Prostaglandin G/H synthase (PGHS)-2 expression in bovine myometrium: influence of steroid hormones and PGHS inhibitors.

Prostaglandins (PGs) are important mediators regulating uterine functions during the reproductive process. The objective of this study was to examine, in myocytes from the circular and longitudinal layers of bovine myometrium, the relative levels of mRNA and proteins corresponding to the gene expression of key enzymes (phospholipase A2; prostaglandin G/H synthase-1 [PGHS-1]; prostaglandin G/H synthase-2 [PGHS-2]; prostaglandin I2 synthase) involved in PG biosynthesis. We examined the influence of estradiol-17beta and progesterone on the expression and activity of these enzymes. Treatment of myocytes with progesterone (P4: 10 nM, 24 h) in the absence or presence of estradiol-17beta (E2: 1 nM, 72 h) suppressed PG biosynthesis by approximately 60% in both myometrial layers. No significant effect was observed after E2 treatment. The combined effect of E2 and P4 on PG accumulation was correlated with the modulation of PGHS-2 protein and mRNA levels in the two myometrial layers without affecting other enzymes of the PG cascade. Selective or nonselective inhibition of PGHS activity with CGP 28238 (PGHS-2-specific; a product from Ciba-Geigy: 6-[2, 4-difluorophenoxy]-5-methyl-sulfonylamino-1-indanone) or indomethacin (PGHS-1 and -2) reduced prostacyclin accumulation (measured as 6-keto-PGF1alpha in the culture medium) in a dose-dependent manner in the two myometrial layers. A significant inhibitory effect was obtained at a low concentration of indomethacin (1 nM, p < 0.05) compared to CGP 28238 (10 nM, p < 0. 05). In both myometrial layers, the maximal effect of indomethacin and/or CGP 28238 on PG accumulation was observed at 100 nM and represented 85% and 65% inhibition, respectively. In the presence of phorbol 12-myristate (100 nM), CGP 28238 (10 nM) significantly suppressed PGHS-2 mRNA level by 44.80 +/- 7.67% (p < 0.01) and 27.83 +/- 7.62% (p < 0.05) in the longitudinal and circular layer, respectively. In contrast, indomethacin did not have any significant effect. These data constitute the first quantitative analysis of key enzymes involved in PG biosynthesis in separated myometrial layers. Furthermore, the results provide interesting information on the CGP 28238 drug modulating both enzymatic activity and mRNA expression of PGHS-2.

Down-regulation of interleukin-1beta production and PGE2 accumulation by an indomethacin-phenylalanine derivative in human monocytes.

This study was initiated to investigate the mechanism of action of a new indomethacin derivative, indomethacin-phenylalanine (indo-Phe) in human monocytes. We determined the effect of indo-Phe on the induction by LPS of prostaglandin-E2 (PGE2) and interleukin-1beta (IL-1beta) production in human monocytes. Indomethacin and indo-Phe inhibited the PGE2 synthesis in treated and untreated IL-1beta or LPS-treated monocytes. Furthermore, in IL-1beta and LPS-treated monocytes, prostaglandin G/H synthase-1 (PGHS-1) protein expression was down-regulated with indomethacin or its indo-Phe analog whereas the level of the inducible protein (PGHS-2) was up-regulated. We analyzed the effect of indomethacin and indo-Phe on the expression of IL-1beta protein in LPS-treated monocytes and found that indo-Phe blocked the LPS-induction of IL-1beta synthesis while indomethacin did not. These differential effects of indomethacin and indo-Phe suggest that two independent ways are involved in the stimulation of monocytes by LPS: the PGHS-2 protein induction and the IL-1beta secretion.

Detection of the pathogenic parasite Toxoplasma gondii by specific amplification of ribosomal sequences using comultiplex polymerase chain reaction.

Amplification of DNA sequences from ribosomal DNA (rDNA) was tested as a specific and sensitive method for the detection of small numbers of Toxoplasma gondii tachyzoite cells. We applied the polymerase chain reaction (PCR) on the basis of detection of the 110-fold repetitive rDNA as a target by using (i) DNA sequences within the small ribosomal subunit known to be universal and conserved in all eukaryotes and (ii) small ribosomal subunit and intergenic spacer rDNA sequences known to be T. gondii species specific. The level of sensitivity obtained from a crude cell lysate allowed the detection of as few as one parasite visualized directly as a specific PCR product in agarose gels. By using a combination of universal and T. gondii species-specific primers, we propose a comultiplex-based PCR approach as a new diagnostic tool. The combination of sensitivity, specificity, and built-in positive and negative PCR controls should make detection of the rDNA sequences by comultiplex PCR a useful clinical test for the diagnosis of toxoplasmosis and for epidemiological studies. Finally, the idea of a built-in positive control to support or counter the T. gondii-specific PCR result is novel and is a notable advance.

Physical and genetic mapping of cloned ribosomal DNA from Toxoplasma gondii: primary and secondary structure of the 5S gene.

The ribosomal DNA (rDNA encoding rRNA) of the obligately intracellular protozoan parasite, Toxoplasma gondii, was identified, cloned, physically mapped, its copy number determined, and the 5S gene sequenced. Using total RNA as a probe, a collection of recombinant lambda phages containing copies of rDNA were isolated from a lambda 2001 tachyzoite genomic library. Northern gel hybridization confirmed specific homology of the 7.5-kb rDNA unit, subcloned into pTZ18R, to T. gondii rRNA. The mapped rDNA found in pTOX1 contained small ribosomal subunit (SS; 18S)- and large ribosomal subunit (LS; 26S)-encoding genes localized using intragenic heterologous probes from the conserved sequences of the SS (18S) and LS (28S) Xenopus laevis genes. the physical mapping data, together with partial digestion experiments and Southern gel hybridization, confirmed a 7.5-kb rDNA unit arranged in a simple head-to-tail fashion that is tandemly repeated. We estimated the rDNA repeat copy number in T. gondii to be 110 copies per haploid tachyzoite genome. Parts of the SS gene and the complete 5S gene were sequenced. The 5S gene was found to be within the rDNA locus, a rare occurrence found only in some fungi and protozoa. Secondary-structure analysis revealed an organization remarkably similar to the 5S RNA of eukaryotes.

[Congestive cardiomyopathies in children. Natural history and studies]. / Les cardiomyopathies congestives chez l'enfant. Histoire naturelle et investigation.

Congestive cardiomyopathies represent a group of diseases having in common an intrinsic abnormality of the myocardial contraction, of which the cause often remains unknown. In children, there is a more marked incidence during the first year of life. At the Cardiology Institute of Quebec, 25 patients have been diagnosed with a congestive cardiomyopathy since January 1967. The mortality remains high at 48 per cent, and the morbidity at 28 per cent. Thus, the chance of total survival at 16 years was 33 percent. The evaluation and the treatment of the cardiac function as well as the search for a specific etiology must be carried out because the etiological treatment is sometimes possible. Biopsy of the skeletal muscle as the study of fatty acids metabolism have become very important since the identification of a carnitine deficiency. On the other hand, biopsy of the endomyocardium remains the only means to make a pathological diagnosis. Since the treatment of persisting myocarditis is feasible, the histological diagnosis is a pre-requisite in children. So, in four patients with congestive cardiomyopathy, biopsy of the endocardium has enabled to demonstrate a chronic inflammation of the myocardium in one patient. In three other cases, two had non specific lesions on histological examination an one had an extensive fibro-elastosis. As the symptomatic treatment is often deceiving, an in-depth investigation is mandatory in a child suffering from a congestive cardiomyopathy in order to identify an etiology. Such an approach will enable to apply a specific treatment, to achieve a better understanding and perhaps modify its natural history.

[Study of tetralogy of Fallot by echocardiography]. / Etude des tétralogies de Fallot par échocardiographie

Twelve patients with a Fallot's tetralogy proved by a haemodynamic and angiographic examination were studied by echography, a non invasive technique. The diameters of the right and left ventricles, of the left atrium and of the aorta were measured. The movements of the interventricular septum and the position of the aorta in relation to it were analysed. Moreover, by scanning of the left ventricle an opening was looked for in the septum. The ventricular septal defect together with a dilatation of the right ventricle and a more or less intense dilatation of the root of the aorta were found in all cases. In 10 cases, an aorta overriding the septum was observed, and this was the more obvious the older the child. In the two post-operative cases studied, the septum was found to be in line with the aorta. There are therefore echocardiographic criteria making it possible to diagnose Fallot's tetralogy. These criteria are the more obvious the older the child.