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R A Harrison

Publications and source records attributed to R A Harrison.

At least 109 records · Page 6Linked to original sources
PubMed1992

An anti-peptide antibody that recognizes a neo-antigen in the CR1 stump remaining on erythrocytes after proteolysis.

Previous studies of erythrocyte CR1 levels in systemic lupus erythematosus (SLE) and other diseases with in vivo complement activation have led to the conclusion that CR1 levels fall because of loss of CR1 from erythrocytes by proteolysis--predominantly in the liver. In order to measure the existence of proteolysed CR1 remnants on erythrocytes an antibody was raised to a peptide corresponding to the CR1 sequence between the proximal standard consensus repeat (SCR) and the transmembrane segment. This antipeptide antibody recognizes a neo-antigen found on trypsinized erythrocytes which has been demonstrated to represent the 'CR1-stump'. The anti-'CR1-stump' antiserum detects proteolysed CR1 on the ex vivo erythrocytes of a patient with cold haemolytic antibody disease (CHAD). However, higher affinity antibodies will be needed to make anti-CR1-stump a satisfactory diagnostic reagent.

Amino Acid Sequence↗
PubMed1992

Short-term interaction of airway and tissue oxygen tensions on ciliary beat frequency in dogs.

The responses of canine tracheal ciliary beat frequency (CBFt) to total lung, tracheal lumen, and peripheral lung hyperoxia, to tracheal lumen anoxia with or without peripheral lung hypoxia, and to isolated tracheal lumen hyperoxia combined with a beta-antagonist were delineated in anesthetized beagle dogs. CBFt was measured using a heterodyne laser light-scattering technique. When oxygen mixtures were delivered to the whole lung, a dose-dependent increase in maximal CBFt was observed from 6.3 +/- 1.0 Hz on air to 13.8 +/- 1.2 Hz on 100% oxygen. When oxygen mixtures were delivered to the isolated tracheal lumen, a dose-dependent increase in maximal CBFt from 6.9 +/- 1.3 Hz on air to 25.7 +/- 6.3 Hz on 100% oxygen was observed. CBFt was unchanged under conditions of peripheral lung hyperoxia. There were no significant changes from room air baseline CBFt of 7.6 +/- 1.5 Hz due to either isolated tracheal anoxia alone or in combination with alveolar hypoxia. CBFt stimulated with 100% oxygen insuffiated to the isolated tracheal lumen decreased from 14.1 +/- 3.2 to 9.5 +/- 1.9 Hz and from 16.5 +/- 1.2 to 9.7 +/- 1.2 Hz in response to 6 and 18 micrograms/kg of intravenous esmolol, respectively. This study demonstrates that short-term, local hyperoxia stimulates CBFt and that a pulmonary or systemically derived factor can be activated to inhibit this stimulation. It indicates that acute airway anoxia and alveolar and blood hypoxia do not suppress ciliary beat frequency. It also suggests that the adrenergic system is involved in the oxygen-induced stimulation of ciliary beat.

Adrenergic beta-Antagonists↗
PubMed1992

Behaviour of ejaculated spermatozoa from bull, boar and ram during thin-layer countercurrent partition in aqueous two-phase systems.

Ejaculated spermatozoa from bulls, boars and rams were subjected to thin-layer countercurrent partition in aqueous two-phase systems composed of dextran T500 and polyethylene glycol 6000 (PEG) in sucrose-based Hepes-buffered media. In the basal system, the great majority of spermatozoa tended to partition with the dextran-rich bottom phase; however, by including very low levels of phosphate, they could be made to partition increasingly with the PEG-rich top phase (complete at 10 mM phosphate). A procedure was developed for carrying out four separations simultaneously under identical conditions, whereby it could be shown that distribution varied with the number of spermatozoa in the sample. In the case of bull, the effect of cell number could be reduced considerably by inclusion of small quantities of seminal plasma in the phase system, but no such effect was found for ram or boar. Considerable variation in distribution pattern was seen between samples, which did not appear to be due to technical inconsistency. Livability in the phase systems was also variable, and we believe that PEG may exert a detergent-like effect on the sperm surface that is exacerbated in highly defined media free of protective proteins.

Animals↗
PubMed1992

Uptake and incorporation of inositol by preimplantation mouse embryos.

The uptake of myo-inositol by preimplantation mouse embryos was investigated using [3H]myo-inositol. Uptake increased about 12-fold between one- and two-cell stages and increased again at the blastocyst stage (> 6-fold compared with the two-cell stage). Uptake at the blastocyst stage was time and temperature dependent; it was stimulated by sodium, inhibited by glucose and appeared to take place mainly via a saturable mechanism. Uptake in the presence of 6.25 mmol inositol l-1 was 1424 fmol inositol per blastocyst per h. About 10% of the [3H]inositol taken up by blastocysts during 8 h in culture was incorporated into lipid. Thin layer chromatography of the lipid showed that most of this inositol was incorporated into lipid material co-migrating with phosphatidylinositol with a small proportion co-migrating with phosphatidylinositol 4-phosphate.

Analysis of Variance↗
PubMed1991

A synthesis of acylphosphonic acids and of 1-aminoalkylphosphonic acids: the action of pyruvate dehydrogenase and lactate dehydrogenase on acetylphosphonic acid.

Acylphosphonic acids, R-CO-PO(OH)2, have been synthesized by the steps [formula: see text] of which the last is new and provides a mild method for de-esterifying acylphosphonic acids. Their reductive amination gives a simple way of making 1-aminoalkylphosphonic acids. Acetylphosphonic acid inhibited NAD+ reduction by pyruvate with the pyruvate dehydrogenases from Escherichia coli and Bacillus stearothermophilus. The inhibition was competitive with pyruvate, with Ki of 6 microM for the E. coli enzyme (pyruvate Km 0.5 mM) and one of 0.4 mM of the B. stearothermophilus enzyme (pyruvate Km 0.1 mM). Acetylphosphonate and its monomethyl ester are substates for pig heart lactate dehydrogenase, with Km values of 15 mM and 10 mM respectively (pyruvate Km 0.05 mM) and specificity constants one thousandth that for pyruvate.

Acetaldehyde↗
PubMed1990

Diacylglycerol and phosphatidate production and the exocytosis of the sperm acrosome.

We have investigated the production of diacylglycerol (DAG) and phosphatidate (PtdOH) during the exocytosis of the sperm acrosome. Ram spermatozoa treated with Ca2+ and the ionophore A23187 experienced a rapid breakdown of the polyphosphoinositides (PPIs), and a rise in [32P]Pi-labelled PtdOH and DAG mass; PtdOH mass, however, was unaffected. Treatment with Ca2+/A23187 and the DAG kinase inhibitor R59022 resulted in a dose-dependent increase in DAG mass and a concomitant decrease in [32P]PtdOH; such treatment showed a dose-dependent stimulation of acrosomal exocytosis. Pre-incubation with exogenous PtdOHs before stimulation with Ca2+/A23187 did not affect the time-course of exocytosis, whereas treatment with Ca2+/A23187 and exogenous DAGs (dioctanoylglycerol, oleoyl-acetyl-glycerol, or dioleoylglycerol) resulted in a dose-dependent stimulation of acrosomal exocytosis. Our results suggest that DAG, rather than PtdOH, is the important metabolite generated upon PPI hydrolysis; however, since spermatozoa lack protein kinase C, the target of DAG in most cells, a role for DAG in acrosomal exocytosis is as yet unclear.

Acrosome↗
PubMed1990

Two-domain structure of the native and reactive centre cleaved forms of C1 inhibitor of human complement by neutron scattering.

The C1 inhibitor component of human complement is a member of the serpin superfamily, and controls C1 activation. Carbohydrate analyses showed that there are seven O-linked oligosaccharides in C1 inhibitor. Together with six N-linked complex-type oligosaccharides, the carbohydrate content is therefore 26% by weight and the molecular weight (Mr) is calculated as 71,100. Neutron scattering gives an Mr of 76,000 (+/- 4000) and a matchpoint of 41.8 to 42.3% 2H2O, in agreement with this carbohydrate and amino acid composition. Guinier plots to determine the radius of gyration RG were biphasic. Neutron contrast variation of C1 inhibitor in H2O-2H2O mixtures gave an overall radius of gyration RG at infinite contrast of 4.85 nm, from analyses at low Q, and a cross-sectional RG of 1.43 nm. The reactive centre cleaved form of C1 inhibitor has the same Mr and structure as the native molecule. The length of C1 inhibitor, 16 to 19 nm, is far greater than that of the putative serpin domain. This is attributed to an elongated structure for the carbohydrate-rich 113-residue N-terminal domain. The radial inhomogeneity of scattering density, alpha, is large at 59 x 10(-5) from the RG data and 28 x 10(-5) from the cross-sectional analysis, and this is accounted for by the high oligosaccharide content of C1 inhibitor. The scattering data were modelled using small spheres. A two-domain structure of length 18 nm based on two distinct scattering densities accounted for all the contrast variation data. One domain is based on the crystal structure of alpha 1 antitrypsin (7 nm x 3 nm x 3 nm). The other corresponds to an extended heavily glycosylated N-terminal domain of length 15 nm, whose long axis is close to the longest axis of the serpin domain. Calculation of the sedimentation coefficient s0(20),w for C1 inhibitor using the hydrodynamic sphere approach showed that a two-domain head-and-tail structure with an Mr of 71,000 and longest axis of 16 to 19 nm successfully reproduced the s0(20),w of 3.7 S. Possible roles of the N-terminal domain in the function of C1 inhibitor are discussed.

Borohydrides↗
PubMed1990

Identification of a new P1 residue mutation (444Arg----Ser) in a dysfunctional C1 inhibitor protein contained in a type II hereditary angioedema plasma.

A new reactive-centre P1 residue mutation (444Arg----Ser), has been identified in a dysfunctional C1 inhibitor protein, C1 inhibitor(Ba), contained in a type II hereditary angioedema plasma. This substitution is compatible with a point mutation of the 444Arg codon (CGC----AGC), and represents the first non-histidine, non-cysteine P1 residue mutant described for C1 inhibitor.

Amino Acid Sequence↗
PubMed1990

Structural comparisons of the native and reactive-centre-cleaved forms of alpha 1-antitrypsin by neutron- and X-ray-scattering in solution.

alpha 1-Antitrypsin is the best-characterized member of the serpin (serine-proteinase inhibitor) superfamily. Its solution structure was studied by high-flux neutron-scattering and synchrotron X-ray-scattering. Neutron data show that its absorption coefficient A1% 280,1cm is 5.4. The neutron radius of gyration RG at infinite contrast for native alpha 1-antitrypsin is 2.61 nm, characteristic of a moderately elongated structure, and its cross-sectional RG is 1.34 nm. The internal inhomogeneity of scattering densities within alpha 1-antitrypsin is high at 29 x 10(-5). The X-ray RG is 2.91 nm, in good agreement with the neutron RG of 2.82 nm in 1H2O. This RG is unchanged in reactive-centre-cleaved alpha 1-antitrypsin. These parameters are also unchanged at pH 8 in sodium/potassium phosphate buffers up to 0.6 M. The neutron and X-ray curves for native alpha 1-antitrypsin were compared with Debye simulation based on the crystal structure of reactive-centre-cleaved (papain) alpha 1-antitrypsin. After allowance for residues not visible in the crystallographic electron-density map, and rejoining the proteolysed site between Met-358 and Ser-359 by means of a relatively minor conformational re-arrangement, good agreement to a structural resolution of 4 nm is obtained with the neutron data in two contrasts and with the X-ray data. The structures of the native and cleaved forms of alpha 1-antitrypsin are thus similar within the resolution of solution scattering. This places an upper limit on the magnitude of the presumed conformational changes that occur in alpha 1-antitrypsin on reactive-centre cleavage, as indicated in earlier spectroscopic investigations of the Met-358-Ser-359 peptide-bond cleavage. Methods for scattering-curve simulations from crystal structures are critically assessed. The RG data lead to dimensions of 7.8 nm x 4.9 nm x 2.2 nm for native alpha 1-antitrypsin. The high internal inhomogeneity and the asymmetric shorter semi-axes of 4.9 nm and 2.2 nm suggest that the three oligosaccharide chains of alpha 1-antitrypsin are essentially freely extended into solvent in physiological conditions. This conclusion is also supported by the Debye simulations, and by modelling based on hydrodynamic parameters.

Binding Sites↗
PubMed1990

Conformational transition between native and reactive center cleaved forms of alpha 1-antitrypsin by Fourier transform infrared spectroscopy and small-angle neutron scattering.

alpha 1-Antitrypsin (alpha 1-AT) is the best-characterized member of the serpin superfamily of plasma proteins. Protease inhibitor members of this family undergo a characteristic reactive-center cleavage during expression of their inhibitory activity. The physical basis of this transition in alpha 1-AT from the stressed native conformation to the more stable reactive center cleaved (split) form was studied by Fourier transform infrared (FT-IR) spectroscopy and neutron scattering. The FT-IR spectra show that, while split alpha 1-AT has three intense well-resolved components associated with the presence of antiparallel beta-sheet and alpha-helix conformations, the amide I band of native alpha 1-AT has only one intense component, associated with the presence of beta-sheet structure. 1H-2H exchange within the polypeptide backbone, studied by FT-IR and NMR spectroscopy, shows that the native form undergoes greater exchange than the split form. Under the same conditions, neutron scattering shows no differences in the radius of gyration RG of the native and the split forms. In contrast, in high concentrations of phosphate approaching those used for crystallization, the native form (unlike the split form) undergoes dimerization. These data indicate that the conformational transition largely involves localized secondary and tertiary structure rearrangements. We propose that the energetically stressed native alpha 1-AT structure is the consequence of a significantly reduced number of hydrogen bonds in secondary structure components and that reactive-site cleavage between Met358 and Ser359 is the key for the development of the fully hydrogen bonded more stable serpin structure.

Fourier Analysis↗
PubMed1990

Structural homologies of component C5 of human complement with components C3 and C4 by neutron scattering.

The complement component C5 is one of a family of structurally related plasma proteins that includes components C3 and C4. Activation of C5 is the initial step in the formation of the membrane attack complex of complement. Analysis of the solution structure of C5 and comparisons with similar analyses of the structures of C3 and C4 are reported here. Neutron solution scattering gave an Mr for C5 of 201,000, which demonstrates that C5 is monomeric in solution. The radius of gyration RG of C5 at infinite contrast is 4.87 nm and corresponds to an elongated structure. The longest length of C5 was determined to be at least 15-16 nm from three calculations on the basis of the RG, the scattering intensity at zero angle I(0), and the indirect transformation of the scattering curve into real space. Comparison of the RG and contrast variation data and indirect transformations of the scattering curves for C3, C4, and C5 show that these have very similar structures. Comparisons of the C5 scattering curve with Debye small-sphere models previously employed for C4 and C3 show that good curve fits could be obtained. Unlike previous studies that have suggested significant differences, these experiments indicate that, while C5 differs from C3 and C4 in its activation and inactivation pathways, significant structural homology exists between the native proteins, as might be predicted from their high (and similar) sequence homology.

Complement C3↗
PubMed1990

Ram spermatozoa produce inositol 1,4,5-trisphosphate but not inositol 1,3,4,5-tetrakisphosphate during the Ca2+/ionophore-induced acrosome reaction.

An investigation was made of the production of inositol tris- and tetrakisphosphates concomitant with ionophore-stimulated breakdown of PtdIns(4,5)P2 in ram spermatozoa. As spermatozoa displayed very low rates of incorporation of [3H]inositol into their phosphoinositides, the studies were carried out using 32P-labelled cells. Using a specially developed procedure, inositol tris- and tetrakisphosphates were isolated, free of labelled ATP and P(i); they were then separated from each other (and from other minor labelled compounds) and analysed, using ionophoresis and HPLC. Levels of 32P-labelled material with the chromatographic characteristics of Ins(1,4,5)P3 were very low in untreated cells, but rose sharply with ionophore treatment, in parallel with rapid PtdInsP2 breakdown. No 32P-labelled material with the characteristics of Ins(1,3,4,5)P4 or Ins(1,3,4)P3 was found, and there was no evidence for phosphorylation of Ins(1,4,5)P3 in sperm homogenates. The implications of our findings are discussed with respect to the physiological modulation of Ca2+ influx that is required to initiate the acrosome reaction at fertilization.

Acrosome↗
PubMed1990

Type II hereditary angioneurotic edema that may result from a single nucleotide change in the codon for alanine-436 in the C1 inhibitor gene.

Identical single-base changes in the C1 inhibitor gene that may result in dysfunctional inhibitor proteins are described in two different families with type II hereditary angioneurotic edema. Initially, a restriction fragment length polymorphism was defined that resulted from loss of a Pst I site within exon VIII, which encodes the region containing the reactive center. Exon VIII from the normal and abnormal allelles was amplified by the polymerase chain reaction. Amplified DNA product was cloned into plasmid pUC18; clones representing normal and mutant allelles were distinguished by the presence and absence, respectively, of the Pst I restriction site. DNA sequence analysis revealed a G----A mutation in the codon for alanine-436, which would result in replacement with a threonine residue. This position is nine amino acid residues amino-terminal to the reactive-center arginylthreonine peptide bond. In contrast, previously defined mutations in type II hereditary angioneurotic edema result in replacement of the reactive-center arginine.

Alanine↗
PubMed1990

Competition for binding sites on C3b by CR1, CR2, MCP, factor B and factor H.

The reaction of radiolabeled C3b-binding proteins with C3b-coated particles has been investigated. CR1 binding was inhibited by factor H and factor B (in the presence of properdin), but not by properdin alone. CR2 and MCP binding were also inhibited by factor H. Therefore factor H, factor B, CR1, CR2 and MCP probably comprise a group of mutually competitive proteins with similar or overlapping binding sites on C3b. These results correlate with their structural homology and suggest that they all evolved from a single C3b-binding molecule. Factor H, CR1 and MCP are also cofactors for the factor-I-mediated cleavage of C3b. A species incompatibility between rat factor I and human CR1 for the cleavage of human C3b suggests the possibility that cofactors may also function by interacting directly with factor I.

Animals↗
PubMed1990

Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa.

Carboxyfluorescein diacetate and propidium iodide were used as fluorescent stains to assess membrane integrity in sperm populations from ram and boar. The living spermatozoa were immobilized with low concentrations of formaldehyde so that individual stained cells could be observed in a suspension with the aid of a fluorescence microscope. Intracellular esterases liberated impermeant-free carboxyfluorescein from the permeant carboxyfluorescein diacetate and caused the product to accumulate and fluoresce green within the acrosome and the mitochondria as well as within the cytoplasm. Most of the spermatozoa (the intact ones) accumulated carboxyfluorescein in all compartments; however, a few cells (those with damaged plasma membranes) accumulated the stain only in the acrosome and/or the mitochondria, while others (all of whose membranes were damaged) remained entirely unstained. The impermeant propidium iodide did not stain any of the (intact) spermatozoa that accumulated carboxyfluorescein throughout their length, but stained all the others (the heads fluoresced red). The technique appeared to provide more reliable estimations of the percentage of functional cells than did motility estimations or assessments of acrosomal integrity (presence of normal apical ridge). The technique also demonstrated the sensitivity of the sperm plasma membrane to cold shock: virtually all cells rapidly became permeable to the stains after such stress. Assessments of boar sperm samples during preparative incubation for in-vitro fertilization indicated a considerable increase in the percentage of cells with damaged plasma membranes as incubation proceeded, in advance of the increase in the percentage of cells with discharged acrosomes.

Animals↗
PubMed1990

Characterization of a decapacitation factor associated with epididymal mouse spermatozoa.

Earlier studies demonstrated that epididymal mouse spermatozoa have a surface-associated factor which inhibits fertilizing ability in a reversible manner. The factor can be removed from uncapacitated spermatozoa by gentle centrifugation, resulting in immediately highly fertile gametes, and it can be added back to capacitated spermatozoa, resulting in poorly fertile cells in which the acrosome reaction has been blocked. Using such inhibition of in-vitro fertilizing ability as an assay, we have carried out experiments to characterize the factor. It appears to be an anionic polypeptide with Mr of approximately 40,000 (according to its behaviour on gel filtration). It is stable to heating at 100 degrees C for 15 min and is not destroyed by proteases at pH 8.0, yet inhibitory activity decreases during sperm incubation in capacitating conditions and is also destroyed in partially purified preparations by endogenous enzyme action during incubation at pH 5.0. Activity is not adsorbed to either concanavalin A-agarose or wheat-germ agglutinin-agarose, suggesting that terminal mannose and N-acetylglucosamine residues are not abundant. The factor causes rapid changes in the patterns of chlortetracycline fluorescence seen on sperm heads, a parameter used to assess the capacitated state. Removal of the factor from uncapacitated cells results in a shift to a predominance of capacitated patterns, while the addition of crude or partially purified factor to capacitated cells inhibits the acrosome reaction and causes a shift to the uncapacitated pattern in acrosome-intact spermatozoa. The factor therefore behaves as a decapacitation factor. However, it appears to differ from other characterized decapacitation factors in terms both of molecular size and of abundance of mannose and N-acetylglucosamine residues.

Acrosome↗
PubMed1990

Phosphoinositides and their products in the mammalian sperm acrosome reaction.

At fertilization, the spermatozoon exocytoses its acrosomal granule in a Ca2(+)-dependent process known as the acrosome reaction. In mammalian spermatozoa, possibly because the acrosome is large and membrane fusion takes place between the outer acrosomal membrane and the overlying plasma membrane extensively over the anterior of the sperm head, the exocytotic process is slow and therefore amenable to biochemical dissection. By prelabelling sperm phospholipids with 32P and inducing the acrosome reaction with Ca2+ and the ionophore A23187, we have been able to show that membrane fusion occurs as the result of a sequence of events following Ca2+ entry; Ca2+ is required for at least 3 of these events. The process is initiated by a large-scale breakdown of polyphosphoinositides that is catalysed by a Ca2(+)-dependent phospholipase C. Of the resultant products, diacylglycerol is the essential one. Although its precise role remains to be established, this compound appears to stimulate a later process; it does not seem to act directly as a fusogen, nor does it act through a metabolite. However, it does not act through protein kinase C. At present we believe that diacylglycerol may simultaneously activate phospholipase A2 and inhibit lysophosphatide acyltransferase, to cause a large-scale build-up of fusogenic lysophospholipids in the acrosomal region; Ca2+ may bring about membrane fusion when the levels of these lipids have risen above a necessary threshold.

Acrosome↗