Expression of neuropeptide Y and its receptors Y1 and Y2 in the rat heart and its supplying autonomic and spinal sensory ganglia in experimentally induced diabetes.

Diabetic cardiomyopathy, involving both cardiomyocytes and the sensory and autonomic cardiac innervation, is a major life-threatening complication in diabetes mellitus. Here, we induced long-term (26-53 weeks) diabetes in rats by streptozotocin injection and analyzed the major cardiac neuropeptide signaling system, neuropeptide Y (NPY) and its receptors Y1R and Y2R. Heart compartments and ganglia supplying sympathetic (stellate ganglion) and spinal sensory fibers (upper thoracic dorsal root ganglia=DRG) were analyzed separately by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Ventricular, but not atrial innervation density by NPY-immunoreactive fibers was diminished, and preproNPY expression was transiently (26 weeks) reduced in left atria, but remained unchanged in sympathetic neurons and was not induced in DRG neurons. In all ganglia and heart compartments, Y1R expression dominated over Y2R, and Y1R-immunoreactivity was observed on cardiomyocytes and neuronal perikarya. Atrial, but not ventricular Y1R expression was up-regulated after 1 year of diabetes. Collectively, these data show that a disturbance of the cardiac NPY-Y1R/Y2R signaling system develops slowly in the course of experimentally induced diabetes and differentially affects atria and ventricles. This is in parallel with the clinically observed imbalances of the cardiac autonomic innervation in diabetic cardiac autonomic neuropathy.

[Ovulatory mucus and its pH, arborization and spermagglutination antibodies in women with fertility disorders]. / Ovulacní hlen a jeho pH, arborizace a spermaglutinacní protilátky u pacientek s poruchou plodnosti.

OBJECTIVE: We studied pH of ovulatory mucus, sperm penetration through capillary filled with ovulatory mucus in one hour and presence of local spermagglutinating antibodies. METHODS: We measured pH, arborization and Kremer test in ovulatory mucus in 127 women with fertility disorder. Indirect mixed antiimunoglobulin reaction test (i-MAR-test for IgG, IgA, IgM and IgE) was used for detection of spermagglutinating antibodies. RESULTS: pH 7.4-9.6 (physiological limit) was found in 94/127 women (74%), pH < 7.4 in 33 women (26%). 60% of 94 patients with physiological pH had Kremer's test above 2cm/hour (normal sperm penetration in ovulatory mucus), in 40% of them reduction of sperm penetration (< 2cm/hour, swelling, shaking, cytotoxicity) was seen. Sperm antibodies in ovulatory mucus in 24% patients with pH < 7.4 and 22% patients with physiological pH were studied. In 111 patients with regular menstrual cycle a classical arborization was found in 81%, in 14% was not proved. In 16 patients with irregular menstrual cycle classical arborization was observed in 11 of them. Local sperm antibodies were detected only in 13% of the total count of patients, it means in 17 patients. Their ovulatory mucus showed classical arborization. 30 healthy fertile women created the control group, only one secretion had pathological findings in all studied parameters owing to latent mycotic infection. SUMMARY: Pathological pH of ovulatory mucus, hormonal dysbalance, and presence of local spermagglutinating antibodies evidently influence penetration of sperm cells through cervix uteri. Otherwise pathological microbial vaginal environment can start a cross reaction with the surface microbes and sperm epitopes. One sign of ovulation, arborization of cervical ovulatory mucus, is not connected directly with the presence of local sperm antibodies, but insufficient estrogen influence is a sign of the reduced immunosuppression in cervix area.

IP3 type 1 receptors in the heart: their predominance in atrial walls with ganglion cells.

Previously we have shown that inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are abundantly expressed in the atria of rat hearts. Since arrangement of atria is very heterogeneous, in this work we focused on the precise localization of IP3 receptors in the left atrium, where the gene expression of the type 1 IP3R was the highest. The mRNA levels of the IP3 type 1 receptors in the left atrium, left ventricle and myocytes were determined using real-time polymerase chain reaction and Taqman probe. For precise localization, immunohistochemistry with the antibody against type 1 IP3Rs was performed. The mRNA of type 1 IP3 receptor was more than three times higher in the left atrium than in the left ventricle, as determined by real-time PCR. Expression of the type 1 IP3 receptor mRNA was higher in the atria, especially in parts containing cardiac ganglion cells. The atrial auricles, which are particularly free of ganglion cells, and the ventricles (wall of the right and left ventricle and ventricular septum) contained four to five times less IP3 receptors than atrial samples with ganglia. IP3R type 1 immunoreactivity detected by a confocal microscope attributed the most condensed signal on ganglionic cells, although light immunoreactivity was also seen in cardiomyocytes. These results show that type 1IP3 receptors predominate in intrinsic neuronal ganglia of cardiac atria.

[Significance of determination of intra-acrosomal proteins and sperm antibodies in human reproduction]. / Význam urcování intra-akrosomálních proteinu a protilátek proti spermiím v lidské reprodukci.

OBJECTIVE: Comparison of the positive intra-acrosomal proteins and spermagglutinating antibodies in human semen samples from various groups of patients. DESIGN: Prospective study. SETTING: Department of Gynecology and Obstetrics and Faculty Hospital, Charles University, Pilsen, Institute of Molecular Genetics, Czech Academy of Science, Prague. METHODS: Monoclonal antibodies Hs-8 and Hs-14 (prepared in the Institute of Molecular Genetics, Prague) were used for detection of intra-acrosomal sperm proteins. Microscopic immunofluorescent methods detected the incidence, the character and the percentage of the spermatozoa specified by above-mentioned monoclonal antibodies. Direct mixed anti-immunoglobulin reactions test (MAR-test) for IgG, IgM, IgA, IgE was used for detection of spermagglutinating antibody. We examined 315 infertile patients from Special Consultation for Immunology of Reproduction and from the IVF programme, and sperm healthy donors (January 2002-March 2003). RESULTS: Native donor's sperm cells had excellent positive intra-acrosomal proteins stained with monoclonal antibodies Hs8 and Hs14 and after thawing as well as. No spermagglutinating antibodies were found. In the group with normal sperm count and light microscopic morphology we found the presence of seminal spermagglutinating antibodies in 11% (IgG), in 14.5% (IgA), in 3.6% (IgM), in 5.2% (IgE). Significant positivity of intra-acrosomal protein stained with Hs8 monoclonal antibody was reached in 68.4%, and with Hs14 monoclonal antibody in even 81.3% of men. On the other hand, in oligoasthenospermatic patients we found significant increasing of spermagglutinating antibodies (for IgG 40.5%, for IgA 28.6%, for IgM 9.5%, for IgE 11.9%). Dominant good staining of intra-acrosomal proteins were seen only in 15.5% of men (for Hs8) and in 20.2% (for Hs14). CONCLUSION: The quantitative detection of intra-acrosomal sperm proteins and spermagglutinating antibodies are used as important properties of human semen and serve for evaluation of acrosomal state, and male fertility together.

Jun oncoproteins do not function as primary transcription factors for the mouse major histocompatibility complex class I H-2 genes in fibroblasts.

There are several reports in the literature focusing on regulation of major histocompatibility complex (MHC) class I genes by transcription factors of the jun family. The methods employed in these reports differed in various respects, and their results are inconsistent. In mouse Lewis lung carcinoma, B16-melanoma and F9-teratocarcinoma cell lines, c-jun was characterized as a transcriptional activator of the murine MHC class I H2-Kb gene, while c-jun was identified as a direct transcriptional repressor of the swine class I PD1 gene, and c-jun stably transfected clones of mouse L-fibroblasts markedly reduced their H-2 class I gene expression. In this study, we attempted to reproduce this last effect by means of transient transfection coupled to Northern hybridization, upon transfecting L-fibroblasts with expression vectors for all jun family members as well as with an array of c-jun-derived dominant negative mutants. No change in H-2 class I expression could be identified. Next, we derived two additional fibroblastic cell lines from the fibrosarcoma of the H2-Kk/v-jun transgenic mouse and transfected them with the two most potent c-jun dominant negative mutants, again without eliciting any change in H-2 class I mRNA level. We conclude that the negative regulation of H-2 class I genes by c-jun in cells of the fibroblastic lineage is not a primary effect.

[Hormonal regulation of gene transcription--specificity and regulation of the hormonal response and results of its dysfunction]. / Hormonální regulace genové transkripce--specificita a regulace hormonální odpovedi a dusledky jejího porusení.

Nuclear hormone receptors act as ligand activated transcription factors. Hormonal response in vivo is, however, modulated at various levels beyond the mere activation of hormone receptors by their respective ligands. There is a great variability of particular arrangements of HRE and receptors of the TR/RAR subfamily use extensive dimerization to produce a particular dimer with optimal affinity for HRE in question. Hormonal response is mediated by complex hormone responsive units (HRU), which integrate HRE together with other regulatory sequences. Some hormone responsive genes may themselves code transcription factors or other regulatory proteins, underlying delayed primary and secondary responses. In addition to transcription activation, nuclear receptors enter repressive interactions with unrelated transcription factors like AP-1 or NF-kappa B. Genes for coding nuclear receptors are subjected to homologous downregulation. Functioning of nuclear receptors might be modulated by phosphorylation, which can underly ligand-independent activation. Nuclear receptors are mutated and/or disregulated in various pathologies, like hormone resistance syndromes of cancer.

Localization of gamma-tubulin in interphase and mitotic cells of a unicellular eukaryote, Giardia intestinalis.

Giardia intestinalis, a bi-nucleated amitochondrial flagellate, possesses a complex cytoskeleton based on several microtubular systems (flagella, adhesive disk, median body, funis, mitotic spindles). MTOCs of the individual systems have not been fully defined. By using monoclonal antibodies against a conserved synthetic peptide from the C-terminus of human gamma-tubulin we investigated occurrence and distribution of gamma-tubulin in interphase and mitotic Giardia cells. On the immunoblots of Giardia cytoskeletal extracts the antibodies bound to a single polypeptide of approximately 50 kDa. Immunostaining of the interphase cell demonstrated gamma-tubulin as four bright spots at the basis of four out of eight flagella. Gamma-tubulin label was associated with perikinetosomal areas of the ventral and posterolateral pairs of flagella which are formed de novo during cell division. Basal body regions of the anterolateral and caudal pairs of flagella which persist during the division and are integrated into the flagellar systems of the daughter cells did not show gamma-tubulin staining. At early mitosis, gamma-tubulin spots disappeared reappearing again at late mitosis in accord with reorientation of parent flagella and reorganization of flagellar apparatus during cell division. The antibody-detectable gamma-tubulin epitope was absent at the poles of both mitotic spindles. Albendazole-treated Giardia, in which spindle assembly was completely inhibited, showed the same gamma-tubulin staining pattern thus confirming that the fluorescent label is exclusively located in the basal body regions. Our results point to a role of gamma-tubulin in nucleation of microtubules of newly formed flagella and indicate unusual mitotic spindle assembly. Moreover, the demonstration of gamma-tubulin in Giardia shows ubiquity of this protein through the evolutionary history of eukaryotes.

[Hormonal regulation of gene transcription--nuclear hormone receptors as ligand-activated transcription factors]. / Hormonální regulace genové transkripce--jaderné receptory jako ligandem-aktivované transkripcní faktory.

Nuclear hormone receptors regulate gene transcription upon recognizing specific regulatory sequences--hormone responsive elements (HRE) in gene promoters, enhancers, and silencers. Receptors for sexual and adrenal steroid hormones, thyroid hormone, retinoic acid and vitamin D, as well as an extensive group of orphan receptors, all exhibit strong homology in structural and functional organization of the molecule and they form together the nuclear receptor superfamily. While classical steroid receptors dissociate upon activation an inhibitory hsp-complex, dimerize and then are able to bind cognate HRE and activate transcription, thyroid and retinoid receptors as well as the vitamin D receptor bind the HRE in question constitutively and activation is represented by dissociating a corepressor and recruiting a coactivator. Similar mode of action applies also for orphan receptors. Some of recently resolved orphan receptors are activated by immediate products of metabolism; the metabolism of cholesterol relies in great part on selective activation of a particular orphan receptors by a particular cholesterol metabolite.

Primary intestinal epithelial cells can be infected with laboratory-adapted strain HIV type 1 NDK but not with clinical primary isolates.

Infectivities of HIV-1 primary isolates and laboratory-adapted strains were compared in primary fetal enterocytes and the colonic epithelial cell line HT29. Infection by two laboratory strains, HIV-1 NDK and HIV-1 NDK(A4), which were adapted on CEM and HT29 cells, respectively, produced significant amounts of virus in both target cell systems. Intestinal cells were resistant to infection with HIV-1 primary isolates regardless of their genetic subtype or SI/NSI phenotype. Biological properties of analyzed viruses rather than differences in cultivation system seem to be responsible for differences between these in vitro and ex vivo results.

Epstein-Barr virus nuclear antigen type 1 binding: electron microscopy.

Epstein-Barr virus (EBV) nuclear antigen type-1 (EBNA-1) was extracted and purified from Raji cells by chromatography on DNA-Sepharose and Blue-dextran Sepharose. Its complexes with plasmid pM765-10 derived from EBV (strain M-ABA) DNA were visualized by electron microscopy. The criteria of specificity were as follows: (1) preferential binding of EBNA-1 to the ori-P region of pM765-10; (2) specific enlargement of EBV DNA/EBNA-1 complexes with anti-EBNA-1 (IR-3) IgG antibody; and (3) resistance of the resulting EBV DNA/EBNA-1/anti-EBNA-1 antibody complexes to treatment with 1.5 M NaCl. The optimal conditions for the formation of EBV DNA/EBNA-1 complexes were 50 to 150 mM NaCl and pH 6.0. A balanced equilibrium of EBNA-1 and pM765-10 was necessary to achieve both a high yield and specificity of EBV DNA/EBNA-1 complexes.

Electron microscopy of binding of Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) to EBV DNA.

Specific binding sites for Epstein-Barr virus (EBV) nuclear antigen (EBNA-1), isolated and semipurified from EBV-transformed nonproductive Raji cells, were visualized on the molecule of EBV DNA by electron microscopy and mapped. Two measures had to be applied to counteract the limited purity of the EBNA-1 preparation: (i) EBV DNA/EBNA-1 complexes were specifically enlarged by binding with anti-EBNA-1 (IR-3) IgG antibody. (ii) DNA-binding proteins that did not react with the anti-EBNA-1 antibody were eluted from EBV DNA with 1.5 M NaCl, taking advantage of the resistance of DNA/EBNA-1/anti-EBNA-1 antibody complexes to the high-salt treatment. EBNA-1 bound at the highest relative frequency (greater than 30%) to the EBV DNA map positions of 8.8 +/- 0.3, 10.3 +/- 0.5, and 46.6 +/- 1.2 kb. It bound with a lower but still statistically significant frequency (16%) to the map positions of 64.5 +/- 1.0, 89.7 +/- 1.6, 129.6 +/- 1.1 kb.

Circular DNA and cardiolipin in hydrogenosomes, microbody-like organelles of trichomonads.

Circular molecules of DNA approximately 3 mum in length were revealed by electron microscopy in deproteinized extracts prepared from purified hydrogenosomal fraction of a protozoan Tritrichomonae foetus. This fraction contained also cardiolipin amounting to approximately 14.4% of its total phospholipids, as detected by thin-layer chromatography and quantitative phosphorus measurement. These characteristics extend a number of biochemical properties of hydrogenosome shared also by mitochondria and by prokaryotic cells.

Structure of herpes simplex virus DNA: topography of the molecule. I. Absence of circularly permuted sequences.

Only linear structures were produced by re-annealing denatured herpes simplex type 1 virus (HSV)-DNA molecules, while T4-DNA molecules (known to be circularly permuted and therefore used as controls in the parallel tests) formed circles. These results suggested that HSV-DNA is a nonpermuted collection of sequences. Unfortunately, most of the linear structures observed after re-annealing were not full-length duplexes, thus making this test not quite satisfactory. Therefore another permutation test, the central region deletion experiment, was employed. Its results indicated that short fragments liberated from the ends of HSV-DNA by enzymatic treatment contained only a portion of the sequences present in the complete molecules. On the other hand, the presence of apparently all sequences was found in the short end-fragments of the control T4-DNA. These data also provide evidence that HSV-DNA molecules are not formed by circularly permuted collections of sequencies.