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1.
Indian J Exp Biol ; 2014 Oct; 52(10): 943-951
Article in English | IMSEAR | ID: sea-153783

ABSTRACT

The anti proliferative potential of siRNA26, targeted to Aurora kinase B, in prostate cancer cells is known from a previous study from our laboratory. Here we first show that siRNA26 cleaves at the same position of the target mRNA in the prostate cancer and hepatocellular carcinoma cell lines, PC3 and HepG2 respectively. Aurorakinase B specific siRNA, but not a control siRNA, inhibited PC3 and HepG2 cell proliferation and cell migration. These effects correlated to RNA silencing of Aurorakinase B in both the cell lines. Intra-tumoral administration of HiPerfect complexed siRNA26 inhibited the growth of HepG2 xenografts in SCID mice. In an orthotopic setting, intravenous administration of HiPerfect encapsulated siRNA26 appeared to reduce the severity of multifocal lesions.


Subject(s)
Animals , Antineoplastic Agents/pharmacology , Aurora Kinase B/genetics , Aurora Kinase B/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Hep G2 Cells , Humans , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/therapy , Male , Mice , Mice, SCID , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/therapy , RNA Interference , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA, Small Interfering/pharmacology , Transfection , Xenograft Model Antitumor Assays
2.
Article in English | WPRIM | ID: wpr-13096

ABSTRACT

Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are capable of differentiating into several lineages and possess immunomodulatory properties. In this study, we investigated the soluble factor-mediated immunomodulatory effects of hAM-MSCs. Mitogen-induced peripheral blood mononuclear cell (PBMC) proliferation was suppressed by hAM-MSCs in a dose-dependent manner as well as hAM-MSC culture supernatant. Moreover, interferon-gamma and interleukin (IL)-17 production significantly decreased from PBMC, whereas IL-10 from PBMCs and transforming growth factor beta (TGF-beta) production from hAM-MSCs significantly increased in co-cultures of hAM-MSCs and PBMCs. Production of several MSC factors, including hepatocyte growth factor (HGF), TGF-beta, prostaglandin E2 (PGE2), and indoleamine 2, 3 dioxygenase (IDO), increased significantly in hAM-MSCs co-cultured with PBMCs. These results indicate that the immunomodulatory effects of hAM-MSCs may be associated with soluble factors (TGF-beta, HGF, PGE2, and IDO), suggesting that hAM-MSCs may have potential clinical use in regenerative medicine.


Subject(s)
Amnion/cytology , Cell Differentiation/immunology , Coculture Techniques , Dinoprostone/genetics , Female , Hepatocyte Growth Factor/genetics , Humans , Immunologic Factors/immunology , Immunophenotyping , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Interferon-gamma/immunology , Interleukin-10/analysis , Interleukin-17/analysis , Leukocytes, Mononuclear/cytology , Mesenchymal Stem Cells/cytology , Pregnancy , RNA, Messenger/chemistry , Regenerative Medicine/methods , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta/genetics
3.
Article in English | WPRIM | ID: wpr-160874

ABSTRACT

The anti-inflammatory effects of an ethanol extract of Angelica gigas (EAG) were investigated in vitro and in vivo using croton oil-induced inflammation models. Croton oil (20 microgram/mL) up-regulated mRNA expression of cyclooxygenase (COX)-I and COX-II in the macrophage cell line, RAW 264.7, resulting in the release of high concentrations of prostaglandin E2 (PGE2). EAG (1~10 microgram/mL) markedly suppressed croton oil-induced COX-II mRNA expression and PGE2 production. Application of croton oil (5% in acetone) to mouse ears caused severe local erythema, edema and vascular leakage, which were significantly attenuated by oral pre-treatment with EAG (50~500 mg/kg). Croton oil dramatically increased blood levels of interleukin (IL)-6 and PGE2 without affecting tumor-necrosis factor (TNF)-alpha and nitric oxide (NO) levels. EAG pre-treatment remarkably lowered IL-6 and PGE2, but did not alter TNF-alpha or NO concentrations. These results indicate that EAG attenuates inflammatory responses in part by blocking the COX-PGE2 pathway. Therefore, EAG could be a promising candidate for the treatment of inflammatory diseases.


Subject(s)
Angelica/immunology , Animals , Cell Line , Cyclooxygenase 1/genetics , Cyclooxygenase 2/genetics , Dinoprostone/genetics , Inflammation/drug therapy , Interleukin-6/blood , Macrophages , Male , Mice , Mice, Inbred ICR , Nitric Oxide/blood , Phytotherapy/methods , Plant Extracts/pharmacology , Plant Roots/immunology , RNA, Messenger/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/blood
4.
Electron. j. biotechnol ; 12(4): 11-12, Oct. 2009. ilus, tab
Article in English | LILACS | ID: lil-558554

ABSTRACT

The systematic study of the genetic fingerprint (genomics) and the biochemistry (metabolites) that goes with a specific cellular process requires the characterization of all the small molecules that form the profile of metabolites and the associated genes. The metabolome represents the collection of all the metabolites during certain process in an organism. The transcriptome represents the gene expression profile, all the messengers RNA in a defined condition. Then to understand the whole process, the studies of metabolites must be accompanied with studies of the gene expression, hence the metabolome must be accompanied by the transcriptome, so we can identify genes and metabolites whose synthesis is induced by a specific process, an infection or stress. Studies of metabolomics generate an enormous amount of data, then they need mathematical and computational tools to establish the correlations between the biochemical and genetic data, and to build up networks that represent the complex metabolic interactions that occur in each case, using tools like Graph and Networks Theory to elucidate the emergent properties inherent to the complex interactions of the metabolic maps. This paper describes the major mathematical tools that can be used for these studies, with emphasis on a semi-qualitative proposal known as the kinetic structural model.


Subject(s)
Humans , Models, Genetic , Metabolism/genetics , /methods , Metabolic Networks and Pathways/genetics , RNA, Messenger/genetics , RNA, Messenger/chemistry , DNA Fingerprinting/methods , /methods
5.
Braz. j. med. biol. res ; 40(11): 1545-1555, Nov. 2007. ilus, tab
Article in English | LILACS | ID: lil-464302

ABSTRACT

Agaricus blazei Murill is a native Brazilian mushroom which functions primarily as an anticancer substance in transplanted mouse tumors. However, the mechanism underlying this function of A. blazei Murill remains obscure. The present study was carried out to investigate the effect of fraction FA-2-b-ß, an RNA-protein complex isolated from A. blazei Murill, on human leukemia HL-60 cells in vitro. Typical apoptotic characteristics were determined by morphological methods using DNA agarose gel electrophoresis and flow cytometry. The growth suppressive effect of fraction FA-2-b-ß on HL-60 cells in vitro occurred in a dose- (5-80 mug/mL) and time-dependent (24-96 h) manner. The proliferation of HL-60 cells (1 x 10(5) cells/mL) treated with 40 mug/mL of fraction FA-2-b-ß for 24-96 h and with 5-80 mug/mL for 96 h resulted in inhibitory rates ranging from 8 to 54.5 percent, and from 4.9 to 86.3 percent, respectively. Both telomerase activity determined by TRAP-ELISA and mRNA expression of the caspase-3 gene detected by RT-PCR were increased in HL-60 cells during fraction FA-2-b-ß treatment. The rate of apoptosis correlated negatively with the decrease of telomerase activity (r = 0.926, P < 0.05), but correlated positively with caspase-3 mRNA expression (r = 0.926, P < 0.05). These data show that fraction FA-2-b-ß can induce HL-60 cell apoptosis and that the combined effect of down-regulation of telomerase activity and up-regulation of mRNA expression of the caspase-3 gene could be the primary mechanism of induction of apoptosis. These findings provide strong evidence that fraction FA-2-b-ß could be of interest for the clinical treatment of acute leukemia.


Subject(s)
Humans , Agaricus/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , RNA, Fungal/chemistry , RNA-Binding Proteins/pharmacology , Antineoplastic Agents/isolation & purification , /analysis , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Electrophoresis, Agar Gel , /drug effects , Microscopy, Electron, Transmission , Reverse Transcriptase Polymerase Chain Reaction , RNA, Fungal/isolation & purification , RNA, Messenger/chemistry , RNA-Binding Proteins/isolation & purification , Time Factors , Telomerase/analysis
6.
J Genet ; 2007 Aug; 86(2): 103-9
Article in English | IMSEAR | ID: sea-114293

ABSTRACT

There is a growing evidence, that antisense transcription might have a key role in a range of human diseases. Although predefined sense-antisense pairs were extensively studied, the antisense expression of the known sense genes is rarely investigated. We retrieved and correlated the expression of sense and antisense sequences of 1182 mouse transcripts to assess the prevalence and to find the characteristic pattern of antisense transcription. We contrasted three Affymetrix MGU74A version 1 mouse genome chips to six MGU74A version 2 chips. For these 1182 transcripts, the version 1 chips contain the antisense sequences of the transcripts presented on the version 2 chips. The original data was taken from the GEO database (GDS431 and GDS432). As the Affymetrix data are semiquantitative, the relative expression levels of antisense partners were analysed. We detected antisense transcription, although the average antisense expression is shifted towards smaller expression values (MGU74A version 1, 516; version 2, 1688). An inverse direct correlation between sense and antisense expression values could be observed at high expression values. At a very high relative expression--above 40,000--the Pearson correlation coefficient is getting closer to -1. Transcripts with high inverse expression ratio may be correlated to the investigated gene (major histocompatibility complex class II trans activator). The ratio of sense to antisense transcripts varied among different chromosomes; on chromosomes 14 and 1 the level of antisense expression was higher than that of sense. We conclude that antisense transcription is a common phenomenon in the mouse genome. The hypothesis of regulatory role of antisense transcripts is supported by the inverse antisense gene expression of highly expressed genes.


Subject(s)
Animals , DNA, Antisense/genetics , Gene Expression Regulation , Mice , RNA Interference/physiology , RNA, Messenger/chemistry , Transcription, Genetic
7.
Indian J Biochem Biophys ; 2005 Dec; 42(6): 350-7
Article in English | IMSEAR | ID: sea-28829

ABSTRACT

Ribosome display was applied in vitro to select single-chain variable fragment (scFv) antibody specific for digoxin from a human non-immune naive scFv library. A cell-free system was used to produce stable antibody-ribosome-mRNA (ARM) complexes to provide the linkage of genotype and phenotype, allowing simultaneous selection of a desired antibody together with its encoding mRNA. The mRNA was then recovered and amplified as DNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Repeating the display cycle enriched the selected molecules, enabling rare species to be isolated. In this study, digoxin-binding segments were selected over four cycles of ARM display and the selected DNA was cloned and expressed as a single-chain variable fragment antibody (the best scFv, A3) in Escherichia coli. The affinity (equilibrium dissociation constant Kd) of digoxin was 8.3 x 10(-8) M for A3, which validated construction of the naïve library and the power of ribosome display lending to the evolution of functional characteristics, such as potency of leading candidate antibodies to provide therapeutic antibodies. A3 was purified using affinity chromatography and determined by Western blot. The results indicate that ribosome display technnique can be efficiently used to isolate specific antibody fragments from a naive library.


Subject(s)
Amino Acid Sequence , Antibody Affinity , Base Sequence , Blotting, Western , Chromatography, Affinity , Cloning, Molecular , Digoxin/immunology , Humans , Immunoglobulin Variable Region/genetics , Molecular Sequence Data , Peptide Library , RNA, Messenger/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Ribosomes
8.
Biol. Res ; 38(2/3): 267-271, 2005. tab, graf
Article in English | LILACS | ID: lil-424730

ABSTRACT

Matrix metalloproteinases (MMPs) comprise a large group of endoproteinases that degrade all protein components of the extracellular matrix. Functionally, MMPs contribute to several different physiological as well as pathological conditions. The number of newly described MMPs has increased in recent years, although current knowledge about their expression pattern in various tissues remains incomplete. Here we analyzed the relative mRNA expression of the most recently described MMPs _ MT5-MMP (MMP-24), MT6-MMP (MMP-25), MMP-27 and epilysin (MMP-28) _ in a broad selection of rat tissues using real time-PCR. MMP-24 mRNA was found to be widely expressed with predominance in the central nervous system. MMP-25 mRNA, in contrast, exhibited peak expression levels in testis, kidney and skeletal muscle, differing from previously described distribution patterns in humans. mRNAs for MMP-27 and MMP-28 were generally expressed at a lower level. All four MMPs studied were detected at higher mRNA levels in bone and kidney, suggesting a possible role of these MMPs in physiological processes within these two organs. The present study highlights the differential distribution pattern of newly described MMPs among different tissues and underlines differences in the mRNA expression between different species.


Subject(s)
Adult , Animals , Infant, Newborn , Rats , Matrix Metalloproteinases/analysis , Matrix Metalloproteinases/biosynthesis , Matrix Metalloproteinases/genetics , Matrix Metalloproteinases/metabolism , Matrix Metalloproteinases/chemistry , RNA, Messenger/isolation & purification , RNA, Messenger/analysis , RNA, Messenger/chemistry , Kidney/chemistry , Testis/chemistry
9.
Article in English | WPRIM | ID: wpr-178957

ABSTRACT

Accurate estimation of the exposure-response relationship between ambient urban particulate matters (PM) and public health is important for regulatory perspective of ambient urban particulate matters (PM). Ambient PM contains various transition metals and organic compounds. PM10 (aerodynamic diameter less than 10 microgram) is known to induce diverse diseases such as chronic cough, bronchitis, chest illness, etc. However, recent evaluation of PM2.5 (aerodynamic diameter less than 2.5 microgram) against health outcomes has suggested that the fine particles may be more closely associated with adverse respiratory health effects than particles of larger size. This study was performed to evaluate PM2.5-induced oxidative stress in rat lung epithelial cell in order to provide basic data for the risk assessment of PM2.5. PM2.5 showed higher cytotoxicity than PM10. Also, PM 2.5 induced more malondialdehyde (MDA) formation than PM10. In Hoechst 33258 dye staining and DNA fragmentation assay, apopotic changes were clearly detected in PM2.5 treated cells in compared to PM10. Expression of catalase mRNA was increased by PM2.5 rather than PM10. PM2.5 induced higher Mth1 mRNA than PM10. In pBR322 DNA treated with PM2.5, production of single strand breakage of DNA was higher than that of PM10. In Western blot analysis, PM2.5 induced more Nrf-2 protein, associated with diverse transcriptional and anti-oxidative stress enzymes, compared to PM10. Our data suggest that PM2.5 rather than PM10 may be responsible for PM-induced toxicity. Additional efforts are needed to establish the environmental standard of PM2.5.


Subject(s)
Air Pollutants/chemistry , Animals , Apoptosis/physiology , Benzimidazoles/metabolism , Blotting, Western , Cell Line , Cell Survival/physiology , DNA Fragmentation/physiology , DNA Repair Enzymes/genetics , DNA-Binding Proteins/metabolism , Epithelial Cells/drug effects , Formazans/metabolism , GA-Binding Protein Transcription Factor , Lipid Peroxides/metabolism , Lung Diseases/chemically induced , Oxidative Stress/physiology , RNA, Messenger/chemistry , Rats , Reverse Transcriptase Polymerase Chain Reaction , Tetrazolium Salts/metabolism , Transcription Factors/metabolism
10.
Article in English | WPRIM | ID: wpr-145919

ABSTRACT

Hepatitis C virus (HCV) is a major causative agent in liver disease. In order to investigate if Korean type HCV core protein and its related mutants, S99Q and S116I, are cytopathic to liver, three types of transgenic mice were established. The expression of transgenes was confirmed by HCV specific RT-PCR and Western immunoblotting. The livers of all wild type core and S116I transgenic lineages remained largely histologically normal. However, the livers of the S99Q transgenic mice showed significant high level of cell dysplasia associated with the transgene expression in hepatocytes largely located around the central veins by in situ hybridization analysis. In conclusion, the mutant HCV core protein at S99Q may contribute to the progress of HCV induced liver disease.


Subject(s)
Amino Acid Sequence , Animals , Base Sequence , Gene Expression , Genetic Vectors/genetics , Hepatitis C/pathology , Hepatitis, Viral, Animal/pathology , Hepatocytes/pathology , Liver/pathology , Mice , Mice, Transgenic , Molecular Sequence Data , Mutation/genetics , RNA, Messenger/chemistry , Transgenes , Viral Core Proteins/analysis
11.
Indian J Exp Biol ; 1997 Sep; 35(9): 1011-3
Article in English | IMSEAR | ID: sea-62744

ABSTRACT

Polypeptides synthesized from polysome-bound and unbound fractions of polyA mRNA were studied by in-vivo cells labelling and cDNA-mRNA hybrid-arrest translation. The polypeptides synthesized from unbound(free) fraction appeared to be significantly reduced in the absence of cycloheximide in L6 myoblast. The cDNA excess-mRNA hybrid-arrest translation exhibited rare polypeptide sequences in the unbound fractions.


Subject(s)
Animals , Chemical Fractionation , Muscles/metabolism , Peptides/analysis , Polyribosomes/chemistry , RNA, Messenger/chemistry , Rats , Ribonucleoproteins/metabolism
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