Inhibition of Pseudomonas aeruginosa ExsA DNA-Binding Activity by N-Hydroxybenzimidazoles.

The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence determinant and a potential target for antivirulence drugs. One candidate target is ExsA, a member of the AraC family of DNA-binding proteins required for expression of the T3SS. A previous study identified small molecules based on an N-hydroxybenzimidazole scaffold that inhibit the DNA-binding activity of several AraC proteins, including ExsA. In this study, we further characterized a panel of N-hydroxybenzimidazoles. The half-maximal inhibitory concentrations (IC50s) for the tested N-hydroxybenzimidazoles ranged from 8 to 45 µM in DNA-binding assays. Each of the N-hydroxybenzimidazoles protected mammalian cells from T3SS-dependent cytotoxicity, and protection correlated with reduced T3SS gene expression in a coculture infection model. Binding studies with the purified ExsA DNA-binding domain (i.e., lacking the amino-terminal self-association domain) confirmed that the activity of N-hydroxybenzimidazoles results from interactions with the DNA-binding domain. The interaction is specific, as an unrelated DNA-binding protein (Vfr) was unaffected by N-hydroxybenzimidazoles. ExsA homologs that control T3SS gene expression in Yersinia pestis, Aeromonas hydrophila, and Vibrio parahaemolyticus were also sensitive to N-hydroxybenzimidazoles. Although ExsA and Y. pestis LcrF share 79% sequence identity in the DNA-binding domain, differential sensitivities to several of the N-hydroxybenzimidazoles were observed. Site-directed mutagenesis based on in silico docking of inhibitors to the DNA-binding domain, and on amino acid differences between ExsA and LcrF, resulted in the identification of several substitutions that altered the sensitivity of ExsA to N-hydroxybenzimidazoles. Development of second-generation compounds targeted to the same binding pocket could lead to drugs with improved pharmacological properties.

Pharmacological characterization and antidiabetic activity of a long-acting glucagon-like peptide-1 analogue conjugated to an antithrombin III-binding pentasaccharide.

AIMS: To examine the biological characteristics of a novel glucagon-like peptide-1 (GLP-1) conjugate, in which an antithrombin III (ATIII)-binding pentasaccharide is conjugated to d-Ala(8) GLP-1 using a tetraethylene glycol linker. METHODS: We assessed GLP-1 receptor binding, cAMP generation and insulin secretory activity of the GLP-1 conjugate in vitro. Circulating half-life, glucose homeostatic and subchronic therapeutic effectiveness were then examined in vivo. RESULTS: The half-life of the GLP-1 conjugate in mice was ∼11 h. In vitro insulin secretion from clonal ß cells and islets was increased (p < 0.001) by the conjugate. The conjugate had half maximum effective concentration values of 1.3 × 10(-7) and 9.9 × 10(-8) M for displacement of (125) I-GLP-1 in competitive GLP-1 receptor binding and cAMP generation, respectively. Glucose tolerance in normal mice, immediately and 4 h after conjugate injection, resulted in significant (p < 0.001) improvements in blood glucose. These effects persisted for >48 h after administration. Daily treatment (21 days) of high-fat-fed and ob/ob mice with 25 nmol/kg conjugate resulted in significant improvement in glucose tolerance (p < 0.001) and reductions in glycated haemoglobin (HbA1c; p < 0.01) equivalent to or better than with exenatide or liraglutide. Treatment of C57BL/KsJ db/db mice for 15 days with 100 nmol/kg conjugate significantly (p < 0.001) reduced glucose and raised plasma insulin. Oral glucose tolerance was significantly (p < 0.001) improved and both 24-h glucose profile (p < 0.001) and HbA1c levels (p < 0.001) were reduced. Islet size (p < 0.001) and pancreatic insulin content were increased without change of islet cell proliferation or apoptosis. CONCLUSION: These data show that d-Ala(8) GLP-1(Lys(37) ) pentasaccharide exerts significant antidiabetic actions and has a projected pharmacokinetic/pharmacodynamic profile that merits further evaluation in humans for a possible once-weekly dosing regimen.

In vitro inhibition of compression-induced catabolic gene expression in meniscal explants following treatment with IL-1 receptor antagonist.

BACKGROUND: Damage to the knee meniscus may result in tears that are difficult or unable to heal, and are often treated by partial removal of the damaged tissue. In vitro, 20% dynamic compressive strains on meniscal tissue explants have resulted in an increase in the release of sulfated glycosaminoglycans (GAG) and nitric oxide (NO) from the tissue explants and increased expression of matrix metalloproteinases (MMP) and interleukin-1α (IL-1α). The objective of this study was to explore the efficacy of IL-1 blockade on the expression of a wide range of genes, as well as NO and GAG release, following dynamic compression of porcine meniscal explants. METHODS: Explants were dynamically compressed for 2 h at 1 Hz to 0, 10, or 20% strain with and without a pre-treatment of 500 ng/ml interleukin-1 receptor antagonist (IL-1RA). Relative changes in gene expression of IL-1α, MMP-1, -3, -13, A Disintegrin and Metalloproteinase with ThromboSpondin 4 (ADAMTS-4), ADAMTS-5, iNOS, aggrecan, and COX-2, as well as changes in NO and GAG release, were measured with standard biochemical assays. RESULTS: Expression of IL-1α, MMP-3, MMP-13, and ADAMTS-4 in superficial explants was significantly downregulated at 20% dynamic strain compared to 10% strain following treatment with IL-1RA. GAG and NO release were not significantly influenced by IL-1RA treatment. CONCLUSIONS: Treatment of meniscal explants with IL-1RA inhibited the expression of many catabolic genes following a single bout of high dynamic strain. IL-1RA may therefore be a potential therapy option during the acute phase of meniscal tear or meniscectomy treatment.

Ampk regulates IgD expression but not energy stress with B cell activation.

Ampk is an energy gatekeeper that responds to decreases in ATP by inhibiting energy-consuming anabolic processes and promoting energy-generating catabolic processes. Recently, we showed that Lkb1, an understudied kinase in B lymphocytes and a major upstream kinase for Ampk, had critical and unexpected roles in activating naïve B cells and in germinal center formation. Therefore, we examined whether Lkb1 activities during B cell activation depend on Ampk and report surprising Ampk activation with in vitro B cell stimulation in the absence of energy stress, coupled to rapid biomass accumulation. Despite Ampk activation and a controlling role for Lkb1 in B cell activation, Ampk knockout did not significantly affect B cell activation, differentiation, nutrient dynamics, gene expression, or humoral immune responses. Instead, Ampk loss specifically repressed the transcriptional expression of IgD and its regulator, Zfp318. Results also reveal that early activation of Ampk by phenformin treatment impairs germinal center formation but does not significantly alter antibody responses. Combined, the data show an unexpectedly specific role for Ampk in the regulation of IgD expression during B cell activation.

Mitogen-activated protein kinase and protein kinase A signaling pathways stimulate cholecystokinin transcription via activation of cyclic adenosine 3',5'-monophosphate response element-binding protein.

Cholecystokinin (CCK) is a potent neuropeptide expressed in the small intestine and in the central nervous system. We have examined the effect of basic fibroblast factor (bFGF) and forskolin on CCK gene transcription and depicted the signaling pathways that lead to promoter activation. bFGF and forskolin stimulated promoter activity via a cAMP response element (CRE)/12-O-tetradecanoylphorbol-13-acetate response element (TRE) located 80 bp upstream from the transcription initiation site. In nuclear extracts from unstimulated as well as stimulated cells, only CRE-binding protein (CREB) and activating transcription factor-1 (ATF-1) bound to the CRE/TRE, and activation was associated with phosphorylation of CREB serine-133 and ATF-1 serine-63. In murine F9 cells, CREB stimulated promoter activity 10-fold in the presence of protein kinase A (PKA), and in SK-N-MC cells activation was inhibited 60-70% by a dominant negative CREB mutant. In contrast, ATF-1 had no effect in F9 cells and exhibited a dominant negative effect in SK-N-MC cells. bFGF stimulation led to phosphorylation of the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase (ERK) MAPK and promoter activation, phosphorylation of CREB, and GAL4-CREB-dependent transcription were selectively prevented by a dominant negative Ras-mutant, the p38 MAPK-specific inhibitor SB203580, and the MAP/ERK kinase 1 (MEK1) inhibitor PD098059. Forskolin stimulation proceeded via the PKA pathway, and to a minor extent via the p38 and ERK MAPK pathways. We conclude that bFGF and forskolin stimulate the CCK gene promoter via the CRE/TRE(-80) in the proximal promoter region. Signaling proceeds through the p38 MAPK, the ERK MAPK, and the PKA-signaling pathways, which leads to cumulative phosphorylation and activation of CREB. We propose that bFGF in combination with neurotransmitters/neuropeptides coupling to the PKA-signaling pathway play an important role in the control of CCK gene expression.

Characterization of quinolone antibacterial-induced convulsions and increases in nuclear AP-1 DNA- and CRE-binding activities in mouse brain.

The quinolone antibacterials enoxacin and norfloxacin (2.5 mg/kg, i.v.) provoked clonic convulsions in mice treated concomitantly with biphenylacetic acid (BPAA, 100 mg/kg, i.p.), a major metabolite of the nonsteroidal anti-inflammatory drug fenbufen. Gel-shift assays showed that enoxacin-induced convulsions resulted in increases in nuclear activator protein 1 (AP-1) DNA- and cyclic AMP responsive element (CRE)-binding activities in the cerebral cortex and hippocampus, but not in other regions, such as the cerebellum and thalamus. In contrast, ofloxacin and levofloxacin, at the same doses, in the presence of BPAA did not evoke convulsions or increase these DNA-binding activities. Administration of these quinolones and BPAA alone elicited neither convulsions nor increases in these DNA-binding activities. These results suggest that the increased nuclear AP-1 DNA- and CRE-binding activities in the cerebral cortex and hippocampus induced by quinolones with BPAA correlated with seizure activities and that these brain regions play pivotal roles in quinolone-induced convulsions.

Genotoxicity of bleomycin in human cell lines differing in catalase activity.

The influence of catalase on the genotoxic effect of bleomycin (BLM) has been evaluated in three cell lines which differ in catalase activity. CRL1307, cells from Xeroderma pigmentosum patient and CLV102, normal embryonic cells have catalase activity 3.5 and 5 times lower then CRL2088, normal skin fibroblasts. Genotoxicity of BLM (0.5-50 micrograms/ml, 2 h treatment) measured with in vitro micronucleus test did not differ in three tested lines. BLM at concentration range from 1 to 25 micrograms/ml (2 h treatment), tested in comet assay, caused similar degree of DNA damage in CLV102 and CRL2088 cells. Exogenous catalase (300 and 900 u/ml) added to the assay medium with BLM did not influence the micronuclei induction. The absence of endo- and exogenous catalase influence on BLM genotoxicity suggests that not hydrogen peroxide but other reactive oxygen species are formed in reaction of activated BLM with molecular oxygen.

Changes of selected morphotic parameters and blood plasma proteins in blood of divers after a single short-time operational heliox exposure.

In the Polish Navy, deep-water dives, performed for the needs of the maritime industry, are conducted using our own national technology and trimix as a breathing medium. In this paper are presented the results obtained during a short-time deep-water diving test using the principles of US Navy technology, combined with the use of diving equipment type AF-2 and heliox-type breathing mixture in the open circuit. In the performed examinations changes in clinical parameters were assessed viz.: blood morphology, hematocrit level, concentration of C3c, C4, IgG, IgA, IgM, CRP, concentration of fibrinogen and factor XII level, obtained 30 minutes prior to commencement, immediately after completion, and 24 hours after termination of the exposure. The results thus generated were subjected to a preliminary analysis by the description of trends observed. It was revealed that the diving technology employed did not generate substantial changes in the examined parameters of blood in divers, and the increase of neutrophils, blood platelets and fibrinogen concentration in the blood plasma immediately after diving is of temporary character, being a typical reaction observed during diving.

The implementation of factor analysis for the evaluation of selected blood parameter changes induced by hyperbaric exposure.

This paper discusses the application of factor analysis when used to compare selected blood parameter (a three-parameter smear, hematocrit, C3c, C4, IgG, IgA, IgM, CRP, fibrinogen and the level of factor XII) properties, just before, and after exposure to pressure changes, and 24-hours after the completion of decompression. To-date the most popular method of statistical analysis was based only on investigation of the significance of the separated individual parameters. This factor analysis that has not been applied previously in the analysis of such problems, enabled the neutral hierarchic evaluation of the significant parameter changes within their chosen range, and mutual relationships. It seems that the application of this method is purposeful and it can be an objective tool for evaluating the significance of changes in blood constituency induced by pressure.

Kinetic studies of cAMP-induced allosteric changes in cyclic AMP receptor protein from Escherichia coli.

Cyclic AMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli. The ability of CRP to bind specific DNA sequences and stimulate transcription is achieved as result of binding of an allosteric ligand: cAMP. Stopped-flow fluorimetry was employed to study the kinetics of the conformational changes in CRP induced by cAMP binding to high and low affinity receptor sites. Results of experiments using CRP labeled at Cys-178 with 1,5-I-AENS indicate change in conformation of the helix-turn-helix, occurring after the formation of CRP-cAMP(2) complex, i.e. after saturation of the high affinity sites. The observed conformational change occurs according to sequential model of allostery and is described by rate constants: k(c) = 9.7 +/- 0.1 s(-1) and k(-c) = 0.31 +/- 0.05 s(-1), for the forward and backward reaction, respectively. Results of experiments monitored using CRP intrinsic fluorescence suggest that conformational change precedes the formation of CRP-cAMP(4) complex and results from displacement of equilibrium between two forms of CRP-cAMP(2), caused by binding of cAMP to low affinity sites of one of these forms only. The observed conformational change occurs according to concerted model of allostery and is described by rate constants: k(on) = 28 +/- 1.5 s(-1) and k(off) = 75.5 +/- 3 s(-1). Results of experiments using single-tryptophan-containing CRP mutants indicate that Trp-85 is mainly responsible for the observed total change in intrinsic fluorescence of wild-type CRP.

Effects of human recombinant growth hormone on donor-site healing in burned adults.

Patients suffering severe burns have an accelerated catabolism with a highly negative nitrogen balance that may worsen their prognosis. Somatropin treatment has been shown to improve this balance in different hypercatabolic situations. Moreover, in children with extensive burns it also reduces the healing time of the skin graft donor site and shortens the hospital stay. In the existing literature there are no controlled prospective clinical trials in adult patients that confirm these data. Our aim was to demonstrate the efficacy of recombinant growth hormone (somatropin) in reducing the healing time of the skin graft donor sites and the length of stay in the burn unit in adult patients with severe burns. A randomized, double-blind, placebo-controlled clinical trial was carried out in 24 adult patients with severe burns (more than 40% of the total body surface burned or more than 15% full-thickness burns). Patients received placebo (n = 11) or somatropin (n = 13) at a dosage of 0.15 mg/kg/day divided into two equal doses (every 12 hours) via intramuscular injection. Treatment was initiated the day the first autograft was performed and terminated the day the patient was discharged from the burn unit. The mean number (+/- SD) of skin grafts per patient was similar between the two groups (4.2 +/- 1.8 vs 3.4 +/- 1.8 in the placebo and somatropin groups, respectively). No reduction in the healing time of the skin graft donor site was observed in the somatropin group compared to the placebo group. Likewise, the time admitted to the burn unit was not significantly different, either in the absolute number of days (36.2 +/- 19.7 vs 30.1 +/- 16.8 days in the placebo and somatropin groups, respectively) or in relation to the percentage of the total body surface burned or the body surface with full-thickness burns. Growth hormone and insulin-like growth factor I (IGF-I) levels were three and five times higher, respectively, in the somatropin group than in the placebo group. Ten of the patients treated with somatropin experienced hyperglycemia, and seven of them required insulin treatment. No other adverse side effect was observed. One patient in the placebo group died as a result of sepsis and multiple organ failure. Somatropin, with the treatment regimen and dosage used in these studies, did not reduce the healing time of the skin graft donor sites or the length of hospitalization in the burn unit in adult patients with severe burns.

Granulocyte-macrophage colony stimulating factor activates proteoglycan, type II collagen, and cAMP production by rat articular chondrocytes through specific binding sites.

OBJECTIVE: To evaluate the effects of granulocyte-macrophage colony stimulating factor (GM-CSF) on rat articular chondrocyte (AC) with respect to DNA synthesis, collagen type II and proteoglycan (PG) synthesis and expression, and cAMP production; to examine these cells for the presence of GM-CSF-specific binding sites; and to study their regulation by growth factors and cytokines. METHODS: First passage monolayers of rat AC were incubated with various concentrations of recombinant human GM-CSF, and then [3H]-thymidine, [3H]-proline, and [35S]SO4 incorporation and cAMP production were measured. The density of GM-CSF-specific binding sites, the effects of growth factors and cytokines on receptor density, and the activation of certain post-receptor signaling pathways were also examined by labeling the cell monolayers with [125I]-GM-CSF. RESULTS: GM-CSF (6-100 U/ml) inhibited (30%) [3H]-thymidine incorporation into DNA, and, in contrast, stimulated up to 3.6- and 2-fold [35S]SO4 and [3H]-proline incorporation into glycosaminoglycan side chains and collagen molecules, respectively. GM-CSF also increased aggrecan and type II collagen (Coll II) transcripts by 2- to 3-fold, respectively. These effects were associated with a concentration-dependent increase in cAMP production. A single class of high affinity (Kd = 98 pM; Bmax = 7.08 pM/microg DNA) binding sites of about 220 kDa were found. The [125I]-GM-CSF binding to the cells was slightly increased with phorbol 12-myristate 13-acetate (PMA), insulin-like growth factor-I, platelet derived growth factor, basic fibroblast growth factor, and tumor necrosis factor-alpha, and decreased with pertussis toxin, cholera toxin, and interleukin-1beta. CONCLUSION: These results suggest that GM-CSF may play a role in the regulation of chondrocyte metabolism as an anabolic agent and may stimulate cartilage healing under pathological conditions.