活性炭分离回收胱氨酸生产废弃物中的酪氨酸

<正> 毛发提取胱氨酸二次母液(pH=3.5),经中和沉淀后,可得到毛发量1—3％的酪氨酸、胱氨酸为主的混合物。其中酪氨酸与胱氨酸的含量各占30％。由于酪氨酸与胱氨酸的等电点、溶解度及其相近,要想从酪氨酸与胱氨酸的比例为1∶1的混合物中     

用酪氨酸苯酚裂合酶催化苯酚和 S-(O-硝基苯)-L-半胱氨酸合成 L-酪氨酸

近年来生物技术学家对酶法合成氨基酸具有浓厚的兴趣。我们对弗氏柠檬酸细菌(ATCC29063)酪氨酸苯酚裂合酶催化高Vmax,低Km 基质S(O—硝基苯)-L-半胱氨酸和苯酚,合成L 酪氨酸进行了研究。用可溶性S(o-硝基苯)-L-半胱氨酸反应迅速,2小时或更短时间反应达到完全,约70%苯酚转化为L-酪氨酸,反应底物所剩无几。这一反应的最适pH 值范围广泛,从6.8到9以上。由于S-(O硝基苯)-L-半胱氨酸易用L-半胱氨酸和对一氟硝基苯制备,所以采用这一反应合成L-酪氨酸及其衍生物具有潜在价值。     

假单胞菌酶法转化DL-ATC合成L-半胱氨酸

采用微生物酶转化法制备L-半胱氨酸具有周期短、成本低、区域和立体选择性强、反应条件容易控制、环境友好等特点,与传统的毛发水解以及化学合成工艺相比显示出明显的优越性。本文从假单胞菌产酶条件和酶学性质、DL-ATC生物转化途径、固定化细胞转化工艺、基因工程菌的研究、以及L-半胱氨酸脱巯基酶的研究等5个方面介绍了国内外关于生物转化DL-2-氨基-Δ2-噻唑啉-4-羧酸(DL-ATC)合成L-半胱氨酸的研究进展。     

角蛋白水解液中L-胱氨酸含量测定方法的比较研究

比较研究了用碘量法和分光光度法测定角蛋白水解液中Ｌ－胱氨酸含量的适用性。结果表明,磺量法不适用于水解液中Ｌ－胱氨酸含量的测定。分光光度法不仅适用于水解液中Ｌ－胱氨酸含量的测定,同时也适用于产品Ｌ－胱氨酸含量的测定,测定结果准确可靠,相对误差为０．２９％～０．８４％。     

L-酪氨酸纯化工艺的优化

采用一步法对不同来源的L-酪氨酸粗品进行了分离纯化。通过对碱溶pH值、分解胱氨酸的温度、分解时间和中和pH值等条件的优化,提高了L-酪氨酸产品的纯度。试制产品主要质量指标均达到日本味之素(AJI92)和中国药典(CP2000)标准。同时,工艺的简化、活性炭的回收利用以及滤液的循环使用,均大幅降低了生产成本。     

Citrobacter freundii休止细胞催化合成L-多巴

以在L-酪氨酸诱导下高效表达酪氨酸酚解酶的菌株Citrobacter freundii 48003-3的休止细胞为生物催化剂,以邻苯二酚、丙酮酸钠、醋酸铵为前体,选择性合成L-DOPA。研究了反应温度、pH和前体浓度等对合成L-DOPA的影响。最优反应条件下,反应12h,L-DOPA的量可达到9．5g／L。     

酶法生产L-苯丙氨酸

<正> 一、前言直到几年前,L—苯丙氨酸(L-phe)主要用作输液,一年不过需要100～200吨。但近来作为人工甜味剂天冬甜精(L-苯丙氨酸甲酯和L-天冬氨酸的肽)的合成原料,对它的需求量显著增加,预测到1990年年需要量近8000吨。L-phe过去主要采用两种方法提供:直     

氨基酸的消旋研究

在纯乙酸溶剂中,以乙酸酐酰化消旋酪氨酸,消旋温度为90℃,L-酪氨酸和乙酸酐的摩尔比为12,每消旋1gL-酪氨酸时,溶剂乙酸的用量为5mL,效果较好。同样的方法应用于L-脯氨酸,L-苏氨酸,L-缬氨酸,L-赖氨酸,L-半胱氨酸等氨基酸,消旋率均可达到100%。     

电解还原法制备L-半胱氨酸盐酸盐

L-半胱氨酸盐酸盐被广泛地用于生物化学和营养学的研究以及组织培养基的制备。因为它的—SH基易被氧化因而被用作还原剂。在医药上可用作肝炎,肝中毒,锑剂中毒,放射性药物中毒等的解毒剂。五十年代末已有人用L-胱氨酸电解还原法制得L-半胱氨酸盐酸盐。我们根据本厂设备条件,建立了L-半胱氨酸盐酸盐的电解还原法制备工艺,并生产了纯度较高的L-半胱氨酸盐酸盐,现介绍如下: 一、原理     

微生物发酵法生产L-丝氨酸及L-半胱氨酸研究进展

L-丝氨酸及L-半胱氨酸在食品、医药和化妆品等行业有着广泛的应用,在植物和微生物中两者从头合成的前体物均为3-磷酸甘油酸。微生物发酵生产这两种氨基酸以其社会、经济及环境效益展现出良好的前景。针对近年来微生物发酵法生产L-丝氨酸及L-半胱氨酸的研究成果,本文综述了当前国内外学者在该领域研究的热点,即代谢途径及其调节、相关氨基酸的转运及运输、菌种及菌株改造、新菌种的开发等。最后结合当前生物技术的新发展,对今后的研究方向进行了展望。     

Growth enhancement induced by prolonged L-dopa administration in rats

To follow growth of rats, in which growth hormone secretion has been chronically stimulated, L-Dopa (5 mg/kg) was injected subcutaneously twice daily for 70 days to growing rats. A control group, matched for sex and sibship, pair fed with the treatment group was given saline injections. At 10-day intervals, the rats were weighed and measured. At 90 days of age the rats were ether anesthesized, bled for growth hormone determination, and internal viscera weighed. Weight gain and length in the L-Dopa-treated group was found to be significantly greater. A mean weight gain of 6% and 12% in the male and female rats, respectively, and a mean length gain of 5% in both male and female rats was observed at 90 days of age. The thymus, thyroid, adrenals, uterus, and gonads all tended to be heavier in the L-Dopa-treated group. Significantly heavier kidneys were found in the L-Dopa group. Serum growth hormone was found to be 8.44 +/- 1.4(SE) ng/ml in the L-Dopa group and 4.6 +/- 0.9 ng/ml in the control group. It is concluded that the continuous administration of L-Dopa produces an increase of circulating serum growth hormone levels, and this in turn enhances growth.     

EFFECTS OF L-DOPA ON CIRCADIAN RHYTHMS OF 6-OHDA STRIATAL LESIONED RATS: A RADIOTELEMETRIC STUDY

Temporal variation in the motor function of Parkinson's disease (PD) patients suggests the potential importance of a chronobiological and chronopharmacological approach in its clinical management. We previously documented the effects of striatal injection of 6-OHDA (as an animal model of PD) on the circadian rhythms of temperature (T), heart rate (HR), and locomotor activity (A). The present work assessed the possible influence of L-Dopa on these same rhythms in the 6-OHDA animal model of PD. The study began after a four-week recovery period following surgical implantation of telemetric devices to monitor the study variables and/or anaesthesia. The study was divided into an initial one-week control period (W1) for baseline measurement of T, HR, and A rhythms. Thereafter, stereotaxic 6-OHDA lesioning was done. and a second monitoring for two weeks followed (W2, W3). Rats were then randomly divided into two groups: eight control rats received, via a mini-osmotic pump implanted subcutaneously, the excipient saline; the other eight rats received L-Dopa (100?mg/kg SC/day). After a seven-day period (W4), the pumps were removed and the T, HR, and A rhythms were monitored for two weeks (W5 and W6). To control for 6-OHDA striatal dopamine-induced depletion, 12 other rats were injected by identical methods (eight rats with 6-OHDA and four controls with saline) and sacrificed at W1, W3, and W5 for dopamine striatal content determination. To verify the delivery of levodopa from the osmotic pumps, plasma levels of levodopa and its main metabolites 3-OMD, DOPAC, and HVA were determined on separate group of rats receiving the drug under the same experimental conditions (osmotic pumps delivering continuously 10 µl/h for seven days, 100?mg/kg/subcutaneously). Our results agree with previously reported rhythmic changes induced by 6-OHDA—loss of circadian rhythmicity or changes in the main parameters of the registered rhythms. When circadian rhythmicity was abolished, L-Dopa treatment improved or accelerated recovery of the circadian rhythms, the effect being more pronounced for the HR rhythm. When circadian rhythms were not abolished but perturbed, L-Dopa treatment did not improve the 6-OHDA-induced changes in the T and A mesor (24?h mean level), while a significant effect was observed for HR. It appears that constant-rate L-Dopa infusion is unable to totally balance dopamine depletion; taking into account the circadian pattern of many structures implicated in drug effect, a sinusoidal delivery of L-Dopa must be evaluated in future experiments. (Author correspondence: bernard.bruguerolle@univmed.fr)     

Caffeine-induced locomotor activity: Possible involvement of GABAergic-dopaminergic-adenosinergic interaction

Caffeine (10–40 mg/kg, p.o.) enhanced locomotor activity (LA). Administration of GABA antagonist, bicuculline (0.5–1.0 mg/kg, i.p.), potentiated this caffeine-induced increase of LA, as well as LA of control rats. Treatment with the GABA agonist, muscimol (0.25–1 mg/kg, i.p.) or dopaminergic antagonist, haloperidol (0.25–1 mg/kg, i.p.) or muscarinic receptor blocker, atropine (3.75–5 mg/kg, i.p.), or inhibitor of acetylcholine esterase physostigmine (0.05–0.30 mg/kg, i.p.) or nicotine (0.5–1.5 mg/kg, i.p.) an nicotinic receptor agonist all decreased the LA of both caffeinetreated and control rats. Haloperidol-induced reduction in caffeine-induced increase in LA was found to be withdrawn with higher dose of caffeine. The dopamine agonist L-Dopa (75–150 mg/kg, p.o.) along with carbidopa (10 mg/kg, p.o.) increased the LA in control rats and potentiated the LA of caffeine treated rats. The haloperidol attenuated the bicuculline-induced increase in LA and atropine or physostigmine attenuated the bicuculline or L-Dopa+carbidopa-induced increase in LA in both caffeine treated and control rats when those drugs were administered concomitantly with bicuculline or L-Dopa+carbidopa. These results suggest that (a) the GABAergic system has direct role in the regulation of LA, and (b) caffeine potentiates LA by antagonism of the adenosine receptor and activation of the dopaminergic system which, in turn, reduces GABAergic activity through the reduction of cholinergic system.     

Restorative effects of glutamate antagonists in experimental parkinsonism

Several compounds with antagonistic actions on N-methyl-D-aspartate (NMDA) receptors were tested for an antiakinesic action in hypoactive MPTP-treated C57 BL/6 mice rendered tolerant to the motor activity enhancing effects of the 20 mg/kg, s.c., dose of L-Dopa; each compound was administered 60 min before the administration of the dopamine precursor. The classes of compounds studied included the noncompetitive NMDA antagonists, memantine, amantadine and MK-801, the competitive NMDA antagonist, CGP 40116, the anticonvulsive and putative anticonvulsive agents, lamotrigine and FCE 26743, with a partial glutamatergic antagonistic action. All six compounds elevated locomotor, rearing and total activity counts of L-Dopa-tolerant mice in co-administration with L-Dopa in dose-specific or dose-dependent manners but only memantine and MK-801 affected motor activity in the control mice, that also received chronic L-Dopa treatment. Thus, the restorative actions of those compounds in suprathreshold L-Dopa-tolerant MPTP-treated mice subjected to "wearing-off" of L-Dopa efficacy were assessed in a series of experiments. Within each class of potentially therapeutic agents a differential restorative efficacy of the motor activity-stimulating effects of hypoactive MPTP mice was obtained, confirming the putative antiparkinsonian applications of compounds with glutamate antagonistic actions.     

酵母氨基酸发酵研究

从32株酵母菌中筛选到丙氨酸产生株产朊假丝酵母(Candida utilisY18),丙氨酸产量为1 mg/ml,发酵最适时间为72小时,最适pH值为6—7。用NTG和DES对菌株Y18进行诱变获得了一些芳香簇氨基酸变异株(FPA~r、T_(rp)~-、phe~-)和赖氨酸类似物抗性变异株(AEC~r)以及其他一些变异菌株。通过对这些变异菌株的氨基酸发酵研究,从芳香簇氨基酸、极性氨基酸和中性氨基酸三个方面,分析和探讨了酵母菌作为氨基酸生产菌的潜力及可行性。     

Non-Healing Ulcer Due to Trichosporon loubieri in an Immunocompetent Host and Review of Published Reports

We report here a case of non-healing ulcer due to Trichosporon loubieri in an apparently immunocompetent female. The identity of isolate was confirmed by DNA sequencing of D1/D2 region of 26S rDNA. The minimum inhibitory concentrations of the isolate were amphotericin B—0.5 μg/ml; fluconazole—4 μg/ml; posaconazole—0.25 μg/ml; voriconazole—0.06 μg/ml. The patient was managed by extensive debridement and oral fluconazole 150 mg daily for 6 weeks. She responded to therapy. To the best of our knowledge, till date, this is the fourth report of human infection due to T. loubieri and the first of its kind in an immunocompetent host. A review of published literature on infections due to T. loubieri is also included.     

Determination of cyclizine and norcyclizine in plasma and urine using gas—liquid chromatography with nitrogen selective detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-a]-[1,4]-benzodiazepine-3-carboxamide [I] and its 4-hydroxy metabolite, 8-chloro-6-(2-chlorophenyl)-4-hydroxy-4H-imidazo-[1,5-a][1,4]-benzodiazepine-3-carboxamide [II] in whole blood, plasma or urine. The assay for both compounds involves extraction into diethyl ether—methylene chloride (70:30) from blood, plasma, or urine buffered to pH 9.0. The overall recoveries of [I] and [II] are 92.0 ± 5.4% (S.D.) and 90.3 ± 4.9% (S.D.), respectively. The sensitivity limit of detection is 50 ng/ml of blood, plasma, or urine using a UV detector at 254 nm. The HPLC assay was used to monitor the blood concentration—time fall-off profiles, and urinary excretion profiles in the dog following single 1 mg/kg intravenous and 5 mg/kg oral doses, and following multiple oral doses of 100 mg/kg/day of compound [I].     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10-μm μBondapak phenyl column with an eluting solvent of water—methanol—1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(d-(-)-α-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 ± 6.3% (S.D.) in the concentration ranges of 0.1–20 μg per 0.2 ml of plasma with a limit of detection equivalent to 0.5 μg/ml plasma. The urine assay was validated over a concentration range of 0.025–5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 μg/ml) using a 0.1-ml urine specimen per assay.The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Chronic Levodopa Administration Followed by a Washout Period Increased Number and Induced Phenotypic Changes in Striatal Dopaminergic Cells in MPTP-Monkeys

In addition to the medium spiny neurons the mammalian striatum contains a small population of GABAergic interneurons that are immunoreactive for tyrosine hydroxylase (TH), which dramatically increases after lesions to the nigrostriatal pathway and striatal delivery of neurotrophic factors. The regulatory effect of levodopa (L-Dopa) on the number and phenotype of these cells is less well understood. Eleven macaques (Macaca fascicularis) were included. Group I (n = 4) received 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) and L-Dopa; Group II (n = 4) was treated with MPTP plus vehicle and Group III (n = 3) consist of intact animals (control group). L-Dopa and vehicle were given for 1 year and animals sacrificed 6 months later. Immunohistochemistry against TH was used to identify striatal and nigral dopaminergic cells. Double and triple labeling immunofluorescence was performed to detect the neurochemical characteristics of the striatal TH-ir cells using antibodies against: TH, anti-glutamate decarboxylase (GAD₆₇) anti-calretinin (CR) anti-dopa decarboxylase (DDC) and anti-dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32). The greatest density of TH-ir striatal cells was detected in the striatum of the L-Dopa treated monkeys and particularly in its associative territory. None of the striatal TH-ir cell expressed DARPP-32 indicating they are interneurons. The percentages of TH-ir cells that expressed GAD67 and DDC was approximately 50%. Interestingly, we found that in the L-Dopa group the number of TH/CR expressing cells was significantly reduced. We conclude that chronic L-Dopa administration produced a long-lasting increase in the number of TH-ir cells, even after a washout period of 6 months. L-Dopa also modified the phenotype of these cells with a significant reduction of the TH/CR phenotype in favor of an increased number of TH/GAD cells that do not express CR. We suggest that the increased number of striatal TH-ir cells might be involved in the development of aberrant striatal circuits and the appearance of L-Dopa induced dyskinesias.     

L-Dopa and the Albino Riddle: Content of L-Dopa in the Developing Retina of Pigmented and Albino Mice

Background

The absence or deficiency of melanin as in albinos, has detrimental effects on retinal development that include aberrant axonal projections from eye to brain and impaired vision. In pigmented retinal pigment epithelium (RPE), dihydroxyphenalanine (L-Dopa), an intermediate in the synthetic path for melanin, has been hypothesized to regulate the tempo of neurogenesis. The time course of expression of retinal L-Dopa, whether it is harbored exclusively in the RPE, the extent of deficiency in albinos compared to isogenic controls, and whether L-Dopa can be restored if exogenously delivered to the albino have been unknown.

Methodology/Principal Findings

L-Dopa and catecholamines including dopamine extracted from retinas of pigmented (C57BL/6J) and congenic albino (C57BL/6J-tyr^c2j) mice, were measured throughout development beginning at E10.5 and at maturity. L-Dopa, but not dopamine nor any other catecholamine, appears in pigmented retina as soon as tyrosinase is expressed in RPE at E10.5. In pigmented retina, L-Dopa content increases throughout pre- and postnatal development until the end of the first postnatal month after which it declines sharply. This time course reflects the onset and completion of retinal development. L-Dopa is absent from embryonic albino retina and is greatly reduced in postnatal albino retina compared to pigmented retina. Dopamine is undetectable in both albino and pigmented retinas until after the postnatal expression of the neuronal enzyme tyrosine hydroxylase. If provided to pregnant albino mothers, L-Dopa accumulates in the RPE of the fetuses.

Conclusions

L-Dopa in pigmented RPE is most abundant during development after which content declines. This L-Dopa is not converted to dopamine. L-Dopa is absent or at low levels in albino retina and can be restored to the RPE by administration in utero. These findings further implicate L-Dopa as a factor in the RPE that could influence development, and demonstrate that administration of L-Dopa could be a means to rescue developmental abnormalities characteristic of albinos.