Biomedical subjects

R Werner

Publications and source records attributed to R Werner.

At least 19 recordsLinked to original sources

PubMed1992

Analysis of carbohydrate-mediated heterogeneity and characterization of N-linked oligosaccharides of glycoproteins by high performance capillary electrophoresis.

Capillary zone electrophoresis (CZE) has been investigated as an alternative method to analyze the carbohydrate moieties of glycoproteins. Carbohydrate-mediated microheterogeneity of the recombinant plasminogen activator (rt-PA) was examined. The glycoprotein was resolved in multiple electrophoretic species using CZE but the separation was complicated by adsorption of the molecules to the wall of the capillary. The influence of several parameters, such as pH, molarity of the buffer and addition of a cationic additive, on the separation of glycopeptides was investigated. High resolution and reproducible separations of rt-PA glycopeptides carrying hybrid and complex type chains were obtained using either a 100 mM phosphate buffer, pH 6.6, or a 100 mM Tricine buffer, pH 8.2, containing 1.25 mM of putrescine. N-Oligosaccharides from fetuin, t-PA and alpha 1-acid glycoprotein were separated within 20 min on the basis of both their sialic acid content and their structure. The use of an oligosaccharide fingerprinting technique, such as the present one, could have many applications in biotechnology to assess, for example, the consistency of production of a glycoprotein or for analytical glycoprotein chemistry.

Animals↗

PubMed1992

Effect of the dose of ultraviolet radiation on the pigment formation by human melanocytes in vitro.

Human melanocytes were cultivated under different conditions with phorbol ester (TPA), or with bovine pituitary extract (BPE). The cells altered their morphology with the different culture conditions. With TPA they were predominantly bipolar, while with BPE most of the cells had a dendritic cell shape. In order to investigate the effect of UV irradiation, the cells were irradiated with 50, 100 and 200 mJ/cm2 UVA/B. After irradiation with 200 mJ/cm2 UVA/B the cells cultured with TPA also showed a dendritic shape. We determined the tyrosinase activity, the cellular melanin content and the cell number 3 days after irradiation. In all cases the number of cells decreased depending on the UVA/B doses. In melanocytes we found a marked increase in tyrosinase activity and melanin content after irradiation with 200 mJ/cm2. The UV-induced effect on tyrosinase activity was higher in melanocytes cultured with BPE than in those cultured with TPA. The results were compared with two human melanoma cell lines. Only little pigment formation could be measured in the tested melanoma cell lines without change after UV irradiation.

Adult↗

PubMed1992

Increased arterial and venous plasma noradrenaline levels in patients with primary hypothyroidism during hypothyroid as compared to euthyroid state.

The use of venous plasma noradrenaline levels as a marker of general sympathetic tone has been questioned as changes in local sympathetic activity may influence the venous levels. To compare arterial and venous plasma noradrenaline levels in patients with primary hypothyroidism, arterial and venous blood were sampled during strictly standardized conditions during hypothyroid and euthyroid states. The patients were hospitalized for 5 days at a metabolic ward on a standardized sodium and potassium intake. On the fourth day catheters were positioned in the axillary artery and vein. Blood samples were drawn simultaneously for noradrenaline and adrenaline determinations during resting conditions. The arterial and venous plasma noradrenaline levels did not differ significantly, neither during hypothyroidism nor during euthyroidism. The arteriovenous difference in plasma adrenaline was similar during hypothyroidism compared to euthyroidism, indicating similar peripheral extraction rate of catecholamines during hypothyroidism as compared to euthyroidism. During hypothyroidism venous and arterial noradrenaline were significantly higher as compared to euthyroidism. In conclusion, there is no difference between arterial and venous noradrenaline levels either in the hypothyroid or the euthyroid state, and the peripheral extraction rate of plasma noradrenaline seems to be similar in hypothyroidism and euthyroidism. The local contribution of noradrenaline from the arm, reflecting local sympathetic nervous activity, is limited during resting conditions. In hypothyroid patients plasma noradrenaline levels are increased as compared to the euthyroid state, indicating increased general sympathetic activity in hypothyroidism.

Adult↗

PubMed1992

Sympathoadrenal activity during helox-cold induced hypothermia in Syrian hamsters.

1. As reflected by increasing plasma concentrations of cortisol, norepinephrine, epinephrine and dopamine, a marked stimulation of the adrenal cortex and of the sympathetic nervous system occurred in Syrian hamsters during moderate hypothermia induced by helium-oxygen atmosphere and cold. 2. A profound hyperglycemia was observed during hypothermia. 3. All effects due to the helium-oxygen atmosphere and cold exposure (helox-cold) disappeared almost completely after rewarming. 4. The results corroborate the hypothesis of an involvement of the adrenal cortex combined with the sympathetic nervous system in the control of acute induced heat production.

Adrenal Cortex↗

PubMed1992

Mutational analysis of gap junction formation.

The paired oocyte cell-cell channel assay was used to investigate the mechanisms involved in the process of formation of gap junction channels. Single oocytes, injected with connexin-specific mRNAs, accumulate a pool of precursors from which cell-cell channels can form rapidly upon pairing. Several lines of evidence, including immunohistochemistry and surface labeling, indicate that part of this precursor pool is located in the cell membrane, probably in the form of closed hemichannels. The homophilic binding of hemichannels to each other can be mimicked by synthetic peptides representing the extracellular loop sequences of connexin32. The peptides specifically suppress channel formation. A crucial role is established for the six cysteines in the extracellular domains that are conserved in all vertebrate gap junction proteins. Change of any of these cysteines into serines results in absolute loss of function of the mutant connexin. The effects of thiol-specific reagents on channel formation suggest that docking and/or opening of channels involves disulfide exchange. Several of the variable amino acids in the extracellular loop sequences were found to determine specificity of connexin-connexin interactions.

Amino Acid Sequence↗

PubMed1991

Liquid chromatographic method for the determination of the carbohydrate moiety of glycoproteins. Application to alpha 1-acid glycoprotein and tissue plasminogen activator.

A rapid procedure is described for the qualitative and quantitative analysis of the carbohydrate composition of glycoproteins by liquid chromatography with light-scattering detection. The analysis was carried out in three steps. First, the glycoprotein samples were purified by a two-step purification on a Sephadex G-25 column with a 90% yield. Second, the selectivity of the separation and the sensitivity of detection of monosaccharides, as methyl glycosides obtained by direct methanolysis of glycoproteins, were improved by modified simplex optimization of the methanolysis parameters (temperature, methanolic hydrochloric acid strength and reaction time) determined at 66 degrees C, 1.2 M and 8.1 h for alpha 1-acid glycoprotein (alpha-AGP) and 73 degrees C, 1.5 M and 12.5 h for tissue plasminogen activator (tPA). Finally, the method was applied to the determination of the carbohydrate moiety of the two N-glycosylated glycoproteins alpha-AGP and tPA.

Acetylglucosamine↗

PubMed1991

Cell/cell channel formation involves disulfide exchange.

The oocyte cell/cell-channel assay was used to identify amino acids involved in the process of cell/cell-channel formation. The expression of the rat liver gap-junction protein, connexin 32, in single oocytes, results in the accumulation of a pool of channel precursors. Upon pairing of such oocytes, cell/cell channels form rapidly from this pool. The rate of formation is affected by thiol-specific reagents and the pH. This suggests the involvement of extracellular cysteine residues in the channel formation process. Two connexin-32 mutants were generated by site-directed mutagenesis in which cysteine residues were replaced by serine. Both mutant connexins were unable to form cell/cell channels. Thus, the cysteine residues appear to play an important role in the channel formation process.

Animals↗

PubMed1991

Gating properties of connexin32 cell-cell channels and their mutants expressed in Xenopus oocytes.

Carboxyl-terminal deletion mutants of the gap junction protein connexin32 were tested in the oocyte cell-cell channel assay. Oocytes expressing a mutant lacking 58 carboxyl terminal amino acids were found to exhibit junctional conductances of the same magnitude as oocytes expressing wild-type connexin32. The gating properties of the channels formed by this mutant of connexin32 with respect to transjunctional voltage and cytoplasmic acidification are indistinguishable from those found with wild-type connexin32 channels. This includes a novel pH-dependent voltage gate. In another mutant, two carboxyl terminal serine residues, Ser233 and Ser240, were replaced by Asn residues. This double mutant has properties indistinguishable from wild-type connexin32, suggesting that phosphorylation of either of these serines is not required for channel opening.

Animals↗

PubMed1991

The solution structure of the lantibiotic gallidermin.

The 21-peptide amide antibiotic gallidermin is a potential therapeutic against acne disease. It belongs to the class of polycyclic lanthionine and alpha,beta-didehydroamino acids containing polypeptides, which were named "lantibiotics." The structural gene of the recently elucidated lantibiotic gallidermin encodes a precursor peptide containing Ser, Thr, and Cys residues in the C-terminal prolantibiotic part, and an unusually hydrophilic leader peptide. The ribosomally synthesized pregallidermin is posttranslationally modified and processed to a complex peptide antibiotic with four sulfide rings and two unsaturated residues. The complete solution structure of gallidermin was determined in trifluoroethanol: water (95:5) and dimethylsulfoxide by two-dimensional 1H-nmr at 500 MHz, using a combination of double quantum filtered correlated spectroscopy, homonuclear Hartman-Hahn, and nuclear Overhauser enhancement spectroscopy experiments. Using a total number of 152 distance constraints from NOEs and 14 torsional constraints, derived from coupling constants, we obtained a screwlike solution structure of gallidermin. Restrained molecular dynamics simulations yielded a set of five converging structures with an atomic rms difference of 1.7 A for the backbone atoms, not dependent on the starting structure. The spatial structure model is in excellent agreement with the amphiphilic and channel-forming properties of gallidermin on membranes and its tryptic cleavage at the exposed site between residues 13 and 14.

Anti-Bacterial Agents↗

PubMed1991

Compressive mononeuropathies of the upper extremity in chronic paraplegia.

Controversy exists with regard to the actual prevalence of compressive mononeuropathies at the wrist which may occur following chronic paraplegia. Thirty one chronic paraplegics, with a mean age of 37.9 years (range 20-68 years), and mean time since injury of 9.7 years (range 1-28 years), were studied with a comprehensive neurologic and electrodiagnostic (EDX) assessment. No patient had any clinical or EDX evidence of a peripheral polyneuropathy. The diagnosis of a median mononeuropathy at the wrist was determined by the following criteria: (a) prolonged median sensory distal latency greater than ipsilateral ulnar sensory distal latency greater than or equal to 0.5 msec; (b) a median mid-palmar sensory latency greater than ipsilateral ulnar mid-palmar sensory latency of greater than or equal to 0.3 msec; or (c) a median motor distal latency greater than or equal to 1.7 milliseconds as compared to the ipsilateral ulnar motor distal latency. Ulnar mononeuropathy at the wrist or across the elbow was also characterised. The EDX criteria for a median mononeuropathy at the wrist was met in 55% of subjects (24% of these with bilateral presentations). The location of ulnar mononeuropathies included: two at the superficial sensory branch at the wrist, one at the deep motor branch at the wrist, and three patients with a conduction block across the elbow. Overall, 67% of all patients tested had evidence of at least one mononeuropathy of the upper extremity. There was no association between prevalence of compressive mononeuropathies and age of the patient or time since onset of injury.

Adult↗

PubMed1991

Cell-cell channel formation and lectins.

The oocyte cell-cell channel assay was used to investigate determinants of the rate of channel formation. After injection of connexin-specific mRNA, oocytes accumulate a pool of precursors from which cell-cell channels can form after oocytes are paired. Channel formation was found to be increased if oocytes are pretreated with lectins before pairing. Several lectins differing in their carbohydrate binding affinities can exert this effect. Lectin-specific sugars suppress the effect on cell-cell channel formation only if the sugar is mixed with the lectin before application to the oocyte. If the lectin is first bound to the oocyte and then the sugar is added, no significant inhibition is seen. The promotion of channel formation by lectins is enhanced by adding an incubation period in regular medium after lectin treatment, before pairing of the oocytes. Electron microscopic studies with gold-conjugated lectins show that the lectin receptors are clustered on the free membrane surface and are taken up in endocytotic vesicles. These data suggest that the observed acceleration of cell-cell channel formation by lectins can be attributed to the removal of steric hindrance, which is a consequence of clustering of the bulky glycoprotein lectin receptors as well as of the removal from the surface by endocytosis.

Animals↗

PubMed1990

The primary structure of rat liver glycogen synthase deduced by cDNA cloning. Absence of phosphorylation sites 1a and 1b.

The cDNA for rat liver glycogen synthase was isolated by screening a rat liver cDNA library constructed in lambda gt11. The cDNA was 2.4 kilobases in length and encoded a protein of 703 amino acid residues with a molecular mass of 80.5 kDa. Comparison of the rat liver and the human muscle sequences show that the amino- and carboxyl-terminal regions are quite divergent as compared to the internal sequences which show an 80% identity. The rat liver carboxyl-terminal region is truncated by 33 residues and has only 46% identity with the muscle sequence but retains the common feature of a low content of hydrophobic amino acids (13%). Phosphorylation sites 1a and 1b, which are the primary targets for phosphorylation by cAMP-dependent protein kinase, are absent in the liver sequence. The presence of these divergent, structurally anomalous carboxyl-terminal regions in liver and muscle glycogen synthase suggests the absence of the requirement that they possess a tertiary structure that is integral to that of the protein core. A model is proposed in which this region interacts with a catalytic core to maintain the I state, and in which phosphorylation serves to uncouple this interaction.

Amino Acid Sequence↗

PubMed1990

[In vitro studies of the adsorption behavior of Wofatit Y 88 in relation to various drugs].

The adsorber Wofatit Y 88 (VEB Chemiekombinat Bitterfeld) was tested regarding its adsorption properties in comparison with Hämoresin (B. Braun, Melsungen, FRG) Hemosorbent SKN-2K (USSR) and the own product Wofatit UH 91. As adsorptives the hypnotics Metaqualon, Pyrithyldion, Crotylbarbital and Phenobarbital were used. The investigation have been performed in a single-pass system in a relation of 1:25 to the clinical practice conditions. The concentration measurements to the estimation of adsorbed amounts were made by UV-VIS spectrometry. It was found that Wofatit Y 88 is superior to Hämoresin with regard to adsorbed amounts and adsorption speed. For all drugs Wofatit Y 88 was superior to UH 91.

Adsorption↗

PubMed1989

Risk factors for median mononeuropathy of the wrist in postpoliomyelitis patients.

There is evidence that chronic cane, crutch, and wheelchair users are at increased risk for developing pain and functional compromise of the wrist and hand. This investigation examined the prevalence and risk factors for median mononeuropathy at the wrist in the postpoliomyelitis population. The medical records of 148 consecutive patients with histories of poliomyelitis were reviewed. Forty-nine percent of this cohort had complaints of wrist pain. The prevalence rate of carpal tunnel syndrome was 22% (n = 33). Age, sex, duration of disability, and work history did not significantly influence the relative risk of developing this disorder. However, the relative risk of developing this compression neuropathy at the wrist by using the combination of a cane and/or a crutch and a wheelchair was 4.86 (95% confidence interval 2.35-10.06). The high risk associated with chronic cane, crutch, and wheelchair use shows the need for developing preventive strategies.

Adult↗

PubMed1989

Factors affecting the sensitivity and specificity of the three-phase technetium bone scan in the diagnosis of reflex sympathetic dystrophy syndrome in the upper extremity.

The three-phase technetium bone scan has been recommended for use in the diagnosis of the reflex sympathetic dystrophy syndrome with a sensitivity and specificity of greater than 90%. A retrospective chart review was conducted of 63 patients who had three-phase technetium bone scan as part of a work-up for unexplained upper extremity pain, to determine the predictive value of the three-phase technetium bone scan in reflex sympathetic dystrophy syndrome, and what factors might affect it. The prevalence of definite or probable reflex sympathetic dystrophy syndrome, as assessed by Kozin's criteria, was 26% in this sample. The 3-hour delayed image demonstrated a sensitivity = 50%; specificity = 92%; positive predictive value = 67%, and negative predictive value = 84%. This was not improved using the data from the blood flow or pool phase. The sensitivity of 50% is lower than previous reports. The predictive value of the three-phase technetium bone scan was affected by the duration of symptoms and the age of the patient. Duration of symptoms less than 6 months, or ages more than 50 years substantially increased the sensitivity and positive predictive value of the three-phase technetium bone scan.

Adult↗

PubMed1989

Formation of hybrid cell-cell channels.

The oocyte cell-cell channel assay was used to demonstrate that connexin-43 is a cell-cell channel-forming protein as previously shown for connexin-32. Expression of connexin-32 in one and connexin-43 in the other oocyte of a pair results in the formation of junctional conductances at rates similar to those observed when only one or the other connexin is expressed in both oocytes of a pair. This suggests that hybrid cell-cell channels form in the oocyte system. Hybrid channels also form when a connexin-43 mRNA-injected oocyte is paired with a noninjected oocyte expressing endogenous connexin. The latter hybrids have properties apparently contributed by both types of hemichannels. Pure connexin-43 channels are not voltage gated, whereas pure oocyte channels are voltage dependent; hybrids of these channels rectify.

Animals↗

PubMed1989

Serum creatine kinase in the post-polio population.

Elevated creatine kinase (CK) levels may be noted in neuromuscular patients as a result of muscle damage and necrosis. We measured the serum CK levels of 62 post-polio patients with chronic neurologic disease (Neuro) and 13 post-polio patients with no evidence of neurologic compromise (Control). Patient groups were comparable for age, gender and years since onset of poliomyelitis. The Neuro group had substantially increased CK levels compared with the Control group, 211 +/- 19.0 IU v 114 +/- 26.2 IU (mean +/- 1 SE; P less than 0.05). Of the Neuro group, 40% had abnormally elevated CK values, compared with 8% of the Control group. Within the Neuro group, there was a robust correlation between elevated CK values and self-reports of strenuous work and level of community ambulation (P less than 0.05). These findings support the hypothesis that chronic muscle overuse may be a contributing factor to the advanced senescence reported by post-polio patients with neuromuscular compromise.

Creatine Kinase↗

PubMed1989

Chronic tolerance to ethanol in the N:NIH rat.

As the basis for selectively breeding rats from the N:NIH stock on the basis of differential development of tolerance to ethanol, chronic tolerance to the motor-disrupting effects of ethanol was examined. Male (n = 50) and female (n = 46) rats were administered 2.5 g/kg ethanol (i.p.) and the blood-ethanol concentration (BEC) at the time of regain of aerial righting reflex (RARR) was measured. The rats were then placed in inhalation chambers for 8 consecutive days and chronically exposed to ethanol. Thirty-two hours following the cessation of chronic ethanol exposure, the rats were again given a 2.5 g/kg dose of ethanol and the BEC at RARR was again measured. The amount of chronic ethanol tolerance developed, as measured by the difference in BEC at RARR prior to and after chronic ethanol administration, was widely and normally distributed for both male and female rats. Control rats, which did not receive chronic ethanol exposure between the two tests of RARR, did not show tolerance on this measure. A negative correlation (r = -0.46) was found between BEC at RARR prior to chronic ethanol treatment and the amount of chronic tolerance developed using the difference in BEC measure. This correlation suggested that the rats who were initially more sensitive to the effects of ethanol were more likely to display the greatest amount of chronic tolerance. However, using the residuals of the regression analysis of post-chronic ethanol BEC at RARR on prechronic BEC at RARR as an index of tolerance, this negative correlation was not found.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcoholism↗