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Biomedical subjects

A Walton

Publications and source records attributed to A Walton.

At least 19 recordsLinked to original sources
PubMed1996

Enhanced infectivity of modified bluetongue virus particles for two insect cell lines and for two Culicoides vector species.

Previous studies (Mertens et al., Virology 157, 375-386, 1987) have shown that removal of the outer capsid layer from bluetongue virus (BTV) significantly reduces (approximately x 10(-4)) the infectivity of the resultant core particle for mammalian cells (BHK 21 cells). In contrast, the studies reported here, using a cell line (KC cells) derived from a species of Culicoides that can act as a vector for BTV (Culicoides variipennis), demonstrated a much higher infectivity of core particles than that in mammalian cells (approximately x 10(3)). This increase resulted in a specific infectivity for cores that was only 20-fold less than that of purified disaggregated virus particles (stored in the presence of 0.1% sodium-N-lauroylsarcosine (NLS)). Removal of this detergent caused intact virus particle aggregation and (as previously reported) resulted in an approximately 1 log10 drop in the specific infectivity of those virus particles which remained in suspension. In consequence the specific infectivity of core particles for the KC cells was directly comparable to that of the intact but aggregated virus. These data are compared with the results from oral infectivity studies using two vector species (C. variipennis and Culicoides nubeculosus), which showed similar infection rates at comparable concentrations of purified cores, or of the intact but aggregated virus particles (NLS was toxic to adult flies). The role of the outer core proteins (VP7) in cell attachment and penetration, as an alternative route of initiation of infection, is discussed. Previous studies (Mertens et al., Virology 157, 375-386, 1987) also showed that the outer capsid layer of BTV can be modified by proteases (including trypsin or chymotrypsin), thereby generating infectious subviral particles (ISVP). The specific infectivity of ISVP for mammalian cells (BHK21 cells) was shown to be similar to that of disaggregated virus particles. In contrast, we report a significantly higher specific infectivity of ISVP but not of the intact virus (approximately x 100) for two insect cell lines (KC cells and C6/36 mosquito cells (derived from Aedes albopictus)). In oral infection studies with adults of the two vector species, ISVP produced the same infection rate at approximately 100-fold lower concentrations than either core particles or the intact but aggregated virus particles. The importance of mammalian host serum proteases, or insect gut proteases, in modification of the intact virus particle to form ISVP and their role in initiation of infection and the vector status of the insect is discussed.

Animals↗
PubMed1996

Ubiquitin expression in degenerating axons of equine cervical compressive myelopathy.

Neuropathologic examination revealed axonal swelling and breakdown leading to Wallerian degeneration of affected myelinated nerve fibers in the spinal cord white matter of four young horses with equine cervical compressive myelopathy. Immunohistochemical reactions for the cell stress protein ubiquitin revealed an enhanced presence in the swollen axons, which may reflect a role for ubiquitin in the neuronal catabolic process of axonal compression and degeneration in this myelopathy.

Animals↗
PubMed1994

Purification and properties of virus particles, infectious subviral particles, cores and VP7 crystals of African horsesickness virus serotype 9.

Methods were developed for the purification, at high yield, of four different particle types of African horsesickness virus serotype 9 (AHSV-9). These products included virus particles purified on CsCl gradients which contain proteins apparently directly comparable to those of bluetongue virus (VP1 to VP7); virus particles purified on sucrose gradients which also contain, as a variable component, protein NS2; infectious subviral particles (ISVPs), containing chymotrypsin cleavage products of VP2; and cores, obtained by treating purified ISVPs with 1 M-MgCl2 to remove the components of the outer capsid layer (VP5 and VP2 cleavage products). Additional protein bands migrating with apparent M(r)s lower than that of VP5 were detected during SDS-PAGE analysis of virus particles. These appear to be conformational variants of VP5 and are identified as VP5' and VP5". BHK-21 cells infected with this strain of AHSV-9 produce large quantities of flat, usually hexagonal crystals of VP7, a major group antigen and core protein; these were also purified. Either 20 mg of virus particles, 20 mg of ISVPs or 10 mg of cores as well as 20 mg of VP7 crystals could be purified from approximately 8 x 10(9) infected cells. None of the preparations of particles or crystals showed any detectable contamination with BHK-21 cell proteins or antigens, as determined by SDS-PAGE or indirect ELISA. Virus particle and ISVP preparations had similar specific infectivities for BHK-21 cells (approximately 1 x 10(9) TCID50/A260 unit) but the infectivity of cores was approximately 10(5)-fold lower.

African Horse Sickness Virus↗
PubMed1993

Design and synthesis of highly potent and selective cyclic dynorphin A analogs. 2. New analogs.

We have designed and synthesized several cyclic disulfide-containing peptide analogs of dynorphin A (Dyn A) which are conformationally constrained in the putative "address" segment of the opioid ligand. Several of these Dyn A analogs exhibit unexpected apparent selectivities for the kappa and mu opioid receptors(s) of the central vs peripheral nervous systems. Thus, incorporation of conformational constraint in the putative "address" segment of Dyn A analogs has resulted in the kappa/mu opioid receptor ligands [L-Pen5,Cys11]Dyn A1-11-NH2 (4), [Cys5,Cys10]Dyn A1-11-NH2 (5), [Cys5,Cys9]DynA1-11-NH2 (6), and [Cys4,Cys9,Arg10]DynA1-11-NH2(7). All of these analogs possess high kappa and mu opioid receptor affinities for the central receptor (guinea pig brain), but effect only weak potency at peripheral kappa and mu opioid receptors (GPI). In fact cyclic dynorphin A analog 4 shows > 19,000-fold differences between central kappa opioid affinity and potency in the guinea pig ileum (GPI). Additionally analog 4 is not an antagonist in the GPI, suggesting possible receptor differences between these sites. Substitution of Tyr1 by Phe1 in the cyclic 1-11 series gave the analog [Phe1,Cys5,Cys11]Dyn A1-11-NH2 (1) that was surprisingly potent in the guinea pig brain binding assay (IC50 = 15.1 nM) at the kappa receptor, but was inactive in the GPI and mouse vas deferens bioassays. D-Ala2 and Tic4 analogs of 1 had lower affinity at brain kappa receptors and had very weak potencies in the GPI and MVD bioassays. On the other hand, [Cys6,Cys10]DynA1-11-NH2 (8), [Cys8,D-Cys13]DynA1-13-NH2 (9), [D-Cys8,D-Cys12]DynA1-13-NH2 (10), and [D-Pro10,Cys5,Cys13]-Dyn A1-13-NH2 (11) were surprisingly potent in the GPI bioassay, though considerable apparent selectivity for central receptors is still retained. The apparent lack of correlation between the pharmacological profiles observed in smooth muscle and in the brain binding assays, particularly with 1 and 4, may suggest the existence of different subtypes of the kappa and mu opioid receptors in the brain and peripheral systems.

Amino Acid Sequence↗
PubMed1993

Automated DNA profiling employing multiplex amplification of short tandem repeat loci.

We have employed automated fluorescence-based technology to detect amplified tri-, tetra-, and pentanucleotide short tandem repeat (STR) loci electrophoresed on denaturing polyacrylamide sequencing gels. The system described incorporates an internal size standard in each sample, allowing the STR-PCR products to be sized automatically with a high degree of precision. By utilizing different fluorescent dye markers for loci that have overlapping allele size ranges, we have developed three multiplex STR systems containing a total of 14 different loci. These multiplex systems were then used to evaluate the usefulness of the 14 loci for the identification of individuals. Allele frequency data were collected from a minimum of 50 individuals from each of three different racial groups: Caucasians, Afro-Caribbeans, and Asians. Of the resulting 42 locus population sets, deviation from Hardy-Weinberg equilibria was detected in only the STR HUMCYARO3-Caucasian data. The probabilities of two unrelated individuals matching by chance (pM) at all 14 loci in the three multiplex reactions was < 1 x 10(14). The combination of multiplex STR-PCR and automatic fluorescence-based detection is thus a rapid and powerful technique for individual identification.

Autoanalysis↗
PubMed1993

Syntheses, opioid binding affinities, and potencies of dynorphin A analogues substituted in positions, 1, 6, 7, 8 and 10.

Structural, stereochemical, stereoelectronic and conformational requirements for biological activity of dynorphin A1-11-NH2 analogues at opioid receptors were explored by substitution of Tyr1, Arg6, Arg7, Ile8 and Pro10 with other amino acid residues. Interestingly, substitution of Tyr1 with N alpha-Ac-Tyr1, D-Tyr1, Phe1 or p-BrPhe1 led to analogues that were quite potent at kappa opioid receptors, and additional substitution of Ile8 with D-Ala8 and/or Pro10 with D-Pro10 retained high potency in brain binding assay: [N alpha-Ac-Tyr1]- (1), [D-Tyr1]-(2) [Phe1]- (3), [Phe1,D-Ala8]- (5), [-BrPhe1, D-Ala8]- (6), [Phe1, D-Pro10]- (7) and [Phe1,D-Ala8, D-Pro10]- Dyn A1-11-NH2 (8) had IC50 (nM) binding affinities of 13.2, 18.6, 1.64, 1.26, 1.84, 2.44 and 1.62 nM, respectively. The D-Phe1 analogue 4, however, was only weakly active (610 nM). All of the analogues except 4 were modestly selective for kappa vs. mu guinea pig brain opioid receptor (11- to 88-fold) and quite selective for kappa vs. delta receptors (65-576). However, all of the analogues appeared to have very low or essentially no activity in the guinea pig ileum and mouse vas deference functional bioassays, and one analogue, 5, appeared to have weak antagonist activities. On the other hand, if constrained amino acids such as beta-methylphenylalanine or 1,2,3,4-tetrahydroisoquinoline carboxylic acid, and hydroxyproline were placed in the 1 position, inactive analogues or analogues with greatly reduced potency and biological activity were obtained (compounds 12-14). It had previously been suggested that the Arg6 and Arg7 residues were critical for biological activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Kinematic gait analysis of the trot in healthy greyhounds.

A noninvasive computer-assisted kinematic gait analysis was used to describe flexion and extension movements of 6 joints in Greyhounds at a trot. Distinct patterns of movements were described for each joint studied. The coxofemoral and carpal joints were characterized by a single peak of maximal extension. The femorotibial, tarsal, scapulohumeral, and cubital joints had 2 peaks of maximal extension, with 1 peak preceding stance phase and a second peak within stance phase. A two-factor repeated-measures ANOVA was used to determine the variance in measurement of joint angles in degrees that was attributable to trial repetitions and to differences between dogs. The coxofemoral, femorotibial, tarsal, scapulohumeral, and cubital joints had a mean variance attributable to trial repetition of 6.6 (range, 1.7 to 12.9), and a mean variance attributable to differences between dogs of 5.1 (range, 0.9 to 9.2). The carpus had more variance, with a mean variance attributable to trial repetition of 16.3 (range, 13.3 to 20.5), and that attributable to differences between dogs of 31.8 (range, 20.5 to 46.7). Kinematic gait analysis provided a reliable description of flexion and extension movements in Greyhounds with minimal variance attributable to trial repetitions and to differences between dogs.

Analysis of Variance↗
PubMed1986

Taenia taeniaeformis: immunoperoxidase localization of metacestode culture product(s) in hyperplastic gastric mucosa.

Rats infected with the hepatic metacestode Taenia taeniaeformis develop an extraordinary gastric hyperplasia. Indirect immunoperoxidase staining localized larval in vitro excretory secretory product specifically in the supranuclear cytoplasm of the epithelial cells lining the pits and glands in the hyperplastic gastric mucosa. The accumulation of this substance in the stomach epithelial cells may be relevant to the gastric hyperplasia induced by tapeworm infection.

Animals↗
PubMed1985

Gossypol in female fertility control: ovum implantation and early pregnancy inhibited in rats.

Intramuscular administration of gossypol to normally cycling female rats induced an irregularity of the cyclic pattern for as long as the treatment was continued. Furthermore, administration of gossypol from days 0 (day of sperm-positive vaginal smear) to 8 of pregnancy prevented the normal maintenance of pregnancy. Serum values of progesterone and estradiol 17 beta in gossypol-treated normally cycling and pregnant rats were significantly lower than the control levels. The supplement of a combination of exogenous progesterone and estradiol 17 beta eliminated the inhibitory effects of gossypol on ovum implantation and the maintenance of pregnancy. Our results indicate that gossypol may have some usefulness in female fertility control.

Animals↗
PubMed1985

Histopathologic changes of uterus following gossypol treatment during early pregnancy in rats.

Intramuscular injections of gossypol acetic acid (25 mg in 10% EtOH/kg/day beginning on day 2 of diestrus) disrupted early pregnancy in rats as determined by light and electron microscopy. As in pregnant controls, in the uteri of treated rats increased glandular secretion, stromal hyperemia, and decidual tissue formation were noted at days 3-5 of pregnancy. At day 6, extreme hyperemia and stromal hemorrhage had occurred around well-developed decidual tissue with foci of denuded mucosal surface. There was extravasation of blood into the uterine lumen, which was absent in controls. At days 5 and 6 of pregnancy, electron microscopy revealed shorter and fewer microvilli on the uterine glandular cells in the treated versus the control uterus. Luminal epithelial cells had not undergone the normal changes of pregnancy. These results imply that gossypol administered under our conditions neither prevented nor delayed implantation and formation of decidual tissue in the rat uterine endometrium but continuing development of the endometrium was disrupted at day 6 of pregnancy. This disruption of pregnancy may have resulted from a luteolytic action by gossypol that would not permit full structural differentiation in the rat uterus after implantation.

Animals↗
PubMed1985

Kinetic investigations in single muscle fibres using luminescent and fluorescent Ca2+ probes.

The Ca2+-sensitive photoprotein aequorin and the Ca2+-dependent fluorescent indicators quin 2 and TnCDANZ have been used to investigate contractile processes in single crustacean muscle fibres. The investigations with quin 2 indicate that the free Ca2+ rises to a maximum value before peak force as with aequorin light (approximately 200 msec delay at 12 degrees C) and subsequently decays more slowly, unlike the majority of the aequorin signal, although an aequorin 'tail' signal remains. The resting quin 2 fluorescence from the cell suggests an upper limit of 348 nM for the resting calcium concentration. Experiments with TnCDANZ indicate that this fluorescence response rises rapidly but then the rate of rise slows to reach a maximum value at a time when peak force is achieved and then the fluorescence signal decays more slowly than force. The latter result implies that Ca2+ is attached to the Ca2+-specific sites of TnC when externally recorded force is small.

Aequorin↗