LDL cholesterol-lowering effects of grape extract used as a dietary supplement on healthy volunteers.

The aim of this study was to evaluate the effects of Eminol®, the polyphenol-rich grape extract supplement (700 mg), on cardiovascular risk and oxidant stress indicators in a sample of volunteers. A randomized, double-blind, placebo-controlled clinical trial was performed over 56 days and included 60 volunteers. Thirty volunteers took 700 mg of the grape extract, Eminol® (E), and 30 took the placebo (P). On comparison of the results, a decrease in total cholesterol (E: 213.77 ± 4.1 mg/dl and P: 245.57 ± 4.1 mg/dl; p = 0.01) and LDL cholesterol (E: 142.17 ± 3.1 mg/dl and P: 165.13 ± 3.1 mg/dl; p = 0.02) levels as well as an increase in antioxidant capacity (E: 65.63 ± 5.8 µmol TE/mg and P: 57.80 ± 7.7 µmol TE/mg; p < 0.01) and vitamin E (E: 11.46 ± 0.5 µg/ml and P: 9.06 ± 0.5 µg/ml; p = 0.018) was observed. This result indicates that the grape extract Eminol® modulated the lipid profile in terms of cardiovascular risk indicators, lowering total blood cholesterol and LDL cholesterol levels.

Two cDNAs coding for the porcine CD51 (αv) integrin subunit: cloning, expression analysis, adhesion assays and chromosomal localization.

CD51 (α(v)) is an integrin chain that associates with multiple ß integrin chains to form different receptor complexes that mediate important human processes. Pigs show substantial physiological, immunological and anatomical similarities to humans, and are therefore a good model system to study immunological and pathological processes. Here we report the cloning and characterization of two cDNAs produced by alternative splicing that encode two different porcine CD51 proteins that differ in five amino acid residues. Pig CD51 cDNAs encode polypeptides of 1046 or 1041 amino acid residues, respectively, that share with other mammalian homologous proteins a high percentage amino acid identity and the functional domains. Expression analysis of CD51 was carried out at two different levels. RT-PCR analysis revealed that both CD51 transcripts were expressed ubiquitously but heterogeneously, with the exception of some platelets in which only the smallest CD51 transcript was detected. A specific monoclonal antibody against a pig CD51 recombinant protein was made and used in the immunohistochemical localization of CD51 proteins. It showed that CD51 was mainly expressed in hematopoietic cells of myeloid linage, epithelial and endothelial cells, osteoclasts, nervous fibers and smooth muscle. Adhesion assays showed that in the presence of Mn(++) pig α(v)-CHO-B2 transfected cells increased their attachment to fibronectin and vitonectin, but not to fibrinogen. Finally, we localized the CD51 gene on the porcine chromosome 15 (SSC15), q23-q26.

Lights and shadows of proteomic technologies for the study of protein species including isoforms, splicing variants and protein post-translational modifications.

Recent reviews pinpointed the enormous diversity of proteins found in living organisms, especially in higher eukaryotes. Protein diversity is driven through three main processes: first, at deoxyribonucleic acid (DNA) level (i.e. gene polymorphisms), second, at precursor messenger ribonucleic acid (pre-mRNA) or messenger ribonucleic acid (mRNA) level (i.e. alternative splicing, also termed as differential splicing) and, finally, at the protein level (i.e. PTM). Current proteomic technologies allow the identification, characterization and quantitation of up to several thousands of proteins in a single experiment. Nevertheless, the identification and characterization of protein species using these technologies are still hampered. Here, we review the use of the terms "protein species" and "protein isoform." We evidence that the appropriate selection of the database used for searches can impede or facilitate the identification of protein species. We also describe examples where protein identification search engines systematically fail in the attribution of protein species. We briefly review the characterization of protein species using proteomic technologies including gel-based, gel-free, bottom-up and top-down analysis and discuss their limitations. As an example, we discuss the theoretical characterization of the two human choline kinase species, α-1 and α-2, sharing the same catalytic activity but generated by alternative splicing on CHKA gene.

Immunohistochemical distribution of the tetraspanin CD9 in normal porcine tissues.

The tetra-membrane-spanning protein, CD9 is a 24-27 kDa cell surface glycoprotein expressed in a wide variety of human cells being involved in a variety of cell processes, including signaling, adhesion, motility, fertilization and tumor cells metastasis. By means of a polyclonal antibody (N1) raised against recombinant swine CD9 protein, we studied the immunohistochemical expression of CD9 on different normal swine tissues. Immunochemistry shows that swine CD9 was distribute in a similar form than in human tissues, being present on epithelial cells of lung, liver, kidney, skin, tonsil, testis (epididymo), gut mucosa, uterus and mama. Furthermore, polyclonal antibody against swine CD9 reacts with white matter from cerebrum and cerebellum, peripheral nerves fibers and Hassal corpuscle from thymus and ovum. Platelets react strongly with our antibody, but monocytes and neutrophils react lightly. These results suggest that CD9 antigen should play a similar functional role in swine and human and therefore studies on CD9 on swine as an animal model would allow new knowledge about its role in adhesion, fertilization and tumor metastasis among other important biomedical processes.

Molecular cloning, expression analysis and chromosome localization of the Tpt1 gene coding for the pig translationally controlled tumor protein (TCTP).

This work describes the cloning and structural analysis of a Tpt1 cDNA coding for the porcine translationally controlled tumor protein (TCTP) molecule and its expression in porcine cells and tissues. Pig Tpt1 cDNA is 842-pb long that displays typical features of translationally controlled mRNAs, including a 5'-UTR containing a 5'-terminal oligopyrimidine tract (5'-TOP), and a 3'-UTR with a high CG-content and one AU rich element (ARE). Both 5'-UTR and 3'-UTR are highly conserved when they are compared with those of other mammals. The pig Tpt1 cDNA contains a 516-b open reading frame that encodes a predicted TCTP protein composed of 172 amino acids that exhibits extensive conservation compared with TCTP sequences from other species and a common structural feature with all the other TCTP proteins analyzed in mammals. Expression analysis demonstrated that Tpt1 mRNA is ubiquitously expressed in normal porcine tissues and cells, showing a higher expression in spleen, lymph nodes and lung, and a lower one in skin and heart. The pig Tpt1 gene localizes on the porcine chromosome 11, region p11.

Molecular characterization and expression analysis of the gene coding for the porcine beta(3) integrin subunit (CD61).

Integrins are heterodimeric cell adhesion molecules with major roles in a variety of biological processes ranging from cell migration to tissue organization, immune and non-immune defense mechanisms and oncogenic transformation. Members of the beta(3) integrin subfamily are composed of a beta(3) subunit (CD61) non-covalently associated with two alpha subunits, alpha(IIb) (CD41) and alpha(v) (CD51), to constitute a group of transmembrane glycoproteins that participate in many physiologically important events. This investigation has focused on the molecular characterization of the cDNA encoding the porcine beta(3) integrin subunit. The deduced 762-amino acid sequence was 93, 92, 91, 89, 79 and 73% homologous to human, dog, rabbit, mouse, chicken and Xenopus laevis CD61 protein, respectively. Porcine CD61 molecule shares many structural features with human CD61, including a region containing a metal ion-dependent adhesion site (MIDAS) folding into an I domain-like structure. Through PCR-SSCP analysis and sequencing, six polymorphic positions were detected in the cDNA sequence of porcine CD61, and their frequencies were observed from a collection of 47 pigs. Expression analysis was done at two different levels: expression of the CD61 mRNA by RT-PCR and localization of the protein by immunohistochemistry. Our results show that CD61 transcripts were detected mainly in platelets and hematopoietic tissues. The immunohistochemical tissue localization of CD61 protein by a specific monoclonal antibody against CD61 recombinant protein showed that CD61 was expressed on vascular and non-vascular smooth muscle, epithelium and myeloid cells, being undetectable in cells of the lymphoid lineage. Furthermore, pulmonary intravascular macrophages (PIM), a subpopulation of macrophages which seem to play an important role in blood clearance, expressed much more CD61 when compared to pulmonary alveolar macrophages (PAM). The knowledge of the structure and distribution of the CD61 provides insight into the physiological function of the porcine beta(3) integrins and should be of importance in understanding the role of this integrin family in biological processes.