Thermodynamic and kinetic destabilization of triosephosphate isomerase resulting from the mutation of conserved and non-conserved cysteines

Several variants of Saccharomyces cerevisiae triosephosphate isomerase (yTIM) were studied to determine how mutations of conserved and non-conserved Cys residues affect the enzyme. Wild-type yTIM has two buried free cysteines: Cys 41 (non-conserved) and the invariant Cys 126. Single-site mutants, containing substitutions of these cysteines with Ala, Val, or Ser (the three most conservative changes for a buried Cys, according to substitution matrices), were examined for stability and enzymatic activity. Neither of the Cys residues was found to be essential for enzyme catalysis. Determination of the global stability of the mutants indicated that, regardless of which Cys was substituted, individual Cys→Ala and Cys→Val mutations, as well as the C41S substitution, all decrease the unfolding free energy of the dimeric protein by less than 23 kJ mol(-1) (at 37 °C, pH 7.4), as compared to the wild-type enzyme. In contrast, a substantially larger destabilization (37 kJ mol(-1)) was found in the C126S mutant. These results suggest that, with the exception of C126S, all of these mutations can be regarded as neutral (i.e., mutations that do not impair the reproductive success of the organism). Accordingly, Cys 126 has remained invariant across evolution because its neutral substitutions by Ala or Val would require a highly unlikely, concerted double mutation at any of the Cys codons. Furthermore, detrimental effects to a cell expressing the C126S TIM mutant more likely arise from the high unfolding rate of this enzyme.     

Nox2 B-loop peptide, Nox2ds, specifically inhibits the NADPH oxidase Nox2

In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2, and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In this study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B-loop of Nox2, would inhibit ROS production by the Nox2-, but not the Nox1- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1-, or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O₂^•−) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance confirmed that Nox2ds inhibits O₂^•− production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production by either Nox1- or Nox4-oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2-oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent.     

A novel clean-up method for urine analysis of low-molecular mass aldehydes by capillary electrophoresis with laser-induced fluorescence detection

A rapid clean-up method using ultra-filtration was developed to remove sample matrix in the determination of low-molecular mass aldehydes in human urine. The ensuing filtrate was derivatized with fluorescein 5-thiosemicarbazide and the labelled aldehydes determined by capillary zone electrophoresis with laser-induced fluorescence detection. Practical aspects related to the effect of the urine sample matrix on the label chemistry and the electrophoretic separation showed that the urine samples must be diluted 20-fold after their ultra-filtration. By using synthetic urine, linear ranges were established in the range of 15-5000 μg/l with limits of detection between 4.5 and 9 μg/l. The intra- and inter-assay precision (relative standard deviation, %) of the aldehydes ranged from 4.1% to 8.4% and 6.1%-9.6%, respectively, and the average specific uncertainty was 149±12 ng. The average recoveries performed on two levels by enriching synthetic urine samples ranged between 94% and 100%. Finally, the proposed method was applied to check low-molecular mass aldehydes in the human urine of a female volunteer to obtain information about the risk in her exposure to these chemicals in the workplace.     

Immunology and probiotic impact of the newborn and young children intestinal microflora

Human body has developed a holistic defence system, which mission is either to recognize and destroy the aggressive invaders or to evolve mechanisms permitting to minimize or restore the consequences of harmful actions. The host immune system keeps the capital role to preserve the microbial intestinal balance via the barrier effect. Specifically, pathogenic invaders such as, bacteria, parasites, viruses and other xenobiotic invaders are rejected out of the body via barriers formed by the skin, mucosa and intestinal flora. In case physical barriers are breached, the immune system with its many components comes into action in order to fence infection. The intestine itself is considered as an “active organ” due to its abundant bacterial flora and to its large metabolic activity. The variation among different species or even among different strains within a species reflects the complexity of the genetic polymorphism which regulates the immune system functions. Additionally factors such as, gender, particular habits, smoking, alcohol consumption, diet, religion, age, gender, precedent infections and vaccinations must be involved. Hormonal profile and stress seems to be associated to the integrity microbiota and inducing immune system alterations. Which bacterial species are needed for inducing a proper barrier effect is not known, but it is generally accepted that this barrier function can be strongly supported by providing benefic alimentary supplements called functional foods. In this vein it is stressed the fact that early intestinal colonization with organisms such as Lactobacilli and Bifidobacteria and possibly subsequent protection from many different types of diseases. Moreover, this benefic microflora dominated but Bifidobacteria and Lactobacilli support the concept of their ability to modify the gut microbiota by reducing the risk of cancer following their capacity to decrease β-glucoronidase and carcinogen levels. Because of their beneficial roles in the human gastrointestinal tract, LAB are referred to as “probiotics”, and efforts are underway to employ them in modern nutrition habits with so-called functional foods. Members of Lactobacillus and Bifidobacterium genera are normal residents of the microbiota in the human gastrointestinal tract, in which they developed soon after birth. But, whether such probiotic strains derived from the human gut should be commercially employed in the so-called functional foods is a matter of debate between scientists and the industrial world. Within a few hours from birth the newborn develops its normal bacterial flora. Indeed human milk frequently contains low amounts of non-pathogenic bacteria like Streptococcus, Micrococcus, Lactobacillus, Staphylococcus, Corynebacterium and Bifidobacterium. In general, bacteria start to appear in feces within a few hours after birth. Colonization by Bifidobacterium occurs generally within 4 days of life. Claims have been made for positive effects of Bifidobacterium on infant growth and health. The effect of certain bacteria having a benefic action on the intestinal ecosystem is largely discussed during the last years by many authors. Bifidobacterium is reported to be a probiotic bacterium, exercising a beneficial effect on the intestinal flora. An antagonism has been reported between B. bifidum and C. perfringens in the intestine of newborns delivered by cesarian section. The aim of the probiotic approach is to repair the deficiencies in the gut flora and restore the protective effect. However, the possible ways in which the gut microbiota is being influenced by probiotics is yet unknown.     

Brucella abortus inhibits IFN-γ-induced FcγRI expression and FcγRI-restricted phagocytosis via toll-like receptor 2 on human monocytes/macrophages

The strategies that allow Brucella abortus to persist for years inside macrophages subverting host immune responses are not completely understood. Immunity against this bacterium relies on the capacity of IFN-γ to activate macrophages, endowing them with the ability to destroy intracellular bacteria. We report here that infection with B. abortus down-modulates the expression of the type I receptor for the Fc portion of IgG (FcγRI, CD64) and FcγRI-restricted phagocytosis regulated by IFN-γ in human monocytes/macrophages. Both phenomena were not dependent on bacterial viability, since they were also induced by heat-killed B. abortus (HKBA), suggesting that they were elicited by a structural bacterial component. Accordingly, a prototypical B. abortus lipoprotein (L-Omp19), but not its unlipidated form, inhibited both CD64 expression and FcγRI-restricted phagocytosis regulated by IFN-γ. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited CD64 expression, indicating that any Brucella lipoprotein could down-modulate CD64 expression and FcγRI-restricted phagocytosis. Pre-incubation of monocytes/macrophages with anti-TLR2 mAb blocked the inhibition of the CD64 expression mediated by HKBA and L-Omp19. These results, together with our previous observations establish that B. abortus utilizes its lipoproteins to inhibit the monocytes/macrophages activation mediated by IFN-γ and to subvert host immunonological responses.     

A kaleidoscopic view of the Arabidopsis core cell cycle interactome

Although protein-protein interaction (PPI) networks have been shown to offer a systems-wide view of cellular processes, only a few plant PPI maps are available. Recently, the core cell cycle of Arabidopsis thaliana has been analyzed by three independent PPI technologies, including yeast two-hybrid systems, bimolecular fluorescence complementation and tandem affinity purification. Here, we merge the three interactomes with literature-curated and computationally predicted interactions, paving the way for a comprehensive picture of the plant core cell cycle machinery. Platform-specific interactions unveil the strengths and weaknesses of each detection method and give insights into the nature of the interactions among cell cycle proteins. Moreover, comparison of the obtained data reveals that a complete interactome can only be obtained when multiple techniques are applied in parallel.     

A genome-wide analysis of FRT-like sequences in the human genome

Efficient and precise genome manipulations can be achieved by the Flp/FRT system of site-specific DNA recombination. Applications of this system are limited, however, to cases when target sites for Flp recombinase, FRT sites, are pre-introduced into a genome locale of interest. To expand use of the Flp/FRT system in genome engineering, variants of Flp recombinase can be evolved to recognize pre-existing genomic sequences that resemble FRT and thus can serve as recombination sites. To understand the distribution and sequence properties of genomic FRT-like sites, we performed a genome-wide analysis of FRT-like sites in the human genome using the experimentally-derived parameters. Out of 642,151 identified FRT-like sequences, 581,157 sequences were unique and 12,452 sequences had at least one exact duplicate. Duplicated FRT-like sequences are located mostly within LINE1, but also within LTRs of endogenous retroviruses, Alu repeats and other repetitive DNA sequences. The unique FRT-like sequences were classified based on the number of matches to FRT within the first four proximal bases pairs of the Flp binding elements of FRT and the nature of mismatched base pairs in the same region. The data obtained will be useful for the emerging field of genome engineering.     

Molecular phylogeny and paclitaxel screening of fungal endophytes from Taxus globosa

We studied the endophytic mycoflora associated with Taxus globosa, the Mexican yew. The study localities; Las Avispas (LA), San Gaspar (SG), and La Mina (LM) were three segments of cloud forest within the range of Sierra Gorda Biosphere Reserve, México. Overall, 245 endophytes were isolated and 105 representative Ascomycota (morphotaxons) were chosen for phylogenetic and genotypic characterization. Maximum likelihood analyses of large subunit of ribosomal RNA (LSU) rDNA showed well-supported clades of Dothideomycetes, Eurotiomycetes, Leotiomycetes, Pezizomycetes, and Sordariomycetes. Analyses of ITS rDNA groups showed 57 genotypes (95% sequence similarity), in general consistent with the phylogenetically delimitated taxa based on LSU rDNA sequences. The endophyte diversity measured by Fisher's α, Shanonn, and Simpson indices was ca. three-fold and ca. two-fold greater in LM than in LA and SG respectively. A screening for paclitaxel using a competitive inhibition enzyme immunoassay showed 16 positive isolates producing between 65 and 250 ng l(-1). The isolates included Acremonium, Botryosphaeria, Fusarium, Gyromitra, Nigrospora, Penicillium, three novel Pleosporales, and Xylaria.     

α-Synuclein expression selectively affects tumorigenesis in mice modeling Parkinson's disease

Alpha Synuclein (α-Syn) is a protein implicated in mechanisms of neuronal degeneration in Parkinson's disease (PD). α-Syn is primarily a neuronal protein, however, its expression is found in various tumors including ovarian, colorectal and melanoma tumors. It has been hypothesized that neurodegeneration may share common mechanisms with oncogenesis. We tested whether α-Syn expression affects tumorigenesis of three types of tumors. Specifically, B16 melanoma, E0771 mammary gland adenocarcinoma and D122 Lewis lung carcinoma. For this aim, we utilized transgenic mice expression the human A53T α-Syn form. We found that the in vivo growth of B16 and E0771 but not D122 was enhanced in the A53T α-Syn mice. The effect on tumorigenesis was not detected in age-matched APP/PS1 mice, modeling Alzheimer's disease (AD), suggesting a specific effect for α-Syn-dependent neurodegeneration. Importantly, transgenic α-Syn expression was detected within the three tumor types. We further show uptake of exogenously added, purified α-Syn, by the cultured tumor cells. In accord, with the affected tumorigenesis in the young A53T α-Syn mice, over-expression of α-Syn in cultured B16 and E0771 cells enhanced proliferation, however, had no effect on the proliferation of D122 cells. Based on these results, we suggest that certain forms of α-Syn may selectively accelerate cellular mechanisms leading to cancer.     

Time-course development of the Cd<Superscript>2+</Superscript> hyper-accumulating phenotype in <Emphasis Type="Italic">Euglena gracilis</Emphasis>

To determine the onset of the Cd²⁺-hyperaccumulating phenotype in Euglena gracilis, induced by Hg²⁺ pretreatment (Avilés et al. in Arch Microbiol 180:1–10, 2003), the changes in cellular growth, Cd²⁺ uptake, and intracellular contents of sulfide, cysteine, γ-glutamylcysteine, glutathione and phytochelatins during the progress of the culture were analyzed. In cells exposed to 0.2 mM CdCl₂, the Cd²⁺-hyperaccumulating phenotype was apparent only after 48 h of culture, as indicated by the significant increase in cell growth and higher internal contents of sulfide and thiol-compounds, along with a higher γ-glutamylcysteine synthetase activity. However, the stiochiometry of thiol-compounds/Cd²⁺ accumulated was similar for both control and Hg²⁺-pretreated cells. Moreover, the value for this ratio was 2.1 or lower after 48-h culture, which does not suffice to fully inactivate Cd²⁺. It is concluded that, although the glutathione and phytochelatin synthesis pathway is involved in the development of the Cd²⁺-hyperaccumulating phenotype in E. gracilis, apparently other pathways and sub-cellular mechanisms are also involved. These may be an increase in other Cd²⁺ chelating molecules such as di- and tricarboxylic acids, phosphate and polyphosphates, as well as Cd²⁺ compartmentation into organelles. César Avilés: In memoriam.