Biomedical subjects

H van den Ende

Publications and source records attributed to H van den Ende.

At least 19 recordsLinked to original sources

PubMed2004

Blue light delays commitment to cell division in Chlamydomonas reinhardtii.

In this study, we describe the effect of red and blue light on the timing of commitment to cell division in Chlamydomonas reinhardtii. The time point and cell size after which cells can complete their cell cycle with one division round were determined for cultures that were exposed to various red and blue light periods. We show that the commitment point of cells grown in blue light is shifted to a later time point and a larger cell size, when compared with cells grown in red light. This shift was reduced when cultures were exposed to shorter blue light periods. Furthermore, this shift occurred only when exposure to blue light started before the cells attained a particular size. We conclude that the critical cell size for cell division, which is the cell size at which commitment to cell division is attained, is dependent on spectral composition.

Animals↗

PubMed2001

Identification and localization of a thylakoid-bound carbonic anhydrase from the green algae Tetraedron minimum (Chlorophyta) and Chlamydomonas noctigama (Chlorophyta).

In order to broaden our understanding of the eukaryotic CO2-concentrating mechanism the occurrence and localization of a thylakoid-associated carbonic anhydrase (EC 4.2.1.1) were studied in the green algae Tetraedron minimum and Chlamydomonas noctigama. Both algae induce a CO2-concentrating mechanism when grown under limiting CO2 conditions. Using mass-spectrometric measurements of 18O exchange from doubly labelled CO2, the presence of a thylakoid-associated carbonic anhydrase was confirmed for both species. From purified thylakoid membranes, photosystem I (PSI), photosystem II (PSII) and the light-harvesting complex of the photosynthetic apparatus were isolated by mild detergent gel. The protein fractions were identified by 77 K fluorescence spectroscopy and immunological studies. A polypeptide was found to immunoreact with an antibody raised against thylakoid carbonic anhydrase (CAH3) from Chlamydomonas reinhardtii. It was found that this polypeptide was mainly associated with PSII, although a certain proportion was also connected to light harvesting complex II. This was confirmed by activity measurements of carbonic anhydrase in isolated bands extracted from the mild detergent gel. The thylakoid carbonic anhydrase isolated from T. minimum had an isoelectric point between 5.4 and 4.8. Together the results are consistent with the hypothesis that thylakoid carbonic anhydrase resides within the lumen where it is associated with the PSII complex.

Animals↗

PubMed2001

Low-expression genes induced by nitrogen starvation and subsequent sexual differentiation in Chlamydomonas reinhardtii, isolated by the differential display technique.

Genes that are expressed upon a shift to nitrogen-free medium, an event that initiates gametogenesis, were identified in Chlamydomonas reinhardtii by using the differential display technique. Ten different cDNAs were isolated and shown to have increased levels of their transcripts upon removal of the nitrogen source. The initial kinetics of RNA accumulation allowed an ordering of the genes with respect to the timing of their expression, with individual genes being expressed very early, early, intermediately, or late after induction. For very early genes, significantly increased transcript levels were detected within 30 min. This fast response suggests that gene expression is rapidly activated after removal of the nitrogen source. The accumulation of transcripts from the very early, early, and intermediate genes preceded the appearance of mating competence. Though transcript levels of several very early genes fluctuated during subsequent incubation in nitrogen-free medium, most of them exhibited maxima when the highest level of mating competence was attained. One of these very early genes was shown to encode a urate oxidase type-II enzyme.

Adaptation, Physiological↗

PubMed2000

Chlamydomonas contains calcium stores that are mobilized when phospholipase C is activated.

Mastoparan induces Ca(2+)-dependent deflagellation of the unicellular green alga Chlamydomonas moewusii Gerloff, as well as the activation of phospholipase C and the production of inositol 1,4, 5-trisphosphate (InsP(3); T. Munnik et al., 1998, Planta 207: 133-145). Even in the absence of extracellular Ca(2+), mastoparan still induces deflagellation (L.M. Quarmby and H.C. Hartzell, 1994, J Cell Biol 124: 807-815; J.A.J. van Himbergen et al., 1999, J Exp Bot, in press) suggesting that InsP(3) mediates Ca(2+) release from intracellular stores. To test this hypothesis, cells were pre-loaded with (45)Ca(2+) and their plasma membranes permeabilized by digitonin. Subsequent treatment of the cells with mastoparan (3.5 microM) induced release of intracellular (45)Ca(2+). Mastoparan also activated phospholipase C in permeabilized cells, as demonstrated by the breakdown of (32)P-phosphatidylinositol 4,5-bisphosphate and the production of diacylglycerol. The mastoparan analogues mas7 and mas17 were also effective and their efficacy was correlated with their biological activity. X-ray microanalysis showed that electron-dense bodies (EDBs) are a major Ca(2+) store in C. moewusii. Analysis of digitonin-permeabilized cells showed that EDBs lost calcium at digitonin concentrations that released radioactivity from (45)Ca(2+)-labelled cells, suggesting that (45)Ca(2+) monitored the content of EDBs. X-ray microanaysis of living cells treated with mastoparan also revealed that calcium was released from EDBs.

Animals↗

PubMed2000

Polar glycerolipids of Chlamydomonas moewusii.

The fatty acid and polar lipid compositions of the unicellular green alga Chlamydomonas moewusii were characterized. Since this organism is an important plant model for phospholipid-based signal transduction, interest was focused on the lipids phosphatidic acid, phosphatidylinositolphosphate and phosphatidylinositolbisphosphate. A phosphatidylinositol:phosphatidylinositolphosphate: phosphatidylinositolbisphosphate ratio of 100:1.7:1.3 was found. The polyphosphoinositides accounted for 0.8 mol% of the total phospholipids and their fatty acid compositions were similar to that of phosphatidylinositol except for the enrichment of linolenic acid in phosphatidylinositol phosphate. Phosphatidic acid accounted for 0.67 mol% of the phospholipids. Major structural glycerolipids were monogalactosyldiacylglycerol (35 mol%), digalactosyldiacylglycerol (15 mol%), sulfoquinovosyldiacylglycerol (10 mol%), diacylglyceryltrimethylhomoserine (16 mol%), phosphatidylglycerol (9 mol%), phosphatidylethanolamine (8 mol%) and phosphatidylinositol (6 mol%). Relative changes in the total fatty acid compositions found during growth on nutrient-limited medium reflected mainly alterations in the compositions of the chloroplast lipids phosphatidylglycerol and monogalactosyldiacylglycerol. [32P]Pi-incorporation studies revealed that it took 6 days before the amount of label in the major phospholipids was proportional to their abundance.

Animals↗

PubMed1999

Cell wall dynamics in yeast.

The yeast Saccharomyces cerevisiae is the first fungus for which the structure of the cell wall is known at the molecular level. It is a dynamic and highly regulated structure. This is vividly illustrated when the cell wall is damaged and a salvage pathway becomes active, resulting in compensatory changes in the wall.

Cell Wall↗

PubMed1999

The Saccharomyces cerevisiae CWH8 gene is required for full levels of dolichol-linked oligosaccharides in the endoplasmic reticulum and for efficient N-glycosylation.

The Saccharomyces cerevisiae mutant cwh8 was previously found to have an anomalous cell wall. Here we show that the cwh8 mutant has an N -glycosylation defect. We found that cwh8 cells were resistant to vanadate and sensitive to hygromycin B, and produced glycoforms of invertase and carboxypeptidase Y with a reduced number of N -chains. We have cloned the CWH8 gene. We found that it was nonessential and encoded a putative transmembrane protein of 239 amino acids. Comparison of the in vitro oligosaccharyl transferase activities of membrane preparations from wild type or cwh8 Delta cells revealed no differences in enzyme kinetic properties indicating that the oligosaccharyl transferase complex of mutant cells was not affected. cwh8 Delta cells also produced normal dolichols and dolichol-linked oligosaccharide intermediates including the full-length form Glc3Man9GlcNAc2. The level of dolichol-linked oligosaccharides in cwh8 Delta cells was, however, reduced to about 20% of the wild type. We propose that inefficient N -glycosylation of secretory proteins in cwh8 Delta cells is caused by an insufficient supply of dolichol-linked oligosaccharide substrate.

Amino Acid Sequence↗

PubMed1998

Identification of a putative alpha-glucan synthase essential for cell wall construction and morphogenesis in fission yeast.

The cell wall protects fungi against lysis and determines their cell shape. Alpha-glucan is a major carbohydrate component of the fungal cell wall, but its function is unknown and its synthase has remained elusive. Here, we describe a fission yeast gene, ags1(+), which encodes a putative alpha-glucan synthase. In contrast to the structure of other carbohydrate polymer synthases, the predicted Ags1 protein consists of two probable catalytic domains for alpha-glucan assembly, namely an intracellular domain for alpha-glucan synthesis and an extracellular domain speculated to cross-link or remodel alpha-glucan. In addition, the predicted Ags1 protein contains a multipass transmembrane domain that might contribute to transport of alpha-glucan across the membrane. Loss of Ags1p function in a temperature-sensitive mutant results in cell lysis, whereas mutant cells grown at the semipermissive temperature contain decreased levels of cell wall alpha-glucan and fail to maintain rod shapes, causing rounding of the cells. These findings demonstrate that alpha-glucan is essential for fission yeast morphogenesis.

Amino Acid Sequence↗

PubMed1998

Loss of the plasma membrane-bound protein Gas1p in Saccharomyces cerevisiae results in the release of beta1,3-glucan into the medium and induces a compensation mechanism to ensure cell wall integrity.

Deletion of GAS1/GGP1/CWH52 results in a lower beta-glucan content of the cell wall and swollen, more spherical cells (L. Popolo, M. Vai, E. Gatti, S. Porello, P. Bonfante, R. Balestrini, and L. Alberghina, J. Bacteriol. 175:1879-1885, 1993; A. F. J. Ram, S. S. C. Brekelmans, L. J. W. M. Oehlen, and F. M. Klis, FEBS Lett. 358:165-170, 1995). We show here that gas1delta cells release beta1,3-glucan into the medium. Western analysis of the medium proteins with beta1,3-glucan- and beta1,6-glucan-specific antibodies showed further that at least some of the released beta1,3-glucan was linked to protein as part of a beta1,3-glucan-beta1,6-glucan-protein complex. These data indicate that Gas1p might play a role in the retention of beta1,3-glucan and/or beta-glucosylated proteins. Interestingly, the defective incorporation of beta1,3-glucan in the cell wall was accompanied by an increase in chitin and mannan content in the cell wall, an enhanced expression of cell wall protein 1 (Cwp1p), and an increase in beta1,3-glucan synthase activity, probably caused by the induced expression of Fks2p. It is proposed that the cell wall weakening caused by the loss of Gas1p induces a set of compensatory reactions to ensure cell integrity.

Blotting, Western↗

PubMed1997

In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae.

Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N-terminal signal sequence for entering the secretory pathway. On further analysis, 51 of these proteins contain a potential glycosyl-phosphatidylinositol (GPI)-attachment signal. Seven additional ORFs were found to belong to this group. Upon examination of the possible GPI-attachment sites, it was found that in yeast the most probable amino acids for GPI-attachment as asparagine and glycine. In yeast, GPI-proteins are found at the cell surface, either attached to the plasma-membrane or as an intrinsic part of the cell wall. It was noted that plasma-membrane GPI-proteins possess a dibasic residue motif just before their predicted GPI-attachment site. Based on this, and on homologies between proteins, families of plasma-membrane and cell wall proteins were assigned, revealing 20 potential plasma-membrane and 38 potential cell wall proteins. For members of three plasma-membrane protein families, a function has been described. On the other hand, most of the cell wall proteins seem to be structural components of the wall, responsive to different growth conditions. The GPI-attachment site of yeast slightly differs from mammalian cells. This might be of use in the development of anti-fungal drugs.

Amino Acid Sequence↗

PubMed1997

An experimental test for synergistic epistasis and its application in Chlamydomonas.

Theoretically, one of the most general benefits of sex is given by its function in facilitating selection against deleterious mutations. This advantage of sex may be deterministic if deleterious mutations affect the fitness of an individual in a synergistic way, i.e., if mutations increase each others' negative fitness effect. We present a new test for synergistic epistasis that considers the skewness of the log fitness distribution of offspring from a cross. We applied this test to data of the unicellular alga Chlamydomonas moewussii. For this purpose, two crosses were made: one between two strains that are presumed to have accumulated slightly deleterious mutations, the other between two strains without a history of mutation accumulation. Fitness was measured by estimating the two parameters of logistic growth in batch culture, the maximum growth rate (r) and the carrying capacity (K). The finding of a negatively skewed distribution for K in the accumulation cross suggests synergism between mutations affecting the carrying capacity, while the absence of skewness for tau in both crosses is consistent with independent effects of mutations affecting this parameter. The results suggest a possible alternative explanation for the general observation that sex is related to constant environments, where selection on K predominates, while asexual reproduction is found in more variable environments, where selection on r is more important.

Animals↗

PubMed1996

The Free Fall Machine--a ground-based facility for microgravity research in life sciences.

A device is described in which a biological specimen is periodically accelerated for a short period. Each event is followed by a variable period of free fall. Assuming that the g-dose (expressed g s) has to surpass a certain minimal value to be perceived by cells, and in addition, there is a minimal time threshold for sensing a change in gravity, it is conceivable that conditions are created in which cells do not detect the periodic acceleration, and only experience the periodic free-fall movement as a long-term weightlessness condition. Using the cell-cycle progression of the unicellular green alga Chlamydomonas as an example, it is shown that with this device effects can be generated which are similar to those observed in satellite flights.

Acceleration↗

PubMed1994

Mating-type differentiation and mate selection in the homothallic Chlamydomonas monoica.

By using combinations of phenotypically-distinct--but sexually-compatible--mutant strains of C. monoica (zym-1, zym-27, and nit-2) and assaying for zygote genotypes in genetically-mixed mating populations (where gametes of the two parental homothallic strains were present at similar frequencies), we have found that matings occur preferentially between cells of the same genotype. Additional support for an hypothesis of non-random mate selection was provided by using an easily-selectable genetic marker (sup-1) to verify the frequent occurrence of matings between cells of identical genotype in populations where the selectable genotype was present at very low relative frequency (10(-2) or 10(-3)) in a mixed mating population. Direct evidence for non-random mate selection was obtained by presenting nitrogen-starved cells with limiting nitrate to synchronize gametic differentiation in wild-type strains. Under these conditions, the four, eight, or 16 mitotic daughters released from the same mother sporangium often immediately established mating pairs within the group. Thus successive mitotic divisions of a single mother cell yielded progeny of opposite expressed mating-type.

Animals↗

PubMed1993

Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae.

The sexual adhesion protein of Saccharomyces cerevisiae MAT alpha cells, alpha-agglutinin, could not be extracted from the cell wall with hot sodium dodecyl sulfate (SDS), but became soluble after digestion of the cell wall with laminarinase. This indicates that it is intimately associated with cell wall glucan. A fusion protein was constructed consisting of the signal sequence of yeast invertase, guar alpha-galactosidase, and the C-terminal half of the alpha-agglutinin. Most of the fusion protein was incorporated in the cell wall. A small amount could be extracted with SDS, but most of it could only be extracted with laminarinase. On the other hand, cells containing a construct consisting of the signal sequence of invertase and alpha-galactosidase released most of the alpha-galactosidase into the medium and all cell wall-associated alpha-galactosidase was released by SDS. Labelling with antibodies showed that the alpha-galactosidase part of the fusion protein was exposed on the surface of the cell wall. The results demonstrate that the C-terminal half of the alpha-agglutinin contains the information needed to incorporate a protein into the cell wall.

Biological Transport↗

PubMed1991

Cell wall glucomannoproteins of Saccharomyces cerevisiae mnn9.

Mannoproteins were isolated from Saccharomyces cerevisiae mnn9 mutant cell walls by laminarinase digestion and purified by affinity and anion-exchange chromatography. The purified mannoprotein fraction contained three predominant proteins with molecular masses of 300 kDa, 220 kDa and 160 kDa. These compounds were absent in an SDS extract of cell walls or in a hot-citrate extract of mnn9 cells. The carbohydrate part of the purified mannoproteins consisted of (N-acetyl)glucosamine, mannose and glucose in a molar ratio of 1:53:4. O-Glycosidically linked chains, containing 70% of the mannose, were released by mild beta-elimination. N-Glycosidically linked chains, representing 80% of the (N-acetyl)glucosamine and 20% of the mannose, were released by peptide N-glycosidase F (PNGase F) digestion. Complete degradation of protein by alkaline hydrolysis released besides the N- and O-glycosidically linked chains, another type of carbohydrate chain containing the residual (N-acetyl)glucosamine, mannose and most of the glucose in a molar ratio of 1:17:18. Glucose was beta-glycosidically linked. The results indicate that beta-glucose is linked to PNGase F-resistant N-linked chains present on cell wall mannoproteins. We propose that these chains are responsible for the linkage between mannoproteins and glucan in the cell wall.

Acetylglucosamine↗

PubMed1990

Increase in calcium triggers mating structure activation in Chlamydomonas eugametos.

For mating Chlamydomonas eugametos gametes to fuse with their partners, they must first lyse part of the anterior cell wall and protrude their mating structures. These responses can be artificially induced by compounds that raise the Cai level, viz. InsP3, A23187, TFP and ethanol. We conclude that calcium should be considered with cAMP to be involved in signal transduction during C. eugametos mating.

Calcimycin↗

PubMed1989

Wheat germ agglutinin induces mating reactions in Chlamydomonas eugametos by cross-linking agglutinin-associated glycoproteins in the flagellar membrane.

Species-specific binding between the flagellar surfaces of mating types plus and minus (mt+ and mt-) gametes of Chlamydomonas eugametos is mediated by mating type-specific agglutinins. Their interaction triggers several mating responses that are necessary for cell fusion, such as flagellar twitching, flagellar tip activation, redistribution of agglutinin molecules to the flagellar tip (tipping), and mating structure activation. Earlier, we reported that a monoclonal antibody (mAb 66.3) can induce mating reactions by cross-linking the agglutinins (Homan, W. L., A. Musgrave, H. de Nobel, R. Wagter, A. H. J. Kolk, D. de Wit, and H. van den Ende. 1988. J. Cell Biol. 107:177-189). Here we report that the lectin wheat germ agglutinin (WGA), which does not bind to the agglutinins, can also invoke all these mating reactions. We show, by immunofluorescence studies using anti-WGA and an agglutinin-specific monoclonal antibody (mAb 66.3), that WGA induces the redistribution of agglutinin to the flagellar tips of mt- gametes. Vice versa, when agglutinin tipping is induced by mAb 66.3, the WGA-binding glycoproteins are also tipped. Under the same conditions, the major flagellar glycoproteins are not redistributed, indicating that membrane transport is limited to a few components. We conclude that each agglutinin is associated with a WGA-binding glycoprotein. When cells lacking agglutinin or cells possessing inactive agglutinins are treated with WGA, mating responses are again elicited. The data suggest that clustering of agglutinin-containing complexes results in the production of intracellular signals, such as cAMP, and the coupling of the complex to a force generating system. In nature, the complexes are clustered via the agglutinins, but artificially they can be clustered by lectins or antibodies directed against other proteins in the complex.

Cell Membrane↗

PubMed1988

Monoclonal antibodies directed against the sexual binding site of Chlamydomonas eugametos gametes.

Monoclonal antibodies were raised against the mt- sexual agglutinin of Chlamydomonas eugametos gametes. Those that blocked the agglutination site were selected. They were divided into two classes dependent upon whether they gave a weak (class A) or clear positive (class B) reaction with mt- flagellar membranes in an ELISA and an indirect immunofluorescence test using glutaraldehyde-fixed mt- gametes. Class A antibodies were shown to be specific for the agglutinin in an extract of mt- gametes, based on results from immunoblotting, immunoprecipitation, affinity chromatography, and the absence of a reaction with nonagglutinable cells. Surprisingly, class A mAbs also recognized two mt+ glycoproteins, one of which is the mt+ agglutinin. Class B antibodies were shown to bind to several glycoproteins in both mt- and mt+ gametes, including the mt- agglutinin. Fab fragments from class A mAbs blocked the sexual agglutination process, but those from class B did not, even though the parent antibody did. We conclude that the class A epitope lies in or close to the agglutination site of the mt- agglutinin, whereas the class B epitope lies elsewhere on the molecule. We also conclude that the mt- agglutinin is the only component on the mt- flagellar surface directly involved in agglutination. Class A mAbs were found to elicit several reactions displayed by the mt+ agglutinin. They bound to the mt- agglutinin on gamete flagella and induced most of the reactions typical of sexual agglutination, with the exception of flagellar tip activation. None of these reactions was induced by Fab fragments. High concentrations of class A mAbs completely repressed the sexual competence of live mt- gametes, but low concentrations stimulated cell fusion.

Agglutination↗