Identification and biochemical characterization of a GDSL-motif carboxylester hydrolase from Carica papaya latex

An esterase (CpEst) showing high specific activities on tributyrin and short chain vinyl esters was obtained from Carica papaya latex after an extraction step with zwitterionic detergent and sonication, followed by gel filtration chromatography. Although the protein could not be purified to complete homogeneity due to its presence in high molecular mass aggregates, a major protein band with an apparent molecular mass of 41 kDa was obtained by SDS-PAGE. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (679 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 1029 bp encoding a protein of 343 amino acid residues, with a theoretical molecular mass of 38 kDa. From sequence analysis, CpEst was identified as a GDSL-motif carboxylester hydrolase belonging to the SGNH protein family and four potential N-glycosylation sites were identified. The putative catalytic triad was localised (Ser³⁵-Asp³⁰⁷-His³¹⁰) with the nucleophile serine being part of the GDSL-motif. A 3D-model of CpEst was built from known X-ray structures and sequence alignments and the catalytic triad was found to be exposed at the surface of the molecule, thus confirming the results of CpEst inhibition by tetrahydrolipstatin suggesting a direct accessibility of the inhibitor to the active site.     

Kinetic and structural characterization of DmpI from Helicobacter pylori and Archaeoglobus fulgidus, two 4-oxalocrotonate tautomerase family members

The tautomerase superfamily consists of structurally homologous proteins that are characterized by a β-α-β fold and a catalytic amino-terminal proline. 4-Oxalocrotonate tautomerase (4-OT) family members have been identified and categorized into five subfamilies on the basis of multiple sequence alignments and the conservation of key catalytic and structural residues. Representative members from two subfamilies have been cloned, expressed, purified, and subjected to kinetic and structural characterization. The crystal structure of DmpI from Helicobacter pylori (HpDmpI), a 4-OT homolog in subfamily 3, has been determined to high resolution (1.8 Å and 2.1 Å) in two different space groups. HpDmpI is a homohexamer with an active site cavity that includes Pro-1, but lacks the equivalent of Arg-11 and Arg-39 found in 4-OT. Instead, the side chain of Lys-36 replaces that of Arg-11 in a manner similar to that observed in the trimeric macrophage migration inhibitory factor (MIF), which is the title protein of another family in the superfamily. The electrostatic surface of the active site is also quite different and suggests that HpDmpI might prefer small, monoacid substrates. A kinetic analysis of the enzyme is consistent with the structural analysis, but a biological role for the enzyme remains elusive. The crystal structure of DmpI from Archaeoglobus fulgidus (AfDmpI), a 4-OT homolog in subfamily-4, has been determined to 2.4 Å resolution. AfDmpI is also a homohexamer, with a proposed active site cavity that includes Pro-1, but lacks any other residues that are readily identified as catalytic ones related to 4-OT activity. Indeed, the electrostatic potential of the active site differs significantly in that it is mostly neutral, in contrast to the usual electropositive features found in other 4-OT family members, suggesting that AfDmpI might accommodate hydrophobic substrates. A kinetic analysis has been carried out, but does not provide any clues about the type of reaction the enzyme might catalyze.     

Disposable superoxide anion biosensor based on superoxide dismutase entrapped in silica sol–gel matrix at gold nanoparticles modified ITO electrode

A novel disposable biosensor based on direct electron transfer of superoxide dismutase (SOD) was fabricated for the determination of superoxide anion. The biosensor was constructed by electrodeposition of gold nanoparticles (GNPs) on the indium tin oxide (ITO) electrode and then immobilization of SOD in silica sol–gel (SG) network in the presence of cysteine on GNPs/ITO modified electrode surface. The distribution of GNPs on ITO electrode surface was examined by scanning electron microscopy (SEM). The immobilized SOD exhibited high catalytical activity towards superoxide anion. Parameters affecting the performance of the biosensor were also investigated. A linear calibration curve was obtained over the range from 0.08 to 0.64 μM with a correlation coefficient of 0.9937. The resulted biosensors were demonstrated to possess striking analytical properties for superoxide anion determination, such as high sensitivity, good accuracy, and long-term stability. It provides a promising platform for the fabrication of disposable biosensors.     

The human pituitary nitroproteome: detection of nitrotyrosyl-proteins with two-dimensional Western blotting, and amino acid sequence determination with mass spectrometry

Nitric oxide is an important mediator that participates in reduction-oxidation (redox) mechanisms and in cellular signal transduction pathways. Two types of post-translational modifications are induced by nitric oxide: S-nitrosylation of cysteine residues and nitration of tyrosine residues. Two-dimensional gel electrophoresis-based Western blotting was used to detect, and liquid chromatography (LC)-tandem mass spectrometry (MS/MS) to determine the amino acid sequence of, several different nitrated proteins in the human pituitary. Proteins from several 2D gel spots, which corresponded to the strongly positive anti-nitrotyrosine Western blot spots, were subjected to in-gel trypsin-digestion and LC-MS/MS analysis. MS/MS, SEQUEST analysis, and de novo sequencing were used to determine the nitration site of each nitrated peptide. A total of four different nitrated peptides were characterized and were matched to four different proteins: synaptosomal-associated protein, actin, immunoglobulin alpha Fc receptor, and cGMP-dependent protein kinase 2. Those nitrotyrosyl-proteins participate in neurotransmission, cellular immunity, and cellular structure and mobility.     

Priming with magnesium-deficient media inhibits preadipocyte differentiation via potential upregulation of tumor necrosis factor-α

The effect of priming stromal-vascular cells in primary cultures with magnesium-deficient (MgD) media on preadipocyte differentiation was studied. Cultures were derived from dorsal subcutaneous fat tissue of young pigs and maintained 3 d in serum-free control or MgD Dulbecco’s modified Eagle’s medium, 3 d in 10% fetal bovine serum and dexamethasone, and 6 d in insulin. At d 12 of culture, immunocytochemical and glycerophosphate dehydrogenase assays indicated depressed adipocyte differentiation in the MgD groups. Cultures were enriched for preadipocytes up to 50% of total cells. On the third day of treatment with control and MgD medium, total cell lysates were isolated and 50 μg of them were run on two-dimensional gel electrophoresis. The separated proteins from both treatment groups showed similar patterns. However, spots of proteins with predicted molecular weight in the range of 25.8–37.4 kDa and pI of 5.39–5.85 were sixfold denser from the MgD 10 groups than from the controls. These proteins migrate similarly to tumor necrosis factor-α (TNF-α). The amount of TNF-α in cell lysates from the MgD group was about 2.35 times greater than controls determined by TNF-α-ELISA. It is likely that proteins upregulated by MgD medium are TNF-α isoforms.     

A Comparison of DNA Extraction Methods using Petunia hybrida Tissues

Extraction of DNA from plant tissue is often problematic, as many plants contain high levels of secondary metabolites that can interfere with downstream applications, such as the PCR. Removal of these secondary metabolites usually requires further purification of the DNA using organic solvents or other toxic substances. In this study, we have compared two methods of DNA purification: the cetyltrimethylammonium bromide (CTAB) method that uses the ionic detergent hexadecyltrimethylammonium bromide and chloroform-isoamyl alcohol and the Edwards method that uses the anionic detergent SDS and isopropyl alcohol. Our results show that the Edwards method works better than the CTAB method for extracting DNA from tissues of Petunia hybrida. For six of the eight tissues, the Edwards method yielded more DNA than the CTAB method. In four of the tissues, this difference was statistically significant, and the Edwards method yielded 27–80% more DNA than the CTAB method. Among the different tissues tested, we found that buds, 4 days before anthesis, had the highest DNA concentrations and that buds and reproductive tissue, in general, yielded higher DNA concentrations than other tissues. In addition, DNA extracted using the Edwards method was more consistently PCR-amplified than that of CTAB-extracted DNA. Based on these results, we recommend using the Edwards method to extract DNA from plant tissues and to use buds and reproductive structures for highest DNA yields.     

A comparative proteomic study of the undeveloped and developed Schistosoma mansoni egg and its contents: The miracidium, hatch fluid and secretions

The schistosome egg is the key agent responsible both for transmission of the parasite from human to molluscan host, and is the primary cause of pathogenesis in schistosomiasis. Characterisation of its proteome is a crucial step in understanding the egg’s interactions with the mammalian host. We devised a scheme to isolate undeveloped eggs from mature schistosome eggs by Percoll gradient and then fractionate the mature egg into miracidial, hatch fluid and secreted protein preparations. The soluble proteins contained within the five preparations were separated by two-dimensional electrophoresis and their spot patterns compared by image analysis. Large numbers of representative spots were then excised and subjected to tandem mass spectrometry to obtain identities. In this way, the principal components of each sub-proteome were established. Chaperones were the most abundant category, with heat shock protein 70 (HSP70) dominant in the undeveloped egg and Schistosoma mansoni protein 40 (Smp-40) in the miracidium. Cytoskeletal proteins were expressed at similar levels in the undeveloped egg and miracidium, with tubulins the most abundant. The proteins of energy metabolism reflected the change from anaerobic to aerobic metabolism as the miracidium developed. None of the above categories was abundant in the hatch fluid but this peri-miracidial compartment was highly enriched for defence proteins such as thioredoxin. Hatch fluid also contained several host proteins and schistosome proteins of unknown function, highlighting its distinct nature and potentially its role. The egg secretions could not be compared with the other preparations due to their unique composition featuring the previously characterised IL-4-inducing principal of S. mansoni eggs (IPSE), Omega-1, egg secreted protein 15 (ESP15), a micro-exon gene 2 (MEG-2) protein and two members of the recently described MEG-3 family. This last preparation contains the subset of egg proteins that probably enables eggs to escape from host tissues and may also initiate granuloma formation, emphasising the need to establish fully the roles of its components in schistosome biology.