1,721,120 research outputs found
NMR Metabolic Profiling of Transgenic Maize with the Cry1A(b) Gene
No full textThe metabolic profiles of seeds from the transgenic maize variety 33P67 and of the corresponding
traditional variety were investigated using one- and two-dimensional NMR techniques. The transgenic
variety carries a functional Cry1A(b) gene, which confers to the plant the ability to produce Bt
insect toxin. About 40 water-soluble metabolites in the maize seed extracts were identified, providing
a more complete 1H and 13C NMR assignment with respect to the assignment reported in the
literature. In particular ethanol, lactic acid, citric acid, lysine, arginine, glycine-betaine, raffinose,
trehalose, R-galactose, and adenine were identified for the first time in the 1H NMR spectrum of
maize seeds extracts. The 1H spectra of transgenic and nontransgenic seed maize samples turned
out to be conservative, showing the same signals and therefore the same metabolites. However, a
higher concentration of ethanol, citric acid, glycine-betaine, trehalose, as well as of another
compound not yet completely identified, was observed in the transgenic extracts than in nontransgenic
samples. So, it was possible to discriminate between transgenic and nontransgenic metabolic
profilings through the use of an appropriate statistical analysis
A mass spectrometry-based targeted metabolomics strategy of human blastocoele fluid: a promising tool in fertility research
No full textEmbryo assessment is currently performed through the analysis of morphology and cleavage rate. Recent studies have sought to identify a correlation between quali-quantitative profiles of small molecules of metabolic interest and the outcome of embryo transfer. Approaches relying on both optical and non-optical spectroscopy have been proposed to non-invasively monitor the embryo culture media. However, the non-invasive approach only offers an indirect strategy to monitor embryos and a turn-around solution to bypass the limits of detection of these analytical techniques. In this paper we pave the way for direct metabolic assessment of embryos through the mass-spectrometry-based analysis of blastocoele fluid, which is withdrawn from the blastocoele cavity prior to cryostorage of blastocysts. We conclude that it is possible to detect most of the metabolites of potential interest right at the very heart of the blastocyst, without disrupting the workflow of a classic laboratory pipeline
Hemocyanin from Palinurus elephas: general properties and effects of heavy metals
No full textThe structural and functional properties of hemocyanin from the lobster Palinurus elephas indicate that this protein is similar to that of Panulirus interruptus, as expected on the basis of the phylogenetic relatedness of the two species. The process of (re)association of subunits into hexamers has been studied by ultracentrifuge and stopped-flow techniques; complete reassociation requires both neutral pH and presence of calcium ions and appears to be a relatively fast process (time-scale of 1 min). The effect of three heavy metals (Hg, Cd and Cr) on the structural and functional properties of this protein has been investigated in order to ascertain whether hemocyanin may be a target of metal poisoning. At low concentrations the three metals are effective in modifying (reversibly) the functional properties of the protein, although each metal induces different modifications. Very high concentrations of Cr and Hg, and long incubation times result in the removal of copper from the active site, while Cd seems unable to do so
Plasma-derived clotting factor VIII: Heterogeneity evaluation in the quest for potential inhibitory-antibody stimulating factors
No full textIn this study, we investigated the heterogeneity and the purity grade of three commercially available plasma-derived clotting factor VIII (FVIII) concentrates, which highly differ with regard to purification strategies, relative concentrations of stabilizers (von Willebrand factor, with or without albumin) and virus inactivation strategies (solvent/detergent and/or heat/pasteurization treatments). Western blot analyses were used to evaluate product-specific variations from Emoclot (R), Alphanate (R) and Haemate (R) both in the presence and absence of reducing agents (dithiotreithol). All the plasma-derived concentrates showed a strong heterogeneity, as they all included a significant amount of truncated forms of the full-length (FL) clotting FVIII protein. The intact protein accounted for the 38% of the total FVIII proteins in Haemate (R) and 29 and 23% in Alphanate (R) and Emoclot (R), respectively. Lower intact FVIII amounts in Emoclot might be mainly due to the low von Willebrand factor dosage and the absence of albumin. Upon addition of thrombin, both the FL and truncated forms of the FVIII protein were almost completely digested. Indeed, after thrombin activation, we could still observe a mixture of B-domain truncated forms of the FL protein along with biologically active digested-A1 forms. Batch-to-batch variation was tested with no evident changes appearing among different batches. Despite the variables in manufacturing processes, inter-product comparisons yielded similar results for all the plasma-derived FVIII considered in this study. However, we could individuate in Emoclot a band that was not digested by thrombin, which we could characterize as the 200 kDa FVIII heavy chain. This investigation prompts new concerns about the strong heterogeneity observed upon thrombin digestion of plasma-derived FVIII, which might contribute to the development of inhibitory antibodies at an early stage of therapy, and to which extent these untoward phenomena could be avoided through direct intervention on routine manufacturing processes
Proteomic analysis of plasma derived from platelet buffy coats during storage at room temperature. An application of ProteoMinerTM technology
No full textThe present study was aimed at revealing new insights into the analysis of storage-related processes occurring in the supernatants of platelet concentrates (PCs) derived from pooled buffy coats suspended in whole plasma. To reduce the dynamic range of plasma protein concentrations and access low-abundance proteins, we made use of a solid-phase combinatorial peptide ligand library, known under the trade name of ProteoMinerTM. Afterwards, two-dimensional electrophoresis (2-DE) was coupled with mass spectrometry (MS) to reveal changes in proteomic profiles. Several storage-induced protein alterations were identified including changes to major plasma proteins. In particular, a precursor of the secretory form of clusterin was shown to accumulate during storage of PC supernatants, together with platelet-derived tropomyosin, suggesting a progressive loss of platelet integrity. Platelet-released proteins following activation have also been detected (alpha-1-B-glycoprotein, kininogen-1, and serpin proteinase inhibitor 8). Moreover, specific protein fragments (vitronectin, plakoglobin, hornerin, and apolipoprotein A-IV) were found to be modulated upon storage, possibly indicating a time-dependent buffy-coat PC deterioration. Globally, our findings provided the disclosure of unique proteins in PC supernatants with respect to previous studies conducted in similar experimental conditions, suggesting ProteoMiner enrichment technology to be a possible complementary tool in the identification of diagnostically relevant proteins as age/quality biomarkers of therapeutic products. © 2011 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted
Proteomic profiling and post-translational modifications in human keratinocytes treated with Mucuna pruriens leaf extract
No full textMucuna pruriens (Mp) is a plant belonging to the Fabaceae family, with several medicinal properties among which its potential to treat diseases where reactive oxygen species (ROS) play an important role in the pathogeneses. The aim was to investigate the effects of Mp leaf methanolic extract (MPME) on human keratinocytes protein expression and its role in preventing proteins oxidation after oxidative stress (OS) exposure.
MATERIAL AND METHODS:
The effects of MPME on HaCaT cells protein expression were evaluated treating cells with different concentrations of MPME, with glucose oxidase (GO, source of OS) and with MPME subsequently treated with GO. The protein patterns of treated HaCaT cells are analyzed by two-dimensional gel electrophoresis (2-DE) and compared with that of untreated HaCaT. Immunoblotting was then used to evaluate the role of MPME in preventing the 4-hydroxynonenal protein adducts (4-HNE PAs) formation (marker of OS).
RESULTS:
Eighteen proteins, identified by mass spectrometry (LC-ESI-CID-MS/MS), were modulated distinctly by MPME in HaCaT. Overall, MPME counteract GO effect, reducing the GO-induced overexpression of several proteins involved in stress response (T-complex protein 1, Protein disulfide-isomerase A3, Protein DJ-1, and Stress-induced-phosphoprotein 1), in cell energy methabolism (Inorganic pyrophosphatase, Triosephosphate isomerase isoform 1, 2-phosphopyruvate-hydratase alpha-enolase, and Fructose-bisphosphate aldolase A isoform 1), in cytoskeletal organization (Cytokeratins 18, 9, 2, Cofilin-1, Annexin A2 and F-actin-capping protein subunit beta isoform 1) and in cell cycle progression (Eukaryotic translation initiation factor 5A-1 isoform B). In addition, MPME decreased the 4-HNE PAs levels, in particular on 2-phosphopyruvate-hydratase alpha-enolase and Cytokeratin 9.
CONCLUSIONS:
Our findings show that MPME might be helpful in the treatment of OS-related skin diseases by preventing protein post-translational modifications (4-HNE PAs)
Biomarkers to Be Used for Decision of Treatment of Hypogonadal Men with or without Insulin Resistance
No full textMale hypogonadism is a result of low testosterone levels, but patients could be insulin-sensitive (IS) or insulin-resistant (IR), showing different impaired metabolic pathways. Thus, testosterone coadministration, which is commonly used to reestablish testosterone levels in hypogonadism, must take into account whether or not insulin is still active. By comparing metabolic cycles recorded in IS and IR plasma before and after testosterone therapy (TRT), it is possible to know what metabolic pathways can be reactivated in the two different groups upon testosterone recovery, and it is possible to understand if antagonism or synergy exists between these two hormones. IS hypogonadism uses glycolysis, while IR hypogonadism activates gluconeogenesis through the degradation of branched-chain amino acids (BCAAs). Upon administration of testosterone, acceptable improvements are observed in IS patients, wherein many metabolic pathways are restored, while in IR patients, a reprogramming of metabolic cycles is observed. However, in both subgroups, lactate and acetyl-CoA increases significantly. In IS patients, lactate is used through the glucose–lactate cycle to produce energy, while in IR patients, both lactate and acetyl-CoA are metabolized into ketone bodies, which are used to produce energy. Thus, in IR patients, an ancestral molecular mechanism is activated to produce energy, mimicking insulin effects. Regarding lipids, in both groups, the utilization of fatty acids for energy (β-oxidation) is blocked, even after TRT; free fatty acids (FFAs) increase in the blood in IS patients, while they are incorporated into triglycerides in those with IR. In both subgroups of hypogonadism, supplementation of useful chemicals is recommended during and after TRT when metabolites are not restored; they are listed in this review
Analysis of Mitochondrial Proteome of Cybrid Cells Harbouring a Truncative Mitochondrial DNA Mutation in Respiratory Complex I
No full textTransmitochondrial cytoplasmic hybrids (cybrids) are a well established model system to reveal the effects of mitochondrial DNA (mtDNA) mutations on cell metabolism excluding the interferences of a different nuclear background. The m.3571insC mutation in MTND1 gene of respiratory complex I (CI) is commonly detected in oncocytic tumors, in which it causes a severe CI dysfunction leading to an energetic impairment when present above 83% mutant load. To assess whether the energetic deficit may alter the mitochondrial proteome, OS-78 and OS-93 cybrid cell lines bearing two different degrees of the m.3571insC mutation (78% and 92.8%, respectively) and control cybrids bearing wild-type mtDNA (CC) were analyzed. Two-dimensional electrophoresis and mass spectrometry revealed significant alterations only in cybrids above the threshold (OS-93). All differentially expressed proteins are decreased. In particular, the level of pyruvate dehydrogenase E1 chain B subunit (E1b), of lipoamide dehydrogenase (E3), the enzyme component of pyruvate and 2-oxoglutarate dehydrogenase complexes and of lactate dehydrogenase B (LDHB) was reduced. Moreover, a significant decrease of the pyruvate dehydrogenase complex activity was found when OS-93 cybrid cells were grown in galactose medium, a metabolic condition that forces cells to use respiration. These results demonstrate that the energetic impairment caused by the almost homoplasmic m.3571insC mutation perturbs cellular metabolism leading to a decreased steady state level of components of very important mitochondrial NAD-dependent dehydrogenases
Na+/K+-ATPase β1-subunit is recruited in Na-K-2Cl co-transporter isoform 2 multiprotein complexes in rat kidneys: Possible role in blood pressure regulation
No full textOBJECTIVE:: The progression from prehypertensive to hypertensive state in spontaneous hypertensive rats (SHRs) is accompanied by a significant increase in membrane expression of Na-K-2Cl co-transporter isoform 2 (NKCC2), suggesting that the altered NKCC2 trafficking and activity are directly related with the development of hypertension in this strain. The aim of this work is to gain insights on the molecular mechanism that underlies this phenomenon. METHODS:: We performed a comparative analysis of NKCC2 multiprotein complexes (MPCs) in the kidney of SHRs versus Wistar Kyoto rats by Blue Native difference gel electrophoresis combined with mass spectrometry. RESULTS:: We found that the recruitment of the β-subunit isoform 1 of the Na+-K +-ATPase (β1NK) in NKCC2 MPCs was significantly increased in the kidneys of SHR compared with Wistar Kyoto rat control strain. Co-immunoprecipitation experiments showed that β1NK actually interacts with NKCC2 in the native tissue. The analysis of the physiological role of β1NK-NKCC2 interaction in human embryonic kidney cells showed that β1NK increased the steady-state membrane expression and activity of NKCC2 enhancing NKCC2 trafficking toward the plasma membrane. CONCLUSION:: We identify a new NKCC2-interacting partner involved in the modulation of NKCC2 intracellular trafficking and possibly involved in the regulation of blood pressure. © 2014 Wolters Kluwer Health Lippincott Williams & Wilkins
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