The effect of sildenafil citrate administration on selected physiological parameters of exercising Thoroughbred horses.

REASONS FOR PERFORMING STUDY: Sildenafil, a phosphodiesterase-5 inhibitor vasodilator, increases cGMP concentrations by inhibiting enzymatic degradation. Marketed to treat erectile dysfunction in men, it also reduces pulmonary arterial pressure (PAP). Because it reduces PAP, sildenafil may enhance performance and/or prevent exercise induced-pulmonary haemorrhage (EIPH). OBJECTIVE: To determine if sildenafil citrate administration altered commonly measured indices of performance or reduced EIPH in exercised horses. METHODS: Thirteen athletically conditioned Thoroughbred horses (2 mares and 11 geldings, age 3-12 years) were administered sildenafil citrate or placebo in 2 crossover design exercise testing studies. In a step-wise test to exhaustion, inspired/expired gas analysis, blood lactate, heart rate, runtime and bronchoalveolar lavage (BAL) cytology were measured. In a 13 m/s test to exhaustion, blood lactate, heart rate, runtime, BAL cytology and pulmonary arterial pressure were measured. Data were analysed with paired and unpaired t tests, one-way ANOVA and Tukey's pair-wise multiple comparison and Friedman repeated measure analysis of variance on ranks. RESULTS: The administration of sildenafil did not alter mean inspired/expired gas measurements, plasma lactate concentrations or acute pulmonary haemorrhage in either exercise test or pulmonary arterial pressure measurement in the 13 m/s trial. Heart rates in both stress tests were significantly different at submaximal speeds and during the early recovery period. Run time was not affected by sildenafil administration in the step-wise trial (P = 0.622) or in the 13 m/s trial (P = 0.059). CONCLUSIONS: Sildenafil did not alleviate pulmonary haemorrhage or enhance performance-related indices in these trials. Sildenafil administration altered cardiovascular adaptation to intense exercise as evidenced by altered heart rates at submaximal speeds and post exercise. The effect of these alterations on other performance perimeters was not evident.

RNA replicons derived from poliovirus are directly oncolytic for human tumor cells of diverse origins.

The failure and/or toxicity of conventional therapies for many types of human cancers underscore the need for development of safe and effective alternative treatments. Toward this goal, we describe the direct oncolytic activity of RNA-based vectors derived from poliovirus, termed replicons, which are genetically incapable of producing infectious virus. These replicons are cytopathic in vitro for human tumor cells originating from brain, breast, lung, ovary, and skin (melanoma). The cytopathic effects in a malignant glioma cell line were associated with nuclear DNA condensation, indicative of cells undergoing apoptosis. Injection of replicons into established xenograft flank tumors in scid mice resulted in oncolytic activity and extended survival. Inoculation of replicons into established intracranial xenograft tumors in scid mice resulted in tumor infection within 8 h and extended survival. Histological analysis revealed that replicons had infected tumor cells at the site of inoculation and, most importantly, diffused to infect tumor cells that had metastasized from the initial site of implantation. The wide spectrum of cytopathic activity for human tumors combined with effective distribution after in vivo inoculation establishes the therapeutic potential of poliovirus replicons for a variety of cancers.

Cytokine production in motor neurons by poliovirus replicon vector gene delivery.

Poliovirus replicon vectors transiently express foreign proteins selectively in motor neurons of the anterior horn of the spinal cord. Here we intraspinally inoculated mice transgenic for the poliovirus receptor (PVR) with replicons encoding murine tumor necrosis factor alpha (mTNF-alpha). We detected high-level expression of mTNF-alpha in the spinal cords of these animals at 8-12 h post inoculation; this returned to background by 72 h. The mice exhibited ataxia and tail atony, whereas animals given a replicon encoding green fluorescent protein (GFP) exhibited no neurological symptoms. Histology of spinal cords from mice given the replicon encoding mTNF-alpha revealed neuronal chromatolysis, reactive astrogliosis, decreased expression of myelin basic protein, and demyelination. These animals recovered with only slight residual damage. This study shows that replicon vectors have potential for targeted delivery of therapeutic proteins to the central nervous system and provide a new approach for treatment of spinal cord trauma and neurological disease.

Safety and efficacy of eptifibatide vs placebo in patients receiving thrombolytic therapy with streptokinase for acute myocardial infarction; a phase II dose escalation, randomized, double-blind study.

AIMS: Thrombolytic therapy restores coronary patency in patients with acute myocardial infarction, although normal perfusion (TIMI 3 flow) is not achieved in all patients. In an attempt to improve TIMI 3 flow, a combination of full-dose streptokinase, aspirin and escalating dosages of a platelet glycoprotein IIb/IIIa receptor blocker, eptifibatide, vs placebo were tested. METHODS AND RESULTS: A bolus of 180 microg. kg(-1)of eptifibatide was administered in each group, followed by a 72 h continuous infusion of 0.75 (44 patients), 1.33 (n=45) and 2.00 microg. kg(-1). min(-1)(n = 30); 62 patients received placebo. Normal perfusion (TIMI 3 flow) at 90 min was observed in 31% of placebo patients compared to 46, 42 and 45% in the ascending eptifibatide groups (44% for combined eptifibatide groups, P = 0.07). Patency (TIMI 2 and 3 flow combined) increased from 61% (placebo) to 78% for the combined eptifibatide groups (P = 0.02). Reocclusion was infrequent. No differences were observed in TIMI flow grades among eptifibatide groups. Major and minor bleeding was increased and occurred mainly at the arterial puncture site. CONCLUSION: A combination of full dose streptokinase with different eptifibatide regimens enhanced coronary perfusion, but bleeding risk was excessive. Additional trials are needed with different dosage regimens to determine the optimal combination of fibrinolytic agents and platelet glycoprotein IIb/IIIa receptor blockers.

ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK.

ATR is a large, > 300 kDa protein containing a carboxy-terminus kinase domain related to PI-3 kinase, and is homologous to the ATM gene product in human cells and the rad3/MEC1 proteins in yeast. These proteins, together with the DNA-PK, are part of a new family of PI-3 kinase related proteins. All members of this family play important roles in checkpoints which operate to permit cell survival following many forms of DNA damage. We have expressed ATR protein in HEK293 cells and purified the protein to near-homogeneity. We show that pure ATR is a protein kinase which is activated by circular single-stranded, double-stranded or linear DNA. Thus ATR is a new member of a sub-family of PIK related kinases, founded by the DNA-PK, which are activated in the presence of DNA. Unlike DNA-PK, ATR does not appear to require Ku proteins for its activation by DNA. We show directly that, like ATM and DNA-PK, ATR phosphorylates the genome surveillance protein p53 on serine 15, a site which is up-regulated in response to DNA damage. In addition, we find that ATR has a substrate specificity similar to, but unique from, the DNA-PK in vitro, suggesting that these proteins have overlapping but distinct functions in vivo. Finally, we find that the kinase activity of ATR in the presence and absence of DNA is suppressed by caffeine, a compound which is known to induce loss of checkpoint control. Our results are consistent with the notion that ATR plays a role in monitoring DNA structure and phosphorylation of proteins involved in the DNA damage response pathways.

Paradoxical activation of Raf by a novel Raf inhibitor.

BACKGROUND: Raf is a proto-oncogene that is activated in response to growth factors or phorbol esters, and is thought to activate MAP kinase kinase-1 (MKK1) and hence the classical MAP kinase (MAPK) cascade. RESULTS: The compound ZM 336372 is identified as a potent and specific inhibitor of Raf isoforms in vitro. Paradoxically, exposure of cells to ZM 336372 induces > 100-fold activation of c-Raf (measured in the absence of compound), but without triggering any activation of MKK1 or p42 MAPK/ERK2. The ZM 336372-induced activation of c-Raf occurs without any increase in the GTP-loading of Ras and is not prevented by inhibition of the MAPK cascade, protein kinase C or phosphatidylinositide 3-kinase. ZM 336372 does not prevent growth factor or phorbol ester induced activation of MKK1 or p42 MAPK/ERK2, or reverse the phenotype of Ras- or Raf-transformed cell lines. The only other protein kinase inhibited by ZM 336372 out of 20 tested was SAPK2/p38. Although ZM 336372 is structurally unrelated to SB 203580, a potent inhibitor of SAPK2/p38, the mutation of Thr106-->Met made SAPK2/p38 insensitive to ZM 336372 as well as to SB 203580. CONCLUSIONS: Raf appears to suppress its own activation by a novel feedback loop, such that inhibition is always counterbalanced by reactivation. These observations imply that some agonists reported to trigger the cellular activation of c-Raf might actually be inhibitors of this enzyme, and that compounds which inhibit the kinase activity of Raf might not be useful as anticancer drugs. The binding sites for ZM 336372 and SB 203580 on Raf and SAPK2/p38 are likely to overlap.

Effect of SB 203580 on the activity of c-Raf in vitro and in vivo.

The inhibition of SAPK2a/p38 (a mitogen activated protein (MAP) kinase family member) by SB 203580 depends on the presence of threonine at residue 106. Nearly all other protein kinases are insensitive to this drug because a more bulky residue occupies this site (Eyers et al., 1998). Raf is one of the few protein kinases that possesses threonine at this position, and we show that SB 203580 inhibits c-Raf with an IC50 of 2 microM in vitro. However, SB 203580 does not suppress either growth factor or phorbol ester-induced activation of the classical MAP kinase cascade in mammalian cells. One of the reasons for this is that SB 203580 also triggers a remarkable activation of c-Raf in vivo (when measured in the absence of the drug). The SB 203580-induced activation of c-Raf occurs without any increase in the GTP-loading of Ras, is not prevented by inhibitors of the MAPK cascade, protein kinase C or phosphatidylinositide 3-kinase, and is not triggered by the binding of this drug to SAPK2a/p38. The paradoxical activation of c-Raf by SB 203580 (and by another structurally unrelated c-Raf inhibitor) suggests that inhibitors of the kinase activity of c-Raf may not be effective as anti-cancer drugs.

Induction of cell death by stimulation of protein kinase C in human epithelial cells expressing a mutant ras oncogene: a potential therapeutic target.

Ras oncogene activation is a key genetic event in several types of human cancer, making its signal pathways an ideal target for novel therapies. We previously showed that expression of mutant ras sensitizes human thyroid epithelial cells to induction of cell death by treatment with phorbol 12-myristate 13-acetate (PMA) and other phorbol esters. We have now investigated further the nature and mechanism of this cell death using both primary and cell line models. The cytotoxic effect of PMA could be blocked by bisindolylmaleimide (GF 109203X), a well-characterized inhibitor of c and n protein kinase C (PKC) isoforms, and by prior down-regulation of PKC, indicating that it is mediated by acute stimulation, rather than down-regulation. Western analysis identified two candidate isoforms--alpha and epsilon--both of which showed PMA-induced subcellular translocation, either or both of which may be necessary for PMA-induced cell death. Immunofluorescence showed that PMA induced a rapid nuclear translocation of p42 MAP kinase of similar magnitude in the presence or absence of mutant ras expression. Cell death exhibited the microscopic features (chromatin condensation, TdT labelling) and DNA fragmentation typical of apoptosis but after a surprising lag (4 days). Taken together with recent models of ras-modulated apoptosis, our data suggest that activation of the MAPK pathway by PMA tips the balance of pro- and anti-apoptotic signals generated by ras in favour of apoptosis. The high frequency of ras mutations in some cancers, such as cancer of the pancreas, which are refractory to conventional chemotherapy, together with the potential for stimulating PKC by cell-permeant pharmacological agents, makes this an attractive therapeutic approach.

Subspecialty distributions of ophthalmologists in the workforce.

OBJECTIVE: To describe the distribution of the supply and requirements for subspecialty ophthalmologists. METHODS: Estimates from the Eye Care Workforce Study were used to provide subspecialty-based assessments of the supply and public health need, as well as market demand, for care provided by subspecialists. Reconciliation with the boundary models (optometry first, ophthalmology first) of the Eye Care Workforce Study and current market status also were performed. RESULTS: Whether subspecialists are in excess depends first on which boundary model most closely approximates the current market conditions. Under an optometry-first model, 70% of all ophthalmologists are in excess, although subspecialists (39%) are relatively less in excess than comprehensive ophthalmologists (91% excess). Under an ophthalmology-first model, no ophthalmologists would be in excess. Extrapolating from current market conditions, a slight excess of ophthalmologists exists, probably proportional across subspecialists and comprehensive ophthalmologists. Future growth in the ophthalmologist supply will be almost entirely among subspecialists. CONCLUSION: Under current market conditions, substantial excesses in subspecialist ophthalmologists are likely to develop and grow worse over time, given current training levels.

Work time estimates for ophthalmic diagnoses and procedures. Results from the Eye Care Workforce Study.

OBJECTIVE: To provide estimates of patient-level annual ophthalmologist work times for the care of common ophthalmic conditions and patient-level global surgical care time for common or important ophthalmic procedures. METHODS: A random sample of the domestic membership (excluding members-in-training) of the American Academy of Ophthalmology, stratified by self-designated practice concentration, was surveyed in 1994 to provide estimates of work times for common ophthalmic services. RESULTS: Comprehensive and subspecialty-specific results were obtained for ophthalmic diagnoses, services, and surgical procedures. For ophthalmic diagnoses and services, initial and follow-up visit work times are reported for comprehensive and subspecialty ophthalmologists separately. For common surgical procedures, aggregate results based on comprehensive and subspecialist survey responses are reported. CONCLUSIONS: These ophthalmology-specific survey results can be used for a variety of purposes, including practice management, "benchmarking," health plan administration and national workforce planning. Such surveys should be repeated as techniques and practice patterns change over time.

Arsenite blocks growth factor induced activation of the MAP kinase cascade, upstream of Ras and downstream of Grb2-Sos.

Pretreatment of cells with 0.5 mM sodium arsenite (but not other activators of stress-activated MAP kinase cascades) prevents the activation of p21Ras and strongly suppresses the activation of c-Raf and the MAP kinase cascade by a variety of growth factors. Arsenite appears to exert its effect by preventing the guanine nucleotide exchange factor mSos from converting Ras to its active GTP-bound state. Exposure to arsenite may be a simple way of assessing whether Ras plays an essential role in mediating activation of the MAP kinase cascade by extracellular signals.

Demand-based assessment of workforce requirements for orthopaedic services.

On the basis of an analysis of the supply of and demand for orthopaedic surgeons, we projected that there will be 21,134 full-time-equivalent orthopaedists in the year 2010 if training continues at current levels. We estimated a demand-based requirement of 17,012 full-time-equivalent orthopaedic surgeons, indicating a surplus of 4122 full-time equivalents. In terms of orthopaedist-to-population ratios, we estimated that there will be 7.5 full-time-equivalent orthopaedists per 100,000 population in 2010 compared with a demand-based requirement of 6.0 full-time equivalents. However, we did not include estimates of the demand for orthopaedic surgeons as assistants in the operating room in our model. If an assistant orthopaedic surgeon is required for all procedures, an additional 3906 full-time-equivalent orthopaedists would be demanded, thus eliminating the surplus. The demand for an assistant orthopaedic surgeon in only half of the procedures would still lead to a sizable reduction in the surplus.

Spinal accessory nerve biopsy as an ante mortem diagnostic test for equine motor neuron disease.

The effectiveness of spinal accessory nerve branch biopsy evaluation as a means to confirm the diagnosis of equine motor neuron disease (EMND) was investigated. Sixteen horses with histories and clinical signs suggestive of EMND and 16 control horses with neither histories nor clinical signs of any neurological disorder, were subjects of the study. Biopsy samples of the ventral branch of the spinal accessory nerve were obtained either surgically, under general anaesthesia or post mortem immediately after euthanasia. Evaluation was done on the spinal cord of all horses to serve as the definitive diagnostic indicator of EMND. Results indicate that biopsy of the ventral branch of the spinal accessory nerve is a reliable ante mortem diagnostic test for EMND. Histological evidence of the degeneration of myelinated axons is present in both acute and arrested cases. The ventral branch of the spinal accessory nerve is easy to approach surgically and biopsy of the nerve causes no disfigurement of the sternocephalicus muscle. The use of semi-thin Epon sections is an excellent method of sample preparation. Formalin fixation and routine paraffin embedment may prove more accessible and provide good quality preparations for reliable interpretation. In the hands of an experienced pathologist, the sensitivity and specificity reliability coefficients for spinal accessory nerve branch biopsy are 94%, making this technique an extremely valuable diagnostic tool for the ante mortem diagnosis of EMND.

Estimating eye care workforce supply and requirements.

PURPOSE: To estimate the workforce supply and requirements for eye care in the United States. METHODS: Three models were constructed for analysis: supply of providers, public health need for eye care, and demand (utilization) for eye care. Ophthalmologists, other physicians, and optometrists were included in the models. Public health need was determined by applying condition-specific prevalence and incidence rates from population-based and other epidemiologic studies. Demand was determined by use of national databases, such as the National Ambulatory Care Survey, National Hospital Discharge Survey, and Medicare Part B. Time requirements for care were obtained through a stratified sample survey of the membership of the American Academy of Ophthalmology. RESULTS: Under modeling assumptions that use a work-time ratio of one between optometrists and ophthalmologists and between specialist and generalist ophthalmologists, a significant excess of eye care providers exists relative to both public health need and demand. Changes in the work-time ratio, work-hours per year per provider, care patterns for the same condition, or other factors could significantly reduce or eliminate the surplus relative to need. CONCLUSION: If optometrists are the preferred primary eye care provider, ophthalmologists would be in excess under all demand scenarios and all need scenarios where the optometrist to ophthalmologist work-time ratio is greater than 0.6. No excess of ophthalmologists would exist if ophthalmologists are the preferred primary eye care provider. Data on the appropriate work time ratio will help refine estimates of the imbalance between supply and requirements.

Neurological manifestation of cholesterinic granulomas in three horses.

Cholesterinic granulomas have been previously reported as an incidental post mortem in horses. Three adult horses with diencephalic dysfunction due to cholesterinic granulomas are described. All the horses exhibited profound depression, somnolence and reluctance to move. One horse experienced generalised seizures. Cerebrosinal fluid was xanthochromic with an elevated total protein in two of the cases evaluated. The large cholesterinic granulomas caused expansion of the lateral ventricle and secondary hydrocephalus due to the build up of cerebrospinal fluid behind the mass. Cholesterinic granulomas are believed to result from choroid plexus congestion and haemorrhage.

A comparison of the relationships of the glucose tolerance test and the glycated haemoglobin assay with diabetic vascular disease in the community. The Islington Diabetes Survey.

We have compared the relationships of fasting and 2 h blood-glucose during a 75 g oral glucose tolerance test, and those of an affinity chromatography assay of glycated haemoglobin, with the presence of vascular complications of diabetes mellitus in 223 subjects without known diabetes aged over 40 years selected from a community screening study population. The subjects included 15 (6.9%) with newly diagnosed diabetes and 52 (24.1%) with impaired glucose tolerance. Employing receiver operating characteristic analysis, the tests were similar in their relationship with three cases of retinopathy, 19 of microalbuminuria and six of peripheral neuropathy. The prevalence of coronary heart disease, defined as angina, myocardial infarction, or electrocardiographic changes of ischaemia, increased linearly across all four quartiles of both 2 h blood glucose and glycated haemoglobin concentration, but using logistic regression analysis, 2 h blood glucose was a better predictor of coronary heart disease than glycated haemoglobin. Receiver operating characteristic analysis also showed that 2 h blood glucose generally performed better than any of four assays of glycated haemoglobin in classifying those subjects with coronary heart disease.

Protein synthesis is required for cholera toxin-induced stimulation of arachidonic acid metabolism.

The molecular events in the mechanism of action of cholera toxin were analyzed using Chinese hamster ovary (CHO) cells. Cholera toxin stimulated both 3',5'-cyclic adenosine monophosphate (cAMP) synthesis and arachidonic acid metabolism in these cells. The turnover of phospholipid by cholera toxin-induced stimulation of phospholipase activity evoked the synthesis of PGE2 and other prostaglandins. Cholera toxin-induced release of both [3H]arachidonic acid and PGE2 was blocked by addition of either cycloheximide or actinomycin D. In contrast, accumulation of cAMP in cholera toxin-treated CHO cells was unaffected by adding these drugs. Further, dibutyryl cAMP or forskolin caused [3H]arachidonic acid release, which also was blocked by cycloheximide and actinomycin D. We concluded that the sequence of molecular events in cholera toxin-treated CHO cells first involved activation of adenylate cyclase, which caused an increase in cAMP. In turn, cAMP promoted transcription of mRNA that encoded either a specific phospholipase or a phospholipase-activating protein. The emerging arachidonic acid metabolites (e.g., PGE2 and PGF2 alpha) might be important mediators of cholera toxin's stimulatory effects on vascular permeability and smooth muscle contraction in the intestine during cholera.

Chloroquine inhibition of cholera toxin.

Cholera toxin (CT) stimulated adenylate cyclase and a phospholipase which elevated cellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) and arachidonic acid (AA). The AA was quickly converted to prostaglandins (PGs) via the cyclo-oxygenase pathway. Chloroquine exerted minimal inhibition of cAMP levels in CT-treated cells, although CT-induced release of [3H]AA and PGs was blocked completely when the drug was added in concentrations as low as 0.1 mM (50 micrograms/ml). Inhibition of [3H]AA release was complete when chloroquine was added before or within 30 min after CT. The capacity of chloroquine to inhibit either phospholipase C (PLC) or phospholipase A2 (PLA2) could explain the antisecretory activity of this drug.

Synthesis of prostaglandins in cholera toxin-treated Chinese hamster ovary cells.

The prostaglandin (PG) and adenosine 3',5'-cyclic monophosphate (cAMP) responses of Chinese hamster ovary (CHO) cells were measured after cholera toxin (CT) exposure to evaluate dose and kinetic relationships. Release of prostaglandin E2 (PGE2) and the accumulation of cAMP were dependent on the dose of CT, with an effective dose of approximately 10-100 ng/ml within 4 h; the PGE2 response was about four- to six-fold more than that of PGE1. CHO cells exposed to CT also released increased amounts of thromboxane B2 (TxB2), PGF2 gamma, and 6-keto PGF1 gamma (a non-enzymatic degradation product of prostacyclin). Kinetic analysis of CT-treated cells revealed that small peaks of cAMP accumulation and of PGE1 and PGE2 release were detected at approximately 30 min, but larger, progressive PG and cAMP responses were measured 2-4 h later. Exposure of the cells to relatively high doses of membrane-permeable derivatives of cAMP (1 mM) and forskolin (10 microM) caused PGE2 release. Concomitantly, exogenous PGE2 (100 microM) increased intracellular levels of cAMP. We have considered the interrelationship of the cyclo-oxygenase and the cyclic nucleotide pathways relative to the molecular mechanism of CT.