Ready-to-use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue

This report summarizes major changes in previously published protocols for DNA extraction to improve the quality of DNA extracted from plants. Here, we highlight the critical modifications in the original protocols. The efficiency of these changes results in high-quality DNA ready to use in a variety of phytogenetically distant plant families, in particular species with mucopolysaccharides. The DNA obtained can be used without further purification in various molecular biology assays, including direct sequencing and AFLP and RAPD (random-amplified polymorphic DNA) analyses. The effectiveness of this method is proven by the amplification and sequencing of PCR products of up to 1 kb with DNA extracted from herbarium tissue ≥60 years old. This versatility is not usually found in DNA extraction protocols. In addition, this method is quick, adaptable to standard laboratories, and most important, safer and more cost-effective.     

Synthesis and biological characterisation of novel dithiocarbamate containing 5-nitroimidazole Tc-complexes as potential agents for targeting hypoxia

With the aim to develop new potential ^99mTc-radiopharmaceuticals for imaging hypoxia based on the formation of Tc-nitrido complexes, two novel dithiocarbamate containing metronidazole derivatives (L1 and L2) have been prepared and characterised. The synthesis of L1 and L2 was achieved in excellent yield and high purity. Labelling with ^99mTc was successfully performed using a low ligand concentration (approximately 2-3 mg) and the desired products were obtained with high radiochemical purity (>90%). Lipophilicity, plasma protein binding, and biodistribution in normal- and tumour-bearing-CD1 mice studies were performed to asses the potentiality for nuclear medicine oncology. According to the physicochemical and biological behaviour both in healthy animals and in animals bearing solid tumours complex dtcTc1 could be considered as a starting point for the development of new radiopharmaceuticals for imaging hypoxia.     

Ferritin, Lipid Peroxidation and Redox-Cycling Xenobiotics

A number of xenobiotics are toxic because they rcdox cycle and generate free radicals. Interaction with iron, either to produce reactive species such as the hydroxyl radical, or to promote lipid peroxidation, is an important factor in this toxicity. A potential biological source of iron is ferritin. The cytotoxic pyrimidines, dialuric acid, divicine and isouramil, readily release iron from ferritin and promote ferritin-dependent lipid peroxidation. Superoxide dismutase and GSH, which maintain the pyrimidines in their reduced form, enhance both iron release and lipid peroxidation. Microsomes plus NADPH can reduce a number of iron complexes, although not ferritin. Reduction of Adriamycin. paraquat or various quinones to their radicals by the microsomes enhances reduction of the iron complexes, and in some cases, enables iron release from ferritin. Adriamycin stimulates iron-dependent lipid peroxidation of the microsomes. Ferritin can provide the iron, and peroxidation is most pronounced at low PO₂. Compiexing agents that supress intraccllular iron reduction and lipid peroxidation may protect against the toxicity of Adriamycin.     

Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications

Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. We present studies on the production of a set of thermostable red- and green-emitting luciferase mutants with bioluminescent properties suitable for dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Starting with the luciferase variant Ser284Thr, we introduced the mutations Thr214Ala, Ala215Leu, Ile232Ala, Phe295Leu, and Glu354Lys to produce a new red-emitting enzyme with a bioluminescence maximum of 610 nm, narrow emission bandwidth, favorable kinetic properties, and excellent thermostability at 37 degrees C. By adding the same five changes to luciferase mutant Val241Ile/Gly246Ala/Phe250Ser, we produced a protein with an emission maximum of 546 nm, providing a set of thermostable enzymes whose bioluminescence maxima were separated by 64 nm. Model studies established that the luciferases could be detected at the attomole level and six orders of magnitude higher. In microplate luminometer format, mixtures containing 1.0 fmol total luciferase were quantified from measurements of simultaneously emitted red and green light. The results presented here provide evidence that it is feasible to monitor two distinct activities at 37 degrees C with these novel thermostable proteins.     

Fruit Flies in Biomedical Research

Many scientists complain that the current funding situation is dire. Indeed, there has been an overall decline in support in funding for research from the National Institutes of Health and the National Science Foundation. Within the Drosophila field, some of us question how long this funding crunch will last as it demotivates principal investigators and perhaps more importantly affects the long-term career choice of many young scientists. Yet numerous very interesting biological processes and avenues remain to be investigated in Drosophila, and probing questions can be answered fast and efficiently in flies to reveal new biological phenomena. Moreover, Drosophila is an excellent model organism for studies that have translational impact for genetic disease and for other medical implications such as vector-borne illnesses. We would like to promote a better collaboration between Drosophila geneticists/biologists and human geneticists/bioinformaticians/clinicians, as it would benefit both fields and significantly impact the research on human diseases.     

Tetravalent Vanadium Releases Ferritin Iron which Stimulates Vanadium-Dependent Lipid Peroxidation

The iron storage protein, ferritin, represents a possible source of iron for oxidative reactions in biological systems. It has been shown that superoxide and several xenobiotic free radicals can release iron from ferritin by a reductive mechanism. Tetravalent vanadium (vanadyl) reacts with oxygen to generate superoxide and pentavalent vanadium (vanadate). This led to the hypothesis that vanadyl causes the release of iron from ferritin. Therefore, the ability of vanadyl and vanadate to release iron from ferritin was investigated. Iron release was measured by monitoring the generation of the Fe²⁺-fcrrozine complex. It was found that vanadyl but not vanadate was able to mobilize ferritin iron in a concentration dependent fashion. Initial rates. and iron release over 30 minutes. were unaffected by the addition of superoxide dismutase. Glutathione or vanadate added in relative excess to the concentration of vanadyl, inhibited iron release up to 45%. Addition of ferritin at the concentration used for measuring iron release prevented vanddyl-induced NADH oxidation. Vanadyl promoted lipid peroxidation in phospholipid liposomes. Addition of ferritin to the system stimulated lipid peroxidation up to 50% above that with vanadyl alone. Fcrritin alone did not promote significant levels of lipid peroxidation.     

Functional Activity of Blood Polymorphonuclear Leukocytes as an Oxidative Stress Biomarker in Human Subjects

In the present work, we studied the role of polymorphonuclear leukocytes (PMN) in aged individuals and coronary heart disease (CHD)-bearing patients, two physiopathological processes associated with overproduction of reactive oxygen species (ROS). The effects of antioxidant supplementation on the functional activity of PMN from CHD patients were also determined. The function of PMNs was evaluated by measuring of phagocytosis, killing activity, and ROS production. Luminol amplified chemiluminescence (CL) was used to estimate ROS production by stimulated PMNs. Total cholesterol and the LDL-cholesterol fraction from CHD patients were found to be higher than those recommended, returning to normal levels after antioxidant therapy. PMN CL of CHD patients was found to be higher than the associated control groups. Antioxidant therapy administrated to CHD patients lead to an increase in the killing activity accompanied by a decrease in PMN CL of these subjects. The study also showed that killing activity of PMN from human subjects over 60 years was significantly lower than the activity measured in younger subjects. PMN CL produced after stimulation was found to be positively correlated with the increasing age of human subjects (r = .946, p < .01).     

Re-infestation of houses by Triatoma dimidiata after intra-domicile insecticide application in the Yucatán peninsula, Mexico

In most countries, Chagas disease transmission control remains based on domestic insecticide application. We thus evaluated the efficacy of intra-domicile cyfluthrin spraying for the control of Triatoma dimidiata, the only Chagas disease vector in the Yucatán peninsula, Mexico, and monitored potential re-infestation every 15 days for up to 9 months. We found that there was a re-infestation of houses by adult bugs starting 4 months after insecticide application, possibly from sylvatic/peridomicile areas. This points out the need to take into account the potential dispersal of sylvatic/peridomestic adult bugs into the domiciles as well as continuity action for an effective vector control.     

1,3-diaryl-propanes and propan-2-ols from Virola species

The trunk woods of two Amazonian Myristicaceae, Virola minutiflora and V. elongata, contain respectively 1-(2-hydroxy-4-methoxyphenyl)-3-(3,4-m     

Diterpenoids from Micrandropsis scleroxylon

The structure of micrandrol-C from Micrandropsis scleroxylon (Euphorbiaceae) is revised to 2,6-dihydroxy-7-methyl-1-methylthiophenanthrene. This and other micrandrols are probably diterpenes in view of their co-occurrence with micrandrol-D, the hemiketal of 1,2,3,4,9,10-hexahydro-6-hydroxy-4a-hydroxymethyl-1,1,7-trimethy-2-oxophenanthrene.