Purification of breast cancer resistance protein ABCG2 and role of arginine-482

Human ABCG2 was efficiently overexpressed in insect cell membranes, solubilized with 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate, and purified through N-terminal hexahistidine tag. Its functionality was assessed by high vanadate-sensitive ATPase activity, and nucleotide-binding capacity. Interestingly, the R482T point mutation increased both maximal hydrolysis rate and affinity for MgATP, and lowered sensitivity to vanadate inhibition. Direct nucleotide binding, as monitored by quenching of intrinsic fluorescence, indicated a mutation-related preference for ATP over ADP. The R482T mutation only produced a limited change, if any, on the binding of drug substrates, indicating that methotrexate, on the one hand, and rhodamine 123 or doxorubicin, on the other hand, bound similarly to wild-type and mutant transporters whether or not they were subject to cellular transport. In addition, the characteristic inhibitors GF120918 and 6-prenylchrysin, which alter mitoxantrone efflux much better for wild-type than mutant ABCG2, bound similarly to purified ABCG2, while the highly-potent Ko143 bound in the nanomolar range also effective in inhibition of drug transport. All results indicate that the role of the arginine-482 mutation on substrate drug transport and inhibitor efficiency is not mediated by changes in drug binding. Received 10 April 2006; received after revision 22 May 2006; accepted 12 June 2006 A. Pozza and J. M. Perez-Victoria contributed equally to this work     

Antimalarial drugs: recent advances in molecular determinants of resistance and their clinical significance

Molecular determinants of antimalarial drug resistance are useful and informative tools that complement phenotypic assays for drug resistance. They also guide the design of strategies to circumvent such resistance once it has reached levels of clinical significance. Established resistance to arylaminoalcohols such as mefloquine and lumefantrine in SE Asia is mediated primarily by gene amplification of the P. falciparum drug transporter, pfmdr1. Single nucleotide polymorphisms in pfmdr1, whether assessed in field isolates or transfection experiments, are associated with changes in IC₅₀ values (to arylaminoalcohols and chloroquine), but not of such magnitude as to influence clinical treatment outcomes. Recently described emerging in vitro resistance to artemisinins in certain areas correlates with mutations in the SERCA-like sequence PfATP6 and supports PfATP6 as a key target for artemisinins. Received 13 February 2006; revised after revision 7 March 2006; accepted 29 March 2006     

Identification of the drug Darvon and its metabolites in the urine of a comatose patient using a gas chromatograph-mass. Spectrometer-computer system

Zusammenfassung Mit Hilfe von hoch- und niederauflösender Massenspektroskopie sowie Daten eines Gas-chromatograph-Massenspektrometer-Computer-Systems wurden die Droge Darvon³ sowie eine Reihe ihrer Metaboliten im Harn eines Überdosispatienten nachgewiesen.

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This publication describes the identification of a drug causing coma in a patient. The drug was identified by analysis of the patient's urine performed at the NIH sponsored Special Research Resource Facility for Mass Spectrometry at M.I.T., under emergency conditions. Most of the experiments, measurements and interpretation reported had to be made within 1 day, thus requiring the simultaneous efforts of many members of M.I.T.'s massspectrometry group.     

Effect of tingenone,a quinonoid triterpene,on growth and macromolecule biosynthesis inTrypanosoma cruzi

Summary Tingenone and horminone, two natural quinonoid substances, inhibited the in vitro growth ofTrypanosoma cruzi, 30 M drug concentration producing total inhibition of growth. Tingenone inhibited total uptake and incorporation of [³H]thymidine, [³H]uridine, L-[³H]leucine into parasite macromolecules. Other quinonoids assayed were either less effective (abruquinone A) or even quite inactive (visminone B and ferruginin B). Investigation of several mechanisms for the cytotoxic action of tingenone pointed to the interaction with DNA as the most likely factor involved. Tingenone also inhibited the growth ofCrithidia fasciculata, but the drug was significantly less active on this organism than onT. cruzi.This work was supported by grants of UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases, Organization of American States (Multinational Programme of Biochemistry) and Programa Nacional de Enfermedades Endémicas (SECYT), República Argentina. A preliminary account was given at the Workshop on Oxidative Damage and Related Enzymes, Frascatti (Italy), 1983.     

The occurrence of arachidonic acid in the venom duct of the marine snailConus textile

Summary A crude extract of the venom of the marine snailConus textile Linné caused a powerful contraction in the guinea-pig isolated ileum. The extract of the venom has been fractionated, and the fractions monitored by the contractile effect. Arachidonic acid was shown to be present as an active substance.Acknowledgment. We are grateful to Mr Z. Nagahama of Okinawa island for suppling specimens ofC. textile and to Dr T. Higashijima and Prof. T. Miyazawa (Department of Biophysics and Biochemistry, Tokyo University) for 270 MHz¹H-NMR-measurements.     

Open-field behavior and acquisition of discriminative response control in Δ9-THC tolerant rats

Résumé La tolérance de l'effet du 1- ⁹-tétrahydrocannabinol ( ⁹-THC) a été examinée par une méthode nouvelle. Les résultats obtenus permettent une interprétation de tolérance pharmacologique pour ⁹-THC.

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Numbering system according to IUPAC rules. The drug was generously supplied by Dr.T. Petrzilka, University of Zürich.     

Protective effect of Shigyaku-to,a traditional Chinese herbal medicine,on the infection of herpes simplex virus type 1 (HSV-1) in mice

The antiviral activity of Shigyaku-to (TJS-109), a traditional Chinese herbal medicine, was investigated in mice infected with herpes simplex virus type 1 (HSV-1). TJS-109 is a combination of the medicinal plant extracts fromZingiberis siccatum rhizoma,Aconiti tuber andGlycyrrhizae radix in a specific proportion. Mice infected with a 10 LD₅₀ dose of HSV-1 were treated with TJS-109 orally at doses of 1.25 to 20 mg/kg 2 days before, and 1 and 4 days after the infection. The treated groups had 80% (1.25 mg/kg), 40% (5 mg/kg) and 23% (20 mg/kg) mortality rates 25 days after the infection as compared with a 100% mortality rate in control mice treated with saline. When HSV-1 infected mice (recipients) received CD8⁺T cell fractions derived from spleens of mice treated with TJS-109 (donors), 70% of recipients survived, as compared with 0% survivors in the groups of mice treated with saline, B cell fractions, CD4⁺ T cell fractions or macrophage-enriched fractions prepared from the same donors. TJS-109 did not show any virucidal activities against HSV-1 or any virostatic activities on the growth of HSV-1 in Vero cells. These results suggest that TJS-109 protected mice exposed to lethal amounts of HSV-1 through the activation of CD8⁺ T cells.     

2-Carboxyethylgermanium sesquioxide,a synthetic organogermanium compound,as an inducer of contrasuppressor T cells

2-Carboxyethylgermanium sesquioxide (Ge-132), a synthesized organogermanium compound with immunomodulaing activities, was shown to be an inducer of anti-suppressor T cells in normal mice. The suppressor cell activity of T6S cells, a clone of burn-induced CD8⁺ IL-4-producing suppressor T cells, was clearly inhibited when a mixed lymphocyte-tumor cell reaction of the clone was conducted with splenic mononuclear cells from mice treated orally with a 100 mg/kg dose of Ge-132. The activity of anti-suppressor cells was demonstrated in spleens of mice 2 days after treatment with Ge-132 and reached its peak on day 3. The anti-suppressor cells induced by the compound were of a contrasuppressor T cell-linage, because they were characterized as CD4⁺ CD28⁺ TCR/⁺ Vicia villosa lectin-adherent T cells. These cells produced IFN- but did not produce IL-2, IL-4, IL-6 or IL-10 in their culture fluids. CD4⁺ anti-suppressor T cells induced by Ge-132 may be different from other subsets of CD4⁺ T cells because Th1 and Th2 cells generated in our laboratory did not adhere toVicia villosa lectin-coated petri dishes, and each produced specific cytokines. Th1 cells produced IFN- and IL-2 while Th2 cells produce IL-4 and IL-10 in vitro. These results suggest that Ge-132 may be useful as an inducer of contrasuppressor T cells in immunocompromised individuals bearing suppressor T cells. To eliminate suppressor T cells from immunocompromised hosts may result in improved resistance from various opportunistic infections.     

Desiccation in the black dragon,Hagenius brevistylus Selys

Summary Hagenius brevistylus lost mass by evaporation in a moderately desiccating environment at the rate of 20.4 mg h^–1, and died of desiccation in less than 1 day at a body mass of 79.8% of their normally hydrated mass. It was estimated thatHagenius minimally would have to consume the equivalent of 60% of its body mass each day to meet its daily water requirements. This amount of food is equivalent to that necessary to power flight of a dragonfly for 4.6h.This research was financed in part by a grant from the American Philosophical Society to C.R.T., and was conducted at the University of Michigan Biological Station, Douglas Lake (USA).     

The partitioning ofΔ 1-tetrahydrocannabinol into erythrocyte membranes in vivo and its effect on membrane fluidity1into erythrocyte membranes in vivo and its effect on membrane fluidity1

Summary ¹-Tetrahydrocannabinol ( ¹-THC) has been quantified directly in erythrocyte membranes from drug-treated mice using gas chromatography/mass spectrometry. Concentrations of approximately 6 ng ¹-THC/mg membrane protein (10^–5 M) were found when effects of the drug on behavior were prevalent. At these concentrations the drug produced a decrease in membrane order as measured by ESR.This work was supported by grants from the Medical Research Council, the Wellcome Trust and the E. P. Abraham Cephalosporin Trust     

Possible involvement of the phloem sealing system in the acceptance of a plant as host by an aphid

Possible reasons for the rejection of some lines ofTriticum monococcum (Tm44 and Tm46) by the aphidSitobion avenae were explored. In allT. monococcum lines studied, whether unfavourable (non-host/resistant plant) or favourable (host/susceptible plant), the concentrations of hydroxamic acids, a family of aphid-resistance factors in cereals, were significantly lower than the levels in the favourable host-plantTriticum aestivum cv. Therefore, hydroxamic acids did not account for the host/non-host patterns observed. Phloem sap was collected by stylectomy from young seedlings of favourable and unfavourable plants. In non-aphid-resistant genotypes, the success in stylectomy, the proportion of amputated stylets resulting in long (>1 min) exudations, the average duration of exudation, and the final volume of sap exuded, were higher than in the aphid-resistant genotypes. It is concluded that aphid interference with the phloem sealing system of the plant is a likely mechanism of rejection ofT. monococcum lines Tm44 and Tm46 as hosts byS. avenae.     

Identification of acrocentric chromosomes involved in the formation of ‘fusion’-metacentrics in mice. proposal for nomenclature ofM. poschiavinus metacentrics

Zusammenfassung Der Befund eines Pentavalents in der Meiose von (M. poschiavinus×AKR/T1Ald) F₁ zeigt, dass homologe akrozentrische Chromosomen bzw. Chromosomenarme unterschiedlich auf die metazentrischen Chromosomen der Tabakmaus (M. poschiavinus) und des AKR-Translokationsstammes T1Ald verteilt sind. Kreuzungsexperimente dieser Art dienen der Identifizierung einzelner Chromosomen oder Chromosomenarme bei der Maus. Für die sieben metazentrischen Chromosomen der Tabakmaus werden die Symbole T1-7 Bnr vorgeschlagen.     

Thyroid activity in response to some gonadal steroids in methallibure-treatedHeteropneustes fossilis (Bloch)

Summary Methallibure treatment is as effective as hypophysectomy in reducing thyroid activity inH. fossilis. Sex steroids (TP and EB) administration restored thyroid activity in methallibure-treated females to normal level, but failed to elicit any response in males. This drug seems to block TSH secretion and thyroid hormone synthesis inH. fossilis.Financial assistance in the form of SRF from ICAR, New Delhi to one of us (R.B.R.), gift of methallibure from ICI Ltd., UK and TSH from NIH, USA, to T.P.S. are gratefully acknowledged.     

Sex pheromone blend of the codling moth,Cydia pomonella: Evidence for a behavioral role of dodecan-1-ol

Summary Pheromone glands and effluvia of the codling moth female containE-8, E-10-dodecadien-1-ol as main component, accompanied by its geometric isomers, the corresponding acetate and aldehyde (both in gland extracts only),E-9-dodecen-1-ol and saturated alcohols of 10 to 18 carbons. Dodecan-1-ol as the most abundant secondary component (about 30% of the female blend) acts in the wind tunnel by widening the dose range over which codling moth males are optimally attracted toE-8, E-10-dodecadien-1-ol.This research was supported by the Swiss National Science Foundation. We thank W. Riggenbach for supplying moths and T. Wildbolz, P. Charmillot and M. Tóth for conducting field trials.     

Molecular characterization of <Emphasis Type="Italic">Arabidopsis</Emphasis> PHO80-like proteins,a novel class of CDKA;1-interacting cyclins

Cyclins are regulatory proteins that interact with cyclin-dependent kinases (CDKs) to control progression through the cell cycle. In Arabidopsis thaliana, 34 cyclin genes have been described, grouped into five different types (A, B, D, H, and T). A novel class of seven cyclins was isolated and characterized in Arabidopsis, designated P-type cyclins (CYCPs). They all share a conserved central region of 100 amino acids (cyclin box) displaying homology to the corresponding region of the PHO80 cyclin from Saccharomyces cerevisiae and the related G1 cyclins from Trypanosoma cruzi and T. brucei. The CYCP4;2 gene was able to partially re-establish the phosphate-dependent expression of the PHO5 gene in a pho80 mutant strain of yeast. The CYCPs interact preferentially with CDKA;1 in vivo and in vitro as shown by yeast two-hybrid analysis and co-immunoprecipitation experiments. P-type cyclins were mostly expressed in proliferating cells, albeit also in differentiating and mature tissues. The possible role of CYCPs in linking cell division, cell differentiation, and the nutritional status of the cell is discussed.Received 9 February 2004; received after revision 18 March; accepted 19 April 2004     

Malaria prophylaxis in travellers: The current position

Summary Malaria prevention is a main challenge for physicians, nurses, health officers and tour operators. The attack rate of malaria in travellers is 1–10/10,000 departures, and the case fatality rate of imported malaria is around 0.5/100. Travellers should be informed about the risk they are going to take, how to protect against mosquito bites, about the antimalarials they will have to take and about what to do when a malaria breakthrough should occur.The 4-aminoquinolines (chloroquine, amodiaquine) remain the drug of choice for the prevention ofPlasmodium vivax and of sensitiveP. falciparum infections. The problem is to find an effective and safe drug combination for travellers to areas whereP. falciparum is either resistant to chloroquine, to Fansidar (the combination of pyrimethamine plus sulfadoxine) or to both. These travellers will probably best be protected by an individually tailored drug combination, which includes amodiaquine or mefloquine as baseline drugs, and a supplementation with Fansidar, Maloprim (the combination of pyrimethamine with dapsone), paludrine or an antibiotic.     

Beeinflussung der ATPase aminspeichernder Granula von Nebennierenmark und Milznerven durch Thallium

Summary ATPases of the amine storing granules from bovine adrenal medulla and splenic nerves are inhibited by Tl⁺⁺⁺ 3×10^–5 and 5×10^–6 M, respectively. Tl⁺ up to 10^–3 M is ineffective. By T1⁺⁺⁺ in concentrations of 10^–4 M or more, proteins are precipitated, so that the enzyme inhibiton by these concentrations is unspecific. If T1⁺ is oxidized to Tl⁺⁺⁺ in the organism, the inhibition of granular ATPase may be responsible for the alterations of the catecholamine metabolism observed in thallium intoxication.     

Expression of multidrug resistance (mdr) gene(s) in primary lymphoid organs of chicken immune system during embryonic development

The presence of a multidrug resistance (MDR) related protein, P-170, in normal and pathological lymphoid cells has been described. The present report evaluates the expression of themdr 1 gene by using the reverse Polymerase Chain Reaction (PCR) on cells obtained from the thymus and bursa of chicken embryos starting from day 12 until hatching. Results show that the thymic cells are positive from day 12 to the end of the observation period. In contrast,mdr mRNA was detected in the bursa from day 14 to day 17 of embryonic life. Possible relationships between the expression ofmdr and the development of T and B lymphocytes are discussed.     

Antimitotic effect of nimbidin — A first report

Summary The antimitotic activity of nimbidin, a drug from the plantMelia azadirecta indica, was assessed by its effect on the meristematic cells of onion root tips. The effect was almost similar to those of colchicine and vinca alkaloids. Recovery trials showed that the drug induces lethal damage in a considerable proportion of treated cells and may hence have applications in cancer chemotherapy.