Inhibitors of adenosine catabolism improve recovery of dog myocardium after ischemia

Summary The effects of inhibitors of adenosine catabolism on contractile function and metabolites were assessed during 15 minutes of ischemia followed by 30 minutes of reperfusion in the open-chest dog heart. As compared to sham treatment, pretreatment with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and dipyridamole (DP) protected contractile function during ischemia, and improved recovery of high energy phosphate content and contractile fucntion during reperfusion following ischemia. Testing EHNA and DP in a free-radical generating system indicated both compounds have some scavenging ability, suggesting the effect of EHNA + DP may not be on adenosine nucleotide metabolism alone. Comparison of end diastolic segment lengths to contractile function indicated the results were not affected by changes in preload resulting from peripheral vasodilation.With the technical assistance of Dennis Dahmen.     

Alpha-factor leader sequence-directed transport of Escherichia coli beta-galactosidase in the secretory pathway of Saccharomyces cerevisiae

Summary The constuction of two fused genes is described. One involves the in-frame fusion of the yeast prepro--factor coding sequence, and the Escherichia coli lac Z gene. The second gene fusion utilizes a 103 bp yeast invertase NH₂-terminal coding sequence at the fusion junction of the hybrid gene described above. The gene fusions, under the control of the -factor promoter, expressed active -galactosidase in haploid yeast cells. The activity could be regulated in a temperature-sensitive sir3 mutant. The incorporation of the invertase coding sequence at the MF1-lacZ fusion junction provided significantly higher levels of -galactosidase activity. A substantial quantity of the hybrid proteins generated from the gene fusions was primarily localized in the intracellular membranes of yeast cells, while a processed form could be secreted into the periplasm.A portion of this work appeared in Biotechnology Progress (Das and Shultz 1986) as proceedings of the symposium on Industrial Scale Protein Purification, held at the annual meeting of the Institute of Chemical Engineers in Miami Beach, Fla, USA on November 4, 1986     

Generation of radical oxygen species by neural crest cells treated in vitro with isotretinoin and 4-oxo-isotretinoin

The effects of isotretinoin (IR) and its primary metabolite (in the human), 4-oxo-isotretinoin (4-OIR or OIR), on isolated chick neural crest cells (NCC's) in culture were studied. NCC's were found to be deficient in both superoxide dismutase (SOD) and catalase, two of the enzymes known to function in the "scavenging" (dismutation) of toxic radical oxygen species (ROS) such as the superoxide anion and hydrogen peroxide. The addition of IR or OIR to the culture medium significantly depressed the viability of the NCC's when compared to untreated cells. OIR was more potent in this regard than IR. In the presence of either IR or OIR, NCC's generated superoxide anions (O2.), hydrogen peroxide (H2O2), and hydroxyl anions (OH.). OIR was again more potent. The cytotoxicity of IR or OIR was demonstrated by the "leakage" of radioactive chromium from prelabeled cells. The latter is suggestive of a primary surface membrane defect, most logically via the induction of lipid peroxides by the retinoids. The latter is accompanied by an increase in membrane permeability and porosity as evidenced by the fact that various fluorescently labeled molecules, including BSA-FITC (MW 69,000), gain entrance into the cytoplasm of the retinoid treated cells. No label was seen in the cytoplasm of similarly treated control cells. When SOD (200 units/ml) or catalase (400 units/ml) was added to the culture media of IR- or OIR-treated NCCs, cell viability was increased and the concentration of the various ROS generated was decreased. Membrane leakiness to chromium and FITC-BSA was also decreased in the presence of these enzymes. Free radicals, when not inactivated (dismutated), are known to be pathobiotic to most cells. Cell membranes are at a particular high risk from ROS which induce structural, physiological, and biochemical alterations in the cell membrane. The latter can have a negative effect on cell permeability, maintenance of normal ionic gradients, membrane enzyme activity, cell-to-cell communication, etc. Such defects can ultimately culminate in hypoplasia, aplasia, and cell necrosis. This study has shown that NCC's may be overtly sensitive to ROS, especially since these undifferentiated cells apparently lack inherent SOD and/or catalase activity. From this study it appears as if both IR and OIR perturb the normal functional state of NCC's by "triggering" the generation of ROS. This may certainly explain the teratogenicity of these drugs as related to the viability of neural crest derived ectomesenchymal cells and normal craniofacial morphogenesis.     

Phosphorylation of smooth muscle myosin by type II Ca2+/calmodulin-dependent protein kinase

Brain type II Ca²⁺/calmodulin-dependent protein kinase was found to phoshorylate smooth muscle myosin, incorporating maximally 2 mol of phosphoryl per mol of myosin, exclusively on the 20,000 dalton light chain subunit. After maximal phosphorylation of myosin or the isolated 20,000 dalton light chain subunit by myosin light chain kinase, the addition of type II Ca²⁺/calmodulin-dependent protein kinase led to no further incorporation indicating the two kinases phosphorylated a common site. This conclusion was supported by two dimensional mapping of tryptic digests of myosin phosphorylated by the two kinases. By phosphoamino acid analysis the phosphorylated residue was identified as a serine. The phosphorylation by type II Ca ²⁺/calmodulin-dependent protein kinase of myosin resulted in enhancement of its actin-activated Mg²⁺-ATPase activity. Taken together, these data strongly support the conclusion that type II Ca²⁺/calmodulin-dependent protein kinase phosphorylates the same amino acid residue on the 20,000 dalton light chain subunit of smooth muscle myosin as is phosphorylated by myosin light chain kinase and suggest an alternative mechanism for the regulation of actin-myosin interaction.Abbreviations SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EGTA Ethylene Glycol Bis (-amino-ethyl ether)-N,N,N,N-Tetraacetic Acid - DTT Dithiothreitol - LC₂₀ Gizzard Smooth Muscle Phosphorylatable 20 kDa Myosin Light Chain - LC₁₇ Gizzard Smooth Muscle, 17 kDa Myosin Light Chain - H Chain Gizzard Smooth Muscle 200 kDa Myosin Heavy Chain - TPCK L-1-Tosylamido-2-Phenylethyl Chloromethyl Ketone - MOPS 3-(N-morpholino) Propanesulfonic Acid     

Circadian Variation in Human Performance Evaluated By the Walter Reed Performance

-As part of a two clock-time (0830 versus 2030) evaluation of administration-time dependent effects of dexedrine (S mg) and triazolam (0.25 mg) on human cognitive performance, placebo (control) studies were conducted on 12 diurnally active (0700-2300) male adults (23-38 yrs) using a double-blind, randomized crossover design. Testing was conducted hourly during a series of sleepless 13-hr spans commencing in the morning or evening, using the Walter Reed computer controlled and scored multi-task cognitive performance assessment battery. For the placebo condition, Single and Group Cosinor analyses documented circadian rhythms in performance for most tasks (reaction time, logical reasoning, serial add/subtract and spatial orientation) both for individuals and the group. Overall, performance was worse overnight, when sleepiness was greatest, and best between 1830 and 2030. It was most variable around 0600-0700. The day-night variation in performance over all cognitive tests amounted to 21% of the 24-hr mean.     

A denervated nonfiltering kidney preparation in the rat: a model for study of renin release

To examine the role of the renal vascular receptor in the control of renin secretion in the rat, a denervated, nonfiltering kidney model (DNFK) was developed. The left kidney was subjected to a 2-hr period of total renal ischemia followed by ureteral ligation and section Denervation was accomplished by stripping all visible nerves and painting the renal vessels with 5% phenol. Forty-eight hours later lissamine green dye was injected iv and failed to appear in either the cortical or medullary tubules, indicating that glomerular filtration had ceased. Histological study of these kidneys revealed diffuse tubular necrosis with extensive intratubular cast formation. Norepinephrine content of the DNFK was reduced 91% compared to the contralateral normal kidney (P less than 0.001). In another group of anesthetized rats with a single DNFK, 15 min of suprarenal aortic constriction (SAC) increased plasma renin activity (PRA) from 3.4 +/- 0.6 to 11.5 +/- 1.6 ng AI/ml/hr; in a time control series, PRA was unchanged. To exclude the influence of adrenal catecholamines in this response, bilateral adrenalectomy was performed in a separate group of animals with a DNFK. In this series, SAC also markedly increased PRA. The present data indicate that in the rat the macula densa, the renal nerves, and adrenal catecholamines were not essential for the hyperreninemia induced by a reduction in renal perfusion pressure.     

Ultraviolet light-induced responses of an mfd mutant of escherichia coli B/r having a slow rate of dimer excision

A mutant of Eschirichia coli B/r designated mfd has drastically reduced ability to exhibit “mutation frequency decline” (MFD) the irreversible loss of potential suppressor mutations which occurs when protein synthesis is briefly inhibited after irradiation with U.V. We have found that the initial rate of thymine dimer excision in the mfd mutant is only about one-third that of its mfd⁺ parent strain after a UV dose of 400 erg/mm². The yield of UV-induced Tyr⁺ revertants is 4–10 times higher in the mfd strain than in the mfd⁺ strain. This is comparable to the level of UV-mutability in the mfd⁺ strain in the presence of caffeine, an inhibitor of dimer excision. UV-mutability, prophage induction and Weigle reactivation of irradiated λ phage occur to a greater extent at low UV doses (10–50 erg/mm²) in the mfd strain compared to the mfd⁺ strain. We propose that the slow excision repair in the mfd mutant results in a shift in the induction threshold for these UV-inducible functions toward lower UV doses.     

The relationship between legal abortion and marriage.

A study using the abortion-birth ratios for residents of each U.S. state for the second half of 1970 and for all of 1971 was done to determine if legal abortions reduced marriages. Data showed that trends were consistent with the hypothesis that a relationship exists between l egalized abortion-birth ratios and trends in crude marriage rates among states between 1967 and 1971 with reduction in crude marriage rates in the states with the relatively high abortion-birth ratios. Change in po licy on induced abortions may be responsible for the increased trend in the U.S. crude marriage rate from 1959-1970 and for its levelling off in 1971 and 1972. Analysis of data also suggested that there is a relation ship between less restrictive abortion policies and a decline in crude marriage rates. An estimation of the number of marriages postponed for at least one year following legal abortion indicated that about 1 abortion in 10 delays a marriage when all the decline in marriage rates were caused by increased abortions and when no legal abortions were subs titutes for illegal abortions. Results were obtained employing general measures for variables, but stronger relationships might be produced if more refined measures which consider race, age, parity, and marital status were introduced.     

Kinetics of light emission by photosynthetic systems: Second order light decay kinetics means elovich kinetics in a solid state reaction; 1.5 order light decay kinetics means second order reaction kinetics

Kinetics of biological light emission processes do not mean what they seem to mean, because measured light intensity is not proportional to reactant concentration but to reaction rate. Therefore, the differential equation for light decay is usually different from that of concentration decay, so that mass action interpretations cannot be applied directly to light intensity decay. An observed second order light decay for Chlorella at 6.5°C, implies Elovich solid state reaction kinetics, which agrees with other evidence for solid state processes in photosynthesis. An observed 1.5 order light decay for Cholorella at 28°C implies second order liquid or solid state reaction kinetics. First ordere light decay implies first order reaction kinetics.