Two categories of lymphocyte unresponsiveness to phytohemagglutinin

Peripheral lymphocytes from healthy subjects, sarcoidosis and influenza patients were studied in vitro by measurement of the tritiated thymidine uptake of unstimulated and phytohemagglutinin. (PHA) stimulated cells. When the mitogen induced metabolic response is defined as the ratio between thymidine uptake by stimulated and unstimulated cells (stimulation index), PHA responsiveness was significantly decreased in both diseases and varied inversely with the level of isotope incorporated by unstimulated cells (p = 0.0002). The uptake of isotope by unstimulated cells from influenza patients was significantly increased (p = 0.0001). Isotope incorporation by mitogen stimulated cells from the same patients did not differ significantly from controls (p = 0.0925). In contrast, the impaired PHA responsiveness of lymphocytes from sarcoidosis patients was associated with levels of isotope incorporation in unstimulated cell cultures similar to those observed in healthy controls (p = 0.6444). These observations suggest that two different mechanisms may be responsible for low lymphocyte PHA stimulation indices associated with disease states. Methods are presented for minimizing variation of replicate observations and identification of both categories of lymphocyte unresponsiveness.     

Ultrastructural Details in Germinating Sporangiospores of Rhizopus stolonifer and Rhizopus arrhizus

Electron microscope examination of sporangiospore sections from Rhizopus stolonifer (Ehrenb. ex Fr.) Lind. and R. arrhizus Fischer revealed details on intracellular organization not previously reported. Aldehyde fixation followed by chromeosmium postfixation permitted clear depiction of ribosomes hitherto unrevealed in these cells. Mitochondria were diversiform. Spore wall structures in the two species were generally similar, but outer contours differed sufficiently to permit easy species identification in examination of sections. The spores of both species abounded in cytosomes, corresponding in size, shape, and heavy-metal "stain" affinities to spherosomes in cells of higher plants. The osmiophilic response of these spherosome-like inclusions was intensified by treatment of sections with thiocarbohydrazide solution and subsequent application of aqueous osmium tetroxide, which strengthens an assumption that they are lipid-rich. The margins of the spherosome-like inclusions in lead citrate-stained sections included dense particles, about 60 A across, whose crystalline-like arrangements suggested that protein as well as lipid was present. Frequent and close associations between the spherosome-like inclusions and various cell membranes suggested that such bodies participate in membrane elaboration during germination.     

Pre-steady-state kinetic studies on cytoplasmic sheep liver aldehyde dehydrogenase

Stopped-flow experiments in which sheep liver cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) was rapidly mixed with NAD(+) and aldehyde showed a burst of NADH formation, followed by a slower steady-state turnover. The kinetic data obtained when the relative concentrations and orders of mixing of NAD(+) and propionaldehyde with the enzyme were varied were fitted to the following mechanism: [Formula: see text] where the release of NADH is slow. By monitoring the quenching of protein fluorescence on the binding of NAD(+), estimates of 2x10(5) litre.mol(-1).s(-1) and 2s(-1) were obtained for k(+1) and k(-1) respectively. Although k(+3) could be determined from the dependence of the burst rate constant on the concentration of propionaldehyde to be 11s(-1), k(+2) and k(-2) could not be determined uniquely, but could be related by the equation: (k(-2)+k(+3))/k(+2) =50x10(-6)mol.litre(-1). No significant isotope effect was observed when [1-(2)H]propionaldehyde was used as substrate. The burst rate constant was pH-dependent, with the greatest rate constants occurring at high pH. Similar data were obtained by using acetaldehyde, where for this substrate (k(-2)+k(+3))/k(+2)=2.3x10 (-3)mol.litre(-1) and k(+3) is 23s(-1). When [1,2,2,2-(2)H]acetaldehyde was used, no isotope effect was observed on k(+3), but there was a significant effect on k(+2) and k(-2). A burst of NADH production has also been observed with furfuraldehyde, trans-4-(NN-dimethylamino)cinnamaldehyde, formaldehyde, benzaldehyde, 4-(imidazol-2-ylazo)benzaldehyde, p-methoxybenzaldehyde and p-methylbenzaldehyde as substrates, but not with p-nitrobenzaldehyde.     

Cardiovascular function in anesthetized miniature swine.

Miniature swine anesthetized with pentobarbital were studied with respect to their cardiovascular function under control conditions and in response to catecholamines, baroreceptor inhibition, bilateral vagotomy and vagal nerve stimulation. Measurements included aortic pressure, heart rate, intraventricular pressure and its maximum rate of rise during contraction, carotid blood flow and resistance, femoral blood flow and resistance, and renal blood flow and resistance. The cardiovascular actions of norepinephrine, epiniphrine and isoproterenol were similar to those in other mammals, and the adrenergic receptor mechanisms also were susceptible to blockade with phentolamine or propranolol. Inhibition of the carotid baroreceptors was accompanied by elevation of aortic pressure, reflex bradycardia and increased femoral and renal resistances. Bileteral vagotomy was followed by hypertension, tachycardia and increased renal resistance. Changes in femoral resistance to these procedures differed between the two strains of miniature swine studied. Stimulation of the peripheral end of either vagus nerve was accompanied by bradycardia without hypotension.     

Kinetic studies on the esterase activity of cytoplasmic sheep liver aldehyde dehydrogenase.

The hydrolysis of 4-nitrophenyl acetate catalysed by cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by steady-state and transient kinetic techniques. NAD+ and NADH stimulated the steady-state rate of ester hydrolysis at concentrations expected on the basis of their Michaelis constants from the dehydrogenase reaction. At higher concentrations of the coenzymes, both NAD+ and NADH inhibited the reaction competitively with respect to 4-nitrophenyl acetate, with inhibition constants of 104 and 197 micron respectively. Propionaldehyde and chloral hydrate are competitive inhibitors of the esterase reaction. A burst in the production of 4-nitrophenoxide ion was observed, with a rate constant of 12 +/- 2s-1 and a burst amplitude that was 30% of that expected on the basis of the known NADH-binding site concentration. The rate-limiting step for the esterase reaction occurs after the formation of 4-nitrophenoxide ion. Arguments are presented for the existence of distinct ester- and aldehyde-binding sites.     

Alcohol dehydrogenase polymorphism in the seaweed fly,Coelopa frigida

Seaweed flies (Coelopa frigida) inhabit piles of decaying seaweed on the seashore. All populations so far studied have been found to be polymorphic at the alcohol dehydrogenase locus (Adh). This article reports an attempt to identify some of the forces of natural selection that may be maintaining this polymorphism. First, the genetic determination of the rather complex isozyme system is described. Several inbred lines homozygous at the Adh locus were derived and the biochemical properties of their allozymes compared. Significant differences in both specific activities and thermal stabilities were found between ADH allozymes. A simple experiment is reported in which individuals with different Adh genotypes were cultured in competition with each other in the presence of elevated levels of ethanol. Although the presence of ethanol resulted in greater mortality, there is no evidence that it was selective with respect to the Adh genotypes. The possible relevance of these results to the maintenance of the Adh polymorphism is discussed.This work was supported by a grant to T. H. D. from the Science Research Council.     

Purification and properties of sheep-liver aldehyde dehydrogenases.

Sheep liver cytoplasmic aldehyde dehydrogenase was purified to homogeneity to give a sample with a specific activity of 380 nmol NADH min(-1) mg(-1). An amino acid analysis of the enzyme gave results similar to those reported for aldehyde dehydrogenases from other sources. The isoelectric point was at pH 5.25 and the enzyme contained no significant amounts of metal ions. On the binding of NADH to the enzyme there is a shift in absorption maximum of NADH to 344 nm, and a 5.6-fold enhancement of nucleotide fluorescence. The protein fluorescence (lambdaexcit = 290 nm, lambdaemisson = 340 nm) is quenched on the binding of NAD+ and NADH. The enhancement of nucleotide fluorescence on the binding of NADH has been utilised to determine the dissociation constant for the enzyme . NADH complex (Kd = 1.2 +/- 0.2 muM). A Hill plot of the data gave a straight line with a slope of 1.0 +/- 0.3 indicating the absence of co-operative effects. Ellman's reagent reacted only slowly with the enzyme but in the presence of sodium dodecylsulphate complete reaction occurred within a few minutes to an extent corresponding to 36 thiol groups/enzyme. Molecular weights were determined for both cytoplasmic and mitochondrial aldehyde dehydrogenases and were 212 000 +/- 8 000 and 205 000 respectively. Each enzyme consisted of four subunits with molecular weight of 53 000 +/- 2 000. Properties of the cytoplasmic and mitochondrial aldehyde dehydrogenases from sheep liver were compared with other mammalian liver aldehyde dehydrogenases.     

Biotin subunits of acetyl CoA carboxylase and pyruvate carboxylase from a thermophilic Bacillus.

Two biotin-containing polypeptides of molecular weights 140,000 and 22,000 have been identified by gel electrophoresis in a sodium dodecyl sulfate-denatured extract of cells of a thermophilic Bacillus. These polypeptides can be separated from each other by either gel filtration through Sepharose 6B or affinity chromatography on a Sepharose-avidin column. The larger polypeptide is renatured under appropriate conditions to yield enzymically active pyruvate carboxylase. Enzyme reconstitution experiments show that the smaller polypeptide is a component of acetyl CoA carboxylase. The biotin subunits of these two carboxylases are thus distinct from, and dissimilar to, each other. The demonstration that a pyruvate carboxylase-deficient mutant of the Bacillus contains the smaller, but not the larger, polypeptide corroborates this conclusion.     

In vitro development of early postimplantation rat embryos

Rat embryos explanted at $\text{7}\text{1}\text{2}$ or $\text{8}\text{1}\text{2}$post coitum were cultured throughout the major stages of organogenesis in a system of rotating bottles containing heat-inactivated, immediately centrifuged (I.C.) serum. About 80% of the $\text{8}\text{1}\text{2}\text{-}\text{day}$ explants and 50% of the $\text{7}\text{1}\text{2}\text{-}\text{day}$ explants developed a blood circulation in the yolk sac; in these embryos, organogenesis and growth rates were similar to those of embryos in vivo. In cultures continued for 4 or 5 days, many of the embryos developed 30–40 somites. There was little difference in the subsequent development of embryos cultured in maternal serum or male serum during the egg-cylinder stage except for a possible decrease in the frequency of normal axial rotation in embryos from the male serum. Development in rotator bottles was much better than in watchglass cultures.