Ascorbic acid as a factor controlling "in vivo" its biosynthetic pathway

The capacity of ascorbic acid biosynthesis in potato tuber tissue is closely correlated with the ascorbic acid content of the cells: the lower the endogenous content of ascorbic acid, the greater its biosynthesis. At the highest level of ascorbic acid found in the cells, the biosynthetic capacity is virtually zero. In these conditions, adding glucose (the first precursor of ascorbic acid) has no effect whatsoever, whereas adding galactono-gamma-lactone (the last precursor) induces a high rate of ascorbic acid synthesis. It is suggested that AA biosynthesis is subject to a regulatory mechanism "in vivo" which controls an initial step in the biosynthetic pathway. The last step in this pathway, catalyzed by galactone oxidase, is never blocked and, moreover, its activity is greater than that of the preceding steps.     

A novel and conserved salt-induced protein is an important determinant of salt tolerance in yeast.

We have isolated a novel yeast gene, HAL1, which upon overexpression improves growth under salt stress. In addition, disruption of this gene decreases salt tolerance. Therefore HAL1 constitutes a rate-limiting determinant for halotolerance. It encodes a polar protein of 32 kDa located in the yeast cytoplasm and unrelated to sequences in data banks. The expression of this gene is increased by high concentrations of either NaCl, KCl or sorbitol. On the other hand, the growth advantage obtained by overexpression of HAL1 is specific for NaCl stress. In cells overexpressing HAL1, sodium toxicity seems to be counteracted by an increased accumulation of potassium. The HAL1 protein could interact with the transport systems which determine intracellular K+ homeostasis. The HAL1 gene and encoded protein are conserved in plants, being induced in these organisms by salt stress and abscisic acid. These results suggest that yeast serves as a convenient model system for the molecular biology of plant salt tolerance.     

C-terminal deletion analysis of plant plasma membrane H(+)-ATPase: yeast as a model system for solute transport across the plant plasma membrane.

The plasma membrane proton pump (H(+)-ATPase) energizes solute uptake by secondary transporters. Wild-type Arabidopsis plasma membrane H(+)-ATPase (AHA2) and truncated H(+)-ATPase lacking 38, 51, 61, 66, 77, 92, 96, and 104 C-terminal amino acids were produced in yeast. All AHA2 species were correctly targeted to the yeast plasma membrane and, in addition, accumulated in internal membranes. Removal of 38 C-terminal residues from AHA2 produced a high-affinity state of plant H(+)-ATPase with a low Km value (0.1 mM) for ATP. Removal of an additional 12 amino acids from the C terminus resulted in a significant increase in molecular activity of the enzyme. There was a close correlation between molecular activity of the various plant H(+)-ATPase species and their ability to complement mutants of the endogenous yeast plasma membrane H(+)-ATPase (pma1). This correlation demonstrates that, at least in this heterologous host, activation of H(+)-ATPase is a prerequisite for proper energization of the plasma membrane.     

Expression of carbohydrate residues in plasma membrane glycoproteins during the differentiation of amphibian epidermal cells

Summary Expression of various sugar residues on the plasma membrane of frog (Rana perezi) epidermal cells at different stages of differentiation has been monitored with the use of a battery of HRP-conjugated lectins. In paraffin-embedded tissue, mannose residues (stained by Concanavalin A) were detected at the keratinocyte cell surface in all epidermal strata. However,Lens culinaris agglutinin (LCA), also specific for mannose, specifically stained the plasma membrane of cells from the stratum germinativum. Expression of N-acetyl-glucosamine (GlcNAc), labelled with wheat germ agglutinin (WGA), was maximum at the cell surface of basal cells and progressively decreased through the stratum spinosum. Galactose (Gal) and N-acetyl-galactosamine (GalNAc) residues, labelled withGriffonia simplicifolia I (GS I) andGlycine max (SBA) agglutinins, respectively, were expressed according to the degree of differentiation in amphibian epidermal cells. Sialic acid-containing glycoproteins, labelled withLimax flavus agglutinin (LFA), were found in the outermost plasma membrane of the replacement cell layer and stratum corneum. Glycoproteins responsible for the observed lectin-binding patterns have been identified by staining on nitrocellulose filters after electrophoresis of solubilized plasma membrane fractions and Western blotting. Changes at the level of glycosylation of plasma membrane glycoproteins as epidermal cells differentiate are discussed on the basis of a progressive addition of Gal residues. Integral membrane proteins have been solubilized with the non-denaturing detergent CHAPS and glycoproteins containing terminal Gal residues, that are expressed according to the degree of differentiation in frog epidermis, have been partially purified by affinity chromatography on a GS I-Sepharose 4 B column. The purified fraction was composed by four acidic glycoproteins with isoelectric points between 4.6 and 5.2 and, in SDS-gels gave five major protein bands with approximate molecular weights of 148, 140, 102, 60, and 52 kDa in SDS-gels. The 102 and 52 kDa bands correspond to the a and subunits of amphibian epidermal Na⁺,K⁺-ATPase as demonstrated by specific staining with a polyclonal antibody against the catalytic subunit of pig kidney proton pump and staining with lectins GS I, GS II, and WGA. Possible relationships between higher molecular weight proteins and the constituents of intramembranous particles from the outermost plasma membranes of the replacement cell layer and the stratum corneum are also discussed.Abbreviations BSA bovine serum albumin - CHAPS (3-[(cholamidopropyl) dimethyl-ammonio] 1-propanesulfonate) - Con A Canavalia ensiformis agglutinin - DTT dithiothreitol - Gal galactose - GalNAc N-acetyl-D-galactosamine - GlcNAc N-acetyl-D-glucosamine - GS I Griffonia simplicifolia agglutinin I - GS II Griffonia simplicifolia agglutinin II - HRP horseradish peroxidase - LFA Limax flavus agglutinin - LCA Lens culinaris agglutinin - NDPAGIF non-denaturing polyacrylamide gel isoelectric focusing - PAGE polyacrylamide gel electrophoresis - PAP peroxidase-antiperoxidase - PBS phosphate buffered saline - PMSF phenyl methyl sulphonyl fluoride - RCL replacement cell layer - SBA soybean agglutinin (Glycine max) - SB stratum basal - SDS sodium dodecyl sulphate - SG stratum granulosum - SS stratum spinosum - UEA I Ulex europaeus agglutinin I - WGA wheat germ (Triticum vulgaris) agglutinin     

Modified plant plasma membrane H+-ATPase with improved transport coupling efficiency identified by mutant selection in yeast

Transport across the plasma membrane is driven by an electrochemical gradient of H⁺ ions generated by the plasma membrane proton pump (H⁺-ATPase). Random mutants of Arabidopsis H⁺-ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H⁺-ATPase (PMA1). A Trp-874-Leu substitution as well as a Trp-874 to Lys-935 deletion in the hydrophilic C-terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1. A Trp-874-Phe substitution in AHA1 was produced by site-directed mutagenesis. The modified enzymes hydrolyzed ATP at 200–500% of wild-type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH. AHA1 did not contribute significantly to H⁺ extrusion by transformed yeast cells. The different species of aha1, however, displayed marked differences in initial rates of net H⁺ extrusion and in their ability to sustain an electrochemical H⁺ gradient. These results provide evidence that Trp-874 plays an important role in auto-inhibition of the plant H⁺-ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H⁺ pumping. Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plants transporters.     

Vitamin C stabilization as a consequence of the plasma membrane redox system

Summary Ascorbate is stabilized in the presence of HL-60 cells. Our results showed that cAMP derivatives and agents that increase cAMP stimulate the ability of HL-60 cells to stabilize ascorbate. On the other hand, tunicamycin, a glycosilation-interfering agent, inhibited this ability. The ascorbate stabilization in the presence of HL-60 cells has been questioned as a simple chemical effect. Further properties and controls about the enzymatic nature of this stabilization are described and discussed. This data, together with hormonal regulation, support the hypothesis that an enzymatic redox system located at the plasma membrane is responsible of the extracellular ascorbate stabilization by HL-60 cells.Abbreviations AFR ascorbate free radicals - FCS fetal calf serum - Sp-cAMPS Sp-cyclic adenosine monophosphothionate - Rp-cAMPS Rp-cyclic adenosine monophosphothionate     

Centrosome linker protein C‐Nap1 maintains stem cells in mouse testes

The centrosome linker component C‐Nap1 (encoded by CEP250) anchors filaments to centrioles that provide centrosome cohesion by connecting the two centrosomes of an interphase cell into a single microtubule organizing unit. The role of the centrosome linker during development of an animal remains enigmatic. Here, we show that male CEP250 ^−/− mice are sterile because sperm production is abolished. Premature centrosome separation means that germ stem cells in CEP250 ^−/− mice fail to establish an E‐cadherin polarity mark and are unable to maintain the older mother centrosome on the basal site of the seminiferous tubules. This failure prompts premature stem cell differentiation in expense of germ stem cell expansion. The concomitant induction of apoptosis triggers the complete depletion of germ stem cells and consequently infertility. Our study reveals a role for centrosome cohesion in asymmetric cell division, stem cell maintenance, and fertility.     

The influence of dietary lipid composition on liver mitochondria from mice following 1 month of calorie restriction

To investigate the role mitochondrial membrane lipids play in the actions of CR (calorie restriction), C57BL/6 mice were assigned to four groups (control and three 40% CR groups) and the CR groups were fed diets containing soya bean oil (also in the control diet), fish oil or lard. The fatty acid composition of the major mitochondrial phospholipid classes, proton leak and H₂O₂ production were measured in liver mitochondria following 1 month of CR. The results indicate that mitochondrial phospholipid fatty acids reflect the PUFA (polyunsaturated fatty acid) profile of the dietary lipid sources. CR significantly decreased the capacity of ROS (reactive oxygen species) production by Complex III but did not markedly alter proton leak and ETC (electron transport chain) enzyme activities. Within the CR regimens, the CR-fish group had decreased ROS production by both Complexes I and III, and increased proton leak when compared with the other CR groups. The CR-lard group showed the lowest proton leak compared with the other CR groups. The ETC enzyme activity measurements in the CR regimens showed that Complex I activity was decreased in both the CR-fish and CR-lard groups. Moreover, the CR-fish group also had lower Complex II activity compared with the other CR groups. These results indicate that dietary lipid composition does influence liver mitochondrial phospholipid composition, ROS production, proton leak and ETC enzyme activities in CR animals.