Preparation of (R) and (S) alpha methyldopa from a chiral hydantoin containing the alpha phenylethyl group

Chiral hydantoin (S)-1 was prepared in good yield from phenyl isocyanate and N-[(S)-alpha-phenylethyl]glycinate, (S)-3. Enolate (S)-1-Li was methylated in high yield and good diastereoselectivity. In contrast, a second alkylation reaction of methylated enolate (S)-4-Li proceeded with essentially no diastereoselectivity. Nevertheless, dialkylated hydantoins, (S,S)-7 and (S,R)-7, could be readily separated by flash chromatography and subsequent hydrolysis of either derivative afforded the desired (S)-L-alpha-methyldopa or (R)-D-alpha-methyldopa in good yield.     

The energy transduction mechanism of Na,K-ATPase studied with iron-catalyzed oxidative cleavage.

This paper extends our recent report on specific iron-catalyzed oxidative cleavages of renal Na,K-ATPase and effects of E1 left arrow over right arrow E2 conformational transitions (Goldshleger, R. , and Karlish, S. J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9596-9601). The experiments indicate that only peptide bonds close to a bound Fe2+ ion are cleaved, and provide evidence on proximity of the different cleavage positions in the native enzyme. A sequence HFIH near trans-membrane segment M3 appears to be involved in Fe2+ binding. Previously we hypothesized that E2 and E1 conformations are characterized by formation or relaxation of interactions within the alpha subunit at or near highly conserved sequences, TGES in the minor cytoplasmic loop and CSDK, MVTGD, and VNDSPALKK in the major cytoplasmic loop. This concept has been tested by examining iron-catalyzed cleavage in both non-phosphorylated and phosphorylated conformations and effects of phosphate, vanadate, and ouabain. The results imply that both E1 left arrow over right arrow E2 and E1P left arrow over right arrow E2P transitions are indeed associated with formation and relaxation of interactions between cytoplasmic domains, comprising the minor loop plus N-terminal tail leading into M1 and major loop, respectively. Furthermore, it appears that either non-covalently or covalently bound phosphate bind near CSDK and MVTGD, and Mg2+ ions may bind to residues within TGES and VNDSPALKK and to bound phosphate. Thus cytoplasmic domain interactions seem to occur within or near the active site. We discuss the relationship between structural changes in the cytoplasmic domain and movements of trans-membrane segments that lead to cation transport. Presumably conformation-dependent formation and relaxation of domain interactions underlie energy transduction in all P-type pumps.     

Synthesis of hydantoin analogues of (2S,3R,4S)-4-hydroxyisoleucine with insulinotropic properties

The first synthesis of an optically pure (2R,3R,4S)-hydantoin 2, analogue of (2S,3R,4S)-4-hydroxyisoleucine, was achieved in two steps in un-optimized 35% overall yield from previously reported aldehyde synthon 1. (2R,3R,4S)-Hydantoin is stable at acidic pH. This solves the major drawback of (2S,3R,4S)-4-hydroxyisoleucine that easily cyclizes into inactive lactone. Furthermore, (2R,3R,4S)-hydantoin stimulates the insulin secretion by 150% at 25 μM compared with 4-hydroxyisoleucine and insulin secretagogue drug repaglinide. In view of its stability and biological activity, (2R,3R,4S)-hydantoin represents a good candidate for type-2 diabetes management and control.     

Physiopathological Effects of the NO Donor 3-Morpholinosydnonimine on Rat Cortical Synaptosomes

The NO donor 3-Morpholinosydnonimine (SIN-1) releases NO in the presence of molecular oxygen. In this study, we evaluated the effect of SIN-1 on mitochondria of rat cortical synaptosomes. We demonstrated in vitro that the amount of ONOO⁻ generated and H₂O₂ formation directly correlated with SIN-1 concentration. The mean oxygen consumption by synaptosomal mitochondria was approximately 3.8 nmol of O₂ min⁻¹ mg⁻¹ protein, which decreased significantly in the presence of SIN-1 1 mM to 2.5 nmol O₂ min⁻¹ mg⁻¹. This decrease was not modified by catalase or Trolox, demonstrating that ONOO⁻ was responsible for the effect. The same concentration of SIN-1 caused a significant decrease of ATP production by synaptosomal mitochondria and depolarized the mitochondrial membrane. Moreover, ROS production increased progressively and was completely inhibited by pre-incubation of synaptosomes with Trolox. Finally, phosphatidylserine was externalized and, at the same time, intrasynaptosomal lactate dehydrogenase decreased confirming both, the external membrane breakdown after the addition of SIN-1 and the damage to the synaptosomes.     

The mechanism of action of tRNA methylases studied with immobilized tRNAs.

Each of the individual tRNAs immobilized on aminohydroxybutyl-cellulose (ABC) through their oxidized 3'-terminal binds affinitively all methylases present in the enzyme extract irrespective of whether this tRNA will be involved in the following step of methylation or not. These data allow to suggest that (a) the formation of a methylase-tRNA complex and the catalytic act of methylation are indeed autonomous processes and (b) the first step of interaction between tRNAs and tRNA methylases is rather unspecific and consists in the recognition of the whole class of tRNA molecules.     

Nociceptin, OP4 receptor ligand in different models of experimental epilepsy

The anticonvulsive activity of nociceptin, endogenous OP4 receptors agonist was investigated in pentylenetetrazole (PTZ), N-methyl D-aspartic acid (NMDA), bicucculine (BCC) and electrically evoked seizure models of experimental epilepsy. Nociceptin, at the dose of 10 nmol, suppressed the clonic seizures induced by PTZ, NMDA and BCC. [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 which has been proposed to be selective antagonist OP4 receptors, did not prevent the action of nociceptin. The effect of [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 on seizures induced by PTZ, NMDA and BCC was very similar to that of nociceptin. These data support the hypothesis that it possesses agonistic properties. Naloxone did not reverse the anticonvulsive action of nociceptin as well as [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 which excludes the participation of opioid receptor in this action. On the other hand in the electroconvulsive model of generalized seizures, nociceptin as well as [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 influenced neither the electroconvulsive threshold nor the maximal electroshock test. The data suggest that nociceptin and [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 can exert anticonvulsive action. These properties depend on OP4 but not opioid receptors activation.     

The mechanism of pentachlorobutadienyl-glutathione nephrotoxicity studied with isolated rat renal epithelial cells

Isolated renal epithelial cells were used to study the mechanism of toxicity of pentachlorobutadienyl-glutathione (PCBG), a nephrotoxic glutathione conjugate of hexachlorobutadiene. The cytotoxicity of PCBG displayed a very steep dose-response relationship; at 10 microM PCBG no toxicity was observed whereas 25, 50, and 100 microM PCBG all resulted in a similar degree of toxicity. In all cases, loss of cell viability was observed only after a 30-min lag period and reached a plateau of 50 to 60% nonviable cells between 90 and 100 min. Toxic doses of PCBG also resulted in the depletion of cellular thiols. Blocking PCBG metabolism by inhibition of gamma-glutamyl transpeptidase [1-gamma-L-glutamyl-2-(2-carboxyphenyl)hydrazine (anthglutin), 2 mM] or renal cysteine conjugate beta-lyase (aminooxyacetic acid, 0.5 mM) resulted in complete protection against PCBG-induced cell damage. Exposure of isolated renal epithelial cells to 100 microM PCBG resulted in the rapid formation of plasma membrane blebs which appeared to be associated with a loss of Ca2+ from the mitochondrial compartment and an elevation of cytosolic Ca2+ concentration as measured by Quin-2. PCBG treatment also resulted in the inhibition of cell respiration and a marked depletion of cellular ATP content, indicating additional mitochondrial effects of the toxin. Our results support a role for renal cysteine conjugate beta-lyase in the metabolic activation of PCBG and suggest that PCBG-induced renal cell injury may be the result of selective effects on mitochondrial function.     

Efficient selective preparation of methyl-1,2,4-tri-O-acetyl-3-O-benzyl-beta-L-idopyranuronate from methyl 3-O-benzyl-L-iduronate

Methyl 1,2,4-tri-O-acetyl-3-O-benzyl-L-idopyranuronate 6beta/6alpha, prepared from methyl 3-O-benzyl-L-iduronate (4), is a key synthon in heparin/heparan sulfate synthesis. The 1H and 13C NMR spectra of the furanose-pyranose mixture of 4, after dissolution and equilibration in d(4)-methanol, were fully assigned allowing to expect that 4 could crystallise in the beta-pyranose form. New acetylation conditions able to trap this form were subsequently devised, allowing the isolation of 83% of pure 6beta by simple crystallisation, along with 9% of the 6beta/6alpha mixture. This represents a major advantage over the previously published procedure, especially on multigram scales.     

大鼠外伤后癫痫动物模型研制方法的探讨

目的:探讨大鼠外伤后癫痫(PTE)动物模型模型的制作方法和稳定性。方法:将50只成年雄性SD大鼠随机分为2组,液压损伤组40只,手术对照组10只。液压损伤组的大鼠在清醒状态下给予液压打击造成其重度颅脑外伤,于致伤后6个月内观察大鼠的行为、脑电图改变,并进行组织病理学分析。结果:液压损伤组在致伤后约有42.86%的大鼠出现典型的癫痫发作症状和脑电图上痫样放电。病理检查见伤侧脑皮质有局部挫伤,蛛网膜下腔及脑实质有出血灶和胶质结节形成等改变。液压损伤组其发作形式、脑电图特征和病理学改变与人类的PTE特征基本一致。结论:颅脑液压冲击伤可建立起清醒状态下大鼠的PTE动物模型,操作简单、稳定性及可靠性较好,有望为PTE的深入研究提供一种更为有效的实验动物模型。