[Loading and strength of single- and multi-unit fixed dental prostheses 2. Strength]. / Op kronen en bruggen uitgeoefende krachten en het weerstaan daarvan 2. Mechanische belastbaarheid.

The ultimate strength of a dental prosthesis is defined as the strongest loading force applied to the prosthesis until afracture failure occurs. Important key terms are strength, hardness, toughness and fatigue. Relatively prevalent complications of single- and multi-unit fixed dental prostheses are porcelain and ceramic fractures. Afactor which also plays a role is the functional loading force from the entire orofacial system. With respect to the strength of multi-unit fixed dental prostheses, the length of the arch span between the abutment teeth, the pontic with the connectors and the possible cantilevers are the critical components. Components of the configuration ofabutment teeth of single- and multi-unit fixed dental prostheses which are relevant for its strength are the convergence angle and the design of(the area above) the (cervical) outline. Finally, the thickness of the porcelain or the ceramic (veneers) ofmetal-ceramic and all-ceramic single- and multi-unit fixed dental prostheses is of importance.

[Loading and strength of single- and multi-unit fixed dental prostheses. 1. Retention and resistance]. / Op kronen en bruggen uitgeoefende krachten en het weerstaan daarvan. 1. Retentie en resistentie.

The degree to which single- and multi-unit fixed dental prostheses are able to withstand loading forces is dependent, among other things, on the quality of their retention and resistance. The quality of the retention and resistance of the configuration of an abutment tooth prepared for a metal and metal-ceramic single-unit fixed dental prosthesis is determined by the configuration's convergence angle, the height, the volume, the interocclusal space, the cervical outline design, the additional preparations, the quality of the (build-up) restoration, and the surface roughness. A silicate ceramic single-unit fixed dental prosthesis is attached through adhesion using a composite cement, but the retention and resistance of an oxide ceramic single-unit fixed dental prosthesis is dependent on the abutment tooth configuration. Most types of multi-unit fixed dental prosthesis have the following additional retention and resistance determining factors: the position in the occlusal system, the number of abutment teeth and their mutual configurations, and the length of (cantilever) pontics. A resin-bonded fixed partial denture's retention and resistance are determined by its bonding as well as its enamel surface coverage and its resistance preparations.

Validation of 70-gene prognosis signature in node-negative breast cancer.

PURPOSE: The 70-gene prognosis signature (van't Veer et al., Nature 415(6871):530-536, 2002) may improve the selection of lymph node-negative breast cancer patients for adjuvant systemic therapy. Optimal validation of prognostic classifiers is of great importance and we therefore wished to evaluate the prognostic value of the 70-gene prognosis signature in a series of relatively recently diagnosed lymph node negative breast cancer patients. METHODS: We evaluated the 70-gene prognosis signature in an independent representative series of patients with invasive breast cancer (N = 123; <55 years; pT1-2N0; diagnosed between 1996 and 1999; median follow-up 5.8 years) by classifying these patients as having a good or poor prognosis signature. In addition, we updated the follow-up of the node-negative patients of the previously published validation-series (Van de Vijver et al., N Engl J Med 347(25):1999-2009, 2002; N = 151; median follow-up 10.2 years). The prognostic value of the 70-gene prognosis signature was compared with that of four commonly used clinicopathological risk indexes. The endpoints were distant metastasis (as first event) free percentage (DMFP) and overall survival (OS). RESULTS: The 5-year OS was 82 +/- 5% in poor (48%) and 97 +/- 2% in good prognosis signature (52%) patients (HR 3.4; 95% CI 1.2-9.6; P = 0.021). The 5-years DMFP was 78 +/- 6% in poor and 98 +/- 2% in good prognosis signature patients (HR 5.7; 95% CI 1.6-20; P = 0.007). In the updated series (N = 151; 60% poor vs. 40% good), the 10-year OS was 51 +/- 5% and 94 +/- 3% (HR 10.7; 95% CI 3.9-30; P < 0.01), respectively. The DMFP was 50 +/- 6% in poor and 86 +/- 5% in good prognosis signature patients (HR 5.5; 95% CI 2.5-12; P < 0.01). In multivariate analysis, the prognosis signature was a strong independent prognostic factor in both series, outperforming the clinicopathological risk indexes. CONCLUSION: The 70-gene prognosis signature is also an independent prognostic factor in node-negative breast cancer patients for women diagnosed in recent years.

The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development.

Recent loss-of-function studies in mice show that the transcription factor GATA6 is important for visceral endoderm differentiation. It is also expressed in early bronchial epithelium and the observation that this tissue does not receive any contribution from Gata6 double mutant embryonic stem (ES) cells in chimeric mice suggests that GATA6 may play a crucial role in lung development. The aim of this study was to determine the role of GATA6 in fetal pulmonary development. We show that Gata6 mRNA is expressed predominantly in the developing pulmonary endoderm and epithelium, but at E15.5 also in the pulmonary mesenchyme. Blocking or depleting GATA6 function results in diminished branching morphogenesis both in vitro and in vivo. TTF1 expression is unaltered in chimeric lungs whereas SPC and CC10 expression are attenuated in abnormally branched areas of chimeric lungs. Chimeras generated in a ROSA26 background show that endodermal cells in these abnormally branched areas are derived from Gata6 mutant ES cells, implicating that the defect is intrinsic to the endoderm. Taken together, these data demonstrate that GATA6 is not essential for endoderm specification, but is required for normal branching morphogenesis and late epithelial cell differentiation.

RET and GDNF gene scanning in Hirschsprung patients using two dual denaturing gel systems.

Hirschsprung disease (HSCR) is a congenital disorder characterised by intestinal obstruction due to an absence of intramural ganglia along variable lengths of the intestine. RET is the major gene involved in HSCR. Mutations in the GDNF gene, and encoding one of the RET ligands, either alone or in combination with RET mutations, can also cause HSCR, as can mutations in four other genes (EDN3, EDNRB, ECE1, and SOX10). The rare mutations in the latter four genes, however, are more or less restricted to HSCR associated with specific phenotypes. We have developed a novel comprehensive mutation detection system to analyse all but three amplicons of the RET and GDNF genes, based on denaturing gradient gel electrophoresis. We make use of two urea-formamide gradients on top of each other, allowing mutation detection over a broad range of melting temperatures. For the three remaining (GC-rich) PCR fragments we use a combination of DGGE and constant denaturing gel electrophoresis (CDGE). These two dual gel systems substantially facilitate mutation scanning of RET and GDNF, and may also serve as a model to develop mutation detection systems for other disease genes. In a screening of 95 HSCR patients, RET mutations were found in nine out of 17 familial cases (53%), all containing long segment HSCR. In 11 of 78 sporadic cases (14%), none had long segment HSCR. Only one GDNF mutation was found, in a sporadic case.

Organization and chromosomal localization of the human ECEL1 (XCE) gene encoding a zinc metallopeptidase involved in the nervous control of respiration.

ECEL1 (endothelin-converting enzyme-like 1; previously known as XCE) is a putative zinc metalloprotease that was identified recently on the basis of its strong identity with endothelin-converting enzyme. Although the physiological function of ECEL1 is unknown, inactivation of the corresponding gene in mice points to a critical role of this protein in the nervous control of respiration. In the present study we have characterized the human ECEL1 gene. It was located to region q36-q37 of chromosome 2 and shown to be composed of 18 exons spanning approx. 8 kb. The structure of the ECEL1 gene displays some striking similarities with those of genes of related metallopeptidases, supporting the hypothesis that they are all derived from a common ancestor. A short phylogenetic study describing the relationship between the various members of this gene family is also presented.

Effect of oxygen concentration on human in-vitro fertilization and embryo culture.

In this prospective randomized study on 1380 consecutive in-vitro fertilization (IVF) treatments, the results were compared of culture of human oocytes and embryos for the first 2 or 3 days of development in microdroplets of medium under oil using a gas phase containing either atmospheric (approximately 20%) or reduced (5%) O2 concentrations. No significant differences were found between the two groups cultured under either 5% or 20% O2 in rates of fertilization (60 versus 61%, respectively), embryo development at day 2 or 3, pregnancy (26.6 versus 25.4%, respectively), and implantation (13.4 versus 14.0%, respectively). Culture of surplus embryos under 5% O2 resulted in a significantly higher mean incidence of blastocyst formation per cycle as compared to the 20% O2 group (25.8 +/- 2.0 versus 20.4 +/- 1.9, respectively). The mean number of cells of embryos classified as blastocysts by microscopic observation of a blastocoel was significantly higher in the 5% O2 group as compared to the 20% O2 group, both in blastocysts fixed on day 5 (39.8 +/- 1.7 versus 31.9 +/- 1.9, respectively), as well as those fixed on day 6 (45.6 +/- 2.6 versus 33.7 +/- 3.4, respectively). This difference was due to the fact that significantly more blastocysts of the 20% O2 group had an abnormal low cell number of < 25 as compared to the 5% O2 group, both in blastocysts fixed on day 5 (39 versus 22%, respectively), as well as those fixed on day 6 (43 versus 22%, respectively). To conclude, although culture under 5% O2 leads to slightly improved preimplantation embryonic viability, this effect is either too marginal to result in higher pregnancy rates, or low O2 concentrations exert an effect during the later stages of preimplantation development only.

A consanguineous family with Hirschsprung disease, microcephaly, and mental retardation (Goldberg-Shprintzen syndrome).

Hirschsprung disease, mental retardation, microcephaly, and specific craniofacial dysmorphism were observed in three children from a large, consanguineous, Moroccan family. A fourth child showed similar clinical features, with the exception of Hirschsprung disease. The association of these abnormalities in these children represents the Goldberg-Shprintzen syndrome (OMIM 235730). Mutation scanning of genes potentially involved in Hirschsprung disease, RET, GDNF, EDN3, and EDNRB, showed a sequence variant, Ser305Asn, in exon 4 of the EDNRB gene in the index patient of this family. The Ser305Asn substitution present in two of the four patients and four healthy relatives and absent in one of the remaining two patients illustrates the difficulties in interpreting the presence of mutations in families with Hirschsprung disease. It is unlikely that the EDNRB variant contributes to the phenotype. This consanguineous family might be useful for the identification of a Goldberg-Shprintzen locus.

Impaired DNA repair capacity in skin fibroblasts from various hereditary cancer-prone syndromes.

Host-cell reactivation (HCR) of UV-C-irradiated herpes simplex virus type 1 (HSV-1) has been determined in skin fibroblasts from the following hereditary cancer-prone syndromes: aniridia (AN), dysplastic nevus syndrome (DNS), Von Hippel-Lindau syndrome (VHL), Li-Fraumeni syndrome (LFS) and a family with high incidence of breast and ovarian cancer. Cells from AN, DNS or VHL patients were found to exhibit heterogeneity in HCR. Cells from individuals belonging to an LFS family show reduced HCR in all cases where the cells were derived from persons carrying one mutated p53 allele, whereas cells derived from members with two wild-type alleles show normal HCR. LFS cells with reduced HCR also reveal reduced genome overall repair, and a slower gene-specific repair of the active adenosine deaminase (ADA) gene, but little if any repair of the inactive 754 gene. In the breast/ovarian cancer family, reduced HCR is observed in skin fibroblasts derived from both afflicted and unaffected individuals. In addition, these cells display lower survival after exposure to UV-C and exhibit higher levels of SCEs than those in normal cells. These observations indicate that various hereditary cancer-prone syndromes, carrying mutations in different tumor-suppressor genes, exhibit an unexplained impairment of the capacity to repair UV-damaged DNA.

The murine homologue of HIRA, a DiGeorge syndrome candidate gene, is expressed in embryonic structures affected in human CATCH22 patients.

A wide spectrum of birth defects is caused by deletions of the DiGeorge syndrome chromosomal region at 22q11. Characteristic features include cranio-facial, cardiac and thymic malformations, which are thought to arise form disturbances in the interactions between hindbrain neural crest cells and the endoderm of the pharyngeal pouches. Several genes have been identified in the shortest region of deletion overlap at 22q11, but nothing is known about the expression of these genes in mammalian embryos. We report here the isolation of several murine embryonic cDNAs of the DiGeorge syndrome candidate gene HIRA. We identified several alternatively spliced transcripts. Sequence analysis reveals that Hira bears homology to the p60 subunit of the human Chromatin Assembly Factor I and yeast hir1p and Hir2p, suggesting that Hira might have some role in chromatin assembly and/or histone regulation. Whole mount in situ hybridization of mouse embryos at various stages of development show that Hira is ubiquitously expressed. However, higher levels of transcripts are detected in the cranial neural folds, frontonasal mass, first two pharyngeal arches, circumpharyngeal neural crest and the limb buds. Since many of the structures affected in DiGeorge syndrome derive from these Hira expressing cell populations we propose that haploinsufficiency of HIRA contributes to at least some of the features of the DiGeorge phenotype.

Detection of aneugenic and clastogenic potential of X-rays, directly and indirectly acting chemicals in human hepatoma (Hep G2) and peripheral blood lymphocytes, using the micronucleus assay and fluorescent in situ hybridization with a DNA centromeric probe.

In human hepatoma (Hep G2) cells and peripheral blood lymphocytes (HPBL) the cytokinesis-blocked micronuclei (MN) and fluorescent in situ hybridization (FISH) assays were applied to study aneugenic and clastogenic potentials of X-rays, directly and indirectly acting chemicals. Induction of MN was studied in vitro following treatment with X-rays, directly acting chemicals, such as methylmeth-anesulphonate (MMS), colchicine (COL), vincristine sulphate (VCS) and vinblastine sulphate (VBS), and indirectly acting agents, such as cyclophosphamide (CP), hexamethylphosphoramide (HMPA), 2-acetylaminofluorene (2-AAF) and 4-acetylaminofluorene (4-AAF). Depending on the presence of the fluorescent signal in the MN following FISH with a human DNA centromeric probe, MN in the binucleated Hep G2 cells and lymphocytes were scored as centromere-positive or centromere-negative, representing an aneugenic and clastogenic event respectively. In the controls approximately 50% of spontaneously occurring MN were centromere-positive. Treatment of human hepatoma cells and HPBL (in vitro) with potent aneugens such as COL, VCS and VBS increased the number of MN in a dose-dependent manner; of these 75-93% were centromere-positive. X-irradiation induced MN in a dose-related manner in binucleated Hep G2 cells and HPBL, of which 33-40% were centromere-positive, which demonstrates the significant aneugenic potentials of X-rays. Strong clastogenic activity was observed with MMS and frequency of centromere-positive MN was low: approximately 20 and 30% for HPBL and Hep G2 cells respectively. In Hep G2 cells significant aneugenic activity was found with indirectly acting promutagens/procarcinogens such as HMPA and 2-AAF, in contrast to CP, which came out as a potent clastogen. The non-carcinogen 4-AAF was not able to induce an increase in the frequency of MN in Hep G2 cells. All indirectly acting chemicals tested came out negative when HPBL were used as targets for DNA damage. The results presented correlate positively with data from in vivo assays and indicate that the Hep G2 cell system is a suitable bioactivation system (in vitro) for evaluating the clastogenic and aneugenic potentials of chemicals which require exogenous metabolic activations in order to exert their mutagenic potential.

A homozygous mutation in the endothelin-3 gene associated with a combined Waardenburg type 2 and Hirschsprung phenotype (Shah-Waardenburg syndrome).

Hirschsprung disease (HSCR) or colonic aganglionosis is a congenital disorder characterized by an absence of intramural ganglia along variable lengths of the colon resulting in intestinal obstruction. The incidence of HSCR is 1 in 5,000 live births. Mutations in the RET gene, which codes for a receptor tyrosine kinase, and in EDNRB which codes for the endothelin-B receptor, have been shown to be associated with HSCR in humans. The lethal-spotted mouse which has pigment abnormalities, but also colonic aganglionosis, carries a mutation in the gene coding for endothelin 3 (Edn3), the ligand for the receptor protein encoded by EDNRB. Here, we describe a mutation of the human gene for endothelin 3 (EDN3), homozygously present in a patient with a combined Waardenburg syndrome type 2 (WS2) and HSCR phenotype (Shah-Waardenburg syndrome). The mutation, Cys159Phe, in exon 3 in the ET-3 like domain of EDN3, presumably affects the proteolytic processing of the preproendothelin to the mature peptide EDN3. The patient's parents were first cousins. A previous child in this family had been diagnosed with a similar combination of HSCR, depigmentation and deafness. Depigmentation and deafness were present in other relatives. Moreover, we present a further indication for the involvement of EDNRB in HSCR by reporting a novel mutation detected in one of 40 unselected HSCR patients.

The distribution and characterization of HNK-1 antigens in the developing avian heart.

The heart originates from splanchnic mesoderm and to a lesser extent from neural crest cells. The HNK-1 monoclonal antibody is a marker for early migrating neural crest cells, but reacts also with structures which are not derived from the neural crest. We investigated whether heart structures are HNK-1 positive before neural crest cells colonize these target tissues. To that end, we determined the HNK-1 antigen expression in the developing avian heart on immunohistochemical sections and on Western blots. The HNK-1 immunoreactivity in the developing chick heart is compared with data from literature on the localization of neural crest cells in chick/quail chimeras. Structures with neural crest contribution, including parts of the early outflow tract and the related endocardial cushions, the primordia of the semilunar valve leaflets and the aorticopulmonary septum were HNK-1 positive. Furthermore, other structures were HNK-1 positive, such as the atrioventricular cushions, the wall of the sinus venosus at stage HH 15 through 21, parts of the endocardium at E3, parts of the myocardium at E6, and the extracellular matrix in the myocardial base of the semilunar valves at E14. HNK-1 expression was particularly observed in morphologically dynamic regions such as the developing valves, the outflow tract cushion, the developing conduction system and the autonomic nervous system of the heart. We observed that atrioventricular endocardial cushions are HNK-1 positive. We conclude that: a HNK-1 immunoreactivity does not always coincide with the presence of neural crest cells or their derivatives; (2) the outflow tract cushions and atrioventricular endocardial cushions are HNK-1 positive before neural crest cells are expected (stage HH 19) to enter the endocardial cushions of the outflow tract; (3) the observed spatio-temporal HNK-1 patterns observed in the developing heart correspond with various HNK-1 antigens. Apart from a constant pattern of HNK-1 antigens during development, stage-dependent HNK-1 antigens were also found.

Regional differences between various axial segments of the avian neural crest regarding the formation of enteric ganglia.

The vagal neural crest adjacent to somites 1-7 gives rise to the enteric ganglia along the entire digestive tract. It is generally assumed that formation of enteric ganglia in preumbilical gut is independent of the axial segment from which the neural crest originates. In post-umbilical gut, however, there is evidence that the axial segment of origin of the neural crest might be relevant to neural differentiation. In this part of the gut, we previously identified a subpopulation of HNK-1-immunoreactive cells within the enteric mesenchyme. This immunoreactivity disappeared upon formation of the enteric nervous system. We studied the interaction between various axial segments of quail neural crest and the microenvironment in a neural chicken hindgut using chorioallantoic membrane cocultures. We found that neural crest cells from various axial segments could migrate into the gut and home to the correct sites. However, whereas vagal neural crest cells differentiated into enteric neurons, neural crest cells from truncal segments mainly differentiated into melanocytes. The HNK-1-immunoreactivity within the enteric mesenchyme only disappeared when neural crest cell colonization was followed by differentiation into enteric neurons and subsequent formation of enteric ganglia. To determine whether differentiation of neural crest cells in chorioallantoic membrane cocultures was influenced by the prolonged presence of the neural tube and notochord, we developed a new coculture system, using neural crest cells cultured in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of alternatively spliced bFGF first coding exons and antisense mRNAs during chicken embryogenesis.

We have identified eight different basic fibroblast growth factor (bFGF or FGF-2) transcripts which can be grouped into three classes expressed during chicken embryogenesis and in specific adult organs. Three of them encode the evolutionarily conserved bFGF proteins. Two additional mRNAs are structurally homologous to the previously isolated Xenopus laevis bFGF antisense transcripts. Their expression during embryogenesis and the inversely proportional amounts of sense and antisense transcripts in several adult organs provide further evidence for a possible regulatory role of the antisense transcripts. Unexpectedly, a novel class of three alternatively spliced bFGF transcripts expressed predominantly during embryogenesis was isolated. Alternative splicing of exon 1 of the open reading frame creates a predicted bFGF isoform containing a completely novel amino-terminal domain. This is the first report describing alternative splicing of the coding region of bFGF. Similar levels of alternatively spliced (alt-bFGF) and canonical bFGF transcripts are expressed during embryogenesis, but in adult organs alt-bFGF transcripts are far less abundant, which suggests roles for the predicted novel bFGF isoform during morphogenesis.