Glycogen-storage disease in rats, a genetically determined deficiency of liver phosphorylase kinase.

Rats from an inbred strain (NZR/Mh) were found to have high concentrations of glycogen in their livers, even after 24 h of starvation. Despite this, blood glucose concentrations were well maintained on starvation for up to 72 h. The primary defect is a deficiency of liver phosphorylase kinase, causing a lack of active glycogen phosphorylase, although total phosphorylase is normal. The intravenous injection of glucagon caused a rapid activation of cyclic AMP-dependent protein kinase in the liver, but no increase in either phosphorylase kinase or phosphorylase a activity. Although total glycogen synthase activity in the livers of affected rats was higher than normal, glycogen synthase in the active form was very low, presumably as a result of the high liver glycogen content. The condition is transmitted as autosomal recessive and, apart from hepatomegaly, the affected rats appear healthy.     

Human erythrocyte acetylcholinesterase in relation to cell age.

Acetylcholinesterase was studied in human red cells that had been fractionated on Ficoll/Triosil density gradients into classes representing different ages in vivo. Reticulocytes have negligible acetylcholinesterase activity; this is rapidly acquired on maturation to the erythrocyte. The activity per cell reaches a maximum and then, after a constant period, declines again towards the end of cell life. The maximum activity and the rates of activity gain and loss per cell are quantitatively different in adults and children. Kinetic studies showed that Vmax. follows the same age/activity profile but Km is unaffected by cell age. The acetylcholinesterase protein content, determined by quantitative crossed immunoelectrophoresis, also shows a profile of increase and then decrease with cell age but the specific activity calculated from the protein estimate shows a reverse picture in which there is a slight decrease from young to mid-age cells followed by an increase again in older cells. These results are interpreted to indicate a complex developmental picture in which the overall cell age against enzyme activity profile is determined partly by the amount of enzyme protein present and partly from the modifying effect on the enzyme activity, of interactions with an aging cell membrane.     

2,4-D-Induced Changes in the K+ Uptake of Wheat Roots at Different pH Values

The effects of an auxin herbicide, 2,4-D, at a concentration of 0.01 mM, on the K⁺ uptake and efflux of excised roots of wheat (Triticum aestivum L. cv. Rannaya) were investigated at different pH values. The K⁺ movement was monitored with a K⁺ (⁸⁶Rb) tracer. In parallel experiments the ATPase activities of microsomal fractions were determined by the inorganic phosphate liberation method. 2,4-D inhibited the K⁺ uptake especially at low pH, irrespective of whether Ca²⁺ was present or not. No marked changes were observed in the K⁺ efflux properties at pH values above 4. The inhibitory effect on K⁺ uptake exhibited a correlation with the hydrocarbon solubility of the herbicide, but not with the 2,4-D-induced decrease of the ATPase activity. It is suggested that 2,4-D exerts a non-specific effect on the lipid-protein interactions, giving rise to a generalized alteration of the transport barrier properties of the plasma membrane even at as low a concentration as 0.01 mM.     

Functional mapping of MHC class II polymorphic residues. The alpha-chain controls the specificity for binding an Ad-versus an A k-restricted peptide and the beta-chain region 65-67 controls T cell recognition but not peptide binding.

MHC proteins are polymorphic cell surface glycoproteins involved in the binding of peptide Ag and their presentation to T lymphocytes. The polymorphic amino acids of MHC proteins are primarily located in the N-terminal domains and are thought to influence T cell recognition both by influencing the binding of peptide Ag and by direct contact with the T cell receptor. In order to determine the relative importance of individual polymorphic amino acids in Ag presentation, a number of groups have taken the approach of interchanging polymorphic amino acids between different alleles of MHC protein in an attempt to define which of the polymorphisms influence peptide binding and which influence T cell recognition by direct contact with the TCR. The peptide OVA323-339 has been previously shown to bind to the MHC class II protein Ad and to have a much lower affinity for Ak, whereas the peptide hen egg lysozyme 46-61 binds well to Ak and poorly to Ad. In the present report, we have analyzed the ability of purified wild-type MHC class II proteins as well as the ability of three different hybrid molecules between Ad and Ak to bind and present these peptides. We find that the alpha-chain of the MHC class II protein plays a critical role in the binding of HEL46-61 and confers the specificity for binding OVA323-339, regardless of which beta-chain is present. We also find that the beta-chain region 65-67 does not control the specificity of peptide binding to the MHC protein, but is important in T cell responses to preformed MHC-peptide complexes, suggesting a role for this region in contacting the TCR.     

Partitioning behaviour of 1-hexanol into lipid membranes as studied by deuterium NMR spectroscopy

Deuterium nuclear magnetic resonance (NMR) spectroscopy was used to study the partitioning behaviour of 1-hexanol specifically deuterated in the alpha-position into model lipid bilayers. In all systems studied, the observed deuterium NMR lineshapes were time-dependent. Initially, 1-hexanol-d2 gave rise to an isotropic deuterium resonance with a different chemical shift from that of aqueous 1-hexanol-d2. After equilibration over a period of days, a broader spectral component characteristic of a spherically-averaged powder-pattern was observed. The quadrupole anisotropy of the 1-hexanol-d2 giving rise to the broad spectrum depended upon the cholesterol content of the membrane. From quantitation of the anisotropic to isotropic deuterium NMR spectra, the partition coefficients of 1-hexanol-d2 in a number of bilayer systems (asolectin and phosphatidylcholine bilayers (the latter with and without cholesterol] were determined. The partitioning of 1-hexanol-d2 into red blood cell membranes, and a suspension of lipids extracted from red blood cell membranes, was also examined. It is suggested that 1-hexanol, and probably other lipophiles, can partition to either the bilayer surface or the bilayer interior in a time-dependent manner.     

Behavioral evaluation of exhibit modifications designed to accommodate an aged diana monkey

Modifications in a zoo exhibit were made to increase the arboreality of an aged female diana monkey, and to increase her use of the central portion of the exhibit where a new food shelf had been added. Branches, which formed ramps to this shelf, also were added. Following these changes, the aged female's arboreality increased slightly, and her use of the central portion of the exhibit increased significantly. Changes in habitat use following these modifications also were noted in the other two diana monkeys in the group. These results suggest that zoo habitats can be made more usable for individuals whose behavioral capabilities might be limited due to age or physical disability.     

Yeast acetyl-CoA carboxylase: in vitro phosphorylation by mammalian and yeast protein kinases

Acetyl-CoA carboxylase (ACC) is regulated in mammalian tissues, in part, by multisite enzyme phosphorylation. Yeast ACC (Y-ACC) has been highly purified from S. cerevisiae by monomeric avidin-Sepharose chromatography, revealing an enzyme subunit species of molecular mass 265,000 Da. Unlike mammalian enzyme, Y-ACC is citrate-independent, and reacts weakly or not at all with a panel of anti-rat liver ACC antibodies. Like rat ACC, Y-ACC is rapidly phosphorylated and inactivated by two mammalian carboxylase kinases, the cAMP-dependent protein kinase and 5'-AMP-stimulated kinase. It is also phosphorylated by rat liver casein kinase II, but without any change in catalytic activity. Three major yeast protein kinases active on ACC have been fractionated; all co-elute with kinases active on casein, but each appears to be a distinct catalytic species. Like the mammalian casein kinases, however, phosphorylation of ACC by these yeast kinases does not alter yeast ACC activity. Taken together, these data indicate that Y-ACC possesses at least two classes of phosphorylation sites, one or more of which acutely regulates enzyme activity. Alterations in Y-ACC phosphorylation in yeast, as in mammalian tissues, could be an important modulator of the rates of fatty acid synthesis.     

Membrane assembly from purified components. I. Isolated M13 procoat does not require ribosomes or soluble proteins for processing by membranes

The coat protein of coliphage M13 is an integral protein of the host-cell cytoplasmic membrane prior to its assembly into virions. It is initially synthesized as procoat, a soluble precursor with a 23 amino acid leader sequence at its amino terminus. ³⁵S-labeled procoat accumulates during an in vitro translation reaction that contains ³⁵S-methionine and RNA from M13-infected cells. Radiochemically pure procoat has been isolated from in vitro translation reactions by extraction into an organic solvent and gel filtration through Sephadex LH-60. Radiochemically pure procoat can be used as substrate in rapid and quantitative assays for leader peptidase and for leader peptide hydrolase, an enzyme that degrades the leader peptide after its release from procoat. Procoat solubility, digestion by leader peptidase and processing by membranes are affected by the presence of Mg²⁺ ion. Isolated procoat is soluble in water at low ionic strength and mildly alkaline pH as well as in detergent solutions. It is cleaved to coat protein by purified E. coli leader peptidase and by inverted E. coli inner-membrane vesicles. These properties of the purified procoat mirror those of the procoat in crude extracts. This suggests that there are no other soluble components that are necessary for the assembly of procoat into the membrane and its conversion to coat; specifically, it provides powerful evidence that protein synthesis is not involved.