PubMed Health⌕ Search

Biomedical subjects

S A Smith

Publications and source records attributed to S A Smith.

At least 19 recordsLinked to original sources
PubMed2006

Effects of rosiglitazone and metformin on pancreatic beta cell gene expression.

AIMS/HYPOTHESIS: Rosiglitazone and metformin are two oral antihyperglycaemic drugs used to treat type 2 diabetes. While both drugs have been shown to improve insulin-sensitive glucose uptake, the direct effects of these drugs on pancreatic beta cells is only now beginning to be clarified. The aim of the present study was to determine the direct effects of these agents on beta cell gene expression. METHODS: We used reporter gene analysis to examine the effects of rosiglitazone and metformin on the activity of the proinsulin and insulin promoter factor 1 (IPF1) gene promoters in the glucose-responsive mouse beta cell line Min6. Western blot and gel retardation analyses were used to examine the effects of both drugs on the regulation of IPF1 protein production, nuclear accumulation and DNA binding activity in both Min6 cells and isolated rat islets of Langerhans. RESULTS: Over 24 h, rosiglitazone promoted the nuclear accumulation of IPF1 and forkhead homeobox A2 (FOXA2), independently of glucose concentration, and stimulated a two-fold increase in the activity of the Ipf1 gene promoter (p<0.01). Stimulation of the Ipf1 promoter by rosiglitazone was unaffected by the presence of the peroxisome proliferator activated receptor gamma antagonist GW9662. No effect of either rosiglitazone or metformin was observed on proinsulin promoter activity. Metformin stimulated IPF1 nuclear accumulation and DNA binding activity in a time-dependent manner, with maximal effects observed after 2 h. CONCLUSIONS/INTERPRETATION: Metformin and rosiglitazone have direct effects on beta cell gene expression, suggesting that these agents may play a previously unrecognised role in the direct regulation of pancreatic beta cell function.

Active Transport, Cell Nucleus↗
PubMed2006

Phospholipid composition controls thromboplastin sensitivity to individual clotting factors.

BACKGROUND: Tissue factor is the active ingredient in thromboplastin reagents used to perform prothrombin time (PT) clotting tests to monitor oral anticoagulant therapy and to screen for clotting factor deficiencies. Thromboplastins are complex mixtures prepared from extracts of brain or placenta, although newer thromboplastins contain recombinant tissue factor incorporated into phospholipid vesicles. Thromboplastins can vary widely in their sensitivity to reductions in the levels of vitamin K-dependent clotting factors. A system to compensate for this, the International Sensitivity Index (ISI) and International Normalized Ratio (INR), has revolutionized the monitoring of oral anticoagulant therapy. The INR system is also sometimes used to monitor coagulopathies in patients with sepsis or liver failure, applications for which it was not originally designed and for which it has not been rigorously validated. OBJECTIVES: To better understand thromboplastin performance, we systematically investigated which properties of recombinant thromboplastins influence their sensitivities to changes in the levels of specific clotting factors. RESULTS: We now report that relative sensitivities to changes in the plasma levels of factors V, VII, X (FV, FVII, FX) and prothrombin are differentially influenced by a recombinant thromboplastin's content of phospholipid and sodium chloride. Furthermore, thromboplastins of similar ISI values may exhibit quite different sensitivities to each of these clotting factors. CONCLUSIONS: Differing sensitivities of thromboplastin reagents to individual clotting factor levels have implications for monitoring of oral anticoagulant therapy and interpreting results of the PT assay.

Adolescent↗
PubMed2006

Traces of factor VIIa modulate thromboplastin sensitivity to factors V, VII, X, and prothrombin.

BACKGROUND: Thromboplastin reagents are used to conduct prothrombin time (PT) clotting tests to monitor oral anticoagulant therapy and screen for clotting factor deficiencies. Thromboplastins made from purified, recombinant tissue factor are generally more sensitive to changes in plasma factor (F) VII levels than are thromboplastins prepared from tissue extracts. This may be problematic as FVII's short plasma half-life can result in day-to-day fluctuation during oral anticoagulant therapy. We hypothesized that trace contamination of tissue-derived thromboplastins with FVII(a) blunts sensitivity to plasma FVII levels. METHODS: Traces of purified FVIIa were added to thromboplastin reagents prepared using recombinant human tissue factor and the effect on sensitivity to individual clotting factors was quantified in PT clotting assays. RESULTS AND CONCLUSIONS: Adding 5-100 pm FVIIa not only decreased thromboplastin sensitivity to plasma FVII, it surprisingly increased sensitivity to plasma levels of FV, FX and prothrombin. In addition, traces of FVIIa interacted with changes in the salt content and phospholipid composition of recombinant thromboplastins to further modulate their sensitivities to individual clotting factors. These results help explain how thromboplastin reagents of differing composition exhibit differing sensitivities to individual clotting factor levels. Implications of our results for monitoring oral anticoagulant therapy and other uses of the PT assay are discussed.

Anticoagulants↗
PubMed2006

Marine mammal neoplasia: a review.

A review of the published literature indicates that marine mammal neoplasia includes the types and distributions of tumors seen in domestic species. A routine collection of samples from marine mammal species is hampered, and, hence, the literature is principally composed of reports from early whaling expeditions, captive zoo mammals, and epizootics that affect larger numbers of animals from a specific geographic location. The latter instances are most important, because many of these long-lived, free-ranging marine mammals may act as environmental sentinels for the health of the oceans. Examination of large numbers of mortalities reveals incidental proliferative and neoplastic conditions and, less commonly, identifies specific malignant cancers that can alter population dynamics. The best example of these is the presumptive herpesvirus-associated metastatic genital carcinomas found in California sea lions. Studies of tissues from St. Lawrence estuary beluga whales have demonstrated a high incidence of neoplasia and produced evidence that environmental contamination with high levels of polychlorinated biphenols and dichlorophenyl trichloroethane might be the cause. In addition, viruses are suspected to be the cause of gastric papillomas in belugas and cutaneous papillomas in Florida manatees and harbor porpoises. While experimental laboratory procedures can further elucidate mechanisms of neoplasia, continued pathologic examination of marine mammals will also be necessary to follow trends in wild populations.

Animals↗
PubMed2006

Activity corrections for ionization constants in defined media.

Correcting ionization constants for activity effects can have a larger impact on some ionic species than correcting for temperature effects. Computer trials compared two pH calculation methods, and showed that an interpolation algorithm found a precise charge balance quickly but was unstable under certain conditions, while a slower pH search method was stable under all test conditions. The optimal calculation strategy was a hybrid of these two approaches. The hybrid calculation method is stable, quick, includes activity and temperature corrections, does not need kinetic rate constants, allows all known ionic species to be included, and allows for easy addition of new chemical species.

Algorithms↗
PubMed2005

Magnetization transfer MRI demonstrates spinal cord abnormalities in adrenomyeloneuropathy.

BACKGROUND: In adrenomyeloneuropathy (AMN) conventional MRI detects only spinal cord atrophy in the late stages. OBJECTIVE: To apply a magnetization transfer-weighted (MTw) imaging to patients with AMN and AMN-like syndrome in order to visualize and quantitatively assess the pathology of white matter tracts in the cervical spinal cord. METHODS: MTw studies were conducted in nine men with AMN, eight symptomatic heterozygous women, and 10 age- and sex-matched controls and compared to the Expanded Disability Status Scale (EDSS) and quantitative tests of vibratory sense and postural sway. MTw data sets were obtained at the level of C1 to C3 using a three-dimensional gradient echo acquisition technique, these images were then standardized between subjects by using the in-slice CSF signal as a normalization reference, allowing a quantitative assessment of the MTw signal. RESULTS: In contrast to conventional MRI, MTw images showed signal hyperintensities in the lateral and dorsal columns of all patients. The MT signal quantified in the dorsal column showed significant differences between patients with AMN, X-linked adrenoleukodystrophy heterozygotes, and controls. MT hyperintensity in the dorsal column correlated with EDSS, vibratory sense, and postural sway. CONCLUSION: Magnetization transfer-weighted imaging is a sensitive modality for the visual and quantitative assessment of spinal cord pathology in adrenomyeloneuropathy, and is a potential tool for evaluation of new therapies.

Adrenoleukodystrophy↗
PubMed2005

Spectroscopic evidence of cerebral axonopathy in patients with "pure" adrenomyeloneuropathy.

BACKGROUND: Adrenomyeloneuropathy (AMN) is the adult variant of X-linked adrenoleukodystrophy. The disease pathology is usually limited to spinal cord and peripheral nerves, and when this is the case, it is referred to as "pure" AMN. Histopathology shows cerebral involvement even in pure AMN; however, not much is known about the nature, extent, and clinical relevance of these findings. OBJECTIVE: To investigate brain involvement in AMN patients with normal MRI, employing multislice MR spectroscopic imaging. METHODS: Twelve men with pure AMN were compared with 19 age-matched healthy volunteers. Metabolite ratios (N-acetylaspartate [NAA]/choline [Cho], NAA/creatine [Cr], and Cho/Cr) were measured from seven brain regions. Global metabolite ratios were generated as an average of these seven regional ratios. The Expanded Disability Status Scale (EDSS) was used for neurologic evaluation. RESULTS: The patients with AMN showed reduced global NAA/Cho (AMN 1.40 +/- 0.16 vs controls 1.75 +/- 0.34; p = 0.003)) and global NAA/Cr (AMN 2.32 +/- 0.13 vs controls 2.62 +/- 0.43; p = 0.03). Regionally, NAA/Cho was lowered in the internal capsule (AMN 1.30 +/- 0.20 vs controls 1.69 +/- 0.37; p = 0.002) and in parieto-occipital white matter (AMN 1.45 +/- 0.19 vs controls 1.78 +/- 0.55; p = 0.04). NAA/Cr was lowered in parieto-occipital white matter (AMN 2.34 +/- 0.31 vs controls 2.83 +/- 0.71; p = 0.04). EDSS demonstrated an inverse association with global NAA/Cr (r = -0.65, p = 0.02) and NAA/Cr in centrum semiovale (r = -0.73, p = 0.006) and in parieto-occipital white matter (r = -0.64, p = 0.02). Cho/Cr was not significantly elevated. CONCLUSIONS: (1)H-MR spectroscopic imaging is able to detect biochemical abnormalities suggestive of axonal damage even in the brains of patients with pure adrenomyeloneuropathy. The axonopathy is most prominent in internal capsule and parieto-occipital white matter and may contribute to clinical disability.

Adrenoleukodystrophy↗
PubMed2004

Rapid and efficient incorporation of tissue factor into liposomes.

Tissue factor (TF), the physiological trigger of the blood clotting cascade, is also the active ingredient in thromboplastin preparations which are widely used in clotting assays such as the prothrombin time (PT) test. A type I integral membrane protein, TF must be incorporated into suitable phospholipid membranes for full procoagulant activity. Several methods exist for incorporating TF into phospholipid vesicles, typically employing the formation of mixed micelles containing detergent, phospholipid and TF, followed by detergent removal or dilution below the critical micelle concentration (CMC). These methods have certain drawbacks: they may take several days to complete, employ expensive detergents, are difficult to scale up, and do not always result in complete detergent removal. In this study we have investigated the use of a variety of detergents [Triton X-100, octaethylene glycol monododecyl ether (C(12)E(8)), cholate, deoxycholate, and n-octyl-beta-D-glucopyranoside], and the use of adsorbent beads (Bio-Beads SM-2) for removing detergent, in processes to incorporate TF into proteoliposomes with high specific activity in coagulation assays. The method we have developed is rapid and readily scalable, yielding thromboplastin preparations with specific activities in plasma clotting assays that are at least as high as those made with detergent dialysis.

Blood Coagulation↗
PubMed2004

Properties of recombinant human thromboplastin that determine the International Sensitivity Index (ISI).

Prothrombin Time (PT) clotting tests are widely used to monitor oral anticoagulation therapy and to screen for clotting factor deficiencies. The active ingredient in PT reagents (thromboplastins) is tissue factor, the integral membrane protein that triggers the clotting cascade through the extrinsic pathway. Several years ago, a system for calibrating and using thromboplastin reagents, known as the International Sensitivity Index (ISI) and the International Normalized Ratio (INR), was developed to standardize monitoring of oral anticoagulant therapy. The ISI/INR method, while revolutionizing the monitoring of coumarin therapy, has been criticized for a number of perceived shortcomings. We have undertaken a series of studies aimed at achieving a detailed understanding of which parameters influence the ISI values of thromboplastin reagents, with an ultimate goal of creating 'designer thromboplastins' whose sensitivities to the various clotting factors can be individually tailored. In this study, we demonstrate that ISI values of thromboplastin reagents based on relipidated, recombinant human tissue factor can be controlled by a combination of changes in the phospholipid content (in particular, the levels of phosphatidylserine and phosphatidylethanolamine) and ionic strength. The sensitivity of a given thromboplastin reagent can be increased (i.e. its ISI value decreased) by decreasing the content of phosphatidylserine and/or increasing the ionic strength. The molar ratio of phospholipid to tissue factor, on the other hand, had essentially no impact on ISI value.

Humans↗
PubMed2004

Lack of N1L gene expression results in a significant decrease of vaccinia virus replication in mouse brain.

Vaccinia virus encodes secretory proteins termed virokines. One of the major virokines encoded by the N1L open reading frame is the 13.8 kDa protein. A recombinant virus, termed vGK5, lacking this protein when injected intracranially into mice, has one of the highest levels of in vivo attenuation achieved by deletion of any single open reading frame of vaccinia virus. Here we show that the 13.8 kDa protein significantly enhances viral replication within brain tissue; however, analysis of histology, neutrophil infiltrate, and nitric oxide synthase activity of brain tissue shows no significant differences between wild-type vaccinia virus and vGK5. Since there is poor growth of vGK5 virus in the brain, the possibility of postvaccinial encephalitis is significantly diminished. Mice injected with vGK5 became resistant to the lethal effects of vaccinia virus, indicating that vGK5 is immunogenic in the brain without being virulent and therefore is a vaccine candidate. This suggests that should vGK5 reach the brain it will not replicate efficiently but still serve as a live vaccine.

Amino Acid Sequence↗
PubMed2004

Evidence that protein kinase Cdelta is not required for palmitate-induced cytotoxicity in BRIN-BD11 beta-cells.

Chronic exposure of pancreatic beta-cells to saturated fatty acids leads to loss of viability, an effect that has been implicated in the process of beta-cell 'lipotoxicity' associated with the progression of type 2 diabetes. The mechanisms involved are unknown but recent evidence has implicated the delta isoform of protein kinase C (PKCdelta) in mediating fatty acid toxicity. We have investigated this proposition in the clonal insulin-secreting cell line, BRIN-BD11. BRIN-BD11 cells were found to undergo apoptosis when exposed to palmitate and this response was attenuated by the purportedly selective inhibitor of PKCdelta, rottlerin. However, activation of PKCdelta with the phorbol ester, phorbol-12-myristate-13-acetate (PMA), failed to promote cell death and down-regulation of PKCdelta did not prevent the cytotoxic effects of palmitate. Moreover, rottlerin remained effective as a blocker of the palmitate response in cells depleted of PKCdelta. Since rottlerin can inhibit various other kinases in addition to PKCdelta, a range of additional kinase inhibitors was also tested. Of these, only the putative Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) inhibitor, KN-62, was found to inhibit palmitate-induced cell death. However, this effect was not reproduced by a more selective pseudo-substrate inhibitor of CaM kinase II. Therefore, the present results reveal that palmitate induces cell death in BRIN-BD11 cells and suggest that this may involve the activation of a rottlerin (and KN-62)-sensitive kinase. However, it is clear that PKCdelta is not required for this response.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2003

Synthesis, pharmacology and pharmacokinetics of 3-(4-aryl-piperazin-1-ylalkyl)-uracils as uroselective alpha1A-antagonists.

Predominance in the urethra and prostate of the alpha(1A)-adrenoceptor subtype, which is believed to be the receptor mediating noradrenaline induced smooth muscle contraction in these tissues, led to the preparation of alpha(1A)-selective antagonists to be tested as uroselective compounds for the treatment of benign prostatic hyperplasia. Thus, a number of selective alpha(1A)-adrenoceptor antagonists were synthesized and assayed in vitro for potency and selectivity. Dog pharmacokinetic parameters of 12 (RO700004) and its metabolite 40 (RO1104253) were established. The relative selectivity of intravenously administered 12, 40 and standard prazosin to inhibit hypogastric nerve stimulation-induced increases in intraurethral prostatic pressure versus phenylephrine-induced increases in diastolic blood pressure in anesthetized dogs was 76, 71 and 0.6, respectively.

Administration, Oral↗
PubMed2003

Central role of the adipocyte in the insulin-sensitising and cardiovascular risk modifying actions of the thiazolidinediones.

Insulin resistance is a key metabolic defect in type 2 diabetes that is exacerbated by obesity, especially if the excess adiposity is located intra-abdominally/centrally. Insulin resistance underpins many metabolic abnormalities-collectively known as the insulin resistance syndrome-that accelerate the development of cardiovascular disease. Thiazolidinedione anti-diabetic agents improve glycaemic control by activating the nuclear receptor peroxisome proliferator activated receptor-gamma (PPARgamma). This receptor is highly expressed in adipose tissues. In insulin resistant fat depots, thiazolidinediones increase pre-adipocyte differentiation and oppose the actions of pro-inflammatory cytokines such as tumour necrosis factor-alpha. The metabolic consequences are enhanced insulin signalling, resulting in increased glucose uptake and lipid storage coupled with reduced release of free fatty acids (FFA) into the circulation. Metabolic effects of PPARgamma activation are depot specific-in people with type 2 diabetes central fat mass is reduced and subcutaneous depots are increased. Thiazolidinediones increase insulin sensitivity in liver and skeletal muscle as well as in fat, but they do not express high levels of PPARgamma, suggesting that improvement in insulin action is indirect. Reduced FFA availability from adipose tissues to liver and skeletal muscle is a pivotal component of the insulin-sensitising mechanism in these latter two tissues. Adipocytes secrete multiple proteins that may both regulate insulin signalling and impact on abnormalities of the insulin resistance syndrome--this may explain the link between central obesity and cardiovascular disease. Of these proteins, low plasma adiponectin is associated with insulin resistance and atherosclerosis--thiazolidinediones increase adipocyte adiponectin production. Like FFA, adiponectin is probably an important signalling molecule regulating insulin sensitivity in muscle and liver. Adipocyte production of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, and angiotensin II secretion are partially corrected by PPARgamma activation. The favourable modification of adipocyte-derived cardiovascular risk factors by thiazolidinediones suggests that these agents may reduce cardiovascular disease as well as provide durable glycaemic control in type 2 diabetes.

Adipocytes↗
PubMed2003

Prolonged retention of vaccinia virus complement control protein following IP injection: implications in blocking xenorejection.

The vaccinia virus complement control protein (VCP) blocks classic and alternate complement pathways by binding to the third and fourth complement components and by blocking the formation of the C3-convertase as well as by accelerating the decay of the C3 and C4 convertase. The therapeutic potential of VCP has been extensively studied for brain injury, xenotransplantation, Alzheimer's disease, and spinal cord injury. We investigated the pharmacokinetic behavior of rVCP in mice. Dosage of rVCP was studied by injecting different concentrations of rVCP. A 25 mg/kg or greater dose injected intraperitoneally was found to be adequate to suppress complement for more than 8 hours.

Animals↗
PubMed2003

Dose-dependent inhibition of complement in baboons by vaccinia virus complement control protein: implications in xenotransplantation.

Vaccinia virus complement control protein (VCP) is a potent inhibitor of both the alternative and the classical complement pathways through its binding to activated third and fourth components. In addition to its complement inhibiting abilities, VCP can bind heparan sulfate on cell surfaces, resulting in further functional activities. Altogether, the multiple functions of VCP have been shown to reduce the inflammatory response of the host, helping the vaccinia virus to evade immune destruction. Recently, we reported that VCP is able to block hyperacute xenograft rejection, significantly prolonging graft survival in two separate in vivo heterotopic cervical cardiac xenograft models. Histopathological examination of the transplanted hearts receiving VCP revealed marked VCP deposition on the endothelium, a significant reduction in cardiac tissue damage, and significantly less C3, IgG and IgM deposition in the tissue. It is concluded that VCP may inhibit hyperacute xenorejection by binding to the endothelial surface, blocking complement fixation activation, thereby preventing xenoantibody attachment. In the current study, the level of serum complement inhibition was evaluated following different bolus dosages of VCP in baboons. The results indicated that to achieve a satisfactory level of complement inhibition higher doses of VCP are needed in baboons, than previously observed in rats. The current observations are critical for future assessment of the role of VCP to suppress hyperacute rejection following pig-to-baboon xenotransplantation.

Animals↗
PubMed2003

Rosiglitazone inhibits the insulin-mediated increase in PAI-1 secretion in human abdominal subcutaneous adipocytes.

OBJECTIVE: The aim of this study was to investigate the effect of insulin and an insulin-sensitizing agent, rosiglitazone (RSG), on the production of plasminogen-activator inhibitor-1 (PAI-1) in isolated subcutaneous abdominal adipocytes. Human tissue-type plasminogen activator (t-PA) was also measured to assess changes in overall thrombotic risk. METHODS: The mean depot-specific expression of PAI-1 and t-PA mRNA (n = 42) in subcutaneous abdominal (n = 21), omental (n = 10) and thigh (n = 11) adipose tissue depots was examined. Furthermore, subcutaneous adipocytes were treated with insulin, RSG and insulin in combination with RSG (10-8 m) for 48 h. Conditioned media were collected and enzyme-linked immunosorbent assays performed for PAI-1 and t-PA (n = 12) antigen. PAI-1 and t-PA mRNA levels were also assessed. RESULTS: PAI-1 mRNA levels were significantly higher in subcutaneous and omental abdominal tissue than in thigh fat (p = 0.037 and p = 0.014). No change in t-PA mRNA expression between the adipose tissue depots was observed. Insulin stimulated PAI-1 protein secretion in a concentration-dependent manner in adipocytes (control: 68.3 +/- 1.2 ng/ml (s.e.m.); 10 nm insulin: 73.7 +/- 3.8 ng/ml upward arrow; 100 nm insulin: 86.8 +/- 4.1 ng/ml upward arrow **; 1000 nm insulin: 102.0 +/- 4.8 ng/ml upward arrow ***; **p < 0.01, ***p < 0.001). In contrast, insulin + RSG (10-8 m) reduced PAI-1 production relative to insulin alone (***p < 0.001), whilst RSG alone reduced PAI-1 protein secretion in a concentration-dependent manner (RSG at 10-10 m: 50.4 +/- 2.87 ng/ml downward arrow ***; RSG at 10-5 m: 30.3 +/- 2.0 ng/ml downward arrow ***; p < 0.001). No difference was observed between control and treatments for t-PA secretion (range 7-11 ng/ml). CONCLUSIONS: Insulin stimulated PAI-1 secretion, whilst RSG reduced both PAI-1 secretion alone and in combination with insulin. These data suggest that adipose tissue may contribute significantly to the elevated circulating PAI-1 in obesity. Therefore, RSG's effects on PAI-1 production in adipose tissue may contribute to the fall in circulating PAI-1 levels observed in patients receiving RSG therapy.

Adipocytes↗
PubMed2003

Cloning and characterization of the human neural cell adhesion molecule, CNTN4 (alias BIG-2).

We report the isolation and characterization of human contactin 4 (CNTN4), a brain-derived, immunoglobulin superfamily molecule-2 (alias BIG-2) as a candidate gene responsible for the differentiation potential of human neuroblastoma cells. Northern blot analysis showed highest CNTN4 expression in testes, thyroid, small intestine, uterus and brain. Induction of CNTN4 mRNA expression in human neuroblastoma tumor cells treated with retinoic acid correlated with a block in retinoid-induced neuritogenesis. Our findings suggest a role for human contactin 4 protein in the response of neuroblastoma cells to differentiating agents.

Amino Acid Sequence↗