A dileucine signal situated in the C-terminal tail of the lysosomal membrane protein p40 is responsible for its targeting to lysosomes

The suicide inactivation mechanism of tyrosinase acting on its substrates has been studied. The kinetic analysis of the proposed mechanism during the transition phase provides explicit analytical expressions for the concentrations of o-quinone against time. The electronic, steric and hydrophobic effects of the substrates influence the enzymatic reaction, increasing the catalytic speed by three orders of magnitude and the inactivation by one order of magnitude. To explain the suicide inactivation, we propose a mechanism in which the enzymatic form E(ox) (oxy-tyrosinase) is responsible for such inactivation. A key step might be the transfer of the C-1 hydroxyl group proton to the peroxide, which would act as a general base. Another essential step might be the axial attack of the o-diphenol on the copper atom. The rate constant of this reaction would be directly related to the strength of the nucleophilic attack of the C-1 hydroxyl group, which depends on the chemical shift of the carbon C-1 (delta(1)) obtained by (13)C-NMR. Protonation of the peroxide would bring the copper atoms together and encourage the diaxial nucleophilic attack of the C-2 hydroxyl group, facilitating the co-planarity with the ring of the copper atoms and the concerted oxidation/reduction reaction, and giving rise to an o-quinone. The suicide inactivation would occur if the C-2 hydroxyl group transferred the proton to the protonated peroxide, which would again act as a general base. In this case, the co-planarity between the copper atom, the oxygen of the C-1 and the ring would only permit the oxidation/reduction reaction on one copper atom, giving rise to copper(0), hydrogen peroxide and an o-quinone, which would be released, thus inactivating the enzyme.     

Expression of peroxisome proliferator-activated receptors alpha and gamma in differentiating human colon carcinoma Caco-2 cells

The expression of peroxisome proliferator-activated receptors alpha (PPARalpha) and gamma (PPARgamma) was studied in the human adenocarcinoma Caco-2 cells induced to differentiate by long term culture (15 days). The differentiation of Caco-2 cells was attested by increases in the activities of sucrase-isomaltase and alkaline phosphatase (two brush border enzymes), fatty acyl-CoA oxidase (AOX) and catalase (two peroxisomal enzymes), by an elevation in the protein levels of villin (a brush border molecular marker), AOX, peroxisomal bifunctional enzyme (PBE), catalase and peroxisomal membrane protein of 70 kDa (PMP70). and by the appearance of peroxisomes. The expression of PPARalpha and PPARgamma was investigated by Western blotting, immunocytochemistry, Northern blotting and S1 nuclease protection assay during the differentiation of Caco-2 cells. The protein levels of PPARalpha, PPARgamma, and PPARgamma2 increased gradually during the time-course of Caco-2 cell differentiation. Immunocytochemistry revealed that PPARalpha and gamma were localized in cell nuclei. The PPARgamma1 protein was encoded by PPARgamma3 mRNA because no signal was obtained for PPARgamma1 mRNA using a specific probe in S1 nuclease protection assay. The amount of PPARgamma3 mRNA increased concomitantly to the resulting PPARgamma1 protein. On the other hand, the mRNA of PPARalpha and PPARgamma2 were not significantly changed, suggesting that the increase in their respective protein was due to an elevation of the translational rate. The role played by the PPAR subtypes in Caco-2 cell differentiation is discussed.     

A cluster of Ankyrin and Ankyrin-TPR repeat genes is associated with panicle branching diversity in rice

The number of grains per panicle is an important yield-related trait in cereals which depends in part on panicle branching complexity. One component of this complexity is the number of secondary branches per panicle. Previously, a GWAS site associated with secondary branch and spikelet numbers per panicle in rice was identified. Here we combined gene capture, bi-parental genetic population analysis, expression profiling and transgenic approaches in order to investigate the functional significance of a cluster of 6 ANK and ANK-TPR genes within the QTL. Four of the ANK and ANK-TPR genes present a differential expression associated with panicle secondary branch number in contrasted accessions. These differential expression patterns correlate in the different alleles of these genes with specific deletions of potential cis-regulatory sequences in their promoters. Two of these genes were confirmed through functional analysis as playing a role in the control of panicle architecture. Our findings indicate that secondary branching diversity in the rice panicle is governed in part by differentially expressed genes within this cluster encoding ANK and ANK-TPR domain proteins that may act as positive or negative regulators of panicle meristem’s identity transition from indeterminate to determinate state.     

Production and validation of CR-39-based dishes for alpha-particle radiobiological experiments

The study of radiobiological effects induced in vitro by low fluences of alpha particles would be significantly enhanced if the precise localization of each particle track in the cell monolayer was known. From this perspective, we developed a new method based on tailor-made UV-radiation-cured CR-39, the production of which is described. Its validation both as a petri dish and as solid-state nuclear track detectors is demonstrated. With respect to the demands on solid-state nuclear track detectors in such experiments, these biologically compatible detectors have a controlled micrometric thickness that allows them to be crossed by the alpha particles. In this study, we present a method for obtaining 10-mum-thick CR-39, its chemical characterization, and its properties as a solid-state nuclear track detector under the environmental conditions of radiobiological experiments. The experimental studies performed with 3.5 MeV alpha particles show that their transmitted energy is sufficient enough to cross the entire cellular volume. Under optimal conditions, etched tracks are clearly defined 2 h after etching. Moreover, the UV-radiation-cured CR-39 represents an essentially zero background that is due to the short time between the production and use of the polymer. Under a confocal microscope, this thin solid-state nuclear track detector allows the precise localization of the impact parameter at the subcellular level.     

Plant mitochondrial rps2 genes code for proteins with a C-terminal extension that is processed

A gene (rps2) coding for ribosomal protein S2 (RPS2) is present in the mitochondrial (mt) genome of several monocot plants, but absent from the mtDNA of dicots. Confirming that in dicot plants the corresponding gene has been transferred to the nucleus, a corresponding Arabidopsis thaliana nuclear gene was identified that codes for mitochondrial RPS2. As several yeast and mammalian genes coding for mt ribosomal proteins, the Arabidopsis RPS2 apparently has no N-terminal targeting sequence. In the maize mt genome, two rps2 genes were identified and both are transcribed, although at different levels. As in wheat and rice, the maize genes code for proteins with long C-terminal extensions, as compared to their bacterial counterparts. These extensions are not conserved in sequence. Using specific antibodies against one of the maize proteins we found that a large protein precursor is indeed synthesized, but it is apparently processed to give the mature RPS2 protein which is associated with the mitochondrial ribosome.     

A Detailed Study of the Amino Acids Produced from the Vacuum UV Irradiation of Interstellar Ice Analogs

We present a detailed analysis of the variety, quantity and distribution of the amino acids detected in organic residues after acid hydrolysis. Such organic residues are produced in the laboratory after the vacuum ultraviolet (VUV) irradiation of several astrophysically relevant ice mixtures containing H(2)O, CO, CO(2), CH(3)OH, CH(4) and NH(3) at low temperature (10-80 K), and subsequent warm-up to room temperature. We explore five experimental parameters: the irradiation time, the temperature, the ice mixture composition, the photon dose per molecule and the substrate for the ice deposition. The amino acids were detected and identified by ex-situ liquid chromatography analysis of the organic residues formed after warming the photolysed ices up to room temperature. This study shows that in all experiments amino acids are formed. Their total quantities and distribution depend slightly on the experimental parameters explored in the present work, the important requirement to form such molecules being that the starting ice mixtures must contain the four elements C, H, O and N. We also discuss the effects of the chemical treatment needed to detect and identify the amino acids in the organic residues. Finally, these results are compared with meteoritic amino acid data from the carbonaceous chondrite Murchison, and the formation processes of such compounds under astrophysical conditions are discussed.     

Bacterial community profiles on feathers during composting as determined by terminal restriction fragment length polymorphism analysis of 16S rDNA genes

Composting is one of the more economical and environmentally safe methods of recycling feather waste generated by the poultry industry, since 90% of the feather weight consists of crude keratin protein, and feathers contain 15% N. However, the keratin in waste feathers is resistant to biodegradation and may require the addition of bacterial inocula to enhance the degradation process during composting. Two keratin-degrading bacteria isolated from plumage of wild songbirds and identified as Bacillus licheneformis (OWU 1411T) and Streptomyces sp. (OWU 1441) were inoculated into poultry feather composts (1.13×10⁸ cfu g^–1 feathers) and co-composted with poultry litter and straw in 200-l compost vessels. Composting temperatures, as well as CO₂ and NH₃ evolution, were measured in these vessels to determine the effects of inoculation on the rate and extent of poultry feather decomposition during composting. Terminal restriction fragment length polymorphisms of 16S rRNA genes were used to follow changes in microbial community structure during composting. The results indicated that extensive carbon conversion occurred in both treatments (55.5 and 56.1%). The addition of the bacterial inocula did not enhance the rate of waste feather composting. The microbial community structure over time was very similar in inoculated and uninoculated waste feather composts.     

Advantages of distinguishing the active fraction in bacterioplankton assemblages: some examples

The difficulty of distinguishing between active and dormant or dead bacterial cells is an important problem for the aquatic microbiologist.Active cells can be detected under the microscope by the presence of an intact electron transport system able to reduce the colourless INT [2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride] to an optically dense intracellular deposit.An improvement of this method has been applied to Lake Geneva and to a fish pond in the Ivory Coast. The portion of INT-reducing bacterial cells ranged from 1 to 71%, depending on place, depth, season and time of the day. In all cases bacterial activity, determined by uptake of ³H Thymidine or ¹⁴C glucose, and frequency of dividing cells were better correlated with the number of INT-reducing cells than with the total number of cells. This means that counts of cells able to reduce INT have a better metabolic significance than total cell counts. Some examples are developed which show the advantages of applying this method in cases where it is useful to distinguish active cells in a bacterial assemblage.     

Rapid identification and typing of <Emphasis Type="Italic">Yersinia pestis</Emphasis> and other <Emphasis Type="Italic">Yersinia</Emphasis> species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry

Background  

Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species.