Induction versus progression of brain tumor development: differential functions for the pRB- and p53-targeting domains of simian virus 40 T antigen.

The ability of simian virus 40-encoded large T antigen to disrupt the growth control of a variety of cell types is related to its ability to interfere with certain cellular proteins, such as p53 and the retinoblastoma susceptibility gene product (pRB). We have used wild-type and mutant forms of T antigen in transgenic mice to dissect the roles of pRB, p53, and other cellular proteins in tumorigenesis of different cell types. In this study, using a cell-specific promoter to target expression specifically to brain epithelium (the choroid plexus) and to B and T lymphoid cells, we characterize the tumorigenic capacity of a T-antigen fragment that comprises only the amino-terminal 121 residues. This fragment (dl1137) retains the ability to interact with pRB and p107 but lacks the p53-binding domain. While loss of the p53-binding region results in loss of the capacity to induce lymphoid abnormalities, dl1137 retains the ability to induce choroid plexus tumors that are histologically indistinguishable from those induced by wild-type T antigen. Tumors induced by dl1137 develop much more slowly, however, reaching an end point at around 8 months of age rather than at 1 to 2 months. Analysis of tumor progression indicates that tumor induction by dl1137 does not require secondary genetic or epigenetic events. Rather, the tumor growth rate is significantly slowed, indicating that the T-antigen C-terminal region contributes to tumor progression in this cell type. In contrast, the pRB-binding region appears essential for tumorigenesis as mutation of residue 107, known to disrupt pRB and p107 binding to wild-type T antigen, abolishes the ability of the dl1137 protein to induce growth abnormalities in the brain.

Use of transgenic mice reveals cell-specific transformation by a simian virus 40 T-antigen amino-terminal mutant.

We have used the multifunctional transforming protein, simian virus 40 T antigen, as a probe to study the mechanisms of cell growth regulation in the intact organism. T antigen appears to perturb cell growth, at least in part, by stably interacting with specific cellular proteins that function to maintain normal cell growth properties. Experiments in cultured cells indicate that at least three distinct regions of simian virus 40 T antigen have roles in transformation. Two regions correlate with the binding of known cellular proteins, p53, pRB, and p107. A third activity, located near the amino terminus, has been defined genetically but not biochemically. By targeting expression of wild-type and mutant forms of T antigen to distinct cell types in transgenic mice, we have begun to systematically determine which activities play a role in tumorigenesis of each cell type. In this study, we sought to determine the role of the amino-terminal transformation function with such an analysis of the T-antigen mutant dl1135. This protein, which lacks amino acids 17 to 27, retains the p53-, pRB-, and p107-binding activities yet fails to transform cells in culture. To direct expression in transgenic mice, we used the lymphotropic papovavirus transcriptional signals that are specific for B and T lymphocytes and the choroid plexus epithelium of the brain. We show here that although defective in cell culture, dl1135 specifically induced the development of thymic lymphomas in the mouse. Expression of the protein was routinely observed in B- and T-lymphoid cells, although B-cell abnormalities were not observed. Choroid plexus tumors were observed only infrequently; however, dl1135 was not consistently expressed in this tissue. Within a given transgenic line, the penetrance of T-cell tumorigenesis was 100% but appeared to require secondary events, as judged from the clonal nature of the tumors. These experiments suggest that the amino-terminal region of T antigen has a role in the transformation of certain cell types (such as fibroblasts in culture and B lymphocytes) but is dispensable for the transformation of T lymphocytes.

Manganese metabolism in cows and goats.

When 54MnCl2 was incubated with fresh bovine or caprine serum for 20 h and the serum subjected to electrophoresis at pH 9.5, the 54Mn bound to transferrin and alpha2-macroglobulin in proportions which varied with the temperature of incubation and the temperature of electrophoresis. Between 0 and 37 degrees C, the higher the temperature of incubation the larger the proportion bound to transferrin and the lower the proportion bound to alpha2-macroglobulin. The temperature at which electrophoresis was performed had little effect on the proportion of 54Mn bound to transferrin, but increasing temperature reduced the proportion of 54Mn bound to alpha2-macroglobulin. Mn2+ did not bind to purified transferrin in vitro in the absence of an oxidising agent. In the presence of permanganate, Mn3+ was formed and chelated by transferrin at physiological pH. In fresh serum this oxidation step may be performed by ceruloplasmin or molecular oxygen. Mn2+ was bound reversibly to alpha2-macroglobulin but this protein played no part in the oxidation of divalent manganese and had no effect on the protein binding of trivalent manganese. Manganese in the divalent state, either free as Mn2+ or bound to alpha2-macroglobulin, is removed from blood plasma very efficiently by the liver. However, the manganic-transferrin complex normally found in circulation is not rapidly removed from plasma. The liver can remove large amounts of excess manganous manganese which it presumably excretes; the small essential fraction of the manganese absorbed is oxidised to the trivalent state and bound to transferrin.

Absorption of N4-D-glucopyranosylsulphamethazine by rat everted intestinal sacs.

Absorption of the N4-D-glucose conjugate of sulphamethazine (glucose-SMZ, 0.5 mM) by isolated everted sacs of the rat small intestine was studied at 37 degrees and pH 6.6. Phlorizin (0.5-2.0 mM) significantly reduced (P < 0.05) both mucosal and serosal transfer of glucose-SMZ and inhibition of mucosal transfer appeared to be concentration-dependent. Phloretin (0.5 mM) and removal of Na+ from the incubation medium also diminished absorption of glucose-SMZ. Furthermore, D-glucose (0.5 and 5.0 mM) inhibited mucosal and serosal transfer of the glycoside. The results suggest the D-glucose/Na+ cotransporter mediates absorption of glucose-SMZ from the small intestine of the rat. Thus, glucose-SMZ might be bioavailable from ingested tissues in which it is present.

Expression of mRNAs coding for the transforming growth factor-beta receptors in brain regions of euthyroid and hypothyroid neonatal rats and in adult brain.

The TGF-beta family of peptides has been postulated to play a role in control of the cell cycle but also may act in the developing brain to influence neuronal differentiation and survival. Because reception of TGF-beta signals requires the simultaneous expression of all three known receptor subtypes, we examined two neonatal rat brain regions in which neurogenesis has been largely completed. mRNA coding for all three receptors was detectable in both the forebrain and brainstem but only the type II receptor in brainstem showed a difference from adult levels of expression. Animals given perinatal PTU treatment to achieve congenital cretinism did not show significant differences in expression of any of the receptor subtypes in either of the regions, despite the fact that the treatment is known to cause anomalies of neuronal differentiation. These results indicate that regions in which neurons are undergoing axonogenesis and synaptogenesis rather than neurogenesis, nevertheless express the mRNAs coding for TGF-beta receptors and are thus likely to be receptive to trophic signals mediated through TGF-beta. However, synthesis and release of TGF-beta, rather than receptor expression per se, is more likely to be the major point for regulation of signaling. The potential roles of TGF-beta in developmental events outside of the cell cycle, such as synaptogenesis and apoptosis, need to be examined.

A novel form of dependency of hepatic extraction ratio of opioids in vivo upon the portal vein concentration of drug: comparison of morphine, diamorphine, fentanyl, methadone and buprenorphine in the chronically cannulated cow.

In the chronically cannulated cow, the hepatic extraction ratio for intravenous boluses of morphine, diamorphine, fentanyl, methadone and buprenorphine increased towards a plateau value as portal vein drug concentration increased. An extraction ratio close to zero for morphine was observed at a portal vein plasma drug concentration of about 200 nanomol per litre, which is within the range for significant pharmacodynamic effects. The similar concentrations extrapolated for the other narcotics would be of less pharmacodynamic importance. The phenomenon did not depend with morphine on the history of drug delivery to the liver; measurement of hepatic blood flow showed the effect was not an artifact of unrepresentative blood sampling, and was not related to any action of the narcotics on hepatic blood flow. The existence of this novel type of concentration dependent hepatic extraction ratio in vivo can explain a number of anomalous observations on narcotic pharmacokinetics, especially for morphine. Furthermore, similar behaviour may be expected for non-opioid drugs having similar pharmacokinetic properties.

Canadian guidelines for cardiac rehabilitation and atherosclerotic heart disease prevention: a summary.

Atherosclerotic heart disease (AHD) is the leading cause of death in Canadian women and men. Cardiac rehabilitation has been repeatedly shown to reduce cardiac morbidity and mortality significantly among patients with documented AHD. The Canadian Association of Cardiac Rehabilitation (CACR) has defined cardiac rehabilitation as "the enhancement and maintenance of cardiovascular health through individualized programs designed to optimize physical, psychological, social, vocational and emotional status. This process includes the facilitation and delivery of secondary prevention through heart hazard (risk factor) identification and modification in an effort to prevent disease progression and the recurrence of cardiac events". This summary presents a limited amount of background information and the majority of clinical practice recommendations contained within the previously published CACR Guidelines. These evidence-based clinical recommendations are intended as guidelines to good clinical practice rather than as standards of care. The key focus of this summary is the need for complete and targeted intervention of all heart hazards in patients at high or very high risk for, or with documented, AHD. To achieve this goal, the CACR Guidelines and this summary present risk stratification strategies designed to determine unambiguously a patient's risk of exercise-related cardiac events (short term absolute risk or disease prognosis) and their risk of recurrent AHD events (long term absolute risk from disease progression). The establishment of the short term and long term absolute AHD risks can then be used to determine heart hazard targets and the type of exercise program prescribed for patients with AHD. Despite the use of evidence-based medical practices, none of the recommendations presented in this document can replace the expert judgment of properly trained and experienced cardiac rehabilitation professionals. Health care providers must always be free to choose where and when clinical practice guidelines are applied, modified or superceded, depending on individual patient circumstances.

Enhancement of tetrathiomolybdate-induced biliary copper excretion in sheep by general anaesthesia and the effect on copper excretion in urine and bile.

To determine whether copper removed from the liver by tetrathiomolybdate (TTM) but not excreted in bile, is excreted in saliva or urine, the effect of an intravenous injection of 100 mg of TTM, as ammonium tetrathiomolybdate, on copper excretion by these routes was measured in conscious sheep and sheep maintained under general anaesthesia. During 4.5 hours the total amount of copper excreted in bile in the untreated, conscious animal was 0.52 +/- 0.04 mumols (mean +/- SE). A dose of 100 mg TTM increased excretion by an average of 11.7-fold to 6.06 +/- 0.29 mumols. Under halothane or barbiturate general anaesthesia copper excretion was increased 3.4-fold to 1.78 +/- 0.30 mumols. In anaesthetised sheep 100 mg TTM produced a 40.7-fold increase to 21.18 +/- 0.95 mumols. Neither anaesthesia nor TTM caused any increase in copper excretion in saliva or urine.

Enhancement of tetrathiomolybdate-induced copper excretion in bile of sheep by the alpha 2-agonistic action of xylazine.

The effect of intravenous doses of cortisol and xylazine on the quantity of copper excreted in response to 100 and 200 mg doses of tetrathiomolybdate (TTM) was studied in seven sheep. Cortisol alone produced a non-significant 1.4-fold increase and had no enhancing action on the response to TTM. Xylazine produced a significant 2.25-fold increase, doubled the quantity of copper excreted in response to both doses of TTM and reduced bile flow by approximately 35 per cent. The alpha 2 antagonist, idazoxan, prevented both the latter effects showing that they were due to xylazine's alpha 2-agonistic action. It is suggested that the combination of an alpha 2-agonist with the intravenous injection of TTM in the treatment of acute copper toxicity in sheep could reduced by 50 per cent the amount of molybdenum needed.

Acute manganese toxicity and the absorption and biliary excretion of manganese in cattle.

The concentration of manganese in the duodenum of three steers was increased by infusing manganese chloride intraduodenally at approximately 100, 515 and 840 mumol per minute for 30 hours and the rate at which manganese was excreted in bile was measured. In one steer an infusion of manganese chloride at 529 mumol per minute was toxic and in two steers infusions at approximately 840 mumol per minute were also toxic, causing inappetence, a reduced bile flow and abdominal discomfort. The maximum rates at which manganese was excreted in bile during the first three hours of the infusions at 515 and 840 mumol per minute were between 2.1 and 3.6 per cent of the rate of infusion. During the last three hours of all nontoxic infusions the rate of excretion of manganese in bile was 0.82 +/- 0.08 per cent of its rate of infusion. Systemic plasma manganese concentrations did not increase during these infusions. In a fourth steer the longer term capacity of the liver to excrete manganese in bile when the manganese chloride was given intravenously was measured, the concentration of manganese in the portal vein being increased by infusions of manganese chloride solution into a mesenteric vein. The rate of infusion was increased at hourly intervals. The liver's capacity to remove all manganese during first pass was only exceeded at an infusion rate of 84 mumol per minute. Infusions could be made into the mesenteric vein at 24 and 35.8 mumol per minute for 53 and 26 hours respectively before signs of toxicity were observed. Following signs of toxicity the excretion of copper in bile was decreased for two days.

A surgical technique for the long term collection of bile in the pig and measurement of the biliary excretion of copper and zinc.

A method is described for surgically modifying the gastrointestinal tract of the pig which enables the long term collection of bile and measurement of bile flow and allows the pig to be kept unrestrained in a large pen. Three surgically prepared gilts grew at an average of 0.79 kg d-1. Bile flow increased with bodyweight from 1.6 ml min-1 at 45 to 50 kg to 3.5 ml min-1 at 124.5 kg bodyweight. Mean output in bile of copper and zinc by the three gilts during the period 08.00 to 12.30 was 161.0 +/- 11.1 and 3.94 +/- 0.41 nmol min-1, respectively. The calculated output during 24 hours represented approximately 5.8 and 0.13 per cent of the daily intake of copper and zinc, respectively.

The absorption of dietary manganese by dairy cows.

Additional manganese, sufficient to increase the dietary concentration to approximately 1000 mg/kg, was administered to one cow for four and another for seven days. Manganese concentrations in portal and mesenteric plasma were increased by a mean of 3.9 microgram/litre above the concentration in systemic plasma. This increment is equivalent to the absorption of 0.54 per cent of the administered manganese. The cow which received manganese for seven days showed after six days larger increases in systemic plasma manganese concentration which may indicate a toxic effect of excess manganese upon the liver.

The half-life of glutamate dehydrogenase in plasma of dry and lactating dairy cows.

The clearance of glutamate dehydrogenase from plasma was measured weekly for three weeks in three dry and three lactating cows. The clearance was exponential with a mean clearance constant of 0.0488/j and means (+/- SE) half-life of 14.2 (+/- 0.77) h. There were variations among cows and among measurements in the same cow but there was no difference between the mean half-lives of GDH in lactating and dry cows. Because of the long half-life the small variations among individual cows are unlikely to affect the interpretation of increased plasma GDH activity.

Maximum capacity of the bovine liver to remove manganese from portal plasma and the effect of the route of entry of manganese on its rate of removal.

The rate of uptake of manganese by the liver was measured during infusions of manganese into either the visceral or the systemic circulation of four cows surgically prepared with cannulae in a mesenteric vein, the portal vein and a hepatic vein and in one carotid artery. In three experiments the maximum capacity of the liver to remove manganese from plasma was measured by infusing manganese chloride solution into a mesenteric vein at increasing rates for six hours. In two experiments the rate of uptake of manganese by the liver was determined during infusions of manganese chloride solution into a jugular vein for six hours at a constant rate. Virtually all the manganese infused into a mesenteric vein was cleared during its first pass through the liver, up to a maximum rate (mean +/- SEM) of 97.1 +/- 14.1 mumol per minute or 12.7 +/- 2.3 mumol per minute per kg of liver weight. However, when manganese was infused into a jugular vein at rates less than this maximum rate of uptake only approximately 50 per cent of the manganese in portal plasma was removed during a single passage through the liver.

Surgical procedure for modifying the duodenum in cattle to measure bile flow and the diurnal variation in biliary manganese, iron, copper and zinc excretion.

A surgical procedure is described for modifying the duodenum of cattle so that bile can be sampled and its rate of flow measured for long periods. In 12 steers, aged three months to three years and weighing between 50 and 650 kg, bile flow ranged from 2 to 12 ml per minute, the rate of flow increasing with bodyweight. The rate of flow expressed as ml per minute per 100 kg bodyweight decreased as bodyweight increased, the regression equation being in (ml per minute per 10 kg) = 1.65-0.0022 x where x = bodyweight (kg), r = 0.871. The mean concentrations of copper, iron, manganese and zinc in bile were 8.2, 6.2, 17.1 and 3.3 mumol per litre respectively. The concentration of iron was the least variable between the steers. The average total cholate concentration was 100 mmol per litre and total solids ranged from 5.4 to 7.2 per cent. Diurnal patterns in bile flow and trace element excretion were measured in four adult steers during a period of 38.5 hours. Although the rate of excretion of iron, zinc and copper and bile flow varied throughout the period, changes could not be associated with feeding or time of day. The concentration of manganese in bile and its excretion rate varied in a reproducible manner which may be related to feeding activity. The mean output of the four trace elements in bile from the four steers during 24 hours was, copper 37.7 mumol, iron 68.0 mumol, manganese 80.3 mumol and zinc 59.6 mumol.

The effect of copper and heliotrope on the composition of bile in sheep.

The effects of interrupting the enterohepatic circulation (EHC) of bile salts for seven hours and of feeding copper and heliotrope alone and combined for 13 weeks, on bile flow and excretion of copper, zinc, iron and alpha-mannosidase were studied in sheep. Interruption of EHC reduced bile flow rate and increased the concentration of copper, zinc, iron and bile acids while alpha-mannosidase's activity remained stable. Changes in concentration were related to changes in bile volume for copper and zinc only. Total output per hour was not changed. Biliary concentration of copper correlated with alpha-mannosidase's activity in control sheep and those given copper or heliotrope, supporting the hypothesis that lysosomes are involved in biliary secretion of copper in sheep. Increasing the intake of copper increased the rate of excretion of copper in bile. Copper output was lower when heliotrope was fed alone.

Bile flow, bile salt secretion and the excretion of iron, copper, zinc and manganese in the bile of calves infected with Fasciola hepatica.

The rate of flow of bile and the concentrations of iron, copper, zinc and manganese in bile were measured in four bull calves, before and for at least 23 weeks after infection with 1000 metacercariae of Fasciola hepatica. Bile flow rate began to increase about 10 weeks after infection and had increased nearly three-fold by 23 weeks but bile salt (total-cholate) secretion rate remained similar to that of two control calves. The excretion of iron in bile increased rapidly eight weeks after infection from a mean of 21.5 nmol per minute to reach 469 nmol per minute at 14 weeks and this increase was accompanied by an increase in gamma-glutamyl transpeptidase activity in plasma. 59Fe studies showed that most of the iron in the bile of infected calves was derived from red blood cells. There were no changes in the excretion of zinc, copper or manganese in bile which could be associated with the infection.

Welfare of calves - 2. Increase in vertebral artery blood flow following exsanguination by neck sticking and evaluation of chest sticking as an alternative slaughter method.

The role of the vertebral arteries in delaying loss of sensibility following neck sticking in slaughter calves was investigated. Vertebral artery blood flow was measured using probes before, during and after electrical stunning and slaughter. Systemic blood pressure, electrocorticogram, visually evoked responses and the occurrence of carotid occlusions were also recorded. When carotid occlusion occurred, the time to onset of brain failure was delayed based on the development of an isoelectric state. In addition, when carotid occlusion occurred the mean arterial blood pressure was sustained for longer following slaughter, and concurrently vertebral artery blood flow could be maintained at about 30% of its initial level for up to 3 min. In some animals vertebral artery flow increased substantially following sticking. When chest sticking was used no occlusion of vessels occurred, mean arterial blood pressure fell promptly (within 8 s) and the onset of an isoelectric state did not extend beyond one minute. In addition, visually evoked responses were not present after 5 s following chest sticking.

The effect of xylazine upon hepatic glucose production and blood flow rate in the lactating dairy cow.

The effect of xylazine (Rompun) upon hepatic glucose production and blood flow rate was measured in two cows. Doses of 0-16 or 0-18 mg/kg bodyweight increased blood glucose concentrations by 200 per cent and hepatic glucose production by 400 per cent. Maximum blood glucose concentrations were reached approximately 40 minutes after dosing and did not start to fall until 185 minutes. Concentrations were near normal 24 hours after dosing. The increase in hepatic glucose production was greatest 20 minutes after dosing and production had returned to control rates 150 minutes after dosing. Visceral glucose utilisation was also increased. Blood flow rates in the hepatic and portal veins were reduced to 50 to 60 per cent of their predosing values. It is concluded that the prolonged hyperglycaemia which persists beyond 150 minutes is produced either by continued glucose production from sites other than liver and viscera or by reduced utilisation of the blood glucose by peripheral tissue.

The choleretic effect of menbutone and clanobutin sodium in steers.

Adult steers were given either clanobutin or menbutone intravenously and the effects on bile flow measured. Doses of 4.3 g of clanobutin or 3.0 g of menbutone had no effect on bile flow. However, when bile flow was previously reduced by reducing the total bile salts in the enterohepatic circulation both compounds were potent choleretics, increasing the volume of bile flow up to four-fold.