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Biomedical subjects

R R Rando

Publications and source records attributed to R R Rando.

At least 127 records · Page 7Linked to original sources
PubMed1983

Effect of light on dopamine turnover and metabolism in rabbit retina.

Some neurochemical responses of dopaminergic neurons in the rabbit retina have been measured during prolonged light or dark adaptation. Light adaptation produced small increases (20%) in dopamine levels but larger increases (50-100%) in the two metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Light also significantly increased tyrosine hydroxylase activity; the increase was more pronounced when activity was measured in vivo than in vitro. Dopamine turnover, was faster in the light than in the dark. All these data support the suggestion that light leads to the activation of dopaminergic neurons in the rabbit retina.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1982

Use of a fluorescent cholesterol derivative to measure lateral mobility of cholesterol in membranes.

N1-Cholesterylcarbamoyl-N8-(4-nitrobenzo-2-oxa-1,3-diazole)-3,6-dioxaoctyl-1,8-diamine (NBD-Chol), a new fluorescent derivative of cholesterol, was incorporated into L-alpha-dimyristoylphosphatidylcholine (Myr2PtdCho)-based liposomes. The lateral mobility of this derivative, as well as that of N-(4-nitrobenzo-2-oxa-1,3-diazole)phosphatidylethanolamine (NBD-PtdEtn), was measured by fluorescence recovery after photobleaching techniques. In Myr2PtdCho liposomes, the diffusion coefficients (D) of the two probes are the same within experimental error below (D, approximately equal to 2 X 10(-10) cm2 X sec-1) and above (D, approximately equal to 2 X 10(-8) cm2 X sec-1) the main phase transition temperature of the bulk lipid (Tm). There is, however, a distinct difference between the mobilities of the derivatives at concentrations of added cholesterol between 5 and 20 mol % at temperatures below the main phase transition. Under these conditions, the diffusion coefficient of NBD-Chol is approximately twice that of NBD-PtdCho, a result consistent with the idea that cholesterol undergoes a lateral phase separation in these membranes at concentrations less than 20 mol %. At cholesterol concentrations greater than 20 mol % or temperatures above the Tm, the D values of the two probes are identical. The lateral mobility of a cholesterol derivative has thus been monitored directly in cholesterol-containing membranes.

4-Chloro-7-nitrobenzofurazan↗
PubMed1982

The differential effects of GABA-transaminase inactivation in the chick retina and brain.

The inactivation of gamma-aminobutyrate (GABA)-transaminase by the highly specific and potent neurotoxin gabaculine leads to different neurochemical consequences in the chick brain as opposed to the chick retina. In the brain, GABA levels continually climb, reaching approximately eightfold increases over control values after 24 h. The elevation in GABA levels leads to a time-dependent and coincident fall in glutamate decarboxylase and cysteine-sulfinate decarboxylase activities, to approximately 50% of control values. On the other hand, in the retina GABA levels only increase to a plateau level two- to threefold that of control after inactivation of GABA-transaminase. Furthermore, although the glutamate decarboxylase activity decreases to about 50% of control values, cysteinesulfinate decarboxylase activity is not affected. These studies show that the processing of GABA in the retina differs from that in the brain, and that cysteinesulfinate and glutamate decarboxylase activity probably reside in different enzyme molecules in the retina, although they may reside in the same enzyme in the brain.

4-Aminobutyrate Transaminase↗
PubMed1981

The chemical labeling of glutamate decarboxylase in vivo.

Mouse brain glutamate decarboxylase(s) was specifically titrated in vivo and in crude brain homogenates by a combination of gabaculine and [alpha-3H]acetylenic gamma-aminobutyric acid. This specific titration is based on the differential spectra of action of these two mechanism-based enzyme inactivators. The specificity of the titration in vitro was demonstrated by showing that the time course of radioactivity incorporation exactly paralleled the time course for glutamate of decarboxylase inactivation. Furthermore, pretreatment of the crude homogenate with aminooxyacetic acid and alpha-methyl-trans-3-dehydroglutamate, two inactivators of glutamate decarboxylase which function by entirely different mechanisms, decreased count incorporation ([alpha-3H]acetylenic gamma-aminobutyric acid) to the same extent as the activity was decreased. Injection of [alpha-3H]acetylenic gamma-aminobutyric acid intraperitoneally after gabaculine injection led to incorporation of 0.46 nmol of inactivator/mouse brain, when approximatley 70% of the enzyme was inactivated. This means that there is approximately 0.66 nmol of glutamate decarboxylase/0.5 g of mouse brain, assuming the stoichiometry of inactivator bound to enzyme is one. This value is similar to the one (0.646 nmol) obtained from a calculation based on the enzyme purification data (Wu, J.-Y. (1974) in gamma-Aminobutyric Acid in Nervous System Function (Roberts, E., Chase, E. N., and Tower, D. B., eds) pp. 7-55, Raven Press, New York).

Alkynes↗
PubMed1981

Irreversible inhibition of glutamate decarboxylase by alpha-(fluoromethyl)glutamic acid.

alpha-(Fluoromethyl)glutamic acid (FMG) was synthesized and shown to be an active site directed irreversible inhibitor of glutamate decarboxylase (EC 4.1.1.15) from Escherichia coli. The KI for the active enantiomer is 1.4 microM, and the kinh = 5.9 X 10(-3) s-1. Substrates for the enzyme, such as L-glutamate, and competitive inhibitors, such as citrate, decrease the rates of FMG-mediated inactivation of the enzyme. A profound change in the ultraviolet spectrum of the enzyme accompanies the inactivation process. When [3H]-FMG is used, it can be shown that the enzyme incorporates radioactivity at the same rate as that of inactivation. There is a 1:1 stoichiometry of [3H]FMG incorporated to pyridoxal phosphate binding subunits of the enzyme. From these and other studies it is concluded that FMG is a substrate for the enzyme and alkylates it as a consequence of this turnover.

Binding, Competitive↗
PubMed1981

The inactivation of gamma-aminobutyric acid transaminase in dissociated neuronal cultures from spinal cord.

It had previously been shown that dissociated cell cultures from chick embryo spinal cord have a high affinity uptake system for the neurotransmitter gamma-aminobutyric acid (GABA) and make functional inhibitory synaptic contacts as determined by electrophysiology (Farb et al., 1979). It is shown here that these cultures can synthesize GABA from added glutamate in a glutamate decarboxylase-dependent reaction. Furthermore, these cultures have a functional GABA transaminase that degrades the neurotransmitter. This enzyme can be specifically and irreversibly blocked with gabaculine. A 15 min incubation with 10(-6) M-gabaculine completely inactivates the enzyme. The inactivation of the enzyme leads to an increase in GABA levels. Long-term incubation (16 days) of gabaculine in the medium does not appear to alter high affinity GABA transport, suggesting that the drug is not toxic to cells capable of accumulating GABA.

4-Aminobutyrate Transaminase↗
PubMed1981

Evidence for a neurotransmitter role for 5-hydroxytryptamine in chick retina.

Evidence to support a neurotransmitter role for 5-hydroxytryptamine in chick retain is presented. Putative 5-hydroxytryptophan and 5-hydroxytryptamine extracted from chick retina have been characterized chromatographically and electrochemically and have been found to behave like the authentic compounds. Retinal tryptophan hydroxylase activity could not be demonstrated in vitro, but chick retina was able to synthesize 5-[3H]hydroxytryptamine from [3H]tryptophan in vivo. Retinal 5-hydroxytryptamine levels were reduced by local injection of alpha-fluoromethyldopa, an inhibitor of aromatic L-amino acid decarboxylase, or reserpine, indicating that his amine is both synthesized and stored in this tissue. The retinae of light-adapted chicks have higher levels of 5-hydroxytryptamine and its major metabolite, 5-hydroxyindoleacetic acid, than dark-adapted animals. The turnover of 5-hydroxytryptamine appears to be greater in the light after inhibition of synthesis. Conversely, the rate of synthesis of 5-hydroxytryptamine appears to be greater in the dark. The possible interaction of dopaminergic neurons and the 5-hydroxytryptamine-containing cells and the effect of alpha-fluoromethyldopa on this relationship are discussed. The data lend support to the notion that 5-hydroxytryptamine in chick retina may have a neurotransmitter function.

5-Hydroxytryptophan↗
PubMed1981

Dopamine metabolism following irreversible inactivation of aromatic amino acid decarboxylase in retina.

The effects of an intravitreal injection of alpha-fluoromethyldopa, an irreversible mechanism-based inactivator of aromatic-L-amino acid decarboxylase, on the retinal dopamine content of light-adapted chicks and rabbits have been examined. A single administration of 10 nmol of alpha-fluoromethyldopa totally inactivates aromatic L-amino acid decarboxylase within 2 hr in vivo in rabbits. By 4 to 6 hr, the level of dihydroxyphenylalanine increased 7-fold and the levels of dopamine and dihydroxyphenylacetic acid fell by 90%. With an injection of 50 nmol, similar results were observed in chicks. The levels of dihyroxyphenylacetic acid began to fall soon after injection when significant (30 to 40%) amounts of dopamine were still present. These results are discussed in relation to the use of tissue dihydroxyphenylacetic acid levels to indicate the level of dopaminergic neuronal activity or dopamine synthesis. Recovery by the retinae of both species was shown by the return of aromatic L-amino acid decarboxylase activity and the resynthesis of dopamine.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1980

Lectin-mediated aggregation of liposomes containing glycolipids with variable hydrophilic spacer arms.

Synthetic glycolipids containing a cholesterol anchor group attached via a spacer group to a sugar moiety can be incorporated into small unilamellar liposomes, rendering them susceptible to agglutination by the appropriate multivalent lectin [Rando, R. R., Orr, G. A., & Bangerter, F. W. (1979) J. Biol. Chem. 254, 8318-8323; Rando, R. R., & Bangerter, F. W. (1979) J. Supramol. Struct. 11, 295-309]. We report here on the role of spacer arm length in rendering these liposomes susceptible to agglutination. In order to eliminate the ambiguities inherent in using hydrophobic or charged spacer groups, we have synthesized a hydrophilic, ethylene glycol based amino acid (8-amino-3,6-dioxaoctanoic acid) for these studies. The Ricinus communis agglutinin (ricin) mediated agglutination of these beta-galactoside-containing glycolipids was studied. A spacer arm length of four atoms will not support agglutination under any conditions. A seven-atom spacer will support agglutination, but only at high phospholipid concentrations (0.24 mumol/mL) with a pseudo-first-order rate of agglutination of 0.0079 min-1. With 13 and 22 atom spacer groups, the pseudo-first-order rate constants were 0.4 min-1 and 1.3 min-1, respectively, at a phospholipid concentration of 0.06 mumol/mL. Under conditions where the liposomes containing the glycolipid with the 13-atom hydrophilic spacer arm were completely agglutinated by ricin, 9.3% of the total available sugar moieties of the liposome were bound to ricin. This means that, on the average, 38 interliposomal bonds were formed in an aggregate.

Agglutination↗
PubMed1980

Functional incorporation of synthetic glycolipids into cells.

Synthetic glycolipids containing an alpha-mannoside group linked by a hydrophilic spacer arm to cholesterol were incorporated into bovine erythrocytes by exchange from glycolipid-containing liposomes. When the distance between the sugar and the cholesterol moieties was approximately 26 A, functional incorporation of these glycolipids could be easily detected, as revealed by the concanavalin A-mediated agglutination of these cells. Bovine erythrocytes are not themselves susceptible to concanavalin A-mediated agglutination. The minimal concentration of concanavalin A required for agglutination of modified erythrocytes, containing 9.15 x 10(6) glycolipid molecules per cell, was 4 microgram/ml. Under these conditions, only approximately 4% of the membrane-bound cholesterol had been exchanged for the synthetic glycolipid. The observed aggregation was reversible in the presence of alpha-methyl mannoside and did not occur when beta-galactosyl-containing glycolipids were used in place of their alpha-mannoside isomers. These studies demonstrate a technique of sugar incorporation into cell membranes which should be of great advantage in studies on the roles of cell surface sugars in biological recognition. Furthermore, they demonstrate that the sugars need only be a short distance (26 A) from the membrane in order to functionally bind concanavalin A.

Animals↗
PubMed1979

Threshold effects on the concanavalin A-mediated agglutination of modified erythrocytes.

Bovine erythrocytes, which are not concanavalin A (ConA)-agglutinable, can be rendered so by attaching alpha-D-mannose residues to their outer membrane. The sugars are incorporated by mildly oxidizing the cells with periodate followed by coupling the liberated aldehyde groups with an alpha-thiomannosyl containing hydrazide (I). The rate and extent of ConA-mediated aggregation of the modified cells are not linearly dependent on the amount of sugar incorporated. For example, treatment of the erythrocytes with 0.075 mM periodate for 5 min followed by I led to the introduction of 1.05 x 10(6) mannosyl residues/erythrocyte. Binding studies with 125I-ConA demonstrated the presence of 66,525 ConA receptors/cell with an average KA = 4.9 X 10(6) M-1 yet the cells failed to aggregate with ConA at concentrations up to 500 microgram ml-1. Treating the cells with 0.1 mM periodate followed by I led to the introduction of 1.42 x 10(6) mannosyl residues/erythrocyte. Binding studies with 125I-ConA indicated the presence of 78,780 binding sites/cell (KA = 5.9 X 10(6) M-1). These cells were readily aggregated by ConA at concentrations greater than or equal to 64 microgram ml-1. We show here that the sugar incorporation technique is random and that no functional differences were detected in the receptors introduced at the different periodate concentrations. Therefore, the ConA-mediated aggregation of these modified erythrocytes is exquisitely sensitive to small changes in functionally identical receptor densities.

Animals↗
PubMed1979

Synthetic glycolipids and the lectin-mediated aggregation of liposomes.

Synthetic mannose-containing glycolipids utilizing the cholesterol nucleus as a lipid anchor, and either the 6-aminohexyl- or the 6-(6-aminohexanamido)hexyl-1-thio-alpha-D-mannopyranosides as the carbohydrate ligands, have been synthesized and incorporated into small unilamellar liposomes. Incorporation of these cholesterol-mannoside derivatives at concentrations up to 14 mol% apparently does not affect the physical characteristics of the liposomes. Addition of concanavalin A to a suspension of liposomes containing the long chain cholesterol-mannose derivative causes an increase in light-scattering at 360 nm. As the increase in absorbance is completely reversed by the addition of alpha-methylmannoside, aggregation rather than fusion of the liposomes appears to be occurring. Liposomes containing 14 mol % of the short chain (6-aminohexyl-) derivative are aggregated by concanavalin A indicating that the lectin can approach to within 10 A of the lipid bilayer. Preliminary results suggest that the aggregation of vesicles containing either the long or short chain derivatives is highly dependent on the density of the sugar in the membrane.

Chemical Phenomena↗
PubMed1979

Threshold effects on the lectin-mediated aggregation of synthetic glycolipid-containing liposomes.

Cholesterol analogs containing sugar residues linked by spacer groups to the cholesterol O can be incorporated into egg yolk lecithin small unilamellar liposomes. The synthetic glycolipid analogs distribute evenly on both sides of the bilayer. These liposomes are aggregated by the appropriate lectin. For example, when the sugar residue is a beta-galactoside the liposomes are aggregated by ricin and when it is an alpha-mannoside they are aggregated by Con A. The lectin-mediated aggregation of these liposomes is reversed by the addition of the appropriate sugar. The rates but not the extents of aggregation of these liposomes are highly sensitive to the amount of glycolipid incorporated. Below approximately 5% glycolipid incorporation the rate of the lectin-mediated aggregation of these liposomes is exceedingly slow, whereas above this level rapid aggregation proceeds. At all concentrations studied the synthetic glycolipids are incorporated in a unimodal fashion so that the observed threshold effects cannot be based on possible differences in the manner in which the glycolipids are incorporated at different concentrations. This conclusion is based on 1) studies with galactose oxidase that show that the percentage of galactose oxidation in a liposome prepared from a galactosyl-containing glycolipid is independent of glycolipid concentration, and 2) studies on the aggregation of liposomes containing mixed glycolipids in which the glycolipids are shown to behave independently. The importance of a critical density of membrane-bound receptors in order for aggregation to occur is discussed.

Chemical Phenomena↗