[Effects of oxygen free radicals on cell growth and productions of alpha-fetoprotein (AFP) and albumin (ALB) by human hepatoma cell line (HH2-6)]. Secreting and producing capacities of alpha-fetoprotein (AFP) and albumin (ALB) by human hepatoma cell line (HH2-6) exposed to oxygen free radicals generated by dihydroxyfumarate (DHF) were studied in vitro. It was found that cell number were declined in proportion to DHF concentrations for 48 hrs culture and cell proliferations were inhibited by DHF in growth curve. The amount of AFP secreted per cell (secreting capacity) was decreased at high concentration of DHF (50 micrograms/ml) for 48 hrs culture, and remarkable elevation of AFP-secreting capacity for growth stage was inhibited even by DHF (10 micrograms/ml). On the other hand, ALB-secreting capacity was not affected with DHF. Producing capacities of AFP and ALB were correlated with secreting capacities. Cu, Zn-superoxide dismutase (Cu, Zn-SOD) interfered these reactions. These results suggest that oxygen free radicals inhibit cell proliferations and suppress AFP-secreting and producing capacities of HH2-6 cells selectively.


[Molecular size heterogeneity of SPan-1 antigen and co-expression with Lewis phenotype determinants in sera from gastrointestinal malignant diseases]. Molecular size of SPan-1 antigen and co-expression between SPan-1 epitope and Lewis phenotype determinants (LPD) on the same molecule in sera from patients with pancreatic, gastric and colon cancer and in culture medium from cancer cell line were studied. Column chromatography study on culture medium and sera showed that SPan-1 immunoreactivity was found in the void volume region as single major peak, or followed by one or two minor peak in the included volume. All of 4 pancreatic cancer cell lines showed molecular size heterogeneity. Co-expression between SPan-1 epitope and LPD on the same molecule was recognized in sera from cancer patients with Lewis a or Lewis b positive, but not in patients with Lewis a-, b-. Molecular carrying SPan-1 epitope had more frequently SPan-1 epitope than LPD in sera from cancer patients. These findings suggest that molecular size heterogeneity of SPan-1 antigen was more often in pancreatic cancer cells and overexpression of SPan-1 epitope on the same molecule seems to be specific for cancer.


[Effects of nutritional status on contents of tryptophan, serotonin and 5-hydroxyindoleacetic acid in rat brain]. Effects of nutritional status on the levels of tryptophan (Trp), serotonin (5HT), and its metabolite, 5-hydroxyindoleacetic acid (5HIAA) in six brain regions of rats were investigated. 1) A low-protein high-carbohydrate diet decreased Trp, 5HT and 5HIAA levels in the cortex and hippocampus, and those of 5HT and 5HIAA in the hypothalamus. This diet did not affect the contents of Trp and 5HT in the midbrain, but decreased 5HIAA. No significant changes of Trp, 5HT and 5HIAA were observed in the pons and medulla and striatum. 2) A low-carbohydrate high-protein diet increased the levels of Trp, 5HT and 5HIAA in the striatum, and 5HT and 5HIAA in the cortex, but showed no effect on the contents of Trp, 5HT and 5HIAA in the hippocampus, midbrain, pons and medulla and hypothalamus. 3) An energy-restriction low-carbohydrate diet increased Trp, 5HT and 5HIAA contents in the striatum, and 5HT and 5HIAA in the cortex and pons and medulla. In the hypothalamus, only 5HIAA was increased by this diet. The diet did not influence Trp, 5HT and 5HIAA contents of the midbrain and hippocampus. These results suggest that i) lowered fat and carbohydrate intakes enhance 5HT synthesis and metabolism in the cortex and that lowered carbohydrate intake enhances them in the striatum, ii) energy restriction enhances the 5HT metabolism in the cortex, pons and medulla and hypothalamus, and 5HT synthesis in the cortex and pons and medulla, iii) lowered protein intake inhibits 5HT metabolism in the cortex, midbrain, hippocampus and hypothalamus, and 5HT synthesis in the cortex, hippocampus and hypothalamus.


Sites of action of 2-thiazoline-2-thiol on biogenesis of thyroid hormones. 2-Thiazoline-2-thiol is an antithyroid agent that strongly reduces thyroid hormone levels. Synthesis of these hormones is catalyzed in vivo by thyroid peroxidase. The interaction of this drug with molecular iodine and its effect on peroxidase activity were investigated. Iodine and 2-thiazoline-2-thiol form a complex of the charge transfer type of 1:1 stoichiometry characterized by a formation constant of 2,527 l.mole-1 at 20 degrees C. This drug was found to inhibit both horseradish peroxidase and lactoperoxidase (used as a model of thyroid peroxidase) in a competitive manner, giving inhibition constants of 5.7 mM and 0.13 mM, respectively. T3 and T4 levels were reduced significantly after a three-week administration of this drug to a group of 10 rats. Histological examination of the thyroid gland showed the presence of a cylindrical epithelium, which is indicative of hyperactivity of the gland. The results indicated that 2-thiazoline-2-thiol acts on both molecular iodine and thyroid peroxidase.


Effects of inhibitors of protein kinase C on the release and synthesis of histamine in rat basophilic leukemia cells (2H3). In rat basophilic leukemia cells (2H3), a tumor analog of mast cells, the aggregation of IgE receptors results in histamine secretion and the increase in histidine decarboxylase activity which synthesizes histamine. Using inhibitors of protein kinases C, we studied the relationships between these events and protein kinase C which is activated by antigens. Histamine release is suppressed by inhibitors of protein kinase C, staurosporine, K252-a and H-7, in this decreasing order of effectiveness; and the IC50 values are 1.5 nM, 29.9 nM and 3.8 microM, respectively. The changes in the intracellular Ca concentration monitored by fura-2 fluorescence is not modified by staurosporine, although the histamine response is suppressed. Meanwhile, the increase of histidine decarboxylase was abolished by inhibitors of protein kinase C; staurosporine was the strongest, K-252a of moderate activity and H-7, the weakest, having IC50 values of 0.8 nM, 100 nM and 11.5 microM, respectively. The inhibitors of protein kinase C suppress both histamine secretion and synthesis. Therefore, the histamine synthesis may be stimulated via activation of protein kinase C to supplement the released histamine.


Sialogogic effects on rat submandibular gland of analogs of the C-terminal hexapeptide of substance P. The sialogogic response of submandibular glands to analogs of the C-terminal hexapeptide of substance P with various amino acids at the N-terminus was investigated in urethane-anesthetized rats. The rank order of potencies was as follows: SP greater than (pGlu6)SP6-11 much greater than (Dab6)SP6-11 greater than (Orn6)SP6-11 greater than (Gln6)SP6-11 greater than (Lys6)SP6-11 much greater than (Ala6)SP6-11. These results suggest that the sialogogic activity of the analogs of the C-terminal hexapeptide is influenced by the steric effects of the N-terminal amino acid, and the nature of its side chain is of particular importance.


Risk factors for dialysis-associated hepatitis C in Venezuela. Utilizing the first and second generation of enzyme immunoassays which detect antibodies to the C virus we investigated the frequency of anti-HCV antibodies in 315 patients undergoing hemodialysis. Other subpopulations at risk were used as reference groups. One hundred and twenty-three samples (39%) from the hemodialysis group repeatedly showed anti-HCV positive antibodies while only 19% and 1% were positive in the reference groups. The rate of anti-HCV reactive patients correlated with time on hemodialysis (less than 1 year, 17%; 1 to 5 years 43%; greater than 5 years, 64%; r = 0.94, P less than 0.001) and with the number of blood transfusions (1 to 10, 40%; greater than 10, 76%; r = 0.97; P less than 0.001). Length of time on hemodialysis was shown to be the major risk factor in thirty-three anti-HCV positive patients who had no previous record of blood transfusions. Co-infection with HBV was demonstrated in 41% out of 123 anti-HCV reactive patients, and increased alanine aminotransferase (ALT) activity was documented in this co-infected group. Our results further extend the observations on the predisposing factors to HCV spread in hemodialysis units, and suggest that in these renal patients co-infection with C and B viruses is a major cause of rising ALT activity.


HLA class II specificities in vasculitis with antibodies to neutrophil cytoplasmic antigens. HLA class II genes were examined in patients with small vessel vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA) using restriction fragment length polymorphism and allele specific oligonucleotide typing. Fifty-nine patients were studied, 34 with Wegener's granulomatosis and 25 with microscopic polyarteritis, and their results were compared with those from 1103 British Caucasoid controls. The frequency of HLA-DQw7 was significantly increased in patients with vasculitis (patients 53%; controls 27.8%, chi 2 17.8, Pc less than 0.0025, relative risk 2.9), and all the DQw7 bearing haplotypes commonly found in Caucasoid populations contributed to the increase. By contrast, the frequency of HLA-DR3 bearing haplotypes was decreased in the patients (patients 6.8%; controls 21.6%, chi 2 6.7, P less than 0.01). HLA specificities were similar in the groups of patients presenting with Wegener's granulomatosis and microscopic polyarteritis and with different types of ANCA assessed by indirect immunofluorescence. However, patients with the DQw7, DR4 haplotype were significantly more likely to have transiently positive tests for ANCA than patients with other DQw7 bearing haplotypes, whereas patients with DR2 bearing haplotypes were more likely to have persistently positive ANCA. These results show that HLA class II genes are associated with small vessel vasculitis and may influence the duration of the associated autoimmune response.


Early increased renal procollagen alpha 1(IV) mRNA levels in streptozotocin induced diabetes. Changes in renal procollagen mRNA levels were measured shortly after the induction of streptozotocin induced diabetes in the rat. "Medullary" procollagen alpha 1(IV) levels seven days after diabetes induction was significantly higher in untreated diabetic rats (DM, N = 12; 244 +/- 57% of the mean control value), than in diabetic rats receiving small doses of insulin insufficient to achieve euglycemia (NPH, N = 10; 87 +/- 12%) and in diluent injected nondiabetic control rats (C, N = 15; 100 +/- 12%; P less than 0.01, DM vs. C and DM vs. NPH). "Medullary" procollagen alpha 1(I) mRNA levels were numerically increased in DM to a lesser degree (141 +/- 5%, ANOVA not significant) compared to C (100 +/- 13%), and this small increment was further normalized by insulin treatment (NPH, 120 +/- 11%). A trend for increased beta-actin mRNA levels in DM did not reach significance (P greater than 0.05). Increases in "medullary" procollagen mRNA levels did not correlate with kidney weight, glomerular tuft volume, creatinine clearance, food intake, or body weight gain, and occurred when renal morphology was normal by light microscopy. Statistically significant but weak correlations were noted between the serum glucose levels and "medullary" procollagen alpha 1(IV) mRNA levels (r = 0.43, P less than 0.05). In addition, weak correlations were noted between glycosuria and "medullary" procollagen alpha 1(I) levels (r = 0.38, P less than 0.05). In situ hybridization studies localized the increased procollagen alpha 1(IV) mRNA levels predominantly in the DM group primarily in the deep cortex and medullary outer stripe of proximal tubules. Glomerular procollagen alpha 1(IV), alpha 1(I), alpha 1(III) and beta-actin mRNA levels were not increased in untreated diabetic rats 7 or 28 days after diabetes induction. Thus, tubular procollagen alpha 1(IV) mRNA levels increased prior to any measurable change in glomerular levels and were ameliorated by insulin administration.


Glomerular size-selectivity and microalbuminuria in early diabetic glomerular disease. Sieving coefficients of uncharged dextrans of graded size (radii 30 to 60 A) were used to characterize barrier size-selectivity in nonazotemic diabetic humans with microalbuminuria (Group 1, N = 11) or macroalbuminuria (Group 2, N = 21). Compared to a non-diabetic control group (N = 21) the low radius end of the sieving profile was depressed, whereas the high radius end was elevated in each diabetic group, more so in Group 2 than Group 1. A heteroporous membrane model revealed the major portion of the glomerular barrier to be perforated by restrictive pores of approximately 56 A radius in all three groups. However, in keeping with a parallel trend for GFR, the relative density of restrictive pores was control greater than Group 1 greater than Group 2. The remaining minor portion of the barrier was perforated by large, shunt-like pores, the relative prominence of which ranked Group 2 greater than Group 1 greater than control. Although the hypothetical, fractional clearance of macromolecules attributable to the shunt-like pores varied directly with fractional clearances of albumin and IgG, the progressive increment in the latter fractional protein clearances in the two diabetic groups was disproportionate. This raises the possibility that factors in addition to barrier size defects contribute to the development, magnitude and composition of proteinuria early in the course of diabetic glomerular disease.(ABSTRACT TRUNCATED AT 250 WORDS)


Mesangial expression of intercellular adhesion molecule-1 in primary glomerulosclerosis. Frozen sections of renal biopsy specimens from eight patients with primary focal segmental glomerulosclerosis (FSGS) and 10 patients with membranous nephropathy (MN) were stained in immuno-peroxidase with the intercellular adhesion molecule-1 (ICAM-1) monoclonal antibody (MoAb), CL203.4. ICAM-1 was expressed by mesangial cells in six patients with FSGS. On the other hand, ICAM-1 was not detected in mesangial cells in patients with MN or in the non-affected portion of tumoral kidneys used to control normal renal expression of ICAM-1. De novo mesangial expression of ICAM-1 in FSGS suggests that sclerosis results from an inflammatory process, possibly associated with local release of cytokines.


Prevention of catecholamine-induced cardiac damage and death with a nucleoside transport inhibitor. The effect of a potent and specific nucleoside transport inhibitor, R 75,231, on catecholamine-induced cardiac toxicity has been studied in rabbits. Epinephrine (1 mg/kg) or norepinephrine (2.5 mg/kg) subcutaneously were lethal in 25 (42%) of 60 control animals, while survivors showed major myocardial damage, as judged from high plasma lactate dehydrogenase (LDH) and its myocardial isoenzyme (LDH1) after 24 h. When a low dose of R 75,231 (0.1 mg/kg) was given intravenously either 1 h before or 1 h after the catecholamine insult, only 1 of 60 animals died. The plasma total LDH and the LDH1 myocardial isoenzyme were low in these animals compared with untreated survivors. Studies ex vivo on isolated perfused hearts confirmed that with treatment using R 75,231, there was both left ventricular nucleoside retention and functional preservation after in vivo exposure of the animals to epinephrine. R 75,231 did not affect the peripheral venous hyperglycemic response to epinephrine. Nucleoside transport inhibition offers a new approach to the prevention and treatment of several cardiac disorders characterized by a pathogenic effect of catecholamines.


Effects of probucol on impaired cardiac performance and lipid metabolism in streptozotocin-induced diabetic rats. Plasma lipids and cardiac performance were studied in diabetic rats treated with probucol. Male Wistar rats were rendered diabetic with a single intraperitoneal injection of streptozotocin (STZ, 75 mg/kg). Nondiabetic control rats received the vehicle alone. Two weeks after STZ or vehicle injection, control and diabetic rats were randomly assigned to probucol-treated or untreated groups. The rats in the two probucol-treated groups (control- and diabetic-probucol groups) were fed a diet containing 1% probucol (w/w) for 4 weeks. Blood was drawn, and then cardiac performance was assessed by the isolated perfused working heart technique. Probucol treatment had no effect on the cardiac performance of the nondiabetic control rats. The peak left ventricular developed pressure and maximum rate of change in left ventricular pressure during systole were significantly greater in the probucol-treated diabetic rats than in the untreated diabetic rats (p less than 0.05), although cardiac performance did not improve to the level in the nondiabetic rats. Plasma cholesterol, free fatty acid, and phospholipid were significantly elevated in the untreated diabetic rats, and probucol treatment decreased significantly the plasma cholesterol and free fatty acid concentrations (p less than 0.05). These data suggest that probucol treatment improves plasma lipids and cardiac performance in rats with experimental diabetes and may prevent diabetic cardiomyopathy.


Suppressive effects of R 56865 on triggered propagated contractions and triggered arrhythmias in rat cardiac trabeculae. Triggered arrhythmias in rat right ventricular trabeculae are induced by triggered propagated contractions (TPCs) that start in damaged regions of the muscle and propagate along the preparation. We analyzed the effects of the Na+/Ca2+ overload inhibiting agents R 56865 on both TPCs and triggered arrhythmias. This compound has been shown to prevent ultrastructural signs of intracellular calcium overload and arrhythmias caused by exposure to toxic concentrations of cardiac glycosides. TPCs were induced by trains of 15 stimuli (2 Hz, 15 s intervals) at 19-21 degrees C and a [Ca2+]o of 1.0-2.5 mM in the superfusate. Force was measured with a silicon strain gauge; length and shortening of sarcomeres were measured at two sites of the muscle using laser diffraction techniques. Exposure to 1.14 x 10(-7) M R 56865 for 30 min decreased the force of the last stimulated twitch (twitch force) to 89.7 +/- 4.7% (mean +/- SEM) of control, the force produced by TPCs to 39.4 +/- 9.8%, and the velocity of propagation of TPCs to 52.8 +/- 6.3%, while TPC latency increased not significantly to 104.7 +/- 2.8% of control. R 56865 suppressed TPC force, for the same small decrease in twitch force (10%), significantly more than 100 nM D-600 did (29.5 +/- 2.0 vs. 12.4 +/- 3.1%). Eventually, TPCs disappeared in 8 of 14 muscles, in 2 of them without any decrease in twitch force. At 5.7 x 10(-7) M, R 56865 abolished TPCs in five additional trabeculae. An increase in [Ca2+]o reintroduced TPCs. During stimulation of 0.5 Hz, 1.14 x 10(-7) M R 56865 increased the stimulus threshold by 21 +/- 4% in 6 of 14 muscles and decreased the twitch force by 26 +/- 3% in 7 of 14 trabeculae. Triggered arrhythmias were induced in six muscles with the use of 0.5 mM caffeine or 5 nM Bay K 8644; R 56865 rapidly terminated these arrhythmias in all muscles. Although the mechanism of the antiarrhythmic effects of R 56865 remains to be determined, we speculate that the drug raises the threshold for both generation of triggered action potentials and calcium-induced calcium release from the sarcoplasmic reticulum.


Electrophysiologic interactions of procainamide and N-acetylprocainamide in isolated canine cardiac Purkinje fibers. The study objective was to characterize the electrophysiologic interactions of procainamide (PA) and its metabolite, N-acetylprocainamide (NAPA), in canine Purkinje fibers. Cell (N = 43) action potentials were measured in Tyrode's solution (K+ = 4.0 mM, 36 degrees C) at a basic cycle length of 1,000 ms using standard microelectrode techniques. Six PA concentrations (0.020-0.32 mM) and six NAPA concentrations (0.010-0.24 mM) were studied alone and in combination. PA caused concentration-dependent decreases in Vmax and APD50 but did not alter APD90, ERP, or RMP. NAPA caused a small but not significant concentration-dependent decrease in Vmax, no change in RMP, and significant concentration-dependent increases in APD50, APD90, and ERP. Low NAPA concentrations increased, intermediate concentrations did not affect, and high NAPA concentrations again increased PA's effect on Vmax. PA-NAPA combinations resulted in concentration-dependent changes in APD50 that were intermediate between the effects of PA or NAPA alone. PA did not significantly alter NAPA's effects on APD90 at NAPA concentrations less than or equal to 0.040 mM, while it antagonized NAPA's effect at higher concentrations. The effects of PA-NAPA combinations on ERP were generally similar to their effects on APD90. The electrophysiologic effects of PA-NAPA combinations in normal canine Purkinje fibers are complex functions of the relative and absolute concentrations of the two compounds.


Temporal alterations in peripheral vascular responsiveness during both the development and recovery from pacing-induced heart failure. The responsiveness of both the in vitro dorsal pedal artery and in vitro saphenous vein to alpha-adrenergic agents and prostaglandin F2 alpha was evaluated at baseline (prior to the onset of rapid ventricular pacing), at 1 week of pacing (mild heart failure), and 4 weeks after pacing when there was recovery from congestive heart failure. Eleven dogs were paced at 250 beats/min for 1 week and 7 dogs were paced for 3 weeks at 250 beats/min and allowed to recover from pacing for a further 4 weeks. At 1 and 4 weeks of recovery, compared to control, the maximal responsiveness of the dorsal pedal artery to norepinephrine was increased from 5.7 +/- 0.9 to 12.9 +/- 3.3 and 18.6 +/- 1.0 g/mm2, respectively. A similar finding was observed in the saphenous vein. The response of the artery to the selective alpha 1-agonist, phenylephrine, was also enhanced at 1 week. Moreover, the response of the artery and vein to phenylephrine was enhanced further at 4 weeks of recovery. In contrast, the response to the selective alpha 2-agonist, BHT 920, remained unaltered at these time points compared to control. Oxymetazoline, an agent used to differentiate between alpha 1-subdivisions, produced a significantly higher maximal response at 4 weeks of recovery (18.4 +/- 1.6 g/mm2 for the artery and 27.7 +/- 2.6 g/mm2 for the vein) compared to control (8.2 +/- 0.5 g/mm2 for the artery and 10.3 +/- 0.8 g/mm2 for the vein). The artery also displayed lower EC50 values for norepinephrine and phenylephrine at 1 week (0.7 and 2.2 microM, respectively) and 4 weeks of recovery (0.9 and 3.1 microM, respectively) compared to control (6.0 and 9.0 microM, respectively). In contrast, the only significant decreases in EC50's in the vein were the norepinephrine and oxymetazoline at 1 week of pacing (0.2 and 0.06 microM, respectively) compared to control (1.5 and 0.09 microM, respectively). The maximal tension developed to PGF2 alpha was enhanced after 1 week of pacing in both vessels and persisted 4 weeks following the cessation of pacing. Compared with control, EC50 values for PGF2 alpha were decreased at 1 and 4 weeks of recovery. We conclude that differences exist in peripheral vascular reactivity to both alpha 1-adrenoceptor agents and PGF2 alpha at 1 week of pacing and 4 weeks of recovery from pacing. Furthermore, the subtle reactivity differences between the artery and vein reflect different populations of alpha 1-adrenoceptors possibly associated with different signal transduction processes.


Comparison of the cardiac and hemodynamic effects of lisinopril and atenolol in patients with hypertension: therapeutic implications. The antihypertensive and hemodynamic effects of lisinopril and atenolol were evaluated in 21 patients with mild-to-moderate essential hypertension. Left ventricular systolic and diastolic performances were assessed prior to and following treatment by first-pass radionuclide cineangiography at rest and during peak upright bicycle exercise. Both lisinopril and atenolol treatment significantly reduced the blood pressure. Lisinopril therapy was associated with a reduction in systemic vascular resistance and left ventricular end-diastolic and end-systolic volumes but no change in stroke volume, cardiac output, peak ejection rate, peak filling rate, time to peak ejection rate, or time to peak filling rate. In contrast, atenolol therapy was associated with an increase in end-diastolic volume and stroke volume but no change in cardiac output; the left ventricular peak ejection and peak filling rates were decreased by atenolol treatment. Although both lisinopril and atenolol each significantly reduced the blood pressure, lisinopril had no effect on left ventricular systolic or diastolic performance; in contrast, atenolol decreased both systolic and diastolic parameters of ventricular performance. Left ventricular function may be affected in significantly different ways despite apparent similarities in blood pressure control in patients who respond to angiotensin converting enzyme inhibition or beta-adrenergic receptor blockade. Differences in hemodynamic response to an antihypertensive agent may be important in the selection of a drug for the treatment of subsets of patients with cardiac function abnormalities.


The effects of aging on the electrophysiologic and hemodynamic responses to nifedipine in isolated perfused hearts. Age effects on responses to calcium channel blockade with nifedipine were studied in isolated Langendorff-perfused Fischer 344 rat hearts. Responses to 25 min of perfusion with nifedipine concentrations of 0, 25, 50, 75, and 100 ng/ml were studied in hearts from 11 mature (6 months) and 13 senescent (23-27 months) male F344 rats. Nifedipine produced significant increases in the atrial cycle length (p less than 0.001), paced atrioventricular (AV) conduction time (p less than 0.001), AV Wenckebach cycle length (p less than 0.001), left ventricular (LV) diastolic pressure (p less than 0.001), and decreases in LV systolic pressure (p less than 0.001) and peak dP/dt (p less than 0.001) in hearts from both mature and senescent rats. Greater decreases in the atrial rate (p less than 0.05) and depression of peak dP/dt (p less than 0.05) were detected in senescent vs. mature rat hearts. No age difference in responses of AV conduction parameters were detected although increases in the AV Wenckebach cycle length appeared to be greater in senescent hearts at concentrations greater than 75 ng/ml.


The lazaroid U74006F, a 21-aminosteroid inhibitor of lipid peroxidation, attenuates myocardial injury from ischemia and reperfusion. U74006F, a novel new 21-aminosteroid inhibitor of lipid peroxidation, has been effective in preventing free-radical-mediated injury in central nervous system models. To assess its ability to diminish myocardial injury due to ischemia and reperfusion, U74006F (n = 11) or its vehicle (n = 11) were administered intravenously to New Zealand white rabbits. After allowing for distribution, the hearts were excised and exposed to 30 min of stop-flow ischemia and 30 min of reperfusion on a nonrecirculating Langendorf apparatus. There was diminished creatine phosphokinase release; improved peak positive dP/dt, developed pressure, and peak negative dP/dt; and diminished diastolic pressure in the group treated with U74006F. Thus, pretreatment with U74006F diminished myocardial injury and enhanced systolic and diastolic functional recovery, probably by protecting the lipid component of cell membranes from peroxidation by reactive oxygen metabolites.


Rapid infusions of bidisomide or disopyramide in conscious dogs: effect of myocardial infarction on acute tolerability. The acute tolerability of rapid infusions of bidisomide or disopyramide was evaluated in normal conscious dogs and in conscious dogs 48 h after the creation of myocardial infarctions (MIs). Both drugs were given in total doses of 15 mg/kg (1.5 x the canine antiarrhythmic dose for each drug). Bidisomide was well tolerated at infusion rates of 3, 5, 11, and 15 mg/kg/min by normal dogs. Disopyramide was well tolerated, except for anticholinergic effects, by normal dogs given infusions at rates of 1.5 and 3 mg/kg/min. Disopyramide caused a ventricular arrhythmia at 4.5 mg/kg/min in one dog, however. Bidisomide (15 mg/kg/min) was well tolerated and antiarrhythmic in dogs with infarctions. Disopyramide (3 and 4.5 mg/kg/min) was lethal in dogs that had myocardial infarctions. A 1 mg/kg/min infusion rate of disopyramide was antiarrhythmic and well tolerated, except for anticholinergic effects, in the post-MI dogs. Both drugs prolonged the ECG lead II P duration, PR interval (bidisomide more so than disopyramide), and QRS duration. Both bidisomide and disopyramide shifted the mean electrical axis of the QRS complex from a right axis deviation to the normal range in dogs with infarctions. The data indicated that the desired cardiac electropharmacologic effects of bidisomide can be achieved in a 1 min infusion. Normal dogs, and especially dogs with infarctions, revealed the potential hazards of rapidly infusing disopyramide.


Effect of calmodulin and protein kinase C inhibitors on globally ischemic rat hearts. Several calmodulin inhibitors have been reported to be cardioprotective, but the ability of these compounds to inhibit protein kinase C (PKC) suggests that calmodulin inhibition may not be the sole mechanism responsible. To distinguish between the effects, we determined the cardioprotective activity of several calmodulin inhibitors with differing PKC inhibitory potencies in isolated globally ischemic rat hearts. Twenty-five minutes of global ischemia caused significant myocardial dysfunction, contracture formation, and lactate dehydrogenase (LDH) release on reperfusion in vehicle-treated hearts. The calmodulin inhibitors trifluoperazine, W-7, calmidazolium, W-13, and CGS 9343B improved postischemic contractile function and/or reduced LDH release. They also reduced preischemic cardiac function, although cardioprotection did not appear to be correlated with cardiodepression. Calmodulin inhibitors increased preischemic coronary flow (CF) and decreased heart rate (HR), but controlling these parameters did not affect the cardioprotection. Pretreatment of ischemic hearts with trifluoperazine was associated with preservation of myocardial ATP. Pretreatment of ischemic rat hearts with the PKC inhibitors staurosporine, calphostin C, polymyxin B, and H-7 did not result in cardioprotection. Thus, calmodulin inhibition causes cardioprotection that appears to be independent of PKC inhibition.


Profile of cardiovascular effects of NKH477, a novel forskolin derivative, assessed in isolated, blood-perfused dog heart preparations: comparison with isoproterenol. Cardiac and coronary vasodilator effects of NKH477, a novel water-soluble forskolin derivative, and isoproterenol, a nonselective beta-adrenoceptor full agonist, were compared in isolated, blood-perfused papillary muscle, sinoatrial (SA) node, and atrioventricular (AV) node preparations of dogs. Both agents were injected intraarterially. The two agents increased the force of contraction of the paced papillary muscle and of the unpaced muscle, and the rate of automaticity of the latter. They increased sinus rate and accelerated AV nodal conduction. In producing these cardiac effects, both agents were similar, although NKH477 was 120-350 times less potent than isoproterenol; however, NKH477 differed distinctly from isoproterenol in that the former increased coronary blood flow more greatly than the latter. Thus, NKH477 is more coronary vasodilatory than positive inotropic, and more positive inotropic than positive chronotropic. Such a cardiovascular profile of NKH477 was similar to that of forskolin, except for the duration of actions; NKH477 was longer-acting than forskolin.


Comparison of the effect of celiprolol and nifedipine on blood pressure and plasma lipids. During a double-blind, randomized study in hypertensive patients, changes in blood pressure (BP) and in plasma lipid and lipoprotein levels during treatment with celiprolol were compared with those occurring during nifedipine treatment. Fifty-three patients (28 men and 25 women) with mild-to-moderate hypertension, aged 20-64 years, were studied. After a 1-month placebo run-in period, patients were randomly assigned to receive either nifedipine (40 mg daily) or celiprolol (200 mg daily) each time using a double dummy technique. After 6 weeks, dosages of each drug could be doubled. Both drugs caused similar reductions in blood pressure but after 12 weeks treatment, the percentage of decrease in diastolic BP (DBP) was more pronounced (p less than 0.01) in the nifedipine group (-18%) than in the celiprolol group (-12%). After 6 weeks, there were no differences in plasma lipids between the two treatment groups. However, the changes after 12 weeks treatment were different (p less than 0.05) between the groups, leading to lower levels of plasma esterified cholesterol, low-density lipoprotein (LDL) cholesterol and apoprotein AI, AII, and B in the celiprolol group. Plasma lecithin cholesterol acyltransferase activity (LCAT) was not modified, suggesting that reverse cholesterol transport was not affected by the drugs. In both treatment groups, a significant positive relationship was observed between changes in LDL cholesterol and apoprotein B. As compared with nifedipine, celiprolol after 12-week therapy had a rather favorable plasma lipid profile. The clinical relevance of such findings, in terms of prevention of cardiovascular complications, has yet to be established.


Vasodilator therapy for acute heart failure: haemodynamic comparison of hydralazine/isosorbide, alpha-adrenoceptor blockade, and angiotensin-converting enzyme inhibition. Haemodynamic comparison of three vasodilation regimens [intravenous (i.v.) hydralazine and isosorbide dinitrate (ISDN) combined, i.v. doxazosin, and i.v. enalaprilat] was undertaken in 36 patients with acute left ventricular (LV) failure due to recent myocardial infarction. Each regimen achieved similar reductions in pulmonary artery occluded pressure (PAOP, preload) and systemic arterial pressures (afterload), with increased cardiac and stroke volume (SV) indexes (p less than 0.01). Only the hydralazine and isosorbide combination induced resting tachycardia. Balanced vasodilatation after selective alpha-adrenoceptor blockade (doxazosin) and angiotensin-converting enzyme (ACE) inhibition (enalaprilat) without increase in heart rate (HR) suggests that these therapies may have definite haemodynamic advantages over the hydralazine/ISDN combination.


Comparison of peptide and nonpeptide receptor-mediated responses in rat tail artery. Chronic uremia and metabolic acidosis impair vascular responses to norepinephrine (NE) and also cause multiple metabolic defects in skeletal muscle. These studies were conducted to determine whether decreased vascular responsiveness resulted from putative second messenger metabolism. Tail arteries were used from rats with metabolic acidosis or nonacidotic uremia and from normal controls. In normal arteries, the maximal responses were the same for arginine vasopressin (AVP), NE, and mixtures of the two agonists, suggesting that the two receptor types use the same transduction mechanism. Nonetheless, qualitative differences exist between AVP- and NE-induced responses: (a) Concentration-response curves are steeper for AVP than for NE, (b) EC50 values are similar for AVP between inositol phosphates (IP assays) and contraction, but not for NE, and (c) arteries from rats with metabolic acidosis or uremia show selective blunting of biochemical and contractile responses to NE but not to AVP. We conclude that these metabolic derangements selectively affect alpha-adrenergic receptors, but not AVP receptors or the transduction mechanism leading to contraction.


Dexamethasone potentiates production of inositol trisphosphate evoked by endothelin-1 in vascular smooth muscle cells. To investigate the possibility of participation of glucocorticoids in the action of endothelin-1 (ET-1), the effect of glucocorticoids on ET-1-induced inositol trisphosphate (IP3) production was studied in cultured vascular smooth muscle cells (VSMC). ET-1 transiently increased IP3 in a dose-dependent manner. Pretreatment with 1 microM dexamethasone for 48 h shifted the dose-response curve of ET-1-induced IP3 production to the left; i.e., it significantly reduced the half-maximal effective concentration of ET-1 (from 50 to 10 nM). This action of dexamethasone required a minimum of 12 h of incubation. A glucocorticoid antagonist, RU 38486, completely blocked this effect. To elucidate the interaction with prostaglandin, we used indomethacin, a potent inhibitor of prostaglandin synthesis. Treatment with 1 microM indomethacin shifted the dose-response curve of ET-1-induced IP3 production to the left, but this shift was significantly less than that observed after treatment with dexamethasone and no additive effect between indomethacin and dexamethasone was noted. Glucocorticoids potentiate the IP3 production response to ET-1 in cultured VSMC, and this action of glucocorticoids depends partially on prostaglandin inhibition. These results suggest that glucocorticoids play an important role in modulation of ET-1 action.


Efficacy and tolerability of isradipine and metoprolol in treatment of hypertension: the Finnish Isradipine Study in Hypertension (FISH). Eight hundred seventy-six men and women with diastolic blood pressure (DBP) of 95-115 mm Hg during a 4-week placebo period were included in a multicenter trial; 479 patients had previously been treated for hypertension. The patients were randomized to receive isradipine or metoprolol; both groups were comparable for age, weight, height, smoking habits, and duration of hypertension. By the end of the placebo period, 79 patients did not fulfill the final entry criteria and were withdrawn. The isradipine group consisted of 398 patients (164 women and 234 men), and the metoprolol group consisted of 399 patients (173 women and 226 men). The initial dose of isradipine was 1.25 mg twice daily (b.i.d.), and the initial dose of metoprolol was 50 mg b.i.d.; the doses were doubled after 4 weeks if DBP had not decreased to less than or equal to 90 mm Hg. After 8 weeks, the isradipine group began combination therapy with metoprolol 50 mg b.i.d. and the metoprolol group began combination therapy with isradipine 1.25 mg b.i.d. if DBP was not less than or equal to 90 mm Hg. After 8 weeks monotherapy, mean BP (MBP) was reduced by 13/11 mm Hg (161/104 to 148/93) in the isradipine group and by 15/12 mm Hg (160/103 to 145/91) in the metoprolol group. Monotherapy with isradipine normalized DBP to less than or equal to 90 mm Hg in 52% with a mean dose of 4.26 mg daily, and monotherapy with metoprolol normalized DBP in 58% with a mean dose of 155 mg daily.1+


Differential effects of quinidine on transmembrane action potentials of normal and infarcted canine Purkinje fibers. Fourteen days after proximal ligation of the left anterior descending coronary artery (LAD) of mongrel dogs, the effects of quinidine on action potentials of normal and infarcted Purkinje fibers were evaluated. The concentration-dependent (10(-7)-3 x 10(-5) M) and frequency-dependent (1 and 3 Hz) actions of quinidine were evaluated by the following parameters: maximum upstroke velocity (Vmax), action potential duration at 50 and 95% repolarization (APD50, APD95), effective refractory period (ERP), resting membrane potential (RMP), and action potential amplitude (APA). Quinidine reduced Vmax in normal and abnormal Purkinje fibers in a concentration- and frequency-dependent manner; these effects were more pronounced in infarcted tissue. The APD50 was shortened significantly at 1 Hz in noninfarcted Purkinje fibers, whereas in infarcted Purkinje fibers quinidine had no effect on APD50. The APD95 was not significantly altered by quinidine in normal Purkinje fibers; in infarcted areas APD95 was significantly prolonged at 1 and 3 Hz. The effective refractory period (ERP) was prolonged in normal and infarcted Purkinje fibers, these effects were more marked in ischemically damaged fibers. No effects were observed on resting membrane potential (RMP). APA was reduced significantly after quinidine at 1 and 3 Hz; there was no difference between normal and infarcted tissue. These data indicate a differential effect of quinidine in normal and infarcted Purkinje fibers which may be an important mechanism of action of quinidine in infarcted tissue.


Twenty-four-hour beta-blockade in stable angina pectoris: a study of atenolol and betaxolol. We examined the importance of a long plasma half-life (t1/2) on the antianginal effects of beta-blockade by comparing equivalent doses of once-daily atenolol 100 mg (t1/2 6-8 h) and betaxolol 20 mg (t1/2 20-22 h) in a double-blind placebo-controlled cross-over study of 20 patients with stable angina pectoris. At 20 h postdose, heart rate (HR) was lower with betaxolol than with atenolol whereas blood pressure (BP) was equally reduced by both drugs. Twenty-four-hour ambulatory HR recording demonstrated that this difference existed for the last 6 h of the dosage cycle. During treadmill exercise, HR remained lower with betaxolol than with atenolol and exercise time was significantly prolonged only by betaxolol. With placebo, radionuclide ventriculography demonstrated that left ventricular ejection fraction (LVEF) decreased during exercise. Betaxolol, but not atenolol, significantly attenuated the exercise-induced decrease in EF. Thus, the long plasma t1/2 of betaxolol is associated with a reduction in exercise-induced ischemia when tested toward the end of the 24-h dosage cycle. Plasma t1/2 therefore is of clinical relevance to the antianginal, but not antihypertensive, actions of beta-blockers.


Cardiac responses after norepinephrine-induced ventricular hypertrophy in rats. The increase in norepinephrine (NE) blood levels in human heart failure correlates with prognosis. In this study, we determined whether continuous NE infusion alters the positive inotropic and chronotropic responses of isolated rat cardiac muscles. Osmotic minipumps were implanted subcutaneously (s.c.) in 43 adult male rats to deliver NE (160 micrograms/kg/h for 14 days); 42 rats were sham-operated. Isolated left and right atria and left and right ventricular (LV, RV) papillary muscles were prepared to measure positive inotropic or chronotropic responses to NE, phenylephrine, forskolin, dibutyryl cyclicAMP (dbcyclicAMP), and calcium chloride. NE infusion caused (a) a 22% increase in LV wet weight without altering atrial or RV wet weights; (b) an 18% decrease in maximal inotropic response to calcium chloride in LV papillary muscles only; (c) a significantly decreased peak response to NE [72 +/- 5 vs. 93 +/- 5% (sham rats) of calcium chloride] but not to forskolin or dbcyclicAMP in RV papillary muscles; (d) an increased incidence of ectopy at low concentrations of NE, forskolin, and dbcyclicAMP in LV papillary muscles; (e) no alteration in papillary muscle responses to phenylephrine but significantly increased left atrial inotropic responses [51 +/- 5 vs. 33 +/- 2% (sham rats) of calcium chloride] and right atrial chronotropic responses [30 +/- 2 vs. 18 +/- 4 (sham rats) beats/min]; and (f) a selective decrease in beta 1-adrenoceptor density in both ventricles. Thus, NE infusion causes selective LV hypertrophy; responses of compounds that increase intracellular cyclicAMP are affected to a greater extent in papillary muscles from the hypertrophied ventricle than in tissues from the other chambers of the heart.


Stereoselective interactions of (R)- and (S)-propafenone with the cardiac sodium channel. The specific interactions of both (R)- and (S)-propafenone with the cardiac sodium channel were studied with patch clamp techniques in the whole-cell recording mode at reduced extracellular Na+ on guinea pig ventricular cells. Both (R)- and (S)-propafenone (10 microM) shifted the membrane potential required for half-maximal steady-state inactivation (E0.5) of the cardiac sodium channel to considerably more negative membrane potentials [E0.5 = -70.8 +/- 2.9 mV for controls vs. -85 +/- 3.1 mV for (R)-propafenone and -91.9 +/- 1.7 mV for (S)-propafenone]. (S)-Propafenone at a concentration of 10 microM is more effective in shifting the h infinity curve of the cardiac sodium channel. Recovery from inactivation of the cardiac sodium current is prolonged by orders of magnitude by both stereoenantiomeric forms [time constants were estimated to be 38 +/- 15 ms at -90 mV vs. 46.5 +/- 14.3 s for (R)-propafenone and 74.2 +/- 37.9 for (S)-propafenone]. Development of block occurs mainly through the inactivated channel conformation for both (R)- and (S)-propafenone. Development of block of inactivated cardiac sodium channels occurs with time constants of 15.9 +/- 3.9 s for (R)-propafenone and 19.7 +/- 7.3 s for (S)-propafenone at 10 microM. Action potential duration and possible stereoselective interaction with ion transport systems other than sodium channels may influence the block developed by either (R)- or (S)-propafenone at a given concentration and beating frequency indirectly through the membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)


Central action of atropine on cardiovascular reflexes in humans. Effects of exciting the central nervous system (CNS) with low doses of atropine on cardiovascular reflexes was investigated in 6 healthy volunteers. Cumulative doses of atropine sulfate (1.43-28.57 micrograms/kg intravenously, i.v.) were used. The ECG parameters used to quantify the nervous activities on the heart were ratios of the longest to the shortest R-R intervals recorded during forced expirations and deep inspirations (respiratory sinus arrhythmia), the ratio of the longest to the shortest R-R intervals which occur immediately after one rises from a low sitting position (30:15 ratio), and the SD of R-R intervals recorded in standing and supine positions. Very low doses of atropine (1-3 micrograms/kg) increased the 30:15 ratios from (means +/- SEM) 1.21 +/- 0.07 to 1.34 +/- 0.04 (60% increase, p less than 0.01) and SD recorded in standing positions from 32.4 +/- 3.5 to 56.0 +/- 11.0 ms (71% increase, p less than 0.01). Respiratory sinus arrhythmia was not affected in either the standing or supine position, nor was SD recorded in supine positions significantly affected by these low doses of atropine. The only indexes increased by low doses of atropine were those that measure cardiovascular responses to active standing, consistent with an increased excitability of the medullary cardiovascular (vasomotor) centre. There was no evidence of central vagal excitation caused by low doses of atropine in the conscious, spontaneously breathing volunteers. The increased excitability of the medullary cardiovascular centre in response to a low dose of atropine may suggest that normal responses of this centre to arterial baroreceptor stimulation is restrained by a cholinergic inhibitory pathway.


Aprotinin decreases release of 6-keto-prostaglandin F1 alpha and increases release of thromboxane B2 in cultured human umbilical vein endothelial cells. Use of the proteinase inhibitor aprotinin significantly improves hemostasis and reduces bleeding after operations in which extracorporeal circulation is used. The mechanism of action, however, has been only partially clarified. In this work we investigated whether aprotinin influenced the production and release of the eicosanoids prostacyclin, measured as the stable metabolite 6-keto-prostaglandin F1 alpha, and thromboxane A2, measured as the stable metabolite thromboxane B2, from endothelial cells. Human umbilical vein endothelial cells were incubated with different concentrations of aprotinin (5.5, 20, 55, and 100 mumol/L). The levels of 6-keto-prostaglandin F1 alpha and thromboxane B2 were measured at baseline and after thrombin stimulation. A concentration-dependent effect of aprotinin on 6-keto-prostaglandin F1 alpha synthesis was demonstrated. After incubation with 100 mumol/L of aprotinin, a 90% reduction in 6-keto-prostaglandin F1 alpha production was seen (31.69 versus 307.44 picograms per million cells; p less than 0.001). Conversely, thromboxane B2 production showed a 345% increase after incubation with aprotinin (287.80 versus 83.82 picograms per million cells; p less than 0.0001). Since 6-keto-prostaglandin F1 alpha inhibits and thromboxane B2 strongly enhances platelet aggregation, it appears that one mechanism of the clinically observed effectiveness of aprotinin lies in the altered ratio of 6-keto-prostaglandin F1 alpha: thromboxane B2 in endothelial cells, which leads to enhanced platelet aggregation and improved vessel sealing.


Limiting edema in neonatal cardiopulmonary bypass with narrow-range molecular weight hydroxyethyl starch. The marked edema observed in neonatal cardiopulmonary bypass is thought to result from pathologic increases in capillary permeability. Pentafraction is a subfraction of hydroxyethyl starch that is thought to be of appropriate size and shape to be retained by leaking capillaries and seal endothelial gaps in capillary basement membranes. To test the hypothesis that pentafraction would reduce edema in neonatal cardiopulmonary bypass, we established a model of edema formation in neonatal bypass in which neonatal piglets underwent 2 hours of normothermic cardiopulmonary bypass with crystalloid prime and no myocardial ischemia. Before initiation of bypass, experimental animals (n = 11) received intravenous pentafraction, 3 gm/kg. Control animals (n = 10) received an equivalent volume of saline. Hemodynamic parameters, animal weight, fluid redistribution, and percent tissue water of individual organs were assessed during and after bypass. Pentafraction treatment resulted in significant differences in (1) lowered percent body weight gain from baseline (11% versus 48%), (2) lowered volume requirement to maintain venous reservoir during cardiopulmonary bypass (148 ml/kg versus 581 ml/kg), (3) less fluid loss from the peritoneum (11 ml/kg versus 115 ml/kg), and (4) lowered percent tissue water of kidney, pancreas, stomach, jejunum, colon, and skeletal muscle (p less than 0.05 by unpaired t test). Pentafraction had no effect on hemodynamic parameters during bypass nor in percent tissue water of heart, lung, liver, spleen, skin, or brain. In summary, pentafraction lessened weight gain and fluid requirements during cardiopulmonary bypass, favorably influencing the percent tissue water of certain organs. If pentafraction functions as proposed, it may have wide applicability not only in cardiopulmonary bypass (or extracorporeal membrane oxygenation) but also in other clinical scenarios with altered capillary permeability.


Age-dependent ventricular response to pressure overload. Considerations for the arterial switch operation. Success with the arterial switch operation for D-transposition of the great arteries and the concept of left ventricular suitability for systemic work stimulated this literature review of the age-dependent mechanisms in normal cardiac growth and pressure-induced left ventricular hypertrophy. Normal postnatal myocardial growth is markedly influenced by hemodynamic factors. It consists of an early hyperplastic phase of both myocytes and capillaries that is followed by a myocyte hypertrophic phase. Similarly, imposition of a pressure overload induces both myocyte hyperplasia/hypertrophy and increased angiogenesis in neonates, but only myocyte hypertrophy at a later age. The functional consequences of ventricular hypertrophy are the result of adaptive and nonadaptive changes resulting from the overload stimulus, for example, induction of protooncogene expression, myosin isoenzyme shifts, degree of coronary perfusion, responsiveness to beta-adrenergic stimulation, and myocyte capacity to re-accumulate or sequester cytosolic calcium. Strikingly, both the capacity and the rapidity of left ventricular hypertrophy decrease with increasing age. This experimental information supports the current use of primary arterial switching for neonates with D-transposition of the great arteries and the use of "rapid" two-stage arterial switching in infants more than 3 to 4 weeks of age; it raises some concern about the practice of late retraining of the left ventricle in cases of failed atrial inversion operation.


Implantation of in vitro endothelialized polytetrafluoroethylene grafts in human beings. A preliminary report. To assess the impact of in vitro endothelialization on prosthetic graft patency, we performed femorotibial reconstruction in four patients. Polytetrafluoroethylene grafts (6 mm), lined with cultivated autologous endothelial cells, harvested from the veins of the forearm, were used. Autologous endothelial cells were harvested enzymatically and characterized by morphology and factor VII staining. After a cultivation period of 17 to 23 days, the cell count increased from 27 +/- 3 x 10(4) endothelial cells to 5.4 +/- 1.1 x 10(6). Endothelial cell seeding on polytetrafluoroethylene prostheses was then performed. To improve endothelial cell attachment to the graft surface, polytetrafluoroethylene grafts (60 to 70 cm; 6 mm diameter) were precoated with fibrin glue containing fibrin and fibronectin and the fibrinolysis inhibitor aprotinin. Seeding density of 49 +/- 10 x 10(3) endothelial cells per square centimeter yielded a preconfluent monolayer immediately after seeding, as demonstrated by scanning electron microscopy. A second cultivation period of 6 days, after seeding and before implantation, was necessary for establishment of a confluent monolayer and to allow for maturation of the endothelial cell cytoskeleton as well as production and excretion of extracellular matrix. Grafts endothelialized in vitro were implanted in four patients requiring femorotibial reconstruction. Scintigraphic studies with indium 111-labeled platelets demonstrated little or no platelet deposition, indicating persistent endothelialization. All grafts remained patent at 3 months after implantation.


Kinetics of diffusion in a spherical cell. II. Solute buffering included. This paper on diffusion kinetics in neurons presents an analysis of diffusion in the presence of solute buffering. Computational rather than theoretical methods are usually necessary since buffering generally precludes an analytical solution to the diffusion equations. As in the companion paper, our methods are illustrated in the context of calcium diffusion in a spherical cell. However, the same methods can be applied to the spread of any second messenger and other geometries. Analytical or computational predictions of the time course of diffusion and buffering may help guide further experiments and simulations. For example, simulations of calcium diffusion in a model of the bullfrog sympathetic ganglion cell show that buffering at depths greater than 5-6 microns is almost instantaneous compared to diffusion from sources at the cell membrane. Since buffering complicates the design of multicompartmental models, we demonstrate that a few compartments designed on the basis of diffusion alone (Carnevale and Rosenthal, 1992) may be a satisfactory framework for a model that includes bimolecular buffering. An analytical solution may be possible if the buffering reaction can be linearized. We describe a method for linearizing bimolecular saturating buffering, i.e., approximating it by a unimolecular non-saturating process that immobilizes solute. The analytical solution for the linearized reactive diffusion problem fits a non-linear model of calcium movement in the bullfrog sympathetic ganglion cell quite well after a few milliseconds.


Biotinylated dextran amine as an anterograde tracer for single- and double-labeling studies. Fluorescent dextran amines have recently been reported to be useful for anterograde pathway tracing. However, fluorescent markers are not always ideal for detailed mapping studies. We therefore evaluated the efficacy of a biotinylated dextran amine (BDA) for anterograde labeling in several different preparations. BDA was visualized with an avidin-biotinylated HRP (ABC) procedure followed by a standard or metal-enhanced diaminobenzidine (DAB) reaction. After iontophoretic injections of BDA into neocortex-like telencephalic regions in pigeons or into visual or somatosensory cortex in rats, there was excellent and abundant labeling of axons and terminals in forebrain, midbrain and hindbrain target areas with 1-week survival times. Large pressure injections of BDA into the avian telencephalon were also found to result in extensive anterograde labeling. We then carried out a series of studies using 2-color DAB double-labeling to determine effective approaches for combining BDA labeling with other labeling methods. Using an isolated embryonic chick spinal cord-hindlimb preparation, we combined BDA labeling with another anterograde labeling method to differentially label two sets of projections. In these studies, sensory neuron and motoneuron projections into the limb from the same segmental level, or motoneuron projections into the limb from two separate segments were differentially labeled by using HRP (visualized first with a blue/black metal-DAB reaction) and BDA (visualized second with a brown DAB reaction). In other double-labeling studies, we combined BDA labeling of axons and terminals with immunohistochemical labeling of neurons. In these experiments, telencephalic neurons in pigeons or rats were labeled immunohistochemically for parvalbumin or substance P (using a brown DAB reaction) and BDA-labeled axons were labeled blue/black (using a metal-intensified DAB reaction). Double-labeling was successful regardless of whether the entire immunohistochemical labeling procedure preceded or followed the BDA labeling procedure. Together, these studies show that BDA is effective for anterograde pathway tracing and can be used in double-label studies with other labeling methods.


Antithyroid drugs and release of inflammatory mediators by complement-attacked thyroid cells. Thyroid cells are exposed to complement attack in Graves' disease and Hashimoto's thyroiditis, but are resistant to killing by homologous complement. We have examined the effects of sublethal complement attack on thyroid cells in vitro. Extracellular reactive oxygen metabolites were produced and prostaglandin E2, interleukin-1 alpha, and interleukin-6 were released after complement attack. Cells pretreated with interferon-gamma and interleukin-1 alpha, which increase expression of CD59, were more resistant to these effects of complement. Conversely, blockade of CD59 with monoclonal antibody increased complement-mediated oxygen radical production and release of prostaglandin E2, interleukin-1 alpha, and interleukin-6. The antithyroid drugs methimazole and propylthiouracil abolished or reduced oxygen radical production by complement-attacked thyroid cells and reduced cytokine release. These results suggest that sublethal complement attack in autoimmune thyroid diseases exacerbates tissue injury by causing thyroid cells to release potent phlogistic mediators, although some degree of protection may be afforded in vivo by cytokine-mediated upregulation of CD59. Antithyroid drugs, concentrated within thyroid cells, will prevent the release of these inflammatory molecules, which may in turn explain the amelioration of thyroiditis and remission of Graves' disease after such treatment.


Induction of cytokines in human peripheral blood mononuclear cells by mycoplasmas. Various species of mycoplasmas were tested for their ability to induce cytokine production in human peripheral blood mononuclear cells (PBMC). Human PBMC were incubated with Mycoplasma pneumoniae, M. hyorhinis, M. arginini, M. salivarium, M. orale, M. gallisepticum or A. laidlawii for 48 hr, and the activities of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN) in the supernatants were determined by ELISA or bioassay. All mycoplasma species induced IL-1 beta, IL-6 and TNF-alpha, although IL-2 was induced only by M. pneumoniae. IFN was induced by 5 of the 7 species, and the IFN produced was antigenically confirmed to be mainly IFN-alpha. On the other hand, mycoplasma-stimulated cultures did not contain detectable amounts of IFN-beta and IL-4 activities. Furthermore, the cytokines were induced by mycoplasmal contaminating cells in human PBMC as well as by mycoplasma alone. These results suggest that many kinds of cytokines induced by mycoplasma contamination in cell culture affect immunological experiments in vitro.


A possible role for nitric oxide in glutamate (MSG)-induced Chinese restaurant syndrome, glutamate-induced asthma, 'hot-dog headache', pugilistic Alzheimer's disease, and other disorders. Endogenous glutamate is thought to be a major neurotransmitter. After binding to a cell membrane receptor there can be a stimulation of what can be called the nitric oxide (NO)-mediated neurotransmission pathway (NO-MNP). The activity of the enzyme that produces NO from arginine, NO synthase, and the level of NO become elevated. NO has little activity within the cell in which it is produced, but it rapidly leaks out of that cell and produces effects in neighboring cells. The NO-MNP can be activated to release NO in endothelial cells which in turn acts on neighboring vascular smooth muscle cells to induce vasodilation. Therefore, we suggest that exogenous, ingested glutamate, like endogenous glutamate, can lead to the same stimulation of the NO-MNP in sensitive individuals which would then cause the symptoms of the Chinese restaurant syndrome and/or glutamate-induced asthma. Further, since ingested nitrite and related compounds can be metabolized to NO, NO may more directly cause the symptoms of 'hot dog headache'. In addition, it has been suggested that NO production can also be controlled in endothelial cells by fluid forces that stimulate pressure receptors. Therefore, elevations of NO and stimulation of the NO-MNP may occur due to sudden, local, alterations of blood pressure during pugilistic activities and play a role in the symptoms of pugilistic Alzheimer's disease. If these ideas are correct, then inhibitors of the NO-MNP and/or temporary reduction of the plasma level of arginine may be useful in preventing at least some of the symptoms of these disorders.


Update: eradication of paralytic poliomyelitis in the Americas. On August 23, 1991, a 2-year-old boy in the district of Junin, Peru, had onset of symptoms of culture-confirmed paralytic poliomyelitis. This is the last case of paralytic poliomyelitis with a wild poliovirus isolate reported to the Pan American Health Organization (PAHO) and the first time since reporting of poliomyelitis began in the Western Hemisphere that no such paralytic disease has been detected for an entire year. This report updates the poliomyelitis eradication effort in the Americas.


Metabotropic glutamate receptors potentiate ionotropic glutamate responses in the rat dorsal horn. The effects of the metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] were examined on responses mediated by the ionotropic glutamate receptor agonists N-methyl D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), and kainic acid (KA), in neurons acutely isolated from the dorsal horn of the rat spinal cord. (1S,3R)-ACPD produced an increase in the intracellular Ca2+ concentration in 50% of acutely isolated dorsal horn neurons, which could be prevented by blockers of voltage-sensitive Ca2+ channels. (1S,3R)-ACPD markedly potentiated increases in the intracellular Ca2+ concentration induced by NMDA, AMPA, and KA but not by 10-50 mM KCl. This potentiation occurred in all cells, required the simultaneous presence of both agonists, and was rapidly reversible. In the spinal cord slice preparation, (1S,3R)-ACPD potentiated the inward currents evoked by pressure application of AMPA, NMDA, and KA, an effect that was also rapidly reversible. These short term effects of (1S,3R)-ACPD may play an important role in the regulation of ionotropic responses mediated by glutamate in the spinal cord.


Biochemical characterization of kainate receptors from goldfish brain. Kainate receptors from goldfish brain were purified by affinity chromatography. Unlike previously published purifications, which have yielded single proteins of 48-50 kDa from frog, chick, and pigeon brain, our preparations contained two polypeptides, of 41 kDa and 45 kDa. In addition, a broad band centered at 120 kDa was present. Some of the 41-kDa and 45-kDa polypeptides were derived from the higher molecular mass protein. All of these proteins were recognized by a monoclonal antibody produced against a purified frog kainate receptor. The distribution of the 41-kDa and 45-kDa proteins varied independently in different major brain regions, suggesting that they can exist as separate independent proteins. A partial amino acid sequence of the 41-kDa polypeptide is very similar (40-60% identity) to specific segments of the frog and chick kainate-binding proteins and the alpha-amino-3-hydroxy-5-methylisoxazolepropionate/kainate ion channels. The characteristics of the 41-kDa and 45-kDa polypeptides suggest that these two proteins are distinct. Photo-affinity labeling with [3H]kainate showed that a [3H]kainate binding site is associated with the 41-kDa polypeptide, and peptide mapping suggests that the two proteins are not identical. In addition, the two peptides do not appear to be related by differential glycosylation or phosphorylation. The 41-kDa and 45-kDa polypeptides, therefore, appear to be distinct and may represent kainate receptor subtypes or, in some cases, possibly two different subunits of a kainate receptor complex.


Histamine increases cytosolic Ca2+ in dibutyryl-cAMP-differentiated HL-60 cells via H1 receptors and is an incomplete secretagogue. Human neutrophils and dibutyryl-cAMP (Bt2cAMP)-differentiated HL-60 cells possess receptors for the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), which mediate activation of phospholipase C, with subsequent increase in cytosolic Ca2+ concentration ([Ca2+]i) and activation of specific cell functions. In many cell types, histamine, via H1 receptors, activates phospholipase C, but it is unknown whether neutrophilic cells possess functional H1 receptors. We compared the effects of histamine with those of fMet-Leu-Phe on activation of these cells. In Bt2cAMP-differentiated HL-60 cells, substances increased [Ca2+]i in the effectiveness order fMet-Leu-Phe greater than histamine greater than betahistine. Pertussis toxin diminished fMet-Leu-Phe-induced rises in [Ca2+]i to a greater extent than those induced by histamine. H1 but not H2 antagonists inhibited histamine- and betahistine-induced rises in [Ca2+]i. fMet-Leu-Phe and histamine activated phospholipase C and increased [Ca2+]i through release of Ca2+ from intracellular stores and sustained influx of Ca2+ from the extracellular space. The substances also induced Mn2+ influx. Ca2+ and Mn2+ influxes were inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl)-1H-imida zole hydrochloride (SK&F 96365). The stimulatory effects of histamine on [Ca2+]i were more sensitive to inhibition by 4 beta-phorbol 12-myristate 13-acetate than were those of fMet-Leu-Phe. Unlike fMet-Leu-Phe, histamine did not activate superoxide anion formation, release of beta-glucuronidase, and tyrosine phosphorylation. In neutrophils, histamine and betahistine did not induce rises in [Ca2+]i. Our data show that (i) in Bt2cAMP-differentiated HL-60 cells, histamine increases [Ca2+]i via H1 receptors coupled to pertussis toxin-sensitive and possibly, pertussis toxin-insensitive heterotrimeric regulatory guanine nucleotide-binding proteins, (ii) histamine activates nonselective cation channels, and (iii) unlike fMet-Leu-Phe, histamine is an incomplete secretagogue.


Histamine increases cytosolic Ca2+ in HL-60 promyelocytes predominantly via H2 receptors with an unique agonist/antagonist profile and induces functional differentiation. Histamine H1 receptors mediate activation of phospholipase C, with subsequent increases in cytosolic Ca2+ concentration ([Ca2+]i), and H2 receptors mediate accumulation of cAMP. HL-60 promyelocytes possess H2 receptors, but it is not known whether these cells also possess H1 receptors. We studied the effects of histamine on [Ca2+]i and the functional importance of histamine receptors in HL-60 promyelocytes. In these cells, histamine and dimaprit increased [Ca2+]i with EC50 values of 15 microM and 30 microM, respectively. Diphenhydramine inhibited the effect of histamine (100 microM) on [Ca2+]i up to 40%, with an IC50 of 100 nM. Famotidine and cimetidine diminished the effect of histamine (100 microM) up to 75%, with IC50 values of 85 nM and 300 nM, respectively. Diphenhydramine plus famotidine abolished histamine-induced rises in [Ca2+]i. Impromidine, with an IC50 of 100 nM, abolished the effect of histamine (100 microM) on [Ca2+]i. Diphenhydramine, famotidine, cimetidine, and impromidine showed marked noncompetitive antagonism with histamine. Histamine-induced increases in [Ca2+]i were largely due to influx of Ca2+ from the extracellular space. Ca2+ influx was inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl)-1H-imida zole hydrochloride (SK&F 96365). Histamine activated phospholipase C. Histamine induced expression of formyl peptide receptors, which effect was abolished by famotidine. In U-937 promonocytes and in the human erythroleukemia cell lines HEL and K-562, histamine did not induce rises in [Ca2+]i. Our data suggest the following. (i) In HL-60 promyelocytes, histamine increases [Ca2+]i predominantly via H2 receptors and to a lesser extent via H1 receptors. (ii) The agonist/antagonist profile of the H2 receptor-mediated increases in [Ca2+]i differs markedly from that for cAMP accumulation, suggesting the involvement of different H2 receptor subtypes. (iii) In HL-60 promyelocytes, histamine activates nonselective cation channels and induces functional differentiation via H2 receptors.


Both enantiomers of 1-aminocyclopentyl-1,3-dicarboxylate are full agonists of metabotropic glutamate receptors coupled to phospholipase C. We tested the effects of two enantiomers of a glutamate analogue, (trans)-1-aminocyclopentyl-1,3-dicarboxylate (t-ACPD), in striatal and cerebellar neurons in primary culture, as well as in Xenopus oocytes injected with cerebellar rat RNA. In the presence of MK-801, to avoid N-methyl-D-aspartate receptor activation, and 3 microM tetrodotoxin, both enantiomers [(1R,3S)- and (1S,3R)-t-ACPD] stimulated inositol phosphate (InsP) formation both in striatal neurons after 9-11 days in vitro [EC50, 3.7 +/- 1.1 microM, three experiments, and 33 +/- 7.5 microM, three experiments; maximal stimulatory effects, 252 +/- 15%, 13 experiments, and 269 +/- 15% of basal InsP formation, 14 experiments, for (1R,3S)- and (1S,3R)-t-ACPD, respectively] and in cerebellar granule cells after 9-11 days in vitro [EC50, 50 +/- 18 microM, four experiments, and 307 +/- 92 microM, four experiments; maximal stimulatory effects, 401 +/- 71%, eight experiments, and 423 +/- 75% of basal InsP formation, eight experiments, for (1R,3S)- and (1S,3R)-t-ACPD, respectively]. These effects were not additive, indicating that both enantiomers acted at the same receptor molecule. When we monitored t-ACPD-induced increases in intracellular Ca2+ concentration ([Ca2+]i) with fura-2 ratio-imaging, we found that both enantiomers could elicit similar increase in [Ca2+]i, in the presence of 1 microM MK-801 and 3 microM tetrodotoxin; these effects were also observed in the absence of external Ca2+. Moreover, in Xenopus oocytes injected with adult rat cerebellar RNA, both drugs elicited oscillatory increases of a Ca(2+)-dependent chloride conductance, with similar efficacy, with (1R,3S)-t-ACPD being the more potent isomer. These data are in contradiction to previous reports showing that, in "immature" cerebellar neurons and adult hippocampal slices, (1S,3R)-t-ACPD was either the only active enantiomer or a full agonist of metabotropic receptors, with (1R,3S)-t-ACPD being ineffective or a partial agonist. However, performing these experiments in immature (2-3 days in vitro) striatal or cerebellar neurons, we found that only (1S,3R)-t-ACPD was active in stimulating [Ca2+]i.


Enalapril reduces the enhanced 1,2-diacylglycerol content and RNA synthesis in spontaneously hypertensive rat hearts before established hypertension. There is evidence that cardiac hypertrophy in spontaneously hypertensive rats (SHR) occurs before the development of hypertension. 1,2-Diacylglycerol, which is thought to be a second messenger activating protein kinase C, is also produced in excess in SHR hearts at 4 weeks of age, before established hypertension. We determined myocardial 1,2-diacylglycerol content in SHR with and without prazosin and enalapril from 3 to 4 weeks of age. Hearts from untreated SHR had greater RNA and DNA synthesis and greater relative weights at 4 weeks of age than those from Wistar-Kyoto (WKY) rats. There was no difference in triglyceride content or phospholipid species between WKY rats and untreated SHR, except for a higher cholesterol content in SHR. Treatment of SHR with enalapril, but not prazosin, lowered not only 1,2-diacylglycerol content but also RNA synthesis to the levels of WKY rats. Moreover, fatty acids involved in 1,2-diacylglycerol were altered by enalapril despite the lack of a difference between WKY rats and untreated SHR. Prazosin did not have any effect on 1,2-diacylglycerol fatty acid composition. Enalapril may decrease cardiac hypertrophy in SHR by lowering myocardial 1,2-diacylglycerol production.


'Run-off' polymerization with digoxigenin labelled nucleotides creates highly sensitive and strand specific DNA hybridization probes: synthesis and application. In this paper the in vitro synthesis and application of non-radioactively labelled strand specific DNA probes is described. The probe is labelled by incorporation of nucleotides with the hapten digoxigenin into single-stranded DNA during a 'run-off' reaction catalyzed by Thermus aquaticus (Taq) DNA-polymerase. The 'run-off' reaction requires a linearized plasmid template and one primer binding site at a defined distance from the restriction site. Single-stranded DNA can be synthesized during repeated cycles of denaturation, annealing, and extension. The conditions for the incorporation of digoxigenin-11-dUTP (dig-11-dUTP) during polymerization were optimized to generate strand specific DNA hybridization probes up to a length of 5000 nt. The strand specificity is demonstrated by a dot-blot, with in vitro-transcribed target RNA of c-sis. The sensitivity of the probe was tested in a Northern blot, and found to be identical to a probe radiolabelled by nick-translation (specific activity 6.5 x 10(8) cpm micrograms-1). The resolution of the signals and speed of development was even superior compared to the radiolabelled probe.


Review of the mutagenicity/genotoxicity of butylated hydroxytoluene. Butylated hydroxytoluene (BHT) is an effective, widely used, low cost antioxidant. A host of studies examining the potential of BHT to cause point mutations have been published. They include in vitro studies on various bacterial species and strains and on various types of mammalian cell lines as well as in vivo studies on Drosophila melanogaster, silk worms and also the mouse specific locus test (involving long-term exposure). Together these studies convincingly show the absence of a potential for BHT to cause point mutations. A great number of studies on many cell types and species have also been carried out to examine the potential of BHT to cause chromosome aberrations. In vitro studies have been published using plant cells and the WI-38, CHL, CHO, and V79 mammalian cell lines. In vivo studies have been carried out on somatic and/or germ cells of Drosophila melanogaster, rats and mice. Nearly all studies, especially those using validated test systems, indicate that BHT lacks clastogenic potential. In vitro studies on bacterial, yeast and various mammalian cell lines including DON, CHO, CHL cells and primary hepatocytes demonstrate the absence of interactions with or damage to DNA. Taking all the existing data into account, the weight of evidence suggests that BHT does not represent a relevant mutagenic/genotoxic risk to man.


Mutagenicity of azo dyes: structure-activity relationships. Azo dyes are extensively used in textile, printing, leather, paper making, drug and food industries. Following oral exposure, azo dyes are metabolized to aromatic amines by intestinal microflora or liver azoreductases. Aromatic amines are further metabolized to genotoxic compounds by mammalian microsomal enzymes. Many of these aromatic amines are mutagenic in the Ames Salmonella/microsomal assay system. The chemical structure of many mutagenic azo dyes was reviewed, and we found that the biologically active dyes are mainly limited to those compounds containing p-phenylenediamine and benzidine moieties. It was found that for the phenylenediamine moiety, methylation or substitution of a nitro group for an amino group does not decrease mutagenicity. However, sulfonation, carboxylation, deamination, or substitution of an ethyl alcohol or an acetyl group for the hydrogen in the amino groups leads to a decrease in the mutagenic activity. For the benzidine moiety, methylation, methoxylation, halogenation or substitution of an acetyl group for hydrogen in the amino group does not affect mutagenicity, but complexation with copper ions diminishes mutagenicity. The mutagenicity of benzidine or its derivatives is also decreased when in the form of a hydrochloride salt with only one exception. Mutagenicity of azo dyes can, therefore, be predicted by these structure-activity relationships.


Review of the genotoxicity of ozone.

Review of the genotoxicity of ozone. Ozone is a powerful oxidant, reactive to biomolecules. In aqueous solution it decomposes to give hydrogen peroxide, superoxide and hydroxy radicals which can take part in secondary reactions. Ozone is a disinfectant that inactivates both viruses and bacteria. Although other reactions are primarily responsible for the inactivation, cellular DNA is also damaged. Ozone is genotoxic to microorganisms, plants and cell cultures in vitro. The results from in vivo cytogenetic studies with laboratory animals after inhalation exposure are contradictory. Chromosome aberrations in lymphocytes, but not SCEs, have been demonstrated in Chinese hamsters but not in mice. Chromatid deletions were induced in pulmonary macrophages in rats. No cytogenetic effects have been reported for bone marrow cells or spermatocytes. The few experimental and epidemiological studies with human subjects do not allow a conclusion on the cytogenetic effects of ozone in lymphocytes in humans. No life-long cancer studies have been performed with ozone. However, after 4 and 6 months of inhalation exposure, lung adenomas were induced in strain A/J mice, but not in Swiss-Webster mice.


Using the polymerase chain reaction to estimate mutation frequencies and rates in human cells. The Polymerase Chain Reaction (PCR) has had a significant impact on molecular studies of human mutagenesis, mainly in the acceleration of molecular characterisation of mutant genes in cells isolated by a phenotypic selection. PCR can also be used to study genetic alterations in cells which have not undergone phenotypic selection. By modifying the standard PCR parameters, the presence of mutations can be assayed in single human cells, creating the potential to determine mutation rates in gametes on a cell-by-cell basis (Section I). Alternatively, PCR can be used to selectively amplify a mutant gene in a pool of normal genomes and thus determine a mutation frequency (Section II). Current applications of these two approaches are summarised and critically reviewed.


Recombinant DNA approaches for the development of metabolic systems used in in vitro toxicology. In the past few years there has been considerable progress in the development of mammalian cell systems for use in genetic toxicology by the stable transfer of genes/cDNAs coding for drug metabolizing enzymes directly into the target cell. Alternative approaches have also been developed in which mammalian cells are transiently transfected with cDNAs coding for drug-metabolizing enzymes and S9 preparations expressing a single metabolizing enzyme isolated and used for metabolic activation. Progress in these areas is reviewed here and the relative merits of the different approaches are discussed. Work to date has focused primarily on the cytochrome P450 family of enzymes, although other enzyme systems involved in xenobiotic metabolism have been used. The central theme of this review is the transfer of genetic information to improve the metabolic capability of cell systems used in genetic toxicology. However, a basic philosophy of the review is that genetic manipulation of cultured mammalian cells has the potential for developing systems to be used to better understand chemically induced toxicological effects.


Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis. Cystic fibrosis is a common, severe autosomal recessive disease caused in a majority of cases by a three-nucleotide deletion (delta F508) in the cystic fibrosis transmembrane regulator gene. Current methods of prenatal diagnosis involve chorionic-villus sampling or amniocentesis. In vitro fertilization and diagnosis during embryonic development before implantation would allow only unaffected embryos to be selected for transfer to the uterus, thereby avoiding the need to terminate a pregnancy. Preimplantation diagnosis of cystic fibrosis was attempted in the cases of three couples, both members of which carried the delta F508 deletion. In vitro fertilization techniques were used to recover oocytes from each woman and fertilize them with her husband's sperm. Three days after insemination, embryos in the cleavage stage underwent biopsy and removal of one or two cells for DNA amplification and analysis. Only two oocytes from one woman were fertilized normally; DNA analysis of one of the embryos failed and cystic fibrosis was diagnosed in the other (i.e., it was homozygous for delta F508), so neither was transferred. The oocytes of each of the other two women produced noncarrier, carrier, and affected embryos. Both couples chose to have one noncarrier embryo and one carrier embryo transferred. One woman became pregnant and gave birth to a girl free of the deletion in both chromosomes. Preimplantation diagnosis of the delta F508 deletion causing cystic fibrosis is possible through in vitro fertilization, biopsy of a cleavage-stage embryo, and amplification of DNA from single embryonic cells. This approach should be equally applicable to other single-gene diseases in which the defect has been identified. Analysis of a series of pregnancies, however, will be required to assess the method adequately.


Effects of cyclic AMP and butyrate on cell cycle, DNA, RNA, and purine synthesis of cultured astrocytes. Dibutyryl cyclic monophosphate (dBcAMP) has been shown to inhibit growth, and alter the morphology of astrocytes. However, the potential contribution of its hydrolytic product, butyrate, in inducing some of the changes that have been attributed to dBcAMP, is not clear. DNA, RNA, and purine synthesis were therefore studied in primary astrocyte cultures after 24 hours of exposure to varying concentrations of butyrate, dBcAMP, and agents that increase intracellular cAMP levels. Progression of cells through cell cycle was also studied by flow cytometry. Dibutyryl cAMP partially arrested cells in Go/G1 phase of cell cycle while sodium butyrate increased the percentage population of cells in G2/M phase. DNA synthesis and de novo purine synthesis were inhibited after treatment with dBcAMP, sodium butyrate, and various drugs that increase intracellular cAMP levels. RNA synthesis was increased with cAMP but was not affected by sodium butyrate. Our study shows that at millimolar concentrations, butyrate is capable of altering the cell cycle and inhibiting DNA synthesis in primary astrocyte cultures, in a manner that is similar although not identical to the effects of dBcAMP.


Vascular endoscopy for intravascular surgery--an experimental study. A combination of endoscope and high-pressure saline irrigation system was tested in the occlusion of experimental aneurysms, and angioplasty and fibrinolysis of vascular occlusive lesions in canine femoral arteries. Endoscopy provided detailed information about the aneurysmal orifice and lumen and aided balloon placement and detachment. Endoscopy visualized the dynamic changes accompanying thrombolysis and the fine intimal injury due to angioplasty invisible on angiography. Vascular endoscopy is valuable for pre-, intra-, and postoperative evaluation of intravascular interventions.


Effect of radiation therapy against intracranial hemangiopericytoma. Seven cases of intracranial hemangiopericytoma were studied retrospectively to investigate the efficacy of radiation therapy. Tumor response evaluated by computed tomography and magnetic resonance imaging was obvious after 20-30 Gy irradiation. The total reduction rate was 80-90% and continued as long as 5-7 months after treatment. In five patients receiving radiation therapy before radical removal, the tumors were easily removed without massive hemorrhage. Histological inspection of specimens after irradiation showed a significant disappearance of tumor cells. Pyknosis frequently occurred in endothelial cells, and proliferating vessels with hyalinoid degeneration were also seen. Reticulin fibers between tumor cells were fewer, split, or absent. Preoperative radiation therapy is useful in the treatment of hemangiopericytoma involving considerable surgical risk. Postoperative radiation therapy should be given even if removal is complete.


New three-dimensional moving field radiation therapy for brain tumors. A new modified rotation radiation method called "three-dimensional moving field radiation therapy" is described. The new method uses rotation in many planes while maintaining the the same isocenter to achieve a good spatial dose distribution. This delivers a high dose to tumors and spares the surrounding normal structures. This easy method can be carried out using the equipment for conventional rotation radiation therapy. The new method was superior to the one plane rotation radiation therapy using a physical phantom with film, a chemical phantom using the iodine-starch reaction, and a new biological model using tumor cells. Treatment of six brain tumors irradiated with total air doses of 50-60 Gy caused no hair loss or radiation necrosis.


"True" posterior communicating artery aneurysm--report of two cases. Two cases of true posterior communicating artery (PcomA) aneurysm are described. A 27-year-old female with subarachnoid hemorrhage demonstrated a saccular aneurysm at the branch of the anterior choroidal artery from the PcomA. The aneurysm was successfully clipped. A 23-year-old male involved in an automobile accident suffered from traumatic subarachnoid hemorrhage. Computed tomography 10 weeks after head injury demonstrated a high-density mass in the right parasellar region, diagnosed as a traumatic aneurysm arising from the PcomA. The aneurysm was successfully clipped. Histological examination demonstrated findings consistent with true aneurysm.


Ruptured cerebral aneurysm associated with coarctation of the aorta--report of two cases. Two cases of ruptured cerebral aneurysm associated with coarctation of the aorta are presented. The aneurysms were successfully clipped in the acute stage prior to correction of the coarctation. Ruptured aneurysm should be treated as early as possible, and unruptured aneurysm should also be treated before aortic repair, if the general condition of the patient allows.


Lymphocytic adenohypophysitis with sudden onset of diabetes insipidus in menopausal female--case report. A rare case of a postmenopausal (60-year-old) female with lymphocytic adenohypophysitis manifesting as a sudden onset of diabetes insipidus is reported. Magnetic resonance imaging with gadolinium-diethylene-triaminepentaacetic acid enhancement showed a spherical lesion, approximately 1 cm in diameter, in the sella turcica and a thickened, deviated pituitary stalk. The abnormal tissue was totally removed. Histological examination showed marked infiltration of lymphocytes and plasma cells. Postoperatively, the pituitary stalk became normal. Preoperative differentiation of lymphocytic adenohypophysitis from pituitary adenoma is extremely difficult, and biopsy is essential.


Recurrent intracranial germinoma refractory to conventional irradiation: effective chemotherapy consisting of cisplatin and etoposide--case report. Recurrent intracranial germinoma with multiple spinal metastases occurred in a 16-year-old male presenting with persistent headache and visual disturbances. Computed tomography revealed enhanced lesions in the pineal region, anterior horn, and infundibulum. Conventional irradiation achieved remission, but local recurrences requiring further irradiation occurred after 23 months. Magnetic resonance imaging showed multiple spinal metastases 11 months later. Partial removal of the spinal lesions gave a histological diagnosis of typical germinoma. Postoperatively, intracranial recurrences were again detected. Chemotherapy consisting of intravenous cisplatin and etoposide achieved remission and no recurrence has occurred for 12 months.


Cerebellar astrocytoma with repeated episodes of fourth ventricle isolation causing peritoneal shunt tube obstruction--case report. A 9-year-old girl underwent total removal of a cerebellar astrocytoma complicated by hydrocephalus after postoperative meningitis, requiring a ventriculoperitoneal shunt. Five months later, headache, vomiting, and gait disturbance appeared and computed tomography detected enlarged fourth ventricle. A fourth ventriculoperitoneal shunt resulted in immediate relief of all symptoms. After 2 months, obstruction of the peritoneal tube required shunt reconstruction. This recurred three times in 8 months. At the last operation, tumor cells were detected in the cerebrospinal fluid and the substance clogging the tube. This suggested that the tumor had recurred and clogging by tumor cells had caused the repeated episodes of isolated fourth ventricle. Radiation therapy prevented further shunt obstruction and achieved remission of all signs and symptoms.


Intracavernous epidermoid tumor presenting with abducens nerve paresis--case report. An unusual case of a pediatric epidermoid tumor entirely located in the cavernous sinus is reported. A 6-year-old boy presented with left abducens nerve paresis which developed over 2 months. Neuroimaging demonstrated a lesion in the left cavernous sinus. Part of the tumor capsule and the pearly contents were removed by the left pterional approach through Dolenc's anterolateral triangle. No bleeding from the cavernous sinus occurred. The tumor was histologically identified as an epidermoid tumor. Postoperatively, the abducens nerve paresis improved. The presence of dural reflection in the lateral wall of the cavernous sinus and displacement of the intracavernous internal carotid artery are useful indicators for intracavernous lesions.


Dorsal column stimulation induces release of serotonin and substance P in the cat dorsal horn. The neurohumoral mechanisms behind the pain-suppressing effect of dorsal column stimulation (DCS) still remain obscure. Experimental observations have indicated an inhibitory role for serotonin and, under certain conditions, also for substance P (SP), on nociceptive transmission in the spinal cord. Furthermore, some observations suggest that these substances might be involved in the effect of DCS. The present series of experiments was undertaken to investigate whether serotonin and SP are released in the dorsal horn by DCS. Twenty-one adult cats, in some experiments anesthetized, in others decerebrated at the midcollicular level, were used. Microdialysis probes were implanted bilaterally in lumbar dorsal horns (L5-L7) and perfused with Krebs' solution. Dialysates were analyzed for serotonin by high-performance liquid chromatography or for SP by radioimmunoassay. DCS was applied at the thoracolumbar junction with current parameters similar to those used clinically in humans. DCS induced a significant release of serotonin in the dorsal horn of decerebrated animals (173 +/- 83% increase; mean +/- standard error; n = 4; P less than 0.01), whereas the levels of the metabolite 5-hydroxyindoleacetic acid were not significantly influenced. In contrast, no release of SP could be recorded in response to DCS in the decerebrated preparation, although peripheral nociceptive stimulation (pinch) and noxious electric dorsal root stimulation induced an elevation of the SP levels. However, in intact animals DCS provoked a marked SP release in the dorsal horn (190 +/- 92% increase; n = 7; P less than 0.01). The release of serotonin and SP after DCS may indicate that these substances participate in the mediation of the pain alleviating effect of DCS.(ABSTRACT TRUNCATED AT 250 WORDS)


Antibodies against Epstein-Barr nuclear antigen (EBNA) in multiple sclerosis CSF, and two pentapeptide sequence identities between EBNA and myelin basic protein. The Epstein-Barr virus (EBV) causes infectious mononucleosis and is linked to several disparate malignancies. Prior studies on patients with multiple sclerosis (MS) showed that 100% are EBV-seropositive and that their blood contains higher antibody titers than those of controls to both transformation and lytic cycle antigens. We performed three different assays for antibodies in CSF to three major EBV antigens from patients with MS and controls. Among 93 patients with MS, 79 (85%) had CSF that reacted with a 70 kD protein, shown to be the nuclear antigen, EBNA-1, whereas only 11 (13%) of 81 EBV-seropositive controls reacted, p less than 0.001. The CSF of all 14 MS patients, unreactive on immunoblots, contained oligoclonal bands on agarose electrophoresis. Together, the two techniques exhibit 100% sensitivity in the confirmatory diagnosis of MS. We also performed amino acid searches of the Protein Identification Resource sequence database for protein homologies to EBNA. Two pentapeptide identities were found between EBNA-1 and myelin basic protein: QKRPS and PRHRD. None of more than 32,000 other proteins in the database contained both pentapeptides. In healthy EBV-seropositive persons, the EBV-specific, MHC-restricted T lymphocytes keep the EBV-containing B lymphocytes locked in the transformed state. However, in the host genetically susceptible to MS, the same population of lymphocytes might recognize and interact with either of the two identified pentapeptides, inadvertently damaging MBP.


Follow-up results from a randomised controlled trial evaluating in- and outpatient pain management programmes. This study reports a 9-18 month follow-up of a randomised controlled trial of pain management programmes for chronic, non-malignant pain. Twenty-two inpatients, 18 outpatients and 12 control subjects completed the follow-up assessments. Significant treatment effects were demonstrated by the inpatient group on pain ratings, the Pain Behaviour Checklist, and General Health Questionnaire, with similar effects demonstrated by the outpatient group on the former 2 measures. The findings were confounded by higher inpatient scores at pretreatment, in comparison with the 2 other conditions. There was a high drop-out rate of subjects, particularly from the control condition which illustrates the limitations of controlled group designs in this area. Analgesic use, activity levels and pain ratings were also evaluated using the criteria for 'success' described by Malec et al. (1981). Results indicated that 68% of inpatients, 61% of outpatients and 21% of control subjects met all 3 criteria. Both treatment programmes were effective in returning patients to paid employment, whilst 3 control group patients gave up work. The cost-benefit implications of these changes are discussed. We conclude that pain management programmes contribute substantially to the rehabilitation of chronic pain sufferers.


Automated 'pain drawing' analysis by computer-controlled, patient-interactive neurological stimulation system. We have developed a new method for the collection and analysis of pain drawings, as part of a computer-controlled, patient-interactive system for use with implanted neurological stimulators. The system has been tested in 44 patients with permanently implanted spinal cord stimulators for the relief of chronic, intractable pain. Patients interact directly with the system, using a graphics tablet, to enter pain drawings and corresponding outlines of their perceptions of stimulation paresthesias, for different stimulating pulse parameters and electrode geometries. Image analysis software has been developed to examine these data quantitatively. This precludes the inter-rater inconsistencies reported for manual pain drawing scoring techniques. A highly significant correlation has been observed between patients' analog ratings of the overlap of pain by paresthesias and the results of our automated analysis of graphic data. This in turn has been found to correlate with clinical observations of pain relief. The contemporary implantable stimulation devices supported by our system permit non-invasive selection of stimulating anodes and cathodes from a linear array of 4 electrodes. The 50 possible electrode combinations have certain geometric features, which we have entered into a multivariate statistical analysis, to determine their relationship with the overlap of pain by paresthesias. One particular configuration (cathode(s) flanked by anode(s) above and below) is significantly better, by this measure, than all the alternatives. This is consistent with prior clinical observations that this configuration is favored by patients whose systems have been adjusted by conventional, manual methods. Pain drawing' entry and analysis by a computerized, patient-interactive system has been useful in this specialized setting and may have broader applications.