Is the central channel of nuclear pore complex really the exclusive gateway for macromolecular transport?

Examination of many nuclear pore complexes revealed in some of them very thin filaments presumably RNA with accompanying proteins, directing from inner nucleus to the edge of the complex. On one representative micrograph it is shown that this strand continues in the same direction through central part of the complex most probably through the peripheral channel. Next route of the strand is through the tunnel of a hollow rod - subunit of the cytoplasmic ring.

Glycoprotein and protein composition of bovine leukemia virus (BLV).

Highly purified preparation of bovine leukemia virus (BLV) was obtained by the method described in this communication. BLV was isolated from cell-free culture medium, harvested from BLV-producing cell-line FLK. Polyacrylamide gel electrophoresis of BLV proteins and glycoproteins, in comparison with avian myeloblastosis virus (AMV), confirmed high purity of BLV preparations. Molecular weights of some BLV-proteins and glycoproteins were calculated. By employing polyacrylamide gradient 5 to 30% it was found that BLV structural protein p15 contains at least two components.

Growth of mammalian cell lines in a spinner culture.

Several mammalian cell lines, derived from embryonic, normal adult and malignant tissues were adapted for long-term growth in agitated fluid medium. A simple method of spinner culture is described, employing a portable magnetic stirrer. Adaptation of cells for growth in spinner culture took a relatively short time, usually a few weeks, depending on the type of cell line used. Characteristics of cell lines maintained in spinner culture are described in details.

Retrovirus-like particles produced by human embryonal cells and cell lines derived from human malignancies.

Very small amounts of retrovirus-like particles were obtained from tissue culture fluids of various human cell lines. The particles were found in almost all cultures of rapidly growing human cells (embryo fibroblasts, various types of leukemias, melanoma, urinary bladder, lung and mammary carcinomas). Morphology and some biochemical characteristics of purified samples of these particles are presented. The particles resemble C-type mammalian retroviruses.

Retrovirus like particles produced by human embryonal cells and cell lines derived from human malignancies. II. Protein structure.

Very small amounts of retrovirus-like particles were isolated from tissue culture media of various types of human cell lines derived from malignant as well as normal cells. The aim of the present study is the characterization of protein profiles of these isolates. The comparison of protein profiles of isolated human virus-like particles with the profiles of well characterized animal as well as human exogenous (LAV/HTLV-III) retroviruses revealed a 25k protein (p25) to be a major protein or at least one of the protein components of human retrovirus-like particles.

Concentration of retroviruses by low-speed centrifugation.

Concentration of retroviruses from volumes of up to 6 liters of medium by low-speed centrifugation is described. In contrast to pelleting, no damage or aggregation of particles was observed. Surface glycoproteins were also fully preserved. This method enables simple handling of relatively large volumes of medium. Highly purified mouse mammary tumor virus (MMTV) was obtained and its transframe protein p30 in SDS-PAGE was recognized as a double band.

Purification and protein composition of endogenous rat viruses.

Endogenous retroviruses are not in the majority of cases the cause of any neoplasia, except for the laboratory conditions. As far as they might serve for the evolution of pathogenic retroviruses more attention should have been paid to them. In this paper we introduce some approaches to the purification of rat endogenous retroviruses to such a degree of purity that enabled satisfactory SDS-PAGE analysis of its structural proteins. Purities of samples obtained by usual purification methods, long-term isopycnic centrifugation at a high gravity force and velocity centrifugation are compared. Protein profile of rat endogenous virus in SDS-PAGE is compared with the ones of other retroviruses. For the first time the evidence was obtained for the striking similarity between electrophoretic protein profile of rat endogenous virus WERC and feline leukemia virus. The major structural proteins of rat endogenous retrovirus and feline leukemia virus cannot be distinguished even when resolution long gradient PAGE had been employed. The accordance of electrophoretic mobilities of major structural proteins in SDS-PAGE can indicate the relatedness of retroviruses.

An attempt to induce formation of antibodies against endogenous retrovirus BP 6 in syngenic rats.

An attempt was made to induce production of antibodies against C-type endogenous rat retrovirus BP 6. Syngenic Lewis rats were immunized with viable tumor cells, induced with benzo(a)pyrene, continuously producing BP 6 virus. By use of immunoblotting technique, neither short- nor long-term immunization did cause formation of any detectable amounts of antibody against structural proteins of the virus. On the other hand, in immunoblotting, antibodies arised in rats immunized with purified mouse leukemia virus have been found to cross-react with endogenous rat retrovirus.

Flow cytometric analysis of some activation/proliferation markers on human thymocytes and their correlation with cell proliferation.

We immunophenotyped cells from ten human thymuses with emphasis on expression of the CD38 and CD71 antigens. These antigens play role in activation cells and increased expression of them was observed in some leukemia. Simultaneously, certain attention has also been devoted to some further activation markers, e.g. CD25, CD26 and HLA-DR. The classification of leukemia is based on comparison of normal and pathological cells. The study of expression of CD38, CD71 and other markers on thymocytes simultaneously with DNA analysis can be useful for answer if expression of CD38 and CD71 on pathologic cells is a sign of their proliferative ability, a part of immature phenotype in some leukemia, or it is a case of aberrant immunophenotype. In our study, 94% thymocytes were CD38+ and only 16% were CD71+. From our immunophenotypic results including MESF (molecules of equivalent soluble fluorochrome) values and analysis of the cell cycle, the conclusion could be drawn that antigen CD71 can participate in regulation of thymocyte development and presence of both -CD38 and CD71 on pathologic cells will be in all probability the case of aberrant phenotype. We observed a clear correlation of the percentage and MESF values of CD71-positive cells with the cell proliferation only after in vitro thymocytes stimulation with PHA and IL-2. In summary, a strong parallelism was observed regarding the positive relationship between the proliferative rate (assessed by the number of S-phase cells) of stimulated thymocytes and the quantitative (% and MESF) values of some markers - CD71, CD25, CD26 and HLA-DR and negative one with CD38 marker values.

High prevalence of circulating antibodies to MuLV p30 antigen in human sera. An autoimmune response?

To study the possible involvement of a murine leukemia virus (MuLV) related agent in human cancer, an extensive immunoblotting analysis of human sera (cancer, autoimmune as well as control normal ones) for the presence of antibodies to MuLV structural proteins was performed. Out of 350 sera, 89 reacted with gag precursor Pr65, 72 reacted with major viral core protein p30 and five with the matrix protein p15. Antibody reactivity to the env-encoded glycoprotein gp70 was detected in 7 cases out of 16 sera tested. There were no significant differences between pathological and normal sera concerning the patterns and the frequency of the reactivity. Sera from patients with various malignancies (mainly with breast cancer) generally displayed more intensive signals to MuLV p30 than normal sera. Epitope mapping revealed that MuLV p30-reactive antibodies recognize an antigenic determinant(s) located at the carboxyterminus of the protein.

Human monoblastoid cell line U-937 cultured in protein-free medium: immunophenotype, cytochemical and biochemical markers.

Human monoblastoid cell line U-937 was adapted to grow in protein-free (protein-free hybridoma--PFH) medium and cloned by limiting dilution. Resulting cell subline (U-937/PF) cultured in protein-free medium was characterized by immunological, cytochemical and biochemical techniques. There were no major differences in immunophenotype (determined by FACS analysis with monoclonal antibodies directed to HLA and CD antigens) and cytochemical markers between the U-937/PF cells cultured in protein-free cell culture medium and parental U-937 cell cultured in serum-supplemented medium. Maximal cell density was slightly decreased in protein-free culture as compared to the parental cell line in FCS-supplemented medium. Cell viability and cell DNA histograms (determined by propidium iodide cytofluorimetry) showed no major differences between parental U-937 and U-937/PF cells. Phorbol ester (TPA)-induction of differentiation-associated cell markers resulted in a proliferation arrest and accumulation of G0/G1 cells in both sublines. All-trans retinoic acid and, to a lesser extent, TPA-stimulated NBT reduction was higher in parental U-937 cells cultured in serum-supplemented medium as compared to U-937/PF cells. Quantitative differences in the expression and inducibility of some cytochemical markers (beta-glucuronidase, chloroacetate esterase) were found between both examined sublines. Described U-937/PF subline cultured in a protein-free cell culture medium (PFH) appeared as a potential tool for studies of in vitro inducing agents and serum components with differentiation promoting (or inhibiting) activities.

An improved colloidal silver staining method of protein blots on nitrocellulose membranes.

A simple method of revealing proteins on nitrocellulose blots is described. It is based on high affinity of colloidal silver for proteins. The sensitivity is about the same as that of colloidal gold and it appears to be about 20 times higher than the presently used staining methods. The whole procedure is very simple and does not take more than 30 min.

Intimate contacts of mitochondria with nuclear envelope as a potential energy gateway for nucleo-cytoplasmic mRNA transport.

The aim of the present study was to show that close contacts of mitochondria with nuclear envelope need not be just an accidental situation in the cell, but that such contacts could serve for flow of energy from a place of origin directly to a place of consumption. Mitochondria in close proximity to the nuclear envelope can be found virtually in all metabolically active cells. We used transmission electron microscopy to demonstrate this entity in different leukemia cells of human origin (patient's blood) and in mouse leukemia cell line. At high resolution, not only close proximity but even fusion of mitochondrial and nuclear membranes can be seen. Based on available data about mRNA transport through the nuclear pore complex and observed contacts of mitochondria with nuclei, we hypothesize that such contacts can provide a gateway for energy delivery to power mRNA export from the nucleus to the cytoplasm. Hence the lumen of the nuclear envelope can serve for transport or even storage of macroergic molecules in a manner similar to sarcoplasmic reticulum in fast-twitch skeletal muscles.

Replication of DNA in UV-irradiated Escherichia coli in the absence of amino acids.

Since pyrimidine dimers are considered to be the cause of the synthesis of short DNA segments, normalization of DNA replication after UV irradiation should be in a temporal correlation with their removal. This correlation holds in exponentially growing excision-proficient Escherichia coli cells. However, when these cells are preincubated and postincubated without amino acids, synthesis of short segments continues although dimers are efficiently excised.

Hydrolysis of ribonucleoside 2',3'-cyclic phosphates by influenza and Newcastle disease viruses.

Two enzymatic activities hydrolysing ribonucleoside 2', 3'-cyclic phosphates (2', 3'-cNMP) to 2'- or 3'- nucleoside monophosphate were found associated with influenza and Newcastle disease viruses. The two enzymatic activities differed from each other by temperature optima and thermoresistance. 2', 3'-Cyclic nucleotide 3'-phosphohydrolase was responsible for splitting of the substrate to 2'-NMP. Splitting of the substrate to 3'-NMP was due either to ribonuclease or to 2', 3'-cyclic nucleotide 2'-phosphohydrolase.

Dependence of DNA dark repair on protein synthesis in Escherichia coli.

We investigated the influence of amino-acidless treatments applied prior and after UV irradiation (AA-irradiated AA+; AA-irradiated A-; AA+ irradiated AA-) on survival, dimer excision, postirradiation DNA degradation, DNA synthesis and sedimentation profiles of parental DNA of E. coli B/r Hcr+ cells. In dependence on the treatment applied, the fluence 50 J/m2 yielded distinctly different fractions of survivors within 0,03-85%. In all cases dimers were completely excised. The rate of DNA degradation was similar during a 30-40 min period after UV during which the bulk of dimers was excised. Degradation ceased, however, earlier in the prestarved cells than in exponentially growing ones; it was prolonged by aminoacidless postincubation. Sedimentation profiles of parental DNA did not differ during the whole period of dimer excision. In AA+ AA- cells DNA synthesis was not restored for several hours after addition of amino acids. In AA- AA- cells addition of amino acids resulted in a fast resumption of DNA synthesis. We conclude that removal of dimers and repair of gaps were similar in all cases. We believe that aminoacidless treatments influence production and repair of damage to the sites of DNA replication. The treatment appears to prevent this damage when applied before UV irradiation, but interferes with its restoration when applied after UV irradiation. Consequently, the former treatment increases survival of cells while the latter produces an opposite effect.

No significant correlation between specific antibodies to mouse mammary tumour virus and human cancer.

To study the possible involvement of mouse mammary tumour virus (MMTV) related agent in human cancer we analysed 300 samples of human sera for the presence of antibodies to MMTV structural proteins. All sera were tested by immunoblotting to achieve high specificity. Out of 300 sera, 22 reacted with transframe protein p30, 16 with the ribonucleoprotein p14, six with the envelope glycoprotein gp52 and three with the major core protein p27. Reactivities to p30 and p14 were observed in sera from cancer patients and healthy controls; reactivities to p27 and gp52 predominated in sera of cancer patients. Sera frequently reacted with a 42 kDa protein which is a cellular contaminant of the virus.