neurokinin 1 (substance P) and neurokinin 2 (neurokinin A) receptors have been exchanged to determine if this region of the neurokinin 1 receptor is involved in its desensitization. When expressed at similar levels in stably transfected Chinese hamster ovary (CHO) cell lines, receptors containing the carboxy-terminal region of the neurokinin 1 receptor desensitized significantly more (as measured by reduction of the inositol 1,4,5-trisphosphate response) when preexposed for 1 min to 1 µ M neurokinin, indicating a role for the carboxy-terminal region of the neurokinin 1 receptor in its desensitization. Measurement of receptor internalization using radiolabeled neurokinins (0.3 n M ) indicated that ∼75–80% of the receptors were internalized in each cell line after 10 min at 37°C, with no observable correlation between neurokinin receptor desensitization and internalization. Measurement of loss of receptor surface sites for cell lines CHO NK1 and CHO NK1NK2 following exposure to 1 µ M substance P also indicated no obvious relationship between the percent desensitization and percent of receptors internalized. Also, two inhibitors of neurokinin 1 receptor internalization, phenylarsine oxide and hyperosmolar sucrose, did not inhibit neurokinin 1 receptor desensitization. The protein kinase inhibitors Ro 31-8220, staurosporine, and Zn²⁺ had no effect on neurokinin 1 receptor desensitization, indicating that the kinases affected by these agents are not rate-limiting in neurokinin 1 receptor desensitization in this system.     

Structure and analysis of phospholipase D gene from Ricinus communis L

Phospholipase D (PLD; EC 3.1.4.4) has been proposed to play a pivotal role in various cellular processes, but molecular understanding of this enzyme is rather limited. This report describes the nucleotide sequence, structure, and genomic organization of a PLD gene from castor bean (Ricinus communis L. cv. Hale). The PLD gene was isolated from a castor bean genomic library using the PLD cDNA as a hybridization probe. Sequence comparison with the PLD cDNA revealed that the PLD gene consisted of four exons and three introns, one of which interrupts the 5-untranslated region. Southern blot analysis indicated that the cloned PLD gene was present as a single-copy gene, and yet there were other PLD or PLD-related sequences in the castor bean genome.     

Energy metabolism and ATP free-energy change of the intertidal wormSipunculus nudus below a critical temperature

The intertidal wormSipunculus nudus was exposed to various temperatures for an analysis of the integrated changes in energy and acid-base status. Animals were incubated in sea water or maintained for up to 8 days at 4 and 0°C while dwelling in the sediment. Cannulation of the animals prior to experimentation allowed the analysis of blood gas parameters ( , and pH). fell to 0 torr within 8 days at 0°C. A simultaneous reduction of ventilatory activity was derived from measurements of the pattern of coelomic fluid pressure changes associated with ventilatory movements. The increase in and an onset of anaerobic metabolism, indicated by the accumulation of end products like acetate and propionate both in the coelomic fluid and the body wall musculature, led to the development of a progressive acidosis and a deviation from the alphastat regulation of intracellular pH seen in unburied animals. The drop in intracellular pH together with the depletion of the adenylates and the phosphagen, phospho-l-arginine, reflect a significant decrease in the Gibb's free-energy change of ATP hydrolysis. These changes are interpreted to indicate lethal cold injuries, because recovery was not possible when the animals were returned to 12°C after more than 2 days of exposure to 0°C. A low critical temperature indicating the onset of cold-induced anaerobiosis is concluded to exist below 4°C owing to the insufficient response of the ventilatory system to the developing hypoxia.     

酸水解蚕蛹制备复合氨基酸的研究

采用硫酸水解法,以蚕蛹制取复合氨基酸产品,得到氨基酸态氮分别为９．０５％和１３．４５％的食用复合氨基粉和精制复合氨基酸粉。食用复合氨基酸粉含有１８种氨基酸,其中必需氨基酸含量为３９．２％。食用复合氨基酸粉的制备方法经工厂小批量生产证实,其工艺简单易行,适合于中小企业采用,该产品的质量优良,生产成本低廉,具有市场竞争力。     

Surfactant Effects on Lipase-Catalyzed Hydrolysis of Olive Oil in AOT/ISOOCTANE Reverse Micelles

The effects of surfactant concentration on the hydrolytic activity of Candida rugosa lipase in AOT/isooctane reverse micelles with olive oil as the substrate has been investigated. A noncompetitive inhibition by the surfactant on the enzyme was observed. Strong dependences of the kinetic constants k_cat and k_M, but not k_I on the water-to-surfactant ratio (R value) have been identified. The benefits of carrying out the hydrolysis at higher surfactant and water concentrations were demonstrated from the improvement of the initial rate and time course of conversion.     

Long lasting anti-adrenergic effect of 7-oxo-prostacyclin in the heart: a cycloheximide sensitive increase of phosphodiesterase isoform I and IV activities

Evidence is accumulating that 7-oxo-prostacyclin (7-oxo-PGI₂) induces a delayed indirect anti-adrenergic and cytoprotective effect on the myocardium, the mechanism of which is still unclear. To demonstrate that a single application of 7-oxo-PGI₂ (50 g/kg i.m.) 48 h prior to starting experiments attenuates the isoprenaline inducible inotropic response and accumulation of cAMP, isolated hearts of pretreated animals were perfused in the Langendorff mode with and without isoprenaline (1 to 100 nM). The late anti-adrenergic effect of the drug was manifested by a significant attenuation in the elevation of cAMP levels as well as in contractile force development. This effect was not due to changes in cAMP generation as there were identical ₁-adrenoceptor densities and affinities (as calculated from [³H]-CGP binding studies), G_i and G_s protein patterns (as taken from Western blots) as well as adenylyl cyclase activity measurements in the hearts studied. The anti-adrenergic potency of 7-oxo-PGI₂, however, was found to be related to a significant rise in cyclic nucleotide hydrolysis by phosphodiesterase (PDE). Using the fast-performance liquid chromatographic separation for PDE isoforms, a significant increase in the activity of PDE isoforms I and IV (260±28 vs 110±12 pmol cGMP/min x enzyme fraction and 77±11 vs 34±3 pmol cAMP/min x enzyme fraction, respectively) was found in the solubilized fraction of cardiac membranes in comparison to untreated controls; PDE IV activity was also increased in the cytosolic fraction (106±14 vs 65±6 pmol cAMP/min x enzyme fraction). The hypothesis that the delayed anti-adrenergic effect of 7-oxo-PGI₂ is initiated by an induction and accelerated synthesis of PDE I and IV in the heart is underlined by the fact that cycloheximide suppresses completely both the rise in PDE activities and the anti-adrenergic effects studied. It is suggested that an inducible predominance of cAMP degradation over its generation may be of relevance in processes related to heart protection.     

Hydrolysis of triglyceride by the whole cell of Pseudomonas putida 3SK in two-phase batch and continuous reactors systems

Batch and continuous hydrolysis of olive oil in an organic-aqueous two-phase system using the live whole cell of Pseudomonas putida 3SK as a source of a lipase is investigated. The strain was not only fully viable and grown well, but also produced extracellular lipase simultaneously. The degree of hydrolysis, depending on olive oil concentration in the solvents, was maximal at 13.5% (w/v) and decreased with the increase of the substrate concentration. At the optimal condition, a degree of hydrolysis higher than 95% was achieved with 24 h at 30 degrees C when the reaction was carried out in a two-phase batch stirred reactor. For long-term operation a continuous stirred reactor was designed. When the reaction was carried out in a continuous stirred reactor, the degree was hydrolysis reached 86% at a dilution rate of 0.2 h(-1). Satisfactory performance of a two-phase bioreactor was obtained in a long-term continous operation, which lasted for at least 30 days by feeding organic solvent containing olive oil and aqueous media separately. (c) 1994 John Wiley & Sons, Inc.     

Enzymatic synthesis of alkyl beta-D-xylosides by transxylosylation and reverse hydrolysis

The Trichoderma reesei beta-xylosidase (EC 3.2.1.37) is used to catalyze the production of alkyl beta-D-xyloside. Two general methods of production are tested and compared using the same enzyme: transglycosylation and reverse hydrolysis. Using both methods, primary, secondary, and tertiary alcohols are studied as acceptors. In kinetically controlled process (transglycosylation), the chosen donor is methyl beta-D-xyloside and primary, secondary, and tertiary alkyl alcohols are accepted. In the equilibrium-controlled synthesis, the donor is xylose whereas acceptors are only primary and secondary alcohols. The influence of the donor concentration is investigated in both processes. The yields of the kinetically controlled reactions are higher compared with those of the equilibrium-controlled synthesis. The specificity of the beta linkage is confirmed by proton nuclear magnetic resonance ((1)H NMR) analysis. (c) 1994 John Wiley & Sons, Inc.     

Polymeric derivatives of plant growth regulators: synthesis and properties

The polymeric formulations of plant growth regulators (PGRs) are high molecular weight systems in which the PGR unit is attached to the polymeric chain by a hydrolysable chemical bond. These polymeric derivatives (esters, ethers, or else) of PGRs are characterised by the ability to release the active compound (PGR) from their solutions (mainly aqueous) in certain conditions. The release of the PGR can be controlled by external factors (pH, temperature, enzymes, solution concentration), and inherent properties of the whole macrosystem chemical structure, such as the type of the hydrolysable bond between PGR unit and the main polymeric chain, the structure of the polymer chain (e.g. molecular weight, level of hydrophilicity, and the content of hydrophobic groups, macromolecular conformation in solution etc.). These controlled (slow) release PGRs display certain advantages over conventional PGR formulations due to their prolonged action, improved efficiency (e.g. wide range of effective concentrations) greater safety to non-target organisms and the applicators. In addition the ability of altering the solubility level and modifying the aplication form is of considerable interest. The biological activity efficiency of polymeric PGRs has been documented and the relation of this efficiency to the PGR unit hydrolytic release ability has been mentioned. Slow release polymeric PGRs are considered to solve certain problems in agriculture.Abbreviations PGR plant growth regulator - C(S)RF controlled (slow) release form - PD polymeric derivative - ACC 1-amino-cyclopropane-1-carboxylic acid - NAA 1-naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - BAP N⁶-benzylaminopurine - ABA abscisic acid - GA gibberellin - LMW low molecular weight - HMW high molecular weight