Biomedical subjects

K J de Vries

Publications and source records attributed to K J de Vries.

10 recordsLinked to original sources

PubMed2003

Caspase-independent exposure of aminophospholipids and tyrosine phosphorylation in bicarbonate responsive human sperm cells.

Only capacitated sperm cells are able to fertilize egg cells, and this process is triggered by high levels of bicarbonate. Bicarbonate renders the plasma membrane more fluid, which is caused by protein kinase A (PKA)-mediated alterations in the phospholipid (PL) bilayer. We studied exposure of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in human sperm cells. Surface exposure of PS and PE on sperm cell activation in vitro was found to be bicarbonate dependent and restricted to the apical area of the head plasma membrane. The PL scrambling in bicarbonate-triggered human sperm was not related to apoptosis, because the incubated cells did not show any signs of caspases or degeneration of mitochondria or DNA. The PL scramblase (PLSCR) gene family has been implicated in this nonspecific, bidirectional PL movement. A 25-kDa isoform of PLSCR was identified that was homogeneously distributed in human sperm cells. We propose that compartment-dependent activation of PKA is required for the surface exposure of aminophospholipids at the apical plasma membrane of sperm cells. Bicarbonate-induced PL scrambling appears to be an important event in the capacitation process, because the entire intact scrambling sperm subpopulation showed extensive tyrosine phosphorylation, which was absent in the nonscrambling subpopulation. The proportion of live cells with PL scrambling corresponded with that showing capacitation-specific chlortetracyclin staining.

Acrosome Reaction↗

PubMed2000

Dynamics of munc18-1 phosphorylation/dephosphorylation in rat brain nerve terminals.

Munc18-1 is a mammalian member of the SEC1 protein family implicated in neuronal secretion. Its sequence contains several consensus sites for phosphorylation by protein kinase C (PKC), a kinase known to enhance secretion. We have characterized the phosphorylation of the synaptic munc18-1 pool by endogenous, presynaptic PKC-isoforms. In isolated rat brain nerve terminals, munc18-1 was almost completely nonphosphorylated. Its phosphorylation state increased by 250% on inhibition of endogenous phosphatases and by 1500% on additional, direct PKC activation using phorbol esters. K+-evoked depolarization also increased munc18-1 phosphorylation, by 50% within 5 s in a Ca2+-dependent manner. Munc18-1 phosphorylation in nerve terminals was blocked by PKC inhibitors. Activation of endogenous PKC in nerve terminals inhibited the interaction of synaptic munc18-1 with its binding partner syntaxin-1A by 50%. Munc18-1 antisera precipitated 80% of native, brain-derived munc18-1 from salt solutions, but only 12% from synaptosomal lysates, together with 6% synaptic syntaxin-1A/B; these amounts were not changed by PKC activation. In this 12%, the phosphate incorporation per mole of munc18 was four-fold lower than the total pool. We conclude that the synaptic munc18-1 pool can be readily and rapidly phosphorylated by endogenous presynaptic PKC isoforms. A high constitutive phosphatase activity keeps its basal phosphorylation state low so that PKC activation can increase the phosphorylation state dramatically. These phosphorylation dynamics and the effects on the interaction with syntaxin-1A make munc18-1 a prominent candidate to account for PKC-dependent enhancement of secretion.

Animals↗

PubMed1997

DOC2 proteins in rat brain: complementary distribution and proposed function as vesicular adapter proteins in early stages of secretion.

DOC2 proteins constitute a novel protein family that may function in secretion and contain a double C2 domain. We have cloned and characterized two DOC2 isoforms in rat brain and studied their interactions with other proteins implicated in secretion. DOC2A was virtually brain specific, DOC2B ubiquitous. Within brain, the isoforms were expressed nonuniformly and complementary within neurons, not astroglia, and copurified with synaptic vesicles. Affinity purification, yeast two-hybrid analysis, and coimmunoprecipitation revealed that DOC2 binds munc18, a protein also implicated in secretion. The first DOC2 C2 domain and most of munc18 are involved in direct interactions. Munc18 may regulate formation of 'core complexes' during vesicle docking, by interacting with syntaxin. We show that DOC2 and syntaxin compete for munc18. Other core complex components shifted the equilibrium between syntaxin-munc18 versus DOC2-munc18. These data suggest that DOC2 proteins are vesicular adapter proteins regulating munc18-syntaxin complexes and herewith synaptic vesicle docking.

Amino Acid Sequence↗

PubMed1995

An isoform of the phosphatidylinositol-transfer protein transfers sphingomyelin and is associated with the Golgi system.

An isoform of the phosphatidylinositol-transfer protein (PI-TP) was identified in the cytosol fraction of bovine brain. This protein, designated PI-TP beta, has an apparent molecular mass of 36 kDa and an isoelectric point of 5.4. The N-terminal amino acid sequence (21 residues) is 90% similar to that of bovine brain PI-TP, henceforth designated PI-TP alpha (molecular mass 35 kDa and pI 5.5). As observed for PI-TP alpha, PI-TP beta has a distinct preference for phosphatidylinositol over phosphatidylcholine. In addition, it expresses a high transfer activity towards sphingomyelin. PI-TP alpha lacks this activity completely. By indirect immunofluorescence we demonstrated that, in Swiss mouse 3T3 fibroblasts, PI-TP beta is preferentially associated with the Golgi system whereas PI-TP alpha is predominantly present in the cytoplasm and the nucleus. In cytosol-depleted HL60 cells, both PI-TP alpha and PI-TP beta were equally effective at reconstituting guanosine 5'-[gamma-thio]triphosphate-mediated phospholipase C beta activity.

3T3 Cells↗

PubMed1995

A sphingomyelin-transferring protein from chicken liver. Use of pyrene-labeled phospholipid.

A phospholipid transfer protein was purified from chicken liver which, in addition to phosphatidylinositol (PI) and phosphatidylcholine (PC), carries sphingomyelin (SM) between membranes. For comparison, the PI-transfer protein from chicken liver only carries PI and PC. Specificity was established by use of phospholipids that carry a pyrene-labeled acyl chain. Based on the N-terminal sequence and Western blot analysis we conclude that this protein is an isoform of the PI-transfer protein. At increasing length of the pyrene-labeled acyl chain, the isoform expresses a high activity toward SM, a low activity toward PI, and virtually no activity toward PC.

Amino Acid Sequence↗

PubMed1994

A novel acidic form of the phosphatidylinositol transfer protein is preferentially retained in permeabilized Swiss mouse 3T3 fibroblasts.

By use of indirect immunofluorescence it was shown that phophatidylinositol transfer protein (PI-TP) remains associated with the Golgi system of Swiss mouse 3T3 fibroblasts after permeabilization with streptolysin O. By Western blot analysis it was demonstrated that intact cells contain the phosphatidylinositol-bound form of PI-TP (pI 5.5) and a novel more acidic form of PI-TP (pI 5.4) in approximately equal amounts. After permeabilization, about 50% of the PI-TP was retained in the cells with an enrichment of the pH 5.4 form relative to the pH 5.5 form; the opposite was observed for the PI-TP released into the medium. Subfractionation of cell homogenates by centrifugation provided evidence that a distinct amount of PI-TP is strongly bound to the membrane fraction with the pH 5.4 form more prominently present than the pH 5.5 form.

3T3 Cells↗

PubMed1992

Environmental regulation of alcohol metabolism in thermotolerant methylotrophic Bacillus strains.

The thermotolerant methylotroph Bacillus sp. C1 possesses a novel NAD-dependent methanol dehydrogenase (MDH), with distinct structural and mechanistic properties. During growth on methanol and ethanol, MDH was responsible for the oxidation of both these substrates. MDH activity in cells grown on methanol or glucose was inversely related to the growth rate. Highest activity levels were observed in cells grown on the C1-substrates methanol and formaldehyde. The affinity of MDH for alcohol substrates and NAD, as well as Vmax, are strongly increased in the presence of a Mr 50,000 activator protein plus Mg(2+)-ions [Arfman et al. (1991) J Biol Chem 266: 3955-3960]. Under all growth conditions tested the cells contained an approximately 18-fold molar excess of (decameric) MDH over (dimeric) activator protein. Expression of hexulose-6-phosphate synthase (HPS), the key enzyme of the RuMP cycle, was probably induced by the substrate formaldehyde. Cells with high MDH and low HPS activity levels immediately accumulated (toxic) formaldehyde when exposed to a transient increase in methanol concentration. Similarly, cells with high MDH and low CoA-linked NAD-dependent acetaldehyde dehydrogenase activity levels produced acetaldehyde when subjected to a rise in ethanol concentration. Problems frequently observed in establishing cultures of methylotrophic bacilli on methanol- or ethanol-containing media are (in part) assigned to these phenomena.

Alcohol Dehydrogenase↗

PubMed1991

Synthesis of phosphatidylinositol 4,5-bisphosphate in the endoplasmic reticulum of Chinese hamster ovary cells.

We have investigated the intracellular localization and synthesis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in Chinese hamster ovary (CHO) cells by analyzing membrane fractions that were obtained by sucrose density gradient centrifugation. After labeling the cells for 24 h with [3H]inositol, the bulk of [3H] PtdInsP2 was found in the plasma membrane fraction, yet this lipid was also distinctly present in the microsomal fraction (20% of total cellular [3H]PtdInsP2). To determine the origin of this microsomal PtdInsP2, gradient fractions from unlabeled CHO cells were incubated with [3H]inositol in the presence of an ATP-generating system. Under these conditions of labeling, [3H]PtdIns was exclusively present in the microsomal fractions and found to be partially converted to [3H] phosphatidylinositol 4-phosphate ([3H]PtdInsP) and phosphatidylinositol 4-phosphate ([3H]PtdInsP) and [3H]PtdInsP2. The ability of microsomes to synthesize PtdInsP and PtdInsP2 was confirmed by assaying the gradient fractions for PtdIns and PtdInsP kinase activity using endogenous substrate and [gamma-32P]ATP. In the presence of exogenous substrate and Triton X-100, PtdInsP kinase activity was particularly high in the plasma membrane fractions. When phosphoinositide synthesis was studied in permeabilized CHO cells under conditions of sustained membrane vesicle flow (Helms, J. B., Karrenbauer, A., Wirtz, K. W. A., Rothman, J. E., and Wieland, F. T. (1990) J. Biol. Chem. 265, 20027-20032), no lag-time could be detected between the synthesis of [3H]PtdIns and the formation of [3H]PtdInsP2. Moreover, when lipid transport pathways were blocked in these permeabilized cells either by omission of membrane-free cytosol, addition of GTP gamma S and brefeldin A, or temperature block, PtdInsP2 formation still occurred at normal levels. These results strongly suggest that PtdInsP2 can be formed at the site of PtdIns synthesis, i.e. the endoplasmic reticulum (ER). The relationship between PtdInsP2, generated in the ER, and PtdInsP2 present in the plasma membrane, remains to be established.

1-Phosphatidylinositol 4-Kinase↗

PubMed1991

Evaluation of a safe sex campaign regarding AIDS and other sexually transmitted diseases among young people in The Netherlands.

During the summer of 1989 a mass media campaign against the spread of AIDS and other sexually transmitted diseases (STDs) was launched in The Netherlands. The campaign was directed at young people and targeted inappropriate beliefs about the transmission of HIV and STDs. These inappropriate beliefs were considered to function as rationalizations and excuses for individuals and their sexual partners not to take preventive measures. To evaluate the campaign, young people who had noticed the campaign were compared with the ones interviewed at the pre-test. The ones that had not noticed the campaign served as a quasi-experimental control group. The campaign not only reached a sizeable majority of young people, but also succeeded in bringing about some of the desired changes. The risk of HIV infection became personally more relevant to the exposed group and several misconceptions or excuses were less often endorsed. This suggests that a mass media campaign does not have to be restricted to mere attention-raising and increasing factual knowledge, but may be of relevance for other psychosocial processes as well.

Acquired Immunodeficiency Syndrome↗

PubMed1990

AIDS in The Netherlands: the effects of several years of campaigning.

One of the primary aims of acquired immunodeficiency syndrome (AIDS) prevention campaigns in the Netherlands has been to inform the public of the facts about AIDS, thereby stimulating informed, but voluntary action to prevent transmission of the human immunodeficiency virus (HIV). By conducting successive population surveys from April 1987 until October 1989, the effects of this approach were assessed. Twice a year, approximately 1000 respondents, a random sample from the general population, were interviewed about AIDS and safe sex. Additionally, condom sales figures and STD incidences were evaluated. It appears that knowledge about the prevention of HIV transmission with condoms has reached 98% of the sample. During the study, there was an increase in the number of people who expressed an intention to use condoms or who already used them. Behaviour, however, appeared to fall short of intention. The observations were confirmed by condom sales figures and STD incidences. We conclude that the AIDS policy in the Netherlands has had beneficial effects, reflected by several indices.

Acquired Immunodeficiency Syndrome↗