Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion

Elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in excitable cells often acts as a negative feedback signal on firing of action potentials and the associated voltage-gated Ca2+ influx. Increased [Ca2+]i stimulates Ca2+-sensitive K+ channels (IK-Ca), and this, in turn, hyperpolarizes the cell and inhibits Ca2+ influx. However, in some cells expressing IK-Ca the elevation in [Ca2+]i by depletion of intracellular stores facilitates voltage-gated Ca2+ influx. This phenomenon was studied in hypothalamic GT1 neuronal cells during store depletion caused by activation of gonadotropin-releasing hormone (GnRH) receptors and inhibition of endoplasmic reticulum (Ca2+)ATPase with thapsigargin. GnRH induced a rapid spike increase in [Ca2+]i accompanied by transient hyperpolarization, followed by a sustained [Ca2+]i plateau during which the depolarized cells fired with higher frequency. The transient hyperpolarization was caused by the initial spike in [Ca2+]i and was mediated by apamin-sensitive IK-Ca channels, which also were operative during the subsequent depolarization phase. Agonist-induced depolarization and increased firing were independent of [Ca2+]i and were not mediated by inhibition of K+ current, but by facilitation of a voltage-insensitive, Ca2+-conducting inward current. Store depletion by thapsigargin also activated this inward depolarizing current and increased the firing frequency. Thus, the pattern of firing in GT1 neurons is regulated coordinately by apamin-sensitive SK current and store depletion-activated Ca2+ current. This dual control of pacemaker activity facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process may also provide a general mechanism for the integration of voltage-gated Ca2+ influx into receptor-controlled Ca2+ mobilization.

Coordinate regulation of lipogenic gene expression by androgens: Evidence for a cascade mechanism involving sterol regulatory element binding proteins

To gain more insight into the molecular mechanisms by which androgens stimulate lipogenesis and induce a marked accumulation of neutral lipids in the human prostate cancer cell line LNCaP, we studied their impact on the expression of lipogenic enzymes. Northern blot analysis of the steady-state mRNA levels of seven different lipogenic enzymes revealed that androgens coordinately stimulate the expression of enzymes belonging to the two major lipogenic pathways: fatty acid synthesis and cholesterol synthesis. In view of the important role of the recently characterized sterol regulatory element binding proteins (SREBPs) in the coordinate induction of lipogenic genes, we examined whether the observed effects of androgens on lipogenic gene expression are mediated by these transcription factors. Our findings indicate that androgens stimulate the expression of SREBP transcripts and precursor proteins and enhance the nuclear content of the mature active form of the transcription factor. Moreover, by using the fatty acid synthase gene as an experimental paradigm we demonstrate that the presence of an SREBP-binding site is essential for its regulation by androgens. These data support the hypothesis that SREBPs are involved in the coordinate regulation of lipogenic gene expression by androgens and provide evidence for the existence of a cascade mechanism of androgen-regulated gene expression.

Coordinate Regulation of G- and C Strand Length during New Telomere Synthesis

We have used the ciliate Euplotes to study the role of DNA polymerase in telomeric C strand synthesis. Euplotes provides a unique opportunity to study C strand synthesis without the complication of simultaneous DNA replication because millions of new telomeres are made at a stage in the life cycle when no general DNA replication takes place. Previously we showed that the C-strands of newly synthesized telomeres have a precisely controlled length while the G-strands are more heterogeneous. This finding suggested that, although synthesis of the G-strand (by telomerase) is the first step in telomere addition, a major regulatory step occurs during subsequent C strand synthesis. We have now examined whether G- and C strand synthesis might be regulated coordinately rather than by two independent mechanisms. We accomplished this by determining what happens to G- and C strand length if C strand synthesis is partially inhibited by aphidicolin. Aphidicolin treatment caused a general lengthening of the G-strands and a large increase in C strand heterogeneity. This concomitant change in both the G- and C strand length indicates that synthesis of the two strands is coordinated. Since aphidicolin is a very specific inhibitor of DNA polα and polδ, our results suggest that this coordinate length regulation is mediated by DNA polymerase.

Coordinate control of translation and localization of Vg1 mRNA in Xenopus oocytes

Vg1, a member of the transforming growth factor-β family involved in mesoderm induction, is translated subsequent to the localization of its mRNA to the vegetal pole of Xenopus oocytes. Whereas the localization of Vg1 mRNA is known to be directed by the 3′ untranslated region (UTR), the basis of its translational regulation is unknown. We show here that the 3′ UTR of Vg1 causes translational repression of two different reporter mRNAs in Xenopus oocytes. A 350-nucleotide region of the 3′ UTR, which is distinct from the localization element, is necessary and sufficient for mediating translational repression and specifically binds to a 38-kDa polypeptide. The translational repression activity is found throughout the oocyte and at all stages of oogenesis. These results suggest that factors colocalized with Vg1 mRNA at the vegetal pole relieve translational repression to allow expression of Vg1 protein.

Coordinate Accumulation of Antifungal Proteins and Hexoses Constitutes a Developmentally Controlled Defense Response during Fruit Ripening in Grape1

During ripening of grape (Vitis labruscana L. cv Concord) berries, abundance of several proteins increased, coordinately with hexoses, to the extent that these became the predominant proteins in the ovary. These proteins have been identified by N-terminal amino acid-sequence analysis and/or function to be a thaumatin-like protein (grape osmotin), a lipid-transfer protein, and a basic and an acidic chitinase. The basic chitinase and grape osmotin exhibited activities against the principal grape fungal pathogens Guignardia bidwellii and Botrytis cinerea based on in vitro growth assays. The growth-inhibiting activity of the antifungal proteins was substantial at levels comparable to those that accumulate in the ripening fruit, and these activities were enhanced by as much as 70% in the presence of 1 m glucose, a physiological hexose concentration in berries. The simultaneous accumulation of the antifungal proteins and sugars during berry ripening was correlated with the characteristic development of pathogen resistance that occurs in fruits during ripening. Taken together, accumulation of these proteins, in combination with sugars, appears to constitute a novel, developmentally regulated defense mechanism against phytopathogens in the maturing fruit.

Association of RNase mitochondrial RNA processing enzyme with ribonuclease P in higher ordered structures in the nucleolus: a possible coordinate role in ribosome biogenesis.

RNase mitochondrial RNA processing enzyme (MRP) is a nucleolar ribonucleoprotein particle that participates in 5.8S ribosomal RNA maturation in eukaryotes. This enzyme shares a polypeptide and an RNA structural motif with ribonuclease P (RNase P), a nuclear endoribonuclease originally described in the nucleus that processes RNA transcripts to generate their mature 5' termini. Both enzymes are also located in mitochondria. This report further characterizes the relationship between RNase MRP and RNase P. Antisense affinity selection with biotinylated 2'-O-methyl oligoribonucleotides and glycerol gradient fractionation experiments demonstrated that small subpopulations of RNase MRP and RNase P associate with each other in vivo in macromolecular complex, possibly 60-80S preribosomes. This latter notion was supported by fluorescence in situ hybridization experiments with antisense oligonucleotides that localized that RNA components of RNase MRP and RNase P to the nucleolus and to discrete cytoplasmic structures. These findings suggest that small subpopulations of RNase MRP and RNase P are physically associated, and that both may function in ribosomal RNA maturation or ribosome assembly.

Localization of the central rhythm generator involved in spontaneous consummatory licking in rats: functional ablation and electrical brain stimulation studies.

Localization of the central rhythm generator (CRG) of spontaneous consummatory licking was studied in freely moving rats by microinjection of tetrodotoxin (TTX) into the pontine reticular formation. Maximum suppression of spontaneous water consumption was elicited by TTX (1 ng) blockade of the oral part of the nucleus reticularis gigantocellularis (NRG), whereas TTX injections into more caudal or rostral locations caused significantly weaker disruption of drinking. To verify the assumption that TTX blocked the proper CRG of licking rather than some relay in its output, spontaneously drinking thirsty rats were intracranially stimulated via electrodes chronically implanted into the oral part of the NRG. Lick-synchronized stimulation (a 100-ms train of 0.1-ms-wide rectangular pulses at 100 Hz and 25-150 microA) applied during continuous licking (after eight regular consecutive licks) caused a phase shift of licks emitted after stimulus delivery. The results suggest that the stimulation has reset the CRG of licking without changing its frequency. The reset-inducing threshold current was lowest during the tongue retraction and highest during the tongue protrusion period of the lick cycle. It is concluded that the CRG of licking is located in the oral part of NRG.

Sterol regulation of acetyl coenzyme A carboxylase: a mechanism for coordinate control of cellular lipid.

Transcription from the housekeeping promoter for the acetyl coenzyme A carboxylase (ACC) gene, which encodes the rate-controlling enzyme of fatty acid biosynthesis, is shown to be regulated by cellular sterol levels through novel binding sites for the sterol-sensitive sterol regulatory element binding protein (SREBP)-1 transcription factor. The position of the SREBP sites relative to those for the ubiquitous auxiliary transcription factor Sp1 is reminiscent of that previously described for the sterol-regulated low density lipoprotein receptor promoter. The experiments provide molecular evidence that the metabolism of fatty acids and cholesterol, two different classes of essential cellular lipids, are coordinately regulated by cellular lipid levels.

Coordinate regulation of Bacillus subtilis peroxide stress genes by hydrogen peroxide and metal ions.

The Bacillus subtilis mrgA gene encodes an abundant DNA-binding protein that protects cells against the lethal effects of H2O2. Transcription of mrgA is induced by H2O2 or by entry into stationary phase when manganese and iron levels are low. We have selected for strains derepressed for transcription of mrgA in the presence of Mn(II). The resulting cis-acting mutants define an operator site just upstream of the mrgA promoter. Similar sequences flank the promoters for the catalase gene, katA, and the heme biosynthesis operon, hemAXCDBL. Like mrgA, transcription of the katA and hem genes is repressed by Mn(II), which thereby potentiates the killing action of H2O2. We identified two classes of trans-acting mutants derepressed for mrgA transcription in the presence of Mn(II): some exhibit a coordinate derepression of MrgA, catalase, heme biosynthesis, and alkyl hydroperoxide reductase and are H2O2 resistant, while others have reduced catalase activity and are H2O2 sensitive. These data indicate that the peroxide stress response of B. subtilis is regulated by a repressor that senses both metal ion levels and H2O2.

Estradiol and the inhibition of hypothalamic gonadotropin-releasing hormone pulse generator activity in the rhesus monkey.

In mammals, gonadal function is controlled by a hypothalamic signal generator that directs the pulsatile release of gonadotropin-releasing hormone (GnRH) and the consequent pulsatile secretion of luteinizing hormone. In female rhesus monkeys, the electrophysiological correlates of GnRH pulse generator activity are abrupt, rhythmic increases in hypothalamic multiunit activity (MUA volleys), which represent the simultaneous increase in firing rate of individual neurons. MUA volleys are arrested by estradiol, either spontaneously at midcycle or after the administration of the steroid. Multiunit recordings, however, provide only a measure of total neuronal activity, leaving the behavior of the individual cells obscure. This study was conducted to determine the mode of action of estradiol at the level of single neurons associated with the GnRH pulse generator. Twenty-three such single units were identified by cluster analysis of multiunit recordings obtained from a total of six electrodes implanted in the mediobasal hypothalamus of three ovariectomized rhesus monkeys, and their activity was monitored before and after estradiol administration. The bursting of all 23 units was arrested within 4 h of estradiol administration although their baseline activity was maintained. The bursts of most units reappeared at the same time as the MUA volleys, the recovery of some was delayed, and one remained inhibited for the duration of the study (43 days). The results indicate that estradiol does not desynchronize the bursting of single units associated with the GnRH pulse generator but that it inhibits this phenomenon. The site and mechanism of action of estradiol in this regard remain to be determined.