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Biomedical subjects

S L Harper

Publications and source records attributed to S L Harper.

At least 19 recordsLinked to original sources
PubMed2001

Role of terminal nonhomologous domains in initiation of human red cell spectrin dimerization.

Human erythrocyte spectrin is an antiparallel heterodimer comprised of a 280 kDa alpha subunit and a 246 kDa beta subunit which further associates into tetramers in the red cell membrane cytoskeleton. Lateral association of the flexible rodlike monomers involves a multiple-step process that is initiated by a high affinity association near the actin-binding end of the molecule (dimer nucleation site). In this study, recombinant alpha and beta proteins comprising two or four "spectrin type" motifs with and without adjacent, terminal nonhomologous domains were evaluated for their relative contributions to dimer initiation, and the thermodynamic properties of these heterodimer complexes were measured. Sedimentation equilibrium studies showed that in the absence of the heterologous subunit, individual recombinant proteins formed weak homodimers (K(d) > 0.3 mM). When 2-motif (alpha20-21 and beta1-2) and 4-motif (alpha18-21 and beta1-4) recombinants lacking the terminal nonhomologous domains were paired with the complementary protein, high affinity heterodimers were formed in sedimentation equilibrium analysis. Both the alpha20-21/beta1-2 complex and the alpha20-21EF/betaABD1-2 complex showed stoichiometric binding with similar binding affinities (K(d) approximately 10 nM) using isothermal titration calorimetry. The alpha20-21/beta1-2 complex showed an enthalpy of -10 kcal/mol, while the alpha20-21EF/betaABD1-2 complex showed an enthalpy of -13 kcal/mol. Pull-down assays using alpha spectrin GST fusion proteins showed strong associations between all heterodimer complexes in physiological buffer, but all heterodimer complexes were destabilized by the presence of Triton X-100 and other detergents. Complexes lacking the nonhomologous domains were destabilized to a greater extent than complexes that included the nonhomologous domains. The detergent effect appears to be responsible for the apparent essential role of the nonhomologous domains in prior reports. Taken together, our results indicate that the terminal nonhomologous domains do not contribute to dimer initiation nor are they required for formation of high affinity spectrin heterodimers in physiological buffers.

Amino Acid Motifs↗
PubMed2001

Probing erectile function: S-(2-boronoethyl)-L-cysteine binds to arginase as a transition state analogue and enhances smooth muscle relaxation in human penile corpus cavernosum.

The boronic acid-based arginine analogue S-(2-boronoethyl)-L-cysteine (BEC) has been synthesized and assayed as a slow-binding competitive inhibitor of the binuclear manganese metalloenzyme arginase. Kinetic measurements indicate a K(I) value of 0.4-0.6 microM, which is in reasonable agreement with the dissociation constant of 2.22 microM measured by isothermal titration calorimetry. The X-ray crystal structure of the arginase-BEC complex has been determined at 2.3 A resolution from crystals perfectly twinned by hemihedry. The structure of the complex reveals that the boronic acid moiety undergoes nucleophilic attack by metal-bridging hydroxide ion to yield a tetrahedral boronate anion that bridges the binuclear manganese cluster, thereby mimicking the tetrahedral intermediate (and its flanking transition states) in the arginine hydrolysis reaction. Accordingly, the binding mode of BEC is consistent with the structure-based mechanism proposed for arginase as outlined in Cox et al. [Cox, J. D., Cama, E., Colleluori D. M., Pethe, S., Boucher, J. S., Mansuy, D., Ash, D. E., and Christianson, D. W. (2001) Biochemistry 40, 2689-2701.]. Since BEC does not inhibit nitric oxide synthase, BEC serves as a valuable reagent to probe the physiological relationship between arginase and nitric oxide (NO) synthase in regulating the NO-dependent smooth muscle relaxation in human penile corpus cavernosum tissue that is required for erection. Consequently, we demonstrate that arginase is present in human penile corpus cavernosum tissue, and that the arginase inhibitor BEC causes significant enhancement of NO-dependent smooth muscle relaxation in this tissue. Therefore, human penile arginase is a potential target for the treatment of sexual dysfunction in the male.

Animals↗
PubMed2001

Amniotic membrane inlay and overlay grafting for corneal epithelial defects and stromal ulcers.

OBJECTIVES: To determine the effect of amniotic membrane transplantation (AMT) on persistent corneal epithelial defects (PEDs) and to compare the efficacy between inlay and overlay techniques. METHODS: Thirty patients (30 eyes) underwent AMT for PED. The use of AMT was restricted to patients in whom all previous measures, including bandage contact lens and tarsorrhaphy, had failed. The amniotic membrane was placed on the surface of the cornea in overlay (group A) or inlay (group B) fashion. RESULTS: The PED healed after the first AMT in 21 eyes (70%) within an average of 25.5 days after surgery and recurred in 6 eyes (29%). Among the 22 eyes treated with an overlay AMT (group A), the PED healed after the first AMT in 14 eyes (64%) within an average of 24.5 days and recurred in 4 eyes (29%). Among the 8 eyes treated with an inlay AMT (group B), the PED healed within an average of 27.4 days after AMT, which did not statistically significantly differ from group A (P = .72). The PED healed after the first AMT in 7 eyes (88%) and recurred in 2 (29%) of 7 eyes. CONCLUSIONS: The AMT can be helpful in the treatment of PED in which all other conventional management has failed. However, the success rate in our study was not as high as that previously reported, and our results showed a high incidence of recurrences of epithelial defects. We did not find any difference between overlay and inlay techniques in terms of healing time and recurrence rate.

Adult↗
PubMed2001

Ontogeny of neurohormonal regulation of the cardiovascular system in the crayfish Procambarus clarkii.

Adult crayfish have a neurogenic heart which is modulated via inputs from the central nervous system and neurohormones, which act on the cardiac ganglion or directly on the myocardium. This study investigates the ontogeny of cardiac regulation by exploring the temporal sequence of cardiac sensitivity to injections of cardioactive neurohormones (proctolin, serotonin and octopamine) and the neurotransmitter gamma-aminobutyric acid. The cardiac response (delta in heart rate, stroke volume, or in cardiac output) to each neurohormone at each developmental stage was assessed. The observed response elicited by each cardioactive drug was stage dependent and changed as the animals progressed from embryonic through larval and juvenile periods. During early developmental stages, octopamine, serotonin, and proctolin (10(-9)-10(-3) M) did not result in a modulation of stroke volume, yet in later developmental stages they caused significant increases in stroke volume, at comparable concentrations. Early developmental stages are capable of regulating cardiac function, however, the mechanisms appear to be quite different from those used by adults. Evidence is also provided to support the hypothesis that cardiac function is initiated prior to the establishment of an adult-like regulatory system.

Adrenergic alpha-Agonists↗
PubMed2001

Sources of variability in collection and preparation of paint and lead-coating samples.

Chronic exposure of children to lead (Pb) can result in permanent physiological impairment. Since surfaces coated with lead-containing paints and varnishes are potential sources of exposure, it is extremely important that reliable methods for sampling and analysis be available. The sources of variability in the collection and preparation of samples were investigated to improve the performance and comparability of methods and to ensure that data generated will be adequate for its intended use. Paint samples of varying sizes (areas and masses) were collected at different locations across a variety of surfaces including metal, plaster, concrete, and wood. A variety of grinding techniques were compared. Manual mortar and pestle grinding for at least 1.5 min and mechanized grinding techniques were found to generate similar homogenous particle size distributions required for aliquots as small as 0.10 g. When 342 samples were evaluated for sample weight loss during mortar and pestle grinding, 4% had 20% or greater loss with a high of 41%. Homogenization and sub-sampling steps were found to be the principal sources of variability related to the size of the sample collected. Analysis of samples from different locations on apparently identical surfaces were found to vary by more than a factor of two both in Pb concentration (mg cm-2 or %) and areal coating density (g cm-2). Analyses of substrates were performed to determine the Pb remaining after coating removal. Levels as high as 1% Pb were found in some substrate samples, corresponding to more than 35 mg cm-2 Pb. In conclusion, these sources of variability must be considered in development and/or application of any sampling and analysis methodologies.

Child↗
PubMed2001

Wegener's granulomatosis: the relationship between ocular and systemic disease.

OBJECTIVE: Wegener's granulomatosis (WG) is an etiologically obscure entity with multiple systemic manifestations. Ocular involvement is present in up to 58% of patients with WG. We describe a series of patients with ocular manifestations of WG to evaluate the presence of ocular lesions in the setting of systemic WG and to determine the value of ocular inflammation in the diagnosis of WG. METHODS: A computerized database was used to generate a list of patients cared for in the Ocular Immunology Service of the Massachusetts Eye and Ear Infirmary during the 10 year period 1988-98 with a diagnosis of Wegener's granulomatosis. A detailed chart review was undertaken to determine demographic characteristics, history, initial manifestation of WG, initial ocular presentation, biopsy results, laboratory testing results, treatment, total followup period, and final outcome. RESULTS: Forty-seven patients diagnosed with WG were identified. Twenty-eight were women (59.6%), 19 were men (40.4%). The average age was 53 years (range 18-90). Patients were divided into 4 groups. Group I included 27 patients (57.4%) who had systemic disease first and who subsequently developed an ocular lesion. Group II included 3 patients (6.3%) who had ocular inflammation first and who then subsequently developed systemic manifestations of WG. Group III included 3 patients (6.3%) who presented due to ocular symptoms but, on initial evaluation by us, were found to have occult systemic manifestations consistent with WG or biopsy evidence of WG. Group IV included 14 patients (30%) with ocular lesions and no history or presence of systemic disease at their last followup visit. CONCLUSION: Ocular inflammation can occur with or without obvious systemic manifestations of WG. It may represent the first sign of WG that enables the knowledgeable physician to diagnose this potentially lethal disease.

Adolescent↗
PubMed2000

Biochemical analysis of the Kruppel-associated box (KRAB) transcriptional repression domain.

The Kruppel-associated box (KRAB) domain is a 75-amino acid transcriptional repressor module commonly found in eukaryotic zinc finger proteins. KRAB-mediated gene silencing requires binding to the RING-B box-coiled-coil domain of the corepressor KAP-1. Little is known about the biochemical properties of the KRAB domain or the KRAB.KAP-1 complex. Using purified components, a combination of biochemical and biophysical analyses has revealed that the KRAB domain from the KOX1 protein is predominantly a monomer and that the KAP-1 protein is predominantly a trimer in solution. The analyses of electrophoretic mobility shift assays, GST association assays, and plasmon resonance interaction data have indicated that the KRAB binding to KAP-1 is direct, highly specific, and high affinity. The optical biosensor data for the complex was fitted to a model of a one-binding step interaction with fast association and slow dissociation rates, with a calculated K(d) of 142 nm. The fitted R(max) indicated three molecules of KAP-1 binding to one molecule of the KRAB domain, a stoichiometry that is consistent with quantitative SDS-polyacrylamide gel electrophoresis analysis of the complex. These structural and dynamic parameters of the KRAB/KAP-1 interaction have implications for identifying downstream effectors of KAP-1 silencing and the de novo design of new repression domains.

Amino Acid Sequence↗
PubMed2000

Initiation of spectrin dimerization involves complementary electrostatic interactions between paired triple-helical bundles.

The spectrin heterodimer is formed by the antiparallel lateral association of an alpha and a beta subunit, each of which comprises largely a series of homologous triple-helical motifs. Initiation of dimer assembly involves strong binding between complementary motifs near the actin-binding end of the dimer. In this study, the mechanism of lateral spectrin association at this dimer nucleation site was investigated using the analytical ultracentrifuge to analyze heterodimers formed from recombinant peptides containing two or four homologous motifs from each subunit (alpha20-21/beta1-2; alpha18-21/beta1-4). Both the two-motif and four-motif dimer associations were weakened substantially with increasing salt concentration, indicating that electrostatic interactions are important for the dimer initiation process. Modeling of the electrostatic potential on the surface of the alpha20 and beta2 motifs showed that the side of the motifs comprising the A and B helices is the most favorable for association, with an area of positive electrostatic potential on the AB face of the beta2 motif opposite negative potential on the AB face of the alpha20 motif and vise versa. Protease protection analysis of the alpha20-21/beta1-2 dimer showed that multiple trypsin and proteinase K sites in the A helices of the beta2 and alpha21 motifs become buried upon dimer formation. Together, these data support a model where complementary long range electrostatic interactions on the AB faces of the triple-helical motifs in the dimer nucleation site initiate the correct pairing of motifs, i.e. alpha21-beta1 and alpha20-beta2. After initial docking of these complementary triple-helical motifs, this association is probably stabilized by subsequent formation of stronger hydrophobic interactions in a complex involving the A helices of both subunits and possibly most of the AB faces. The beta subunit A helix in particular appears to be buried in the dimer interface.

Animals↗
PubMed2000

Intraocular lens explantation in uveitis.

Visual rehabilitation after cataract surgery has been improved with the development of IOLs. These lenses are well tolerated in many uveitis patients when complete control of preoperative inflammation is achieved. However, in some patients, IOL placement after cataract extraction results in chronic inflammation, deposition of inflammatory cells and debris on the IOL surface, and inflammatory membrane formation despite antiinflammatory coverage. Patients with systemic diseases characterized by chronic inflammation, such as sarcoidosis and JRA, and those with chronic ocular inflammatory conditions or inflammation involving the intermediate segment of the eye may be at high risk for these complications. In patients in whom antiinflammatory therapy fails, adequate control of inflammation may be achieved after lens explantation.

Cataract Extraction↗
PubMed2000

Choroidopathy of systemic lupus erythematosus.

PURPOSE: To describe the ocular and systemic manifestations associated with systemic lupus erythematosus (SLE) choroidopathy. METHODS: Three new cases of choroidopathy in patients with active SLE were described. Twenty-five published cases of lupus choroidopathy were summarized. RESULTS: There have been 28 cases of lupus choroidopathy (47 involved eyes) that have been reported in the English literature since 1968, including the three current cases. Only two of the patients were male. The choroidopathy was bilateral in 19 patients (68%). All 28 patients (100%) had active systemic vascular disease at the onset of their choroidopathy; 18 (64%) had nephropathy and 10 (36%) had central nervous system (CNS) lupus vasculitis. All but one of the patients had a known diagnosis of SLE at the onset of choroidopathy. 30 of the 47 involved eyes had presenting visual acuity of 20/40 or better; 14 eyes showed improvement in visual acuity with therapy. 23 patients (82%) had resolution of their choroidopathy when their systemic disease was brought under control. Despite treatment, 4 of the 28 patients (14%) died from complications of SLE. CONCLUSIONS: Although less known than retinopathy, lupus choroidopathy may be more common than generally appreciated. It usually serves as a sensitive indicator of lupus activity. The presence of SLE choroidopathy is generally indicative of coexistent (although sometimes occult) nephropathy, CNS vasculitis, and other SLE visceral lesions. Immunomodulation of the systemic disease can lead to improvement and resolution of the systemic vasculitis as well as the choroidopathy.

Adolescent↗
PubMed2000

Implementing a syphilis elimination and importation control strategy in a low-incidence urban area: San Diego County, California, 1997-1998.

OBJECTIVES: This study assessed a strategy designed to contain imported cases of syphilis and prevent reestablishment of ongoing transmission. METHODS: Reported syphilis cases during an endemic period (1990-1992) and an elimination period (1997-1998) were compared in San Diego, Calif. The elimination strategy, which focuses on rapid reporting of infectious syphilis cases by clinicians, prompt partner and sexual network management, outreach to marginalized populations, and implementation of an outbreak containment plan, was evaluated. RESULTS: Infectious syphilis incidence rates declined from 18.3 per 100,000 in 1998 to 1.0 per 100,000 in 1998. Of the 46 cases involving probable infection during 1997-1998, 19 (41%) were imported, mostly (79%) from Mexico. Outbreak containment procedures were implemented successfully for 2 small clusters. Outreach workers provided sexually transmitted disease information to a large number of individuals; however, no cases of infectious syphilis were identified, suggesting that syphilis transmission was not occurring among marginalized groups. CONCLUSIONS: This syphilis elimination and importation control strategy will require monitoring and adjustments. Controlling syphilis along the US-Mexico border is a necessary component of syphilis elimination in the United States.

California↗
PubMed1998

The ocular manifestations of rheumatoid disease.

The inflammatory arthropathies that affect the eye most commonly are RA, JRA, and the seronegative spondyloarthropathies. These conditions not only cause devastating systemic findings but can be the source of damaging ocular disease. The inflammatory nature of these entities, with the accompanying liberation of mediators of inflammation, can result in a cycle of tissue destruction that culminates in blindness. The diseases reviewed can present first with systemic or ocular findings; thus, all physicians must be equipped with the appropriate knowledge to make accurate and timely diagnoses so that appropriate management strategies can be employed. The successful recognition and treatment of these conditions can prevent their associated systemic and ocular morbidity.

Adolescent↗
PubMed1997

The exon 46-encoded sequence is essential for stability of human erythroid alpha-spectrin and heterodimer formation.

Human erythroid alpha-spectrin alleles responsible for hereditary elliptocytosis (alphaHE alleles) undergo increased incorporation into red blood cell membranes when the polymorphism alphaLELY (LELY: Low Expression LYon) occurs in trans. The alphaLELY polymorphism is characterized by a mutation in exon 40 at codon 1857 (CTA --> GTA, Leu --> Val) and the partial (50%) skipping of exon 46, which encodes residues 2177-2182 (Wilmotte et al, J Clin Invest 91:2091, 1993). Both of these peptide sequence alterations are located within the region of the alpha-chain involved in initiating heterodimer assembly, and either or both mutations could potentially contribute to decreased incorporation of alpha-chains from the alphaLELY allele in heterozygotes into red blood cell membranes. These possibilities were evaluated by testing the protease resistance and in vitro binding properties of normal and mutant recombinant 4-motif alpha subunit peptides containing the dimer initiation region. The two forms of alpha spectrin produced by alternative mRNA splicing of the alphaLELY allele were represented by alpha18-21(1857), a peptide with the codon 1857 mutation and retaining the exon 46 encoded sequence, and alpha18-21(1857-Delta46), a peptide carrying both the 1857 codon mutation and the exon 46 deletion. The properties of these two recombinant peptides were compared with alpha18-21, a peptide with the normal sequence at codon 1857 and retaining the exon 46 encoded sequence. The codon 1857 mutation does not adversely affect dimer formation, but it is responsible for the increased trypsin cleavage between the alphaIV and alphaV domains that was the characteristic feature initially used to identify the alphaLELY (SpalphaV/41) polymorphism (Alloisio et al, J Clin Invest 87:2169, 1991). Deletion of the six amino acids encoded by exon 46 perturbs folding of the alpha21 motif, because this region of the alpha18-21(1857-Delta46) peptide is rapidly degraded and this recombinant peptide is unusually prone to self-aggregation. Exon 46 deletion reduces, but does not eliminate, dimerization. Comparison of mild trypsin proteolytic products from an alphaLELY homozygote and the two alphaLELY recombinant peptides strongly suggests that little, if any, of the 50% of the alpha chains from the alphaLELY allele that contain the exon 46 deletion are incorporated into the mature erythroid membrane. Based on the in vitro analysis of recombinant alphaLELY peptides, the inability of detectable amounts of exon 46(-) alpha chains to assemble into the mature membrane skeleton in vivo is probably due to a combination of decreased dimer binding affinity and increased proteolytic degradation of these mutant chains.

Alleles↗
PubMed1997

Physical properties of a single-motif erythrocyte spectrin peptide: a highly stable independently folding unit.

Spectrin is a long flexible rod-like actin cross-linking protein mostly comprised of many tandem homologous 106-residue motifs. In this study, the conformational stability and physical properties of a single homologous motif peptide, alpha1, were evaluated and compared to intact spectrin monomers and alphabeta heterodimers. It is interesting that while spectrin dimers elongate by about 3-fold in low ionic strength buffers relative to their size in physiological buffers, the single-motif peptide does not show significant changes in secondary structure in 10 mM phosphate buffer compared with isotonic buffer. This single-motif peptide is monomeric in physiological buffer as demonstrated by equilibrium sedimentation studies, and its hydrodynamic radius determined by gel filtration and dynamic light scattering of about 2.2 nm is consistent with an elongated rod-like shape. Unfolding of the single-motif peptide in urea solutions was similar to unfolding of intact heterodimers. Differential scanning calorimetry analyses showed that this single motif undergoes a reversible two-state transition with a Tm of 53 degrees C and an enthalpy of 65 kcal/mol in physiological buffer. Thermal stability was unaffected by ionic strength changes, but was decreased below physiological pH. These data show that this 13 kDa spectrin motif is a monomeric, highly stable, triple-helical, independently folding protein building block with physical characteristics that define many of the structural properties of the 526 kDa spectrin heterodimer. In contrast, interactions between adjacent motifs are probably responsible for spectrin's molecular flexibility and elasticity.

Calorimetry, Differential Scanning↗
PubMed1991

Non-neoplastic ovarian cysts with ectopic pregnancy.

Thirty non-neoplastic ovarian cysts, discovered at laparotomy in association with 27 cases of tubal ectopic pregnancy, were examined to document changes seen in early pregnancy and possibly to identify precursor lesions to the luteinized cysts of later pregnancy. The ages of the patients ranged from 16 to 40 years, and length of gestation varied between 5 and 11 weeks. Of the 30 cysts, 12 were corpus luteum cysts; 10 were called lutein cysts, nonspecific, and had a lining of luteinized cells of a single cell type; three cysts were luteinized follicles; four cysts were unlined or lined by a single layer of cells. The origin of these four was not obvious, and they were called "simple cysts." The last was a granulosa-theca lutein cyst. Corpus luteum cysts are assumed to arise from the corpus luteum, and simple cysts appear to arise from both corpus luteum cysts and other follicular structures. The nonspecific lutein cysts appeared to arise from structures other than the corpus luteum or corpus luteum cysts; these nonspecific lutein cysts may be precursors to the so-called large solitary luteinized follicle cyst of pregnancy.

Adolescent↗
PubMed1987

Effects of the antihypertensive prostaglandin analog Ro 22-1327 on regional blood flows in the spontaneously hypertensive rat.

The hemodynamic effects of Ro 22-1327 [nat-(15R,16R)-16-fluoro-15-hydroxy-9-oxoprosta-5(Z),1 3(E)-dienoic acid], a prostaglandin E2 analog, were evaluated in anesthetized spontaneously hypertensive rats (SHR). Within 3-4 h of topical application (1 and 3 mg/kg), a dose-dependent decrease in mean arterial blood pressure was observed. At 6 h following drug application, steady-state mean arterial pressure was 79-87% of control (pretreatment) values. Increments in blood flow, as well as reductions in vascular resistance, were apparent in the kidneys, skin, and the majority of the splanchnic organs. The magnitude of the drug-induced hyperemia decreased from the duodenum to the ileum. Significant elevations in colonic blood flow were not apparent with either dose, suggesting a proximal-to-distal gradient of response of the intestinal vasculature to the drug. The results of this study indicate that Ro 22-1327 is an effective antihypertensive agent when applied topically in SHR. Our results also imply that the splanchnic vascular bed contributes heavily to the antihypertensive activity of the compound.

Animals↗