Amino acid neurotransmission: Dynamics of vesicular uptake

Glutamate, GABA and glycine, the major neurotransmitters in CNS, are taken up and stored in synaptic vesicles by a Mg²⁺-ATP dependent process. The main driving force for vesicular glutamate uptake is the membrane potential, whereas both the membrane potential and the proton gradient contribute to the uptake of GABA and glycine. Glutamate is taken up by a specific transporter with no affinity for aspartate. Evans blue and related dyes are competitive inhibitors of the uptake of glutamate. GABA, β-alanine, and glycine are taken up by the same family of transporter molecules. Aspartate, taurine, and proline are not taken up by any synaptic vesicle preparations. It is suggested that vesicular uptake and release are characteristics that identify these amino acids as neurotransmitters. We also discuss that “quanta” in the brain are not necessarily related the content of neurotransmitter in the synaptic vesicles, but rather to postsynaptic events. Special issue dedicated to Dr. Herman Bachelard.     

Submerged fermentation in wheat substrates for production of Monascus pigments

Cereal grains are normally used as solid substrates for the production of Monascus metabolites. However, solid fermentation in these substrates requires complex control systems, whereas in liquid culture the control of the fermentation is simpler and consequently significant reductions in fermentation times can be achieved. In the same way, the use of submerged culture can benefit the production of many secondary metabolites and decrease production costs by reducing the labour involved in solid-state methods. A flour composed of a mixed variety of Canadian hard wheat was used as sole nutrient source to produce the pigments of Monascus purpureus Went (IMI 210765). Supplementation with NH₄Cl promoted biomass and orange dye formation, whereas the use of zinc sulphate favoured red dyes production. In submerged fermentations significant differences in final pigment yields were observed in the use of wheat-based broth at different concentrations in the presence of bran particles and/or gluten protein. It has been found that the viscosity of the broth had a significant effect on the growth morphology and production of pigments. Gluten-free wheat flour at concentrations of 3–5% was found to be the most suitable for liquid Monascus culture. The subsequent use of passive immobilization of Monascus served to enhance red pigment yields and to facilitate the downstream processing of the dyes.     

Effect of diabetes on levels and uptake of putative amino acid neurotransmitters in rat retina and retinal pigment epithelium

Free amino acid levels and high affinity uptake of glutamate, aspartate γ-aminobutyrate, glycine and taurine were studied in retina and retinal pigment epithelium of streptozotocin diabetic rats. Results show that experimental diabetes produces a generalized fall in the content of free amino acids in both retina and retinal pigment epithelium. With regard to the high affinity uptake, in the two tissues of diabetic animals showed decreased aspartate uptake, enhanced taurine and γ-aminobutyrate uptake, whereas that of glycine and glutamate was unchanged. These results might suggest that diabetes causes alterations of specific amino acid transport systems and/or alterations of some cell populations.     

α-Amino acid behaves differently from β- or γ-amino acids as treated by trimetaphosphate

Summary. The condensation reactions of sodium trimetaphosphate with single amino acids, namely glycine, L-alanine, β-alanine and γ-aminobutyric acid or pairs of these amino acids were reinvestigated by electrospray ion-trap mass spectrometry and high performance liquid chromatography. It was found when mixtures were treated by sodium trimetaphosphate only in the presence of α-amino acid dipeptides were formed. Without addition of α-amino acids, the β-amino acid or γ-aminobutyric acid could not form peptide either by themselves or with their mixtures under the same conditions. From the data it is concluded that phosphate might select α-amino acids to produce the peptides being important precursors for the origin of life. Authors’ address: Dr. Pengxiang Xu, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemistry, Xiamen University, Xiamen 361005, China     

The cone visual pigments of an Australian marsupial,the tammar wallaby (Macropus eugenii): sequence,spectral tuning,and evolution

Studies on marsupial color vision have been limited to very few species. There is evidence from behavioral, electroretinographic (ERG), and microspectrophotometric (MSP) measurements for the existence of both dichromatic and trichromatic color vision. No studies have yet investigated the molecular mechanisms of spectral tuning in the visual pigments of marsupials. Our study is the first to determine the mRNA sequence, infer the amino acid sequence, and determine, by in vitro expression, the spectra of the cone opsins of a marsupial, the tammar wallaby (Macropus eugenii). This yielded some information on mechanisms and evolution of spectral tuning of these pigments. The tammar wallaby retina contains only short-wavelength sensitive (SWS) and middle-wavelength sensitive (MWS) pigment mRNAs. This predicts dichromatic color vision, which is consistent with conclusions from previous behavioral studies ( Hemmi 1999). We found that the wallaby has a SWS1 class pigment of 346 amino acids. Sequence comparison with eutherian SWS pigments predicts that this SWS1 pigment absorbs maximally (lambdamax) at 424 nm and, therefore, is a blue rather than a UV pigment. This (lambdamax) is close to that of the in vitro-expressed wallaby SWS pigment (lambdamax of 420 +/- 2 nm) and to that determined behaviorally (420 nm). The difference from the mouse UV pigment (lambdamax of 359 nm) is largely accounted for by the F86Y substitution, in agreement with in vitro results comparing a variety of other SWS pigments. This suggests that spectral tuning employing F86Y substitution most likely arose independently in the marsupials and ungulates as a result of convergent evolution. An apparently different mechanism of spectral tuning of the SWS1 pigments, involving five amino acid positions, evolved in primates. The wallaby MWS pigment has 363 amino acids. Species comparisons at positions critical to spectral tuning predict a lambdamax near 530 nm, which is close to that of the in vitro-expressed pigment (529 +/- 1 nm), but quite different from the value of 539 nm determined by microspectrophotometry. Introns interrupt the coding sequences of the wallaby, mouse, and human MWS pigment sequences at the same corresponding nucleotide positions. However, the length of introns varies widely among these species.     

Degradation of proteins and amino acids by Caloramator proteoclasticus in pure culture and in coculture with Methanobacterium thermoformicicum Z245

This study investigated the degradation of proteins and amino acids by Caloramator proteoclasticus, an anaerobic thermophilic (55 °C) fermentative bacterium isolated from an anaerobic bioreactor. Experiments were performed in the presence and absence of Methanobacterium thermoformicicum Z245, a methanogen that can use both hydrogen and formate for growth. Higher production rates and yields of the principal fermentation products from gelatin were observed in methanogenic coculture. The specific proteolytic activity in coculture tripled the value obtained in pure culture. C. proteoclasticus fermented glutamate to acetate, formate, hydrogen and alanine. In methanogenic coculture, a shift towards higher amounts of acetate and hydrogen with no alanine production was observed. Extracts of glutamate-grown cells possessed high activities of β-methylaspartase, a key enzyme of the mesaconate pathway leading to acetate. The presence of two enzymes (alanine-α-ketoglutarate aminotransferase and NADH-dependent alanine dehydrogenase) usually involved in the biosynthesis of alanine from pyruvate was also detected. The fermentation of amino acids known to be oxidatively deaminated (leucine and valine) was improved in the presence of both methanogenesis and glycine, a known electron acceptor in the Stickland reaction. Culture conditions seem to be very important in the way C. proteoclasticus disposes of reducing equivalents formed during the degradation of amino acids. Received: 29 March 1999 / Received revision: 2 July 1999 / Accepted: 1 August 1999     

Visibly healthy corals exhibit variable pigment concentrations and symbiont phenotypes

Understanding the natural variability of photosynthetic pigment ranges and distributions in healthy corals is central to evaluating how useful these measurements are for assessing the health and bleaching status of endosymbiotic reef-building corals. This study examined the photosynthetic pigment variability in visibly healthy Porites lobata and Porites lutea corals from Kaneohe Bay, Hawaii and explored whether pigment variability was related to the genetic identity or phenotypic characteristics of the symbionts. Concentrations of the photosynthetic pigments chlorophyll a, peridinin, chlorophyll c ₂ , diadinoxanthin, diatoxanthin, β,β-carotene and dinoxanthin were quantified using high-performance liquid chromatography (HPLC). Pigment concentrations were found to range 1.5–10 fold in colonies of each species at similar depths (0–2, 2–4, 10–15 and 19–21 m). Despite the high pigment variability, pigment ratios for each species were relatively conserved over the 0–21 m depth gradient. The genetic identity of the symbiont communities was examined for each colony using 18S nuclear ribosomal DNA (nrDNA) restriction fragment length polymorphisms. All colonies contained symbionts belonging to clade C. The density and phenotypic characteristics of the symbionts were explored using flow cytometry, and fluorescence and side scatter (cell size) properties revealed phenotypically distinct symbiont subpopulations in every colony. The symbiont subpopulations displayed pigment trends that may be driven by acclimatization to irradiance microenvironments within the host. These results highlight the biological complexity of healthy coral–symbiont associations and the need for future research on pigments and symbiont subpopulation dynamics.     

Cloning,production and secretion of β-galactosidase inAcetobacter pasteurianus

The gene coding for β-galactosidase fromEscherichia coli was cloned into plasmid pACT71 containing the replicon from plasmid pAC1 fromAcetobacter pasteurianus. E. coli MC4100,E. coli JM105,E. coli LE392.23 andA. pasteurianus 3614 were transformed with the recombinant plasmid pACB815. Cells were cultivated in LB, YPG and M media supplemented with glucose, glycerol, lactose or ethanol and β-galactosidase activity was detected in the cells and in the cultivation medium. The best substrate for production of β-galactosidase was lactose. To release β-galactosidase fromA. pasteurianus cells amino acids were added to the cultivation medium. The highest secretory activity was achieved using 1.5% glycine after 36 h of cultivation in the M medium.     

Expression of pigmentation genes following electroporation of albino<Emphasis Type="Italic"> Monascus purpureus</Emphasis>

A UV-induced albino strain of Monascus purpureus was subjected to electroporation in the presence of genomic DNA from a wild-type red strain of the fungus. Eight colonies expressed color after several weeks of growth. The growth rates of all eight color variants were significantly greater than the recipient and donor strains under some culture conditions. Spectrophotometric analysis of the pigments extracted from the color variants revealed the pigments had absorbance spectra different from the DNA donor strain. These color variants may have resulted from transformation with wild-type DNA, mutation reversion, or activation of alternative pathway(s)—i.e., new mutations—that resulted in pigment production.     

Low-specificity l-threonine aldolase of Pseudomonas sp. NCIMB 10558: purification, characterization and its application to β-hydroxy-α-amino acid synthesis

Low-specificity l-threonine aldolase, catalyzing the reversible cleavage/condensation reaction between l-threonine/l-allo-threonine and glycine plus acetaldehyde, was purified to homogeneity from Pseudomonas sp. NCIMB 10558. The enzyme has an apparent molecular mass of approximately 145 kDa and consists of four identical subunits with a molecular mass of 38 kDa. The enzyme, requiring pyridoxal- 5′-phosphate as a coenzyme, is strictly l-specific at the α position, whereas it can not distinguish between threo and erythro forms at the β position. Besides the reversible cleavage/condensation of threonine, the enzyme also catalyzes the reversible interconversion between glycine plus various aldehydes and l-β-hydroxy-α-amino acids, including l-β-(3,4-dihydroxyphenyl)serine, l-β-(3,4-met‐hylenedioxyphenyl)serine and l-β-phenylserine, providing a new route for the industrial production of these important amino acids. Received: 10 November 1997 / Received revision: 7 January 1998 / Accepted 30 January 1998     

Crude bacterial extracts of two new Streptomyces sp. isolates as bio-colorants for textile dyeing

Renewed demand for incorporation of natural dyes (bio-colorants) in textile industry could be met through biotechnological production of bacterial pigments. Two new Streptomyces strains (NP2 and NP4) were isolated for the remarkable ability to produce diffusible deep blue and deep red pigment into fermentation medium. Crude mycelial extracts of both strains were used as bio-colorants in conventional textile dyeing procedures avoiding downstream purification procedures. The yields of bio-colorants obtained in this way were 62 and 84 mg per g of mycelia for Streptomyces sp. NP2 and Streptomyces sp. NP4, respectively. Through nuclear magnetic resonance analysis of crude extracts before and after dyeing procedures, it was shown that both extracts contained prodigiosin-like family of compounds that exhibited different dyeing capabilities towards different textile fibers. Polyamide and acrylic fibers were colored to the deepest shade, polyester and triacetate fibers to a noticeable, but much lower shade depth, while cotton and cellulosic fibers stained weakly. These results confirmed that crude bacterial extracts had the characteristics similar to those of ionic and disperse dyes, which was consistent with the identified polypyrrolic prodigiosin-like structures.     

栀子兰色素可能为栀子粉末外用抗炎消肿时的活性物质

传统中药山栀子中的环烯醚萜成分及尼平（ｇｅｎｉｐｉｎ）与甘氨酸反应可形成兰色素,初步药理实验显示,该兰色色素对小鼠具有抗炎和镇痛作用,推测栀子兰色素可能为民间将栀子粉末外用抗炎消肿时的活性物质。     

Design, chemical synthesis, and expression of genes for the three human color vision pigments.

Color vision in humans is mediated by three pigments from retinal cone photoreceptor cells: blue, green, and red. We have designed and chemically synthesized genes for each of these three pigments. The genes were expressed in COS cells, reconstituted with 11-cis-retinal chromophore, and purified to homogeneity using an immunoaffinity procedure. To facilitate the immunoaffinity purification, each pigment was modified at the carboxy terminus to contain an additional eight amino acid epitope for a monoclonal antibody previously used to purify bovine rhodopsin. The spectra for the isolated pigments had maxima of 424, 530, and 560 nm, respectively, for the blue, green, and red pigments. These maxima are in excellent agreement with the maxima previously observed by microspectrophotometry of individual human cone cells. The spectra are the first to be obtained from isolated human color vision pigments. They confirm the original identification of the three color vision genes, which was based on genetic evidence [Nathans, J., Thomas, D., & Hogness, D.S. (1986) Science 232, 193].     

Microbial decolorization and degradation of synthetic dyes: a review

The synthesis of dyes and pigments used in textiles and other industries generate the hazardous wastes. A dye is used to impart color to materials of which it becomes an integral part. The waste generated during the process and operation of the dyes commonly found to contain the inorganic and organic contaminant leading to the hazard to ecosystem and biodiversity causing impact on the environment. The amount of azo dyes concentration present in wastewater varied from lower to higher concentration that lead to color dye effluent causing toxicity to biological ecosystem. The physico-chemical treatment does not remove the color and dye compound concentration. The decolorization of the dye takes place either by adsorption on the microbial biomass or biodegradation by the cells. Bioremediation takes place by anaerobic and/or aerobic process. The anaerobic process converts dye in toxic amino compounds which on further treatment with aerobic reaction convert the intermediate into CO₂ biomass and inorganics. In the present review the decolorization and degradation of azo dyes by fungi, algae, yeast and bacteria have been cited along with the anaerobic to aerobic treatment processes. The factors affecting decolorization and biodegradation of azo dye compounds such as pH, temperature, dye concentration, effects of CO₂ and Nitrogen, agitation, effect of dye structure, electron donor and enzymes involved in microbial decolorization of azo dyes have been discussed. This paper will have the application for the decolorization and degradation of azo dye compound into environmental friendly compounds.     

基于花青素代谢培育蓝色花卉的研究进展

花色是植物吸引昆虫传播花粉的主要因素,对于植物在自然界的生存必不可少,也是观赏植物最重要的性状之一。在蓬勃发展的花卉产业中,色彩各异花卉的培育,可以弥补自然花色的匮乏,但是令人垂涎的蓝色花比较难培育。花色的多样性主要是由花青素及其衍生物的种类和含量等因素决定的,飞燕草色素的合成是形成蓝色花的关键因素,许多植物体内缺少合成飞燕草色素的结构基因。近年来,利用基因工程技术培育蓝色花的研究也时有报道。文中以常见的观赏植物为例,基于花青素代谢调控,从影响飞燕草色素合成的关键因素和不同分子改良途径培育蓝色花等几个方面对植物花朵呈色的机制进行了综述,并展示不同分子育种策略可能在其他领域的应用,为其他植物或经济作物的色泽改良如彩色棉蓝色纤维的培育等提供参考和技术支持。     

2-Oxo-2H-benzo[h]benzopyran as a new light sensitive protecting group for neurotransmitter amino acids

Aiming at the development of new benzopyran-based photocleavable protecting groups, novel chloromethylated and hydroxymethylated 2-oxo-2H-benzo[h]benzopyran derivatives bearing a methoxy substituent were designed and used in the synthesis of a series of fluorescent bioconjugates, by linking through an ester or urethane bond to several model neurotransmitter amino acids (glycine, alanine, β-alanine and γ-aminobutyric acid, GABA). The resulting fluorescent bioconjugates with emission in the visible range and high fluorescent quantum yields, were subjected to photocleavage reaction in methanol/HEPES buffer (80:20) solution at different wavelengths of irradiation (250, 300, 350 and 419 nm) and photocleavage kinetic data were obtained.     

Synthesis of β-galactosyl-(hydroxy amino acid) derivatives using β-galactosidase activity ofAchatina achatina digestive juice

Summary The transglycosylation activity of β-galactosidase fromAchatina achatina digestive juice was tested for glycosylating protected hydroxy amino acids. Attractive yields of β-galactosyl-(Z-Ser-OMe) (35%) and β-galactosyl-(Z-Hyp-OMe) (28%) could be obtained using lactose as glycosyl donor and the corresponding amino acid methyl esters N-protected by a benzyloxycarbonyl group (Z) as glycosyl acceptors.     

Membrane processing for purification and concentration of β-glycosidases from barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>)

Tangential flow ultrafiltration with a polyethersulfone (100 kDa) membrane was used for the purification and concentration of β-glycosidases (β-galactosidase and β-glucosidase) from aqueous extract of barley. The performances of mode 1 (concentration followed by diafiltration) and mode 2 (diafiltration followed by concentration) were compared. In mode 1 activity recoveries of 91.44 and 88% as well as purifications of 1.84 and 1.77-fold for β-galactosidase and β-glucosidase, respectively, were obtained in a total processing time of about 9 h. In mode 2 activity recoveries of 95.68 and 91.76% with purifications of 4.56 and 4.38-fold for β-galactosidase and β-glucosidase, respectively, were obtained in a total processing time of about 6 h. The removal of total carbohydrates and protein was 56.74 and 50.73%, respectively, in mode 1, whereas it was 81.46 and 79.04%, respectively, in mode 2. The diafiltration volume and volume concentration of 3 were maintained in both mode 1 and 2. Flux decline was severe in mode 1 and led to a long processing time of about 9 h. These results indicate that mode 2 was better than mode 1 for purification of β-glycosidases.     

Scaled-up biotechnological production of individual betalains in a microbial system

The recent interest in plant pigment betalains as bioactive compounds and chemopreventive agents has led to the search for a reliable and scalable process to obtain them. The cloning of the novel and efficient enzyme 4,5-DOPA-extradiol dioxygenase from Gluconacetobacter diazotrophicus in an expression vector, and the subsequent heterologous expression in Escherichia coli cultures has led to the start-up of a biotechnological production system of individual pigments. The aim of this study was to search for the optimal conditions for the production of betalamic acid in microbial factories and the scaled-up obtention of the derived pigments. Four different betaxanthins and two betacyanins were obtained after the addition of non-transformable amines and amino acids and their condensation with the betalamic acid produced by the dioxygenase. The scaled-up obtention and purification of betalains improved the yields of the previous methodologies reaching quantities by up to 150 mg of pure compounds.     

The extracellular products of Anabaena cylindrica Lemm. I. Isolation of a macromolecular pigment-peptide complex and other components

The extracellular products of Anabaena cylindrica Lemm. comprise a large variety of compounds including peptides, brownish pigments and substances fluorescing white and blue in ultraviolet light. A number were separated or isolated using techniques of gel filtration, ion exchange and paper chromatography. Serine and threonine comprised over 90% of the amino acids in a group of complex pigmented and fluorescent compounds. One of these accounted for a large proportion of the peptide and pigment present. It contained a large pigment moiety of molecular weight > 5 000 which formed a firm complex with more than 10% of the iron supplied in the culture medium. The anti-polymyxin activity described by Whitton was not associated with any of the major pigments or peptides present.