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Biomedical subjects

M D Ryan

Publications and source records attributed to M D Ryan.

At least 37 records · Page 2Linked to original sources
PubMed1998

1,4-Cyclohexanecarboxylates: potent and selective inhibitors of phosophodiesterase 4 for the treatment of asthma.

Evaluation of a variety of PDE4 inhibitors in a series of cellular and in vivo assays suggested a strategy to improve the therapeutic index of PDE4 inhibitors by increasing their selectivity for the ability to inhibit PDE4 catalytic activity versus the ability to compete for high affinity [3H]rolipram-binding sites in the central nervous system. Use of this strategy led ultimately to the identification of cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxyl ic acid (1, SB 207499, Ariflo), a potent second-generation inhibitor of PDE4 with a decreased potential for side effects versus the archetypic first generation inhibitor, (R)-rolipram.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Heterotypic recognition of recombinant FMDV proteins by bovine T-cells: the polymerase (P3Dpol) as an immunodominant T-cell immunogen.

In this study we have examined the recognition of VP0, VP1, VP2, VP3 and P3Dpol by PBMC and CD4+ T-cells from infected, vaccinated-challenged, and multiply-vaccinated (O1, A24, C1 or ASIA1) cattle using recombinant proteins of an O1 serotype virus. The structural protein VP1 was recognised in an homotypic context whereas VP2, VP3, VP4 and P3Dpol were also recognised by T-cells from animals exposed to heterotypic viruses. Only the non-structural protein P3Dpol was consistently recognised by T-cells from the majority of animals examined and heterotypic recognition correlated with the presence of serologically detectable P3Dpol in purified virus. Thus, P3Dpol is a major cross-reactive immunodeterminant of FMDV, eliciting heterotypic T-cell responses and, therefore, with possible potential for inclusion in a subunit vaccine.

Animals↗
PubMed1998

Differentiating infection from vaccination in foot-and-mouth disease using a panel of recombinant, non-structural proteins in ELISA.

A profiling ELISA was developed to detect antibody to the non-structural (NS) proteins Lb, 2C, 3A, 3D, and the polyprotein 3ABC, of foot-and-mouth disease virus (FMDV). The assay was used to examine panels of sera from naive cattle, and from experimentally infected or vaccinated animals. All sera from cattle experimentally infected with any of the seven serotypes of FMDV were positive for antibody to 2C, 3A, 3D and 3ABC, and the majority were positive for Lb. The three categories of sera could be differentiated on the basis of the presence or absence of antibody to the structural and/or NS proteins of FMDV. The assay is simple, rapid and reproducible and can be used to identify previous infection in animals which are seropositive for antibody to the structural proteins of the virus. Validating the assay with field sera demonstrated that antibody to 3ABC, and usually one or more of the other non-structural proteins, was detected only in animals reported to have shown clinical signs of FMD. Vaccinated cattle which had received less than five vaccinations, were frequently positive for antibody to 3D but were negative for antibody to 3ABC. Occasional animals which had received more than ten vaccinations had NS protein antibody profiles which were similar to those seen following infection.

Animals↗
PubMed1997

The cleavage activities of aphthovirus and cardiovirus 2A proteins.

The primary 2A/2B polyprotein cleavage of aphtho-and cardioviruses is mediated by their 2A proteins cleaving C-terminally. Whilst the aphthovirus 2A region is only 16 aa (possibly 18 aa) long, the cardiovirus 2A protein is some 150 aa. We have previously shown that foot-and-mouth disease virus (FMDV) 2A is able to mediate cleavage in an artificial (chloramphenicol acetyltransferase/FMDV 2A/beta-glucuronidase [CAT-2A-GUS]) polyprotein system devoid of any other FMDV sequences with high (approximately 85%), although not complete, cleavage. In this paper we show that insertion of upstream FMDV capsid protein 1 D sequences increases the activity. In addition, we have demonstrated that the cardiovirus Theiler's murine encephalomyelitis virus(TME) 2A protein, when linked to GUS in a single ORF, is able to cleave at its own C terminus with high efficiency--if not completely. The C-terminal 19 aa of TME 2A, together with the N-terminal proline residue of protein 2B, were inserted into the CAT/GUS artificial polyprotein system (in a single ORF). This recombinant [CAT-deltaTME2A-GUS] polyprotein was able to mediate cleavage with high (approximately 85%) efficiency--directly comparable to the activity observed when FMDV 2A was inserted. A similar insertion into [CAT-GUS] of the C-terminal 19 aa of the cardiovirus encephalomyocarditis virus (EMC) 2A, together with the N-terminal proline residue of protein 2B, produced a [CAT-delta EMC2A-GUS] polyprotein which also mediated cleavage at approximately 85%. Analysis of the products of expression of these artificial polyproteins in a prokaryotic translation system did not, apparently, reveal any GUS cleavage product.

Amino Acid Sequence↗
PubMed1997

Role of conserved histidines in catalytic activity and inhibitor binding of human recombinant phosphodiesterase 4A.

To identify critical amino acids within the central conserved region of recombinant human cAMP-specific phosphodiesterase 4 subtype A (rhPDE4A), we engineered the expression of point mutants in a fully active rhPDE4A/Met201-886. When histidine residues at positions 433, 437, 473, and 477, which are highly conserved among all PDE families, were changed independently to serine residues, cAMP hydrolyzing activities were substantially reduced or abolished. The ability of these mutants to bind prototypical PDE4 inhibitors [3H]-(R)-rolipram or [3H]RP 73401 was also decreased in parallel with the loss of catalytic activity. The parallel loss of catalytic activity and inhibitor binding suggests that these changes resulted from non-localized perturbations in the structure of the enzyme. More interesting results were obtained when histidine residues at positions 505 and 506 were changed independently to aspar agines. The K(m) value for cAMP increased 3-fold in H505N (K(m) = 11 +/- 3 microM) and 11-fold in H506N (K(m) = 44 +/- 6 microM) compared with the wild-type protein (K(m) = 4 +/- 1 microM). These mutant proteins bound [3H]-(R)-rolipram and [3H]RP 73401 with K(d) values of 1.8 +/- 0.4 and 0.3 +/- 0.1 nM, respectively, for H505N, and 3.9 +/- 0.9 and 0.5 +/- 0.1 nM, respectively, for H506N. These values are nearly identical to those obtained with the wild-type rhPDE4A/Met201-886. In contrast, the IC50 values for cAMP competition with either [3H]-(R)-rolipram or [3H]RP 73401 binding increased approximately 2-fold in H505N and approximately 13-fold in H506N compared with the wild type protein. These increases are virtually identical to the changes in the K(m) value for cAMP in these mutants. We conclude that His506 and, perhaps, His505 are involved in binding of cAMP to PDE4A/Met201-886 but not in binding of PDE4-selective inhibitors.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Symptomatic ossification of the tibiofibular syndesmosis in professional football players: a sequela of the syndesmotic ankle sprain.

Ankle syndesmosis sprains are common injuries in collegiate and professional football. Several reports have documented that patients with syndesmosis injuries require a longer time to return to full athletic participation than patients with lateral ankle sprains. Here we present the cases of two professional football players with ankle pain secondary to syndesmosis ossification following documented syndesmosis ankle sprains. Both patients eventually required resection of the heterotopic ossification to allow a pain-free return to football. We conclude that syndesmosis ossification may be symptomatic in some patients, and surgical excision of the ossification may be required to allow an asymptomatic return to sports.

Ankle↗
PubMed1994

L4-5 degenerative spondylolisthesis in monozygous twins.

OBJECTIVE: The author reports symptomatic spinal canal stenosis due to degenerative spondylolisthesis at L4-5 in identical twins. SUMMARY OF BACKGROUND DATA: The patient reported his brother had a similar condition. METHODS: Monozygosity was established by phenotyping. CONCLUSIONS: Spinal morphology and degenerative disease may be genetically determined.

Aged↗
PubMed1994

Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein.

We describe the construction of a plasmid (pCAT2AGUS) encoding a polyprotein in which a 19 amino acid sequence spanning the 2A region of the foot-and-mouth disease virus (FMDV) polyprotein was inserted between the reporter genes chloramphenicol acetyl transferase (CAT) and beta-glucuronidase (GUS) maintaining a single, long open reading frame. Analysis of translation reactions programmed by this construct showed that the inserted FMDV sequence functioned in a manner similar to that observed in FMDV polyprotein processing: the CAT2AGUS polyprotein underwent a cotranslational, apparently autoproteolytic, cleavage yielding CAT-2A and GUS. Analysis of translation products derived from a series of constructs in which sequences were progressively deleted from the N-terminal region of the FMDV 2A insertion showed that cleavage required a minimum of 13 residues. The FMDV 2A sequence therefore provides the opportunity to engineer either whole proteins or domains such that they are cleaved apart cotranslationally with high efficiency.

Amino Acid Sequence↗
PubMed1994

Foot-and-mouth disease virus particles contain replicase protein 3D.

An antibody against the Escherichia coli-expressed RNA polymerase of foot-and-mouth disease virus (FMDV) reacts with the virus in ELISA and radioimmunoprecipitation experiments and with a protein of the disrupted virus particle in an immunoblot analysis. Treatment of the virus with trypsin, which cleaves capsid protein VP1 and a 56-kDa polypeptide present in trace amount in the particles, reduces the level of the reaction in ELISA and radioimmunoprecipitation and eliminates the immunoblot reaction. Electron microscopy showed that only approximately 20% of the virus particles reacted with the anti-polymerase antibody, whereas most reacted with an antibody against the immunodominant G-H loop of the virus. In the presence of ammonium ions, the expressed polymerase degrades the RNA of the virus into molecules sedimenting at approximately 12 S, indicating that it can act as a hydrolytic as well as a polymerizing enzyme. Moreover, the RNA in trypsin-treated virus particles is degraded when incubated at 37 degrees C, suggesting that the cleaved 56-kDa protein still possesses hydrolytic activity. In addition, the anti-polymerase antibody, which inhibits the polymerase activity of the E. coli-expressed protein, also partially inhibits the hydrolytic activity of the previously described endonuclease of the virus particle, suggesting that this enzyme is identical with the polymerase or forms part of it.

Antibodies, Viral↗
PubMed1994

Function of minor polypeptides in foot-and-mouth disease virus and poliovirus.

Foot-and-mouth disease virus and poliovirus each contain several minor polypeptides, in addition to the four structural proteins. One of these, the viral RNA polymerase, can also act as a nuclease, hydrolysing the RNA and thus destroying viral infectivity. It is tightly bound to the RNA and may be the packaging signal for assembly of the particle.

Aphthovirus↗
PubMed1993

Extrapulmonary tuberculosis in children.

OBJECTIVE: To investigate an apparent increase in the number of children admitted to the Royal Alexandra Hospital for Children with extrapulmonary tuberculosis (TB). Further, to highlight both the seriousness of this disease and the difficulty of its diagnosis, and to draw attention to factors such as ethnic origin in identifying children at risk. DESIGN: Clinical and microbiological data were collected for all children admitted to the hospital with a confirmed diagnosis of TB. A standardised incidence ratio (SIR) was used to analyse the number of children admitted with extrapulmonary TB in 1990-1991 compared with 1982-1989, and in 1987-1991 compared with 1982-1986. RESULTS: Eleven children (five of these in 1990 and 1991) had extrapulmonary TB (4, central nervous system; 3, osteomyelitis; 2, cervical lymphadenitis; 2, abdominal). One was Aboriginal and 10 were from families of overseas origin. Twenty-one others had pulmonary TB and 17 of these were from families of overseas origin. The apparent increase in the number of admissions for extrapulmonary TB was not statistically significant. CONCLUSION: There has been a recent increase in the number of children hospitalised with extrapulmonary TB but when this is compared with the increase in children hospitalised for all causes, the increase is not significant. Immigration and the continuing contact of children with infected adults appear to account for most cases of TB in this series.

Adolescent↗
PubMed1993

Odontoid fractures in the elderly.

A review of 35 patients > 60 years of age with odontoid fractures showed that a type II injury (Anderson and D'Alonso) was the most common fracture pattern (82% of cases). On the basis of mechanism of injury and sex incidence, we deduced that osteopenia is a contributory factor in the occurrence of odontoid fractures. A fall in a domestic setting was the cause in 53%. The peg was posteriorly displaced in 88% of type II fractures, and primary union occurred in 23%. The incidence of concomitant spinal cord injury with type II fractures was higher in older patients than it was in those < 60 years of age. The outcome for these patients was largely determined by their neurological status at presentation. Myelopathy as a late complication of nonunion was not observed in nine patients with an average follow-up of 21 months. The data suggest that vigorous attempts to secure both primary union and a sound arthrodesis for non-union are questionable in the elderly except in unusual circumstances.

Aged↗