Tumor Necrosis Factor-α +489G/A gene polymorphism is associated with chronic obstructive pulmonary disease

Background  

Chronic obstructive pulmonary disease (COPD) is characterized by a chronic inflammatory process, in which the pro-inflammatory cytokine Tumor Necrosis Factor (TNF)-α is considered to play a role. In the present study the putative involvement of TNF-α gene polymorphisms in pathogenesis of COPD was studied by analysis of four TNF-α gene polymorphisms in a Caucasian COPD population.     

A simple method for studying whole sections of rice grain

We developed a new and simple method to collect sections of a whole brown rice kernel for investigation of histological properties. A single kernel of rice was dehydrated through a graded ethanol series, transferred to xylene, and embedded in paraffin. During sectioning of the blocks using a rotary microtome, we used a special adhesive tape to collect and place the sections on slides so they remained flat. The use of the adhesive tape technique combined with autofluorescence characteristics allowed us to visualize cell walls throughout an entire longitudinal or transverse section of a whole rice kernel. We obtained scanning electron microscopy images of the sections to determine section quality.     

Quantifying cumulative phenotypic and genomic evidence for procedural generation of metabolic network reconstructions

Genome-scale metabolic network reconstructions (GENREs) are valuable tools for understanding microbial metabolism. The process of automatically generating GENREs includes identifying metabolic reactions supported by sufficient genomic evidence to generate a draft metabolic network. The draft GENRE is then gapfilled with additional reactions in order to recapitulate specific growth phenotypes as indicated with associated experimental data. Previous methods have implemented absolute mapping thresholds for the reactions automatically included in draft GENREs; however, there is growing evidence that integrating annotation evidence in a continuous form can improve model accuracy. There is a need for flexibility in the structure of GENREs to better account for uncertainty in biological data, unknown regulatory mechanisms, and context-specificity associated with data inputs. To address this issue, we present a novel method that provides a framework for quantifying combined genomic, biochemical, and phenotypic evidence for each biochemical reaction during automated GENRE construction. Our method, Constraint-based Analysis Yielding reaction Usage across metabolic Networks (CANYUNs), generates accurate GENREs with a quantitative metric for the cumulative evidence for each reaction included in the network. The structuring of CANYUNs allows for the simultaneous integration of three data inputs while maintaining all supporting evidence for biochemical reactions that may be active in an organism. CANYUNs is designed to maximize the utility of experimental and annotation datasets and to ultimately assist in the curation of the reference datasets used for the automatic construction of metabolic networks. We validated CANYUNs by generating an E. coli K-12 model and compared it to the manually curated reconstruction iML1515. Finally, we demonstrated the use of CANYUNs to build a model by generating an E. coli Nissle CANYUNs model using novel phenotypic data that we collected. This method may address key challenges for the procedural construction of metabolic networks by leveraging uncertainty and redundancy in biological data.     

Decisive structural determinants for the interaction of proline derivatives with the intestinal H+/peptide symporter.

To elucidate the decisive structural factors relevant for dipeptide-carrier interaction, the affinity of short amide and imide derivatives for the intestinal H+/peptide symporter (PEPT1) was investigated by measuring their ability to inhibit Gly-Sar transport in Caco-2 cells. Dipeptides with proline or alanine in the C-terminal position displayed affinity constants (Ki) of 0.15-1.2 mM and 0.08-9.5 mM, respectively. There was no clear relationship between hydrophobicity, size or ionization status of the N-terminal amino acid and the affinity of the dipeptides. However, analyzing the individual peptide bond conformations of Xaa-Pro dipeptides, a striking correlation between the cis/trans ratios (trans contents 24-70%) and the affinity constants was observed. After correcting the Ki values for the incompetent cis isomers, the Ki corr values of most dipeptides were in a small range of 0.1-0.16 mM. This result revealed the decisive role of peptide bond conformation even for a transport protein that is quite promiscuous in substrate translocation. When measuring affinity constants of Xaa-Pro and Xaa-Sar dipeptides, the cis/trans ratios cannot be ignored. Lower affinities of Lys-Pro, Arg-Pro and Pro-Pro indicate that additional molecular factors affect their binding at PEPT1. The Ki values obtained for the corresponding Xaa-Ala dipeptides support this conclusion. Potential substrates or inhibitors of peptide transport were found among Xaa-piperidides and Xaa-thiazolidides. Dipeptides with N-terminal proline displayed a very diverse affinity profile. However, in contrast to current knowledge, several Pro-Xaa dipeptides such as Pro-Leu, Pro-Tyr and Pro-Pro are recognized by PEPT1 with appreciable affinities. Binding seems mainly determined by the hydrophobicity of the C-terminal amino acid and the rigidity of the structure.     

1H NMR conformational study on n-terminal nonapeptide sequences of HIV-1 Tat protein: a contribution to structure–activity relationships

On the basis of our recent results, the N-terminal sequence of HIV-1 Tat protein as a natural competitive inhibitor of dipeptidyl peptidase IV (DP IV) is supposed to interact directly with the active site of DP IV hence mediating its immunosuppressive effects via specific DP IV interactions. Of special interest is the finding that amino acid substitutions of the Tat(1–9) peptide (MDPVDPNIE) in position 5 with S-isoleucine and in position 6 with S-leucine led to peptides with strongly reduced inhibitory activity suggesting differences in the solution conformation of the three analogues. Therefore, ¹H NMR techniques in conjunction with molecular modelling have been used here to determine the solution structure of Tat(1–9), I⁵-Tat(1–9) and L⁶-Tat(1–9) and to examine the influence of amino acid exchanges on structural features of these peptides. The defined structures revealed differences in the conformations what might be the reason for different interactions of these Tat(1–9) analogues with certain amino acids of the active site of DP IV. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Development of a Biosensor for Detection of Pleural Mesothelioma Cancer Biomarker Using Surface Imprinting

Hyaluronan-linked protein 1 (HAPLN1) which has been shown to be highly expressed in malignant pleural mesotheliomas (MPM), was detected in serum using an electrochemical surface-imprinting method. First, the detection method was optimized using Bovine serum albumin (BSA) as a model protein to mimic the optimal conditions required to imprint the similar molecular weight protein HAPLN1. BSA was imprinted on the gold electrode with hydroxyl terminated alkane thiols, which formed a self-assembled monolayer (SAM) around BSA. The analyte (BSA) was then washed away and its imprint (empty cavity with shape-memory) was used for detection of BSA in a solution, using electrochemical open-circuit potential method, namely potentiometry. Factors considered to optimize the conditions include incubation time, protein concentration, limit of detection and size of electrode. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used to confirm selectivity of imprints. With the obtained imprinting control parameters, HAPLN1 was imprinted in duplicate and the detection of spiked HAPLN1 was successfully conducted in serum.     

Pseudotype Formation of Moloney Murine Leukemia Virus with Sendai Virus Glycoprotein F

Mixed infection of cells with both Moloney murine leukemia virus (MoMLV) and related or heterologous viruses produces progeny pseudotype virions bearing the MoMLV genome encapsulated by the envelope of the other virus. In this study, pseudotype formation between MoMLV and the prototype parainfluenza virus Sendai virus (SV) was investigated. We report for the first time that SV infection of MoMLV producer cells results in the formation of MoMLV(SV) pseudotypes, which display a largely extended host range compared to that of MoMLV particles. This could be associated with SV hemagglutinin-neuraminidase (SV-HN) glycoprotein incorporation into MoMLV envelopes. In contrast, solitary incorporation of the other SV glycoprotein, SV fusion protein (SV-F), resulted in a distinct and narrow extension of the MoMLV host range to asialoglycoprotein receptor (ASGP-R)-positive cells (e.g., cultured human hepatoma cells). Since stably ASGP-R cDNA-transfected MDCK cells, but not parental ASGP-R-negative MDCK cells, were found to be transduced by MoMLV(SV-F) pseudotypes and transduction of ASGP-R-expressing cells was found to be inhibited by ASGP-R antiserum, a direct proof for the ASGP-R-restricted tropism of MoMLV(SV-F) pseudotypes was provided. Cultivation of ASGP-R-positive HepG2 hepatoma cells on Transwell-COL membranes led to a significant enhancement of MoMLV(SV-F) titers in subsequent flowthrough transduction experiments, thereby suggesting the importance of ASGP-R accessibility at the basolateral domain for MoMLV(SV-F) pseudotype transduction. The availability of such ASGP-R-restricted MoMLV(SV-F)-pseudotyped vectors opens up new perspectives for future liver-restricted therapeutic gene transfer applications.     

Derivatives of beta-casomorphins with high analgesic potency

Beta-casomorphin (5) Tyr-Pro-Phe-Pro-Gly, a partial sequence of bovine beta-casein with moderate opioid properties and mu-receptor affinity, was modified by substituting for the natural L-amino acids their D-analogs, and D-pipecolic acid, as well as by amidation of the C-terminal. Substitution of D-Pro or D-pipecolic acid for L-Pro4 considerably increased the analgesic action and the potency on guinea-pig ileum of beta-casomorphin (5) as well as of casomorphin [4] amide. The resulting D-Pro4 analogs Deprolorphin and Deproceptin which showed high analgesic potency after both intracerebroventricular and intravenous administrations. Also, the substitution of D-Phe for L-Phe3 enhanced, even though to a lesser degree, the antinociceptive action. Both naltrexone and naloxone completely blocked the effects in vivo and in vitro. The substitution of D-Pro for L-Pro2 abolished the opioid-like actions, while substituting D-pipecolic acid for L-Pro2 resulted in an increased analgesic effect of remarkably long duration. The correlation of analgesic action with the effects on isolated organs separates the L-Pro4-substituted derivatives and D-Phe3-CM(5) from the other modified casomorphins and morphine, indicating that the analgesic potency of the former was about ten times that of the latter group in the case of identical GPI-potency. This may involve different subpopulations of opiate mu-receptors.     

Are diprotin A (Ile-Pro-Ile) and diprotin B (Val-Pro-Leu) inhibitors or substrates of dipeptidyl peptidase IV?

Dipeptidyl peptidase IV preferably hydrolyzes peptides and proteins with a penultimate proline residue. Umezawa and co-workers (Umezawa et al. (1984) J. Antibiotics 37, 422-425) reported that diprotin A (Ile-Pro-Ile) and diprotin B (Val-Pro-Leu) are inhibitors for dipeptidyl peptidase IV. We could show that both compounds as well as other tripeptides with a penultimate proline residue are substrates for dipeptidyl peptidase IV. An apparent competitive inhibition by those compounds is a kinetic artifact due to the substrate-like structure of such tripeptides.     

Dipeptidyl peptidase IV in the immune system. Effects of specific enzyme inhibitors on activity of dipeptidyl peptidase IV and proliferation of human lymphocytes.

Dipeptidyl peptidase IV (DP IV) is a membrane peptidase playing a significant role in the process of activation and proliferation of human thymus-derived lymphocytes. This conclusion is drawn from (1) the induction of this enzyme on mitogen-activated T lymphocytes (cf. Sch?n, E. & Ansorge, S. (1990) Biol. Chem. Hoppe-Seyler 371, 699-705) and (2) the impairment of different functions of activated T cells in the presence of specific inhibitors and antibodies against DP IV (Sch?n, E. & al. (1987) Eur. J. Immunol 17, 1821-1826). This paper is aimed at testing new active site-specific peptide inhibitors for their efficiency as inhibitors of lymphocyte DP IV and DNA synthesis of mitogen-stimulated lymphocytes. These inhibitors comprise (i) diacylhydroxylamine derivatives of Xaa-Pro or Xaa-Ala peptides, (ii) different oligopeptides with N-terminal Xaa-Pro-sequences, and (iii) amino-acid amides of the pyrrolidide and the thiazolidide type. The thiazolidides of epsilon-(4-nitrobenzyloxycarbonyl)-L-lysine and of L-isoleucine as well as Ala-Pro-nitrobenzoylhydroxylamine are the most effective inhibitors in both test systems, yielding half-maximal inhibitory concentrations in the micromolar range. Cell viability was not impaired in this effective concentration range. Other inhibitors of DP IV are one to two orders of magnitude less efficient in the suppression of lymphocyte proliferation.