Calcium-Dependent Release of Tyrosine in Brain Elicited by Stimulation of Neuropeptide Receptors

We sought to establish whether the endogenous opiate-receptor agonist Met-enkephalin (m-ENK) selectively modulates the release of endogenous tyrosine (Tyr) from brain slices prepared from the corpus striatum (CS). Amino acids (AAs) released from slices of CS and, for comparison, cerebral cortex (Cx) were measured by HPLC. Incubation of slices with m-ENK (1-10 microM) increased the basal release of Tyr (up to 293% of control) from CS, but not Cx, whereas other nonneurotransmitter AAs, phenylalanine (Phe) and valine (Val), were unchanged. The release of the putative neurotransmitter AAs glutamate (Glu), taurine (Tau), and glycine (Gly) were similarly increased by 50-150% with m-ENK in slices of CS, but not Cx. The enhanced release of AAs by m-ENK was prevented by removal of extracellular Ca2+ or by preincubation with the opiate receptor antagonist naloxone. Neuronal depolarization by potassium (5-55 mM) in the presence of Ca2+ did not affect the release of Tyr, whereas release of neurotransmitter AAs such as gamma-aminobutyric acid (GABA) were markedly increased. The increase in basal Tyr release by m-ENK was not the result of a decreased uptake of Tyr. Relative to slices, the basal release of Tyr, Phe, and Val from a synaptosomal (P2) preparation of CS was small (8-51%) compared to that of GABA, Gly, Glu, and Tau (49-123%). Nonetheless, m-ENK (10 microM) markedly increased the release of Tyr (to 833%), but not Glu, Gly, and Tau from the P2 fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chloroplast photooxidation inhibits the expression of a set of nuclear genes

Summary Mutations or herbicides which inhibit the accumulation of carotenoid pigments in higher plants also result in the arrest of chloroplast development at a very early stage. The cause is extensive photooxidative damage within the chloroplast in the absence of protective carotenoids. Because the extent of photooxidation is dependent upon light intensity, normal chloroplast development can occur when carotenoid-deficient seedlings are grown in very dim light. Normal accumulation of chloroplastic and cytosolic mRNAs encoding chloroplast proteins proceeds only under permissive dim light conditions. Illumination with higher intensity light causes rapid chlorophyll photooxidation and the loss of two cytosolic mRNAs coding for proteins destined for the chloroplast, but does not affect another light-regulated cytosolic mRNA encoding a cytosolic protein. This experimental system may have uncovered a mechanism which coordinates the expression of genes in different cellular compartments.Abbreviations LHCP light-harvesting chlorophyll a/b protein - SSu small subunit - RuBP fibulose 1,5-bisphoshate - PEP phosphoenolpyruvate     

Mutations that increase the mitotic stability of minichromosomes in yeast: characterization of RAR1

Summary In an attempt to identify proteins involved in the initiation of DNA replication, we have isolated a series of Saccharomyces cerevisiae mutants in which the function of putative replication origins is affected. The phenotype of these Rar^- (regulation of autonomous replication) mutants is to increase the mitotic stability of plasmids whose replication is dependent on weak ARS elements. These mutations are generally recessive and complementation analysis shows that mutations in several genes may improve the ability of weak ARS elements to function. One mutation (rar1-1) also confers temperature-sensitive growth, and thus an essential gene is affected. We have determined the DNA sequence of the RAR1 gene, which reveals an open reading frame for a 48.5 kDa protein. The RAR1 gene is linked to rna1 on chromosome XIII.     

Isolation of 3 beta,20 alpha-hydroxysteroid oxidoreductase from sheep fetal blood

3 beta,20 alpha-Hydroxysteroid oxidoreductase has been isolated from ovine fetal blood by a 2,370-fold purification scheme of ammonium sulfate fractionation, calcium phosphate gel adsorption, affinity chromatography, and fast performance liquid chromatography. A new high performance liquid chromatography-based assay for measuring 20 alpha-reductase activity is described. The enzyme is a monomer with a molecular weight of 35,000 and uses NADPH as a cofactor for reductase activity. It reduces progesterone to 4-pregnen-20 alpha-ol-3-one or 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol with kinetic characteristics of Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1 or Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1, respectively. 5 alpha-Dihydrotestosterone competitively inhibits 20 alpha-reductase activity with a Ki value of 102 microM.     

Inhibition of progesterone secretion and decrease of ovarian blood flow by isoxazole in pregnant rats

Pregnant rats were treated on day 11 of pregnancy with 0.62, 1.25, 2.5 or 5.0 mg of isoxazole per 100 g of body weight (bw) (Experiment I) or with 2.5 mg of isoxazole per 100 g of bw (Experiment II). In Experiment I animals were anaesthetized with pentobarbital at 48 hr. In experiment II animals were similarly anaesthetized at 0, 12, 24, or 48 hr following administration of isoxazole. Blood was collected separately from the ovary or from the uterus via a thin cannula which was inserted into the utero-ovarian vein. Blood was also collected from the vena cava inferior. The concentrations of progesterone (P), 17 beta-oestradiol (E2), and corticosterone were measured in blood samples by RIA. All of the animals exhibited reduced rates of ovarian venous blood flow and rates of P secretion when pregnancy was terminated by isoxazole (2.5 mg/100 g bw or greater). The concentration of P was found to be correspondingly reduced by 90% in ovarian venous blood and by 50% in peripheral blood, relative to the controls. After isoxazole had been administered, the rate of P secretion and also the concentration of P in peripheral blood continued to decrease rapidly and to remain at low levels for at least 48 hr. By contrast, the concentration of corticosterone in peripheral blood decreased during the first 12 hr but then it returned to the control levels by 48 hr. The ovarian secretion rate of E2 and the concentration of E2 in peripheral blood remained unchanged during the same 48 hr period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-dependent intermolecular subunit communication between isolated alpha beta heterodimeric insulin receptor complexes

The dissociation of the purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex into an alpha beta heterodimeric state was found to occur in a pH- and dithiothreitol (DTT)-dependent manner. Formation of the alpha beta heterodimeric complex, under conditions which preserved tracer insulin binding and protein kinase activities (pH 8.75 for 25 min followed by 2.0 mM DTT for 5 min) occurred with an approximate 50% efficiency. The resulting nondissociated alpha 2 beta 2 heterotetrameric complexes could then be separated effectively by Bio-Gel A-1.5m gel filtration chromatography at neutral pH. The isolated DTT-treated but nondissociated alpha 2 beta 2 heterotetrameric complex was resistant to any further dissociation by a second round of DTT and alkaline pH treatment, whereas the isolated alpha beta heterodimeric complex was stable to spontaneous reassociation for at least 72 h at pH 7.60. Kinetic analyses of the insulin receptor protein kinase activity demonstrated that the insulin stimulation of glutamic acid:tyrosine (4:1) synthetic polymer phosphorylation for both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes occurred via an increase in Vmax without any significant change in Km. Examination of beta subunit autophosphorylation of the alpha beta heterodimeric complex, in the presence but not in the absence of insulin, demonstrated the appearance of the covalent 32P-labeled alpha 2 beta 2 heterotetrameric complex. Further, the initial rate of insulin-stimulated beta subunit autophosphorylation in the isolated alpha beta heterodimeric complex occurred in a dilution-dependent (intermolecular) manner. These data demonstrate that the isolated alpha beta heterodimeric insulin receptor complex is fully capable of expressing insulin-dependent activation of the beta subunit protein kinase domain with the covalent reassociation of the alpha beta heterodimeric complex into an alpha 2 beta 2 heterotetrameric disulfide-linked state.     

l-Alanine uptake in brush border membrane vesicles from the gill of a marine bivalve

Summary Brush border membrane vesicles were prepared from mussel gills using differential and sucrose density gradient centrifugation. These vesicles contained both the maximal Na⁺-dependent alanine transport activity found in the gradient and the maximal activities of -glutamyl transpeptidase and alkaline phosphatase. Electron micrographs showed closed vesicles of approximately 0.1–0.5 m diameter. Transport experiments using these vesicles demonstrated a transient 18-fold overshoot in intravesicular alanine concentration in the presence of an inwardly directed Na⁺ gradient, but not under Na⁺ equilibrium conditions. A reduced overshoot (10-fold) was seen with an inwardly directed K⁺ gradient. Further studies revealed a broad cation selectivity, with preference for Na⁺, which was characteristic of alanine transport but not glucose transport in these membranes. The apparent amino acid specificity of the uptake pathway(s) was similar to that of intact gills and supported the idea of at least four separate pathways for amino acid transport in mussel gill brush border membranes. The apparent Michaelis constant for alanine uptake was approximately 7m, consistent with values forK _t determined with intact tissue.     

bcd: A mutation affecting the width of organelle domains in the cortex of Tetrahymena thermophila

Summary A single-gene recessive mutation, bcd (broadened cortical domains), of Tetrahymena thermophila is characterized by a variable broadening of the spatial domains within which cortical organelles, including both the contractile vacuole pores (CVP) and oral apparatus (OA), are formed. The phenotype is not temperature-sensitive. During the development of the organelles of the mutant prior to cell division, extra CVPs and extra oral primordia (OP) appear near ciliary rows adjacent to the rows at which these structures normally form. In the later stages of development, some, but not all, of these extra structures are resorbed, or in the case of the oral domain, multiple adjacent OPs may be completely or partially integrated into a single enlarged OA. When multiple OAs persist, one or more of these may display a reversed orientation reminiscent of those encountered in janus mutants. However, unlike janus, bcd cells do not express any sign of a mirror-image global organization.Our results can best be accounted for by postulating that the bcd mutation affects some common determinant of the widths of both CVP and OA domains. Studies are in progress which explore the relationship between this width-determining mechanism(s) and the mechanism(s) determining the location of cortical organelles around the cell circumference.     

Transposon Mutants of Bradyrhizobium japonicum Altered in Attachment to Host Roots

Transposon mutants of Bradyrhizobium japonicum 110 ARS were produced and screened for changes in attachment ability. Mutant CFK4 produced twice as many piliated cells, attached in 2.5-fold-higher numbers to soybean root segments, and colonized roots in about 2-fold-higher numbers than did the parental strain, 110 ARS. Mutants CFK35 and CFK38 were reduced in their attachment about 2-fold and 3.5-fold, respectively. This corresponded to reductions in piliated cells in their populations, reduced reaction with anti-pilus antiserum, and reduced hydrophobic attachment. Mutants CFK4 and CFK38 nodulated soybeans at about the same level as the parent strain, but CFK35 induced only pseudonodules. Two-dimensional gel analyses of the proteins from the mutants showed relatively few changes in proteins.