Biomedical subjects
Publications and source records attributed to R Nishimura.
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Prostaglandin E1 (PGE1) is a kind of prostaglandin that produces vasodilation of the peripheral vessels and platelet disaggregation, similar to prostacyclin (PGI2). The effects of PGE1 administered intraarterially or intravenously on peripheral vascular disease have been reported by many authors. In this investigation, 2 experiments were designed to assess the effect of intravenous infusion of PGE1 on the experimental skin flaps in rabbits. In the first experiment, the blood flow in the flap was measured before and after the administration of PGE1 by using the technique of clearance of hydrogen gas, which was generated by electrolysis. The result indicated a biphasic effect of PGE1 on the blood flow in the flap. That is, the low dose (3 micrograms per kilogram per hour) increased the blood flow on the critical line of the dye distance, while the high dose (20 micrograms per kilogram per hour) decreased it. The second experiment, in which the survival length was measured, indicated the practical effect of intravenous infusion of PGE1 on flap survival.
A free skin graft about 12 cm in diameter transplanted after excision of a Bowen's carcinoma on the back totally survived for a long period on seroma and was confirmed to have revascularization from the host skin margin. Repeated evacuations of the fluid and subsequent pressure dressings failed to cause adherence of the graft to the bed, even on the thirty-ninth postoperative day. Histologic examination of the graft and the bed revealed partial epithelialization on the face-to-face surfaces, to which no adherence was attributed. The incomprehensible phenomenon in this unusual clinical case evokes a new interest in the mechanism of free skin graft survival, particularly in the phase of serum imbibition.
Free alpha subunit (PU alpha) was extracted and purified from the urine of normal term pregnant women, and examined for molecular weight, electric charge, affinity to lectins (ConA, RCA and PNA) and the ability to combine with hCG beta in comparison with hCG alpha dissociated from hCG in vitro. The molecular weight of PU alpha was greater than that of hCG alpha in gel chromatography, but smaller in SDS-PAGE. However the free alpha subunits from the urine of patients with cancer were estimated to be larger than those of hCG alpha by both methods. In isoelectric focusing, while hCG alpha exhibited a neutral charge, PU alpha exhibited a negative charge. After treatment with sialidase, both hCG alpha and PU alpha were shifted to the basic region, indicating that they contained terminal sialic acid residues. The affinity to lectins indicated that PU alpha may contain both asparagine-linked and O-linked oligosaccharides, while hCG alpha contains asparagine-linked oligosaccharide only. PU alpha scarcely showed any combining activity with hCG beta, whereas hCG alpha combined actively. The O-linked oligosaccharide, which is not present in hCG alpha, may cause PU alpha to fail to combine with hCG beta.
Human chorionic gonadotropin (hCG) from urine of patients with trophoblastic diseases was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blotting using specific antibodies. Western blotting using anti-hCG beta carboxy-terminal peptide (CTP) revealed that the molecular weights of the beta subunits of the three molar hCG samples were identical to that of standard hCG beta, but those of choriocarcinoma hCG samples were individually different. In the five choriocarcinoma hCG samples, the beta subunits of three samples were apparently larger than standard hCG beta, while those of two samples were smaller than standard hCG beta but larger than desialylated standard hCG beta. These results suggest that structural changes of carbohydrates of hCG, which may be induced by malignant transformation of the trophoblast, can be observed as differences in molecular weight. In SDS-PAGE under nonreducing conditions. Western blotting using anti-hCG beta-CTP revealed a single band corresponding to hCG beta. Under reducing conditions with dithiothreitol (DTT), however, a second low-molecular-weight material (called here CTP') could be observed with anti-hCG beta-CTP. All five choriocarcinoma hCG samples were much more susceptible than standard hCG to cleavage of CTP' by DTT. We conclude that estimations of molecular weight and susceptibility to DTT reduction of urinary hCG by Western blotting using specific antibodies are useful in the diagnosis of choriocarcinoma.
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Human choriogonadotropin [hCG] has two dissimilar noncovalently associated subunits, designated alpha and beta. An ectopically secreted hCG alpha subunit that fails to associate with the beta subunit and displays an anomalously high molecular weight on molecular sieve chromatography but not on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been sequenced. A single substitution of Glu56 by Ala56 has been found in the altered subunit. No evidence for conformational differences between normal and ectopic alpha could be found using circular dichroism or intrinsic fluorescence as measures of secondary and tertiary structure, respectively. Hydrophobicity profiles as determined by the method of Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132) predicted, however, that the hydrophilic segment, Thr54-Ser55-Glu56-Ser57-Thr58, becomes an extension of the preceding hydrophobic segment when Glu56 is substituted with Ala. This solitary hemoglobin S-like mutation may lead to an altered tertiary structure, self dimerization, or an alteration in glycosylation that could be responsible for the ectopic alpha subunit's failure to associate with the beta subunit.
Human choriogonadotropin (hCG) is a heterodimeric hormone composed of an alpha and a beta subunit. hCG and its asialo (ashCG) and deglycosylated (dghCG) forms vary in their ability to stimulate hormone responsive adenylate cyclase. ashCG is a partial agonist, and dghCG is an antagonist. Photoactivatable moieties were coupled to hCG, ashCG, and dghCG, and the derivatives were radioiodinated. Competitive binding studies indicate that all of the derivatives had a similar affinity for the gonadotropin receptor on porcine granulosa cell membranes. Radiolabeled derivatives were used to photoaffinity label the gonadotropin receptor. Radiolabeled complexes were separated by NaDodSO4/PAGE. All of the derivatives produced similar autoradiographic patterns, except that dghCG produced an additional 48-kDa complex. To investigate the structure of the complexes further, peptide mapping of proteolytic digests was used. All, except for the 48-kDa complex, generated similar peptide maps indicating a relationship between those complexes in which the smaller components are part of the larger. The 48-kDa complex contained both subunits of 40-kDa dghCG. Therefore, this complex is expected to contain an additional component of 8 kDa. The complex was generated whether the hormone-receptor complex was photoaffinity labeled on cells, on isolated membranes, or after solubilizing in detergent. Formation was blocked by excess hCG and did not occur in the absence of UV irradiation. We conclude that the hCG derivatives are able to photoaffinity label the hCG receptor but that the dghCG derivative can photoaffinity label an additional component that was not observed when derivatives of hCG or ashCG were used to label the receptor.
A rare case of adenocarcinoma of the sweat glands of the vulva producing isolated alpha-subunit of glycoprotein hormones is reported. By an immunohistochemical method, eccrine gland adenocarcinoma tissues obtained from the right vulva and from its metastatic lesion in the right subclavicular lymph nodes were found to react with human chorionic gonadotropin (hCG), alpha-hCG but not beta-hCG. The concentrations of alpha-subunit in the serum and urine were markedly elevated to 6000 ng/mL and 55,000 ng/mL, respectively, just before the death of the patient.
Human chorionic gonadotropin (hCG) was effectively separated into alpha and beta subunits by treating with 8 M guanidine hydrochloride, followed by high performance gel permeation chromatography (GPC) on a 2.5 X 60 cm TSK G 3000 SW column in 6 M guanidine hydrochloride. Their molecular weights estimated by GPC were about 29,000 daltons for the beta subunit and 19,000 daltons for the alpha subunit. Both subunits were examined for SDS-polyacrylamide gel electrophoresis, amino acid analyses and radioimmunoassay, respectively. Neither subunit showed significant binding to the hCG-receptor fraction of rat Leydig cell in vitro. However, the separated subunits easily recombined and recovered about 90% of receptor binding activity of intact hCG. The present results indicate that the GPC technique is an effective, simple and non-denaturing method for the separation of hCG into alpha and beta subunits.
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To examine whether plasminogen activator reflects the functional state of estrogen receptors in human breast cancer, the enzyme activities were determined in extracts prepared from 160 breast cancer specimens and compared on qualitative and quantitative bases with the levels of steroid receptors, such as cytoplasmic estrogen receptor (ERC), progesterone receptor (PgR) and nuclear estrogen receptor (ERN). With any receptor, plasminogen activator activity was significantly higher in receptor-positive tumors than in receptor-negative tumors. When these breast tumors were categorized into 8 groups in terms of combinations of receptor status, breast cancers which were positive for all these receptors were found to contain the highest plasminogen activator activity. Furthermore, quantitative analyses demonstrated positive correlations of the enzyme activity with either ERC content (correlation coefficient +0.37, P less than 0.001) or PgR content (correlation coefficient +0.45, P less than 0.001). These results strongly suggest that plasminogen activator can be used as an effective functional marker for hormone dependence in human breast cancer.
Activity of transforming growth factor (TGF) in urine was examined by the use of cloned BALB/3T3 A31-1-1 indicator cells. When urine was extracted by an acid-ethanol method, fractionated on a Bio-Gel P-100 column, and analyzed for TGF activity, samples from healthy persons showed no activity, but 80% of the samples from advanced cancer patients had the activity. Activity observed in tumor-bearing patients was eliminated after therapy. Further analysis was carried out in a much simpler way by the fractionation of urine samples after only dialysis and lyophilization. An extensive survey of TGF activity in urine using BALB/3T3 indicator cells revealed weak or intermediate (19%) activity in healthy persons and high (56%) or intermediate (31%) activity in advanced cancer patients. When urine samples were analyzed and compared using both BALB/3T3 cells and NRK 49F cells, the BALB/3T3 cells were shown to form colonies in response to beta-type TGF but not to alpha-type TGF. The present results are the first demonstration of an elevated level of beta-type TGF in urine from cancer patients.
Serum levels of CA15-3, a mammary tumor associated antigen recognized by two different murine monoclonal antibodies (115D8 and DF3), were investigated in patients with mammary carcinoma and other benign or malignant diseases. The reference value of the serum CA15-3 level was obtained as 24 units/ml at the 99% confidence limit among healthy individuals (n = 462). Elevation of serum CA15-3 levels was observed in 24.3% of overall patients with mammary carcinoma. Serum CA15-3 levels in breast cancer patients correlated with the clinical stage; higher percentages of positivity were observed in those with advanced breast cancer (stage IV, 64.7%, recurrent, 52.4% and metastatic, 70.3%). Furthermore, elevated serum CA15-3 levels in breast cancer patients responded well to the effect of therapy. Although the serum CA15-3 test gave percentages of positivity of breast cancer similar to those found by the serum CEA test, the serum CA15-3 test revealed lower percentages of positivity than the serum CEA test among patients with benign breast lesions, liver cirrhosis or other carcinomas. These results suggest that the serum CA15-3 antigen level provides a very useful marker for diagnosis and clinical monitoring of patients with breast cancer.
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We carried out studies to determine whether the Tritiated Thymidine Labeling Index (TLI) would correlate with hormone receptors as well as with clinical and histological data. Sixty-four patients with breast cancer were the subjects studied. TLI showed no relationship to age, menopausal status, T-factor, n-factor, stage, or histological type. However, compared to tubule formation, nuclear pleomorphism, and mitotic activity, TLI showed a positive correlation with each and accordingly there was a positive correlation between TLI and Bloom's histological grading. The cumulative disease-free rate at three years was higher in case of a TLI below 4.0 (median TLI value). TLI significantly correlated inversely with the binding sites not only of cytoplasmic estrogen receptors (ERC), but also of cytoplasmic progesterone receptors (PRC) and nuclear estrogen receptors (ERN). Cancers negative for all three receptors indicated the highest TLI, while cancers positive for all three receptors showed the lowest TLI. The results suggest that breast cancers positive for hormone receptors were of low malignancy because lower TLI related to a lower proliferative activity. Thus, TLI is an useful parameter for assessing the clinical status of breast cancers.