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  • Effects of RNAi-mediated knockdown of histone methyltransferases on the sex-specific mRNA expression of Imp in the silkworm Bombyx mori. 24758924

    Sexual differentiation in Bombyx mori is controlled by sex-specific splicing of Bmdsx, which results in the omission of exons 3 and 4 in a male-specific manner. In B. mori, insulin-like growth factor II mRNA-binding protein (Imp) is a male-specific factor involved in male-specific splicing of Bmdsx. Male-specific Imp mRNA results from the male-specific inclusion of exon 8. To verify the link between histone methylation and alternative RNA processing in Imp, we examined the effects of RNAi-mediated knockdown of several histone methyltransferases on the sex-specific mRNA expression of Imp. As a result, male-specific expression of Imp mRNA was completely abolished when expression of the H3K79 methyltransferase DOT1L was repressed to less than 10% of that in control males. Chromatin immunoprecipitation-quantitative PCR analysis revealed a higher distribution of H3K79me2 in normal males than in normal females across Imp. RNA polymerase II (RNAP II) processivity assays indicated that RNAi knockdown of DOT1L in males caused a twofold decrease in RNAP II processivity compared to that in control males, with almost equivalent levels to those observed in normal females. Inhibition of RNAP II-mediated elongation in male cells repressed the male-specific splicing of Imp. Our data suggest the possibility that H3K79me2 accumulation along Imp is associated with the male-specific alternative processing of Imp mRNA that results from increased RNAP II processivity.
    Document Type:
    Reference
    Product Catalog Number:
    12-370
    Product Catalog Name:
    Normal Rabbit IgG

  • The let-7-Imp axis regulates ageing of the Drosophila testis stem-cell niche. 22660319

    Adult stem cells support tissue homeostasis and repair throughout the life of an individual. During ageing, numerous intrinsic and extrinsic changes occur that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. In the Drosophila testis, ageing results in a marked decrease in the self-renewal factor Unpaired (Upd), leading to a concomitant loss of germline stem cells. Here we demonstrate that IGF-II messenger RNA binding protein (Imp) counteracts endogenous small interfering RNAs to stabilize upd (also known as os) RNA. However, similar to upd, Imp expression decreases in the hub cells of older males, which is due to the targeting of Imp by the heterochronic microRNA let-7. In the absence of Imp, upd mRNA therefore becomes unprotected and susceptible to degradation. Understanding the mechanistic basis for ageing-related changes in stem-cell behaviour will lead to the development of strategies to treat age-onset diseases and facilitate stem-cell-based therapies in older individuals.
    Document Type:
    Reference
    Product Catalog Number:
    17-700
    Product Catalog Name:
    Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit

  • Different fear states engage distinct networks within the intercalated cell clusters of the amygdala. 21451049

    Although extinction-based therapies are among the most effective treatments for anxiety disorders, the neural bases of fear extinction remain still essentially unclear. Recent evidence suggests that the intercalated cell masses of the amygdala (ITCs) are critical structures for fear extinction. However, the neuronal organization of ITCs and how distinct clusters contribute to different fear states are still entirely unknown. Here, by combining whole-cell patch-clamp recordings and biocytin labeling with full anatomical reconstruction of the filled neurons and ultrastructural analysis of their synaptic contacts, we have elucidated the cellular organization and efferent connections of one of the main ITC clusters in mice. Our data showed an unexpected heterogeneity in the axonal pattern of medial paracapsular ITC (Imp) neurons and the presence of three distinct neuronal subtypes. Functionally, we observed that the Imp was preferentially activated during fear expression, whereas extinction training and extinction retrieval activated the main ITC nucleus (IN), as measured by quantifying Zif268 expression. This can be explained by the IPSPs evoked in the IN after Imp stimulation, most likely through the GABAergic monosynaptic innervation of IN neurons by one subtype of Imp cells, namely the medial capsular-projecting (MCp)-Imp neurons. MCp-Imp neurons also target large ITC cells that surround ITC clusters and express the metabotropic glutamate receptor 1α. These findings reveal a distinctive participation of ITC clusters to different fear states and the underlying anatomical circuitries, hence shedding new light on ITC networks and providing a novel framework to elucidate their role in fear expression and extinction.
    Document Type:
    Reference
    Product Catalog Number:
    AB5060
    Product Catalog Name:
    Anti-Substance P Receptor Antibody, pain

  • Connexin29 expression, immunocytochemistry and freeze-fracture replica immunogold labelling (FRIL) in sciatic nerve. 12372015

    The recently discovered connexin29 (Cx29) was reported to be present in the central and peripheral nervous systems (CNS and PNS), and its mRNA was found in particular abundance in peripheral nerve. The expression and localization of Cx29 protein in sciatic nerve were investigated using an antibody against Cx29. The antibody recognized Cx29 in HeLa cells transfected with Cx29 cDNA, while nontransfected HeLa cells were devoid of Cx29. Immunoblotting of sciatic nerve homogenate revealed monomeric and possibly higher molecular weight forms of Cx29. These were distinguished from connexin32 (Cx32), which also is expressed in peripheral nerve. Double immunofluorescence labelling for Cx29 and Cx32 revealed only partial colocalization of the two connexins, with codistribution at intermittent, conical-shaped striations along nerve fibers. By freeze-fracture replica immunogold labelling (FRIL), Cx32 was found in gap junctions in the outermost layers of myelin, whereas Cx29-immunogold labelling was found only in the innermost layer of myelin in close association with hexagonally arranged intramembrane particle (IMP) 'rosettes' and gap junction-like clusters of IMPs. Although both Cx32 and Cx29 were detected in myelin of normal mice, only Cx29 was present in Schwann cell membranes in Cx32 knockout mice. The results confirm that Cx29 is a second connexin expressed in Schwann cells of sciatic nerve. In addition, Cx29 is present in distinctive IMP arrays in the inner most layer of myelin, adjacent to internodal axonal plasma membranes, where this connexin may have previously unrecognized functions.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1567
    Product Catalog Name:
    Anti-Myelin Associated Glycoprotein Antibody, clone 513

  • Metabolic adaptation to a disruption in oxygen supply during myocardial ischemia and reperfusion is underpinned by temporal and quantitative changes in the cardiac proteo ... 22352837

    Despite decades of intensive research, there is still no effective treatment for ischemia/reperfusion (I/R) injury, an important corollary in the treatment of ischemic disease. I/R injury is initiated when the altered biochemistry of cells after ischemia is no longer compatible with oxygenated microenvironment (or reperfusion). To better understand the molecular basis of this alteration and subsequent incompatibility, we assessed the temporal and quantitative alterations in the cardiac proteome of a mouse cardiac I/R model by an iTRAQ approach at 30 min of ischemia, and at 60 or 120 min reperfusion after the ischemia using sham-operated mouse heart as the baseline control. Of the 509 quantified proteins identified, 121 proteins exhibited significant changes (p-value<0.05) over time and were mostly clustered in eight functional groups: Fatty acid oxidation, Glycolysis, TCA cycle, ETC (electron transport chain), Redox Homeostasis, Glutathione S-transferase, Apoptosis related, and Heat Shock proteins. The first four groups are intimately involved in ATP production and the last four groups are known to be important in cellular antioxidant activity. During ischemia and reperfusion, the short supply of oxygen precipitates a pivotal metabolic switch from aerobic metabolism involving fatty acid oxidation, TCA, and phosphorylation to anaerobic metabolism for ATP production and this, in turn, increases reactive oxygen species (ROS) formation. Therefore the implication of these 8 functional groups suggested that ischemia-reperfusion injury is underpinned in part by proteomic alterations. Reversion of these alterations to preischemia levels took at least 60 min, suggesting a refractory period in which the ischemic cells cannot adjust to the presence of oxygen. Therefore, therapeutics that could compensate for these proteomic alterations during this interim refractory period could alleviate ischemia-reperfusion injury to enhance cellular recovery from an ischemic to a normoxic microenvironment. Among the perturbed proteins, Park7 and Ppia were selected for further investigation of their functions under hypoxia. The results show that Park7 plays a key role in regulating antioxidative stress and cell survival, and Ppia may function in coping with the unfolded protein stress in the I/R condition.
    Document Type:
    Reference
    Product Catalog Number:
    05-505

  • Large-scale quantitative LC-MS/MS analysis of detergent-resistant membrane proteins from rat renal collecting duct. 18596208

    In the renal collecting duct, vasopressin controls transport of water and solutes via regulation of membrane transporters such as aquaporin-2 (AQP2) and the epithelial urea transporter UT-A. To discover proteins potentially involved in vasopressin action in rat kidney collecting ducts, we enriched membrane "raft" proteins by harvesting detergent-resistant membranes (DRMs) of the inner medullary collecting duct (IMCD) cells. Proteins were identified and quantified with LC-MS/MS. A total of 814 proteins were identified in the DRM fractions. Of these, 186, including several characteristic raft proteins, were enriched in the DRMs. Immunoblotting confirmed DRM enrichment of representative proteins. Immunofluorescence confocal microscopy of rat IMCDs with antibodies to DRM proteins demonstrated heterogeneity of raft subdomains: MAL2 (apical region), RalA (predominant basolateral labeling), caveolin-2 (punctate labeling distributed throughout the cells), and flotillin-1 (discrete labeling of large intracellular structures). The DRM proteome included GPI-anchored, doubly acylated, singly acylated, cholesterol-binding, and integral membrane proteins (IMPs). The IMPs were, on average, much smaller and more hydrophobic than IMPs identified in non-DRM-enriched IMCD. The content of serine 256-phosphorylated AQP2 was greater in DRM than in non-DRM fractions. Vasopressin did not change the DRM-to-non-DRM ratio of most proteins, whether quantified by tandem mass spectrometry (LC-MS/MS, n=22) or immunoblotting (n=6). However, Rab7 and annexin-2 showed small increases in the DRM fraction in response to vasopressin. In accord with the long-term goal of creating a systems-level analysis of transport regulation, this study has identified a large number of membrane-associated proteins expressed in the IMCD that have potential roles in vasopressin action.
    Document Type:
    Reference
    Product Catalog Number:
    05-184

  • Hydroxamic acid derivatives of mycophenolic acid inhibit histone deacetylase at the cellular level. 18838793

    Mycophenolic acid (MPA, 1), an inhibitor of IMP-dehydrogenase (IMPDH) and a latent PPARgamma agonist, is used as an effective immunosuppressant for clinical transplantation and recently entered clinical trials in advanced multiple myeloma patients. On the other hand, suberoylanilide hydroxamic acid (SAHA), a non-specific histone deacetylase (HDAC) inhibitor, has been approved for treating cutaneous T-cell lymphoma. MPA seemed to bear a cap, a linker, and a weak metal-binding site as a latent inhibitor of HDAC. Therefore, the hydroxamic acid derivatives of mycophenolic acid having an effective metal-binding site, mycophenolic hydroxamic acid (MPHA, 2), 7-O-acetyl mycophenolic acid (7-O-Ac MPHA, 3), and 7-O-lauroyl mycophenolic hydroxamic acid (7-O-L MPHA, 4) were designed and synthesized. All these compounds inhibited histone deacetylase with IC50 values of 1, 0.9 and 0.5 microM, and cell proliferation at concentrations of 2, 1.5 and 1 microM, respectively.
    Document Type:
    Reference
    Product Catalog Number:
    06-559

  • The addition of monosodium glutamate and inosine monophosphate-5 to high-protein meals: effects on satiety, and energy and macronutrient intakes. 19267954

    In a fed and orally stimulated state, whether the addition of monosodium glutamate (MSG) (alone or in combination with inosine monophosphate-5 (IMP-5)) to a high-protein (HP) meal leads to early satiety and a difference in energy intake at a second course was investigated. Ten men and twelve women consumed, in random order, a first-course meal consisting of: (1) water (control); (2) a HP meal with 0.6% MSG and 0.25% IMP-5; (3) a HP meal with no additives; (4) a HP meal with MSG only; (5) a sham-fed meal 2 (oral-stimulation). Appetite perceptions, plasma concentrations of glucagon-like peptide 1 (GLP-1), glucose and insulin, and energy intake at a buffet (i.e. a second course) were measured before and after each condition. Changes in appetite, and in GLP-1, glucose and insulin, were similar for the three fed HP conditions and all were greater (post hoc all P < 0.01) than the control and sham conditions. Energy intake was not different following the HP+MSG+IMP (1.86 (SEM 0.3) MJ) as compared with the HP+MSG-only (2.24 (SEM 0.28) MJ) condition (P = 0.08), or for the HP+MSG+IMP compared with the HP no-additives condition (1.60 (SEM 0.29) MJ) (P = 0.21). Following the HP+MSG-only condition, 0.64 (SEM 0.20) MJ more energy was consumed compared with the HP no-additives condition (P = 0.005). We conclude that the addition of MSG to a HP meal does not influence perceptions of satiety and it may increase energy intake at a second course. Cephalic responses after the sham condition were of similar magnitude to the control and therefore just tasting food is not enough to influence appetite and energy intake.
    Document Type:
    Reference
    Product Catalog Number:
    EGLP-35K
    Product Catalog Name:
    Glucagon Like Peptide-1 (Active) ELISA

  • Greater superficial petrosal nerve transection in rats does not change unconditioned licking responses to putatively sweet taste stimuli. 18635557

    The greater superficial petrosal nerve (GSP), innervating taste buds in the palate, is known to be exceptionally responsive to sucrose, especially compared with the responsiveness of the chorda tympani nerve (CT). However, whereas transection of the CT (CTX) alone has little or no effect on unconditioned licking responses to many "sweet" stimuli, the impact of GSP transection (GSPX) alone is equivocal. To further examine the role of the GSP on licking responses to putatively sweet-tasting substances, brief-access taste tests were conducted in nondeprived rats before and after sham surgery (SHAM) or CTX or GSPX. A range of concentrations of sucrose, L-alanine, glycine, and L-serine, with and without 1.0 mM inosine monophosphate (IMP) added, were used. All groups showed significant concentration-dependent increases in licking to all stimuli presurgically and postsurgically. CTX decreased licking responses relative to SHAM rats in the first sucrose test. There was also a group x concentration interaction for L-alanine, but post hoc tests did not reveal its basis. Other than this, there were no significant differences among the surgical groups. Interestingly, rats with GSPX tended to initiate fewer trials than SHAM rats. Overall, after GSPX, the remaining gustatory nerves are apparently sufficient to maintain concentration-dependent licking responses to all stimuli tested here. The disparity between our results and others in the literature where GSPX reduced licking responses to sucrose is possibly related to differences in surgical technique or test trial duration.
    Document Type:
    Reference
    Product Catalog Number:
    5001

  • IMP1 promotes choriocarcinoma cell migration and invasion through the novel effectors RSK2 and PPME1. 23911878

    Oncofetal protein insulin-like growth factor II mRNA-binding protein 1 (IMP1) regulates cellular proliferation and migration. Expression of IMP1 is limited to a few adult human tissues. However, it commonly expresses in a variety of cancers. Our objective was to study the regulatory mechanism of IMP1 on the cellular functions of choriocarcinoma (CC) JAR cells.IMP1 protein levels were measured in CC tissues via immunohistochemistry. Specific siRNAs were used to down-regulate gene expressions. The abilities of migration and invasion were estimated by wound-healing and Matrigel chamber assays. The profile of IMP1-binding genes was investigated with an Agilent microarray. RT-qPCR, RNA immunoprecipitation, and IMP1 rescue experiments were performed to confirm the association between IMP1 and its binding genes. Gene expression was further analyzed by using RT-PCR and Western blotting.Strong IMP1 expressions were frequently detected in CC tissues. Knockdown of IMP1 expression in JAR cells inhibited cell migration and invasion, but did not affect cellular proliferation and morphology. Microarray and RNA-immunoprecipitation results revealed several candidate genes regulated by IMP1. Among them, ribosomal protein S6 kinase (RSK2) and protein phosphatase methylesterase 1 (PPME1) were confirmed to be down-regulated in IMP1-depleted JAR cells. Re-expression of IMP1 into the cells restored the expressions of RSK2 and PPME1. Furthermore, the depletion of RSK2 or PPME1 decreased the migration and invasion of JAR cells.Our results suggest that IMP1 plays an essential role in the regulation of migration and invasion of human CC cells, possibly through the novel effectors RSK2 and PPME1.
    Document Type:
    Reference
    Product Catalog Number:
    17-700
    Product Catalog Name:
    Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit