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Plasma levels of thrombomodulin (TM), fibrinogen, antithrombin III (ATIII) and thrombin ATIII complex (TAT) were studied in healthy young subjects (group A), healthy elderly subjects (group B) and patients with level of TM in group B tended to be higher than that in group A. Levels of TM, fibrinogen and TAT in group C suggested the presence of a hypercoagulable state. When group C was further divided into those with and without diabetes mellitus (DM), the TM level in the former tended to be higher than that in the latter. Furthermore, among the patients with DM, those with diabetic retinopathy showed significantly higher levels of TM than those without retinopathy. Thus, high TM levels indicate the presence of endothelial injury. In groups B and C, TM correlated positively with fibrinogen, and negatively with ATIII, which also indicates that a high TM level is related to a hypercoagulable state. In conclusion, the TM level is considered to be a potential marker of the presence of endothelial injury.

The authors immunohistochemically assessed the presence of estrogen receptor (ER) in formalin-fixed, paraffin-embedded tissue sections of 68 breast carcinomas by an automated method using Pronase (CalBiochem, La Jolla, CA) predigestion and alkaline phosphatase detection (Method 1). These results were compared with those obtained by an automated peroxidase-antiperoxidase method with DNAse pretreatment of fixed embedded sections (Method 2), with ER immunostain on frozen sections (Method 3), and with biochemical results (dextran-coated charcoal cytosolic [DCC] assay). Compared with the DCC assay, Methods 1, 2, and 3 gave sensitivities of 54%, 25%, and 89%, respectively. The sensitivity for Method 1 was increased to 74% in those cases with DCC results showing greater than 50 fmol/mg protein. These findings indicate that ER immunohistochemical studies on formalin-fixed paraffin-embedded tissues (as assayed by Method 1) provide useful clinical information when the results are positive. A negative result, especially if surrounding normal elements are not positive, may indicate no receptors, receptor levels less than 50 fmol/mg protein, or improper tissue preservation. In the absence of fresh tissue for ER assay by DCC assay or of frozen sections for immunostaining, and with an understanding of its limitations, this method may be useful.

Oestrogen receptor (ER) concentrations were measured in 100 breast cancer cytosols using both the dextran coated charcoal assay (DCC with 6--point scatchard plot) and a newly developed enzyme linked immunoassay (EIA) using a monoclonal antibody against ER. The efficiency of the two methods was compared. A highly significant correlation was observed between the ER levels determined by DCC and EIA methods (r = 0.94, p less than 0.001). The mean ER-EIA value (43.25 +/- 74.77) was, however, significantly higher than the mean ER-DCC value (18.00 + 37.27) (p less than 0.001); in both pre- menopausal (p less than 0.001) and post-menopausal (p less than 0.001) groups. Using a cut-off point at 10 fmo/mg protein for ER-EIA and 3 fmol/mg protein for ER-DCC/to distinguish ER +ve and -ve tumours, a 97% concordance between the two assays was achieved. The EIA method appears to be preferable to the DCC method because it is easy to perform, rapid, requires less tissue and does not involve the use of radioactive substances.

Eighty six plasma samples from 41 patients with suspected disseminated intravascular coagulation (DIC) were divided into 3 groups as follows: Group A, 53 samples from established DIC; Group B, 19 samples from possible DIC; and Group C, 14 samples from probable DIC. The following parameters of coagulation and fibrinolysis were evaluated: thrombin/antithrombin III complex (TAT), plasmin/alpha 2 plasmin inhibitor complex (PIC), D-dimer (D-D) and fibrin monomer (FM). In Group A, TAT and PIC were significantly elevated, being 29.5 +/- 20.7 micrograms/l and 7.2 +/- 6.1 mg/l respectively, suggesting marked hypercoagulability and accelerated fibrinolysis. There were no correlations between antithrombin III (ATIII) and TAT, between alpha 2 plasmin inhibitor (alpha 2PI) and PIC, or between TAT and PIC, showing the clinical diversity of patients with DIC. In all groups abnormal TAT, PIC and D-D findings were observed in many samples (86-100%), and FM was present in 83%, 63%, and 29% of samples in Groups A, B, and C respectively. In Groups B and C, abnormal findings for TAT, PIC, D-D, FM and alpha 2PI apparently indicated hypercoagulability and accelerated fibrinolysis, even though fibrinogen and platelet count were within normal limits.

Three cis-acting domains that contribute to the basal promoter activity of the human c-fos gene were identified. One encompasses the serum response element and has been previously described. Another spans an NF1-like site situated at -170. Mutations and in vitro protein binding assays pinpoint this site as the sole basal element of the medial domain. The third, or promoter-proximal, domain can be divided into several distinct sites, one containing a directly repeated GC-rich element and the other consisting of partially overlapping recognition sites for transcription factors ATF/CREB and MLTF/USF. Each of these sites contributes to basal activity as assayed by transient transfections and by in vitro transcription. Consistent with this, several complexes could be visualized between this region and nuclear proteins in vitro and genomic footprinting demonstrated that both elements are constitutively bound in vivo. On the basis of these results, we conclude that all three domains are necessary for full c-fos promoter function.

Genes that act inside the cell to negatively regulate proliferation are of great interest because of their implications for such processes as development and cancer, but these genes have been difficult to clone. This report details the cloning and analysis of cDNA for prohibitin, a novel mammalian antiproliferative protein. Microinjection of synthetic prohibitin mRNA blocks entry into S phase in both normal fibroblasts and HeLa cells. Microinjection of an antisense oligonucleotide stimulates entry into S phase. By sequence comparison, the prohibitin gene appears to be the mammalian analog of Cc, a Drosophila gene that is vital for normal development.

To determine whether dietary sodium intake modifies the generation of adrenal-produced angiotensins and/or their relative proportions, Sprague-Dawley rats were maintained on a low (0.02%), normal (0.4%), or high (1.5%) sodium intake for 5 days. The animals were then killed by decapitation at 0900 h, and their adrenal glands were removed and dissected into two parts: capsular tissue, containing the zona glomerulosa (ZG), and the decapsulated adrenal gland. The tissue was frozen in liquid nitrogen and extracted, and the individual angiotensins [angiotensin-II (AII), angiotensin-III (AIII), angiotensin-I (AI), and Des-Asp-angiotensin-I (Des-Asp-AI)] were separated by HPLC and quantitated by RIA. On a normal sodium intake, the molar contents of the four angiotensins were similar in ZG, ranging from 3.1-6.6 pmol/g, although AII was present in a 60-70% higher concentration than AIII. In the decapsulated adrenal, the concentrations of the various angiotensins were again similar, but the absolute levels (per g tissue) were significantly (P less than 0.02) less than those in the ZG layer. With sodium restriction, the AII content increased more than 2-fold in the ZG, but not in the decapsulated adrenal tissue. In contrast, both AI and Des-Asp-AI significantly (P less than 0.01) decreased with sodium restriction, so that their contents on the low salt diet were only 15-20% of those observed on the high sodium diet. Thus, there was an inverse correlation (P less than 0.001) between the salt content of rat chow and the AII content of the ZG. The correlation between salt intake and AI as well as Des-Asp-AI levels was direct and significant (P less than 0.02). The AIII level in the ZG was similar on all diets. After a lag period, ZG AII increased sharply between 16-48 h of sodium restriction. These data document that sodium intake has a profound effect on the angiotensin content of the ZG, with sodium restriction substantially increasing the levels of AII while reducing the level of its substrate, AI. This also appears to be unique for glomerulosa cells, as in the decapsulated adrenal gland there is little if any change with sodium restriction. We conclude that these sodium-mediated changes in tissue AII production may be involved in the increased responsiveness of glomerulosa cells to aldosterone secretagogues during sodium restriction.

Based on transfection into cells in culture or natural transduction into retroviruses, proto-ras genes seem to derive transforming function either from heterologous promoters or from point mutations. Here we ask how such different events could achieve the same results. To identify homologous regulatory elements, about 3 kilobases of rat DNA upstream of the first untranslated proto-Ha-ras exon was sequenced. Surprisingly, the sequence shares at -1858 a homology of 148 nucleotides with Harvey (Ha) sarcoma virus, 5' of viral ras, signaling possibly a second untranslated proto-Ha-ras exon. In addition the sequence contains a perfect repeat of 25 CA dinucleotides at -2655. A retroviral promoter, even from upstream of the poly(CA), conferred transforming function on proto-Ha-ras and increased transcription greater than 100-fold compared with that of unrearranged proto-ras. Point mutations were not necessary for transforming function of rat and human proto-Ha-ras genes with retroviral promoters but did enhance it greater than 10-fold. A unifying hypothesis proposes that proto-ras genes depend on high expression from heterologous promoters or enhancers for transforming function, which is modulated by ras point mutations. The hypothesis makes two testable predictions. (i) Unrearranged proto-ras genes with point mutations, which occur in some cancers, have no transforming function. Indeed, tumors with mutated proto-ras genes, even those that also lack hypothetical tumor-suppressor genes, are indistinguishable from counterparts with normal proto-ras genes. (ii) Proto-ras genes in transfected cells derive transforming function from heterologous promoters or enhancers acquired via illegitimate recombination from vector DNAs and particularly from viral helper genes that must be cotransfected for transformation of primary cells. Indeed, expression of exogenous proto-ras genes in cells transformed by transfection is as high as for viral ras genes and is much higher than in the cells of origin.

The heparin affinity of normal and two P1 variants of antithrombin-III (AT) was studied by gradient elution with NaCl in Tris buffer on heparin-Sepharose. At pH 7.4 normal AT eluted at [Na+] 0.78 mol/l and the variants both showed increased affinity with AT Pescara eluting at [Na+] 0.86 mol/l and AT Glasgow at [Na+] 0.92 mol/l. We have earlier proposed a model for heparin activation in which the native state of AT maintains a salt bridge involving the P1 Arg-393 residue. Binding of heparin induces a higher heparin affinity conformation in which the salt bridge is disrupted to reveal the reactive centre for inhibition of thrombin. The Glasgow and Pescara variants, lacking a reactive centre P1 basic residue, would be unable to form this salt bridge, and we suggested that the high affinity conformation which they adopt as their native state would resemble the heparin induced conformation. To examine this model, we measured the heparin induced fluorescence of two P1 variants and tested the susceptibility of their reactive loops to catalytic cleavage. Both variants had fluorescence spectra indistinguishable from normal AT. In the absence of heparin, neither variant was more susceptible than normal to catalytic cleavage by human neutrophil elastase. These findings suggest that the conformation of these P1 variants is different to that of fully heparinized normal AT.

Blood coagulation and platelet aggregation were assessed in children with nephrotic syndrome who were divided into the following groups: (1) relapse without treatment: (2) relapse on steroids; (3) early remission; (4) late remission and (5) steroid resistant. The renal histological findings were also recorded. Plasma anti-thrombin III (ATIII) levels were markedly reduced in groups 1 and 2, below normal in group 3 and were normal in groups 4 and 5. There was significant urinary loss of ATIII in groups 1 and 2 as well as in group 5. Plasma fibrinogen fluctuations exhibited the expected negative correlations with plasma ATIII. Reptilase time showed significant prolongation in groups 1, 2 and 3, and was near normal in groups 4 and 5. Platelet aggregation in response to arachidonic acid exhibited aggregation followed by disaggregation in groups 1, 2, 4 and 5, and was normal in group 3. Hyperaggregation in response to decreasing doses of ADP was noted in all patient groups as well as controls with no relationship to serum albumin levels. Aggregation responses to collagen and ristocetin were normal. It is concluded that: 1. The fluctuations in ATIII levels in childhood nephrotic syndrome are determined by the response to steroids and not by the renal histology per se. 2. An acquired fibrin polymerization defect (dysfibrinogenaemia) and an abnormality of the prostaglandin pathway of platelet activation, both reversible, are yet other haemostatic abnormalities in childhood nephrosis. 3. The discrepancies in the literature on haemostatic parameters, specially ATIII in childhood nephrosis, would not have arisen if their fluctuation in relation to steroid therapy as well as the renal histological features of nephrotic syndrome had been documented simultaneously.

Disseminated intravascular coagulation (DIC) or renal damage associated with septicemia was induced in rats by ligating the cecum or by injecting endotoxin. In the septicemia model, the number of E. coli and Bacteroides spp in the blood increased concomitantly with an increase of endotoxin. In this model the development of hypercoagulability with mild fibrinolysis was observed. Histopathologic findings in the kidneys, including the formation of microthrombi in the glomeruli and the vacuolization and dilatation of renal tubular cells, suggest the development of mild DIC. In the endotoxin-induced DIC model, both remarkable state of hypercoagulability and fibrinolysis were observed with fibrin thrombi in glomeruli. The administration of the platelet-activating factor antagonist, CV-6209, or of human antithrombin III, ameliorated DIC significantly by limiting the increases in prothrombin time, activated partial thromboplastin time and fibrin degradation products. These agents significantly reduced the deposition of fibrin in the glomeruli and significantly prolonged the survival time of the endotoxin injected rats. These observations suggest that the PAF antagonist CV-6209 and ATIII merit clinical evaluation in the management of DIC caused by septisemia.