High expression level of a gene coding for a chloroplastic amino acid selective channel protein is correlated to cold acclimation in cereals

A cold-regulated gene (cor tmc-ap3) coding for a putative chloroplastic amino acid selective channel protein was isolated from cold-treated barley leaves combining the differential display and the 5-RACE techniques. Cor tmc-ap3 is expressed at low level under normal growing temperature, and its expression is strongly enhanced after cold treatment. A positive correlation between the expression of cor tmc-ap3 and frost tolerance was found both among barley cultivars and among cereal species. The COR TMC-AP3 protein was expressed in vitro, purified and used to raise a polyclonal antibody. Western analysis showed that the cor tmc-ap3 gene product is localized to the chloroplastic outer envelope fraction, supporting its putative function. The frost-resistant winter cultivar Onice accumulated COR TMC-AP3 more rapidly and at a higher level than the frost-susceptible spring cultivar Gitane. After 28 days of cold acclimation the winter cultivar had about 2-fold more protein than the spring genotype. All these results suggest that an increased amount of a chloroplastic amino acid selective channel protein could be required for cold acclimation in cereals. Hypotheses about the role of COR TMC-AP3 during the hardening process are discussed.     

Detecting Resistance to Organophosphates and Carbamates in the Cattle Tick Boophilus Microplus, with a Propoxur-based Biochemical Test

Rapid and sensitive detection of resistance to insecticides in arthropods is needed. In the cattle tick, Boophilus microplus, resistance to a variety of acaricides is widespread. The most commonly used assay for resistance, the larval packet test, takes at least two, but generally six weeks for a one-host tick like B. microplus to complete and may take up to three months to complete for three-host ticks. Here we describe a test for resistance to organophosphate acaricides that can be used on larvae and adult ticks which takes less than 24 hours. The test measures the difference in acetylcholinesterase (AChE) activity in homogenates of ticks in the presence and absence of propoxur, a carbamate acaricide. We found clear discrimination of organophosphate-susceptible and organophosphate-resistant adults with 100 M propoxur. AChE from susceptible ticks had almost no activity at this concentration of propoxur whereas AChE from resistant ticks had 67% of its potential activity. AChE from heterozygote ticks could also be distinguished from AChE from homozygous-susceptible and homozygous-resistant ticks. This is the first biochemical test for resistance to an acaricide. Rapid, sensitive tests like ours will allow resistance to organophosphates to be detected soon after it develops in the field, thus, the spread of resistance might be slowed and the useful life of acaricides extended.     

Effects of permethrin at different temperatures on pyrethroid-resistant and susceptible strains of Anopheles

The influence of temperature (16, 22, 28, 37 degrees C) on effects of permethrin was investigated for susceptible and pyrethroid-resistant strains of the mosquitoes Anopheles gambiae and An. stephensi (Diptera: Culicidae). Young unfed female adult mosquitoes were exposed to 0.25% permethrin test papers or to polyester netting treated with permethrin 500mg a.i./m2. The time to 50% knockdown (KT50) declined as temperature increased, i.e. there was a positive temperature coefficient of this effect of the pyrethroid. Resistance ratios (comparing KT50 values) between resistant and susceptible An. stephensi ranged between 2.5 and 4.4 at the different temperatures. Comparative tests of pyrethroid tolerance of different strains would be valid over the 22-28 degrees C range but, when using a discriminating dose to detect resistance, more precise temperature control is desirable. Mortality 24h after exposure to 0.25% permethrin of both susceptible and resistant strains of An. stephensi showed a negative correlation with temperature between 16 and 22 degrees C and a positive correlation at higher temperatures. In An. gambiae, however, the correlation was positive over the whole range. Irritancy of permethrin-treated netting to Anopheles females (measured as time lapse until first flight take-off, and the number of take-offs during 7.5 min exposure) was positively correlated with temperature in all four strains and was much greater for the susceptible than the resistant strains.     

刺激面神经核背内侧区及腹内侧区对颏舌肌和膈肌肌电活动的影响

在４４只氨基甲酸乙酯麻醉、断双侧迷走神经的健康成年家兔上、观察电、化学刺激面神经核背内侧区和腹内侧区对颏舌肌和膈肌肌电活动的影响。结果如下：（１）长串电脉冲刺激ｄＭＮＦ引起颏舌肌和膈肌肌电活动明显增强;（２）短串电脉冲刺激ｄＭＦＮ,当刺激落用于吸气相时,引起颏舌肌和膈肌在呼气相的肌是有终止;（３）长串电脉冲刺激ｖＭＮＦ使颏舌肌和膈肌肌电活动明显被抑制;（４）短中电脉冲刺激ｖＭＮＦ,当刺激落位有气相     

Transformation of the nematode-trapping fungus Arthrobotrys oligospora

The nematode-trapping fungus Arthrobotrys oligospora was transformed to hygromycin resistance using the hygromycin-B phosphotransferase gene from Escherichia coli under the control of various heterologous fungal promoters. Plasmid DNA was introduced into fungal protoplasts by polyethylene glycol/CaCl2 treatment. Transformation frequencies varied between 1-6 transformants per microgram DNA. Seven out of 13 integration events analyzed from transformants were single copy integrations, whereas the remaining were multiple and more complex integrations. The addition of restriction enzymes during transformations increased the frequency of single copy integrations. Co-transformation, using the E. coli uidA gene encoding the beta-glucuronidase reporter gene under the control of an Aspergillus nidulans promoter, occurred at frequencies of up to 63%.     

PI-3-kinase/NF-kappaB mediated response of Jurkat T leukemic cells to two different chemotherapeutic drugs, etoposide and TRAIL

Jurkat T leukemic cells respond to Etoposide, antineoplastic agent which targets the DNA unwinding enzyme, Topoisomerase II, and TNF-Related-Apoptosis-Inducing-Ligand (TRAIL), 34 kDa transmembrane protein, which displays minimal or no toxicity on normal cells and tissues, not only disclosing the occurrence of apoptosis but also a kind of resistance. A similar rate of viability upon the exposure to these two drugs up to 24 h has been evidenced, followed by the occurrence of a rescue process against TRAIL, not performed against Etoposide, along with an higher number of dead cells upon Etoposide exposure, in comparison with TRAIL treatment. These preliminary results let us to speculate on the possible involvement of PI-3-kinase in TRAIL resistance disclosed by surviving cells (20%), may be phosphorylating Akt-1 and, in parallel, IkappaB alpha on both serine and tyrosine residues. On the other hand, in Etoposide Jurkat exposed cells Ser 32-36 phosphorylation of IkappaB alpha is not sufficient to overbalance the apoptotic fate of the cells, since Bax increase, IAP decrease, and caspase-3 activation determine the persistence of the apoptotic state along with the occurrence of cell death by necrosis. Thus, the existence of a balance between apoptotic and rescue response in 20% of cells surviving to TRAIL suggests the possibility of pushing it in favor of cell death in order to improve the yield of pharmacological strategies.     

Efficiency of a second-generation HIV-1 protease inhibitor studied by molecular dynamics and absolute binding free energy calculations

A subnanomolar inhibitor of human immunodeficiency virus type 1 (HIV-1) protease, designated QF34, potently inhibits the wild-type and drug-resistant enzyme. To explain its broad activity, the binding of QF34 to the wild-type HIV-1 protease is investigated by molecular dynamics simulations and compared to the binding of two inhibitors that are used clinically, saquinavir (SQV) and indinavir (IDV). Analysis of the flexibility of protease residues and inhibitor segments in the complex reveals that segments of QF34 were more mobile during the dynamics studies than the segments of SQV and IDV. The dynamics of hydrogen bonding show that QF34 forms a larger number of stable hydrogen bonds than the two inhibitors that are used clinically. Absolute binding free energies were calculated with molecular mechanics-generalized Born surface area (MM-GBSA) methodology using three protocols. The most consistent results were obtained using the single-trajectory approach, due to cancellation of errors and inadequate sampling in the separate-trajectory protocols. For all three inhibitors, energy components in favor of binding include van der Waals and electrostatic terms, whereas polar solvation and entropy terms oppose binding. Decomposition of binding energies reveals that more protease residues contribute significantly to the binding of QF34 than to the binding of SQV and IDV. Moreover, contributions from protease main chains and side chains are balanced in the case of QF34 (52:48 ratio, respectively), whereas side chain contributions prevail in both SQV and IDV (main-chain:side-chain ratios of 41:59 and 45:55, respectively). The presented results help explain the ability of QF34 to inhibit multiple resistant mutants and should be considered in the design of broad-specificity second-generation HIV-1 protease inhibitors.     

The biological functions of polyamine oxidation products by amine oxidases: Perspectives of clinical applications

Summary. The polyamines spermine, spermidine and putrescine are ubiquitous cell components. If they accumulate excessively within the cells, due either to very high extracellular concentrations or to deregulation of the systems which control polyamine homeostasis, they can induce toxic effects. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper containing amine oxidases. Polyamine concentrations are high in growing tissues such as tumors. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. These enzymes catalyze the oxidative deamination of biogenic amines and polyamines to generate the reaction products H₂O₂ and aldehyde(s) that are able to induce cell death in several cultured human tumor cell lines. H₂O₂ generated by the oxidation reaction is able to cross the inner membrane of mitochondria and directly interact with endogenous molecules and structures, inducing an intense oxidative stress. Since amine oxidases are involved in many crucial physiopathological processes, investigations on their involvement in human diseases offer great opportunities to enter novel classes of therapeutic agents.     

The microbiology of a pharmaceutical effluent and its public health implications

The effluent of a pharmaceutical company was examined microbiologically. Its bacterial count was 2.15 × 10⁵ c.f.u./ml and there was evidence of faecal contamination with MPN of > 1800. The organisms encountered included Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Serratia marcescens and Pseudomonas aeruginosa. The resistances of the 25 bacterial strains isolated from the effluent to the commonly used antibiotics were studied. About 80% of the isolates were resistant to Amoxycillin, 76% to Nitrofurantoin, 64% to Cotrimoxazole and Augmentin, 60% were resistant to Nalidixic acid, 52% were resistant to Tetracycline and Ofloxacin, while resistance of 12% was obtained for Gentamicin. Among the eight antibiotics tested, seven patterns of drug resistance were obtained and all of them were multiple-drug resistance with the number of antibiotics ranging from 2–8. All the strains of E. coli and S. aureus had high MIC values for Cloxacillin and Amoxycillin. In all, 13 strains of the bacterial isolates had evidence for the production of -lactamases. The potential of the effluent in spreading drug resistance and the public health implications are discussed.