Functional replacement of the oligomerization domain of H-NS by the Hha protein of Escherichia coli

Members of the H-NS family of proteins play a relevant role as modulators of gene expression in gram-negative bacteria. Interaction of these proteins with members of the Hha/YmoA family of proteins has been previously reported. It has been hypothesized that the latter proteins are functionally equivalent to the N-terminal domain of H-NS-like proteins. In this report we test this assumption by replacing the N-terminal domain of Escherichia coli H-NS by Hha. It has been possible to obtain a functional protein that can compensate for some of the hns-induced phenotypes. These results highlight the relevance of H-NS-Hha interactions to generate heterooligomeric complexes that modulate gene expression in gram-negative bacteria.     

Protein kinase D3 activation and phosphorylation by signaling through G alpha q

PKD is the founding member of a novel protein kinase family that also includes PKD2 and PKD3. PKD has been the focus of most studies up to date, but little is known about the mechanisms that mediate PKD3 activation. Here, we demonstrate that PKD3 immunoprecipitated from COS-7 cells transfected with a constitutively active G alpha q subunit (alpha(q)Q209L) exhibited a marked increase in basal activity. Addition of aluminum fluoride to cells co-transfected with PKD3 and wild type G alpha(q) also induced PKD3 activation. G alpha(q)-mediated PKD3 activation is associated with persistent translocation of PKD3 from both cytosol and nucleus to plasma membrane. Expression of a COOH-terminal fragment of G alpha q that acts in a dominant-negative fashion attenuated PKD3 activation in response to bombesin receptor stimulation. Our results indicate that G alpha q activation is sufficient to stimulate sustained PKD3 activation and show that the endogenous G alpha q is a major component in the signaling pathway that mediates bombesin-induced PKD3 activation.     

Lectin cytochemical characterization of the N- and O-linked oligosaccharides in the human rectum

The oligosaccharides of the mucus glycoproteins of the human rectum are important for the lubricant and protective role suggested for the rectal mucus. Changes in oligosaccharide composition are observed in several colon diseases, and some of these changes could be used as diagnostic and prognostic indicators. Thus, a previous knowledge of the normal mucus glycoproteins is necessary. The aim of the present study is the characterization of the oligosaccharides of the goblet cells and enterocytes of the human rectum. For this, a battery of 15 lectins, in combination with chemical and enzymatic deglycosylation procedures, was used. Our results suggest the presence of N-acetylglucosamine (GlcNAc), Man, Glc, N-acetylneuraminic acid (Neu5Ac)(2–6)- and Neu5Ac(2–3)-linked, N-acetylgalactosamine (GalNAc) and Gal(1–3)GalNAc in the oligosaccharides of the goblet cells. Moreover, N-linked oligosaccharides specifically contained Gal(1–4)GlcNAc, while AAA-positive Fuc was only detected in O-linked oligosaccharides. Some of these carbohydrates were only visualized after removal of N- or O-linked oligosaccharides, suggesting a high level of approximation between the oligosaccharide chains, that render the carbohydrate inaccessible to the lectins. Differences in the labelling pattern between the goblet cells of the surface epithelium and the upper half of the crypts, and those of the lower half of the crypts suggests a maturation process for the goblet cells, which modifies the oligosaccharide composition of the secreted glycoproteins, as they ascend throughout the crypts. This maturation process includes the incorporation of new carbohydrates (GlcNAc), and the masking (Neu5Ac(2–3)-linked) or unmasking (Glc and GalNAc) of others.     

Lectin histochemical identification of the carbohydrate moieties on N- and O-linked oligosaccharides in the duct cells of the testis of an amphibian urodele, the spanish newt (Pleurodeles waltl)

The aim of the present work was to study the carbohydrate moieties present on N- and O-linked oligosaccharides of duct cells of a urodele amphibian testis, by means of lectin histochemistry. It was found that duct cells have a carbohydrate composition that includes ensuremath (1,3)-, (1,4)- or (1,6)-linked Fuc and Man on N-linked oligosaccharides, Gal and GlcNAc on O-linked oligosaccharides, and DBA-positive GalNAc, (1,2)-linked Fuc and Neu5Ac(2,3)Gal (1,4)GlcNAc on both N- and O-linked oligosaccharides. All the duct cells showed the same lectin labelling pattern, the only exception being some sparse duct cells that showed the sequence Neu5Ac(2,6)Gal/GalNAc. The possible roles of duct cells in sperm maturation and the hypothesis for a common origin of duct and follicle (Sertoli) cells in the urodele testis are discussed.     

Glycan residues of N- and O-linked oligosaccharides in the premeiotic spermatogenetic cells of the urodele amphibian Pleurodeles waltl characterize by means of lectin histochemistry

The aim of this work was the characterization of the glycoconjugates of the premeiotic spermatogenetic cells of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. In the cytoplasm of the primordial germ cells, primary and secondary spermatogonia and primary spermatocytes, a granular structure can be observed close to the nucleus. These granules contain four types of sugar chains according to their appearance during the differentiation process: 1. some oligosaccharides that are identified in all the four cell types above mentioned, which include N-linked oligosaccharides with Fuc, Gal beta1,4GlcNAc and Neu5Ac alpha2,3Gal beta1,4GlcNAc and O-linked oligosaccharides with Gal beta1,4GlcNAc and Neu5Ac alpha2,3Gal beta1,4GlcNAc; 2. other glycan chains that are not present in the primary spermatocytes (N-linked oligosaccharides with DBA-positive GalNAc, GlcNAc, and a slight amount of Neu5Ac alpha2,6Gal/GalNAc and O-linked oligosaccharides with WGA-positive GlcNAc); 3. the sugar chains that are not in the earliest step of spermatogenesis (formed by both N-linked and O-linked oligosaccharides with Glc); and 4. other that appear at the earliest and latest stages, but not in the intermediate ones, (N-linked oligosaccharides with Man and O-linked oligosaccharides with SBA- and HPA-positive GalNAc and PNA-positive Gal beta1,3GalNAc). This structure could be related with the Drosophila spectrosome and fusome, unusual cytoplasmic organelles implicated in cystic germ cell development. Data from the present work, as compared with those from mammals and other vertebrates, suggest that, although no dramatic changes in the glycosylation pattern are observed, some cell glycoconjugates are modified in a predetermined way during the early steps of the spermatogenetic differentiation process.     

Self-design of fish diets by means of self-feeders: validation of procedures

In this paper, we show the results from four different experiments in which sea bass, maintained under laboratory conditions, could choose between two or three different diets through self-feeders, which gradually increased the complexity and potential range of selection, to design their own diet in accordance with their requirements. At first, sea bass were allowed to select between two complete diets differing in the proportion of protein (52-58%): this showed their capacity to distinguish between two diets made of the same ingredients. Next, two incomplete diets, containing a fixed amount of protein (56%) and lacking either fat or carbohydrate, were made available. Three mixed diets made up of pairs of macronutrients (protein-carbohydrate, protein-fat or fat-carbohydrate) were tested in the next experiment and, finally, three diets containing only one macronutrient (protein fat or carbohydrate) were made available to fish. Taking into account selection made by the fish in the first three experiments, in which macronutrient selection was statistically different, protein was the main macronutrient chosen by fish (278.15 kJ/kgBW/day, on average), followed by fat and carbohydrate (162.85 and 64.56 kJ/kgBW/day, respectively). In conclusion, the results reveal the ability of sea bass to select an appropriate diet from experimental diets containing two or three macronutrients and suggest that the proposed methodology is a powerful tool for studying the differing nutritional needs of different species of fish.     

Vanadium(V) complexes with salicylaldehyde semicarbazone derivatives bearing in vitro anti-tumor activity toward kidney tumor cells (TK-10): crystal structure of [VVO2(5-bromosalicylaldehyde semicarbazone)

As a contribution to the development of novel vanadium complexes with pharmacologically interesting moieties, new dioxovanadium(V) semicarbazone complexes with the formula cis-VO(2)L, where L=5-bromosalicylaldehyde semicarbazone and 2-hydroxynaphtalen-1-carboxaldehyde semicarbazone have been synthesized and characterized by (1)H and (13)C NMR, Raman and FTIR spectroscopies. Results were compared with those previously reported for other three analogous complexes of this series. The five complexes were tested in three different human tumor cell lines for bioactivity as potential anti-tumor agents, showing selective cytotoxicity on TK-10 cell line. Results showed that structural modifications on the semicarbazone moiety could have a significant effect on the anti-tumor activity of the vanadium complexes. In addition, the electrochemical behavior of all the complexes was studied. No apparent correlation could be demonstrated between reduction potentials of the complexes and their anti-tumor activities. The molecular structure of the novel [V(V)O(2)(5-bromosalicylaldehyde semicarbazone)] complex was solved by X-ray diffraction methods. The vanadium atom shows a distorted square pyramidal coordination sphere. The (VO(2))(+) cation is coordinated to a nearly planar (L)(-) anion acting as a tridentate ligand through both oxygen and one nitrogen atoms.