Klebsiella pneumoniae subverts the activation of inflammatory responses in a NOD1-dependent manner

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Subversion of inflammation is essential for pathogen survival during infection. Evidence indicates that K. pneumoniae infections are characterized by lacking an early inflammatory response although the molecular bases are currently unknown. Here we unveil a novel strategy employed by a pathogen to counteract the activation of inflammatory responses. K. pneumoniae attenuates pro-inflammatory mediators-induced IL-8 secretion. Klebsiella antagonizes the activation of NF-?B via the deubiquitinase CYLD and blocks the phosphorylation of mitogen-activated protein kinases (MAPKs) via the MAPK phosphatase MKP-1. Our studies demonstrate that K. pneumoniae has evolved the capacity to manipulate host systems dedicated to control the immune balance. To exert this anti-inflammatory effect, Klebsiella engages NOD1. In NOD1 knock-down cells, Klebsiella neither induces the expression of CYLD and MKP-1 nor blocks the activation of NF-?B and MAPKs. Klebsiella inhibits Rac1 activation; and inhibition of Rac1 activity triggers a NOD1-mediated CYLD and MKP-1 expression which in turn attenuates IL-1ß-induced IL-8 secretion. A capsule (CPS) mutant does not attenuate the inflammatory response. However, purified CPS neither reduces IL-1ß-induced IL-8 secretion nor induces the expression of CYLD and MKP-1 thereby indicating that CPS is necessary but not sufficient to attenuate inflammation.

Klebsiella pneumoniae outer membrane protein A is required to prevent the activation of airway epithelial cells

Outer membrane protein A (OmpA) is a class of proteins highly conserved among the Enterobacteriaceae family and throughout evolution. Klebsiella pneumoniae is a capsulated Gram-negative pathogen. It is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by a lack of an early inflammatory response. Data from our laboratory indicate that K. pneumoniae CPS helps to suppress the host inflammatory response. However, it is unknown whether K. pneumoniae employs additional factors to modulate host inflammatory responses. Here, we report that K. pneumoniae OmpA is important for immune evasion in vitro and in vivo. Infection of A549 and normal human bronchial cells with 52OmpA2, an ompA mutant, increased the levels of IL-8. 52145-?wca ompA, which does not express CPS and ompA, induced the highest levels of IL-8. Both mutants could be complemented. In vivo, 52OmpA2 induced higher levels of tnfa, kc, and il6 than the wild type. ompA mutants activated NF-?B, and the phosphorylation of p38, p44/42, and JNK MAPKs and IL-8 induction was via NF-?B-dependent and p38- and p44/42-dependent pathways. 52OmpA2 engaged TLR2 and -4 to activate NF-?B, whereas 52145-?wca ompA activated not only TLR2 and TLR4 but also NOD1. Finally, we demonstrate that the ompA mutant is attenuated in the pneumonia mouse model. The results of this study indicate that K. pneumoniae OmpA contributes to attenuate airway cell responses. This may facilitate pathogen survival in the hostile environment of the lung. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional expression of KCNQ (Kv7) channels in guinea-pig bladder smooth muscle and their contribution to spontaneous activity

Background and Purpose: The aim of the study was to determine whether KCNQ channels are functionally expressed in bladder smooth muscle cells (SMC) and to investigate their physiological significance in bladder contractility. 

Experimental Approach: KCNQ channels were examined at the genetic, protein, cellular and tissue level in guinea pig bladder smooth muscle using RT-PCR, immunofluorescence, patch-clamp electrophysiology, calcium imaging, detrusor strip myography, and a panel of KCNQ activators and inhibitors. 

Key Results: KCNQ subtypes 1-5 are expressed in bladder detrusor smooth muscle. Detrusor strips typically displayed TTX-insensitive myogenic spontaneous contractions that were increased in amplitude by the KCNQ channel inhibitors XE991, linopirdine or chromanol 293B. Contractility was inhibited by the KCNQ channel activators flupirtine or meclofenamic acid (MFA). The frequency of Ca2+-oscillations in SMC contained within bladder tissue sheets was increased by XE991. Outward currents in dispersed bladder SMC, recorded under conditions where BK and KATP currents were minimal, were significantly reduced by XE991, linopirdine, or chromanol, and enhanced by flupirtine or MFA. XE991 depolarized the cell membrane and could evoke transient depolarizations in quiescent cells. Flupirtine (20M) hyperpolarized the cell membrane with a simultaneous cessation of any spontaneous electrical activity. 

Conclusions and Implications: These novel findings reveal the role of KCNQ currents in the regulation of the resting membrane potential of detrusor SMC and their important physiological function in the control of spontaneous contractility in the guinea pig bladder.

Role of central dopaminergic pathways in the neural control of growth hormone secretion in normal men:Studies with metoclopramide

The aim of this study was to gain further insight into the role that central dopaminergic pathways play in GH neuroregulation in man. Our experimental hypothesis was based on the possibility that most of the controversies on DA role could be due to the fact that the hypothalamic somatotroph rhythm (HSR) was not taken into account when interpreting the GH responses after pharmacological manipulations on dopaminergic pathways. In 10 normal subjects we monitored the effect of central dopaminergic blockade, achieved with metoclopramide (MCP; 10 mg, i.v. Bolus), on the pattern of spontaneous GH secretion and the GH responses to a GHRH challenge (GRF , 1 µg/kg, i.v. bolus) administered together with MCP or 60 min after this drug was given. The study of HSR was made according to our previous postulate. Our results indicate that MCP administration, either prior to or together with the GHRH bolus, significantly increased GHRH-induced GH release during a refractory HSR phase; but not when the GHRH challenge took place during a spotaneous secretory phase. The strong relationship between pre-GHRH plasma GH values and GHRH-elicited GH peaks was lost when MCP was given. These data indicate that MCP was able to disrupt the intrinsic HSR by inhibiting the hypothalamic release of somatostain (SS). While a main conclusion would be that central DA is a secretagogue for SS secretion, our results also suggest that this role could be dependent on its effects on the adrenergic inputs to SS neurons.

The role of sexual steroids in the modulation of growth hormone (GH) secretion in humans

Sex steroids contribute to modulate GH secretion in man. However, both the exact locus and mechanism by which their actions are exerted still remain not clearly understood. We undertook a number of studies designed to ascertain: (1) whether or not sudden or chronic changes in circulating gonadal steroids may affect GH secretion in normal adults; and (2) the reason(s) for gender-related dimorphic pattern of GH release. The pituitary reserve of GH, as evaluated by means of a GHRH challenge, was similar in women with anorexia nervosa and in normally menstruating women. Estrogenic receptor blockade with tamoxifen (TMX) did not significantly change GHRH-induced GH response in these normal women. Therefore, acute or chronic hypoestrogenism apparently had no important effects at level of somatotrophs. In another group of normal women we tested the possibility that changes in circulating estrogens might induce changes in the hypothalamic-somatotroph rhythm (HSR). GHRH challenges were performed throughout a menstrual cycle, and again after having achieved functional ovarian blockade with a GnRH agonist treatment. Short-term ovarian blockade did not significantly affect the parameters of GH response to GHRH, although it was accompanied by an increase in the number of women ina refractory HSR phase at testing. This suggested a low potentiating effect on the basic pattern of somatostatin (SS) release occurring as a consequence of the decrease in circulating estrogens. In normal men, neither the GH response to GHRH nor the HSR were affected by functional testicular blockade (after GnRH agonist treatment). However, the administration of testosterone enanthate (250 mg) to another group of men increased both the GHRH-induced GH release and the number of subjects in a spontaneous secretory HSR phase at testing; these were reversed by estrogenic receptor blockade with TMS. In another group of normal men, the fraction of GH secreted in pulses (FGHP) during a nocturnal sampling period was significantly decreased by testicular blockade. Other parameters of GH secretion, such as the number of GH pulses and their mean amplitude (A), and the mean plasma GH concentration (MCGH), showed a slight, although not significant, decrease following the lack of androgens. The administration of testosterone enanthate (500 mg) reversed these parameters to values similar to those in the basal study. Interestingly, when tamoxifen was given after testosterone enanthate, A, MCGH and FGHP increased to values significantly higher than in any other experimental condition in that study. In all, these data suggest that 17ß-estradiol may participate in GH modulation by inhibiting the hypothalamic release of somatostatin, while testosterone stimulates it. The results obtained after estrogenic receptor blockade appear to indicate that the effect of testosterone in such a modulation is dependent on its aromatization to 17ß-estradiol. The differential levels of this steroid in both sexes might account for the sexual dimorphic pattern of GH secretion. From other data in the literature, obtained in rats, and our preliminary data in children with constitutional delay of growth and puberty, it is tempting to speculate that the effect of 17ß-estradiol may be exerted by modifying the functional activity of a-2 adrenergic pathways involved in the negative modulation of SS release.

Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules

Dynamic microtubules (MTs) are required for neuronal guidance, in which axons extend directionally toward their target tissues. We found that depletion of the MT-binding protein Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) or treatment with the MT drug Taxol reduced axon outgrowth in spinal cord neurons. To quantify the dynamic distribution of MTs in axons, we developed an automated algorithm to detect and track MT plus ends that have been fluorescently labeled by end-binding protein 3 (EB3). XCLASP1 depletion reduced MT advance rates in neuronal growth cones, very much like treatment with Taxol, demonstrating a potential link between MT dynamics in the growth cone and axon extension. Automatic tracking of EB3 comets in different compartments revealed that MTs increasingly slowed as they passed from the axon shaft into the growth cone and filopodia. We used speckle microscopy to demonstrate that MTs experience retrograde flow at the leading edge. Microtubule advance in growth cone and filopodia was strongly reduced in XCLASP1-depleted axons as compared with control axons, but actin retrograde flow remained unchanged. Instead, we found that XCLASP1-depleted growth cones lacked lamellipodial actin organization characteristic of protrusion. Lamellipodial architecture depended on XCLASP1 and its capacity to associate with MTs, highlighting the importance of XCLASP1 in actin-microtubule interactions.

Phosphorylation of plant actin-depolymerising factor by calmodulin-like domain protein kinase

actin-depolymerising factor (ADF)/cofilin group of proteins are stimulus-responsive actin-severing proteins, members of which are regulated by reversible phosphorylation. The phosphorylation site on the maize ADF, ZmADF3, is Ser-6 but the kinase responsible is unknown [Smertenko et al,, Plant J. 14 (1998) 187-193]. We have partially purified the ADF kinase(s) and found it to be calcium-regulated and inhibited by N-(6-aminohesyl)-[H-3]5-chloro-1-naphthalenesulphonamide. Immunoblotting reveals that calmodulin-like domain protein kinase(s) (CDPK) are enriched in the purified preparation and addition of anti-CDPK to in vitro phosphorylation assays results in the inhibition of ADF phosphorylation, These data strongly suggest that plant ADP is phosphorylation by CDPK(s), a class of protein kinases unique to plants and protozoa. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Glycation does not alter LDL-induced secretion of tissue plasminogen activator and plasminogen activator inhibitor-1 from human aortic endothelial cells

Diabetes may induce both quantitative and qualitative changes in lipoproteins, especially low-density lipoprotein (LDL). Effects of LDL glycation on endothelial cell secretion of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) have not been fully elucidated. Human aortic endothelial cell (HAEC) tPA and PAI-1 production were determined after incubation with LDL (50 to 500 microg/mL protein, 24 h) from three sources: (1) nondiabetic LDL (N-LDL) modified in vitro to form six preparations: native, nonmodified (N); glycated (G); minimally oxidized (MO); minimally oxidized and glycated (MOG); heavily oxidized (HO); and heavily oxidized and glycated (HOG); (2) in vivo glycated and relatively nonglycated LDL subfractions from type 1 diabetic patients; (3) LDL from type 1 diabetic patients and matched controls, which was subfractionated using density gradient ultracentrifugation. In experiments using LDL modified in vitro, the rate of tPA release by HAECs incubated with N-LDL (83 +/- 4 ng/mg cell protein/24 h) did not differ significantly from those incubated with G-LDL (73 +/- 7), MO-LDL (74 +/- 13), or MOG-LDL (66 +/- 15) and was not influenced by LDL concentration. The rate of PAI-1 release was similar in HAECs incubated with N-LDL (5.7 +/- 0.6 mug/mg cell protein/24 h), G-LDL (5.7 +/- 0.7), MO-LDL (5.5 +/- 0.8), or MOG-LDL (5.7 +/- 0.9) and was not influenced by LDL concentration. In contrast, tPA release was significantly decreased in cells incubated with LDL (10 microg/mL) modified extensively by oxidation, and averaged 45.2 +/- 5.0 and 43.7 +/- 9.9 ng/mg/24 h for HO-LDL and HOG-LDL, respectively, and was further decreased with increasing concentrations of the heavily oxidized LDL preparations. PAI-1 release was not significantly decreased relative to N-LDL in cells incubated with low concentrations (5 to 50 microg/mL) of HO-LDL and HOG-LDL, but was decreased to 3.2 +/- 0.5 and 3.1 +/- 0.7 microg/mg/24 h for HO-LDL and HOG-LDL at 200 microg/mL, respectively. Results using in vivo glycated versus nonglycated LDL showed that tPA and PAI-1 release did not differ between subfractions. Release of tPA averaged 5.11 +/- 0.6 and 5.12 +/- 0.7 ng/mg/24 h, whereas release of PAI-1 averaged 666 +/- 27 ng/mg/24 h and 705 +/- 30 ng/mg/24 h for nonglycated and glycated LDL subfractions, respectively. Using LDL of different density subclasses, tPA and PAI-1 release in response to LDL from diabetic patients compared with control subjects did not differ when HAECs were incubated with LDLs of increasing density isolated from each subject pair. We conclude that oxidation of LDL, but not glycation, may contribute to the altered fibrinolysis observed in diabetes.

Apolipoprotein C-III protein concentrations and gene polymorphisms in Type 1 diabetes:associations with microvascular disease complications in the DCCT/EDIC cohort

We investigated the associations of apolipoprotein C-III (apoCIII) protein and apoCIII gene variation with microvascular disease complications in Type 1 diabetes.

Apolipoprotein C-III protein concentrations and gene polymorphisms in type 1 diabetes:associations with lipoprotein subclasses

Serum apolipoprotein C-III (apoCIII) concentration and apoCIII gene polymorphisms have been shown to be a risk factor for cardiovascular disease; however, the underlying mechanisms remain unclear. In addition, no studies have been performed that address these issues in type 1 diabetes. The current study investigated apoCIII protein and apoCIII gene variation in a normotriglyceridemic (82 +/- 57 mg/dL) population of patients with type 1 diabetes, the Diabetes Control and Complications Trial/Epidemiology of Diabetes Intervention and Complications (DCCT/EDIC) cohort. Blood samples were obtained in 409 patients after an overnight fast. Serum apoCIII concentration was highly correlated with multiple changes in lipids and lipoproteins that resulted in an adverse cardiovascular disease risk profile. Higher apoCIII concentrations were associated (P <.0001) with increased triglycerides (r = 0.78), total (r = 0.61) and low-density lipoprotein (LDL) (r = 0.40) cholesterol, apoA-I (r = 0.26), and apoB (r = 0.50), and these relationships persisted after controlling for age, gender, body mass index (BMI), and hemoglobin A1c (HbA1c). Nuclear magnetic resonance (NMR) lipoprotein subclass analyses demonstrated that apoCIII was correlated with an increase in very-low-density lipoprotein (VLDL) subclasses (P = .0001). There also was a highly significant positive relationship between serum apoCIII concentration and the LDL particle concentration in both men (r = 0.49, P = .001) and women (r = 0.40, P = .001), and a highly significant negative relationship between serum apoCIII levels and average LDL particle size in both men (r = -0.37, P = .001) and women (r = -0.22, P = .001) due primarily to an augmentation in the small L1 subclass (r = 0.42, P = .0001). Neither the T(-455) --> C polymorphism affecting an insulin response element in the apoCIII gene promoter nor a SacI polymorphism in the 3'UTR were associated with any alterations in circulating apoCIII concentrations, serum lipids, apolipoprotein concentrations, lipoprotein composition, or parameters measured by NMR lipoprotein subclass analyses. In summary, elevated apoCIII concentration was associated with risk factors for cardiovascular disease in normolipidemic type 1 diabetic patients through associated changes in lipoprotein subfraction distributions, which were independent of apoCIII genotype.

The Arabidopsis RNA-binding protein FCA requires a lysine-specific demethylase 1 homolog to downregulate FLC.

A repressor of the transition to flowering in Arabidopsis is the MADS box protein FLOWERING LOCUS C (FLC). FCA, an RNA-binding protein, and FY, a homolog of the yeast RNA 3' processing factor Pfs2p, downregulate FLC expression and therefore promote flowering. FCA/FY physically interact and alter polyadenylation/3' processing to negatively autoregulate FCA. Here, we show that FCA requires FLOWERING LOCUS D (FLD), a homolog of the human lysine-specific demethylase 1 (LSD1) for FLC downregulation. FCA also partially depends on DICER-LIKE 3, involved in chromatin silencing. fca mutations increased levels of unspliced sense FLC transcript, altered processing of antisense FLC transcripts, and increased H3K4 dimethylation in the central region of FLC. These data support a close association of FCA and FLD in mediating H3K4 demethylation and thus transcriptional silencing of FLC and reveal roles for antisense RNA processing and DCL3 function in this regulation.

Detection of Protein-Protein Interactions Using Protein-Fragment Complementation Assays (PCA)

Protein-protein interactions play a central role in many cellular processes. Their characterisation is necessary in order to analyse these processes and for the functional identification of unknown proteins. Existing detection methods such as the yeast two-hybrid (Y2H) and tandem affinity purification (TAP) method provide a means to answer rapidly questions regarding protein-protein interactions, but have limitations which restrict their use to certain interaction networks; furthermore they provide little information regarding interaction localisation at the subcellular level. The development of protein-fragment complementation assays (PCA) employing a fluorescent reporter such as a member of the green fluorescent protein (GFP) family has led to a new method of interaction detection termed Bimolecular Fluorescent Complementation (BiFC). These assays have become important tools for understanding protein interactions and the development of whole genome interaction maps. BiFC assays have the advantages of very low background signal coupled with rapid detection of protein-protein interactions in vivo while also providing information regarding interaction compartmentalisation. Modified forms of the assay such as the use of combinations of spectral variants of GFP have allowed simultaneous visualisation of multiple competing interactions in vivo. Advantages and disadvantages of the method are discussed in the context of other fluorescence-based interaction monitoring techniques.

Ranachensinin: A Novel Aliphatic Tachykinin from the Skin Secretion of the Chinese Brown Frog, Rana chensinensis

Amphibian skin secretions contain a plethora of pharmacologically-active substances and represent established sources of bioactive peptides, including tachykinins. Tachykinins are one of the most widely-studied peptide families in animals and are found in neuroendocrine tissues from the lowest vertebrates to mammals. They are characterized by the presence of a highly-conserved C-terminal pentapeptide amide sequence motif (-FXGLM-amide) that also constitutes the bioactive core of the peptide. Amidation of the C-terminal methioninyl residue appears to be mandatory in the expression of biological activity. Here, we describe the isolation, characterization and molecular cloning of a novel tachykinin named ranachensinin, from the skin secretion of the Chinese brown frog, Rana chensinensis. This peptide, DDTSDRSN QFIGLM-amide, contains the classical C-terminal pentapeptide amide motif in its primary structure and an Ile (I) residue in the variable X position. A synthetic replicate of ranachensinin, synthesized by solid-phase Fmoc chemistry, was found to contract the smooth muscle of rat urinary bladder with an EC50 of 20.46 nM. However, in contrast, it was found to be of low potency in contraction of rat ileum smooth muscle with an EC50 of 2.98 µM. These data illustrate that amphibian skin secretions continue to provide novel bioactive peptides with selective effects on functional targets in mammalian tissues.