Effects of Sulfanilamide and Methotrexate on 13C Fluxes through the Glycine Decarboxylase/Serine Hydroxymethyltransferase Enzyme System in Arabidopsis1

In C3 plants large amounts of photorespiratory glycine (Gly) are converted to serine by the tetrahydrofolate (THF)-dependent activities of the Gly decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT). Using 13C nuclear magnetic resonance, we monitored the flux of carbon through the GDC/SHMT enzyme system in Arabidopsis thaliana (L.) Heynh. Columbia exposed to inhibitors of THF-synthesizing enzymes. Plants exposed for 96 h to sulfanilamide, a dihydropteroate synthase inhibitor, showed little reduction in flux through GDC/SHMT. Two other sulfonamide analogs were tested with similar results, although all three analogs competitively inhibited the partially purified enzyme. However, methotrexate or aminopterin, which are confirmed inhibitors of Arabidopsis dihydrofolate reductase, decreased the flux through the GDC/SHMT system by 60% after 48 h and by 100% in 96 h. The uptake of [α-13C]Gly was not inhibited by either drug class. The specificity of methotrexate action was shown by the ability of 5-formyl-THF to restore flux through the GDC/SHMT pathway in methotrexate-inhibited plants. The experiments with sulfonamides strongly suggest that the mitochondrial THF pool has a long half-life. The studies with methotrexate support the additional, critical role of dihydrofolate reductase in recycling THF oxidized in thymidylate synthesis.

The synthesis, discovery, and development of a highly promising class of microtubule stabilization agents: Curative effects of desoxyepothilones B and F against human tumor xenografts in nude mice

We have evaluated two synthetic epothilone analogues lacking the 12,13-epoxide functionality, 12,13-desoxyepothilone B (dEpoB), and 12,13-desoxyepothilone F (dEpoF). The concentrations required for 50% growth inhibition (IC50) for a variety of anticancer agents were measured in CCRF-CEM/VBL1000 cells (2,048-fold resistance to vinblastine). By using dEpoB, dEpoF, aza-EpoB, and paclitaxel, the IC50 values were 0.029, 0.092, 2.99, and 5.17 μM, respectively. These values represent 4-, 33.5-, 1,423- and 3,133-fold resistance, respectively, when compared with the corresponding IC50 in the parent [nonmultiple drug-resistant (MDR)] CCRF-CEM cells. We then produced MDR human lung carcinoma A549 cells by continuous exposure of the tumor cells to sublethal concentrations of dEpoB (1.8 yr), vinblastine (1.2 yr), and paclitaxel (1.8 yr). This continued exposure led to the development of 2.1-, 4,848-, and 2,553-fold resistance to each drug, respectively. The therapeutic effect of dEpoB and paclitaxel was also compared in vivo in a mouse model by using various tumor xenografts. dEpoB is much more effective in reducing tumor sizes in all MDR tumors tested. Analysis of dEpoF, an analog possessing greater aqueous solubility than dEpoB, showed curative effects similar to dEpoB against K562, CCRF-CEM, and MX-1 xenografts. These results indicate that dEpoB and dEpoF are efficacious antitumor agents with both a broad chemotherapeutic spectrum and wide safety margins.

Glucocorticoids have state-dependent stimulant effects on the mesencephalic dopaminergic transmission.

An increase in the activity of mesencephalic dopaminergic neurons has been implicated in the appearance of pathological behaviors such as psychosis and drug abuse. Several observations suggest that glucocorticoids might contribute to such an increase in dopaminergic activity. The present experiments therefore analyzed the effects of corticosterone, the major glucocorticoid in the rat, both on dopamine release in the nucleus accumbens of freely moving animals by means of microdialysis, and on locomotor activity, a behavior dependent on accumbens dopamine. Given that glucocorticoids have certain state-dependent neuronal effects, their action on dopamine was studied in situations differing in dopaminergic tonus, including during the light and dark phases of the circadian cycle, during eating, and in groups of animals differing in their locomotor reactivity to novelty. Dopaminergic activity is increased in the dark period, further increased during food-intake, and is higher in rats defined as high responders to novelty than in low responders. Corticosterone, peripherally administered in a dose that approximates stress-induced plasma concentrations, increased extracellular concentrations of dopamine, and this increase was augmented in the dark phase, during eating, and in high responder rats. Corticosterone had little or no effects in the light phase and in low responder rats. Corticosterone also stimulated locomotor activity, an effect that paralleled the release of dopamine and was abolished by neurochemical (6-hydroxydopamine) depletion of accumbens dopamine. In conclusion, glucocorticoids have state-dependent stimulant effects on mesencephalic dopaminergic transmission, and an interaction between these two factors might be involved in the appearance of behavioral disturbances.

Hemodynamic effects of epidermal growth factor in conscious rats and monkeys.

The therapeutic application of growth factors to human disease has become closer to reality with the advent of faster means of synthesizing these molecules and novel drug delivery strategies. Epidermal growth factor (EGF) belongs to a large family of molecules with the ability to modulate growth. Purified extracts of EGF have been used clinically to modulate gastrointestinal secretion of hormones and accelerate healing. EGF is also reported to have both vascular smooth muscle contractile and relaxing activity Cardiovascular studies were performed with the bioactive 48-amino acid fragment of human EGF in rodents and primates to determine the effects of EGF on blood pressure and heart rate in conscious animals. Intravenous infusion of EGF induced an initial pressor response in rats followed by a prolonged decrease in blood pressure. In contrast, in monkeys, EGF had dose-related blood pressure-lowering effects only; significant hypotension was observed at doses ranging from 3 to 300 microg/kg i.v. Hypotension was associated with modest tachycardia in both species. To our knowledge, this is the first report of hemodynamic effects of EGF in primates, and it clearly documents that the mitogenic role of growth factors such as EGF is but one aspect of their physiology.

Molecular determinants of drug access to the receptor site for antiarrhythmic drugs in the cardiac Na+ channel.

The clinical efficacy of local anesthetic and antiarrhythmic drugs is due to their voltage- and frequency-dependent block of Na+ channels. Quaternary local anesthetic analogs such as QX-314, which are permanently charged and membrane-impermeant, effectively block cardiac Na+ channels when applied from either side of the membrane but block neuronal Na+ channels only from the intracellular side. This difference in extracellular access to QX-314 is retained when rat brain rIIA Na+ channel alpha subunits and rat heart rH1 Na+ channel alpha subunits are expressed transiently in tsA-201 cells. Amino acid residues in transmembrane segment S6 of homologous domain IV (IVS6) of Na+ channel alpha subunits have important effects on block by local anesthetic drugs. Although five amino acid residues in IVS6 differ between brain rIIA and cardiac rH1, exchange of these amino acid residues by site-directed mutagenesis showed that only conversion of Thr-1755 in rH1 to Val as in rIIA was sufficient to reduce the rate and extent of block by extracellular QX-314 and slow the escape of drug from closed channels after use-dependent block. Tetrodotoxin also reduced the rate of block by extracellular QX-314 and slowed escape of bound QX-314 via the extracellular pathway in rH1, indicating that QX-314 must move through the pore to escape. QX-314 binding was inhibited by mutation of Phe-1762 in the local anesthetic receptor site of rH1 to Ala whether the drug was applied extracellularly or intracellularly. Thus, QX-314 binds to a single site in the rH1 Na+ channel alpha subunit that contains Phe-1762, whether it is applied from the extracellular or intracellular side of the membrane. Access to that site from the extracellular side of the pore is determined by the amino acid at position 1755 in the rH1 cardiac Na+ channel.

Dynamics of active lamellae in cultured epithelial cells: effects of expression of exogenous N-ras oncogene.

We examined the functional consequences of cellular transformation of rat IAR-2 epithelial cells, by a mutant N-ras oncogene, on the dynamics of active lamellae, structures that play an important role in cell motility, adhesion, and surface-receptor capping. Lamellar activity was assessed by measuring the rate of outer-edge pseudopodial activity and by analyzing the motility of Con A-coated beads placed on lamellar surfaces with optical tweezers. Although transformation dramatically affected the shape and size of active cellular lamellae, there was little detectable effect on either pseudopodial activity or bead movement. To investigate the potential relationship between functional lamellar activity and the microtubule cytoskeleton, lamellar activity was examined in nontransformed and transformed cells treated with the microtubule-disrupting drug nocodazole. In the absence of microtubules, transformed cells were less polarized and possessed decreased rates of pseudopodial and bead motility. On the basis of these observations, it is suggested that ras-induced transformation of epithelial cells consists of two cytoskeletal modifications: overall diminished actin cytoskeletal dynamics in lamellae and reorganization of the microtubule cytoskeleton that directs pseudopodial activity to smaller polarized lamellae.

Molecular and cytotoxic effects of camptothecin, a topoisomerase I inhibitor, on trypanosomes and Leishmania.

Parasites pose a threat to the health and lives of many millions of human beings. Among the pathogenic protozoa, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani are hemoflagellates that cause particularly serious diseases (sleeping sickness, Chagas disease, and leishmaniasis, respectively). The drugs currently available to treat these infections are limited by marginal efficacy, severe toxicity, and spreading drug resistance. Camptothecin is an established antitumor drug and a well-characterized inhibitor of eukaryotic DNA topoisomerase I. When trypanosomes or leishmania are treated with camptothecin and then lysed with SDS, both nuclear and mitochondrial DNA are cleaved and covalently linked to protein. This is consistent with the existence of drug-sensitive topoisomerase I activity in both compartments. Camptothecin also inhibits the incorporation of [3H]thymidine in these parasites. These molecular effects are cytotoxic to cells in vitro, with EC50 values for T. brucei, T. cruzi, and L. donovani, of 1.5, 1.6, and 3.2 microM, respectively. For these parasites, camptothecin is an important lead for much-needed new chemotherapy, as well as a valuable tool for studying topoisomerase I activity.

Iontophoresis for modulation of cardiac drug delivery in dogs.

Cardiac arrhythmias are a frequent cause of death and morbidity. Conventional antiarrhythmia therapy involving oral or intravenous medication is often ineffective and complicated by drug-associated side effects. Previous studies from our laboratory have demonstrated the advantages of cardiac drug-polymer implants for enhanced efficacy for cardiac arrhythmia therapy compared with conventional administration. However, these studies were based on systems that deliver drugs at a fixed release rate. Modulation of the drug delivery rate has the advantage of regulating the amount of the drug delivered depending upon the disease state of the patient. We hypothesized that iontophoresis could be used to modulate cardiac drug delivery. In this study, we report our investigations of a cardiac drug implant in dogs that is capable of iontophoretic modulation of the administration of the antiarrhythmic agent sotalol. We used a heterogeneous cation-exchange membrane (HCM) as an electrically sensitive and highly efficient rate-limiting barrier on the cardiac-contacting surface of the implant. Thus, electric current is passed only through the HCM and not the myocardium. The iontophoretic cardiac implant demonstrated in vitro drug release rates that were responsive to current modulation. In vivo results in dogs have confirmed that iontophoresis resulted in regional coronary enhancement of sotalol levels with current-responsive increases in drug concentrations. We also observed acute current-dependent changes in ventricular effective refractory periods reflecting sotalol-induced refractoriness due to regional drug administration. In 30-day dog experiments, iontophoretic cardiac implants demonstrated robust sustained function and reproducible modulation of drug delivery kinetics.

Electrohydrodynamic Atomization for Improved Macromolecular Drug Delivery

With advances in drug research, the use of biological therapeutics is becoming a reality. Unfortunately, methods for processing and delivering these fragile macromolecules often limit their therapeutic potential. For this dissertation, we explore the aerosolization of macromolecules by way of electrohydrodynamic atomization (EHDA) and how this method can be used to process and deliver therapeutics. EHDA employs a high voltage to break a column of liquid into drops. It was unknown if or how the residual charge left of the resulting droplets would affect lung cells. An in vitro experiment was conducted to spray aerosolized DNA, by way of EHDA, onto human derived lungs cells to test for immunogenic and toxic effects. The lung cells displayed no immunogenic or toxic response to the DNA or high voltage. Previous researchers have used EHDA to aerosolize proteins with mixed results. This work sets forth a simplified thermodynamic theory and provides recommendations to pharmaceutical companies on how to design more stable protein formulations for aerosol processing or delivery. Finally, a new method of producing liposomes was created. It constructs the liposome one layer at a time. The inside of the liposome is sprayed by EHDA, with the lipid and drug in solution together. As the sprayed monolayer passes through a pool containing a solution of lipid in water, the second part of the bilayer attaches to the inner layer creating a complete bilayer liposome.

Profound activity of the anti-cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes.

A library of 426 FDA-approved drugs was screened for in vitro activity against E. multilocularis metacestodes employing the phosphoglucose isomerase (PGI) assay. Initial screening at 20 µM revealed that 7 drugs induced considerable metacestode damage, and further dose-response studies revealed that bortezomib (BTZ), a proteasome inhibitor developed for the chemotherapy of myeloma, displayed high anti-metacestodal activity with an EC50 of 0.6 µM. BTZ treatment of E. multilocularis metacestodes led to an accumulation of ubiquinated proteins and unequivocally parasite death. In-gel zymography assays using E. multilocularis extracts demonstrated BTZ-mediated inhibition of protease activity in a band of approximately 23 kDa, the same size at which the proteasome subunit beta 5 of E. multilocularis could be detected by Western blot. Balb/c mice experimentally infected with E. multilocularis metacestodes were used to assess BTZ treatment, starting at 6 weeks post-infection by intraperitoneal injection of BTZ. This treatment led to reduced parasite weight, but to a degree that was not statistically significant, and it induced adverse effects such as diarrhea and neurological symptoms. In conclusion, the proteasome was identified as a drug target in E. multilocularis metacestodes that can be efficiently inhibited by BTZ in vitro. However, translation of these findings into in vivo efficacy requires further adjustments of treatment regimens using BTZ, or possibly other proteasome inhibitors.

Epileptic networks are strongly connected with and without the effects of interictal discharges

OBJECTIVE Epilepsy is increasingly considered as the dysfunction of a pathologic neuronal network (epileptic network) rather than a single focal source. We aimed to assess the interactions between the regions that comprise the epileptic network and to investigate their dependence on the occurrence of interictal epileptiform discharges (IEDs). METHODS We analyzed resting state simultaneous electroencephalography-functional magnetic resonance imaging (EEG-fMRI) recordings in 10 patients with drug-resistant focal epilepsy with multifocal IED-related blood oxygen level-dependent (BOLD) responses and a maximum t-value in the IED field. We computed functional connectivity (FC) maps of the epileptic network using two types of seed: (1) a 10-mm diameter sphere centered in the global maximum of IED-related BOLD map, and (2) the independent component with highest correlation to the IED-related BOLD map, named epileptic component. For both approaches, we compared FC maps before and after regressing out the effect of IEDs in terms of maximum and mean t-values and percentage of map overlap. RESULTS Maximum and mean FC maps t-values were significantly lower after regressing out IEDs at the group level (p < 0.01). Overlap extent was 85% ± 12% and 87% ± 12% when the seed was the 10-mm diameter sphere and the epileptic component, respectively. SIGNIFICANCE Regions involved in a specific epileptic network show coherent BOLD fluctuations independent of scalp EEG IEDs. FC topography and strength is largely preserved by removing the IED effect. This could represent a signature of a sustained pathologic network with contribution from epileptic activity invisible to the scalp EEG.

The pragmatics of computer-mediated communication between South African and Mexican drug traffickers

South Africa and Mexico are ripe with drug trafficking. The gangs and syndicates running the drug businesses in these two countries collaborate occasionally. Communication between these international drug business partners takes place on social media. Their main language of communication is English, mixed with some limited use of Spanish and Afrikaans. The key purpose of the interactions between the South African and Mexican parties is the organisation of their business activities. This study aims at examining how the drug traffickers position each other and themselves regarding their common business interest and how their relationship evolves throughout their interactions. Moreover, it is of interest to look at how these people make use of different social media and their affordances. For this a qualitative analysis of the interaction between two drug traffickers (one South African and one Mexican) on Facebook, Threema and PlayStation 4 was performed. Computer-mediated communication between these two main informants was studied at various stages of their relationship. Results show that at first the interaction between the South African and Mexican drug traffickers consists of interpersonal negotiations of power. The high risk of the drug business and gang/syndicate membership paired with intercultural frictions causes the two interlocutors to be extremely cautious and at the same time to mark their position. As their relationship develops and they gain trust in each other a shift to interpersonal negotiations of solidarity takes place. In these discursive practices diverse linguistic strategies are employed for creating relational effects and for positioning the other and the self. The discursive activities of the interactants are also identity practices. Thus, the two drug traffickers construct identities through these social practices, positioning and their interpersonal relationship.

The biological effects of ionizing radiation ; an overview /

Mode of access: Internet.

Symposium on Biological Effects and Measurement of Radio Frequency/Microwaves : proceedings of a conference held in Rockville, Maryland, February 16-18, 1977/

Includes bibliographical references.

Teratologic effects of long-term exposure to diesel exhaust emissions (rabbits) /

"Contract no. 68-03-2652."