Amino Acid Analysis Demonstrates that Increased Plasma Free Tryptophan Causes the Increase of Brain Tryptophan During Exercise in the Rat

Rats were trained to run on a horizontal treadmill for 2 h at 20 m/min. This activity considerably increased plasma free tryptophan (TRP) (+70%) but did not alter plasma total TRP levels and had little or no effect on plasma concentrations of the other large neutral amino acids (LNAAs) that compete with TRP for entry into the brain. Brain TRP levels increased by 80%. The only other brain LNAA to be affected by exercise was threonine, which rose moderately. The results indicate that increased plasma free TRP was specifically responsible for the increase of brain TRP after 2 h of exercise. Brain lysine was also increased whereas glycine, alanine, and gamma-aminobutyric acid were decreased. The differences between the present findings and those previously obtained following 2 h immobilization stress are discussed.     

Coupling of a loop diuretic-sensitive Na+ influx with the net loop diuretic-sensitive K+ efflux in mouse NIH 3T3 cells

Mouse 3T3 fibroblasts have a loop diuretic sensitive Na+ transport system, responsible for more than 50% of the total Na+ influx. This transport system is dependent on the simultaneous presence of all three ions; Na+, K+, (Rb+) and Cl- in the extracellular medium. The same requirement for these three ions was also found for the loop diuretic-sensitive K+ efflux. In addition, the sensitivities of Na+ influx and Rb+ efflux for the two loop diuretics, furosemide and bumetanide were found to be similar. The similar ionic requirement and sensitivity towards loop diuretics of the two fluxes, support the hypothesis, that this loop diuretic-sensitive Na+ influx in mouse 3T3 cells, is accompanied by the net loop diuretic-sensitive K+ efflux.     

Distribution of Alginate Lyase Activity among Strains of Bacillus circulans

Strains from four different DNA relatedness groups of Bacillus circulans showed apparent alginate lyase activity; the activity of three strains examined had mannuronidase specificity. A representative strain of group 4 also produced apparent inducible unsulfated chrondroitin lyase activity.     

Temporal Profiles of Proteins Responsive to Transient Ischemia

The responses of long and short half-lived proteins to ischemia were measured in rat brain during 6 days of recovery from 30 min of transient forebrain ischemia produced by four-vessel occlusion. At the end of the ischemic interval, the neocortical activities of four vulnerable enzymes [ornithine (ODC) and S-adenosylmethionine (SAMDC) decarboxylases, and RNA polymerases I and II] were unchanged, but within 30 min of reperfusion, their activities dropped by 25-50%. The loss of substance P in the striatum and substantia nigra was slower, reaching about 50% by 12 h. On the other hand, the activities of 5 long half-lived enzymes did not change in the neocortex at 5 and 15 h of reperfusion and regional protein concentrations were essentially unaffected over 6 days survival. The rate and extent of normalization of the amounts or activities of the vulnerable proteins varied. RNA polymerase II and ODC activities were restored within 4 h, and ODC showed a biphasic increase in activity, with peaks at 10 h and 2-3 days. RNA polymerase I and SAMDC activities were restored by 18 h and 5 days, respectively, whereas substance P concentrations did not completely recover, even at 6-15 days. The greater the regional reduction of blood flow during ischemia, the larger the net change (gain or loss) of SAMDC or ODC activity and the longer the time required to normalize the activities of these enzymes. The average rate of proteolysis, assessed by measuring the rate of clearance of 14C from protein prelabeled with [14C]bicarbonate, was abnormal during the first 2 days of reperfusion. Postischemic changes in both protein synthesis and degradation could affect the amounts of some of the proteins responsive to transient ischemia.     

Deprenyl Protects Dopamine Neurons from the Neurotoxic Effect of 1-Methyl-4-Phenylpyridinium Ion

1-Methyl-4-phenylpyridinium ion (MPP+) is the product of the metabolic oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by monoamine oxidase (MAO). MPP+ is toxic to 3,4-dihydroxyphenylethylamine (dopamine, DA) neurons in explant cultures of rat embryonic midbrain. Addition of 2.5 microM MPP+ to the feeding medium for 6 days results in significant reduction of the DA levels in the cultures (to 19% of control) as well as in the uptake of [3H]DA (to 32% of control). When the cultures are treated with the MAO inhibitor deprenyl (10 microM) 24 h prior to and during exposure to MPP+, the DA neurons are protected from the toxicity of the drug. In the combined deprenyl plus MPP+ treatment, the levels of DA in the cultures remain at the control range and the [3H]DA uptake is reduced to only 73% of control. These results indicate that MAO is involved in the toxicity of MPP+ on DA neurons.     

Radiation effects on diamine oxidase activities in intestine and plasma of the rat

Diamine oxidase (DAO; EC 1.4.3.6) activity was measured in plasma and in ileal tissue homogenates prepared from male Sprague-Dawley rats euthanized at 1-15 days after acute whole-body irradiation with 14.5-MeV electrons. Animals irradiated with 1 Gy showed no diminution in plasma and ileal DAO activities through Day 13 relative to nonirradiated controls. Animals irradiated with 5, 10, and 12 Gy displayed marked declines in ileal DAO activity, with levels reaching a nadir on Day 3. This was paralleled by a decrease in plasma DAO activity in all three dose groups. Recovery of ileal and plasma DAO levels was later seen as early as Day 4 in animals irradiated with 5- and 10-Gy doses, but animals receiving 12 Gy did not survive beyond Day 3. The relationship between radiation dose and levels of plasma and ileal DAO on Day 3, the time of maximum decrease at all doses, was also investigated. Ileal DAO activity decreased almost linearly between 2 and 8 Gy. Plasma DAO activity closely paralleled the dose dependency of the ileal levels. These data suggest that plasma DAO activity might be useful as a biologic marker of intestinal epithelial injury and recovery after acute radiation exposure.     

Purine nucleoside phosphorylase synthesis and turnover in human lymphoid cells

We have studied the turnover and synthesis of purine nucleoside phosphorylase by using a polyclonal rabbit antiserum to this protein. The turnover of purine nucleoside phosphorylase was studied in the B lymphoblast cell, WI-L2, by specific immunoprecipitation of [3H]leucine-labeled proteins. The half-lives for total protein and purine nucleoside phosphorylase were 14.5 and 14.1 hr, respectively. For cells cultured in the presence of inosine the half-life of purine nucleoside phosphorylase was reduced to 11.2 hr. The synthesis of purine nucleoside phosphorylase was analyzed during phytohemagglutinin-stimulated T cell transformation by pulse labeling cells with [35S]methionine. Purine nucleoside phosphorylase synthesis increased greater than 10-fold during the first 12 hr of transformation and continued to a maximum of 30-fold. The relative rate of purine nucleoside phosphorylase labeled to total proteins was 0.04% in unstimulated T cells and increased to 0.18% 12 hr after stimulation. These studies identify some preferential synthesis of purine nucleoside phosphorylase during the early stages of T cell transformation.     

Rapid changes in the phospholipid composition of gill membranes during thermal acclimation of the rainbow trout,Salmo gairdneri

Summary The phospholipid composition of gill tissue was determined in rainbow trout (Salmo gairdneri) undergoing thermal acclimation between 5°C and 20°C for a period of up to 28 days. Proportions of phosphatidylethanolamine (PE) and cardiolipin (CL) increased during cold acclimation and decreased during warm acclimation; proportions of phosphatidylcholine (PC) changed in the opposite direction (i.e., decreased during cold acclimation). In contrast, levels of phosphatidylserine,-inositol, and sphingomyelin did not vary significantly. Thermal modulation of headgroup composition occurred rapidly as reflected by changes in the ratio of PC-to-PE, which rose significantly from 2.40±0.09 to 2.92±0.09 within 72 h of transfer from 5 to 20°C; adaptation to 5°C was equally rapid. Proportions of PE changed more rapidly than those of PC during cold adaptation, whereas the opposite was true during warm acclimation. Both the time course and the direction of the observed changes in phospholipid composition suggest that such adjustments may contribute to the homeoviscous regulation of membrane properties, particularly during the initial stages of thermal adaptation.     

A critique of the utility of the prediction of protein secondary structure

The Chou-Fasman predictive algorithm for determining the secondary structure of proteins from the primary sequence is reviewed. Many examples of its use are presented which illustrate its wide applicability, such as predicting (a) regions with the potential for conformational change, (b) sequences which are capable of assuming several conformations in different environments, (c) effects of single amino acid mutations, (d) amino acid replacements in synthesis of peptides to bring about a change in conformation, (e) guide to the synthesis of polypeptides with definitive secondary structure,e.g. signal sequences, (f) conformational homologues from varying sequences and (g) the amino acid requirements for amphiphilicα-helical peptides.     

Isolation of the gene encoding yeast DNA polymerase I

A yeast genomic DNA expression library in lambda gt11 antibody prepared against yeast DNA polymerase I were used to isolate the gene encoding DNA polymerase I. The identity of the DNA polymerase I gene was determined by several criteria. First, the clone-encoded protein is immunologically related to DNA polymerase I. Second, cells containing the gene cloned in the high copy number plasmid YEp24 overproduce the polymerase activity 4- to 5-fold as measured in yeast extracts. Finally, insertion of the gene downstream from a bacteriophage T7 promoter allows synthesis of yeast DNA polymerase I in Escherichia coli. Gene disruption and Southern hybridization experiments show that the polymerase is encoded by an essential, single copy gene. Examination of the germinated spores containing the disrupted gene reveals a defect in nuclear division and a terminal phenotype typical of replication mutants.