Biomedical subjects

C Visser

Publications and source records attributed to C Visser.

At least 19 recordsLinked to original sources

PubMed2001

Differential effects of the allosteric enhancer (2-amino-4,5-dimethyl-trienyl)[3-trifluoromethyl) phenyl]methanone (PD81,723) on agonist and antagonist binding and function at the human wild-type and a mutant (T277A) adenosine A1 receptor.

The 2-amino-benzoylthiophene derivative PD81,723 [(2-amino-4,5-dimethyl-trienyl)[3-(trifluoromethyl) phenyl]methanone] has been shown to allosterically enhance agonist binding and function at the adenosine A(1) receptor. The aim of the present study was to elucidate the effects of PD81,723 both as an allosteric enhancer and as an antagonist on the adenosine A(1) receptor. We investigated its effect on the human wild-type in relation to a mutant (T277A) adenosine A(1) receptor for which agonists have a greatly diminished affinity. Binding (saturation and displacement experiments) and functional adenosine 3',5'-cyclic monophosphate studies were performed, and differential effects of allosteric enhancer PD81,723 on agonists and antagonists were observed on the wild-type (wt) and mutant adenosine A(1) receptor. Our results showed opposite effects of PD81,723 on the binding of agonists and antagonists. Within the concept of a simplified two-state receptor model, it is possible that the effects of PD81,723 are mainly "allosteric", enhancing the binding of adenosine A(1) agonists and inhibiting the binding of antagonists/inverse agonists. However, the suggestion that PD81,723 acts as an allosteric inhibitor of DPCPX (1,3-dipropyl-8-cyclopentylxanthine) binding cannot be confirmed by kinetic studies, since PD81,723 does not seem to affect the dissociation kinetics of [(3)H]DPCPX. Nevertheless, our results show that the action of PD81,723 on DPCPX binding is due to more than mere competitive antagonistic activity, i.e. binding to the ligand-binding site and competing with the binding of DPCPX, as suggested previously. The effect of PD81,723 on the mutant receptor was much less pronounced. Mutation of Thr277 to Ala not only decreased agonist affinity but also inhibited the effects of PD81,723. Insensitivity of the mutT277A to PD81,723 may be linked to the fact that this mutant appears to be uncoupled from G proteins. It further supported a differential binding mode of PD81,723 compared to competitive antagonists for the adenosine A(1) receptor.

Alanine↗

PubMed2001

Quantitative assessment of mechanical prosthetic valve area by 3-dimensional transesophageal echocardiography.

OBJECTIVE: The goal of this study was to assess the geometric orifice area of mechanical valve prostheses by transesophageal 3-dimensional echocardiographic planimetry. METHODS AND RESULTS: Currently used Doppler methods for prosthetic assessment (orifice area-Doppler) were compared with 3D planimetry for orifice area (orifice area-3D) and with manufacturer's values (orifice area-manufacturer) for the corresponding prosthesis types and sizes and with historical controls provided by Doppler literature (orifice area-literature). Twenty-four mechanical valve prostheses (in 22 patients) were studied: 13 in mitral position and 11 in aortic position. Orifice area-manufacturer, orifice area-Doppler, orifice area-literature, and orifice area-3D were 3.6 +/- 1.1 cm(2), 2.3 +/- 0.9 cm(2), 2.4 +/- 0.9 cm(2), and 2.6 +/- 0.7 cm(2), respectively. Orifice area-manufacturer values were significantly larger. Correlation coefficients between orifice area-3D and orifice area-manufacturer, and between orifice area-3D and orifice area-Doppler and orifice area-literature were 0.83, 0.90, and 0.73, respectively (all P < .0001). CONCLUSION: Three-dimensional transesophageal echocardiography is feasible and has good correlation with orifice area-Doppler (in aortic position) and good correlation with orifice area-manufacturer (in aortic and mitral positions) methods.

Adult↗

PubMed2000

From gene to product in yeast: production of fungal cutinase.

In the mid-1970s, information technology and recombinant DNA technology were considered as the breakthrough technologies of the final quarter of the 20th century. Now, about 25 years later, information technology has penetrated deeply into our society and nearly everyone uses this technology. Compared to the formidable success of information technology, the progress in the commercialization of recombinant DNA technology is moderate, even when taking into account that all that is related to the technological application of biological sciences needs extensive safety testing. However, there are signs that the speed of this commercialization will increase in the first decade of the 21st century. Moreover, new breakthroughs in our understanding of the complete genetic make up of eukaryotes will contribute to this increase in speed. An important aspect of the commercialization of this technology is the development of cells as factories for the production of valuable and/or useful molecules. Lower eukaryotes, such as yeasts and molds, are the most promising candidates to become the factories of the future, but at present these factories still contains a lot of process lines that may be superfluous under the well controlled conditions in fermentors. On the other hand, the speed and yield of these cellular production lines can be increased by eliminating the rate-determining steps of these process lines. In this contribution to the European Union symposium from Cell to Factory, some steps in the improvement of S. cerevisiae as cell factories for (heterologous) hydrophobic molecules are presented.

Journal Article↗

PubMed2000

Introduction of an N-glycosylation site increases secretion of heterologous proteins in yeasts.

Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama V(HH) antibody fragments by S. cerevisiae or Pichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion.

Animals↗

PubMed2000

Apolipoprotein H, a new mediator in the inflammatory changes ensuring in jeopardised human myocardium.

AIM: To investigate the presence of membrane "flip flop" in ischaemic human myocardium, we assessed depositions of apolipoprotein H (apoH; beta 2-glycoprotein 1) in ischaemic myocardium. Serum protein apoH can bind to negatively charged phospholipids and can also inhibit blood coagulation in vitro. We hypothesised that, because of its affinity for phosphatidyl serine, apoH might bind to "flip flopped" cells and would therefore be useful as a marker for membrane flip flop in vivo. METHODS: Myocardial tissue specimens were obtained from patients who had died within 14 days of acute myocardial infarction. RESULTS: Immunohistochemical analysis of these specimens revealed that apoH was selectively deposited in infarcted areas of human myocardium of at least one day's duration. Depositions of apoH were not found in non-ischaemic myocardial tissue samples obtained from patients who died from other (extracardial) causes. In vitro experiments with the human leukaemia T cell line Jurkat, subjected to apoptosis by etoposide, showed that apoH was bound to the membrane of apoptotic cells. However, these experiments also indicated that flip flop itself is not sufficient for apoH binding. In addition, Jurkat cells that bound apoH were positive for activated complement complexes, as was also found in the human heart. CONCLUSIONS: These results suggest that apoH is involved in the inflammatory processes that occur in ischaemic myocardium.

Apolipoproteins↗

PubMed1997

Two human homologs of Rad23 are functionally interchangeable in complex formation and stimulation of XPC repair activity.

XPC-hHR23B protein complex is specifically involved in nucleotide excision repair (NER) of DNA lesions on transcriptionally inactive sequences as well as the nontranscribed strand of active genes. Here we demonstrate that not only highly purified recombinant hHR23B (rhHR23B) but also a second human homolog of the Saccharomyces cerevisiae Rad23 repair protein, hHR23A, stimulates the in vitro repair activity of recombinant human XPC (rhXPC), revealing functional redundancy between these human Rad23 homologs. Coprecipitation experiments with His-tagged rhHR23 as well as sedimentation velocity analysis showed that both rhHR23 proteins in vitro reconstitute a physical complex with rhXPC. Both complexes were more active than free rhXPC, indicating that complex assembly is required for the stimulation. rhHR23B was shown to stimulate an early stage of NER at or prior to incision. Furthermore, both rhHR23 proteins function in a defined NER system reconstituted with purified proteins, indicating direct involvement of hHR23 proteins in the DNA repair reaction via interaction with XPC.

DNA Repair↗

PubMed1997

Clinical evaluation of six hollow-fibre membrane oxygenators.

In a clinical evaluation to study the performance of hollow-fibre membrane oxygenators, we compared the gas exchange characteristics and the production of plasma free haemoglobin for different oxygenators. In this study, the data of the Univox, Cobe-Optima, Capiox-SX, Affinity, Safe II and Sarns Turbo 440 oxygenators were evaluated during cardiac surgery in comparable patient groups. Thirteen patients scheduled for elective surgery were enrolled in each group. In all groups, cardiopulmonary bypass (CPB) was conducted using pulsatile blood flow during fibrillation and the period in which the aorta was crossclamped. Arterial and venous blood gases were determined from which oxygen transfer, carbon dioxide transfer, oxygen gradient, shunt fraction and diffusing capacity were calculated. Blood samples were taken 5, 30, 60 and 90 min after beginning CPB. As a result of our measurements we found that the gas exchange capacities of all six oxygenators are within clinically acceptable limits, although the data considering oxygen and carbon dioxide transfer showed a significantly higher capacity for the Sarns Turbo 440 oxygenator (p < 0.05) in comparison with the other oxygenators. Plasma free haemoglobin was, however, significantly higher in the Univox and Sarns Turbo 440 groups (p < 0.005). This difference was already present after 30 min of bypass and increased with time, and is considered as a negative aspect, in relation to optimal patient care.

Adult↗

PubMed1996

XPC and human homologs of RAD23: intracellular localization and relationship to other nucleotide excision repair complexes.

The xeroderma pigmentosum syndrome complementation group C (XP-C) is due to a defect in the global genome repair subpathway of nucleotide excision repair (NER). The XPC protein is complexed with HHR23B, one of the two human homologs of the yeast NER protein, RAD23 (Masutani at al. (1994) EMBO J. 8, 1831-1843). Using heparin chromatography, gel filtration and native gel electrophoresis we demonstrate that the majority of HHR23B is in a free, non-complexed form, and that a minor fraction is tightly associated with XPC. In contrast, we cannot detect any bound HHR23A. Thus the HHR23 proteins may have an additional function independent of XPC. The fractionation behaviour suggests that the non-bound forms of the HHR23 proteins are not necessary for the core of the NER reaction. Although both HHR23 proteins share a high level of overall homology, they migrate very differently on native gels, pointing to a difference in conformation. Gel filtration suggests the XPC-HHR23B heterodimer resides in a high MW complex. However, immunodepletion studies starting from repair-competent Manley extracts fall to reveal a stable association of a significant fraction of the HHR23 proteins or the XPC-HHR23B complex with the basal transcription/repair factor TFIIH, or with the ERCC1 repair complex. Consistent with a function in repair or DNA/chromatin metabolism, immunofluorescence studies show all XPC, HHR23B and (the free) HHR23A to reside in the nucleus.

Amino Acid Sequence↗

PubMed1996

Leakage across hollow-fibre membranes in oxygenators: a pilot study.

The purpose of this study was to create an in vitro system to quantify the fluid loss over two different types of hollow-fibre membrane oxygenators. In the first stage of the study, the circuit was primed with 0.9% NaCl and the line pressure was 200 mmHg; in the second stage, the line pressure was 400 mmHg. The experiment was repeated using bovine blood. A clear difference (p < 0.005) was found in the first stage, accumulating to 47.46 ml (SD 0.41) in the Capiox-SX group and 72.33 ml (SD 3.77) in the Univox group after 4 h. This difference persisted when using bovine blood. Surprisingly, no differences in volume loss occurred when the line pressure was increased to 400 mmHg. In conclusion, leakage in hollow-fibre microporous polypropylene membrane oxygenators can be quantified and the amount of leakage appears to be dependent on the type of membrane and the manufacturing process.

Animals↗

PubMed1993

An accessory gene, lipB, required for the production of active Pseudomonas glumae lipase.

Pseudomonas glumae PG1 is able to secrete lipase into the extracellular medium. The lipase is produced as a precursor protein, with an N-terminal signal sequence. A second open reading frame (ORF) was found immediately downstream of the lipase structural gene, lipA, a situation found for the lipases of some other Pseudomonas species. Inactivation of this ORF resulted in a lipase-negative phenotype, indicating its importance in the production of active extracellular lipase. The ORF, lipB, potentially encodes a protein of 353-amino-acid residues, having a hydrophobic N-terminal (amino acids 1 to 90) and a hydrophilic C-terminal part. As a first step in determining the role of LipB, its subcellular location was determined. The protein was found to fractionate with the inner membranes. The expression of fusions of lipB fragments with phoA revealed an N(in)-C(out) topology for the LipB protein, which was confirmed by protease accessibility studies on EDTA-permeabilized cells and on inverted inner membrane vesicles. These and other results indicate that most of the LipB polypeptide is located in the periplasm and anchored to the inner membrane by an N-terminal transmembrane helix, located between amino acids 19 and 40.

Amino Acid Sequence↗

PubMed1992

Detection of acute myocardial infarction with digital image processing of two-dimensional echocardiograms.

We have previously described the ability of a computer-based image digitizing system to assess early textural changes in acute canine myocardial infarction. To determine whether this technique could be applied to human beings, we studied 12 patients with a first acute transmural myocardial infarction and five normal subjects. Two-dimensional echocardiograms were performed on days 1 and 8 in normal subjects and on days 1 (day of admission), 2, 3, 5, and 14 in the patient population. All recording parameters on the echocardiography machine were kept identical for serial studies. The mean period between hospitalization and first echocardiogram was 11.1 hours (range 4 to 20 hours). End-diastolic frames from the two-dimensional echocardiographic images were digitized and displayed on a monitor. The mean pixel intensity (MPI) (+/- SD) in the region of asynergy (area of myocardial infarction) and a normal area were determined. In normal volunteers, no significant change in MPI was noted between anteroseptal and lateral areas on two separate two-dimensional echocardiographic studies, which were performed 7 days apart (anteroseptal: MPI, 21.6 +/- 1.1 vs 21.8 +/- 0.4, p = not significant) and (lateral: MPI, 21.5 +/- 1.2 vs 21.4 +/- 1.4, p = not significant). In patients with myocardial infarction, a significant increase in MPI was noted on the first day of myocardial infarction between normal and infarcted myocardium (20.4 +/- 2.0 vs 24.3 +/- 2.3, p less than 0.05) and progressively increased thereafter until day 14 (20.5 +/- 1.7 vs 31.9 +/- 3.7, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Echocardiography↗

PubMed1992

Morphoregulatory role of thidiazuron : substitution of auxin and cytokinin requirement for the induction of somatic embryogenesis in geranium hypocotyl cultures.

Somatic embryogenesis was induced in hypocotyl explants of geranium (Pelargonium x hortorum) cultured on media supplemented with various concentrations of N-phenyl-N'-1,2,3-thiadiazol-5-ylurea (thidiazuron). In less than 2 weeks, somatic embryos were observed in treatments containing levels of thidiazuron (TDZ) ranging from 0.2 to 1.0 micromolar. The use of N(6)-benzylaminopurine in combination with indole-3-acetic acid also evoked embryogenesis, but the efficiency of somatic embryo production was significantly lower than that obtained with TDZ. Hypocotyl culture for only 2 days on TDZ-supplemented medium before transfer to a basal medium was sufficient for inducing somatic embryogenesis. This distinction between the induction and expression of embryogenesis may provide an experimental system for studying the developmental biology of somatic embryogenesis. Substitution of the auxin-cytokinin requirement for the induction of somatic embryogenesis by TDZ suggests the possibility of a novel mode of its action by modulation of endogenous growth regulators.

Journal Article↗

PubMed1992

Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues.

The lipA gene encoding the extracellular lipase produced by Pseudomonas glumae PG1 was cloned and characterized. A sequence analysis revealed an open reading frame of 358 codons encoding the mature lipase (319 amino acids) preceded by a rather long signal sequence of 39 amino acids. As a first step in structure-function analysis, we determined the Ser-Asp-His triad which makes up the catalytic site of this lipase. On the basis of primary sequence homology with other known Pseudomonas lipases, a number of putative active site residues located in conserved areas were found. To determine the residues actually involved in catalysis, we constructed a number of substitution mutants for conserved Ser, Asp, and His residues. These mutant lipases were produced by using P. glumae PG3, from which the wild-type lipase gene was deleted by gene replacement. By following this approach, we showed that Ser-87, Asp-241, and His-285 make up the catalytic triad of the P. glumae lipase. This knowledge, together with information on the catalytic mechanism and on the three-dimensional structure, should facilitate the selection of specific modifications for tailoring this lipase for specific industrial applications.

Amino Acid Sequence↗

PubMed1989

Echocardiography in diagnosis of aortic dissection.

In a multicentre study the accuracy of echocardiography was measured in 164 consecutive patients with suspected aortic dissection. The diagnosis was subsequently proven (82 patients) on the basis of transthoracic and transoesophageal echocardiography and additional diagnostic procedures, including computed tomography (CT), aortic angiography, and surgery and/or necropsy. The sensitivity and specificity of echocardiography were 99% and 98%, respectively, with positive and negative predictive values of 98% and 99%. For CT sensitivity was 83%, specificity 100%, and positive and negative predictive values 100% and 86%, respectively. For aortography sensitivity and specificity were 88% and 94%, and the positive and negative predictive values 96% and 84%, respectively. Echocardiography, including the transoesophageal route, can provide accurate diagnosis of aortic dissection within 15 min. Diagnostic difficulties occur only in a few type II dissections, when complementary diagnostic procedures, including CT or angiography, may be needed. To clarify branch involvement angiography is required.

Acute Disease↗

PubMed1985

Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula polymorpha.

A gene library from the methanol utilizing yeast Hansenula polymorpha, constructed in a lambda Charon4A vector, was used to clone the gene encoding a key methanol assimilating enzyme, dihydroxyacetone synthase (DHAS) by differential plaque hybridization. The nucleotide sequence of the 2106 bp structural gene and the 5' and 3' non-coding regions was determined. The deduced amino acid sequence of the protein is in agreement with the apparent molecular weight and amino acid composition of the purified protein. The codon bias is not so pronounced as in some Saccharomyces cerevisiae genes.

Aldehyde-Ketone Transferases↗

PubMed1985

Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha.

The structural gene and the regulatory DNA sequence of the yeast Hansenula polymorpha methanol oxidase have been isolated. According to the nucleotide sequence data obtained, the structural gene encodes a 664 amino acids long protein, contains no intervening sequences, and the 5'- and 3'-non-coding region contains several sequences implicated in transcription initiation and termination in the yeast Saccharomyces cerevisiae. Although the methanol oxidase is translocated to the peroxisomes, no cleavable signal sequence was found at the N-terminus of the protein.

Alcohol Oxidoreductases↗

PubMed1984

Synthesis and processing of the plant protein thaumatin in yeast.

Various maturation forms of the plant protein thaumatin were expressed in yeast, using a promoter fragment of the glyceraldehyde- 3P -dehydrogenase (GAPDH) gene. Plasmids encoding preprothaumatin were shown to direct the synthesis of a processed form of the plant protein. The important role of signal sequences in the expression of the plant protein in yeast was indicated by the observation that plasmids encoding processed thaumatin forms were only poorly expressed, if at all. Nucleotide sequence analysis of the 843 nucleotide GAPDH promoter fragment revealed a characteristic structure with two regions of dyad symmetry containing translational starts of GAPDH and a putative 38 amino acid peptide. A promoter fragment from which the upstream region was deleted proved to be less efficient in thaumatin expression.

Base Sequence↗

PubMed1982

Cloning of cDNA encoding the sweet-tasting plant protein thaumatin and its expression in Escherichia coli.

The structural gene of the sweet-tasting plant protein (prepro)thaumatin was cloned and expressed in Escherichia coli. Expression was effected under control of lac and trp promoter/operator systems and through the use of bacterial ribosome-binding sites. The naturally occurring thaumatin II represents a processed form. The primary translation product, preprothaumatin, of the cloned mRNA-derived cDNA contains extensions at both the amino terminus and the carboxy terminus. The amino terminal extension of 22 amino acids is hydrophobic and very much resembles an excretion-related signal sequence. The six amino acids-long carboxy terminal extension is very acidic in character, in contrast to the overall highly basic thaumatin molecule. The possible role of such an acidic tail with respect to compartmentalization is discussed.

Base Sequence↗