Expression and secretion of the beta subunit of human chorionic gonadotropin by bladder carcinoma in vivo and in vitro.

Expression and secretion of the beta subunit of human chorionic gonadotropin (hCG) by bladder carcinoma cell lines were investigated in vitro and in vivo. As an in vitro study, immunoreactive hCG beta (IR-hCG beta) secreted into the culture media of two bladder transitional cell lines (KoTCC-1 and HT-1197) was analyzed using three kinds of enzyme immunoassays which were specific for intact hCG, free hCG beta, and beta core fragment (beta-CF). Both of the cell lines were determined to secrete IR-hCG beta into the media, which consisted principally of free hCG beta, but detectable levels of intact hCG and beta-CF were not present in the media. Northern blot analysis revealed that the hCG beta gene was expressed in both KoTCC-1 and HT-1197 cells where the sizes of mRNA from these cells were smaller than those from placental and NJG choriocarcinoma cells. As an in vivo study, distribution of IR-hCG beta was analyzed in the tumor tissues, sera, and urine of the mice and the rats transplanted with KoTCC-1 cells. By the immunohistochemical study, the IR-hCG beta was clearly observed in transitional cell carcinoma cells of the transplanted tumor. High levels of IR-hCG beta were detected in both the serum and urine from the animals, but there were quantitative and qualitative differences between serum and urinary IR-hCG beta. Quantitatively, the concentrations of IR-hCG beta in the urine were consistently much higher than those in the serum. Qualitatively, free hCG beta was exclusively detected in the serum whereas high levels of beta-CF in addition to free hCG beta were found in the urine. Intact hCG could not be detected in the serum and urine. These distributions of IR-hCG beta in the animals transplanted with KoTCC-1 cells were completely analogous to those in a patient with hCG beta-producing bladder carcinoma. The present study shows that the same metabolic pathway of IR-hCG beta is operating in mice and rats as in humans, indicating that IR-hCG beta found in patients with bladder carcinoma originates from the tumor and it may be recognized as a tumor marker when beta-CF is measured in the patient's urine.

Comparative nucleotide and amino acid sequence analysis of the sequence-specific RNA-binding rotavirus nonstructural protein NSP3.

NSP3, an acidic nonstructural protein, encoded by gene 7 has been implicated as the key player in the assembly of the 11 viral plus-strand RNAs into the early replication intermediates during rotavirus morphogenesis. To date, the sequence of NSP3 from only three animal rotaviruses (SA11, SA114F, and bovine UK) has been determined and that from a human strain has not been reported. To determine the genetic diversity among gene 7 alleles from group A rotaviruses, the nucleotide sequence of the NSP3 gene from 13 strains belonging to nine different G serotypes, from both humans and animals, has been determined. Based on the amino acid sequence identity as well as phylogenetic analysis, NSP3 from group A rotaviruses falls into three evolutionarily related groups, i.e., the SA11 group, the Wa group, and the S2 group. The SA11/SA114F gene appears to have a distant ancestral origin from that of the others and codes for a polypeptide of 315 amino acids (aa) in length. NSP3 from all other group A rotaviruses is only 313 aa in length because of a 2-amino-acid deletion near the carboxy-terminus. While the SA114F gene has the longest 3' untranslated region (UTR) of 132 nucleotides, that from other strains suffered deletions of varying lengths at two positions downstream of the translational termination codon. In spite of the divergence of the nucleotide (nt) sequence in the protein coding region, a stretch of about 80 nt in the 3' UTR is highly conserved in the NSP3 gene from all the strains. This conserved sequence in the 3' UTR might play an important role in the regulation of expression of the NSP3 gene.

Involvement of H1 and other chromatin proteins in the formation of DNA-protein cross-links induced by visible light in the presence of methylene blue.

The formation of DNA-protein cross-links (DPCs), induced by irradiation with visible light, was studied in methylene blue-treated (MB-treated) chromatin and H1-depleted chromatin. The effects of the MB concentration and radiation dose were studied using sodium dodecylsulphate-chloroform-isoamyl alcohol assay and sodium dodecylsulphate-polyacrylamide gel electrophoresis. Under identical experimental conditions, DPC formation was less in H1-depleted chromatin (70%) than in chromatin (92%). The non-histone proteins and core proteins of chromatin contributed towards DPC formation. Of the core proteins, H2A was more cross-linked than H4, whereas the bands for H2B and H3 melted into one in chromatin and H1-depleted chromatin. In both cases, the gel pattern showed the appearance of two new protein bands with approximate molecular weights of 27 kDa and 29 kDa as a result of histone-histone cross-linking. Viscometric studies showed that the dissociation of the compact structure of chromatin in 2 M NaCl was more extensive in irradiated, MB-treated, H1-depleted chromatin than in irradiated, MB-treated chromatin, indicating a reduction in the amount of DPC formation in H1-depleted chromatin.

Case report: successful use of antifibrinolytic therapy in acquired factor VIII deficiency.

Acquired factor VIII deficiency is a rare immunologic disorder characterized by severe bleeding due to an antibody inhibitor directed against factor VIII. Treatment of this coagulopathy often is ineffective and costly. The authors report a case of acquired factor VIII deficiency in a patient who developed severe recurrent epistaxis. Antifibrinolytic therapy with epsilon aminocaproic acid (EACA) was used to control the epistaxis with excellent results. To the authors' knowledge, this is the first report of the efficacy of EACA therapy in acquired factor VIII deficiency. Use of antifibrinolytic therapy may represent a relatively safe, effective, and inexpensive approach to treating factor VIII inhibitors.

Visible light induced DNA-protein crosslinking in DNA-histone complex and sarcoma-180 chromatin in the presence of methylene blue.

Formation of DNA-protein cross-links by the action of visible light in the presence of methylene blue was studied in calf thymus DNA-calf thymus histone complex and sarcoma-180 chromatin. The extent of cross-link formation decreases with a decrease in the histone to DNA ratio in the DNA-histone complex. In chromatin, it is at a maximum (93%) at a dye to DNA nucleotide ratio (D/P ratio) of 0.04 and is appreciable even at a very low dye concentration (75% at a D/P ratio of 0.0033). Sepharose 4B-CL column chromatography indicates that methylene blue acts as a mediator in the cross-linking process, but not as a linker in the DNA-protein cross-link. Dodecylsulphate-polyacrylamide gel electrophoresis patterns reveal that both histone and non-histone proteins are involved in cross-linking, but to a varied extent. Competition experiments with ethidium bromide demonstrated the necessity of intercalative binding of methylene blue in the formation of DNA--protein cross-links. Viscometric studies in 2 M NaCl indicate that the compact structure of chromatin is stabilized by cross-linking.