Subdiffusion as a model of transport through the nuclear pore complex

Cargo transport through the nuclear pore complex continues to be a subject of considerable interest to experimentalists and theorists alike. Several recent studies have revealed details of the process that have still to be fully understood, among them the apparent nonlinearity between cargo size and the pore crossing time, the skewed, asymmetric nature of the distribution of such crossing times, and the non-exponentiality in the decay profile of the dynamic autocorrelation function of cargo positions. In this paper, we show that a model of pore transport based on subdiffusive particle motion is in qualitative agreement with many of these observations. The model corresponds to a process of stochastic binding and release of the particle as it moves through the channel. It suggests that the phenylalanine-glycine repeat units that form an entangled polymer mesh across the channel may be involved in translocation, since these units have the potential to intermittently bind to hydrophobic receptor sites on the transporter protein. (C) 2011 American Institute of Physics. [doi:10.1063/1.3651100]

Intestinal Cell Proliferation and Senescence Are Regulated by Receptor Guanylyl Cyclase C and p21

Guanylyl cyclase C (GC-C) is expressed in intestinal epithelial cells and serves as the receptor for bacterial heat-stable enterotoxin (ST) peptides and the guanylin family of gastrointestinal hormones. Activation of GC-C elevates intracellular cGMP, which modulates intestinal fluid-ion homeostasis and differentiation of enterocytes along the crypt-villus axis. GC-C activity can regulate colonic cell proliferation by inducing cell cycle arrest, and mice lacking GC-C display increased cell proliferation in colonic crypts. Activation of GC-C by administration of ST to wild type, but not Gucy2c(-/-), mice resulted in a reduction in carcinogen-induced aberrant crypt foci formation. In p53-deficient human colorectal carcinoma cells, ST led to a transcriptional up-regulation of p21, the cell cycle inhibitor, via activation of the cGMP-responsive kinase PKGII and p38 MAPK. Prolonged treatment of human colonic carcinoma cells with ST led to nuclear accumulation of p21, resulting in cellular senescence and reduced tumorigenic potential. Our results, therefore, identify downstream effectors for GC-C that contribute to regulating intestinal cell proliferation. Thus, genomic responses to a bacterial toxin can influence intestinal neoplasia and senescence.

Differential expression of estrogen receptor alpha in the embryonic adrenal-kidney-gonadal complex of the oviparous lizard, Calotes versicolor (Daud.)

Estrogen signalling is critical for ovarian differentiation in reptiles with temperature-dependent sex determination (TSD). To elucidate the involvement of estrogen in this process, adrenal-kidney-gonadal (AKG) expression of estrogen receptor (ER alpha) was studied at female-producing temperature (FPT) in the developing embryos of the lizard, Calotes versicolor which exhibits a distinct pattern of TSD. The eggs of this lizard were incubated at 31.5 +/- 0.5 degrees C (100% FPT). The torso of embryos containing adrenal-kidney-gonadal complex (AKG) was collected during different stages of development and subjected to Western blotting and immunohistochemistry analysis. The ER alpha, antibody recognized two protein bands with apparent molecular weight similar to 55 and similar to 45 kDa in the total protein extracts of embryonic AKG complex of C. versicolor. The observed results suggest the occurrence of isoforms of ER alpha. The differential expression of two different protein isoforms may reveal their distinct role in cell proliferation during gonadal differentiation. This is the first report to reveal two isoforms of the ER alpha in a reptile during development. Immunohistochemical studies reveal a weak, but specific, cytoplasmic ER alpha immunostaining exclusively in the AKG during late thermo-sensitive period suggesting the responsiveness of AKG to estrogens before gonadal differentiation at FPT. Further, cytoplasmic as well as nuclear expression of ER alpha in the medulla and in oogonia of the cortex (faint activity) at gonadal differentiation stage suggests that the onset of gonadal estrogen activity coincides with sexual differentiation of gonad. Intensity and pattern of the immunoreactions of ER alpha in the medullary region at FPT suggest endogenous production of estrogen which may act in a paracrine fashion to induce neighboring cells into ovarian differentiation pathway. (C) 2014 Elsevier Inc. All rights reserved.

Exploration of new drug delivery pathways: I. Mechanism of folate uptake in cultured human cells. II. Role of nuclear localization signal peptides in the delivery of oligonucleotide into reconstituted nuclei

The roles of the folate receptor and an anion carrier in the uptake of 5- methyltetrahydrofolate (5-MeH_4folate) were studied in cultured human (KB) cells using radioactive 5-MeH_4folate. Binding of the 5-MeH_4folate was inhibited by folic acid, but not by probenecid, an anion carrier inhibitor. The internalization of 5-MeH_4folate was inhibited by low temperature, folic acid, probenecid and methotrexate. Prolonged incubation of cells in the presence of high concentrations of probenecid appeared to inhibit endocytosis of folatereceptors as well as the anion carrier. The V_(max) and K_M values for the carrier were 8.65 ± 0.55 pmol/min/mg cell protein and 3.74 ± 0.54µM, respectively. The transport of 5-MeH4folate was competitively inhibited by folic acid, probenecid and methotrexate. The carrier dissociation constants for folic acid, probenecid and methotreate were 641 µM, 2.23 mM and 13.8 µM, respectively. Kinetic analysis suggests that 5-MeH_4folate at physiological concentration is transported through an anion carrier with the characteristics of the reduced-folate carrier after 5-MeH_4folate is endocytosed by folate receptors in KB cells. Our data with KB cells suggest that folate receptors and probenecid-sensitive carriers work in tandem to transport 5-MeH_4folate to the cytoplasm of cells, based upon the assumption that 1 mM probenecid does not interfere with the acidification of the vesicle where the folate receptors are endocytosed.

Oligodeoxynucleotides designed to hybridize to specific mRNA sequences (antisense oligonucleotides) or double stranded DNA sequences have been used to inhibit the synthesis of a number of cellular and viral proteins (Crooke, S. T. (1993) FASEB J. 7, 533-539; Carter, G. and Lemoine, N. R. (1993) Br. J. Cacer 67, 869-876; Stein, C. A. and cohen, J. S. (1988) Cancer Res. 48, 2659-2668). However, the distribution of the delivered oligonucleotides in the cell, i.e., in the cytoplasm or in the nucleus has not been clearly defined. We studied the kinetics of oligonucleotide transport into the cell nucleus using reconstituted cell nuclei as a model system. We present evidences here that oligonucleotides can freely diffuse into reconstituted nuclei. Our results are consistent with the reports by Leonetti et al. (Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 2702-2706, April 1991), which were published while we were carrying this research independently. We also investigated whether a synthetic nuclear localization signal (NLS) peptide of SV40 T antigen could be used for the nuclear targeting of oligonucleotides. We synthesized a nuclear localization signal peptide-conjugated oligonucleotide to see if a nuclear localization signal peptide can enhance the uptake of oligonucleotides into reconstituted nuclei of Xenopus. Uptake of the NLS peptide-conjugated oligonucleotide was comparable to the control oligonucleotide at similar concentrations, suggesting that the NLS signal peptide does not significantly enhance the nuclear accumulation of oligonucleotides. This result is probably due to the small size of the oligonucleotide.

RNAi suppression of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) mediated differentially expressed genes involved in Toll-like receptor signaling pathway and caused increased susceptibility to Flavobacterium columnare

In Drosophila, Toll signaling cascade, which resembles the mammalian Toll-like receptor (TLR)/IL-1R signaling pathways and regulates the expression of anti-microbial peptide genes, mainly relies on peptidoglycan recognition proteins (PGRPs) for the detection of bacterial pathogens. To explore the effect of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) on Toll-like receptor signaling pathway, RNA interference (siRNA) and real time quantitative PCR (RQ-PCR) methods were used to identify differentially expressed genes regulated by zfPGRP6. The target genes included TLR2, TLR3, TLR5, TLR7, TLR8, IL1R, Sterile-alpha and Armadillo motif containing protein (SARM), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-kappa B2 (p100/p52). The results of RQ-PCR showed that RNAi-mediated Suppression of zfPGRP6 significantly down-regulated the expression of TLR2, TLR5, IL1R, SARM, MyD88 and p100/p52. The expression of beta-defensin-1 was also down-regulated in those embryos silenced by zfPGRP6. In challenge experiments to determine the anti-bacterial response to Gram-negative bacteria, RNAi knock-down of zfPGRP6 markedly increased susceptibility to Flavobacterium columnare. (C) 2008 Elsevier B.V. All rights reserved.

Androgen receptor targets NFKB and TSPI to suppress prostate tumor growth in vivo

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti-tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR-expressing cells formed tumors in male mice at a much lower rate than the AR-negative controls. Moreover, the AR-expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti-angiogenic protein, thrombospondin-1. AR activation caused a decrease in RelA, a subunit of the pro-survival transcription factor NF kappa B, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti-apoptotic targets, Bcl-2 and IL-6. Increased apoptosis within AR-expressing tumors was likely due to the NF kappa B suppression, since it was restricted to the cells lacking nuclear (active) NF kappa B. Thus we for the first time identified combined decrease of NF kappa B and increased TSP1 as molecular events underlying the AR anti-tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early-stage prostate cancer. (C) 2007 Wiley-Liss, Inc.

The role of glucocorticoid-induced tumour necrosis factor receptor in developing mouse sympathetic neurons

Hereditary sensory autonomic neuropathy IV (HSAN IV) is an autosomal recessive disorder characterised by inability to feel pain and anhidrosis and is a consequence of defective NGF/TrkA signalling and growth of sensory and sympathetic neurons. Glucocortiocoid-induced tumour necrosis factors receptor (GITR), a transmembrane protein, activated by its specific ligand, GITRL, is well known for its role in the regulation of innate and acquired immune system responses. Recently, GITR was found to be required for NGF-dependant and extracellular signal-related kinase 1/2 (ERK1/2)-induced neurite growth and target innervation in the developing sympathetic nervous system (SNS). Given this novel role of GITR, it is possible that strategies targeting GITR have potential therapeutic benefit in promoting neurite growth in autonomic neuropathies such as HSAN IV. Using P1 mouse SCG neurons as a model, in addition to various SCG cell treatments, knock down models and transfection methods, we investigated whether GITR increases the sensitivity of sympathetic neurons to NGF; the region of GITR required for the enhancement of NGF-promoted growth, the signalling pathways downstream of GITR and how extensively GITR is involved in regulating peripheral innervation of the SNS. Results indicate that the region responsible for the growth promoting effects of GITR lies in its juxtamembrane intracellular region (here termed the growth promoting domain (GPD)) of GITR. The GPD of GITR activates ERK1/2 and inhibits nuclear factor kappa B (NF-κB) in an inverse fashion to provide an optimal cellular growth environment for P1 SCG neurons. While deleting the GPD of GITR had no effect on TrkA expression, constitutive phosphorylation of specific sites in the GPD reduced TrkA expression indicating a possible role for GITR in increasing the sensitivity of SCG neurons to NGF by the regulation of these sites, TrkA expression and subsequent NGF/TrkA binding. GITR appears to be heterogeneously required for NGF-promoted target innervation of SCG neurons in some organs, implying additional factors are involved in extensive NGF-target innervation of the SNS. In conclusion, this study answers basic biological questions regarding the molecular mechanism behind the role of GITR in the development of the SNS, and provides a basis for future research if GITR modulation is to be developed as a strategy for promoting axonal growth.

beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1.

beta-arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether beta-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) beta-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous beta-arrestin-1 was knocked down by transfection of beta-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT beta-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both beta-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) beta-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) beta-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on beta-arrestin and the amino terminus of beta-arrestin-1 are required for this effect of beta-arrestin on IRS-1 degradation; and (iv) inhibition of beta-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.

Distribution of the Receptor for Advanced Glycation End Products (RAGE) in the Human Male Reproductive Tract. Prevalence in Men with Diabetes Mellitus.

BACKGROUND: Diabetics have a significantly higher percentage of sperm with nuclear DNA (nDNA) fragmentation and increased levels of advanced glycation end products (AGEs), in their testis, epididymis and sperm. As the receptor for AGEs (RAGE) is important to oxidative stress and cell dysfunction, we hypothesise, that it may be involved in sperm nDNA damage. METHODS: Immunohistochemistry was performed to determine the presence of RAGE in the human testis and epididymis. A comparison of the receptor's incidence and localisation on sperm from 10 diabetic and 11 non-diabetic men was conducted by blind semi-quantitative assessment of the immunostaining. ELISA analysis ascertained RAGE levels in seminal plasma and sperm from 21 diabetic and 31 non-diabetic subjects. Dual labelling immunolocalisation was employed to evaluate RAGE's precise location on the sperm head. RESULTS: RAGE was found throughout the testis, caput epididymis, particularly the principle cells apical region, and on sperm acrosomes. The number of sperm displaying RAGE and the overall protein amount found in sperm and seminal plasma were significantly higher in samples from diabetic men (p

Estrogen Receptor-beta Affects the Prognosis of Human Malignant Mesothelioma

Malignant pleural mesothelioma is an asbestos-related neoplasm with poor prognosis, refractory to current therapies, the incidence of which is expected to increase in the next decades. Female gender was identified as a positive prognostic factor among other clinical and biological prognostic markers for malignant mesothelioma, yet a role of estrogen receptors (ERs) has not been studied. Our goal was to investigate ERs expression in malignant mesothelioma and to assess whether their expression correlates with prognosis. Immunohistochemical analysis revealed intense nuclear ER beta staining in normal pleura that was reduced in tumor tissues. Conversely, neither tumors nor normal pleura stained positive for ER alpha. Multivariate analysis of 78 malignant mesothelioma patients with pathologic stage, histologic type, therapy, sex, and age at diagnosis indicated that FRO expression is an independent prognostic factor of better survival. Moreover, studies in vitro confirmed that treatment with 17 beta-estradiol led to an ER beta-mediated inhibition of malignant mesothelioma cell proliferation as well as p21(CIP1) and p27(KIP1) up-regulation. Consistently cell growth was suppressed by ER beta overexpression, causing a G(2)-M-phase cell cycle arrest, paralleled by cyclin B1 and survivin down-regulation. Our data support the notion that ER beta acting as a tumor suppressor is of high potential relevance to prediction of disease progression and to therapeutic response of malignant mesothelioma patients. [Cancer Res 2009;69(11):4598-604]

Klebsiella pneumoniae targets an EGF receptor-dependent pathway to subvert inflammation

The NF-kB transcriptional factor plays a key role governing the activation of immune responses. Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by lacking an early in?ammatory response. Recently, we have demonstrated that Klebsiella antagonizes the activation of NF-kB via the deubiquitinase CYLD. In this work, by applying a high-throughput siRNA gain-of-function screen interrogating the human kinome, we identi?ed 17 kinases that when targeted by siRNA restored IL-1b-dependent NF-kB translocation in infected cells. Further characterization revealed that K. pneumoniae activates an EGF receptor (EGFR)- phosphatidylinositol 3-OH kinase (PI3K)–AKT–PAK4–ERK–GSK3b signalling pathway to attenuate the cytokine-dependent nuclear translocation of NF-kB. Our data also revealed that CYLD is a downstream effector of K. pneumoniae-induced EGFR–
PI3K–AKT–PAK4–ERK–GSK3b signalling pathway. Our efforts to identify the bacterial factor(s) responsible for EGFR activation demonstrate that a capsule (CPS) mutant did not activate EGFR hence
suggesting that CPS could mediate the activation of EGFR. Supporting this notion, puri?ed CPS did activate EGFR as well as the EGFR-dependent PI3K–AKT–PAK4–ERK–GSK3b signalling pathway. CPS-mediated EGFR activation was dependent on a TLR4–MyD88–c-SRC-dependent pathway. Several promising drugs have been developed to antagonize this cascade. We propose that agents targeting this signalling pathway might provide selective alternatives for the management of K. pneumoniae pneumonias.

Lipoprotein subclass profiles of hyperlipidemic diabetic mice measured by nuclear magnetic resonance spectroscopy

Dyslipidemia accelerates vascular complications of diabetes. Nuclear magnetic resonance (NMR) analysis of lipoprotein subclasses is used to evaluate a mouse model of human familial hypercholesterolemia +/- streptozotocin (STZ)-induced diabetes. A double knockout (DKO) mouse (low-density lipoprotein receptor [LDLr] -/-; apolipoprotein B [apoB] mRNA editing catalytic polypeptide-1 [Apobec1] -/-) was studied. Wild-type (WT) and DKO mice received sham or STZ injections at age 7 weeks, yielding control (WT-C, DKO-C) and diabetic (WT-D, DKO-D) groups. Fasting serum was collected when the mice were killed (age 40 weeks) for Cholestech analysis (Cholestech Corp, Hayward, CA) and NMR lipoprotein subclass profile. By Cholestech, fasting triglyceride and total cholesterol increased in DKO-C versus WT-C. Diabetes further increased total cholesterol in DKO. High-density lipoprotein cholesterol (HDL-C) was similar among all groups. NMR revealed that LDL in all groups was present in a subclass the size of large human LDL and was increased 48-fold in DKO-C versus WT-C animals, but was unaffected by diabetes. HDL was found in a subclass equivalent to large human HDL, and was similar among groups. In conclusion, NMR analysis reveals lipoprotein subclass distributions and the effects of genetic modification and diabetes in mice, but lack of particles the size of human small LDL and small HDL may limit the relevance of the present animal model to human disease.

The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense

Toll-like receptors (TLRs) are crucial in the innate immune response to pathogens, in that they recognize and respond to pathogen associated molecular patterns, which leads to activation of intracellular signaling pathways and altered gene expression. Vaccinia virus (VV), the poxvirus used to vaccinate against smallpox, encodes proteins that antagonize important components of host antiviral defense. Here we show that the VV protein A52R blocks the activation of the transcription factor nuclear factor kappa B (NF-kappa B) by multiple TLRs, including TLR3, a recently identified receptor for viral RNA. A52R associates with both interleukin 1 receptor-associated kinase 2 (IRAK2) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two key proteins important in TLR signal transduction. Further, A52R could disrupt signaling complexes containing these proteins. A virus deletion mutant lacking the A52R gene was attenuated compared with wild-type and revertant controls in a murine intranasal model of infection. This study reveals a novel mechanism used by VV to suppress the host immunity. We demonstrate viral disabling of TLRs, providing further evidence for an important role for this family of receptors in the antiviral response.

Macrophages Control Vascular Stem/Progenitor Cell Plasticity Through Tumor Necrosis Factor-α-Mediated Nuclear Factor-κB Activation

Objective: Vascular lineage differentiation of stem/progenitor cells can contribute to both tissue repair and exacerbation of vascular diseases such as in vein grafts. The role of macrophages in controlling vascular progenitor differentiation is largely unknown and may play an important role in graft development. This study aims to identify the role of macrophages in vascular stem/progenitor cell differentiation and thereafter elucidate the mechanisms that are involved in the macrophage- mediated process.

Approach and Results: We provide in vitro evidence that macrophages can induce endothelial cell (EC) differentiation of the stem/progenitor cells while simultaneously inhibiting their smooth muscle cell differentiation. Mechanistically, both effects were mediated by macrophage-derived tumor necrosis factor-α (TNF-α) via TNF-α receptor 1 and canonical nuclear factor-κB activation. Although the overexpression of p65 enhanced EC (or attenuated smooth muscle cell) differentiation, p65 or TNF-α receptor 1 knockdown using lentiviral short hairpin RNA inhibited EC (or rescued smooth muscle cell) differentiation in response to TNF-α. Furthermore, TNF-α–mediated EC differentiation was driven by direct binding of nuclear factor-κB (p65) to specific VE-cadherin promoter sequences. Subsequent experiments using an ex vivo decellularized vessel scaffold confirmed an increase in the number of ECs and reduction in smooth muscle cell marker expression in the presence of TNF-α. The lack of TNF-α in a knockout mouse model of vein graft decreased endothelialization and significantly increased thrombosis formation.

Conclusions: Our study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-α–mediated TNF-α receptor 1 and nuclear factor-κB activation that is likely required for endothelial repair in vascular diseases such as vein graft.

Nuclear translocation and functions of growth factor receptors

Cellular signal transduction in response to environmental signals involves a relay of precisely regulated signal amplifying and damping events. A prototypical signaling relay involves ligands binding to cell surface receptors and triggering the activation of downstream enzymes to ultimately affect the subcellular distribution and activity of DNA-binding proteins that regulate gene expression. These so-called signal transduction cascades have dominated our view of signaling for decades. More recently evidence has accumulated that components of these cascades can be multifunctional, in effect playing a conventional role for example as a cell surface receptor for a ligand whilst also having alternative functions for example as transcriptional regulators in the nucleus. This raises new challenges for researchers. What are the cues/triggers that determine which role such proteins play? What are the trafficking pathways which regulate the spatial distribution of such proteins so that they can perform nuclear functions and under what circumstances are these alternative functions most relevant?