Normal Cell Protein Research Articles

Mechanism of selective anticancer activity of isothiocyanates relies on differences in DNA damage repair between cancer and healthy cells

PurposeIsothiocyanates (ITCs) are compounds derived from Brassica plants with documented anticancer activity. Molecular mechanisms of their selective activity against cancer cells are still underexplored. In this work, the impact of ITC on DNA replication and damage was compared between PC-3 prostate cancer cells and HDFa normal fibroblasts as well as PNT2 prostate epithelial cells.MethodsCells were treated with sulforaphane or phenethyl isothiocyanate. [3H]thymidine incorporation and the level of histone γH2A.X were estimated as indicators of DNA replication and double-strand breaks (DSB), respectively. Levels of HDAC3, CtIP, and p-RPA were investigated by immunoblotting. Comet assay was performed to visualize DNA damage.ResultsITCs inhibited DNA replication in all tested cell lines, and this activity was independent of reactive oxygen species of mitochondrial origin. It was followed by DSB which were more pronounced in cancer than noncancerous cells. This difference was independent of HDAC activity which was decreased in both cell lines when treated with ITCs. On the other hand, it correlated with faster removal of DSB, and thus, transient activation of repair proteins in normal cells, while in PC-3 prostate cancer, cell DNA repair was significantly less effective.ConclusionDNA damage induced by ITCs is a consequence of the block in DNA replication which is observed in both, cancer and normal cells. Selective antiproliferative activity of ITCs towards cancer cells results from less efficient DNA repair in cancer cells relative to normal cells.

Effect of JAG2 on migration and invasion in colorectal cancer cell by PRAF2, independent of epithelial-mesenchymal transition.

e15107 Background: Our previous studies revealed the increased expression of Jagged 2 (JAG2) in most intestinal cancer tissues. In colon cancer cell lines, JAG2 involved in the regulation of migration and invasion without affecting cell proliferation. This study further explored the mechanisms of how JAG2 promotes migration and invasion of colorectal cancer cells. Methods: We analyzed the expression of JAG2 mRNA and protein in normal human colon tissue cells and colorectal cancer cells. The promotive role of JAG2 in migration and invasion was tested by JAG2 siRNA and JAG2 overexpression in various colon cancer cell lines. To understand the mechanisms, we first treated HT29 cells with LY2157299, a TGF-β signaling pathway inhibitor, and Slug siRNA, to identify the cross-talk between JAG2 and EMT pathway. In addition, co-expression status of JAG2 and TGF-β-induced epithelial-mesenchymal transition (EMT) markers was analyzed. Finally, by using siRNA and proteomics technology, co-expressed proteins of JAG2 in colorectal cancer cells were identified. Results: JAG2 was abnormally expressed in colorectal cancer tissues and directly related with clinical stages. Similar to the findings in human tissues, the expression of both JAG2 mRNA and protein was significantly increased in the colorectal cancer cell lines compared with that of normal colorectal cell line CCD18-Co. Interestingly, the promotion of JAG2 in migration and invasion was independent of EMT pathway. Furthermore, we found that the expression of JAG2 was correlated with PRAF2 (PRA1 Domain Family Member 2), a protein involved in the formation of exosome-like vesicles. In the presence of PRAF2, JAG2-rich exosome promoted migration and invasion. JAG2 might regulate the migration and invasion of colon cell through PRAF2. Conclusions: This is the evidence supporting the biological function of JAG2 in migration and invasion through non-EMT-dependent pathways and also the first exploration of the role of PRAF2 in colorectal cancer cells. These findings provide the theoretical basis for potential targeted therapy against JAG2/PRAF2.

Nectin-4 and p63 immunohistochemical expression in canine prostate tumourigenesis.

Nectin-4 is an E-cadherin-based adherens junction protein of normal epithelial cells, as well as a potent mediator of anchorage-independent cancer colony formation. It is considered a tumour-associated histological and serological marker in various human cancers. The transcription factor p63 is a basal cell marker in the normal prostate, involved in cell adhesion, as well as in the formation and survival of circulating tumour cell clusters. The aim of this study was to evaluate Nectin-4 and p63 immunohistochemical expression in 42 canine prostate tissues including 2 normal prostates, 10 benign prostatic hyperplasias (BPHs), 30 prostatic carcinomas (PCs), 1 pulmonary and 1 lymph node metastasis. From normal to neoplastic tissues, Nectin-4 showed a progressive switching from membranous (m-Nectin-4) to cytoplasmic (c-Nectin-4), regardless of the histological subtypes, except for lack of expression in solid PCs. Metastatic cells exhibited both strong membranous and cytoplasmic positivity. c-Nectin-4 expression was significantly (P < 0.0001) increased in PCs/metastasis compared to BPHs cases and a decrease (P < 0.05) of nuclear p63 immunostaining was also detected in the two groups. Furthermore, data showed a significant association (P < 0.05) between p63 and m-Nectin-4 distribution, although their colocalization was detected only in scattered cells by double immunofluorescence. Our results suggest the involvement of m-Nectin-4 in canine prostate tumourigenesis and metastatic potential, while the exact role of c-Nectin-4 expression detectable in primary PCs requires further investigations.

Toxicological exploration of peptide-based cationic liposomes in siRNA delivery.

The toxicology of cationic liposomes was explored to advance clinical trials of liposome-mediated gene therapy through the analysis of a peptide cationic liposome with DOTAP as a positive control. We first investigated the delivery of luciferase siRNA by several peptide liposomes in mice bearing lung cancer A549 cell xenografts. Of these, a cationic liposome (CDO14) was selected for further investigation. CDO14 efficiently mediated IGF-1R-siRNA delivery and inhibited the growth of the A549 cell xenografts. The in vivo toxicity and toxicological mechanisms of the selected liposome were evaluated to assess its potential utility for gene delivery. Specifically, the effects of CDO14 on mouse body weight, hematology, urine, serum biochemical indices, and histopathology were measured in acute toxicity and subchronic toxicity tests. CDO14 showed limited toxicological effects at low dosages although it induced pulmonary inflammation and liver injury at higher dosages. The toxicity of CDO14 was lower than that of DOTAP, and the toxicity of CDO14 did not change when complexed with siRNA. The pulmonary inflammation induced by CDO14 occurred via expressional up-regulation of the pro-inflammatory cytokines TNF-α and IL-6, and expressional down-regulation of the anti-inflammatory cytokine IL-10. Liver injury induced by CDO14 was mediated by the JAK2-STAT3 signaling pathway. Lastly, CDO14 did not affect the expression of apoptosis-related proteins in normal liver cells, suggesting that it did not induce apoptosis of normal cells. The toxicological results demonstrate that peptide-based headgroups in lipids are superior to those with quaternary ammonium headgroups that are used as gene vectors for cancer therapy.

Autoimmune inflammatory meningoencephalitis in a patient negative for glial fibrillary acidic protein-specific immunoglobulin G

BackgroundIn this study, we describe clinical findings in a patient with autoimmune inflammatory meningoencephalitis who was negative for antibodies against glial fibrillary acidic protein (GFAP-IgG). MethodsSerum and cerebral spinal fluid (CSF) samples were collected from the patient as part of a study of 520 patients with neurological syndromes. Antibodies against GFAP and other proteins associated with neurological disorders were measured by rat brain- and cell-based indirect immunofluorescence assays. ResultsA 42-year-old female was diagnosed with autoimmune inflammatory meningoencephalitis. She experienced a subacute and relapsing course with decreased vision, fever, headache, ataxia, hemiplegia, and disturbance of consciousness. Brain magnetic resonance imaging showed extensive lesions in the white matter along the ventricle, brainstem, right internal capsule, and meninges. The patient responded well to steroid treatment. Examination of CSF revealed a normal white blood cell count and protein level. Serum and CSF were negative for GFAP-specific antibodies and all other autoantibodies tested. Immunohistochemical staining of a brain biopsy collected during relapse revealed chronic inflammation and severe edema. Extensive and strong staining of CD163+ macrophages were evident throughout the lesions; however, CD3+ cells were rare and CD138+ and CD20+ cells were absent. ConclusionWe describe a case of subacute corticosteroid-responsive nonvasculitic autoimmune inflammatory meningoencephalitis in the absence of GFAP-IgG. The pathological features were distinct from those of patients with GFAP-IgG-positive meningoencephalitis, suggesting that nonvasculitic autoimmune inflammatory meningoencephalitis is a heterogeneous neurological syndrome.

The Impact of EVI1 on Carbonic Anhydrase III Expression and the Sensitivity of Rat1 Fibroblasts to H2O2-induced Apoptosis

EVI1 is a transcriptional factor with two distinct zinc finger domains and encoded in human chromosome 3q26. In human, the protein is believed to have role in cell proliferation, organogenesis and it was detected in kidneys and lungs. EVI1 overexpression has been associated with various conditions such as myelodysplastic syndrome (MDS), juvenile myelomonocytic leukaemia (JMML) and acute myeloid leukaemia (AML). In the present study, Carbonic Anhydrase III (CaIII) was expressed at much lower levels in EVI1 overexpressing fibroblast cells compared to the wild type indicating an inhibitory role of EVI1 against CaIII. Our results were further confirmed by western blot in which CaIII protein in normal fibroblast cells was more abundance than that in cells with EVI1 enforced expression. Furthermore, Luciferase reporter assay showed repressed promotor activity in EVI1 overexpressing cells resulted from EVI1 interfering with CaIII promotor by direct or indirect binding. Fibroblast cells with repressed CaIII showed higher sensitivity to apoptosis induced by hydrogen peroxide, this is attributed to the downregulation of CaIII gene which has a defensive role against the oxidative damage. It has been reported that the CaIII overexpression increases the cell resistance against the oxidative stress caused by exposure to H2O2. Thus, elevated EVI1 levels repress CaIII expression leading to increased cell sensitivity to H2O2-induced apoptosis. These findings seem to be constant with previous studies, and might introduce a novel approach to treat leukaemia depending on H2O2-induced apoptosis.

Inhibitor of DNA Binding 2 (ID2) Plays a Key Tumor Suppressor Role in Promoting Oncogenic Transformation in Multiple Myeloma

Dysregulation of transcriptional control is a common phenomenon associated with oncogenesis. Inhibitors of DNA binding (ID) proteins are critical actors in lymphopoiesis, acting as regulators of transcription through a helix-loop-helix (HLH) domain which enables heterodimerization with basic HLH (bHLH) proteins inhibiting their binding to DNA. ID proteins have been implicated in malignant transformation, but their role in multiple myeloma (MM) is unknown. Here, we evaluated the role of ID proteins in biology and transcriptional dysregulation in MM.We first evaluated the expression of the four ID proteins in normal and malignant plasma cells using RNA sequencing data from a cohort of 360 newly diagnosed MM patients and 16 normal plasma cells. We observed significant downregulation of ID2 in primary patient MM cells in comparison to normal plasma cells (p 0.0013).To study ID2 function in MM cells, we next overexpressed ID2 in 2 MM cell lines (MM1S and NCIH929) and observed a significant decrease in proliferation rate, together with G0/G1 phase cell cycle arrest. We performed RNA-sequencing to evaluate the transcriptomic changes following ID2 overexpression. Gene set enrichment analysis (GSEA) revealed significant downregulation of genes involved in E2F pathway and significant changes in pathways related to immune response, regulation of cell death and cell proliferation. In addition, analysis of upstream cis-regulatory motifs of genes significantly dysregulated in both cell lines (>1.5 fold change) showed a highly significant enrichment for bHLH class I transcription factors (E proteins) binding motifs. Conversely, stable ID2 knockdown in 4 MM cell lines (MM1S, NCIH929, RPMI8226 and KMS11) expressing intermediate levels of ID2, showed an increased proliferation rate, assessed by cell counting, H3-thymidine incorporation and ATP production. RNA-sequencing after ID2 knockdown in MM1S and NCIH929 cells showed 600 common genes upregulated in both cell lines (>1.5 fold change). GSEA revealed upregulation of pathways involved in inflammatory response and epithelial-to-mesenchymal transition, while upstream cis regulatory motifs analysis showed a highly significant enrichment for binding motifs of bHLH class I transcription factors E proteins, in particular Tcf3 (p <0.0001).Next, we sought to investigate the mechanisms involved in ID2 downregulation in MM. Since the role of the microenvironment is critical in myelomagenesis, we evaluated the impact of BM microenvironment on ID2 expression in a co-culture system. Using bone marrow stromal cells (BMSC) derived from MM patients and stromal cell line (HS5) in co-culture with various MM cell lines, we observed that both cell-cell interactions and soluble factors secreted by BMSC or HS5 were able to significantly downregulate ID2 expression at the RNA and protein level. Furthermore, ID2 overexpression in MM cell lines (MM1S and NCIH929) abrogated the impact of BMSC on MM cell proliferation. Next, we evaluated ID2 promoter methylation profile and binding motifs using Sequenom mass array and the assay for transposase-accessible chromatin sequencing (ATAC-seq), respectively. While we didn't observe any increase in methylation of CpG islands located in ID2 promoter in co-culture, explaining ID2 downregulation, we identified several binding motifs corresponding to known driver transcription factors in MM. Especially, we identified SP1 binding motif and we confirmed SP1 binding to ID2 promoter by ChIP-sequencing in MM1S, NCIH929 and U266.These data demonstrate that in MM, ID2 acts as a tumor suppressor by promoting major transcriptomic changes and cell cycle arrest. Bone marrow stromal cells further induce significant downregulation of ID2 in myeloma cells suggesting that ID2/bHLH axis and other ID2 related pathways represent a potential new therapeutic target in myeloma. DisclosuresAnderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy. Munshi:OncoPep: Other: Board of director.

Nuclear Factor I Represses the Notch Effector HEY1 in Glioblastoma

Glioblastomas (GBMs) are highly aggressive brain tumors with a dismal prognosis. Nuclear factor I (NFI) is a family of transcription factors that controls glial cell differentiation in the developing central nervous system. NFIs have previously been shown to regulate the expression of astrocyte markers such as glial fibrillary acidic protein (GFAP) in both normal brain and GBM cells. We used chromatin immunoprecipitation (ChIP)–on-chip to identify additional NFI targets in GBM cells. Analysis of our ChIP data revealed ~400 putative NFI target genes including an effector of the Notch signaling pathway, HEY1, implicated in the maintenance of neural stem cells. All four NFIs (NFIA, NFIB, NFIC, and NFIX) bind to NFI recognition sites located within 1 kb upstream of the HEY1 transcription site. We further showed that NFI negatively regulates HEY1 expression, with knockdown of all four NFIs in GBM cells resulting in increased HEY1 RNA levels. HEY1 knockdown in GBM cells decreased cell proliferation, increased cell migration, and decreased neurosphere formation. Finally, we found a general correlation between elevated levels of HEY1 and expression of the brain neural stem/progenitor cell marker B-FABP in GBM cell lines. Knockdown of HEY1 resulted in an increase in the RNA levels of the GFAP astrocyte differentiation marker. Overall, our data indicate that HEY1 is negatively regulated by NFI family members and is associated with increased proliferation, decreased migration, and increased stem cell properties in GBM cells.

Mechanism of TRIM25 mediated ubiquitination of metastasis associated protein (MTA) 1 in normal liver cells

Ninety percent of all cancer related deaths happen due to metastatic progression. One important protein facilitating metastatic progression in hepatocellular carcinoma (HCC) is the metastasis associated 1 protein (MTA-1). We have earlier shown that in the context of HCC and normal liver cell lines, HuH6 and THLE-2, respectively. MTA-1 protein is actively stabilized in HCC cell lines and actively degraded in normal liver cells. We had also shown that TRIM25 is the E3 ligase that interacts with and degrades MTA-1 protein in normal liver cells. However, the exact mechanism by which TRIM25 degrades MTA-1 protein has still not been elucidated. In the study, we used both in situ prediction algorithms and mass spectrometry based post-translational modification analysis to map the lysine residues in MTA-1 that are polyubiquitinated. Whereas UbPred algorithm revealed a combination of medium and low confidence sites, it revealed only one high confidence lysine (K98) residue. The hCKSAAP_UbSite algorithm also predicted K98 site. Mass spectrometry analysis also showed that K98 has ubiquitin modification. Immunofluorescence analysis showed that in normal liver cell line, THLE-2, which has high expression of TRIM25, ectopically expressed FLAG-tagged wild-type MTA-1 was actively degraded, but the K98R mutant MTA-1 was not. In vitro ubiquitination assay using recombinant wild-type and K98R mutant MTA-1 confirmed that MTA-1 is poly-ubiquitinated at K98 residue by TRIM25. The K98R mutant had a longer half-life than wild-type MTA-1 protein in an in vitro protein stability assay. We establish that TRIM25 ubiquitinates MTA-1 at lysine 98 and degrades it normal liver cells.

Structural Alterations in Human Fibroblast Growth Factor Receptors in Carcinogenesis.

Fibroblast growth factor (FGF) plays an important role in human embryogenesis, angiogenesis, cell proliferation, and differentiation. Carcinogenesis is accompanied by aberrant constitutive activation of FGF receptors (FGFRs) resulting from missense mutation in the FGFR1-4 genes, generation of chimeric oncogenes, FGFR1-4 gene amplification, alternative splicing shift toward formation of mesenchymal FGFR isoforms, and FGFR overexpression. Altogether, these alterations contribute to auto- and paracrine stimulation of cancer cells and neoangiogenesis. Certain missense mutations are found at a high rate in urinary bladder cancer and can be used for non-invasive cancer recurrence diagnostics by analyzing urine cell pellet DNA. Chimeric FGFR1/3 and amplified FGFR1/2 genes can predict cell response to the targeted therapy in various oncological diseases. In recent years, high-throughput sequencing has been used to analyze exomes of virtually all human tumors, which allowed to construct phylogenetic trees of clonal cancer evolution with special emphasis on driver mutations in FGFR1-4 genes. At present, FGFR blockers, such as multi-kinase inhibitors, specific FGFR inhibitors, and FGF ligand traps are being tested in clinical trials. In this review, we discuss current data on the functioning of the FGFR family proteins in both normal and cancer cells, mutations in the FGFR1-4 genes, and mechanisms underlying their oncogenic potential, which might be interesting to a broad range of scientists searching for specific tumor markers and targeted anti-cancer drugs.

RHO GTPases in cancer: known facts, open questions, and therapeutic challenges.

RHO GTPases have been traditionally associated with protumorigenic functions. While this paradigm is still valid in many cases, recent data have unexpectedly revealed that RHO proteins can also play tumor suppressor roles. RHO signaling elements can also promote both pro- and antitumorigenic effects using GTPase-independent mechanisms, thus giving an extra layer of complexity to the role of these proteins in cancer. Consistent with these variegated roles, both gain- and loss-of-function mutations in RHO pathway genes have been found in cancer patients. Collectively, these observations challenge long-held functional archetypes for RHO proteins in both normal and cancer cells. In this review, I will summarize these data and discuss new questions arising from them such as the functional and clinical relevance of the mutations found in patients, the mechanistic orchestration of those antagonistic functions in tumors, and the pros and cons that these results represent for the development of RHO-based anticancer drugs.

NRAGE confers poor prognosis and promotes proliferation, invasion, and chemoresistance in gastric cancer

Neurotrophin receptor-interacting melanoma antigen-encoding protein (NRAGE) is a type II melanoma-associated antigen that plays an essential role in various processes, including cell differentiation and apoptosis. NRAGE has been shown to act as a cancer-related protein, with complex and apparently contradictory functions in a variety of cancers. In the current study, we examined the expression of NRAGE protein in 169 gastric cancer samples. NRAGE upregulation was correlated with advanced TNM stage, local invasion, and poor survival. Importantly, NRAGE could serve as an independent prognostic factor in patients with gastric cancer. We also examined the expression of NRAGE protein in GES-1 normal gastric epithelial cells and in six gastric cancer cell lines. Inhibition of NRAGE expression by transfection with small interfering RNA reduced the proliferation and invasion of MGC-803 and HGC-27 cells, as demonstrated by CCK-8 and Matrigel invasion assays. NRAGE depletion also sensitized HGC-27 and MGC-803 cells to cisplatin, as shown by CCK-8 and Annexin V/propidium iodide analyses. Western blot analysis also showed that NRAGE depletion negatively regulated Bcl-2 and p-ERK and upregulated ZO-1 and p27 expression levels. In conclusion, our results suggest that NRAGE acts as a tumor promoter in gastric cancer by facilitating cancer invasion and chemoresistance, possibly through regulation of p-ERK and Bcl-2.

Neurosyphilis as a Cause of Transverse Myelitis in a Teenage Girl

BackgroundSyphilis is a sexually transmitted infection that was nearly eradicated in 2001 but is now making a resurgence. It has a wide range of clinical manifestations depending on disease stage. Neurosyphilis is an infrequently seen infectious disease with central nervous system involvement that can occur in either early- or late-stage syphilis. The diagnosis of neurosyphilis is challenging, primarily because Treponema pallidum, the infecting organism, cannot be cultured in vitro. This article describes a patient with neurosyphilis and reviews the epidemiology and clinical manifestations, diagnostics, and treatment of neurosyphilis. Case ReportIn compliance with the request of the Privacy Board of our institution, the numerical age of this patient has been omitted. A sexually active teenage girl who was treated for primary syphilis 2 years earlier presented to a tertiary children's hospital with paresthesia and weakness of her right leg, left arm, and neck. Magnetic resonance imaging revealed cervical intramedullary cord edema consistent with transverse myelitis. Serum studies showed positive syphilis enzyme immunoassay, T. pallidum particle agglutination assay, and fluorescent treponemal antibody absorption. A serum rapid plasma reagin test was negative. A lumbar puncture was performed with normal cell count and protein. A cerebrospinal fluid Venereal Disease Research Laboratory test was negative. She was diagnosed with neurosyphilis and treated with intravenous steroids and penicillin G, with near complete resolution of symptoms. Why should an Emergency Physician Be Aware of This?The Centers for Disease Control and prevention has noted a steady rise of the incidence of syphilis since 2002. Emergency physicians should be familiar with the spectrum of the clinical manifestations of syphilis, challenges in diagnostics, and appropriate treatment course.

Differential mRNA expression of the main apoptotic proteins in normal and malignant cells and its relation to in vitro resistance

BackgroundApoptosis plays an important role in the development and homeostasis of multicellular organisms and its deregulation may result in many serious diseases, including cancer. Now it is clear that some oncogenic mutations disrupt apoptosis, leading to tumour initiation, progression or metastasis. Here, expression of apoptotic genes in context of drug resistance was investigated.MethodsWe examined total of 102 samples from leukemic patients (n = 60) and patients with solid tumours (n = 42). We used RT-PCR to determine the levels of mRNA expression and the in vitro chemoresistance of leukemic cells was evaluated using the MTT assay.ResultsWe found statistically significant increase in mRNA expression of all investigated proteins (p53, BAX, Bcl-2 and Bcl-XL) between the leukemia samples and leukocytes from healthy volunteers. We did not find any significant difference in mRNA levels among the solid tumour samples. Notably, we showed a significant positive correlation in both leukemic and solid tumour patient groups between p53 and BAX mRNA. We found that the highest values for the Bcl-2/BAX ratio were in solid tumours in comparison to leukemic cells or normal leukocytes. Moreover, we assessed the impact of p53 and BAX mRNA levels on the sensitivity of the leukemic cells to selected cytostatics.ConclusionsElevated levels of p53 and BAX mRNA may indicate cellular response to possible changes in genomic DNA integrity associated with malignant transformation. We suggest that the BAX gene is regulated by the p53 protein but the initiation of apoptosis through the transcription activation of BAX is blocked by the high levels of Bcl-2. Given that the apoptosis resistance mechanisms are different among oncological patients as well as stages of identical malignancy cases, personalized and specific combination therapy is proposed to be more effective in clinical application.

Graphene and graphene oxide as asolid matrix for extraction of membrane and membrane-associated proteins.

The extraction of membrane proteins remain a challenge due to innate hydrophobicity, dynamic discrepancy, and restrain effect of membrane lipids. Nanomaterials with high surface area have competency of hydrophobic-hydrophobic lipid interactions. It is shown here that both graphene and graphene oxide dissolved in solubilization buffer are viable sorbents for efficient extraction of membrane proteins. LC-MS/MS analysis further revealed that graphene (50-200nm) and graphene oxide (50-200nm) can enrich more kinds of membrane proteins than a commercially available kit. Graphene was further applied to the enrichment of membrane proteins of normal cells as well as cancer cells, and 1079 and 872 proteins were identified, respectively, among which 56.5% and 60.5% were membrane proteins. In particular, 241 proteins were significantly regulated in cancer cells. Gene expression of 15 proteins was verified by qRT-PCR, and 4 of them were further quantified by immunoassay. These data collectively demonstrate that graphene has great potential to improve membrane protein extractions and thus can serve downstream cancer proteomics. Graphical abstract Two dimensional carbon nanomaterials, including graphene and graphene oxide, were employed as solid matrix to avoid lipid bilayer interference and enhance the extraction efficiency of membrane and membrane associated proteins. The strategy will benefit downstream membrane proteomics analysis.

Болезнь Крейтцфельдта-Якоба. Случай из клинической практики

Creutzfeldt-Jakob disease is a rare degenerative, prion disease of the brain. The causative agent of prion diseases is an infectious prion protein Prion Protein-scarpie (PrPSc), formed as a result of changes in the normal (non - infectious) cell protein Prion Protein-cellular (Rgrs). Accumulation of the pathological form of PrPSc leads to mass death of nerve cells. The main clinical manifestations of the disease include rapidly progressive cognitive impairment, myoclonus, dystonia, kinetically syndrome, spasticity, hyperreflexia, ataxia, visual disturbances, in the later stages of the disease akinetic mutism. About a third of the cases are epileptic seizures. The average life expectancy is about 5 months. More than 90% of patients die within 1 year due to aspiration pneumonia in the state of akinetic mutism. The complexity of diagnosis of CDR is associated with the rarity of the disease, clinical polymorphism and lack of awareness of doctors.

Immunomodulatory Role of Complement Proteins in the Neuropathology Associated with Opiate Abuse and HIV-1 Co-Morbidity

ABSTRACTThe complement system which is a critical mediator of innate immunity plays diverse roles in the neuropathogenesis of HIV-1 infection such as clearing HIV-1 and promoting productive HIV-1 replication. In the development of HIV-1 associated neurological disorders (HAND), there may be an imbalance between complement activation and regulation, which may contribute to the neuronal damage as a consequence of HIV-1 infection. It is well recognized that opiate abuse exacerbates HIV-1 neuropathology, however, little is known about the role of complement proteins in opiate induced neuromodulation, specifically in the presence of co-morbidity such as HIV-1 infection. Complement levels are significantly increased in the HIV-1-infected brain, thus HIV-induced complement synthesis may represent an important mechanism for the pathogenesis of AIDS in the brain, but remains underexplored. Anti-HIV-1 antibodies are able to initiate complement activation in HIV-1 infected CNS cells such as microglia and astrocytes during the course of disease progression; however, this complement activation fails to clear and eradicate HIV-1 from infected cells. In addition, the antiretroviral agents used for HIV therapy cause dysregulation of lipid metabolism, endothelial, and adipocyte cell function, and activation of pro-inflammatory cytokines. We speculate that both HIV-1 and opiates trigger a cytokine-mediated pro-inflammatory stimulus that modulates the complement cascade to exacerbate the virus-induced neurological damage. We examined the expression levels of C1q, SC5b-9, C5L2, C5aR, C3aR, and C9 key members of the complement cascade both in vivo in post mortem brain frontal cortex tissue from patients with HAND who used/did not use heroin, and in vitro using human microglial cultures treated with HIV tat and/or heroin. We observed significant expression of C1q and SC5b-9 by immunofluorescence staining in both the brain cortical and hippocampal region in HAND patients who abused heroin. Additionally, we observed increased gene expression of C5aR, C3aR, and C9 in the brain tissue of both HIV-1 infected patients with HAND who abused and did not abuse heroin, as compared to HIV negative controls. Our results show a significant increase in the expression of complement proteins C9, C5L2, C5aR, and C3aR in HIV transfected microglia and an additional increase in the levels of these complement proteins in heroin-treated HIV transfected microglia. This study highlights the a) potential roles of complement proteins in the pathogenesis of HIV-1-related neurodegenerative disorders; b) the combined effect of an opiate, like heroin, and HIV viral protein like HIV tat on complement proteins in normal human microglial cells and HIV transfected microglial cells. In the context of HAND, targeting selective steps in the complement cascade could help ameliorating the HIV burden in the CNS, thus investigations of complement-related therapeutic approaches for the treatment of HAND are warranted.

Effects of calcium channel on ovarian cancer cells.

The aim of the present study was to examine the effects of calcium channel protein on ovarian cancer cells. The expression of calcium channel protein in normal ovarian cells and ovarian cancer cells was detected by fluorescence quantitative PCR. Subsequently, the ovarian cancer cells were added to calcium channel protein activator media at various concentrations of 0, 1, 4, 8, 12, 16 and 20 mmol/l. The concentration of calcium ion in different samples was produced, and using an MTT assay, ovarian cancer cell activity in various samples was detected. Finally, a flow cytometer was used to explore the apoptosis rate. It was found that there was a significant difference between the expression of calcium channel protein in normal ovarian tissue and ovarian cancer cells (P<0.05), as well as a significant difference of calcium concentration among various samples (P<0.05). When the concentrations of calcium channel activator were 1, 4, 8, 12, 16 and 20 mmol/l, the values of the ovarian cancer cell inhibition rates were 4.6, 21.3, 48.3, 67.9, 52.8 and 31.8%, respectively. It showed that the calcium channel activator inhibited the proliferation of ovarian cancer cells to a certain extent, in a dose-dependent manner, especially when the concentration was at 12 mmol/l at which the intracellular calcium concentration was similar to that in normal ovarian cells. In conclusion, calcium ions play an important role in promoting cell proliferation of ovarian cancer cells, and they were involved in apoptosis of ovarian cancer cells to some extent, which regulates apoptosis by controlling the content of intracellular calcium.

Polyclonal antibody preparation against candidate tumour suppressor protein MIP for detection of its expression and localization in hepatocellular carcinoma

ABSTRACTMIP is a candidate tumour suppressor protein found in hepatocellular carcinoma. It can interact with different proteins in different subcellular locations. However, antibodies against MIP for testing purposes are still not available and this has hindered the research on MIP protein. In this study, the full coding sequence of MIP protein was cloned into the prokaryotic expression vector pET-28a. The recombinant protein his-MIP was expressed by IPTG (isopropyl β-D-1-thiogalactopyranoside) induction and purified by nickel-affinity chromatography, then used to immunize New Zealand rabbits to obtain a polyclonal antibody against MIP. The sensitivity and specificity of the antibody were evaluated by enzyme-linked immunosorbent assay and western blot. We used the obtained antibody to detect endogenous MIP protein in hepatocellular carcinoma (HCC) cells and normal human hepatic cells L-O2 and found decreased MIP expression in HCC cells. The immunohistochemistry results further showed that the expression level of MIP protein in HCC tissues was lower than that in the corresponding adjacent non-cancerous tissues. The MIP protein was distributed mainly in the cytoplasm in HCC. In adjacent tissues, however, high expression of MIP was detected in the nucleus in addition to the cytoplasm. The prepared polyclonal antibody against MIP provides a useful tool for further research on the functions of MIP.

O32 Curcumin induces expression of 15-hydroxyprostaglandin dehydrogenase in normal gastric mucosa RGM-1 cells and mouse stomach in vivo: A potential role of AP-1

Overproduction of prostaglandin E 2 (PGE 2 ) has been reported to be implicated in carcinogenesis. The intracellular level of PGE 2 is regulated not only by its biosynthesis, but also by the degradation process. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme that catalyzes the inactivation of PGE 2 . In the present study, we found that curcumin, a yellow coloring agent present in the rhizome of Curcuma longa Linn (Zingiberaceae), induced expression of 15-PGDH at both the protein and mRNA levels in normal rat gastric mucosa cells (RGM-1). By using deletion constructs of the 15-PGDH promoter, we have found that activator protein-1 (AP-1) is the most essential transcription factor responsible for curcumin-induced upregulation of 15-PGDH expression. In addition, oral administration of curcumin increased the expression of 15-PGDH in the mouse stomach. Curcumin enhanced the expression of c-Jun and c-Fos in the nuclear fraction of RGM-1 cells and the mouse stomach in vivo . Knockdown of c- jun , a gene encoding one of the major components of AP-1, suppressed curcumin-induced expression of 15-PGDH. In addition, curcumin increased phosphorylation of ERK1/2 and JNK. Curcumin-induced 15-PGDH expression was abrogated by the pharmacological inhibition of the aforementioned kinases. In contrast, tetrahydrocurcumin which lacks the α , β -unsaturated carbonyl group failed to induce expression of 15-PGDH, suggesting that the electrophilic α , β -unsaturated carbonyl group of curcumin is essential for its induction of 15-PGDH expression in RGM-1 cells. Taken together, our study suggests that curcumin-induced upregulation of 15-PGDH in RGM-1 cells and mouse stomach through activation of AP-1 may contribute to chemopreventive effects of this phytochemical on inflammation-associated gastric carcinogenesis.