Effect of lipid composition on the calcium/adenosine 5'-triphosphate coupling ratio of the Ca2+-ATPase of sarcoplasmic reticulum

The Ca2+-ATPase of sarcoplasmic reticulum was purified and depleted of proteolipids by solubilization in Triton X-100 and by fractionation on a DE-52 column. The protein reconstituted by deoxycholate-cholate dialysis at low lipid to protein ratios (2-5 mg of lipid/mg of protein), with either dioleoylphosphatidylethanolamine or monogalactosyldiglyceride, exhibited high initial rates of ATP-dependent Ca2+ uptake [300-900 nmol min-1 (mg of protein)-1] and coupling ratios (Ca2+ transported/ATP hydrolyzed) up to 1.2. Ca2+-ATPase reconstituted with lipids of increasing degrees of methylation (dioleoylphosphatidylethanolamine, dioleoylmonomethylphosphatidylethanolamine, dioleoyldimethylphosphatidylethanolamine and dioleoylphosphatidylcholine) or increasing degrees of glycosylation (monogalactosyldiglyceride and digalactosyldiglyceride) revealed a progressive decrease in both ATP-dependent Ca2+-uptake and coupling ratios. The rate and extent of Ca2+ uptake decreased as the dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine or monogalactosyldiglyceride/dioleoylphosphatidylcholine molar ratios in the reconstituted vesicles were reduced. Vesicles reconstituted with high molar ratios of dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine or monogalactosyldiglyceride/dioleoylphosphatidylcholine and at a high lipid to protein ratio became leaky and released the Ca2+ accumulated inside the vesicles when the temperature of the incubation mixture was increased (e.g., from 20 to 37 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of pectinases byAspergillus sp using differently pretreated lemon peel as the carbon source

Summary Aspergillus sp strains from decaying lemons were tested for extracellular pectinase production, testing differently pretreated lemon peel as the carbon source instead of pectin. It was found that the production of extracellular polygalacturonase was about the same and that of pectinesterase substantially higher when unwashed fresh lemon peel was used instead of pectin. The culture filtrate obtained showed a clarifying capacity similar to that of a commercial pectinase preparation, but the vitamin C of the juice was less affected by the treatment.     

A new synaptic anomaly: irregular synaptonemal complexes

Summary In this paper we describe a new synaptic anomaly characterized by the presence of irregular synaptonemal complexes (SCs) in two sterile patients.     

Effects of oral meso-2,3-dimercaptosuccinic acid (DMSA) administration on late gestation and postnatal development in the mouse

The present study was conducted to evaluate the effects of meso-2,3-dimercaptosuccinic acid (DMSA) on late gestation and postnatal viability and growth in the mouse. DMSA was given po to four groups of pregnant Swiss mice at 0, 200, 400, and 800 mg/kg/day from day 14 of pregnancy until postnatal day 21. At birth, the following data were recorded: length of gestation, number of live, dead, and abnormal pups, sex, and individual pup weights. Each pup was weighed again on day 4, 14, and 21 of lactation. Pinna detachment, incisor eruption and eye opening were also monitored. No treatment-related signs of toxicity were noted in any of the dams during the study. No adverse effects on offspring survival or development were evident in the 200 or 400 mg DMSA/kg/day groups. However, on days 14 and 21 of lactation a significant decrease in pup body weight was observed in the 800 mg/kg/day group. Also, a significant increase in the relative weight of the brain was seen in this group. The "no observable effect level" (NOEL) for health hazards to the developing pup was greater than 400 mg/kg/day. This dose is higher than the amounts of DMSA usually given in the treatment of human heavy metal intoxications.     

Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues

Gestational exposure to nicotine has been shown to affect development of noradrenergic activity in both the central and peripheral nervous systems. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants. After birth, offspring of the nicotine-infused dams exhibited marked increases in basal adenylate cyclase activity in membranes prepared from kidney and heart, as well as supersensitivity to stimulation by either a beta-adrenergic agonist, isoproterenol, or by forskolin. The altered responses were not accompanied by up-regulation of beta-adrenergic receptors: in fact, [125I]pindolol binding was significantly decreased in the nicotine group. These results indicate that fetal nicotine exposure affects enzymes involved in membrane receptor signal transduction, leading to altered responsiveness independently of changes at the receptor level.     

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     

Dilute passage promotes expression of genetic and phenotypic variants of human immunodeficiency virus type 1 in cell culture.

We have studied the extent of genetic and phenotypic diversification of human immunodeficiency virus type 1 (HIV-1) upon 15 serial passages of clonal viral populations in MT-4 cell cultures. Several genetic and phenotypic modifications previously noted during evolution of HIV-1 in infected humans were also observed upon passages of the virus in cell culture. Notably, the transition from non-syncytium-inducing to syncytium-inducing phenotype (previously observed during disease progression) and fixation of amino acid substitutions at the main antigenic loop V3 of gp120 were observed in the course of replication of the virus in MT-4 cell cultures in the absence of immune selection. Interestingly, most genetic and phenotypic alterations occurred upon passage of the virus at a low multiplicity of infection (0.001 infectious particles per cell) rather than at a higher multiplicity of infection (0.1 infectious particles per cell). The degree of genetic diversification attained by HIV-1, estimated by the RNase A mismatch cleavage method and by nucleotide sequencing, is of about 0.03% of genomic sites mutated after 15 serial passages. This value is not significantly different from previous estimates for foot-and-mouth disease virus when subjected to a similar process and analysis. We conclude that several genetic and phenotypic modifications of HIV-1 previously observed in vivo occur also in the constant environment provided by a cell culture system. Dilute passage promotes in a highly significant way the expression of deviant HIV-1 genomes.     

Behavioural differences in starving herring Clupea harengus L. larvae correlate with body levels of essential fatty acids

Twenty days after hatching, a single stock of Atlantic herring ( Clupea harengus L.) larvae, cultured in the presence of rotifers and Artemia nauplii but showing little or no active feeding behaviour, displayed clear signs of starvation. Three groups of fish were distinguished: group I was generally pinhead-shaped, tended to swim with a spinning motion and floated vertically; group 2 lay moribund on the bottom of the tank; group 3 showed normal, active swimming behaviour. Fatty acids of total lipid extracted from groups 1 and 2 differed from group 3 in having markedly reduced percentages of 20:5n-3, 22:6n-3, 20:1 and 22:1. We conclude that individuals within a single stock of cultured herring larvae respond differently to starvation and that this generates well defined behavioural differences which correlate with levels of n-3 polyunsaturated fatty acids (PUFA) in body lipid. The implications of these observations are discussed.     

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