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Biomedical subjects

R A Smith

Publications and source records attributed to R A Smith.

At least 109 records · Page 6Linked to original sources
PubMed1999

Expression and characterization of the intact N-terminal domain of streptokinase.

Proteolytic studies have enabled two of the three putative domains of the fibrinolytic protein streptokinase to be isolated and characterized (Conejero-Lara F et al., 1996, Protein Sci 5:2583-2591). The N-terminal domain, however, could not be isolated in these experiments because of its susceptibility to proteolytic cleavage. To complete the biophysical characterization of the domain structure of streptokinase we have overexpressed, purified, and characterized the N-terminal region of the protein, residues 1-146. The results show this is cooperatively folded with secondary structure content and overall stability closely similar to those of the equivalent region in the intact protein.

Calorimetry, Differential Scanning↗
PubMed1999

A new point mutation (P157S) in the reverse transcriptase of human immunodeficiency virus type 1 confers low-level resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine.

A P157S mutation in the reverse transcriptase (RT) of human immunodeficiency virus type 1 conferred fivefold resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine in cell culture. Interestingly, the P157S mutation resulted in increased sensitivity (two- to threefold) to 3'-azido-3'-deoxythymidine (AZT) and to (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). A similar increase in susceptibility to AZT and to PMPA was also conferred by the M184V mutation in RT.

Anti-HIV Agents↗
PubMed1999

Health of finishing steers: effects on performance, carcass traits, and meat tenderness.

The impact of respiratory disease during a 150-d feedlot finishing period on daily gain, carcass traits, and longissimus tenderness was measured using 204 steer calves. Feedlot health status was monitored in two ways. First, clinical signs of respiratory infection were evaluated each day; treatment with antibiotic was based on degree of fever (if rectal temperature exceeded 40 degrees C then calves were treated). Steers that were treated (n = 102) had lower (P<.05) final live weights, ADG, hot carcass weights (HCW), less external and internal fat, and more desirable yield grades. Steers that were treated had a higher prevalence of carcasses that graded U.S. Standard than steers that were never treated. Second, as an alternative index of health status, lungs of all steers were evaluated at the processing plant using a respiratory tract lesion classification system; this health index included presence or absence of preexisting pneumonic lesions in the anterioventral lobes plus activity of the bronchial lymph nodes (inactive vs active). Lung lesions were present in 33% of all lungs and were distributed almost equally between treated (37%) and untreated cattle (29%). Steers with lesions (n = 87) had lower (P<.05) daily gains, lighter HCW, less internal fat, and lower marbling scores than steers without lesions. Compared to steers with lesions but inactive bronchial lymph nodes (n = 78), steers with lung lesions plus active lymph nodes had lower (P<.01) ADG and dressing percentage. Longissimus shear force values for steaks aged 7 d were lower (P = .05) from steers without lung lesions than those for steaks from steers with lung lesions. Overall, morbidity suppressed daily gains and increased the percentage of U.S. Standard carcasses. Compared to health assessment by clinical appraisal (based on elevated body temperature), classification based on respiratory tract lesions at slaughter proved more reliable statistically and, thereby, more predictive of adverse effects of morbidity on production and meat tenderness.

Animals↗
PubMed1999

Principles of successful cancer screening.

Screening for cancer is the application of various tests to apparently healthy individuals in order to identify who among them has occult disease, which may be either invasive disease or a precursor lesion. For any given cancer site, the potential of screening to reduce morbidity and mortality is based on well-defined criteria for the evaluation of screening effectiveness and on acceptable performance to be realized in the average community setting. Screening programs are most successful when they are organized into a system that leads to high rates of participation, high quality, and constant surveillance and evaluation. If the elements that contribute to a successful screening program are not well organized and integrated, then the fullest potential of screening will not be realized.

Community Participation↗
PubMed1998

Structural and dynamical characterization of a biologically active unfolded fibronectin-binding protein from Staphylococcus aureus.

A 130-residue fragment (D1-D4) taken from a fibronectin-binding protein of Staphylococcus aureus, which contains four fibronectin-binding repeats and is unfolded but biologically active at neutral pH, has been studied extensively by NMR spectroscopy. Using heteronuclear multidimensional techniques, the conformational properties of D1-D4 have been defined at both a global and a local level. Diffusion studies give an average effective radius of 26.2 +/- 0.1 A, approximately 75% larger than that expected for a globular protein of this size. Analysis of chemical shift, 3JHNalpha coupling constant, and NOE data show that the experimental parameters agree well overall with values measured in short model peptides and with predictions from a statistical model for a random coil. Sequences where specific features give deviations from these predictions for a random coil have however been identified. These arise from clustering of hydrophobic side chains and electrostatic interactions between charged groups. 15N relaxation studies demonstrate that local fluctuations of the chain are the dominant motional process that gives rise to relaxation of the 15N nuclei, with a persistence length of approximately 7-10 residues for the segmental motion. The consequences of the structural and dynamical properties of this unfolded protein for its biological role of binding to fibronectin have been considered. It is found that the regions of the sequence involved in binding have a high propensity for populating extended conformations, a feature that would allow a number of both charged and hydrophobic groups to be presented to fibronectin for highly specific binding.

Adhesins, Bacterial↗
PubMed1998

The effect of extracellular matrix on the growth of mouse olfactory tissue in vitro.

The aim of this study was to compare the capacity of extracellular matrix (ECM) components to support the growth and differentiation of olfactory tissue in vitro. Reconstituted basement membrane matrix (RBM) supported profuse neurite outgrowth from explants of olfactory bulb (OB), with no non-neuronal cell migration. In contrast, nasal tissue (NT) explants cultured on RBM produced profuse fibroblastic outgrowth, but no neurite outgrowth from olfactory sensory neurones (OSN). Three-dimensional collagen gels, on the other hand, supported extensive outgrowth of both non-neuronal and neurite outgrowth from OB and NT explants, mature OSNs in the latter being identified using antibodies against olfactory marker protein (OMP). These culture systems represent useful tools for investigating potential tropic influences of target tissue.

Animals↗
PubMed1998

Recombination breakpoints in the human beta-globin gene cluster.

The human beta-globin gene complex spans a region of 70 kb and contains numerous sequence variants. These variant sites form a 5' cluster (5' beta-haplotype) and a 3' cluster (3' beta-haplotype) with strong linkage disequilibrium among the sites within each cluster, but not between the two clusters. The 9-kb region between the 5' and 3' clusters has been estimated to have rates of recombination that are 3 to 30 times normal, and the region has therefore been proposed as a 'hotspot' of recombination. We describe three families with evidence of meiotic recombination within this 'hotspot' of the beta-globin gene cluster and in which the cross-over breakpoints have been defined at the sequence level. In one family, the recombination has occurred in the maternal chromosome within a region of 361 bp between positions -911 and -550 5' to the beta-globin gene. In the other two families, the recombination has occurred in the paternal chromosome within a region of approximately 1,100 bp between positions -542 and +568 relative to the beta-globin gene cap site. Both regions occur within the 2-kb region of replication initiation (IR) in the beta-globin gene domain with no overlap. The IR region contains a consensus sequence for a protein (Pur), which binds preferentially to single-stranded DNA, a role implicated in recombination events.

Chromosome Mapping↗
PubMed1998

Solid-phase synthesis of a library of piperazinediones and diazepinediones via Kaiser oxime resin.

A combinatorial library of piperazinediones has been prepared by automated parallel solid-phase synthesis. The five-step reaction protocol makes use of Kaiser oxime resin, to enable cleavage from the polymeric support concomitant with an intramolecular displacement reaction, under very mild conditions. The methodology was also successfully extended to the preparation of the seven-membered ring homologs, diazepinediones.

Azepines↗
PubMed1998

Protection against hippocampal kainate excitotoxicity by intracerebral administration of an adenosine A2A receptor antagonist.

We have previously shown that the peripheral administration of an A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680) protected the hippocampus against kainate-induced excitotoxicity. The present study utilises the intrahippocampal route to further investigate CGS 21680-mediated protection as well as examining the role of adenosine and both A1 and A2A receptors in kainate-induced excitotoxicity. Injections were made directly into the hippocampus of anaesthetised male Wistar rats. Following surgery and the administration of 0.25 nmol kainate in 1 microl of solution, the animals were left to recover for seven days before perfusion and brain slicing. Haematoxylin and eosin staining revealed substantial damage to the CA3 region. Co-administration of the A2A receptor agonist CGS 21680 over a range of doses did not protect the region to any degree. Similarly neither the A1 receptor agonist R-phenylisopropyladenosine (R-PIA), nor adenosine itself reduced kainate-induced damage. The intrahippocampal injection of the selective A2A receptor antagonist, 4-(2-[7-amino-2-¿2-furyl¿¿1,2, 4¿triazolo¿2,3-a¿¿1,3,5¿triazin-5-yl-amino]ethyl)phenol (ZM241385) however, significantly decreased kainate damage to the CA3 region. These results show that adenosine A2A receptor-induced protection is most likely to be mediated peripherally and is probably not due to activation of A2A receptors within the hippocampus. The lack of protection observed with either R-PIA or adenosine may be due to an inhibitory action of the A2A receptor on the neuroprotective A1 receptor. Importantly, this study also questions the role of endogenously released adenosine in protecting the hippocampus from excitotoxic damage.

Adenosine↗
PubMed1998

Nitric oxide synthase inhibitors L-NAME and 7-nitroindazole protect rat hippocampus against kainate-induced excitotoxicity.

The role of nitric oxide in cerebral insults remains controversial. While numerous studies have used models of ischaemia and hypoxia, few have examined nitric oxide in the kainate model of excitotoxicity. Kainate (10 mg/kg) was administered to rats via the intraperitoneal (i.p.) route to induce submaximal damage to the CA1, CA2 and CA3a regions of the hippocampus after 7 days. Systemic injections of the nitric oxide synthase (NOS) inhibitors N(G)-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI), both at a dose of 5 mg/kg, reduced cell death in all three regions. As 7-NI selectively inhibits the neuronal form of NOS, this study suggests that nitric oxide produced from a neuronal and not epithelial source may contribute to neuronal damage in this model.

Analysis of Variance↗
PubMed1998

Changes in neuropeptide immunoreactivity in cultured adult mouse sensory neurons following methylmercury chloride treatments.

Changes in the neuropeptide expression of sensory neurons, related to functional modulation, have been widely reported both following physical injury in vivo, and after toxic insult in vitro and in vivo. The current immunocytochemical study aimed to monitor the neuropeptide status of neuronal cultures prepared from adult mouse dorsal root ganglia (DRG), and to ascertain whether changes occurred following treatments with 0.1-1 microM methylmercury (MeHg). Proportions of both substance P (SP) and calcitonin gene related peptide (CGRP) containing neurons increased significantly, and were maintained throughout the 24 h exposure period. In contrast the numbers of somatostatin (SOM)-ir neurons decreased. Substance P- and CGRP-ir neuron increases may be related to nociceptive responses, whereas the decreases in SOM containing neurons could reflect a differential loss in this subset of sensory neurons.

Animals↗
PubMed1998

Retroviral recombination is nonrandom and sequence dependent.

Sequence variation plays a significant role in the pathogenesis and persistence of retroviral infections and is a major obstacle in the development of vaccines as well as therapies against lethal diseases caused by retroviruses. Recombination is one means by which sequence variation is generated. However, the basic molecular mechanisms of recombination are not adequately understood. In the present study, a spleen necrosis virus (SNV) recombination system was used to ask whether a known hot spot for mutation was also a hot spot for retroviral recombination. The system consisted of a pair of SNV vectors expressing two drug-resistance genes, constructed so that recombinants could be selected by a double resistant phenotype. Restriction enzyme site differences engineered into the vectors were used to map the location of recombination sites within relatively small intervals (55 to 420 bp). The vectors were modified to create two pairs that differed only by the presence of runs of identical nucleotides. The runs of identical nucleotides had been shown previously to be hot spots for frameshift mutations during SNV reverse transcription. Each vector pair was introduced into DSDh helper cells by infection. Viruses were harvested from doubly infected DSDh helper cells and used to infect D-17 target cells. Proviral sequences from 228 cell clones were analyzed by polymerase chain reaction and restriction enzyme digestion. Significant differences in the patterns of recombination were found between the two pairs of vectors. In particular, the frequency of recombination was higher than expected in the interval immediately following the runs. For both pairs of vectors, the overall pattern of recombination was nonrandom and one region was refractory toward recombination.

Animals↗
PubMed1998

Adenosine receptor-mediated inhibition of neurite outgrowth from cultured sensory neurons is via an A1 receptor and is reduced by nerve growth factor.

Adult dorsal root ganglion (DRG) cells are capable of neurite outgrowth in vitro as well as in vivo. We have investigated the influence of adenosine and analogs on the potential of cultured adult mouse DRG neurons to produce neurites in the presence and absence of nerve growth factor (NGF) which is a well-established trophic factor of sympathetic and sensory neurons during development. It is also believed to be essential for the maintenance or regulation of differentiated phenotypes of mature peripheral neurons. The results demonstrate that DRG neurons are modulated by purines in the absence of exogenous NGF. The addition of 100 microM adenosine to neurite-bearing DRG neurons inhibited neurite growth by 47% after 2-day exposures in vitro and by 50% after 5 days whereas in the presence of NGF this inhibition was reduced to 28% and 32%, respectively. 100 microM CHA (N(6)-cyclohexyl adenosine) alone reduced neurite total length by 47% after 2 days and by 48% after 5 days. 100 microM CGS21680 (2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamido adenosine hydrochloride) alone also reduced neurite total length by 46% after 2 days and by 58% after 5 days which was reduced to 21% and 37%, respectively, in the presence of 100 ng/ml NGF. The antagonist studies revealed that activation of A1 adenosine receptors is primarily responsible for the effect on neuritogenesis since the inclusion of 1 or 10 microM CPX (8-cyclopentyl-1,3-dipropyl xanthine) fully prevented the inhibitory activity of adenosine or CHA whereas DMPX (3,7-dimethyl-1-propargyl xanthine) did not prevent inhibition by CHA. The converse experiment yielded the consistent result that inhibition by the A2 receptor agonist CGS21680 could be prevented by CPX, but not DMPX.

Animals↗