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Biomedical subjects

R A Harrison

Publications and source records attributed to R A Harrison.

At least 73 records · Page 4Linked to original sources
PubMed1998

Expression of factor I-resistant mutants of the human complement component C3 in heterologous systems.

Complement plays a major role in hyperacute rejection of xenografts. In order to overcome this, we are developing, by minimal mutagenesis, a modified C3 molecule that, like cobra venom factor (CVF), escapes normal complement regulatory processes and inhibits complement-mediated responses by systemic depletion of C3. Unlike CVF, this protein should have little or no immunogenicity and be suitable for repeat administrations. As an initial step in this process, we have modified human C3 to make it resistant to inactivation by factor I. The factor I resistant C3 is capable of forming an active C3 convertase. Preincubation with normal human serum abrogated subsequent complement-mediated cytolysis by both the classical and alternative pathways, while wild-type (wt) C3 was inactive. The modified human C3 also blocked complement activity of guinea-pig serum. For economical and rapid production, we have developed expression of recombinant C3 wt and mutant proteins in the Baculovirus system. Large quantities are also being produced from stably transfected CHO cell lines. In addition, we have developed a fast C3 purification method by engineering a 6XHIS tag into the C3a portion of the molecule, thereby avoiding the need for subsequent separation of the tag from active C3b molecules.

Animals↗
PubMed1998

Comparative mortality of patients diagnosed with localized and in situ melanomas.

BACKGROUND: The rates for incidence and mortality from melanoma are continuing to rise, although long term survival experience for patients with in situ and localized melanomas is favorable. RESULTS: Excess mortality varied with age at diagnosis, sex, duration since diagnosis and tumor thickness. CONCLUSIONS: Compared to the general white population in the United States, patients with localized melanoma experience a mild increase in Excess Death Rates (EDRs) the first year following diagnosis. For 1-5 years following diagnosis the EDRs are markedly increased across all age groups and decline thereafter. At 15-20 years following diagnosis the EDRS are not elevated except among patients < 40 years of age at diagnosis. Patients with tumors > 1.5 mm thick have mild to moderate increases in EDR for the first year following diagnosis, and extremely high EDRs up to 5 years. There is no elevation in mortality ratios or EDR for patients diagnosed with in situ melanomas.

Adult↗
PubMed1997

Difficulties in the ascertainment of C9 deficiency: lessons to be drawn from a compound heterozygote C9-deficient subject.

A group of patients with long-surviving mismatched kidney allografts were investigated for complement function using haemolytic assays in agarose gels. One patient was found to have no alternative pathway activity but a low normal classical pathway. Surprisingly, investigation revealed that the patient's complement was normal for all components except C9, which was functionally absent. The patient was shown to be heterozygous for DNA markers in the C6, C7 and C9 region of chromosome 5 and therefore appears to be a compound heterozygote for two uncharacterized C9 deficiency genes. Serological analysis by ELISA revealed that he has trace concentrations of a non-functional C9 molecule. Western blot analysis was not sufficiently sensitive to permit detection of this molecule. We hypothesize that the patient is heterozygous for a complete deficiency of C9 and for a gene directing hyposynthesis of a defective C9. We also suggest that C9 deficiency may be more common among Caucasians than has been reported.

Complement C8↗
PubMed1997

Molecular defects in hereditary angioneurotic edema.

Thirty-eight previously unreported, unrelated patients with hereditary angioneurotic edema were studied, and each was found to have a single mutation in the C1 inhibitor gene. On the basis of serine protease inhibitor crystal structure, these and published mutations affect critical domains in the reactive center loop, alpha-helices A, B, C, E, and F, and beta-sheets A and C. Almost all mutations, other than in the reactive center loop, occur at residues that are highly conserved among serine protease inhibitors, and the others are likely to interfere with molecular movement. These mutations begin to identify residues critical for molecular function of the C1 inhibitor molecule.

Angioedema↗
PubMed1997

Distribution of astigmatism among Sioux Indians in South Dakota.

BACKGROUND: Previous studies have reported a greater prevalence of high astigmatism and vision disorders among Native Americans than occurs in the general U.S. population. The majority of these studies, however, have focused on Native American schoolchildren. This study assessed the distribution of astigmatism in a general clinic population at the Rosebud Indian Reservation in South Dakota. METHODS: From examination of 174 patients, demographic information, visual acuities, refractive error, binocular vision status, and eye health were recorded. RESULTS: The data showed more high astigmatism than would be expected in a general U.S. population. Three or more diopters of astigmatism were found in 9.2% of the right eyes and 10.8% of the left eyes in the study population. High astigmatism was noted less frequently among those ages 40 years and older (3.3% right eyes, 6.3% left eyes). Most of the astigmatism was with-the-rule, but a shift toward against-the-rule and oblique astigmatism was found in older individuals. No significant gender differences were noted. CONCLUSIONS: The results suggest an ethnic difference in astigmatism and emphasize thee need for continued and expanded vision services for Native American populations.

Adolescent↗
PubMed1997

Sperm plasma membrane characteristics and boar semen fertility.

Much effort is being made to establish relationships between the molecular events that take place in spermatozoa under fertilizing conditions and actual sperm function during fertilization. During capacitation, the process that 'primes' spermatozoa for interaction with the egg, components of the sperm's environment, notably bicarbonate, provoke various specific changes in the architecture and functioning of the sperm plasma membrane in a large number of cells. The individual changes have been found to proceed on different time scales, and may therefore represent sequential stages in the capacitation process. However, each change takes place at different rates in individual cells, revealing considerable functional heterogeneity within the sperm population. Recent work on membrane changes provoked by cooling has indicated similarities with capacitational changes. The effect of cooling may therefore be to induce premature capacitation (and destabilization). Such an effect would greatly compromise sperm fertilizing potential. A pig sperm-egg interaction model was used to examine quantitative details of zona binding and zona penetrating abilities within capacitated sperm populations, and sperm behaviour was found not to accord with generally held beliefs. In particular, individual spermatozoa that have bound to the zona pellucida show great variation in the delay before penetrating: no evidence has been found for a specially competent subgroup. Even in sperm samples incubated to undergo maximal capacitational membrane changes, cells with actual penetrating potential represent less than 15% of the total number that attach initially to the zona pellucida. Thus detection of capacitational membrane changes appears greatly to overestimate zona penetrating capability. Future studies linking sperm membrane characteristics with semen fertility in the field will need to consider differences between in vitro and in vivo conditions. The need for survival in the female tract may require much slower sperm responses than are considered optimal for in vitro fertilization.

Animals↗
PubMed1996

Bicarbonate/CO2, an effector of capacitation, induces a rapid and reversible change in the lipid architecture of boar sperm plasma membranes.

Bicarbonate/CO2 is believed to be the key in vitro effector of sperm capacitation, a process which induces major changes in the sperm plasma membrane in preparation for fertilization. In a flow cytometric study, we examined the effect of bicarbonate on boar spermatozoa using merocyanine, an impermeant lipophilic probe which binds to plasma membranes with increasing affinity as their lipid components become more disordered. We found that bicarbonate causes a rapid increase in the ability of live boar spermatozoa to bind merocyanine. First detected about 100 sec after exposure to bicarbonate and largely complete by 300 sec, this increase appears to result from individual cells within the sperm population switching from a low merocyanine-binding state to a high binding state. The majority of live spermatozoa are capable of responding in this way, and do so in proportion to bicarbonate concentration, half-maximal response being induced by about 3 mM bicarbonate; however, overall population response varies greatly between ejaculates. Increased merocyanine stainability is observed over the whole surface area of the cell, and is reversible both with respect to temperature (it is only manifested above 30 degrees C) and with respect to presence of bicarbonate. A similar effect can be induced by phosphodiesterase inhibitors such as isobutylmethylxanthine, and enhanced by a permeant cyclic nucleotide analogue. We conclude that bicarbonate causes a major alteration in sperm plasma membrane lipid architecture, apparently by perturbing enzymic control processes. This novel action of bicarbonate may represent an initial permissive event in the capacitation sequence.

Animals↗
PubMed1996

Homologous and heterologous protective immunity to Egyptian strains of Schistosoma mansoni and S. haematobium induced by ultraviolet-irradiated cercariae.

C57BL/6 and Balb/c mice were immunized with ultraviolet-irradiated cercariae of Egyptian strains of Schistosoma mansoni and S. haematobium, challenged with nonirradiated cercariae of the homologous or heterologous species, and assayed for protection against challenge infection by comparing the adult worm burdens of immunized and non-immunized mice. Homologous protection (per cent reduction in worm recovery) ranged from 56% to 69% for S. mansoni and 88% to 99% for S. haematobium. Significant heterologous protection was consistently induced against S. haematobium by immunization with S. mansoni, but not against S. mansoni by immunization with S. haematobium. These results are discussed in relation to those of previous studies and in terms of implications for vaccine development.

Animals↗
PubMed1996

Capacitation mechanisms, and the role of capacitation as seen in eutherian mammals.

Capacitation, the process whereby spermatozoa are rendered capable of interacting with and fertilizing the egg, was discovered more than 40 years ago. However, our understanding of it is still far from satisfactory. Several factors conspire to obfuscate studies of capacitation mechanisms: the inherent functional heterogeneity of sperm populations, the range of functions used as parameters of capacitation (whence the endpoint of the process has become conceptually uncertain), and the several profound differences between model in vitro fertilization (IVF) systems and the situation in vivo in the female reproductive tract. Recent investigations in the author's laboratory have shown that bicarbonate/CO2, an essential component for successful IVF, causes rapid changes in lipid architecture of the sperm plasma membrane and slower changes in surface coating. These changes are accompanied by membrane destabilization and cell death. Evidence suggests that bicarbonate's actions are mediated through cyclic nucleotide signalling. Of particular note is the heterogeneity in rate of response to bicarbonate shown by individual cells in the sperm populations. Taken together with other observations, the findings suggest that capacitation is a series of positive destabilizing events that eventually lead to cell death. The 'capacitated' state would then be a window of destabilization within which spermatozoa can undergo a zona-induced acrosome reaction and display hyperactivated motility. Further along the destabilization pathway, spontaneous acrosome reactions would occur before total membrane degeneration. In vivo, capacitation would be a conflict between destabilization and sperm survival. Concentrations of bicarbonate are maintained low in the cauda epididymidis, where sperm survive for long periods, and one may speculate that hormonal control of local bicarbonate/CO2 in oviducal 'storage' sites in the female tract could allow 'safe' sequestering of live spermatozoa until around the time of ovulation; the environment may then change to produce a 'capacitating' effect, whence, due to the inherent functional heterogeneity of the sequestered population, small numbers of capacitated spermatozoa are released sequentially. In this way, a succession of spermatozoa in the correct physiological state may be provided for the freshly ovulated egg.

Animals↗
PubMed1996

Transmission of full-length echocardiographic images over ISDN for diagnosing congenital heart disease.

OBJECTIVES: To study the feasibility of transmitting full-length diagnostic-quality echocardiograms with video conferencing systems linked by ISDN. METHODS: Forty-three previously video-recorded full-length echocardiograms (22 complete two-dimensional Doppler studies, 21 separate M-mode studies) were transmitted from a site 30 miles distant using desktop video conferencing systems linked with one ISDN line (128 kbps). Blinded interpretations of the original recorded and transmitted studies were made by two pediatric cardiologists (two-dimensional Doppler) and a pediatric sonographer (M-mode). RESULTS: Diagnoses of normal (N = 12) and abnormal (N = 10) conditions were made from the original tapes; the same interpretations were made from the transmitted studies. Twenty-one specific abnormalities in the 10 abnormal studies were seen on both original and transmitted videotapes. These abnormalities ranged from simple congenital heart defects, including ventricular septal defect, atrial septal defect, and patent ductus arteriosus, to complex ones, such as coronary artery fistula, double-outlet right ventricle, and complete atrioventricular canal. Qualitatively, there was mild degradation in gray scale, brightness, and contrast of the images. Six M-mode variables from the transmitted images had excellent concordance with the original tracings (P = 0.506 to 0.838; r = 0.86 to 0.97). CONCLUSIONS: Although our sample size was small, this preliminary experience indicates that video conferencing equipment utilizing ISDN technology is a reliable method for transmitting full-length diagnostic-quality echocardiographic studies from remote sites. This technology may have a significant impact on the management of pediatric patients with suspected heart disease. Further evaluation is warranted.

Child↗
PubMed1995

A rapid method for the isolation of analogues of human CD59 by preparative SDS-PAGE: application to pig CD59.

A method for the rapid isolation of functionally active analogues of human CD59 from erythrocytes (E) is described. The method, here applied to pig E, is based on the fractionation of a butanol extract of E ghosts by preparative SDS-PAGE followed by gel filtration on Superose 12. Purification was monitored using a functional complement inhibition assay. SDS-PAGE analysis of the product of this procedure indicated a single protein band with apparent M(r) of 20 kDa under reducing and non-reducing conditions. The preparation could be incorporated into guinea pig E to inhibit both CVF-reactive lysis and lysis through C8 and C9 using preformed C5b-7 sites, demonstrating that it contained a CD59-like activity. PIPLC treatment of the isolated protein abolished the inhibition. In contrast to SDS-PAGE analysis, amino-terminal sequence analysis of the preparation showed that it comprised two components. One was identified from databank searches as a fragment of pig glycophorin. These two components could not be separated by either standard or affinity chromatographic techniques. The second component was novel and had high sequence homology with human CD59, identifying it as the pig analogue. Further functional studies showed that the pig analogue of human CD59 was effective in the protection of guinea pig E against lysis by serum from a variety of species, including human.

Amino Acid Sequence↗
PubMed1995

Flow cytometric detection of bicarbonate-induced changes in lectin binding in boar and ram sperm populations.

Boar and ram spermatozoa were incubated in Tyrode's medium in the presence or absence of bicarbonate/CO2, a component believed essential for capacitation. At intervals, samples were stained with a range of FITC-lectins to detect changes in surface glycoconjugates, using a rapid staining technique to avoid problems of lectin toxicity. The samples were then analysed directly by flow cytometry, using propidium iodide to distinguish dead cells. In the presence of bicarbonate, a live subpopulation of spermatozoa developed, which in both animal species showed higher binding affinities towards Phaseolus Vulgaris Agglutinin (PHA-E), Sophora Japonica Agglutinin (SJA), and Soybean Agglutinin (SBA), and lower binding affinity towards Erythrina Cristagalli Lectin (ECL). In boar samples, the modified subpopulation reached a maximum after 3 hr incubation, whereas in ram samples it maximized after 1.5 hr. No changes were seen when spermatozoa were incubated in bicarbonate-free medium. The bicarbonate-induced changes in lectin binding were not due to the onset of acrosome reactions, because spermatozoa induced to undergo acrosome reactions with the ionophore A23187 displayed very different lectin-binding patterns. Tested on boar spermatozoa, seminal plasma not only inhibited but reversed the bicarbonate-induced development of the modified subpopulation. EGTA also inhibited development of boar sperm subpopulations; excess Ca2+ was unable to overcome this inhibition, suggesting that multivalent metal ions might be involved in bicarbonate's action. We conclude that bicarbonate causes a loss of surface coating material with affinity for ECL and an unmasking of binding sites for SBA, SJA and PHA-E. A modified subpopulation of live spermatozoa is thereby established, which appears to maximize at a rate in accord with reported capacitation times.

Animals↗
PubMed1995

Schistosoma haematobium in the baboon (Papio anubis): assessment of protection levels against either a single mass challenge or repeated trickle challenges after vaccination with irradiated schistosomula.

Baboons vaccinated intramuscularly with three times 9000 20 krad irradiated Schistosoma haematobium schistosomula at monthly intervals were exposed percutaneously to either a single mass challenge of 3000 (VMC) or ten, weekly trickle challenges of 300 (VTC) normals S. haematobium cercariae. Unvaccinated mass (MCC) or trickle (TCC) challenge controls were exposed simultaneously. Faecal and urine egg production was delayed in the vaccinated groups which also had reduced adult, particularly female, worm recoveries. Total faecal, urine and tissue eggs were lower in the vaccinated groups, as also were the size of granulomata and the gross pathology and severity of inflammatory responses in the bladder and ureters, except for an increased proportion of tissue eggs in the livers of vaccinated animals. Differences in pathology between groups were less marked in the other organs. Most indices were reduced in the trickle versus the mass challenge control groups. Some of these trends were statistically significant, mostly in the trickle vaccination group (VTC), but others were not. Compared with the appropriate unvaccinated controls, the percentage reduction for the trickle challenge (VTC) group (73%) was three times greater than that of the mass challenge (VMC) group (23%). Overall, the protective effect of vaccination was more clearly demonstrated in the trickle than in the mass challenge groups. This conclusion is based on a single experiment. Nevertheless, because trickle infections probably approximate more closely to what humans receive naturally, it is recommended that they should be used for future testing of all potential vaccines in baboons.

Animals↗
PubMed1995

Monitoring respiratory mechanics.

Emphasis is on the mechanical properties of the spontaneously breathing patient. The occasional reference to mechanically ventilated patients is used to clarify and provide additional insight to the topic of spontaneously breathing individuals. Reference to the basic principles of mechanics are to establish the foundation for an integrated description of lung mechanics. Finally, a brief review of the "state of the art" respiratory monitoring parameters shown to have direct clinical applications.

Critical Care↗
PubMed1995

Acid-base balance.

This article emphasizes the interactions between the respiratory and metabolic sources of acid and their clearance. It includes the chemistry of acid-based balance as expressed by the Henderson-Hasselbach equation, the principles and importance of biologic buffering systems, and a brief review of renal regulation of acid-based balance. Finally, the reader is given a set of guidelines for a systematic approach to the analysis of acid-base data.

Acid-Base Equilibrium↗
PubMed1994

Molecular cloning of the rat analogue of human CD59: structural comparison with human CD59 and identification of a putative active site.

We have previously described the purification and partial characterization of the rat analogue of the human complement regulatory molecule CD59 [Hughes, Piddlesden, Williams, Harrison and Morgan (1992) Biochem. J. 284, 169-176]. We present here the molecular cloning and full sequence analysis of this molecule. A PCR-based approach utilizing primers designed from the amino-terminal protein sequence was used to isolate a full-length cDNA clone from a rat kidney cDNA library. This clone encoded a 92 bp 5'-flanking sequence, a 66 bp signal peptide and a 315 bp coding region containing putative glycosylation and GPI-anchor signals. The 3' untranslated flanking region was approximately 1.1 kbp long and included the poly-A tail and a CATA repeating sequence. The coding region was 58% identical with the human cDNA at the nucleotide level and 44% identical at the amino acid level. Despite this relatively low overall sequence conservation, several highly conserved stretches were apparent, particularly in the N-terminal portion of the molecule, in the cysteine-rich region immediately preceding the site of glycolipid attachment and in the C-terminal peptide removed during glycolipid attachment. An N-glycosylation site was identified at Asn-16 and a putative glycosylphosphatidylinositol anchor addition site at Asn-79, indicating that the mature processed protein was two residues longer than human CD59. Comparison of the sequences of rat and human CD59, together with consideration of the published three-dimensional structure of human CD59 and functional data, implicates specific regions of the protein in interactions with C-8 and/or C-9.

Amino Acid Sequence↗