Purification, structure and immunobiological activity of an arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds

The structure and bioactivity of a polysaccharide extracted and purified from a 4M KOH + H3BO3 solution from Prunus dulcis seed cell wall material was studied. Anion-exchange chromatography of the crude extract yielded two sugar-rich fractions: one neutral (A), the other acidic (E). These fractions contain a very similar monosaccharide composition: 5:2:1 for arabinose, uronic acids and xylose, respectively, rhamnose and galactose being present in smaller amounts. As estimated by size-exclusion chromatography, the acidic fraction had an apparent molecular mass of 762 kDa. Methylation analysis (from the crude and fractions A and E), suggests that the polysaccharide is an arabinan-rich pectin. In all cases, the polysaccharides bear the same type of structural Ara moieties with highly branched arabinan-rich pectic polysaccharides. The average relative proportions of the arabinosyl linkages is 3:2:1:1 for T-Araf:(1-->5)-Araf:(1-->3,5)-Araf:(1-->2,3,5)-Araf. The crude polysaccharide extract and fractions A and E induced a murine lymphocyte stimulatory effect, as evaluated by the in vitro and in vivo expression of lymphocyte activation markers and spleen mononuclear cells culture proliferation. The lymphocyte stimulatory effect was stronger on B- than on T-cells. No evidence of cytotoxic effects induced by the polysaccharide fractions was found.     

Nitric oxide-induced carbonylation of Bcl-2, GAPDH and ANT precedes apoptotic events in insulin-secreting RINm5F cells

Generation of high levels of nitric oxide (NO) following induction of NOS2 by interleukin-1 beta (IL-1beta) triggers beta cell apoptosis in insulin-secreting RINm5F cells. Mitochondrial and nuclear events such as downregulation of the antiapoptotic protein Bcl-2, activation of the pore responsible for the permeability transition (PT) and DNA fragmentation are involved in the process. We report in the present paper that exposure of insulin-producing RINm5F cells to NO donors and to IL-1beta leads to oxidative carbonylation of both Bcl-2 and the adenine nucleotide translocator (ANT) component of the mitochondrial PT pore. When the effect of endogenous generation of high concentrations of NO following exposure of cells to IL-1beta was studied, carbonylation of Bcl-2 preceded downregulation of the protein. Overexpression of Mn-SOD decreases substantially the extent of Bcl-2 carbonylation in SIN-1-exposed cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition, carbonylation and translocation from cytoplasm to nucleus and DNA fragmentation were also induced by DETA/NO exposure. DETA/NO-induced carbonylation of Bcl-2 and ANT proteins takes place 6 h before apoptotic release of histone-associated DNA to cytoplasm. Time course studies also reveal a close parallel between GAPDH translocation to nucleus and carbonylation. Inhibitors of lipooxidation end products formation such as piridoxamine (PM) and aminoguanidine (AG) block NO-triggered carbonylation of Bcl-2, ANT and GAPDH, prevent NO-induced GAPDH enzyme inhibition and nuclear translocation and DNA fragmentation. Our results support the notion that the oxidative carbonylation of proteins plays a role in the control of NO-induced apoptosis.     

The erythrocyte skeletons of beta-adducin deficient mice have altered levels of tropomyosin, tropomodulin and EcapZ

The erythrocyte membrane cytoskeleton is organized as a polygonal spectrin network linked to short actin filaments that are capped by adducin at the barbed ends. We have constructed a mouse strain deficient in beta-adducin having abnormal erythrocytes. We show here that the levels of several skeletal proteins from beta-adducin mutant erythrocytes are altered. In fact, CapZ, the main muscle actin-capping protein of the barbed ends that in the erythrocytes is cytoplasmic, is 9-fold upregulated in mutant skeletons of erythrocytes suggesting a compensatory mechanism. We also detected upregulation of tropomodulin and downregulation of alpha-tropomyosin and actin. In addition, purified adducin can be re-incorporated into adducin-deficient ghosts.     

Sex-specific meiotic drive and selection at an imprinted locus

We present a one-locus model that breaks two symmetries of Mendelian genetics. Whereas symmetry of transmission is breached by allowing sex-specific segregation distortion, symmetry of expression is breached by allowing genomic imprinting. Simple conditions for the existence of at least one polymorphic stable equilibrium are provided. In general, population mean fitness is not maximized at polymorphic equilibria. However, mean fitness at a polymorphic equilibrium with segregation distortion may be higher than mean fitness at the corresponding equilibrium with Mendelian segregation if one (or both) of the heterozygote classes has higher fitness than both homozygote classes. In this case, mean fitness is maximized by complete, but opposite, drive in the two sexes. We undertook an extensive numerical analysis of the parameter space, finding, for the first time in this class of models, parameter sets yielding two stable polymorphic equilibria. Multiple equilibria exist both with and without genomic imprinting, although they occurred in a greater proportion of parameter sets with genomic imprinting.     

A novel yeast mutation, rad52-L89F, causes a specific defect in Rad51-independent recombination that correlates with a reduced ability of Rad52-L89F to interact with Rad59

We isolated a novel rad52 mutation, rad52-L89F, which specifically impairs recombination in rad51Delta cells. rad52-L89F displays phenotypes similar to rad59Delta and encodes a mutant protein impaired in its ability to interact with Rad59. These results support the idea that Rad59 acts in homologous recombination via physical interaction with Rad52.     

Group I metabotropic glutamate receptors mediate a dual role of glutamate in T cell activation

Metabotropic glutamate receptors (mGluR) are present in cells of the nervous system, where they are activated by one of the main neurotransmitters, glutamate. They are also expressed in cells outside the nervous system. We identified and characterized two receptors belonging to group I mGluR, mGlu1R and mGlu5R, in human cell lines of lymphoid origin and in resting and activated lymphocytes from human peripheral blood. Both are highly expressed in the human Jurkat T cell line, whereas mGlu5R is expressed only in the human B cell line SKW6.4. In blood lymphocytes, mGlu5R is expressed constitutively, whereas mGlu1R is expressed only upon activation via the T cell receptor-CD3 complex. Group I receptors in the central nervous system are coupled to phospholipase C, whereas in blood lymphocytes, activation of mGlu5R does not trigger this signaling pathway, but instead activates adenylate cyclase. On the other hand, mGlu5R does not mediate ERK1/2 activation, whereas mGlu1R, which is coupled neither to phospholipase C nor to calcium channels and whose activation does not increase cAMP, activates the mitogen-activated protein kinase cascade. The differential expression of mGluR in resting and activated lymphocytes and the different signaling pathways that are triggered when mGlu1Rs or mGlu5Rs are activated point to a key role of glutamate in the regulation of T cell physiological function. The study of the signaling pathways (cAMP production and ERK1/2 phosphorylation) and the proliferative response obtained in the presence of glutamate analogs suggests that mGlu1R and mGlu5R have distinct functions. mGlu5R mediates the reported inhibition of cell proliferation evoked by glutamate, which is reverted by the activation of inducible mGlu1R. This is a novel non-inhibitory action mechanism for glutamate in lymphocyte activation. mGlu1R and mGlu5R thus mediate opposite glutamate effects in human lymphocytes.     

Characterization of a small metal binding protein from Nitrosomonas europaea

A small metal-binding protein (SmbP) with no known similarity to other proteins in current databases was isolated and characterized from the periplasm of Nitrosomonas europaea. The primary structure of this small (9.9 kDa) monomeric protein is characterized by a series of 10 repeats of a seven amino acid motif and an unusually high number of histidine residues. The protein was isolated from N. europaea with Cu(II) bound but was found to be capable of binding multiple equivalents of a variety of divalent and trivalent metals. The protein was overexpressed in Escherichia coli and used for the study of its metal-binding properties by UV/vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopy and equilibrium dialysis and isothermal titration calorimetry. The protein was found to bind up to six Cu(II) atoms with dissociation constants of approximately 0.1 microM for the first two metal ions and approximately 10 microM for the next four. Binding of Cu(II) resulted in spectroscopic features illustrating two distinctive geometries, as determined by EPR spectroscopy. The levels of SmbP in the periplasm were found to increase by increasing the levels of copper in the growth media. This protein is proposed to have a role in cellular copper management in the ammonia-oxidizing bacterium N. europaea.     

The Pseudomonas putida Crc global regulator controls the expression of genes from several chromosomal catabolic pathways for aromatic compounds

The Crc protein is involved in the repression of several catabolic pathways for the assimilation of some sugars, nitrogenated compounds, and hydrocarbons in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium (catabolic repression). Crc appears to be a component of a signal transduction pathway modulating carbon metabolism in pseudomonads, although its mode of action is unknown. To better understand the role of Crc, the proteome profile of two otherwise isogenic P. putida strains containing either a wild-type or an inactivated crc allele was compared. The results showed that Crc is involved in the catabolic repression of the hpd and hmgA genes from the homogentisate pathway, one of the central catabolic pathways for aromatic compounds that is used to assimilate intermediates derived from the oxidation of phenylalanine, tyrosine, and several aromatic hydrocarbons. This led us to analyze whether Crc also regulates the expression of the other central catabolic pathways for aromatic compounds present in P. putida. It was found that genes required to assimilate benzoate through the catechol pathway (benA and catBCA) and 4-OH-benzoate through the protocatechuate pathway (pobA and pcaHG) are also negatively modulated by Crc. However, the pathway for phenylacetate appeared to be unaffected by Crc. These results expand the influence of Crc to pathways used to assimilate several aromatic compounds, which highlights its importance as a master regulator of carbon metabolism in P. putida.