Role of antioxidants in the survival of normal and vitiliginous avian melanocytes.

Mutant feather melanocytes from Barred Plymouth Rock (BPR) and White Leghorn (WL) chickens are currently being used as avian models of vitiligo. Feather melanocytes in BPR and WL chickens die prematurely in vivo due to low (50-66%) antioxidant glutathione and superoxide dismutase levels when compared to the wild type Jungle Fowl (JF) melanocytes. Excess superoxide anions, generated by xanthine:xanthine oxidase (X:XO), caused a 15-20% increase in mortality after 1 and 2 hrs. in all three genotypes of in vitro melanocytes as compared to control values that received no X:XO. Overall, the JF wild type melanocytes had the lowest mortality rate, WL melanocytes had the highest mortality rate and the BPR melanocytes had an intermediate mortality rate. Superoxide anion and hydroxyl radical production in the WL feather were double the production in the JF wild type feather. The production of reactive oxygen species in BPR was intermediate to the other two genotypes. In an effort to mimic the low antioxidant levels of the BPR and WL feathers in the JF feather, JF in vitro feather melanocytes were treated with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor. With BSO added to the medium, the JF mortality rates increased by 20-25%, reaching the mortality levels of the mutant BPR melanocytes. The addition of iron to the JF melanocyte X:XO medium increased their mortality rate by 20%, probably via the Fenton reaction. Thus, antioxidants play an extremely important role in both the viability of normal avian melanocytes and the premature death of the vitiliginous avian melanocytes. A working hypothesis, supported in part by the current results, is that the premature death of the mutant melanocytes could be precipitated in the poorly vascularized feather by low antioxidant protection due to both low turnover of tissue fluids which contain SOD and to genetically determined low levels of internal antioxidant protection in these melanocytes. This same mechanistic hypothesis could apply as "a" cause of premature melanocyte cell death in human vitiligo wherein the vitiliginous melanocytes may have a genetic defect in their antioxidant protection system and blood flow to an area may be restricted.     

A codon window in mRNA downstream of the initiation codon where NGG codons give strongly reduced gene expression in Escherichia coli

The influences on gene expression by codons at positions +2, +3, +5 and +7 downstream of the initiation codon have been compared. Most of the +2 codons that are known to give low gene expression are associated with a higher expression if placed at the later positions. The NGG codons AGG, CGG, UGG and GGG, but not GGN or GNG (where N is non-G), are unique since they are associated with a very low gene expression also if located at positions +2, +3 and +5. All codons, including NGG, give a normal gene expression if placed at positions +7. The negative effect by the NGG codons is true for both the lacZ and 3A′ model genes. The low expression is suggested to originate at the translational level, although it is not the result of mRNA secondary structure or a lowered intracellular mRNA pool.     

IL-10 inhibits vascular smooth muscle cell activation in vitro and in vivo

The anti-inflammatory cytokine IL-10 inhibits intimal hyperplasia after stent implantation via a powerful inactivation of monocytes. We tested the hypothesis that IL-10 may also inhibit vascular smooth muscle cell (SMC) activation via the inhibition of the NF-kappaB/I-kappaB system. The IL-10 receptor was detected in rat SMCs in vitro and in vivo. In LPS-stimulated rat SMCs, 1 ng/ml recombinant murine IL-10 (mIL-10) reduced I-kappaBalpha and I-kappaBbeta degradation, NF-kappaB activation, as well as the expression of the NF-kappaB-dependent gene IL-6 by 32%, 31%, 75%, and 19%, respectively (P < 0.05 for all). Similar results were obtained in vivo 6 h and 4 days after balloon abrasion of the rat aorta, a model in which intimal hyperplasia results essentially from SMC activation. Moreover, mIL-10 reduced SMC proliferation and migration in vitro (by 60% for both, P < 0.0001), resulting in reduced SMC proliferation and intimal growth 14 days after balloon abrasion of the rat aorta (by 76% and 75%, respectively; P < 0.005). In conclusion, mIL-10 has a direct inhibitory effect on SMCs in vitro and in vivo. This effect is mediated in part by NF-kappaB inactivation and may participate in the overall protective effect of IL-10 on postangioplasty restenosis.     

Interaction between clonal plasma cells and the immune system in plasma cell dyscrasias

The term "monoclonal gammopathy" (MG) includes a group of clonal plasma cell disorders, which show heterogeneous clinical behavior. While multiple myeloma (MM) and plasma cell leukemia (PCL) are incurable malignant diseases, most patients with MG of undetermined significance (MGUS) show an indolent/benign clinical course. Evidence has accumulated which supports the role of the bone marrow microenvironment in MG. Accordingly, the survival, drug-resistance and proliferation of MM cells have been shown to be largely dependent on a supportive microenvironment. Among the different environment-associated parameters, those related to the status/activity of the immune system are particularly relevant. This review focuses on the different ways clonal plasma cells (PC) interact with the immune system in different models of MG, to characterize crucial events in the development and progression of MG. These advances may support the design of novel therapeutic approaches in patients with MG.     

Comparison of two alternative dominant selectable markers for wine yeast transformation

Genetic improvement of industrial yeast strains is restricted by the availability of selectable transformation markers. Antibiotic resistance markers have to be avoided for public health reasons, while auxotrophy markers are generally not useful for wine yeast strain transformation because most industrial Saccharomyces cerevisiae strains are prototrophic. For this work, we performed a comparative study of the usefulness of two alternative dominant selectable markers in both episomic and centromeric plasmids. Even though the selection for sulfite resistance conferred by FZF1-4 resulted in a larger number of transformants for a laboratory strain, the p-fluoro-DL-phenylalanine resistance conferred by ARO4-OFP resulted in a more suitable selection marker for all industrial strains tested. Both episomic and centromeric constructions carrying this marker resulted in transformation frequencies close to or above 10(3) transformants per microg of DNA for the three wine yeast strains tested.     

Somatic embryogenesis and plant regeneration through leaf culture in <Emphasis Type="Italic">Arachis glabrata (Leguminosae)</Emphasis>

Plants of two accessions of Arachis glabrata were regenerated via somatic embryogenesis. Embryogenic calli were initiated from leaflet explants on Murashige and Skoog medium supplemented with picloram alone or picloram in combination with 6-benzylaminopurine. Leaflets of accession A6138 induced the highest percentage of somatic embryos in media composed of 10 mg dm⁻³ and 15 mg dm⁻³ picloram. In contrast, 5 mg dm⁻³ picloram with 0.1 mg dm⁻³ 6-benzylaminopurine was one of the most effective combinations in accession AF385. MS medium supplemented with 2 g dm⁻³ activated charcoal (AC) used for 30 days was the most effective for embryo maturation. After 20 days of culture on MS medium devoid of growth regulators, 6 % of embryos converted into plantlets in accession A6138.     

Contribution of noncentrosomal microtubules to spindle assembly in Drosophila spermatocytes

Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.     

Assessment of potential (inhalation and dermal) and actual exposure to acetamiprid by greenhouse applicators using liquid chromatography-tandem mass spectrometry

New analytical methods based on liquid chromatography with electrospray tandem mass spectrometry (LC-MS/MS) have been developed and validated for assessing the exposure of greenhouse workers to acetamiprid. Both ambient (potential inhalation and dermal exposure) and internal dose (biological monitoring of urine samples) measurements were carried out. Potential inhalation exposure was assessed using Chromosorb 102 cartridges connected to air personal samplers. Potential dermal exposure was estimated by using whole body dosimetry. The measurement of actual exposure was done by analyzing the parent compound in urine samples of the applicators, after a solid-phase extraction (SPE) step. The methods showed a good accuracy (72-92%), precision (2-13%) and lower limits (few microg l(-1)). The validated approaches have been applied to assess potential and actual exposure of agricultural workers spraying acetamiprid in greenhouses. The results shown the need to wear personal protective equipment (suits) in order to reduce the absorbed dose of acetamiprid.     

Enantiospecific syntheses of C(5)-functionalized precursors of artemisinin derivatives

Enantiomerically pure compounds as precursors for the synthesis of hydroxylated derivatives of artemisinin/arteether have been prepared from (+)-(S)-carvone and (+)-car-2-ene.     

Effects of reducing agents on glutathione metabolism and the function of carotid body chemoreceptor cells

Two current hypotheses of O2 sensing in the carotid body (CB) chemoreceptors suggest participation of oxygen reactive (ROS) species, but they are mechanistically opposed. One postulates that hypoxia decreases ROS levels; the other that hypoxia increases them. Yet, both propose that the ensuing alteration in the cellular redox environment is the key signal triggering hypoxic chemoreception. Since the glutathione redox pair is the main cellular buffer for ROS and the main determinant of the general redox environment of the cells, a way to test whether ROS participate in chemoreception is to determine glutathione levels and to correlate them with the activity of CB chemoreceptor cells. We found that hypoxia does not alter the glutathione reduction potential but that it activates chemoreceptor cell neurosecretion. Incubation of tissues with reduced glutathione increases the glutathione-reducing potential but does not activate chemoreceptor cells in normoxia nor does it modify hypoxic activation. Like reduced glutathione, N-acetylcysteine promoted a general reducing environment in the cells without alteration of chemoreceptor cell activity. N-(mercaptopropionyl)-glycine, like the two previous agents, increases the reduction potential of glutathione. In contrast, the compound activated chemoreceptor cells in normoxia, promoting a dose- and Ca(2+)-dependent neurosecretion and a potentiation of the hypoxic responses. The existence of multiple relationships between glutathione reduction potential in the cells and their activity indicates that the general cellular redox environment is not a factor determining chemoreceptor cell activation. It cannot be excluded that the local redox environments of restricted microdomain(s) in the cells with specific regulating mechanisms are important signals for chemoreceptor cell activity.