Efficient bioconversion of lactose in milk and whey: immobilization and biochemical characterization of a beta-galactosidase from the dairy <em>Streptococcus thermophilus</em> LMD9 strain

International audienceThe gene encoding beta-galactosidase from dairy Streptococcus thermophilus strain LMD9 was cloned, sequenced and expressed in Escherichia coli. The recombinant enzyme was purified and showed high specific activity of 464 U/mg. This protein displays a homotetrameric arrangement composed of four 118 kDa monomers. Monitoring of the activity showed that this enzyme was optimally active at a wide range of temperatures (25-40 degrees C) and at pH from 6.5 to 7.5. Immobilization of the recombinant E. coli in alginate beads clearly enhanced the enzyme activity at various temperatures, including 4 and 50 degrees C, and at pH values from 4.0 to 8.5. Stability studies indicated that this biocatalyst has high stability within a broad range of temperatures and pH. This stability was improved not only by addition of 1 mM of Mn(2+) and 1.2 mM Mg(2+), but essentially through immobilization. The remarkable bioconversion rates of lactose in milk and whey at different temperatures revealed the attractive catalytic efficiency of this enzyme, thus promoting its use for lactose hydrolysis in milk and other dairy products

A chemical proteomics approach for the search of pharmacological targets of the antimalarial clinical candidate albitiazolium in Plasmodium falciparum using photocrosslinking and click chemistry.

Plasmodium falciparum is responsible for severe malaria which is one of the most prevalent and deadly infectious diseases in the world. The antimalarial therapeutic arsenal is hampered by the onset of resistance to all known pharmacological classes of compounds, so new drugs with novel mechanisms of action are critically needed. Albitiazolium is a clinical antimalarial candidate from a series of choline analogs designed to inhibit plasmodial phospholipid metabolism. Here we developed an original chemical proteomic approach to identify parasite proteins targeted by albitiazolium during their native interaction in living parasites. We designed a bifunctional albitiazolium-derived compound (photoactivable and clickable) to covalently crosslink drug-interacting parasite proteins in situ followed by their isolation via click chemistry reactions. Mass spectrometry analysis of drug-interacting proteins and subsequent clustering on gene ontology terms revealed parasite proteins involved in lipid metabolic activities and, interestingly, also in lipid binding, transport, and vesicular transport functions. In accordance with this, the albitiazolium-derivative was localized in the endoplasmic reticulum and trans-Golgi network of P. falciparum. Importantly, during competitive assays with albitiazolium, the binding of choline/ethanolamine phosphotransferase (the enzyme involved in the last step of phosphatidylcholine synthesis) was substantially displaced, thus confirming the efficiency of this strategy for searching albitiazolium targets

Intratumoral and peritumoral expression of CCR2 in pancreatic adenocarcinoma and its impact on prognosis.

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A simple and rapid protocol to non-enzymatically dissociate fresh human tissues for the analysis of infiltrating lymphocytes.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, isnow accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytesplay in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cellsfrom normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanicaldissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues.Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoidcells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/orprotein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefitof this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines,immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues andshould be applicable to a wide variety of normal and abnormal tissues.The video component of this article can be found at http://www.jove.com/video/52392/info:eu-repo/semantics/publishe

CXCL13-producing extrafollicular Tfh-like CD4+ T cells in human breast cancer

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Parasite proteins identified in UA1936 treated parasites under competition with albitiazolium.

<p>Parasites were incubated in the presence of the UA1936 bifunctional compound alone or in co-incubation with albitiazolium. Subsequently UA1936-interactiing proteins were photocrosslinked and clicked to an alkyne resin to be identified by mass spectrometry. Means of peptide spectrum matches (PSM) of two independent experiments are indicated for each experimental condition together with the standard deviaton (SD).</p><p>*This ratio was calculated by dividing the mean spectral counts of samples incubated with UA1936 by the mean spectral counts of samples incubated with both UA1936 and albitiazolium across two independent replicates.</p><p>Parasite proteins identified in UA1936 treated parasites under competition with albitiazolium.</p

Protein interaction network.

<p>Protein interaction networks were built in Cytoscape 2.5.1 using the PlasmoID plugin. Three clusters of UA1936-interacting proteins were found based on the following biological process terms: “phospholipid metabolism” depicted in pink, “vesicle-mediated transport” in blue and “transport” in violet. A fourth cluster of unknown or unrelated functions was created and named “miscellaneous”, as depicted in orange. For each protein (node), the PlasmoDB ID accession number is indicated in a colored rectangle followed by the putative name of the gene. Proteins in dark-colored rectangles were found in the three replicated experiments and proteins in light-colored rectangles were found in two replicates. The proteins depicted in ellipses are interacting proteins identified in the PlamsoMap interactome analysis with their respective likelihood scores. The colors on the edges in the network represent: Codes for (black), Interacts with (blue), and Participates in (orange). The p values for the gene ontology terms found to be significantly enriched are presented for genes having those annotations in the biological process.</p

Structure of albitiazolium and photoactivable analogs.

<p>(A) The clinical antimalarial candidate albitiazolium, (B) the bifunctional bis-thiazolium derivative UA1936 and (C) the pharmacologically inactive bifunctional derivative UA2050 are depicted. The albitiazolium pharmacophore consists of two cationic thiazolium heads linked by a hydrophobic flexible spacer. The bifunctional compounds UA1936 and UA2050 incorporate a phenyl azido group as photoactivable moiety and a benzy azido group as “clickable” function.</p

Flow chart for the UA1936-target fishing approach using photocrosslinking and click chemistry.

<p>Free parasites were obtained by saponin treatment of <i>P. falciparum</i>-infected red blood cells (IRBC) and incubated at 37°C with 100 µM UA1936 or 100 µM UA2050 for 1 h in HEPES-buffered RPMI 1640 medium. Control experiments were also conducted without compound. In competitive experiments, free parasites were first incubated with 100 µM albitiazolium for 30 min and then with 100 µM of UA1936. Parasites were then irradiated at 254 nm for 2.5 min. After centrifugation and wash, parasites were lysed and 10 mg of parasite proteins were used for click chemistry reactions with the alkyne agarose resin. After stringent washes, the resin-bound proteins were digested with trypsin overnight. The peptides were analyzed by mass spectrometry in a LTQ-Orbitrap VELOS mass spectrometer. After spectral data analysis, the identified parasite proteins were clustered based on gene ontology annotations using different bioinformatics packages.</p