A simple, fast and reliable method for obtaining dopamine histofluorescence from mesencephalic cultures

A modified glyoxylic acid technique for obtaining dopamine histofluorescence from cultured mesencephalic cells is described. This method requires only two solutions: one contains glyoxylic acid, sucrose and monobasic potassium phosphate and is used at room temperature, the other is a Hepes buffered solution used at 37 C. Relatively high concentrations of a monoamine oxidase inhibitor and dopamine are added to the cultures to load dopaminergic neurons; the cell bodies and their processes take up and hold dopamine quickly and evenly. The cultures are dipped in a glyoxylic acid solution, dried in air, heated for 5 min and coverslipped with mineral oil. Since the cultures remain in their culture dishes, the entire procedure takes less than 2 hr. The green histofluorescence characteristic of dopamine is seen when the cultures are viewed by standard fluorescence microscopy. Various cell body types and sizes can be distinguished, as well as the complete extent of their processes and varicosities.     

A Simple, Fast and Reliable Method for Obtaining Dopamine Histofluorescence from Mesencephalic Cultures

A modified glyoxylic acid technique for obtaining dopamine histofluorescence from cultured mesencephalic cells is described. This method requires only two solutions: one contains glyoxylic acid, sucrose and monobasic potassium phosphate and is used at room temperature, the other is a Hepes buffered solution used at 37 C. Relatively high concentrations of a monoamine oxidase inhibitor and dopamine are added to the cultures to load dopaminergic neurons; the cell bodies and their processes take up and hold dopamine quickly and evenly. The cultures are dipped in a glyoxylic acid solution, dried in air, heated for 5 min and coverslipped with mineral oil. Since the cultures remain in their culture dishes, the entire procedure takes less than 2 hr. The green histofluorescence characteristic of dopamine is seen when the cultures are viewed by standard fluorescence microscopy. Various cell body types and sizes can be distinguished, as well as the complete extent of their processes and varicosities.     

The use of glyoxylic acid for the fluorescence histochemical demonstration of peripheral stores of noradrenaline and 5-hydroxytryptamine in whole mounts.

The reactions of glyoxylic acid with peripheral stores of noradrenaline and 5-hydroxytryptamine to provide a fluorescence histochemical method for their localization have been investigated. Incubation in glyoxylic acid, followed by drying and heating of whole mount preparations gives an intense and well localized reaction. For incubation, a concentration of 2% glyoxylic acid, buffered to pH 7 at room temperature for 30 minutes gives ideal results. The method is equally good if the pH is varied in the range 6 to 9 or if the tissue is stored in the incubation mixture for up to 6 hours. Ideal development of the fluorophore requires an initial excess of moisture in the tissue, that this moisture is driven off during development, and that the tissue is protected from further moistening. A suitable method of achieving these ends is to heat partially dried tissue at 100 degrees C for 4 minutes and then cover it with paraffin oil. 5-hydroxytryptamine can be readily distinguished from noradrenaline because it forms a fluorophore after reaction at pH 3.5, whereas noradrenaline does not. Both amines can be visualized after incubation at neutral pH. Comparison with the formaldehyde vapour technique reveals three main advantages (and no disadvantages) of the glyoxylic acid method: (1) it gives a finer localization with higher fluorescence yield, (2) the glyoxylic acid method is less susceptible to variations in procedure and, (3) it is both simpler and quicker to apply.     

A method for the determination of the pKa of alpha-N-benzoyl-L-arginine in the presence of an impurity.

A sensitive and specific assay method for allantoin is described together with its application to rat plasma. The method consists of two main successive steps: hydrolytic formation of glyoxylic acid from allantoin, and fluorometric measurement of NAD produced from the reaction of glyoxylic acid and NADH, catalyzed by glyoxylate reductase. As little as 0.1 μg of allantoin can be determined. For the determination of endogeneous allantoin levels in rat plasma, 50 μl of the plasma is sufficient. Due to the higher sensitivity and specificity, the proposed method has advantages over the colorimetric method which has long been used for the determination of allantoin.     

Catecholamine-containing neurons in cultures of fetal rat hypothalamus: distribution,morphology, and maturation

The distribution, morphology, and maturation of catecholamine (CA) neurons have been studied in hypothalamic explants from late-gestation rats. CA-containing neurons were identified using the glyoxylic acid technique. CA-containing processes were present from all hypothalamic areas except the preoptic region. Several fiber types were identified. CA neurons in vitro resemble CA neurons in adult hypothalamus. This tissue culture system may be useful in the study of a number of properties of hypothalamic CA neurons.     

Localization of catecholamine-containing nerve fibres in the branchial heart and cardiac ganglion of the common cuttlefish Sepia officinalis L. (cephalopoda)

The innervation of the branchial heart of Sepia officinalis was examined using TEM and glyoxylic acid induced fluorescence. In the cardiac ganglion and in cardiac nerves bluish-green fluorophores were seen associated with perikarya and varicose nerve fibres. Microspectrofluorometric analysis provided clear evidence that monoaminergic neurons in the branchial heart contain only catecholamines. Considering pharmacological data, it is more than likely that 5-hydroxytryptamine (serotonin) is not present in this system.     

产乙醇酸氧化酶的重组毕赤酵母的构建及催化合成乙醛酸的研究

乙醇酸氧化酶（Go）是植物光呼吸途径中的一种关键酶,可以催化乙醇酸生产乙醛酸。从新鲜菠菜叶中提取总RNA,利用RT-PCR技术获得编码GO基因的cDNA片断。通过基因重组将GO基因克隆到载体pA0815中,构建了胞内表达载体pA0815／GO,重组质粒经电转整合至甲醇营养酵母GS115染色体。在混合碳源为10g/L山梨醇和0．5g/L甲醇的培养条件下,细胞的GO酶活达到474IU／g（DCW）。利用该重组毕赤酵母作为催化剂生产乙醛酸,结果表明：在乙醇酸浓度为0．25mol／L,重组酵母湿菌体为10dL,黄素单核苷酸（FMN）浓度为0．01mmol／L,pH8．0,20℃,反应18h后乙醛酸的产率达到51．8％。     

Enzyme-catalysed peptide amidation. Isolation of a stable intermediate formed by reaction of the amidating enzyme with an imino acid

A series of hydrazones and semicarbazones of glyoxylic acid were shown to have a potent inhibitory effect on the enzyme-catalysed conversion of D-Tyr-Val-Gly to D-Tyr-Val-NH2. Among the derivatives tested, the inhibitory activity was increased by the presence of hydrophobic substituents and decreased by polar substituents. The inhibition produced by glyoxylic acid phenylhydrazone was shown to be competitive. No inhibition was obtained with pyruvic acid phenylhydrazone, which possesses a methyl group in place of the alpha-H of glyoxylic acid phenylhydrazone. The inhibitory potencies of these non-peptide substances are in accord with the specificity exhibited by the amidating enzyme in its reaction with peptide substrates. The inhibition produced by the glyoxylic acid derivatives was shown to be due to their ability to act as substrates for the peptide-amidating enzyme. The product formed from [14C]glyoxylic acid phenylhydrazone was identified as oxalic acid phenylhydrazide by co-chromatography in three chromatographic systems. The results demonstrate that the enzyme-catalysed oxidation of glyoxylic acid phenylhydrazone takes place by a mechanism involving hydroxylation. It is implicit that peptide amidation catalysed by the same enzyme proceeds by a similar mechanism.     

Cytophysiology of neurosecretory axon terminals in the brain of an annelid (Ophryotrocha puerilis,Polychaeta)

Summary In the posterior part of the brain of the protandric polychaete Ophryotrocha puerilis neurosecretory cells form prominent axon terminals. The terminals are arranged in two complexes. The perikarya of these presumably monopolar neurons are scattered in the anterior part of the cerebral perikaryal layer. In females the terminals store large amounts of neurosecretory material. It has been suggested earlier that neurosecretions of the terminals may play a role during sex reversal from females to males. Application of histamine caused the release of neurosecretory material from the respective terminals in females. However, this discharge was not followed by sex reversal. Application of reserpine had no influence on the terminals. Neither by in vivo observation nor by ultrastructural analysis any effect of reserpine on the terminal complexes could be observed. In isolated terminals filled with neurosecretory material from females, catecholamines could not be detected by HPLC. Also, polyclonal antibodies against dopamine did not stain the terminal complexes. Furthermore, the complexes did not develop any fluorescence after glyoxylic acid treatment. Therefore, the present results contradict the hypothesis that the neurosecretory material of the respective axon terminals is catecholaminergic and that it is involved in sex differentiation. The function of the secretory neurons studied here remains unclear.Abbreviations AT axon terminal - CA catecholamine(s) - DA dopamine - DAB diaminobenzidine - GA glyoxylic acid - GIF glyoxylic acid-induced fluorescence - LY Lucifer Yellow - MB methylene blue - NSM neurosecretory material - OTH ootropic hormone - TC terminal complex     

Development of dopamine-containing neurons and dopamine uptake in embryos of Hirudo medicinalis

The embryonic development of neurons which contain or take up dopamine was studied with glyoxylic acid histofluorescence in Hirudo medicinalis. Beginning at the time of the formation of the tail ganglion, one pair of dopamine-containing neurons was stained per segmental ganglion. The normal outgrowth of the cell bodies into the anterior roots was prevented in isolated and cultured chains of embryonic ganglia. Preincubation of intact embryos in dopamine led to the staining of additional neurons at certain developmental stages. These neurons presumably are the precursors of serotonin-containing cells, which have a temporary capability of taking up and storing dopamine.     

Catecholamine-containing neurons in the peripheral nervous system of Aplysia

Numerous green-fluorescent neurons have been revealed by means of the glyoxylic acid histochemical method in cryostat sections of several organs of two Adriatic aplysiid gastropods, Aplysia depilans and A. fasciata. Catecholamine-containing perikarya and their processes have been found to be especially abundant in the vaginal portion of the large hermaphrodite duct, in the penis and its sheath, and in the gill. In the reproductive organs, two subpopulations of catecholamine-containing neurons could be distinguished according to their size and location. Axons of larger neurons formed bundles which seemed to project at the CNS.     

Fluorescent properties of monoamine neurons following glyoxylic acid treatment of intact leech ganglia

Summary The SPG modification (de la Torre and Surgeon 1976) of the glyoxylic acid method for amine condensation is a straightforward procedure which can be used upon intact ganglia from the leech. Intense fluorescence of the neurosomata of identified neurons which contain either indoleamine (serotonin, 5-HT) or catecholamine (CA) is obtained in less than 30 min. The fluorescence of the 5-HT containing neurons is yellow (518–526 nm) and decays more rapidly than the dominant blue emission (478–480 nm) of the CA neurons. Most importantly, the SPG technique greatly enhances the visibility of the axonal processes of neurons which contain both classes of amines over the fluorescence produced by formaldehyde condensation techniques. Both blue and yellow fluorescent varicosities are easily distinguished in the longitudinal connectives and lateral roots of the leech C.N.S. Because of its simplicity and high fluorescence yields, the SPG method for histochemical fluorescence should contribute to investigations of amine functions in invertebrate nervous systems.Supported by NIH research grant No. NS14481-03 and NSF research grant BNS-7915/08     

The fluorimetric microdetermination of glyoxylic acid in blood, urine and bacterial extracts

1. A spectrophotofluorimetric method for the determination of glyoxylic acid in biological materials is described. 2. The method is based on the reaction between glyoxylic acid and resorcinol in acid solution, a fluorescent complex being obtained on the subsequent addition of alkali. 3. The reaction was found to be sensitive and highly specific, the minimum detectable amount of glyoxylic acid being 1·35×10⁻⁸ mole. 4. The urinary excretion of glyoxylic acid by ten normal adults ranged from 1·4 to 4·7mg./24hr. Small but measurable amounts of glyoxylic acid were found in cell-free extracts of Pseudomonas oxalaticus OX1 grown on oxalic acid as a source of carbon. No glyoxylic acid was detected in human serum.     

Development of the nervous system of the pluteus larva of Strongylocentrotus droebachiensis

Summary Development of the nervous system of the pluteus larva of Strongylocentrotus droebachiensis was investigated using indirect immunofluorescence with antibodies against dopamine, GABA, and serotonin, and glyoxylic acid-induced fluorescence of catecholamines. Serotonergic cells first appear in full gastrulae; dopaminergic and GABAergic cells are present in early four-arm plutei. The number of neurons and the complexity of the nervous system increases through development of the pluteus. In the pluteus the dopaminergic component of the nervous system includes a ganglion in the lower lip of the mouth and a pair of ganglia at the base of the post-oral arms which extend axons along the base of the circumoral ciliary band. The distribution of cells visualized by glyoxylic acid-induced fluorescence is similar to that of dopaminergic cells. GABAergic neurons occur in the upper lip and in the wall of the esophagus. Serotonergic neurons are present in the lower lip; the pre-oral hood contains an apical ganglion which extends axons along the base of the epidermis overlying the blastocoel. The dopaminergic and GABAergic components of the nervous system are associated with effectors involved in feeding and swimming. The serotonergic component is not associated with any apparent effectors but may have a role in metamorphosis.     

A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: The SPG method

Summary A modified approach of the glyoxylic acid (GA) condensation reaction for the visualization of biogenic amines in tissue is described.Cryostat sections are used from brain or extracerebral tissue in dog, monkey, rat and mouse and exposed for 3 s to a room temperature solution containing sucrose-potassium phosphate-glyoxylic acid (SPG). The tissues are air dried and heated in an oven for 5 min. The complete processing time from fresh tissue to microscopic examination takes 18 min. Morphologically sharp and brightly fluorescent monoamine-containing neurons, pre-and terminal axons are seen against a dark parenchymal background without drug pre-treatment. The SPG method retains the high specific sensitivity for monoamines previously described in the original technique but is, in addition, more rapid and simple and is easily accessible as a research tool to investigators inexperienced in histofluorescence techniques.     

Glutathione transferase zeta-catalyzed biotransformation of deuterated dihaloacetic acids.

Glutathione transferase zeta (GSTZ) catalyzes the biotransformation of alpha-haloalkanoic acids. Treatment of rats or humans with dichloroacetic acid prolongs its elimination half-life, and preliminary studies in this laboratory show that fluorine-lacking, but not fluorine-containing dihaloacetic acids inactivate GSTZ. In the present study, the GSTZ-catalyzed biotransformation of unlabeled and deuterated dihaloacetic acids was investigated. With [(2)H]dichloroacetic acid and [(2)H]chlorofluoroacetic acid as substrates, the deuterium present in the [(2)H]dihaloacetic acid was retained in the [(2)H]glyoxylic acid formed. This finding indicates that the enol of the dihaloacetic acid does not serve as the substrate for the enzyme. The data afford an explanation of the failure of fluorine-containing dihaloacetic acids to inactivate GSTZ: dichloroacetic acid is converted to glyoxylic acid and inactivates GSTZ, whereas chlorofluoroacetic acid is biotransformed to glyoxylic acid, but produces negligible inactivation. Mechanisms are presented indicating that this difference may be attributed to the nucleofugicity of the leaving group.     

Amine-containing neurons in the brain of Lymnaea stagnalis: distribution and effects of precursors

Glyoxylic acid-induced fluorescence in whole-brain preparations of the central nervous system of the freshwater pond snail, Lymnaea stagnalis, was used to map the distribution of serotonin-and dopamine-containing neurons. Serotonin and dopamine were easily distinguishable by differences in color of fluorescence. Serotonin-containing neurons were consistently found in the cerebral, pedal, right parietal and visceral ganglia. Dopamine-containing neurons were found in the pedal, and buccal ganglia. Prior incubation of brains in 5-hydroxytryptophan (5-HTP), the immediate precursor to serotonin, produced serotonin-like fluoresence in neurons which do not normally fluoresce. These neurons thus probably possess specific uptake mechanisms for 5-HTP. Since 5-HTP itself fluoresces yellow, the glyoxylic acid technique cannot determine if these neurons contain the enzyme aromatic amino acid decarboxylase, which converts 5-HTP to serotonin, or merely fluoresce because of the 5-HTP taken into the cells.     

Effects of dbcAMP and theophylline on rat adrenal medulla grown in tissue culture.

Explants of rat adrenal medulla were grown in tissue culture. The effects of various doses of dbcAMP ranging from 0.001 mM up to 1 mM and equimolar amounts of theophylline were recorded by phase contrast optics and catecholamine histochemistry (glyoxylic acid method) over six days. There was a dose-dependent inhibition of the normally occurring outgrowth of Schwann cells, "chromaffin" cells and axons from the explants. Maintenance of glyoxylic acid-induced fluorescence in "chromaffin" cells was dose-dependent, too. Since theophylline is known to enhance intracellular levels of cAMP only, these effects are probably due to the action of cAMP. cAMP obviously maintains the degree of differentiation of chromaffin cells. Thus it could be argued that a certain degree of dedifferentiation is a prerequisite for the formation of axons from these cells.     

In search of a correlation between the electron affinity and the radiosensitizing activity of some glyoxylic compounds.

The electron affinity of some glyoxylic compounds with radiosensitizing properties was evaluated using different approaches. The half-wave reduction potentials were measured by polarography, and the molecular structures in solution were determined by NMR spectroscopy. The electron affinity of the unhydrated and hydrated molecules was theoretically evaluated by means of a semi-empirical quantum mechanical procedure, the MNDO. The possible correlations between these values and the radiosensitizing efficiency of these glyoxylic compounds is discussed.     

Histofluorescence studies of central monoamines. Comparison of Falck, cryostat and SPG methods.

The aim of the presented paper was to compare the Falck-Hillarp histofluoresce method for monoamines with its cryostat modification and the glyoxylic acid SPG method. The sensitivity and specificity of these methods and also the occurence of unspecific fluorescence is discussed.