Development of new immunoradiometric assay for CA 125 antigen using two monoclonal antibodies produced by immunizing lung cancer cells

CA 125 is an antigen associated with non-mucinous epithelial ovarian cancer, which is defined by OC 125 antibody developed by immunizing ovarian cancer cells. We have produced two monoclonal antibodies, 130-22 and 145-9, by using the human lung adenocarcinoma cell line PC-9. Both 130-22 and 145-9 antibodies recognized CA 125 antigen. However, the binding sites seemed to be separate from those of OC 125. Testing by 9 immunoradiometric assays (IRMA), using different combinations of the 3 monoclonal antibodies 130-22, 145-9 and OC 125 demonstrated that the best standard curve for detecting CA 125 could be obtained by a "simultaneous sandwich" assay based on a mixture of 125I-labeled OC 125 and 130-22 or 145-9 coated beads. One-step IRMA, using 130-22 as a tracer and 145-9 as an immunoadsorbent, also showed good reproducibility and sensitivity for measuring CA 125. Antigens were detectable in the culture supernatants of PC-9 cells and 5 of 6 ovarian cancer and endometrial adenocarcinoma cells. These results indicate that one-step IRMA using 130-22 and 145-9 is useful for detecting CA 125 antigen.     

Diazepam reduces both arterial blood pressure and muscle sympathetic nerve activity in human

It is known that benzodiazepines have a hypotensive effect, but the mechanism has not been well elucidated yet. To clarify whether this effect is due to central or peripheral mechanism, we administered 5 mg of diazepam or saline intravenously to healthy volunteers and assessed the change in blood pressure, heart rate, muscle sympathetic nerve activity and heart rate variability. After diazepam administration, systolic and mean blood pressure decreased significantly. Muscle sympathetic nerve activity was also significantly reduced but heart rate did not change, whereas the variables of spectral analysis of heart rate variability did not show significant change. We concluded that the hypotensive effect of diazepam in human is mainly due to the central mechanism.     

Chymase participates in chronic dermatitis by inducing eosinophil infiltration

An epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) to a mouse ear caused a transient skin swelling, and the repetition of the challenge enlarged the contact dermatitis. The repeated challenge with DNFB also induced eosinophil infiltration on the application site. Administration of a chymase inhibitor significantly inhibited the ear swelling as well as eosinophil accumulation. An intradermal injection of human chymase to the mouse ear also elicited transient skin swelling and eosinophil infiltration, both of which were augmented in proportion to the number of injections. Human serum albumin and heat-inactivated chymase failed to induce such skin reactions, suggesting the participation of proteolytic activity of the enzyme. In addition, chymase stimulated eosinophil migration in vitro in a concentration-dependent manner. Taken together, these observations suggest that mast cell chymase may contribute to development of the DNFB-induced dermatitis, probably by promoting eosinophil infiltration. It is therefore possible that chymase plays a role in pathogenesis of chronic dermatitis such as atopic dermatitis.     

Regulation by Ca2+-calmodulin of the actin-bundling activity ofPhysarum 210-kDa protein

Summary From the plasmodia of a lower eukaryote,Physarum polycephalum, we have previously purified a 210-kDa protein that showed similar properties to those of smooth muscle caldesmon. Further characterization of the 210-kDa protein revealed that it bundled actin filaments. This bundling activity was inhibited by calmodulin in the presence of Ca²⁺. Unlike smooth muscle caldesmon, the 210-kDa protein bundled actin filaments whether or not a reducing agent, such as dithiothreitol, was present. The protein was shown to have two (or more) different actin-binding sites which were classified into salt-sensitive and salt-insensitive sites. Electron microscopy revealed that the 210-kDa protein was an elongated molecule (mean length, 97 ± 25 nm) which was bent in the middle. The Stokes radius and sedimentation coefficient of the 210-kDa protein were 130 Å and 2.9 S, respectively. An immunofluorescence study revealed that the 210-kDa protein colocalized with the bundles of actin filaments in thin-spread preparations ofPhysarum plasmodia, suggesting that the 210-kDa protein was regulating the appearance and disappearance of the actin bundles that are associated with the contraction-relaxation cycle of the plasmodia.     

HISTOPATHOLOGICAL STUDIES ON THROMBOTIC MICROANGIOPATHY WITH SPECIAL REFERENCES TO THREE CASES ASSOCIATED WITH ACUTE PROMYELOCYTIC LEUKAEMIA

Five autopsy cases of thrombotic microangiopathy, including 3 cases associated with acute promyelocytic leukaemia, were examined macroscopically, light-and electronmicroscopically.
The so-called hyaline thrombi In thrombotic microangiopathy were composed of fibrin and its degenerative products. Thrombocytes and other blood cells were not seen in the thrombi.
At the site of the formation of a thrombus, there was no conspicuous change in the walls of the capillaries and arterioles. It was considered, therefore, that the intravascular deposition of fibrin was the primary event in the development of thrombotic microangiopathy.
In regard to the distribution and morphologic findings, there was no basic difference between the microthrombi in cases associated with acute promyelocytic leukaemia and those without it.
The bone marrow and some other organs in cases of thrombotic microangiopathy associated with acute promyelocytic leukaemia macroscopically revealed a green colour. Many thrombi composed of leukaemic cells and fibrin were found in the pulmonary arteries of these cases. Furthermore, prominent erythrophagocytosis in the bone marrow and lymph nodes was a common finding in these cases.     

COX-2+ myofibroblasts may play a key role in marked stromal fibrosis in strictured colorectal carcinomas

AIMS: To elucidate the mechanism of marked stromal fibrosis in strictured colorectal carcinomas (SC) that cause complete ileus. METHODS AND RESULTS: Sixteen cases of SC and 29 cases of non-strictured colorectal carcinoma (NSC) were studied. These carcinomas showed similar clinicopathological features except for bowel stricture. The stricture index (SI) showing the degree of bowel stricture was 59.8 +/- 12.1% in SC versus 20.8 +/- 24.6% in NSC (P < 0.001). The fibrosis index (FI), defined to reflect the extent of stromal fibrosis, was 56.3 +/- 8.8% in SC versus 21.9 +/- 10.6% in NSC (P < 0.001). COX-2+ myofibroblasts were detected in 13 cases (81.3%) in SC versus eight cases (27.6%) in NSC (P < 0.01). The COX-2+ myofibroblast density was 276.7 +/- 181.1 cells/mm(2) in SC versus 26.6 +/- 52.7 cells/mm(2) in NSC (P < 0.001). When all cases were divided into two groups with and without COX-2+ myofibroblasts, the SI was 48.8 +/- 19.1% in those with COX-2+ myofibroblasts versus 24.8 +/- 29.3% in those with COX-2- myofibroblasts (P < 0.001). CONCLUSION: COX-2+ myofibroblasts may play an important role in extensive bowel stricture in colorectal carcinomas.     

Cytokeratin expression in normal human thymus at different ages

Subsets of thymic epithellal cells were examined Immuno-histochemically to determine whether or not their pheno-types change during thymic growth and at early involution in terms of cytokeratin (CK) expression. Five monoclonal antibodies specific for CK4, CK8, CK13, CK18 and CK19 were used and applied for 16 neonatal, three Infantile and one adult thymus speeimen, which had been obtained at autopsy, that were normal macroscopically and microscopicaily. CK4, CK8, CK13, CK18 and CK19 were expressed simultaneously in the cortex, medulla and subcapsular area with the exception of CK4, which showed expression on the adult thymus. Light and electron microscopy showed that CK8 and CK19 expression was overlapped. Thus, It was thought that CK8 and CK19 formed complexes in the cytoplasm of thymic epithelial cells. The Immunoreactivity to CK4, CK13 and CK18 were attenuated or disappeared In the subcapsular area during the early involution stage. Interestingly, two patterns of CK18 expression were observed in the neonatal and Infantile thymus tissues, which Indicated that the thymic microenvironment was changeable even under normal conditions.     

Two case reports of childhood liver cell adenomas harboring beta-catenin abnormalities

The benign epithelial neoplasm liver cell adenoma is rare, especially in childhood. We report 2 such cases, 1 of which was associated with Prader-Willi syndrome. Differential diagnosis of the liver cell adenomas on the basis of histopathologic findings proved difficult and was based on the absence of cellular and nuclear atypia, mitotic activity, and invasive growth. In both cases, immunohistochemical staining demonstrated the nuclear accumulation of beta-catenin, and in 1 case, the tumor cells carried a mutation of the beta-catenin gene. Recently, disregulation of the Wnt/beta-catenin pathway, attributable to abnormalities of the beta-catenin gene, has been reported to be a major event in the development of hepatocellular carcinomas and hepatoblastomas. Our report may be the first to describe the beta-catenin abnormalities in childhood liver cell adenoma. These findings imply that abnormalities of beta-catenin can be an early initiating event in human liver tumorigenesis.     

Isolation, characterization, and disruption of dnr1, the areA/nit-2-like nitrogen regulatory gene of the zoophilic dermatophyte, Microsporum canis.

A homolog of the major nitrogen regulatory genes areA from Aspergillus nidulans and nit-2 from Neurospora crassa was isolated from the zoophilic dermatophyte, Microsporum canis. This gene, dnr1, encodes a polypeptide of 761 amino acid residues containing a single zinc-finger DNA-binding domain, which is almost identical in amino acid sequence to the zinc-finger domains of AREA and NIT-2. The functional equivalence of dnr1 to areA was demonstrated by complementation of an areA loss-of-function mutant of A. nidulans with dnr1 cDNA. To further characterize this gene, dnr1 was disrupted by gene replacement based on homologous recombination. Of 100 transformants analyzed, two showed the results expected for replacement of dnr1. The growth properties of the two dnr1(-) mutant strains on various nitrogen sources were examined. Unlike the A. nidulansareA(-) mutant, these dnr1(-) mutants showed significantly reduced growth on ammonia, a preferred nitrogen source for fungi. These mutant strains were also able to utilize various amino acids for growth. In comparison with wild-type M. canis, the two dnr1(-) mutants showed reduced growth on medium containing keratin as the sole nitrogen source. This is the first report describing successful production of targeted gene-disrupted mutants by homologous recombination and their phenotypic analysis in dermatophytes.