[Does coxitis fugax predispose for later Perthes' disease?--first results of an insurance data-based study]. / Prädisponiert die Coxitis fugax für einen späteren Morbus Perthes?--Erste Ergebnisse einer auf Versicherungsdaten basierenden Untersuchung.

INTRODUCTION: For decades, it has been a matter of debate whether coxitis fugax (CF) may trigger the onset of Perthes' disease (PD). However, the low incidence of both conditions limits the validity of clinical studies. As a novel approach, an analysis of patient data provided by a private health insurance (PHI) was performed. After calculation of the frequencies of CF and PD possible correlations were statistically assessed. We hypothesised that CF predisposes to the development of PD. MATERIALS: A retrospective database analysis was conducted based on insurance data of patients aged between 1 and 14 years covering an observation period of 7 years. Cases of CF and PD were detected by a search algorithm based on the International Classification System of Diseases (ICD) encoding the ICD codes M12.85 to CF and M91.1 to PD, respectively. Cases where CF was followed by PD were separately assessed for plausibility considering the clinical course and the length of the symptom-free interval. Statistical analysis was performed by using the chi-square test with a significance level set at 5 %. RESULTS: Among a cohort of 407,875 children 960 cases of CF were detected. Of these, 876 (91.3 %) had one single event of CF whereas 84 (8.7 %) children had two or more episodes. The average incidence of CF was 0.24 % per year. The frequency of PD was calculated to be 15.7 cases per 100, 000 children per year. In eleven cases (all male) CF was found to be followed by PD, however, after checking for plausibility only three cases remained. Statistical analysis revealed that the incidence of PD in male children with a previous CF episode was 21-times higher compared to children without CF (p < 0.0001). DISCUSSION: The results of the hitherto largest study including more than 400 ,000 children showed a significantly higher rate of PD in male children with previous CF compared to boys without CF. However, different patterns of age distribution and the observation that multiple CF episodes do not trigger the development of PD contradict the assumption of a possible correlation between these two diseases. In two of the three cases where CF was followed by PD a so-called "late onset PD" was evident suggesting a misdiagnosed PD at initial presentation. The chosen study design using patient data provided by a PHI allows the acquisition and evaluation of large numbers of cases which may help to elucidate possible correlations between different medical conditions. To unambiguously answer the hypothesis of this study, the inclusion of additional insurance data is necessary.

Coronary hemodynamics in endothelial NO synthase knockout mice.

For the specific analysis of endothelial NO synthase (eNOS) function in the coronary vasculature, we generated a mouse homozygous for a defective eNOS gene (eNOS-/-). Western blot as well as immunohistochemical staining revealed the absence of eNOS protein in eNOS-/- mice. Aortic endothelial cells derived from eNOS-/- mice displayed only background levels of NOx formation compared with wild-type (WT) cells (88 versus 1990 pmol NOx x h-1/mg protein-1). eNOS-/- mice were hypertensive (mean arterial pressure, 135 +/- 15 versus 107 +/- 8 mm Hg in WT) without the development of cardiac hypertrophy. Coronary hemodynamics, analyzed in Langendorff-perfused hearts, showed no differences either in basal coronary flow or in maximal and repayment flow of reactive hyperemia. Acute NOS inhibition with Nomega-nitro-L-arginine methyl ester (L-NAME) in WT hearts substantially reduced basal flow and reactive hyperemia. The coronary response to acetylcholine (ACh) (500 nmol/L) was biphasic: An initial vasoconstriction (flow, -35%) in WT hearts was followed by sustained vasodilation (+190%). L-NAME significantly reduced vasodilation in WT hearts (+125%) but did not alter the initial vasoconstriction. In eNOS-/- hearts, the initial vasoconstriction was augmented (-70%), whereas the ACh-induced vasodilation was not affected. Inhibition of cyclooxygenase with diclofenac converted the ACh-induced vasodilation into vasoconstriction (-49% decrease of basal flow). This effect was even more pronounced in eNOS-/- hearts (-71%). Our results demonstrate that (1) acute inhibition of eNOS reveals a role for NO in setting the basal coronary vascular tone as well as participation in reactive hyperemia and the response to ACh; (2) chronic inhibition of NO formation in eNOS-/- mutant mice induces no changes in basal coronary flow and reactive hyperemia, suggesting the activation of important compensatory mechanisms; and (3) prostaglandins are the main mediators of the ACh-induced vasodilation in both WT and eNOS-/- mice.

The c-ros tyrosine kinase receptor controls regionalization and differentiation of epithelial cells in the epididymis.

The c-ros gene was originally identified in mutant form as an oncogene. The proto-oncogene encodes a tyrosine kinase receptor that is expressed in a small number of epithelial cell types, including those of the epididymis. Targeted mutations of c-ros in the mouse reveal an essential role of the gene in male fertility. Male c-ros -/- animals do not reproduce, whereas the fertility of female animals is not affected. We demonstrate that c-ros is not required in a cell autonomous manner for male germ cell development or function. The gene, therefore, does not affect sperm generation or function in a direct manner. The primary defect in the mutant animals was located in the epididymis, showing that c-ros controls appropriate development of the epithelia, particularly regionalization and terminal differentiation. The epididymal defect does not interfere with production or storage of sperm but, rather, with sperm maturation and the ability of sperm to fertilize in vivo. Interestingly, sperm isolated from c-ros -/- animals can fertilize in vitro. Our results highlight the essential role of the epididymis in male fertility and demonstrate a highly specific function of the c-ros receptor tyrosine kinase during development of distinct epithelial cells.

Cloning of the rat P2u receptor and its potential role in coronary vasodilation.

We cloned and sequenced the cDNA as well as the genomic DNA of the P2u receptor gene from the rat. The coding region of the gene is not interrupted by introns. P2u is expressed in a variety of rat organs with pronounced differences of expression intensities. Highest expression was found in liver and testis, while no expression could be detected in the brain. High P2u expression was found in primary microvascular endothelial cells from the rat heart, but not in cardiac myocytes. By in situ analysis, we localized P2u expression in epithelial cells of esophagus and bronchi. Functional analysis revealed that, in isolated perfused rat hearts, the P2u ligands UTP and ATP induce a pronounced vasodilation of coronary blood vessels. In contrast, UMP and uridine, the degradative products of UTP, act as potent vasoconstrictors. Our experiments suggest that, in the rat heart, endothelial P2u receptors are involved in the ATP/UTP-mediated vasodilation of coronary blood vessels.

Biochemical and functional characterization of the murine ros protooncogene.

The ros gene was originally found because it can, when mutated, induce malignant transformation. The protooncogene encodes an orphan receptor tyrosine kinase. We report here the isolation and characterization of the mouse c-ros cDNA and, in addition, the biochemical characterization of the receptor. Both, the endogenous c-ros protein from embryonal tissues and the recombinant protein are glycosylated molecules with an apparent molecular weight of 260,000. Pulse-chase analysis in Sf9 cells demonstrates that the c-ros protein is synthesized as a single chain, uncleaved molecule. Since the specific ligand of c-ros is not known, a hybrid receptor (trk/c-ros) which transmits c-ros-specific signals in response to nerve growth factor (NGF) was used to study the biological activities. In NIH3T3 cells, this trk/c-ros hybrid induces growth, a fusiform cell shape, and loss of contact inhibition of growth. However, the active hybrid receptor cannot replace IL-3 as survival factor in 32D myeloid cells. Compared to other receptors, the active c-ros tyrosine kinase domain displays thus overlapping, but not identical signalling specificities.

Identification of sequences responsible for transcriptional regulation of the strongly expressed methanol oxidase-encoding gene in Hansenula polymorpha.

The methylotrophic yeasts have been the subject of intensive studies, because of their highly regulated methanol metabolism and the biogenesis of peroxisomes. We investigated the 5' regulatory region of the MOX gene from the yeast, Hansenula polymorpha, encoding the peroxisomal methanol oxidase, the key enzyme of methanol metabolism. This tightly regulated yeast promoter of approximately 1.5 kb is unusually large, and also of remarkable strength under inducing conditions, belonging to the strongest yeast promoters yet described. Deletion analyses revealed a complex promoter structure composed of several sequence elements with positive and negative regulatory effects on reporter gene expression and a pronounced cooperation between the elements. Specific binding of several factors was detected in vitro by gel retardation and DNase I footprinting experiments. On the basis of deletion data, two binding sites could be identified as upstream activation sequences (UAS1 and UAS2) and one binding site as an upstream repressing sequence (URS1).

Cloning and sequencing of the ura3 locus of the methylotrophic yeast Hansenula polymorpha and its use for the generation of a deletion by gene replacement.

The ura3 gene of Hansenula polymorpha was cloned, sequenced and used to generate a ura3 mutant from the wild-type strain of this yeast via integrative mutagenesis. The Tn5 neomycin-resistance marker (neo) under control of the ADH1 promoter from Saccharomyces cerevisiae served as a transformation marker. The results show that gene replacement can be achieved in H. polymorpha, a yeast with a high level of non-homologous integration.