Aggressive and social behavior after alprazolam withdrawal: experimental therapy with Ro 19-8022

A 4-year-old male, miniature Schnauzer dog showed two large masses in the mesentery at necropsy. Histological examination of both masses revealed plain smooth muscle tumour cells intermingled with thick-walled blood vessels. The bundles of tumour cells often extended from the periphery of the vessels. Mitotic figures were rare. From these findings, the tumour was diagnosed as a vascular leiomyoma (angiomyoma), previously unreported in animals. The term, vascular leiomyoma, was proposed to describe this tumour in order to avoid confusion with hamartomatous angiomyoma.     

Prophylactic Greenfield filters: acute complications and long-term follow-up

The efficacy of prophylactic vena caval filters (VCF) in reducing morbidity and mortality from pulmonary embolism (PE) in high-risk trauma patients has been shown, but minimal follow-up data is currently available. VCFs were prophylactically placed in 110 patients between August 1991 and June 1995. There was an early VCF complication rate of 7%. Twenty-two patients died; the remaining 88 patients formed the basis for the follow-up study. Forty-five patients were located and interviewed by phone, and 30 of these patients (34%) returned for evaluation. The mean follow-up time was 18 months (range, 4-42 months). There was no incidence of caval thrombosis on follow-up. Eleven patients had physical findings, and duplex evidence consistent with postphlebitic syndrome. An additional three patients had evidence of old deep venous thrombosis (DVT) by duplex, but no significant symptomatology. VCF are effective in preventing PE related deaths and have few major complications. The long-term morbidity associated with posttraumatic venous thrombosis is significant. This morbidity is related not to PE or VCF, but to the underlying DVT. Improved strategies against DVT are necessary.     

In vivo modulation of iododeoxyuridine metabolism and incorporation into cellular DNA by 5'-amino-5'-deoxythymidine in normal mouse tissues and two human colon cancer xenografts

This in vivo study examines the ability of 5'-amino-5'-deoxythymidine (5'-AdThd) to modulate 5-iododeoxyuridine (IdUrd) cellular metabolism in two human colon cancer xenografts (HT 29 and HCT-116), two actively proliferating normal mouse tissues (bone marrow and intestine), and a quiescent normal mouse tissue (liver). 5'-AdThd is a thymidine analogue that at low concentrations (<30 micrometer) can increase thymidine kinase activity, which is the rate-limiting enzyme for activation of IdUrd. We reported recently that the in vitro incubation of HT 29 and HCT-116 cells in 5'-AdThd + IdUrd resulted in an enhancement of 5-iodo-2'-dUTP pools, IdUrd DNA incorporation, and subsequent radiosensitization compared with incubation with IdUrd alone (Clin. Cancer Res., 1: 407-416, 1995). These in vitro effects were more significant in the radioresistant cell line HT 29. Using a 6-day continuous infusion of IdUrd (50 or 100 mg/kg/day) and/or 5'-AdThd (200 mg/kg/day), no increase in systemic toxicity (percentage of body weight loss) was observed in athymic nude mice with 5'-AdThd alone or when combined with IdUrd. There was significant dose-dependent, systemic toxicity with IdUrd, which was reversible within 3 days of completing the lower-dose IdUrd infusion. However, a comparison of plasma levels during the 6-day continuous infusion of IdUrd +/- 5'-AdThd showed a significant interaction of IdUrd and 5'-AdThd, resulting in higher plasma levels by day 6 of both compounds and the principal metabolites, iodouracil and deoxyuridine, which is consistent with nonlinear saturating effects on dihydrouracil dehydrogenase. Coadministration of IdUrd and 5'-AdThd resulted in an increase in the percentage of IdUrd DNA incorporation in the two proliferating normal tissues, which was significant only with the lower IdUrd dose. No effect on IdUrd DNA incorporation was found in normal liver at either IdUrd dose +/- 5'-AdThd. Similar to our in vitro data, the continuous infusion of IdUrd and 5'-AdThd showed a significant effect by increasing the percentage of IdUrd DNA incorporation in HT-29 xenografts at both IdUrd doses, whereas coadministration of 5'-AdThd had no such effect in HCT-116 xenografts.     

Functional characterization of human gamma-aminobutyric acidA receptors containing the alpha 4 subunit

The alpha subunits are an important determinant of the pharmacology of gamma-aminobutyric acidA (GABAA) receptors with respect to agonists, antagonists, and modulatory compounds, particularly the benzodiazepines. The alpha 4 subunit is the least abundant subunit in the brain and the most similar in deduced primary amino acid sequence to the alpha 6 subunit. We demonstrate that the human alpha 4 subunit forms a functional receptor when expressed with beta gamma 2, demonstrating some properties similar to alpha 6 beta gamma 2 and some properties more akin to alpha 1 beta gamma 2. It also exhibited some properties that were unlike any other alpha subunit-containing receptor. GABA affinity seemed to be identical to that of the alpha 1 beta 1 gamma 2 receptor; however, the partial agonists 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol and piperidine-4-sulfonic acid showed lower efficacy than at either alpha 1 beta 1 gamma 2 or alpha 6 beta 1 gamma 2. Benzodiazepine pharmacology of alpha 4-containing receptors was similar to that of alpha 6-containing receptors with the exception of dimethoxy-4-ethyl-beta-carboline-3-carboxylate, which behaved as a partial inverse agonist. Pentobarbital potentiated alpha 4 beta 1 gamma 2 receptor GABA responses to a level comparable with alpha 6 beta 1 gamma 2 (approximately 700% of EC20); however, unlike alpha 6 beta 1 gamma 2 receptors, it did not elicit any direct activation of the receptor. Propofol also potentiated alpha 4 beta 1 gamma 2 GABA responses but to a level more comparable to that of alpha 1 beta 1 gamma 2, suggesting that these compounds act via different sites. Unlike other subunit combinations, propofol did not elicit a direct activation of the receptor. These results suggest that the mechanism for direct activation of the GABAA receptor by pentobarbital and propofol is absent on alpha 4-containing receptors. Furosemide, which non-competitively inhibits the GABAA receptor, showed 700-fold selectivity for alpha 6 beta 3 gamma 2 receptors over alpha 1-, alpha 2-, alpha 3-, and alpha 5-containing receptors and exhibited selectivity for alpha 4 beta 3 gamma 2 receptors (> 50-fold). These experiments reveal a unique pharmacology for alpha 4-containing receptors with some similarities to both alpha 6- and alpha 1-containing receptors.     

Synthesis and biological evaluation of 4-chloro-3, 5-dinitrobenzotrifluoride analogues as antileishmanial agents

Desnitro analogues of 4-chloro-3,5-dinitrobenzotrifluoride (chloralin) (2), an in vitro microtubule inhibitor of several Leishmania species, have been synthesized from 2-halo-5-(trifluoromethyl)benzenesulfonyl chlorides 4 and 5. The analogues exhibited moderate to excellent activity when tested against Leishmania donovani amastigotes in vitro. Two representative compounds, 7f and 8, were tested against the Khartoum strain of L. donovani in a hamster model using chloralin (2) and Glucantime (one of the current therapeutics of choice in the treatment of Leishmania) as standards, the results of which will be discussed herein.     

Purification of a WD repeat protein, EMAP, that promotes microtubule dynamics through an inhibition of rescue

The major microtubule-associated protein in echinoderms is a 77-kDa, WD repeat protein, called EMAP. EMAP-related proteins have been identified in sea urchins, starfish, sanddollars, and humans. We describe the purification of sea urchin EMAP and demonstrate that EMAP binding to microtubules is saturable at a molar ratio of 1 mol of EMAP to 3 mol of tubulin dimer. Unlike MAP-2, MAP-4, or tau proteins, EMAP binding to microtubules is not lost by cleavage of tubulin with subtilisin. In addition to binding to the microtubule polymer, EMAP binds to tubulin dimers in a 1:1 molar ratio. The abundance of EMAP in the egg suggests that it could function to regulate microtubule assembly. To test this hypothesis, we examined the effects of EMAP on the dynamic instability of microtubules nucleated from axoneme fragments as monitored by video-enhanced differential interference contrast microscopy. Addition of 2.2 microM EMAP to 21 microM tubulin results in a slight increase in the elongation and shortening velocities at the microtubule plus ends but not at the minus ends. Significantly, EMAP inhibits the frequency of rescue 8-fold without producing a change in the frequency of catastrophe. These results indicate that EMAP, unlike brain microtubule-associated proteins, promotes microtubule dynamics.     

Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver

Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (10(12) to 10(13) particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1alpha promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 x 10(10) particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1alpha-F. IX of 2.7 x 10(11) particles resulted in plasma levels of 700 to 3, 200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1alpha-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.