HPLC isolation and GC-MS characterization of a compound strongly cross reacting with tetrahydroaldosterone antiserum

Urines from patients with hypertension and elevated aldosterone levels, i.e. primary aldosteronism due to adrenal adenoma or hyperplasia or carcinoma were extracted, paper chromatographed and thereafter chromatographed repeatedly with normal phase and repeatedly with reversed phase HPLC systems in an attempt to find new metabolites of aldosterone. Specific 3 alpha-hydroxy-5 beta-tetrahydroaldosterone antiserum was used in a radioimmunoassay system to detect possible aldosterone metabolites in the HPLC fractions after each isolation step. The immunoactive HPLC fractions were derivatized and analysed by GC-MS. A relatively nonpolar compound, 11 beta:18(S),18:20 alpha-diepoxy-5 beta-pregnan-3 alpha-ol, was isolated and identified in this manner. This material was originally described by Kelly et al., in 1962 after loading human subjects with huge amounts (25-160 mg) of exogenous aldosterone. This material has not yet been described from endogenously produced aldosterone. Very small amounts, if any, were similarly isolated from the urine of a control subject. Therefore, this compound could prove to be a new marker for hypertension due to hyper-production of aldosterone.     

Diurnal variations in plasma arginine vasotocin (AVT) concentrations in the ovine fetus

Previously we have demonstrated the presence of AVT in the blood of fetal sheep. The source is not clear, but AVT has been identified in fetal pineal and pituitary glands. In view of the circadian secretory pattern of the pineal gland, we questioned whether fetal plasma AVT levels might vary diurnally. Plasma samples from five chronically catheterized ovine maternal ewes and fetal lambs 129-135 days' gestation were obtained at 3-19 hourly intervals for 1-2 days (mean +/- S.E.M. = 35 +/- 6 hours. Plasma AVT levels were determined by radioimmunoassay. Results were analyzed by nonlinear curve fitting procedures to relate hormone levels with time of day. Plasma AVT values for maternal ewes did not vary during the day in response to light/dark periods. The curve for mean fetal plasma AVT plotted against time showed oscillations with a period of about 25 hours (p less than 0.05). Peak fetal AVT levels were observed at 1600 hours and minimal levels at 0400 hours. These results indicate that ovine fetal AVT secretion varies diurnally. The site of AVT secretion may be the pineal gland; however, confirmation of this and identification of the physiological stimuli for secretion of fetal plasma AVT require further information.     

Effects of beta-adrenergic stimulation on 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine-mediated vasoconstriction and glycogenolysis in the perfused rat liver

The beta-adrenergic agonist isoproterenol inhibited the glycogenolytic response of platelet-activating factor (AGEPC, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) in perfused livers derived from fed rats. AGEPC-stimulated hepatic vasoconstriction, measured by increases in portal vein pressure, also was inhibited by prior isoproterenol infusion. Isoproterenol-mediated inhibition of these hepatic responses to AGEPC was not apparent when isoproterenol (10 microM) was coinfused with the beta-receptor antagonist propranolol (75 microM) or when isoproterenol was replaced with the alpha-adrenergic agonist phenylephrine (10 microM). alpha-Agonist-induced glycogenolysis and vasoconstriction in the perfused liver was unaffected by isoproterenol infusion. Glucagon (2.3 nM) had no effect on the glycogenolytic or vasoconstrictive responses of the liver to AGEPC despite the fact that glucagon increased hepatic cAMP levels to a far greater extent than isoproterenol. Additionally, inhibition of the hepatic responses to AGEPC by isoproterenol occurred in perfused livers from mature rats (i.e. greater than 300 g) in which liver parenchymal cells lack functional beta-adrenergic receptors. The data presented in this study illustrate a specific inhibition of AGEPC-induced hepatic glycogenolysis and vasoconstriction by beta-adrenergic stimulation of the perfused liver. This inhibition appears to be mediated by interaction of isoproterenol with nonparenchymal cells within the liver. These findings are consistent with the concept that AGEPC stimulates hepatic glycogenolysis by an indirect mechanism involving hepatic vasoconstriction.     

Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells

We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.     

Synthesis of phosphatidylcholine by rat lung during choline deficiency

The effect of choline deficiency on the de novo pathway for phosphatidylcholine (PC) synthesis in the lung was investigated in rats fed a washed soy protein (lipotrophic) diet deficient in choline and methionine for 2-3 wk. Lungs from lipotrophic rats showed a decreased content of choline and choline-phosphate (P less than 0.05) compared with control but no change in content of cytidine 5'-diphosphocholine or PC. Isolated perfused lungs from lipotrophic rats were evaluated for choline and fatty acid utilization for PC synthesis. Lipotrophic lungs perfused with 5 microM [14C-methyl]-choline chloride showed increased incorporation into PC while there was no significant effect at saturating levels of choline (100 microM). There was increased incorporation of [1-14C]-palmitic acid into PC and diglyceride and increased incorporation of D-[U-14C]glucose into fatty acids of PC. Increased choline and glucose incorporation was not due to alteration of intracellular specific activity of these substrates. This study indicates the utilization of choline and fatty acid for PC synthesis is stimulated as a result of choline deficiency while lung CDP-choline concentration is maintained, possibly through regulation of choline phosphate cytidyl transferase activity. These mechanisms compensate for decreased choline availability to maintain the PC content of lungs.     

Effects of 24-hour fast on cycling endurance time at two different intensities

Ten competitive cyclists were exercised to exhaustion to test the potential of a 24-h fast for increasing endurance. One group (n = 4) was tested at an initial intensity of 86% maximum O2 uptake (VO2max) (HI) and a second group (n = 6) at 79% VO2max (MI). Both groups repeated test rides in fasted and normal-diet conditions. Time to fatigue was designated at two points: fatigue 1 occurred when pedal frequency could not be maintained at the initial percent VO2max; fatigue 2 occurred when pedal frequency could not be maintained at a workload of approximately 65% VO2max. In both HI and MI the 24-h fast had no effect on resting muscle glycogen stores but significantly increased plasma free fatty acid (FFA) levels. Despite the increased FFA availability, time to fatigue was reduced in the fasted groups. Fatigue 1 and 2 times (mean +/- SE) for HI-fasted were 42.0 +/- 6.2 and 170.0 +/- 20.4 min, respectively, compared with those of the HI-normal diet of 115.3 +/- 25.6 and 201.0 +/- 14.8 min. Fatigue 1 and 2 times for MI-fasted were 142.0 +/- 19.6 and 167.5 +/- 10.5 min compared with those of the MI-normal diet of 191.3 +/- 25.0 and 214.3 +/- 18.9 min. The cause of fatigue at fatigue 1 was not readily apparent. Fatigue 2 in all groups seemed to be related to hypoglycemia as well as muscle glycogen depletion.     

Identification of zona binding proteins of rabbit, pig, human, and mouse spermatozoa on nitrocellulose blots

Mammalian fertilization is a multi-step process with different requirements for specificity at each step. In the present report we have examined the binding of spermatozoa to homologous and heterologous zonae pellucidae. The homologous zona binding proteins (ZBP) of ejaculated rabbit, pig and human spermatozoa and epididymal mouse spermatozoa have been identified. The rabbit's ZBPs have relative molecular weights (MW) of 32K, 18K, 16K and 14K; the pig's major ZBP is 16K while human spermatozoa bind human zona protein at 17K and 18K. Mouse sperm ZBPs are 19K, 18K and 16K.     

Production and characterization of a monoclonal antibody that binds Reed-Sternberg cells

A murine monoclonal antibody, termed HeFi-1, was produced after immunization with the L428 Hodgkin's disease tissue culture cell line. HeFi-1 selectively stained only the Reed-Sternberg or Hodgkin's cells in 18 of 18 cases of Hodgkin's disease, including the nodular sclerosis, mixed cellularity, and lymphocyte-depleted histologic subtypes. HeFi-1 did not stain any cells in normal lung, brain, salivary gland, thyroid, gall bladder, pancreas, liver, testis, breast, endometrium, or kidney. Rare large cells at the edge of the lymphoid follicles were stained in normal tonsil, colon, and hyperplastic thymus. There was no staining of any cells in 14 cases of B cell non-Hodgkin's lymphoma; however, the malignant cells in three of 11 cases of non-Hodgkin's lymphoma which appeared to express T cell markers were also stained with HeFi-1. Tissue culture cell lines including the T cell acute lymphocytic leukemia lines MOLT4 and CEM, the histiocytic cell line U-937, and the amniotic cell line WISH were not stained. Seven Epstein Barr virus (EBV)-positive lymphoblastoid cell lines were stained with HeFi-1, but there was no staining of three EBV+ African Burkitt's lymphoma cell lines or three EBV- American Burkitt's cell lines. HeFi-1 did not block the ability of the L428 cells to stimulate a mixed lymphocyte reaction or function as accessory cells for mitogen-induced human T cell proliferative responses. Modulation of the HeFi-1 cell surface antigen on the L428 cells was not observed. HeFi-1 specifically immunoprecipitated a cell surface protein of approximately 120,000 daltons from both the L428 and EBV+ lymphoblastoid cell lines. HeFi-1 monoclonal antibody should prove useful not only in the diagnosis, staging, and potential therapy of Hodgkin's disease, but also for determining the cell of origin of the Reed-Sternberg cell.     

Studies of in vitro activation and differentiation of human B lymphocytes. I. Phenotypic and functional characterization of the B cell population responding to anti-Ig antibody

Investigation of the activation of splenic B cells by anti-immunoglobulin (Ig) antibody has enabled us to characterize the anti-Ig-responsive B cell and to analyze the phenotypic changes which accompany proliferation and differentiation. The anti-Ig antibody-responsive B cell population was characterized by the expression of high levels of the B2 antigen and represented approximately 40% of splenic B cells. Brisk mitogenesis which peaked at 3 to 4 days was induced by anti-Ig antibody. The proliferative phase was characterized phenotypically by a dramatic decline in B2 antigen expression, with most cells showing no detectable B2 by 4 days post-activation. The other hallmark of this phase was de novo expression of a group of "activation antigens." These included the B cell-restricted antigens B-LAST 1, BB1, and B5, and the T cell-associated interleukin 2 receptor and T12 antigens. Concomitantly, B1, B4, and Ia expression increased, the increase being roughly proportional to the increase in cell size. After day 4, the mitogenic response progressively diminished, while Ig synthesis increased. During this differentiation phase, cell surface antigens again displayed a distinct sequence of changes. The five activation antigens and the B1, B4, and Ia antigens began to decrease. However, two markers, T10 and PCA-1, which are found on plasmacytomas, appeared and their level of expression steadily increased. These changes and the appearance of morphologically identifiable plasma cells required the presence of T cells in this system. T cell supernatants alone induced Ig secretion but did not induce expression of PCA-1 or the appearance of cells with plasma cell morphology. The culture system developed in this study has allowed us to analyze the antigenic changes following activation by anti-Ig antibody. This sequence of changes has not only permitted the identification of antigens which, by their appearance at distinct stages may have an important role in proliferation and differentiation of B cells, but also provides us with the means of studying the function of each antigen.