Fluorophotometric enzyme immunoassay of thyroid-stimulating hormone.

A method for enzyme immunoassay of thyroid-stimulating hormone (TSH) is described, TSH was conjugated with horseradish peroxidase according to periodate oxidation method. Separation of the bound and free was obtained by double-antibody solid-phase technique using Sepharose 4B-anti-rabbit immunogiobulin G (IgG)-geat IgG. The fluorescence reaction using tyramine and hydrogen peroxide as substrates was used for the determination of enzyme activity in order to increase the sensitivity of enzyme immunoassay. The standard curve for serum TSH was satisfactory to recognize TSH concentrations as 0.06 μU/tube. TSH values obtained by this method correlated well with those obtained by radioimmunoassay (r, 0.96). The coefficients of variation were 1.8 to 5.3% (within assay) and 5.1 to 10.5% (between assay). The method is about equal to radioimmunoassay with respect to sensitivity. Since it requires minimal equipment and is less expensive than radioimmunoassay, it is possible to perform routine assays even in laboratories with limited facilities.     

Appearance of a Cell Surface Antigen Associated with the Activation of Peritoneal Macrophages in Mice

A relatively large population of murine peritoneal exudate macrophages induced with viable BCG or heat-killed Corynebacterium parvum was stained by the antiserum prepared against purified gangliotetraosyl ceramide (asialo GM1), while only a small population of peritoneal resident macrophages or peritoneal exudate macrophages induced with proteose peptone was stained. The cytotoxicity assay of those macrophages with anti-asialo GM1 plus complement supported these results. Peritoneal macrophages induced with BCG or C. parvum showed strong cytotoxicity for EL4 cells in vitro, while resident or peptone-induced peritoneal macrophages showed no cytotoxicity. BCG- or C. parvum-induced peritoneal cells contained both NK cells and cytotoxic macrophages, and either in vivo or in vitro pretreatment of the cells with anti-asialo GM1 and complement abolished the activities of both types of cells. Peptone-induced peritoneal macrophages incubated with lymphokines (LK) or lipopolysaccharide (LPS) were cytotoxic for EL4 cells and contained an increased number of cells stained by anti-asialo GM1. The cytotoxicity of these in vitro activated macrophages was reduced by treatment with anti-asialo GM1 plus complement. When peptone-induced peritoneal macrophages were incubated with LK, the number of cells stained by anti-Ia antiserum increased, but the number did not increase when the macrophages were incubated with LPS. Pretreatment of peptone-induced macrophages with anti-asialo GM1 plus complement did not affect the ability of the macrophages to be activated by LK. These results taken together strongly suggest that the antigen (s) reactive with anti-asialo GM1 is expressed on the cell surface of cytotoxic peritoneal macrophages in mice.     

Atrial natriuretic peptide correlates with pulmonary arterial pressure and cardiac output

Correlations between plasma atrial natriuretic polypeptide (ANP) levels and hemodynamic parameters were studied in the central circulation of 12 patients with angina pectoris. The average plasma ANP level determined in the aorta was found to be 619 +/- 140 pg/ml. The plasma ANP levels showed a significant positive correlation with mean pulmonary arterial (PA) pressure, right ventricular pressure, and with cardiac index. In contrast, there was no significant correlation between plasma ANP levels and other hemodynamic variables including atrial pressure. These results suggest that hemodynamics other than the atrial pressure may have some role in modulating ANP secretion in certain pathological states.     

Atrial natriuretic factor is biologically active also in the absence of vasopressin: studies on Brattleboro-strain diabetes insipidus rats

Since atrial natriuretic factor (ANF) has been shown to inhibit vasopressin secretion, the role of this effect in the acute biological actions of ANF was investigated using Brattleboro-strain diabetes insipidus (DI) rats. Under thiobarbital anesthesia, synthetic rat ANF of a 25 amino acid sequence was administered intravenously as a bolus (8 micrograms/kg) into the jugular vein. The urine volume, urinary sodium and potassium concentration, blood pressure, and heart rate were determined. It was found that ANF administered exogenously can exhibit its diuretic, natriuretic and vasorelaxant activities even in the absence of vasopressin. This indicates that the inhibition of vasopressin secretion is not an indispensible mechanism for acute biological effects of ANF.     

Atrial natriuretic factor inhibits vasopressin secretion from rat posterior pituitary

The effects of synthetic atrial natriuretic factor (ANF) were studied in superfused rat posterior pituitary gland. ANF (10(-6)M, 10(-10)M) significantly inhibited basal as well as KC1 (50 mM) or angiotensin II-stimulated immunoreactive arginine vasopressin secretion. The magnitude of inhibition was greater at 10(-6)M than at 10(-10)M. ANF also decreased cAMP secretion and increased cGMP secretion from the posterior pituitary. These results suggest that ANF directly acts on the posterior pituitary to inhibit arginine vasopressin secretion and that this effect is, at least, partly mediated by the changes in cyclic nucleotide production.     

The production of cloned fish in the medaka (Oryzias latipes)

The measurement of cellular DNA content by DNA microfluorometry revealed that medaka embryos that were fertilized with normal sperm and exposed to heat shock (41 degrees C for 3 min) or hydrostatic pressure (700 kg/cm2 for 10 min) at 85-95 min after insemination were tetraploid. Embryos fertilized with normal sperm and exposed to heat shock (41 degrees C for 2 min at 2-3 min after insemination) were triploid. These results suggest that heat shock or hydrostatic pressure at 85-95 min after insemination arrests the first cleavage, while heat shock at 2-3 min after insemination arrests the second meiotic division. Medaka clones have been produced by the following method: Eggs from orange-red or variegated variety were activated by UV-irradiated, genetically impotent sperm of wild-type fish (UV sperm). The haploid eggs obtained were diploidized by preventing the first cleavage with heat shock or hydrostatic pressure to produce homozygous females. Each of the two homozygous females was mated with vasectomized male in isotonic balanced salt solution to collect unfertilized eggs. The collected eggs were activated with UV sperm and converted from haploid to diploid by arrest of the second meiotic division with heat shock. Hatched fry of each homozygous diploid (all females) were fed with a methyltestosterone-containing diet (40 micrograms/gm diet) to produce sex-reversed males, which were mated with brood females, and thus two cloned lines were obtained.     

Prevention of polydactyly manifestation in Polydactyly Nagoya (Pdn) mice by administration of cytosine arabinoside during pregnancy

Male mice heterozygous for the dominant polydactyly gene Pdn (Polydactyly Nagoya) were crossed with normal or heterozygous females of the same strain. Pregnant females were treated with 5 mg/kg of cytosine arabinoside (Ara-C) on day 12 of gestation. The offspring were removed on day 18 of gestation and examined for external malformations, and the fore- and hindlimbs were examined by means of bone- and cartilage-stained cleared specimens. In +/+ x Pdn/+ matings, Pdn/+ fetuses, bearing preaxial polydactyly of the distal phalangeal type in the hindlimb and deformity of the 1st digit in the forelimb, were obtained in about 50% of the nontreated group. In treated fetuses, however, the incidence of polydactyly and deformity of the 1st digit decreased to 1.4 and 10.1%, respectively. Nontreated Pdn/Pdn fetuses exhibited preaxial polydactyly of the duplicated or triplicated metacarpal/metatarsal type both in the fore- and hindlimbs. In the treated Pdn/Pdn fetuses, the number of preaxial extra digits decreased in both limbs. Some hindlimbs of the treated Pdn/Pdn fetuses exhibited five metatarsals, normally. In the vitally stained specimens at 6 and 24 hours after injection of Ara-C, preaxial marginal necrotic zones (fMI) were observed in almost all of the treated embryos from +/+ x Pdn/+ matings. However, approximately half of the embryos did not exhibit fMI in the nontreated control group at the same stage. Those embryos deficient in fMI were regarded as Pdn/+. These findings indicated that a subteratogenic dose of Ara-C prevented the genetic expression of polydactyly in almost all Pdn/+ and some cases of Pdn/Pdn mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereochemical Control in Microbial Reduction. Part 10. Asymmetric Reduction of Alkyl 3-Methyl-2-Oxobutanoate with Immobilized Bakers' Yeast in Hexane

Esters of 3-methyl-2-oxobutanoic acid are reduced with bakers' yeast by three methods: free bakers' yeast in water, immobilized bakers' yeast in water, and immobilized bakers' yeast in hexane. Although (R)-hydroxy esters are obtained in all cases, the enantiomeric excess varies from 3% (reduction of the methyl ester with free bakers' yeast in water) to 93% (reduction of the butyl ester with immobilized bakers' yeast in hexane) depending on the structure of substrate and on the reaction conditions. The mechanism of the present stereochemical control is discussed.