Escherichia coli biotin protein ligase: characterization and development of a high-throughput assay

The rapid rise in pathogenic bacteria resistant to current treatments, coupled with the paucity of new therapeutic agents in the pipeline, has resulted in a significant need for new antibiotics. One strategy to overcome resistance requires new chemical entities that inhibit key enzymes in essential metabolic processes that have not been previously targeted and for which there is no preexisting drug resistance. Biotin protein ligase (BPL), required to complete acetyl CoA carboxylase’s capability for fatty acid biosynthesis, is one target that has not yet been fully explored. However, its application in large-scale compound screens has been limited due to the lack of a truly high-throughput assay for enzyme activity. Here we report a novel assay system for BPL from Escherichia coli (BirA). This assay employs fluorescence polarization technology together with a unique peptide substrate for BirA. Additionally, the multiple handling steps and requirement for radiolabeled ligands associated with previous assays have been eliminated. Kinetic analysis of MgATP (K_m 0.25 ± 0.01 mM) and biotin (K_m 1.45 ± 0.15 μM) binding produced results consistent with published data. Inhibition studies with end products of the BPL reaction, AMP and pyrophosphate, further validated the assay. Statistical analysis, performed upon both intraassay and interassay results (n = 30), showed the coefficient of variance to be <10% across all data sets. Furthermore, Z′ factors between 0.5 and 0.8 demonstrated the utility of this technology in high-throughput applications.     

The mycolyltransferase 85A,a putative drug target of Mycobacterium tuberculosis: Development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall

The enzymes of the antigen 85 complex (Ag85A, B, and C) possess mycolyltransferase activity and catalyze the synthesis of the most abundant glycolipid of the mycobacterial cell wall, the cord factor. The cord factor (trehalose 6,6′-dimycolate, TDM) is essential for the integrity of the mycobacterial cell wall and pathogenesis of the bacillus. Thus, TDM biosynthesis is regarded as a potential drug target for control of Mycobacterium tuberculosis infections. Trehalose 6,6′-dimycolate (TDM) is synthesized from two molecules of trehalose-6′-monomycolate (TMM) by antigen 85A. We report here a novel enzyme assay using the natural substrate TMM. The novel colorimetric assay is based on the quantification of glucose from the degradation of trehalose, which is the product from catalytic activity of antigen 85A. Using the new assay, K_m and K_cat were determined with values of 129.6 ± 8.1 µM and 65.4 ± 4.1 min^− 1, respectively. This novel assay is also suitable for robust high-throughput screening (HTS) for compound library screening against mycolyltransferase (antigen 85A). The assay is significantly faster and more convenient to use than all assays currently in use. The assay has a very low coefficient of variance (0.04) in 96-well plates and shows a Z′ factor of 0.67–0.73, indicating the robustness of the assay. In addition, this new assay is highly suitable for the quantification of total TMM of the mycobacterial cell envelope.     

Deprotonation of β-cyclodextrin in alkaline solutions

Variable pH ¹³C NMR and ¹H NMR spectroscopic studies of the β-cyclodextrin (β-CD) in alkaline aqueous solutions revealed that β-CD does not deprotonate at pH < 12.0. Further increase in solution pH results in the deprotonation of OH-groups adjacent to C-2 and C-3 carbon atoms of β-CD glucopyranose units, whereas the deprotonation of OH-groups adjacent to C-6 carbon atoms is expressed less markedly. The pK_a values for β-CD OH-groups adjacent to C-2 and C-3 carbon atoms are rather close, pK_a1,2 being 13.5 ± 0.2 (22.5 °C).     

Oxadiazolone derivatives,new promising multi-target inhibitors against M. tuberculosis

A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc²6230. The encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX compounds displayed a diversity of action and were found to act either on extracellular M. tb growth only with moderated MIC₅₀, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 18 potential candidates, all being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings support the assumption that OX derivatives may represent a novel class of multi-target inhibitors leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes involved in various important physiological processes.     

Chemical evaluation of fatty acid desaturases as drug targets in Trypanosoma cruzi

Four positional isomers of Thiastearate (TS) and Isoxyl (Thiocarlide) were assayed as fatty acid desaturase inhibitors in Trypanosoma cruzi epimastigotes. 9-TS did not exert a significant effect on growth of T. cruzi, nor on the fatty acid profile of the parasite cells. One hundred micromolars of 10-TS totally inhibited growth, with an effective concentration for 50% growth inhibition (EC₅₀) of 3.0 ± 0.2 μM. Growth inhibition was reverted by supplementing the culture media with oleate. The fatty acid profile of treated cells revealed that conversion of stearate to oleate and palmitate to palmitoleate were drastically reduced and, as a consequence, the total level of unsaturated fatty acids decreased from 60% to 32%. Isoxyl, a known inhibitor of stearoyl-CoA Δ9 desaturase in mycobacteria, had similar effects on T. cruzi growth (EC₅₀ 2.0 ± 0.3 μM) and fatty acid content, indicating that Δ9 desaturase was the target of both drugs. 12- and 13-TS were inhibitors of growth with EC₅₀ values of 50 ± 2 and 10 ± 3 μM, respectively, but oleate or linoleate were unable to revert the effect. Both drugs increased the percentage of oleate and palmitate in the cell membrane and drastically reduced the content of linoleate from 38% to 16% and 12%, respectively, which is in agreement with a specific inhibition of oleate Δ12 desaturase. The absence of corresponding enzyme activity in mammalian cells and the significant structural differences between trypanosome and mammalian Δ9 desaturases, together with our results, highlight these enzymes as promising targets for selective chemotherapeutic intervention.     

A direct spectrophotometric γ-glutamyltransferase inhibitor screening assay targeting the hydrolysis-only mode

γ-Glutamyltransferase (GGT, E.C. 2.3.2.2) catalyzes the hydrolysis and transpeptidation of extracellular glutathione. Due to its central role in maintaining mammalian glutathione homeostasis, GGT is now believed to be a valuable drug target for a variety of life-threatening diseases, such as cancer. Unfortunately, however, effective tools for screening GGT inhibitors are still lacking. We report here the synthesis and evaluation of an α-phenylthio-containing glutathione peptide mimic that eliminates thiophenol upon GGT-catalyzed hydrolysis of the γ-glutamyl peptide bond. The concurrent, real-time spectrophotometric quantification of the released thiophenol using Ellman’s reagent creates a GGT assay format that is simple, robust, and highly sensitive. The versatility of the assay has been demonstrated by its application to the kinetic characterization of equine kidney GGT, and enzyme inhibition assays. The ability of the glutathione mimic to behave as an excellent donor substrate (exhibiting Michaelis-Menten kinetics with a K_m of 11.3 ± 0.5 μM and a k_cat of 90.1 ± 0.8 nmol mg⁻¹ min⁻¹), coupled to the assay’s ability to study the hydrolysis-only mode of the GGT-catalyzed reaction, make our approach amenable to high-throughput drug screening platforms.     

Flow cytometric screening for anti-leishmanials in a human macrophage cell line

High-throughput drug screening methods against the intracellular stage of Leishmania have been facilitated by the development of in vitro models of infection. The use of cell lines rather than primary cells facilitates these methods. Peripheral blood mononuclear cell (PBMC) derived macrophages and THP-1 cells were infected with stationary phase egfp transfected Leishmania amazonensis parasites and then treated with anti-leishmanial compounds. Drug activity was measured using a flow cytometric approach, and toxicity was assessed using either the MTT assay or trypan blue dye exclusion. Calculated EC₅₀’s for amphotericin B, sodium stibogluconate, and miltefosine were 0.1445 ± 0.0005 μg/ml, 0.1203 ± 0.018 mg/ml, and 26.71 μM using THP-1 cells, and 0.179 ± 0.035 μg/ml, 0.1948 ± 0.0364 mg/ml, and 13.77 ± 10.74 μM using PBMC derived macrophages, respectively. We conclude that a flow cytometric approach using egfp transfected Leishmania species can be used to evaluate anti-leishmanial compounds against the amastigote stage of the parasite in THP-1 cells with excellent concordance to human PBMC derived macrophages.     

The influence of β-cyclodextrin on acid-base and tautomeric equilibrium of fluorescein dyes in aqueous solution

The protolytic equilibrium of fluorescein in aqueous solutions was studied in the presence of cycloheptaamylose (β-cyclodextrin, or β-CD). The constants of stepwise ionization of the dye (), K_a0, K_a1, and K_a2 were determined using vis-spectroscopy at ionic strength 0.05 M (NaCl + buffer) and 25 °C. In the presence of 0.0086 M β-CD, the indices of ionization constants are as follows: pK_a0 = 1.21 ± 0.12, pK_a1 = 5.08 ± 0.03, pK_a2 = 6.35 ± 0.02. The changes in these pK_as, as compared with the values determined without cyclodextrin, are unequal. Namely, the pK_a0 value decreases by 1.0, while the pK_a1 value increases by 0.7. Thus, the introduction of β-CD allows to govern the ratios K_a0/K_a1 and K_a1/K_a2, which are equal to, respectively, 141 and 151 in water, and 7.4 × 10³ and 18.6 with cyclodextrin added. Rationalization of the observed phenomenon is possible taking into account the detailed scheme of protolytic equilibrium. Conclusions concerning tautomerism of dye molecules were deduced from absorption spectra; the fractions of tautomers, tautomerization constants, and microscopic ionization constants were evaluated. These data allow concluding that the main reason for the aforementioned pK_a alterations is the binding of H₂R by the cyclodextrin cavity accompanied by turning these neutral species into the colorless lactone. The host-guest interaction of neutral species of fluorescein isothiocyanate, 2,7-dichlorofluorescein, and 3′,4′,5′,6′-tetrachlorofluorescein also results in the cyclodextrin-assisted shift of tautomeric equilibrium. Such nature of interactions is proved by the addition of competing agents, camphor-4-carboxylic acid and sodium n-nonylsulfonate, which results in the removing of neutral dye species from the cycloheptaamylose cavity.     

Estrogen receptor expression and vessel density in the vagina wall in postmenopausal women with prolapse

After menopause, critically estrogen low levels result in modifications in vaginal wall. This cross-sectional study aims to determine whether there is a change in the number of vessels in the lamina propria of the vagina after menopause in parallel to the ER-alpha expression on the vaginal wall. Twelve women who underwent a genital surgery for genital prolapse up to grade II were selected. They were divided into two groups: a premenopausal group (PG) consisting of six women who were 18–40 years old with FSH levels =12 mIU/ml and regular cycles, and a menopausal group (MG) consisting of six women at least one year after menopause who were <65 years old with FSH levels =40 mIU/ml. Slides were stained for ER-alpha immunohistochemistry, and an endothelial cell marker CD3 was used to label vessels which were identified by using a system for morphometry. The number of vessels was significantly higher in the PG than in the MG both on the anterior wall (PG: 1.055 ± 145.8 vessels/mm², MG: 346.6 ± 209.9 vessels/mm², p < 0.0001) and on the posterior wall (PG: 1064 ± 303.3 vessels/mm², MG: 348.6 ± 167.3 vessels/mm², p = 0.0005). The ER-alpha score was significantly higher in the PG than the score for the MG on both the anterior and posterior walls (PG: 6.0 ± 0.52, MG: 2.5 ± 0.89, p = 0.007; PG: 5.8 ± 0.79, MG: 2.7 ± 0.95, p = 0.03, respectively). There was a positive correlation between the ER-alpha score and the vessel concentration on the anterior (r = 0.6656, p = 0.018) and posterior (r = 0.6738, p = 0.016) vaginal walls. Age was strongly negatively correlated with vessel concentration on the vaginal walls (respectively r = -0.9033, p < 0.0001, r = -0.7440, p = 0.0055). Therefore, postmenopausal women with genital prolapse have a smaller number of vessels on the vaginal wall compared to normoestrogenic controls with the same pathological condition. Hypoestrogenism and advancing age are factors that are associated to these changes.     

Sequential interactions of silver–silica nanocomposite (Ag–SiO2NC) with cell wall,metabolism and genetic stability of Pseudomonas aeruginosa,a multiple antibiotic‐resistant bacterium

The study was carried out to understand the effect of silver–silica nanocomposite (Ag–SiO₂NC) on the cell wall integrity, metabolism and genetic stability of Pseudomonas aeruginosa, a multiple drug‐resistant bacterium. Bacterial sensitivity towards antibiotics and Ag–SiO₂NC was studied using standard disc diffusion and death rate assay, respectively. The effect of Ag–SiO₂NC on cell wall integrity was monitored using SDS assay and fatty acid profile analysis, while the effect on metabolism and genetic stability was assayed microscopically, using CTC viability staining and comet assay, respectively. Pseudomonas aeruginosa was found to be resistant to β‐lactamase, glycopeptidase, sulfonamide, quinolones, nitrofurantoin and macrolides classes of antibiotics. Complete mortality of the bacterium was achieved with 80 μg ml^?1 concentration of Ag–SiO₂NC. The cell wall integrity reduced with increasing time and reached a plateau of 70% in 110 min. Changes were also noticed in the proportion of fatty acids after the treatment. Inside the cytoplasm, a complete inhibition of electron transport system was achieved with 100 μg ml^?1 Ag–SiO₂NC, followed by DNA breakage. The study thus demonstrates that Ag–SiO₂NC invades the cytoplasm of the multiple drug‐resistant P. aeruginosa by impinging upon the cell wall integrity and kills the cells by interfering with electron transport chain and the genetic stability.

Significance and Impact of Study

Although the synthesis, structural characteristics and biofunction of silver nanoparticles are well understood, their application in antimicrobial therapy is still at its infancy as only a small number of microorganisms are tested to be sensitive to nanoparticles. A thorough knowledge of the mode of interaction of nanoparticles with bacteria at subcellular level is mandatory for any clinical application. The present study deals with the interactions of Ag–SiO2NC with the cell wall integrity, metabolism and genetic stability of Pseudomonas aeruginosa, which would contribute substantially in strengthening the therapeutic applications of silver nanoparticles.     

Protection and Polyfunctional T Cells Induced by Ag85B-TB10.4/IC31? against Mycobacterium tuberculosis Is Highly Dependent on the Antigen Dose

Background

Previously we have shown that Ag85B-TB10.4 is a highly efficient vaccine against tuberculosis when delivered in a Th1 inducing adjuvant based on cationic liposomes. Another Th1 inducing adjuvant, which has shown a very promising profile in both preclinical and clinical trials, is IC31®. In this study, we examined the potential of Ag85B-TB10.4 delivered in the adjuvant IC31® for the ability to induce protection against infection with Mycobacterium tuberculosis. In addition, we examined if the antigen dose could influence the phenotype of the induced T cells.

Methods and Findings

We found that vaccination with the combination of Ag85B-TB10.4 and IC31® resulted in high numbers of polyfunctional CD4 T cells co-expressing IL-2, IFN-γ and TNF-α. This correlated with protection against subsequent challenge with M.tb in the mouse TB model. Importantly, our results also showed that both the vaccine induced T cell response, and the protective efficacy, was highly dependent on the antigen dose. Thus, whereas antigen doses of 5 and 15 µg did not induce significant protection against M.tb, reducing the dose to 0.5 µg selectively increased the number of polyfunctional T cells and induced a strong protection against infection with M.tb. The influence of antigen dose was also observed in the guinea pig model of aerosol infection with M.tb. In this model a 2.5 fold increase in the antigen dose reduced the protection against infection with M.tb to the level observed in non-vaccinated animals.

Conclusions/Significance

Small changes in the antigen dose can greatly influence the induction of specific T cell subpopulations and the dose is therefore a crucial factor when testing new vaccines. However, the adjuvant IC31® can, with the optimal dose of Ag85B-TB10.4, induce strong protection against Mycobacterium tuberculosis. This vaccine has now entered clinical trials.     

A high-throughput assay based on fluorescence polarization for inhibitors of the polo-box domain of polo-like kinase 1

The serine/threonine kinase polo-like kinase 1 (Plk1) is critically involved in multiple mitotic processes and has been established as an adverse prognostic marker for tumor patients. Plk1 localizes to its substrates and its intracellular anchoring sites via its polo-box domain (PBD), which is unique to the family of polo-like kinases. Therefore, inhibition of the Plk1 PBD has been suggested as an approach to the inhibition of Plk1 that circumvents specificity problems associated with the inhibition of the conserved adenosine triphosphate (ATP) binding pocket. Here we report on the development of a high-throughput assay based on fluorescence polarization that allows the discovery of small-molecule inhibitors of the Plk1 PBD. The assay is based on binding of the Plk1 PBD to a phosphothreonine-containing peptide comprising its optimal binding motif with a K_d of 26 ± 2 nM. It is stable with regard to dimethyl sulfoxide (DMSO) and time, and it has a Z′ value of 0.73 ± 0.06 in a 384-well format.     

A continuous assay for α-methylacyl-coenzyme A racemase using circular dichroism

α-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular dichroism (CD)-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was subcloned into a pET15b vector, and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2R)- or (2S)-ibuprofenoyl-CoA were determined by following the change in ellipticity at 279 nm in the presence of octyl-β-d-glucopyranoside (0.2%). MCR exhibited slightly higher affinity for (2R)-ibuprofenoyl-CoA (K_m = 48 ± 5 μM, k_cat = 291 ± 30 s⁻¹), but turned over (2S)-ibuprofenoyl-CoA (K_m = 86 ± 6 μM, k_cat = 450 ± 14 s⁻¹) slightly faster. MCR expressed as a fusion protein bearing an N-terminal His₆-tag had a catalytic efficiency (k_cat/K_m) that was reduced 22% and 47% in the 2S → 2R and 2R → 2S directions, respectively, relative to untagged enzyme. The continuous CD-based assay offers an economical and efficient alternative method to the labor-intensive, fixed-time assays currently used to measure AMACR activity.     

Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation

For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (Me₂SO). Me₂SO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60 ± 2%. In addition, the number of metabolically active cells 24 h post-thaw, normalised to that before freezing, was found to be between 103 ± 4% and 91 ± 5%. This was found to be comparable to cells frozen using Me₂SO. Although reduced (by 22 ± 2%, p = 0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using Me₂SO, where the doubling time was significantly reduced (by 41 ± 4%, p = 0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications.     

Colorectal cancer metastases affect the biochemical characteristics of the human liver β-adrenoceptor-G-protein-adenylate cyclase system

The sympathetic-catecholamine system is involved in the regulation of hepatic metabolic pathways mainly through cAMP-linked β₂-adrenoceptors (β₂-ARs) in humans and to a lesser extent through cAMP-independent mechanisms, but no information is available about the possible biochemical changes of β₂-ARs and their signalling pathways in human colorectal cancer (CRC) and colorectal cancer hepatic metastases (CRCHM). Changes in density and distribution of β-ARs as well as in post-receptor signalling components were studied in membranes of human liver with CRCHM, and for comparison, in membranes of nonadjacent, non-metastatic human liver (NA-NM) obtained from 13 patients, using binding and competition binding studies. Studies were also carried out using normal and cancerous human colon tissues. In CRCHM, the density of β-ARs (B_max) was significantly reduced, compared to NA-NM liver tissues (40.09 ± 2.83 vs. 23.09 ± 3.24 fmol/mg protein; P < 0.001). A similar decrease in the β-AR density was observed in the colon with primary colorectal cancer compared to healthy colon (37.6 ± 2.2 vs. 23.8 ± 3.5 fmol/mg protein), whereas the affinity of ICYP binding to the receptor remained unaffected. Desensitized β-ARs were uncoupled from stimulatory G-protein (G_S), as total density of β-adrenoceptors in the high affinity state was significantly reduced. Concomitantly, CRCHM elicited decrease in the catalytic adenylate cyclase (AC) activity (cAMP formation) in response to isoproterenol plus GTP or forskolin or NaF. In NA-NM and CRCHM liver, the inhibition–concentration curves of ICI 118.551 showed the presence of a homogeneous population of the β₂-AR subtypes. Neither the binding patterns nor the inhibition constant (K_i) of ICI 118.551 were altered in CRCHM. In CRCHM, the hepatic β-AR-G-protein(s)-AC signalling system was markedly impaired, thus, these changes may well influence β-AR-mediated functions in both organs.     

Allergen-specific B cell subset responses in cow’s milk allergy of late eczematous reactions in atopic dermatitis

B cells have regulatory functions in immune responses. Antigen-specific responses of B cell subsets by allergen stimulation ex vivo were examined in milk allergy of late eczematous reactions. Eight milk allergy subjects and 13 milk tolerant subjects were selected by DBPCFC. PBMCs were stimulated by casein ex vivo and stained for B cell subsets using monoclonal antibodies. CD19+ B cells unchanged from 8.7 ± 3.8% to 8.0 ± 5.1% (p = 0.504, n = 8) in the milk allergy group and decreased in the milk tolerant group from 8.5 ± 3.2% to 5.0 ± 1.6% (p = 0.001, n = 13). The fraction of apoptotic B cells in B cells significantly decreased 4.4 ± 3.1% to 1.3 ± 0.4% (p = 0.027, n = 4) in the allergy group and insignificantly increased from 2.8 ± 0.6% to 5.4 ± 2.6% (p = 0.059, n = 6) in the milk tolerant group. CD5+ regulatory B1 cell% in B cells decreased in milk allergy subjects from 36.2 ± 5.0% to 31.0 ± 5.7% (p = 0.010) and unchanged in milk tolerant subjects from 41.6 ± 10.2% to 43.8 ± 10.0% (p = 0.413). IL-10 producing CD19+CD5+ regulatory B cell% in CD19+CD5+ regulatory B cells significantly decreased from 24.9 ± 6.5% to 13.8 ± 5.6% (p = 0.002, n = 5) by casein stimulation in milk allergy group and unchanged from 44.8 ± 11.3% to 43.9 ± 10.0% (p = 0.297, n = 5) in the milk tolerant group. B cell subset responses to IL-4 and IL-5 were also similar in both groups. B cell subset changes seemed to have diagnostic value. Exact immunologic roles of regulatory CD5+ B1 cells need further investigation.     

A novel class of antagonists for the FFAs receptor GPR40

The free fatty acid receptor, GPR40, is implicated in the pathophysiology of type 2 diabetes, and is a new potential drug target for the treatment of type 2 diabetes. Its antagonist is thought to be not only a useful chemical probe for further exploring the function of GPR40 but also a lead structure for drug development. With virtual screening based on a homology model followed by a cell-based calcium mobilization assay, we found that sulfonamides are a new class of small organic antagonists for GPR40. One of the compounds, DC260126, dose-dependently inhibited GPR40-mediated Ca²⁺ elevations stimulated by linoleic acid, oleic acid, palmitoleic acid and lauric acid (IC₅₀: 6.28 ± 1.14, 5.96 ± 1.12, 7.07 ± 1.42, 4.58 ± 1.14 μM, respectively), reduced GTP-loading and ERK1/2 phosphorylation stimulated by linoleic acid in GPR40-CHO cells, suppressed palmitic acid potentiated glucose-stimulated insulin secretion, and negatively regulated GPR40 mRNA expression induced by oleic acid in Min6 cells.     

A high-throughput assay of NK cell activity in whole blood and its clinical application

Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as ⁵¹Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.     

Determination of the membrane permeability characteristics of Pacific oyster, Crassostrea gigas, oocytes and development of optimized methods to add and remove ethylene glycol

In order for cryopreservation to become a practical tool for aquaculture, optimized protocols must be developed for each species and cell type. Knowledge of a cell’s osmotic tolerance and membrane permeability characteristics can assist in optimized protocol development. In this study, these characteristics were determined for Pacific oyster oocytes and modified methods for loading and unloading ethylene glycol (EG) were tested. Oocytes were found to behave as ideal osmometers and their osmotically inactive fraction (V_b) was calculated to be 0.48. Oocytes exposed to NaCl solutions of 0.6 to 2.3 Osm fertilized at rates equivalent to oocytes left in seawater. This corresponds to volume changes of +27.3 and −38.1 ± 1.2%. The permeability of the oocytes to water (L_p) was determined to be 3.8 ± 0.4 × 10⁻², 5.7 ± 0.8 × 10⁻², and 13.2 ± 1.3 × 10⁻² μm min⁻¹ atm⁻¹, when measured at temperatures of 5, 10 and 20 °C. The respective EG permeability values (P_s) were 9.5 ± 0.1 × 10⁻⁵, 14.6 ± 1.2 × 10⁻⁵, and 41.7 ± 2.4 × 10⁻⁵ cm min⁻¹. The activation energies for L_p and P_s were determined to be 14.5 and 17.5 kcal mol⁻¹, respectively. Different models for EG loading and unloading from oocytes were developed and tested. Post-thaw fertilization did not differ significantly between a published step addition method and single step addition at 20 °C. This represents a considerable reduction in handling. The results of this study demonstrate that the cryobiological characteristics of a given cell type should be taken into account when developing cryopreservation methods.     

NMR study on the hydroxy protons of the Lewis X and Lewis Y oligosaccharides

The ¹H NMR chemical shifts and NOEs of hydroxy protons in Lewis X trisaccharide, β-d-Galp-(1 → 4)[α-l-Fucp-(1 → 3)]-β-d-GlcpNAc, and Lewis Y tetrasaccharide, α-l-Fucp-(1 → 2)-β-d-Galp-(1 → 4)[α-l-Fucp-(1 → 3)]-β-d-GlcpNAc, were obtained for 85% H₂O/15% (CD₃)₂CO solutions. The OH-4 signal of Galp in Lewis X and OH-3, OH-4 signals of Galp, and OH-2 signal of Fucp linked to Galp in Lewis Y had chemical shifts which indicate reduced hydration due to their proximity to the hydrophobic face of the Fucp unit linked to GlcpNAc. The inter-residue NOEs involving the exchangeable NH and OH protons confirmed the stacking interaction between the Fucp linked to the GlcpNAc and the Galp residues in Lewis X and Lewis Y.