Syntaxin 7 complexes with mouse Vps10p tail interactor 1b, Syntaxin 6, vesicle-associated membrane protein (VAMP)8, and VAMP7 in B16 melanoma cells

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7, Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha -synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMPS to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.

Intramolecular electronic energy transfer in bichromophoric macrocyclic complexes

Efficient intramolecular electronic energy transfer (EET) has been demonstrated for three novel bichromophoric compounds utilizing a macrocyclic spacer as the bridge between the electronic energy donor and acceptor fragments. As their free base forms, emission from the electronically excited donor is absent and the acceptor emission is reductively quenched via photoinduced oxidation of proximate amine lone pairs. As their Zn(II) complexes, excitation of the donor results in sensitization of the electronic acceptor emission.

An XPS study of an isomorphous trivalent lanthanoid series

X-ray photoelectron spectroscopy has been used to investigate the core electron binding energies within an isomorphous series of lanthanoid complexes; [LnL(1)] where Ln=La-Lu (except Pm) and H3L is the heptadentate ligand 2,2',2-tris(salicylideneimino)triethylamine. This study also examines spin-orbit coupling of the trivalent lanthanoids bound to this organic ligand and addresses the issue of satellite peaks in the spectra. (C) 2002 Elsevier Science B.V. All rights reserved.

Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae

Picornavirus RNA replication requires the formation of replication complexes (RCs). consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited. Since BFA inhibits COPI-dependent steps in endoplasmic reticulum (ER)-Golgi transport, we tested a hypothesis that different picornaviruses may have differential requirements for COPI in the formation of their RCs. Using immunofluorescence and cryo-immunoelectron microscopy we examined the association of a COPI component, beta-COP, with the RCs of EMCV, ParV1, and EV11 EMCV RCs did not contain beta-COP. In contrast, beta-COP appeared to be specifically distributed to the RCs of EV11 In ParV1-infected cells beta-COP was largely dispersed throughout the cytoplasm, with some being present in the RCs. These results suggest that there are differences in the involvement of COPI in the formation of the RCs of various picornaviruses, corresponding to their differential sensitivity to BFA. EMCV RCs are likely to be formed immediately after vesicle budding from the ER, prior to COPI association with membranes. ParV1 RCs are formed from COPI-containing membranes but COPI is unlikely to be directly involved in their formation, whereas formation of EV11 RCs appears to be dependent on COPI association with membranes.

From molecular complexes to zwitterions and final products. Reactions between C3O2 and amines

The formation of molecular complexes (prereactive intermediates) between C3O2 and amines (ammonia, dimethylamine, trimethylamine, and 4-(dimethylamino)pyridine) as well as the subsequent transformation of the complexes into C3O2-amine zwitterions in cryogenic matrixes (ca. 40 K) has been observed. In the case of dimethylamine, the formation of tetramethylmalonamide has also been documented. Calculations using density functional theory (B3LYP/6-31G(2d, p)) are used to assign all above species and are in excellent agreement with the IR spectra.

New W-isotope evidence for rapid terrestrial accretion and very early core formation

The short-lived Hf-182-W-182-isotope system is an ideal clock to trace core formation and accretion processes of planets. Planetary accretion and metal/silicate fractionation chronologies are calculated relative to the chondritic Hf-182-W-182-isotope evolution. Here, we report new high-precision W-isotope data for the carbonaceous chondrite Allende that are much less radiogenic than previously reported and are in good agreement with published internal Hf-W chronometry of enstatite chondrites. If the W-isotope composition of terrestrial rocks, representing the bulk silicate Earth, is homogeneous and 2.24 epsilon(182W) units more radiogenic than that of the bulk Earth, metal/silicate differentiation of the Earth occurred very early. The new W-isotope data constrain the mean time of terrestrial core formation to 34 million years after the start of solar system accretion. Early terrestrial core formation implies rapid terrestrial accretion, thus permitting formation of the Moon by giant impact while Hf-182 was still alive. This could explain why lunar W-isotopes are more radiogenic than the terrestrial value. Copyright (C) 2002 Elsevier Science Ltd.

Syntheses of polycationic dendrimers on lipophilic peptide core for complexation and transport of oligonucleotides

Synthesis of novel polycationic lipophilic peptide core(s) was accomplished and these agents successfully transfected human retinal pigment epithelium cells with ODN1 upon complexation with the oligonucleotide. The level of transfection was indirectly measured by the decreased production of the protein hVEGF (human vascular endothelial growth factor) in comparison to the transfection agent cytofectin GSV(TM). (C) 2002 Elsevier Science Ltd. All rights reserved.

A lipid core peptide construct containing a conserved region determinant of the group a streptococcal M protein elicits heterologous opsonic antibodies

The study reported here investigated the immunogenicity and protective potential of a lipid core peptide (LCP) construct containing a conserved region determinant of M protein, defined as peptide J8. Parenteral immunization of mice with LCP-J8 led to the induction of high-titer serum immunoglobulin G J18-specific antibodies when the construct was coadministered with complete Freund's adjuvant (CFA) or administered alone. LCP-J8 in CFA had significantly enhanced immunogenicity compared with the monomeric peptide J8 given in CFA. Moreover, LCP-J8/CFA and LCP-J8 antisera opsonized four different group A streptococcal (GAS) strains, and the antisera did not cross-react with human heart tissue proteins. These data indicate the potential of an LCP-based M protein conserved region GAS vaccine in the induction of broadly protective immune responses in the absence of a conventional adjuvant.

Enhancing the immunogenicity and modulating the fine epitope recognition of antisera to a helical group A streptococcal peptide vaccine candidate from the M protein using lipid-core peptide technology

A conserved helical peptide vaccine candidate from the M protein of group A streptococci, p145, has been described. Minimal epitopes within p145 have been defined and an epitope recognized by protective antibodies, but not by autoreactive T cells, has been identified. When administered to mice, p145 has low immunogenicity. Many boosts of peptide are required to achieve a high antibody titre (> 12 800). To attempt to overcome this low immunogenicity, lipid-core peptide technology was employed. Lipid-core peptides (LCP) consist of an oligomeric polylysine core, with multiple copies of the peptide of choice, conjugated to a series of lipoamino acids, which acts as an anchor for the antigen. Seven different LCP constructs based on the p145 peptide sequence were synthesized (LCP1-->LCP7) and the immunogenicity of the compounds examined. The most immunogenic constructs contained the longest alkyl side-chains. The number of lipoamino acids in the constructs affected the immunogenicity and spacing between the alkyl side-chains increased immunogenicity. An increase in immunogenicity (enzyme-linked immunosorbent assay (ELISA) titres) of up to 100-fold was demonstrated using this technology and some constructs without adjuvant were more immunogenic than p145 administered with complete Freund's adjuvant (CFA). The fine specificity of the induced antibody response differed for the different constructs but one construct, LCP4, induced antibodies of identical fine specificity to those found in endemic human serum. Opsonic activity of LCP4 antisera was more than double that of p145 antisera. These data show the potential for LCP technology to both enhance immunogenicity of complex peptides and to focus the immune response towards or away from critical epitopes.

Core outcome domains for chronic pain clinical trials: IMMPACT recommendations

Objective. To provide recommendations for the core outcome domains that should be considered by investigators conducting clinical trials of the efficacy and effectiveness of treatments for chronic pain. Development of a core set of outcome domains would facilitate comparison and pooling of data, encourage more complete reporting of outcomes, simplify the preparation and review of research proposals and manuscripts, and allow clinicians to make informed decisions regarding the risks and benefits of treatment. Methods. Under the auspices of the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT), 27 specialists from academia. governmental agencies, and the pharmaceutical industry participated in a consensus meeting and identified core outcome domains that should be considered in clinical trials of treatments for chronic pain. Conclusions. There was a consensus that chronic pain clinical trials should assess outcomes representing six core domains: (1) pain, (2) physical functioning, (3) emotional functioning, (4) participant ratings of improvement and satisfaction with treatment, (5) symptoms and adverse events, (6) participant disposition (e.g. adherence to the treatment regimen and reasons for premature withdrawal from the trial). Although consideration should be given to the assessment of each of these domains, there may be exceptions to the general recommendation to include all of these domains in chronic pain trials. When this occurs, the rationale for not including domains should be provided. It is not the intention of these recommendations that assessment of the core domains should be considered a requirement for approval of product applications by regulatory agencies or that a treatment must demonstrate statistically significant effects for all of the relevant core domains to establish evidence of its efficacy. (C) 2003 International Association for the Study of Pain.

Amino acids 1-20 of the hepatitis C virus (HCV) core protein specifically inhibit HCV IRES- dependent translation in Hep G2 cells, and inhibit both HCV IRES- and cap-dependent translation in HuH7 and CV-1 cells.

A self-modulating mechanism by the hepatitis C virus (HCV) core protein has been suggested to influence the level of HCV replication, but current data on this subject are contradictory. We examined the effect of wild-type and mutated core protein on HCV IRES- and cap-dependent translation. The wild-type core protein was shown to inhibit both IRES- and cap-dependent translation in an in vitro system. This effect was duplicated in a dose-dependent manner with a synthetic peptide representing amino acids 1-20 of the HCV core protein. This peptide was able to bind to the HCV IRES as shown by a mobility shift assay. In contrast, a peptide derived from the hepatitis B virus (HBV) core protein that contained a similar proportion of basic residues was unable to inhibit translation or bind the HCV IRES. A recombinant vaccinia-HCV core virus was used to examine the effect of the HCV core protein on HCV IRES-dependent translation in cells and this was compared with the effects of an HBV core-recombinant vaccinia virus. In CV-1 and HuH7 cells, the HCV core protein inhibited translation directed by the IRES elements of HCV, encephalomyocarditis virus and classical swine fever virus as well as cap-dependent translation, whereas in HepG2 cells, only HCV IRES-dependent translation was affected. Thus, the ability of the HCV core protein to selectively inhibit HCV IRES-dependent translation is cell-specific. N-terminal truncated (aa 1-20) HCV core protein that was expressed from a novel recombinant vaccinia virus in cells abrogated the inhibitory phenotype of the core protein in vivo, consistent with the above in vitro data.

Use of a thermodilution catheter to measure core body temperature, central venous pressure and cardiac output during anaesthesia and surgery in the dog

The use of thermodilution and other methods of monitoring in dogs during surgery and critical care was evaluated. Six Greyhounds were anaesthetised and then instrumented by placing a thermodilution catheter into the pulmonary artery via the jugular vein. A catheter in the dorsal pedal artery also permitted direct measurement of arterial pressures. Core body temperature (degreesC) and central venous pressure (mmHg) were measured, while cardiac output (mL/min/kg) and mean arterial pressure (mmHg) were calculated. A mid-line surgical incision was performed and the physiological parameters were monitored for a total of two hours. All physiological parameters generally declined, although significant increases (P<0.05) were noted for cardiac output following surgical incision. Central venous pressure was maintained at approximately 0mmHg by controlling an infusion of sterile saline. Core body temperature decreased from 37.1+/-0.6degreesC (once instrumented) to 36.6+/-0.60degreesC (at the end of the study), despite warming using heating pads. Physiological parameters indicative of patient viability will generally decline during surgery without intervention. This study describes an approach that can be undertaken in veterinary hospitals to accurately monitor vital signs in surgical and critical care patients.

Synthesis and characterization of mono- and bis-ligand zinc(II) and cadmium(II) complexes of the di-2-pyridylketone Schiff base of S-benzyl dithiocarbazate (Hdpksbz) and the X-ray crystal structures of the [Zn(dpksbz)2] and [Cd(dpksbz)NCS]2 complexes

New mono- and bis-chelated zinc(II) and cadmium(II) complexes of formula, [M(dpksbz)NCS] (dpksbz = anionic form of the di-2-pyridylketone Schiff base of S-benzyldithiocarbazate) and [M(dpksbz)(2)] (M = Zn-II, Cd-II) have been prepared and characterized. The structure of the bis-ligand complex, [Zn(dpksbZ)(2)] has been determined by X-ray diffraction. The complex has a distorted octahedral geometry in which the ligands are coordinated to the zinc(II) ion as uninegatively charged tridentate chelates via the thiolate sulfur atoms, the azomethine nitrogen atoms and the pyridine nitrogen atoms. The distortion from a regular octahedral geometry is attributed to the restricted bite angles of the Schiff base ligands. X-ray structural analysis shows that the [Cd(dpksbz)NCS](2) complex is a centrosymmetric dimer in which each of the cadmium(II) ions adopts a five-coordinate, approximately square-pyramidal configuration with the Schiff base acting as a tetradentate chelating agent coordinating a cadmium(II) ion via one of the pyridine nitrogen atoms, the azomethine nitrogen atom and the thiolate sulfur atom; the second pyridine nitrogen atom is coordinated to the other cadmium(II) ion of the dimer. The fifth coordination position around each cadmium(II) is occupied by an N-bonded thiocyanate ligand. (C) 2003 Elsevier Science Ltd. All rights reserved.

Synthesis of optically complex core-shell colloidal suspensions: Pathways to multiplexed biological screening

The ability to generate enormous random libraries of DNA probes via split-and-mix synthesis on solid supports is an important biotechnological application of colloids that has not been fully utilized to date. To discriminate between colloid-based DNA probes each colloidal particle must be 'encoded' so it is distinguishable from all other particles. To this end, we have used novel particle synthesis strategies to produce large numbers of optically encoded particle suitable for DNA library synthesis. Multifluorescent particles with unique and reproducible optical signatures (i.e., fluorescence and light-scattering attributes) suitable for high-throughput flow cytometry have been produced. In the spectroscopic study presented here, we investigated the optical characteristics of multi-fluorescent particles that were synthesized by coating silica 'core' particles with up to six different fluorescent dye shells alternated with non-fluorescent silica 'spacer' shells. It was observed that the diameter of the particles increased by up to 20% as a result of the addition of twelve concentric shells and that there was a significant reduction in fluorescence emission intensities from inner shells as an increasing number of shells were deposited.