Permeabilization of yeast for in situ determination of α-glucosidase

A quantitative in situ assay of yeast α-glucosidase involving permeabilization of the cells by freezing and thawing is described. The assay was applied to different strains in different physiological states and was shown to give results comparable to those obtained with total cell homogenates. The primary advantage of the in situ assay was the possibility of analyzing a large number of samples from the same culture during a growth curve using a very reduced cell mass.     

Influence of benzo[a]pyrene diol epoxide chirality on solution conformations of DNA covalent adducts: the (-)-trans-anti-[BP]G.C adduct structure and comparison with the (+)-trans-anti-[BP]G.C enantiomer.

Benzo[a]pyrene (BP) is an environmental genotoxin, which, following metabolic activation to 7,8-diol 9,10-epoxide (BPDE) derivatives, forms covalent adducts with cellular DNA. A major fraction of adducts are derived from the binding of N2 of guanine to the C10 position of BPDE. The mutagenic and carcinogenic potentials of these adducts are strongly dependent on the chirality at the four asymmetric benzylic carbon atoms. We report below on the combined NMR-energy minimization refinement characterization of the solution conformation of (-)-trans-anti-[BP]G positioned opposite C and flanked by G.C base pairs in the d(C1-C2-A3-T4-C5-[BP]G6-C7-T8-A9-C10-C11).d(G12-G13-T14++ +-A15-G16-C17- G18-A19-T20-G21-G22) duplex. Two-dimensional NMR techniques were applied to assign the exchangeable and non-exchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid in the modified duplex. These results establish Watson-Crick base pair alignment at the [BP]G6.C17 modification site, as well as the flanking C5.G18 and C7.G16 pairs within a regular right-handed helix. The solution structure of the (-)-trans-anti-[BP]G.C 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOE buildup curves as constraints in energy minimization computations. The BP ring spans both strands of the duplex in the minor groove and is directed toward the 3'-end of the modified strand in the refined structure. One face of the BP ring of [BP]G6 stacks over the C17 residue across from it on the partner strand while the other face is exposed to solvent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exploring the Factor Structure of Neurocognitive Measures in Older Individuals

Here we focus on factor analysis from a best practices point of view, by investigating the factor structure of neuropsychological tests and using the results obtained to illustrate on choosing a reasonable solution. The sample (n=1051 individuals) was randomly divided into two groups: one for exploratory factor analysis (EFA) and principal component analysis (PCA), to investigate the number of factors underlying the neurocognitive variables; the second to test the “best fit” model via confirmatory factor analysis (CFA). For the exploratory step, three extraction (maximum likelihood, principal axis factoring and principal components) and two rotation (orthogonal and oblique) methods were used. The analysis methodology allowed exploring how different cognitive/psychological tests correlated/discriminated between dimensions, indicating that to capture latent structures in similar sample sizes and measures, with approximately normal data distribution, reflective models with oblimin rotation might prove the most adequate.     

Use of prey and non-prey food by the ladybird beetle Eriopis connexa (Coleoptera: Coccinellidae) under laboratory-rearing conditions

Prey for predators can fluctuate in abundance and in quality over time requiring predator strategies to cope with food shortage. Coccinellinae are often associated with sap-sucking pests that exhibit high population unpredictability such as aphids and psyllids. Eriopis connexa (Germar) (Coleoptera: Coccinellidae) is a predator with potential for biological control, especially a well-studied population which is resistant to pyrethroids used to control insect defoliators. Both larvae and adult E. connexa were provided ad libitum prey and non-prey foods (pollen and honey water solution) at increasing intervals from 1 to 10 days. Neonate larvae of E. connexa required eating prey daily to develop into adults. However, non-prey food such as honey water solution did prolong larval and adult survival but neither fulfilled larval development nor adult reproduction. Honey water solution promoted 100% adult survival up to 25 days in the adult stage without prey with oviposition returning after daily feeding on prey. Females subjected to increased feeding intervals over four days reduced oviposition and lived longer, but 10-day feeding intervals correlated with risk to adult survival. These results indicate the importance of non-food sources in E. connexa maintenance and the ability of larvae and adult females to compensate for prey scarcity.     

Geographic differences in the allele frequencies of the human Y-linked tetranucleotide polymorphism DYS19

We have studied the allele frequency distribution of the microsatellite locus DYS 19 in several populations with different geographical origins worldwide. Three new alleles were found. In addition, remarkable geographic and ethnic differences were observed in the allele frequency profiles and DNA marker (gene) diversity among populations and major ethnic groups. Amerindians showed an overwhelming predominance of the A allele, while in Caucasians the B allele was modal, and in Greater Asians and Africans allele C became predominant. Even within these geographic regions there were significant gradients, as exemplified by the decreasing frequency profile of the B allele from Great Britain over Germany to Slovakia. Thus, DYS 19 emerges as a useful tool for studying the structure and dynamics of human populations.     

Correlation between cell survival,clonogenic activity and micronuclei induction in DMBA-OC-1R cells treated with immunospecific albumin microspheres containing cisplatin and 5-fluorouracil

An ideal chemotherapeutic strategy would be to deliver a high concentration of drug that would be released in sustained small amounts from targeted microspheres to effectively kill only the tumour cells and thus reduce toxicity to normal tissue. Clonogenic and cell survival growth curve assays, as well as the micronucleus assay, were used to determine the feasibility of employing targeted immunomicrospheres in the treatment of cancer. Cells of a rodent ovarian carcinoma cell line, were exposed to cisplatin and 5-fluorouracil, either as free drug or encapsulated in albumin microspheres that were either conjugated to monoclonal antibodies or not. In cell survival growth curve assays, cell survival was reduced to 1.2% of the control when cells were treated with drug-containing immunomicrospheres. 3.2-fold more micronuclei were found in those cells that had been exposed to the drugs in immunomicrospheres than in those subjected to untargeted microspheres. All three assays demonstrated that the targeted immunomicrospheres were more effective in delivering cisplatin and 5-fluorouracil directly to the cells than the unconjugated microspheres, thus suggesting that targeted chemotherapy might be a more effective option in the treatment of cancer.     

Inhibitory actions of eugenol on rat isolated ileum

The effects of eugenol (1-2000 microM) on rat isolated ileum were studied. Eugenol relaxed the basal tonus (IC50 83 microM) and the ileum precontracted with 60 mM KCl (IC50 162 microM), an action unaltered by 0.5 microM tetrodotoxin, 0.2 mM N(G)-nitro-L-arginine methyl ester, 0.5 mM hexamethonium, and 1 microM indomethacin. Eugenol did not alter the resting transmembrane potential (Em) of the longitudinal muscle layer under normal conditions (5.0 mM K+) or in depolarised tissues. Eugenol reversibly inhibited contractions induced by submaximal concentrations of acetylcholine (ACh) and K+ (40 mM) with IC50 values of approximately 228 and 237 microM, respectively. Eugenol blocked the component of ACh-induced contraction obtained in Ca(2+)-free solution (0.2 mM EGTA) or in the presence of nifedipine (1 microM). Our results suggest that eugenol induces relaxation of rat ileum by a direct action on smooth muscle via a mechanism largely independent of alterations of Em and extracellular Ca2+ influx.     

Thermococcus kodakarensis Mutants Deficient in Di-myo-Inositol Phosphate Use Aspartate To Cope with Heat Stress

Many of the marine microorganisms which are adapted to grow at temperatures above 80°C accumulate di-myo-inositol phosphate (DIP) in response to heat stress. This led to the hypothesis that the solute plays a role in thermoprotection, but there is a lack of definitive experimental evidence. Mutant strains of Thermococcus kodakarensis (formerly Thermococcus kodakaraensis), manipulated in their ability to synthesize DIP, were constructed and used to investigate the involvement of DIP in thermoadaptation of this archaeon. The solute pool of the parental strain comprised DIP, aspartate, and α-glutamate. Under heat stress the level of DIP increased 20-fold compared to optimal conditions, whereas the pool of aspartate increased 4.3-fold in response to osmotic stress. Deleting the gene encoding the key enzyme in DIP synthesis, CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, abolished DIP synthesis. Conversely, overexpression of the same gene resulted in a mutant with restored ability to synthesize DIP. Despite the absence of DIP in the deletion mutant, this strain exhibited growth parameters similar to those of the parental strain, both at optimal (85°C) and supraoptimal (93.7°C) temperatures for growth. Analysis of the respective solute pools showed that DIP was replaced by aspartate. We conclude that DIP is part of the strategy used by T. kodakarensis to cope with heat stress, and aspartate can be used as an alternative solute of similar efficacy. This is the first study using mutants to demonstrate the involvement of compatible solutes in the thermoadaptation of (hyper)thermophilic organisms.Hyperthermophilic bacteria and archaea isolated from saline environments accumulate unusual organic solutes in response to osmotic as well as heat stress. Mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, mannosyl-di-myo-inositol phosphate (DIP), diglycerol phosphate, and glycero-phospho-myo-inositol are examples of compatible solutes highly restricted to thermophiles and hyperthermophiles (27, 31). Our team has, over several years, examined the compatible solute composition in a large number of hyperthermophiles and their accumulation under stressful conditions. The data reveal a trend toward specialization of roles in thermoadaptation and osmoadaptation. Indeed, mannosylglycerate and diglycerol phosphate typically accumulate in response to increased NaCl concentration in the growth medium, whereas the levels of DIP and derivatives consistently increase at supraoptimal growth temperatures (11, 16, 17, 27, 31).DIP is widespread among extreme archaeal hyperthermophiles, such as Methanotorris igneus, Aeropyrum pernix, Stetteria hydrogenophila, Pyrodictium occultum, Pyrolobus fumarii, Archaeoglobus spp., and all the members of the Thermococcales examined thus far, except Palaeococcus ferrophilus (5, 7, 11, 13, 16, 18, 31). This organic solute has also been found in representatives of the two hyperthermophilic bacterial genera, Aquifex and Thermotoga (14, 17, 22).The specific chemical nature of solutes encountered in hyperthermophiles, together with their accumulation in response to elevated temperatures, led to the hypothesis that they play a role in thermoprotection of cellular components in vivo. However, there is a lack of convincing experimental evidence, such as that obtained with suitable mutants. Progress toward understanding the physiological functions of these solutes critically depends on two conditions: the availability of genetic tools to manipulate hyperthermophilic organisms and knowledge about the genes and enzymes implicated in the synthesis of these unusual solutes.Thermococcus kodakarensis (formerly Thermococcus kodakaraensis) is a member of the order Thermococcales with an optimal growth temperature of 85°C and is able to grow at temperatures up to 94°C in batch cultures. The NaCl concentration for optimal growth matches that of seawater (1). T. kodakarensis is the only marine hyperthermophile for which a number of genetic tools have been developed, including Escherichia coli-T. kodakarensis shuttle vectors and a reliable gene disruption system (19, 29, 32, 34). The genome of T. kodakarensis possesses a gene encoding CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase (IPCT/DIPPS), a key enzyme in DIP synthesis (2, 25, 26). This enzyme catalyzes the synthesis of CDP-inositol from CTP and inositol-1-phosphate as well as the transfer of the inositol group from CDP-inositol to a second molecule of inositol-1-phosphate to yield a phosphorylated form of DIP (2). Therefore, we set out to investigate whether DIP was involved in thermoadaptation of T. kodakarensis. A DIP-deficient mutant was constructed by deleting the IPCT/DIPPS gene; subsequently, this strain was complemented in this activity by inserting the gene under the control of a constitutive promoter, resulting in a construct with restored ability to synthesize DIP. The effects of heat and osmotic stress on the pattern of solute accumulation and on the growth profiles of the two mutants provided evidence for the involvement of DIP in thermoprotection.