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Biomedical subjects

R B Harris

Publications and source records attributed to R B Harris.

At least 73 records · Page 4Linked to original sources
PubMed1996

Heparins designed to specifically inhibit platelet interactions with von Willebrand factor.

BACKGROUND: Platelet interactions with the injured vessel wall may contribute significantly to the early and late failures of many cardiovascular interventions; the adhesive protein von Willebrand factor (vWF) is thought to play an important role. Previously, we demonstrated that heparin interfered with platelet/vWF hemostatic mechanisms by binding to vWF within the proteins's domain responsible for binding the platelet vWF receptor, glycoprotein Ib. The purpose of the present study was to develop and refine heparins with greater potency to inhibit platelet/vWF interactions. METHODS AND RESULTS: Immobilized synthetic peptides based on a known heparin-binding domain of vWF were used to yield novel fractions of standard heparin that demonstrated a sevenfold increase in their ability to inhibit vWF-dependent platelet agglutination and vWF/platelet binding. The high vWF affinity heparin showed enhanced anti-factor Xa activity but comparable activated partial thromboplastin time activity. Chemical modification of a standard heparin by periodate oxidation and borohydride reduction enhanced its ability to inhibit platelet/vWF interactions by threefold, while eliminating more than 90% of its activated partial thromboplastin time and anti-factor Xa activity. Affinity chromatography of the chemically modified heparin yielded a heparin with an eightfold higher inhibitory potency than the original heparin. CONCLUSIONS: Subspecies of heparin can be developed with significantly enhanced potency to inhibit vWF/platelet interactions. The vWF-inhibiting property of heparin can be dissociated from its antithrombin-binding activity. Based on a growing understanding of heparin/vWF interactions, combinations of affinity separations and chemical modifications could be designed to yield heparins uniquely suitable for prevention of arterial thrombosis.

Anticoagulants↗
PubMed1996

The role of neutrophils in peritoneal adhesion formation.

The most common cause of intraperitoneal adhesions is previous abdominal surgery. Postoperative adhesion formation results from a fibroproliferative inflammatory reaction that begins with an influx of polymorphonuclear leukocytes (PMNs) into the peritoneal cavity. Adherence of the PMNs to the endothelial cells (EC) is necessary for PMN migration into the tissue in response to a stimulus. Several receptor-counterreceptor pairs of ligands such as CD11/CD18 on the PMN and ICAM-1 (CD54) on EC have been identified. Monoclonal antibody against CD11/CD18 (R15.7) inhibits PMN adherence and migration and consequently protects against PMN-induced tissue injuries. We therefore studied the effect of preventing PMN-EC adherence, using anti-CD18 monoclonal antibody, on postoperative adhesion formation in rabbits. Group 1 was a control receiving physiologic saline, and group 2 received anti-CD18 antibody (R15.7, 2 mg/kg). The treatment was administered iv at the end of surgery and repeated on the first and second postoperative days. Peritoneal adhesions were induced at laparotomy by repairing two peritoneal defects, by oversewing the defect (model 1), and by resuturing the removed parietal peritoneum in its place as an ischemic graft (model 2). Adhesions were evaluated blindly at 10 days after operation by measuring the percentage of the suture line covered with adhesions (model 1) or by a scoring system (model 2). All control animals developed intraperitoneal adhesions and the percentage of the suture line covered with adhesions was 25 +/- 5.9% (mean +/- SEM) and the mean score in model 2 was 0.9 +/- 0.2. Anti-CD18 antibody, R15.7, increased the degree of postoperative adhesion formation in both models, but the results were significant only in model 2. Also, anti-CD18 antibody significantly decreased peritoneal neutrophils from 11.1 x 10(7) +/- 1.8 x 10(7) to 2.2 x 10(7) +/- 0.4 x 10(7) (P < 0.001) on the first postoperative day. It is concluded that inhibition of PMN-EC adherence does influence the postoperative adhesion formation. These results might suggest that PMNs have a role in modulating postoperative adhesion formation.

Animals↗
PubMed1996

Antibody characteristics for a continuous response fiber optic immunosensor for theophylline.

A self-contained fiber optic immunosensor was developed to measure continuously theophylline concentrations. The analytical signal was derived from the non-radiative energy transfer quench of fluorescence following binding of a fluorescence donor, B-phycoerythrin labeled theophylline, to an energy acceptor, Texas Red labeled antibody. Increases in free theophylline analyte concentrations resulted in a shift in antibody binding equilibrium between labeled and unlabeled theophylline that elicited a proportional increase in the fluorescence. The selection criteria for a monoclonal antibody as a molecular recognition element, and the optimization of labeling conditions to maximize the dynamic range and minimize sensor response time are described. Under one or more Texas Red labeling conditions, five antibody clones exhibited significant quenching when mixed with labeled analyte and also demonstrated 95% or greater reversible binding to labeled analyte. Two clones failed to exhibit fluorescence quenching when mixed with labeled analyte. The response time of the indicator chemistry system was dependent on the dissociation rate constant of the antibody. The equilibrium response time of intact sensors was limited by analyte diffusion across the containment membrane.

Antigen-Antibody Reactions↗
PubMed1996

Physician advice and tobacco use: a survey of 1st-year college students.

First-year college students were surveyed by telephone to determine their current level of tobacco use and find out what advice they had previously received from physicians regarding tobacco products. Current tobacco use reported in this 1st-year population was 19% in men and 17% in women. Although 99.6% of the students reported having had a medical visit within the last 5 years and 89% reported a visit within the past 12 months, only 26% remembered being asked at the last visit about their use of tobacco. Women were significantly more likely than men to have been asked about tobacco (31% compared with 21%), perhaps because of oral contraceptive counseling and the women's medical history. It appeared that healthcare providers are not fully using the opportunities available to them to educate young adults about using tobacco.

Adolescent↗
PubMed1996

Structure/activity studies of anti-inflammatory peptides based on a conserved peptide region of the lectin domain of E-, L- and P-selectin.

Previously, it was established that the peptide YYWIGIRK-NH2 inhibits both myeloid cell adhesion to selectins in vitro and neutrophil influx into inflammatory sites in vivo (Briggs et al., 1995). Initial structure/activity studies revealed that at least one Y residue at the N-terminus of the peptide was essential for these bioactivities but that the C-terminal K residue was unnecessary for inhibitory activity. We have now synthesized a new series of peptides which contain single residue substitutions at each position of the reference peptide, YYWIGIR-NH2, and have tested these peptides for inhibitory activity in a selectin cell binding assay. In addition, peptides containing single D-amino acids at selected positions, or an all D-configured reference peptide sequence, or the retro-inverso version (rigiwyy-NH2) of the reference peptide sequence have also been analyzed for inhibitory activity in the same assays. Finally, the ability of the reference peptide and a specifically designed control sequence (YY(AIB)IGIR-NH2) to discriminate between potential synthetic saccharide ligands, including sialyl-Lewis x, Lewis x, and sialyl-N-acetyl-lactosamine, was investigated using isothermal titration calorimetry. The results of these studies demonstrate that whereas many single amino acid substitutions are tolerated in the peptide without complete loss of inhibitory activity, substitution at some positions (e.g., the W residue) results in relatively inactive compounds, clearly pointing to the importance of these residues in making critical contacts with the appropriate saccharide ligand. Titration calorimetry revealed that the reference peptide does not discriminate between Lewis x or sialyl-Lewis x in vitro, but binds these saccharides with nearly 40-fold higher affinity (KD 25 microM) than the nonfucosylated trisaccharide, sialyl-N-acetyl-lactosamine. We can infer from these studies that the presence of a sialyl group per se, is not a requisite for complex formation between the reference peptide and its saccharide ligand. Substitution of single D-amino acid residues at various positions in the reference peptide sequence reduces or eliminates all inhibitory properties. However, the all D-configured peptide or the retro-inverso peptide sequence have greater activity than the all L-configured reference peptide in the in vitro biological assays, and each was an effective inhibitor of neutrophil infiltration in a thioglycolate-induced mouse peritonitis model. These results, combined with the results of titration, allow us to conclude that binding between the reference peptide and its saccharide ligand, which affords its inhibitory properties, is mediated by the presence of a contiguous, nonpolar surface, or face, presented at the N-terminus of the reference peptide, likely encompassing the sequence YYWI. Furthermore, the W plays a critical role in binding, probably through formation of an essential hydrogen bond with a suitably juxtaposed group carried on the saccharide ligand.

Amino Acid Sequence↗
PubMed1996

The effect of preexisting long-term diabetes on the outcome after islet transplantation in rats.

Studies of islet transplantation conducted immediately following diabetes induction may not accurately reflect the clinical situation. Long term preexisting diabetes with generalized microvasculature complication might adversely affect the outcome after islet transplantation. The present study testing this hypothesis by evaluating the effect of long-term preexisting diabetes on glucose-induced insulin secretion up to 6 months after transplantation of two different quantities of islets. One thousand two hundred or 2,400 islets were isotransplanted into the left renal subcapsular space at 10 days (acute diabetes), 3 months (chronic diabetes), or 6 months (long-term diabetes) after diabetes induction by streptozotocin in the rat. In addition, one group of diabetic rats in which normoglycemia was maintained with exogenous insulin treatment for 6 months was then transplanted with 1,200 islets. Intravenous glucose tolerance tests were performed at 10, 90, and 180 days after islet transplantation. Islet transplantation normalized the basal blood glucose levels within 24-48 h in all transplanted groups that remained normal for the entire study period of 6 months, with no differences among acute, chronic, or long-term diabetes. Basal plasma insulin levels were also normalized in all transplanted groups. Diabetic (acute, chronic, or long-term) rats transplanted with 2,400 islets achieved normal glucose-induced insulin secretion at 10 and 90 days after transplantation. In contrast, glucose-induced insulin secretion was impaired in rats transplanted with only 1,200 islets, with no differences among acute, chronic, and long term diabetes. However, at 180 days after transplantation, long term diabetic rats transplanted with 2,400 islets had impaired insulin secretion compared to normal controls. Insulin-treated long-term diabetic rats transplanted with 1,200 islets had normal glucose-induced insulin secretion at 10 days after transplantation. However, at 90 and 180 days after transplantation, insulin-treated long-term diabetic rats had impaired glucose-induced insulin secretion which was not different from nontreated transplanted long-term diabetic rats. It is concluded that long-term preexisting diabetes has no impact on the early outcome after islet transplantation. However, it may adversely affect the long-term function of the transplanted islets. Also, transplantation of a sufficient islet mass is the critical factor in achieving complete glucose homeostasis.

Animals↗
PubMed1995

Preparations of psi-peptide bond and peptide-aldehyde inhibitors of atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor.

Pseudo-peptide bond inhibitors (psi-bond inhibitors) and peptide-aldehyde inhibitors of atrial granule serine proteinase, the candidate processing enzyme of pro-atrial natrieuretic factor, are prepared in high yield and purity by novel synthetic routes. The psi-bond compounds retain essential residues for enzyme binding, but place the enzyme inhibition site in the midst of the peptide sequence. Thus, Bz-APR-psi-LR and Bz-APR-psi-SLRR can be considered "readthrough inhibitors" of atrial granule serine proteinase. The most potent psi-peptide, Bz-APR-psi-SLRR (IC50=250 microM), is about fivefold less potent than the best peptide-aldehyde inhibitor (EACA-APR-CHO), and both the psi-bond and peptide-aldehyde compounds are competitive, reversible inhibitors of the enzyme. The psi-bond peptides containing two C-terminal Arg residues are three- to tenfold more potent than the analogous compounds containing only one C-terminal Arg residue, confirming the importance of both Arg residues in the enzyme processing recognition site. As expected, because of their moderate potencies, the psi-peptides are not useful affinity ligands for purification of atrial granule serine proteinase, but both peptide aldehydes are effective affinity ligands.

Aldehydes↗
PubMed1995

N-terminal sequence analysis of atrial granule serine proteinase purified by affinity chromatography.

Atrial granule serine proteinase is considered the leading candidate endoproteolytic processing enzyme of pro-atrial natriuretic factor. Its cleavage specificity is directed toward a monobasic amino acid processing site, and as such, the atrial enzyme is distinguished from the family of prohormone convertases which act at dibasic amino acid processing sites. To delineate the molecular mechanisms which distinguish monobasic from dibasic amino acid-directed processing enzymes, pure atrial enzyme is needed for sequence determination leading to molecular cloning, and for preparation of antisera. An affinity chromatography purification scheme seemed a logical modification of our established procedures to yield suitable amounts of enzyme for further studies [Damodaran and Harris (1995, J. Protein Chem., this issue] formed ineffective affinity ligands, even though these compounds contain essential residues on either side of what would be the scissile bond in a peptide substrate. On the other hand, tripeptide aldehydes (based on the substrate recognition sequence of the atrial enzyme) linked to Sepharose formed effective affinity matrices, permitting purification of the enzyme in a single step from a subcellular fraction enriched for atrial granules and lysosomes. Hence, the enzyme was purified 2000-fold in 90% overall yield, and subjected to N-terminal sequence analysis through 26 residues. The sequence determined, XXPEAAGLPG[R,L]GNPVP[F,G]R[Q,I]XY[G,E]XR(N,A]V, indicates that the atrial enzyme is unique, showing little sequence homology to other proteins in the database.

Amino Acid Sequence↗
PubMed1995

Dissociation between food intake, diet composition, and metabolism in parabiotic partners of obese rats.

When one member of a parabiosed pair of rats is overfed, its ad libitum-fed partner loses body fat in the absence of a statistically significant decrease in food intake. Three experiments investigated the relationships between food intake, metabolism, and body composition in this model. In vivo measurement of lipogenesis confirmed that loss of fat is associated with decreased fat deposition. When partners of overfed rats were compared with food-restricted single rats, proportional changes in body composition and metabolism were similar for the two treatments, although there was no significant change in the food intake of parabiotic rats, whereas restricted rats received only 60% of the intake of their controls. The final experiment demonstrated that changes in body composition of partners of overfed rats were independent of dietary composition. These results suggest that, when a rat is made obese by overfeeding, a circulatory factor is released that inhibits fat deposition and disrupts regulatory mechanisms that normally stimulate food intake during a period of negative energy balance.

Adipose Tissue↗
PubMed1994

Heparin binding domain peptides of antithrombin III: analysis by isothermal titration calorimetry and circular dichroism spectroscopy.

The serine proteinase inhibitor antithrombin III (ATIII) is a key regulatory protein of intrinsic blood coagulation. ATIII attains its full biological activity only upon binding polysulfated oligosaccharides, such as heparin. A series of synthetic peptides have been prepared based on the proposed heparin binding regions of ATIII and their ability to bind heparin has been assessed by CD spectrometry, by isothermal titration calorimetry, and by the ability of the peptides to compete with ATIII for binding heparin in a factor Xa procoagulant enzyme assay. Peptide F123-G148, which encompasses both the purported high-affinity pentasaccharide binding region and an adjacent, C-terminally directed segment of ATIII, was found to bind heparin with good affinity, but amino-terminal truncations of this sequence, including L130-G148 and K136-G148 displayed attenuated heparin binding activities. In fact, K136-G148 appears to encompass only a low-affinity heparin binding site. In contrast, peptides based solely on the high-affinity binding site (K121-A134) displayed much higher affinities for heparin. By CD spectrometry, these high-affinity peptides are chiefly random coil in nature, but low microM concentrations of heparin induce significant alpha-helix conformation. K121-A134 also effectively competes with ATIII for binding heparin. Thus, through the use of synthetic peptides that encompass part, if not all, of the heparin binding site(s) within ATIII, we have further elucidated the structure-function relations of heparin-ATIII interactions.

Amino Acid Sequence↗
PubMed1994

Localization of the binding site on plasma kallikrein for high-molecular-weight kininogen to both apple 1 and apple 4 domains of the heavy chain.

The C-terminal end of the heavy chain of human plasma prekallikrein or kallikrein contains a binding site for high-molecular-weight kininogen, the nonenzymatic procofactor of contact activation. To further map this binding site, a series of overlapping peptides were synthesized. The amount of kallikrein that bound to kininogen-coated microtiter plate wells in the presence of increasing concentrations of each peptide was determined by kallikrein amidolytic activity. A peptide encompassing Lys266-Gly295 of kallikrein, conformationally constrained by a disulfide bond, displayed the lowest Kd value (approximately 67 microM). The linear peptide, Leu262-Gly295, displayed lower affinity (129 microM). N-terminal or C-terminal truncation/extension peptides of this sequence diminished binding activity. Since the closely related protein, factor XI, has been shown to bind kininogen, a kallikrein-based peptide (Phe56-Gly86) homologous to the binding domain of FXI, was examined and found to possess less, but significant, binding affinity for kininogen (Kd 530 microM). Isothermal titration calorimetry was used to assess binding between the kallikrein-based peptides and a peptide encompassing the kallikrein binding domain in kininogen (Ser565-Lys595). Leu262-Gly295 possesses potent binding activity (Kd 52 microM), while Phe56-Gly86 displays poorer binding activity (Kd 400 microM). These interactions are endothermic and entropically favored, suggesting that a conformational rearrangement takes place upon binding. We conclude that the binding site for kininogen within prekallikrein is composed of discontinuous linear segments that form a contiguous surface in the folded protein.

Amino Acid Sequence↗
PubMed1994

Habitual sleep durations and health in a 50-65 year old population.

Several studies have suggested that individuals with long or short sleep durations are at greater risk for adverse outcomes relative to individuals sleeping 7-8 hours a night. The mechanisms leading to these results have never been fully explained, but individual differences in how long an individual sleeps are usually considered to reflect lifestyle rather than disease. Alternatively, individuals may sleep a particular amount because of characteristics of their sleep physiology. In this study, we examined population-based data on the associations between sleep duration and several symptoms of sleep-related disease, reported snoring and daytime sleepiness. Results from 1877 independently living individuals between the ages of 50 and 65 years suggested that long, but not short, sleep durations were related to greater reported snoring. Higher levels of reported snoring and daytime sleepiness, but not habitual sleep duration, were related to measures of disease and lower psychosocial function. We suggest that future epidemiologic studies use such additional items as potential indicators of sleep-related disease.

Cardiovascular Diseases↗
PubMed1994

Effect of recruitment strategy on types of subjects entered into a primary prevention clinical trial.

Clinical trials typically recruit subjects through referrals or media promotion, with generalizability of the results often uncertain. As part of a primary prevention trial to evaluate strategies for increasing physical activity in sedentary men and women, two recruitment sources, a random-digit-dial telephone survey and a community media campaign, were used to identify subjects. Baseline characteristics of 357 randomized men and women aged 50 to 65 years were compared by recruitment source. Whereas there were few differences between recruitment sources for demographic variables, telephone survey recruitment was particularly successful in recruiting smokers and persons with other cardiovascular risk factors into the trial. Counter to expectations, subsequent exercise adherence rates did not differ by recruitment source. The results suggest that the survey method, while more expensive, may be particularly useful for locating higher-risk subjects who could especially benefit from increases in physical activity but who rarely are recruited through more traditional approaches.

Cardiovascular Diseases↗
PubMed1994

Effect of the number of islets on the outcome after transplantation in streptozotocin-induced diabetic rats.

This study tested the hypothesis that normalization of glucose homeostasis after islet transplantation is correlated to the number of islets, and by increasing this number a complete normalization of glucose homeostasis could be achieved, 1,200 or 2,400 islets were transplanted into the left kidney subcapsular space in streptozotocin-induced diabetic rats. Intravenous glucose tolerance tests were performed at 10 days, 3 and 6 months after transplantation. Transplantation of both 1,200 and 2,400 islets normalized the basal blood glucose levels within 24-48 hours, which remained normal for the entire study period of 6 months. Basal plasma insulin levels and body weight were also normalized in both transplanted groups. Transplantation of 2,400 islets achieved normal glucose-induced insulin secretion at 10 days after transplantation and for the following 6 months. In contrast, glucose intolerance was present in rats transplanted with only 1,200 islets. It is concluded that complete glucose homeostasis after islet transplantation is dependent on the number of transplanted islets and can be achieved by increasing this number.

Animals↗
PubMed1994

Deleterious effect of cyclosporins on the ischemic kidney in the rat and the protection by the calcium antagonist verapamil.

Cyclosporine A (CsA) nephrotoxicity has been suggested to be aggravated in the presence of ischemia, as occurs after renal transplantation. Cyclosporine G (CsG) may be less nephrotoxic than CsA. This study evaluated in the rat (1) the effect of CsA and CsG on blood flow and the function of the kidney subjected to 60 min of warm ischemia and (2) the protective effect of the calcium antagonist verapamil (VP). After left nephrectomy, ischemia was induced in the right kidney by the clamping of the kidney pedicle for 60 min, which resulted in a significant increase in serum creatinine (SCr) to 2.30 +/- 0.25 mg/dL by Day 1 with 25% mortality by Day 7. The administration of CsA or CsG (20 mg/kg i.v. daily for 7 days) after 60 min of renal ischemia significantly increased SCr and mortality compared with ischemia alone. In another set of experiments, 60 min of warm ischemia was applied to the right kidney and RBF was measured in both kidneys with a laser Doppler flowmeter. Blood flow in the ischemic kidney returned to the preischemic level by 15 min after the removal of the vascular clamp in the control animals. In contrast, in animals treated with CsA, a significant decrease in RBF was seen in both kidneys; however, blood flow in the ischemic kidney was significantly lower than that in the nonischemic kidney. CsG also decreased RBF in both kidneys, although in the left (nonischemic) kidney, RBF remained significantly higher with CsG than with CsA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Factors influencing energy intake of rats fed either a high-fat or a fat mimetic diet.

The aim of this study was to compare the effects of chronic feeding of a high-fat diet and a diet containing a fat mimetic on energy intake, body composition and tissue metabolism of mature female Sprague Dawley rats. Rats were fed a control, 25% kJ fat, diet for 10 days. Preference for this diet compared with a high-fat, 45% kJ fat, or fat mimetic, 25% kJ fat, diet was determined by offering rats two diets, in random order, for a period of 13.5 hours on three different occasions at two day intervals. Animals were then divided into three groups, receiving one of the three diets for 42 days. Dietary preference was tested again. Hepatic and muscle glucose and fatty acid utilization were measured in vitro and body composition was determined. Most of the rats preferred the mimetic diet over either control or high-fat diet, but there was no correlation between preference for a diet and intake of that diet during the experimental period. Animals fed either the high-fat or mimetic diet had greater energy intakes and body fat contents than control rats. Stepwise multiple regression determined which combination of variables correlated with energy intake of animals in each group. In rats fed high-fat diet, energy intake = 7.2 hepatic fatty acid oxidation (FAO) -0.2 hepatic glycogen - 131 muscle glycogen -0.9 hepatic fatty acid esterification (FAE) + 1.3 hepatic fatty acid synthesis (FAS) (R2 = 0.67). In rats fed mimetic diet, energy intake = 77.3 initial weight + 4.5 hepatic FAS -2.4 serum free fatty acids + 68.4 serum insulin (R2 = 0.67). These data suggest that obesity can be induced by changing the orosensory properties of a diet without changing macronutrient composition.

Adipose Tissue↗