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Sixty-two selected breast cancers were used to compare the conventional dextran-coated charcoal (DCC) assay with a new method of separating progesterone receptor by isoelectric focusing in flat beds of agarose gel. Ninety assays were performed. Isoelectric focusing indicated correctly the presence of receptor in 92% and the absence of receptor in 86% of assays, when compared with the DCC assay. The relationship between the results of the two methods was linear. Isoelectric focusing underestimated receptor to a variable extent, finding relatively less receptor at higher absolute levels of binding than at lower levels. The lower limit of sensitivity of isoelectric focusing was 30 fmol/ml cytosol. The protein concentrations of cytosols prepared from 46 needle biopsy samples (mean weight 25 mg) ranged from 0.5 to 30 g/l (median 4 g/l, 10th percentile 0.75 g/l). Isoelectric focusing is a satisfactory method of progesterone receptor measurement and can be applied to samples too small for conventional techniques.

Direct and extensive sequencing of RSV RNA genome is reported. More than 10,000 nt of the T1 RNase resistant RSV RNA fragments (1) have been sequenced and shown to cover 3900 nt of RSV genome. The frequent sequence variations found indicate that RSV supports a very high incidence of spontaneous mutations in the course of replication, one very probable cause of the genetic diversity among the avian retroviruses. Sequences of the structured RSV RNAs allowed us also to precisely characterize the structured domains of the retroviral genome and show that the src gene is not structured.

With current technological advances, it is now possible to measure in less than 50 microL of plasma picomolar amounts of circulating products of platelet activation, products of protease activation related to coagulation and fibrinolytic pathways, and prostaglandin metabolites formed during a physiologic or pathologic process. Most of these markers, which circulate in blood in nanogram or picogram amounts per milliliter during or after pathologic activation, provide pertinent information on the status of a patient in terms of specificity and early detection, and will be of crucial value in the diagnosis of hemostatic defects and the management of newer antithrombotic drugs that cannot be monitored by currently available assays. Currently, 125I- and 3H-based simple radioimmunoassays are available for platelet factor 4, beta-thromboglobulin, fibrinopeptide A, B beta 15-42 related peptides, thromboxane B2, and the prostaglandins 6-keto-PGF1 alpha and PGE2. Nonisotopic methods such as enzyme-linked immunosorbent assays and fluoroimmunoassays are being developed. Serotonin and ADP, products of platelet activation, are measurable by liquid-chromatographic, immunoenzymatic, and spectrophotofluorometric methods. Analytical methods for fibrin split products (fragments D and E) and serine protease inhibitor complexes such as thrombin-antithrombin-III, factor Xa-antithrombin-III, and kallikrein-C1-esterase are also being developed. We have evaluated all of these methods and found them to be very sensitive to those components of endogenous activation of the hemostatic system listed above.

The synthesis of the C-terminal nonapeptide of bovine insulin B-chain is described. 4-(Bromomethyl)-3-nitrobenzoylglycyl-poly(ethylene glycol) Mr = 15,000) was used as soluble support. The C-terminal alanine was first converted to Boc-Ala-O-(2-nitro-4-carboxy) benzyl ester which was then coupled to Gly-PEG via DCC activation. The synthesis was performed using the in situ symmetrical anhydride coupling method. Cleavage of the protected peptide from the polymeric support was achieved by photolysis. The product was then chromatographed on a column of Sephadex LH-20. All the protecting groups of a sample were removed with liquid HF and the unprotected crude peptide was purified by ion-exchange chromatography on CM-Sephadex to obtain an electrophoretically and chromatographically pure peptide. The identity of this peptide was confirmed by field desorption mass spectrometry and amino acid analysis. Circular dichroism measurement suggests that the free nonapeptide possesses a disordered conformation. The nonapeptide was tested for the racemization of the individual amino acids by gas chromatography and the results showed that no residue was significantly racemized.

Gabexate mesilate (FOY), a synthetic serine proteinase inhibitor, has an anticoagulant activity in the absence of antithrombin-III. We investigated FOY therapy for the treatment of disseminated intravascular coagulation (DIC) associated with sepsis in 15 patients (group F), and compared it with heparin therapy in 8 patients (group H). Successful treatment was observed in 13 patients in group F and in 4 patients in group H. The efficacy of the therapy in both groups was not significantly different. However, in patients whose antithrombin-III values were less than 20 mg/dl at the initiation of the therapy, FOY therapy was successful in 6 of 7 patients, whereas heparin therapy was not at all successful in 4 patients (rho less than 0.05). We conclude that FOY can be used as effectively as heparin for the treatment of DIC, and that FOY therapy is superior to heparin therapy in DIC associated with decreased antithrombin-III.

The Alaska salmon industry conducted 9 recalls of 7 3/4-oz cans of salmon in 1982 after a 7 3/4-oz can of Alaskan salmon was implicated in illness and one death in Belgium from Clostridium botulinum type E toxin. By the code number on the can, the Food and Drug Administration (FDA), Seattle District, traced it to a specific salmon packer. Subsequently, the FDA received a report about a defect in the can. Investigation of the salmon packer's plant by the Agency revealed that the equipment used at the plant to reform the cans--which arrived at the cannery in a nearly flattened state--might have been responsible for the defect. The death and illness in Belgium, combined with the results of the FDA inspection of the plant implicated in the Belgian incident, provided strong evidence of the existence of a hazardous situation that might have widespread adverse health effects. The Food and Drug Administration therefore requested the firm to recall its 1980 and 1981 production of salmon packaged in 7 3/4-oz cans. The Agency then began an investigation of all U.S. salmon packed inn cans of this size that had been reformed on the equipment implicated in the can defect. Of 300,000 cans examined, 22 with the defect were found. As additional firms were identified as having used the defective cans, subsequent recalls were initiated.

Two hundred and seventy-four patients with hemochromatosis and 1005 controls were HLA-typed, and HLA haplotypes were determined for 163 patients and 123 controls. The increased frequency of antigen A3 and haplotypes A3, B7 and A3, B14 in patients with hemochromatosis was confirmed. After correction for the space taken up by A3, a significant increase in All was found. This increase could not be explained by cross reaction between A3 and All. All showed a phenotype association and a haplotype link with Bw35. The genetic significance of this increased All frequency is discussed.

A patient with coexistent hemochromatosis and alpha-1-antitrypsin deficiency which led to cirrhosis and death despite adequate therapy for hemochromatosis is reported. Evaluation of the family revealed first degree relatives with iron overload and others with alpha-1-antitrypsin deficiency but none with both conditions. The role of family studies in the early recognition and possible prevention of overt clinical disease in individuals with either of these two genetic diseases is discussed.

The non-steroidal antiestrogens LY117018 and monohydroxytamoxifen are partial agonists in the 3-day immature rat uterine weight test. Both compounds stimulated increases in uterine progesterone receptor concentration (as determined by DCC assay and SDG analysis) and luminal epithelial cell height. However, LY117018 was much less estrogenic than monohydroxytamoxifen. The study also showed that the antiestrogenic effects of LY117018 and monohydroxytamoxifen could be reversed in vivo by increasing doses of estradiol. The partial uterotrophic effect of monohydroxytamoxifen and full uterotrophic effects of estradiol were both inhibited by high doses of LY117018 at an approximate dosage ratio of 1:24, w/w. This result suggests a common mechanism for the action of estradiol and the hydroxylated antiestrogens. Since LY117018 reversed the binding of [3H]estradiol and estrogen-competable [3H]monohydroxytamoxifen binding in the rat uterus in vivo, the effects of nonsteroidal antiestrogens on gross uterine wet weight can be explained by competitive interaction with estradiol via the estrogen receptor mechanism. However, the weakly estrogenic antiestrogen LY117018 was unable to inhibit estrogen-stimulated rises in luminal epithelium cell height and progesterone receptor levels. These data suggest a differential interaction by LY117018 in the rat uterus so that only selected estrogen stimulated effects i.e.: uterine wet weight, are antagonized by this particular "antiestrogenic" dose of the drug.

The mechanisms of cytotoxicity by porcine pulmonary alveolar macrophages (PAM) involved in antibody-dependent cellular cytotoxicity (ADCC) and immobilized immune complex-dependent cellular cytotoxicity (IIC-DCC) were investigated. The results indicate that IIC-DCC was inhibited by both catalase and thioglycollate broth whereas these peroxide scavengers had no effect on ADCC in an 18-hr chromium-release assay. Furthermore, it was found that when the PAM and red blood cell targets were cross-linked with PHA, catalase still completely eliminated IIC-DCC and had no effect on ADCC, which suggests that catalase is able to penetrate the lytic site when the effector and targets are cross-linked as in ADCC. The presence of cytochalasin B, which inhibits internalization of immune complexes by PAM and presumably prevents intracellular killing, also had no effect on the differential susceptibility of IIC-DCC and ADCC to catalase. Finally, it is shown that the nonspecific cytotoxicity generated by exposing PAM to immune complexes in suspension in conjunction with cytochalasin B, so that the immune complex-bound Fc receptor (FcR) cannot be internalized, also was susceptible to catalase. These data show that the lytic mechanism involved in the nonspecific cytotoxicity generated by exposing PAM to immobilized immune complexes or immune complexes in suspension in conjunction with cytochalasin B, both of which prevent the internalization of immune complex-bound FcR, is mediated solely by peroxide whereas the lytic mechanism involved in ADCC operates, at least partially, through a peroxide-independent mechanism.