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R Graf

Publications and source records attributed to R Graf.

At least 181 records · Page 10Linked to original sources
PubMed1990

A GCN4 protein recognition element is not sufficient for GCN4-dependent regulation of transcription in the ARO7 promoter of Saccharomyces cerevisiae.

The gene ARO7 encodes the monofunctional enzyme chorismate mutase, a branch point enzyme in the aromatic amino acid biosynthetic pathway in Saccharomyces cerevisiae. We investigated the transcription of the ARO7 gene. Three 5' ends at positions -36, -56 and -73 and the 3' end of the transcripts 146 bp downstream of the translational stop codon were mapped. As in the promoters of other aromatic amino acid biosynthetic genes, a recognition element for the GCN4 transcriptional activator of amino acid biosynthesis is located 425 base pairs (bp) upstream of the first transcriptional start point. This element binds GCN4 specifically in vitro. Northern analysis and determination of the specific enzyme activity reveals however, that the element is not sufficient to mediate transcriptional regulation by GCN4 in vivo. We thus suggest that in addition to a consensus sequence capable of binding the GCN4 protein other factors like, for example, chromatin structure, determine whether a recognition site for a transcription factor functions as an upstream activation sequence.

Blotting, Northern↗
PubMed1990

G protein coupling of receptors to ionic channels and other effector systems.

1. Four questions raised by previous studies that had shown activation of K+ channels by alpha subunits of the type 3 Gi protein are addressed in the present communication: a) are K+ channels specific for one Gi? b) are there more ionic channels under direct G protein control? c) can we confirm using recombinant G alpha s the results obtained with biochemically resolved G alpha s and continue ascribing the regulatory effector to this part of the alpha beta gamma holo-G protein? and d) can we confirm that a single G alpha, Gs alpha in this case, is able to affect more than one type of effector function? 2. We found Gi alpha s are isoforms, that there exist also Gi-insensitive, Go-responsive K+ channels and that G alpha s can be multifunctional. Thus, a single receptor will elicit cellular responses that will depend on the endogenous G protein as well as the type of effector function expressed in it. 3. In another set of experiments we found that G beta gamma s, be they derived from human erythrocytes, human placenta, bovine brain or bovine retina, all inhibit Gk-gated K+ channel activity as seen in inside out membrane patches with GTP as the driving nucleotide. In addition we noted that inhibition was much more effective under basal (no agonist in the pipette) than agonist stimulated conditions, as reported in earlier experiments in which beta-adrenoceptors, Gs and catalytic unit of adenylyl cyclase had been incorporated into phospholipid vesicles. 4. We propose that one of the roles of G beta gamma s in membranes is to quench ligand independent G protein activation by unoccupied receptors. Other roles of G beta gamma s are: a) by re-associating with GDP-G alpha s, to promote interaction with receptors, and b) by dissociating from activated R.G alpha *GTP.beta gamma, to allow for receptor dissociation from GTP-activated G alpha s, which is required to satisfy the catalytic mode of receptor action.

GTP-Binding Proteins↗
PubMed1990

Differences in ischemia-induced accumulation of amino acids in the cat cortex.

It is well established that excitatory amino acid neurotransmitters are extensively liberated during ischemia and that they have neurotoxic properties contributing to neuronal injury. To study changes in the liberation of excitatory and other amino acids during cerebral ischemia, we measured their extracellular concentrations and related them to blood flow levels and electrophysiologic activity (electrocorticogram and auditory evoked potentials) before and for up to 2 hours after multiple cerebral vessel occlusion in 14 anesthetized cats. Blood flow levels between 0 and 43 ml/100 g/min were reached. Concentrations of the excitatory amino acid neurotransmitters increased most (aspartate 10-fold, glutamate 30-fold, and gamma-aminobutyric acid 300-fold compared with control values) below a blood flow threshold of 20 ml/100 g/min. The total power of the electrocorticogram and the amplitude of the auditory evoked potentials were affected below the same blood flow threshold. In contrast, concentrations of the nontransmitter amino acids taurine, alanine, asparagine, serine, and glutamine increased 1.5-5-fold as blood flow decreased, while concentrations of the essential amino acids phenylalanine, valine, leucine, and isoleucine did not change during cerebral ischemia. The great increases in concentrations of the excitatory amino acid neurotransmitters below a blood flow threshold close to that for functional disturbance is in accordance with the role of these amino acids in ischemic cell damage. Their release at blood flow levels compatible with cell survival and the increase in their concentrations with severity and duration of cerebral ischemia imply that excitotoxic antagonists may have potential as therapeutic agents.

Amino Acids↗
PubMed1990

Functional impairment due to white matter ischemia after middle cerebral artery occlusion in cats.

We recorded regional cerebral blood flow, somatosensory evoked potentials, and auditory evoked potentials in the thalamic relay nuclei (ventral posterior lateral nucleus and medial geniculate body) and in the somatosensory and auditory cortices during and after 1 hour of transient left middle cerebral artery occlusion in nine cats. Regional cerebral blood flow was also measured in the thalamocortical tracts of five of these cats. Additionally, the integrity of thalamocortical connections was tested by retrograde labeling of the thalamic nuclei with horseradish peroxidase in eight cats (three of which experienced no ischemia). Regional cerebral blood flow was severely reduced during middle cerebral artery occlusion in the left primary auditory cortex (8.5 ml/100 g/min) and in white matter pathways (6.4-7.6 ml/100 g/min). In contrast, regional cerebral blood flow did not change significantly in the somatosensory cortex or in either thalamic nucleus. Evoked potentials were abolished in both cortices but remained unchanged in the thalamic nuclei. Cortical somatosensory evoked potentials disappeared 5-8 minutes later than auditory evoked potentials. Recirculation after 1 hour of ischemia resulted in rapid and almost complete recovery (94%) of somatosensory evoked potentials and little recovery (18.4%) of auditory evoked potentials. We conclude that in the auditory pathway both cortical and fiber tract ischemia are (perhaps synergistically) responsible for dysfunction, while in the somatosensory cortex evoked potentials are abolished due to white matter ischemia. The delayed disappearance and better recovery of somatosensory than of auditory evoked potentials indicate that ischemic tolerance is higher in fiber tracts than in cortex.

Animals↗
PubMed1990

Roles of G proteins in coupling of receptors to ionic channels and other effector systems.

Guanine nucleotide binding (G) proteins are heterotrimers that couple a wide range of receptors to ionic channels. The coupling may be indirect, via cytoplasmic agents, or direct, as has been shown for two K+ channels and two Ca2+ channels. One example of direct G protein gating is the atrial muscarinic K+ channel K+[ACh], an inwardly rectifying K+ channel with a slope conductance of 40 pS in symmetrical isotonic K+ solutions and a mean open lifetime of 1.4 ms at potentials between -40 and -100 mV. Another is the clonal GH3 muscarinic or somatostatin K+ channel, also inwardly rectifying but with a slope conductance of 55 pS. A G protein, Gk, purified from human red blood cells (hRBC) activates K+ [ACh] channels at subpicomolar concentrations; its alpha subunit is equipotent. Except for being irreversible, their effects on gating precisely mimic physiological gating produced by muscarinic agonists. The alpha k effects are general and are similar in atria from adult guinea pig, neonatal rat, and chick embryo. The hydrophilic beta gamma from transducin has no effect while hydrophobic beta gamma from brain, hRBCs, or retina has effects at nanomolar concentrations which in our hands cannot be dissociated from detergent effects. An anti-alpha k monoclonal antibody blocks muscarinic activation, supporting the concept that the physiological mediator is the alpha subunit not the beta gamma dimer. The techniques of molecular biology are now being used to specify G protein gating. A "bacterial" alpha i-3 expressed in Escherichia coli using a pT7 expression system mimics the gating produced by hRBC alpha k.

Animals↗
PubMed1990

Enhancement of immunoreactivity of von Willebrand factor in vascular endothelial cells of rat organs after glucocorticoid administration.

Our study showed that synthetic glucocorticoids are potent mediators of vWF-reactivity in rat fetal placental, liver and lung vascular elements. However, it is not yet possible to draw final conclusions from these results obtained only by immunohistochemical studies using antibody against vWF, since the mechanisms in factor VIII/vWF are highly complex. Further investigations for example with anti-vW AgII and measurements of blood vWF levels after treatment with synthetic glucocorticoids may deliver more insight into the mechanisms of action of these drugs. Investigations after treatment of pregnant rats with methylprednisolone might help, in addition, to find out whether the increased vWF-reactivity in caused by special substituents in the steran ring of the glucocorticoids.

Animals↗
PubMed1990

[Sonographic neonatal screening of the hip joint--luxury or necessity?].

Based on the ultrasound investigation of 19,666 babies (1981-1988), 6341 of them directly at the maternity ward, we picked out the group of sonographically unstable or decentered hip joints, which must be treated instantly. Almost 50% of these hips did not show any clinical signs of instability! At follow-up the subgroup of sonographically screened hip joints showed optimal results after consequent earliest treatment: 100% physiological results of CE-angle (Wiberg and Engelhardt), no femoral head necrosis. Ultrasound screening is the only tool to detect definitely all newborn hips requiring therapy. Earliest treatment is the pre-condition of anatomical healing up. Thus--a general sonographic newborn screening is indispensable.

Follow-Up Studies↗
PubMed1990

Enzyme histochemical and histological changes in the adult rat kidney after prenatal gentamicin exposure.

Treatment of rats between day 15 and 20 of gestation with gentamicin caused histological as well as enzyme histochemical lesions in the kidney of the one year old offspring (F1 generation). Other organs were not significantly affected. Primarily in the female kidney dilated convoluted proximal tubulus with reduced or absent staining for brush border and lysosomal proteases, phosphatases and glycosidases and mitochondrial dehydrogenases were observed. In comparison, glomeruli were less frequently damaged and contained fewer capillary loops or irregularly arranged tissue elements with lowered or no activities for plasma membrane-associated proteases as well as specific and non-specific phosphatases. In addition, the activities of proteases and phosphatases in cortical and medullary endothelial cells of capillaries were reduced or even abolished in the kidney of the female and male F1 generation after treatment of their mothers with gentamicin.

Animals↗
PubMed1989

Splice variants of the alpha subunit of the G protein Gs activate both adenylyl cyclase and calcium channels.

Signal transducing guanine nucleotide binding (G) proteins are heterotrimers with different alpha subunits that confer specificity for interactions with receptors and effectors. Eight to ten such G proteins couple a large number of receptors for hormones and neurotransmitters to at least eight different effectors. Although one G protein can interact with several receptors, a given G protein was thought to interact with but one effector. The recent finding that voltage-gated calcium channels are stimulated by purified Gs, which stimulates adenylyl cyclase, challenged this concept. However, purified Gs may have four distinct alpha-subunit polypeptides, produced by alternative splicing of messenger RNA. By using recombinant DNA techniques, three of the splice variants were synthesized in Escherichia coli and each variant was shown to stimulate both adenylyl cyclase and calcium channels. Thus, a single G protein alpha subunit may regulate more than one effector function.

Adenylyl Cyclases↗
PubMed1989

Recombinant alpha i-3 subunit of G protein activates Gk-gated K+ channels.

G proteins, particularly those sensitive to pertussis toxin, are difficult to separate biochemically, creating uncertainty in functional assignments. For this reason the cDNAs encoding G alpha i-3 and two of the G alpha s splice variants were expressed as fusion proteins in Escherichia coli using a T7 promoter-based expression system. These proteins were denoted r alpha i-3 and r alpha s (short and long) and accumulated in bacteria to as much as 5-10% of total cellular protein, of which 5-10% was soluble in lysates. Soluble r alpha subunits were tested for stimulation of K+ channel activity in inside-out atrial membrane patches and for reconstitution of cyc- adenylyl cyclase activity. r alpha i-3, activated either by guanosine 5'-(3-thio)triphosphate (GTP gamma S) or AlF-4, stimulated in a concentration-dependent manner single channel K+ currents in isolated atrial membrane patches of three species: guinea pigs, neonatal rats, and embryonic chick. In contrast, GTP gamma S-activated r alpha s did not. In agreement with a similar study by Graziano et al. (Graziano, M. P., Casey, P. J. and Gilman, A. G. (1987) J. Biol. Chem. 262, 11375-11381), both r alpha s forms reconstituted GTP gamma S-stimulated cyc- adenylyl cyclase activity, albeit at concentrations 50-100 times higher than those needed with native Gs. The concentrations of r alpha i-3 needed to stimulate the K+ channels were also higher than needed with native human erythrocyte Gk, in this case 30-50 times. Single K+ channel currents stimulated by r alpha i-3 were indistinguishable from those stimulated by the natural effector acetylcholine. Thus, bacterial expression of G alpha subunits provided the means to demonstrate unequivocally that Gi-3 has intrinsic Gk activity.

Adenylyl Cyclases↗
PubMed1989

Placental toxicity in rats after administration of synthetic glucocorticoids. A morphological, histochemical and immunohistochemical investigation.

Administration of the synthetic glucocorticoids dexamethasone and triamcinolone to pregnant rats between gestational day (GD) 16 and 20 caused dose-dependent placental lesions on GD 21 and 22 which were detected by morphological, histochemical and immunohistochemical means. Maternal blood spaces, trophoblast layer and fetal blood vessels were altered primarily in the centre of the placental labyrinth. Less severe changes were found in the junctional zone, chorionic plate and intraplacental yolk sac. On GD 21, low doses increased the amount of glycogen, while high doses induced a loss of glycogen. gamma-glutamyl transpeptidase activity was increased in the spongiotrophoblast and the labyrinthic trophoblast and dipeptidyl peptidase IV activity in fetal capillary endothelium, whereas alpha-glutamyl aminopeptidase and microsomal alanyl amino-peptidase were not affected. Additionally, in the fetal capillary endothelium an increase of immunoreactivity for the von Willebrand factor occurred. These data suggest that synthetic glucocorticoids affect placental tissues at different and rather specific levels, which may in turn disturb placental function and contribute to fetal maldevelopment.

Animals↗
PubMed1989

Analysis of hepatotoxicity in maternal and fetal rats after glucocorticoid administration by lipid histochemistry and thin layer chromatography.

Changes in lipid metabolism of fetal and maternal rat livers were investigated on day 20 of pregnancy after administration of either 3 mg/kg or 24 mg/kg triamcinolone-acetonide or 124 mg/kg hydrocortisone in crystalline suspension to the mothers on day 15 of pregnancy. Sudan black B and Nile red as well as the UV-Schiff reaction and thin layer chromatography were used to study qualitatively the response of lipids to these glucocorticoids. Generally, after application of triamcinolone-acetonide fetal livers accumulated more lipids as toxic response to this glucocorticoid than the maternal organ; the degree of lipid accumulation was clearly dose-dependent in the fetuses. After hydrocortisone treatment, lipids in maternal livers were slightly, those in the fetuses were not affected. Histochemistry and thin layer chromatography revealed an accumulation of neutral lipids, especially of triglycerides and fatty acids which both contained increased amounts of ethylene bonds after treatment with triamcinolone-acetonide. The results also show that using combined histochemistry and thin layer chromatography, the analysis of hepatic lipids is a promising tool for the assessment of toxic effects of glucocorticoids on fetal and maternal hepatocytes in rats.

Animals↗
PubMed1989

Enhancement of gentamicin-induced nephrotoxicity by Mg deficiency in non-pregnant rats. Morphological, enzyme histochemical and immunohistochemical studies.

Non-pregnant rats fed an Mg-deficient diet showed some degenerated and calcified proximal tubules in the inner region of the medullary rays accompanied by reduced or absent enzyme activities. After gentamicin treatment some damaged convoluted proximal tubules occurred. Enzyme histochemistry revealed decreased activities for brush border and lysosomal hydrolases; using immunohistochemistry lesions were detectable for the cytoskeletal proteins keratin and vimentin. Administration of gentamicin to Mg-deficient rats led to a further decrease of hydrolase activities in obviously intact proximal tubules and drastic structural and enzymatic defects as well as alterations of the cytoskeletal proteins in the convoluted and straight segments of other proximal tubules and to a lesser degree also in glomeruli and further portions of the tubular apparatus including the collecting ducts.

Animals↗
PubMed1989

Lesions and repair of cells of maternal mice after valproic acid (VPA) treatment on day 8 of pregnancy: an enzyme histochemical analysis.

Mice received a single teratogenic dose of the antiepileptic drug valproic acid (VPA; 500 mg/kg i.p.) on 8th d of pregnancy. The effects of VPA were studied primarily by enzyme histochemical means over a period of 48 h. Plasma membrane-associated hydrolases were more affected than lysosome-associated ones. Lesions were not found in the spleen, but a slight response, without morphological damages, was observed in the lung and thymus. Comparatively more severe injuries, which were not accompanied by clear-cut structural changes either, occurred in the liver. The most severe damages including morphological lesions were noticed especially in the proximal tubules of the kidney. The first damages were already seen after 3 h and concerned primarily the liver. After 12 h, a maximal response to VPA was detected in all organs. Repair of most of the enzymatic lesions set in after 24 h. After 48 h, lesions were not longer seen in the lung and thymus. In contrast, enzymatically and morphologically injured proximal renal tubules could still be found after this period as a response to a single dose of VPA; in the liver only enzymatic lesions were present at this stage.

Alkaline Phosphatase↗
PubMed1989

Ischemic flow threshold for extracellular glutamate increase in cat cortex.

Extracellular glutamate (Glu), cerebral blood flow (CBF), and auditory-evoked potentials (AEPs) were measured concurrently using microdialysis and hydrogen clearance in the auditory cortex of anesthetized cats during global ischemia of various severities. A threshold-type relationship was observed between extracellular Glu and CBF: Glu increased at CBF levels below about 20 ml/100 g/min. The Glu increase was related to the impairment of AEPs. The results suggest that Glu neurotoxicity is an important factor for ischemic neuronal injury even in penumbra.

Animals↗
PubMed1989

Crustacean peptidergic neurons in culture show immediate outgrowth in simple medium.

The survival and outgrowth of neurons in culture has usually required conditioning factors. We now report that crustacean neurons, taken from the peptidergic neurosecretory system of the eyestalk of crabs (Cardisoma carnifex) and lobsters (Panulirus marginatus), show immediate outgrowth, sustained for a week or more, in defined medium as simple as physiological saline with glucose and glutamine. The neurons show peptide hormone immunoreactivity that is prominent at growth cones, exhibit differences in form correlated with their immunoreactivity, release peptides to the medium, and have voltage-dependent currents, including a well-sustained Ca current. Cd blocks secretion, growth, and the Ca current. Peptidergic secretory neurons may be able to utilize existing membrane from their store of granules and already active synthetic, transport, and secretory mechanisms for immediate outgrowth.

Animals↗