Recombination and Chromosome Segregation during the Single Division Meiosis in SPO12–1 and SPO13–1 Diploids

This paper reports a study of chromosome segregation and recombination during sporulation of spo12–1 and spo13–1 diploid strains of S. cerevisiae. These strains undergo a single division to form asci containing two diploid or near-diploid spores. The segregation of centromere-linked markers in the two-spored (dyad) products indicates that the division is generally equational. However, in a small percentage of the spo12–1 and spo13–1 cells, it appears that a meiosis I-like division occurs. Aberrant segregation of the MAT locus on chromosome III, yielding a monosomic and a trisomic spore pair, occurs in 12% of all dyads. The segregation patterns of markers at various distances from their centromeres and several pairs of markers on the same chromosome indicate that recombination takes place in both strains at nearly standard meiotic levels.     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

Localization of immunoreactive prolactin in ependyma and circumventricular organs of rat brain

Summary Immunoreactive prolactin (IMP) has been localized in the male rat brain using the soluble peroxidase-anti-peroxidase (PAP) technique. In normal untreated animals, reaction product was seen in choroid plexus (CP) and in ependymal cells of the ventricular lining with heaviest concentrations of positively staining cells in the 3rd ventricle near the subcommisural organ (SCO), in the lateral ventricles near the subfornical organ (SFO), and in the 4th ventricle near the area postrema (AP). IMP was also present in numerous ependymal cells resembling tanycytes in the cerebral aqueduct, central canal of the spinal cord at the level of the AP, the organum vasculosum of the lamina terminalis (OVLT) and the floor of the infundibular recess. Immunoreactive cells resembling neurons were localized within the substance of the AP, SCO, and OVLT. IMP was also present in fibers of the zona externa of the median eminence and infundibular stalk; a few cells of the pars tuberalis contained reaction product. Hypophysectomized rats and bromocriptine-treated rats exhibited a similar staining pattern except that bromocriptine treatment eliminated IMP from most CP cells. Hypophysectomy, bromocriptine or estrogen treatment enhanced staining for IMP in cells of the pars tuberalis; estrogen treatment or hypophysectomy produced an increase in the number and distribution of immunoreactive cells as well as increased density of reaction product in cells of the medial habenular nucleus. The functional relevance of prolactin in these locations in the brain, the possible routes of transport of prolactin from the pituitary gland to the central nervous system, and the strong suggestion of extra-pituitary sites of synthesis of a prolactin-like hormone are discussed.     

Uraniosomes produced in cultured rabbit kidney cells by uranyl acetate

Cultured rabbit kidney cells were exposed to uranyl acetate. This produced single-membrane-bound presumably lysosomal bodies (called 'uraniosomes') containing electron-dense crystals in the cultured cells. Similar crystalline deposits were seen in extracellular locations also. All uraniosomes and extracellular uranium deposits analyzed by electron-probe x-ray analysis were found to contain uranium, potassium, calcium and phosphorus. Traces of sulphur were detected in some but not all uraniosomes and extracellular uranium deposits.     

Cooperative interaction of reduced pyridine nucleotides with estrogen synthetase of human placental microsomes

The estrogen synthetase present in human placental microsomes appears to be dependent on the cooperative interaction of the reduced cofactors NADPH and NADH for optimal activity. Using steady-state concentrations of either cofactor, it was found that while the estrogen synthetase activity followed hyperbolic saturation kinetics with NADPH (K_mapp = 14 μM), the enzyme followed sigmoidal saturation kinetics when the cofactor was NADH, with the half-maximum velocity attained at a cofactor concentration of 1.1 mm. The maximum velocity obtained with NADPH as the cofactor was greater than with corresponding concentrations of NADH. Estrogen synthetase activity in the presence of NADH was not due to NADPH contamination. NADH, in the presence of small concentrations of NADPH (0.5 to 5 μm), stimulated significantly the rate of estrogen formation from androstenedione by placental microsomes and, in addition, the enzyme saturation kinetics changed from sigmoidal to hyperbolic, thus mimicking the effect of NADPH. Estrogen synthetase activity, measured in the presence of 1 mm NADH, was stimulated in a dose-dependent manner by NADPH (K_mapp = 0.4 μM NADPH) and, when the enzyme was measured in the presence of 5 μm NADPH, the activity was stimulated in a dose-dependent manner by NADH (K_mapp = 45 μM NADH). Estrogen synthetase activity measured in the presence of NADH, without and with NADPH (1 μm) remained linear both with time of incubation for approximately 15 min and with microsomal protein concentration up to 3 mg/ml. The apparent K_m of estrogen synthetase for androstenedione, when measured in the presence of NADH, was 1 μm. The synergistic interaction between NADH and NADPH in stimulating placental estrogen synthetase activity observed in vitro may, conceivably, take place in vivo in the intact placenta.     

The rise and fall of pineal N-acetyltransferase in vitro: Neural regulation in the developing rat

In rats, the pineal gland has a rhythm in the activity of the enzyme, N-acetyltransferase (NAT), which is thought responsible for daily cycles of melatonin synthesis. Neonatal rat pineal glands, but not those of adult rats, have a single cycle that is observed in vitro during the first day of organ culture. The neural regulation of the cycle was investigated using neonatal rats with adult rats used for comparison. Prior treatment of rat pups with constant light did not abolish the cycle in vitro though it did abolish the in vivo rhythm. Removal of the superior cervical ganglia did not abolish the in vivo rhythm that was measured the first day after surgery, but ablation of the ganglia did abolish the rhythm if several days or more elapsed after surgery. Extirpation of the superior cervical ganglia abolished the in vitro NAT cycle in pup pineal glands as did the pharmacological equivalent, injection of 6-hydroxydopamine. Propranolol, a beta blocking agent, prevented the occurrence of the cycle in vitro.     

Angiotensin II potentiates responses of the rabbit basilar artery to adrenergic nerve stimulation

Angiotensin II has little contractile effect on the isolated rabbit basilar artery; however, it markedly potentiates contractile responses to adrenergic nerve stimulation. This is not a post-synaptic effect of angiotensin, as responses to exogenous norepinephrine are not altered. Angiotensin increases stimulation-evoked release of norepinephrine, and this effect probably accounts for the increased response to adrenergic nerve stimulation. Since sympathetic stimulation may protect the cerebral circulation from hypertensive damage, increased responsiveness to adrenergic nerve activity produced by angiotensin may have a beneficial effect.     

An ultrastructural search for lectin-binding sites on surfaces of spinach leaf organelles

Organelles isolated from leaves of spinach (Spinacia oleracea L.) were prefixed in glutaraldehyde and then incubated with ferritin conjugates of four lectins — Concanavalin A (Con A), Ricinus communis L. agglutinin, MW 120,000 (RCA), soybean agglutinin (SBA), and wheat germ agglutinin (WGA) — in order to probe their cytoplasmic surfaces for saccharide residues. In each case the major leaf organelles, including microbodies, mitochondria and chloroplast derivatives, failed to exhibit labeling when examined with the electron microscope. Tobacco (Nicotiana tabacum L.) leaf protoplasts, incubated simultaneously with and under identical conditions to the spinach organelles, showed specific labeling of their plasma membranes with all four lectin conjugates, thus establishing the efficacy of the procedure for demonstrating the presence of binding sites when they exist. Further attempts to show binding of one of the lectins, Con A, by labeling with fluorescein-Con A and by organelle agglutination, yielded results consistent with the absence of ultrastructural labeling. It is concluded that no saccharide residues recognized by the four lectins are present on the cytoplasmic surfaces of organelles and that those residues reported to be constituents of intracellular membranes, therefore, are most likely exposed on the luminal (extracytoplasmic) surfaces.Abbreviations Con A Concanavalin A - RCA Ricinus communis agglutinin, MW 120,000 - SBA soybean agglutinin - WGA wheat germ agglutinin     

DNA and protein content of mouse sperm: Implications regarding sperm chromatin structure

A variety of biochemical and histochemical techniques have been used to compare the composition of chromatin in sperm nuclei isolated from the epididymides of five mouse strains. The DNA content was determined by phosphorus analysis, deoxyribose analysis, absorption spectroscopy at 260 nm, and cytomorphometry following gallocyanine chrome alum staining. All four methods indicate that the mouse sperm nucleus contains approx. 3.3 pg DNA and that the DNA content does not vary significantly among the strains tested. Three different techniques, quantitative amino acid analysis, absorption spectroscopy at 230 nm, and sperm head density analysis in cesium chloride, were used to determine the protein content. Sperm nuclei from each strain of mouse were found to have a protein to DNA ratio of 0.9 and a chromatin protein content of 3 pg/nucleus. Comparisons of the basic proteins by disc gel electrophoresis demonstrate that the sperm nuclei contain only protamine and lack significant levels of somatic histones or transition proteins. The sperm from each strain contained both mouse protamine variants and the relative distribution of the two proteins did not appear to differ among strains. Using this information, we have been able to draw certain conclusions regarding DNA-protamine interactions and the mode of DNA packaging in the sperm nucleus. The most important of these is that the DNA in the mouse sperm nucleus cannot be packaged in nucleosomes. The protamines in sperm chromatin do not function as structural proteins, providing a subunit core around which the DNA is wrapped, but appear to completely neutralize the phosphodiester backbone of the DNA molecule, thereby minimizing the repulsion between neighboring segments of DNA and allowing it to be condensed into a biochemically inactive particle of genetic information.