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Biomedical subjects

J J Cassiman

Publications and source records attributed to J J Cassiman.

At least 19 recordsLinked to original sources
PubMed2002

Viability of cultured periodontal pocket epithelium cells and Porphyromonas gingivalis association.

OBJECTIVES: Porphyromonas gingivalis, one of the key pathogens in the development of periodontitis, produces a number of virulence factors that might explain its pathogenicity. One of them is the ability to adhere and invade pocket epithelium. The aim of this study was to follow, over time, the association of P. gingivalis and consequent morphological changes of the pocket epithelium cells. MATERIAL AND METHODS: The association capacity of four P. gingivalis serotypes [K1, K2, K4, K- (nonencapsulated)] with in vitro cultured mono-layers from periodontal pocket epithelial cells of patients with periodontitis, was followed by fluorescence microscopy and bacterial culture. The contact time between bacteria and epithelium cells ranged from 45 min to 8 h. The microscopic evaluation allowed differentiation between dead and living cells (bacteria as well as epithelium) and description of the morphological changes after association. RESULTS: A highly significant difference in the number of associating bacteria was found between dead and living epithelium cells, and between non-capsulated and capsulated strains. A significant increase in the proportion of dead pocket epithelium cells was found with prolonged association time. The morphological changes (rounding of the epithelial cell, detachment from the glass cover-slip and loss of intercellular contact) occurred faster for mono-layers inoculated with the non-encapsulated P. gingivalis strain. CONCLUSIONS: This study indicates that dead pocket epithelium cells harbor more P. gingivalis cells, and that a positive correlation exists between contact time and cell death. For the P. ginigvalis species, non-encapsulated strains associate in higher number. As a result, the damage they cause to the host cell seems to occur faster than occurs in encapsulated strains. As such, cell death can be seen as the end-result of bacterial association.

Bacterial Adhesion↗
PubMed2001

Y-chromosomal STR haplotypes in three major population groups in Bulgaria.

A total of 280 unrelated males from the three largest population groups in Bulgaria: Bulgarians, Bulgarian Turks and Gypsies, were analyzed for seven Y-chromosome STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392 and DYS393). Comparison of the allele frequency distributions revealed significant differences between the three ethnic groups which were confirmed with haplotype analysis. This permits us to suggest that population differentiation should be taken into account in forensic case analysis and paternity testing in Bulgaria.

Bulgaria↗
PubMed2001

Tcf-3 expression and beta-catenin mediated transcriptional activation in aggressive fibromatosis (desmoid tumour).

Aggressive fibromatosis harbours mutations resulting in beta-catenin protein stabilization. Primary cell cultures demonstrate constitutive tcf activation in aggressive fibromatosis. Expression and co-immunoprecipitation studies suggest that beta-catenin binds and activates tcf-3 in this tumour. This is the first demonstration of tcf-3 activation by beta-catenin stabilization in a human neoplastic process.

Cytoskeletal Proteins↗
PubMed2001

Functional interaction between TRP4 and CFTR in mouse aorta endothelial cells.

BACKGROUND: This study describes the functional interaction between the putative Ca2+ channel TRP4 and the cystic fibrosis transmembrane conductance regulator, CFTR, in mouse aorta endothelium (MAEC). RESULTS: MAEC cells express CFTR transcripts as shown by RT-PCR analysis. Application of a phosphorylating cocktail activated a Cl- current with characteristics similar to those of CFTR mediated currents in other cells types (slow activation by cAMP, absence of rectification, block by glibenclamide). The current is present in trp4 +/+ MAEC, but not in trp4 -/- cells, although the expression of CFTR seems unchanged in the trp4 deficient cells as judged from RT-PCR analysis. CONCLUSIONS: It is concluded that TRP4 is necessary for CFTR activation in endothelium, possibly by providing a scaffold for the formation of functional CFTR channels.

Animals↗
PubMed2001

Pitfalls in the analysis of mitochondrial DNA from ancient specimens and the consequences for forensic DNA analysis: the historical case of the putative heart of Louis XVII.

Amplification of mtDNA D-loop fragments with a length of 200 bp or more from ancient and even from fairly recent biological samples, can lead to erroneous results. This was clearly illustrated in our investigation of the putative heart of Louis XVII. By selecting different sets of primers which amplified shorter fragments of mtDNA (length 109 bp-201 bp), authentic polymorphisms could be visualised which remained undetected with the more classical primers for fragment sizes > 210 bp. Here we have extended those findings to other biological materials. A competitive PCR assay for quantitation of the amount of mtDNA for different fragment lengths, using a 10 bp deletion construct, was applied to ancient material and on a set of hairs of various ages of sampling (1966 up to the present). The results showed that DNA degradation started a few years after sampling. In the DNA extracts of the older hair shafts (1983-1995), the proportion of the number of short fragments to the number of long fragments is on average 4 in contrast to the most recent hair shafts. The numbers of amplifiable mtDNA copies for the hairs from 1975 and older were too small to show a clear difference. Use of long PCR fragments in such cases can yield misleading results. Use of short PCR fragments for the analysis of mtDNA from shed hair, in combination with a competitive PCR assay to determine the state of degradation, should improve the reliability of forensic mtDNA analysis considerably.

Base Sequence↗
PubMed2001

Functional integrity of the vesicle transporting machinery is required for complete activation of cFTR expressed in xenopus laevis oocytes.

We expressed the human cystic fibrosis transmembrane conductance regulator (CFTR) in oocytes of the South African clawed frog Xenopus laevis. We performed simultaneous and continuous recording of membrane current (Im), conductance (Gm) and capacitance (Cm), the latter being a direct measure of membrane surface area. A cAMP-cocktail containing cAMP and isobutylmethylxanthine (IBMX) increased all parameters, demonstrating that CFTR activation was partly achieved by exocytotic delivery and insertion of preformed CFTR molecules into the plasma membrane. CFTR currents after cAMP-cocktail were correlated with the capacitance of the oocytes: oocytes with larger Cm exhibited larger currents. Expression of CFTR itself did not change the Cm of the oocytes. However, activation of CFTR with cAMP-cocktail increased Im and Gm 15- and 20-fold, respectively while membrane surface area increased by about 7%, indicating the functional insertion of preformed CFTR into the plasma membrane. While cAMP-cocktail yielded maximal CFTR stimulation, IBMX alone, but not caffeine or theophylline, was sufficient to stimulate more than half of the increases in Im and Gm as observed with cAMP-cocktail. Since Cm was not significantly stimulated by IBMX, we conclude that IBMX alone activated the CFTR channels already present in the oocyte membrane. CFTR stimulation by cAMP-cocktail was independent of external Ca2+ and ATP had no additional activating potency. The role of protein trafficking in the activation of CFTR evoked by increases of cytoplasmic cAMP was assessed by measuring the effects of brefeldin A (BFA), nocodazole and primaquine on the bioelectric parameters and membrane surface area. All these compounds that interfere with the protein trafficking machinery at different stages prevented the translocation of CFTR from intracellular pools to the plasma membrane. These data confirm and extend our previous observations that CFTR expressed in Xenopus laevis oocytes is activated via dual pathways including direct activation of CFTR already present in the membrane and exocytotic insertion of preformed CFTR channels into the membrane. Furthermore, we show that complete activation of CFTR requires an intact protein trafficking machinery.

1-Methyl-3-isobutylxanthine↗
PubMed2001

The C-terminal part of the R-domain, but not the PDZ binding motif, of CFTR is involved in interaction with Ca(2+)-activated Cl- channels.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits Ca(2+)-activated Cl- channels (CaCC) by an unknown mechanism. This inhibition does not require CFTR activation (activity-independent inhibition), but is potentiated when CFTR is activated (activity-dependent inhibition). In this study, we evaluated, in endothelial cells, possible structural determinants for this interaction. Bovine pulmonary artery endothelium (CPAE) cells, which do not express CFTR, were transfected transiently with three hybrid CFTR constructs. The functional interaction between CaCC and CFTR was assessed using the patch-clamp technique in the whole-cell configuration. CaCC was stimulated by application of adenosine 5'-triphosphate (ATP) to the bath solution. CFTR currents were evoked by application of a forskolin/3-isobutyl-l-methylxanthine (IBMX) cocktail. The inhibitory effect of CFTR was conserved when the PDZ (PSD-95/Discs large/ZO-1) binding motif was deleted (CFTR-delta PDZ). In contrast, both the CFTR activity-independent and -dependent inhibition of CaCC were abolished when the C-terminal part of the regulatory (R)-domain of CFTR was deleted (CFTR-delta R780-830). The activity-dependent inhibition of CaCC, but not the activity-independent inhibition, could be rescued by introducing the multiple drug resistance (MDR)-1 mini-linker in place of the deletion (CFTR-delta R-linker). It is concluded that the C-terminal part of the R-domain is an important determinant for CFTR-CaCC interaction.

Amino Acid Motifs↗
PubMed2001

Different activation mechanisms of cystic fibrosis transmembrane conductance regulator expressed in Xenopus laevis oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP sensitive Cl- channel that is defective in cystic fibrosis (CF). The most frequent mutation, namely deltaF508-CFTR, accounts for 66% of CF. Here we show that cAMP-activation of CFTR occurs via at least two distinct pathways: activation of CFTR molecules already present in the plasma membrane and protein kinase A (PKA)-mediated vesicular transport of new CFTR molecules to the plasma membrane and functional insertion into the membrane. We investigated the mechanisms that are responsible for these activation pathways using the Xenopus laevis oocytes expression system. We expressed CFTR and recorded continuously membrane current (Im), conductance (Gm) and capacitance (Cm), which is a direct measure of membrane surface area. Expression of CFTR alone did not change the plasma membrane surface area. However, activation of CFTR with cAMP increased Im, Gm and Cm while deltaF508-CFTR-expressing oocytes showed no response on cAMP. Inhibition of protein kinase A or buffering intracellular Ca2+ abolished the cAMP-induced increase in Cm while increases of Im and Gm were still present. ATP or the xanthine derivative 8-cyclopentyl-1,3-dipropylxanthine (CPX) did not further activate CFTR. Insertion of pre-formed CFTR into the plasma membrane could be prevented by compounds that interfere with intracellular transport mechanisms such as primaquine, brefeldin A, nocodazole. From these data we conclude that cAMP activates CFTR by at least two distinct pathways: activation of CFTR already present in the plasma membrane and exocytotic delivery of new CFTR molecules to the oocyte membrane and functional insertion into it.

Adenosine Triphosphate↗
PubMed2001

Mitochondrial DNA analysis of the putative heart of Louis XVII, son of Louis XVI and Marie-Antoinette.

According to official historiography, the 10-year-old Louis XVII died in the Temple of Paris on June 8, 1795. However, public rumour spread the theory that Louis XVII escaped and that his descendants would be alive today. One such putative 'Louis XVII' was Carl Wilhelm Naundorff, who died in 1845 in Delft (the Netherlands). Comparative mitochondrial DNA (mtDNA) analysis gave evidence that his remains could not be identified as those of Louis XVII. In the present study, mtDNA analysis was performed on the heart of the young boy who died in the prison of Paris in 1795. In order to obtain the strongest evidence possible, two laboratories independently analysed the heart. The results showed that the consensus mtDNA sequence of the heart was identical to that of the maternal relatives of Louis XVII.

DNA, Mitochondrial↗
PubMed2001

Linkage and association of HLA class II genes with vitiligo in a Dutch population.

BACKGROUND: Serological typing of HLA has shown discrepancies in HLA associations with vitiligo in different ethnic populations. OBJECTIVES: To perform genotyping of HLA class II genes on a Dutch vitiligo population in order clearly to identify susceptible and protective HLA alleles in vitiligo. METHODS: HLA typing was carried out by amplifying genomic DNA by polymerase chain reaction (PCR) followed by dot-blot hybridization with sequence-specific oligonucleotides (SSO). Fifty Dutch vitiligo probands, and their parents (150 individuals) and 204 healthy controls were studied. RESULTS: Family-based case-control association studies and linkage disequilibrium analysis showed the linkage and association of DRB4*0101 allele with vitiligo (P(c) = 0.0016, relative risk = 2.21). The family-based association study also provided evidence for linkage and association of DQB1*0303 allele with vitiligo (chi(2) = 7.36, P = 0.006). We measured the clinical relevance of the test by calculating the prevalence corrected positive predictive values (PcPPV). The PcPPV of disease for the DRB4*0101 allele was 0.017 and for the DRB4*0101/0101 genotype was 0.0358. In other words, a DRB4*0101/0101 genotype carries a 3.58% risk of developing vitiligo. CONCLUSIONS: Both DRB4*0101 and DQB1*0303 alleles provide significant susceptibility for vitiligo.

Adolescent↗
PubMed2001

Adhesion of Porphyromonas gingivalis strains to cultured epithelial cells from patients with a history of chronic adult periodontitis or from patients less susceptible to periodontitis.

BACKGROUND: The present study aimed to explain the interindividual variation in periodontitis susceptibility by differences in the initial adhesion rate of Porphyromonas gingivalis to the pocket epithelium of these individuals, and/or by inter-P. gingivalis strain differences in association capacity (adhesion and internalization). METHODS: Adhesion assays were performed on epithelial monolayers (cultured in vitro from pocket epithelium belonging to patients who were less or more susceptible to chronic adult periodontitis) using 11 genetically different clinical strains of P. gingivalis. RESULTS: Both the disease category (less susceptible versus susceptible) and the interstrain variation were found to have a significant effect (both P <0.05) on the initial bacterial association. The chronic adult periodontitis group showed significantly more association of P. gingivalis when compared to less susceptible patients (4.2 x 10(6) versus 3.5 x 10(6)). Also, the interstrain variation was significant, with strains Pg 4 and 5 representing the least and best associating bacteria (1.8 x 10(6) colony forming units for Pg 4, 9 x 10(6) for Pg 5). CONCLUSIONS: These results indicate that periodontitis susceptibility is influenced by both the interindividual differences in pocket epithelium (allowing more adhesion of P. gingivalis) or by the strain type by which the patient is infected (intra-species differences in adhesion capacity).

Adult↗
PubMed2001

[Is cancer a hereditary or a degenerative disease?].

All malignancies, whether they are solid tumors or leukemias, always originate from modifications of the genetic information of the cells. In most cases these changes occur in a single cell, which will then generate a whole series of malignant cells through cell division. In these cases the cancer is sporadic and therefore not hereditary. In a small percentage of the cases the DNA defects are inherited from one of the parents. All cells of the body will then carry this defect, but only a few tissues will become cancerous later in life based on this defect. These forms of cancer, such as some forms of breastca, colonca, thyroidca a.o. are hereditary. Nevertheless the malignant process will not be initiated in all individuals who carry these defects and the age at onset cannot be predicted with precision. In some malignancies, whether sporadic or hereditary, the first gene that carries a defect will be essential for normal cellular function. As a result, the malignant behavior of the cells becomes irreversible. Gradually, additional defects will accumulate in other genes in these cells leading to an increasing malignant behavior of the cancer. In other cases the consequences of the first defect remain limited but the accumulation of additional defects in other genes is facilitated resulting in an increased aggressive behavior of the cells. The environmental factors that cause or facilitate the occurrence of the first DNA defect in the cells, or that facilitate the initiation of hereditary cancers remain largely unknown. Chance plays of course an important role since we accumulate continuously defects in our DNA. Nevertheless evidence exists for the presence of components in our atmosphere, our food or beverages that facilitate the accumulation of defects in our DNA. The identification of these components provides an important starting point for improved prevention. In the mean time, making genetic tests available to individuals at risk for hereditary cancers remains an important, be it delicate task, since they can potentially influence their chances of survival. The improving insights into the mechanisms involved in the pathogenesis of cancers guarantees a better and more efficient treatment in the future.

Age of Onset↗
PubMed2000

Primary role of the HLA class II DRB1*0301 allele in Graves disease.

The association of the Graves disease (GD) with HLA DR3 and DQA1*0501 in Caucasians has been described previously. From these studies it could not be determined whether one specific locus was primarily involved. Using a case-control study design, we have examined the role of HLA class II gene polymorphisms in the predisposition for GD in a group of Belgian subjects. We demonstrated that both DRB1*0301 and DQA1*0501 alleles conferred significant susceptibility in the DRB1*0301-DQA1*0501 haplotype. The DRB1*0301 allele was the primary susceptibility allele for GD, however, because the susceptibility provided by DQA1*0501 was most likely due to it being in linkage disequilibrium with DRB1*0301. The DRB1*0701/x and DQA1*0201/x genotypes and the DRB1*0701-DQA1*0201 haplotype provided protection with an equal RR of 0.29. Predictive value calculations showed that testing for DRB1*0301 gave the highest positive predictive value for GD in females and males. This was, however, 10 times higher in females and predicted a 3.63% risk for a random female to develop GD.

Alleles↗
PubMed2000

Novel syndromic form of X-linked complicated spastic paraplegia.

This study presents a family with a syndromic form of X-linked mental retardation in which four males in two generations present severe mental retardation, slowly progressive spastic paraplegia, facial hypotonia, and maxillary hypoplasia. Multipoint linkage analysis with 24 highly polymorphic markers indicated two possible candidate regions: Xp21.1-Xq21.3 (flanking markers DXS1214 and DXS990) and Xq23-Xq27.1 (flanking markers DXS8020 and DXS984). The two known loci for X-linked mental retardation and spastic paraplegia are excluded: proteolipid protein in Xp21 and L1 cell adhesion molecule in Xq28. Therefore, the syndrome in this family appears to represent a previously undescribed X-linked spastic paraplegia-mental retardation syndrome.

Adolescent↗
PubMed2000

Morphological changes in the vas deferens and expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in control, deltaF508 and knock-out CFTR mice during postnatal life.

The morphology of the mouse vas deferens still undergoes major changes from birth to 40 days of age, such as differentiation of the mesenchymal cells into fibroblasts and muscle cells, differentiation of the epithelium into basal and columnar epithelial cells, development of stereocilia, and the appearance of smooth endoplasmic reticulum organised in fingerprint-like structures or parallel, flattened saccules. In mutant homozygous DeltaF508 (DeltaF/DeltaF) and knock-out (cf/cf) CFTR mice, strain 129/FvB and 129/C57BL-6, respectively, a similar development occurred until the age of 20 days. At 40 days, however, the lumen was filled with eosinophilic secretions, and sperm cells were absent in the majority of the animals examined, although sperm production in testis and epididymis appeared to be normal. CFTR was localised in the apical membrane and cytoplasm of the vas deferens epithelium from 40 days on but could not be detected in the vas deferens before 20 days or in mutant adult CFTR mice as expected. Western blots of membrane preparations showed that the mature form of CFTR was present in vas deferens and testis but absent in seminal vesicles. Our results suggest that the function of CFTR is probably essential after 20 days in the vas deferens and that its absence or dysfunction may result in a vas deferens with a differentiated epithelium but a collapsed lumen, which could at least temporarily delay the transport of spermatozoa. These observations contrast with those made in the overall majority of CF patients. Mol. Reprod. Dev. 55:125-135, 2000.

Animals↗
PubMed2000

Genetic testing and quality control in diagnostic laboratories.

To evaluate the quality of genetic testing for cystic fibrosis, 136, 145 and 159 laboratories participated in a European study in 1996, 1997 and 1998, respectively. We sent six purified DNA samples carrying the more common CFTR mutations with the request to test them using routine protocols. A panel of experts reviewed the results together with the raw data.

Cystic Fibrosis↗
PubMed2000

Recommendations for quality improvement in genetic testing for cystic fibrosis. European Concerted Action on Cystic Fibrosis.

These recommendations for quality improvement of cystic fibrosis genetic diagnostic testing provide general guidelines for the molecular genetic testing of cystic fibrosis in patients/individuals. General strategies for testing as well as guidelines for laboratory procedures, internal and external quality assurance, and for reporting the results, including the requirements of minimal services in mutation testing, the nomenclature for describing mutations, procedures to control false-positive amplification reactions and to validate tests, and guidelines to implement a quality system in a molecular diagnostic laboratory are reviewed.

Accreditation↗