Steric aspects of dopaminergic drugs.

There are a variety of molecule species of dopamine avaialable at physiological pH. The predominant form available at physiological pH is the phenolic ammonium salt. However, at the present time the molecular form that is optimum for producing dopaminergic activity is unknown. In attempting to delineate the conformational requirements of dopaminergic agonists, a variety of conformationally restricted analogs and complex molecules possessing a dopaminergic segment have been investigated. It appears at this time that the trans extended form of dopamine is the optimum form for binding to dopamine receptors. The rotameric forms of dopamine are also important considerations when examining a molecule for dopaminergic agonist activity. A high degree of stereospecificity has been shown in different dopaminergic systems.     

Mechanism-based inhibitors of dopamine beta-hydroxylase

The copper-containing monooxygenase dopamine beta-hydroxylase catalyzes the hydroxylation of dopamine at the benzylic position to form norepinephrine. Mechanism-based inhibitors for dopamine beta-hydroxylase have been used as probes of the mechanism of catalysis. The variety of such inhibitors that have been developed for this enzyme can be divided into three groups: (i) those in which the inactivating species is formed by abstraction of a hydrogen atom to form a radical intermediate; (ii) those in which the inactivating species is formed by abstraction of an electron to form an epoxide-like intermediate; and (iii) those in which the product is the inactivating species. A mechanism consistent with inactivation by all three groups of inhibitors which proposes that hydroxylation of dopamine by dopamine beta-hydroxylase involves formation of a benzylic radical has been developed. The benzylic radical is formed by abstraction of a hydrogen atom from the substrate by a high-potential copper-oxygen species.     

Stress responses and the mesolimbic dopamine system: social contexts and sex differences

Organisms react to threats with a variety of behavioral, hormonal, and neurobiological responses. The study of biological responses to stress has historically focused on the hypothalamic-pituitary-adrenal axis, but other systems such as the mesolimbic dopamine system are involved. Behavioral neuroendocrinologists have long recognized the importance of the mesolimbic dopamine system in mediating the effects of hormones on species specific behavior, especially aspects of reproductive behavior. There has been less focus on the role of this system in the context of stress, perhaps due to extensive data outlining its importance in reward or approach-based contexts. However, there is steadily growing evidence that the mesolimbic dopamine neurons have critical effects on behavioral responses to stress. Most of these data have been collected from experiments using a small number of animal model species under a limited set of contexts. This approach has led to important discoveries, but evidence is accumulating that mesolimbic dopamine responses are context dependent. Thus, focusing on a limited number of species under a narrow set of controlled conditions constrains our understanding of how the mesolimbic dopamine system regulates behavior in response to stress. Both affiliative and antagonistic social interactions have important effects on mesolimbic dopamine function, and there is preliminary evidence for sex differences as well. This review will highlight the benefits of expanding this approach, and focus on how social contexts and sex differences can impact mesolimbic dopamine stress responses.     

Effect of dopamine on amylase secretion from guinea pig pancreatic acinar cells in vitro

Dopamine has been shown to effect pancreatic flow, protein output and amylase secretion in a variety of species. However, there is conflicting evidence regarding the role of dopamine on amylase release in vitro. Specific studies were conducted to evaluate the effect of dopamine and to compare its effects with other substances on basal- and secretagogue-stimulated amylase secretion in a guinea pig dispersed pancreatic acinar cells preparation. Dopamine (10(-6) M) induced a small, but significant (P less than 0.05) increase of amylase secretion. Established secretagogues (10(-6) M) including bombesin, cholecystokinin-octapeptide (CCK-8) and carbachol as anticipated induced significantly larger responses. Other substances tested (10(-6) M) including thyrotropin-releasing hormone (TRH) and muscimol were without effect. Complete dose-response studies (10(-11)-10(-3) M) in the presence of bombesin, CCK-8 and carbachol revealed that dopamine does not affect amylase release in response to these secretagogues. These findings suggest that dopamine is a weak stimulant of amylase secretion in vitro, and that it may therefore play a minor role in regulation of pancreatic enzyme secretion. Several factors including vascular, hormonal and neural have been implicated in regulation of pancreatic exocrine secretion. In particular, autonomic nervous system activity, notably cholinergic, has been shown to affect the secretory status of the pancreatic acinar cell. In addition, several biologically active peptides including bombesin, cholecystokinin (CCK), secretin, vasoactive intestinal peptide (VIP), substance P, gastrin and stimulation of cholinergic (muscarinic) receptors with carbachol have been shown to stimulate pancreatic enzyme secretion both in vivo and in vitro. Certain controversy regarding the role of the sympathetic nervous system in regulation of pancreatic exocrine secretion does exist. For example, several studies with agonists and antagonists of noradrenergic and dopaminergic receptor subtypes suggest a stimulatory effect on pancreatic fluid, electrolyte and enzyme secretion. However, these responses are species-specific and variations inherent to the model have been described. Dopamine administration has been shown to stimulate pancreatic bicarbonate and enzyme secretion in a variety of species including mice, dogs, and man. Radioligand binding studies with 3H-dopamine have revealed the presence of high- and low-affinity dopamine binding sites in dog pancreatic acinar cells. Stimulation of these receptors has been correlated with dose-dependent increases in intracellular cAMP levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective vulnerability of dopaminergic neurons to microtubule depolymerization

Parkinson disease (PD) is characterized by the specific degeneration of dopaminergic (DA) neurons in substantia nigra and has been linked to a variety of environmental and genetic factors. Rotenone, an environmental PD toxin, exhibited much greater toxicity to DA neurons in midbrain neuronal cultures than to non-DA neurons. The effect was significantly decreased by the microtubule-stabilizing drug taxol and mimicked by microtubule-depolymerizing agents such as colchicine or nocodazole. Microtubule depolymerization disrupted vesicular transport along microtubules and caused the accumulation of dopamine vesicles in the soma. This led to increased oxidative stress due to oxidation of cytosolic dopamine leaked from vesicles. Inhibition of dopamine metabolism significantly reduced rotenone toxicity. Thus, our results suggest that microtubule depolymerization induced by PD toxins such as rotenone plays a key role in the selective death of dopaminergic neurons.     

Refinement of the AMBER force field for nucleic acids: improving the description of alpha/gamma conformers

We present here the parmbsc0 force field, a refinement of the AMBER parm99 force field, where emphasis has been made on the correct representation of the alpha/gamma concerted rotation in nucleic acids (NAs). The modified force field corrects overpopulations of the alpha/gamma = (g+,t) backbone that were seen in long (more than 10 ns) simulations with previous AMBER parameter sets (parm94-99). The force field has been derived by fitting to high-level quantum mechanical data and verified by comparison with very high-level quantum mechanical calculations and by a very extensive comparison between simulations and experimental data. The set of validation simulations includes two of the longest trajectories published to date for the DNA duplex (200 ns each) and the largest variety of NA structures studied to date (15 different NA families and 97 individual structures). The total simulation time used to validate the force field includes near 1 mus of state-of-the-art molecular dynamics simulations in aqueous solution.     

Characterization of metal ligand mutants of tyrosine hydroxylase: insights into the plasticity of a 2-histidine-1-carboxylate triad

The amino acid ligands to the active site iron in the aromatic amino acid hydroxylase tyrosine hydroxylase are two histidines and a glutamate. This 2-histidine-1-carboxylate motif has been found in a number of other metalloenzymes which catalyze a variety of oxygenase reactions. As a probe of the plasticity of this metal binding site, each of the ligands in TyrH has been mutated to glutamine, glutamate, or histidine. The H336E and H336Q enzymes show dramatic decreases in iron affinity but retain substantial activity for both tyrosine hydroxylation and tetrahydropterin oxidation. The H331E enzyme shows a lesser decrease in iron affinity and is unable to hydroxylate tyrosine. Instead, this enzyme oxidizes tetrahydropterin in the absence of added tyrosine. The E376H enzyme has no significant activity, while the E376Q enzyme hydroxylates tyrosine at about 0.4% the wild-type rate. When dopamine is bound to either the H336Q or H331E enzymes, the position of the long wavelength charge-transfer absorbance band is consistent with the change in the metal ligand. In contrast, the H336E enzyme does not form a stable binary complex with dopamine, while the E376H and E376Q enzymes catalyze dopamine oxidation.     

Emerging concepts: novel integration of in vivo approaches to localize the function of nicotinic receptors

Nicotinic acetylcholine receptors (nAChRs) are important targets of the neuromodulator acetylcholine (ACh) and the drug nicotine. The role of their different subunits has been analysed for a decade by the creation of knock-out (KO) mice using homologous recombination. This technique shows that a given subunit is necessary for a given function. However, for ubiquitously expressed genes, it cannot demonstrate the localization for a given subunit in which its expression is sufficient, especially for behavioural phenotypes. Sufficient in this context means that the brain region requiring the expression of the gene product has been localized. Novel strategies have therefore been developed to re-express, region specifically, nAChR subunits on a KO background using lentiviral vectors. Localized regeneration of fully functional high-affinity nAChRs in defined brain regions has proven that these receptors are sufficient to restore a variety of functions: nicotine-induced dopamine release, nicotine self-administration in mice, dopamine neuron firing patterns, and exploratory and locomotor behaviours in a sequential locomotor task testing executive function were thus defined as depending exclusively on the 'knock-back' of beta2*-nAChRs into the ventral tegmental area. These analyses highlight the important role of endogenous cholinergic regulation of a variety of functions. The novel integrated use of restricted re-expressed nAChR subunits with in vivo electrophysiology and automated quantitative behavioural analysis enables the further analysis of defined neuronal circuits in nicotine addiction and higher cognitive function.     

GDNF重组腺病毒增加MN9D细胞多巴胺合成与释放

有表达ＧＤＮＦ的重组腺病毒直接感染中脑多巴胺能神经元来源的ＭＮ９Ｄ细胞并经神经毒素ＭＰＰ＋处理．感染３６ｈ分别收获细胞及其培养基,用反相高效液相色谱方法测定多巴胺含量．结果显示,经ＧＤＮＦ重组腺病毒感染的ＭＮ９Ｄ细胞内及其培养基中的多巴胺水平分别增加５０．７％和５３．５％．给予神经毒素ＭＰＰ＋损伤后,细胞内多巴胺含量降低５３．５％,但同时给予ＧＤＮＦ腺病毒则可抑制这种降低,并使多巴胺水平增加１４１．３％．以上结果提示ＧＤＮＦ腺病毒可提高细胞内的多巴胺水平并促进其释放,同时还具有对抗神经毒素ＭＰＰ＋损伤作用,表明ＧＤＮＦ重组腺病毒用于帕金森氏病基因治疗具有良好的前景．     

基因序列在蚤蝇科分子系统学研究中的应用

概述基因序列在双翅目蚤蝇科分子系统学研究中的应用。对蚤蝇科已测序的分类单元和基因序列进行了总结,12S rDNA和16S rDNA应用最广泛,涉及蚤蝇科17个属;获得基因序列最多的是Melaloncha属。蚤蝇科分子系统学研究内容为高级阶元系统发育分析、物种鉴定和隐存种发现。今后蚤蝇科分子系统学研究应增加蚤蝇标本的种类与数量,选择标准化基因。     

Plasmid pLTRpoly: a versatile high-efficiency mammalian expression vector.

Plasmid pLTRpoly is an expression vector enabling high-level expression of introduced genes in a variety of cell types. A large multiple cloning site (MCS) and the availability of the full-length nucleotide sequence facilitate the generation of constructs using this vector. Here, constructs made with pLTRpoly have been tested in NIH3T3 mouse fibroblasts.     

Role of adenosine in drug-induced catatonia in mice

Parkinson's disease is one of the most common neurodegenerative disorders affecting large majority of population who are older than age of 65. Apart from dopamine, acetylcholine and glutamate, adenosinc has also been identified in the basal ganglia. Adenosine modulates the release of a variety of neurotransmitters including dopamine. In order to establish adenosine-dopamine interactions in drug-induced catatonia we studied the effect of adenosine in drug-induced catatonia in mice. In the present study adenosine dose dependently produced catatonia when assessed on rota-rod and bar tests in mice. Adenosine also potentiated the catatonic effect of perphenazine. L-dopa plus carbidopa or OR-486 (a potent centrally acting COMT inhibitor) completely reversed adenosine-induced catatonia. Since reversal by scopolamine of adenosine-induced catatonia was not to the same extent as with l-dopa and OR-486 it appears that catecholamines particularly dopamine rather than cholinergic modulation is more important in adenosine induced catatonia. The motor dysfunction (catatonia) could be easily assessed using rota-rod test apparatus in mice.     

Vaccinia virus: a versatile tool for molecular biologists.

Continued advances in genetic engineering have made possible the high-level expression of correctly processed cellular, viral and bacterial polypeptides. This article focuses on viral expression vectors and, more specifically, the vaccinia virus expression system. Vaccinia virus has been used to express a variety of proteins with useful immunogenic, catalytic or pharmaceutical properties. We discuss briefly the biology of vaccinia and its significance in the use of vaccinia as an expression vector, the variety of vaccinia systems currently in use and, finally, we summarize some recent developments which bode well for future applications of vaccinia virus technology.     

Effects of Chloral Hydrate, γ-Butyrolactone, and Equithesin on the Efflux of Dopamine and 5-Hydroxytryptamine Metabolites into Cerebroventricular Perfusates of Rats

Chloral hydrate, gamma-butyrolactone, and Equithesin are general anesthetics which have been employed in a variety of neurochemical and electrophysiological experiments. The effects of these anesthetics on the efflux of dopamine and 5-hydroxytryptamine metabolites into lateral cerebroventricular perfusates were determined in rats bearing chronically implanted cannulas. Chloral hydrate and Equithesin increased, whereas gamma-butyrolactone decreased, the efflux of the dopamine metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid. None of the anesthetics had a consistent effect on the efflux of the 5-hydroxytryptamine metabolite 5-hydroxyindoleacetic acid.     

The functional impact of mental imagery on conscious perception

Mental imagery has been proposed to contribute to a variety of high-level cognitive functions, including memory encoding and retrieval, navigation, spatial planning, and even social communication and language comprehension. However, it is debated whether mental imagery relies on the same sensory representations as perception, and if so, what functional consequences such an overlap might have on perception itself. We report novel evidence that single instances of imagery can have a pronounced facilitatory influence on subsequent conscious perception. Either seeing or imagining a specific pattern could strongly bias which of two competing stimuli reach awareness during binocular rivalry. Effects of imagery and perception were location and orientation specific, accumulated in strength over time, and survived an intervening visual task lasting several seconds prior to presentation of the rivalry display. Interestingly, effects of imagery differed from those of feature-based attention. The results demonstrate that imagery, in the absence of any incoming visual signals, leads to the formation of a short-term sensory trace that can bias future perception, suggesting a means by which high-level processes that support imagination and memory retrieval may shape low-level sensory representations.     

Non-conventional yeasts as hosts for heterologous protein production.

Yeasts are an attractive group of lower eukaryotic microorganisms, some of which are used in several industrial processes that include brewing, baking and the production of a variety of biochemical compounds. More recently, yeasts have been developed as host organisms for the production of foreign (heterologous) proteins. Saccharomyces cerevisiae has usually been the yeast of choice, but an increasing number of alternative non-Saccharomyces yeasts has now become accessible for modern molecular genetics techniques. Some of them exhibit certain favourable traits such as high-level secretion or very strong and tightly regulated promoters, offering significant advantages over traditional bakers' yeast. In the present work, the current status of Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris (the best-known alternative yeast systems) is reviewed. The advantages and limitations of these systems are discussed in relation to S. cerevisiae.     

Peripheral receptor populations involved in the regulation of gastrointestinal motility and the pharmacological actions of metoclopramide-like drugs

This minireview is concerned with a re-examination of the locus of action and the possible peripheral mechanisms involved in the gastrointestinal (GI) stimulant effects of metoclopramide. Such a re-evaluation is opportune given the increasing use of this drug in the therapy of certain GI tract disorders. To provide an orientation on this subject the location in the GI tract and function of several relevant receptor types have been reviewed. In the past metoclopramide has been reported to enhance contractions of a variety of GI preparations to electrical stimulation, acetylcholine, carbachol and ganglion stimulants, to inhibit responses to alpha 2-adrenoreceptor agonists and 5-hydroxytryptamine, as well as blocking those to dopamine. Also in such preparations metoclopramide facilitates the release of acetylcholine to transmural stimulation. One important question is whether this effect is mediated via a specific prejunctional receptor. In this respect 2 suggestions have been made. Firstly that the drug may act as a preferential, prejunctional muscarinic antagonist thus inhibiting the negative feedback inhibition of acetylcholine release and secondly that metoclopramide may be a prejunctional agonist (partial) at 5-hydroxy-tryptamine receptors. Although the latter possibility appears most tenable at present, the involvement of a specific receptor remains to be confirmed. The important finding that dopamine receptors are probably not involved in the local stimulant effects of metoclopramide has important implications for future research orientated towards the discovery of a new generation of GI drugs lacking the side effects associated with central dopamine receptor blockade. Several compounds (cinitapride, BRL 20627A and cisapride) are now in the early stages of clinical evaluation.     

Role of endogenous Cyclo(His-Pro) in voluntary alcohol consumption by alcohol-preferring C57BL mice

PRASAD, C. Role of Endogenous Cyclo(His-Pro) in Voluntary Alcohol Consumption by C57BL Mice. PEPTIDES —-. Cyclo (His-Pro) or CHP is a cyclic dipeptide endogenous to the brain of a variety of animal species including man. Administration of exogenous peptide to rodents has been shown to exhibit a variety of biological activities some of which appear to be mediated via a dopaminergic mechanism. Since a hypodopaminergic state has been associated with excessive drinking in animal models as well as man, we have explored the potential role of CHP in alcohol-preferring C57BL mice. The results of this study show that the level of CHP, a peptide that mimics dopamine in many of its pharmacologic actions, is lower in brains of alcohol-preferring C57BL mice compared to alcohol non-preferring DBA2 mice. Furthermore, administration of exogenous CHP to C57BL mice caused a pronounced decrease in their voluntary alcohol consumption. In conclusion, endogenous CHP may play a role in risk for developing excessive alcohol use by modulating central dopaminergic tone.     

Gas chromatographic/mass spectrometric methodology for simultaneous assay of salsolinol, dopamine, norepinephrine, dihydroxyphenylacetic acid and dihydroxyphenylethanol

Synthesis of deuterated (2H4)salsolinol from (2H4)dopamine via a Pictet-Spengler condensation is described. This (2H4)salsolinol is an ideal internal standard to determine picomole (ng) amounts of salsolinol (SAL) in a variety of sample types including urine, plasma, beverages and fruits. The deuterated standard is completely free of contamination by the non-deuterated species. The extraction procedure described is fast, highly efficient and does not lead to artifactual salsolinol formation even in the face of high dopamine concentrations. As well as SAL the method described allows simultaneous determination of norepinephrine (NE), dopamine (DA) and its two metabolites dihydroxyphenylacetic acid (DOPAC) and dihydroxyphenylethanol (DOPET). Each of the analytes is measured as its trifluoroacetyl derivative. Using trifluoroacetic anhydride in conjunction with trifluoroethanol allows simultaneous one-step derivatization of the acid function of DOPAC. All compounds were measured in the single ion monitoring (SIM) mode and quantified using appropriate deuterated internal standards. SAL, DA, DOPET, DOPAC and NE have been quantified in a variety of food and beverage sources. Soy sauce and dried banana have been identified as rich dietary sources of SAL. These data suggest diet should be considered a potentially important source of 'mammalian alkaloids' such as SAL, and the presence of SAL in mammalian systems is not necessarily evidence for an in vivo Pictet-Spengler condensation.