PVA和PEG(6000)预处理对大豆胚轴生长、蛋白质合成和ATP含量的影响

在低温吸胀阶段,经PVA(聚乙烯醇)和PEG(聚乙二醇6000)预处理的大豆胚轴蛋白质合成和ATP含量均比对照高。在萌发阶段,胚轴生长增快,蛋白质合成明显加快,ATP迅速被消耗,而对照胚轴则相反。试验结果表明,预处理大豆种子萌发和生长与其蛋白质合成、ATP水平和消耗能力有密切关系。     

Increased Neostriatal Tyrosine Hydroxylation During Stress: Role of Extracellular Dopamine and Excitatory Amino Acids

Abstract: We examined the regulation of neostriatal tyrosine hydroxylation during acute stress, testing the hypothesis that excitatory amino acids (EAAs) contribute to the stress-evoked increase in dopamine (DA) synthesis. Dialysis probes implanted into neostriatum permitted delivery of drugs and sampling of extracellular fluid. Rats were exposed to 30 min of intermittent tail shock during infusion of an inhibitor of aromatic amino acid decarboxylase (AAAD), NSD-1015 (100 µM), and DOPA was measured in the dialysate. Tail shock was applied beginning either 15 min after the onset of NSD-1015 treatment (the initial rate of DOPA accumulation) or 75 min after the onset of treatment (when DOPA had approached steady state). Tail shock increased the steady-state levels of extracellular DOPA in neostriatum (+40%). However, there was no change in the initial rate of DOPA accumulation unless animals also received the D₂ receptor antagonist eticlopride (50 nM), in which case an increase was observed (+228%). The impact of tail shock on the steady-state level of DOPA was attenuated by the D₂ agonist quinpirole (100 µM), or by 2-amino-5-phosphonovalerate (APV) (100 µM) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (100 µM), EAA antagonists acting at NMDA or d ,l -α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) receptors, respectively. These data suggest that acute stress normally has little effect on tyrosine hydroxylation in neostriatum due to the inhibitory influence of DA in the extracellular fluid. However, when that influence is absent (e.g., during extended inhibition of DOPA decarboxylation or blockade of DA receptors), stress increases tyrosine hydroxylation via EAAs acting on NMDA and AMPA receptors. Thus, EAAs released from corticostriatal projections may stimulate DA synthesis and thereby restore dopaminergic activity under conditions in which the availability of DA for release has been compromised.     

Prebiotic synthesis of diaminopyrimidine and thiocytosine

The reaction of guanidine hydrochloride with cyanoacetaldehyde gives high yields (40–85%) of 2,4-diaminopyrimidine under the concentrated conditions of a drying lagoon model of prebiotic synthesis, in contrast to the low yields previously obtained under more dilute conditions. The prebiotic source of cyanoacetaldehyde, cyanoacetylene, is produced from electric discharges under reducing conditions. The effect of pH and concentration of guanidine hydrochloride on the rate of synthesis and yield of diaminopyrimidine were investigated, as well as the hydrolysis of diaminopyrimidine to cytosine, isocytosine, and uracil. Thiourea also reacts with cyanoacetaldehyde to give 2-thiocytosine, but the pyrimidine yields are much lower than with guanidine hydrochloride or urea. Thiocytosine hydrolyzes to thiouracil and cytosine and then to uracil. This synthesis would have been a significant prebiotic source of 2-thiopyrimidines and 5-substituted derivatives of thiouracil, many of which occur in tRNA. The applicability of these results to the drying lagoon model of prebiotic synthesis was tested by dry-down experiments where dilute solutions of cyanoacetaldehyde, guanidine hydrochloride, and 0.5m NaCl were evaporated over varying periods of time. The yields of diaminopyrimidine varied from 1 to 7%. These results show that drying lagoons and beaches may have been major sites of prebiotic syntheses.     

Evolution of structure and substrate specificity ind-alanine:d-Alanine ligases and related enzymes

Thed-alanine:d-alanine-ligase-related enzymes can have three preferential substrate specificities. Usually, these enzymes synthesized-alanyl-d-alanine. In vancomycin-resistant Gram-positive bacteria, structurally related enzymes synthesized-alanyl-d-lactate or Dalanyl-d-serine. The sequence of internal fragments of eight structurald-alanine:d-alanine ligase genes from enterococci has been determined. Alignment of the deduced amino acid sequences with those of other related enzymes from Gram-negative and Gram-positive bacteria revealed the presence of four distinct sequence patterns in the putative substrate-binding sites, each correlating with specificity to a particular substrate (d-alanine:d-lactate ligases exhibited two patterns). Phylogenetic analysis showed different clusters. The enterococcal subtree was largely superimposable on that derived from 16S rRNA sequences. In lactic acid bacteria, structural divergence due to differences in substrate specificity was observed. Glycopeptide resistance proteins VanA and VanB, the VanC-type ligases, and Dd1A and DdlB from enteric bacteria andHaemophilus influenzae constituted separate clusters. Correspondence to: P. Courvalin     

Global Brain Ischemia and Reperfusion: Modifications in Eukaryotic Initiation Factors Associated with Inhibition of Translation Initiation

Abstract: We used in vitro translation and antibodies against phosphoserine and the eukaryotic initiation factors eIF-4E, eIF-4G, and eIF-2α to examine the effects of global brain ischemia and reperfusion on translation initiation and its regulation in a rat model of 10 min of cardiac arrest followed by resuscitation and 90 min of reperfusion. Translation reactions were performed on postmitochondrial supernatants from brain homogenates with and without aurintricarboxylic acid to separate incorporation due to run-off from incorporation due to peptide synthesis initiated in vitro. The rate of leucine incorporation due to in vitro-initiated protein synthesis in normal forebrain homogenates was ∼0.4 fmol of leucine/min/µg of protein and was unaffected by 10 min of cardiac arrest, but 90 min of reperfusion reduced this rate 83%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blots of these homogenates showed that neither 10 min of global brain ischemia nor 90 min of reperfusion induced significant alterations in the quantity or serine phosphorylation of eIF-4E. However, we observed in all 90-min-reperfused samples eIF-4G fragments that also bound eIF-4E. The amount of eIF-2α was not altered by ischemia or reperfusion, and immunoblotting after isoelectric focusing did not detect serine-phosphorylated eIF-2α in normal samples or in those obtained after ischemia without reperfusion. However, serine-phosphorylated eIF-2α was uniformly present after 90 min of reperfusion and represented 24 ± 3% of the eIF-2α in these samples. The serine phosphorylation of eIF-2α and partial fragmentation of eIF-4G observed after 90 min of reperfusion offer an explanation for the inhibition of protein synthesis.     

Effects of the Phosphatase Inhibitors Calyculin A and Okadaic Acid on Acetylcholine Synthesis and Content of Rat Hippocampal Formation

Abstract: The biochemical mechanisms involved in the regulation of acetylcholine (ACh) turnover are poorly understood. In the experiments reported here, we examined whether inhibition of the serine/threonine phosphatases 1 and 2A by calyculin A or okadaic acid alters ACh synthesis by rat hippocampal preparations. With hippocampal slices, calyculin A (50 n M ) and okadaic acid (50 n M ) reduced significantly ( p < 0.01) the synthesis of [³H]ACh from [³H]choline. Both calyculin A and okadaic acid produced significant depletion of endogenous tissue ACh in a concentration-dependent manner ( p < 0.01). This depletion was not the result of a drug-induced increase of spontaneous ACh release, which was not changed significantly ( p > 0.7) by either drug. Choline acetyltransferase (ChAT) activity from tissue exposed to calyculin A or okadaic acid was reduced in a concentration-dependent manner ( p < 0.05), but these phosphatase inhibitors did not act directly on ChAT in vitro; i.e., enzymatic activity was not altered significantly ( p > 0.4) in the presence of calyculin A or okadaic acid. Both high-affinity and low-affinity [³H]choline uptake by hippocampal synaptosomes were reduced significantly in a concentration-dependent manner in the presence of calyculin A or okadaic acid; these agents reduced V _max values for high- and low-affinity choline uptake ( p < 0.01) with no significant change in K _m values ( p > 0.1), indicating a noncompetitive inhibition. Taken together, these data suggest that phosphatase activity plays a role in presynaptic central cholinergic nerve terminal function, in particular in the modulation of ACh synthesis.     

Regulation of and intervention into the oxidative pentose phosphate pathway and adenine nucleotide metabolism in the heart

The capacity of the oxidative pentose phosphate pathway (PPP) in the heart is limited, since the activity of glucose-6-phosphate dehydrogenase (G-6-PD), the first and regulating enzyme of this pathway, is very low. Two mechanisms are involved in the regulation of this pathway. Under normal conditions, G-6-PD is inhibited by NADPH. This can be overcome in the isolated perfused rat heart by increasing the oxidized glutathione and by elevating the NADP⁺/NADPH ratio. Besides this rapid control mechanism, there is a long-term regulation which involves the synthesis of G-6-PD. The activity of G-6-PD was elevated in the rat heart during the development of cardiac hypertrophy due to constriction of the abdominal aorta and in the non-ischemic part of the rat heart subsequent to myocardial infarction. The catecholamines isoproterenol and norepinephrine stimulated the activity of myocardial G-6-PD in a time- and dose-dependent manner. The isoproterenol-induced stimulation was cAMP-dependent and due to increased new synthesis of enzyme protein. The G-6-PD mRNA was elevated by norepinephrine. As a consequence of the stimulation of the oxidative PPP, the available pool of 5-phosphoribosyl-l-pyrophosphate (PRPP) was expanded. PRPP is an important precursor substrate for purine and pyrimidine nucleotide synthesis. The limiting step in the oxidative PPP, the G-6-PD reaction, can be bypassed with ribose. This leads to an elevation of the cardiac PRPP pool. The decline in ATP that is induced in many pathophysiological conditions was attenuated or even entirely prevented by i.v. infusion of ribose. In two in vivo rat models, the overloaded and catecholamine-stimulated heart and the infarcted heart, the normalization of the cardiac adenine nucleotide pool by ribose was accompanied by an improvement of global heart function. Combination of ribose with adenine or inosine in isoproterenol-treated rats was more effective to restore completely the cardiac ATP level within a short period of time than either intervention alone. (Mol Cell Biochem 160/161: 101–109, 1996)     

Co-purification,co-imniunoprecipitation,and coordinate expression of acetyl-coenzyme A carboxylase activity,biotin carboxylase,and biotin carboxyl carrier protein of higher plants

Acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a regulatory enzyme of fatty acid synthesis, and in some higher-plant plastids is a multi-subunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protein (BCCP), and carboxyl transferase (CT). We recently described a Nicotiana tabacum L. (tobacco) cDNA with a deduced amino acid sequence similar to that of prokaryotic BC. We here provide further biochemical and immunological evidence that this higher-plant polypeptide is an authentic BC component of ACCase. The BC protein co-purified with ACCase activity and with BCCP during gel permeation chromatography of Pisum sativum L. (pea) chloroplast proteins. Antibodies to the Ricinus communis L. (castor) BC co-precipitated ACCase activity and BCCP. During castor seed development, ACCase activity and the levels of BC and BCCP increased and subsequently decreased in parallel, indicating their coordinate regulation. The BC protein comprised about 0.8% of the soluble protein in developing castor seed, and less than 0.05% of the protein in young leaf or root. Polypeptides cross-reacting with antibodies to castor BC were detected in several dicotyledons and in the monocotyledons Hemerocallis fulva L. (day lily), Iris L., and Allium cepa L. (onion), but not in the Gramineae species Hordeum vulgare L. (barley) and Panicum virgatum L. (switchgrass). The castor endosperm and pea chloroplast ACCases were not significantly inhibited by long-chain acyl-acyl carrier protein, free fatty acids or acyl carrier protein. The BC polypeptide was detected throughout Brassica napus L. (rapeseed) embryo development, in contrast to the multi-functional ACCase isoenzyme which was only detected early in development. These results firmly establish the identity of the BC polypeptide in plants and provide insight into the structure, regulation and roles of higherplant ACCases.Abbreviations ACCase acetyl-CoA carboxylase - ACP acyl carrier protein - BC biotin carboxylase - BCCP biotin carboxyl carrier protein - CT carboxyl transferase - MF multi-functional - MS multi-subunit We thank our colleagues Nicki Engeseth and Vicki Eccleston for advice on fatty acid analysis and Sarah Hunter for providing the developing Iris seed. This work was supported in part by grant MCB 9406466 from NSF. Acknowledgement is also made to the Michigan Agriculture Experiment Station for its support of this research.     

Metabolism of glucose-6-phosphate and utilization of multiple metabolites for fatty acid synthesis by plastids from developing oilseed rape embryos

The aim of this work was to investigate the partitioning of imported glucose 6-phosphate (Glc6P) to starch and fatty acids, and to CO₂ via the oxidative pentose phosphate pathway (OPPP) in plastids isolated from developing embryos of oilseed rape (Brassica napus L.). The ability of the isolated plastids to utilize concurrently supplied substrates and the effects of these substrate combinations on the Glc6P partitioning were also assessed. The relative fluxes of carbon from Glc6P to starch, fatty acids, and to CO₂ via the OPPP were close to 2∶1∶1 when Glc6P was supplied alone. Under these conditions NADPH generated via the OPPP was greater than that required by the concurrent rate of fatty acid synthesis. Fatty acid synthesis was unaffected by the presence or absence of exogenous NADH and/or NADPH and the requirement of fatty acid synthesis for reducing power is therefore met entirely by intraplastidial metabolism. When Glc6P was supplied in the presence of either pyruvate or pyruvate and acetate, the total flux from these metabolites to fatty acids was up to threefold greater than that from either Glc6P or pyruvate when they were supplied singly. In these experiments there was little competition between Glc6P and pyruvate in fatty acid synthesis and the flux to starch was unchanged. This implies that the starch and fatty acid biosynthesis pathways did not compete for the exogenously supplied ATP on which they were strongly dependent. When Glc6P and pyruvate were provided together, the NADPH generated by the OPPP pathway was less than that required by the concurrent rate of fatty acid synthesis. This suggests that the metabolism of exogenous Glc6P via the OPPP can contribute to the NADPH demand created during fatty acid synthesis but it also indicates that other intraplastidial sources of reducing power must be available under the in-vitro conditions used.