The development potential of ethanol-induced monosomic and trisomic conceptuses in the mouse

The development potential of fertilized embryos isolated from female mice previously given a single dose of either a dilute solution of ethanol or distilled water (controls) by mouth was studied. Exposure to ethanol occurred at various times during the cycle leading to ovulation and shortly after fertilization. The chromosome constitution of all preimplantation embryos isolated from these females was determined either at the first cleavage mitosis or at the morula stage. The incidence of aneuploidy in the ethanol-exposed groups at these times was approximately 19% and 13.5%, respectively, with a similar number of monosomic and trisomic conceptuses observed at these times. In addition, about 2% of all conceptuses examined were triploid. Further females were autopsied on the 10th or 11th day of gestation, though the chromosome constitution of only the morphologically abnormal or developmentally retarded embryos was determined. Eight embryos out of a total of 16 studied in the ethanol-exposed group were either aneuploid or triploid, whereas in the control group only one out of 11 examined proved to be aneuploid. The triploids and ethanol-induced aneuploid conceptuses appeared to be capable of surviving to the morula stage but generally failed to survive to the 10th/11th day. No monosomics were in fact observed in the postimplantation series. The present findings are briefly discussed with reference to the possible pathogenesis of spontaneous abortions in man, which often possess similar types of chromosomal anomalies.     

Enzymatic basis of macrophage activation. Kinetic analysis of superoxide production in lysates of resident and activated mouse peritoneal macrophages and granulocytes

To compare the kinetics of the O-2-generating enzyme in nonactivated and activated macrophages and granulocytes from the mouse peritoneal cavity, we sought conditions in which the activity of this enzyme in cell lysates was comparable to that in intact cells. Pretreatment of macrophages with 10 mM diethyldithiocarbamate inhibited endogenous superoxide dismutase by 70% and enhanced O-2 secretion up to 15-fold, so that it was comparable to H2O2 secretion. O-2 secretion was terminated by detergent lysis and reconstituted by addition of NAD(P)H to the lysates. Optimal detection of O-2 production in lysates depended on prior stimulation of the respiratory burst, lysis with 0.05% deoxycholate rather than any of 4 other detergents or sonication, acetylation of the cytochrome c used as an indicator, and addition of NADPH rather than NADH. Kinetic analysis using NADPH-reconstituted deoxycholate lysates, together with spectra of oxidized and reduced cells, failed to reveal either marked differences in the Vmax of the O-2-generating enzyme or correlations between O-2 secretion and cytochrome b559 content among 5 macrophage populations whose H2O2 secretion ranged from 0 to 365 nmol/90 min/mg of protein. In contrast, the Km of the oxidase for NADPH varied markedly and inversely with the capacity of the intact cells to secrete O-2 or H2O2: J774G8 histiocytoma cells, 1.43 mM; resident macrophages, 0.41 mM; proteose peptone-elicited macrophages, 0.20 mM; casein-activated macrophages, 0.05 mM; NaIO4-activated macrophages, 0.05 mM; and granulocytes, 0.04 mM. These results suggest that macrophage activation, a process that enhances oxygen-dependent antitumor and antimicrobial functions, may equip the cell to secrete increased amounts of reactive oxygen intermediates largely by increasing the affinity of the oxidase for NADPH.     

Ovulation and fertilization of primary and secondary oocytes in LT/Sv strain mice

In this study, the chromosome constitution of both unfertilized oocytes and fertilized eggs isolated from the oviducts of LT/Sv strain mice were analyzed. Air-dried chromosome preparations from unfertilized oocytes revealed that about one-third of those examined were ovulated as primary oocytes. These were arrested at metaphase of the first meiotic division and exhibited the characteristic “tetrad” chromosome configuration. The remaining two-thirds of the unfertilized oocytes were ovulated at metaphase of the second meiotic division. The fertilized eggs were isolated from the oviducts of LT/Sv females previously mated to (C57BL × CBA) F1 hybrid males. Analysis of the fertilized eggs at metaphase of their first cleavage mitosis revealed that about one-third of the eggs examined were digynic triploids, whereas the remaining two-thirds had the normal diploid chromsome constitution. In the triploids, the 40 female chromosomes present (mouse, n = 20) were derived from a single diploid pronucleus formed after the extrusion of a first polar body, and following the monospermic fertilization of primary oocytes. The female pronuclear-derived chromosomes invariably exhibited “homologous pairing,” and these were associated at their centromeres. The ovulation, penetration, and subsequent fertilization of primary oocytes is an extremely unusual phenomenon in mammals and only appears to occur on a regular basis in LT/Sv mice. The premature “cytoplasmic maturation” of these oocytes is of interest, as they clearly have the same developmental capacity as secondary oocytes. The significance of these observations in relation to folliculogenesis and litter size in LT/Sv mice is discussed.     

Inhibition of aortic chemoreceptor discharge by pressor response to muscular contraction

We have examined the effect of static contraction of the hindlimb muscles on the discharge of aortic chemoreceptors in chloralose-anesthetized cats. The responses of the chemoreceptors to contraction were dependent on the arterial pressure response to this maneuver. When contraction reflexly evoked a pressor response of at least 20 mmHg, the discharge of 26 chemoreceptors was reduced from control levels by 53% (P less than 0.01). The contraction-induced inhibition of chemoreceptor discharge was prevented by phentolamine, an alpha-adrenergic antagonist that also attenuated the contraction-induced pressor response. In addition, the inhibition evoked by contraction was simulated by injection of phenylephrine and inflation of an aortic balloon, both of which evoked pressor responses. However, when contraction failed to significantly change arterial pressure, the discharge of 20 aortic chemoreceptors was not significantly changed from control levels. We conclude that the reflex pressor response to static contraction inhibits the discharge of aortic chemoreceptors. This inhibition of discharge needs to be considered when interpreting the effects of aortic barodenervation on the cardiovascular responses to exercise.     

Evidence for transcription and potential translation of the human 1.9 kb HindIII repetitive element.

Recombinant cDNA clones corresponding to the human 1.9kb HindIII repetitive element have been isolated from a cDNA library of liver cytoplasmic polyadenylated RNA. These cDNAs share 95% homology with the reported genomic DNA sequence and a similar amount of homology at the amino acid level with putative coding sequences (see preceding article by Mottez et al). They were isolated as two of four false positives from a human cDNA library in lambda gt11 and were selected with an antibody to an unrelated enzyme. These results provide direct evidence that this repetitive element is transcribed to form poly(A)+ RNA which could be translatable. Also, these observations may add to our understanding of the sources of false positives which are frequently observed in screens of cDNA libraries with antibodies as probes.     

Stimulation of splanchnic afferents reflexly relaxes tracheal smooth muscle in dogs

Although chemical stimulation of abdominal visceral afferents has been shown to reflexly increase cardiovascular and ventilatory function, the effect of stimulating these afferents on airway smooth muscle is unknown. Therefore, we recorded transverse smooth muscle tension from an innervated segment of trachea in chloralose-anesthetized dogs while we topically applied capsaicin (200 micrograms/ml) and bradykinin (0.01-10 micrograms/ml) to the serosal surfaces of the stomach, small intestine, and gallbladder. Application of these irritant substances to the stomach and small intestine decreased tracheal tension and increased mean arterial pressure. However, application of capsaicin and bradykinin to the gallbladder had only small effects on both of these variables. Cutting the splanchnic nerves abolished or greatly attenuated the decreases in tension and increases in mean arterial pressure, whereas cutting the vagi had no effect on them. We conclude that stimulation of splanchnic afferent endings in the stomach and small intestine reflexly relaxes tracheal smooth muscle in dogs. This effect may be one component of the constellation of autonomic responses reflexly evoked by abdominal visceral pain and inflammation.     

Unique acceptors for poly(ADP-ribose) in resting,proliferating and DNA-damaged human lymphocytes

Acceptor proteins for poly(adenosine diphosphoribosyl)ation were determined in resting human lymphocytes, in lymphocytes with N-methyl-N′-nitro-N-nitrosoguanidine-induced DNA damage and in lymphocytes stimulated to proliferate by phytohemagglutinin. Kinetic studies showed that the increase in ADP-ribosylation which occurred in response to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment was greater in magnitude but more transient in duration than that which occurred in phytohemagglutinin-stimulated cells. Gel electrophoretic analyses revealed that MNNG treatment and phytohemagglutinin stimulation both caused an increase in ADP-ribosylation of poly(ADP-ribose) polymerase and core histones. In MNNG-treated cells, an increase in ADP-ribosylation of histone H1 was also observed. In contrast, phytohemagglutinin-stimulated cells showed no increase in ADP-ribosylation of histone H1. In MNNG-treated cells there was also ADP-ribosylation of a protein of molecular weight 62 000, while in phytohemagglutinin-stimulated cells there was a marked increase in ADP-ribosylation of a protein of molecular weight 96000. MNNG treatment of phytohemagglutinin-stimulated cells produced a pattern of ADP-ribosylation that appeared to be due to the combined effects of the individual treatments. 3-Aminobenzamide effectively inhibited ADP-ribosylation under all treatment conditions.     

Freeze-thaw-refreeze cycle to prepare lymphocytes for HLA antibody detection or tissue typing

Human lymphocytes stored in the frozen state may be thawed, placed on cytotoxicity plates, refrozen, rethawed and used for screening sera or tissue-typing of the cells. The simple procedure described uses only a ?90 °C refrigerator for both freezing and storage of the cells. The technique permits a laboratory to collect a variety of cells over a long period, so that a set of test plates with cells from 10 to 20 donors can be prepared when a convenient number of donor cells are available. Also, the refrozen cells in cytotoxicity test plates may be warmed to the temperature of dry ice for 24 hr, returned to the refrigerator set at a slightly lower temperature, and at a later time, these cells may be thawed and used for serum screening. In view of these results, it appears possible to ship the refrozen cells from one laboratory to another using simple dry ice storage during the transfer. Negative reactions due to soluble antigens in the suspending sera can be obviated by washing out these sera and replacing them with medium 199 or alternatively, fetal calf serum can be used to replace the human serum in the suspending media.