Macrophage inhibitory cytokine-1/growth differentiation factor-15 as a predictor of colonic neoplasia.

BACKGROUND: Serum macrophage inhibitory cytokine-1 (MIC-1/GDF15) concentration has been associated with colonic adenomas and carcinoma. AIMS: To determine whether circulating MIC-1/GDF15 serum concentrations are higher in the presence of adenomas and whether the level decreases after excision. METHODS: Patients were recruited prospectively from a single centre and stratified into five groups: no polyps (NP); hyperplastic polyps (HP); sessile serrated ademona (SSA); adenomas (AP); and colorectal carcinoma (CRC). Blood samples were collected immediately before and 4 weeks after colonoscopy. MIC-1/GDF15 serum levels were quantified using ELISA. RESULTS: Participants (n=301) were stratified as: NP; n=116 (52%), HP; n=37 (12%), SSA; n=19 (7%), AP; n=68 (23%); and CRC; n=3 (1%). Patients were excluded from the study due to nondiagnostic pathology (n=9, 3%) and exclusion criteria (n=20, 6%). In the 272 remaining subjects (M=149; F=123), age (P=.005), history of colonic polyps (P=.003) and family history of colonic polyps (P=.002) were associated with presence of adenomas. Baseline median MIC-1/GDF15 serum levels increased significantly from NP 609 (460-797) pg/mL, HP 582 (466-852) pg/mL, SSA 561 (446-837) pg/mL to AP 723 (602-1122) pg/mL and CRC 1107 (897-1107) pg/mL; (P<.001). In the pre- and postpolypectomy paired adenoma samples median MIC-1/GDF15 reduced significantly from 722 (603-1164) pg/mL to 685 (561-944) pg/mL (P=.002). A ROC analysis for serum MIC-1/GDF15 to identify adenomatous polyps indicated an area under the curve of 0.71. CONCLUSIONS: Our data suggest that serum MIC-1/GDF15 has the diagnostic characteristics to increase the detection of colonic neoplasia and improve screening.

Cyclometallated platinum(II) complexes: oxidation to, and C-H activation by, platinum(IV).

A series of cyclometallated phenylpyridine platinum(II) complexes have been synthesised with a systematic variation in both the phenylpyridine and the ancillary ligand. Oxidation of one of the cyclometallated species leads to a number of isomeric platinum(IV) complexes, all of which eventually isomerize to a single compound. The route to these new compounds has been demonstrated to involve an initial slow oxidation followed by a rapid C-H activation to give doubly cyclometallated complexes. The solid state structures of a number of both the platinum(II) and the platinum(IV) species have been solved; many of the structures exhibited extended interactions that result in complex three dimensional packing.

Magnetic, spectroscopic, and structural studies of dicobalt hydroxamates and model hydrolases.

The cobalt(II) urease model complex [Co(2)(mu-OAc)(3)(urea)(tmen)(2)][OTf] (2) prepared from the cobalt model hydrolase [Co(2)(mu-H(2)O)(mu-OAc)(2)(OAc)(2)(tmen)(2)] (1) undergoes facile reaction with acetohydroxamic acid (AHA) to give the monobridged hydroxamate complex [Co(2)(mu-OAc)(2)(mu-AA)(urea)(tmen)(2)][OTf]( )()(3) while 1 gives the dibridged hydroxamate complex [Co(2)(mu-OAc)(mu-AA)(2)(tmen)(2)][OTf] (4). The structures and Co-Co distances of the hydroxamate derivatives of 1 and 2 are very close to those of their nickel analogues and suggest that hydroxamic acids can also inhibit cobalt-based hydrolases as well as inhibiting urease. 1 also reacts with glutarodihydroxamic acid (gluH(2)A(2)) to eliminate hydroxylamine with formation of [Co(2)(mu-OAc)(2)[mu-O(N) (OC)(2)(CH(2))(3)](tmen)(2)][OTf] (5), the structure of which is very close to that of its nickel analogue. Both 1 and 3 show weak antiferromagnetic coupling. Oxidation of 1 with H(2)O(2) gives three dicobalt(III) hydroxy complexes (7-9), the first of which [Co(2)(mu-OAc)(2)(OAc)(2)(mu-OH)(tmen)(2)][OTf] (7) contains a bridging hydroxyl and the second [Co(2)(mu-OAc)(2)(OAc)(mu-OH)(OH)(tmen)(2)][OTf] (8) containing both a bridging and terminal hydroxyl, while the third [Co(2)(mu-OAc) (OAc)(2)(mu-OH)(2)(tmen)(2)][OTf] (9) contains two bridging OH groups with mixed-valence Co(II)/(Co(III) intermediates.

Subcellular localisation of the X protein in HBV infected hepatocytes.

Hepatitis B virus X protein (HBx) is a multifunctional protein that exerts its effects primarily by acting as a transcriptional transactivator of viral and multiple host cell genes. HBx is thought to be essential for maintaining viral replication and has been implicated in the development of hepatocellular carcinoma in patients chronically infected with hepatitis B virus. Very little is known about its functional mechanisms and although interactions with several nuclear and cytoplasmic proteins have been demonstrated in vitro, there is no clear consensus as to where HBx localises in infected hepatocytes. In this study, the expression and intracellular distribution of HBx were examined in human liver biopsies using an anti-HBx rabbit polyclonal antiserum. HBx was detected in a high proportion (69%) of samples from patients with chronic HBV infection. Detection of HBx correlated with the absence of cirrhosis and the presence of serum e-antigen. HBx was detected predominantly in the cytoplasm; however, it was also found in the nuclei of up to 20% of positively stained hepatocytes, either exclusively nuclear or localised both in the nucleus and cytoplasm within the same cell. Furthermore, the intracellular distribution of HBx was analysed in transfected Huh-7 cells by confocal microscopy, using the monoclonal antibody 16F1. In these experiments, a substantial nuclear detection was confirmed in a significant proportion of HBx expressing cells. The data indicate a high functional significance of nuclear HBx, consistent with the concept that transactivation may involve interactions with nuclear proteins.

Subcellular localisation of NS3 in HCV-infected hepatocytes.

Hepatitis C virus (HCV) NS3 is a multifunctional protein with both protease and helicase activities and has been shown to interact with host cell proteins. It is shown that NS3 is present in the hepatocytes from patients with chronic HCV infection by using anti-NS3 antisera. NS3 is detectable in approximately 4% of the hepatocytes from these patients. In most infected cells, NS3 is present in the cytoplasm; however, in a minority of HCV-infected cells, both the cytoplasm and the nucleus or the nucleus on its own are positive for NS3. The presence of NS3 in the nuclei of hepatocytes in chronically infected patients indicates that the protein may play a role other than in virus replication, such as in persistence of HCV infection.

Synthesis of E- and Z-pyrazolylacrylonitriles and their evaluation as novel antioxidants.

A facile synthesis of (Z)- and (E)-2-(5-arylpyrazol-3-yl)-3-(pyrrol-2-yl)acrylonitriles and (Z)-2-(1,3-diarylpyrazol-5-yl)-3-pyrrol-2-yl)acrylonitriles, and isomerisation of (Z)-2-(5-arylpyrazolyl)acrylonitriles to (E)-2-(5-arylpyrazolyl)acrylonitriles under basic conditions have been reported. (Z)-2-(1,3-Diarylpyrazolyl)acrylonitriles did not undergo isomerisation under the similar conditions. New compounds were identified on the basis of their spectral data (1H-, 13C-, 1H-1H COSY, NOESY, NOE, HMQC NMR, IR, UV and EI mass). The structures of one acrylonitrile and five of their precursor 6-arylpyran-2-ones and cyanomnethylpyrazoles were confirmed by X-ray crystallographic studies. Effects of pyrazolylacrylonitriles and their precursors on rat liver-microsomal lipid peroxidation were evaluated in vitro with a view to establish structure activity relationship and to identify a lead compound.

Characterization and mutational analysis of the helicase and NTPase activities of hepatitis C virus full-length NS3 protein.

The non-structural protein 3 (NS3) of hepatitis C virus (HCV) possesses three activities which are likely to be essential for virus replication; a serine protease located in the N terminus and helicase and NTPase activities located in the C terminus. Sequence analysis of the helicase/NTPase domain has identified motifs indicative of the DEAD-box family of helicases. Here we present the characterization of the helicase and NTPase activities of full-length NS3, expressed as a His-tagged fusion protein in E. coli, and make comparisons with published data of NS3 helicase domain alone. The helicase and NTPase activities of full-length NS3 have been demonstrated and we have characterized the effects of amino acid substitutions on conserved motifs of NS3 helicase. Helicase and NTPase activities were dependent on Mg2+ and ATP and inhibited by monovalent cations. NS3 was able to hydrolyse all four NTPs and dNTPs to drive DNA duplex unwinding but with differing abilities. NTPase activity was stimulated by all polynucleotides tested, with poly(U) having the greatest effect. Mutational analysis of conserved motifs of NS3 helicase showed all conserved residues to be required for optimal activity. These results are in accord with a recently proposed model for NS3 helicase activity.

Lipase-catalysed selective deacetylation of phenolic/enolic acetoxy groups in peracetylated benzyl phenyl ketones.

Highly chemo- and regioselective de-esterification has been observed in the deacetylation of peracetylated enolic forms of polyphenolic benzyl phenyl ketones by lipase from porcine pancreas (PPL) suspended in tetrahydrofuran (THF). The enzyme selectively deacetylates the enolic acetoxy over the phenolic acetoxy group(s) and continuation of the reaction resulted, in addition the regioselective deacetylation of acetoxy function para to the nuclear carbonyl group.

Assembly of recombinant Newcastle disease virus nucleocapsid protein into nucleocapsid-like structures is inhibited by the phosphoprotein.

A recombinant baculovirus expressing the nucleocapsid gene (NP) of Newcastle disease virus (NDV), a member of the genus Rubulavirus, has been generated and shown to express the native protein to high levels in insect cells. In contrast to the NP protein of the rubulavirus human parainfluenza virus 2, the NDV protein has been demonstrated by electron microscopy and caesium chloride gradient analysis to be capable of self-assembly in vivo to form nucleocapsid-like structures in the absence of other NDV proteins. These structures, which contained RNA that was resistant to micrococcal nuclease digestion, were also observed when the protein was expressed in E. coli, a phenomenon which was not inhibited by the presence of a 40 amino acid fusion region at the amino terminus of the protein. Further, the formation of these structures was inhibited by the co-expression of the phosphoprotein (P). Therefore, we conclude that the P protein acts as a chaperone, preventing uncontrolled encapsidation of non-viral RNA by NP protein.

A diagnostic immunoassay for Newcastle disease virus based on the nucleocapsid protein expressed by a recombinant baculovirus.

A recombinant baculovirus containing a cDNA clone encoding the nucleocapsid (NP) protein of Newcastle disease virus (strain Ulster 2C) has been used to infect insect cells (Spodoptera frugiperda). High levels of overexpressed NP protein were observed, comprising up to 40% of total cellular protein, which were subsequently shown to be antigenic. Nucleoprotein derived from the crude soluble lysate of infected insect cells has been used in an indirect ELISA to detect the presence of anti-NDV antibodies in a cohort of chicken sera. Data produced from these tests indicated a good correlation between ELISA titre and haemagglutination inhibition test data. The test was not affected by interference from background cellular proteins nor by cross-reactivity with non-NDV poultry pathogens. Additionally, the test did not generate false-positive readings.

The Newcastle disease virus V protein binds zinc.

The V protein of Newcastle disease virus (NDV) is produced by the insertion of a single nontemplated G residue at a specific point during transcription of the phosphoprotein (P) gene, accessing a new reading frame upon translation. The V protein, in common with its counterpart in other paramyxoviruses contains a highly cysteine rich motif near the carboxyl terminus, suggestive of a zinc-binding domain. By constructing E. coli overexpression plasmids for the NDV P and V proteins, and monitoring the binding of 65ZnCl2 to proteins electroblotted onto nitrocellulose membranes, we have demonstrated that the V protein strongly binds zinc.