Transition-metal complexes of isatin-beta-thiosemicarbazone. X-ray crystal structure of two nickel complexes

Manganese, iron, cobalt, nickel, copper and zinc complexes of isatin-beta-thiosemicarbazone (H2L) have been synthesized and spectroscopically characterized The X-ray crystal structures of two nickel complexes, namely [Ni(HL)2]. EtOH (1) and [Ni(HL)2]. 2DMF (2), reveal a distorted octahedral coordination with the monodeprotonated ligand that behaves as an O,N,S terdentate. Different packing interactions are determined by the presence of different crystallization solvents, i.e., ethanol in 1 and dimethylformamide (DMF) in 2. 1H and 13C NMR studies of the ligand and zinc complexes in solution were carried out and a complete assignment for the ligand was made by homodecoupling, gradient assisted 2D 1H-13C HMQC and HMBC NMR spectroscopy. Biological studies, carried out in vitro on human leukaemic cell lines U937, have shown that the free ligand and the copper (II) complex are more active in the inhibition of cell proliferation than the nickel complexes. No compound was able to induce apoptosis.     

Acute insulin infusion decreases plasma ghrelin levels in uncomplicated obesity

BACKGROUND: Plasma ghrelin levels have been shown to decrease after insulin infusion in lean subjects. Nevertheless, the mechanism of the suggested inhibitory effect of insulin on ghrelin is still unclear and no data about the effect of acute insulin infusion on plasma ghrelin concentration in obese subjects are available. OBJECTIVE: We sight to evaluate plasma ghrelin concentration during an hyperinsulinemic euglycemic clamp in uncomplicated obese subjects. METHODS: 35 uncomplicated obese subjects, body mass index (BMI) 43.3+/-10.1 kg/m(2), 33 women and 2 men, mean age 34.9+/-10, with a history of excess fat of at least 10 years underwent euglycemic hyperinsulinemic clamp. Blood samples for ghrelin were performed at baseline and steady state of euglycemic insulin clamp. RESULTS: Ghrelin concentrations decreased over time to 10.6+/-15% (range 2-39%) of baseline, from a mean of 205.53+/-93.79 pg/ml to 179.03+/-70.43 pg/ml during the clamp (95% CI, 10.69 to 36.44, P<0.01). In a univariate linear regression analysis baseline plasma ghrelin levels were inversely correlated to BMI (r=-0.564, P=0.04). A linear positive trend between whole body glucose utilization (M(FFMkg) index) and ghrelin reduction during the clamp was found (chi(2) 3.05, p=0.05). CONCLUSIONS: Our data seem to suggest that hyperinsulinemia during a euglycemic clamp is able to suppress plasma ghrelin concentrations in uncomplicated obesity. This effect appears to be positively related to insulin sensitivity.     

Toxicity of melanin-free ink of Sepia officinalis to transformed cell lines: identification of the active factor as tyrosinase

The melanin-free ink of the cephalopod Sepia officinalis is shown to contain a heat labile proteinaceous component toxic to a variety of cell lines, including PC12 cells. Gel filtration chromatography indicated that the toxic component was concentrated in those fractions eluted at a molecular weight higher than 100 kDa and exhibiting the highest tyrosinase activity. SDS-PAGE analysis of the active fractions displayed a single major band migrating at an approximate molecular weight of 100 kDa, identical with that of the single tyrosinase band in the melanin-free ink. These data unambiguously demonstrated the identity of the toxic component with tyrosinase. Treatment of purified Sepia as well as of mushroom tyrosinase with an immobilized version of proteinase K resulted in a parallel loss of tyrosinase activity and cytotoxicity. Sepia apotyrosinase was ineffective in inducing cytotoxicity in PC12 cells. Purified Sepia tyrosinase was found to induce a significant increase in caspase 3 activity in PC12 cells, leading eventually to an irreversible apoptotic process. Overall, these results disclose a hitherto unrecognized property of tyrosinase that may lead to a reappraisal of its biological significance beyond that of a mere pigment producing enzyme.     

Synthesis,characterization and X-ray structures of new antiproliferative and proapoptotic natural aldehyde thiosemicarbazones and their nickel(II) and copper(II) complexes

Synthesis and characterization of new thiosemicarbazones derived from natural aldehydes (1–9) have been investigated in order to develop a research program aimed at the development of compounds with antiviral, antibacterial, and antitumor properties. These substances contain both a chain with N and S nucleophilic centers with tuberculostatic activity, and an alkyl or terpenic moiety. In addition, a few nickel(II) and copper(II) complexes (10–18), derived also from the previously studied ligands, were synthesized and characterized by means of NMR and IR techniques. The trans-2-octenal N¹-phenylthiosemicarbazone and its nickel complex were also characterized by X-ray diffractometry. Biological studies, performed with some of these compounds, have involved both inhibition of cell proliferation and apoptosis tests in vitro on human leukemia cell line U937 to deepen our knowledge on the way these substances interfere with biological processes in leukemic cells.     

Sex- and tissue-specific expression of aspartic proteinases in Danio rerio (zebrafish)

Full-length zebrafish cDNAs encoding two aspartic proteinases were cloned and sequenced. One of the two cDNAs was a 1708 bp product with an open reading frame of 398 amino acid residues corresponding to a cathepsin D. The other was a 1383 bp product encoding a polypeptide chain of 416 amino acids homologous to nothepsin, an aspartic proteinase first identified by us in the liver of Antarctic Notothenioidei. Gene expression assessed by RT–PCR and northern blot hybridization of RNA from different tissues showed that the expression was tissue- and sex-specific. Whereas the cathepsin D gene was expressed in all the tissues examined independently of the sex, the nothepsin gene was expressed exclusively in female livers.     

Conformational Dynamics of the Bovine Mitochondrial ADP/ATP Carrier Isoform 1 Revealed by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

The mitochondrial adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in cellular energy metabolism. During the transport mechanism the carrier switches between two different conformations that can be blocked by two toxins: carboxyatractyloside (CATR) and bongkrekic acid. Therefore, our understanding of the nucleotide transport mechanism can be improved by analyzing structural differences of the individual inhibited states. We have solved the three-dimensional structure of bovine carrier isoform 1 (bAnc1p) in a complex with CATR, but the structure of the carrier-bongkrekic acid complex, and thus, the detailed mechanism of transport remains unknown. Improvements in sample processing in the hydrogen/deuterium exchange technique coupled to mass spectrometry (HDX-MS) have allowed us to gain novel insights into the conformational changes undergone by bAnc1p. This paper describes the first study of bAnc1p using HDX-MS. Results obtained with the CATR-bAnc1p complex were fully in agreement with published results, thus, validating our approach. On the other hand, the HDX kinetics of the two complexes displays marked differences. The bongkrekic acid-bAnc1p complex exhibits greater accessibility to the solvent on the matrix side, whereas the CATR-bAnc1p complex is more accessible on the intermembrane side. These results are discussed with respect to the structural and biochemical data available on Ancp.     

Natural variation in the appendicular skeleton of Triturus carnifex (Amphibia: Salamandridae)

Intraspecific variation in the appendicular skeleton of two geographically isolated populations of Triturus carnifex, one from northern Italy (Rosate, Milano) and one from central Italy (Bagnaia, Perugia), has been studied. A total of 1,746 forelimbs and 830 hindlimbs were examined. Forelimb skeletal variability was much greater in the Rosate than the Bagnaia population. Skeletal variants were present in 36.3% and 13.5% of the forelimbs, respectively, or in 54.7% and 22.7% of the netws (P < 0.0001). There were no predominant skeletal variants in Bagnaia, while in the Rosate population, the majority of the variants consisted of fusion of radiale and prepollicis and of phalangeal formula 1-2-3-2. Hindlimb skeletal variability was similar in the two populations and appeared to be much lower than that of the forelimb, with highly significant differences in the frequency of basipodium variants within the Rosate population and in the frequency of acropodium variants in both populations. Skeletal variants were present in about 9% of the hindlimbs, or in about 12% of the newts from either population. At present, no conclusion can be drawn about the mechanisms, genetic and/or epigenetic, underlying the skeletal variability observed in the Triturus carnifex from northern and central Italy. © 1996 Wiley-Liss, Inc.     

Plant stress proteins of the thaumatin-like family discovered in animals

Thaumatin-like proteins (TLPs) are polypeptides of about 200 residues synthesized by plants in response to fungal infection. In addition to the exceptionally strong sweet taste exhibited by some members, they are also reported to be endowed with endo-beta-1,3-glucanase activity and alpha-amylase inhibiting properties. However, the detailed mechanism of their antifungal action is not completely understood. So far, TLPs have only been described in plants, with several members of the family expressed in the same species. Here, for the first time in animals, we report the identification of two genes encoding members of the thaumatin-like proteins family in the desert locust Schistocerca gregaria and show their expression in different parts of the body. Southern blot and Western blot experiments revealed the presence of orthologous genes and their expression products in the related species Locusta migratoria. A search through the available genomes yielded similar sequences in the nematode Caenorhabditis but not in Drosophila and other insects. A three-dimensional model of S. gregaria TLP suggests a glucanase function. As in plants, TLPs could play a defense role in insects against pathogens.