Biomedical subjects

T Boone

Publications and source records attributed to T Boone.

At least 55 records · Page 3Linked to original sources

PubMed1993

Comparison of the biophysical characteristics of human brain-derived neurotrophic factor, neurotrophin-3, and nerve growth factor.

The structural properties of human brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were studied using sedimentation equilibrium and circular dichroism (CD), fluorescence and Fourier-transform infrared spectroscopies, and compared with those of human nerve growth factor (NGF). Both the far UV CD and infrared spectra indicate that these three proteins have similar, but not identical, secondary structures which contain primarily beta-sheet and irregular structures. NGF appears to contain the most beta-sheet while NT-3 contains a small fraction of alpha-helix. The near UV CD spectra appear to indicate that the three proteins contain disulfide bonds in similar environments, suggesting a resemblance in tertiary structure. The fluorescent tryptophans found in the molecules are relatively solvent exposed, while Trp102 found only in NT-3 is possibly quenched. The fluorescent Trp(s) in NGF are significantly quenched relative to those in the other two neurotrophic factors. Both NT-3 and BDNF have very hydrophilic surfaces at neutral pH, as indicated by a low binding affinity to a hydrophobic probe, anilinonaphthalenesulfonate. Sedimentation equilibrium showed that BDNF, NT-3, and NGF exist as strongly associated dimers in phosphate-buffered saline, pH 7.1. Fits of the observed fringe displacements to various association models suggested that the BDNF, NT-3, and NGF samples contain, in addition to the principal dimeric species, some oligomers, and that NT-3 contains a small fraction of incompetent monomer.

Amino Acid Sequence↗

PubMed1993

Sites of iodination in recombinant human brain-derived neurotrophic factor and its effect on neurotrophic activity.

Recombinant human brain-derived neurotrophic factor (BDNF) is now under extensive investigation because of its potential clinical applications. Radioactively labeled proteins are usually required to study receptor binding and pharmacokinetic properties of proteins. This study was undertaken to see if iodination affects the biological and conformational properties of a recombinant BDNF. BDNF was iodinated using a stoichiometric amount of nonradioactive cold NaI to minimize multiple iodinations. Of the four tyrosines present in BDNF--Tyr-52, Tyr-54, Tyr-63, and Tyr-86--only Tyr-63 and Tyr-86 were iodinated under the experimental conditions used. Iodination of Tyr-63 resulted in modification without alteration of the biological activity, whereas iodination of Tyr-86 resulted in a molecule with highly compromised biological activity. Similar inactivation was observed if both Tyr-63 and Tyr-86 were iodinated. These modified proteins exhibited conformation and dimerization apparently identical to those of the native protein, as demonstrated by analytical ultracentrifugation, gel filtration, light scattering, and circular dichroism. From these results, we concluded that Tyr-52 and Tyr-54 are not accessible to the reagent and are probably buried in the hydrophobic core, whereas Tyr-63 and Tyr-86 are exposed on the surface of the molecule; of the two exposed residues, only Tyr-86 contributes to the biological activity.

Amino Acid Sequence↗

PubMed1993

Effects of qigong on cardiorespiratory changes: a preliminary study.

Qigong, a special form of breathing exercise, was investigated to examine its effect on cardiorespiratory changes. Ten volunteers (five males and five females) participated in a 20-minute group instructional session for 10 consecutive days before testing of its treatment effects. The testing protocol followed a C1-T-C2 design, where C1, T, and C2 represented the first, treatment, and second control period, respectively. Each period consisted of a 5-minute interval, and thus each testing session consisted of 15 minutes. The results indicated there were no statistically significant differences (p > 0.05) in heart rate or tidal volume for the three 5-minute periods. There was a significant decrease (p < 0.05) in respiratory exchange ratio between T and C2. A significant increase in ventilatory efficiency for carbon dioxide production was found between C1 and T. Statistically significant differences (p < 0.05) were found in the volume of oxygen consumed and carbon dioxide produced, frequency of breath, expired ventilation, and ventilatory efficiency for oxygen produced between the T and the two control periods. This preliminary study of Qigong demonstrates that the subjects were able to learn the technique in a short period of time. The data also suggest that, with an improvement of nearly 20% in ventilatory efficiency for oxygen uptake and carbon dioxide production, this technique may have useful therapeutic value.

Adult↗

PubMed1992

Role of glycosylation on the secretion and biological activity of erythropoietin.

The erythropoietin (EPO) molecule contains four carbohydrate chains. Three contain N-linkages to asparagines at positions 24, 38, and 83, and one contains an O-linkage to a serine at position 126. We constructed human EPO variants that eliminated the three N-glycosylation sites by replacing the asparagines with glutamines singly or in combination. The O-linked carbohydrate chain was removed by replacing the serine with glutamine, valine, histidine, or alanine. A variant with a double mutation (Gln38,83) and another with a triple mutation (Gln24,38,83) were secreted poorly from COS1 and CHO cells even though RNA encoding these variants was present. All other variants with mutations in N-linked glycosylation sites were secreted normally. Removal of any of the N-glycosylation sites reduced the in vivo but not the in vitro biological activity of the EPO molecule. All the mutations at Ser126, the O-glycosylation site, were secreted normally. In vitro activity was also unaffected except for Ala126 which had a 50-fold decrease. The Val126 variant was tested in vivo, and its specific activity was only slightly less than that of the native EPO, which indicates that the O-linked carbohydrate is not essential for activity.

Animals↗

PubMed1992

Effect of endotoxin on serum granulocyte and granulocyte-macrophage colony-stimulating factor levels in dogs.

The biologic effects of endotoxin are attributed to the release of several cytokines, including interleukin-1, interleukin-6, tumor necrosis factor, and the colony-stimulating factors. To investigate the mechanism of endotoxin-induced neutrophilia in dogs, several cell lines known to proliferate selectively in response to recombinant human colony-stimulating factors were examined to determine their responses to recombinant canine granulocyte colony-stimulating factor (rcG-CSF) or recombinant canine granulocyte-macrophage colony-stimulating factor (rcGM-CSF). The murine cell line NFS-60 was found to respond well to rcG-CSF and the human cell line TALL-101 to rcGM-CSF, and these responses were neutralized by antibodies to these recombinant proteins. These bioassays were then used to determine G-CSF and GM-CSF levels in dogs after intravenous endotoxin administration. G-CSF levels increased by 2 h, peaked at 4 h, and had not returned to normal by 24 h after endotoxin. In contrast, GM-CSF was not detectible before or after endotoxin administration.

Animals↗

PubMed1992

Hematologic and bone marrow changes after short- and long-term administration of two recombinant bovine granulocyte colony-stimulating factors.

Colony-stimulating factors are a category of glycoproteins that are instrumental in the regulation of hematopoiesis and inflammation. This investigation documented the clinical bone marrow and peripheral blood responses to short-term and long-term administration of a recombinant bovine granulocyte colony-stimulating factor (rb-GCSF) and an analog, where the cysteine at position 17 was substituted with a serine (rb-GCSF ser17). The colony-stimulating factors produced the expected changes in the hematologic findings of the bovine subjects in the study, and there was a cell-specific response to the compounds. The sustained neutrophilia in the long-term study indicates that the bovine species can tolerate the administration of recombinant forms of bovine GCSF for extended periods of time without detectable adverse side effects. The neutrophils from the short-term study revealed no apparent fluctuation, either as enhanced or reduced capability to reduce nitro blue tetrazolium as compared to pretreatment neutrophils. The administration of both recombinant forms of GCSF produced large increases in the bone marrow myeloid:erythroid (M:E) ratio concomitantly with the neutrophilias. This is the first preliminary report documenting the bone marrow response of cattle to the native and recombinant (rb-GCSF ser17) forms of bovine GCSF.

Animals↗

PubMed1991

Novel fold and putative receptor binding site of granulocyte-macrophage colony-stimulating factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the development of and the cytotoxic activity of white blood cells. Recombinant human GM-CSF has proven useful in the treatment of blood disorders. The structure of GM-CSF, which was determined at 2.4 angstrom resolution by x-ray crystallography, has a novel fold combining a two-stranded antiparallel beta sheet with an open bundle of four alpha helices. Residues implicated in receptor recognition, which are distant in the primary sequence, are on adjacent alpha helices in the folded protein. A working model for the receptor binding site is presented.

Amino Acid Sequence↗

PubMed1991

The effect of carbohydrate on the structure and stability of erythropoietin.

Erythropoietin is a glycoprotein hormone that stimulates the maturation of late erythroid progenitor cells. It has three N-linked and one O-linked carbohydrates which play an important role in the biosynthesis and biological activities of the protein. To determine the role the carbohydrate might have in maintaining the conformational stability of the protein, the protein expressed in mammalian cells (fully glycosylated), the asialo mammalian-expressed protein, and the protein expressed in Escherichia coli (no carbohydrate) were compared for their stability to guanidine HCl, pH, and temperature. Circular dichroism was used to follow protein unfolding. Both the intact and asialo mammalian-expressed proteins unfolded with a cooperative transition in guanidine HCl, with a midpoint at 1.75 M guanidine HCl. The E. coli-expressed material unfolded with a midpoint of 1.2 M guanidine HCl, and a delta G of unfolding which was 1.4 kcal/mol less than that of the two glycosylated molecules. The E. coli-derived protein was also significantly less stable to pH-induced conformational changes, showing a cooperative transition in 35% glycerol with a midpoint at pH 4.4, while both the intact and asialo mammalian-expressed molecules had a transition midpoint of pH 3.75 in the absence of glycerol, and approximately pH 3 in the presence of 35% glycerol. The E. coli-expressed molecule unfolded and precipitated upon heating to 44 degrees C, while the asialo and intact mammalian-expressed proteins remained soluble, with a Tm of 56 degrees C. From these experiments, the carbohydrate appears to play a critical role in stabilizing the erythropoietin molecule to denaturing conditions, and this increased stability does not depend on the presence of sialic acid.

Animals↗

PubMed1991

Low-resolution structure of recombinant human granulocyte-macrophage colony stimulating factor.

A recombinant form of human granulocyte-macrophage colony stimulating factor (GM-CSF) which contains no carbohydrate has been crystallized. Multiple isomorphous replacement analysis using five heavy-atom derivatives has yielded an image of the structure at 6 A resolution that showed two molecules per asymmetric unit and allowed determination of the non-crystallographic symmetry transformation. The 6 A resolution result shows that the core of GM-CSF consists of four helices. The angles at which the helices pack together distinguishes this structure from known antiparallel four-helix bundle proteins. Consideration of the amino acid sequence properties and previous structural characterizations of GM-CSF leads to an assignment of the probable protein segments that form the helices.

Granulocyte-Macrophage Colony-Stimulating Factor↗

PubMed1991

Molecular cloning and in vivo evaluation of canine granulocyte-macrophage colony-stimulating factor.

Canine granulocyte-macrophage colony-stimulating factor (caGM-CSF) was cloned and expressed to allow further investigation of GM-CSF in a large animal model. The cDNA is 850 base pairs (bp) long and encodes a peptide of 144 amino acids. The nucleotide and amino acid sequence homology between caGM-CSF and human GM-CSF (hGM-CSF) is 80% and 70%, respectively. A mammalian expression vector pCMV/CAGM was constructed and used to transfect COS cells for expression of caGM-CSF. Supernatant from transfected COS cells enriched with caGM-CSF was shown to have significant stimulating activity in granulocyte-macrophage colony forming unit (CFU-GM) assays of canine marrow. caGM-CSF, expressed from bacteria, was used to treat seven dogs at varying doses twice daily subcutaneously (sc) for 14 to 16 days. Circulating blood neutrophils and monocytes increased significantly. The increase in circulating eosinophils was variable. Thrombocytopenia developed during administration of caGM-CSF but corrected rapidly after cessation of treatment. Evaluation of survival times of 51Cr-labeled autologous platelets suggested increased consumption as the primary reason for thrombocytopenia. A species-specific GM-CSF will be a useful tool for hematologic or immunologic studies in dogs.

Amino Acid Sequence↗

PubMed1991

Effects of venous return on respiratory response.

This study reevaluated the link between the rate of CO2 flow to the pulmonary circulation and changes in cardiac output to the respiratory responses following leg lifting in normal man subjects. Thirty healthy subjects (mean age = 22.2 years) were subjected to the experimental maneuver which increased venous return to the right heart. Cardiac output was significantly increased and with it a significant increase in CO2 output by the lungs was found. These results were associated with a significant increase in expired ventilation. The rapidity of the ventilatory response to the increase in cardiac output suggests the existence of pressoreceptors localized in the pulmonary circulation which, when activated, results in the elicitation of respiratory reflexes. The end result is an adjustment in the respiratory response in accordance with venous return.

Adult↗

PubMed1991

Blood pressure responses to different arm positions during vertical head-down ankle suspension.

This study compares the effects of different arm positions on blood pressure during vertical head-down ankle suspension. Eighteen healthy volunteers (10 men and 8 women, aged 20-30 years) were placed first in the upright sitting position for 4 min, and second in the head-down suspension position for 4 min. In the latter position, the arms were first alongside the head and then raised alongside the body at the end of the 4th min. Blood pressure was measured non-invasively during the last 30 s of the 4th min of upright sitting and head-down suspension with the arms alongside the head, and then immediately following the movement of the arms alongside the body. In relation to the upright sitting position, head-down suspension with the arms dangling alongside the head resulted in a significant increase in brachial artery blood pressure (121/78 mm Hg vs. 146/91 mm Hg, respectively). This finding may be explained on the basis of a variable hydrostatic component. Following the immediate movement of the arms to alongside the body while still suspended, there was a significant decrease in blood pressure to 124/79 mm Hg. No significant change in blood pressure occurred from upright sitting to full head-down suspension with the arms alongside the body (121/78 mm Hg vs. 124/79 mm Hg, respectively). Based on previous disparate reports of pressure during head-down suspension, the findings of the present study indicate a need to standardize the method of blood pressure measurement during this procedure.

Adaptation, Physiological↗

PubMed1990

Identification of interleukin 6 as a synergistic factor for the differentiation-inducing effect of TNF on leukemic ML-1 cells.

Recombinant TNF was capable of inducing differentiation of human leukemic ML-1 cells in the monocytic pathway. Recombinant interleukin 6 did not have the activity but it could significantly increase the activity of recombinant TNF. Both of these molecules were found to play a similar role in PWM-induced conditioned medium from human lymphocytes (LCM). The differentiation inducing effect of LCM could be partially neutralized by antibody to interleukin 6. Fractionation of LCM also identified interleukin 6 as the factor that synergizes with TNF.

Acid Phosphatase↗

PubMed1990

Effect of povidone-iodine and saline soaking on bacterial counts in acute, traumatic, contaminated wounds.

It is common practice to soak acute traumatic wounds in dilute povidone-iodine solution before definitive wound cleaning and debridement. The effectiveness of soaking wounds is unknown. Using quantitative wound bacterial counts as a measure of efficacy, we compared wounds soaked in 1% povidone-iodine solution or in normal saline with wounds receiving no treatment. Adult patients were eligible for the study if they were seen in the emergency department with visibly contaminated traumatic wounds within 12 hours of injury. Patients were excluded if they refused consent, were allergic to iodine, or had taken antibiotics within five days; if the wound did not require debridement; if the wound had been previously cleaned; or if subsequent cultures were sterile. Thirty-three heavily contaminated acute traumatic wounds in 29 patients were included in the study. Wounds were randomly assigned to one of three groups. Tissue samples were taken before and after a ten-minute period of soaking in either povidone-iodine or saline; controls were covered with gauze during the ten-minute period. The mean bacterial count per gram of tissue decreased 6.4 x 10(5) (standard deviation, 1.68 x 10(6)) after no soak, increased 3.39 x 10(7) (1.05 x 10(8)) after saline soak, and decreased 9.19 x 10(6) (1.72 x 10(7)) after povidone-iodine soak. Wounds with counts of less than 10(5) bacteria/g tissue are unlikely to become infected. Multiple regression analysis was used to analyze the changes in bacterial count after treatment as a function of experimental group and initial bacterial count. There was no significant difference between the control and povidone-iodine groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Topical↗

PubMed1990

Interleukin-4 induces expression of the CD45RA antigen on human thymocyte subpopulations.

Interleukin-4 (IL-4) is a multifunctional lymphokine which promotes the growth and/or maturation of multiple cell types. We have examined the ability of IL-4 to promote the phenotypic maturation of subsets of human thymocytes. When cultured in serum-free medium supplemented with recombinant IL-4, a subset of immature CD3-CD45RA- human thymocytes ceased to express the CD1 common thymocyte antigen and acquired phenotypic features characteristic of relatively mature thymocytes, such as high-density expression of the CD3 antigen and de novo expression of the CD45RA isoform of the common leukocyte antigen family. These changes, which were not seen in cells cultured in medium alone, occurred over an 8-9 day period and were accompanied by a significant increase in cell size. The CD45RA+ cells that derived from these immature CD3-CD45RA- precursors were mainly CD4-CD8- or CD8+ cells, and a significant proportion of these cells expressed the T cell receptor delta chain. IL-4 also increased expression of the CD45RA antigen on the more mature CD3+ thymocyte population. However, the CD45RA+ cells derived from IL-4 stimulated CD3+ thymocyte precursors expressed either the CD4 or the CD8 antigen, and virtually all expressed alpha/beta TCR chains. Studies of cell viability and cell growth indicated that these findings were due to direct changes in the phenotype of responsive cells rather than the growth or selective survival of a small number of mature thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗

PubMed1990

Sexual issues and research in counseling the postmyocardial infarction patient.

This article reviews the literature relevant to the postmyocardial infarction (MI) patient and sexual activities. It begins with an analysis of the published accounts of the physiologic effects of sexual activity (including intercourse) and the anticipated concerns associated with coital deaths. The article includes an examination of the need for sexual counseling in any treatment and rehabilitation setting. In addition, general sexual adjustment guidelines for the uncomplicated post-MI patient are presented.

Fear↗

PubMed1989

Selective stimulation of human thymocyte subpopulations by recombinant IL-4 and IL-3.

Human thymocytes and thymocyte subsets were examined for their proliferative response to recombinant interleukin-4 (IL-4) and interleukin-3 (IL-3) in serum-free cultures. IL-4 induced marked proliferation of thymocytes after PHA and TPA stimulation, in contrast to the marginal response of T cells from adult peripheral blood. However, depletion of thymocytes bearing the CD3 antigen diminished the IL-4-induced proliferation of thymocytes, indicating that the response of thymocytes to IL-4 is mainly mediated by the CD3-positive cells. Phenotypic changes after culture with IL-4 showed an increase in the percentage of total thymocytes expressing mature T cell antigens (CD3, CD5, and TCR-1) and a decrease in CD1-positive cells. In addition there was an increase in the percentage of CD4+8- cells in both nylon wool-separated thymocytes and CD3-depleted cells with the disappearance of most of the CD4+8+ cells. However, an increase in the percentage of CD4-8- cells was also observed. The IL-4-responding cells do, however, express the mature T cell antigen, CD5, in high density. The effect of IL-3 on the proliferation of human thymocytes was very low and detected only when the thymocytes were cultured in serum-free medium. Depletion of CD3-positive cells did not diminish the IL-3-mediated proliferation of thymocytes, indicating that IL-3-responsive thymocytes are more immature than the subset of thymocytes which responds to IL-4. These results suggest that IL-4 and IL-3 play different roles in the development of human T cells.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1989

Action of interleukin-3, G-CSF, and GM-CSF on highly enriched human hematopoietic progenitor cells: synergistic interaction of GM-CSF plus G-CSF.

Purified preparations of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and interleukin 3 (IL-3 or multi-CSF) alone and in combination, have been compared for their stimulatory effects on human granulocyte-macrophage colony forming cells (GM-CFC). In cultures of unseparated normal human bone marrow, the combinations of G-CSF plus IL-3 and GM-CSF plus IL-3 stimulated additive numbers of GM colonies, while GM-CSF plus G-CSF stimulated greater than additive numbers of GM colonies, compared with the sum of the colony formation obtained with each factor alone. Cultures of unseparated bone marrow, harvested from patients four to six days after administration of 5-fluorouracil (5-FU), resulted in additive GM colony formation with GM-CSF plus G-CSF, GM-CSF plus IL-3, and G-CSF plus IL-3. In order to address the possibility of secondary factor involvement in the synergistic interaction of GM-CSF and G-CSF, CD33+/CD34+ colony forming cells were separated from normal and post FU marrow by two color fluorescence activated cell sorting. In cultures of CD33+/CD34+ cells the combination of GM-CSF plus G-CSF stimulated a synergistic increase in GM colonies while GM-CSF plus IL-3 stimulated additive numbers of colonies. These results suggest that GM-CSF, G-CSF, and IL-3 stimulate distinct populations of GM-CFC. Furthermore GM-CSF and G-CSF interact synergistically and this action is a direct effect on progenitor cells not stimulated by GM-CSF or G-CSF alone.

Antigens, CD↗