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Biomedical subjects

R Swanson

Publications and source records attributed to R Swanson.

At least 19 recordsLinked to original sources
PubMed2000

Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel.

We report here a characterization of two families of calcium-activated K(+) channel beta-subunits, beta2 and beta3, which are encoded by distinct genes that map to 3q26.2-27. A single beta2 family member and four alternatively spliced variants of beta3 were investigated. These subunits have predicted molecular masses of 27. 1-31.6 kDa, share approximately 30-44% amino acid identity with beta1, and exhibit distinct but overlapping expression patterns. Coexpression of the beta2 or beta3a-c subunits with a BK alpha-subunit altered the functional properties of the current expressed by the alpha-subunit alone. The beta2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the beta3a-c subunits resulted in only partial inactivation of the current, and the beta3b subunit conferred an apparent inward rectification. Furthermore, unlike the beta1 and beta2 subunits, none of the beta3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these beta-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.

Alternative Splicing↗
PubMed2000

A viable ubiquitin-activating enzyme mutant for evaluating ubiquitin system function in Saccharomyces cerevisiae.

Ligation of proteins to ubiquitin requires activation of ubiquitin by E1, the ubiquitin-activating enzyme. Mutant alleles of E1 in mammalian cells have been crucial for dissecting the contribution of the ubiquitin system to cell function. Comparable mutants have been unavailable for Saccharomyces cerevisiae. Here we describe the isolation and characterization of a hypomorphic allele of S. cerevisiae E1. Protein modification by ubiquitin is strongly impaired in the mutant, inhibiting degradation of ubiquitin-proteasome pathway substrates as well as ubiquitin-dependent but proteasome-independent degradation of membrane receptors. This allele will be a useful tool for evaluating the ubiquitin-dependence of cellular processes in yeast, even those in which the proteasome is not involved.

Base Sequence↗
PubMed2000

Role of arginine 129 in heparin binding and activation of antithrombin.

The contribution of Arg(129) of the serpin, antithrombin, to the mechanism of allosteric activation of the protein by heparin was determined from the effect of mutating this residue to either His or Gln. R129H and R129Q antithrombins bound pentasaccharide and full-length heparins containing the antithrombin recognition sequence with similar large reductions in affinity ranging from 400- to 2500-fold relative to the control serpin, corresponding to a loss of 28-35% of the binding free energy. The salt dependence of pentasaccharide binding showed that the binding defect of the mutant serpin resulted from the loss of approximately 2 ionic interactions, suggesting that Arg(129) binds the pentasaccharide cooperatively with other residues. Rapid kinetic studies showed that the mutation minimally affected the initial low affinity binding of heparin to antithrombin, but greatly affected the subsequent conformational activation of the serpin leading to high affinity heparin binding, although not enough to disfavor activation. Consistent with these findings, the mutant antithrombin was normally activated by heparin for accelerated inhibition of factor Xa and thrombin. These results support an important role for Arg(129) in an induced-fit mechanism of heparin activation of antithrombin wherein conformational activation of the serpin positions Arg(129) and other residues for cooperative interactions with the heparin pentasaccharide so as to lock the serpin in the activated state.

Antithrombins↗
PubMed2000

The human inward rectifier K(+) channel subunit kir5.1 (KCNJ16) maps to chromosome 17q25 and is expressed in kidney and pancreas.

A novel human Kir5.1 (inward rectifier K+ channel subunit, gene name KCNJ16) was identified through database searches. This human KCNJ16 was mapped to chromosome 17q25. The full-length cDNA was identified and its genomic structure was determined. Tissue distribution studies showed that human KCNJ16 is significantly expressed in human kidney, pancreas and thyroid gland. In situ hybridization revealed expression in convoluted tubule cells of kidney and in the acinar and ductal cells of pancreas. These suggest that human Kir5.1 may be involved in the regulation of fluid and pH balance, thus making it a potential therapeutic target for hypertension, renal failure, or pancreatic disease.

Amino Acid Sequence↗
PubMed1999

The Saccharomyces cerevisiae ubiquitin-proteasome system.

Our studies of the yeast ubiquitin-proteasome pathway have uncovered a number of general principles that govern substrate selectivity and proteolysis in this complex system. Much of the work has focused on the destruction of a yeast transcription factor, MAT alpha 2. The alpha 2 protein is polyubiquitinated and rapidly degraded in alpha-haploid cells. One pathway of proteolytic targeting, which depends on two distinct endoplasmic reticulum-localized ubiquitin-conjugating enzymes, recognizes the hydrophobic face of an amphipathic helix in alpha 2. Interestingly, degradation of alpha 2 is blocked in a/alpha-diploid cells by heterodimer formation between the alpha 2 and a1 homeodomain proteins. The data suggest that degradation signals may overlap protein-protein interaction surfaces, allowing a straightforward steric mechanism for regulated degradation. Analysis of alpha 2 degradation led to the identification of both 20S and 26S proteasome subunits, and several key features of proteasome assembly and active-site formation were subsequently uncovered. Finally, it has become clear that protein (poly) ubiquitination is highly dynamic in vivo, and our studies of yeast de-ubiquitinating enzymes illustrate how such enzymes can facilitate the proteolysis of diverse substrates.

Amino Acid Sequence↗
PubMed1999

Efficacy and safety of ampicillin/sulbactam and cefuroxime in the treatment of serious skin and skin structure infections in pediatric patients. UNASYN Pediatric Study Group.

BACKGROUND: Pediatric skin and skin structure infections are often polymicrobial and require empiric therapy effective against pathogens that may be resistant to many antimicrobial agents. The present study tested the efficacy and safety of a parenteral beta-lactam/beta-lactamase inhibitor combination, ampicillin/sulbactam, and a beta-lactamase-stable cephalosporin, cefuroxime, in serious pediatric skin and skin structure infections requiring hospitalization and parenteral antimicrobial therapy. METHODS: This was a multicenter, randomized, prospective, comparative open label trial that enrolled patients 3 months through 11 years of age. Patients received 150 to 300 mg/kg/day ampicillin/sulbactam in equally divided intravenous doses every 6 h. Cefuroxime was given in a dosage of 50 to 100 mg/kg/day either intravenously or intramuscularly in equally divided doses every 6 or 8 h. Maximum treatment was not to exceed 14 days. Patients could receive subsequent oral antimicrobial treatment at the investigator's discretion. RESULTS: At final evaluation for clinical efficacy, 78.0% (n = 46) of the 59 evaluable patients who received ampicillin/sulbactam were cured and 22.0% (n = 13) were improved. The respective values for the 39 evaluable patients treated with cefuroxime were 76.9% (n = 30) and 23.1% (n = 9). At the end of treatment all pathogens were eradicated from 93.2% (n = 55) of 59 patients treated with ampicillin/sulbactam and from 100% of 39 who received cefuroxime. There were no significant differences between treatments in clinical or bacteriologic efficacy. Both ampicillin/sulbactam and cefuroxime were well-tolerated. CONCLUSION: Both ampicillin/sulbactam and cefuroxime provide safe and effective parenteral antibiotic therapy in pediatric patients with serious skin and skin structure infections.

Ampicillin↗
PubMed1998

Degradation signal masking by heterodimerization of MATalpha2 and MATa1 blocks their mutual destruction by the ubiquitin-proteasome pathway.

Proteolysis by the ubiquitin-proteasome pathway is often regulated, but the mechanisms underlying such regulation remain ill-defined. In Saccharomyces cerevisiae, cell type is controlled by the MAT transcription factors. The alpha2 repressor is a known ubiquitin pathway substrate in alpha haploid cells. We show that a1 is rapidly degraded in a haploids. In a/alpha diploids, alpha2 and a1 are stabilized by heterodimerization. Association depends on N-terminal coiled-coil interactions between a1 and alpha2. Residues in alpha2 important for these interactions overlap a critical determinant of an alpha2 degradation signal, which we delimit by extensive mutagenesis. Our data provide a detailed description of a natural ubiquitin-dependent degradation signal and point to a molecular mechanism for regulated turnover in which proteolytic signals are differentially masked in alternative multiprotein complexes.

Amino Acid Sequence↗
PubMed1998

Role of allosteric: zinc interdomain region of the regulatory subunit in the allosteric regulation of aspartate transcarbamoylase from Escherichia coli.

The hydrophobic interface between the allosteric and the zinc domains of the regulatory subunit of aspartate transcarbamoylase has previously been implicated in the heterotropic ATP activation of the enzyme. The present work shows that this interface also affects CTP and CTP-UTP inhibition and proposes a structural explanation for the effects. Mutant enzymes derived from nonselective mutagenesis of residues r101-r106 (residues that contribute part of the interface) displayed a variety of homotropic and heterotropic effects. The cooperative behavior of the enzymes was affected, as indicated by reduced aspartate S0.5 values and apparent Hill coefficient values for V106L, V106L/N105S, and I103F/R102C. In addition, both ATP activation and CTP inhibition were significantly reduced and CTP+UTP synergistic inhibition was decreased in these mutants. The D104G mutant enzyme was subject to inhibition by CTP andCTP+UTP, but was not activated by ATP. Finally, the I103T mutant enzyme had an increased S0.5 value of 11.5 mM and displayed altered effector responses: ATP acted as an inhibitor, and the CTP+UTP synergistic inhibition was reduced. Most of these allosteric variations can be explained in terms of perturbations to the "tongue and groove" hydrophobic interface between the allosteric and the zinc domains and a consequent impact on a second interface ("reg1:cat4") between regulatory and catalytic subunits.

Adenosine Triphosphate↗
PubMed1998

Characterization of rhesus cytomegalovirus genes associated with anti-viral susceptibility.

Studies were initiated to determine whether rhesus cytomegalovirus (RhCMV)-infected macaques could serve as an animal model for evaluating anti-CMV compounds, as macaques have a naturally occurring CMV that is similar to human CMV (HCMV). Utilizing plaque reduction assays, RhCMV was tested to anti-viral susceptibility. By these assays. RhCMV displayed anti-viral susceptibility to ganciclovir at a 50% effective dose (ED50) of 0.8 microM, acyclovir at an ED50 of 15 microM, and foscarnet at an ED50 of 250 microM. By Southern blot analysis with HCMV-UL97 (phosphotransferase) and DNA polymerase (pol) genes as probes, we isolated viral DNA fragments that strongly hybridized. DNA sequence analysis of these DNA fragments revealed two open reading frames with homology to HCMV UL97 and DNA polymerase. Steady-state RNA analysis revealed that the RhCMV UL97 homologue and pol genes are transcribed as early late and early genes, respectively. Comparison against HCMV showed the RhCMV UL97 homologue exhibits 54.4% amino acid (aa) sequence identity to HCMV UL97 and the RhCMV DNA polymerase 59.2% aa sequence identity to HCMV DNA polymerase. Results from anti-viral assays and molecular characterization of these two viral genes suggest that RhCMV-infected rhesus macaques should serve as an excellent animal model for evaluating future anti-CMV compounds.

Acyclovir↗
PubMed1998

Paradigms and expectations: the nature of research and diagnostics.

Researchers are judged by their best work. No matter what disasters happen at the researcher's bench, there is time to repeat experiments, discard ambiguous data and work steadily toward a clearer understanding of the research problem. The work practices of researchers are matters of personal choice, because the process of peer review prior to publication considers only the work that is reported and not all work done by the researcher. On the other hand, the diagnostician's credibility is directly linked to every experiment performed, because a customer is waiting for the result. This paper contrasts aspects of the work of researchers and diagnosticians and concludes that researchers could benefit from understanding the framework within which diagnosticians operate.

Animals↗
PubMed1998

[A method for intermittent hypoxic exposures in the combined treatment of bronchial asthma patients].

The method of intermittent increasing normobaric isocapnic hypoxia was used for the treatment of bronchial asthma. The parameters of respiration, metabolism, free-radical processes and immune system were monitored before and after training. The therapeutic diagnostic complex "Hypotron" (Ukraine), which allowed to determine the individual reactivity of the patient's respiratory system, tolerance to hypoxia, and to choose an optimal program of treatment, was used. The hypoxic training resulted in considerable increase of lung vital capacity, maximal ventilation and forced expired velocity. Normalization of initially increased free radical processes, accompanied by a decrease in the lipid peroxidation products was observed. The hypoxic training positively influenced specific and nonspecific immunological status, and appeared to be associated with a far better stimulation of lymphocytes and neutrophils.

Adult↗
PubMed1997

Apparent formation of sodium dodecyl sulfate-stable complexes between serpins and 3,4-dichloroisocoumarin-inactivated proteinases is due to regeneration of active proteinase from the inactivated enzyme.

Protein proteinase inhibitors of the serpin family were recently reported to form SDS-stable complexes with inactive serine proteinases modified at the catalytic serine with 3, 4-dichloroisocoumarin (DCI) that resembled the complexes formed with the active enzymes (Christensen, S., Valnickova, Z., Thogersen, I. B. , Pizzo, S. V., Nielsen, H. R., Roepstorff, P., and Enghild, J. J. (1995) J. Biol. Chem. 270, 14859-14862). The discordance between these findings and other reports that similar active site modifications of serine proteinases block the ability of serpins to form SDS-stable complexes prompted us to investigate the mechanism of complex formation between serpins and DCI-inactivated enzymes. Both neutrophil elastase and beta-trypsin inactivated by DCI appeared to form SDS-stable complexes with the serpin, alpha1-proteinase inhibitor (alpha1PI), as reported previously. However, several observations suggested that such complex formation resulted from a reaction not with the DCI enzyme but rather with active enzyme regenerated from the DCI enzyme by a rate-limiting hydrolysis reaction. Thus (i) complex formation was blocked by active site-directed peptide chloromethyl ketone inhibitors; (ii) the kinetics of complex formation indicated that the reaction was not second order but rather showed a first-order dependence on DCI enzyme concentration and zero-order dependence on inhibitor concentration; and (iii) complex formation was accompanied by stoichiometric release of a peptide having the sequence SIPPE corresponding to cleavage at the alpha1PI reactive center P1-P1' bond. Quantitation of kinetic constants for DCI and alpha1PI inactivation of human neutrophil elastase and trypsin and for reactivation of the DCI enzymes showed that the observed complex formation could be fully accounted for by alpha1PI preferentially reacting with active enzyme regenerated from DCI enzyme during the reaction. These results support previous findings of the critical importance of the proteinase catalytic serine in the formation of SDS-stable serpin-proteinase complexes and are in accord with an inhibitory mechanism in which the proteinase is trapped at the acyl intermediate stage of proteolysis of the serpin as a substrate.

Animals↗
PubMed1997

Effect of individual carbohydrate chains of recombinant antithrombin on heparin affinity and on the generation of glycoforms differing in heparin affinity.

Two major glycoforms of recombinant antithrombin which differ 10-fold in their affinity for the effector glycosaminoglycan, heparin, were previously shown to be expressed in BHK or CHO mammalian cell lines (I. Björk, et al., 1992, Biochem. J. 286, 793-800; B. Fan et al., 1993, J. Biol. Chem. 268, 17588-17596). To determine the source of the glycosylation heterogeneity responsible for these different heparin-affinity forms, each of the four Asn residue sites of glycosylation, residues 96, 135, 155, and 192, was mutated to Gln to block glycosylation at these sites. Heparin-agarose chromatography of the four antithrombin variants revealed that Gln 96, Gln 135, and Gln 192 variants still displayed the two functional heparin-affinity forms previously observed with the wild-type inhibitor, whereas the Gln 155 variant showed only a single functional high heparin affinity form. These results demonstrate that heterogeneous glycosylation of Asn 155 of recombinant antithrombin is responsible for generating the low heparin affinity glycoform. Analysis of heparin binding to the higher heparin affinity forms of the four variants showed that all exhibited increased heparin affinities of two- to sevenfold compared to wild-type higher heparin affinity form or to plasma antithrombin, with the Gln 135 variant showing the largest effect on this affinity. The extent of heparin-affinity enhancement was correlated with the distance of the mutated glycosylation site to the putative heparin-binding site in the X-ray structure of antithrombin. All variants displayed normal kinetics of thrombin inhibition in the absence and presence of saturating heparin, indicating that the carbohydrate chains solely affected heparin binding and not heparin-activation or proteinase-binding functions. These results indicate that all carbohydrate chains of recombinant antithrombin adversely affect heparin-binding affinity to an extent that correlates with their relative proximity to the putative heparin-binding site in antithrombin.

Animals↗
PubMed1997

A fosmid-based genomic map and identification of 474 genes of the hyperthermophilic archaeon Pyrobaculum aerophilum.

We have constructed a physical map of the approximately 1.7-Mb genome of the hyperthermophilic archaeon Pyrobaculum aerophilum. Derived from a 12x coverage genomic fosmid library with an average insert size of 36 Kb, the map consists of a single circular contig of 96 overlapping fosmid clones with 211 markers ordered along them. One hundred of the sequence markers have strong similarities to known genes. Many overlaps were also checked using restriction fingerprint analysis. This map is an important step in the elucidation of the sequence of the entire genome of Pyrobaculum aerophilum. To this end we have determined more than 95% of the genome with 15,000 random sequences. Each sequence has been screened against the public sequence databases to identify similarities to known genes. We report here a list of the 474 putative genes we have identified.

Animals↗
PubMed1997

Strychnine-like multifocal myoclonus and seizures in extremely high-dose opioid administration: treatment strategies.

While occasional myoclonic jerks are prevalent in cancer patients receiving opioids, severe myoclonic jerks and seizures due to opioids are uncommon. In this retrospective case series, we describe five cancer patients with refractory cancer pain and severe neuroexcitatory toxicity associated with extremely high-dose opioid therapy to characterize better the syndrome, its treatment, and its outcome. Two patients died following seizures, but three patients recovered following prompt treatment with parenteral midazolam infusions and rotation to alternative opioids. Possible mechanisms and treatment options for this potentially lethal clinical syndrome are reviewed. The authors conclude that severe multifocal myoclonus and seizures associated with extremely high-dose opioid therapy are life-threatening, and respond to parenteral midazolam infusion, rotation to alternative opioids, and aggressive supportive care.

Adult↗