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Biomedical subjects

R A Harrison

Publications and source records attributed to R A Harrison.

At least 19 recordsLinked to original sources
PubMed2000

cAMP-dependent protein kinase control of plasma membrane lipid architecture in boar sperm.

Bicarbonate/CO(2), a physiological effector of sperm capacitation, has been shown to induce a rapid and reversible change in the lipid architecture of the plasma membrane of live boar sperm: the change is detectable as an increase in the cells' ability to bind the fluorescent dye merocyanine, a characteristic which implied an increase in lipid packing disorder (Harrison et al. 1996. Mol Reprod Dev 45:378-391). Evidence suggested that cAMP may act as a second messenger in the system, and we have therefore investigated this cAMP-dependency in more detail. Bicarbonate stimulates cAMP levels within 1 min in a dose-dependent fashion, prior to parallel increases in merocyanine binding. Although the potent somatic cell adenylyl cyclase activator forskolin is unable to induce significant increases in cAMP or merocyanine binding, increases in merocyanine binding are inducible in a dose-dependent fashion by 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphothioate, a cAMP analogue highly specific in its ability to stimulate protein kinase A; moreover, the bicarbonate-induced membrane change is inhibited by H89, a specific protein kinase A inhibitor. Neither bisindolylmaleimide I (protein kinase C inhibitor) nor lavendustin A (protein tyrosine kinase inhibitor) are inhibitory. In the presence of low levels of the potent phosphodiesterase inhibitor papaverine, increases in merocyanine binding are enhanced by okadaic acid and (more effectively) by calyculin (both protein phosphatase inhibitors). We conclude that boar sperm plasma membrane lipid architecture is controlled via a target protein that is dynamically phosphorylated by cAMP-dependent protein kinase and dephosphorylated by protein phosphatase type 1. Mol. Reprod. Dev. 55:220-228, 2000.

Adenylyl Cyclase Inhibitors↗
PubMed2000

DNA immunization with Onchocerca volvulus genes, Ov-tmy-1 and OvB20: serological and parasitological outcomes following intramuscular or GeneGun delivery in a mouse model of onchocerciasis.

The objective of this study was to evaluate whether the distinct immune responses invoked by epidermal and intramuscular DNA immunization could be harnessed to improve upon the levels of protection to Onchocerca volvulus infective larvae achieved previously by recombinant protein immunization. Intramuscular (IM) and epidermal (GeneGun) routes of DNA immunization generally drive T helper1 and Th2 dominant responses, respectively. This dichotomy was used in an attempt to further define the nature of host-protective immunity in a mouse model of onchocerciasis. Mice were immunized with DNA plasmids expressing the O. volvulus antigens, Ov-TMY-1 (tropomyosin) and OvB20 (a nematode specific gene product). While, IM and GeneGun immunization of mice with Ov-tmy-1 induced expected Th1/Th2-associated IgG isotype profiles, mice responded to OvB20 immunization with a Th2 dominant response, irrespective of the delivery route. Despite inducing potent serological responses, neither DNA construct promoted statistically significant levels of protection to L3 challenge infection. We conclude that DNA immunization has good potential for induction of humoral responses against nematode infections and that serological responses alone do not predict vaccination efficacy under the conditions used here to measure host resistance to parasite challenge.

Animals↗
PubMed2000

The capacitating agent bicarbonate induces protein kinase A-dependent changes in phospholipid transbilayer behavior in the sperm plasma membrane.

A flow cytometric procedure was used to follow the effect of bicarbonate, a key inducer of sperm capacitation in vitro, on the transbilayer behavior of C6NBD-phospholipids in the plasma membrane of living acrosome-intact boar spermatozoa under physiological conditions. In the absence of bicarbonate, 97% of C6NBD-phosphatidylserine and 78% of C6NBD-phosphatidylethanolamine was rapidly translocated from the outer leaflet to the inner, whereas relatively little C6NBD-phosphatidylcholine and C6NBD-sphingomyelin was translocated (15% and 5%, respectively). Inclusion of 15 mM bicarbonate/5%CO(2) markedly slowed down the rates of translocation of the aminophospholipids without altering their final distribution, whereas it increased the proportions of C6NBD-phosphatidylcholine and C6NBD-sphingomyelin translocated (30% and 20%, respectively). Bicarbonate activated very markedly the outward translocation of all four phospholipid classes. The changes in C6NBD-phospholipid behavior were accompanied by increased membrane lipid disorder as detected by merocyanine 540, and also by increased potential for phospholipase catabolism of the C6NBD-phospholipid probes. All three changes were mediated via a cAMP-dependent protein phosphorylation pathway. We suspect that the changes result from an activation of the non- specific bidirectional translocase ('scramblase'). They have important implications with respect to sperm fertilizing function.

4-Chloro-7-nitrobenzofurazan↗
PubMed1999

DNA immunisation with Onchocerca volvulus chitinase induces partial protection against challenge infection with L3 larvae in mice.

The parasitic nematode, Onchocerca volvulus is a major cause of blindness and dermal pathology in tropical regions. A vaccine directed to infective larvae would provide a valuable control tool alongside the current methods of chemotherapy and vector control. Previously we have described the identification of a chitinase cDNA that is expressed in a stage specific manner by O. volvulus infective third stage (L3) larvae. To evaluate its host protective potential, the complete open reading frame was cloned into the eukaryotic expression plasmid pJW4303 and used to vaccinate mice by DNA immunisation with the Accell GeneGun. The survival of challenge infective larvae was monitored using implanted micropore chambers. In the first trial, mice immunised 3 times over 4 months with 1 microg O. volvulus chitinase DNA responded with modest antibody responses dominated by IgG2a and exhibited a 36% (p=0.189, NS) reduction in parasite survival compared with challenge controls. In the second trial, an increased dose of DNA (5 microg) and more frequent immunisations (5 times over 6 months) stimulated an IgG1 dominant response and a 53% reduction in parasite survival (p=0.042). Antibodies from the vaccinated mice reacted with the cuticle of post-infective L3 larvae, implying that this may be the site of immune attack following secretion of chitinase.

Animals↗
PubMed1999

Protective immune responses induced by vaccination with an expression genomic library of Leishmania major.

To develop an effective vaccine against the intracellular protozoan parasite Leishmania spp., we investigated the feasibility of expression library immunization (ELI) in the mouse. Genomic expression libraries of L. major were constructed and used to immunize mice. One of the three libraries (L1, with 10(5) clones) induced a significant protective immune response and delayed the onset of lesion development in highly susceptible BALB/c mice after i.m. immunization, compared with control mice immunized with the empty vector (EV). L1 was then divided into five sublibraries of approximately 2 x 10(4) clones each. Mice immunized with one of the sublibraries (SL1A) developed an even stronger protective effect than that induced by L1. SL1A was further divided into 20 sublibraries (SL2) of approximately 10(3) clones each. One of the SL2 libraries (SL2G) induced a strong protective effect against L. major infection. In direct comparative studies, the protective effect of the sublibraries was in the order of SL2G > SL1A > L1. Lymphoid cells from mice vaccinated with SL2G produced more IFN-gamma and NO, compared with cells from control mice injected with EV. Serum from the vaccinated mice also contained more parasite-specific IgG2a Ab, compared with controls. Therefore, these data demonstrate that ELI is feasible against this complex intracellular parasitic infection, by preferentially inducing the development of Th1 responses. Furthermore, by sequential division of the libraries, this approach may be used to enrich and identify protective genes for effective gene vaccination against other parasitic infections.

Animals↗
PubMed1999

Molecular cloning and characterization of the polypeptide backbone of Schistosoma mansoni circulating cathodic antigen.

One of the candidate schistosome antigens for the development of a circulating antigen detection diagnostic assay is the circulating cathodic antigen (CCA). Detection of CCA in urine provides a non-invasive assay with high sensitivity. Previously we reported that CCA is secreted in patients' urine as a small molecular weight material which is probably of a proteinaceous nature. In an attempt to further characterize the secreted component of CCA, we used a monoclonal antibody (MAb) reactive with urine-CCA to isolate an adult worm cDNA clone (SmN3-1) that encodes the polypeptide backbone of CCA. The sequence, gene organization and expression of SmN3-1 were analyzed. The 1.6 kb nucleotide and 347 amino acid sequences of SmN3-1 showed no significant homology to any published sequence. The size and antigenic properties of the expression product of SmN3-1 in Escherichia coli greatly resembled the CCA molecule excreted in urine, suggesting that the latter is primarily composed of the protein element of CCA.

Amino Acid Sequence↗
PubMed1999

Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: elimination of labeling artifacts.

Reliable protocols were established for investigating asymmetric distributions of 6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-caproyl (C6NBD) phospholipids in the plasma membrane of boar sperm cells under physiological conditions. A method based on fluorescence resonance energy transfer was used to ensure that incorporation of the fluorescent phospholipids into the sperm proceeded via monomeric transfer. The total amount of incorporated phospholipid fluorescence and the proportion of translocated phospholipid fluorescence were determined by flow cytometric analysis before, and after, dithionite destruction of outer leaflet fluorescence. Catabolism of incorporated fluorescent phospholipids was blocked with phenylmethylsulfonyl fluoride. Membrane-damaged cells were detected with impermeant DNA stains, thereby enabling their exclusion from subsequent analyses of the flow cytometric data, whence it could be demonstrated that the labeled phospholipids were incorporated only via the outer plasma membrane leaflet in living sperm cells. Phospholipid uptake and internalization was followed at 38 degrees C. After 1 hr of labeling, about 96% of the incorporated C6NBD-phosphatidylserine, 80% of C6NBD-phosphatidylethanolamine, 18% of C6NBD-phosphatidylcholine, and 4% of C6NBD-sphingomyelin were found to have moved across the plasma membrane bilayer to the interior of the spermatozoa. These inward movements of fluorescent phospholipids were ATP-dependent and could be blocked with sulfhydryl reagents. Movements from the inner to the outer leaflet of the sperm plasma membrane were minimal for intact fluorescent phospholipids, but were rapid and ATP-independent for fluorescent lipid metabolites. The described method enables, for the first time, assessment of changes in lipid asymmetry under fertilizing conditions.

4-Chloro-7-nitrobenzofurazan↗
PubMed1999

Associations of MHC genes with levels of caries-inducing organisms and caries severity in African-American women.

The aim of this investigation was to evaluate the relationship between MHC alleles at the HLA-DRB1, DQB1 and TNFa microsatellite loci and levels of oral bacteria that play a role in the etiology of dental caries, and the DMFS index in 186 AA primparous women. The average age of the cohort was 20.8+/-3.7 years. The median DMFS index was 9 (range 0-68). High levels of S. mutans were positively associated with DRB1*3 and DRB1*4 presence (p < or = 0.005). DRB1*8 was positively associated with higher levels of S. mutans as a percentage of total Streptococci (p = 0.04). DRB1*1 was positively associated with high levels L. casei (p = 0.04). DQB1 alleles were not observed associated with oral bacterial levels. TNFa allele 103 was negatively associated (p = 0.04), and TNFa 117 was positively associated (p = 0.007), with high levels of L. acidophilus. No significant associations were observed between any DRB1, DQB1 or TNFa allele and the DMFS index. These results support an hypothesis of an association between host HLA class II and TNFa genetic profile and colonization of S. mutans, L. casei, and L. acidophilus thought to be pathogens involved in the etiology of dental caries.

Adolescent↗
PubMed1999

A mixed model for factors predictive of pain in AIDS patients with herpes zoster.

A unifying model of herpes zoster pain presents considerable analytical challenges due to the requirement for prospective data collection and the varying rates of pain resolution reported by individual patients. Demographic, clinical, and quality-of-life measures were collected on 166 human immunodeficiency virus (HIV)-infected patients enrolled in a randomized, controlled trial of antiviral therapy of herpes zoster comparing acyclovir with sorivudine. A "mixed model" was used to assess factors predictive of pain severity, activity impairment, and sleep interruption. The average rate of change in acute pain was -0.04 unit pain per day for the first month. Chronic pain decreased -0.12 per month for months 1-12. Acute pain severity was positively correlated with number of new skin vesicles, analgesic use, and baseline pain, and negatively related to percentage of lesion healing and crusting. Postherpetic neuralgia was correlated with baseline pain, pain at 1 month, and duration of lesions. Treatment group, gender, race, and CD4 count were not related to change in pain severity. These analyses verify the significance of baseline pain as a significant predictor of pain resolution and average pain severity as a predictor of return to normal daily activities and sleep. The severity of acute pain at presentation and at 1 month are significant predictors of chronic pain.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Understanding meta-analysis in cancer epidemiology: dietary fat and breast cancer.

Meta-analyses of the relationship between dietary fat and breast cancer risk using different methodologies have reported conflicting results. This investigation compares methodologic aspects of meta-analyses of patient data (MAP) with meta-analyses of data from the literature (MAL), and computes relative risk (RR) estimates from a random effects model using 28 published studies of dietary fat and breast cancer. MAP and MAL results compare closely when homogeneity is verified. When statistical homogeneity is rejected, a random effects model adjusting for study design and location is appropriate. The highest RR was found for case-control studies of European women (RR: 1.46), followed by North American case-control studies (RR: 1.25), case-control studies of women on other continents (RR: 1.23), cohort studies in Europe (RR: 1.20), and cohort studies in North America (RR: 1.02). The overall risk estimate in a MAL with heterogeneous studies should be interpreted only in a conditional model.

Breast Neoplasms↗
PubMed1999

Sperm subpopulations in boar (Sus scrofa) and gazelle (Gazella dama mhorr) semen as revealed by pattern analysis of computer-assisted motility assessments.

The aim of this study was to test the suitability of "pattern analysis" for the exploration of data provided by computer-assisted semen analysis methods. Data sets derived from the examination of boar sperm responses to bicarbonate and caffeine (measurements on 3208 spermatozoa) and from studies of semen cryopreservation in Mohor gazelles (7278 spermatozoa) were reanalyzed. A nonhierarchical classification method was used to generate initial subgroups of spermatozoa (9 for boar, 13 for gazelle). The subgroup centroids were fused, yielding three boar sperm subpopulations and four gazelle sperm subpopulations distinguished by sperm behaviors. Bicarbonate and caffeine both induced major transitions (p < 0.0001) of boar sperm behavior, detected as shifts in group membership (from group 2, i.e., active but nonlinear movement, into group 1, i.e., linear, rapid movement). Some spermatozoa (approximately 3%) were refractory to both caffeine and bicarbonate. The gazelle sperm subpopulation structure was affected by the inclusion of equex (sodium triethanolamine lauryl sulfate) in the cryoprotective diluents. Equex suppressed the appearance of spermatozoa with erratic behavior (p < 0.0001; high curvilinear velocity, low linearity, low straight-line velocity) after cryopreservation. The proportion of these erratic spermatozoa was positively correlated with animal age (r = 0.68, p = 0.029). Pattern analysis revealed novel aspects of the data not seen in the original investigations and usefully supplemented the more standard data analysis approaches.

Animals↗
PubMed1998

Factors affecting survival following local, regional, or distant recurrence from localized melanoma.

BACKGROUND AND OBJECTIVES: Approximately one third of all melanoma patients will experience disease recurrence. Factors that affect patient survival following local, regional, or distant first recurrences of localized melanoma are the subject of this investigation. METHODS: Survival times for a total of 1,085 first recurrences from 4,568 localized melanoma patients were examined in relationship to patient and disease factors by Cox regression. Nearly half (48.8%) of all first recurrences were regional, 21.8% were local, and 29.4% were distant recurrences. RESULTS: Survival following recurrence differed significantly by site of recurrence (local, regional, or distant; P < 0.0001). Within each site, the median survival time did not differ by time of recurrence following diagnosis. Significant tumor factors for survival following local recurrence included tumor thickness (P = 0.0263) and lesion location (P < 0.0001). For regional recurrences, survival was significantly related to ulceration (P = 0.0105) and whether the recurrence was combined with a local recurrence (P = 0.0429). Survival following distant metastasis was related to number of distant sites (P < 0.0001) and whether a visceral site was involved (P < 0.0001). CONCLUSIONS: Patient and tumor characteristics predict survival following recurrence. Regardless of disease-free interval, long-term follow-up of melanoma patients is necessary. Patients experiencing distant metastasis have the shortest median survival time compared to patients experiencing local or regional recurrences.

Disease-Free Survival↗
PubMed1998

Clinical outcomes of localized melanoma of the foot: a case-control study.

The controversy over whether melanoma of the foot has a poorer prognosis than melanoma of the leg remains unresolved. This investigation used a case-control design to address this issue. This design consisted of a survival analysis of 119 cases with localized melanoma of the foot and 238 controls with localized melanoma of the leg that were matched on prognostic factors including tumor thickness, ulceration, surgical treatment, gender, year of diagnosis, and age. There was a statistically significant difference between the survival rates of cases and controls. The 5-year survival rate for cases was 74.3% compared to 85.2% for controls. At 10 years, the survival rate was 63.6% for cases and 77.2% for controls. Cases experienced a higher percentage of distant recurrences than controls. These results imply that patients with melanoma of the foot have a poorer survival than patients with melanoma of the leg after controlling for prognostic factors.

Adult↗
PubMed1998

Socioeconomic characteristics and melanoma incidence.

PURPOSE: This investigation examines the relationship between socioeconomic status (SES) and melanoma incidence in counties included in the Surveillance, Epidemiology, and End Results Registry (SEER) in the United States from 1973 to 1993. METHODS: Cases included whites, aged at least 15 years, with a morphologic diagnosis of malignant melanoma, residing in one of 199 counties at the time of diagnosis. County level measures of SES including median household income, percentage of high school graduates, and percentage of families below poverty were abstracted from the 1950, 1960, 1970, 1980, and 1990 U.S. Census data. The relationship between SES factors and melanoma rates was examined by hierarchical Poisson regression. RESULTS: The percentage of high school graduates was significantly and positively associated with the incidence of melanoma (relative risk (RR), 1.28; 95% confidence interval (CI), 1.21-1.35), after controlling for age at diagnosis, gender, time period, latitude, and percentage of Hispanics in the county. Percentage of families below poverty was significantly inversely associated with the incidence of melanoma (RR, 0.66; 95% CI, 0.55-0.78). When education and poverty were included in the same model, both the positive effects of education (RR, 1.23; 95% CI, 1.16-1.31) and the negative effects of poverty (RR, 0.85; 95% CI, 0.74-0.98) persisted. In contrast, median household income was not associated with melanoma incidence in a similar multivariable model (RR, 1.00; 95% CI, 0.99-1.00). CONCLUSION: Whether the effect of education on incidence of melanoma reflects lifestyle behaviors that modify exposure to sunlight or some other factor remains unclear. Nonetheless, the findings of this study suggest that the determinant is primarily related to education, not income.

Adolescent↗
PubMed1998

Enhanced binding of zona pellucida proteins to the acrosomal region of the intact boar spermatozoa in response to fertilizing conditions: a flow cytometric study.

In this investigation we sought to determine whether sperm capacitation in vitro is accompanied by changes in the functional presence of zona binding sites on the plasma membrane of boar spermatozoa. During sperm incubation at 39 degrees C in various modifications of a Tyrode's-based in vitro fertilization medium, the zona binding ability of individual spermatozoa was assessed with fluorescein-conjugated solubilized zona pellucida proteins, using a flow cytometer. Propidium iodide was routinely included to allow simultaneous assessment of membrane integrity; rhodamine-conjugated peanut agglutinin was used to assess acrosomal status. During incubation in the fertilization medium, a subpopulation of live acrosome-intact spermatozoa developed enhanced binding of the fluorescein-conjugated solubilized zona proteins. Microscopy revealed that the increase in cytometrically detected zona binding was paralleled by an increase in the area on the sperm head to which zona proteins bound, from the apical region to the whole of the acrosomal region. The changes were accelerated by phosphodiesterase inhibitors, were attenuated by omission of bicarbonate, and were completely inhibited by addition of EGTA. In the fertilization medium, numbers of sperm showing enhanced zona binding maximized after 60-90 min. This time course is somewhat similar to that reported by others for development of egg-penetrating ability in vitro. We suggest that the observed changes in zona binding ability bring about optimal sperm-egg attachment; they may also relate to induction of the acrosome reaction by zona pellucida components. In consequence, the zona binding changes may be an important part of the process by which the sperm acquires fertilizing ability as a result of capacitation.

Acrosin↗
PubMed1998

Use of stored pig eggs to assess boar sperm fertilizing functions in vitro.

Methodology for studying sperm-zona interaction in the pig was established using cryopreserved cumulus-free immature eggs obtained in large numbers from gilts' ovaries. Boar sperm were preincubated in a medium known to support in vitro fertilization (IVF) and coincubated briefly with groups of eggs, and the resultant sperm-egg complexes were transferred to fresh sperm-free medium so that the behavior of the egg-associated sperm sample could be assessed during further incubation ("postincubation"). Complexes were passaged repeatedly through a wide-bore pipette tip to rinse off loosely bound sperm and leave tightly bound sperm; alternatively, they were passaged repeatedly through a narrow-bore pipette tip to strip off sperm bound to the zona surface, whence residual zona-penetrated sperm could be counted. Zona-binding ability was present in the sperm populations very soon after the start of preincubation, although it increased during the following 3 h; considerable binding was also noted in a medium that did not support IVF. Zona-penetrating ability was absent at the start of preincubation, increased slowly to a maximum after 3 h, and declined thereafter; penetration was insignificant in the medium that did not support IVF. Associated sperm numbers remained constant during postincubation of sperm-egg complexes. However, numbers of penetrated sperm rose slowly in a curvilinear fashion to maximize after some 3 h of postincubation, when they constituted less than about 15% of the bound sperm. No rapidly penetrating cohort was detectable, and the proportion of sperm that became tightly bound to the zona was unaffected by either preincubation or postincubation. It was concluded that 1) the strength of sperm-zona attachment reflected the area of the sperm head in contact with the zona rather than any physiologically specific binding and 2) zona attachment was not a functional or temporal indicator of zona penetration.

Acrosome↗