Rate constants and branching ratios for the dissociative recombination of C3D(+)7 and C4D(+)9.

Product branching ratios and thermal rate coefficients for the dissociative recombination of C3D(+)7 and C4D(+)9 have been measured in the ion storage ring CRYRING. The results for C3D(+)7 are believed to be slightly more accurate than those obtained earlier for C3H(+)7. Only the C-C bond breaking channels could be measured for C4D(+)9 and were found to be in excellent agreement with earlier data.

First observation of four-body breakup in electron recombination: C2D+5.

We report the first observation of four-body breakup in electron dissociative recombination of a molecular ion: C2D+5. In an ion storage ring experiment, the branching ratio for the process C2D+5 + e(-)-->C2D2 + D + D + D was determined to be 13%. This means that three covalent chemical bonds are broken as a result of the action of a single electron. This is the first time a four-body breakup of chemical bonds has been observed in a low-energy binary reaction.

Transparahippocampal selective amygdalohippocampectomy in children and adolescents: efficacy of the procedure and cognitive morbidity in patients.

OBJECT: Unilateral resection of the hippocampus and amygdala can be used to treat medically intractable mesial temporal lobe seizures. To date seizure outcome and the extent of cognitive morbidity have been unknown in children following the transparahippocampal variation of selective amygdalohippocampectomy (TSA), which prompted the present prospective study. METHODS: Preoperative examinations and outcomes in 22 consecutive children and adolescents who underwent TSA were studied. Cognitive and psychological morbidity were assessed using standard neuropsychological instruments. The authors evaluated relationships between seizure control and cognitive morbidity and 13 and nine clinical variables, respectively. Seizure control was achieved in 11 (65%) of 17 patients (>2 years follow up). Among 13 clinical variables, the only preoperative finding that had a significant bearing on seizure control was the presence of unilateral hypometabolism, which could be observed on [18F]fluorodeoxyglucose-positron emission tomography scans (p<0.001). Patients with seizure control showed significant improvements in verbal and full scale intelligence quotients (both p = 0.05). Patients with longer preoperative durations of seizures exhibited more cognitive impairment that persisted postoperatively. Cognitive outcome analysis based on nine clinical factors revealed no significant difference in cognitive parameters postoperatively, except that significant improvement occurred in rote verbal memory scores among patients who underwent right-sided TSA (p = 0.01). Individually, 81% of the children achieved significant improvement in at least one of seven cognitive parameters, and 52% had stable or improved scores in all parameters. CONCLUSIONS: The results indicate that TSA is a safe effective approach for the treatment of medically intractable mesial temporal lobe seizures in children with minimum effect on cognitive morbidity. Given that the literature suggests that children suffer progressive cognitive morbidity from persistent seizures, the results of this study support early surgical intervention for this group of children.

Serum autoantibodies to brain in Landau-Kleffner variant, autism, and other neurologic disorders.

OBJECTIVE: Etiologically unexplained disorders of language and social development have often been reported to improve in patients treated with immune-modulating regimens. Here we determined the frequency of autoantibodies to brain among such children. DESIGN: We collected sera from a cohort of children with (1) pure Landau-Kleffner syndrome (n = 2), (2) Landau-Kleffner syndrome variant (LKSV, n = 11), and (3) autistic spectrum disorder (ASD, n = 11). None had received immune-modulating treatment before the serum sample was obtained. Control sera (n = 71) were from 29 healthy children, 22 with non-neurologic illnesses (NNIs), and 20 children with other neurologic disorders (ONDs). We identified brain autoantibodies by immunostaining of human temporal cortex and antinuclear autoantibodies using commercially available kits. RESULTS: IgG anti-brain autoantibodies were present in 45% of sera from children with LKSV, 27% with ASD, and 10% with ONDs compared with 2% from healthy children and control children with NNIs. IgM autoantibodies were present in 36% of sera from children with ASD, 9% with LKSV, and 15% with ONDs compared with 0% of control sera. Labeling studies identified one antigenic target to be endothelial cells. Antinuclear antibodies with titers >/=1:80 were more common in children with ASD and control children with ONDs. CONCLUSION: Children with LKSV and ASD have a greater frequency of serum antibodies to brain endothelial cells and to nuclei than children with NNIs or healthy children. The presence of these antibodies raises the possibility that autoimmunity plays a role in the pathogenesis of language and social developmental abnormalities in a subset of children with these disorders.

Epilepsy in children.

Childhood epilepsies comprise a broad range of disorders which vary from benign to progressive and disabling. Accurate diagnosis of epilepsy type and determination of aetiology, when possible, are essential for appropriate treatment. The most common seizure type encountered in children is febrile seizures. These represent a benign condition which is not, in fact, epilepsy and usually does not require antiepileptic medication. When partial seizures occur in childhood, benign syndromes with spontaneous remission, such as rolandic epilepsy, must be distinguished from symptomatic epilepsies which may be refractory to medical management. Complex partial seizures in young children may appear different than in adults. The adverse effect profiles and dosing regimens of antiepileptic drugs in children are also different than in adults, and influence the choice of treatment. Epilepsy surgery should be considered for some children with intractible partial seizures. Generalized epilepsies also have a broader spectrum in children. The idiopathic generalized absence epilepsies are usually easy to control with medication. They range from childhood absence epilepsy which tends to remit in adolescence to juvenile myoclonic epilepsy which is a lifelong condition. In contrast, the seizures of West syndrome and Lennox-Gastaut syndrome are difficult to control, and treatment involves therapeutic modalities rarely used in adults such as ACTH and the ketogenic diet. Many childhood epilepsy syndromes have a familial predisposition, and the genetic bases for several disorders have been described.

P-glycoprotein-independent mechanism of resistance to VP-16 in multidrug-resistant tumor cell lines: pharmacokinetic and photoaffinity labeling studies.

The interaction of etoposide (VP-16), Vinca alkaloids, and verapamil with the P-glycoprotein (P-gp) was studied in human breast (MCF-7) and Chinese hamster lung (DC3F) cell lines and the corresponding multidrug-resistant MCF-7/ADR and DC3F/ADX tumor cell lines, selected for resistance to Adriamycin and actinomycin D, respectively, and overexpressing P-gp. Verapamil (10 microM) markedly reversed resistance to vincristine (11-fold in DC3F/ADX and 125-fold in MCF-7/ADR; 1-hr exposure), but it had a very modest effect on resistance to VP-16 (3- to 4-fold; 1-hr exposure). Resistant cells accumulated 2- to 4-fold less VP-16 and vincristine than the parental cell lines. Verapamil (10 microM) significantly increased accumulation and retention of vincristine, but not of VP-16, in resistant cell lines. Photoaffinity labeling of resistant cell lines with radioactive analogs of verapamil [N(p-azido-3-125I-salicyl)-N'-beta-aminoethylverapamil (NASVP)] and vinblastine[N-(p-azido-3-125I-salicyl)-N'-beta-aminoethylvindesine (NASV)] showed distinctly labeled P-gp bands in both resistant cell lines, compared with wild-type cells. Excess nonradioactive vinblastine or verapamil effectively competed with the P-gp photolabeling by either NASVP or NASV, with IC50 levels of 0.6 and 10 microM, respectively. In contrast, nonradioactive VP-16 was 100- to 500-fold less potent than vinblastine in competing with P-gp photolabeling, suggesting that VP-16 has significantly lower affinity for P-gp than Vinca alkaloids have. Taken together, our data indicate that P-gp glycoprotein by itself may not be important in the transport/efflux of VP-16 and, thus, in the mechanism of resistance to VP-16 in these cells.

Mechanisms of multidrug resistance in HL60 cells. Analysis of resistance associated membrane proteins and levels of mdr gene expression.

HL60 cells isolated for resistance to Adriamycin do not contain P-glycoprotein, as determined with immunological probes. These cells, however, are multidrug resistant and defective in the cellular accumulation of drug. In view of these findings, we have examined in greater detail certain properties of the HL60/Adr cells and have compared these properties to an HL60 drug-resistant isolate (HL60/Vinc) which contains high levels of P-glycoprotein. The results of these studies demonstrated that verapamil induces a major increase in cellular drug accumulation in both HL60/Adr and HL60/Vinc isolates. An 125I-labeled photoaffinity analog of verapamil labeled P-glycoprotein contained in membranes of HL60/Vinc cells. In contrast, this agent did not label any protein selectively associated with drug resistance in membranes of the HL60/Adr isolate. The photoactive dihydropyridine calcium channel blocker [3H]azidopine and [125I]NASV, a photoaffinity analog of vinblastine, labelled P-glycoprotein in membranes from HL60/Vinc cells, whereas in experiments with the HL60/Adr isolate there was no detectable labeling of a drug resistance associated membrane protein. Additional studies have been carried out to analyze membrane proteins of HL60/Adr cells labeled with the photoaffinity agent 8-azido-alpha-[32P]ATP (AzATP32). The results demonstrate that this agent labeled a resistance associated membrane protein of 190 kilodaltons (P190). P190 is essentially absent in membranes of drug-sensitive cells. Labeling of P190 with AzATP32 in membranes of resistant cells was blocked completely when incubations were carried out in the presence of excess unlabeled ATP. Additional studies were carried out to analyze mdr gene amplification and expression in sensitive and resistant cells. Experiments carried out with human 5',mdr1 (1.1 kb) and mdr3 (1.0 kb) cDNAs demonstrate that both of these sequences were highly amplified in the HL60/Vinc isolate. Only the mrd1 gene sequence however, was overexpressed. In contrast, there was no detectable amplification or overexpression of mdr1 or mdr3 sequences in HL60/Adr cells. The results of this study thus identify a new nucleotide binding protein which is overexpressed in membranes of HL60 cells isolated for resistance to Adriamycin. P190, which exhibits properties distinct from P-glycoprotein, possibly functions in the energy-dependent drug efflux system contained in the HL60/Adr resistant isolate.

High affinity binding of an N-terminal myristoylated p60src peptide.

N-Myristoyl and non-myristoyl peptides corresponding to the N terminus of p60src were used to examine whether N-myristoylation facilitates the binding of p60src to specific protein sites at the plasma membrane. We discovered high affinity protein acceptor sites (Kd = 2.7 nM) to a 15-amino acid N-myristoylated N-terminal p60src peptide in red cell membrane vesicles. Binding was not competed by the non-myristoylated analog of the peptide nor by shorter N-myristoyl src peptides and peptides homologous to the N terminus of other N-myristoylated proteins. Binding was not evident after treatment of vesicles with proteolytic enzymes. Raising the salt concentration of the buffer to 50 mM NaCl caused an apparent inhibition of binding. However, no significant effect of salt was observed on the off-rate of bound ligand under these conditions. The results indicate the existence of N-myristoyl-dependent p60src protein acceptor sites at or near the plasma membrane/skeleton interface of red cells which could be responsible for the localization of p60src to this region and may represent new regulatory components for p60src-mediated tyrosine kinase activity.

Arabinosyl-5-azacytosine: mechanisms of native and acquired resistance.

Factors influencing the activity of the nucleoside analogue arabinosyl-5-azacytosine (ara-AC) were studied in P388 murine lymphoblasts in vitro and in vivo, in variants of these cells with artificially acquired resistance, in the naturally resistant colon 38 carcinoma in vivo, and in a panel of six human tumors maintained in continuous culture. Differences were noted not only between the sensitive and artificially developed resistant variants of P388, but also between the naturally sensitive (P388) and naturally resistant (colon 38) tumors. The artificially developed resistant P388 cell lines showed an inhibited capacity to accumulate nucleotides derived from ara-AC and deoxycytidine, whereas the accumulation of cytidine nucleotides remained unchanged. Studies of the initial velocity of facilitated diffusion of ara-AC showed only minor differences between parental and resistant lines, while the nucleotide formation rates from both ara-AC and deoxycytidine were markedly depressed in the latter cells. It is concluded, therefore, that the failure of resistant P388 cells to accumulate these compounds results not from a transport deficit per se but rather from a failure to convert the nucleosides to nondiffusible (i.e., phosphorylated) species inside the cell. This failure was accompanied by a substantial reduction in the incorporation of a radiolabeled product derived from deoxycytidine into the nucleic acids of the resistant clones. The common factor responsible for the resistance of P388 variants toward ara-AC appears to be a markedly decreased level of deoxycytidine kinase activity. The naturally resistant colon 38 carcinoma, on the other hand, in addition to a decrease in the activity of its deoxycytidine kinase, showed a lower level of activity of all its purine and pyrimidine kinases, along with a notably elevated nucleoside triphosphatase activity (with ATP as substrate) when compared to P388. These differences were reflected in lower endogenous nucleoside triphosphate pool sizes in colon 38, and in a lower level of ara-AC-5'-triphosphate accumulation in colon 38 than in P388 after comparable drug exposure. In the six human tumor lines, a positive correlation was established between sensitivity to ara-AC (as determined by its median inhibitory concentration) and cellular content of deoxycytidine kinase. It is concluded that this latter enzyme is a generally important determinant of sensitivity to arabinosyl-5-azacytosine.

Adenosine export from the liver: oxygen dependency.

The liver is believed to be an important regulator of purine concentrations in the circulation. It has been an accepted notion that the liver exports the riboside adenosine to the circulation for use by other tissues. This phenomenon was reexamined using an artificial oxygen carrier to perfuse the isolated rat liver and high-performance liquid chromatography (HPLC) to analyze the purine content. It was found that perfusion with Krebs-Ringer-bicarbonate at a physiological flow rate as had been used by previous investigators results in a mildly hypoxic preparation, and it is this hypoxia that results in the export of adenosine. In a liver perfused with Fluosol-43, little (less than 0.05 microM) or no adenosine was released. However, if the oxygen content of Fluosol-43 was decreased, the liver could be induced to release adenosine at 1.0 microM levels. In addition, the liver was found to release adenine under control conditions; thus it is this purine base that is likely to be the important purine released by the liver for extrahepatic salvage, not adenosine as previously thought.

The disposition and metabolism of tiazofurin in rodents, rabbits, and dogs.

The pharmacokinetics and metabolism of tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) have been examined in the mouse, rat, rabbit, and dog using tritiated drug as a marker. In all four species, tiazofurin, given as a single bolus iv injection, is removed from the circulation in a triphasic manner, with a generally prolonged terminal half-life. In all cases, the mean concentration of unchanged drug prevailing during this terminal phase was well within the cytotoxic range (IC50 vs. P388 cells is 2 microM in vitro). Urinary excretion accounted for between approximately 40 and 90% of the administered dose in all four species, with only minor quantities (less than 3%) of drug-derived radioactivity detected in the feces. The metabolism of tiazofurin was examined in mice and rats: although no evidence was uncovered for hydroxylation of tiazofurin at carbon atom 5 of the thiazole ring, phosphorylation of the drug at its 5'-hydroxyl was demonstrable in nearly every organ of both species, but, liver, striated muscles, and kidney were the only tissues catalyzing the synthesis of thiazole-4-carboxamide adenine dinucleotide to any prominent degree. This synthesis did not appear to be a saturated process, even at doses as high as 8000 mg/m2. Since rodent skeletal muscle accumulated high concentrations of tiazofurin phosphates in vivo, it is suggested that musculature may serve as a reservoir for the drug, and contribute to its rather protracted terminal half-life in plasma.

Local and systemic toxicity resulting from large-volume ip administration of doxorubicin in the rat.

Doxorubicin was administered to rats in a simulated "belly bath" protocol. Fifty milliliters of various concentrations of drug solution was administered ip and was allowed to remain in situ for either 4 or 36 hours prior to removal. Animals were analyzed at 2, 14, and 60 days after treatment. Doses ranged from lethal (75 and 150 micrograms/ml for 4 hours; 12 and 24 micrograms/ml for 36 hours) to nontoxic (5 micrograms/ml for 4 hours). The most common lesion in surviving animals was chronic fibrosing peritonitis. Grossly, there were large volumes of peritoneal fluid in animals exposed to low concentrations (12 and 24 micrograms/ml) for 36 hours, but peritoneal adhesions were the most commonly observed finding when higher concentrations (20-150 micrograms/ml) were used for 4 hours. Commonly observed systemic toxic effects (bone marrow, gastrointestinal tract, and heart) were not seen in this study. Vehicle-treated control animals were negative for all histologic lesions and gross observations.

Platinate toxicity: past, present, and prospects.

Using traditional toxicologic methods, four species were studied for their qualitative and quantitative predictiveness of the toxic effects of cis-dichlorodiammineplatinum(II) in man. Of the four species studied, mouse, monkey, rat, and dog, the latter two gave the best overall results. Using an in vivo rat model, it was found that except for chloroplatinic acid, eight of the tested analogs were less nephrotoxic than the parent drug, cis-dichlorodiammineplatinum(II). The in vitro renal toxicity screen using flounder tubules showed that of the 26 compounds studied, about half were less toxic than the parent compound. This in vitro mini-tox system can be performed about 30 times faster and at one fiftieth the cost of the in vivo model. The in vitro studies also provided evidence that the biochemical site of toxicity of platihates is on ATPases. The latter studies suggested a basis for unifying the mechanistic interpretation of the toxic actions on such disparate target organs as the kidney, nerve, stomach, and inner ear.