Hydrogel substrate amendment alleviates drought effects on young citrus plants

Water deficits affect citrus physiology, yield, fruit size and quality. Citrus can respond to drought stress conditions through endogenous hormonal regulation of water status and leaf abscission. In this work, we assayed the efficiency of an amendment to soilless media in delaying the drought stress effect in young citrus seedlings and trees. Substrate amendment promoted plant survival of citrus seedlings subjected to several cycles of drought stress and rehydration. In budded trees, the amendment increased substrate water content, leaf water potential, leaf number, root biomass, CO₂ assimilation and stomatal conductance over that of control plants growing in non-amended substrates. We conclude that the substrate amendment reduced the damaging effects of drought stress in citrus plants. The longer survival of seedlings in the amended treatment together with the reduction in leaf abscission and the improvement of physiological parameters, can account for a higher vigour of citrus grown under water stress conditions.     

Male fertility in natural populations of red deer is determined by sperm velocity and the proportion of normal spermatozoa

Male reproductive success is determined by the ability of males to gain sexual access to females and by their ability to fertilize ova. Among polygynous mammals, males differ markedly in their reproductive success, and a great deal of effort has been made to understand how selective forces have shaped traits that enhance male competitiveness both before and after copulation (i.e., sperm competition). However, the possibility that males also may differ in their fertility has been ignored under the assumption that male infertility is rare in natural populations because selection against it is likely to be strong. In the present study, we examined which semen traits correlate with male fertility in natural populations of Iberian red deer (Cervus elaphus hispanicus). We found no trade-offs between semen traits. Our analyses revealed strong associations between sperm production and sperm swimming velocity, sperm motility and proportion of morphologically normal spermatozoa, and sperm viability and acrosome integrity. These last two variables had the lowest coefficients of variation, suggesting that these traits have stabilized at high values and are unlikely to be related to fitness. In a fertility trial, our results show a large degree of variation in male fertility, and differences in fertility were determined mainly by sperm swimming velocity and by the proportion of morphologically normal sperm. We conclude that male fertility varies substantially in natural populations of Iberian red deer and that, when sperm numbers are equal, it is determined mainly by sperm swimming velocity and sperm morphology.     

Application of the yeast Yarrowia lipolytica as a model to analyse human pathogenic mutations in mitochondrial complex I (NADH:ubiquinone oxidoreductase)

While diagnosis and genetic analysis of mitochondrial disorders has made remarkable progress, we still do not understand how given molecular defects are correlated to specific patterns of symptoms and their severity. Towards resolving this dilemma for the largest and therefore most affected respiratory chain enzyme, we have established the yeast Yarrowia lipolytica as a eucaryotic model system to analyse respiratory chain complex I. For in vivo analysis, eYFP protein was attached to the 30-kDa subunit to visualize complex I and mitochondria. Deletions strains for nuclear coded subunits allow the reconstruction of patient alleles by site-directed mutagenesis and plasmid complementation. In most of the pathogenic mutations analysed so far, decreased catalytic activities, elevated K(M) values, and/or elevated I(50) values for quinone-analogous inhibitors were observed, providing plausible clues on the pathogenic process at the molecular level. Leigh mutations in the 49-kDa and PSST homologous subunits are found in regions that are at the boundaries of the ubiquinone-reducing catalytic core. This supports the proposed structural model and at the same time identifies novel domains critical for catalysis. Thus, Y. lipolytica is a useful lower eucaryotic model that will help to understand how pathogenic mutations in complex I interfere with enzyme function.     

Immobilization of enzymes in microtiter plate scale

Different immobilization methods were adapted to the 96-well microtiter plate scale using esterases as model enzymes. The methods tested were based on adsorption, coprecipitation, aggregation and covalent bonding. The protein covered microcrystals proved to be the best method in terms of yield and expressed activity for the test reaction, which was the alcoholysis of p-nitrophenyl acetate with 1-propanol under anhydrous conditions.     

Mammalian Mitochondrial DNA Replication Intermediates Are Essentially Duplex but Contain Extensive Tracts of RNA/DNA Hybrid

We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mitochondrial DNA replication intermediates are essentially duplex throughout their length but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mitochondrial DNA replication involving RNA incorporation throughout the lagging strand.     

MAP4K3 regulates body size and metabolism in Drosophila

The TOR pathway mediates nutrient-responsive regulation of cell growth and metabolism in animals. TOR Complex 1 activity depends, amongst other things, on amino acid availability. MAP4K3 was recently implicated in amino-acid signaling in cell culture. We report here the physiological characterization of MAP4K3 mutant flies. Flies lacking MAP4K3 have reduced TORC1 activity detected by phosphorylation of S6K and 4EBP. Furthermore MAP4K3 mutants display phenotypes characteristic of low TORC1 activity and low nutrient availability, such as reduced growth rate, small body size, and low lipid reserves. The differences between control and MAP4K3 mutant animals diminish when animals are reared in low-nutrient conditions, suggesting that the ability of TOR to sense amino acids is most important when nutrients are abundant. Lastly, we show physical interaction between MAP4K3 and the Rag GTPases raising the possibility they might be acting in one signaling pathway.     

Trypanosoma cruzi strains isolated from human, vector, and animal reservoir in the same endemic region in Mexico and typed as T. cruzi I, discrete typing unit 1 exhibit considerable biological diversity

In this study, three strains of Trypanosoma cruzi were isolated at the same time and in the same endemic region in Mexico from a human patient with chronic chagasic cardiomyopathy (RyC-H); vector (Triatoma barberi) (RyC-V); and rodent reservoir (Peromyscus peromyscus) (RyC-R). The three strains were characterized by multilocus enzyme electrophoresis, random amplified polymorphic DNA, and by pathological profiles in experimental animals (biodemes). Based on the analysis of genetic markers the three parasite strains were typed as belonging to T. cruzi I major group, discrete typing unit 1. The pathological profile of RyC-H and RyC-V strains indicated medium virulence and low mortality and, accordingly, the strains should be considered as belonging to biodeme Type III. On the other hand, the parasites from RyC-R strain induced more severe inflammatory processes and high mortality (> 40%) and were considered as belonging to biodeme Type II. The relationship between genotypes and biological characteristics in T. cruzi strains is still debated and not clearly understood. An expert committee recommended in 1999 that Biodeme Type III would correspond to T. cruzi I group, whereas Biodeme Type II, to T. cruzi II group. Our findings suggest that, at least for Mexican isolates, this correlation does not stand and that biological characteristics such as pathogenicity and virulence could be determined by factors different from those identified in the genotypic characterization.     

Clinical forms of Trypanosoma cruzi infected individuals in the chronic phase of Chagas disease in Puebla, Mexico

In Mexico, despite the relatively high seroprevalence of Trypanosoma cruzi infection in humans in some areas, reported morbidity of Chagas disease is not clear. We determined clinical stage in 71 individuals seropositive to T. cruzi in the state of Puebla, Mexico, an area endemic for Chagas disease with a reported seroprevalence of 7.7%. Diagnosis of Chagas disease was made by two standardized serological tests (ELISA, IHA). Individuals were stratified according to clinical studies. All patients were submitted to EKG, barium swallow, and barium enema. Groups were identified as indeterminate form (IF) asymptomatic individuals without evidence of abnormalities (n = 34 cases); those with gastrointestinal alterations (12 patients) including symptoms of abnormal relaxation of the lower esophageal sphincter and absent peristalsis in the esophageal body, grade I megaesophagus, and/or megacolon; patients with clinical manifestations and documented changes of chronic Chagas heart disease who were subdivided as follows: mild (8 patients)--mild electrocardiographic changes of ventricular repolarization, sinus bradychardia); moderate (6 patients)--left bundle branch block, right bundle branch block associated with left anterior fascicular block); severe (8 patients)--signs of cardiomegaly, dilated cardiomyopathy); and the associated form (3 cases) that included presence of both cardiomyopathy and megaesophagus. These data highlight the importance of accurate evaluation of the prevalence and clinical course of Chagas disease in endemic and non-endemic areas of Mexico.     

Substrate specificity of human kallikrein 6: salt and glycosaminoglycan activation effects

Human kallikrein 6 (hK6) is abundantly expressed in the central nervous system and is implicated in demyelinating disease. This study provided biochemical data about the substrate specificity and activation of hK6 by glycosaminoglycans and by kosmotropic salts, which followed the Hofmeister series. The screening of fluorescence resonance energy transfer (FRET) peptide families derived from Abz-KLRSSKQ-EDDnp resulted in the finding that Abz-AFRFSQ-EDDnp (where Abz is ortho-aminobenzoic acid and EDDnp is N-[2,4-dinitrophenyl]ethylenediamine)) is the best synthetic substrate described so far for hK6 (kcat/Km 38,667 s(-1) mm(-1)). It is noteworthy that the AFRFS sequence was found as a motif in the amino-terminal domain of seven human ionotropic glutamate receptor subunits. We also examined the hK6 hydrolytic activity on FRET peptides derived from human myelin basic protein, precursor of the Abeta amyloid peptide, reactive center loop of alpha1-antichymotrypsin, plasminogen, and maturation and inactivation cleavage sites of hK6, which were described earlier as natural substrates for hK6. The best substrates were derived from myelin basic protein. The hK6 maturation cleavage site was poorly hydrolyzed, and no evidence was found to support a two-step self-activation process reported previously. Finally, we assayed FRET peptides derived from sequences that span the cleavage sites for activation of protease-activated receptors (PAR) 1-4, and only the substrate with the PAR 2 sequence was hydrolyzed. These results further supported the hypothesis that hK6 expressed in the central nervous system is involved in normal myelin turnover/demyelination processes, but it is unlikely to self-activate. This report also suggested the possible modulation of ionotropic glutamate receptors and activation of PAR 2 by hK6.