The use of lectins for determination of absolute configurations of small amounts of sugars eluted from chromatograms

A simple procedure for the determination of the absolute configuration (i.e., assignment to the D- or L-enantiomeric series) of glucose, mannose, galactose, fucose, arabinose, and rhamnose is described, based on inhibition by these sugars of 125I-labeled lectin binding to the glycoconjugates immobilized on the wells of plastic microculture plates. The method works well with 10 to 100-micrograms amounts of the sugars isolated after paper chromatography of the glycoprotein or polysaccharide hydrolysates.     

Histidine residues in elongation factor EF-tu from Escherichia coli protected by aminoacyl-tRNA against photo-oxidation

Complexes of Escherichia coli elongation factor EF-Tu with GTP or GTP and aminoacyl-tRNA were photo-oxidized by irradiation with visible light in the presence of rose bengal dye. EF-Tu was isolated, digested with trypsin, the resulting tryptic peptides were separated by high-performance liquid chromatography (HPLC), and the position of most of the peptides on the chromatogram was determined. Irradiation of complexes resulted in the inactivation of the factor (as tested by its capacity to interact with aminoacyl-tRNA) and was accompanied by the loss of its histidine residues (as revealed by amino acid analysis) and by the decrease in the amount of some tryptic peptides (as detected by HPLC). Aminoacyl-tRNA, bound to EF-Tu during the irradiation, protected the protein from inactivation, from the loss of histidine residues and some of its peptides from photo-oxidative degradation. Comparison of quantities of individual tryptic peptides recovered from the irradiated EF-Tu X GTP X aminoacyl-tRNA complex with those from the irradiated EF-Tu X GTP complex revealed that histidine-containing peptides T12 and T15 as well as methionine-containing peptide T14 were in the ternary complex markedly protected against the photo-oxidative degradation. This finding suggests that their histidines, i.e. His-66 and His-118 respectively and at least one of the methionines (Met-91, 98 or 112) present in peptide T14 are located near to or at the binding site of EF-Tu for aminoacyl-tRNA and could be involved in the interaction between aminoacyl-tRNA and the factor.     

Dynamic structure of metallothionein

Molecular sieve chromatography of rabbit liver metallothionein at different electrolyte concentrations revealed that this protein undergoes an increase in Stokes radius from 1.50 to 1.78 nm when the ionic strength is lowered from 0.5 to 0.015 indicating a change in molecular shape and/or hydration. The variation in ionic strength also affects the far-UV circular dichroism of metallothionein reflecting a conformational transition in the protein. The effects are attributed to changes in intramolecular repulsion between the strongly negatively charged metal-thiolate clusters of the protein. It is suggested that metallothionein exists in at least two interchangeable conformational states which differ in hydrodynamic properties and whose equilibrium concentrations are determined by the electrostatic free energy of the system.     

Effect of theophylline and theobromine on cell respiration and calcium transport in isolated rat liver mitochondria

The influence of theophylline and theobromine on cellular respiration and on membrane transport of calcium has been studied in isolated rat liver mitochondria, using oxygen and Ca2+ selective electrodes. A linear decrease in respiratory coefficients, in the total amount and rate of "extra" oxygen consumption induced by ADP is observed with drug concentration. Theobromine does not show any appreciable effect on these respiratory parameters, but this result is similar to that observed with theophylline for the same concentration range. Calcium uptake coupled to respiration is inhibited by both drugs depending on their concentrations. Theobromine is more effective than theophylline. Calcium saturation of the mitochondria takes place in all cases after 36 +/- 2 s but only a 20% of the maximum calcium uptake observed in the absence of the drugs is determined in the presence of 15 mM theophylline or only 1.8 mM theobromine. Comparative studies show direct correlation between the pharmacological activities as stimulants of caffeine, theophylline and theobromine and their behaviour as inhibitors of calcium uptake coupled to respiration by mitochondria.     

Antilipolytic activity in vitro of various analogs of nicotinic acid. Structure-activity relationship

The antilipolytic activity of a series of N aryl-nicotinamides and of alpha picolinic acid, has been tested in vitro. Lipolysis was stimulated by epinephrine (20 micrograms/ml of incubation medium) using rat's epididymal adipose tissue slices. Only N(2-carboxy methyl phenyl) nicotinamide showed antilipolytic effect comparable to that of nicotinic acid at similar concentrations (2 X 10(-5) M). Picolinic acid (10(-4) M) showed no antilipolytic effect. These results, together with those of the literature, are discussed in regard to the relations between structure and antilipolytic activity.     

Effects of salicylate on insulin and glucagon secretion by the isolated and perfused rat pancreas

The effects of sodium salicylate, a prostaglandin synthesis inhibitor, on glucose-induced secretion of insulin and glucagon by the isolated perfused rat pancreas have been studied. Sodium salicylate inhibited both basal (2.8 mM glucose) and stimulated (16.7 mM glucose) insulin release in a dose dependent manner (1, 5 and 10 mM). This inhibition is not interpretable in terms of a simple inhibition of cyclooxygenase by sodium salicylate. Basal glucagon release was not changed by 1 mM sodium salicylate but the latter partially blocked its inhibition by 16.7 mM glucose. Higher doses of sodium salicylate (5 and 10 mM) inhibited basal glucagon secretion without affecting its response to 16.7 mM glucose. These findings suggest a predominant stimulatory action of endogenous prostaglandins on glucagon release.     

Incorporation of (U-14C)-glucose into glycogen in normal rat pancreatic islets

The incorporation of glucose into glycogen was determined in pancreatic islets isolated from normal rats and incubated with glucose (5 or 20 mM) and compounds known to affect glycogen metabolism in other tissues. Incubation of pancreatic islets with glucose (20 mM) induced a marked increase in radioactive glycogen. Exposure to epinephrine in the presence of glucose (20 mM) slightly increased incorporation of glucose into glycogen. In contrast the incorporation of glucose into glycogen was not affected when isolated islets were exposed to glucagon or insulin, whereas anti-insulin serum in the incubation medium decreased radioactive glycogen formation.     

Possible effect of bromocriptine on the insulin receptor

The administration of 0.00011 mg/g weight/day of bromocriptine (CB154) for 7 days to Wistar rats, improved the peripheral glucose uptake without significant changes in plasma insulin level, during the intravenous glucose tolerance test (0.33 g/kg). The mode of the bromocriptine action on binding of 125I insulin to erythrocyte insulin receptors has been evaluated. The total number of sites was greater with bromocriptine (513.1 +/- 124.1 pM/1,CB154 815.6 +/- 107.9 pM/l) (p less than 0.01). The high affinity/low capacity compound of insulin receptor, in CB154 rats (51.8 +/- 16.8 pM/l) was higher than in normal rats (18.3 +/- 8.9 pM/l) (p less than 0.005). Additional studies indicated that CB154 had no effect on the rate of association and dissociation of 125I insulin from rats erythrocyte insulin receptors. The degradation of insulin or the erythrocyte receptor sites do not change, after the treatment with CB154.     

Effects of divalent cations and functionally related substances on the yeast to mycelium transition in Sporothrix schenckii

In a minimal basal medium with glucose at pH 4.0 and 25 degrees C, a lowering of the magnesium and zinc concentrations or increase in the calcium concentration of the medium favoured the yeast-mycelium transition in Sporothrix schenckii. Addition of zinc (1 and 10 mM) inhibited mycelial development and induced reversion to a yeast-like morphology. EDTA and EGTA also delayed germ tube formation, possibly by their calcium-chelating effects or by altering intracellular concentrations of this or other ions. Ionophore X537A also caused a delay in germ tube formation, possibly by interfering with magnesium metabolism in these cells.     

Phospholipid methylation in pancreatic islets

Glucose provokes a transient stimulation of phospholipid methylation in rat pancreatic islets, possibly by increasing phospholipid methyltransferase activity. The association of DL-homocysteine and 3-deazaadenosine inhibits phospholipid methylation. The methylation of phospholipids may play a role in the stimulus-secretion coupling for glucose-induced insulin release.