Heparanase-enhanced Shedding of Syndecan-1 and Its Role in Driving Disease Pathogenesis and Progression

Both heparanase and syndecan-1 are known to be present and active in disease pathobiology. An important feature of syndecan-1 related to its role in pathologies is that it can be shed from the surface of cells as an intact ectodomain composed of the extracellular core protein and attached heparan sulfate and chondroitin sulfate chains. Shed syndecan-1 remains functional and impacts cell behavior both locally and distally from its cell of origin. Shedding of syndecan-1 is initiated by a variety of stimuli and accomplished predominantly by the action of matrix metalloproteinases. The accessibility of these proteases to the core protein of syndecan-1 is enhanced, and shedding facilitated, when the heparan sulfate chains of syndecan-1 have been shortened by the enzymatic activity of heparanase. Interestingly, heparanase also enhances shedding by upregulating the expression of matrix metalloproteinases. Recent studies have revealed that heparanase-induced syndecan-1 shedding contributes to the pathogenesis and progression of cancer and viral infection, as well as other septic and non-septic inflammatory states. This review discusses the heparanase/shed syndecan-1 axis in disease pathogenesis and progression, the potential of targeting this axis therapeutically, and the possibility that this axis is widespread and of influence in many diseases.     

Characterization of mice deficient in interleukin-1β converting enzyme

Interleukin-1β converting enzyme (ICE) processes the inactive proIL-1β to the proinflammatory mature IL-1β. ICE belongs to a family of cysteine proteases that have been implicated in apoptosis. To address the biological functions of ICE, we generated ICE-deficient mice through gene targeting technology. ICE-deficient mice developed normally, appeared healthy, and were fertile. Peritoneal macrophages from ICE-deficient mice underwent apoptosis normally upon ATP treatment. Thymocytes from young ICE-deficient mice also underwent apoptosis when triggered by dexamethasone, gamma irradiation, or aging. ICE-deficient mice had a major defect in the production of mature IL-1β and had impaired IL-1α production on LPS stimulation in vitro and in vivo. ICE-deficient mice were resistant to LPS-induced endotoxic shock. J. Cell. Biochem. 64:27–32. © 1997 Wiley-Liss, Inc.     

RACK1 identified as the PCBP1‐interacting protein with a novel functional role on the regulation of human MOR gene expression

Poly C binding protein 1 (PCBP1) is an expressional regulator of the mu‐opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co‐regulator modifying human MOR gene expression by protein–protein interaction with PCBP1. A human brain cDNA library was screened using the two‐hybrid system with PCBP1 as the bait. Receptor for activated protein kinase C (RACK1) protein, containing seven WD domains, was identified. PCBP1‐RACK1 interaction was confirmed via in vivo validation using the two‐hybrid system, and by co‐immunoprecipitation with anti‐PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co‐immunoprecipitation suggested that RACK1‐PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 over‐expression resulted in a dose‐dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock‐down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT‐PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by ³H‐diprenorphine binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in neuronal NMB cells.     

Regulatory role of miR‐2909 in cell‐mediated immune response

Keeping in view the micromanagement of immune response by micro RNAs, the present study was directed to explore the role of miR‐2909 in the differentiation and maturation of T‐lymphocytes within the population of normal human peripheral blood mononuclear cells maintained in in vitro culture. The results of such a study revealed that miR‐2909 had the inherent capacity to significantly increase Treg (CD4+CD25+Foxp3+) cell population and dominant Th1‐type cytokine (especially with decrease in IL‐4 level and higher levels of INF‐β and INF‐γ) profile. Based upon these results, we propose that miR‐2909 may modulate native immunity in general and help in providing protective immunity against viral infections in particular. Copyright © 2012 John Wiley & Sons, Ltd.     

N/OFQ system in brain areas of nerve‐injured mice: its role in different aspects of neuropathic pain

Several studies showed that chronic pain causes reorganization and functional alterations of supraspinal brain regions. The nociceptin‐NOP receptor system is one of the major systems involved in pain control and much evidence also suggested its implication in stress, anxiety and depression. Therefore, we investigated the nociceptin‐NOP system alterations in selected brain regions in a neuropathic pain murine model. Fourteen days after the common sciatic nerve ligature, polymerase chain reaction (PCR) analysis indicated a significant decrease of pronociceptin and NOP receptor mRNA levels in the thalamus; these alterations could contribute to the decrease of the thalamic inhibitory function reported in neuropathic pain condition. Nociceptin peptide and NOP mRNA increased in the anterior cingulate cortex (ACC) and not in the somatosensory cortex, suggesting a peculiar involvement of this system in pain regulating circuitry. Similarly to the ACC, an increase of nociceptin peptide levels was observed in the amygdala. Finally, the pronociceptin and NOP mRNAs decrease observed in the hypothalamus reflects the lack of hypothalamus‐pituitary‐adrenal axis activation, already reported in neuropathic pain models. Our data indicate that neuropathic pain conditions affect the supraspinal nociceptin‐NOP system which is also altered in regions known to play a role in emotional aspects of pain.     

The role of thiol redox systems in the peroxide stress response of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The effect of mutations in the genes encoding glutathione, glutaredoxin, thioredoxin, and thioredoxin reductase on the response of growing Escherichia coli to oxidative stress was studied. The gshA mutants defective in glutathione synthesis had the lowest resistance to high doses of H₂O₂, whereas the trxB mutants defective in thioredoxin reductase synthesis had the highest resistance to this oxidant, exceeding that of the parent strain. Among the studied mutants, the trxB cells demonstrated the highest basic levels of catalase activity and intracellular glutathione; they were able to rapidly reach the normal GSH level after oxidative stress. At the same time, these bacteria showed high frequency of induced mutations. The expression of the katG and sulA genes suggests that, having different sensitivity to high oxidant concentrations, the studied mutants differ primarily in their ability to induce the antioxidant genes of the OxyR and SOS regulons.     

Cell‐type‐specific role of lamin‐B1 in thymus development and its inflammation‐driven reduction in thymus aging

Cellular architectural proteins often participate in organ development and maintenance. Although functional decay of some of these proteins during aging is known, the cell‐type‐specific developmental role and the cause and consequence of their subsequent decay remain to be established especially in mammals. By studying lamins, the nuclear structural proteins, we demonstrate that lamin‐B1 functions specifically in the thymic epithelial cells (TECs) for proper thymus organogenesis. An up‐regulation of proinflammatory cytokines in the intra‐thymic myeloid immune cells during aging accompanies a gradual reduction of lamin‐B1 in adult TECs. We show that these cytokines can cause senescence and lamin‐B1 reduction of the young adult TECs. Lamin‐B1 supports the expression of TEC genes that can help maintain the adult TEC subtypes we identified by single‐cell RNA‐sequencing, thymic architecture, and function. Thus, structural proteins involved in organ building and maintenance can undergo inflammation‐driven decay which can in turn contribute to age‐associated organ degeneration.     

Dynorphinergic system alterations in the corticostriatal circuitry of neuropathic mice support its role in the negative affective component of pain

The dynorphinergic system is involved in pain transmission at spinal level, where dynorphin exerts antinociceptive or pronociceptive effects, based on its opioid or non‐opioid actions. Surprisingly, little evidence is currently available concerning the supraspinal role of the dynorphinergic system in pain conditions. The present study aimed to investigate whether neuropathic pain is accompanied by prodynorphin (Pdyn) and κ‐opioid receptor (Oprk1) gene expression alterations in selected mouse brain areas. To this end, mice were subjected to chronic constriction injury of the right sciatic nerve and neuropathic pain behavioral signs were ascertained after 14 days. At this interval, a marked increase in Pdyn mRNA in the anterior cingulate cortex (ACC) and prefrontal cortex (PFC) was observed. Oprk1 gene expression was increased in the PFC, and decreased in the ACC and nucleus accumbens (NAc). No changes were observed in the other investigated regions. Because of the relationship between dynorphin and the brain‐derived neurotrophic factor, and the role of this neurotrophin in chronic pain‐related neuroplasticity, we investigated brain‐derived neurotrophic factor gene (Bdnf) expression in the areas showing Pdyn or Oprk1 mRNAs changes. Bdnf mRNA levels were increased in both the ACC and PFC, whereas no changes were assessed in the NAc. Present data indicate that the dynorphinergic system undergoes quite selective alterations involving the corticostriatal circuitry during neuropathic pain, suggesting a contribution to the negative affective component of pain. Moreover, parallel increases in Pdyn and Bdnf mRNA at cortical level suggest the occurrence of likely interactions between these systems in neuropathic pain maladaptive neuroplasticity.     

Accumulation of stem sugar and its remobilisation in response to drought stress in a sweet sorghum genotype and its near‐isogenic lines carrying different stay‐green loci

• Near isogenic lines (NILs) of sweet sorghum genotype S35 into which individual stay green loci were introgressed, were used to understand the contribution of Stay green loci to stem sugar accumulation and its remobilization under drought stress exposure.
• Sugar and starch content, activities of sugar metabolism enzymes and levels of their expression were studied in the 3rd (source) leaf from panicle and the 5th (sugar storing) internode of the three lines, in irrigated plants and in plants exposed to a brief drought exposure at the panicle emergence stage. Annotation of genes in the respective Stay green loci introgressed in the NILs was carried out using bioinformatics tools.
• The leaves of NILs accumulated more photoassimilates and the internodes accumulated more sugar, as compared to the parent S35 line. Drought stress exposure led to a decrease in the starch and sugar levels in leaves of all three lines, while an increase in sugar levels was observed in internodes of the NILs. Sugar fluxes were accompanied by alterations in the activities of sugar metabolizing enzymes as well as the expression of genes related to sugar metabolism and transport.
• Remobilization of sugars from the stem internodes was apparent in the NILs when subjected to drought stress, since the peduncle, which supports the panicle, showed an increase in the sugar content, even when photoassimation in source leaves was reduced. Several genes related to carbohydrate metabolism were located in the Stay green loci, which probably contributed to variation in the parameters studied.

     

Temperature‐dependent study reveals that dynamics of hydrophobic residues plays an important functional role in the mitochondrial Tim9–Tim10 complex

Protein–protein interaction is a fundamental process in all major biological processes. The hexameric Tim9–Tim10 (translocase of inner membrane) complex of the mitochondrial intermembrane space plays an essential chaperone‐like role during import of mitochondrial membrane proteins. However, little is known about the functional mechanism of the complex because the interaction is weak and transient. This study investigates how electrostatic and hydrophobic interactions affect the conformation and function of the complex at physiological temperatures, using both experimental and computational methods. The results suggest that, first, different complex conformational states exist at equilibrium, and the major difference between these states is the degree of hydrophobic interactions. Second, the conformational change mimics the biological activity of the complex as measured by substrate binding at the same temperatures. Finally, molecular dynamics simulation and detailed energy decomposition analysis provided supporting evidence at the atomic level for the presence of an excited state of the complex, the formation of which is largely driven by the disruption of hydrophobic interactions. Taken together, this study indicates that the dynamics of the hydrophobic residues plays an important role in regulating the function of the Tim9–Tim10 complex. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Structure‐function analysis of npr1 alleles in Arabidopsis reveals a role for its paralogs in the perception of salicylic acid

Salicylic acid (SA) is necessary for plant defence against some pathogens, whereas NPR1 is necessary for SA perception. Plant defence can be induced to an extreme by several applications of benzothiadiazole (BTH), an analogue of SA. Thus, plants that do not perceive BTH grow unaffected, whereas wild‐type plants grow stunted. This feature allows us to screen for mutants in Arabidopsis thaliana that show insensitivity to BTH in a high‐throughput fashion. Most of the mutants are npr1 alleles, with similar phenotypes in plant weight and pathogen growth. The mutations are clustered in the carboxyl‐terminal part of the protein, and no obvious null alleles were recovered. These facts have prompted a search for knockouts in the NPR1 gene. Two of these KO alleles identified are null and have an intermediate phenotype. All the evidence presented lead us to propose a redundancy in SA perception, with the paralogs of NPR1 taking part in this signalling. We show that the mutations recovered in the screening genetically interact with the paralogs preventing their function in SA signalling.     

The role of thiol redox systems in the response of <Emphasis Type="Italic">Escherichia coli</Emphasis> to far-UV irradiation

Escherichia coli mutants deficient in glutathione (gshA), glutaredoxin (grxA), thioredoxin (trxA), and thioredoxin reductase (trxB) synthesis were studied with respect to their resistance to far-UV (UV₂₅₄) exposure. The trxA, trxB, and grxA mutants subjected to a short-term UV exposure were found to be more resistant to UV irradiation than the parent cells. Under the same conditions, the trxA and trxB mutants demonstrated a high level of induction of the sulA gene, a component of the SOS regulon. The mutagenic effect of long-term UV exposure of all the mutants with redox deficiencies was more pronounced than in the case of the parent strain, and the trxA and trxB mutants were found to be the least viable microorganisms. Pretreatment of the cells with low concentrations of the thiol-oxidizing agent diamide enhanced the sulA gene expression; however, high concentrations of diamide inhibited sulA expression. The data obtained indicate that the thiol redox systems of E. coli are involved in its response to far-UV irradiation.     

Prevention of inflammation‐mediated neurotoxicity by butylidenephthalide and its role in microglial activation

Microglial cells are the prime effectors in immune and inflammatory responses of the central nervous system (CNS). During pathological conditions, the activation of these cells helps restore CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory molecules and neurotoxins. Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target neurodegeneration, such as that in Alzheimer's and Parkinson's diseases. The rhizome of Ligusticum chuanxiong Hort. (Ligusticum wallichii Franch) has been widely used for the treatment of vascular diseases in traditional oriental medicine. Butylidenephthalide (BP), a major bioactive component from L. chuanxiong, has been reported to have a variety of pharmacological activities, including vasorelaxant, anti‐anginal, anti‐platelet and anti‐cancer effects. The aim of this study was to examine whether BP represses microglial activation. In rat brain microglia, BP significantly inhibited the lipopolysaccharide (LPS)‐induced production of nitric oxide (NO), tumour necrosis factor‐α and interleukin‐1β. In organotypic hippocampal slice cultures, BP clearly blocked the effect of LPS on hippocampal cell death and inhibited LPS‐induced NO production in culture medium. These results newly suggest that BP provide neuroprotection by reducing the release of various proinflammatory molecules from activated microglia. Copyright © 2013 John Wiley & Sons, Ltd.     

NDRG1 is down‐regulated in the early apoptotic event induced by camptothecin analogs: The potential role in proteolytic activation of PKCδ and apoptosis

We previously reported that NSC606985, a new camptothecin analog, induces apoptosis of acute myeloid leukemic cells, which is triggered by proteolytic activation of protein kinase C delta (PKCδ). Here, we performed quantitative proteomic analysis of NSC606985‐treated and untreated leukemic U937 cells with two‐dimensional fluorescence difference gel electrophoresis (2‐D DIGE) in combination with matrix‐assisted laser desorption/ionization time‐of‐flight/time‐of‐flight tandem mass spectrometry. Thirty‐three proteins were found to be deregulated. Then, we focused on N‐myc downstream regulated gene 1 (NDRG1) down‐regulated during apoptosis induction. The results demonstrated that the down‐regulation of NDRG1 protein but not its mRNA was an early event prior to proteolytic activation of PKCδ in U937 cells under treatments of NSC606985 as well as other camptothecin analogs. With the ectopic expression of NDRG1, the proteolytic activation of PKCδ in NSC606985‐treated leukemic cells was delayed and the cells were less sensitive to apoptosis. On the contrary, the suppression of NDRG1 expression by specific small interfering RNA significantly enhanced NSC606985‐induced activation of PKCδ and apoptosis of U937 cells. In summary, our study suggests that the down‐regulation of NDRG1 is involved in proteolytic activation of PKCδ during apoptosis induction, which would shed new light on the understanding the apoptotic process initiated by camptothecin.