Functional analysis of the glycero-manno-heptose 7-phosphate kinase domain from the bifunctional HldE protein, which is involved in ADP-L-glycero-D-manno-heptose biosynthesis

The core oligosaccharide component of the lipopolysaccharide can be subdivided into inner and outer core regions. In Escherichia coli, the inner core consists of two 3-deoxy-d-manno-octulosonic acid and three glycero-manno-heptose residues. The HldE protein participates in the biosynthesis of ADP-glycero-manno-heptose precursors used in the assembly of the inner core. HldE comprises two functional domains: an N-terminal region with homology to the ribokinase superfamily (HldE1 domain) and a C-terminal region with homology to the cytidylyltransferase superfamily (HldE2 domain). We have employed the structure of the E. coli ribokinase as a template to model the HldE1 domain and predict critical amino acids required for enzyme activity. Mutation of these residues renders the protein inactive as determined in vivo by functional complementation analysis. However, these mutations did not affect the secondary or tertiary structure of purified HldE1, as judged by fluorescence spectroscopy and circular dichroism. Furthermore, in vivo coexpression of wild-type, chromosomally encoded HldE and mutant HldE1 proteins with amino acid substitutions in the predicted ATP binding site caused a dominant negative phenotype as revealed by increased bacterial sensitivity to novobiocin. Copurification experiments demonstrated that HldE and HldE1 form a complex in vivo. Gel filtration chromatography resulted in the detection of a dimer as the predominant form of the native HldE1 protein. Altogether, our data support the notions that the HldE functional unit is a dimer and that structural components present in each HldE1 monomer are required for enzymatic activity.

Activation of the cGMP/PKG pathway inhibits electrical activity in rabbit urethral interstitial cells of Cajal by reducing the spatial spread of Ca2+ waves.

In the present study we used a combination of patch clamping and fast confocal Ca2+ imaging to examine the effects of activators of the nitric oxide (NO)/cGMP pathway on pacemaker activity in freshly dispersed ICC from the rabbit urethra, using the amphotericin B perforated patch configuration of the patch-clamp technique. The nitric oxide donor, DEA-NO, the soluble guanylyl cyclase activator YC-1 and the membrane-permeant analogue of cGMP, 8-Br-cGMP inhibited spontaneous transient depolarizations (STDs) and spontaneous transient inward currents (STICs) recorded under current-clamp and voltage-clamp conditions, respectively. Caffeine-evoked Cl- currents were unaltered in the presence of SP-8-Br-PET-cGMPs, suggesting that activation of the cGMP/PKG pathway does not block Cl- channels directly or interfere with Ca2+ release via ryanodine receptors (RyR). However, noradrenaline-evoked Cl- currents were attenuated by SP-8-Br-PET-cGMPs, suggesting that activation of cGMP-dependent protein kinase (PKG) may modulate release of Ca2+ via IP3 receptors (IP3R). When urethral interstitial cells (ICC) were loaded with Fluo4-AM (2 microm), and viewed with a confocal microscope, they fired regular propagating Ca2+ waves, which originated in one or more regions of the cell. Application of DEA-NO or other activators of the cGMP/PKG pathway did not significantly affect the oscillation frequency of these cells, but did significantly reduce their spatial spread. These effects were mimicked by the IP3R blocker, 2-APB (100 microm). These data suggest that NO donors and activators of the cGMP pathway inhibit electrical activity of urethral ICC by reducing the spatial spread of Ca2+ waves, rather than decreasing wave frequency.

Janus kinase inhibition and its effect upon the therapeutic landscape for myelofibrosis:from palliation to cure?

Following the discovery of the Janus kinase (JAK) 2 V617F mutation in 2005 the explosion of research and drug development activity has not only advanced our understanding of the pathogenesis of myeloproliferative neoplasms (MPNs) but also triggered debate about classification, allowed revised diagnostic and response criteria, provided a target for treatment and a mode of monitoring its success. These changes and the resultant clinical research are discussed in this article where we argue that discovery of the JAK2 V617F mutation has signalled the much delayed change in therapeutic paradigm for myelofibrosis and possibly other MPNs from palliation and allowing us to move closer to, but not yet attain, a cure.

Abnormal changes in voltage-gated sodium channels NaV1.1, NaV1.2, NaV1.3, NaV1.6 and in Calmodulin/calmodulin-dependent protein kinase II, within the brains of spontaneously epileptic rats and tremor rats

Voltage-gated sodium channels (VGSCs) play a crucial role in epilepsy. The expressions of different VGSCs subtypes are varied in diverse animal models of epilepsy that may reflect their multiple phenotypes or the complexity of the mechanisms of epilepsy. In a previous study, we reported that NaV1.1 and NaV1.3 were up-regulated in the hippocampus of the spontaneously epileptic rat (SER). In this study, we further analyzed both the expression and distribution of the typical VGSC subtypes NaV1.1, NaV1.2, NaV1.3 and NaV1.6 in the hippocampus and in the cortex of the temporal lobe of two genetic epileptic animal models: the SER and the tremor rat (TRM). The expressions of calmodulin (CaM) and calmodulin-dependent protein kinase II (CaMKII) were also analyzed with the purpose of assessing the effect of the CaM/CaMKII pathway in these two models of epilepsy. Increased expression of the four VGSC subtypes and CaM, accompanied by a decrease in CaMKII was observed in the hippocampus of both the SERs and the TRM rats. However, the changes observed in the expression of VGSC subtypes and CaM were decreased with an elevated CaMKII in the cortex of their temporal lobes. Double-labeled immunofluorescence data suggested that in SERs and TRM rats, the four subtypes of the VGSC proteins were present throughout the CA1, CA3 and dentate gyrus regions of the hippocampus and temporal lobe cortex and these were co-localized in neurons with CaM. These data represent the first evidence of abnormal changes in expression of four VGSC subtypes (NaV1.1, NaV1.2, NaV1.3 and NaV1.6) and CaM/CaMKII in the hippocampus and temporal lobe cortex of SERs and TRM rats. These changes may be involved in the generation of epileptiform activity and underlie the observed seizure phenotype in these rat models of genetic epilepsy.

Mammalian triokinase and dihydroxyacetone kinase are the same enzyme

It is widely accepted that the ATP-dependent phosphorylation of D-glyceraldehyde in the fructokinase pathway of fructose metabolism requires the enzyme “triokinase”. However, experimental data on this enzyme are remarkably scarce. The enzyme has been purified from a variety of sources and peptides derived from the pig kidney enzyme show high similarity to human dihydroxyacetone kinase – an enzyme which also has FMN cyclase activity in high manganese ion concentrations. The properties of the two enzymes are also highly similar. Therefore it is proposed that mammalian triokinase and dihydroxyacetone kinase are, in fact, the same enzyme. This has consequences for investigations of normal and aberrant fructose metabolism and for the teaching of biochemistry in medical and science courses.

Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia

AtsR is a membrane-bound hybrid sensor kinase of Burkholderia cenocepacia that negatively regulates quorum sensing and virulence factors such as biofilm production, type 6-secretion and protease secretion. Here, we elucidate the mechanism of AtsR phosphorelay by site-directed mutagenesis of predicted histidine and aspartic acid phosphoacceptor residues. We demonstrate by in vitro phosphorylation that histidine-245 and aspartic acid-536 are conserved sites of phosphorylation in AtsR, and we also identify the cytosolic response regulator AtsT (BCAM0381) as a key component of the AtsR phosphorelay pathway. Monitoring the function of AtsR and its derivatives in vivo by measuring extracellular protease activity and swarming motility confirmed the in vitro phosphorylation results. Together, we find that the AtsR receiver domain plays a fine-tuning role in determining the levels of phosphotransfer from its sensor kinase domain to the AtsT response regulator.

Reduced phospholipase A2 activity is not accompanied by reduced arachidonic acid release

Arachidonic acid release in cells highly over expressing cytosolic phospholipase A2 has been attributed to mitogen-activated protein kinase phosphorylation of cytosolic phospholipase A2 on serine-505. To investigate the role of cytosolic phospholipase A2 in cellular physiology, we attempted to inhibit cytosolic phospholipase A2 in the intact cell employing an antisense RNA strategy. Swiss 3T3 cells were stably transfected with an antisense cytosolic phospholipase A2 expression vector. A clone of cells with reduced immunodetectable cytosolic phospholipase A2, compared to a vector transfected cell line, was identified by Western blotting and a corresponding decrease in phospholipase A2 activity was confirmed by enzymatic assay in cell free extracts. However, arachidonic acid release from intact cells in response to agonists was not different between antisense and control cell lines. Thus, arachidonic acid release in intact cells with decreased cytosolic phospholipase A2 activity is likely to be modulated by rate limiting factors that are extrinsic to cytosolic phospholipase A2.

Synergistic effects of proteasome inhibitor carfilzomib in combination with tyrosine kinase inhibitors in imatinib-sensitive and -resistant chronic myeloid leukemia models

The tyrosine kinase inhibitor (TKI) imatinib has transformed the treatment and outlook of chronic myeloid leukemia (CML); however, the development of drug resistance and the persistence of TKI-resistant stem cells remain obstacles to eradicating the disease. Inhibition of proteasome activity with bortezomib has been shown to effectively induce apoptosis in TKI-resistant cells. In this study, we show that exposure to the next generation proteasome inhibitor carfilzomib is associated with a decrease in ERK signaling and increased expression of Abelson interactor proteins 1 and 2 (ABI-1/2). We also investigate the effect of carfilzomib in models of imatinib-sensitive and -resistant CML and demonstrate a potent reduction in proliferation and induction of apoptosis in a variety of models of imatinib-resistant CML, including primitive CML stem cells. Carfilzomib acts synergistically with the TKIs imatinib and nilotinib, even in imatinib-resistant cell lines. In addition, we found that the presence of immunoproteasome subunits is associated with an increased sensitivity to carfilzomib. The present findings provide a rational basis to examine the potential of carfilzomib in combination with TKIs as a potential therapy for CML, particularly in imatinib-resistant disease.

Phosphorylated polyols, pyrophosphates, and derivatives thereof having biological activity

The present invention provides phosphorylated and pyrophosphate derivatives of polyols, and structural derivatives of these compounds, and provides pharmaceutical compositions comprising the same. The compounds and compositions disclosed herein have various biological activities, including for example, as allosteric effectors of hemoglobin and/or as kinase inhibitors. The present invention further provides methods for therapy in human or mammalian patients, and methods for synthesis of biologically active compounds and their intermediates.

Adult cardiac fibroblast proliferation is modulated by calcium/calmodulin-dependent protein kinase II in normal and hypertrophied hearts

Increased adult cardiac fibroblast proliferation results in an increased collagen deposition responsible for the fibrosis accompanying pathological remodelling of the heart. The mechanisms regulating cardiac fibroblast proliferation remain poorly understood. Using a minimally invasive transverse aortic banding (MTAB) mouse model of cardiac hypertrophy, we have assessed fibrosis and cardiac fibroblast proliferation. We have investigated whether calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) regulates proliferation in fibroblasts isolated from normal and hypertrophied hearts. It is known that CaMKIIδ plays a central role in cardiac myocyte contractility, but nothing is known of its role in adult cardiac fibroblast function. The MTAB model used here produces extensive hypertrophy and fibrosis. CaMKIIδ protein expression and activity is upregulated in MTAB hearts and, specifically, in cardiac fibroblasts isolated from hypertrophied hearts. In response to angiotensin II, cardiac fibroblasts isolated from MTAB hearts show increased proliferation rates. Inhibition of CaMKII with autocamtide inhibitory peptide inhibits proliferation in cells isolated from both sham and MTAB hearts, with a significantly greater effect evident in MTAB cells. These results are the first to show selective upregulation of CaMKIIδ in adult cardiac fibroblasts following cardiac hypertrophy and to assign a previously unrecognised role to CaMKII in regulating adult cardiac fibroblast function in normal and diseased hearts.

Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions

Ischaemic strokes evoke blood-brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho-kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho-kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil- versus vehicle-treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post-ischaemia or 4 h post-ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress- and tight junction-related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen-glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin-5. Cotreatment of cells with Y-27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho-kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions. Inhibition of Rho-kinase (ROCK) activity in a mouse model of human ischaemic stroke significantly improved functional outcome while reducing cerebral lesion and oedema volumes compared to vehicle-treated counterparts. Studies conducted with brain microvascular endothelial cells exposed to OGD ± R in the presence of Y-27632 revealed restoration of intercellular junctions and suppression of prooxidant NADPH oxidase activity as important factors in ROCK inhibition-mediated BBB protection.

PKC-β exacerbates in vitro brain barrier damage in hyperglycemic settings via regulation of RhoA/Rho-kinase/MLC2 pathway

Stroke patients with hyperglycemia (HG) develop higher volumes of brain edema emerging from disruption of blood-brain barrier (BBB). This study explored whether inductions of protein kinase C-β (PKC-β) and RhoA/Rho-kinase/myosin-regulatory light chain-2 (MLC2) pathway may account for HG-induced barrier damage using an in vitro model of human BBB comprising human brain microvascular endothelial cells (HBMEC) and astrocytes. Hyperglycemia (25 mmol/L D-glucose) markedly increased RhoA/Rho-kinase protein expressions (in-cell westerns), MLC2 phosphorylation (immunoblotting), and PKC-β (PepTag assay) and RhoA (Rhotekin-binding assay) activities in HBMEC while concurrently reducing the expression of tight junction protein occludin. Hyperglycemia-evoked in vitro barrier dysfunction, confirmed by decreases in transendothelial electrical resistance and concomitant increases in paracellular flux of Evan's blue-labeled albumin, was accompanied by malformations of actin cytoskeleton and tight junctions. Suppression of RhoA and Rho-kinase activities by anti-RhoA immunoglobulin G (IgG) electroporation and Y-27632, respectively prevented morphologic changes and restored plasma membrane localization of occludin. Normalization of glucose levels and silencing PKC-β activity neutralized the effects of HG on occludin and RhoA/Rho-kinase/MLC2 expression, localization, and activity and consequently improved in vitro barrier integrity and function. These results suggest that HG-induced exacerbation of the BBB breakdown after an ischemic stroke is mediated in large part by activation of PKC-β.

Small GTPase RhoA and its effector rho kinase mediate oxygen glucose deprivation-evoked in vitro cerebral barrier dysfunction

BACKGROUND AND PURPOSE: Enhanced vascular permeability attributable to disruption of blood-brain barrier results in the development of cerebral edema after stroke. Using an in vitro model of the brain barrier composed of human brain microvascular endothelial cells and human astrocytes, this study explored whether small GTPase RhoA and its effector protein Rho kinase were involved in permeability changes mediated by oxygen-glucose deprivation (OGD), key pathological phenomena during ischemic stroke.

METHODS: OGD increased RhoA and Rho kinase protein expressions in human brain microvascular endothelial cells and human astrocytes while increasing or unaffecting that of endothelial nitric oxide synthase in respective cells. Reperfusion attenuated the expression and activity of RhoA and Rho kinase in both cell types compared to their counterparts exposed to equal periods of OGD alone while selectively increasing human brain microvascular endothelial cells endothelial nitric oxide synthase protein levels. OGD compromised the barrier integrity as confirmed by decreases in transendothelial electric resistance and concomitant increases in flux of permeability markers sodium fluorescein and Evan's blue albumin across cocultures. Transfection of cells with constitutively active RhoA also increased flux and reduced transendothelial electric resistance, whereas inactivation of RhoA by anti-RhoA Ig electroporation exerted opposite effects. In vitro cerebral barrier dysfunction was accompanied by myosin light chain overphosphorylation and stress fiber formation. Reperfusion and treatments with a Rho kinase inhibitor Y-27632 significantly attenuated barrier breakdown without profoundly altering actin structure.

CONCLUSIONS: Increased RhoA/Rho kinase/myosin light chain pathway activity coupled with changes in actin cytoskeleton account for OGD-induced endothelial barrier breakdown.

Vasoactivity of Rucaparib, a PARP-1 Inhibitor, is a Complex Process that Involves Myosin Light Chain Kinase, P2 Receptors, and PARP Itself

Therapeutic inhibition of poly(ADP-ribose) polymerase (PARP), as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK) 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation.

Insulin and insulin-like growth factor signaling increases proliferation and hyperplasia of the ovarian surface epithelium and decreases follicular integrity through upregulation of the PI3-kinase pathway

BACKGROUND: The ovarian surface epithelium responds to cytokines and hormonal cues to initiate proliferation and migration following ovulation. Although insulin and IGF are potent proliferative factors for the ovarian surface epithelium and IGF is required for follicle development, increased insulin and IGF activity are correlated with at least two gynecologic conditions: polycystic ovary syndrome and epithelial ovarian cancer. Although insulin and IGF are often components of in vitro culture media, little is known about the effects that these growth factors may have on the ovarian surface epithelium morphology or how signaling in the ovarian surface may affect follicular health and development.

METHODS: Ovaries from CD1 mice were cultured in alginate hydrogels in the presence or absence of 5 μg/ml insulin or IGF-I, as well as small molecule inhibitors of IR/IGF1R, PI 3-kinase signaling, or MAPK signaling. Tissues were analyzed by immunohistochemistry for expression of cytokeratin 8 to mark the ovarian surface epithelium, Müllerian inhibiting substance to mark secondary follicles, and BrdU incorporation to assess proliferation. Changes in gene expression in the ovarian surface epithelium in response to insulin or IGF-I were analyzed by transcription array. Extracellular matrix organization was evaluated by expression and localization of collagen IV.

RESULTS: Culture of ovarian organoids with insulin or IGF-I resulted in formation of hyperplastic OSE approximately 4-6 cell layers thick with a high rate of proliferation, as well as decreased MIS expression in secondary follicles. Inhibition of the MAPK pathway restored MIS expression reduced by insulin but only partially restored normal OSE growth and morphology. Inhibition of the PI 3-kinase pathway restored MIS expression reduced by IGF-I and restored OSE growth to a single cell layer. Insulin and IGF-I altered organization of collagen IV, which was restored by inhibition of PI 3-kinase signaling.

CONCLUSIONS: While insulin and IGF are often required for propagation of primary cells, these cytokines may act as potent mitogens to disrupt cell growth, resulting in formation of hyperplastic OSE and decreased follicular integrity as measured by MIS expression and collagen deposition. This may be due partly to altered collagen IV deposition and organization in the ovary in response to insulin and IGF signaling mediated by PI 3-kinase.