Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding site.
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cdc25C induces mitosis by activating the cdc2-cyclin B complex. The intracellular localization of cyclin B1 is regulated in a cell cycle-specific manner, and its entry into the nucleus may be required for the initiation of mitosis. To determine the cellular localization of cdc25C, monoclonal antibodies specific for cdc25C were developed and used to demonstrate that in human cells, cdc25C is retained in the cytoplasm during interphase. A deletion analysis identified a 58-amino-acid region (amino acids 201 to 258) in cdc25C that was required for the cytoplasmic localization of cdc25C. This region contained a specific binding site for 14-3-3 proteins, and mutations in cdc25C that disrupted 14-3-3 binding also disrupted the cytoplasmic localization of cdc25C during interphase. cdc25C proteins that do not contain a binding site for 14-3-3 proteins showed a pancellular localization and an increased ability to induce premature chromosome condensation. The cytoplasmic localization of cdc25C was not altered by gamma irradiation or treatment with the nuclear export inhibitor leptomycin B. These results suggest that 14-3-3 proteins may negatively regulate cdc25C function by sequestering cdc25C in the cytoplasm. (+info)
Expression of Recombinant bovine L-selectin in Escherichia coli and insect cells.
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Bovine L-selectin was expressed in bacteria using pGEX vector and in insect cells infected with recombinant baculovirus in order to obtain recombinant protein for preparation of specific antiserum and its functional studies. In bacterial expression, L-selectin fusion protein with glutathione S-transferase was detected in the insoluble fraction with the expected molecular weight of 60 kDa by SDS-PAGE and reacted with anti-bovine CD62L monoclonal antibody in immunoblot analysis. In insect cells infected with recombinant baculovirus, a band corresponding to L-selectin was not observed in SDS-PAGE with protein staining, but they apparently reacted with anti-bovine CD62L monoclonal antibody in immunoblot analysis. Furthermore, the indirect immunofluorescence test revealed that bovine L-selectin was efficiently expressed on the surface of Sf9 cells infected with recombinant baculovirus, and flow cytometric analysis showed that the percentage of CD62L positive cells in bovine PBMC was about 66% and that most Sf9 cells infected with recombinant baculovirus had specific immunofluorescence. (+info)
Intracellular localization and loss of copper responsiveness of Mnk, the murine homologue of the Menkes protein, in cells from blotchy (Mo blo) and brindled (Mo br) mouse mutants.
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Menkes disease is an X-linked copper deficiency disorder that results from mutations in the ATP7A ( MNK ) gene. A wide range of disease-causing mutations within ATP7A have been described, which lead to a diversity of phenotypes exhibited by Menkes patients. The mottled locus ( Mo, Atp7a, Mnk ) represents the murine homologue of the ATP7A gene, and the mottled mutants exhibit a diversity of phenotypes similar to that observed among Menkes patients. Therefore, these mutants are valuable models for studying Menkes disease. Two of the mottled mutants are brindled and blotchy and their phenotypes resemble classical Menkes disease and occipital horn syndrome (OHS) in humans, respectively. That is, the brindled mutant and patients with classical Menkes disease are severely copper deficient and have profound neurological problems, while OHS patients and the blotchy mouse have a much milder phenotype with predominantly connective tissue defects. In this study, in an attempt to understand the basis for the brindled and blotchy phenotypes, the copper transport characteristics and intracellular distribution of the Mnk protein were assessed in cultured cells from these mutants. The results demonstrated that the abnormal copper metabolism of brindled and blotchy cells may be related to a number of factors, which include the amount of Mnk protein, the intracellular location of the protein and the ability of Mnk to redistribute in elevated copper. The data also provide evidence for a relationship between the copper transport function and copper-dependent trafficking of Mnk. (+info)
Primary biliary cirrhosis associated with painless thyroiditis.
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A case of anti-mitochondrial antibody (AMA)-negative primary biliary cirrhosis (PBC) associated with painless thyroiditis is reported in a 47-year-old woman who diagnosed as PBC based on her elevated serum gamma-glutamyl transpeptidase and immunoglobulin M levels, as well as histological findings of destroyed bile ducts surrounded by mononuclear infiltrates in the biopsied liver. She was negative for AMA and had a depressed level of thyroid-stimulating hormone accompanied by increased free thyrosine, thyroxine and triiodothyronine levels and low titers of anti-microsomal and anti-thyroid peroxidase antibodies. Her thyroid disorder corresponded with painless thyroiditis. An association between PBC and hyperthyroidism is rare. Furthermore, an association between AMA-negative PBC and hyperthyroidism due to painless thyroiditis has not previously been reported. (+info)
Expression of tissue type and urokinase type plasminogen activators as well as plasminogen activator inhibitor type-1 and type-2 in human and rhesus monkey placenta.
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The distribution of mRNAs and antigens of tissue type (t) and urokinase type (u) plasminogen activators (PA) plus their corresponding inhibitors, type-1 (PAI-1) and type-2 (PAI-2) were studied in human and rhesus monkey placentae by in situ hybridisation and immunocytochemistry. Specific monkey cRNA and antibodies against human tPA, uPA, PAI-1 and PAI-2 were used as probes. The following results were obtained. (1) All the molecules tPA, uPA, PAI-1 and PAI-2 and their mRNAs were identified in the majority of the extravillous cytotrophoblast cells of the decidual layer between Rohr's and Nitabuch's striae and in cytotrophoblast cells of the chorionic plate, basal plate, intercotyledonary septae and cytotrophoblast cells of the chorionic villous tree. (2) Expression of uPA and PAI-2 was noted in villous trophoblast whereas tPA and PAI-1 were mainly concentrated where detachment from maternal tissue occurs. (3) No expression of tPA, uPA, PAI-1 and PAI-2 was observed in the basal plate endometrial stromal cells, chorionic plate connective tissue cells, septal endometrial stromal cells or villous core mesenchyme. (4) The distribution of probes observed following in situ hybridisation is generally consistent with the immunofluorescence pattern of the corresponding antigens and no significant interspecies differences were noted. It is possible that both decidual and extravillous trophoblast cells of placentae of human and rhesus monkey are capable of producing tPA, uPA, PAI-1 and PAI-2 to differing extents. Coordinated expression of these genes in the tissue may play an essential role in the maintenance of normal placentation and parturition. The differences in distribution we observed are consistent with the suggestion that coordinated expression of tPA and its inhibitor PAI-1 may play a key role in fibrinolytic activity in the early stages of placentation and separation of placenta from maternal tissue at term. On the other hand, uPA with its inhibitor PAI-2 appears mainly to play a role in degradation of trophoblast cell-associated extracellular matrix, and thus may be of greatest importance during early stages of placentation. (+info)
Anti-perinuclear factor compared with the so called "antikeratin" antibodies and antibodies to human epidermis filaggrin, in the diagnosis of arthritides.
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OBJECTIVE: Antiperinuclear factor (APF), "antikeratin antibodies" ("AKA"), and antibodies to human epidermis filaggrin (AFA), are highly specific serological markers of rheumatoid arthritis (RA), which recognise epitopes on various isoforms of (pro)filaggrin. It was proposed that these antibodies are globally named antifilaggrin autoantibodies. Here the diagnostic value of the detection of each one is compared and the overlap between the three tests evaluated. METHODS: 492 serum samples were tested, including 279 RA serum samples, taken from patients in France and Belgium. APF and "AKA" titres were estimated by indirect immunofluorescence, and AFA titres by immunoblotting on filaggrin enriched human epidermis extracts. RESULTS: By a convenient choice of the positivity thresholds, the diagnostic sensitivity and specificity of the tests were shown to be similar (0.52 and 0.97, respectively). Although the antibody titres were strongly correlated, the associations APF-AFA or AFA-"AKA" permitted more than 52% or 55% of RA to be diagnosed, with a specificity of 0.99. CONCLUSION: APF, "AKA", and AFA detection have a similar diagnostic value. However, because the three tests do not totally overlap, associating APF with "AKA" or AFA with "AKA" can improve diagnostic sensitivity. None of the three antigens used bear all the epitopes recognised by antifilaggrin autoantibodies. (+info)
Comparative study of viral encephalopathy and retinopathy in juvenile sea bass Dicentrarchus labrax infected in different ways.
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The transmission of viral encephalopathy and retinopathy (VER) was investigated in juvenile sea bass (3 g) Dicentrarchus labrax by using cell culture supernatant (SSN-1 cell line) containing nodavirus. Five methods of infection were tested: intramuscular injection (IM), intraperitoneal injection (IP), oral infection, bath exposure and cohabitation of healthy fish with infected fish. Some differences were observed in time of disease onset and severity of symptoms depending on the mode of infection used. Clinical symptoms such as whirling swimming and lethargic or hyperactive behaviour were generally reproduced, except for fish infected via oral and IP infection. First mortalities occurred 3 d after IM and IP infection and 6 d after for the other modes of infection. Cumulative mortalities were also variable: 100% after IM infection, 10% after IP infection, 32% for bath exposure, 43% after cohabitation and 24% via oral infection. Histopathologically, vacuolation was observed in the central nervous tissues and in the retina. The observed lesions were more or less severe depending on the mode of infection, the sampling time and the organs: lesions on the surviving fish (42 days post infection, d p.i.) seemed to be generally more conspicuous in the retina than in the brain of the same fish. In most cases, the presence of nodavirus was confirmed in the same samples of brain and retina by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). The virus was not detected in other organs examined. The present results suggest that 2 forms of VER can be induced: IM injection leads to an acute form (severe nervous disorders with high and fast mortality) whereas oral infection, bath exposure and cohabitation induce a subacute form (less severe disorders and weak daily mortality). This experiment demonstrates experimentally induced horizontal transmission of VER in sea bass for the first time. (+info)
Localization of pestiviral envelope proteins E(rns) and E2 at the cell surface and on isolated particles.
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The glycoproteins E(rns) of classical swine fever virus (CSFV) and E(rns) and E2 of bovine viral diarrhoea virus (BVDV) are shown to be located at the surface of infected cells by the use of indirect immunofluorescence and by cytofluorometric analysis. The positive immunostaining of the cell surface was further analysed by immunogold electron microscopy and it could be shown that only extracellular virions were labelled. Gold granules were not seen at the cellular plasma membrane. In contrast to BVDV E2, the CSFV E2 of virions sticking to the plasma membrane was not accessible to the respective monoclonal antibodies. However, CSFV particles isolated from culture supernatant were able to bind both monoclonal anti-E(rns) and anti-E2 antibodies. For CSFV and BVDV, binding of anti-E(rns) antibodies to the virions was more pronounced than that of anti-E2. This finding was unexpected since E2 is considered to be the immunodominant glycoprotein. (+info)