Biomedical subjects
R Swanson
Publications and source records attributed to R Swanson.
[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.
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.
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.
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.
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.
The human P2x1 receptor: molecular cloning, tissue distribution, and localization to chromosome 17.
A human urinary bladder cDNA library was screened with a rat P2x purinoceptor probe. A full-length cDNA was isolated, and like its rat homologue, the deduced protein consists of 399 amino acids (M(r) = 44980 Da), contains two hydrophobic, putative transmembrane, domains flanking a large presumed extracellular loop, and represents the P2x1 subtype of this multigene family (approximately 89% amino acid sequence identity to rat P2x1, but only 40-50% identity to rat P2x2-4). Expression of the P2x1 gene in human bladder was confirmed by Northern analysis, which demonstrated a major transcript of approximately 2.9 kb. Transcripts were also found in a variety of other tissues including adult peripheral leukocytes, pancreas, spleen, prostate, small intestine, colon, testis, and ovary, and in fetal liver. The gene encoding the human P2x1 receptor was localized to chromosome 17 by Southern analysis of DNAs isolated from a panel of somatic cell hybrids. The results support a role for P2x purinoceptors in the regulation of human bladder function.
Algorithms for finding the axis of a helix: fast rotational and parametric least-squares methods.
Several methods for finding the axis of a helix are presented and compared. The most accurate determines the helix axis as the axis of rotation necessary to map point i to point i + 1 of the helix. The fastest method calculates the helix axis as the best-fit line through the coordinates by a three-dimensional parametric linear least-squares algorithm, taking advantage of the sequential nature of the data.
Preoperative angiography and embolization of the site of intermittent acute small bowel bleeding with a radiopaque microcoil: facilitated precise surgical excision of the source.
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A new approach to chronic pain in the ED.
Patients presenting to emergency departments (EDs) for primary management of chronic or recurrent nonmalignant pain conditions and their physicians frequently report dissatisfaction, in part because of the impressions created by a small percentage of such patients that frequently visit EDs requesting opioids. Treating such patients with opioids is contrary to many published guidelines, but refusing them increases dissatisfaction. Narcotic registers serve to label patients who are suspected of seeking drugs, thus creating anxiety and often distrust in health care professionals treating them. The four Calgary adult EDs have developed a system that will attempt to remove labels associated with some of these patients, insure communication between patients, their family doctors, and ED staff, and facilitate optimal care of the patients' real problems, be they difficult home management of pain, drug dependence or addiction, or other social issues. Emphasis will be shifted to home management and the family doctor's office. If successful, the system will minimize ED visits by frequent attendees seeking medication for pain control, and should also decrease overall expenditure to the health care system.
IK of rabbit ventricle is composed of two currents: evidence for IKs.
The delayed rectifier K+ current (IK) in rabbit heart has long been thought to consist of only a single, rapidly activating, dofetilide-sensitive current, IKr. However, we find that IK of rabbit ventricular myocytes actually consists of both rapid and slow components, IKr and IKs, respectively, that can be isolated pharmacologically. Thus, after complete blockade of IKr with dofetilide, the remaining current, IKs, is homogeneous as judged by an envelope of tails test. IKs activates and deactivates slowly, continues to activate during sustained depolarizations, has a half-activation potential of 7.0 +/- 0.8 mV and slope factor of 11.0 +/- 0.7 mV, reverses at -77.2 +/- 1.3 mV (extracellular K+ concentration = 4 mM), is increased by removing extracellular K+, and is enhanced by isoproterenol and stocked by azimilide. Northern analysis demonstrates that the minK (IsK) gene, which encodes a subunit of the channel that underlies the IKs current, is expressed in rabbit heart. Expression of the rabbit protein in Xenopus oocytes elicits a slowly activating, voltage-dependent current, IsK, similar to those expressed previously from mouse, rat, guinea pig, and human genes. The results demonstrate that IKs is present in rabbit ventricle and therefore contributes to cardiac repolarization in this species.
Functional role of the beta subunit of high conductance calcium-activated potassium channels.
Mammalian high conductance, calcium-activated potassium (maxi-K) channels are composed of two dissimilar subunits, alpha and beta. We have examined the functional contribution of the beta subunit to the properties of maxi-K channels expressed heterologously in Xenopus oocytes. Channels from oocytes injected with cRNAs encoding both alpha and beta subunits were much more sensitive to activation by voltage and calcium than channels composed of the alpha subunit alone, while expression levels, single-channel conductance, and ionic selectivity appeared unaffected. Channels from oocytes expressing both subunits were sensitive to DHS-I, a potent agonist of native maxi-K channels, whereas channels composed of the alpha subunit alone were insensitive. Thus, alpha and beta subunits together contribute to the functional properties of expressed maxi-K channels. Regulation of co-assembly might contribute to the functional diversity noted among members of this family of potassium channels.
Duodenal duplication cyst with massive gastrointestinal bleeding.
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Efficient infection from cDNA clones of cucumber mosaic cucumovirus RNAs in a new plasmid vector.
Full-length cucumber mosaic cucumovirus (CMV) cDNAs were cloned into a new plasmid vector containing a modified plant virus promoter designed to transcribe the inserted sequence from its first nucleotide. cDNA copies of CMV strain Q (Q-CMV) genomic RNAs 1, 2 and 3 cloned into this vector were infectious when inoculated together, producing symptoms indistinguishable from those caused by wildtype Q-CMV infection. The infectivity of the clones could be substantially increased by excision of the viral insert together with the transcriptional promoter and terminator prior to inoculation. A diagnostic but silent mutation was introduced into the RNA 2 cDNA and found to be stably maintained in viral infection, allowing distinction of the recombinant virus from native contaminants. The infectious cDNA clones supported the replication of CMV satellite RNA when co-inoculated with biologically active Q-CMV satellite RNA transcripts. Using the infectious cDNAs described, it was found that a newly-identified overlapping gene (2b) encoded by Q-CMV RNA 2 was not essential for either systemic viral infection of Nicotiana glutinosa or replication of the satellite RNAs.
Selective suppression of cytochrome P-450 gene expression by interleukins 1 and 6 in rat liver.
Inflammatory stimuli suppress constitutive hepatic expression of the CYP2C11 and CYP2C12 genes in male and female rat livers, respectively. We have shown previously that injection of interleukin-1 (IL1), but not interleukin-6 (IL6), to female rats also suppresses CYP2C12. In the present study, we examined the effects of these cytokines on CYP2C12 expression in rat hepatocyte cultures, and their in vivo effects on expression of multiple cytochrome P-450 (P450) gene products in male rat livers. IL1 suppressed the expression of CYP2C12 mRNA and protein in hepatocytes cultured on Matrigel in the presence of growth hormone. No consistent effect of IL6 was observed. Maximal suppression of CYP2C12 mRNA after 24 h of IL1 treatment reached 12 and 32% of control levels in two separate experiments. The approximate ED50 for IL1 was 5 ng/ml. CYP2C12 protein was suppressed to 28% of control levels as early as 12 h after IL1 treatment. Injection of IL1, low doses of dexamethasone, or both, in male rats produced decreases in total P450, and in CYP3A2 and CYP2C11 mRNA and protein expression similar to effects previously seen for CYP2C12 expression in females. CYP2E1 mRNA and protein was significantly suppressed only by the combination of IL1 and dexamethasone. IL6 treatment of male rats down-regulated the CYP2C11 and CYP2E1 mRNAs at a dose of 4.5 micrograms/kg, which was lower than that required to induce haptoglobin mRNA, a prototype acute phase gene product. CYP2C11 protein content of the microsomes was also decreased by IL6 treatment, with a slower time-course than for suppression of its mRNA. No significant effects of IL6 treatment were seen on CYP3A2 mRNA or CYP3A2/1 proteins. These results demonstrate that IL1 and IL6 treatments in vivo differentially affect subsets of P450 gene products in rat liver.
Confirmation of the assignment of the gene encoding Kv1.3, a voltage-gated potassium channel (KCNA3) to the proximal short arm of human chromosome 1.
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Primary sequence and immunological characterization of beta-subunit of high conductance Ca(2+)-activated K+ channel from smooth muscle.
The charybdotoxin receptor, purified from bovine tracheal smooth muscle, consists of two subunits (alpha and beta) and, when reconstituted into planar lipid bilayers, forms functional high conductance Ca(2+)-activated K+ channels. Amino acid sequence, obtained from proteolytic fragments of the beta-subunit, was used to design oligonucleotide probes with which cDNAs encoding this protein were isolated. The cDNAs encode a protein of 191 amino acids that contains two hydrophobic (putative transmembrane) domains and bears little sequence homology to subunits of other known ion channels. Site-directed antisera, raised against putative extracellular epitopes of this protein, specifically immunoprecipitated 125I-labeled Bolton-Hunter beta-subunit as well as [125I]charybdotoxin-cross-linked beta-subunit. Under nondenaturing conditions, however, these anti-beta sera immunoprecipitated a complex consisting of both the alpha- and beta-subunits. The data demonstrate that, in vivo, the high conductance Ca(2+)-activated K+ channel exists as a multimer containing both alpha- and beta-subunits, and this cDNA represents the first beta-subunit of a potassium channel cloned to date. Furthermore, we demonstrate that the cloned protein is the subunit to which charybdotoxin is specifically and covalently incorporated when cross-linked to the channel.
K+ currents expressed from the guinea pig cardiac IsK protein are enhanced by activators of protein kinase C.
We have isolated cardiac cDNA and genomic clones encoding the guinea pig IsK protein. The deduced amino acid sequence is approximately 78% identical to the rat, mouse, and human variants of this channel, and the structure of the gene encoding the protein is also similar to that in other species. For example, the gene is present only once in the haploid genome, the protein-coding sequence is present on a single uninterrupted exon, an intron exists in the 5' untranslated domain, and multiple alternative polyadenylation sites are used in processing the transcript. Expression of the guinea pig protein in Xenopus oocytes results in a slowly activating, voltage-dependent K+ current, IsK, similar to those expressed previously from the rat, mouse, and human genes. However, in sharp contrast to the rat and mouse currents, activation of protein kinase C with phorbol esters increases the amplitude of the guinea pig IsK current, analogous to its effects on the endogenous IKs current in guinea pig cardiac myocytes. Mutagenesis of the guinea pig cDNA to alter four cytoplasmic amino acid residues alters the phenotype of the current response to protein kinase C from enhancement to inhibition, mimicking that of rat and mouse IsK currents. This mutation is consistent with reports that phosphorylation of Ser-102 by protein kinase C decreases the current amplitude. These data explain previously reported differences in the regulatory properties between recombinant rat or mouse IsK channels and native guinea pig IKs channels and provide further evidence that the IsK protein forms the channels that underlie the IKs current in the heart.