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R Swanson

Publications and source records attributed to R Swanson.

At least 19 recordsLinked to original sources
PubMed2001

The pH dependence of serpin-proteinase complex dissociation reveals a mechanism of complex stabilization involving inactive and active conformational states of the proteinase which are perturbable by calcium.

Serpin family protein proteinase inhibitors trap proteinases at the acyl-intermediate stage of cleavage of the serpin as a proteinase substrate by undergoing a dramatic conformational change, which is thought to distort the proteinase active site and slow deacylation. To investigate the extent to which proteinase catalytic function is defective in the serpin-proteinase complex, we compared the pH dependence of dissociation of several serpin-proteinase acyl-complexes with that of normal guanidinobenzoyl-proteinase acyl-intermediate complexes. Whereas the apparent rate constant for dissociation of guanidinobenzoyl-proteinase complexes (k(diss, app)) showed a pH dependence characteristic of His-57 catalysis of complex deacylation, the pH dependence of k(diss, app) for the serpin-proteinase complexes showed no evidence for His-57 involvement in complex deacylation and was instead characteristic of a hydroxide-mediated deacylation similar to that observed for the hydrolysis of tosylarginine methyl ester. Hydroxylamine enhanced the rate of serpin-proteinase complex dissociation but with a rate constant for nucleophilic attack on the acyl bond several orders of magnitude slower than that of hydroxide, implying limited accessibility of the acyl bond in the complex. The addition of 10-100 mm Ca(2+) ions stimulated up to 80-fold the dissociation rate constant of several serpin-trypsin complexes in a saturable manner at neutral pH and altered the pH dependence to a pattern characteristic of His-57-catalyzed complex deacylation. These results support a mechanism of kinetic stabilization of serpin-proteinase complexes wherein the complex is trapped as an acyl-intermediate by a serpin conformational change-induced inactivation of the proteinase catalytic function, but suggest that the inactive proteinase conformation in the complex is in equilibrium with an active proteinase conformation that can be stabilized by the preferential binding of an allosteric ligand such as Ca(2+).

Amino Acid Sequence↗
PubMed2001

The antithrombin P1 residue is important for target proteinase specificity but not for heparin activation of the serpin. Characterization of P1 antithrombin variants with altered proteinase specificity but normal heparin activation.

Heparin has been proposed to conformationally activate the serpin, antithrombin, by making the reactive center loop P1 arginine residue accessible to proteinases. To evaluate this proposal, we determined the effect of mutating the P1 arginine on antithrombin's specificity for target and nontarget proteinases in both native and heparin-activated states of the serpin. As expected, mutation of the P1 arginine to tryptophan, histidine, leucine, and methionine converted the specificity of antithrombin from a trypsin inhibitor (k(assoc) = 2 x 10(5) M(-1) s(-1)) to a chymotrypsin inhibitor (k(assoc) = 10(3)-10(5) M(-1) s(-1)). However, heparin pentasaccharide activation increased the reactivity of the P1 variants with chymotrypsin or of the wild-type inhibitor with trypsin only 2-6-fold, implying that the P1 residue had similar accessibilities to these proteinases in native and activated states. Mutation of the P1 arginine greatly reduced k(assoc) for antithrombin inhibition of thrombin and factor Xa from 40- to 5000-fold, but heparin normally accelerated the reactions of the variant antithrombins with these enzymes to make them reasonably efficient inhibitors (k(assoc) = 10(3)-10(4) M(-1) s(-1)). Fluorescence difference spectra of wild-type and P1 tryptophan variant antithrombins showed that the P1 tryptophan exhibited fluorescence properties characteristic of a solvent-exposed residue which were insignificantly affected by heparin activation. Moreover, all P1 variant antithrombins bound heparin with approximately 2-3-fold higher affinities than the wild type. These findings are consistent with the P1 mutations disrupting a P1 arginine-serpin body interaction which stabilizes the native low-heparin affinity conformation, but suggest that this interaction is of low energy and unlikely to limit the accessibility of the P1 residue. Together, these findings suggest that the P1 arginine residue is similarly accessible to proteinases in both native and heparin-activated states of the serpin and contributes similarly to the specificity of antithrombin for thrombin and factor Xa in the two serpin conformational states. Consequently, determinants other than the P1 residue are responsible for enhancing the specificity of antithrombin for the two proteinases when activated by heparin.

Animals↗
PubMed2001

Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin.

Heparin activates the primary serpin inhibitor of blood clotting proteinases, antithrombin, both by an allosteric conformational change mechanism that specifically enhances factor Xa inactivation and by a ternary complex bridging mechanism that promotes the inactivation of thrombin and other target proteinases. To determine whether the factor Xa specificity of allosterically activated antithrombin is encoded in the reactive center loop sequence, we attempted to switch this specificity by mutating the P6-P3' proteinase binding sequence excluding P1-P1' to a more optimal thrombin recognition sequence. Evaluation of 12 such antithrombin variants showed that the thrombin specificity of the serpin allosterically activated by a heparin pentasaccharide could be enhanced as much as 55-fold by changing P3, P2, and P2' residues to a consensus thrombin recognition sequence. However, at most 9-fold of the enhanced thrombin specificity was due to allosteric activation, the remainder being realized without activation. Moreover, thrombin specificity enhancements were attenuated to at most 5-fold with a bridging heparin activator. Surprisingly, none of the reactive center loop mutations greatly affected the factor Xa specificity of the unactivated serpin or the several hundred-fold enhancement in factor Xa specificity due to activation by pentasaccharide or bridging heparins. Together, these results suggest that the specificity of both native and heparin-activated antithrombin for thrombin and factor Xa is only weakly dependent on the P6-P3' residues flanking the primary P1-P1' recognition site in the serpin-reactive center loop and that heparin enhances serpin specificity for both enzymes through secondary interaction sites outside the P6-P3' region, which involve a bridging site on heparin in the case of thrombin and a previously unrecognized exosite on antithrombin in the case of factor Xa.

Allosteric Regulation↗
PubMed2001

Differential expression of voltage-gated K(+) channel genes in arteries from spontaneously hypertensive and Wistar-Kyoto rats.

Voltage-gated K(+) currents play an important role in determining membrane potential, intracellular Ca(2+), and contraction in arterial smooth muscle. In this study, the expression of genes encoding voltage-gated K(+) channels of the Kv1.X family was compared in arteries from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Expression of Kv1.X in thoracic aorta, mesenteric arteries, tail artery, and heart was determined, both qualitatively and quantitatively, by reverse transcription-polymerase chain reaction. Our results demonstrate distinct but overlapping patterns of expression in vascular tissues. In general, Kv1.2 and Kv1.5 were most highly represented, and the levels of Kv1.2 were significantly larger in all tissues from SHR. Levels of Kv1.5 in arteries did not differ significantly between strains but were greater in SHR heart. Moderate levels of Kv1.3 and Kvbeta1.1 expression were also found in all tissues and were larger in SHR. Kv1.1 expression was not different between the 2 strains, and no significant expression of Kv1.4 (except in heart and aorta), Kv1.6, or Kvbeta2.1 was observed in either strain. Kv1.2 and Kv1.5 transcripts represent approximately 1 to 2 parts/10(5) of total mesenteric arterial RNA with approximately 2- to 5-fold lower levels in aorta and tail artery. Whole-cell voltage-gated K(+) channel currents, recorded from mesenteric arterial myocytes, were larger in SHR than WKY (eg, at 0 mV: 7.3+/-0.8 versus 10.9+/-1.2 pA/pF). The voltage dependence of activation was more negative in SHR (V(0.5): -20+/-4 mV versus -32+/-3 mV) but that of availability was not different. These results indicate that Kv1.X genes are differentially expressed between WKY and SHR (especially Kv1.2 and Kvbeta1.1). These differences in gene expression are associated with a greater voltage-gated K(+) channel current density in SHR and shifted voltage-dependent activation compared with WKY. These differences may be a compensatory mechanism related to the membrane potential depolarization in SHR or some manifestation thereof.

Animals↗
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↗