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Biomedical subjects

S Ott

Publications and source records attributed to S Ott.

At least 55 records · Page 3Linked to original sources
PubMed1993

Inactivation of PR8 influenza virus through the octadecylrhodamine B chloride membrane marker.

The octadecylrhodamine B chloride (R18) membrane marker was incorporated into PR8 influenza viruses and virus receptor (GD1a-) containing small unilamellar vesicles (SUV). Both were tested in a fusion/lipid transfer assay [Wunderli-Allenspach, H., & Ott, S. (1990) Biochemistry 29, 1990-1997] to find out whether incorporation into artificial and biological membranes yields equivalent results. The R18 assay is based on incorporation of quenched concentrations of the label into donor membranes and monitoring of the dequenching upon its dilution into unlabeled acceptors. With PR8 viruses and R18-labeled SUV, a fast, hemagglutinin-specific fusion takes place at pH 5.3, independently of the initial quenching. At neutral pH, a slow, nonspecific R18 transfer occurs. Both processes follow second-order kinetics. Upon incubation of R18-labeled PR8 viruses with unlabeled SUV or LUV in neutral buffer, transfer is also found. At pH 5.3, a complex dequenching curve best described with superposition of two second-order functions was encountered: a fast, hemagglutinin-specific component and a slow, nonspecific component. A decreasing proportion of the fast fusion was found with increasing initial quenching of labeled virus. Extrapolation showed that full fusion activity is obtained only with low initial quenching (20-30%). The gradual inactivation of the virus by increasing amounts of R18 was confirmed with biological assays (e.g., infectivity). The R18 surface density is much higher in viruses than in liposomes to obtain the same initial quenching. Analysis with the Stern-Volmer plot revealed that R18 monomers and dimers contribute to the quenching in labeled PR8 viruses, whereas only dimers determine the quench curve in liposomes.

Biomarkers↗
PubMed1993

Effects of diclofenac on isradipine pharmacokinetics and platelet aggregation in volunteers.

In this open, two-period crossover study in 18 healthy male volunteers, a single oral dose of 50 mg diclofenac was administered alone and on day 7 of multiple oral dosing of 5 mg b.i.d. isradipine to assess a possible pharmacokinetic interaction. The effect of these drugs on ex vivo platelet function was also determined. Serial blood samples were obtained over 12-hour periods on three occasions: after the single diclofenac dose; after the morning dose of isradipine on day 6 and after co-administration of both drugs on day 7 of steady-state isradipine administration. Additional samples were taken at 2 h post dose for determination of ex vivo platelet aggregation. Isradipine plasma concentrations were determined by a gas chromatographic method and diclofenac plasma concentrations by an HPLC method. The pharmacokinetic characteristics of diclofenac were unaltered during co-administration. The maximum plasma concentration of isradipine was increased 19.6% during co-administration from 5.06 to 6.05 ng.ml-1. This is not expected to be of clinical importance. Isradipine's apparent total body clearance and steady-state AUC remained unchanged. Ex vivo induced platelet aggregation was not affected by any of the treatments.

Adult↗
PubMed1993

Effects of in utero administration of alcohol on glutathione levels in brain and liver.

Previous studies in our laboratory have shown that the ontogenic development of gamma-glutamyltranspeptidase (gamma-GTP) activity is delayed by the in utero administration of alcohol. gamma-GTP is responsible for the degradation and recycling of glutathione (GSH) via the gamma-glutamyl cycle. In this study, we examined the effects of the in utero administration of alcohol on GSH levels in gestational age 21-day-old (g21) rats. Pregnant rats were placed on a liquid diet containing either 35% ethanol-derived calories (35% EDC) or a pair-fed (PF) diet or a lab chow (LC) diet starting on day 1 of gestation and maintained on their respective diets until gestational day 21. On gestational day 21, the pups were delivered by Cesarean section and brains and livers removed and prepared for analysis of GSH, gamma-GTP, or gamma-glutamyl-cysteine synthetase (gamma-GCSyn). GSH levels in brain and liver were found to be significantly lower in the offspring of the 35% EDC-treated mothers than from the PF and LC controls. gamma-GTP activity was higher in brain and liver of the 35% EDC group than the PF group. gamma-GCSyn, the enzyme involved in the rate-limiting step of GSH synthesis, was not affected in liver, but was found to be decreased in brain of the 35% EDC and PF groups when compared with the LC group. GSH is involved in many cellular reactions that appear to protect the cell from damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

[Assessing the quality of the speaking voice after therapy of T1 and T2 vocal cord cancers].

All studies which analysed voice quality after T1 and T2 glottic cancer employed sustained vowels for evaluation. Sustained vowels can often be produced more clearly than expected in a severe degree of hoarseness in running speech. In this investigation voice quality was analysed by measuring the signal-to-noise-ratio (SNRc) in running speech. For evaluation these data were correlated to the scores from an auditive evaluation and from a clinical examination of the larynx considering functional parameters. 39 patients were examined after different therapies (primary radiation, chordectomy, frontolateral partial resection). The grading of the three different methods for characterisation of the voice quality correlated well. Therefore the SNRc is presumed to be a valuable additional method to the psychoacoustic voice evaluation. Comparison of voice quality after different therapies provided no significant difference between radiotherapy and chordectomy. The functional results after frontolateral partial resection were significantly less satisfying as expected.

Aged↗
PubMed1990

Kinetics of fusion and lipid transfer between virus receptor containing liposomes and influenza viruses as measured with the octadecylrhodamine B chloride assay.

Octadecylrhodamine B chloride (R18) and ganglioside GD1a (virus receptor) were incorporated into small unilamellar liposomes [Hoekstra et al. (1984) Biochemistry 23, 5675-5681]. Upon interaction of these liposomes with PR8 influenza viruses without prebinding, two types of dequenching were observed at 37 degrees C, both second-order processes: a fast reaction at pH 5.3, 2k = 17.53 x 10(-3) (Q.s)-1, and a slow reaction at pH 7.4, 2k = 0.335 x 10(-3) (Q.s)-1. The maximal level of dequenching was the same for both. Upon prebinding of liposomes to PR8 viruses (30 min, 0 degrees C, pH 7.4) at high concentrations, a very fast dequenching occurred when the prebinding mixture was diluted into prewarmed (37 degrees C) 10 mM PBS, pH 5.3. For the initial phase, a first-order rate constant of 0.5 s-1 could be extrapolated. After a quick drop in velocity during the first 30 s, the reaction was kinetically indistinguishable from the one found without prebinding. A second-order process with 2k = 16.52 x 10(-3) (Q.s)-1 became rate-limiting. The fast reactions at pH 5.3 can be abolished by inactivation or removal of the virus hemagglutinin. We conclude that the reaction at pH 5.3 reflects the hemagglutinin-dependent fusion process known to occur between influenza viruses and partner membranes at low pH; however, second-order kinetics indicate that specific binding rather than fusion is the rate-limiting step. For the slow dequenching, which is not affected by prebinding, the rate constant is 20 times lower than for the fast reaction, and the process is independent of viral hemagglutinin.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence↗
PubMed1989

Guanine nucleotide-mediated inhibition of opioid agonist binding. Modulatory effects of ions and of receptor occupancy.

We have analysed the potency of GTP, GDP and their analogues in reducing [3H]DADLE binding to opioid receptors in NG 108-15 cell membranes. Under conditions where non-specific hydrolysis and transphosphorylation is inhibited, the following rank order of potency was found: GDP greater than or equal to GTP gamma S greater than GTP greater than GDP beta S greater than or equal to GDPNH2 greater than GppNHp much greater than GMP. Remarkably, the slopes for the inhibition curves of GTP, GDP and their thiosubstituted analogues, but not of GDPNH2 and GppNHp, were extremely shallow, indicating either negative cooperativity or the existence of two states for the guanine nucleotide binding proteins, that both can mediate the effect of nucleotides on agonist receptor binding. The potencies of the different guanine nucleotide analogues, except that of GppNHp, were increased by the presence of sodium or chloride ions in the assay medium. Magnesium also affected GTP-mediated inhibition of opioid agonist binding since it decreased the IC50 of the nucleotide and steepened the slope of the inhibition curve. The IC50s of nucleotides and the slopes of their inhibition curves were also dependent on the extent of receptor occupancy by the agonist. From these data we conclude that (1) either diphospho- or triphosphonucleotides can regulate agonist binding. (2) Magnesium, sodium and chloride, by acting at different components of the receptor/G protein complex produce similar effects on nucleotide mediated regulation of agonist binding. (3) A mutual influence exists between receptor occupancy by agonists and G protein-mediated guanine nucleotide effect on the receptor.

Cell Line↗
PubMed1989

Opioid receptors of neuroblastoma cells are in two domains of the plasma membrane that differ in content of G proteins.

Opioid receptors of NG 108-15 cell membranes are distributed in two membrane fractions sedimenting at 20,000 g (P2) and 200,000 g(P3). The number of receptors is identical in P2 and P3, but in P2 all sites are present in one high-affinity state (2 nM), whereas in P3 60% of these receptors display lower affinity (150 nM). Upon addition of GTP or pretreatment with pertussis toxin, 80% of the sites exist in low affinity in both P2 and P3. Therefore, the effect of GTP and pertussis toxin on agonist binding appears to be smaller in P2 than in P3. In contrast, sodium inhibits agonist binding in P2 and P3 to the same extent and with identical potency. Opioid-mediated stimulation of GTPase is much greater in P2 than in P3, whereas inhibition of adenylate cyclase does not differ in the two fractions. Using site-specific antibodies and pertussis toxin-catalyzed ADP-ribosylation, we found that the amount of G proteins in P3 is only 30-50% of that in P2. Treatment of intact cells with the hydrophilic protein-modifying agent sulfosuccinimido-biotin results in biotinylation of proteins from both fractions and in a similar reduction of opioid binding in P2 and P3. Likewise, exposure of intact cells to the alkylating opioid antagonist, chlornaltrexamine, produces identical degrees of receptor inactivation in P2 and P3. The rate of in vivo pertussis toxin-mediated modification of G proteins is not different in the two fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1989

Enzymatic degradation of GTP and its "stable" analogues produce apparent isomerization of opioid receptors.

GTP reduces the affinity of agonists to opioid receptors in membranes from NG 108-15 cells. This can be demonstrated using a short incubation time in equilibrium binding and in dissociation experiments: GTP decreases equilibrium binding, increases the percentage of fast dissociating binding and produces a conversion of high into low affinity receptors. However, upon prolonged incubation time (more than 60 min) these effects of GTP are gradually lost. This loss of guanine nucleotide effect is also observed with other nucleotides, namely GDP, GTP gamma S and GppNHp. Incubation of radioactive GTP with membranes and subsequent analysis of the guanine nucleotides by TLC (thin layer chromatography) reveals that GTP is rapidly hydrolyzed to GDP and thereupon to the inactive nucleotide GMP and guanosine. The so-called "hydrolysis-resistant" analogues of GTP, GTP gamma S and GppNHp are also degraded under these experimental conditions. The hydrolysis is apparent with membranes from different tissues and occurs with half times of less than 5 min for GTP and of 10 - 30 min for GDP and "stable" GTP-analogues. Conditions that reduce the degradation of guanine nucleotides during incubation with membranes are described.

Animals↗
PubMed1988

Sodium modulates opioid receptors through a membrane component different from G-proteins. Demonstration by target size analysis.

The target size for opioid receptor binding was studied after manipulations known to affect the interactions between receptor and GTP-binding regulatory proteins (G-proteins). Addition of GTP or its analogs to the binding reaction, exposure of intact cells to pertussis toxin prior to irradiation, or treatment of irradiated membranes with N-ethylmaleimide did not change the target size (approximately equal to 100 kDa) for opioid receptors in NG 108-15 cells and rat brain. These data suggest that the 100-kDa species does not include an active subunit of a G-protein or alternatively that GTP does not promote the dissociation of the receptor-G-protein complex. The presence of Na+ (100 mM) in the radioligand binding assay induced a biphasic decay curve for agonist binding and a flattening of the monoexponential decay curve for a partial agonist. In both cases the effect was explained by an irradiation-induced loss of the low affinity state of the opioid receptor produced by the addition of Na+. This suggests that an allosteric inhibitor that mediates the effect of sodium on the receptor is destroyed at low doses of irradiation, leaving receptors which are no longer regulated by sodium. The effect of Na+ on target size was slightly increased by the simultaneous addition of GTP but was not altered by pertussis toxin treatment. Thus, the sodium unit is distinct from G-proteins and may represent a new component of the opioid receptor complex. Assuming a simple bimolecular model of one Na+ unit/receptor, the size of this inhibitor can be measured as 168 kDa.

Animals↗
PubMed1988

Purification of murine IgG3 and IgM monoclonal antibodies by euglobulin precipitation.

We describe a simple and efficient non-chromatographic method for the purification of murine IgG3 and IgM monoclonal antibodies (MAbs) which takes advantage of their euglobulin properties. Following filtration, ascitic fluid is dialysed against demineralized water and centrifuged at 22,000 X g for 30 min. The resulting precipitate is dissolved in a high salt buffer (0.1 M Tris-HCl, 1 M NaCl, pH 8). A second cycle of dialysis and centrifugation yields a product of high purity. Nine IgG3 MAbs and eight IgM MAbs were purified by this procedure. Recovery was greater than 90% for seven of nine IgG3 MAbs. It was less reproducible for IgM MAbs and ranged from 40% to greater than 90% depending on antibody and batch. Purity was assessed by SDS-polyacrylamide gel electrophoresis. The purified immunoglobulin was essentially free of albumin, transferrin, and other mouse ascites proteins. No loss of antibody function was observed.

Animals↗
PubMed1987

Bacteriological effects of anti-Pseudomonas aeruginosa chemotherapy in cystic fibrosis.

Strains of Pseudomonas aeruginosa resistant to clinically relevant antibiotics (beta-lactams, quinolones, aminoglycosides) were detectable in sputa of cystic fibrosis patients. Correlations between in vitro susceptibility and bacteriological results in vivo were demonstrated at a quantitative level. P. aeruginosa strains susceptible prior to antibiotic therapy were observed to become resistant towards each of the compounds used for treatment. We conclude that antibiotic therapy in cystic fibrosis has to be optimized by culture specific selection of the drugs and consecutive bacteriological follow-ups.

Anti-Bacterial Agents↗
PubMed1987

Progressive pulmonary calcification complicating successful renal transplantation.

Metastatic pulmonary calcification occurs in a chronic form in patients with malignancy, chronic renal failure, and primary hyperparathyroidism. A rapidly progressive form is associated with renal transplant failure. This case report describes chronic progressive pulmonary calcification after successful transplant with no obvious underlying cause.

Adenoma↗
PubMed1987

Prevention of peritoneal adhesions in the rat with sustained intraperitoneal dexamethasone delivered by a novel therapeutic system.

The rat caecal crush model for the production of intraperitoneal adhesions was optimized with respect to an objective adhesion grading system based on the determination of the incidence of adhesions and the caecal adherent surface area. Intraperitoneal saline solution given before closure of the peritoneum significantly reduced the severity but not the incidence of adhesions. The addition of dexamethasone crystal suspension led to somewhat lower incidence of adhesions but the mean adherent surface area values were not different from the saline regimen. However, sustained release of dexamethasone by use of intraperitoneal biodegradable poly(lactide-co-glycolide) microparticles as a novel therapeutic system further reduced both the incidence and the severity of adhesions. Despite of the differences in therapeutic effects glucocorticoid-associated local and systemic adverse effects were similar in both regimens. As effective adhesion prophylaxis with glucocorticoids appears to be a balance between benefit and adverse reactions, optimal drug delivery is mandatory. The novel biodegradable therapeutic system is superior to the intraperitoneal crystal suspension regimen in the rat and should be tested further in clinical trials.

Animals↗
PubMed1986

Bone mineral density after resumption of menses in amenorrheic athletes.

Amenorrheic athletes have been found to have a lower vertebral bone mineral density (BMD) than matched groups of eumenorrheic athletes. This study reports changes in BMD over a 15.5 month period in athletes who regained menses, athletes who remained amenorrheic, and athletes with regular cycles. The BMD was measured at two sites on the radius and at the lumbar vertebrae (L-1 through L-4), using single- and dual-photon densitometry, respectively. Changes in vertebral BMD were significant for the amenorrheic group (+6.3%), but not for cyclic women (-0.3%). A slight increase in radial density at S-1 and S-2 was not significant for either group. Two athletes who remained amenorrheic during this period continued to lose bone (-3.4%). We conclude that resumption of menses was the primary factor for the significant increase in the vertebral BMD of the formerly amenorrheic athletes.

Adult↗
PubMed1986

Target size analysis of opioid receptors. No difference between receptor types, but discrimination between two receptor states.

Target size analysis of opioid receptor is complicated by the presence of multi-exponential inactivation curves. Irradiation of intact frozen tissue proved essential to eliminate such artifacts, due to indirect irradiation effects. Upon irradiation condition, opioid binding activity was inactivated in a single mono-exponential manner. Identical inactivation curves were obtained for mu, delta and kappa binding activities in brain membranes from rat, guinea-pig and frog and in NG 108-15 cells: the molecular mass obtained was 98 +/- 2 kDa. However, when opioid binding was assayed in the presence of Na+, Mg2+ and GTP, the molecular mass was found to be only 56 +/- 4.4 kDa. We suggest that the opioid recognition site comprises a unit of 56 kDa and that in the absence of Na+, Mg2+ and GTP an additional membrane component of 40-44 kDa is necessary for high-affinity opioid binding.

Animals↗
PubMed1986

Effects of sodium and GTP on the binding kinetics of [3H]diprenorphine in NG 108-15 cell membranes.

Equilibrium binding isotherms of [3H]diprenorphine in membranes from NG 108-15 cells are consistent with a homogeneous population of binding sites. Upon addition of Na+, Mg2+ and GTP, only a 2-fold reduction in affinity with a minor decrease in the number of sites is observed. Dissociation curves of [3H]diprenorphine, however, are clearly biphasic: in the absence of Na+, Mg2+ and GTP, 80% of the bound ligand dissociates slowly with a t1/2 of 100 min, and only 20% rapidly (t1/2 4.5 min). In the presence of Mg2+, nearly all the binding is found in the slowly dissociating form. Upon the addition of either Na+ or GTP, 20-30% of the binding dissociates more rapidly. The rate constant of the rapidly dissociating form generated by Na+, however, is 2.5 times greater than that induced by the presence of GTP. Thus, the addition of both, Na+ and GTP, converts about 80% of the receptor into a very fast dissociating form (t1/2 1.7 min). Exposure of intact cells to pertussis toxin (10 ng/ml) or treatment of membranes with N-ethyl maleimide (500 microM), strongly reduces the proportion of the slowly dissociating component. Following these treatments, the effect of GTP is reduced or abolished, but that of Na+ remains unaffected. We conclude from these data that the effects of Na+ and GTP are not only distinct in site but also in mechanism of action and that there are three forms of opioid receptors that can be differentiated by their kinetic properties. The slowly dissociating receptor form requires a functional N unit.

Animals↗
PubMed1983

The molecular size of multiple opiate receptors.

The functional size of different types of opiate receptors was determined in situ by the radiation inactivation technique. The Mr of opiate binding sites was estimated to be about 90,000 daltons; the data revealed no significant differences between the molecular sizes of either mu-, delta- or kappa-sites in rat brain membranes. An identical molecular size was found also for the delta-sites in the neuroblastoma X glioma hybrid cell clone, NG 108-15.

Animals↗