Molecular and enzymatic analysis of ammonium assimilation in woody plants

Ammonium is assimilated into amino acids through the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) enzymes. This metabolic pathway is driven by energy, reducing power and requires the net supply of 2-oxoglutarate that can be provided by the reaction catalysed by isocitrate dehydrogenase (IDH). Most studies on the biochemistry and molecular biology of N-assimilating enzymes have been carried out on annual plant species and the available information on woody models is far more limited. This is in spite of their economic and ecological importance and the fact that nitrogen is a common limiting factor for tree growth. GS, GOGAT and IDH enzymes have been purified from several woody species and their kinetic and molecular properties determined. A number of cDNA clones have also been isolated and characterized. Although the enzymes are remarkably well conserved along the evolutionary scale, major differences have been found in their compartmentation within the cell between angiosperms and conifers, suggesting possible adaptations to specific functional roles. The analysis of the gene expression patterns in a variety of biological situations such as changes in N nutrition, development, biotic or abiotic stresses and senescence, suggest that cytosolic GS plays a central and pivotal role in ammonium assimilation and metabolism in woody plants. The modification of N assimilation efficiency has been recently approached in trees by overexpression of a cytosolic pine GS in poplar. The results obtained, suggest that an increase in cytosolic GS might lead to a global effect on the synthesis of nitrogenous compounds in the leaves, with enhanced vegetative growth of transgenic trees. All these data suggest that manipulation of cytosolic GS may have consequences for plant growth and biomass production.     

Depletion of the high-abundance plasma proteins

Summary. Body fluids, like plasma and urine, are comparatively easy to obtain and are useful for the detection of novel diagnostic markers by applying new technologies, like proteomics. However, in plasma, several high-abundance proteins are dominant and repress the signals of the lower-abundance proteins, which then become undetectable either by two-dimensional gels or chromatography. Therefore, depletion of the abundant proteins is a prerequisite for the detection of the low-abundance components. We applied affinity chromatography on blue matrix and Protein G and removed the most abundant human plasma proteins, albumin and the immunoglobulin chains. The plasma proteins, prior to albumin and immunoglobulin depletion, as well the eluates from the two chromatography steps were analyzed by two-dimensional electrophoresis and the proteins were identified by MALDI-TOF-MS. The analysis resulted in the identification of 83 different gene products in the untreated plasma. Removal of the high-abundance proteins resulted in the visualization of new protein signals. In the eluate of the two affinity steps, mostly albumin and immunoglobulin spots were detected but also spots representing several other abundant plasma proteins. The methodology is easy to perform and is useful as a first step in the detection of diagnostic markers in body fluids by applying proteomics technologies.Current address: Foundation for Biomedical Research of the Academy of Athens, Greece     

Calcitriol inhibits interleukin-10 expression in cultured human trophoblasts under normal and inflammatory conditions

Preeclampsia is associated with systemic inflammation and increased expression of placental Th1-cytokines. IL-10 and calcitriol inhibit proinflammatory cytokines expression in human placenta helping to fetal allograft toleration. Regulation of placental IL-10 by calcitriol and Th-1 cytokines has not yet been fully elucidated. Since it is believed that calcitriol promotes a shift from a Th1- to a Th2 profile, we hypothesized that it would stimulate IL-10 in a normal and an inflammatory scenario to conjointly restrain inflammation. Therefore, we investigated calcitriol effects upon IL-10 expression in cultured human trophoblasts obtained from normal (NT) and preeclamptic (PE) pregnancies. Similar studies in the presence of TNF-α (as an inflammatory stressor) were also performed. Calcitriol dose-dependently inhibited IL-10 expression in NT, PE and TNF-α-challenged trophoblasts (P<0.05). This effect was prevented by a vitamin D receptor (VDR) antagonist. IL-10 expression was significantly stimulated by TNF-α and IL-1β, inhibited by IFN-γ and was not affected by IL-6. Finally, calcitriol inhibited TNF-α and IL-1β stimulation upon IL-10. In summary, in cultured human trophoblasts, calcitriol down-regulates IL-10 expression under normal as well as under natural and experimental inflammatory conditions. This effect is mediated by the VDR and might involve direct inhibition of TNF-α. In view of these and previous results it seems that in placenta calcitriol suppresses both Th1- and Th2 cytokines while undertakes the anti-inflammatory effects of IL-10 by itself, since both factors exert this task redundantly. The regulation of IL-10 by IFN-γ suggests that this cytokine could be a viable candidate to explain low IL-10 levels in preeclampsia.     

Tau function and dysfunction in neurons: its role in neurodegenerative disorders

Alzheimer’s disease (AD) is the most usual neurodegenerative disorder leading to dementia in the aged human population. It is characterized by the presence of two main brain pathological hallmarks: senile plaques and neurofibrillary tangles (NFTs). NFTs are composed of fibrillar polymers of the abnormally phosphorylated cytoskeletal protein tau.     

Acid and neutral hydrolases in Trypanosoma cruzi. Characterization and assay.

Twelve acid hydrolases, 4 near-neutral hydrolases, and alkaline phosphatase were demonstrated in 0.34 M sucrose homogenates of Trypanosoma cruzi strain Y: p-nitrophenylphosphatase and alpha-naphthylphosphatase, with optimum pH at approximately 6.0; alpha=ga;actpsodase. beta=ga;actpsodase. beta=g;icpsodase, N-acetyl-beta-glucosaminidase, cathepsin A and peptidase I and III, with optimum pH between 5.0 and 6.0; and arylsulfatase, cathepsin D, alpha-arabinase and alpha-mannosidase with optimum pH at approximately 4.0. alpha-Glucosidase, glucose-6-phosphatase and peptidase II had optimum pH at approximately 7.0. beta-Glycerophosphatase had a broad pH-activity curve from 4,0 to 7.4, with maximum activity at pH 7.0. The main kinetic characteristics of these enzymes and their quantitative assay methods were studied. No activity was detected for alpha-fucosidase, beta-xylosidase, beta-glucuronidase, elaidate esterase, acid lipase, and alkaline phosphodiesterase.     

Implication of brain cdc2 and MAP2 kinases in the phosphorylation of tau protein in Alzheimer's disease.

Brain tau protein is phosphorylated in vitro by cdc2 and MAP2 kinases, obtained through immunoaffinity purification from rat brain extracts. The phosphorylation sites are located on the tau molecule both upstream and downstream of the tubulin-binding motifs. A synthetic peptide comprising residues 194-213 of the tau sequence, which contains the epitope recognized by the monoclonal antibody tau-1, is also efficiently phosphorylated in vitro by cdc2 and MAP2 kinases. Phosphorylation of this peptide markedly reduces its interaction with the antibody tau-1, as it has been described for tau protein in Alzheimer's disease. Both cdc2 and MAP2 kinases are present in brain extracts obtained from Alzheimer's disease patients. Interestingly, the level of cdc2 kinase may be increased in patient brains as compared with non-demented controls. These results suggest a role for cdc2 and MAP2 kinases in phosphorylating tau protein at the tau-1 epitope in Alzheimer's disease.     

Serine hydroxymethyltransferase activity in Trypanosoma cruzi, Trypanosoma rangeli and American Leishmania spp

Serine hydroxymethyltransferase (SHMT) was studied in several American trypanosomatids, Trypanosoma cruzi epimastigotes displaying, in contrast with T. rangeli, high enzymatic activity. Several Leishmania spp. members, including L. braziliensis, L. mexicana and L. garnhami promastigotes, under identical assay conditions, showed low enzymatic activity. The T. cruzi and leishmanial enzymes presented several different kinetic properties, and thus apparent Km for THF was 0.30 mM for the trypanosomal SHMT vs 0.60 mM for the leishmanial enzyme, while the apparent Km for serine was 0.40 mM for trypanosomal SHMT vs 0.15 mM for leishmanial enzyme. There were significant variations in the specific activity of SHMT between the several different trypanosomatids strains studied, but the meaning of these results is not clear because they showed no correlation either with taxonomy or infectivity.     

Cytochemical characterization of mucosubstances in the chick glandular stomach during embryonary and postnatal development

The chick stomach is composed of two parts: glandular or proventriculus and muscular or gizzard which are morphological and functionally different. Since characterization of the mucosubstances in the adult stomach and its comparison with the embryonary stomach have not been made, we performed the study of the cytochemical characteristics of mucosubstances in the chick glandular stomach during the embryonic and post-natal periods, to obtain information on changes produced in these components during functional differentiation. In this work we established that during development, the epithelial cells of the superficial layer content predominantly glycoproteins and the glycosaminoglycans of the glands decrease when they begin to secrete other compounds, such as proteolytic enzymes necessary for digestion. This sequence of mucosubstances appearance is concordant with the increase of carbonic anhydrase, which reaches its highest specific activity from 15 to 20 days of incubation. In this period hydrochloric acid secretion increases and therefore, glycoprotein secretion becomes necessary to protect the mucous membranes.