Fish and mammalian metallothioneins: a comparative study

Structural studies show that fish and mammalian metallothioneins are endowed of distinctive features. In particular, the ninth cysteine residue present in the alpha domain of fish metallothionein is shifted of two positions with respect to the mammalian metallothionein, introducing a conformational modification in the protein structure. In addition, the fish metallothionein is less hydrophobic and more flexible than its mammalian counterpart. Our previous studies showed that the hydropathy of piscine and mammalian metallothioneins is significantly correlated with organismal temperature. In the present paper we have performed phylogenetic comparative analysis on metallothioneins of 24 species of fish and mammals. The results of such analysis failed to indicate that metallothionein hydropathy is an adaptive response to the thermal regime of the species. We concluded that metallothionein hydropathy is a trait that did not evolve in association with environmental changes.     

5-Arylidene-2-imino-4-thiazolidinones: design and synthesis of novel anti-inflammatory agents

The synthesis and pharmacological activity of 5-arylidene-2-imino-4-thiazolidinones (3a-8a) are described. All derivatives exhibited significant activity levels in models of acute inflammation such as carrageenan-induced paw and pleurisy edema in rats. In particular, 5-(3-methoxyphenylidene)-2-phenylimino-3-propyl-4-thiazolidinone (3a) displayed high levels of carrageenan-induced paw edema inhibition comparable to those of indomethacin. In addition the ability of such a new class of anti-inflammatory agents to inhibit COX isoforms was assessed in murine monocyte/macrophage J774 cell line assay. 5-(4-Methoxyphenylidene)-2-phenylimino-3-propyl-4-thiazolidinone (6a), the most interesting compound in such an experiment, was docked in the known active site of COX-2 protein and showed that its 4-methoxyarylidene moiety can easily occupy the COX-2 secondary pocket considered as the critical interaction for COX-2 selectivity.     

Structure-activity relationships and molecular modelling of 5-arylidene-2,4-thiazolidinediones active as aldose reductase inhibitors

The structure-activity relationships (SARs) of 5-arylidene-2,4-thiazolidinediones active as aldose reductase inhibitors (ARIs) were extended by varying the substitution pattern on the 5-arylidene moiety and on N-3. In particular, the introduction of an additional aromatic ring or an H-bond donor group on the 5-benzylidene ring enhanced ALR2 inhibitory potency. Moreover, the presence of a carboxylic anionic chain on N-3 was shown to be an important, although not essential, structural requisite to produce high levels of ALR2 inhibition. The length of this carboxylic chain was critical and acetic acids 4 were the most effective inhibitors among the tested derivatives. Molecular docking simulations into the ALR2 active site accorded with the in vitro inhibition data. They allowed the rationalization of the observed SARs and provided a pharmacophoric model for this class of ARIs.     

Ceramide in nitric oxide inhibition of glioma cell growth. Evidence for the involvement of ceramide traffic

The treatment of C6 glioma cells with the nitric oxide donor, PAPANONOate ((Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate), resulted in a dose-dependent inhibition of cell proliferation. This was associated to a rapid and significant increase of ceramide levels and was mimicked by treatments that augment cellular ceramide. Metabolic experiments with radioactive sphingosine, serine, and choline showed that nitric oxide strongly reduced the utilization of ceramide for the biosynthesis of both sphingomyelin and glucosylceramide. Nevertheless, nitric oxide did not modify the activity of different enzymes of ceramide metabolism. The possibility that nitric oxide impairs the availability of ceramide for sphingolipid biosynthesis was then investigated. The metabolism of N-hexanoyl-[(3)H]sphingosine demonstrated that nitric oxide did not affect the biosynthesis of N-hexanoyl-[(3)H]sphingolipids but inhibited the metabolic utilization of long chain [(3)H]ceramide, synthesized in the endoplasmic reticulum (ER) from the recycled [(3)H]sphingosine. Moreover, results obtained with fluorescent ceramides, brefeldin A, ATP depletion, as well as in a ceramide transport assay indicate that nitric oxide impairs the traffic of ceramide from ER to Golgi apparatus. All this supports that, in glioma cells, the modulation of ceramide traffic can contribute to the regulation of its intracellular levels and participate in the nitric oxide-activated signaling pathway involved in the control of cell proliferation.     

CiC3-1a-mediated chemotaxis in the deuterostome invertebrate Ciona intestinalis (Urochordata)

Deuterostome invertebrates possess complement genes, and in limited instances complement-mediated functions have been reported in these organisms. However, the organization of the complement pathway(s), as well as the functions exerted by the cloned gene products, are largely unknown. To address the issue of the presence of an inflammatory pathway in ascidians, we expressed in Escherichia coli the fragment of Ciona intestinalis C3-1 corresponding to mammalian complement C3a (rCiC3-1a) and assessed its chemotactic activity on C. intestinalis hemocytes. We found that the migration of C. intestinalis hemocytes toward rCiC3-1a was dose dependent, peaking at 500 nM, and was specific for CiC3-1a, being inhibited by an anti-rCiC3-1a-specific Ab. As is true for mammalian C3a, the chemotactic activity of C. intestinalis C3-1a was localized to the C terminus, because a peptide representing the 18 C-terminal amino acids (CiC3-1a(59-76)) also promoted hemocyte chemotaxis. Furthermore, the CiC3-1a terminal Arg was not crucial for chemotactic activity, because the desArg peptide (CiC3-1a(59-75)) retained most of the directional hemocyte migration activity. The CiC3-1a-mediated chemotaxis was inhibited by pretreatment of cells with pertussis toxin, suggesting that the receptor molecule mediating the chemotactic effect is G(i) protein coupled. Immunohistochemical analysis with anti-rCiC3-1a-specific Ab and in situ hybridization experiments with a riboprobe corresponding to the 3'-terminal sequence of CiC3-1, performed on tunic sections of LPS-injected animals, showed that a majority of the infiltrating labeled hemocytes were granular amebocytes and compartment cells. Our findings indicate that CiC3-1a mediates chemotaxis of C. intestinalis hemocytes, thus suggesting an important role for this molecule in inflammatory processes.     

Interaction of the ADP-ribosylating enzyme from the hyperthermophilic archaeon S. solfataricus with DNA and ss-oligo deoxy ribonucleotides

The DNA-binding ability of the poly-ADPribose polymerase-like enzyme from the extremely thermophilic archaeon Sulfolobus solfataricus was determined in the presence of genomic DNA or single stranded oligodeoxyribonucleotides. The thermozyme protected homologous DNA against thermal denaturation by lowering the amount of melted DNA and increasing melting temperature. The archaeal protein induced structural changes of the nucleic acid by modifying the dichroic spectra towards a shape typical of condensing DNA. However, enzyme activity was slightly increased by DNA. Competition assays demonstrated that the protein interacted also with heterologous DNA. In order to characterize further the DNA binding properties of the archaeal enzyme, various ss-oligodeoxyribonucleotides of different base composition, lengths (12-mer to 24-mer) and structure (linear and circular) were used for fluorescence titration measurements. Intrinsic fluorescence of the archaeal protein due to tryptophan (excitation at 295 nm) was measured in the presence of each oligomer at 60 degrees C. Changes of tryptophan fluorescence were induced by all compounds in the same range of base number per enzyme molecule, but independently from the structural features of oligonucleotides, although the protein exhibited a slight preference for those adenine-rich and circular. The binding affinities were comparable for all oligomers, with intrinsic association constants of the same order of magnitude (K=10(6) M(-1)) in 0.01 M Na-phosphate buffer, pH 8.0, and accounted for a "non-specific" binding protein. Circular dichroism analysis showed that at 60 degrees C the native protein was better organized in a secondary structure than at 20 degrees C. Upon addition of oligonucleotides, enzyme structure was further stabilized and changed towards a beta-conformation. This effect was more marked with the circular oligomer. The analysed oligodeoxyribonucleotides slightly enhanced enzyme activity with the maximal increase of 50% as compared to the control. No activation was observed with the circular oligomer.     

Characterization of serum immunoglobulin M of the Antarctic teleost Trematomus bernacchii

Trematomus bernacchii immunoglobulin M concentration was determined in the serum by ELISA; the mean concentration value was 2.7 mg/ml corresponding to 9.6% of the total serum proteins. Purified IgM was analyzed by SDS-polyacrylamide gel electrophoresis, isoelectrofocusing and 2D electrophoresis. The relative molecular mass of the polymeric form was 830 kDa; that of separated H and L chains was, respectively, 78 and 25 kDa. The isoelectric points of the entire molecule ranged from 4.4 to 6.5, that of isolated H chains was between 4.0 and 6.0. Separated H chains were shown to reaggregate in tetrameric form. The cleavage site of trypsin was at the end of the CH1 domain, as confirmed by the N-terminal amino acid sequence of one of the resultant peptides. Immunoblotting was used to detect carbohydrates in the H and L chains labeled with digoxigenin. Glycosyl residues were detected only in the H chain. The carbohydrate content was evaluated to be 12.8% of the entire chain. Purified Igs were hydrolyzed by N-glycosidase F at different conditions and at least four different hydrolytic sites were revealed by limited deglycosylation. T. bernacchii IgM was also compared to those of five other polar fish species.     

Linkage disequilibrium analysis of the human adenosine deaminase (ada) gene provides evidence for a lack of correlation between hot spots of equal and unequal homologous recombination

The linkage disequilibrium (LD) pattern within the adenosine deaminase (ADA) gene was analyzed by studying 13 polymorphic loci in 137 families from two European and three African populations. Evidence for the presence of a 12-kb meiotic crossover hot spot, spanning part of the first and the second intron and flanked by regions of reduced recombination activity, was obtained. Moreover, segregation analysis of 113 informative meioses revealed two recombination events that are internal or overlap the 12-kb region, thus suggesting a recombination rate for the hot-spot region about 50-fold higher than the mean rate across the human genome. Within the hot spot, a 144-bp palindromic sequence was also identified and its possible involvement in the recombination process is discussed. The 12-kb region characterized by the low degree of LD does not include the 3.2-kb region that is deleted, as a result of recurrent unequal homologous recombination between two Alu elements, in patients affected by autosomal severe combined immunodeficiency. This observation provides the first evidence for an absence of correlation between hot spots of equal and unequal homologous recombination.     

Equilibria and kinetics of the intercalation of Pt-proflavine and proflavine into calf thymus DNA

The interaction of the cis-platinum derivative of proflavine [[PtCl(tmen)(2)][HNC(13)H(7)(NHCH(2)CH(2))(2)]](+) (PRPt) with CT-DNA is investigated by spectrophotometry and T-jump relaxation in 0.11M NaCl, pH 7.0, and 25 degrees C. The DNA-proflavine (PR) system is investigated under the same conditions. Static measurements indicate that base-dye interactions prevail and their analysis reveals that the site size for PRPt (n=2.6) is twice that found for PR (n=1.3). One relaxation effect is observed for the DNA/PR system and two effects for the DNA/PRPt system, the faster of them being similar to that of DNA/PR. The kinetics of the process are discussed in terms of the three-step sequence D+S <= => DS(I) <= => DS(II) <= => DS(III), where PR and the aromatic residues of PRPt intercalate into DNA by the same mechanism. The third step represents the penetration of platinum residues between base-pairs and is associated to remarkable enthalpy and entropy changes. Further mechanistic details are discussed.