Design, synthesis and in vitro antitumor activity of new trans 2-[2-(heteroaryl)vinyl]-1,3-dimethylimidazolium iodides

The design, the synthesis, and the in vitro antitumor activities of trans 2-[2-(heteroaryl)vinyl]-1,3-dimethylimidazolium iodides versus MCF7 (human mammary carcinoma) and LNCap (prostate carcinoma) cell lines are reported. The design indicates trans 2-[2-[5-(2-chlorophenyl)furan-2-yl]vinyl]-1, 3-dimethylimidazolium iodide 5 and trans 2-[2-[5-(4-bromophenyl)furan-2-yl]vinyl]-1, 3-dimethylimidazolium iodide 6 as highly active compounds in the series. The synthesis of the above new derivatives and in vitro antitumor tests, confirm their significant antiproliferative and cytotoxic activities.     

Single mutation in the linker domain confers protein flexibility and camptothecin resistance to human topoisomerase I

DNA topoisomerase I relaxes supercoiled DNA by the formation of a covalent intermediate in which the active-site tyrosine is transiently bound to the cleaved DNA strand. The antineoplastic agent camptothecin specifically targets DNA topoisomerase I, and several mutations have been isolated that render the enzyme camptothecin-resistant. The catalytic and structural dynamical properties of a human DNA topoisomerase I mutant in which Ala-653 in the linker domain was mutated into Pro have been investigated. The mutant is resistant to camptothecin and in the absence of the drug displays a cleavage-religation equilibrium strongly shifted toward religation. The shift is mainly because of an increase in the religation rate relative to the wild type enzyme, indicating that the unperturbed linker is involved in slowing religation. Molecular dynamics simulation indicates that the Ala to Pro mutation increases the linker flexibility allowing it to sample a wider conformational space. The increase in religation rate of the mutant, explained by means of the enhanced linker flexibility, provides an explanation for the mutant camptothecin resistance.     

Diagnostic criteria for diabetes revisited: making use of combined criteria

Background  

Existing cut-offs for fasting plasma glucose (FPG) and post-load glucose (2hPG) criteria are not equivalent in the diagnosis of diabetes and glucose intolerance. Adjusting cut-offs of single measurements have not helped so we undertook this project to see if they could be complementary.     

Caspase-1 levels in biological fluids from patients with multiple sclerosis and from patients with other neurological and non-neurological diseases

In multiple sclerosis (MS), pathological white matter damage in the central nervous system is sustained by immune-inflammatory response. Caspase-1 plays a pivotal role in immune-mediated inflammation, as it regulates the cellular export of IL-1beta and IL-18. We carried out a preliminary in vitro study of the kinetics of extracellular caspase-1 release. We then measured caspase-1 levels in paired serum and cerebrospinal fluid (CSF) samples of 75 MS patients, 15 healthy subjects, and patients with other neurological diseases. Paired synovial fluid and serum samples of patients with juvenile idiopathic arthritis, and paired sputum and serum samples of asthma patients were also studied. Mean serum caspase-1 concentrations did not differ between groups. Caspase-1 was detected in the CSF of patients with acute, but not stable, MS [7.5 +/- (SEM) 0.9 pg/ml; test's sensitivity, 56% and specificity, 100%]. Its levels correlated with pleocytosis. The highest mean caspase-1 levels were found in the arthritic synovial fluids (945.5 +/- 126.6 pg/ml, which correlated with erythrocyte sedimentation rate), and in the sputum samples (370.1 +/- 71.0 pg/ml, which correlated with the number of macrophages in the sputum). On condition that caspase-1 is determined in the fluids pertaining to the disease-specific inflammatory sites, its level is a reliable marker of ongoing immune-inflammatory response. The enzyme measurement in CSF can also help define state-trait in MS.     

The small GTPase Rab13 regulates assembly of functional tight junctions in epithelial cells

Junctional complexes such as tight junctions (TJ) and adherens junctions are required for maintaining cell surface asymmetry and polarized transport in epithelial cells. We have shown that Rab13 is recruited to junctional complexes from a cytosolic pool after cell-cell contact formation. In this study, we investigate the role of Rab13 in modulating TJ structure and functions in epithelial MDCK cells. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delayed the formation of electrically tight epithelial monolayers as monitored by transepithelial electrical resistance (TER) and induced the leakage of small nonionic tracers from the apical domain. It also disrupted the TJ fence diffusion barrier. Freeze-fracture EM analysis revealed that tight junctional structures did not form a continuous belt but rather a discontinuous series of stranded clusters. Immunofluorescence studies showed that the expression of Rab13Q67L delayed the localization of the TJ transmembrane protein, claudin1, at the cell surface. In contrast, the inactive Rab13T22N mutant did not disrupt TJ functions, TJ strand architecture nor claudin1 localization. Our data revealed that Rab13 plays an important role in regulating both the structure and function of tight junctions.     

Taurocholate feeding prevents CCl4-induced damage of large cholangiocytes through PI3-kinase-dependent mechanism

Bile acids are cytoprotective in hepatocytes by activating phosphatidylinositol-3-kinase (PI3-K) and its downstream signal AKT. Our aim was to determine whether feeding taurocholate to CCl(4)-treated rats reduces cholangiocyte apoptosis and whether this cytoprotective effect is dependent on PI3-K. Cholangiocyte proliferation, secretion, and apoptosis were determined in cholangiocytes from bile duct ligation (BDL), CCl(4)-treated BDL rats, and CCl(4)-treated taurocholate-fed rats. In vitro, we tested whether CCl(4) induces apoptosis and whether loss of cholangiocyte proliferation and secretion is dependent on PI3-K. The CCl(4)-induced cholangiocyte apoptosis and loss of cholangiocyte proliferation and secretion were reduced in CCl(4)-treated rats fed taurocholate. CCl(4)-induced cholangiocyte apoptosis, loss of cholangiocytes secretion, and proliferation were prevented by preincubation with taurocholate. Taurocholate cytoprotective effects were ablated by wortmannin. Taurocholate prevented, in vitro, CCl(4)-induced decrease of phosphorylated AKT protein expression in cholangiocytes. The cytoprotective effects of taurocholate on CCl(4) effects on cholangiocyte proliferation and secretion were abolished by wortmannin. Taurocholate protects cholangiocytes from CCl(4)-induced apoptosis by a PI3-K-dependent mechanism. Bile acids are important in the prevention of drug-induced ductopenia in cholangiopathies.     

Solid state structural analysis of the cyclooctapeptide cyclo- (Pro1-Pro-Phe-Phe-Ac6c-Ile-D-Ala-Val8)

A solid state analysis of the cyclic octapeptide c(-Pro(1)-Pro-Phe-Phe-Ac(6)c-Ile-D-Ala-Val(8)-) (C8-CLA), containing the Pro-Pro-Phe-Phe sequence, followed by the bulky helicogenic C(alpha,alpha)-dialkylated 1-aminocyclohexane-1-carboxylic acid (Ac(6)c) residue and a D-Ala residue in position 7, has been carried out by x-ray diffraction.The crystals, grown from a DMSO solution, are monoclinic, space group P2(1) with a = 13.458(3) A, b = 19. 404(5) A, c = 21.508(4) A, and beta = 90.83(6) degrees, with two independent cyclic molecules in the asymmetric unit, two DMSO molecules, and three water molecules. The structure has been solved using the half and bake procedure by Sheldrick, and refined to final R1 and wR2 indices of 0.0613 and 0.1534 for 9867 reflections with I > 2sigma(I).This cyclic peptide, a deletion analogue of the naturally occurring cyclic nonapeptide cyclolinopeptide A [c(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val), CLA] has been designed to study the influence of the ring size reduction on the conformational behavior of CLA and more in general to obtain structural information on asymmetric cyclic octapeptides.The compound exhibits, in the solid state, a "banana-twisted" conformation with a cis peptide bond located between the two proline residues. Five intramolecular H bonds stabilize the structure: one type VIa beta-turn, two consecutive type III/I beta-turns, one gamma-turn, and one C(16) bend.The structure has also been compared with either the solution structure previously reported by us and obtained by nmr and computational analysis, and with solid state structural data reported in the literature on cyclic octapeptides.     

Inhibition of ErbB-2 mitogenic and transforming activity by RALT, a mitogen-induced signal transducer which binds to the ErbB-2 kinase domain

The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen employing the ErbB-2 juxtamembrane and kinase domains as bait. This interaction was reproduced in vitro with a glutathione S-transferase fusion protein spanning positions 282 to 395 of the 459-residue gene 33 protein. Activation of ErbB-2 catalytic function was required for ErbB-2-gene 33 physical interaction in living cells, whereas ErbB-2 autophosphorylation was dispensable. Expression of gene 33 protein was absent in growth-arrested NIH 3T3 fibroblasts but was induced within 60 to 90 min of serum stimulation or activation of the ErbB-2 kinase and decreased sharply upon entry into S phase. New differentiation factor stimulation of mitogen-deprived mammary epithelial cells also caused accumulation of gene 33 protein, which could be found in a complex with ErbB-2. Overexpression of gene 33 protein in mouse fibroblasts inhibited (i) cell proliferation driven by ErbB-2 but not by serum, (ii) cell transformation induced by ErbB-2 but not by Ras or Src, and (iii) sustained activation of ERK 1 and 2 by ErbB-2 but not by serum. The gene 33 protein may convey inhibitory signals downstream to ErbB-2 by virtue of its association with SH3-containing proteins, including GRB-2, which was found to associate with gene 33 protein in living cells. These data indicate that the gene 33 protein is a feedback inhibitor of ErbB-2 mitogenic function and a suppressor of ErbB-2 oncogenic activity. We propose that the gene 33 protein be renamed with the acronym RALT (receptor-associated late transducer).