Cytosine-block telomeric type DNA-binding activity of hnRNP proteins from human cell lines

Following the observation of the presence in mammalian nuclear extracts of a DNA binding activity quite specific for the single-stranded C-rich telomeric motif, we have isolated from the K562 human cell line by affinity chromatography and identified by mass spectrometry a number of proteins able to bind to this sequence. All of them belong to different heterogeneous nuclear ribonucleoprotein subgroups (hnRNP). Whereas many of them, namely hnRNP K, two isoforms of hnRNP I, and the factor JKTBP, appear to bind to this sequence with limited specificity after isolation, an isoform of hnRNP D (alias AUF1) and particularly hnRNP E1 (alias PCBP-1) show a remarkable specificity for the (CCCTAA)n repeated motif. Both have been obtained also as recombinant proteins expressed in Escherichia coli and have been shown to retain their binding specificity toward the C-block repeated sequence. In the light of the current knowledge about these proteins, their possible involvement in telomere functioning is discussed.     

Construct validity of the Short Form-36 Health Survey and its relationship with BMI in obese outpatients

Objective: To investigate the construct validity of the Short Form‐36 (SF‐36) Health Survey questionnaire in obese patients. Research Methods and Procedures: Our series consisted of 1735 obese patients (age, 44.7 ± 11.0 years; 1346 women) consecutively enrolled in the QUOVADIS study, an observational multicenter study of obese treatment‐seeking outpatients. The construct validity of the SF‐36 was assessed by main component analysis. Age‐, gender‐, and education‐adjusted general linear models were used to investigate the relationship between BMI and SF‐36 domains or factors identified by main component analysis. Results: BMI was significantly associated with poor health‐related quality of life in all eight SF‐36 domains, and the strongest association was observed with physical activity. Main components analysis generated a six‐factor solution explaining 59% of the observed variance. BMI was strongly associated with factors based on the loading of items regarding the physical activity domain and factors based on role‐physical and role‐emotional items or general health and bodily pain items. In contrast, mental health‐, vitality‐, and social functioning‐based factors were not related to BMI. Discussion: In obese treatment‐seeking outpatients, the clustering of SF‐36 items in main components is not significantly different from the domain‐based approach generally used, thus confirming the robustness of such a generic questionnaire in this specific condition. However, the peculiar clustering of some SF‐36 items and their relationship with BMI suggest that the health‐related quality of life profile of subjects belonging to that population may be better described with alternative aggregations of the SF‐36 items or with disease‐tailored questionnaires.     

Over-expression of GTP-binding proteins and GTPase activity in mouse astrocyte membranes in response to Theiler's murine encephalomyelitis virus infection

Intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV) induces a demyelinating disease that resembles human multiple sclerosis. In order to delineate the early events in this virus-induced neuroinflammatory disease, we have analyzed global GTPases gene activation following TMEV infection of murine brain astrocytes. DNA hybridization microchip analysis demonstrated that 10 sequences described as GTPbinding proteins and GTPases in different protein databases were over-expressed, in response to this infectious agent in astroglial cells. We have first characterized both the GTP-binding and GTPase activities in uninfected astrocyte membranes from a biochemical point of view. The increase in such activities was further validated in TMEV-infected astrocytes, peaking 2-4 h after infection. Over-expression is also induced by the inflammation-related chemokines interleukin-6 and interferon-gamma but not by interleukin-1alpha or tumor necrosis factor-alpha. From the many GTPases that could be over-expressed we have studied two, because of its biological significance; Ras p21 and the subunit alphai2 of G proteins. Western blots revealed increases in both proteins after infection with TMEV, in accordance with the previous enzymologic results. An increase in the active form of Ras (the GTP bound form) in cell lysates was also confirmed by affinity binding to a glutathione-S-transferase-fusion protein, following TMEV infection. A final demonstration of physiological up-regulation is provided by UV cross-linking of membrane proteins with the hydrolysis-resistant GTP agonist GTP [gamma-(35)S]. This technique allow us to detect, after SDS-PAGE, the increase of two further majoritary GTPbinding proteins with MW of 62 and 49 KDa. A quantitative analysis of four selected genes coding for p21 ras, Galphai2 subunit of protein G, Munc-18 and protein interacting with C kinase 1, was performed by real-time RT-PCR to verify the microarray results. The study of GTPase activity and of the above genes by RT-PCR in brains of sick mice, demonstrated a significative increase in mRNA coding for p21ras and protein interacting with C kinase 1 in vivo. Here we demonstrate that one of the mechanisms triggered by TMEV infection of astrocytes is the up-regulation of proteins related to GTP metabolism, one important signal transduction system in mammalian cells.     

Galectin-1 in cartilage: Expression, influence on chondrocyte growth and interaction with ECM components

Galectin-1 is a 14 kDa beta-galactoside binding protein, capable of forming lattice-like structures with glycans of cellular glycoconjugates and inducing intracellular signaling. The expression of Galectin-1 in porcine cartilage is described in this work for the first time. Immunocytochemical methods revealed distinct distribution patterns for both articular and growth plate cartilage. In articular cartilage, the highest reactivity for Galectin-1 was found in all chondrocytes at the superficial zone and in most of those at the lower layer of the middle zone. In the growth plate, marked reactivity was seen in chondrocytes at the proliferative zone and reached a maximum level for the column-forming cells at the hypertrophic zone. In addition, different Galectin-1 distribution patterns were observed at the subcellular level. With regards to the metabolic effects of Galectin-1, the results in vitro seem to indicate an inhibitory effect of Galectin-1 on articular chondrocyte anabolism (i.e. inhibition of cell proliferation and anabolic gene expression) and a stimulation of catabolic processes (i.e. induction of matrix degradation and hypertrophy marker expression). These data represent a starting point for the understanding the molecular mechanisms underlining ECM-Galectin-1 interaction and the subsequent signaling-cell transduction processes involving cartilage formation and maturation.     

Di-organocobalt complexes of macrocyclic ligands

Three new di-metallorganic cobalt complexes of the type trans-(Bz)₂Co(chel), where Bz is a benzyl group σ-bonded to cobalt atom and chel is an equatorial chelating system constituted by an amino-oximic ligand and its conjugated base, were synthesised. The protonated and the unprotonated ligands interact through an O-H ? O bridge stabilising the entire structure. The complexes differ in the equatorial moiety which is derived from the following ligands: HLN-py=3-[(2-pyridyl)ethylimino]-butan-2-one oxime), HLN-Ph=3-[(2-phenyl)ethylimino]-butan-2-one oxime and the analogous HLN-PhCl=3-[(2-chlorophenyl)ethylimino]-butan-2-one oxime. Two of these compounds, namely those derived from HLN-py and HLN-PhCl were structurally characterised by means X-ray diffractometry. Data reveal that each complex is characterised by the presence of two unusually long cobalt-carbon bonds which are 2.120(4) Å (mean value) in complex with HLN-py ligand and 2.119(4) Å (mean value) in complex with HLN-PhCl. These data are consistent with a strong mutual trans-influence exerted by one ligand on the other.     

Different Arginine Transfer Ribonucleic Acid Species Prevalent in Shaken and Unshaken Cultures of Neurospora

When the arginyl-transfer ribonucleic acid (tRNA) species isolated from unshaken and from shaken cultures of Neurospora were compared by co-chromatography, a marked change in the relative abundance of the two main tRNA(arg) species was found. The two arginine tRNA species had different codon responses in ribosome binding assays. The tRNA(arg) eluting first (prevalent in shaken cultures) bound strongly to polyadenylic-guanylic acid [poly(A,G)] and to a lesser extent to polycytidylic-guanylic-adenylic acid [poly(C,G,A)]. The second tRNA(arg) species (prevalent in unshaken cultures) bound to poly(C,G,A) but not to poly(A, G). The possible significance of these observations is briefly discussed. Several modifications that improve the yield of tRNA from Neurospora were introduced in a standard isolation procedure.     

Biological Responses of Human Gingival Fibroblasts (HGFs) in an Innovative Co-Culture Model with Streptococcus mitis to Thermosets Coated with a Silver Polysaccharide Antimicrobial System

This study sought to evaluate the in vitro biological response of human gingival fibroblasts (HGFs) co-coltured with Streptococcus mitis to bisphenol A glycidylmethacrylate/triethylene glycol dimethacrylate (BisGMA/TEGDMA) thermosets coated with Chitlac-nAg, a nanocomposite system with antimicrobial properties. To avoid bacterial adhesion to dental devices and to reduce cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilizing silver nanoparticles, which have well-known antimicrobial properties, with a polyelectrolyte solution containing Chitlac. Cytotoxicity, cell morphology, cell migration and inflammatory interleukine-6 (IL-6) and prostaglandin E₂ (PGE₂) secretion were evaluated. Our results showed that the cytotoxicity exerted on HGFs by our nanocomposite material was absent in our co-culture model, where fibroblasts are able to adhere and migrate. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release rises up 20%. Moreover the presence of S. mitis reduced this release in a greater amount with Chitlac-nAg coated thermosets. The secretion of IL-6 was significant in both Chitlac and Chitlac-nAg coated thermosets, but PGE₂ production was minimal, suggesting that the IL-6 production was not related to an inflammatory response. Co-culture and the addiction of saliva did not influence IL-6 and PGE₂ secretion. Data obtained in the present work suggest that Chitlac n-Ag coated thermosets could significantly improve the success rates of restorative dentistry, since they limit bacterial adhesion and are not toxic to HGFs.     

Neurospora Mutant Deficient in Tryptophanyl-Transfer Ribonucleic Acid Synthetase Activity

A tryptophan auxotroph of Neurospora crassa, trp-5, has been characterized as a mutant with a deficient tryptophanyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.2) activity. When assayed by tryptophanyl-tRNA formation, extracts of the mutant have less than 5% of the wild-type specific activity. The adenosine triphosphate-pyrophosphate exchange activity is at about half the normal level. In the mutant derepressed levels of anthranilate synthetase and tryptophan synthetase were associated with free tryptophan pools equal to or higher than those found in the wild type. We conclude that a product of the normal tryptophanyl-tRNA synthetase, probably tryptophanyl-tRNA, rather than free tryptophan, participates in the repression of the tryptophan biosynthetic enzymes.     

Regulation of Purine Metabolism in Intact Leaves of Coffea arabica

The capacity of Coffea arabica leaves (5- x 5-mm pieces) to synthesize de novo and catabolize purine nucleotides to provide precursors for caffeine (1,3,7-trimethylxanthine) was investigated. Consistent with de novo synthesis, glycine, bicarbonate, and formate were incorporated into the purine ring of inosine 5[prime]-monophosphate (IMP) and adenine nucleotides ([sigma]Ade); azaserine, a known inhibitor of purine de novo synthesis, inhibited incorporation. Activity of the de novo pathway in C. arabica per g fresh weight of leaf tissue during a 3-h incubation period was 8 [plus or minus] 4 nmol of formate incorporated into IMP, 61 [plus or minus] 7 nmol into [sigma]Ade, and 150 nmol into caffeine (the latter during a 7-h incubation). Coffee leaves exhibited classical purine catabolism. Radiolabeled formate, inosine, adenosine, and adenine were incorporated into hypoxanthine and xanthine, which were catabolized to allantoin and urea. Urease activity was demonstrated. Per g fresh weight, coffee leaf squares incorporated 90 [plus or minus] 22 nmol of xanthine into caffeine in 7 h but degraded 102 [plus or minus] 1 nmol of xanthine to allantoin in 3 h. Feedback control of de novo purine biosynthesis was contrasted in C. arabica and Cucurbita pepo, a species that does not synthesize purine alkaloids. End-product inhibition was demonstrated to occur in both species but at different enzyme reactions.