Cellular Localization Patterns Research Articles

Delta-like 1 homolog in Capra hircus: molecular characteristics, expression pattern and phylogeny.

To research the molecular characteristics, expression pattern and phylogeny of the Delta-like 1 homolog gene (Dlk1) in goats. Dlk1 transcripts were identified in the Jianyang Da'er goats by reverse-transcription polymerase chain reaction (RT-PCR). Phylogenetic trees were constructed by Bayesian inference and neighbor-joining methods. Quantitative real-time PCR (qPCR), western blotting and in situ hybridization were performed to analyze the expression pattern of Dlk1. Five alternatively transcripts were identified in different tissues and designated as Dlk1-AS1, 2, 3, 4 and 5. Compared with the normal transcript Dlk1-AS1, Dlk1-AS4 and Dlk1-AS5 retained the identical open reading frame (ORF) and encoded proteins with truncated epidermal-growth-factor like repeats of 121 and 83 amino acids, respectively. Using the Bayesian inference method, the consensus phylogenetic tree indicated that caprine Dlk1 had a closer relationship with bovine Dlk1 than with Dlk1 from pigs, humans and mice. qPCR revealed high expression levels of Dlk1 in the kidney (P<0.01). However, mRNA and protein levels presented an inconsistent correlation, possibly because of post-transcriptional regulation. RNA in situ hybridization indicated that Dlk1 mRNA was localized in the interlobular bile duct and alongside the hepatocyte nuclei, in the epithelial cells of proximal and distal convoluted tubules and in the connective region between the mesothelium and myocardium in the heart. The Dlk1 gene in goats produces alternatively spliced transcripts, with specific expression and cellular localization patterns. These findings would lay the foundation for further study.

Abstract A19: Cytoplasmic ARID1A and poor outcome in ovarian cancer patients.

Abstract Objective: Frequent loss of function mutations in the chromatin remodeling protein and tumor suppressor, AT-rich interactive domain-containing protein 1A (ARID1A), have been noted in ovarian cancers, particularly clear-cell and mucinous histotypes. We initially investigated the relationship between differential ARID1A protein levels and patient survival in a well-annotated ovarian cancer tissue microarray. Our analyses revealed distinct cellular localization patterns for ARID1A using moderately stringent assay conditions and we observed a relationship between cytoplasmic staining of ARID1A and poor disease outcome. We further investigated the relevance of cytoplasmic ARID1A in vitro in a cell line model of ARID1A-mutated, clear-cell ovarian cancer and found that ectopic expression of a cytoplasm-localized variant of ARID1A enhances cellular proliferation relative to wild type ARID1A restored cells. Methods: Tissue microarrays representing a natural distribution of ovarian cancer histotypes (N=259) were stained for ARID1A and scored. Four patient subgroups were further defined based on the absence or presence of distinct ARID1A staining in nuclear and/or cytoplasmic tumor cell compartments. Relationships between ARID1A staining pattern and patient outcome were investigated using univariate and multivariate Cox regression modeling with Wald testing and Kaplan-Meier method with log-rank testing. Nuclear localization sequence variants of ARID1A were generated and phenotypic assessments of cellular proliferation were performed following ectopic expression of cytoplasm-localized versus wild type, i.e. nuclear-localized, ARID1A in a cell line model of ARID1A-mutated, clear cell ovarian cancer cells (TOV21G). Results: Using a moderately stringent IHC assay for ARID1A, loss of nuclear ARID1A was observed in 7% of ovarian cancer patients, and women with versus without loss of nuclear ARID1A had similar survival. Prevalent cytoplasmic staining was observed in 14% of cases overall and was more common in mucinous (50%), clear cell (42%) and serous (11%) adenocarcinomas than other histotypes (p=0.002). Prevalent cytoplasmic ARID1A was not only associated with an increased risk of death in the entire cohort (HR=1.67, 95% CI=1.137-2.454, p=0.009), but was also an independent predictor of worse survival (adjusted HR=1.530, 95% CI=1.017-2.301, p=0.041) after adjusting for patient age at diagnosis, site of disease, stage, cell type and grade. Comparative in vitro analyses of cytoplasmic and wild type ARID1A variants, ectopically-expressed in clear-cell ovarian cancer cells revealed that cytoplasmic ARID1A confers an increased proliferative phenotype relative to wild type ARID1A restored cells. Conclusions: ARID1A cellular localization and abundance varied across ovarian cancer histotypes and cytoplasmic localization was indicative of worse outcome. Our in vitro studies suggest cytoplasmic ARID1A increases the proliferative potential of clear-cell ovarian tumor cells and provides phenotypic evidence supporting the poor disease outcomes we observe in patients exhibiting cytoplasmic ARID1A. These findings support further investigations combining assessments of ARID1A cellular localization and abundance patterns towards the identification of ovarian cancer patients with an elevated risk to experience poor disease outcome. Citation Format: Nicholas W. Bateman, Kevin A. Byrd, Kelly A. Conrads, John I. Risinger, Carl Morrison, Kunle Odunsi, Chad A. Hamilton, G. Larry Maxwell, Kathleen M. Darcy, Thomas P. Conrads. Cytoplasmic ARID1A and poor outcome in ovarian cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A19.

NFI-C2 temporal-spatial expression and cellular localization pattern during tooth formation.

Currently, little is known regarding critical signaling pathways during later stages of tooth development, especially those associated with root formation. Nfi-c null mice, lacking molar roots, have implicated the transcription factor NFI-C as having an essential role in root development. Previously, we identified three NFI-C isoforms expressed in dental tissues with NFI-C2 being the major transcript. However, the expression pattern of the NFI-C2 protein is not characterized. In this study we performed in situ hybridization and immunohistochemistry using isoform specific probes. We show the production of a NFI-C2 peptide antibody, its characterization, the temporal-spatial expression pattern of the NFI-C2 protein during odontogenesis and sub-cellular localization in dental cells. Moderate NFI-C2 staining, as early as bud stage, was detected mostly in the condensing dental ectomesenchyme. This staining intensified within the dental pulp at later stages culminating in high expression in the dentin producing odontoblasts. The dental epithelium showed slight staining until cytodifferentiation of enamel organ into ameloblasts and stratum intermedium. During root formation NFI-C2 expression was high in the Hertwig's epithelial root sheath and later was found in the fully developed root and its supporting tissues. NFI-C2 cellular staining was cytosolic, associated with the Golgi, and nuclear. These data suggest a broader role for NFI-C during tooth formation than limited to root and periodontal ligament development.

Receptor Tyrosine Kinase Expression Profiles in Canine Cutaneous and Subcutaneous Mast Cell Tumors.

The receptor tyrosine kinase (RTK) KIT is a major focus of current research into canine mast cell tumors (MCTs). Little is known about the role of other RTKs, such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs). These RTKs are dysregulated in many human and animal cancers and are key regulators of tumor angiogenesis. The aims of this study were to assess the expression and activation (phosphorylation) status of KIT, VEGFR2, and PDGFR (α and β) in canine MCTs and to examine associations with various clinical outcomes. c-KITmutational status and KIT cellular localization pattern were also evaluated for these tumors. Twenty-seven MCTs, consisting of 5 subcutaneous and 22 cutaneous tumors, from 25 dogs were evaluated. MCT biopsies, cultured mast cells, and skin from the surgical margin were analyzed through Western blotting. MCT biopsies were also used for KIT immunohistochemical labeling and polymerase chain reaction for c-KITmutational analysis. MCT had heterogeneous expression profiles for all 3 RTKs, which varied in intensity and activation status. Statistical analyses showed phosphorylated KIT, VEGFR2, and KIT cellular localization to be predictive of decreased survival time, disease-free interval, and increased metastatic rate. Expression of VEGFR2 and KIT diffuse cytoplasmic labeling were also significantly associated with increased rate of local recurrence. The results of the study show that phosphorylated KIT, KIT, VEGFR2, and PDGFRβ, in addition to KIT localization, may be valuable prognostic determinants in MCTs and should be further studied to improve diagnostic and therapeutic modalities.

Structure and Mechanism of RNA Mimics of Green Fluorescent Protein.

RNAs have highly complex and dynamic cellular localization patterns. Technologies for imaging RNA in living cells are important for uncovering their function and regulatory pathways. One approach for imaging RNA involves genetically encoding fluorescent RNAs using RNA mimics of green fluorescent protein (GFP). These mimics are RNA aptamers that bind fluorophores resembling those naturally found in GFP and activate their fluorescence. These RNA-fluorophore complexes, including Spinach, Spinach2, and Broccoli, can be used to tag RNAs and to image their localization in living cells. In this article, we describe the generation and optimization of these aptamers, along with strategies for expanding the spectral properties of their associated RNA-fluorophore complexes. We also discuss the structural basis for the fluorescence and photophysical properties of Spinach, and we describe future prospects for designing enhanced RNA-fluorophore complexes with enhanced photostability and increased sensitivity.

In silico simulation modeling reveals the importance of the Casparian strip for efficient silicon uptake in rice roots.

Silicon (Si) uptake by the roots is mediated by two different transporters, Lsi1 (passive) and Lsi2 (active), in rice (Oryza sativa). Both transporters are polarly localized in the plasma membranes of exodermal (outer) and endodermal (inner) cells with Casparian strips. However, it is unknown how rice is able to take up large amounts of Si compared with other plants, and why rice Si transporters have a characteristic cellular localization pattern. To answer these questions, we simulated Si uptake by rice roots by developing a mathematical model based on a simple diffusion equation that also accounts for active transport by Lsi2. In this model, we calibrated the model parameters using in vivo experimental data on the Si concentrations in the xylem sap and a Monte Carlo method. In our simulation experiments, we compared the Si uptake between roots with various transporter and Casparian strip locations and estimated the Si transport efficiency of roots with different localization patterns and quantities of the Lsi transporters. We found that the Si uptake by roots that lacked Casparian strips was lower than that of normal roots. This suggests that the double-layer structure of the Casparian strips is an important factor in the high Si uptake by rice. We also found that among various possible localization patterns, the most efficient one was that of the wild-type rice; this may explain the high Si uptake capacity of rice.

Role of Inhibitors of Apoptosis Proteins in Testicular Function and Male Fertility: Effects of Polydeoxyribonucleotide Administration in Experimental Varicocele.

Neuronal apoptosis inhibitory protein (NAIP) and survivin might play an important role in testicular function. We investigated the effect of PDRN, an agonist of adenosine A2A receptor, on testicular NAIP and survivin expression in an experimental model of varicocele. After the creation of experimental varicocele (28 days), adolescent male Sprague-Dawley rats were randomized to one of the following treatments lasting 21 days: vehicle, PDRN (8 mg/kg i.p., daily), PDRN + 3,7-dimethyl-propargylxanthine (DMPX, a specific adenosine A2A-receptor antagonist, 0.1 mg/kg i.p., daily), varicocelectomy, and varicocelectomy + PDRN (8 mg/kg i.p., daily). Sham-operated animals were used as controls. Animals were then euthanized and testis expression of NAIP and survivin was evaluated through qRT-PCR, western blot, and immunohistochemical analysis. Spermatogenetic activity was also assessed. NAIP and survivin expressions were significantly reduced following varicocele induction when compared to sham animals whereas PDRN-treated rats showed an increase in NAIP and survivin levels. Immunohistochemistry revealed an enhanced expression of NAIP and survivin with a characteristic pattern of cellular localization following PDRN treatment. Moreover, administration of PDRN significantly restored spermatogenic function in varicocele rats. PDRN may represent a rational therapeutic option for accelerating recovery from depressed testicular function through a strategic modulation of apoptosis in experimental varicocele.

Evidence for multiple, distinct ADAR-containing complexes in Xenopus laevis.

ADAR (adenosine deaminase acting on RNA) is an RNA-editing enzyme present in most metazoans that converts adenosines in double-stranded RNA targets into inosines. Although the RNA targets of ADAR-mediated editing have been extensively cataloged, our understanding of the cellular function of such editing remains incomplete. We report that long, double-stranded RNA added to Xenopus laevis egg extract is incorporated into an ADAR-containing complex whose protein components resemble those of stress granules. This complex localizes to microtubules, as assayed by accumulation on meiotic spindles. We observe that the length of a double-stranded RNA influences its incorporation into the microtubule-localized complex. ADAR forms a similar complex with endogenous RNA, but the endogenous complex fails to localize to microtubules. In addition, we characterize the endogenous, ADAR-associated RNAs and discover that they are enriched for transcripts encoding transcriptional regulators, zinc-finger proteins, and components of the secretory pathway. Interestingly, association with ADAR correlates with previously reported translational repression in early embryonic development. This work demonstrates that ADAR is a component of two, distinct ribonucleoprotein complexes that contain different types of RNAs and exhibit diverse cellular localization patterns. Our findings offer new insight into the potential cellular functions of ADAR.

Identification and functional comparison of seven-transmembrane G-protein-coupled BILF1 receptors in recently discovered nonhuman primate lymphocryptoviruses.

Coevolution of herpesviruses with their respective host has resulted in a delicate balance between virus-encoded immune evasion mechanisms and host antiviral immunity. BILF1 encoded by human Epstein-Barr virus (EBV) is a 7-transmembrane (7TM) G-protein-coupled receptor (GPCR) with multiple immunomodulatory functions, including attenuation of PKR phosphorylation, activation of G-protein signaling, and downregulation of major histocompatibility complex (MHC) class I surface expression. In this study, we explored the evolutionary and functional relationships between BILF1 receptor family members from EBV and 12 previously uncharacterized nonhuman primate (NHP) lymphocryptoviruses (LCVs). Phylogenetic analysis defined 3 BILF1 clades, corresponding to LCVs of New World monkeys (clade A) or Old World monkeys and great apes (clades B and C). Common functional properties were suggested by a high degree of sequence conservation in functionally important regions of the BILF1 molecules. A subset of BILF1 receptors from EBV and LCVs from NHPs (chimpanzee, orangutan, marmoset, and siamang) were selected for multifunctional analysis. All receptors exhibited constitutive signaling activity via G protein Gαi and induced activation of the NF-κB transcription factor. In contrast, only 3 of 5 were able to activate NFAT (nuclear factor of activated T cells); chimpanzee and orangutan BILF1 molecules were unable to activate NFAT. Similarly, although all receptors were internalized, BILF1 from the chimpanzee and orangutan displayed an altered cellular localization pattern with predominant cell surface expression. This study shows how biochemical characterization of functionally important orthologous viral proteins can be used to complement phylogenetic analysis to provide further insight into diverse microbial evolutionary relationships and immune evasion function. Epstein-Barr virus (EBV), known as an oncovirus, is the only human herpesvirus in the genus Lymphocryptovirus (LCV). EBV uses multiple strategies to hijack infected host cells, establish persistent infection in B cells, and evade antiviral immune responses. As part of EBV's immune evasion strategy, the virus encodes a multifunctional 7-transmembrane (7TM) G-protein-coupled receptor (GPCR), EBV BILF1. In addition to multiple immune evasion-associated functions, EBV BILF1 has transforming properties, which are linked to its high constitutive activity. We identified BILF1 receptor orthologues in 12 previously uncharacterized LCVs from nonhuman primates (NHPs) of Old and New World origin. As 7TM receptors are excellent drug targets, our unique insight into the molecular mechanism of action of the BILF1 family and into the evolution of primate LCVs may enable validation of EBV BILF1 as a drug target for EBV-mediated diseases, as well as facilitating the design of drugs targeting EBV BILF1.

Localization of anionic phospholipids in Escherichia coli cells.

Cardiolipin (CL) is an anionic phospholipid with a characteristically large curvature and is of growing interest for two primary reasons: (i) it binds to and regulates many peripheral membrane proteins in bacteria and mitochondria, and (ii) it is distributed asymmetrically in rod-shaped cells and is concentrated at the poles and division septum. Despite the growing number of studies of CL, its function in bacteria remains unknown. 10-N-Nonyl acridine orange (NAO) is widely used to image CL in bacteria and mitochondria, as its interaction with CL is reported to produce a characteristic red-shifted fluorescence emission. Using a suite of biophysical techniques, we quantitatively studied the interaction of NAO with anionic phospholipids under physiologically relevant conditions. We found that NAO is promiscuous in its binding and has photophysical properties that are largely insensitive to the structure of diverse anionic phospholipids to which it binds. Being unable to rely solely on NAO to characterize the localization of CL in Escherichia coli cells, we instead used quantitative fluorescence microscopy, mass spectrometry, and mutants deficient in specific classes of anionic phospholipids. We found CL and phosphatidylglycerol (PG) concentrated in the polar regions of E. coli cell membranes; depletion of CL by genetic approaches increased the concentration of PG at the poles. Previous studies suggested that some CL-binding proteins also have a high affinity for PG and display a pattern of cellular localization that is not influenced by depletion of CL. Framed within the context of these previous experiments, our results suggest that PG may play an essential role in bacterial physiology by maintaining the anionic character of polar membranes.

Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes.

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial signalling molecule produced by diguanylate cyclases of the GGDEF-domain family. Elevated c-di-GMP levels or increased GGDEF protein expression is frequently associated with the onset of sessility and biofilm formation in numerous bacterial species. Conversely, phosphodiesterase-dependent diminution of c-di-GMP levels by EAL- and HD-GYP-domain proteins is often accompanied by increased motility and virulence. In this study, we individually overexpressed 23 predicted GGDEF, EAL or HD-GYP-domain proteins encoded by the phytopathogen Pectobacterium atrosepticum strain SCRI1043. MS-based detection of c-di-GMP and 5'-phosphoguanylyl-(3'-5')-guanosine in these strains revealed that overexpression of most genes promoted modest 1-10-fold changes in cellular levels of c-di-GMP, with the exception of the GGDEF-domain proteins ECA0659 and ECA3374, which induced 1290- and 7660-fold increases, respectively. Overexpression of most EAL domain proteins increased motility, while overexpression of most GGDEF domain proteins reduced motility and increased poly-β-1,6-N-acetyl-glucosamine-dependent flocculation. In contrast to domain-based predictions, overexpression of the EAL protein ECA3549 or the HD-GYP protein ECA3548 increased c-di-GMP concentrations and reduced motility. Most overexpression constructs altered the levels of secreted cellulases, pectinases and proteases, confirming c-di-GMP regulation of virulence in Pe. atrosepticum. However, there was no apparent correlation between virulence-factor induction and the domain class expressed or cellular c-di-GMP levels, suggesting that regulation was in response to specific effectors within the network, rather than total c-di-GMP concentration. Finally, we demonstrated that the cellular localization patterns vary considerably for GGDEF/EAL/HD-GYP proteins, indicating it is a likely factor restricting specific interactions within the c-di-GMP network.

Inhibitors of apoptosis proteins in experimental benign prostatic hyperplasia: effects of serenoa repens, selenium and lycopene.

BackgroundThe apoptosis machinery is a promising target against benign prostatic hyperplasia (BPH). Inhibitors of apoptosis proteins (IAPs) modulate apoptosis by direct inhibition of caspases. Serenoa Repens (SeR) may be combined with other natural compounds such as Lycopene (Ly) and Selenium (Se) to maximize its therapeutic activity in BPH. We investigated the effects of SeR, Se and Ly, alone or in association, on the expression of four IAPs, cIAP-1, cIAP-2, NAIP and survivin in rats with experimental testosterone-dependent BPH. Moreover, caspase-3, interleukin-6 (IL-6) and prostate specific membrane antigen (PSMA) have been evaluated.Rats were administered, daily, with testosterone propionate (3 mg/kg/sc) or its vehicle for 14 days. Testosterone injected animals (BPH) were randomized to receive vehicle, SeR (25 mg/kg/sc), Se (3 mg/kg/sc), Ly (1 mg/kg/sc) or the SeR-Se-Ly association for 14 days. Animals were sacrificed and prostate removed for analysis.ResultsBPH animals treated with vehicle showed unchanged expression of cIAP-1 and cIAP-2 and increased expression of NAIP, survivin, caspase-3, IL-6 and PSMA levels when compared with sham animals. Immunofluorescence studies confirmed the enhanced expression of NAIP and survivin with a characteristic pattern of cellular localization. SeR-Se-Ly association showed the highest efficacy in reawakening apoptosis; additionally, this therapeutic cocktail significantly reduced IL-6 and PSMA levels. The administration of SeR, Se and Ly significantly blunted prostate overweight and growth; moreover, the SeR-Se-Ly association was most effective in reducing prostate enlargement and growth by 43.3% in treated animals.ConclusionsThe results indicate that IAPs may represent interesting targets for drug therapy of BPH.

Towards Matched Pairs of Porphyrin–ReI/99mTcI Conjugates that Combine Photodynamic Activity with Fluorescence and Radio Imaging

We recently prepared two novel water soluble porphyrins bearing a single peripheral chelator, either diethylenetriamine (1) or bipyridyl (2), tethered to one meso position. The preparation of their conjugates with a fac-{(99m) Tc(CO)3 }(+) fragment and the potential of these resulting conjugates as fluorescence and radio imaging tools were also described. In this work, we focused on the corresponding non-radioactive analogues that bear the fac-{Re(CO)3 }(+) fragment (diethylenetriamine 3 and bipyridyl 4). We report on the uptake, in vitro PDT activity, and cellular localization of Re(I) conjugates 3 and 4 in comparison to the parent porphyrins 1 and 2. Compounds 1-4 have modest or negligible cytotoxicity in the dark against HeLa human cervical cancer cells but become remarkably cytotoxic after exposure to moderate doses of red visible light (590-700 nm). This phototoxicity was found to be directly proportional to the total light dose. Although the four compounds show distinct uptake patterns, they have comparable PDT activity. Confocal fluorescence measurements showed that porphyrin 1 and its Re(I) conjugate 3 have different cellular localization patterns in HeLa cells.

Advances in Understanding the Expression and Function of Dipeptidyl Peptidase 8 and 9

DPP8 and DPP9 are recently identified members of the dipeptidyl peptidase IV (DPPIV) enzyme family, which is characterized by the rare ability to cleave a post-proline bond two residues from the N-terminus of a substrate. DPP8 and DPP9 have unique cellular localization patterns, are ubiquitously expressed in tissues and cell lines, and evidence suggests important contributions to various biological processes including: cell behavior, cancer biology, disease pathogenesis, and immune responses. Importantly, functional differences between these two proteins have emerged, such as DPP8 may be more associated with gut inflammation whereas DPP9 is involved in antigen presentation and intracellular signaling. Similarly, the DPP9 connections with H-Ras and SUMO1, and its role in AKT1 pathway downregulation provide essential insights into the molecular mechanisms of DPP9 action. The recent discovery of novel natural substrates of DPP8 and DPP9 highlights the potential role of these proteases in energy metabolism and homeostasis. This review focuses on the recent progress made with these post-proline dipeptidyl peptidases and underscores their emerging importance.

NRMT2 is an N-terminal monomethylase that primes for its homologue NRMT1

NRMT (N-terminal regulator of chromatin condensation 1 methyltransferase) was the first eukaryotic methyltransferase identified to specifically methylate the free α-amino group of proteins. Since the discovery of this N-terminal methyltransferase, many new substrates have been identified and the modification itself has been shown to regulate DNA-protein interactions. Sequence analysis predicts one close human homologue of NRMT, METTL11B (methyltransferase-like protein 11B, now renamed NRMT2). We show in the present paper for the first time that NRMT2 also has N-terminal methylation activity and recognizes the same N-terminal consensus sequences as NRMT (now NRMT1). Both enzymes have similar tissue expression and cellular localization patterns. However, enzyme assays and MS experiments indicate that they differ in their specific catalytic functions. Although NRMT1 is a distributive methyltransferase that can mono-, di- and tri-methylate its substrates, NRMT2 is primarily a monomethylase. Concurrent expression of NRMT1 and NRMT2 accelerates the production of trimethylation, and we propose that NRMT2 activates NRMT1 by priming its substrates for trimethylation.

Spinal astrocytes produce and secrete dynorphin neuropeptides

Dynorphin peptide neurotransmitters (neuropeptides) have been implicated in spinal pain processing based on the observations that intrathecal delivery of dynorphin results in proalgesic effects and disruption of extracellular dynorphin activity (by antisera) prevents injury evoked hyperalgesia. However, the cellular source of secreted spinal dynorphin has been unknown. For this reason, this study investigated the expression and secretion of dynorphin-related neuropeptides from spinal astrocytes (rat) in primary culture. Dynorphin A (1–17), dynorphin B, and α-neoendorphin were found to be present in the astrocytes, illustrated by immunofluorescence confocal microscopy, in a discrete punctate pattern of cellular localization. Measurement of astrocyte cellular levels of these dynorphins by radioimmunoassays confirmed the expression of these three dynorphin-related neuropeptides. Notably, BzATP (3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate) and KLA (di[3-deoxy-d-manno-octulosonyl]-lipid A) activation of purinergic and toll-like receptors, respectively, resulted in stimulated secretion of dynorphins A and B. However, α-neoendorphin secretion was not affected by BzATP or KLA. These findings suggest that dynorphins A and B undergo regulated secretion from spinal astrocytes. These findings also suggest that spinal astrocytes may provide secreted dynorphins that participate in spinal pain processing.

Inhibition of Cdc42-Interacting Protein 4 (CIP4) Impairs Osteosarcoma Tumor Progression

In patients with Osteosarcoma (OS), larger primary tumor size correlates with higher metastasis incidence and lower overall survival. Identifying the mechanisms that control primary tumor growth can lead to the new therapeutic approaches and improve prognosis. Cytoskeleton regulatory molecules play an important role in tumor cells proliferation and motility. In the current study, we investigated the role of scaffolding protein Cdc42-interacting protein 4 (CIP4) in OS.Murine OS cells, DLM8 were stably transfected with shCIP4 plasmid and their tumorigenic activity was studied in vitro and in vivo. The effect of CIP4 downregulation on cytoskeleton was studied by immunohistochemical analysis.In vivo studies revealed that downregulation of CIP4 in OS tumor cells can inhibit primary subcutaneous tumor growth and delay spontaneous metastases growth in the animal lungs. In vitro studies confirmed that inhibiting CIP4 expression significantly reduced tumor cells invasiveness and migration. Changes in CIP4 expression altered its cellular localization pattern and tumor cell morphology, which could explain changes in OS cell behavior in vivo and in vitro. Cells with downregulated CIP4 showed lower levels of actin in cells along with fewer perinuclear actin stress filaments and actin bridges to the lipid bilayer of the cell membrane than control cells.This study presents compelling evidence that inhibition of CIP4 expression in OS cells impairs metastatic behavior of cells in vitro and prolongs survival of animals by inhibiting primary tumor growth. Although the lack of CIP4 in tumor cells did not prevent formation of metastasis, it did substantially delay their formation in the animals' lungs.

Hyperbaric oxygenation alters temporal expression pattern of superoxide dismutase 2 after cortical stab injury in rats

AimTo evaluate the effect of hyperbaric oxygen therapy (HBOT) on superoxide dismutase 2 (SOD2) expression pattern after the cortical stab injury (CSI).MethodsCSI was performed on 88 male Wistar rats, divided into control, sham, lesioned, and HBO groups. HBOT protocol was the following: pressure applied was 2.5 absolute atmospheres, for 60 minutes, once a day for consecutive 3 or 10 days.‎ The pattern of SOD2 expression and cellular localization was analyzed using real-time polymerase chain reaction, Western blot, and double-label fluorescence immunohistochemistry. Neurons undergoing degeneration were visualized with Fluoro-Jade®B.ResultsCSI induced significant transient increase in SOD2 protein levels at day 3 post injury, which was followed by a reduction toward control levels at post-injury day 10. At the same time points, mRNA levels for SOD2 in the injured cortex were down-regulated. Exposure to HBO for 3 days considerably down-regulated SOD2 protein levels in the injured cortex, while after 10 days of HBOT an up-regulation of SOD2 was observed. HBOT significantly increased mRNA levels for SOD2 at both time points compared to the corresponding L group, but they were still lower than in controls. Double immunofluorescence staining revealed that 3 days after CSI, up-regulation of SOD2 was mostly due to an increased expression in reactive astrocytes surrounding the lesion site. HBOT attenuated SOD2 expression both in neuronal and astroglial cells. Fluoro-Jade®B labeling showed that HBOT significantly decreased the number of degenerating neurons in the injured cortex.ConclusionHBOT alters SOD2 protein and mRNA levels after brain injury in a time-dependent manner.

Inhibition of Cdc42-Interacting Protein 4 (CIP4) Impairs Osteosarcoma Tumor Progression

In patients with Osteosarcoma (OS), larger primary tumor size correlates with higher metastasis incidence and lower overall survival. Identifying the mechanisms that control primary tumor growth can lead to the new therapeutic approaches and improve prognosis. Cytoskeleton regulatory molecules play an important role in tumor cells proliferation and motility. In the current study, we investigated the role of scaffolding protein Cdc42-interacting protein 4 (CIP4) in OS. Murine OS cells, DLM8 were stably transfected with shCIP4 plasmid and their tumorigenic activity was studied in vitro and in vivo. The effect of CIP4 downregulation on cytoskeleton was studied by immunohistochemical analysis. In vivo studies revealed that downregulation of CIP4 in OS tumor cells can inhibit primary subcutaneous tumor growth and delay spontaneous metastases growth in the animal lungs. In vitro studies confirmed that inhibiting CIP4 expression significantly reduced tumor cells invasiveness and migration. Changes in CIP4 expression altered its cellular localization pattern and tumor cell morphology, which could explain changes in OS cell behavior in vivo and in vitro. Cells with downregulated CIP4 showed lower levels of actin in cells along with fewer perinuclear actin stress filaments and actin bridges to the lipid bilayer of the cell membrane than control cells. This study presents compelling evidence that inhibition of CIP4 expression in OS cells impairs metastatic behavior of cells in vitro and prolongs survival of animals by inhibiting primary tumor growth. Although the lack of CIP4 in tumor cells did not prevent formation of metastasis, it did substantially delay their formation in the animals' lungs.

Evolutionary adaptation of plant annexins has diversified their molecular structures, interactions and functional roles

Annexins are an homologous, structurally related superfamily of proteins known to associate with membrane lipid and cytoskeletal components. Their involvement in membrane organization, vesicle trafficking and signaling is fundamental to cellular processes such as growth, differentiation, secretion and repair. Annexins exist in some prokaryotes and all eukaryotic phyla within which plant annexins represent a monophyletic clade of homologs descended from green algae. Genomic, proteomic and transcriptomic approaches have provided data on the diversity, cellular localization and expression patterns of different plant annexins. The availability of 35 complete plant genomes has enabled systematic comparative analysis to determine phylogenetic relationships, characterize structures and observe functional specificity between and within individual subfamilies. Short amino termini and selective erosion of the canonical type 2 calcium coordinating sites in domains 2 and 3 are typical of plant annexins. The convergent evolution of alternate functional motifs such as 'KGD', redox-sensitive Cys and hydrophobic Trp/Phe residues argues for their functional relevance and contribution to mechanistic diversity in plant annexins. This review examines recent findings and advances in plant annexin research with special focus on their structural diversity, cellular and molecular interactions and their potential integrated functions in the broader context of physiological responses.