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Biomedical subjects

W Busby

Publications and source records attributed to W Busby.

8 recordsLinked to original sources
PubMed2001

Emulsion-derived foams (PolyHIPEs) containing poly(epsilon-caprolactone) as matrixes for tissue engineering.

The preparation of PolyHIPE foams containing poly(epsilon-caprolactone) from macromonomers by free radical homo- or copolymerization is described. The macromonomers are synthesized from PCL diols and are polymerized in the continuous phase of high internal phase emulsions (HIPEs). Subsequent drying yields low-density foams with cell diameters of 5-100 microm. Foam morphology, as determined by scanning electron microscopy, depends on the type of diluent (styrene, methyl methacrylate, or toluene) added to the emulsion organic phase and on the PCL content. Increasing the latter increases the continuous phase viscosity to a point where emulsion formation is impeded. Foam swelling in toluene, 2-propanol, and water was investigated by solvent imbibition and increased with increasing solvent hydrophobicity. Furthermore, it was found generally to decrease with increasing PCL content, due to increasing cross-link density. Swelling generally increased when higher molar mass PCL macromonomer was used due to the formation of a less tightly cross-linked network. One type of foam sample was shown to support the growth of human fibroblasts over a period of 2.5 days.

Bioartificial Organs↗
PubMed1997

Insulin-like growth factor binding protein-5 binds to plasminogen activator inhibitor-I.

Insulin-like growth factor binding protein-5 (IGFBP-5) has been shown to bind to the extracellular matrix (ECM) of both fibroblasts and smooth muscle cells. The ECM-IGFBP-5 interaction is mediated in part by binding to heparan sulfate containing proteoglycans. Because proteoglycans may not be the only components of ECM that bind to IGFBP-5, we have determined its ability to bind to other ECM proteins. When a partially purified mixture of the proteins that were present in fibroblast conditioned medium was purified by IGFBP-5 affinity chromatography, a 55-kDa protein was eluted. Amino acid sequencing of the amino terminal 28 amino acids showed that it was human plasminogen activator inhibitor-1 (PAI-1). To determine if this interaction was specific, purified human PAI-1 was incubated with IGFBP-5 and the IGFBP-5/PAI-1 complex immunoprecipitated with anti-PAI-1 antiserum. When the precipitate was analyzed by immunoblotting using anti-IGFBP-5 antiserum, the intensity of the IGFBP-5 band was substantially increased compared with controls that did not contain human PAI-1. A synthetic IGFBP-5 peptide that contained the amino acid sequence between positions 201 and 218 inhibited IGFBP-5/PAI-1 interaction. Coincubation of IGFBP-5 mutants that contained substitutions for specific basic residues located between positions 201 and 218 with PAI-1 indicated that some of these amino acids were important for binding. Two mutants that contained neutral substitutions for specific basic amino acids within the glycosaminoglycan binding domain had reduced binding to PAI-1. In contrast, three other mutants that also had substitutions for charged residues in the same region had no reduction in binding. Heparin and heparan sulfate inhibited the IGFBP-5/PAI-1 interaction; however, several other glycosaminoglycans had no effect. PAI-1 was determined to be an important ECM component for binding because approximately 27% of total ECM binding could be inhibited with anti-PAI-1 antiserum. Competitive binding studies with unlabeled IGFBP-5 showed that the dissociation constant of PAI-1 for IGFBP-5 was 9.1 x 10(-8) M. In summary, IGFBP-5 binds specifically to plasminogen activator inhibitor-1. Because this is present in the extracellular matrix of several cell types, it may be one of the important binding components of ECM. PAI-1 binding partially protects IGFBP-5 from proteolysis, suggesting that it is one of the ECM components that is involved in mediating this effect.

Animals↗
PubMed1996

Substitution of specific amino acids in insulin-like growth factor (IGF) binding protein 5 alters heparin binding and its change in affinity for IGF-I response to heparin.

Heparin binding to insulin-like growth factor (IGF)-binding protein 5 (IGFBP-5) leads to a 17-fold decrease in its affinity for IGF-I, and a region that contains several basic amino acids (Arg201-Arg218) may be involved in this affinity shift. In the present study, mutagenesis was used to analyze the effect of substitutions for basic amino acids in the Arg201-Arg218 region of IGFBP-5 on heparin-binding and the heparin-induced affinity shift. Nine mutant forms were prepared. Their association constants (Ka) for IGF-I were similar to native IGFBP-5. When 10 microg/ml of heparin was added, the Ka of native IGFBP-5 decreased 17-fold, and the Ka of the K134A/R136A mutant decreased 16-fold. In contrast, substitutions for specific basic amino acids in the Arg2O1-Arg218 region decrease the affinity shift to 1.1-3.2-fold. Lys 211 was especially important. When a mutant containing that single substitution was tested, heparin caused only a 2.5-fold reduction in IGF-I affinity. Affinity cross-linking studies showed that heparin was equipotent in inhibiting the formation of 125I-IGF-I-K134A/Rl36A mutant complexes compared to native IGFBP-5. In contrast, heparin had minimal effects on the formation of complexes between 125I-IGF-I and the other mutants. The heparin-binding activity of each mutant was determined. Four mutants, R201A/K202N, K202A/K206A/R207A, R201A/K202N/K206N/K208N, and K211N/R214A/K217A/R218A, had reduced heparin binding compared to native IGFBP-5. The other five mutants, including the K21IN mutant, showed no change in heparin binding. The four mutants with reduced heparin binding could be dissociated from heparin-Sepharose with much lower NaCl concentrations, indicating that they had reduced affinity. These findings suggest that Arg201 Lys202, LysS206, and Arg214 are important for heparin binding. In contrast, LyS211 is not important for the binding of IGFBP-5 to heparin, but substitution for it reduced the heparin-induced affinity shift.

Affinity Labels↗
PubMed1996

Binding of insulin-like growth factor (IGF) I or II to IGF-binding protein-2 enables it to bind to heparin and extracellular matrix.

Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) is secreted by several cell types that also secrete IGF-I or IGF-II. The binding of IGF-I or IGF-II to IGFBP-2 has been shown to alter their actions. Although IGFBP-2 is not an abundant component of the extracellular matrix (ECM), it is easily localized by immunohistochemical staining in basement membranes of several epithelial cell types. We have previously shown that IGFBP-5 associates with glycosaminoglycans and binds to proteoglycans that are contained in ECM and basement membranes. In those studies we were unable to demonstrate an association of IGFBP-2 with glycosaminoglycans. In this study we report that IGFBP-2 binds to heparin if IGF-I or IGF-II is also included in the incubation buffer. IGFBP-1, -3, -4, or -5 did not have increased heparin binding in the presence of IGF-I or IGF-II. The binding of IGFBP-2 to heparin was detectable using an IGF-I to IGFBP-2 molar ratio of 2:1. Binding to heparin-Sepharose could be inhibited by soluble heparin or heparan sulfate, but not by glycosaminoglycans that do not contain O-linked sulfate groups in the 2 or 3 position of the iduronic acid ring. Binding was also inhibited by a synthetic IGFBP-5 peptide that contained a heparin-binding domain, but not by a peptide with an identical charge to mass ratio that does not bind to heparin. Binding appeared to be physiologically significant, as IGFBP-2 bound to human fibroblast ECM if IGF-I or IGF-II was added. IGF-II was more potent than IGF-I in facilitating the binding interaction, and des(1-3)-IGF-I or insulin had no effect, suggesting that IGF binding to IGFBP-2 is required for this effect to be detected. In summary, IGFBP-2 binding to glycosaminoglycans is dependent upon binding of IGF-I and IGF-II to IGFBP-2. This suggests that IGFBP-2 undergoes a conformational change that exposes a glycosaminoglycan-binding domain. This could provide a mechanism for focally concentrating IGFBP-2 in the pericellular environment.

Amino Acid Sequence↗
PubMed1994

Heparin, heparan sulfate, and dermatan sulfate regulate formation of the insulin-like growth factor-I and insulin-like growth factor-binding protein complexes.

The mechanisms by which insulin-like growth factor-I (IGF-I) is released from insulin-like growth factor binding proteins (IGFBPs) and then binds to its receptor have not been defined. This study was designed to determine the role of glycosaminoglycans in altering the formation of the IGF-I.IGFBP complexes. Heparin inhibited formation of the IGF-I.IGFBP-5 complex and also separated preformed IGF-I.IGFBP-5 complexes. Heparin also inhibited formation of the IGF-I.IGFBP-3 complex; however, it did not inhibit formation of complexes between IGF-I and IGFBP-1, -2, or -4. Heparin exposure was associated with a 17-fold decrease in the affinity of IGFBP-5 for IGF-I. A synthetic peptide that contains residues from Arg221 to Arg238 of IGFBP-5, and a heparin binding domain prevented the inhibitory effects of heparin on formation of the IGF-I.IGFBP-5 complex. It did not directly compete with IGF-I for binding to IGFBP-5, suggesting that heparin binding to this region of IGFBP-5 resulted in a conformational change in IGFBP-5 which lowered its affinity for IGF-I. Other glycosaminoglycans that contained O-linked sulfates in the 2 or 3 carbon positions of iduronic acid, e.g. heparan sulfate and dermatan sulfate, also inhibited the IGF-I.IGFBP-5 complex formation, whereas those that did not, such as keratan sulfate or hyaluronic acid, had minimal effects. Anionic polysaccharides that contained O-sulfate groups in the 2 or 3 positions, such as dextran sulfate, pentosan polysulfate, and fucoidan, also had inhibitory activity. The findings suggest a role for these compounds in inhibiting IGF-I.IGFBP interactions, thus making IGF-I available to bind to its receptor.

Amino Acid Sequence↗
PubMed1989

Comparison of intraosseous, intramuscular, and intravenous administration of succinylcholine.

The intraosseous route of vascular access has been popularized recently for resuscitation of children. Intraosseous succinylcholine use has been anecdotally reported for airway management. This drug could be utilized for control of the airway in pediatric burns, status epilepticus, or combative head-injured patients. No series quantifying the efficacy of the intraosseous route has been reported for succinylcholine. To accomplish this goal, six sheep serving as their own controls were anesthetized with halothane and subsequently intubated. Succinylcholine (1 mg/kg) was given. The time to respiratory arrest and 100% loss of the forefoot twitch upon stimulation of the anterior tibial nerve were noted. Each animal was successively studied using the intravenous (IV), intraosseous (IO), and intramuscular (IM) routes of administration, with a minimum of seven days separating trials. The average time from administration to respiratory arrest in seconds was 30.8 +/- 7.3 (IV), 57.5 +/- 10.3 (IO), and 230 +/- 106 (IM). The average time from administration to 100% loss of forefoot twitch in seconds was 93.3 +/- 34.0 (IV), 100.8 +/- 24.2 (IO), and 291 +/- 109 (IM). All groups were statistically significantly different using the t-test for the difference of means, with a p value less than 0.0015. We conclude that the intraosseous route of administration of succinylcholine in this series of sheep is comparable to the intravenous route and superior to the intramuscular route.

Animals↗
PubMed1988

Pulmonary embolism in the elderly.

A retrospective study was made of a consecutive series of patients over 65 years of age diagnosed as having a pulmonary embolus by either ventilation-perfusion lung scan or at necropsy. No clinical features were particularly helpful in making the diagnosis though signs of deep-vein thrombosis were present in 35%. Anticoagulants were well tolerated by all patients to whom they were prescribed. Postmortem findings showed a decreasing frequency of pulmonary emboli in the elderly which may indicate a reduction in predisposing factors.

Aged↗