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R Graf

Publications and source records attributed to R Graf.

At least 163 records · Page 9Linked to original sources
PubMed1993

Analysis of feedback-resistant anthranilate synthases from Saccharomyces cerevisiae.

The initial step of tryptophan biosynthesis is catalyzed by the enzyme anthranilate synthase, which in most microorganisms is subject to feedback inhibition by the end product of the pathway. We have characterized the TRP2 gene from a mutant Saccharomyces cerevisiae strain coding for an anthranilate synthase that is unresponsive to tryptophan. Sequence analysis of this TRP2(Fbr) (feedback-resistant) allele revealed numerous differences from a previously published TRP2 sequence. However, TRP2(Fbr) was found to differ in only one single-point mutation from its own parent wild type, a C-to-T transition resulting in a serine 76-to-leucine 76 amino acid substitution. Therefore, serine 76 is a crucial amino acid for proper regulation of the yeast enzyme. We constructed additional feedback-resistant enzyme forms of the yeast anthranilate synthase by site-directed mutagenesis of the conserved LLES sequence in the TRP2 gene. From analysis of these variants, we propose an extended sequence, LLESX10S, as the regulatory element in tryptophan-responsive anthranilate synthases from prokaryotic and eukaryotic organisms.

Amino Acid Sequence↗
PubMed1993

A randomized double-blind comparison of epidural sufentanil versus intravenous sufentanil or epidural fentanyl analgesia after major abdominal surgery.

This randomized double-blind study compared epidural sufentanil (SEPI) with intravenous sufentanil (SIV) or epidural fentanyl (FEPI) analgesia in 45 patients after major abdominal operations. On first complaint of severe postoperative pain, SIV patients were given a 15-micrograms bolus and then a 5 micrograms/h infusion of sufentanil intravenously. SEPI patients were given the same bolus and infusion, but epidurally. FEPI patients had a 60-micrograms bolus and 20 micrograms/h infusion of fentanyl epidurally. All patients also received a bolus injection and then an infusion of coded saline via the alternate route. Analgesic requirements were tailored continuously to individual needs by patient-controlled supplementary boluses of 3.1 micrograms of sufentanil or 12.5 micrograms of fentanyl, or by 50% reduction in opiate infusion rate at predetermined intervals. Pain scores, circulatory variables, and respiratory rate did not differ between groups. Mean opiate dose requirements (+/- SD) to maintain analgesia for 24 h were 202 +/- 43 micrograms (SIV), 149 +/- 45 micrograms (SEPI), and 627 +/- 226 micrograms (FEPI). The relative analgesic potencies (AP) calculated from the equianalgesic dose requirement ratios were 1.4 for AP-sufentanil IV/EPI and 4.2 for AP-epidural F/S. SIV patients required more supplementary boluses than SEPI patients, were more sedated during the entire treatment, and had higher PaCO2 and higher serum sufentanil concentrations within the first 3 h of treatment. In addition, severe respiratory depression occurred in four SIV patients soon after the start of treatment, despite serum sufentanil concentrations of less than 0.3 ng/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Abdomen↗
PubMed1993

[The effect of ultrasonography screening of hips in newborn infants on femur head necrosis and the rate of surgical interventions].

We compared two similarly sized groups of sonographically unstable or decentered hips with comparable initial findings, both treated by standardized conservative primary treatment. The results show that an early definite diagnosis improves the prognosis. If built on a secure initial diagnosis rationally grounded and efficient conservative therapy can be initiated and followed through on. Since the treatment is started extremely early, the hip can benefit from the considerably greater potential for spontaneous development present in the first three months of life. In this way we can shorten the treatment time considerably and also complete it at a younger age. Finally, early treatment also reduces the risk of the complication of necrosis of the femoral head and significantly decreases the rate of surgical procedures. Only a sufficiently well executed and comprehensively organized sonographic neonatal screening program can produce a definite very early diagnosis and with it almost 100% successful early treatment of all grades of disturbances of hip maturity. Sonographic screening of the newborn is a very important piece in the jigsaw of prophylaxis and primary prevention in our public health care program.

Acetabulum↗
PubMed1992

A truncated recombinant alpha subunit of Gi3 with a reduced affinity for beta gamma dimers and altered guanosine 5'-3-O-(thio)triphosphate binding.

The baculovirus-based expression system was adapted to express alpha subunits of the complete (alpha i3) and an amino-terminally truncated (alpha i3') form of Gi3 and of two complete forms of Gs (alpha s-L and alpha s-S). Subunits encoded in full length cDNAs were obtained with yields of 40-60 mg of recombinant protein/liter of cells, of which alpha i3 was between 30 and 50% soluble, but alpha s subunits were only 5-10% soluble. Only the complete alpha i3 was myristoylated. alpha i3 was purified in four steps. The purified protein bound 0.8-0.9 mol of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) per mol of protein and had one predominant contaminant which was identified as a truncated form that begins with methionine 18 instead of methionine 1. Both the full length alpha i3 and the truncated alpha i3' formed trimers with human erythrocyte beta gamma as seen by their migration in sucrose density gradients and by an increased rate of ADP ribosylation by pertussis toxin, but compared to alpha i3, alpha i3' interacted with beta gamma with a reduced affinity and dissociated upon warming. At 32 degrees C, only full length alpha i3 was ADP-ribosylated; at 4 degrees C, alpha i3 and alpha i3' were both ADP-ribosylated, with the truncated form requiring approximately 200-fold higher concentrations of beta gamma. A genetically engineered alpha i3' (alpha i3[18-354]) was also expressed in Sf9 cells. Yields, assessed as saturable GTP gamma S binding sites, were 3-5 mg per liter. Scatchard analysis showed that truncation of the amino terminus interferes with the ability of Mg2+ to promote high affinity binding of GTP gamma S. We conclude that the G protein alpha subunit amino terminus is not essential for interaction with beta gamma dimers, but rather is important in determining the affinity of the alpha subunit for both the beta gamma dimers and guanine nucleotide.

Amino Acid Sequence↗
PubMed1992

Studies on the interaction of alpha subunits of GTP-binding proteins with beta gamma dimers.

The interaction of several preparations of purified beta gamma dimers with two types of guanosine-nucleotide-binding-regulatory-(G)-protein alpha subunits, a recombinant bv alpha i3, made in Sf9 Spodoptera frugiperda cells by the baculovirus (bv) expression system, and alpha s, either purified from human erythrocyte Gs-type GTP-binding protein, and activated by NaF/AlCl3, or unpurified as found in a natural membrane, were studied. The beta gamma dimers used were from bovine rod outer segments (ROS), bovine brain, human erythrocytes (hRBC) and human placenta and contained distinct ratios of beta subunits that, upon electrophoresis, migrated as two bands with approximate M(r) of 35,000 and 36,000, as well as distinct complements of at least two gamma subunits each. When tested for their ability to recombine at submaximal concentrations with bv alpha i3, ROS, brain, hRBC and placental beta gamma dimers exhibited apparent affinities that were the same within a factor of two. When bovine brain, placental and ROS beta gamma dimers were tested for their ability to promote deactivation of Gs, brain and placental beta gamma dimers were equipotent and at least 10-fold more potent than that of ROS beta gamma dimers; likewise, brain beta gamma and placental dimers were equipotent in inhibiting GTP-activated and GTP-plus-isoproterenol-activated adenylyl cyclase, while ROS beta gamma dimers were less potent when assayed at the same concentration. The possibility that different alpha subunits may distinguish subsets of beta gamma dimers from a single cell was investigated by analyzing the beta gamma composition of three G proteins, Gs, Gi2 and Gi3, purified to near homogeneity from a single cell type, the human erythrocyte. No evidence for an alpha-subunit-specific difference in beta gamma composition was found. These findings suggests that, in most cells, alpha subunits interact indistinctly with a common pool of beta gamma dimers. However, since at least one beta gamma preparation (ROS) showed unique behavior, it is clear that there may be mechanisms by which some combinations of beta gamma dimers may exhibit selectivity for the alpha subunits they interact with.

Adenosine Diphosphate Ribose↗
PubMed1992

Lipid interactions of the hemagglutinin HA2 NH2-terminal segment during influenza virus-induced membrane fusion.

Fusion of influenza viruses with target membranes is induced by acid and involves complex changes in the viral fusion protein hemagglutinin (HA) and in the contact sites between viruses and target membranes (Stegmann, T., White, J. M., and Helenius, A. (1990) EMBO J. 9, 4231-4241). At 0 degrees C, in a first, kinetically distinct step, target membranes irreversibly adhere to the viruses. Fusion itself starts only after a lag-phase of several minutes (X-31 strain viruses) or after raising the temperature (PR8/34 strain viruses). We now provide evidence that the initial conformational change resulting in virus-target membrane adhesion is restricted to a (minor) subpopulation of the HA molecules. These molecules become susceptible to bromelain digestion, and they could be labeled with the photoactivatable reagent [3H]PTPC/11, a nonexchangeable lipid present in the target lipid bilayer (Harter, C., Bächi, T., Semenza, G., and Brunner, J. (1988) Biochemistry 27, 1856-1864). Only the HA2 subunit was labeled, and analyses of 2-nitro-5-thio-cyanobenzoic acid fragments derived thereof indicate that the HA2 NH2-terminal segment (fusion peptide) inserted into the target membrane bilayer. When the temperature was raised to trigger fusion of PR8/34 viruses, labeling of HA2 increased by a factor of 130. Most (74%) of that label was incorporated into the COOH-terminal membrane anchor region, but there was also a strong increase (about 30-fold) of NH2-terminal fusion peptide labeling. This suggests that fusion is preceded., or accompanied, by further changes in HA which lead to additional extensive lipid insertions of HA2 fusion peptides.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Differential stimulation of the GTPase activity of G-proteins by polylysine.

Polylysine, polyornithine and, to a lesser extent, polyarginine were found to stimulate the GTPase activity of the purified recombinant alpha subunit of the human G(i)-3 transducing protein alpha i-3. Optimal stimulation of 4- to 5-fold was obtained with polylysine concentrations between 1 and 20 microM, higher concentrations being inhibitory. Polylysine at similar concentrations stimulated by 50% the GTPase of transducin (GT), the vision transducing protein, but had only a very slight effect on the GTPase of the p21 product of the H-ras protooncogene. The stimulation of the alpha i-3 GTPase caused by polylysine was due to a reduction of the apparent Km for GTP from 3.8 to 1.3 microM. The stimulation by polylysine was observed at free Mg2+ concentrations below 1 microM. These results indicate that polylysine acts in a fashion similar to mastoparan and substance P in mimicking the action of an agonist-bound receptor on G-proteins.

Enzyme Activation↗
PubMed1992

Flow thresholds for extracellular purine catabolite elevation in cat focal ischemia.

Ischemic glutamate excitotoxicity may be counteracted by adenosine which appears extracellularly during ischemia as an intermediate purine catabolite and has the potential to modulate glutamate release and its receptor action. The present study was conducted to evaluate the flow threshold for purine catabolite accumulation in relation to that for glutamate elevation in focal ischemia which was induced by middle cerebral artery (MCA) occlusion in halothane anesthetized cats. Assemblies of platinum electrodes and microdialysis probes were inserted into the somatosensory (SF, n = 13) and the auditory (A, n = 9) cortices to assess local cerebral blood flow (CBF) using hydrogen clearance and purine catabolite (adenosine, inosine and hypoxanthine) as well as glutamate concentrations in the dialysate using high-performance liquid chromatography (HPLC). In both investigated areas, purine catabolites were elevated if CBF fell below 25 ml/100 g/min, while glutamate increased at a flow threshold below 20 ml/100 g/min. Maximum elevations of adenosine, inosine and hypoxanthine were 76-, 29- and 11-fold, respectively, that of glutamate was 24-fold. In the range between 20 and 25 ml/100 g/min, significant increases of adenosine (5-15-fold) were measured, while glutamate did not markedly increase. The elevation of adenosine was transient whereas that of inosine, hypoxanthine and glutamate persisted over an ischemic period of 3 h. The higher flow threshold for adenosine may reflect an inherent but time limited protective mechanism against glutamate excitotoxicity.

Adenosine Triphosphate↗
PubMed1992

Sequence-specific initiator elements focus initiation of transcription to distinct sites in the yeast TRP4 promoter.

Transcription from the yeast TRP4 promoter initiates at two basal (i127 and i76) and three GCN4 dependent (i31, i25 and i12) initiator elements. All of these elements contain not more than one deviation from the earlier proposed initiator consensus sequence PuPuPyPuPu, a pyrimidine nucleotide flanked on either side by two purine nucleotides. A point mutation analysis of these elements in various combinations was performed and revealed that the central pyrimidine nucleotide and at least one of the 3' flanking purine nucleotides of the PuPuPyPuPu consensus sequence are essential but alone not sufficient to define a functional initiator element. Multiple cryptic transcription start sites, which function independently whether they are located on the coding or the non-coding strand, can replace the function of mutated initiator elements and therefore the overall level of transcription initiation is not affected. The sequence specificity is identical for basal and GCN4 dependent initiator elements demonstrating that they are functionally homologous. These findings imply that the role of initiator elements is to 'focus' the start point(s) of transcription to distinct sites located in the region between the site(s) of the assembly of the transcriptional complex and the start codon of translation.

Anthranilate Phosphoribosyltransferase↗
PubMed1992

Placental morphology and concentrations of glutathione (GSH and GSSG) and lipid peroxides (LPO) in two models of disturbed pregnancy of Uje:WIST rats.

Treatment of pregnant rats with heat-denaturated bacterial material (endotoxin-model) or exposure to chronic restraint of prenatally lithium-treated pregnant dams (stress-model) were used as two models of disturbed pregnancy, both causing decreased fetal body mass. The quotient of fetal/placental mass was lowered in the endotoxin-group only. Placental mass and protein content were not changed significantly in both experimental groups, although a tendency to smaller placentae was noticed in the stress-group. Placental histology of the stress-group did not differ from untreated controls. In the endotoxin-group an altered structure of the placental barrier was observed. Small decreases of GSH and GSSG in the endotoxin-group and of GSSG in the stress-group without significant changes of the GSH/GSSG relationship were measured in homogenates of the placental labyrinth. Moderate enhancement of LPO concentration occurred in the endotoxin-group and more distinctly in two litters of the stress-group, the latter being connected with high GSSG concentrations and low fetal/placental mass-quotients.

Animals↗
PubMed1992

Interference of substrate quenching with the kinetics of placental peptidases.

The study was undertaken to clarify the putative presence and influence of substrate inhibition and substrate quenching on the kinetic analysis of placental peptidases using naphthylamide substrates in a fluorometric assay. Using the appropriate naphthylamide substrates, we analysed microsomal alanine aminopeptidase (EC 3.4.11.2) and angiotensinase A (EC 3.4.11.7) in the presence or absence of inhibitors (1,10'-phenanthroline, EDTA) or activators (calcium) in the human and rat placenta. All substrates were shown to quench the fluorescence of the enzymatically released unspecific moiety. Since Michaelis-Menten-derived kinetic equations do not include quenching phenomena, we deduced a mathematical model integrating enzyme kinetics and quenching kinetics, thereby allowing simultaneous analysis of both processes from the same raw data. Curves derived from this expanded kinetic model fitted significantly better to velocity data than those derived from Michaelis-Menten-equation. According to the presented data, we assume that substrate inhibition for both enzymes, as previously proposed in the literature, in contrast may be due to formerly unconsidered substrate quenching. The presented kinetic model could serve as a useful tool in the study of putative physiological substrates of the investigated placental peptidases, characterizing the physiologic substrates by their competitive interaction with the synthetic naphthylamides. Additionally, this model may not only be important in fluorometric analysis of peptidases, but also of other enzymes which are analysed fluorometrically with synthetic substrates.

Aminopeptidases↗
PubMed1992

Peptide inhibitors of ADP-ribosylation by pertussis toxin are substrates with affinities comparable to those of the trimeric GTP-binding proteins.

Pertussis toxin (PTX) ADP-ribosylates alpha subunits of GTP-binding proteins (G proteins) when they are in association with beta gamma dimers, and free alpha subunits are thought not to be substrates under standard assay conditions. We now report the rather unexpected discovery that synthetic peptides encompassing the last 10-20 amino acids of alpha subunits of PTX-sensitive G proteins are substrates for PTX by themselves and in the absence of beta gamma dimers. As determined for G13, the Km of PTX for the 20-amino acid carboxyl-terminal peptide is 10-fold higher than that for the trimeric G protein. Interestingly, PTX ADP-ribosylates the free full length alpha 13 subunit with a Km not different from that of the trimer but with a Vmax that is only 1% of that with which it ADP-ribosylates the trimer. It follows that the primary role of beta gamma dimers in ADP-ribosylation of G proteins is one of increasing the Vmax of the reaction without affecting the Km of the substrate for the toxin. Mutant peptides lacking the ADP-ribose acceptor site act as competitive inhibitors.

Adenosine Diphosphate Ribose↗
PubMed1992

Hip sonography--how reliable? Sector scanning versus linear scanning? Dynamic versus static examination?

In Europe, the use of ultrasonography has become a routine procedure for the detection of hip dislocations and dysplasias; clinical and historical data alone are not fully reliable. Under the right conditions, ultrasonographic examination of the hip can reliably detect hip joints in need of treatment. Furthermore, results are reproducible without the use of roentgenograms; thus the danger of "overtreatment" can be avoided. The use of sector scanners for examinations of hip joints leads to distortions in the geometry of the picture. As a result, the use of linear transducer is presently indispensable. Thanks to a reference level, static examinations of the hip joint provide us with the possibility of measuring and comparing individual hip joints. With dynamic examinations, we can quantify the data and assess hip stability. In fact, the static and dynamic approaches do not exclude but, rather, complement each other. The ultrasonographic method must be learned from experts. Given the above-mentioned conditions, all developmental abnormalities of the hip can be diagnosed in the first six weeks of life, and healthy hip joints can be clearly distinguished from those requiring follow-up examination. Based on observations on 8530 cases, hip joints of infants at least 4.5 months of age can be successfully treated in a conservative manner. Femoral head necroses have disappeared altogether and the need for later surgery has decreased dramatically.

Hip Dislocation, Congenital↗
PubMed1992

[Ultrasound diagnosis of hip dysplasia--current status and future perspectives].

Hip sonography provides a safe pathoanatomical assessment of a newborn hip joint at the earliest possible moment. Based on this safe diagnosis an adequate biomechanical treatment can be started instantly. The mean age of healing even of originally decentered hip joints is 7.5 months, if earliest sonographic diagnosis and adequate biomechanical treatment are performed correctly. Secondary hip surgery can be reduced to a minimum. Cost-benefit-analysis, too, supports the institution of a general sonographic screening of all newborn hip joints.

Austria↗
PubMed1991

Structural diversity of trypsin from different mosquito species feeding on vertebrate blood.

Mosquito trypsin was purified using a combination of ion exchange and affinity chromatography with the ligand soybean trypsin inhibitor. Three Aedes and three Anopheles species were tested, all of which are specialized in the digestion of vertebrate blood. Amino-terminal sequences of HPLC-purified trypsins from Aedes aegypti and Anopheles quadrimaculatus revealed homologies of 30-40% with vertebrate and other invertebrate proteases previously identified as serine-proteases. The purified mosquito trypsins have molecular masses between 25 kDa and 36 kDa, as determined by denaturing polyacrylamide electrophoresis, and are heterogeneous in size and number in the various species. The number of SDS-bands varies between 3 and 6 in Aedes and between 1 and 3 in Anopheles. The specific activities, determined with the substrate TAME, range from 240 U/mg in Aedes aegypti to 1065 U/mg in Anopheles quadrimaculatus. All mosquito trypsins tested have acidic isoelectric points between pH 3.5 and pH 5.4. No alkaline proteases were detected. Polyclonal antisera against Aedes aegypti and Anopheles albimanus trypsin do not cross-react with bovine trypsin. Cross-reactivity of the two sera with trypsin from six mosquito species suggests the presence of at least 2 enzyme families.

Aedes↗
PubMed1990

Three GCN4 responsive elements act synergistically as upstream and as TATA-like elements in the yeast TRP4 promoter.

The yeast TRP4 promoter contains three responsive elements (GCREs) for the 'general control' transcriptional activator GCN4, which are arranged in two upstream elements, UAS1 (GCRE1) and UAS2 (GCRE2 and GCRE3). A point mutation analysis of these elements revealed that all three GCREs are required for GCN4-dependent transcription, but none are involved in basal transcription. Basal transcription and GCN4-dependent transcription use completely different initiator elements in the TRP4 promoter. UAS1 acts synergistically with UAS2 to activate the GCN4-dependent transcription of TRP4. A consensus TATA box can functionally replace the UAS2 element to allow normal GCN4-dependent transcription, suggesting that UAS2 is analogous to the TATA element of other promoters. GCN4 might therefore activate transcription by exhibiting two alternative functions within the natural TRP4 promoter.

Alleles↗