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Biomedical subjects

M Cornelissen

Publications and source records attributed to M Cornelissen.

At least 37 records · Page 2Linked to original sources
PubMed2000

Virtually full-length subtype F and F/D recombinant HIV-1 from Africa and South America.

For reliable classification of HIV-1 strains appropriate reference sequences are needed. The HIV-1 genetic subtype F has a wide geographic spread, causing significant epidemics in South America, Africa, and some regions of Europe. Previously only two full-length sequences of each of the HIV-1 subtype F subclusters F1 and F2 have been described. To extend the knowledge of subtype F variation on a complete genome level, three new virtually full-length F1 sequences were cloned and sequenced, two from Africa and one from South America. Comparison of the new and previously described sequences showed that monophyletic clustering of the subcluster F1 of subtype F is consistent and highly supported in all genome regions. Two additional full-length strains were shown to be mosaics of subtypes F and D. These epidemiologically unrelated F/D sequences showed similar chimeric structure, suggesting that they may represent a previously undescribed circulating recombinant form (CRF). This was supported by partial sequences from three additional unlinked F/D recombinants. Genetic distances in the phylogenetic trees suggest that the recombination event leading to the putative CRF occurred relatively long ago, close to the divergence of the F1 and F2 subclusters. Furthermore, all five F/D recombinants are linked to the Democratic Republic of Congo, suggesting that the original recombination event took place in central Africa.

Africa↗
PubMed2000

Subtype-specific sequence variation of the HIV type 1 long terminal repeat and primer-binding site.

We studied sequence differences in regulatory elements of the long terminal repeat (LTR) and primer-binding site (PBS) among various human immunodeficiency virus type 1 (HIV-1) subtypes. Phylogenetic sequence analysis of a fragment of 729 base pairs (bp) covering the Gag-coding region for half of p24 and all of p17 revealed the gag subtype of all 60 viruses included in the study: A (n = 20), B (n = 12), C (n = 7), D (n = 10), E (n = 3), F (n = 4), G (n = 3), and H (n = 1). The subtype was also determined by analysis of a 689-bp fragment comprising the LTR and the PBS motif. Comparison of the LTR versus gag sequences showed a mosaic genome for seven isolates. After analysis of all sequences, we could describe subtype-specific differences in sequences encompassing the regulatory elements of the LTR and the PBS motif.

Base Sequence↗
PubMed2000

Genetic evidence of multiple transmissions of HIV type 1 subtype F within Romania from adult blood donors to children.

We studied the phylogeny of HIV-1 subtype F viruses from children and adults in Romania in order to (1) clarify whether the Romanian subtype F epidemic was caused by one or several virus introductions and (2) gain insight into the route of spread of the HIV-1 subtype F virus among children and adults in Romania. env (V3), gag (p17/half p24), and pol (prot/half RT) sequences were obtained from three districts in Romania: Tirgu Mures (n = 9, children), Craiova (n = 15, children), and Bucharest (n = 13, adults). Of 37 HIV V3 sequences from Romania, 35 belonged to the genetic subtype F in the neighbor-joining tree, whereas 2 sequences from adults clustered with subtypes A and C. Within the subtype F cluster, no bootstrap-supported subclusters were observed according to geographic area in Romania. Two of the adult V3 sequences that clustered with the children were obtained from individuals who tested HIV seropositive in 1989 and 1990, showing that the subtype F virus was present among adults when the HIV epidemic began among children in Romania. The HIV-1 subtype F viruses obtained from children showed a mean pairwise V3 nucleotide distance of 7.9% and maximum distances of between 18 and 19%; both are higher than previously described. The mean V3 distances (overall, synonymous, and nonsynonymous) were significantly higher for adults than for children. One V3 sequence from the Democratic Republic of Congo clustered within the Romanian sequences, suggesting that the subtype F virus in Romania may originate from this area. Our data also suggest that HIV-1 subtype F was present among Romanian adults before it appeared in 1989 among institutionalized children. The juvenile population was most likely infected with the HIV-1 subtype F virus on more than one occasion, presumably through HIV-contaminated blood (products) obtained from adults.

Adolescent↗
PubMed2000

Structural and rheological properties of methacrylamide modified gelatin hydrogels.

Dynamic shear oscillation measurements at small strain were used to characterize the viscoelastic properties and related differences in the molecular structure of hydrogels based on gelatin methacrylamide. Gelatin was derivatized with methacrylamide side groups and was subsequently cross-linked by radical polymerization via photoinitiation. The light treatment of methacrylamide gelatin solutions resulted in the production of hydrogel films with high storage modulus (G'). Mechanical spectra and thermal scanning rheology of the obtained hydrogels are described. The temperature scan of the network below and above melting point of gelatin allowed us to identify the respective contributions of chemical and physical cross-linkage to the hydrogel elastic modulus. The results indicate that the rheological properties of the gelatin-based hydrogels can be controlled by the degree of substitution, polymer concentration, initiator concentration, and UV irradiation conditions.

Acrylamides↗
PubMed2000

Influence of aging on the synthesis and morphology of the aggrecans synthesized by differentiated human articular chondrocytes.

OBJECTIVE: Synthesis rates of aggrecans by phenotypically stable human articular chondrocytes and the immobilization of these aggrecans in large aggregates were used as variables reflecting the capability of these cells of restoring the extracellular matrix of articular cartilage in vivo in an aging population. DESIGN: Human articular chondrocytes were isolated from articular cartilage obtained from 33 different donors at autopsy. The chondrocytes were cultured in gelled agarose. Synthesis of aggrecans was investigated using Na(2)(35)SO(4)as a radioactive precursor after a 2-week culture period. Electron microscopic study of aggrecan aggregates was done on the macromolecules accumulated over 3 weeks in culture by the chondrocytes obtained from eight other donors with increasing ages. RESULTS: Sulfate incorporation rates into aggrecans correlated inversely with the age of the donor. The value of sulfate incorporation in aggrecans for chondrocytes obtained from mature cartilage of a 20-year-old individual in this system drops to 50% and 25% for chondrocytes obtained from 45- and 69-year-old individuals respectively. Electron microscopic study of aggrecan aggregates showed that the 'de novo' synthesized hyaluronan molecules were fully loaded with aggrecans. Mature human articular cartilage cells were found to synthesize an aggrecan aggregate which carried an average number of 11.7 to 13.1 aggrecans. Cells obtained from immature donors synthesized aggrecan aggregates of which the hyaluronan chain carried twice the amount of aggrecans. These immature human articular cartilage cells were also found to synthesize significant proportions of large aggrecan aggregates with 20 to over 100 aggrecans immobilized on a single hyaluronan chain. The proportions of these large aggrecan aggregates decreased with increasing age of the donors of the chondrocytes. CONCLUSION: The declining aggrecan synthesis rates and the decreased capability of assembling large molecular size aggregates with increasing age in humans illustrates a progressive failure of the repair function of articular cartilage cells in humans.

Adolescent↗
PubMed2000

Entamoeba histolytica disturbs the tight junction complex in human enteric T84 cell layers.

Entamoeba (E.) histolytica trophozoites initiate amebiasis through invasion into the enteric mucosa. It was our aim to understand the molecular interactions between amebic trophozoites and enterocytes during the early steps of invasion. Trophozoites of E. histolytica strain HM1:IMSS were seeded on the apical side of enteric T84 cell layers, which were established on filters in two-compartment culture chambers. Cocultures were analyzed for paracellular permeability by measurement of transepithelial electrical resistance (TER) and for the tight junction proteins ZO-1, ZO-2, occludin, and cingulin by immunocytochemistry and immunoprecipitation. On direct contact with the apical side of the enteric cells, trophozoites caused an increase in paracellular permeability as evidenced by a decrease of TER associated with an increase in [(3)H]mannitol flux. Immunoprecipitation of cocultures revealed dephosphorylation of ZO-2, loss of ZO-1 from ZO-2, and degradation of ZO-1 but less so of ZO-2 and none of occludin or E-cadherin. In conclusion, trophozoite-associated increase in paracellular permeability of enteric cell layers is ascribed to disturbance of the molecular organization of tight junction proteins.

Animals↗
PubMed2000

Spread of distinct human immunodeficiency virus type 1 AG recombinant lineages in Africa.

To identify new subtype G human immunodeficiency virus type 1 (HIV-1) strains and AG recombinant forms, we collected 28 serum samples from immigrants to the Netherlands from 12 countries throughout Africa. Based on the gag sequences 22 isolates were identified as subtype A or G. Phylogenetic analysis of discontinuous regions of the gag (726 nt), pol (1176 nt) and env (276 nt) genes revealed 13 AG recombinants with the mosaic structure A(gag)/G(pol)/A(env), three with A(gag)/G(pol)/G(env) and one other with A(gag) /G(pol)/G(env), in addition to 'pure' subtypes A(gag)/A(pol)/A(env) (n=1) and G(gag)/G(pol)/G(env) (n=4). To analyse the crossover points in more detail, a new RT-PCR was developed resulting in a large contiguous sequence of 2600 nt from the gag region to half the pol region. All the 13 A(gag)/G(pol)/A(env) recombinants appeared to belong to the circulating recombinant form (CRF) AG (IbNG). The three A(gag)/G(pol) /G(env) recombinants differed from the CRF AG (IbNG) subtype, suggesting the identification of a new CRF subtype. The recovery of AG recombinants from African countries a thousand miles apart indicates the active spread of new recombinants.

Africa↗
PubMed1999

Apoptosis induced by gamma irradiation in peripheral blood mononuclear cells is not mediated by cytochrome-c release and only partially involves caspase-3-like proteases.

Caspase 3 has been shown to be actively involved in the apoptotic process in thymocytes after gamma-irradiation. We examined caspase 3 activation in mature peripheral blood lymphocytes (PBL) after gamma irradiation. Since the activation of caspase 3 is generally prceded by a decrease in mitochondrial membrane potential (delta psi m) and cytochrome c release, these two parameters were also examined. Apoptosis in PBL after a 5-Gy gamma irradiation, is characterized by a decrease in delta psi m, but surprisingly no release of cytochrome-c and only a weak caspase 3 activation was noticed. In contrast, staurosporin treated PBL showed a decrease in delta psi m with cytochrome-c release and a clear caspase 3 activation. We were unable to block the decrease in delta psi m with the caspase-inhibitors zVAD-fmk or zDEVD-fmk after gamma irradiation, but DNA fragmentation as measured by the TUNEL assay was partially inhibited. Therefore, in gamma irradiated mature PBL, caspase-dependent and -independent pathways, but not cytochrome c, seem to be involved in the apoptotic process.

Apoptosis↗
PubMed1999

Posttranscriptional gene silencing of gn1 in tobacco triggers accumulation of truncated gn1-derived RNA species.

Posttranscriptional silencing of basic beta-1,3-glucanase genes in the tobacco line T17 is manifested by reduced transcript levels of the gn1 transgene and homologous, endogenous basic beta-1,3-glucanase genes. An RNA ligation-mediated rapid amplification of cDNA ends (RLM-RACE) technique was used to compare the 3' termini of gn1 RNAs present in expressing (hemizygous and young homozygous) and silenced (mature homozygous) T17 plants. Full-length, polyadenylated gn1 transcripts primarily accumulated in expressing plants, whereas in silenced T17 plants, mainly 3'-truncated, nonpolyadenylated gn1 RNAs were detected. The relative abundance of these 3'-truncated gn1 RNA species gradually increased during the establishment of silencing in homozygous T17 plants. Similar 3'-truncated, nonpolyadenylated gn1 RNA products were observed in an independent case of beta-1,3-glucanase posttranscriptional gene silencing. This suggests that these 3'-truncated gn1 RNAs are a general feature of tobacco plants showing posttranscriptional silencing of the gn1 transgene.

Base Sequence↗
PubMed1999

The effect of caspase-inhibitors on radiation induced apoptosis in human peripheral blood lymphocytes: an electron microscopic approach.

Resting lymphocytes are sensitive to radiation damage and die by apoptosis. We investigated the effect of caspase-inhibitors on radiation induced apoptosis in human peripheral blood lymphocytes. Lymphocytes were irradiated in vitro with 5 Gy 60 Co-gamma-rays and cultured for 24 hours in the presence or absence of the caspase-inhibitors zVAD-fmk and zDEVD-fmk. Cell death was evaluated by electron microscopy. Irradiation in the absence of the inhibitors resulted in about 30% dead cells, almost all showing typical apoptotic morphologies. Addition of either one of the inhibitors could not rescue cells from death. Part of the dead lymphocytes (about 65%) still showed typical nuclear characteristics of apoptotic cells: sharply marginated, condensed chromatin, clumped into one sphere or into a crescent shaped mass. The remaining part of the dead cells had ultrastructural characteristics, aberrant from apoptotic cells: clumping of the chromatin was less pronounced and less sharply marginated. Irregular clumps were formed. Data indicate that part of the lymphocytes go in apoptosis in a caspase-independent way. The other part shows caspase-dependent apoptosis with respect to the nuclear events.

Journal Article↗
PubMed1999

Sequences throughout the basic beta-1,3-glucanase mRNA coding region are targets for homology dependent post-transcriptional gene silencing.

In the transgenic tobacco line T17, plants homozygous for the gn1 transgene display developmentally regulated post-transcriptional silencing of basic beta-1,3-glucanase genes. Previously, it has been shown that silencing involves a markedly increased turnover of silencing-target glucanase mRNAs. Using a two-component viral reporter system facilitated a comparison, in a quantitat- ive manner, of the relative silencing efficiencies of various sequences derived from the gn1 transgene. The results show that target sites for the silencing mechanism are present throughout the coding region of the gn1 mRNA. Similar-sized coding region sequences along the entire gn1 mRNA display a similar susceptibility to the silencing mechanism. The susceptibility to silencing increases as the coding region elements increase in size. Relative to internal sequences, the 5' and 3' terminal regions of the gn1 mRNA are inefficient targets for the silencing machinery. Importantly, sequences of the gn1 transgene that are not part of the mature gn1 mRNA are not recognized by the silencing machinery when expressed in chimeric viral RNAs. These results show that the glucanase silencing mechanism in T17 plants is primarily directed against gn1 mRNA-internal sequences and that terminal sequences of the gn1 mRNA are relatively unaffected by the silencing mechanism.

Journal Article↗
PubMed1999

Influence of polysulfated polysaccharides on aggrecans synthesized by differentiated human articular chondrocytes.

OBJECTIVE: Synthesis rates of aggrecans by phenotypically stable human articular chondrocytes and the immobilization of these aggrecans in large aggregates have been used as variables reflecting the capability of these cells to restore the extracellular matrix of articular cartilage in vitro. The effects of heparin, chondroitin polysulfate, and xylosan polysulfate on these variables were investigated. METHODS: Human articular chondrocytes were maintained in culture in gelled agarose. After 2 weeks of culture, the media were supplemented for one week with 10 microg/ml each of heparin, chondroitin polysulfate, and xylosan polysulfate. Synthesis of aggrecans was investigated using Na2(35SO4) as a radioactive precursor during the last 24 h of exposure to polysaccharides. Size exclusion chromatography was used to assay the proportions of aggrecans immobilized in aggregates. Native aggrecan aggregates accumulated during culture were liberated in associating conditions by agarase digestion of the artificial agarose matrix and studied by electron microscopy. Hyaluronan synthesis was studied in monolayer cultured chondrocyte derived fibroblasts. The cells were exposed to 3H glucosamine for 24 h after the nutrient media were supplemented with 3 polysaccharides for one week. Size exclusion chromatography was used to assess the length of the hyaluronan filaments. RESULTS: Xylosan polysulfate and chondroitin polysulfate but not heparin significantly increased total 35S incorporation rates in aggrecan. Electron microscopy study of aggrecan aggregates showed that xylosan and chondroitin polysulfate but not heparin significantly increased aggrecan aggregate sizes. The same polysulfated polysaccharides increased the synthesis of high molecular weight hyaluronan by chondrocyte derived fibroblast-like cells. CONCLUSION: Polysulfated polysaccharides significantly increased the synthesis rates and the accumulation of aggrecan in aggregates in the extracellular environment in this in vitro system.

Adult↗
PubMed1998

Silencing of beta-1,3-glucanase genes in tobacco correlates with an increased abundance of RNA degradation intermediates.

Post-transcriptional gene silencing of beta-1,3 glucanase genes in the transgenic tobacco line T17 is characterised by an increased turnover and, as a consequence, reduced levels of gn1 transgene and endogenous beta-1,3 glucanase mRNAs. Here, additional gn1 RNAs, both larger and smaller than the full-length messenger, are shown to accumulate in silenced plants of the transgenic tobacco line T17. The longer-than-full-length gn1 RNAs are the result of cryptic processing of the gn1 messenger. The small gn1 RNAs in silenced plants correspond to distal and proximal parts of the mature gn1 messenger. The proximal RNA products are intact at their 5' extremity, but terminate at different positions at the 3'-end. The distal RNA products contain a poly(A) tail and are truncated to various positions at the 5'-end. These observations indicate that degradation of the mature gn1 transcript does not start at the 5'- or 3'-end, but rather are consistent with degradation of the gn1 transcript starting with an endonucleolytic cleavage followed by internal exonuclease digestion. Importantly, the truncated products are more abundant in silenced plants than in expressing plants. This suggests, together with the previously reported silencing-related increased gn1 mRNA turnover and the similar rates of gn1 transcription in silenced and expressing T17 plants, that the predominant decay route for the gn1 transcripts differs between silenced and expressing conditions.

Chromosome Mapping↗
PubMed1998

Cathepsin B-mediated activation of the proinflammatory caspase-11.

Members of the caspase (CASP) family of cysteine proteases can be subdivided in proapoptotic caspases and proinflammatory caspases. Whereas the apical activation pathways for the caspases that are involved in the execution of the apoptotic process are beginning to be understood, the pathways that lead to the activation of proinflammatory caspases are still largely unknown. Analysis of subcellular fractions for their ability to process and activate several caspases in vitro led to the identification of lysosomes as the source for a protease that could proteolytically activate the proinflammatory CASP-11. Although this lysosomal activity was sensitive to caspase inhibitors, affinity purification with the biotinylated broad spectrum caspase inhibitor z-VAD.fmk revealed the CASP-11 activating protease as cathepsin B. Activation of CASP-11 by cathepsin B as well as its sensitivity to several caspase inhibitors was further confirmed with purified proteases. Similar to the role of mitochondrial factors in the activation of proapoptotic caspases, our results suggest a potential role for lysosomes and cathepsin B as activators of specific proinflammatory caspases. In addition, the aspecific inhibition of cathepsin B by so-called specific caspase inhibitors implicates that results obtained with these inhibitors should be interpreted with care.

Animals↗
PubMed1998

Flow cytometric scoring of apoptosis compared to electron microscopy in gamma irradiated lymphocytes.

One of the early events occurring at the cell membrane during apoptosis is the translocation of phosphatidylserine from the inner side of the plasma membrane to the outer layer. These phosphatidylserine groups can be bound by fluorescein isothiocyanate (FITC)-labelled annexin V. The aim of this study was to evaluate the power of the annexin V flow cytometric assay in detecting apoptosis in gamma irradiated peripheral blood lymphocytes and in differentiating between apoptosis and primary necrosis in these cells. Therefore, 5 Gy and 20 Gy gamma irradiated peripheral blood mononuclear cells (PBMCs) were examined after a 24-h culture period. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique was performed as well. A comparison with an electron microscopic (EM) evaluation was made. EM is based on established morphological criteria allowing the classification of cells into four groups: viable, early apoptotic, secondary necrotic and primary necrotic cells. EM performed on annexin V positive sorted cells proved that a 5 Gy gamma irradiation of PBMCs mainly causes apoptosis, whereas a 20 Gy gamma irradiation mainly induces primary necrosis. Neither the annexin V flow cytometric assay nor the TUNEL assay were able to distinguish between primary and secondary necrotic cells. These results illustrate that if quantification of apoptosis is required, one should be careful in interpreting flow cytometric results obtained by annexin V or TUNEL staining in peripheral blood lymphocytes. Although in general primary necrotic cells show an increased forward scatter due to cellullar swelling, both early apoptotic and necrotic (primary or secondary) lymphocytes show a decreased forward scatter signal. Moreover, both primary and secondary necrotic lymphocytes are annexin V and propidium iodide (PI) positive and therefore indistinguishable. We conclude that if a new experiment focusing on apoptosis is set up, an initial EM evaluation is mandatory. If EM shows that the apoptosis inducing agent used in the design of the experiments is not causing primary necrosis, than the annexin V flow cytometric assay can provide rapid and quantitative information about apoptosis.

Adult↗
PubMed1998

Features of the autonomous function of the translational enhancer domain of satellite tobacco necrosis virus.

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a cap and a poly(A) tail. Translation of STNV RNA in vitro is promoted by a 120-nt translational enhancer domain (TED) in the 3'-untranslated region. TED also stimulates translation of heterologous mRNAs. In this study, we show that TED stimulates translation of a cat mRNA by increasing translation efficiency to the level of capped mRNA. This stimulatory activity is not impaired by translation through TED. TED stimulates translation efficiency from different positions within the mRNA, varying from the 5' end to 940 nt downstream of the coding region. Duplication of TED has an additive effect on translation stimulation only when located at both ends of the mRNA. On dicistronic RNAs, TED stimulates translation of both cistrons to the same extent. These data suggest that TED acts primarily by recruiting the translational machinery to the RNA.

3' Untranslated Regions↗
PubMed1998

5'- and 3'-sequences of satellite tobacco necrosis virus RNA promoting translation in tobacco.

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap and a 3' poly(A) tail. The STNV trailer contains an autonomous translational enhancer domain (TED) that promotes translation in vitro by more than one order of magnitude when combined with the 5'-terminal 173 nt of STNV RNA. We now show that the responsible sequence within the 5' region maps to the first 38 nt of the STNV RNA. Mutational analysis indicated that the primary sequence of the STNV 5' 38 nt and TED is important for translation stimulation in vitro, but did not reveal a role for the complementarity between the two. Translation of chimeric STNV-cat RNAs in tobacco protoplasts showed that TED promotes translation in vivo of RNAs lacking a cap and/or a poly(A) tail. Similar to in vitro, TED-dependent translation in tobacco was stimulated further by the STNV 5' 38 nt.

Base Sequence↗