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R W Scott

Publications and source records attributed to R W Scott.

At least 37 records · Page 2Linked to original sources
PubMed1997

From expressed sequence tags to 'epigenomics': an understanding of disease processes.

Expressed sequence tags (ESTs) are at the forefront of technological change that is sweeping the biomedical research community. ESTs provide a high throughput means for identifying gene transcripts and monitoring complex gene expression patterns. EST-based technologies coupled with sophisticated computer analysis tools enable the informational content and output of the genome to be accessed and evaluated on a scale immensely larger than previously possible. EST-based technologies are being used to understand disease processes and to find better disease treatments, and will allow biology to move from single gene to multigene, or even more complex epigenetic, explanations for disease.

Gene Expression↗
PubMed1996

Enhanced amyloidogenic processing of the beta-amyloid precursor protein in gene-targeted mice bearing the Swedish familial Alzheimer's disease mutations and a "humanized" Abeta sequence.

The processing of the beta-amyloid precursor protein (APP) in vivo has been characterized in a novel animal model that recapitulates, in part, the APP genotype of a familial form of Alzheimer's disease (AD). A gene-targeting strategy was used to introduce the Swedish familial AD mutations and convert mouse Abeta to the human sequence. The mutant APP is expressed at normal levels in brain, and cleavage at the mutant beta-secretase site is both accurate and enhanced. Furthermore, human Abeta production is significantly increased to levels 9-fold greater than those in normal human brain while nonamyloidogenic processing is depressed. The results on Abeta production extend similar findings obtained in cell culture to the brain of an animal and substantiate Abeta as a etiological factor in Swedish familial AD. These animals provide several distinguishing features over others created by conventional transgenic methodologies. The spatial and temporal expression patterns of human Abeta are expected to be faithfully reproduced because the gene encoding the mutant APP remains in its normal chromosomal context. Thus, the neuropathological consequences of human Abeta overproduction can be evaluated longitudinally in the absence of potential mitigating effects of APP overexpression or presence of the mouse Abeta peptide.

Alzheimer Disease↗
PubMed1996

Biologically active monomeric and heterodimeric recombinant human calpain I produced using the baculovirus expression system.

Calpain I is a heterodimeric protein that is part of a family of calcium-activated intracellular cysteine proteases presumed to play a role in mediating signals transduced by calcium. Expression of bioactive recombinant human calpain I has been achieved using the baculovirus expression system, by either co-infection with two viruses, each expressing one of the subunits, or infection with a single virus containing both subunits. The approximately 80 kDa catalytic subunit exhibited calcium-dependent proteolytic activity when expressed alone or with the approximately 30 kDa regulatory subunit. Baculoviral recombinant calpain I appeared fully active in that the catalytic subunit in unpurified cell extracts exhibited calcium-dependent autocatalytic cleavage at the correct locus. The amount of approximately 80 kDa subunit accumulated at steady state was greatly increased by co-expression of the approximately 30 kDa subunit, suggesting a possible role for enzyme stabilization by the latter subunit. The recombinant human calpain I was purified to near homogeneity and compared with purified native human erythrocyte calpain I. The recombinant and native enzymes had equivalent inhibition constants for structurally diverse calpain inhibitors, identical calcium activation profiles, and similar specific activities, demonstrating the suitability of using the recombinant protein for studies of the native enzyme.

Amino Acid Sequence↗
PubMed1996

Can strong back extensors prevent vertebral fractures in women with osteoporosis?

OBJECTIVE: To determine the influence of back extensor strength on vertebral fractures in 36 women with osteoporosis. DESIGN: We conducted a cross-sectional study of female patients with osteoporosis by assessing anthropometric variables, bone mineral density, muscle strength, level of physical activity, and radiographic findings in the spine. MATERIAL AND METHODS: The 36 study subjects with osteoporosis, who ranged from 47 to 84 years of age, satisfied specific inclusion and exclusion criteria that minimized confounding factors related to pathophysiologic features, diet, and medications. A physical activity score was determined for each subject on the basis of daily physical activities relating to homemaking, occupation, and sports. RESULTS: The range of the physical activity scores-from 2 to 13-indicated that no subject was involved in unusually demanding physical activities. Bone mineral density values ranged from 0.49 to 0.92 g/cm2. Thoracic kyphosis ranged from 31.0 to 84.0 degrees. Isometric strength of the back extensor muscles ranged from 7.3 to 34.0 kg. Statistical analysis demonstrated a significant negative correlation between the strength of the back extensor muscles and thoracic kyphosis. Significant negative correlations were also found between back extensor strength and the number of vertebral compression fractures and between bone mineral density and the number of vertebral fractures. CONCLUSION: The negative association between back extensor strength and both kyphosis and number of vertebral fractures suggests that increasing back strength may prove to be an effective therapeutic intervention for the osteoporotic spine. In persons with stronger back muscles, the risk of vertebral fractures will likely decrease.

Aged↗
PubMed1996

Strategies to alter the progression of Alzheimer's disease.

Recent advances in the genetics of familial Alzheimer's disease provide direction for therapeutic strategies to alter the progressive neurodegeneration. The rationale is particularly strong for targeting the deposition of amyloid into neuritic plaques, but attention has also turned to abnormalities in apoptosis and signal-transduction processes.

Alzheimer Disease↗
PubMed1996

Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury.

The discovery that some cases of familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) has focused much attention on the function of SOD1 as related to motor neuron survival. Here we describe the creation and characterization of mice completely deficient for this enzyme. These animals develop normally and show no overt motor deficits by 6 months in age. Histological examination of the spinal cord reveals no signs of pathology in animals 4 months in age. However Cu/Zn SOD-deficient mice exhibit marked vulnerability to motor neuron loss after axonal injury. These results indicate that Cu/Zn SOD is not necessary for normal motor neuron development and function but is required under physiologically stressful conditions following injury.

Animals↗
PubMed1996

Proteasome inhibition enhances the stability of mouse Cu/Zn superoxide dismutase with mutations linked to familial amyotrophic lateral sclerosis.

Point mutations occurring within the Cu/Zn superoxide dismutase (SOD1) gene have been implicated in the etiology of some cases of familial amyotrophic lateral sclerosis (FALS). In order to better understand the functional consequences of these mutations, we have introduced FALS mutations into the mouse SOD1 gene and studied the expression of the mutant templates in stably transformed cell lines. Pulse-chase analyses of lysates derived from cell lines stably expressing the Cu/Zn SOD isoforms indicate that the FALS mutant Cu/Zn SOD proteins are turned over more rapidly than wild-type SOD. Protease inhibitors specific for the major intracellular proteolytic activities were used to characterize the degradative pathways involved in the turnover of mutant Cu/Zn SOD. Inhibition of the chymotrypsin-like activity of the proteasome (also known as multicatalytic proteinase or ubiquitin, ATP-dependent proteinase) by a synthetic dipeptide aldehyde led to a significant increase in levels of the mutant Cu/Zn SOD implicating this proteolytic pathway in the turnover of the FALS mutant SOD proteins.

Amyotrophic Lateral Sclerosis↗
PubMed1995

Protection against endotoxic shock by bactericidal/permeability-increasing protein in rats.

Bactericidal/permeability-increasing protein (BPI) is a neutrophil primary granule protein that inhibits effects of LPS in vitro. The current study examined the effects of BPI on hemodynamics, mortality, and circulating endotoxin and cytokines in conscious rats with endotoxic shock. Catheters were implanted into the right femoral artery and vein. 1 d later, human recombinant BPI (10 mg/kg) or vehicle was intravenously injected immediately, 30 min, or 2 h after intravenous injection of LPS (7.5 mg/kg). Mean arterial pressure (MAP) and heart rate were monitored and blood was collected before and after injection. BPI given immediately or 30 min after LPS prevented the LPS-induced reduction in MAP at 4-8 h and markedly reduced mortality. BPI given 2 h after LPS injection had no protective effect. BPI treated immediately after LPS reduced the circulating levels of endotoxin and IL-6 but increased the circulating levels of TNF. We propose that BPI exerts its protective effect through a TNF-independent mechanism, by inhibiting endotoxin-stimulated production of IL-6.

Animals↗
PubMed1994

Relative concentrations of endotoxin-binding proteins in body fluids during infection.

Endotoxin initiates the systemic inflammatory response, haemodynamic changes, and multi-organ failure that may occur as a consequence of systemic gram-negative bacterial infection. The serum protein lipopolysaccharide-binding protein (LBP) binds to the lipid A component of bacterial endotoxin and facilitates its delivery to the CD14 antigen on the macrophage, where inflammatory cytokines are released and a cascade of host mediators is initiated. The neutrophil granular protein bactericidal/permeability-increasing protein (BPI) competes with LBP for endotoxin binding and functions as a molecular antagonist of LBP-endotoxin interactions. We have measured concentrations of both proteins in body fluids from 49 consecutive patients. In 16 of 17 samples of fluid from closed-space infections, BPI was present in greater concentration than LBP (median BPI/LBP ratio 7.6 [95% CI 2.32-22.1]). The ratio of BPI and LBP was not significantly different from 1.0 in abdominal fluid from 10 patients with peritonitis (ratio 0.235 [0.18-0.47]), whereas the BPI/LBP ratio was low in 22 non-infected body fluids (0.01 [0.001-0.04]) and concentrations of both proteins approached those in normal human plasma. BPI concentrations were directly correlated with the quantity of neutrophils within clinical samples (rs = 0.81, p < 0.0001). Thus, within abscess cavities BPI is available in sufficient quantities for effective competition with LBP for endotoxin. BPI may attenuate the local inflammatory response and the systemic toxicity of endotoxin release during gram-negative infections.

Abscess↗
PubMed1994

Bactericidal/permeability-increasing protein and lipopolysaccharide (LPS)-binding protein. LPS binding properties and effects on LPS-mediated cell activation.

We have previously shown that human bactericidal/permeability-increasing protein (BPI) is able to inhibit serum-dependent lipopolysaccharide (LPS)-mediated activation of human monocytes and neutrophils in vitro, and to counteract the lethal effects of LPS challenge in vivo. Lipopolysaccharide-binding protein (LBP) is a serum protein which participates in LPS-mediated activation of cells (Tobias, P. S., Mathison, J., Mintz, D., Lee, J. D., Kravchenko, V., Kato, K., Pugin, J., and Ulevitch, R. J. (1992) Am. J. Respir. Cell. Mol. Biol. 7, 239-245). We have proposed that BPI functions in a negative feedback loop which opposes this activation (Marra, M. N., Wilde, C. G., Collins, M. S., Snable, J. L., Thornton, M. B., and Scott, R. W. (1992) J. Immunol. 148, 532-537). We have now cloned and expressed recombinant forms of human BPI and LBP. Here we demonstrate that purified recombinant human LBP can replace the serum requirement for both LPS binding to human monocytes and LPS-mediated secretion of tumor necrosis factor alpha from these cells. These activities of LBP are inhibited by a neutralizing anti-CD14 monoclonal antibody. We further demonstrate that purified recombinant human BPI can inhibit LBP-mediated LPS binding to cells and their subsequent activation. Comparison of the LPS binding properties of BPI and LBP in enzyme-linked immunosorbent type assays and in the Limulus amebocyte lysate assay suggest that BPI has a stronger affinity for LPS than does LBP. Direct competition between BPI and LBP for LPS may explain the inhibition by BPI of the proinflammatory effects of LBP in the presence of LPS.

Acute-Phase Proteins↗
PubMed1994

Changes in polymorphonuclear leukocyte surface and plasma bactericidal/permeability-increasing protein and plasma lipopolysaccharide binding protein during endotoxemia or sepsis.

OBJECTIVE: To evaluate changes in levels of polymorphonuclear leukocyte surface bactericidal/permeability-increasing protein (BPI), plasma BPI, and plasma lipopolysaccharide (LPS) binding protein (LBP) in normal human volunteers administered Escherichia coli LPS and in patients with sepsis and gram-negative infections. DESIGN: Survey; case series. SETTING: Clinical research center and surgical intensive care unit of a medical school and an associated tertiary care hospital. PATIENTS OR OTHER PARTICIPANTS: Volunteers (n = 10) screened prior to study by history and physical examination to exclude those with underlying diseases or hematologic abnormalities. Consecutive sample of surgical intensive care unit patients (n = 10) meeting criteria for sepsis syndrome with gram-negative infection. An additional patient with systemic inflammatory response syndrome but no gram-negative infection. All patients were studied on meeting the criteria. Three of the patients with sepsis syndrome and the patient with systemic inflammatory response syndrome were evaluated on recovery (approximately 25 days after initial study). Because these studies in volunteers and patients overlapped temporally, the control values were those of volunteers evaluated prior to LPS administration. No matching was employed. MEASUREMENTS AND RESULTS: Compared with controls, LPS-challenged volunteers and patients with sepsis both exhibited significant granulocytosis (P < .01) and increased concentrations of polymorphonuclear leukocyte surface BPI (P < .01) and of plasma LBP (P < .01). Plasma BPI concentrations were increased (P < .01) in volunteers following LPS administration. There was a trend toward increased concentrations of plasma BPI in patients, but this was not significant relative to controls. Maximum concentrations of plasma LBP were approximately 250- and 3000-fold higher than plasma BPI concentrations in endotoxemic volunteers and in patients, respectively. CONCLUSIONS: Circulating polymorphonuclear leukocytes increase expression of BPI in response to LPS or gram-negative sepsis. Subsequently, concentrations of plasma BPI and LBP increase. Because both LBP and BPI bind to LPS, it is suggested that endogenously derived plasma levels of BPI are likely to be inadequate to compete for LPS binding to the much more abundant LBP in the circulation.

Acute-Phase Proteins↗
PubMed1994

The role of bactericidal/permeability-increasing protein in the treatment of primate bacteremia and septic shock.

Human neutrophil azurophilic granules contain an approximately 55-kDa protein, known as bactericidal/permeability-increasing protein (BPI), which possesses a high-affinity binding domain for the lipid A component of lipopolysaccharide (LPS). The in vivo LPS neutralizing activity of exogenous BPI was studied in a model of lethal Escherichia coli bacteremia. Five baboons were treated with BPI (5 mg/kg bolus injection followed by a 95 micrograms/kg/min BPI infusion over 4 hr), while four additional animals received a genetically engineered variant of BPI (NCY103). Five animals received a placebo treatment and served as controls. Both wild-type rhBPI and NCY103 significantly (P < 0.05) decreased blood levels of LPS throughout an 8-hr evaluation period following live bacterial challenge. Two hours following E. coli administration, LPS levels peaked in the controls, at 6.86 +/- 3.22 ng/ml, whereas LPS levels were 3.39 +/- 2.1 ng/ml in the BPI group and 2.04 +/- 1.18 ng/ml in the NCY103 group. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 levels likewise were attenuated in the treatment groups, whereas circulating sTNFR I was significantly (P < 0.05) reduced only in the BPI group. Leukocytopenia and granulocytopenia were significantly (P < 0.02) lessened in the BPI group, by an average of 59% leukocytopenia and 65% granulocytopenia, respectively. This study supports the concept of E. coli LPS neutralization by BPI in vivo and demonstrates that a moderate (70%) reduction in peak LPS-LAL activity is sufficient to alter some hematologic and cytokine manifestations of bacteremia.

Animals↗
PubMed1994

Production and characterization of recombinant mouse brain-derived neurotrophic factor and rat neurotrophin-3 expressed in insect cells.

Bioactive brain-derived neurotrophic factor (BDNF) and neurotrophin-3 were produced using the baculovirus expression system and purified to homogeneity using ion-exchange and reversed-phase chromatography. Yields of purified neurotrophin-3 (300-500 micrograms/L) were similar to levels reported for baculovirus-expressed nerve growth factor (NGF), whereas initial yields of BDNF were significantly lower (20-50 micrograms/L). Improved production of BDNF (150-200 micrograms/L) was achieved by expressing BDNF from a chimeric prepro-NGF/mature BDNF construct using the Trichoplusia ni insect cell line. Tn-5B1-4. Examination of the distribution of BDNF protein from both the non-chimeric prepro-BDNF and the chimeric prepro-NGF/mature BDNF viruses in Sf-21- and Tn-5B1-4-infected cells suggests a specific deficiency in the Tn-5B1-4 cells in processing the nonchimeric precursor. In addition, the vast majority of the BDNF protein at 2 days after infection was intracellular and insoluble. N-terminal amino acid sequencing of purified recombinant BDNF and neurotrophin-3 demonstrated that the insect cells processed their precursors to the correct N-terminus expected for the mature protein. Bioactivity was characterized in vitro on primary neuronal cultures from the CNS and PNS.

Animals↗
PubMed1994

Cloning and characterization of human tissue inhibitor of metalloproteinases-3.

The tissue inhibitors of metalloproteinases (TIMPs) comprise a family of proteins, of which two members have so far been described in humans. We have cloned and sequenced a third human TIMP (hTIMP-3) from phorbol ester-differentiated THP-1 cells stimulated with bacterial lipopolysaccharide. The open reading frame encodes a 211-amino-acid precursor including a 23-residue secretion signal. The mature polypeptide has a calculated molecular weight of 21.6 kD and includes an N-linked glycosylation site near the carboxyl terminus. The protein is quite basic, having a predicted isoelectric point of 9.04. We have mapped the single gene encoding human TIMP-3 to chromosome 22. By Northern analysis, transcripts for TIMP-3 were identified in a broad cross-section of tissues examined from both embryonic and adult origin. In all tissues except the placenta, the predominant transcript was 5.0 kb in size, with minor bands around 2.4 and 2.6 kb comprising no more than about 10% of the signal. In the placenta, the smaller bands accounted for close to 50% of the signal. Human TIMP-3 shows slightly closer amino acid sequence similarity to TIMP-2 (44.3%) than to TIMP-1 (38.4%), but is most closely related to a recently reported chicken TIMP, chIMP-3 (80.8% amino acid; 77.7% nucleic acid similarity.

Amino Acid Sequence↗
PubMed1994

Human neutrophil bactericidal/permeability-increasing protein reduces mortality rate from endotoxin challenge: a placebo-controlled study.

OBJECTIVES: To study the toxicology and pharmacology of the endotoxin-neutralizing agent, bactericidal/permeability-increasing protein. DESIGN: Prospective, randomized, placebo-controlled laboratory study. SETTING: Academic research laboratory. SUBJECTS: CD-1 mice (n = 259); Sprague Dawley rats (n = 26); New Zealand White rabbits (n = 19). INTERVENTIONS: Pharmacokinetics of intravenously injected bactericidal/permeability-increasing protein was assessed in mice. Toxicology was tested in mice and rats. Efficacy of intravenously administered bactericidal/permeability-increasing protein as an endotoxin-neutralizing agent was tested in mice, rats, and rabbits. MEASUREMENTS AND MAIN RESULTS: Administration of a single 10-mg/kg bolus injection of bactericidal/permeability-increasing protein resulted in no alterations in hematologic, renal, or hepatic function, activity level, or weight gain in animals observed over a 7-day study period. A single bolus injection (10 mg/kg) of bactericidal/permeability-increasing protein protected 15 of 16 mice from a lethal endotoxin challenge (mortality rate 1/16 [6.25%]) compared with a 100% (16/16) mortality rate in the saline-treated controls (p < .001). Bactericidal/permeability-increasing protein administered up to 1 hr after endotoxin provided significant protection against lethal endotoxin challenge. Furthermore, bactericidal/permeability-increasing protein reduced the induration and dermal necrosis observed in the localized dermal Shwartzman reaction. CONCLUSIONS: Bactericidal/permeability-increasing protein is a potent antiendotoxin that neutralizes endotoxin in vivo and prevents mortality in animal models of lethal endotoxemia.

Animals↗
PubMed1994

Endotoxin-binding and -neutralizing properties of recombinant bactericidal/permeability-increasing protein and monoclonal antibodies HA-1A and E5.

OBJECTIVE: To compare the endotoxin-binding and -neutralizing properties of bactericidal/permeability-increasing protein, the human monoclonal antiendotoxin antibody HA-1A, and the murine antiendotoxin antibody E5. DESIGN: Prospective, randomized, placebo-controlled laboratory study. SETTING: Biotechnology company research laboratory. SUBJECTS: Female CD-1 mice. INTERVENTIONS: Recombinant bactericidal/permeability-increasing protein, HA-1A, a human immunoglobulin M monoclonal antibody raised against Escherichia coli J5 (Rc) endotoxin, and E5, a murine immunoglobulin M monoclonal antibody raised against E. coli J5 endotoxin, were compared in the following assays: a) binding to rough lipopolysaccharide immobilized onto microtiter plates; b) inhibition of lipopolysaccharide activity in the limulus amebocyte lysate assay; c) inhibition of lipopolysaccharide-induced cytokine release in whole blood; and d) protection against lethal endotoxin challenge in CD-1 mice. MEASUREMENTS AND MAIN RESULTS: The binding affinity of bactericidal/permeability-increasing protein for immobilized lipopolysaccharide is apparently greater than the binding affinity of HA-1A or E5. Bactericidal/permeability-increasing protein neutralized lipopolysaccharide activity in the chromogenic limulus amebocyte lysate assay, while neither monoclonal antibody inhibited lipopolysaccharide activity. Similarly, bactericidal/permeability-increasing protein reduced lipopolysaccharide-mediated tumor necrosis factor production in human whole blood in vitro, whereas monoclonal antibodies had slight (HA-1A) or no (E5) effect on lipopolysaccharide activity in this system. Administration of bactericidal/permeability-increasing protein gave > 90% protection against an LD60 dose of endotoxin in CD-1 mice, while treatment with HA-1A or E5 did not improve survival rate. CONCLUSIONS: Neither monoclonal antibody was as effective as bactericidal/permeability-increasing protein at binding or neutralizing endotoxin in vitro or in vivo. The potent endotoxin-binding and -neutralizing properties of bactericidal/permeability-increasing protein indicate that it might be useful in the treatment of endotoxin-related disorders in humans.

Animals↗
PubMed1993

Processing of the beta-amyloid precursor. Multiple proteases generate and degrade potentially amyloidogenic fragments.

Proteolytic processing of the beta-amyloid precursor proteins (APP) is required for release of the beta/A4 protein and its deposition into the amyloid plaques characteristic of aging and Alzheimer's disease. We have examined the involvement of acidic intracellular compartments in APP processing in cultured human cells. The use of acidotropic agents and inhibitors to a specific class of lysosomal protease, coupled with metabolic labeling and immunoprecipitation, revealed that APP is degraded within an acidic compartment to produce at least 12 COOH-terminal fragments. Nine likely contain the entire beta/A4 domain and, therefore, are potentially amyloidogenic. Treatment with E64 or Z-Phe-Ala-CHN2 irreversibly blocked activities of the lysosomal cysteine proteases cathepsins B and L but did not inhibit the lysosomal aspartic protease cathepsin D and did not alter the production of potentially amyloidogenic fragments. Instead, the inhibitors prevented further degradation of the fragments. Thus, large numbers of potentially amyloidogenic fragments of APP are routinely generated in an acidic compartment by noncysteine proteases and then are eliminated within lysosomes by cysteine proteases. Immunoblot and immunohistochemical analyses confirmed that chronic cysteine protease inhibition leads to accumulation of potentially amyloidogenic APP fragments in lysosomes. The results provide further support for the hypothesis that an acidic compartment may be involved in amyloid formation and begin to define the proteolytic events that may be important for amyloidogenesis.

Amyloid beta-Protein Precursor↗