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An immunocytochemical assay using monoclonal anti-progesterone receptor (PR-ICA) was performed in nonmalignant (N = 57) and malignant (N = 200) breast disorders. The results were analyzed with a computerized system of image analysis referred to as SAMBA and correlated with binding assays (DCC), and with standard histopathologic findings. It was shown that there was a correlation between 1) the PR-ICA and the binding assays (91.5%), and 2) between the binding assays and the multiparametric computerized (SAMBA) analysis of the PR-ICA. It was also shown that SAMBA provides an accurate, reliable, and reproducible evaluation of PR-ICA that is complementary to binding assays and constitutes a standardized method of evaluating the heterogeneity of the progesterone receptor (PR) distribution in tumors. It is concluded that SAMBA analysis of both the PR-ICA and estrogen receptor (ER-ICA) should improve the prognostic evaluation and the prediction of responsiveness to endocrine therapy in breast carcinomas.

A better understanding of the structure and function of C1 requires knowledge of the regions (domains) of the subcomponents that are responsible for Ca2+-dependent assembly. Toward this end, C1-s was digested with trypsin in the presence of Ca2+, a treatment that rapidly degraded the B chain, leaving a 56-kDa fragment comprised of a complete A chain disulfide linked to a small (less than 4-kDa) residual piece of the B chain. The purified fragment, referred to as C1-s-A, was shown by fast exclusion chromatography to be similar to C1-s in its ability to (1) reversibly dimerize in the presence of Ca2+, (2) substitute for C1-s in the formation of C1-r2-s2 tetramers, and (3) associate with C1-r and C1q to form macromolecular C1. Although C1-s-A was itself catalytically and hemolytically inactive, it competitively inhibited the expression of the hemolytic activity of C1-s in a reconstitution assay. When heated in the absence of Ca2+, C1-s exhibited a low-temperature transition (LTT) near 31 degrees C and a high-temperature transition (HTT) near 51 degrees C, similar to those previously observed in the homologous protein C1-r [Busby, T. F., & Ingham, K. C. (1987) Biochemistry 26, 5564-5571]. The midpoint of the LTT was shifted to 58 degrees C in 5 mM Ca2+ whereas the HTT was unaffected by Ca2+. C1-s-A exhibited only a LTT whose midpoint and Ca2+ dependence were similar to those of the LTT in C1-s. The HTT, which was accompanied by a loss of esterolytic activity, was reproduced in a plasmin-derived fragment representing the catalytic domain. These results provide strong support for the structural and functional independence of the catalytic and interaction domains of C1-s and strengthen current models regarding the role of these domains in various interactions. They also provide direct proof for the occurrence of Ca2+ binding sites on the A chain and demonstrate that all or most of the sites on C1-s that are responsible for its interaction with C1-r and C1q are located on the A chain.

<h4>Unlabelled</h4>It is well known that many pathophysiological findings in toxemia of pregnancy are explained by imbalance of coagulation and fibrinolysis system. The purpose of this study is to elucidate a precise role of coagulation and fibrinolysis system in pathogenesis of toxemia of pregnancy.<h4>Subjects and methods</h4>1) Classification of toxemia of pregnancy. Three hundred and thirty seven of toxemia of pregnancy are classified based on the onset period, and incidence of severity of disease and IUGR, rate of genetic factor of hypertension are compared in each group. 2) Platelet factor 4 (pf4) and beta-thromboglobulin (beta-TG), Fibrinopeptide A (FPA), thrombin-ATIII complex, ATIII fibrinopeptide B beta 15-42, D dimer FDP and plasmin-alpha 2 PI complex are assayed. The levels of PGI2, tissue plasminogen activator (tPA) and thrombomodulin (TM) are measured after venous occlusion. Immunoreactivity and biological activity of TM in urine are analyzed. 3) Aminoacid sequence of TM from normal and toxemia of pregnancy are determined by analyzing cDNA for TM. Moreover, TM are synthesized from recombined DNA and enzymological properties of TM obtained from normal and toxemia of pregnancy are compared. 4) Release of PGI2, tPA and TM by addition of thrombin are observed using monolayer culture of endothelial cells from cord. Enzymological properties of purified placental TM are analyzed.<h4>Results</h4>1) The incidence of severe type, IUGR and the rate of patients who possess genetic factors for hypertension are higher in early onset type, suggesting that hypertension is the predominant characteristics in early onset type and that genetic factors for hypertension are tightly involved. 2) All parameters such as platelets, coagulation and fibrinolysis system are elevated in toxemia of pregnancy compared to those in normal pregnancy. Coagulation index that consists of above parameters is well correlated with clinical index that consists of clinical findings (r = 0.7006, p less than 0.0001). The net increase of PGI2, tPA and TM by venous occlusion are decreased along with severity of toxemia of pregnancy. The potency of production of PGI2 from endothelial cells in maternal omentum is impaired in the severe toxemia of pregnancy. Purified TM in urine from normal pregnancy and toxemia of pregnancy has 63K dalton of single band on SDS-PAGE. However, bioactivity/immunoreactivity ratio of TM in early onset type is lower than those in late onset type, and affinity of TM for thrombin and protein C is decreased in early onset type.(ABSTRACT TRUNCATED AT 400 WORDS)

When grown at high osmotic pressure, some strains of Escherichia coli K-12 synthesized substantial levels of free sugar and accumulated proline if it was present in the growth medium. The sugar was identified as trehalose by chemical reactivity, gas-liquid chromatography, and nuclear magnetic resonance spectroscopy. Strains of E. coli K-12 could be divided into two major classes with respect to osmoregulation. Those of class A showed a large increase in trehalose levels with increasing medium osmolarity and also accumulated proline from the medium, whereas those in class B showed no accumulation of trehalose or proline. Most class A strains carried suppressor mutations which arose during their derivation from the wild type, whereas the osmodefective strains of class B were suppressor free. When amber suppressor mutations at the supD, supE, or supF loci were introduced into such sup0 osmodefective strains, they became osmotolerant and gained the ability to accumulate trehalose in response to elevated medium osmolarity. It appears that the original K-12 strain of E. coli carries an amber mutation in a gene affecting osmoregulation. Mutants lacking ADP-glucose synthetase (glgC) accumulated trehalose normally, whereas mutants lacking UDP-glucose synthetase (galU) did not make trehalose and grew poorly in medium of high osmolarity. Trehalose synthesis was repressed by exogenous glycine betaine but not by proline.

Endothelial cells were cultured from bovine adipose microvessels, pulmonary arteries, and aortas. The effects of insulin, IGF-I, and IGF-II (MSA) on glucose uptake, neutral amino acid (AIB) uptake, and thymidine incorporation into DNA by the endothelial cells were determined. Each hormone markedly stimulated all three processes in the microvessel endothelial cells but had no effect on the larger-vessel endothelial cells. In the microvessel cells the monoclonal antiinsulin receptor antibody, Ab 47-9, was observed to specifically inhibit insulin binding in the bovine microvessel cells without having intrinsic activity on the three biologic processes that were stimulated by insulin and the IGFs. When insulin binding was first inhibited by Ab 47-9, dose-response curves for insulin were markedly shifted to the right for glucose uptake, AIB uptake, and thymidine incorporation into DNA. Similar antibody treatment had no effect on dose-response curves of IGF stimulation of any of the three processes. These data further extend the biologic actions of insulin and the IGFs in cultured microvessel endothelial cells. They also suggest that certain functions mutually stimulated by both insulin and the IGFs, ie, glucose uptake, AIB uptake, and thymidine incorporation into DNA, are substantially mediated through homologous receptors.

Male and female C3H/HeNCrl mice were divided into test groups and fed either a purified diet (AIN-76A) or a natural ingredient diet (NIH-07). Lesions of dystrophic cardiac calcinosis (DCC) were found to be more prevalent and more severe in mice fed the purified diet. The cardiac changes, which were similar in nature in both groups of mice, consisted of randomly distributed foci of myocardial mineralization and fibrosis. These lesions were not associated with clinical disease or significant alterations in serum calcium, lactic dehydrogenase, aspartate aminotransferase, alkaline phosphatase or creatine kinase levels. We conclude that AIN-76A purified diet should be utilized with caution in toxicology studies which use mice as experimental subjects, especially if the heart is a potential target organ.

A dysfunctional antithrombin III (ATIII) gene encoding a qualitatively and quantitatively abnormal anticoagulant molecule is responsible for hereditary thrombosis in a Utah kindred [Bock et al. (1985) Am. J. Hum. Genet. 37, 32-41]. Nucleotide sequencing of the entire protein-encoding portion of the cloned ATIII-Utah gene revealed a C to T transitional mutation which converts proline-407 to leucine. Proline-407 is located 14 amino acids C-terminal to the reactive site arginine of ATIII in a core region of the molecule that has been highly conserved during evolution of the serine protease inhibitor (serpin) gene family. The location of this proline in the crystal structure of the homologous serpin alpha 1-antitrypsin suggests that the leucine substitution in ATIII-Utah may interfere with correct folding of the mutant gene product, leading to its rapid turnover and the low antithrombin levels observed in patient plasmas. The Pro-407 to Leu mutation does not interfere with binding of antithrombin III to heparin. Patient antithrombin III, isolated by affinity chromatography on heparin-Sepharose, was reacted with purified thrombin. ATIII encoded by the patient's normal gene formed protease-inhibitor complexes with thrombin, whereas the product of the ATIII-Utah gene did not. The Pro-407 to Leu mutation destroys a restriction site for the enzyme StuI, permitting rapid diagnosis of affected members of the Utah kindred by Southern blotting of genomic DNA.