Direct radioimmunoassay of specific urinary estrogen glucosiduronates in normal men and nonpregnant women.

Direct radioimmunoassay are described for the measurement of each of three specific estrogen glucosiduronates: estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate and estriol-16 alpha-glucosiduronate in urine. Each assay utilizes a specific antiserum prepared by complexing the carboxylic acid group of the appropriate glucosiduronate to the epsilon-amino group of lysine in bovine serum albumin or bovine thyroglobulin. The antisera showed little or no cross reactivity toward other estrogens that might be present in significant amounts in urine. These antisera were used for the direct assay of the conjugates in urine from normal men and nonpregnant women without prior extraction or chromatography. The values were similar to those obtained after extraction, chromatographic purification on DEAE-Sephadex and subsequent immunoassay; The following mean values +/- SE (microgram/g creatinine) were obtained: estrone glucosiduronate, male 10.1 +/- 0.6, follicular phase female 17.3+/- 1.6, luteal phase female 31.8 +/- 2.5; 17 beta-estradiol-17-glucosiduronate, male 1.7 +/- 0.3, follicular phase female 2.4 +/- 0.1, luteal phase female 4.2 +/- 0.4; estriol-16 alpha-glucosiduronate, male 1.8 +/- 0.2, follicular phase female 4.7 +/- 0.9, luteal phase female 10.0 +/- 1.6.     

Isocratic separation of estrogen conjugates on DEAE-sephadex.

Mixtures of estrogen conjugates containing estrone-3-glucosiduronate, 17β-estradiol-3-glucosiduronate, 17β-estradiol-17-glucosiduronate, estrone-3-sulfate and 17β-estradiol-3-sulfate have been separated on DEAE-Sephadex resin by isocratic elution using NaCI concentrations ranging from 0.05M to 0.4M. The results indicate that an NaCI gradient is not necessary for the Chromatographic separation of these estrogen conjugates. An NaCl concentration of 0.05M was adequate to separate the various monoglucosiduronates and sulfates. Isocratic elution of the columns with or without a possible stepup in the salt concentration was shown to give a higher resolution of estrogen conjugates in a more convenient volume than a gradient elution. For ideal chromatography of estrogen conjugates on a DEAE-Sephadex column, isocratic elution with 0.05 or 0. IM NaCl is preferred for the separation of monoglucosiduronates and 0.2 or 0.25M NaCl for the separation of sulfate conjugates. Contrary to current expectations, the molarity at which a particular conjugate elutes in a gradient mode does not bear a consistent relationship to the structure of the conjugate. However, the holdback volume in the isocratic mode may be used for identification purposes. When holdback volume was plotted against molarity, separate curves were obtained for each of the above mentioned conjugates. Tests of fit were carried out using a number of models. The best fit was obtained using the simple model $\text{y}\text{=}\text{a}\text{+}\text{b}\text{1}\text{x}$ where the independent variable, x, is the molarity; the dependent variable, y, is the volume and a and b are the intercept and slope respectively. Each curve fitted the model, but the values for a and b were significantly different. Using this model, a simple and predictable relationship between molarity and holdback volume can be demonstrated for each of the estrogen conjugates. The advantages of the isocratic mode of elution over gradient elution are discussed.     

Splanchnic and intestinal uptake and formation of estriol and estriol conjugates in the dog in vivo.

A loading dose of 3H-estriol was given to male dogs followed by a constant infusion. The concentrations of total radioactivity, conjugated estriol metabolites, estriol, estriol-o-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-3-sulfate and estriol-3-sulfate, 16alpha-glucosiduronate were determined in plasma from the femoral artery(A), hepatic vein(HV) and superior mesenteric vein (SMV). From these values the splanchnic (100[1-HV/A]) and intestinal (100[1-SMV/A]) extractions were calculated. The mean splanchnic extraction of total radioactivity was positive (23, SE 3, P less than .01), indicating net uptake by the splanchnic area, possibly due to biliary excretion. The mean splanchnic extraction of estriol was 77, SE 1, P less than .01, also indicating net uptake. The splachnic extractions of estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate and estriol-3-sulfate were negative (-15, SE 3, P less than .01; -23, SE 6, P less than .01; -31, SE 8, P less than .01 respectively) indicating net formation of these conjugates for release into the systemic circulation. The mean intestinal extraction of estriol was 12, SE 4, P less than .01, indicating net uptake by the intestine. This net uptake was associated with mean negative intestinal extractions of estriol-3-glucosiduronate (-15, SE 7, P approximately .05), estriol-3-sulfate (-33, SE 10, P less than .01) and estriol-3-sulfate, 16alpha-glucosiduronate (-53, SE 13, P less than .01), indicating net formation of these conjugates by the intestine.     

Identification of conjugated estrogen metabolites in dog plasma following administration of estriol-2,4,6,7-3H.

Following the constant infusion of 2,4,6,7-3H-estriol in male dogs for a period of 90 minutes, the radioactive metabolites present in arterial plasma were separated by solvent partition, DEAE-Sephadex, Celite partition and thin layer chromatography. The identities of the individual estrogens and estrogen conjugates were confirmed by specific activity determinations after chromatography in several different solvent systems, enzyme hydrolysis and chromatography of the unconjugated steroids and their derivatives.     

Factors in the cryopreservation of bone marrow cells from children with acute lymphocytic leukemia.

Bone marrow cells from 42 children with acute lymphocytic leukemia in remission and 19 normal adults were preserved in liquid nitrogen for periods of up to 50 weeks. Many factors in the process of cryopreservation were investigated in an attempt to optimize the recovery of the bone marrow colony forming cells. The effect of cryopreservation on the cells which produce colony stimulating activity also was investigated. With optimization of this technique, it was observed that approximately 100% of the bone marrow nucleated cells were recovered and approximately 50% of the total colony forming cells were viable after 50 weeks in liquid nitrogen.     

Separation of estrogen conjugates by high pressure liquid chromatography.

High pressure liquid chromatography using a prepacked commercial strong anion exchanger column (mu Partisil 10 SAX, 25 cm x 4.6 mm) was used to separate a mixture of eight estrogen conjugates. Chromatographic conditions using a 0.01 M potassium phosphate or 0.1 M NaCl as solvent in the isocratic mode are described for the separation of estrone glucosiduronate, 17beta-estradiol-3-glucosiduronate, 17beta-estradiol-17-glucosiduronate, estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-17-glucosiduronate, estrone sulfate and 17beta-estradiol-3-sulfate. This system gives high resolution of the estrogen conjugates in small eluent volumes in less than 30 min. The advantages of this high pressure liquid chromatographic system over other methods of separation are discussed.     

Structural changes in light- and dark-adapted compound eyes of the australian earwig Labidura riparia truncata (dermaptera)

The apposition acone eye of Labidura is relatively small—550–600 facets—with a thick corneal lens and shallow retina. The retinula cell columns are each formed of six peripheral cells plus two central cells, a partially fused rhabdom, and dense pigment in two or three cell types. Upon adaptation from light to dark, the most striking photomechanical response is a proximal broadening of the cone cells, which results in a 38-fold increase in cross-sectional area of the aperture. While longitudinal rhabdom movement is small, microvillar diameters swell in response to light and contract in the dark. Irregularities of facet pattern and shape, and in ommatidial alignment were found, particularly towards eye margins. Three types of interommatidial sense organs on the eye surface are described, one of which has not been previously reported. An argument is presented to explain how the field of view and sensitivity are both apparently decreased in the acone eye by exposure to light.     

Evidence for a shift from IgD to IgA synthesis in the maturation of human B lymphocytes.

A case of chronic lymphatic leukemia (CLL) is described in which IgD and IgA are the copredominant membrane immunoglobulins. Since CLL represents malignant proliferations of B lymphocytes arrested at discrete points during maturation, the findings in this case suggest that at least some of the developing cells destined to synthesize IgA for secretion pass through a stage in which immunoglobulins D and A are present together on the cell membrane.     

Estrogenic regulation of uterine cyclic amp metabolism

Ovariectomized and ovariectomized, estrogen-treated (48 h) rats were injected intravenously with increasing doses of epinephrine. Uteri were frozen in situ 30 s later. Estrogen pre-treatment significantly increased the sensitivity of both cyclic AMP and phosphorylase to epinephrine. The cyclic AMP response to intravenous injection of the pure β-agonist, isoproterenol, was enhanced by estrogen pre-treatment (48 h) and the cyclic AMP response of isolated uteri treated with epinephrine in vitro was also enhanced by in vivo estrogen pre-treatment (48 h). Other groups of ovariectomized rats were treated with estrogen and cyclic AMP levels were estimated at various times after estrogen treatment. 6 h after intraperitoneal injection and 48 h after subcutaneous injection, estrogen caused 20 and 30% increases in cyclic AMP. Estrogen had no effect on cyclic AMP 30 s after intravenous injection or 15 min after intraperitoneal injection. The was also no change in uterine catecholamine sensitivity 30 s after intravenous estrogen injection.The uterine site(s) at which estrogen acts to alter uterine cyclic AMP metabolism could be uterine β-adrenergic receptors, adenyl cyclase, and/or phosphodiesterase.     

A comparison between the synthesis of contractile proteins and the accumulation of their translatable mRNAs during calf myoblast differentiation

The morphogenesis of muscle spindles in the mouse extensor digitorum longus was studied in closely spaced, serial, ultrathin transverse sections, permitting evaluation of the developing spindles' three-dimensional cytoarchitecture. Afferent nerve terminals are identifiable as early as 15 days in utero, and the adult number of spindles is present at 17 days in utero. By establishing continuity between the clearly identifiable equatorial regions and the polar regions, which are morphologically indistinguishable from surrounding extrafusal fibers, it could be determined that fusimotor innervation was present by the 19th day of the in utero development. In all spindles examined, the efferent innervation occurred extracapsularly. At birth, the adult number (four or five) of intrafusal fibers are found in each spindle, and fusimotor innervation is frequently intracapsular. Evidence is presented supporting the hypothesis that successive generations of intrafusal myotubes are formed by the fusion of mononucleated cells. Newly formed myotubes are preferentially distributed in close relationship to the sensory-innervated region of the primary intrafusal myotube. Analyses of growth parameters indicate that the difference in length and diameter between intrafusal and extrafusal fibers is principally a postnatal phenomenon.     

Early degranulation of human neutrophils: immunocytochemical studies of surface and intracellular phagocytic events.

Degranulation of azurophil and specific granules after phagocytic challenge with E. coli for 5 sec to 10 min was investigated in the human polymorphonuclear neutrophil (PMN). PMN were stained simultaneously with fluorescein and rhodamine-labeled monospecific antisera to myeloperoxidase (MPO) and lactoferrin (LF) to identify azurophil and specific granules, respectively, within single cells. Fixation was designed to preserve or disrupt differential permeability of cell membrane to fluorescent conjugates in order to study granule translocation. Within 5 sec after phagocytic challenge, MPO and LF appeared on the cell surface coating the bacteria as granule contents leaked from the incompletely formed phagolysosomes. The phagocytic cup, shown by scanning electron microscopy as large and circular, appeared by immunofluorescent markers to be outlined by curvilinear staining for both granule markers, and was always coincident with bacterial localization. MPO and LF appeared singly or simultaneously on the cell surface, suggesting that degranulation to the surface was random. Sequential phagocytic events were demonstrated by comparing staining intensities for each granule marker on the surface and intracellularly within single cells. LF sometimes appeared on the cell surface independent of the nascent phagosome, suggesting that perturbation of the cell membrane by bacteria may cause some specific granule extrusion not limited to the phagosome. These results imply that bacteria make contact with granule-associated anti-microbial substances within 5 sec after phagocytosis is initiated and that free communication of granule constituents occurs between the newly forming phagolysosome and the extracellular space.     

Isolation of the Ochromanas danica plasma membrane and identification of several membrane enzymes

Ochromonas danica cell homogenate can be fractionated by differential centrifugation into chloroplast, mitochondrial, ribosome, lysosomal, plasma membrane and soluble fractions. The plasma membrane fraction was further purified by discontinuous sucrose density gradient centrifugation and was found to be enriched 4–16-fold in the following enzymes: β-galactosidase, acid phosphatase, alkaline phosphatase, 5′-nucleotidase, and (Na⁺, K⁺)-ATPase. The role of plasma membrane phosphatase in the phosphate metabolism of plants is discussed.     

Evidence for seasonal variation in aggressive behaviour by Macaca mulatta

Observations were made on the Macaca mulatta colony on the island of Cayo Santiago over a period that included two reproductive cycles. The frequency of aggressive behaviour was found to fluctuate cyclically as a function of the colony's reproductive state. Males were wounded and died significantly more frequently during the mating season than at any other time of year. Females were wounded about as often during the mating season as during the birth season, and more died during the birth season than during the mating season, although not significantly so. There was no significant difference between sexes in age at death, nor was there a significant departure from expectation of number of deaths per age class. However, mortality among the reproductively mature males was greater than among mature females. It was suggested that differential injury and mortality rates among adult males might be an indirect result of high levels of androgens which could conceivably alter threshold levels for stimuli initiating severe aggressive interactions.     

Trypanosoma cruzi: the T-cell dependence of the primary immune response and the effects of depletion of T cells and Ig-bearing cells on immunological memory

The effects of T-cell depletion on primary infection with Trypanosoma cruzi and on immunological memory to this parasite were studied in a syngeneic mouse system. Exacerbation of T. cruzi infections occurred in thymectomized, irradiated, bone marrow-reconstituted (TX) C57BL/6J mice compared to sham thymectomized, irradiated, bone marrow-reconstituted (STX) mice. Reconstitution of TX mice with thymocytes restored the resistance to a level equivalent to that of STX mice. Immunological memory against T. cruzi present in spleen cells in mice recovered from T. cruzi infections could be ablated by treatment with rabbit anti-brain-associated theta serum but not with rabbit anti-mouse immunoglobulin serum prior to adoptive transfer of immune spleen cells into TX mice. These experiments suggest that modulation of the primary immune response and memory against T. cruzi depends largely on the thymus-derived lymphocyte. The possible implications of this T-cell regulation on previously reported effector mechanisms againt this parasite are discussed.     

In vitro biosynthesis of radioactive estradiol-17 beta, 17-glucosiduronate by rhesus monkey liver.

A method for the preparation of radioactive estradiol-17 beta, 17-glucosiduronate by incubating 3H-estradiol with rhesus monkey liver microsomal preparation in the presence of uridine diphosphoglucuronic acid is described. Small but significant amounts of the conjugate were also obtained from the 150,00 pellet and cytosol fractions. The addition of NADPH to the incubation media increased the yield of radioactive-estradiol-17 beta, 17-glucosiduronate perhaps by preserving the integrity of the C-17-hydroxyl group. As expected, the effect of the reduced nucleotide was more pronounced in the fractions other than the microsome. The biosynthesized conjugate was isolated and purified by multiple column chromatography and the structure was confirmed by derivative formation, enzyme hydrolysis and crystallization of the aglycone.     

Proteins foretelling head or abdomen development in the embryo of Smittia spec. (Chironomidae,Diptera)

The development of the segment pattern in Smittia embryos can be manipulated experimentally. Centrifugation during intravitelline cleavage leads to a mirror image duplication of most of the head in the absence of abdominal segments (“double cephalons”). Conversely, mirror image duplications of abdominal segments in the absence of head and thorax (“double abdomens”) can be generated by UV-irradiation of the anterior pole before blastoderm formation. By subsequent exposure to blue light, UV-irradiated embryos can be reprogrammed for normal development (photoreversal). We have characterized an “anterior indicator” protein (designated AI₁; M_r ? 35,000; IEP ? 4.9). Its synthesis was restricted to anterior fragments of embryos during a late blastoderm stage (Bl_VI). This protein was synthesized, however, in both anterior and posterior fragments of prospective double cephalons. Conversely, this protein was synthesized neither in anterior nor in posterior fragments of UV-induced double abdomens. Upon photoreversal, the protein was synthesized again in anterior fragments. Thus, synthesis of this protein in a given fragment always indicated development of head and thorax there. Likewise, we have characterized a “posterior indicator protein” (designated PI₁, M_r ? 50,000, IEP ? 5.5). Its synthesis during early blastoderm stages (Bl_I and Bl_II) was restricted to posterior fragments but not to pole cells in normal embryos. In UV-induced double abdomens, PI_I was synthesized in both anterior and posterior fragments at stage Bl_II. Photoreversal again led to restriction of PI_I synthesis to posterior fragments. Thus, the synthesis of PI_I in a given fragment at stage Bl_II always foreshadowed the formation of an abdomen several hours before this can be discerned morphologically. The synthesis of two other proteins (designated a₁ and p₁) was also restricted, during certain blastoderm stages, to anterior or posterior fragments, respectively. However, UV-irradiation or centrifugation had little or no effect on the synthesis of these proteins. Conversely, programming embryos for double abdomen development by UV-irradiation caused a set of reproducible, and mostly photoreversible, changes in the pattern of proteins synthesized in anterior embryonic fragments. However, the synthesis of most of the affected proteins was not region-specific in normal embryos.     

Nucleus-associated RNA in measles virus-infected cells

Measles virus RNA was found in the nuclear fraction of infected Vero cells. 24-hr labeling periods revealed heterogeneously sedimenting 15–50 S RNA associated with a membrane-containing particulate. Viral RNA isolated after shorter labeling periods was larger in size (30–50 S) and associated with both nucleoplasmic and particulate fractions.     

Ultrastructure-function correlative studies for cardiac cryopreservation. V. Absence of a correlation between electrolyte toxicity and cryoinjury in the slowly frozen,cryoprotected rat heart

Hearts were frozen to ?17 °C in the initial presence of 2.1 m DMSO. Attempts were made to prevent or minimize the consequences of an osmotic shock based on Lovelock's classical hypothesis of freezing injury. Substitution of mannitol or potassium for NaCl before freezing did not improve the results, nor did perfusion of thawed hearts with hyperosmotic perfusate. It was found that freezing and thawing resulted in a significant attenuation of coronary flow and that, as a result of this, DMSO was apparently retained within the heart after thawing. DMSO was also difficult to remove at 30 °C in the absence of prior freezing and caused a significant drop in coronary flow upon institution of DMSO washout with balanced salt solution. The blanching of freezing and thawing was also seen, in milder form, in nonfrozen hearts. For both frozen-thawed and nonfrozen hearts, the blanching was associated with DMSO washout with balanced salt solution. Flow was improved by perfusion with hyperosmotic perfusate in both nonfrozen and in frozen-thawed hearts, but the improvement was largely temporary. Evidence from earlier studies indicates that electrolyte concentrations during freezing cannot be correlated with cardiac cryoinjury, in support of the present findings. It is suggested instead that cryoprotectant toxicity may be the chief agent of injury under the conditions studied.