hRad9 is a cell cycle checkpoint gene that is up-regulated in breast cancer. We have previously shown that the mRNA up-regulation correlated with tumor size and local recurrence. Immunohistochemical studies were made to better define the role of hRad9 in breast carcinogenesis. Localisation of hRad9 protein were performed on paired tumor and normal breast tissues. Immunoblotting with and without dephosphorylation was used to define the protein isolated from breast cancer cells. Increased hRad9 protein was observed in breast cancer cells nucleus compared to non-tumor epithelium. This nuclear protein existed in hyperphosphorylated forms which may be those of the hRad9-hRad1-hHus1 complex. Finding of hyperphosphorylated forms of hRad9 in the nucleus of cancer cells is in keeping with its function in ameliorating DNA instability, whereby it inadvertently assists tumor growth.
Factor VIII related antigen (VIII-RAG) can be demonstrated in cultured human endothelial cells (EC) from umbilical cord by immunofluorescence. 75Se-Methionine tracer added onto culture medium showed that radioactive 75Se was incorporated into VIII-RAG as evidenced by Laurell Crossed Immunoelectrophoresis (CIE) and autoradiograph. This indicates that EC synthesizes VIII-RAG in culture, confirming the previous findings of Jaffe et al (1973) and Shearn et al (1977). 75Se- VIII in medium had similar CIE pattern as normal plasma VIII, normal ristocetin cofactor activity (RCoF), but no procoagulant (VIII-C) activity. However, 75Se-VIII in cell sonicate (EC-VIII) migrated faster on electrophoresis and had no RCoF or VIII-C activities. The EC-VIII was compared to factor VIII dissociated into subunits by cleavage of SH bonds. It can be concluded that VIII-RAG is synthesized as monomers in EC and polymers are formed on secretion. Hence, defect in VIII-RAG may arise from defective synthesis or defective assembly mechanism in the EC.
Heparin antithrombin III binding was studied by crossed immunoelectrophoresis. In plasma and purified antithrombin III standard, multicomponent patterns were obtained with low concentrations of mucosal heparin. There is evidence that antithrombin III may bind more than one heparin molecule. At high heparin concentration (greater than 16 U/ml), single symmetrical peaks were obtained. Serum samples showed two antithrombin III peaks due to a decreased heparin binding of the slower peak (2.1-3.9 times), which was probably antithrombin III-activated procoagulant complexes. Heparin analogue (A 73025) also bound antithrombin III in vitro but the mobility of the peak was slower than with mucosal heparin and only a single peak was obtained in serum samples. Radioimmunoassay showed a decreased binding of antithrombin III antibody to heparin-antithrombin III complex. Venous occlusion to the forearm resulted in a slow second peak in the plasma. Heparin therapy gave rise to a double peak in the plasma antithrombin III profile and with continuous infusion, quantitative decreases were noted in all subjects studied, two of whom rethrombosed at the end of 7 days therapy.
Tight junction (TJ) constitutes the barrier by controlling the passage of ions and molecules via paracellular pathway and the movement of proteins and lipids between apical and basolateral domains of the plasma membrane. Claudins, occludin, and junctional adhesion molecules are the major three transmembrane proteins at TJ. This study focuses a newly identified mammalian TJ gene, claudin‐19, in kidneys. Mouse claudin‐19 composes of 224 amino acids and shares 98.2% and 95% amino acid homology with rat and human, respectively; the most evolutionary‐related claudins are claudin‐1 and ‐7, which share ∼75% DNA sequence homology with claudin‐19. Claudin‐19 is abundantly expressed in the mouse and rat kidneys among the organs examined by Northern blots, and to a much less extent, also found in brain by RT‐PCR. Claudin‐19 and zonula occludens‐1 (ZO‐1) are localized at junctional regions of Madin–Darby canine kidney (MDCK) cells by immunofluorescent microscopy. In addition, ZO‐1 is found in the claudin‐19‐associated protein complexes in MDCK cells by co‐immunoprecipitation. Using aquaporin‐1 and aquaporin‐2 antibodies as markers for different renal segment, strong expression of claudin‐19 was observed in distal tubules of the cortex as well as in the collecting ducts of the medulla. To less extent, claudin‐19 is also present in the proximal tubules (cortex) and in the loop of Henle (medulla). Furthermore, intense claudin‐19 immunoreactivity is found co‐localized with the ZO‐1 in kidneys from postnatal day 15, day 45, and adult rats and mice. Similar localizations of claudin‐19 and ZO‐1 are also observed in human kidneys. Since these renal segments are mainly for controlling the paracellular cation transport, it is suggested that claudin‐19 may participate in these processes. In human polycystic kidneys, decreased expression and dyslocalization of claudin‐19 are noticed, suggesting a possible correlation between claudin‐19 and renal disorders. Taken together, claudin‐19 is a claudin isoform that is highly and specifically expressed in renal tubules with a putative role in TJ homeostasis in renal physiology.
Five men and three women with active acromegaly were treated with bromocriptine. After three months' therapy (30 mg/day) mean GH during the day decreased by 50% in six out of eight subjects. In the remaining two subjects (non-responders) GH was persistently over 100 micrograms/l. Mean GH during glucose tolerance test were not significantly decreased in three out of the eight subjects, of whom two were the nonresponders. The minimum dose of bromocriptine required to achieve maximum GH suppression ranged from 7.5 to 20 mg/day. In contrast, serum prolactin (PRL) throughout the day suppressed significantly in all subjects after 5 mg/day bromocriptine. Decreases in clinical symptoms, hand volume, urinary hydroxyproline and calcium excretion were seen in about half of the subjects. Three of the four subjects with diabetes mellitus showed improvement in glucose tolerance. Although minor side effects were uncommon, one patient died because of massive gastrointestinal haemorrhage from a duodenal ulcer.
A clonal-specific polymerase chain reaction technique to detect T-cell receptor delta gene rearrangement in acute lymphoblastic leukaemia (ALL) and non-Hodgkin's lymphoma (NHL) was evaluated. It was applied to detect minimal residual disease. A sensitive and specific technique to detect minimal residual disease for T-cell malignancies was explored. Southern analysis and polymerase chain reaction (PCR) were used to detect the rearranged V-D-J segment of T-cell receptor delta (TCR delta) gene from malignant cell specimens of patients with leukemia and lymphoma of T-cell lineage. The PCR product was sequenced and from the DNA sequences of the V-D-J region, a 3' anti-sense primer was designed and synthesized for clonal specific PCR (CS-PCR). T-cell receptor delta (TCR-delta) gene rearrangement was studied in 40 cases of acute leukaemia and lymphoma of T-cell lineage at diagnosis. Using Southern analysis, the positive rates were 28 and 32% for the 18 T-lymphoma and 22 T-ALL, respectively. A one stage Polymerase Chain Reaction (PCR) technique was used to detect the rearrangement in Southern positive cases and the PCR positive rates were 80 and 86%, respectively. The PCR technique had a sensitivity of 0.1%. Serial follow-up marrow specimens were available from 4 T-ALL patients following chemotherapy for monitoring of minimal residual disease. Their PCR products were DNA sequenced. A 3' primer was designed for each case for a clonal specific (CS) PCR. The technique had a sensitivity of 0.003%. It was applied to detect minimal residual disease in serial follow-up marrow samples. The first patient had persistent negative CS-PCR results and enjoyed continuous remission for more than 3 years. The second patient with negative one stage PCR but positive CS-PCR results had eventual relapse of leukaemia. The other two patients never achieved a morphological remission. These preliminary results appeared to support the usefulness of these PCR techniques in detecting minimal residual disease and predicting relapses for ALL. However, further clinical correlation in larger populations of patients is necessary.
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