Isolation of a heat-stable maize endosperm ADP-glucose pyrophosphorylase variant

Heat stress reduces maize yield and several lines of evidence suggest that the heat lability of maize endosperm ADP-glucose pyrophosphorylase (AGPase) contributes to this yield loss. AGPase catalyzes a rate-limiting step in starch synthesis. Herein, we present a novel maize endosperm AGPase small subunit variant, termed BT2-TI that harbors a single amino acid change of residue 462 from threonine to isoleucine. The mutant was isolated by random mutagenesis and heterologous expression in a bacterial system. BT2-TI exhibits enhanced heat stability compared to wildtype maize endosperm AGPase.The TI mutation was placed into another heat-stable small subunit variant, MP. MP is composed of sequences from the maize endosperm and the potato tuber small subunit. The MP-TI small subunit variant exhibited greater heat stability than did MP. Characterization of heat stability as well as kinetic and allosteric properties suggests that MP-TI may lead to increased starch yield when expressed in monocot endosperms.     

Membrane protein synthesis in Micrococcus lysodeikticus and selective effect of chloramphenicol.

Micrococcus lysodeikticus cytoplasmic membranes labeled with ]-14C]arginine plus [-14C]-threonine were prepared and subjected to mild washing treatments to fractionate membrane proteins. Polyacrylamide gel electrophoresis of total membranes, in the presence of sodium dodecyl sulfate, results in the separation of 28-30 bands of labeled protein. Three peaks of protein show higher specific radioactivity than the others. Chloramphenicol at 100 mug/ml inhibits the incorporation of labeled precursors into membrane proteins by 45-70 percent, some of them being more affected by the antibiotic. From all available results, we suggest that the partial inhibitory effect shown by this antibiotic could be due to the existence of specific biosynthetic sites for some membrane proteins, which are differently affected by chloramphenicol.     

Lysosomal cystine transport. Effect of intralysosomal pH and membrane potential

The regulation of lysosomal cystine transport was studied using cystine dimethyl ester-loaded lysosomes isolated from human diploid fibroblasts. Net efflux from normal fibroblast lysosomes was compared to that from lysosomes of cystinotic fibroblasts, which contain an inherited mutation decreasing lysosomal cystine transport. This exodus of cystine from normal fibroblast lysosomes was greater than from cystinotic fibroblast lysosomes. When lysosomes were incubated with both 5 mM MgCl2 and 2 mM ATP (Mg/ATP), the amount of lysosomal cystine lost from normal lysosomes doubled, but the amount of cystine lost from cystinotic lysosomes remained small. This effect of Mg/ATP on cystine loss from lysosomes isolated from normal fibroblasts was abolished when either carbonyl cyanide m-chlorophenylhydrazone or N-ethylmaleimide was present, suggesting that the effect of Mg/ATP was mediated by the action of a lysosomal proton-translocating ATPase. Addition of KCl, RbCl, or NaCl to normal lysosomes caused smaller increases in cystine exodus. A variety of experimental conditions altered lysosomal pH, membrane potential, and the cystine lost from normal fibroblast lysosomes. These same experimental conditions produced similar alterations in the lysosomal pH and membrane potential of cystinotic fibroblast lysosomes without a comparable alteration in cystine loss. These results have led us to propose a model in which the transport of cystine out of the normal lysosome is regulated by both the lysosomal membrane potential gradient and the transmembrane pH gradient.     

Short and plump physique of Mexican-American children

Mexican-American children are shorter but relatively heavier than non-Hispanic whites and blacks. The objectives of this paper are to assess the extent to which this "short and plump" physique occurs in data collected in two national surveys (HANES I and II); to determine variations by age, sex, and socioeconomic status; and to investigate the anthropometric characteristics that may account for the overweight. Three groups, defined on the basis of reported ancestry and observed race, are studied: Mexican-Americans (MEXAME), non-Hispanic Whites, (EURAME), and blacks (BLACK). Short stature was clearly associated with the poverty index (PI) in all three groups. MEXAMEs with a PI greater than 1.6 were similar in stature to EURAMEs at the same income level at ages 1-11 years but not at 12-17 years. On the other hand, MEXAMEs were shorter than BLACKs at all ages and income levels. The body mass index (kg/cm2) and poverty were unrelated. With respect to the anthropometric characteristics examined that are related to the body mass index, MEXAMEs and EURAMEs were similar in sitting height as a proportion of total height, arm muscle and fat areas, and triceps skinfold but different in the following ways: MEXAMEs had narrower elbow but broader bitrochanteric breadths and larger chest circumferences and subscapular skinfolds. Greater upper body dimensions and fatfolds seem to best describe the physique of MEXAMEs. However, in multiple regressions, these anthropometric characteristics failed to account fully for the greater relative weight of MEXAMEs as compared to EURAMEs.     

Techniques in molecular biology

Book Review

Techniques in molecular biologyJ.M. Walker and W. Gaastra (Eds.), vol. 2. London: Croom Helm, 1987. iv + 332 pages. £14.95. ISBN 0-7099-3673-7     

Phorbol esters and diacylglycerol inhibit vasopressin-induced increases in cytoplasmic-free Ca and Ca efflux in Swiss 3T3 cells

Vasopressin increased intracellular free calcium concentration [Ca²⁺]_i in quin-2-loaded quiescent Swiss 3T3 cells. This effect of vasopressin was rapidly inhibited by biologically active tumour promoters including phorbol dibutyrate (PBt₂) and by the synthetic diacylglycerol 1-oleoyl-2-acetyl-glycerol (OAG). Prolonged pretreatment of Swiss 3T3 cells with PBt₂ causes a loss of protein kinase C activity (Rodriguez-Pena & Rozengurt, Biochem biophys res commun 120 (1984) 1053) [28]. This pretreatment abolished the inhibition by PBt₂ or OAG of vasopressin-mediated increases in Ca²⁺]_i. Vasopressin also stimulated ⁴⁵Ca²⁺ efflux from cells pre-loaded with the isotope. This effect of the hormone was also inhibited by PBt₂. Prolonged pretreatment with PBt₂ prevented the inhibition of vasopressin-stimulated ⁴⁵Ca²⁺ release by PBt₂. Thus, protein kinase C stimulation inhibits vasopressin-mediated increases in [Ca²⁺]_i and ⁴⁵Ca²⁺ efflux apparently by blocking the increased release of Ca²⁺ from an intracellular store caused by the hormone. These findings suggest that activation of protein kinase C may act as a feedback inhibitor to modulate ligand-mediated increases in [Ca²⁺]_i.     

Sugar utilization by developing wild type and shrunken-2 maize kernels

To characterize the movement of sugars during kernel development in maize, a newly devised in vitro kernel development scheme was utilized. Viable seeds of wild type maize (Zea mays L.) as well as the mutant shrunken-2 (sh2) were found to mature when grown in culture with reducing sugars or sucrose as the carbon source. However, wild type and sh2 kernels had greater germination, starch content, and seed weight when sucrose, rather than reducing sugars, was the carbon source. By the use of labeled sucrose it was shown that sucrose can move into endosperm tissue without intervening degradation and resynthesis. These results show that when grown in vitro the maize seed can utilize reducing sugars for development, but it prefers sucrose.     

The cell coat of the olfactory epithelium proper and vomeronasal neuroepithelium of the rat as revealed by means of the Ruthenium-red reaction

Summary The apical cell coat of the olfactory epithelium proper and the vomeronasal neuroepithelium of the rat was investigated electronmicroscopically by means of the Ruthenium-red reaction. In the olfactory epithelium proper, the cilia of receptor cells and microvilli of supporting cells possess a cell coat measuring approximately 10 nm in thickness. In the vomeronasal neuroepithelium, the apical cell coat is thicker than in the olfactory epithelium proper. On microvilli of vomeronasal receptor cells the cell coat varies in thickness from 15 to 20 nm, and on microvilli of supporting cells it measures approximately 75 nm. The functional implications of these findings are discussed.A portion of this study was presented at the 6^th European Anatomical Congress in Hamburg. This publication is dedicated to Prof. E. KlikaSupported by the Deutsche Forschungsgemeinschaft (Br 358/5-1).