猪脑谷氨酸脱羧酶的分离纯化以及生物学活性鉴定

L-谷氨酸脱羧酶是γ-氨基丁酸合成的关键限速酶,广泛的存在于脊椎动物神经细胞以及β-胰腺细胞,是胰岛素依赖型糖尿病(IDDM)病人以及僵硬综合症(SMS)病人血清的关键抗原。运用sephamryl S-200以及DEAEsepharose可以从猪脑中分离纯化出谷氨酸脱羧酶。纯化的GAD在变性条件下电泳,经考马斯亮蓝R250染色以及Western-Blot鉴定主要有两条带,分子量分别为67kD和44kD。根据L-谷氨酸脱羧酶能够分解谷氨酸产生γ-氨基丁酸和CO2的特性,通过测定产物γ-氨基丁酸推断酶活。以上实验结果表明从猪脑中分离纯化到的是具有生物学活性以及免疫原性的谷氨酸脱羧酶,可进一步改良为IDDM检测试剂盒,用于IDDM的预防和预测。     

Expression of Rice Glutamate Decarboxylase in <Emphasis Type="Italic">Bifidobacterium Longum</Emphasis> Enhances γ-Aminobutyric Acid Production

Bifidobacteria are important for the production of fermented dairy products and probiotic formulas but have a low capacity for γ-aminobutyric acid (GABA) production. To develop a Bifidobacterium strain with an enhanced GABA production, we transformed Bifidobacterium longum with a rice glutamate decarboxylase (OsGADC⁻) gene by electroporation. When the transformed strain was cultured in medium containing monosodium glutamate, the amount of GABA increased significantly compared with those of untransformed Bifidobacterium. Thus, by introducing a plant derived GAD gene, a Bifidobacterium strain has been genetically engineered to produce high levels of GABA from glutamate.     

外源性铜对SD鼠脑内铜稳态与γ-氨基丁酸、谷氨酸含量影响的实验研究

目的：探讨脑内铜稳态与γ-氨基丁酸、谷氨酸含量之间的关联性,了解脑内铜代谢参与神经疾病的作用机制.方法：把实验动物分为8组：对照组,戊巴比妥组,腹腔注射CuCl2组（剂量分别为2 mg/kg、10 mg/kg、50 mg/kg）,CuCl2-戊巴比妥混合组（先腹腔注射CuCl2,再注射戊巴比妥）.检测SD鼠血清和海马内不同形态铜、γ-氨基丁酸和谷氨酸含量.结果：单一铜胁迫下发现随着外源铜升高血清和海马总铜及血清非蛋白结合铜和海马内谷氨酸含量显著升高,在50 mg/kg注射剂量下达到最高（P＜0.02）;海马非蛋白结合铜和神经递质γ-氨基丁酸含量在2 mg/kg注射剂量下达到最高（P＜0.02）,并随着外源铜浓度升高而降低.单一注射戊巴比妥后海马游离铜含量明显降低（P＜0.02）.铜胁迫1小时后注射1％戊巴比妥结果表明,戊巴比妥能明显降低铜胁迫下海马及血清总铜和游离铜含量（P＜0.05）.结论：外源铜能改变体内铜稳态;铜稳态失衡导致神经递质γ-氨基丁酸、谷氨酸含量变化,γ-氨基丁酸含量与非蛋白结合铜呈正相关.     

Duplication of the Rdl GABA receptor subunit gene in an insecticide-resistant aphid, Myzus persicae

Resistance to cyclodiene insecticides is associated with replacements of a single amino acid (alanine 302) in a γ-aminobutyric acid (GABA) receptor subunit encoded by the single-copy gene Resistance to dieldrin (Rdl). Alanine 302 is predicted to reside within the second membrane-spanning region of the Rdl receptor, a region that is thought to line the integral chloride ion channel pore. In all cyclodiene-resistant insects studied to date, this same alanine residue is replaced either by a serine, or, in some resistant strains of Drosophila simulans, a glycine residue. Therefore, individuals can carry only two different Rdl alleles. In contrast, here we report the presence of up to four different Rdl-like alleles in individual clones of the green peach aphid, Myzus persicae. In addition to the wild-type copy of Rdl gene (encoding A302 or allele A), M. persicae carries three other alleles with the following amino acid replacements: A302 → Glycine (allele G), A302 → Serine^TCG (allele S) and A302 → Serine^AGT (allele S′). Evidence from direct nucleotide sequencing and Single Stranded Conformational Polymorphism (SSCP) analysis shows that at least three of these different Rdl alleles (i.e. A, G and S) are commonly present in individual aphids or aphid clones. Southern analysis using allele-specific probes and analysis of sequences downstream of the exon containing the resistance-associated mutation confirm the presence of two independent Rdl-like loci in M. persicae. One locus carries the susceptible alanine (A) and/or resistant glycine (G) allele while the other carries the two serine alleles (S or S′). Whereas resistance levels are correlated with the glycine replacement, the S allele was present in all aphid clones, regardless of their resistance status. These results suggest that target site insensitivity is associated with replacements at the first (A/G) but not the second (S/S′) locus. Phylogenetic analysis of nucleotide sequences indicates that both putative aphid Rdl loci are monophyletic with respect to other insect Rdl genes and may have arisen through a recent gene duplication event. The implications of this duplication with respect to insecticide resistance and insect GABA receptor subunit diversity are discussed. Received: 10 March 1998 / Accepted: 21 July 1998     

Differences in Quality of Life,Anxiety and Depression in Patients with Paroxysmal Atrial Fibrillation and Common Forms of Atrioventricular Reentry Supraventricular Tachycardias

Introduction

The aim of this study was to evaluate the differences in quality of life and psychosocial stress parameters among patients with paroxysmal atrial fibrillation (AF) and common forms of atrioventricular reentry supraventricular tachycardias (SVTs).

Methods and Results

The total study population included 106 patients, 54 patients with paroxysmal AF (32 males, age 56.64±12.50 years) and 52 with SVTs (25 males, age 40.46±14.96 years). General health (p<0.01), physical function (p=0.004), role emotion (p=0.002) and role physical (p<0.01) scores were lower in patients who suffered AF. SF-36 physical and mental health summary measures were also significantly lower in the AF group compared to those in SVT group (p<0.01 and p=0.001, respectively). Lower SF-36 total score was observed in patients with AF compared to those with SVTs (p<0.01). Comparing the anxiety and depression scores all the values were higher in patients with AF. Higher STAI-state scores (p<0.01), STAI-trait scores (p=0.039) and BDI scores (p=0.077) were seen in patients who suffered AF comparing to those with SVTs.

Conclusions

Quality of life is significantly impaired and the level of anxiety is significantly higher in patients with AF comparing to those with common forms of SVTs.     

Oxidative Activation of K-Cl Cotransport by Diamide in Erythrocytes from Humans with Red Cell Disorders,and from Several Other Mammalian Species

Red blood cells (RBCs) from different mammalian species were investigated for the presence of diamide-induced oxidative activation of K-Cl cotransport reported to be present in sheep but absent in human RBCs. K efflux was measured in RBCs from human with hemoglobin (Hb) A or S, glucose-phosphate dehydrogenase (G6PDH) and a cytoskeletal deficiency, and from rat, mouse and rabbit. RBCs were incubated with diamide (0–1.0 mm) in K-free Cl or NO₃ media of variable osmolalities (200–450 mOsM). Cl-dependent K efflux or K-Cl cotransport (estimated as the difference between K efflux rate constants in Cl and NO₃) was activated by diamide in a sigmoidal fashion. Relative maximum K-Cl cotransport followed the sequence: human HbA (1) < rabbit (1.8) < sheep (6.9) < human HbS (9.5) ∼ rat (9.7). Relative diamide concentrations for half maximal activation of K-Cl cotransport followed the sequence: sheep (1.9) > human Hb A (1) > rabbit (0.75) > human HbS and rat (0.67). Cell swelling in 200 mOsM doubled K-Cl cotransport in diamide, both in human HbA and S cells but reduced that in rat RBCs. In contrast, cell shrinkage at 450 mOsM obliterated K-Cl cotransport in human HbA and S but not in rat RBCs. Human RBCs with G6PDH and a cytoskeleton deficiency behaved like HbA RBCs. In mouse RBCs, diamide-activated K-Cl cotransport was 30% higher in isotonic than in hypotonic medium. In human HbA and S, and in low or high K sheep RBCs fractionated by Percoll density gradient, diamide increased the activity of K-Cl cotransport, an effect inversely correlated with cell density. Analysis of pooled data reveals that K-Cl cotransport accounted for about 80% of all K flux in Cl. There was a statistically significant correlation between K-Cl cotransport and K efflux in Cl (P < 0.00001) and in NO₃ (P < 0.00001). In conclusion, a diamide-activated K-Cl cotransport was present in human RBCs and in all other mammalian RBCs tested, with a large inter-, and for human and sheep, intraspecies variability for its maximum activity. Received: 5 June 1996/Revised: 4 October 1996     

Neurosteroid Biosynthesis Regulates Sexually Dimorphic Fear and Aggressive Behavior in Mice

The neurosteroid allopregnanolone is a potent positive allosteric modulator of GABA action at GABA_A receptors. Allopregnanolone is synthesized in the brain from progesterone by the sequential action of 5α-reductase type I (5α-RI) and 3α-hydroxysteroid dehydrogenase (3α-HSD). 5α-RI and 3α-HSD are co-expressed in cortical, hippocampal, and olfactory bulb glutamatergic neurons and in output neurons of the amygdala, thalamus, cerebellum, and striatum. Neither 5α-RI nor 3α-HSD mRNAs is expressed in glial cells or in cortical or hippocampal GABAergic interneurons. It is likely that allopregnanolone synthesized in principal output neurons locally modulates GABA_A receptor function by reaching GABA_A receptor intracellular sites through lateral membrane diffusion. This review will focus on the behavioral effects of allopregnanolone on mouse models that are related to a sexually dimorphic regulation of brain allopregnanolone biosynthesis. Animal models of psychiatric disorders, including socially isolated male mice or mice that receive a long-term treatment with anabolic androgenic steroids (AAS), show abnormal behaviors such as altered fear responses and aggression. In these animal models, the cortico-limbic mRNA expression of 5α-RI is regulated in a sexually dimorphic manner. Hence, in selected glutamatergic pyramidal neurons of the cortex, CA3, and basolateral amygdala and in granular cells of the dentate gyrus, mRNA expression of 5α-RI is decreased, which results in a downregulation of allopregnanolone content. In contrast, 5α-RI mRNA expression fails to change in the striatum medium spiny neurons and in the reticular thalamic nucleus neurons, which are GABAergic. By manipulating allopregnanolone levels in glutamatergic cortico-limbic neurons in opposite directions to improve [using the potent selective brain steroidogenic stimulant (SBSS) S-norfluoxetine] or induce (using the potent 5α-RI inhibitor SKF 105,111) behavioral deficits, respectively, we have established the fundamental role of cortico-limbic allopregnanolone levels in the sexually dimorphic regulation of aggression and fear. By selectively targeting allopregnanolone downregulation in glutamatergic cortico-limbic neurons, i.e., by improving the response of GABA_A receptors to GABA, new therapeutics would offer appropriate and safe management of psychiatric conditions, including impulsive aggression, irritability, irrational fear, anxiety, posttraumatic stress disorders, and depression. Special issue article in honor of Dr. Ji-Sheng Han.     

Fluoxetine Partly Exerts its Actions Through GABA: A Neurochemical Evidence

Fluoxetine, as a serotonin re-uptake inhibitor augments serotonin concentration within the synapse by inhibiting the serotonin transporter. The contribution of amino acids has also been shown in depression. We hypothesized that fluoxetine exerts its actions at least in part by intervening brain signaling operated by amino acid transmitters. Therefore the aim of this study is to supply neurochemical evidence that fluoxetine produces changes in amino acids in cerebrospinal fluid of rats. Sprague-Dawley rats were anesthetized and concentric microdialysis probes were implanted stereotaxically into the right lateral ventricle. Intraperitoneal fluoxetine (2.5 or 5 mg/kg) or physiological saline was administered and the probes were perfused with artificial cerebrospinal fluid at a rate of 1 μl/min. In the chronic fluoxetine group, the rats were treated daily with oral fluoxetine solution or inert syrup for 3 weeks. The microdialysis probes were placed on the 21st day and perfused the next day. Fluoxetine was ineffective in changing the cerebrospinal fluid GABA levels at the dose of 2.5 mg/kg but produced a significant increase in the perfusates following injection of 5 mg/kg of fluoxetine (P < 0.05). Oral fluoxetine administration (5 mg/kg) for 21 days also elevated the CSF GABA levels by approximately 2-fold (P < 0.05). l-glutamic acid levels were not affected in all groups. These neurochemical findings show that fluoxetine, a selective serotonin re-uptake inhibitor affects brain GABA levels indirectly, and our results suggest that acute or chronic effects may be involved in beneficial and/or adverse effects of the drug.     

Cloning and expression of a full-length glutamate decarboxylase gene fromLactobacillus brevis BH2

A bacterium (BH2) that was found to produce a large amount of γ-aminobutyric acid (GABA) was isolated fromKimchi, a traditional fermented food in Korea. Phylogenetic analysis based on the 16S rDNA sequence and biochemical studies indicated that BH2 belonged to the genusLactobacillus brevis. Under controlled conditions in MRS broth (Difco) with 5% monosodium glutamate, this strain produced GABA at a concentration of 194 mM with a 73% GABA conversion rate after 48 h. A full-length glutamate decarboxylase (gad) gene was cloned by the rapid amplification of cDNA ends (RACE) PCR. The open reading frame (ORF) of thegad gene was composed of 1,407 nucleotides and encoded a protein (468 amino acids) with a predicted molecular weight of 53.5 kDa. The deduced amino acid sequence of GAD fromL. brevis showed 97.5 and 82.7% identities to theL. brevis OPK-3 GAD andL. plantarum WCFS1 GAD, respectively. Thegad gene was expressed inEscherichia coli cells and the expression was confirmed by SDS-PAGE analysis and enzyme activity studies.     

Changes in carotenoids, tocopherols and diterpenes during drought and recovery, and the biological significance of chlorophyll loss in Rosmarinus officinalis plants

Two-year-old rosemary (Rosmarinus officinalis L.) plants were subjected to severe stress by exposure to prolonged drought during a Mediterranean summer. Severely stressed plants recovered completely after the autumn rainfalls although the relative water content remained below 35% for 3 months and the chlorophyll content of leaves was reduced by up to 85% during the drought. In severe stress: (i) α-tocopherol increased 9-fold per g dry weight and 20-fold per unit of chlorophyll; (ii) lutein and β-carotene contents decreased on a dry-weight basis, but an 80% increase in lutein and constant levels of β-carotene were observed on a chlorophyll basis; (iii) there were transient and sustained increases in the de-epoxidation state of the xanthophyll cycle; and (iv) the highly oxidised abietane diterpene isorosmanol increased 8-fold as a result of the oxidation of carnosic acid. With the autumn rainfalls, water status, α-tocopherol and violaxanthin recovered first and the levels of photosynthetic pigments and abietane diterpenes increased later. The photoprotection conferred by the xanthophyll cycle and the antioxidant function of tocopherols, lutein and diterpenes may help to avoid irreversible damage in severe drought, making possible the recovery of functional membranes after the autumn rainfalls. Besides, chlorophyll loss reduces the amount of photons absorbed by leaves, which enhances the photoprotective and antioxidant capacity of leaves per amount of photons absorbed, since the ratios of xanthophylls, α-tocopherol and abietane diterpenes to chlorophyll increase. Received: 12 July 1999 / Accepted: 25 November 1999     

Correlation of trace mineral concentrations with fructose,gamma-glutamyl transpeptidase,and acid phosphatase in seminal plasma of different categories of infertile men

The study describes the concentrations of zinc, magnesium, copper, iron, and biochemical markers of seminal vesicle and prostate in seminal plasma of different categories of infertile men. The zinc level in oligasthenospermic subjects was significantly higher than in azoospermic, asthenospermic, and oligospermic subjects. The γ-glutamyl transpeptidase activity in both oligoasthenospermic and azoospermic subjects were significantly lower than in asthenospermic and oligospermic individuals. There was no significant difference in iron, copper, and magnesium levels and acid phosphatase activity among the different infertile categories. A positive correlation between zinc and acid phosphatase (r=0.84, r=0.73; p<0.05), and between copper and fructose (r=0.81, r=0.72; p<0.05) was observed in oligoasthenospermic and azoospermic men, respectively. On the basis of our results, it may be postulated that there is a direct effect of the trace elements zinc and copper on acid phosphatase and fructose, respectively, in oligoasthenospermic and azoospermic subjects.     

A meta‐review of “lifestyle psychiatry”: the role of exercise,smoking, diet and sleep in the prevention and treatment of mental disorders

There is increasing academic and clinical interest in how “lifestyle factors” traditionally associated with physical health may also relate to mental health and psychological well‐being. In response, international and national health bodies are producing guidelines to address health behaviors in the prevention and treatment of mental illness. However, the current evidence for the causal role of lifestyle factors in the onset and prognosis of mental disorders is unclear. We performed a systematic meta‐review of the top‐tier evidence examining how physical activity, sleep, dietary patterns and tobacco smoking impact on the risk and treatment outcomes across a range of mental disorders. Results from 29 meta‐analyses of prospective/cohort studies, 12 Mendelian randomization studies, two meta‐reviews, and two meta‐analyses of randomized controlled trials were synthesized to generate overviews of the evidence for targeting each of the specific lifestyle factors in the prevention and treatment of depression, anxiety and stress‐related disorders, schizophrenia, bipolar disorder, and attention‐deficit/hyperactivity disorder. Standout findings include: a) convergent evidence indicating the use of physical activity in primary prevention and clinical treatment across a spectrum of mental disorders; b) emerging evidence implicating tobacco smoking as a causal factor in onset of both common and severe mental illness; c) the need to clearly establish causal relations between dietary patterns and risk of mental illness, and how diet should be best addressed within mental health care; and d) poor sleep as a risk factor for mental illness, although with further research required to understand the complex, bidirectional relations and the benefits of non‐pharmacological sleep‐focused interventions. The potentially shared neurobiological pathways between multiple lifestyle factors and mental health are discussed, along with directions for future research, and recommendations for the implementation of these findings at public health and clinical service levels.     

RFLP mapping of quantitative trait loci controlling abscisic acid concentration in leaves of drought-stressed maize (Zea mays L.)

 Abscisic acid (ABA) concentration in leaves of drought-stressed plants is a quantitatively inherited trait. In order to identify quantitative trait loci (QTLs) controlling leaf ABA concentration (L-ABA) in maize, leaf samples were collected from 80 F_3:4 families of the cross Os420 (high L-ABA)×IABO78 (low L-ABA) tested under drought conditions in field trials conducted over 2 years. In each year, leaf samples were collected at stem elongation and near anthesis. The genetic map obtained with 106 restriction fragment length polymorphism (RFLP) loci covered 1370 cM, which represented approximately 85% of the UMC maize map. Sixteen different QTLs with a LOD>2.0 were revealed in at least one sampling. Across samplings, only four QTLs significantly influenced L-ABA, accounting for 66% of the phenotypic variation and 76% of the genetic variation among families. At these QTLs, the alleles which increased L-ABA were contributed by Os420. The two most important QTLs were mapped on chromosome 2 near csu133 and csu109a. The effects associated with the QTL near csu133 were more pronounced near anthesis. The support intervals of the four primary QTLs for L-ABA did not overlap the presumed map position of mutants impaired in ABA biosynthesis. Received: 27 January 1998 / Accepted: 22 April 1998     

Light-harvesting complex II pigments and proteins in association with Cbr, a homolog of higher-plant early light-inducible proteins in the unicellular green alga Dunaliella

 Like higher plants, unicellular green algae of the genus Dunaliella respond to light stress by enhanced de-epoxidation of violaxanthin and accumulation of Cbr, a protein homologous to early light-inducible proteins (Elips) in plants. Earlier studies indicated that Cbr was associated with the light-harvesting complex of photosystem II (LHCII) and suggested it acted as a zeaxanthin-binding protein and fulfilled a photo-protective function (Levy et al. 1993, J. Biol. Chem. 268: 20892–20896). To characterize the protein-pigment subcomplexes containing Cbr in greater detail than attained so far, thylakoid membranes from Dunaliella salina grown in high light or normal light were solubilized with dodecyl maltoside and fractionated by isoelectric-focusing. Analysis of the resolved LHCII subcomplexes indicated preferred associations among the four LHCIIb polypeptides and between them and Cbr: subcomplexes including Cbr contained one or two of the more acidic of the four LHCIIb polypeptides as well as large amounts of lutein and zeaxanthin relative to chlorophyll a/b. After sucrose gradient centrifugation, Cbr free of LHCIIb polypeptides was detected together with released pigments; this Cbr possibly originated in subcomplexes dissociated in the course of the analysis. These results agree with the conclusion that Cbr is part of the network of LHCIIb protein-pigment complexes and suggest that the role played by Cbr involves the organization and/or stabilization of assemblies highly enriched in zeaxanthin and lutein. Such assemblies may function to protect PSII from photodamage due to overexcitation. Received: 6 August 1999 / Accepted: 23 November 1999     

The puzzle of the Krebs citric acid cycle: Assembling the pieces of chemically feasible reactions,and opportunism in the design of metabolic pathways during evolution

The evolutionary origin of the Krebs citric acid cycle has been for a long time a model case in the understanding of the origin and evolution of metabolic pathways: How can the emergence of such a complex pathway be explained? A number of speculative studies have been carried out that have reached the conclusion that the Krebs cycle evolved from pathways for amino acid biosynthesis, but many important questions remain open: Why and how did the full pathway emerge from there? Are other alternative routes for the same purpose possible? Are they better or worse? Have they had any opportunity to be developed in cellular metabolism evolution? We have analyzed the Krebs cycle as a problem of chemical design to oxidize acetate yielding reduction equivalents to the respiratory chain to make ATP. Our analysis demonstrates that although there are several different chemical solutions to this problem, the design of this metabolic pathway as it occurs in living cells is the best chemical solution: It has the least possible number of steps and it also has the greatest ATP yielding. Study of the evolutionary possibilities of each one-taking the available material to build new pathways-demonstrates that the emergence of the Krebs cycle has been a typical case of opportunism in molecular evolution. Our analysis proves, therefore, that the role of opportunism in evolution has converted a problem of several possible chemical solutions into asingle-solution problem, with the actual Krebs cycle demonstrated to be the best possible chemical design. Our results also allow us to derive the rules under which metabolic pathways emerged during the origin of life.     

Copper-induced changes in the growth, oxidative metabolism, and saponin production in suspension culture roots of Panax ginseng in bioreactors

Roots of Panax ginseng exposed to various concentrations of Cu (0.0, 5, 10.0, 25.0, and 50.0 μM) accumulated high amounts of Cu in a concentration-dependent and duration-dependent manner. Roots treated with 50 μM Cu resulted in 52% and 89% growth inhibition after 20 and 40 days, respectively. Saponin synthesis was stimulated at a Cu concentration between 5 and 25 μM but decreased at 50 μM Cu. Malondialdehyde content (MDA), lipoxygenase activity (LOX), superoxide ion (O₂ ^•−) accumulation, and H₂O₂ content at 5 and 10 μM Cu-treated roots were not increased but strongly increased at 50 μM Cu resulting in the oxidation of ascorbate (ASC) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively indicating a clear oxidative stress. Seven well-resolved bands of superoxide dismutase (SOD) were detected in the gel and an increase in SOD activity seemed to be mainly due to the induction of Fe-SOD 3. Five to 10 μM Cu slightly induced activity of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR), guaiacol peroxidase (G-POD) but inhibited monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) enzyme activities. No changes in catalase (CAT) activity and in activity gel were found up to 25 μM Cu, but both G-POD and CAT activities were inhibited at 50 μM Cu. Glutathione metabolism enzymes such as γ-glutamylcysteine synthetase (γ-GCS), glutathione-S-transferase (GST), and glutathione peroxidase activities (GPx) were activated at 5 and 10 μM Cu but were strongly inhibited at 50 μM Cu due to the Cu accumulation in root tissues. The strong depletion of GSH at 50 μM Cu was associated to the strong induction of γ-glutamyltranspeptidase (γ-GGT) activity. These results indicate that plant could grow under Cu stress (5–25 μM) by modulating the antioxidant defense mechanism for combating Cu induced oxidative stress.