Taurine supplementation preserves hypothalamic leptin action in normal and protein-restricted mice fed on a high-fat diet.

Malnutrition programs the neuroendocrine axis by disruption of food-intake control, leading to obesity. Taurine (Tau) is neuroprotective and improves anorexigenic actions in the hypothalamus. We evaluated the hypothalamic gene-expression profile and food-intake control in protein-restricted mice submitted to a high-fat diet (HFD) and Tau supplementation. Mice were fed on a control (14 % protein-C) or a protein-restricted diet (6 % protein-R) for 6 weeks. Thereafter, mice received, or not, HFD for 8 weeks (CH and RH) with or without 5 % Tau supplementation (CHT and RHT). Protein restriction led to higher food intake, but calories were matched to controls. Excessive calorie intake occurred in HFD mice and this was prevented by Tau supplementation only in the CH group. Additionally, RH and CH mice developed hypothalamic leptin resistance, which was prevented by Tau. Global alterations in the expressions of genes involved in hypothalamic metabolism, cellular defense, apoptosis and endoplasmic reticulum stress pathways were induced by dietary manipulations and Tau treatment. The orexigenic peptides NPY and AgRP were increased by protein restriction and lowered by the HFD. The anorexigenic peptide Pomc was increased by HFD, and this was prevented by Tau only in CH mice. Thus, food intake was disrupted by dietary protein restriction and obesity. HFD-induced alterations were not enhanced by previous protein deficiency, but the some beneficial effects of Tau supplementation upon food intake were blunted by protein restriction. Tau effects upon feeding behavior control are complex and involve interactions with a vast gene network, preventing hypothalamic leptin resistance.

Stable SET knockdown in head and neck squamous cell carcinoma promotes cell invasion and the mesenchymal-like phenotype in vitro, as well as necrosis, cisplatin sensitivity and lymph node metastasis in xenograft tumor models.

BACKGROUND: SET/I2PP2A is a multifunctional protein that is up-regulated in head and neck squamous cell carcinoma (HNSCC). The action of SET in HNSCC tumorigenicity is unknown. METHODS: Stable SET knockdown by shRNA (shSET) was established in three HNSCC cell lines: HN12, HN13, and Cal27. Protein expression and phosphorylated protein levels were determined by Western blotting and immunofluorescence, cell migration and invasion were measured by functional analysis, and PP2A activity was determined using a serine/threonine phosphatase assay. A real-time PCR array was used to quantify 84 genes associated with cell motility. Metalloproteinase (MMP) activity was assessed by zymographic and fluorometric assays. HN12shSET xenograft tumors (flank and tongue models) were established in Balb/c nude mice; the xenograft characteristics and cisplatin sensitivity were demonstrated by macroscopic, immunohistochemical, and histological analyses, as well as lymph node metastasis by histology. RESULTS: The HN12shSET cells displayed reduced ERK1/2 and p53 phosphorylation compared with control. ShSET reduced HN12 cell proliferation and increased the sub-G1 population of HN12 and Cal27 cells. Increased PP2A activity was also associated with shSET. The PCR array indicated up-regulation of three mRNAs in HN12 cells: vimentin, matrix metalloproteinase-9 (MMP9) and non-muscle myosin heavy chain IIB. Reduced E-cadherin and pan-cytokeratin, as well as increased vimentin, were also demonstrated as the result of SET knockdown. These changes were accompanied by an increase in MMP-9 and MMP-2 activities, migration and invasion. The HN12shSET subcutaneous xenograft tumors presented a poorly differentiated phenotype, reduced cell proliferation, and cisplatin sensitivity. An orthotopic xenograft tumor model using the HN12shSET cells displayed increased metastatic potential. CONCLUSIONS: SET accumulation has important actions in HNSCC. As an oncogene, SET promotes cell proliferation, survival, and resistance to cell death by cisplatin in vivo. As a metastasis suppressor, SET regulates invasion, the epithelial mesenchymal transition, and metastasis.

Linker for activation of T-cell family member2 (LAT2) a lipid raft adaptor protein for AKT signaling, is an early mediator of alkylphospholipid anti-leukemic activity.

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 µm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 µm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five µm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.

Inhibition of neutrophil migration by hemopexin leads to increased mortality due to sepsis in mice.

RATIONALE: The reduction of neutrophil migration to the bacterial focus is associated with poor outcome in sepsis. OBJECTIVES: The objective of this study was to identify soluble substances in the blood of septic mice that inhibit neutrophil migration. METHODS: A pool of serum obtained from mice 2 hours after the induction of severe sepsis by cecal ligation and puncture inhibited the neutrophil migration. The proteins with inhibitory activity on neutrophil migration were isolated by Blue-Sepharose chromatography, high-performance liquid chromatography, and electrophoresis, and identified by mass spectrometry. MEASUREMENTS AND MAIN RESULTS: Hemopexin was identified as the serum component responsible for the inhibition of neutrophil migration. In sepsis, the pretreatment of wild-type mice with hemopexin inhibited neutrophil migration to the focus of infection and decreased the survival rate from 87.5 to 50.0%. Hemopexin-null mice subjected to severe sepsis presented normal neutrophil migration, low bacteremia, and an improvement of 40% in survival rate. Moreover, hemopexin inhibited the neutrophil chemotaxis response evoked by C5a or macrophage inflammatory protein-2 and induced a reduction of CXCR2 and L-selectin as well as the up-regulation of CD11b expression in neutrophil membranes. The inhibitory effect of hemopexin on neutrophil chemotaxis was prevented by serine protease inhibitors or ATP. In addition, serum levels of ATP were decreased 2 hours after severe sepsis. CONCLUSIONS: These data demonstrate for the first time the inhibitory role of hemopexin in neutrophil migration during sepsis and suggest that the therapeutic inhibition of hemopexin or its protease activity could improve neutrophil migration to the focus of infection and survival in sepsis.

New insights into trypanosomatid U5 small nuclear ribonucleoproteins.

Several protozoan parasites exist in the Trypanosomatidae family, including various agents of human diseases. Multiple lines of evidence suggest that important differences are present between the translational and mRNA processing (trans splicing) systems of trypanosomatids and other eukaryotes. In this context, certain small complexes of RNA and protein, which are named small nuclear ribonucleoproteins (U snRNPs), have an essential role in pre-mRNA processing, mainly during splicing. Even though they are well defined in mammals, snRNPs are still not well characterized in trypanosomatids. This study shows that a U5-15K protein is highly conserved among various trypanosomatid species. Tandem affinity pull-down assays revealed that this protein interacts with a novel U5-102K protein, which suggests the presence of a sub-complex that is potentially involved in the assembly of U4/U6-U5 tri-snRNPs. Functional analyses showed that U5-15K is essential for cell viability and is somehow involved with the trans and cis splicing machinery. Similar tandem affinity experiments with a trypanonosomatid U5-Cwc21 protein led to the purification of four U5 snRNP specific proteins and a Sm core, suggesting U5-Cwc-21 participation in the 35S U5 snRNP particle. Of these proteins, U5-200K was molecularly characterized. U5-200K has conserved domains, such as the DEAD/DEAH box helicase and Sec63 domains and displays a strong interaction with U5 snRNA.

Analysis of detergent-insoluble and whole cell lysate fractions of resting neutrophils using high-resolution mass spectrometry.

Neutrophilic granulocytes play a major role in the initiation and resolution of the inflammatory response, and demonstrate significant transcriptional and translational activity. Although much was known about neutrophils prior to the introduction of proteomics, the use of MS-based methodologies has provided an unprecedented tool to confirm and extend previous findings. In the present study, we performed a Gel-LC-MS/MS analysis of neutrophil detergent insoluble and whole cell lysate fractions of resting neutrophils. We achieved a set of identifications through the use of high-resolution mass spectrometry and validation of its data. We identified a total of 1249 proteins with a wide range of intensities from both detergent-insoluble and whole cell lysate fractions, allowing a mapping of proteins such as those involved in intracellular transport (Rab and Sec family proteins) and cell signaling (S100 proteins). These results represent the most comprehensive proteomic characterization of resting human neutrophils to date, and provide important information relevant for further studies of the immune system in health and disease. The methods applied here can be employed to help us understand how neutrophils respond to various physiologic and pathophysiologic conditions and could be extended to protein quantitation after cell activation.

Proteomic analysis of total cellular proteins of human neutrophils.

BACKGROUND: Neutrophils are the most abundant leukocytes in peripheral blood and represent one of the most important elements of innate immunity. Recent subcellular proteomic studies have focused on the identification of human neutrophil proteins in various subcellular membrane and granular fractions. Although there are relatively few studies dealing with the analysis of the total extract of human neutrophils, many biological problems such as the role of chemokines, adhesion molecules, and other activating inputs involved in neutrophil responses and signaling can be approached on the basis of the identification of the total cellular proteins. RESULTS: Using gel-LC-MS/MS, 251 total cellular proteins were identified from resting human neutrophils. This is more than ten times the number of proteins identified by an initial proteome analysis of human neutrophils and almost five times the number of proteins identified by the first 2-DE map of extracts of rat polymorphonuclear leukocytes. Most of the proteins identified in the present study are well-known, but some of them, such as neutrophil-secreted proteins and centaurin beta-1, a cytoplasmic protein involved in the regulation of NF-kappaB activity, are described here for the first-time. CONCLUSION: The present report provides new information about the protein content of human neutrophils. Importantly, our study resulted in the discovery of a series of proteins not previously reported to be associated with human neutrophils. These data are relevant to the investigation of comparative pathological states and models for novel classes of pharmaceutical drugs that could be useful in the treatment of inflammatory disorders in which neutrophils participate.

Low molecular weight squash trypsin inhibitors from Sechium edule seeds.

Nine chromatographic components containing trypsin inhibitor activity were isolated from Sechium edule seeds by acetone fractionation, gel filtration, affinity chromatography and RP-HPLC in an overall yield of 46% of activity and 0.05% of protein. The components obtained with highest yield of total activity and highest specific activity were sequenced by Edman degradation and their molecular masses determined by mass spectrometry. The inhibitors contained 31, 32 and 27 residues per molecule and their sequences were: SETI-IIa, EDRKCPKILMRCKRDSDCLAKCTCQESGYCG; SETI-IIb, EEDRKCPKILMRCKRDSDCLAKCTCQESGYCG and SETI-V, CPRILMKCKLDTDCFPTCTCRPSGFCG. SETI-IIa and SETI-IIb, which differed by an amino-terminal E in the IIb form, were not separable under the conditions employed. The sequences are consistent with consensus sequences obtained from 37 other inhibitors: CPriI1meCk_DSDCla_C_C_G_CG, where capital letters are invariant amino acid residues and lower case letters are the most preserved in this position. SETI-II and SETI-V form complexes with trypsin with a 1:1 stoichiometry and have dissociation constants of 5.4x10(-11)M and 1.1x10(-9)M, respectively.

Preliminary functional characterization, cloning and primary sequence of Fastuosain, a cysteine peptidase isolated from fruits of Bromelia fastuosa.

The present work reports the characterization of Fastuosain, a novel cysteine protease of 25kDa, purified from the unripe fruits of Bromelia fastuosa, a wild South American Bromeliaceae. Proteolytic activity, measured using casein and synthetic substrates, was dependent on the presence of thiol reagents, having maximum activity at pH 7.0. The present work reports cDNA cloning of Fastuosain; cDNA was amplified by PCR using specific primers. The product was 1096pb long. Mature fastuosain has 217 residues, and with the proregion has a total length of 324 residues. Its primary sequence showed high homology with ananain(74%), stem bromelain (66%) and papain (44%).

Lymph node or perineural invasion is associated with low miR-15a, miR-34c and miR-199b levels in head and neck squamous cell carcinoma.

BACKGROUND: MicroRNAs (miRNAs or miRs) are post-transcriptional regulators of eukaryotic cells and knowledge of differences in miR levels may provide new approaches to diagnosis and therapy. METHODS: The present study measured the levels of nine miRs in head and neck squamous cell carcinomas (HNSCC) and determined whether clinical pathological features are associated with differences in miR levels. SET (I2PP2A) and PTEN protein levels were also measured, since their levels can be regulated by miR-199b and miR-21, respectively. Nine miRs (miR-15a, miR-21, miR-29b, miR-34c, miR-100, miR-125b, miR-137, miR-133b and miR-199b) were measured by real time qRT-PCR in HNSCC samples from 32 patients and eight resection margins. SET (I2PP2A) and PTEN protein levels were estimated by immunohistochemistry in paired HNSCC tissues and their matched resection margins. RESULTS: In HNSCC, the presence of lymph node invasion was associated with low miR-15a, miR-34c and miR-199b levels, whereas the presence of perineural invasion was associated with low miR-199b levels. In addition, miR-21 levels were high whereas miR-100 and miR-125b levels were low in HNSCC compared to the resection margins. When HNSCC line HN12, with or without knockdown of SET, were transfected with miR-34c inhibitor or miR-34c mimic, the miR-34c inhibitor increased cell invasion capacity while miR-34c mimic decreased the cell invasion. CONCLUSIONS: We showed that the levels of specific miRs in tumor tissue can provide insight into the maintenance and progression of HNSCC. GENERAL SIGNIFICANCE: MiRNAs are up- or down-regulated during cancer development and progression; they can be prognosis markers and therapeutic targets in HNSCC.

Topology of the substrate-binding site of a Lys49-phospholipase A2 influences Ca2+-independent membrane-damaging activity.

BthTx-I (bothropstoxin-I) is a myotoxic Lys49-PLA2 (phospholipase A2 with Lys49) isolated from Bothrops jararacussu venom, which damages liposome membranes by a Ca2+-independent mechanism. The highly conserved Phe5/Ala102/Phe106 motif in the hydrophobic substrate-binding site of the Asp49-PLA2s is substituted by Leu5/Val102/Leu106 in the Lys49-PLA2s. The Leu5/Val102/Leu106 triad in BthTx-I was sequentially mutated via all single- and double-mutant combinations to the Phe5/Ala102/Phe106 mutant. All mutants were expressed as inclusion bodies in Escherichia coli, and the thermal stability (Tm), together with the myotoxic and Ca2+-independent membrane-damaging activities of the recombinant proteins, were evaluated. The far-UV CD profiles of the native, wild-type recombinant and the L106F (Leu106-->Phe) and L5F/F102A/L106F mutant proteins were identical. The L5F, V102A, L5F/V102A and V102A/L106F mutants showed distorted far-UV CD profiles; however, only the L5F and L5F/V102A mutants showed significant decreases in Tm. Alterations in the far-UV CD spectra correlated with decreased myotoxicity and protein-induced release of a liposome-entrapped marker. However, the V102A/L106F and L5F/V102A/L106F mutants, which presented high myotoxic activities, showed significantly reduced membrane-damaging activity. This demonstrates that the topology of the substrate-binding region of BthTx-I has a direct effect on the Ca2+-independent membrane damage, and implies that substrate binding retains an important role in this process.

Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops jararacussu snake venom.

An acidic (pI approximately 4.5) phospholipase A(2) (BthA-I-PLA(2)) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on a CM-Sepharose column followed by reverse phase chromatography on an RP-HPLC C-18 column. It is an approximately 13.7kDa single chain Asp49 PLA(2) with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 1SLWQFGKMINYVM-GESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC(51). Crystals of this acidic protein diffracted beyond 2.0A resolution. These crystals are monoclinic and have unit cell dimensions of a=33.9, b=63.8, c=49.1A, and beta=104.0 degrees. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3-4 times more active than BthTX-II, a basic D49 myotoxic PLA(2) from the same venom and other Bothrops venoms. Although it showed no toxic activity, it was able to induce time-independent edema, this activity being inhibited by EDTA. In addition, BthA-I-PLA(2) caused a hypotensive response in the rat and inhibited platelet aggregation. Catalytic, antiplatelet and other activities were abolished by chemical modification with 4-bromophenacyl bromide, which is known to covalently bind to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA(2), while anti-Asp49-BthTX-II recognized it weakly and anti-Lys49-BthTX-I showed the least cross-reaction. These data confirm that myotoxicity does not necessarily correlate with catalytic activity in native PLA(2) homologues and that either of these two activities may exist alone. BthA-I-PLA(2), in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies.

Determination and reoxidation of the disulfide bridges of a squash-type trypsin inhibitor from Sechium edule seeds.

The determination of the disulfide pairings of SETI-II, a trypsin inhibitor isolated from Sechium edule, is described herein. The inhibitor contains 31 amino acid residues per mol, 6 of which are cysteine. Forty-five nmol (160 microg) of SETI-II was hydrolyzed with 20 microg thermolysin for 48 hr at 45 degrees C, and peptides were separated by reverse phase high performance liquid chromatography (RP-HPLC). The major products were identified by amino acid composition, Edman degradation, and on the basis of the sequence of the inhibitor. The disulfide bridge pairings and (yields) are: Cys1-Cys4 (79%), Cys2-Cys5 (21%) and Cys3-Cys6 (43%). When the reduced inhibitor was reoxidized with glutathione reduced form (GSH)/glutathione oxidized form (GSSG) at pH 8.5 for 3 hr, full activity was recovered. These data show that disulfide bridge pairing and oxidation can be determined at nanomole levels and that sensitive and quantitative Edman degradation can eliminate the final time- and material-consuming step of disulfide determinations by eliminating the need to purify and cleave each peptide containing a disulfide bridge.

Maturation of human iDCs by IL-18 plus PGE2, but not by each stimulus alone, induced migration toward CCL21 and the secretion of IL-12 and IFN-γ.

Dendritic cells (DCs) are potent antigen-presenting cells that initiate the primary immune response and whose functional properties in vivo depend on the maturation stimulus. We describe the functional properties of human monocyte-derived DCs after the maturation of immature DCs (iDCs) for 2 days with LPS (100 ng/ml), PGE2 (1 µg/ml), CD40L (1 µg/ml) or IL-18 (200 ng/ml) and with CD40L+PGE2 and IL-18+PGE2 mixtures at the same concentrations as above. Neither IL-18 nor PGE2 alone stimulated IL-12 or IFN-γ secretion. When administered simultaneously to 1×10(6)iDCs/ml, IL-18+PGE2 induced the secretion of 131.4±6.7 pg IL-12/ml and 355±87 pg IFN-γ/ml but there was no detectable IL-10 secretion. However, PGE2 alone stimulated the secretion of 208±89 pg IL-10/ml whereas IL-18 alone did not stimulate the secretion of IL-10, IL-12, TNF-α or INF-γ. When the mixture of CD40L+PGE2 was used, only migration toward CCL19 and CCL21 was induced. CD40L did not stimulate the secretion of IL-10, IL-12, TNF-α or IFN-γ and did not stimulate migration toward CCL19 or CCL21. The extent of stimulation of T cell proliferation was essentially the same for all stimuli at the concentrations given above. New properties such as IL-12 and INF-γ secretion and migration toward CCL21 emerged when a mixture of IL-18+PGE2 was employed. These data show that when the pairs of stimuli reported here were used simultaneously their effect was not additive. This system can be used to prepare mDCs with properties useful for cell therapy and also as a model to investigate the mechanisms of cytokine secretion and cell migration.

Expression of human protein S100A7 (psoriasin), preparation of antibody and application to human larynx squamous cell carcinoma.

BACKGROUND: Up-regulation of S100A7 (Psoriasin), a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag) was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray. RESULTS: The rS100A7 (His-tag) protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag) rabbit serum (polyclonal antibody anti-rS100A7). The molecular weight of rS100A7 (His-tag) protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da). Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue. CONCLUSIONS: The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.

Adaptive shifts in osmoregulatory strategy and the invasion of freshwater by brachyuran crabs: evidence from Dilocarcinus pagei (Trichodactylidae).

To evaluate putative adaptive changes underpinning the invasion of freshwater by the Brachyura, this investigation examines anisosmotic extra and isosmotic intracellular osmoregulatory capabilities in Dilocarcinus pagei, a neotropical, hololimnetic crab, including its embryonic and juvenile phases. All ontogenetic stages show a remarkable ability to survive a high salinity medium (25 per thousand, 750 mOsm/kg H2O, 350 mm Na+, 400 mM Cl-). Adults hyper-regulate hemolymph osmolality up to isosmoticity at 744 mOsm kg/H2O (24 per thousand), [Na+] and [Cl-] becoming isoionic at 449 (22 per thousand) and 256 mM (16 per thousand), respectively. Hemolymph (420+/-39 mOsm/kg H2O) and urine (384+/-44 mOsm/kg H2O) are isosmotic in adults held in freshwater, and after 5-days exposure to 25 per thousand (787+/-9 mOsm/kg H2O and 777+/-43 mOs/kg H2O, respectively); D. pagei does not produce dilute urine. Total free amino acid (FAA) concentrations in embryos (14.9+/-1.2), juveniles (32.8+/-0.1) and adult muscle (10.9+/-2.1 mmol/kg wet weight) in freshwater are 30-fold less than in brackish/marine Crustacea, suggesting that FAA constitute a useful parameter to evaluate adaptation to freshwater. On acclimation to 25 per thousand, total FAA increase by approximately 100% in embryos and in adult muscle and nerve tissue and hemolymph, owing to large increases in proline, arginine and/or alanine. However, effective FAA contribution to intracellular osmolality increases only in embryos, from 3 to 4.5%. These findings suggest that gill-based, anisosmotic extracellular regulation has supplanted isosmotic intracellular regulatory mechanisms during the conquest of freshwater by the Brachyura, and indicate that D. pagei may be an old, well-adapted inhabitant of this biotope.

Efficient constitutive expression of Bacillus subtilis xylanase A in Escherichia coli DH5alpha under the control of the Bacillus BsXA promoter.

Xylanase A (XynA) is a class G/11 xylanase secreted by Bacillus subtilis. XynA was purified to homogeneity from B. subtilis strain 168 culture supernatants by ethanol precipitation and cation-exchange chromatography. The DNA fragment encoding the XynA together with the BsXA promoter region was amplified by PCR from B. subtilis 168 genomic DNA, and cloned into the plasmid pT7T3 to give the plasmid pT7BsXA. After transformation of Escherichia coli DH5alpha with pT7BsXA, a 19-fold increase in the levels of the secreted XynA was detected in the supernatant as compared with the B. subtilis culture. Correct post-translation modification of the recombinant protein was confirmed by N-terminal amino acid sequencing and MS analyses. The pH- and temperature-dependences of the native and recombinant proteins were identical, indicating that the pT7BsXA may be useful for the constitutive expression of heterologous protein in E. coli.

Expression in E. coli and purification of the nucleoside diphosphate kinase b from Leishmania major.

Leishmaniasis is considered by the World Health Organization to be the second most important disease caused by a protozoan parasite. Biochemical and molecular biology studies can help in the understanding of the biology of the Leishmania parasite. All protozoan parasites, including Leishmania, are unable to synthesize purines de novo, and nucleoside diphosphate kinases (NDK) are involved in the salvage pathway by which free purines are converted to nucleosides and subsequently to nucleotides. In this report, we describe the cloning of NDK coding-sequence from Leishmania major, the expression of the enzyme containing a His(6)-tag in Escherichia coli, and purification of the catalytically active native protein by affinity chromatography using Ni-NTA resin.

Subproteomic analysis of soluble proteins of the microsomal fraction from two Leishmania species.

Parasites of the genus Leishmania are the causative agents of a range of clinical manifestations collectively known as Leishmaniasis, a disease that affects 12 million people worldwide. With the aim of identifying potential secreted protein targets for further characterization, we have applied two-dimensional gel electrophoresis and mass spectrometry methods to study the soluble protein content of the microsomal fraction from two Leishmania species, Leishmania L. major and L. L. amazonensis. MALDI-TOF peptide mass fingerprint analysis of 33 protein spots from L. L. amazonensis and 41 protein spots from L. L. major identified 14 proteins from each sample could be unambiguously assigned. These proteins include the nucleotide diphosphate kinase (NDKb), a calpain-like protease, a tryparedoxin peroxidase (TXNPx) and a small GTP-binding Rab1-protein, all of which have a potential functional involvement with secretion pathways and/or environmental responses of the parasite. These results complement ongoing genomic studies in Leishmania, and are relevant to further understanding of host/parasite interactions.

Proteomic and SAGE profiling of murine melanoma progression indicates the reduction of proteins responsible for ROS degradation.

Using 2-DE of total cell protein extracts, we compared soluble proteins from murine melanoma lines Tm1 and Tm5 with proteins from the nontumoral cell melan-a from which they were derived. Seventy-one of the 452 spots (average) detected with CBB were differentially accumulated, i.e., increased or decreased twofold. Forty-four spots were identified by PMF/MALDI-TOF, 15 with increased and 29 with decreased protein levels. SAGE showed that 17/34 (50%) of the differentially accumulated proteins, pI range 4-7, presented similar differences at the mRNA level. Major reductions in protein were observed in tumor cells of proteins that degrade reactive oxygen species (ROS). Decreases of > or = twofold in GST, superoxide dismutase, aldehyde dehydrogenase, thioredoxin, peroxiredoxin 2, and peroxiredoxin 6 protein were observed. SAGE indicated the reduction of other proteins involved in ROS degradation. As expected, the accumulation of exogenous peroxides was significantly higher in the tumor cells while the levels of glutathionylation were two times lower in the tumor cells compared to melan-a. The differential accumulation of proteins involved in oncogene/tumor suppressor pathways was observed. Melanoma cells can favor survival pathways activated by ROS by inhibiting p53 pathways and activation of Ras and c-myc pathways.