Gegen Qinlian decoction ameliorates TNBS-induced ulcerative colitis by regulating Th2/Th1 and Tregs/Th17 cells balance, inhibiting NLRP3 inflammasome activation and reshaping gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese herbal medicine Gegen Qinlian Decoction (GQD) has been clinically shown to be an effective treatment of ulcerative colitis (UC) in China. However, the underlying mechanism of GQD's anti-ulcerative colitis properties and its effect on gut microbiota still deserve further exploration. AIM OF THE STUDY: This study observed the regulatory effects of GQD on Th2/Th1 and Tregs/Th17 cells balance, the NOD-like receptor family pyrin domain containing 3 (NLRP3) infammasome and gut microbiota in TNBS-induced UC in BALB/c mice. MATERIALS AND METHODS: 61 main chemical compounds in the GQD were determined by UPLC-Q-TOF/MS. The UC BALB/c model was established by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and GQD was orally administered at low and high dosages of 2.96 and 11.83 g/kg/day, respectively. The anti-inflammatory effects of GQD for ulcerative colitis were evaluated by survival rate, body weight, disease activity index (DAI) score, colonic weight and index, spleen index, hematoxylin-eosin (HE) staining and histopathological scores. Flow cytometry was used to detect the percentage of CD4, Th1, Th2, Th17 and Tregs cells. The levels of Th1-/Th2-/Th17-/Tregs-related inflammatory cytokines and additional proinflammatory cytokines (IL-1ß, IL-18) were detected by CBA, ELISA, and RT-PCR. The expressions of GATA3, T-bet, NLRP3, Caspase-1, IL-Iß, Occludin and Zonula occludens-1 (ZO-1) on colon tissues were detected by Western blot and RT-PCR. Transcriptome sequencing was performed using colon tissue and 16S rRNA gene sequencing was performed on intestinal contents. Fecal microbiota transplantation (FMT) was employed to assess the contribution of intestinal microbiota and its correlation with CD4 T cells and the NLRP3 inflammasome. RESULTS: GQD increased the survival rate of TNBS-induced UC in BALB/c mice, and significantly improved their body weight, DAI score, colonic weight and index, spleen index, and histological characteristics. The intestinal barrier dysfunction was repaired after GQD administration through promoting the expression of tight junction proteins (Occludin and ZO-1). GQD restored the balance of Th2/Th1 and Tregs/Th17 cells immune response of colitis mice, primarily inhibiting the increase in Th2/Th1 ratio and their transcription factor production (GATA3 and T-bet). Morever, GQD changed the secretion of Th1-/Th2-/Th17-/Tregs-related cytokines (IL-2, IL-12, IL-5, IL-13, IL-6, IL-10, and IL-17A) and reduced the expressions of IL-1ß, IL-18. Transcriptome results suggested that GQD could also remodel the immune inflammatory response of colitis by inhibiting NOD-like receptor signaling pathway, and Western blot, immunohistochemistry and RT-PCR further revealed that GQD exerted anti-inflammatory effects by inhibiting the NLRP3 inflammasome, such as down-regulating the expression of NLRP3, Caspase-1 and IL-1ß. More interestingly, GQD regulated gut microbiota dysbiosis, suppressed the overgrowth of conditional pathogenic gut bacteria like Helicobacter, Proteobacteria, and Mucispirillum, while the probiotic gut microbiota, such as Lactobacillus, Muribaculaceae, Ruminiclostridium_6, Akkermansia, and Ruminococcaceae_unclassified were increased. We further confirmed that GQD-treated gut microbiota was sufficient to relieve TNBS-induced colitis by FMT, involving the modulation of Th2/Th1 and Tregs/Th17 balance, inhibition of NLRP3 inflammasome activation, and enhancement of colonic barrier function. CONCLUSIONS: GQD might alleviate TNBS-induced UC via regulating Th2/Th1 and Tregs/Th17 cells Balance, inhibiting NLRP3 inflammasome and reshaping gut microbiota, which may provide a novel strategy for patients with colitis.

Transient receptor potential melastatin 2 is involved in trinitrobenzene sulfonic acid-induced acute and chronic colitis-associated fibrosis progression in mice.

Crohn's disease, a chronic and recurrent gastrointestinal disease, frequently causes intestinal fibrosis. Transient receptor potential melastatin 2 (TRPM2), a non-selective cation channel, is activated by reactive oxygen species. This study investigated the role of TRPM2 in acute colitis and chronic colitis-associated fibrosis progression. Acute colitis and chronic colitis-associated fibrosis were induced in TRPM2-deficient (TRPM2KO) and wild-type (WT) mice through single and repeated intrarectal injections of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Bone marrow-derived macrophages (BMDMs) from WT and TRPM2KO mice were stimulated using H2O2. In WT mice, a single TNBS injection induced acute colitis with upregulated inflammatory cytokines/chemokines and Th1/Th17-related cytokines, while repeated TNBS injections induced chronic colitis-associated fibrosis with upregulation of fibrogenic factors and Th2-related cytokines. Acute colitis and chronic colitis-associated fibrosis with cytokines/chemokine upregulation and fibrogenic factors were considerably suppressed in TRPM2KO mice. Treating BMDMs with H2O2 increased cytokine/chemokine expression and JNK, ERK, and p38 phosphorylation; however, these responses were significantly less in TRPM2KO than in WT mice. These findings suggest that TRPM2 contributes to acute colitis progression via Th1/Th17-mediated immune responses. Furthermore, TRPM2 may be directly involved in colitis-associated fibrosis induction, likely due to the regulation of Th2/TGF-ß1-mediated fibrogenesis in addition to a consequence of acute colitis progression.

Catechol-O-Methyltransferase Loss Drives Cell-Specific Nociceptive Signaling via the Enteric Catechol-O-Methyltransferase/microRNA-155/Tumor Necrosis Factor α Axis.

BACKGROUND & AIMS: The etiology of abdominal pain in postinfectious, diarrhea-predominant irritable bowel syndrome (PI-IBS-D) is unknown, and few treatment options exist. Catechol-O-methyltransferase (COMT), an enzyme that inactivates and degrades biologically active catecholamines, plays an important role in numerous physiologic processes, including modulation of pain perception. Our objective was to determine the mechanism(s) of how decreased colonic COMT in PI-IBS-D patients contributes to the chronic abdominal pain phenotype after enteric infections. METHODS: Colon neurons, epithelial cells, and macrophages were procured with laser capture microdissection from PI-IBS-D patients to evaluate cell-specific colonic COMT, microRNA-155 (miR-155), and tumor necrosis factor (TNF) α expression levels compared to recovered patients (infection cleared: did not develop PI-IBS-D) and control individuals. COMT-/-, colon-specific COMT-/-, and miR-155-/- mice and human colonoids were used to model phenotypic expression of COMT in PI-IBS-D patients and to investigate signaling pathways linking abdominal pain. Citrobacter rodentium and trinitrobenzene sulfonic acid animal models were used to model postinflammatory changes seen in PI-IBS-D patients. RESULTS: Colonic COMT levels were significantly decreased and correlated with increased visual analog scale abdominal pain ratings in PI-IBS-D patients compared to recovered patients and control individuals. Colonic miR-155 and TNF-α were increased in PI-IBS-D patients with diminished colonic COMT. COMT-/- mice had significantly increased expression of miR-155 and TNF-α in both colon tissues and dorsal root ganglia. Introduction of cV1q antibody (anti-TNF-α) into mice reversed visceral hypersensitivity after C rodentium and trinitrobenzene sulfonic acid. CONCLUSIONS: Decreased colonic COMT in PI-IBS-D patients drives abdominal pain phenotypes via the COMT/miR-155/TNF-α axis. These important findings will allow new treatment paradigms and more targeted and personalized medicine approaches for gastrointestinal disorders after enteric infections.

Matrix metalloproteinase 7 contributes to intestinal barrier dysfunction by degrading tight junction protein Claudin-7.

Background: Previous studies implicated matrix metalloproteinases (MMPs), such as MMP-7, in inflammatory bowel diseases (IBD) by showing increased activity during inflammation of the gut. However, the pathophysiological roles of MMP-7 have not been clearly elucidated. Methods: The expression of MMP-7 was assessed in colonic biopsies of patients with ulcerative colitis (UC), in rodents with experimental colitis, and in cell-based assays with cytokines. Wild-type and MMP-7-null mice treated with dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid were used for determining the pro-inflammatory function(s) of MMP-7 in vivo. Results: MMP-7 was highly expressed in patients with UC and in rodents with experimental colitis. IL-1ß, IL-4, IL-13, TNFα, or lipopolysaccharide enhanced MMP-7 expression in human colonic epithelial cells, rat colonic smooth muscle cells, and THP-1-derived macrophages. Active MMP-7 degraded tight junction protein Claudin-7 in epithelial cells, cleaved recombinant Claudin-7 in cell-free system, and increased Caco-2 monolayer permeability. Immunostaining of colon biopsies revealed up-regulation of MMP-7 and reduction of Claudin-7 in UC patients. Compared to wild-type mice, Mmp7 -/- mice had significantly less inflammation in the colon upon DSS insult. DSS-induced alterations in junction proteins were mitigated in Mmp7 -/- mice, suggesting that MMP-7 disrupts the intestinal barrier. MMP-7 antibody significantly ameliorated colonic inflammation and Claudin-7 reduction in 2 different rodent models of colitis. Summary: MMP-7 impairs intestinal epithelial barrier by cleavage of Claudin-7, and thus aggravating inflammation. These studies uncovered Claudin-7 as a novel substrate of MMP-7 in the intestinal epithelium and reinforced MMP-7 as a potential therapeutic target for IBD.

Tangeretin Inhibits IL-12 Expression and NF-κB Activation in Dendritic Cells and Attenuates Colitis in Mice.

In the preliminary study, tangeretin (5,6,7,8,4'-pentamethoxy flavone), a major constituent of the pericarp of Citrus sp., inhibited TNF-α, IL-12, and IL-23 expression and nuclear factor kappa-B activation in lipopolysaccharide-stimulated dendritic cells; however, it did not affect IL-10 expression. Furthermore, tangeretin (5, 10, and 20 µM) suppressed the activation and translocation of nuclear factor kappa-B (p65) into the nuclei in vitro by inhibiting the binding of lipopolysaccharide on dendritic cells. Oral administration of tangeretin (10 and 20 mg/kg) suppressed the inflammatory responses, such as nuclear factor kappa-B and mitogen-activated protein kinase activation and myeloperoxidase activity, in the colon of mice with 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Tangeretin increased 2,4,6-trinitrobenzene sulfonic acid-suppressed expression of tight junction proteins occludin, claudin-1, and ZO-1. Tangeretin also inhibited 2,4,6-trinitrobenzene sulfonic acid-induced differentiation of Th1 and Th17 cells as well as the expression of T-bet, RORγt, interferon-γ, IL-12, IL-17, and TNF-α. However, tangeretin increased 2,4,6-trinitrobenzene sulfonic acid-suppressed differentiation of regulatory T cells as well as the expression of Foxp3 and IL-10. These results suggest that oral administration of tangeretin may attenuate colitis by suppressing IL-12 and TNF-α expression and nuclear factor kappa-B activation through the inhibition of lipopolysaccharide binding on immune cells such as dendritic cells.

Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca2+-ATPase in the absence of Ca2+.

Trinitrophenyl derivatives of adenine nucleotides are widely used for probing ATP-binding sites. Here we describe crystal structures of Ca(2+)-ATPase, a representative P-type ATPase, in the absence of Ca(2+) with bound ATP, trinitrophenyl-ATP, -ADP, and -AMP at better than 2.4-Šresolution, stabilized with thapsigargin, a potent inhibitor. These crystal structures show that the binding mode of the trinitrophenyl derivatives is distinctly different from the parent adenine nucleotides. The adenine binding pocket in the nucleotide binding domain of Ca(2+)-ATPase is now occupied by the trinitrophenyl group, and the side chains of two arginines sandwich the adenine ring, accounting for the much higher affinities of the trinitrophenyl derivatives. Trinitrophenyl nucleotides exhibit a pronounced fluorescence in the E2P ground state but not in the other E2 states. Crystal structures of the E2P and E2 ∼ P analogues of Ca(2+)-ATPase with bound trinitrophenyl-AMP show that different arrangements of the three cytoplasmic domains alter the orientation and water accessibility of the trinitrophenyl group, explaining the origin of "superfluorescence." Thus, the crystal structures demonstrate that ATP and its derivatives are highly adaptable to a wide range of site topologies stabilized by a variety of interactions.

Toxicity of trinitrotoluene to sheepshead minnows in water exposures.

Lethal effects of trinitrotoluene (TNT) to juvenile sheepshead minnows (JSHM) (Cyprinodon variegatus) were assessed in ten-day water exposures. Ten-day median lethal concentrations (LC50s) were 2.3 and 2.5 mg L(-1), the 10-d median lethal residue value (LR50) was 26.1 micromol kg(-1) wet weight (ww), and bioconcentration factors (BCFs) ranged from 0.7 to 2.4 L kg(-1). The lethal effects of TNT and its transformation products 2-aminodinitrotoluene (2-ADNT), 2,4-diaminonitrotoluene (2,4-DANT) and trinitrobenzene (TNB) to JSHM were compared in 5-d static-renewal exposures. Nitroreduction decreased the toxicity of TNT to SHM, as the 5-d LC50 for 2-ADNT was 8.6 mg L(-1) and the lowest lethal concentration of 2,4-DANT was 50.3 mg L(-1). TNB (5-d LC50=1.2 mg L(-1)) was more toxic than TNT to SHM. The 5-d LR50s were 4.3 mg kg(-1)ww (20.4 micromol kg(-1)) for SumTNT (TNT exposure) and 54.2 mg kg(-1)ww (275.3 micromol kg(-1)) for 2-ADNT and significant mortality occurred at 47.4 mg kg(-1)ww (283.6 micromol kg(-1)). The range of BCF values was from 1.8 to 2.4, 5.6 to 8.0, and 0.6 to 0.9Lkg(-1) for TNT, 2-ADNT, and 2,4-DANT, respectively.

Modulating catalytic activity by unnatural amino acid residues in a GSH-binding loop of GST P1-1.

The loop following helix alpha2 in glutathione transferase P1-1 has two conserved residues, Cys48 and Tyr50, important for glutathione (GSH) binding and catalytic activity. Chemical modification of Cys48 thwarts the catalytic activity of the enzyme, and mutation of Tyr50 generally decreases the k(cat) value and the affinity for GSH in a differential manner. Cys48 and Tyr50 were targeted by site-specific mutations and chemical modifications in order to investigate how the alpha2 loop modulates GSH binding and catalysis. Mutation of Cys48 into Ala increased K(M)(GSH) 24-fold and decreased the binding energy of GSH by 1.5 kcal/mol. Furthermore, the protein stability against thermal inactivation and chemical denaturation decreased. The crystal structure of the Cys-free variant was determined, and its similarity to the wild-type structure suggests that the mutation of Cys48 increases the flexibility of the alpha2 loop rather than dislocating the GSH-interacting residues. On the other hand, replacement of Tyr50 with Cys, producing mutant Y50C, increased the Gibbs free energy of the catalyzed reaction by 4.8 kcal/mol, lowered the affinity for S-hexyl glutathione by 2.2 kcal/mol, and decreased the thermal stability. The targeted alkylation of Cys50 in Y50C increased the affinity for GSH and protein stability. Characterization of the most active alkylated variants, S-n-butyl-, S-n-pentyl-, and S-cyclobutylmethyl-Y50C, indicated that the affinity for GSH is restored by stabilizing the alpha2 loop through positioning of the key residue into the lock structure of the neighboring subunit. In addition, k(cat) can be further modulated by varying the structure of the key residue side chain, which impinges on the rate-limiting step of catalysis.

SAP expression in T cells, not in B cells, is required for humoral immunity.

SAP (also named SH2D1A) is an intracellular adaptor molecule expressed in T cells, natural killer (NK) cells, and some B cells. The SAP gene is mutated in X-linked lymphoproliferative (XLP) disease, a human immunodeficiency characterized by a faulty immune response to Epstein-Barr virus infection. Previous reports documented severe defects in antibody production and germinal center (GC) formation in SAP-deficient humans and mice genetically engineered to lack SAP expression. However, in vitro studies and adoptive transfer experiments provided conflicting data as to whether this phenotype is caused by a functional defect resulting from SAP deficiency in T cells, B cells, or both. Here, we ascertained which cell types are responsible for this humoral immunity defect by using a conditional gene targeting approach. We also thoroughly examined the expression pattern of SAP in normal immune cells by using intracellular flow cytometry. The results showed that expression of SAP in T cells, but not in B cells or NK cells, is required and sufficient for SAP-dependent antibody production and GC formation. These data provide a critical insight into the mechanism by which SAP regulates humoral immunity. They also help elucidate the basis of a severe human immunodeficiency.

Environmental process descriptors for TNT, TNT-related compounds and picric acid in marine sediment slurries.

Process descriptors were determined for picric acid, TNT, and the TNT-related compounds 2,4DNT, 2,6DNT, 2ADNT, 4ADNT, 2,4DANT, 2,6DANT, TNB and DNB in marine sediment slurries. Three marine sediments of various physical characteristics (particle size ranging from 15 to >90% fines and total organic carbon ranging from <0.10 to 3.60%) were kept in suspension with 20ppt saline water. Concentrations of TNT and its related compounds decreased immediately upon contact with the marine sediment slurries, with aqueous concentrations slowly declining throughout the remaining test period. Sediment-water partition coefficients could not be determined for these compounds since solution phase concentrations were unstable. Kinetic rates and half-lives were influenced by the sediment properties, with the finer grained, higher organic carbon sediment being the most reactive. Aqueous concentrations of picric acid were very stable, demonstrating little partitioning to the sediments. Degradation to picramic acid was minimal, exhibiting concentrations at or just above the detection limit.

Conformational and functional characterization of trapped complexes of the P-glycoprotein multidrug transporter.

The Pgp (P-glycoprotein) multidrug transporter couples ATP hydrolysis at two cytoplasmic NBDs (nucleotide-binding domains) to the transport of hydrophobic compounds. Orthovanadate (V(i)) and fluoroaluminate (AlF(x)) trap nucleotide in one NBD by forming stable catalytically inactive complexes (Pgp-M2+-ADP-X), which are proposed to resemble the catalytic transition state, whereas the complex formed by beryllium fluoride (BeF(x)) is proposed to resemble the ground state. We studied the trapped complexes formed via incubation of Pgp with ATP (catalytically forward) or ADP (reverse) and V(i), BeF(x) or AlF(x) using Mg2+ or Co2+ as the bivalent cation. Quenching of intrinsic Pgp tryptophan fluorescence by acrylamide, iodide and caesium indicated that conformational changes took place upon formation of the trapped complexes. Trapping with V(i) and ATP led to a 6-fold increase in the acrylamide quenching constant, K(SV), suggesting that large conformational changes take place in the Pgp transmembrane regions on trapping in the forward direction. Trapping with V(i) and ADP gave only a small change in quenching, indicating that the forward- and reverse-trapped complexes are different. TNP (trinitrophenyl)-ATP/TNP-ADP interacted with all of the trapped complexes, however, the fluorescence enhancement differed for the trapped states, suggesting a change in polarity in the nucleotide-binding sites. The nucleotide-binding site of the BeF(x)-trapped complex was much more polar than that of the V(i) and AlF(x) complexes. Functionally, all the trapped complexes were able to bind drugs and TNP-nucleotides with unchanged affinity compared with native Pgp.

H2O2 intensifies CN(-)-induced apoptosis in pea leaves.

H2O2 intensifies CN(-)-induced apoptosis in stoma guard cells and to lesser degree in basic epidermal cells in peels of the lower epidermis isolated from pea leaves. The maximum effect of H2O2 on guard cells was observed at 10(-4) M. By switching on non-cyclic electron transfer in chloroplasts menadione and methyl viologen intensified H2O2 generation in the light, but prevented the CN--induced apoptosis in guard cells. The light stimulation of CN- effect on guard cell apoptosis cannot be caused by disturbance of the ribulose-1,5-bisphosphate carboxylase function and associated OH* generation in chloroplasts with participation of free transition metals in the Fenton or Haber-Weiss type reactions as well as with participation of the FeS clusters of the electron acceptor side of Photosystem I. Menadione and methyl viologen did not suppress the CN(-)-induced apoptosis in epidermal cells that, unlike guard cells, contain mitochondria only, but not chloroplasts. Quinacrine and diphenylene iodonium, inhibitors of NAD(P)H oxidase of cell plasma membrane, had no effect on the respiration and photosynthetic O2 evolution by leaf slices, but prevented the CN(-)-induced guard cell death. The data suggest that NAD(P)H oxidase of guard cell plasma membrane is a source of reactive oxygen species (ROS) needed for execution of CN(-)-induced programmed cell death. Chloroplasts and mitochondria were inefficient as ROS sources in the programmed death of guard cells. When ROS generation is insufficient, exogenous H2O2 exhibits a stimulating effect on programmed cell death. H2O2 decreased the inhibitory effects of DCMU and DNP-INT on the CN(-)-induced apoptosis of guard cells. Quinacrine, DCMU, and DNP-INT had no effect on CN(-)-induced death of epidermal cells.

Ghrelin stimulates interleukin-8 gene expression through protein kinase C-mediated NF-kappaB pathway in human colonic epithelial cells.

Ghrelin, a newly identified gastric peptide, is known for its potent activity in growth hormone (GH) release and appetite. Although ghrelin is involved in several other responses such as stress and intestinal motility, its potential role in intestinal inflammation is not clear. Here, we show that expression of ghrelin and its receptor mRNA is significantly increased during acute experimental colitis in mice injected intracolonically with trinitrobenzene sulfate (TNBS). We found by PCR that ghrelin receptor mRNA is expressed in non-transformed human colonic epithelial NCM460 cells. Exposure of NCM460 cells stably transfected with ghrelin receptor mRNA to ghrelin, increased IkappaBalpha phosphorylation and its subsequent degradation. In addition, ghrelin stimulated NF-kappaB-binding activity and NF-kappaB p65 subunit phosphorylation, and induced IL-8 promoter activity and IL-8 protein secretion. Furthermore, our data show that ghrelin-induced IkappaBalpha and p65 phosphorylation was markedly reduced by pharmacological inhibitors of intracellular calcium mobilization (BAPTA/AM) and protein kinase C (GF 109203X). Pretreatment with BAPTA/AM or GF109203X also significantly attenuated ghrelin-induced IL-8 production. Together, our results strongly suggest that ghrelin may be a proinflammatory peptide in the colon. Ghrelin may participate in the pathophysiology of colonic inflammation by inducing PKC-dependent NF-kappaB activation and IL-8 production at the colonocyte level.

Fluorescent and colored trinitrophenylated analogs of ATP and GTP.

Fluorescent and colored trinitrophenylated (TNP) analogs of ATP and GTP can interact with nucleotide-requiring enzymes and proteins as a substitute for the parent nucleotide. These analogs have strong binding affinities for most nucleotide-requiring systems. Their bindings are easily detected by absorption and fluorescence changes in the visible region. Recent years have seen dramatic developments in the application of the TNP nucleotide analogs as spectroscopic probes for the study on the nucleotide-interacting properties of various enzymes and proteins including their mutants. This review is intended as a broad overview of currently extensively used applications of the nucleotide analogs in various biological systems.

The Methanocaldococcus jannaschii protein Mj0968 is not a P-type ATPase.

The Methanocaldococcus jannaschii (formerly Methanococcus jannaschii) protein Mj0968 has been reported to represent a soluble P-type ATPase [Ogawa et al., FEBS Lett. 471 (2000) 99-102]. In this study, we report that the heterologously expressed Mj0968-His(10) protein exhibits high rates of phosphatase activity, whereas only very low ATPase activity was measured. Replacement of the aspartate residue in the DSAGT motif (D7A), which becomes phosphorylated during the reaction cycle of P-type ATPases, does not affect the V(max), but only the K(M) of the reaction. Labeling studies with [gamma-(32)P]ATP and [alpha-(32)P]ATP revealed that the previously reported labeling experiments [Ogawa et al., 2000] do not necessarily show phosphorylation of Mj0968, but rather point to ATP binding. Binding studies with trinitrophenyl adenosine nucleotides showed low apparent K(d) values for those molecules. These results provide evidence that the native function of Mj0968 seems to be that of a phosphatase, rather than that of an ATP-hydrolyzing enzyme.

Stoichiometry and affinity of nucleotide binding to P-glycoprotein during the catalytic cycle.

Drug transport mediated by P-glycoprotein (Pgp) is driven by hydrolysis of ATP at the two cytosolic nucleotide binding domains. However, little is currently known concerning the stoichiometry of nucleotide binding and how both stoichiometry and binding affinity change during the catalytic cycle of the transporter. To address this issue, we used fluorescence techniques to measure both the number of nucleotides bound to P-glycoprotein during various stages of the catalytic cycle and the affinity of nucleotide binding. Results showed that resting state P-glycoprotein bound two molecules of the fluorescent nucleotide derivative, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), whereas the vanadate-trapped transition state bound only one nucleotide molecule. Both resting and transition state P-glycoprotein showed similar affinity for TNP-ATP/TNP-ADP and unlabeled ATP/ADP. Following binding of various drugs, resting state P-glycoprotein displayed a higher affinity for nucleotides, up to 4-fold depending on the compound used. In contrast, the transition state showed substantially lower (up to 3-fold) nucleotide binding affinity when the drug binding site(s) is/are occupied. These results indicate that both nucleotide binding domains of P-glycoprotein are likely to be occupied with either ATP (or ADP) in the resting state and the transition state in the absence of transport substrates. Drugs alter the binding affinity to favor association of ATP with P-glycoprotein at the start of the catalytic cycle and release of ADP from the transition state following nucleotide hydrolysis.

BOB.1/OBF.1 deficiency affects marginal-zone B-cell compartment.

Marginal-zone (MZ) B cells represent a first line of defense against particulate blood-borne antigens. Together with the B1 cells, they are responsible for the early response against type II T-independent antigens. The molecular pathways controlling the development of MZ B cells are only poorly understood. We found that these cells are virtually absent in mice deficient in the BOB.1/OBF.1 coactivator. Loss of these B cells was demonstrated by the lack of cells showing the appropriate cell surface phenotype but also by histological analyses and tri-nitro-phenol-Ficoll capturing. The lack of these cells is a B-cell-intrinsic defect, as shown by bone marrow complementation experiments. We also show that the expression of BOB.1/OBF.1 in peripheral B cells is required for the development of MZ B lymphocytes. Our analysis of BOB.1/OBF.1-deficient splenic B cells reveals alterations in cell motility, tumor necrosis factor receptor expression, and B-cell receptor (BCR) signaling. These changes could contribute to the loss of MZ B lymphocytes by altering the maturation of the cells. Interestingly, development of and BCR signaling in B1 B cells are completely normal in BOB.1/OBF.1 mutant mice.

The antibody site in Atlantic salmon; phage display and modeling of scFv with anti-hapten binding ability.

Cloning and sequencing the cDNA of around 50 VH (VDJ) and 15 VL genes in Atlantic salmon demonstrated nine VH families (above 80% identity within each family) and one dominating but relatively diverse VL family in this species. The highest variability of the VH was seen in the CDR3, but CDR2 also expressed a modest variability. The 'whole' antibody repertoire was expressed as single chain Fv (scFv) in a phage display library by combining 12 VH and two VL specific primers (FR1/microl and FR1/CL, respectively). The PCR products (VH and VL) were ligated (with a G-rich spacer) into the lambda Surf-Zap (Stratagene) vector and expressed as a surface fusion protein on the M13 phage. Anti-TNP and anti-FITC specific scFv clones were isolated by panning using hapten-coated magnetic beads and the coding DNA sequenced. The specificities of the anti-TNP and anti-FITC clones were similar to mouse monoclonal antibodies. 3D-models of the active sites (CDRs) of the anti-TNP and anti-FITC clones suggest hapten-interacting structures of the salmon antibody site similar to mammalian antibodies.

The chemical modification of alpha-chymotrypsin with both hydrophobic and hydrophilic compounds stabilizes the enzyme against denaturation in water-organic media.

We considered alpha-chymotrypsin (CT) in homogeneous water-organic media as a model system to examine the influence of enzyme chemical modification with hydrophilic and hydrophobic substances on its stability, activity and structure. Both types of modifying agents may lead to considerable stabilization of the enzyme in water-ethanol and water-DMF mixtures: (i) the range of organic cosolvent concentration at which enzyme activity (Vm) is at least 100% of its initial value is broadened and (ii) the range of organic cosolvent concentration at which the residual enzyme activity is observed is increased. We found that for both types of modification the stabilization effect can be correlated with the changes in protein surface hydrophobicity/hydrophilicity brought about by the modification. Circular dichroism studies indicated that the effects of these two types of modification on CT structure and its behavior in water-ethanol mixtures are different. Differential scanning calorimetry studies revealed that after modification two or three fractions or domains, differing in their stability, can be resolved. The least stable fractions (or domains) have properties similar to native CT.

Effect of ionic strength on the conformation of myosin subfragment 1-nucleotide complexes.

The effect of ionic strength on the conformation and stability of S1 and S1-nucleotide-phosphate analog complexes in solution was studied. It was found that increasing concentration of KCl enhances the reactivity of Cys(707) (SH1 thiol) and Lys(84) (reactive lysyl residue) and the nucleotide-induced tryptophan fluorescence increment. In contrast, high KCl concentration lowers the structural differences between the intermediate states of ATP hydrolysis in the vicinity of Cys(707), Trp(510) and the active site, possibly by increasing the flexibility of the molecule. High concentrations of neutral salts inhibit both the formation and the dissociation of the M**.ADP.Pi analog S1.ADP.Vi complex. High ionic strength profoundly affects the structure of the stable S1.ADP.BeF(x) complex, by destabilizing the M*.ATP intermediate, which is the predominant form of the complex at low ionic strength, and shifting the equilibrium to favor the M**.ADP.Pi state. The M*.ATP intermediate is destabilized by perturbation of ionic interactions possibly by disruption of salt bridges. Two salt-bridge pairs, Glu(501)-Lys(505) in the Switch II helix and Glu(776)-Lys(84) connecting the catalytic domain to the lever arm, seem most appropriate to consider for participating in the ionic strength-induced transition of the open M*.ATP to the closed M**.ADP.Pi state of S1.