Mutation screening of the BRCA1 gene in Slovak patients.

Breast cancer is the most commonly observed malignancy in women of the western world. The family history is the strongest risk factor for the disease. Two major genes, BRCA1 and BRCA2 that are involved in the familial breast and ovarian cancer have been described. Germ-line mutations of the BRCA1 gene have been linked to 85% of all hereditary breast and ovarian cancers. We performed a mutation screening ofthe entire codingregion of the BRCA1 gene in 29 Slovak families suspected of having inherited predisposition to breast cancer. For the analysis we used a combination of a single strand conformation polymorphism (SSCP), denaturing high-performance liquid chromatography (DHPLC) and sequencing. Genetic alterations were consistently indicated by SSCP and DHPLC and consequently confirmed by DNA sequencing as previously described pathogenic mutations. The patients with inherited BRCA1 mutations will undergo genetic counseling and cancer prevention health care program.

[Effect of pilocarpine in tissue structures of the anterior eye segment from the aspect of its interaction with free amino acids]. / Mechanizmus úcinku pilokarpínu v tkanivových struktúrach predného segmentu oka z hl'adiska jeho interakcie s vol'nými aminokyselinami.

On the basis of the proofs of pilocarpine interaction with free amino acids, the mechanism of this alkaloid effect in the tissue structures of the eye anterior segment is explained. Differences of colour reactions of amino acids mixtures with pilocarpine, decrease of radioactivity of Na[I125]tyrosine mixture with pilocarpine, unsteadiness of radioactivity of Na[I125]protein A mixture with pilocarpine and rise of 16 radioactivity fractions after adding pilocarpine to Na[I125]proteins A a) show the specificity of interaction of each amino acid with pilocarpine; b) give evidence of a strong covalent bond between pilocarpine and free amino acid and give rise to new biologically active metabolite; c) show non-cholinergic pathway of pilocarpine effect; d) for ophthalmo-physiological practice it is said that with respect to the rate between pilocarpine quantity and concentration and free amino acid quantity and concentration requested therapeutical effect by lower pilocarpine dose in glaucoma disease treatment might be achieved. Thus free amino acids being reactants can be identified as mechanism by which pilocarpine becomes biologically active.

[Bioactivity and effect of a mixture of pilocarpine and lysine on the pupil and intraocular pressure in rabbits]. / Bioaktivita zmesi pilokarpínu s lyzínom na pupilu a vnútroocný tlak ocí králikov.

Based on experiments with rabbit eyes (strain New Zealand white) the authors submit evidence that the metabolite which is formed as a result of interaction of 2% pilocarpine and L-lysine 2 HCl.2H2O has a significantly greater and more rapid effect on the development of miosis than 2% pilocarpine alone. The amino acid lysine does not have a miotic effect. The authors explain the bioactivity of the new metabolite, its effect during the development of miosis and reduction of intraocular pressure. They emphasize a greater and more rapid bioactivity of the new metabolite as compared with pilocarpine. These results provide evidence that during treatment of primary glaucoma it is important to consider not only the effect on reduction of intraocular pressure but also physiological processes which develop in tissue structures after administration of different chemotherapeutic agents.

Immunosuppressive activity of lymphocyte mitogenesis by breast cancer-associated p43.

Placental isoferritin (PLF), an acidic isoform of ferritin, and its unique superheavy chain of 43 kDa (p43) has been described to be synthesized by human breast cancer cells. Physiologically, p43 PLF produced by the placenta is involved in immune suppression of maternal lymphocytes aimed at fetal antigens. A study was carried out to elucidate a paradigm of p43 occurrence in breast cancer patients. Immunosuppression of cytotoxic CD8+ lymphocytes was measured via inhibition of blast transformation in concanavalin A (ConA) stimulated peripheral blood lymphocytes (PBL) using 3H-thymidine uptake in vitro. PBLs were cultivated from 29 women having benign lesions in the breast as well as from 41 patients with breast adenocarcinoma. In breast cancer patients addition of p43 significantly inhibited the activation of lymphocytes proliferation by ConA compared to women with benign tumors. The addition of indomethacin or levamisole did not influence this inhibitory effect of p43 in breast cancer patients. Presence of interleukin-2 in cultures was able to overcome the inhibitory effect of p43 on CD8+ lymphocytes proliferation from women having breast adenocarcinomas and to increase its value in patients with benign lesions.

Expression of p43 associated placental isoferritin (PLF) correlates inversely with cell growth in breast cancer cell lines MCF-7 and T47-D.

We have previously demonstrated that the expression of the recently described immunosuppressive antigen p43 in breast cancer patients correlates with early stages of the disease and a low degree of proliferation of the tumors. Attempts were made to evaluate the expression of p43 in two breast cancer cell lines (MCF-7 and T47-D) stimulated to proliferation by 17-beta estradiol and fetal bovine serum (FBS). p43 expression was determined by RIA technique using the new monoclonal antibody CM-H-9, the rate of proliferation was assessed by [3H]thymidine incorporation during 72 hours of incubation. Induction of proliferation by addition of 17-beta estradiol and FBS to serum-free tissue culture medium correlated with a decrease of p43 synthesis in both cell lines. The level of p43 expression in nonstimulated cells was low in comparison to that in cells cultivated routinely (15% FBS, no estrogen). However, the drop of p43 synthesis was significantly stronger in cell lines with estrogen stimulated proliferation. Our in vitro results confirmed previous clinical observations describing an inverse correlation between p43 synthesis and degree of proliferation and differentiation in breast cancer for the first time. However, the pathologic mechanisms leading to this phenomenon need to be elucidated.

Comparison of two defective hepatitis A virus strains adapted to cell cultures.

The replication of two defective hepatitis A virus strains in cell culture was examined. The w.t. HAS-15 strain growing in FRhK-4 cells produced infectious icosahedral virions 27 nm in size as well as round shaped particles with lipids attached to their surface. The morphogenesis of HAV was membrane-dependent and the detected particles were in various degree of maturation. The MBB 11/5 strain growing in PLC/PRF/5 cells produced mainly noninfectious empty procapsids without RNA genome. The translation of viral proteins was uninhibited in both strains. The reason for restricted replication competence of both strains seemed to be different. In HAS-15, highly efficient encapsidation of the progeny RNA positive-strand lowered the formation of replicative intermediate forms. In MBB 11/5, nearly exclusive empty procapsid production gave evidence for the failure of the VPg primer protein attachment to viral RNA. Changes in the efficacy of viral genome replication were a result of the adaptation of HAV to propagation in vitro.

Radioimmunoscintigraphy of human malignant melanoma. I. Pharmacokinetics and localization of 125I-labeled monoclonal antibody RG-12 in thymectomized mice bearing renal melanoma xenotransplants.

The novel RG-12 monoclonal antibody (MoAb) recognizing a high-molecular-weight antigen of human melanoma cells was radioiodinated and its biodistribution and tumor imaging was determined in immunosuppressed mice bearing xenografted human malignant melanoma HMB-2. Control and tumor-bearing mice were injected with 6 micrograms of 125I-labeled RG-12 IgG (8.9 MBq 125I-IgG/animal). Clearance of the MoAb from plasma had a mean half life of 20.6 hours. At day 2 after injection, radiolabeled RG-12 IgG localized in the tumor was 1.43% of the injected dose bound per gram tissue (ID/g), whereas the localization in the healthy kidney was below 0.5%. Tumor to tissue ratio of MoAb accumulation was low for hepatic tissue (1.25) but high for spleen (3.30) and kidney (3.25), respectively. Scanning with a gamma camera localized tumor mass in the right kidney and implanted peritoneal metastases.

Morphologic and antigenic properties of mouse mammary tumor virus produced in a hormone-responsive fashion by C57Bl/10 mammary tumors of non-viral origin.

Bl-MaTU/A1 mouse mammary tumor cells, derived from a C57B1/10 mammary adenocarcinoma induced by dimethylbenzanthracene and mammotropic hormones, express virus particles and proteins related to mouse mammary tumor virus (MMTV). Immunocytochemical analysis by means of monospecific and monoclonal anti-gp52 sera revealed a different localization of the main structural proteins of MMTV in Bl-MaTU/A1 and GR cells (the latter used as a positive virus-producing control). Immunoelectron microscopy of B-type particles budding from the microvilli of dexamethasone-stimulated Bl-MaTU/A1 cells showed remarkably weak reactivity of the viral envelope with anti-gp52-protein A-gold complexes as compared with that of dexamethasone-stimulated GR cells. Since Bl-MaTU/A1-associated MMTV originates from the amplified unit II of endogenous MMTV, which is altered probably within the env gene, the observed antigenic difference in the Bl-MaTU/A1-associated MMTV may be due to altered synthesis of gp52 glycoprotein in these cells.

Biological and immunological properties of the HMB-2 human melanoma cell line.

The HMB-2 human melanoma cell line derived from a lymph node metastatis is described. After long-term cultivation in vitro the cells retained their morphology, high growth rate, xenotransplantability into immunosuppressed mice and genotypic and phenotypic markers of human melanoma cells. Upon infection of the HMB-2 cells with temperature-sensitive mutant vesicular stomatitis virus (VSVts045) at nonpermissive temperature, a complemented virus pseudotype (VSV(HMB-2] was produced carrying assembled melanoma-associated antigens. Monoclonal antibodies prepared against HMB-2 cell membrane proteins and proteins of purified VSV(HMB-2) particles showed different reactivity with various human tumor cell lines and tissues: While the RG-12 anti-HMB-2 monoclonal antibody recognized a class II tumor-associated antigen present in melanoma and carcinoma tissues, the B-6 anti-VSV(HMB-2) antibody showed selective reactivity with melanoma cells.

[Expression of virus-specific functions in 3-methylcholanthrene-induced leukemia in rats]. / Ekspressiia virusspetsificheskikh funktsii pri leikoze u krys, indutsirovannom 3-metilkholantrenom.

It was isolated and identified MR-Le virus from Wistar rats with myelogenous leukemia. The comparison of isolated in vivo and in vitro MR-Le virus with endogenous oncovirus RaLV in protein profile as well as results of molecular hybridization pointed out that RaLV did not participate in the induction of MR leukemia. The question about the role of MR-Le virus in the etiology of MR leukemia is opened now.

Expression of retrovirus-related functions in the methylcholanthrene-induced rat myelogenous leukemia.

The rat MR-leukemia (MR-Le) induced in Wistar rats by methylcholanthrene and whole-body irradiation, has been shown to be transmitted by means of cell-free filtrates of spleen and liver extracts. These earlier results lead us to determine the expression of retroviral functions by MR-Le myeloblasts in vivo and in vitro. DNA polymerase activity associated with particulate material purified from plasma of rats carrying MR-Le and from MR-Le tissue culture fluid, increased with the number of MR-Le myeloblasts. Interspecies-specific antigenic determinants, common to some mammalian retroviruses, are expressed on the surface of MR-Le cells. Electrophoretic pattern of the MR-Le viral material purified from leukemic plasma and tissue culture fluid revealed the presence of polypeptides with molecular weights related to proteins of mammalian retroviruses which were not identical with main structural proteins of the endogenous rat C-type helper virus (RaLV). The results strongly support the proposition that RaLV-unrelated rat retrovirus is involved in the onset of MR-Le.