A Combination of Proteomic Approaches Identifies A Panel of Circulating Extracellular Vesicle Proteins Related to the Risk of Suffering Cardiovascular Disease in Obese Patients

Plasma‐derived extracellular vesicles (EVs) have been extensively described as putative biomarkers in different diseases. Interestingly, increased levels of EVs subpopulations are well known to associate with obesity. The goal of this study is to identify EVs‐derived biomarkers in plasma from obese patients in order to predict the development of pathological events associated with obesity. Samples are obtained from 22 obese patients and their lean‐matched controls are divided into two cohorts: one for a 2D fluorescence difference gel electrophoresis (2D‐DIGE)‐based study, and the other one for a label free LC–MS/MS‐based approach. EVs are isolated following a serial ultracentrifugation protocol. Twenty‐two and 23 differentially regulated features are detected from 2D‐DIGE and label free LC–MS/MS, respectively; most of them involve in the coagulation and complement cascades. Remarkably, there is an upregulation of complement C4, complement C3, and fibrinogen in obese patients following both approaches, the latter two also validated by 2D‐western‐blotting in an independent cohort. These results correlate with a proinflammatory and prothrombotic state of those individuals. On the other hand, a downregulation of adiponectin leading to an increased risk of suffering cardiovascular diseases has been shown. The results suggest the relevance of plasma‐derived‐EVs proteins as a source of potential biomarkers for the development of atherothrombotic events in obesity.     

The peptide nucleic acid targeted to a regulatory sequence of the translocated c-myc oncogene in Burkitt's lymphoma lacks immunogenicity: follow-up characterization of PNAEmu-NLS

The present study aims to evaluate the antigenicity of a PNA complementary to the Emu sequence (PNAEmu) with cancer therapeutic potential properties in Burkitt's lymphoma (BL). In BL cells, the c-myc oncogene is repositioned next to the Emu enhancer of the immunoglobulin (Ig) locus, due to chromosomal translocation, and up-regulated. PNAEmu linked to a nuclear localization signal peptide was shown specifically to block c-myc hyperexpression by inhibiting cell growth in vitro and in vivo. Recently, we reported that the administration of PNAEmu to mice, following inoculation with BL cells, hinders tumor growth without toxic effects. To investigate the potential use of PNAEmu in clinical applications further, we tested its antigenicity. Mice were inoculated with an emulsion of free PNA or PNA crosslinked to the immunogenic carrier keyhole limpet hemocyanin (KLH) with Freund's adjuvant. Antibodies to free PNA were undetected, whereas both IgG and IgM antibodies to PNA-KLH were detected in mouse serum 28 and 38 days after inoculation.     

Absolute configuration of phomactatriene diterpenoids obtained by Wagner‐Meerwein rearrangement of epimeric verticillols

The epimeric diterpenes (+)‐(1S,3E,7E,11S,12S)‐verticilla‐3,7‐dien‐12‐ol ( 1 ), isolated from Bursera suntui, and (+)‐(1S,3E,7E,11S,12R)‐verticilla‐3,7‐dien‐12‐ol ( 2 ), isolated from Bursera kerberi, gave the same Wagner‐Meerwein rearrangement product (?)‐(1E,4Z,8Z,11S,12R)‐phomacta‐1,(15)4,8‐triene ( 3 ). The Et₂O:BF₃‐induced transformations evidence that verticillenes and phomactanes, both containing the bicyclo[9.3.1]pentadecane skeleton, are biogenetically related through the verticillen‐12‐yl cation ( A ⁺ ), which also is a key intermediate in the biosynthetic pathways to generate antitumor taxanes. Molecular modeling using the Monte Carlo protocol, followed by density functional theory (DFT) geometry optimization employing the hybrid functionals B3LYP and B3PW91, both with the DGDZVP basis set, secured the configuration of 3 as followed from the good agreement between the calculated and experimental vibrational circular dichroism spectra. Similar DFT calculations allowed determining the absolute configuration of (+)‐(1R,4R,5R,8S,9S,11S,12R,15R)‐1,15:4,5:8,9‐triepoxyphomactane ( 9 ), which surprisingly derives from epoxidation of the second minimum energy conformer of 3 .     

Variable specific activity of Escherichia coli beta-galactosidase in bacterial cells

Escherichia coli lacZ is a frequently employed reporter gene for the monitoring of gene expression and recombinant protein production due the simple determination of beta-galactosidase activity in both qualitative and quantitative assays. In the absence of either total or recombinant protein synthesis, we observed a lack of correlation between protein amount and enzymatic activity in both engineered and native beta-galactosidases in Escherichia coli cells. A delayed fading of beta-galactosidase activity compared with the rapid degradation of intact protein suggests a progressive increase in enzyme-specific activity during the life of the protein. This intriguing event does not involve solubilization from major protein aggregates and it occurs both in vivo and in cell extracts, but not in solutions of purified protein. Possible explanations for this activation are examined in the context of the assisted protein folding network and proteolytic degradation of misfolded proteins.     

Helminths in the wolf, Canis lupus, from north-western Spain

Fifteen helminth species were collected from 47 wolves (Canis lupus ) which were surveyed from 1993 to 1999 in northwestern Spain. These included the trematode Alaria alata (2.1%); the cestodes Taenia hydatigena (44.7%), T. multiceps (29.8%), T. serialis (2.1%), Dipylidium caninum (6.4%) and Mesocestoides sp. aff. litteratus (4.2%); and the nematodes Pearsonema plica (7.4%), Trichuris vulpis (10.6%), Trichinella britovi (12.8%), Ancylostoma caninum (8.5%), Uncinaria stenocephala (51.1%), Toxocara canis (6.4%) Toxascaris leonina (4.2%), Angiostrongylus vasorum (2.1%) and Dirofilaria immitis (2.1%). Only two wolves were not infected. A single infection occurred in 28.9% of the cases, but the commonest infracommunity (31.1%) involved three species. The helminths Alaria alata, Taenia hydatigena, Mesocestoides sp. aff. litteratus, P. plica, Trichuris vulpis, and Ancylostoma caninum parasitizing C. lupus are reported for the first time in Spain. Taenia serialis and D. immitis are reported for the first time in wolves in Europe. Angiostrongylus vasorum represents a new host record for wolves. The helminth fauna of Spanish wolves is compared with that of other European wolf populations. Some epidemiological considerations of the helminth fauna of wolves in Spain and the health risk to humans are also discussed.     

Fingerprinting of Mixed Bacterial Strains and BIOLOG Gram-Negative (GN) Substrate Communities by Enterobacterial Repetitive Intergenic Consensus Sequence-PCR (ERIC-PCR)

PCR-based genomic fingerprinting by use of enterobacterial repetitive intergenic consensus primers (ERIC-PCR) was evaluated for its use in fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG Gram-negative (GN) microplate substrate communities. ERIC-PCR fingerprints of six different pure bacterial strains and a combined mixture of the strains were compared with fingerprints obtained by two more established methods: amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA analysis (RAPD-PCR). The ERIC-PCR fingerprint of the mixed strains was highly reproducible and was more species-specific and representative of the individual strain fingerprints than the ARDRA and RAPD-PCR fingerprints, respectively. ERIC-PCR fingerprinting of model and rhizosphere BIOLOG GN substrate communities also provided clearly distinguishable fingerprints. Results of this study suggest that ERIC-PCR represents a rapid and highly discriminating method for fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG GN substrate communities. Received: 11 September 1998 / Accepted: 29 October 1998     

Antioxidant effects of water- and lipid-soluble nitroxide radicals in liposomes

Liposomes are today useful tools in different fields of science and technology. A lack of stability due to lipid peroxidation is the main problem in the extension of the use of these formulations. Recent investigative works have reported the protective effects of stable nitroxide radicals against oxidative processes in different media and under different stress conditions. Our group has focused its attention on the natural aging of liposomes and the protection provided by the water- and lipid-soluble nitroxide radicals 2,2,6,6-tetramethylpiperdine-1-oxyl (TEMPO) and doxylstearic acids (5-DSA, 12-DSA, and 16-DSA), respectively. Unilamellar liposomes were incubated under air atmosphere at 37°C, both in the absence and in the presence of these radicals. Conjugated dienes, lipid hydroperoxides, TBARS, membrane fluidity, and nitroxide ESR signal intensity were followed as a function of time. Our results demonstrated that doxylstearic acids were more efficient than TEMPO in retarding lipid peroxidation at all the concentrations tested. The inhibition percentages, depending on the total nitroxide concentration, were not proportional to the lipid–water partition coefficient. Furthermore, time-course ESR signals showed a slower decrease for doxylstearic acids than for TEMPO. No significant differences were found among 5-DSA, 12-DSA, and 16-DSA. We concluded that the nitroxide radical efficiency as antioxidant directly depends on both nitroxide concentration and lipophilicity.     

Efficient Transformation of <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> by Electroporation and Conjugation with <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The conjugative self-transmissible plasmid pHT73, harbored in Bacillus thuringiensis var. kurstaki, was demonstrated to be transferred to Cellulomonas flavigena, a cellulolytic bacterium. Both conjugation and transformation procedures yielded resistant colonies; however, chromosomal integration was observed only when bacterial conjugation occurred. The efficiency of conjugation was 10% of recipient strain, which is considered a very efficient process. When the plasmid pHT73 was introduced by transformation, erythromycin-resistant cells contained the plasmid as an episome with no arrangements, as assayed by Southern blot analysis. In contrast, conjugated-resistant cells harbor the plasmid integrated into the chromosome. These data suggest a common mechanism of cell communication between nonrelated bacterial species with similar ecological habitats, and also that both electroporation and conjugation can be used to transform C. flavigena efficiently.