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Biomedical subjects

R T Swank

Publications and source records attributed to R T Swank.

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

Inherited abnormalities in platelet organelles and platelet formation and associated altered expression of low molecular weight guanosine triphosphate-binding proteins in the mouse pigment mutant gunmetal.

Gunmetal (gm/gm) is a recessively inherited mouse pigment dilution mutant that has high mortality and poor reproductive rates. In these studies, several hematologic defects were found associated with the mutation, including prolonged bleeding times, together with thrombocytopenia and increased platelet size. A unique feature is the presence of simultaneous abnormalities in two platelet organelles, dense granules and alpha-granules. The dense granule component serotonin is present at about half the normal concentration, as are visible dense granules. Three alpha-granule components (fibrinogen, platelet factor 4, and von Willebrand factor) are also significantly reduced. Thus, in several respects the gunmetal mutant resembles the human gray platelet syndrome. A novel abnormality in expression of low molecular weight guanosine triphosphate (GTP)-binding proteins occurs in platelets of gunmetal. In Western blot assays, two additional GTP-binding proteins of 28.5 and 25 Kd were detected. The abnormal expression of GTP-binding proteins is, like the hematologic defects, genetically recessive and is tissue specific. Liver, kidney, brain, spleen, macrophages, and neutrophils have normal GTP-binding protein expression. The additional GTP-binding proteins are soluble. The data indicate that platelet formation and platelet organelle biogenesis are under common genetic control and that abnormal regulation of GTP-binding proteins may affect one or both processes.

Animals↗
PubMed1991

Platelet storage pool deficiency associated with inherited abnormalities of the inner ear in the mouse pigment mutants muted and mocha.

Several inherited human syndromes have combined platelet, auditory, and/or pigment abnormalities. In the mouse the pallid pigment mutant has abnormalities of the otoliths of the inner ear together with a bleeding abnormality caused by platelet storage pool deficiency (SPD). To determine if this association is common, two other mouse pigment mutants, muted and mocha, which are known to have inner ear abnormalities, were examined for hematologic abnormalities. Both mutants had prolonged bleeding times accompanied by abnormalities of dense granules as determined by whole mount electron microscopy of platelets and by labeling platelets with mepacrine. When mutant platelets were treated with collagen, there was minimal secretion of adenosine triphosphate and aggregation was reduced. Lysosomal enzyme secretion in response to thrombin treatment was partially reduced in muted platelets and markedly reduced in mocha platelets. Similar reductions in constitutive lysosomal enzyme secretion from kidney proximal tubule cells were noted in the two mutants. These studies show that several mutations that cause pigment dilution and platelet SPD are associated with abnormalities of the inner ear. Also, these mutants, like previously described mouse pigment mutants, are models for human Hermansky-Pudlak syndrome and provide additional examples of single genes that simultaneously affect melanosomes, lysosomes, and platelet dense granules.

Animals↗
PubMed1991

Synthesis, isolation, and characterization of endogenous beta-galactoside-binding lectins in human leukocytes.

Galaptin, a beta-galactoside-binding lectin, was isolated from human buffy coat cells (peripheral leukocytes) and spleen by affinity chromatography. The molecular weight (32K) of the native buffy coat galaptin was similar to that for splenic galaptin. Their subunit molecular weight (14.5K), pI (4.60-4.85), and amino acid composition were identical. Both galaptins showed the presence of a single polypeptide when subjected to reversed-phase HPLC. Monospecific rabbit polyclonal antiserum raised against the 14.5-kDa subunit of splenic galaptin reacted with a 14.5-kDa polypeptide present in buffy coat cells, Epstein-Barr virus-immortalized B lymphoblastoid cells, and HL-60 promyelocytic leukemia cells. However, galaptin was not synthesized in vitro by buffy coat cells. Rather, a monomeric beta-galactoside-binding protein of Mr 15.5-16.5K that is immunologically distinct from galaptin was synthesized. This galactoside-binding protein was separable from galaptin by polyacrylamide gel electrophoresis and by anion-exchange chromatography. In contrast, immunoprecipitation experiments confirmed that galaptin was synthesized by the B lymphoblastoid cells. cDNA corresponding to the B lymphoblastoid cell mRNA encoding galaptin was amplified by the polymerase chain reaction. The amplified product was partially sequenced, and 299 nucleotides were identified. The derived amino acids corresponded to residues 6-65, 84-114, and 118-126 found to be present in human splenic galaptin. Immunohistochemical analyses revealed that galaptin was distributed throughout the cytoplasm of B lymphoblastoid cells rather than being localized to the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Expression of egasyn-esterase in mammalian cells. Sequestration in the endoplasmic reticulum and complexation with beta-glucuronidase.

Mouse egasyn cDNA was inserted into expression vector pCDpoly and transfected into mammalian cell lines. Transfected human HepG2 cells, monkey COS-1 cells, and mouse L cells expressed egasyn-esterase catalytic activity. Within COS-1 cells, egasyn was localized to the endoplasmic reticulum. Although individual cells produced large amounts of egasyn, no secretion was observed. No beta-glucuronidase-egasyn complexes were formed in transfected HepG2 or COS-1 cells. However, these complexes were readily detected in transfected L cells. Although the signal for retention of egasyn in the endoplasmic reticulum appears to be species independent, the signal for association with beta-glucuronidase is species restricted.

Animals↗
PubMed1991

Characterization and functional expression of a cDNA encoding egasyn (esterase-22): the endoplasmic reticulum-targeting protein of beta-glucuronidase.

Egasyn (esterase-22), a member of the nonspecific carboxylesterase multigene family (E.C. 3.1.1.1), is the endoplasmic reticulum (ER)-targeting protein of beta-glucuronidase. We utilized the polymerase chain reaction (PCR) in the eventual isolation of murine egasyn cDNAs. PCR primers were based upon: (1) partial amino acid sequences derived from egasyn peptides and (2) a conserved active site region shared by carboxylesterases. The amino acid sequence deduced from the PCR product matched that obtained from egasyn protein. This product was utilized as a probe to screen a cDNA library. Two cDNAs whose composite sequence encoded an open reading frame of 562 amino acids were isolated. A message size of 1700-2000 bp was revealed by RNA blot hybridization analysis. S1 nuclease protection analyses detected mRNA in liver, kidney, lung, and submandibular gland, but not in spleen, brain, and testes. Genetic mapping confirmed the location of an egasyn cDNA fragment in cluster 1 of the esterase region on chromosome 8. Transfection of COS cells with the 2022-bp cDNA resulted in the expression of esterase activity, which comigrated on native gels with liver esterase-22. The features of the deduced amino acid sequence of the egasyn cDNA are compared with previously characterized carboxylesterases and with other lumenal ER proteins.

Amino Acid Sequence↗
PubMed1991

Sandy: a new mouse model for platelet storage pool deficiency.

Sandy (sdy) is a mouse mutant with diluted pigmentation which recently arose in the DBA/2J strain. Genetic tests indicate it is caused by an autosomal recessive mutation on mouse Chromosome 13 near the cr and Xt genetic loci. This mutation is different genetically and hematologically from previously described mouse pigment mutations with storage pool deficiency (SPD). The sandy mutant has diluted pigmentation in both eyes and fur, is fully viable and has prolonged bleeding times. Platelet serotonin levels are extremely low although ATP dependent acidification activity of platelet organelles appears normal. Also, platelet dense granules are extremely reduced in number when analysed by electron microscopy of unfixed platelets. Platelets have abnormal uptake and flashing of the fluorescent dye mepacrine. Secretion of lysosomal enzymes from kidney and from thrombin-stimulated platelets is depressed 2- and 3-fold, and ceroid pigment is present in kidney. Sandy platelets have a reduced rate of aggregation induced by collagen. The sandy mutant has an unusually severe dense granule defect and thus may be an appropriate model for cases of human Hermansky-Pudlak syndrome with similarly extreme types of SPD. It represents the tenth example of a mouse mutant with simultaneous defects in melanosomes, lysosomes and/or platelet dense granules.

Animals↗
PubMed1990

The RIIIS/J inbred mouse strain as a model for von Willebrand disease.

Mice of the RIIIS/J inbred strain have prolonged bleeding times (greater than 15 minutes) after experimental injury when compared with normal C57BL/6J mice (1.8 minutes) and other strains of mice. The prolonged bleeding time was accompanied by normal platelet counts. Platelet aggregation with collagen and agglutination with ristocetin were not significantly altered in RIIIS/J mice. Also, platelets from RIIIS/J mice had normal serotonin content and normal numbers of dense granules by electron microscopy. Thus, the bleeding abnormality is not due to platelet storage pool deficiency as has been found in several other mouse mutants. The activated partial thromboplastin time (APTT) which plasma from RIIIS/J mice was prolonged compared with normal mice, and factor VIII:C activity and von Willebrand antigen levels were one half to one third that of normal mouse plasma. Factor XI activity was also significantly deficient (levels at 42% to 64% of normal). Plasma of RIIIS/J mice contained the full complement of multimers of von Willebrand factor, although each multimer was lower in concentration compared with that in normal mice. Platelet alpha-granule von Willebrand antigen levels were similar to those of normal mice. The prolonged bleeding time of RIIIS/J mice was corrected by treatment with desmopressin. Heterozygous C57BL/6J x RIIIS/J F1 animals had low plasma von Willebrand antigen levels like the RIIIS/J parent and had variable bleeding times. Inheritance of the bleeding tendency was as an incomplete dominant, autosomal trait. These data indicate the RIIIS/J strain is a suitable animal model for type IA von Willebrand disease.

Animals↗
PubMed1990

Functional status after coronary artery bypass grafting and percutaneous transluminal coronary angioplasty.

Two cohorts of consecutive patients of comparable age with similar preprocedure cardiac function who underwent either coronary artery bypass grafting (CABG; n = 106) or percutaneous transluminal coronary angioplasty (PTCA; n = 64) were entered into a prospective comparison study examining functional status and return to work during the first year of recovery. Patients were evaluated using standardized functional status instruments for activities of daily living, work performance, social activity, mental health and quality of social interaction at 1, 6 and 12 months after the procedure. Within the CABG group, statistically significant improvements of functional status on every subscale were noted over the 1-year follow-up. Patients undergoing PTCA demonstrated significant improvement in all dimensions except for the quality of interaction at 1 year as compared with baseline. When the 2 groups were compared, the PTCA group demonstrated greater participation than the CABG group in routine daily physical and social activities at 1 and 6 months, but this apparent advantage disappeared by 1 year. Measures of psychological functioning were better after CABG than after PTCA. A reduction in the number of those with employment occurred in both the CABG and PTCA groups, independent of physical functional status measures, which improved in both groups after the procedures. For those with employment, the CABG group reported the greatest improvement in work performance.

Activities of Daily Living↗
PubMed1990

Ending smoking at the Johns Hopkins Medical Institutions. An evaluation of smoking prevalence and indoor air pollution.

An empiric evaluation of a policy ending smoking in a large urban medical center was conducted. The study included a prospective cohort tracking of employees to measure changes in smoking behavior, environmental fires, smoking-related litter, and environmental tobacco pollution exposure. A 25% decrease in employee smoking prevalence was found (21.7% vs 16.2% before vs after policy implementation, respectively). The daily number of cigarettes reportedly smoked by employees who continued smoking and the total number smoked at work decreased across all occupational categories by an average of 25%. Significant reductions were noted in the level of public smoking and the amount of cigarette remnants. Nicotine vapor concentrations decreased significantly in all areas except restrooms. These findings suggest that visible smoking and environmental tobacco smoke exposure can be markedly decreased by instituting a policy eliminating smoking in a large medical center.

Air Pollutants, Occupational↗
PubMed1990

The propeptide of beta-glucuronidase. Further evidence of its involvement in compartmentalization of beta-glucuronidase and sequence similarity with portions of the reactive site region of the serpin superfamily.

A significant portion of murine hepatocyte beta-glucuronidase is maintained within the endoplasmic reticulum (ER) by complex formation with the esterase active site of the protein egasyn. The carboxyl-terminal propeptide of the precursor form of glucuronidase appears important in localization of glucuronidase to the ER since a naturally occurring mutation in it is associated with decreased levels of ER glucuronidase. A sequence similarity was noted between the carboxyl-terminal propeptide and portions of the conserved sequences of the reactive site region of members of the serpin (serine proteinase inhibitor) superfamily. Also, previous studies had shown that a synthetic peptide, corresponding to the propeptide region, was a specific and potent inhibitor of the esterase activity of purified egasyn. Taken together, these results suggest that (a) the egasyn-glucuronidase system may use a novel mechanism related to that of serine proteinases and their inhibitors in complex formation and in subsequent localization of glucuronidase within the ER and that (b) a possible function of ER glucuronidase is to modulate the esterase activity of egasyn.

Amino Acid Sequence↗
PubMed1990

von Willebrand's variant (type II Buffalo). Thrombocytopenia after desmopressin but absence of in vitro hypersensitivity to ristocetin.

Von Willebrand's disease is categorized into types and subtypes based on multimeric analysis of plasma von Willebrand's factor. Such categorization is of value because both the mode of inheritance and the choice of therapeutic material differ between subtypes. The Type IIB variant is characterized by hypersensitivity in vitro to ristocetin and thrombocytopenia after administration of desmopressin (DDAVP). Hypersensitivity to ristocetin has also been described in Type I variants but without thrombocytopenia after DDAVP. This report describes a new Type II variant characterized by the converse situation, absence of hypersensitivity to ristocetin in vitro but transient thrombocytopenia after intravenous administration of DDAVP.

Deamino Arginine Vasopressin↗
PubMed1990

Effects of mixed chimeric bone marrow repopulation on platelet storage pool-associated bleeding defects in mouse mutants.

We have previously shown mouse platelet storage pool deficiency (SPD) to be associated with lesions at eight different genetic loci, each of which is sufficient to produce murine SPD. We have also shown that normal bleeding times and normal platelet functions are restored when mice with SPD are transplanted with marrow from normal mice. Conversely, when normal mice are transplanted with mutant marrow, they present symptoms of SPD. In order to determine the amount of normal platelets needed to prevent the prolonged bleeding times associated with SPD, we established stable mixed chimeric mice by transplanting various ratios of normal and mutant marrow into lethally irradiated host animals. The proportion of normal input marrow correlated well with the proportion of normal peripheral red blood cells and platelets determined in chimerae 100 days after transplantation using direct morphology and electrophoretic variants of glucose phosphate isomerase to identify normal and mutant cell populations. The proportions of normal input marrow were also reflected in the proportions of platelets with normal and mutant platelet morphology in the chimerae. This confirms that the platelet abnormality in SPD is intrinsic to the stem cell population from which the platelets are derived. When bleeding times were determined in the mixed chimeric mice, a surprisingly high percentage of normal platelets (greater than 50% and sometimes greater than 75%) were needed to stop bleeding. These results suggest that the mutant platelets in the mixed chimeric mice may interfere with normal platelet aggregation patterns. They also raise some important considerations in devising treatment for SPD. Bleeding episodes in human SPD are normally treated by platelet transfusion. The results suggest that, at least in some cases, transfusions may not be effective. Also, in future gene therapy of this disease, it is like that a functional gene will have to be present in greater than 50% of stem cells for therapy to be effective.

Animals↗
PubMed1990

Factors related to functional status after coronary artery bypass surgery.

We studied physical functioning and social and leisure functioning of 125 men before, 1 month after, and 6 months after coronary artery bypass surgery. Relationships between functional status outcomes and selected psychosocial and physical variables were examined. Although functional status outcomes improved significantly 6 months after surgery, 13% of patients continued to report important functional status disabilities, and 45% reported difficulty in or no participation in moderately vigorous activities. The variable most predictive of functional status was the patient's estimation a priori of his ability to carry out specific activities (self-efficacy). Other psychosocial and physical factors, other than postoperative treadmill performance, did not add significantly to the prediction of the outcomes. Findings suggest that rehabilitative programs attempting to facilitate physical and social reintegration of the patient after coronary artery bypass surgery should test the effects of interventions to increase self-efficacy.

Activities of Daily Living↗
PubMed1989

Involvement of the carboxyl-terminal propeptide of beta-glucuronidase in its compartmentalization within the endoplasmic reticulum as determined by a synthetic peptide approach.

The proenzyme form of beta-glucuronidase is compartmentalized in large quantities within the endoplasmic reticulum by binding to the esterase, egasyn. Also, the propeptide of the proenzyme form of beta-glucuronidase is likely located at the carboxyl terminus. We have, therefore, tested if this carboxyl-terminal peptide is important in binding to egasyn. A polyclonal antibody to a 30-mer synthetic peptide, corresponding to the carboxyl-terminal 30 amino acids of pro-beta-glucuronidase, provided evidence that egasyn binds to the carboxyl terminus of beta-glucuronidase. This antibody interacted with proenzyme beta-glucuronidase-egasyn complexes in which one, two, or three egasyn molecules were bound to the beta-glucuronidase tetramer, but not with those complexes (M4) which contained four egasyn molecules. We interpret these results as indicating that all available carboxyl termini of the beta-glucuronidase proenzyme tetramer are shielded by egasyn in the M4 complexes. The same antibody did not recognize the mature lysosomal form of beta-glucuronidase, indicating that only the proenzyme form of microsomal beta-glucuronidase contains the original carboxyl terminus. Also, the synthetic 30-mer was found to be a specific and potent inhibitor (50% inhibition at 1.3 microM) of the esterase activity of purified egasyn but exhibited little inhibitory activity toward other purified esterases including a rat trifluoroacetylated esterase or egasyn esterase from another species. Together, these data describe a potent interaction of the exposed carboxyl terminus of precursor glucuronidase with the esterase catalytic site of egasyn, which in turn results in the specific localization of glucuronidase within the lumen of the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1989

Inhibitors of elastase and cathepsin G in Chédiak-Higashi (beige) neutrophils.

Previous studies have established that mature neutrophils from the peritoneal cavity, blood, and bone marrow of beige (Chédiak-Higashi syndrome) mice essentially lack activities of two lysosomal proteinases: elastase and cathepsin G. There are, however, significant levels of each enzyme in early neutrophil precursors in bone marrow. In the present experiments, it was found that the addition of extracts from mature beige neutrophils to extracts of normal neutrophils or to purified human neutrophil elastase and cathepsin G resulted in a significant inhibition of elastase and cathepsin G G activities. 125I-Labeled human neutrophil elastase formed high molecular mass complexes at 64 and 52 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis when added to beige neutrophil extracts. The molecular masses of the inhibitor-125I-elastase complexes suggested that the molecular masses of the inhibitors are approximately 36 and 24 kDa, respectively. These results were confirmed by gel filtration on Superose 12 under nondenaturing conditions. Cathepsin G was inhibited only by the 36-kDa component. The inhibitors formed a covalent complex with the active sites of elastase and cathepsin G. No inhibitory activity was present in mature neutrophil extracts of genetically normal mice or in extracts of bone marrow of beige mice. These results thus represent an unusual example of an enzyme deficiency state caused by the presence of excess inhibitors. Inactivation of neutrophil elastase and cathepsin G in mature circulating and tissue neutrophils may contribute to the increased susceptibility of Chédiak-Higashi patients to infection.

Animals↗
PubMed1988

Synthesis and processing of lysosomal enzymes of mouse kidney after treatment with [3H]fucose.

[3H]Fucose, intravenously injected into androgen-treated mice, was incorporated at high rates into the immunopurified kidney lysosomal enzymes beta-glucuronidase and beta-galactosidase. Initially, label was incorporated into high molecular weight proenzyme forms. Processing of fucose-labeled proenzyme forms to lower molecular weight mature forms was very rapid, being detectable at 30 min, and complete by 90 min, compared with the several hours required for processing of lysosomal enzymes labeled with amino acids. This result is consistent with addition of fucose residues within the Golgi apparatus just before transfer of lysosomal proenzyme forms to the lysosome where maturation is thought to occur. The combination of the high rates of incorporation of [3H]fucose and the known metabolic stability of this precursor sugar suggests that the mouse kidney system is advantageous for studies of the synthesis, processing, and degradation of fucose-containing complex oligosaccharides of lysosomal enzymes and, by extension, of other kidney glycoproteins.

Animals↗