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PubMed2005

13C and 18O isotopic analysis to determine the origin of L-tartaric acid.

Due to the ever-increasing amount of attention paid to the 'naturalness' of ingredients in food and beverages by both consumers and controlling authorities, the search for suitable methods for the characterisation of origin is of primary importance. Within the European Community the wine production industry is often faced with the problem of origin control of tartaric acid. This has led to the decision that only L-tartaric acid extracted from grapes (therefore natural) should be used. In order to implement these regulations, a screening of different techniques has been carried out to assess the methodology that best identifies the origin of the tartaric acid. It has already been indicated in previous scientific literature that isotope ratio mass spectrometry is an ideal technique for this type of identification. In this paper we present the results obtained for the measurement of the isotope ratios of carbon-13 and oxygen-18 of natural and synthetic samples of L-tartaric acid considering also natural samples of different geographical origin and years of production.

Carbon Isotopes↗
PubMed2005

Determination of proanthocyanidins in fresh grapes and grape products using liquid chromatography with mass spectrometric detection.

Fresh grapes and grape products, such as grape wine and grape juice, were analyzed for proanthocyanidins (PACs) using liquid chromatography with electrospray ionization mass spectrometric (MS) detection. PACs were successfully separated and analyzed on the basis of their protonated molecules, allowing the identification of PACs in different degrees of polymerization from monomers to oligomers (up to 7 units), and in various isomeric forms. Using reversed-phase high-performance liquid chromatography (HPLC) combined with MS detection, the PAC monomers, (+)-catechin (C), (-)-epicatechin (EC), (-)-catechin gallate (CG), and (-)-epicatechin gallate (ECG), were successfully quantified using selected ion monitoring (SIM) mode. Standard curves were fitted for each PAC ranging from 43.8 to 5600 ng/mL for C, from 42.2 to 5400 ng/mL for EC, from 36.7 to 4700 ng/mL for CG, and from 39.8 to 5100 ng/mL for ECG. Good linearity (r2>0.999) was achieved for each analyte. The accuracy and precision (RSD) were within 10% (n=8) at the limit of detection. This method allows direct quantification of monomeric PACs in fresh grapes and grape-derived products. Additionally, flow injection analysis (FIA) was applied to estimate the concentration levels of PAC oligomers by comparing their FIA-MS peak areas, which were well correlated (r2=0.936) to the total concentrations of PAC monomers.

Chromatography, Liquid↗
PubMed1998

Efficient antibody generation using histone H1 subfractions purified from western blots.

Linker histones from grapevine were purified by electrophoretic methods (SDS-PAGE and electrotransfer onto a nitrocellulose sheet). Individual linker histones were recovered after nitrocellulose solubilization by acetone. The proteins precipitated after this treatment were used as antigen for subsequent immunizations of mice. Such purified immunogens were injected into mice with dimethyl sulfoxide, as the presence of nitrocellulose-protein complexes in the antigen pellets after the acetone treatment was suspected. The resulting antisera were specific to the injected antigens after only the first immunization and could be used as specific probes in immunohistological studies. Our approach seems more efficient (in using less antigen and obtaining a faster response) than the classical procedure recommended for histones in general (B. D. Stollar and M. Ward, 1970, J. Biol. Chem. 245, 1261-1266) and other methods that use free linker histone as immunogen.

Animals↗
PubMed2001

Chiral analysis of amino acids using electrochemical composite bienzyme biosensors.

The construction and performance of bienzyme amperometric composite biosensors for the selective determination of l- or d-amino acids is reported. D- or L-Amino acid oxidase, horseradish peroxidase, and the mediator ferrocene were coimmobilized by simple physical inclusion into the bulk of a graphite-70% Teflon electrode matrix. Working conditions including amino acid oxidase loading and pH were optimized. Studies on the repeatability of the amperometric response obtained at +0.00 V, with and without regeneration of the electrode surface by polishing, on the useful lifetime of one single biosensor and on the reproducibility in the fabrication of different biosensors illustrate the robustness of the bioelectrodes design. Calibration plots by both amperometry in stirred solutions and flow injection with amperometric detection were obtained for L-arginine, L-phenylalanine, L-leucine, L-methionine, L-tryptophan, D-leucine, D-methionine, D-serine, and D-valine. Differences in sensitivity were discussed in terms of the hydrophobicity of the substrate and of the electrode surface. The bienzyme composite electrode was applied to the determination of L- and D-amino acids in racemic samples, as well as to the estimation of the L-amino acids content in muscatel grapes.

Amino Acid Oxidoreductases↗
PubMed1998

Induction of systemic lupus erythematosus-like disease in mice by immunization with heparan sulfate.

Experimental systemic lupus erythematosus (SLE)-like disease was induced in BALB/c mice by immunization with heparan sulfate, the major glycosaminoglycan of glomerular basement membrane. Following booster injections with heparan sulfate (HS), high levels of anti-HS, anti-dsDNA, and anti-cardiolipin antibodies were detected in the sera of the immunized mice. An enzyme-linked immunospot (ELISPOT) assay indicted that IgG anti-HS and anti-dsDNA antibody-secreting cells were present in the kidneys and most likely contributed to antibody localization. Antibodies eluted from the kidneys of immunized mice were found to react strongly with HS and dsDNA when tested in vitro. The HS-immunized mice developed moderate to severe levels of proteinuria. Histologic examination of kidneys from HS-immunized mice revealed deposition of immunoglobulin in the kidneys. Our results describe the induction of SLE-like disease in normal mice following immunization with HS. This experimental model may be useful for understanding the immunologic basis for autoimmunity to HS.

Animals↗
PubMed1996

Isolation and functional characterization of IL-2 responsive T cell clones from NZB x NZW F1 mice.

Autoantigen-reactive T cells might play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Autoantigen-reactive T cell clones were generated from spleens of NZB x NZW F1 (BWF1) and normal control BALB/c mice with interleukin-2 (IL-2), a procedure that selects for in vivo activated antigen-reactive T cells. The antigen-specificity of the T cell clones was tested by using a panel of candidate autoantigens. The T cell clones from BWF1 mice but not those from BALB/c mice proliferated against heparan sulfate, the major glycosaminoglycan of glomerular basement membrane. None of the clones proliferated against dsDNA or cardiolipin. All the heparan sulfate-reactive T cell clones had the ability to selectively augment the production of IgG anti-dsDNA autoantibodies. When cultured with either heparan sulfate or Concanavalin A, the T cell clones produced high levels of IL-4 and IL-5 with no detectable IL-2 or IFN-gamma. In contrast, T cell clones derived from BALB/c mice augmented the production of total polyclonal IgG but not the production of anti-dsDNA antibodies. These studies indicate the existence of heparan sulfate-reactive T cells in BWF1 mice. Characterization of heparan sulfate-reactive T cells that could selectively augment anti-dsDNA production will permit the design of targeted and antigen-specific therapy.

Animals↗
PubMed1999

Suppression of systemic lupus erythematosus disease in mice by oral administration of kidney extract.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of antibodies reactive with a variety of self and non-self antigens. A number of immunomodulatory therapies have been investigated for the disease process. Intragastric administration of low dose kidney extract (KE) three times weekly for 5 weeks and then weekly until 6 months of age in SLE mice, showed decreased anti-dsDNA antibody levels, less kidney damage and significantly prolonged survival compared with control phosphate buffered saline (PBS)-fed mice. The KE-fed mice also exhibited reduced T cell proliferative response to KE in comparison with PBS-fed controls. Serum isotype distribution of the anti-dsDNA antibodies revealed a marked reduction of IgG1 and IgG3 responses in the KE-fed mice. While the renal inflammatory cell infiltration and expression of interleukin-4 (IL-4) and IL-10 were markedly suppressed, no local enhancement of transforming growth factor-beta (TGF-beta) was detected. Oral administration of low dose KE, however, upregulated expression of IL-2, IFN-gamma and TNF-alpha in the kidneys and suppressed glomerulonephritis. These findings suggest that oral KE affects the disease process in SLE and raise the possibility that oral administration of KE or other potential autoantigens may provide a new approach for the treatment of SLE.

Administration, Oral↗
PubMed1999

A strategy for the isolation of catalytic activities from repertoires of enzymes displayed on phage.

We have aimed at developing a general methodology for the isolation of enzymatic activities from large repertoires of protein displayed on the surface of a filamentous phage. When selecting for protein binders by phage display, phage particles with suitable specificities are physically isolated by affinity capture and amplified by bacterial infection. Selection for catalysis mediated by enzymes displayed on filamentous phage is more difficult, as reaction products (which represent the biochemical memory of the reaction catalysed by the phage particle) diffuse away after the reaction is complete. We reasoned that if we were able to anchor the reaction products on the phage surface, the catalytically active phages could then be physically isolated using specific anti-product affinity reagents. We achieve the conditional anchoring of reaction substrates and products on phage by displaying enzyme-calmodulin chimeric proteins on filamentous phage as gene III fusions. Such phage particles can be targeted in a stable fashion (koff<10(-4) s(-1)) by chemical derivatives of a calmodulin-binding peptide. The peptide-phage complexes are stable in purification procedures such as capture with magnetic beads and polyethylene glycol precipitation, and can be conditionally dissociated by addition of calcium chelators. Glutathione-S-transferase and an endopeptidase were used in model selection experiments to demonstrate that it is possible to isolate catalytic activities from calmodulin-tagged enzymes displayed on filamentous phage, with enrichment factors >50 per round of selection.

Amino Acid Sequence↗
PubMed2001

A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold.

We report the design, construction and use of an antibody bacteriophage display library built on the scaffold of a single-chain variable fragment (scFv) previously proven to be functionally expressed in the reducing environment of both bacterial and plant cytoplasm and endowed with intrinsic high thermodynamic stability. Four amino acid residues of the third hypervariable loop (CDR3) of both VH and VL were combinatorially mutated, generating a repertoire of approximately 5x10(7) independent scFvs, cloned in a phagemid vector. The ability of the antibody phage library to yield specific binders was tested by biopanning against several antigens. Successful selection of fully active scFvs was obtained, confirming the notion that combinatorial mutagenesis of few amino acid residues centrally located in the antigen-binding site is sufficient to provide binding specificities against virtually any target. High yields of both soluble and phage antibodies were obtained in Escherichia coli. Maintenance of the cognate scFv antibody stability in the newly selected scFv fragments was demonstrated by guanidinium chloride denaturation/renaturation studies and by soluble antibody expression in the bacterial cytoplasm. The antibody library described here allows the isolation of new stable binding specificities, potentially exploitable as immunochemical reagents for intracellular applications.

Amino Acid Sequence↗
PubMed1997

High-Temperature Rotational Transitions of Water in Sunspot and Laboratory Spectra

Assignments are presented for spectra of hot water obtained in absorption in sunspots (T approximately 3000&deg;C and 750 </= nu; </= 1010 cm-1) and in emission in the laboratory (T approximately 1550&deg;C and 370 </= nu; </= 930 cm-1). These assignments are made using variational nuclear motion calculations based on a high-level ab initio electronic surface, with allowance for both adiabatic and nonadiabatic corrections to the Born-Oppenheimer approximation. Some 3000 of the 4700 transitions observed in the laboratory spectrum are assigned as well as 1687 transitions observed in the sunspot spectrum. All strong lines are now assigned in the sunspot measurements. These transitions involve mostly high-lying rotational levels within the (0,0,0), (0,1,0), (0,2,0), (1,0,0), and (0,0,1) vibrational states. Transitions within the (0,3,0), (0,4,0), (1,1,0), (0,1,1), (0,2,1), (1,1,1), (1,2,0), and (1,0,1) states are also assigned. For most bands the range of Ka values observed is significantly extended, usually doubled. New features observed include numerous cases where the closely degenerate levels JKaKc and JKaKc+1 with high Ka are split by Coriolis interactions. Comparisons are made with the recent line list of Partridge and Schwenke (1997, J. Chem. Phys. 106, 4618). Copyright 1997 Academic Press. Copyright 1997Academic Press

Journal Article↗
PubMed1998

Water Vapor Line Assignments in the Near Infrared.

The high-resolution spectrum of water vapor between 13 200 and 16 500 cm-1 recorded by J.-Y. Mandin, J-P. Chevillard, C. Camy-Peyret, J.-M. Flaud, and J. W. Brault (1986. J. Molec. Spectrosc., 116, 167) is analyzed using high-accuracy linelists obtained using ab initio calculations and spectroscopically determined potential. Assignments to H216O transitions are presented for 663 of the 795 unassigned lines presented in the original paper. In addition, 38 lines are reassigned. The majority of these assignments and reassignments are confirmed by combination differences. These assignments significantly extend the measured data for the 4nu and 4nu + delta polyads and provide the first information on the (240), (033), (160), (170), and (071) bands. It is likely that a significant fraction of the remaining unassigned lines belong to H218O. Copyright 1998 Academic Press.

Journal Article↗
PubMed2002

Genetic variability among flavescence dorée phytoplasmas from different origins in Italy and France.

Flavescence dorée is a devastating disease of grapevine widespread in several countries in EU such as France, Italy and Spain. Genetic variability among 17 Italian and 3 French FD strains was investigated by RFLP analyses based on a fragment of the ribosomal protein operon and on the non-ribosomal DNA fragment FD9. RFLP analysis of the PCR amplified ribosomal protein fragment, coding for the 3' end of rpl22 and the entire rps3 genes, differentiated 4 rp-subgroups among the FD strains and 4 subgroups among the reference strains belonging to elm yellows group (16SrV). Sequencing and phylogenetic analysis of the same ribosomal protein DNA fragment validated the delineation of 4 distinct FD strain types derived by RFLP analyses. The results supported the differentiation based on analysis of the non-ribosomal DNA fragment FD9. The phylogenetic analysis further revealed relationships and a probable evolutionary trend among the FD strains and the other representatives of elm yellows group. All the FD strains together with the reference strains ALY, RuS and JWB formed a cluster very well distinct from the EY/ULW cluster. Moreover, ALY was shown to be more closely related to three FD strain types: the Lombardia/Piemonte, the French FD70, and the French FD88/Italian FD-D strain clusters.

DNA, Bacterial↗
PubMed2001

Involvement of RNA2-encoded proteins in the specific transmission of Grapevine fanleaf virus by its nematode vector Xiphinema index.

The nepovirus Grapevine fanleaf virus (GFLV) is specifically transmitted by the nematode Xiphinema index. To identify the RNA2-encoded proteins involved in X. index-mediated spread of GFLV, chimeric RNA2 constructs were engineered by replacing the 2A, 2B(MP), and/or 2C(CP) sequences of GFLV with their counterparts in Arabis mosaic virus (ArMV), a closely related nepovirus which is transmitted by Xiphinema diversicaudatum but not by X. index. Among the recombinant viruses obtained from transcripts of GFLV RNA1 and chimeric RNA2, only those which contained the 2C(CP) gene (504 aa) and 2B(MP) contiguous 9 C-terminal residues of GFLV were transmitted by X. index as efficiently as natural and synthetic wild-type GFLV, regardless of the origin of the 2A and 2B(MP) genes. As expected, ArMV was not transmitted probably because it is not retained by X. index. These results indicate that the determinants responsible for the specific spread of GFLV by X. index are located within the 513 C-terminal residues of the polyprotein encoded by RNA2.

Animals↗
PubMed2002

Potential health benefits from the flavonoids in grape products on vascular disease.

In the dog, monkey, a nd human we have shown that 5 ml/kg of red wine or 5-10 ml/kg of purple grape juice but not orange or grapefruit juice inhibits platelet activity, and protects against epinephrine activation of platelets. Red wine and purple grape juice enhances platelet and endothelial production of nitric oxide (Fitzpatrick et al., 1993, Parker et al., 2000). This is thought to be one of the mechanisms whereby purple grape juice significantly improved endothelial function in 15 patients with coronary artery disease. The consumption of purple grape juice by the patients also offered increased protection against LDL cholesterol oxidation, even though all the patients were also taking another antioxidant vitamin E, 400 IU/day. The number of people and animals in these studies was small; however, each one acted as their own control as measurements were made in each before, and then after consumption of red wine or purple grape juice. Thus these studies are thought to be significant. We feel that the results of these studies are encouraging and justify further research on larger numbers of subjects. This suggests that the flavonoids in purple grape juice and red wine may inhibit the initiation of atherosclerosis by one or more of the mechanisms described above. It will take years to fully characterize the potential benefits of daily consumption of red wine or purple grape juice for maintaining a healthy heart. Based on the existing evidence of antiplatelet and antioxidant benefits and improved endothelial function from red wine and purple grape juice, it seems reasonable to suggest that moderate amounts of red wine or purple grape juice be included among the 5-7 daily servings of fruits and vegetables per day as recommended by the American Heart Association to help reduce the risk of developing cardiovascular disease.

Adult↗