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PubMed2004

[Quantitative analysis of sigma genes expression in Mycobacterium tuberculosis cultures exposed to rifampicin and isoniazid].

The aim of the study was quantitative analysis of five genes encoding Mycobacterium tuberculosis sigma factors sigA, sigE, sigF, sigH, and sigI as well as the 85B reference gene known as the mycobacterial viability marker, in cultures exposed to rifampicin and isoniazid. The mRN levels were assessed using QRT-PCR technique, in the automated system of real time quantification with the ABI PRISM 7700 Sequence Detector System (TaqMan). The number of each analyzed gene transcript copies was expressed as a number of mRNA per 1 eg of isolated total RNA. In cultures exposed to the tested chemicals the number of 85B mRNA copies declined as compared to the controls (without tested chemicals). There was no detectable expression of sigA and sigI in the control cultures. Both, rifampicin and isoniazid induced expression of sigA and sigI genes. The sigE gene expression increased during exposure to isoniazid and decreased under rifampicin exposure conditions. The sigF mRNA was detected neither in the control culture, nor in cultures exposed to rifampicin or isoniazid. Both tested chemicals caused decrease of sigH expression.

Antibiotics, Antitubercular↗

PubMed2005

Recreational music-making modulates the human stress response: a preliminary individualized gene expression strategy.

BACKGROUND: A central component of the complex human biological stress response is the modulation of the neuro-endocrine-immune system with its intricate feedback loops that support homeostatic regulation. Well-documented marked gene expression variability among human and animal subjects coupled with sample collection timing and delayed effects, as well as a host of molecular detection challenges renders the quest for deciphering the human biological stress response challenging from many perspectives. MATERIAL/METHODS: A novel Recreational Music-Making (RMM) program was used in combination with a new strategy for peripheral blood gene expression analysis to assess individualized genomic stress induction signatures. The expression of 45 immune response-related genes was determined using a multiplex preamplification step prior to conventional quantitative Real Time Polymerase Chain Reaction (qRT-PCR) mRNA analysis to characterize the multidimensional biological impact of a 2-phase controlled stress induction/amelioration experimental protocol in 32 randomly assigned individuals. RESULTS: In subjects performing the RMM activity following a 1-hour stress induction protocol, 19 out of 45 markers demonstrated reversal with significant (P = 0.05) Pearson correlations in contrast to 6 out of 45 markers in the resting control group and 0 out of 45 in the ongoing stressor group. CONCLUSIONS: The resultant amelioration of stress-induced genomic expression supports the underlying premise that RMM warrants additional consideration as a rational choice within our armamentarium of stress reduction strategies. Modulation of individualized genomic stress induction signatures in peripheral blood presents a new opportunity for elucidating the dynamics of the human stress response.

Adolescent↗

PubMed2005

[Biological effects of nano-hydroxyapatite/polyamide 66 on the dental pulp cells].

OBJECTIVE: To investigate the biological effects of the new nano-hydroxyapatite/polyamide 66 biological composites (nHA-PA66) on the dental pulp cells. METHODS: After interaction with the nHA-PA66 eluate, the growth, proliferation, and function of the in vitro cultured human dental pulp cells were studied by cell culture technique, inverted phase-contrast microscope observation, MTT assay, flow cytometry, ALP activity assay and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) analysis. RESULTS: The cultured pulp cells grew well and showed no morphological variation. Moreover, this material had no negative effects on the proliferation, cell cycle, ALP activity and the expression of dentin sialophosphoprotein (DSPP) mRNA of the pulp cells. CONCLUSION: As a new nano-biomaterial, nHA-PA66 has good biocompatibility to the pulp cells, but no obvious bioactivity.

Biocompatible Materials↗

PubMed2004

[Overexpression of thymosin beta4 in the cochlea of senescence-accelerated mouse].

OBJECTIVE: To develop gene expression profiles of young and old senescence-accelerated mouse (SAM) cochlea and identify genes responsible for aging-related hearing loss. METHODS: Gene micro-array slides containing 1101 mouse genes were hybridized to cDNA micro-arrays (Atlas Glass Array Mouse 1.0) that were synthesized using total RNAs from the cochlea of 2 mounts and 12 mounts mouse. Hybridization signals were visualized with cyanine-3 fluorescent reporter molecules, and the fluorescence intensities of the images were analyzed. Real-time quantitative reverse transcription PCR (qRT-PCR) was performed to validate the micro-array results. Immunofluorescence was used to identify the located region of the protein encoding by the candidate gene in the cochlea. RESULTS: Expression of a majority of the 1101 genes represented on the micro-array slides was not altered during aging; nonetheless, changes in the expression of 3 genes were detected between young and old mouse cochlea. RT-PCR results confirmed the changes in expression of thymosin beta4 of 3 genes examined. Through the using of immunofluorescence, it was shown that thymosin beta4 was primarily located in the tectorial membrane and the supporting cells of outer hair cell. CONCLUSIONS: Using commercially available slide micro-arrays, the results show that aging of the mouse cochlea is associated with changes in patterns of gene expression. This analysis suggests that thymosin beta4 may play a role in aging-related hearing loss. These studies lay the foundation for future studies defining the genetic basis of aging-related hearing loss.

Aging↗

PubMed2005

[Effect of ERK1/2 on low shear stress-induced expression of IL-8 mRNA in human endothelial cells].

Fluid shear stress plays an important role in many physiological and pathophysiological processes of the cardiovascular system. It modulates vascular function and structure via stimulating mechanosensitive endothelial cell signal events. Previous studies have identified that the exposure of vascular endothelial cells to fluid mechanical forces can modulate the expressions of many genes, including IL-8 gene. In order to gain an insight into the role of extracellular signal regulated kinase (ERK1/2) signal pathway in the expression of IL-8 mRNA in human umbilical vein endothelial cells (HUVECs) under the stimulation by low shear stress (4.20 dyne/cm2), we employed Western blot to measure phosphorylation of ERK1/2 and used quantitative reversal transcription-polymerase chain reaction (qRT-PCR) to assay the expression of IL-8 mRNA. The results showed: (1) Shear stress could activate ERK1/2 with a rapid, biphasic time course (maximum by 10 min and basal by 2 h); the treatment of HUVECs with Genistein (a highly specific inhibitor of tyrosine protein kinase, TPK) or PD98059 (the inhibitor of mitogen-activated protein/extracellular signal regulated kinase kinase, MEK) culd prevent shear-dependent activation of ERK1/2; (2) When treated with Genistein or PD98059, significant inhibition of IL-8 mRNA expression induced by low shear stress was observed in HUVECs. This in vitro study demonstrates that ERK1/2 plays an important role in IL-8 mRNA expression induced by low shear stress.

Cells, Cultured↗

PubMed2005

Effect of mosquito midgut trypsin activity on dengue-2 virus infection and dissemination in Aedes aegypti.

The effect of mosquito midgut trypsins in dengue serotype 2 flavivirus (DENV-2) infectivity to Aedes aegypti was studied. Addition of soybean trypsin inhibitor (STI) in a DENV-2 infectious blood meal resulted in a 91-97% decrease in midgut DENV-2 RNA copies (qRT-PCR analysis). STI treatment also resulted in slower DENV-2 replication in the midgut, less DENV-2 E protein expression, and decreased dissemination to the thorax and the head. A second uninfected blood meal, 7 days after the STI-treated infectious meal, significantly increased DENV-2 replication in the midgut and recovered oogenesis, suggesting that the lower viral infection caused by STI was in part due to a nutritional effect. Mosquitoes fed DENV-2 digested in vitro with bovine trypsin (before STI addition) exhibited a transient increase in midgut DENV-2 4 days postinfection. Blood digestion and possibly DENV-2 proteolytic processing, mediated by midgut trypsins, influence the rate of DENV-2 infection, replication, and dissemination in Ae. aegypti.

Aedes↗

PubMed2005

[Biological effects of nano-hydroxyapatite/polyamide 66 on the osteoblast].

OBJECTIVE: To evaluate the biological effects of nano-hydroxyapatite/polyamide 66(nHA-PA66) on the growth and activity of osteoblast. METHODS: MTT assay was used to determine the growth of osteoblast, enzymatic measure was used to determine the activity of ALP and quantitative RT-PCR (QRT-PCR) to evaluate the changes of osteoclacin mRNA expression in osteoblasts treated by DMEM eluate of nHA-PA66. RESULTS: Osteoblasts of different test groups demonstrated relative proliferation rate ranging from 98% - 106% without dose-dependent effect. The ALP activity and osteocalcin mRNA expression were similar in test and control groups (P > 0.05). CONCLUSION: nHA-PA66 has no negative effects on the osteoblast and its osteoblast-compatibility is proved.

Durapatite↗

PubMed2005

Galectin-9 as a regulator of cellular adhesion in human oral squamous cell carcinoma cell lines.

Oral squamous cell carcinomas (OSCCs), a major public health problem worldwide, are the most common neoplasms of the head and neck. The most important prognostic indicator for patients with OSCC is metastasis to cervical lymph nodes or distant organs. Galectin-9 is correlated with cellular adhesion and aggregation in melanoma cells. To investigate expression levels of galectin-9 mRNA and protein, we performed qRT-PCR and Western blot analyses on OSCC cell lines (Ca9-22, HSC-2, and HSC-3) and normal oral keratinocytes (NOKs). Galectin-9 mRNA and protein were commonly down-regulated in OSCC cell lines compared with NOKs. We further analyzed Ca9-22, which had the lowest expression of galectin-9. We then transfected the galectin-9 cDNA into Ca9-22 cells to examine whether overexpression of galectin-9 increases cellular adhesion in vitro. An adhesion assay using a fibronectin and collagen I coating plate revealed an increased cellular adherence ratio in overexpressed galectin-9 cells compared with nontransfected cells (p < 0.05). The data suggest that galectin-9 is correlated with oral cancer cell-matrix interactions and may therefore play an important role in the metastasis of OSCCs.

Blotting, Western↗

PubMed2005

[Intestinal tissue kallikrein-kinin system in patients with ulcerative colitis during treatment with cyclosporine A].

Kinins are mediators of inflammation and proliferation and act by activating their specific B1 and B2 receptors. In the present study we evaluate the tissue kallikrein-kinin system during cyclosporine A treatment in patients with ulcerative colitis (UC). Six severe active UC patients were treated with cyclosporine A, i.v. 4 mg/kg/d by 7 days with clinical improvement. During sigmoidoscopy biopsy specimens were taken in 0, 5, 7 day of the treatment. The genes expression for tissue kallikrein, kallistatin, B1 and B2 receptors were evaluated by QRT-PCR analysis using TaqMan detector. There was no statistically significant difference in the expression of these genes during treatment with cyclosporine A. In addition, genes encoding kallistatin as well as B1 and B2 receptors were investigated in CaCo cells cultures after exposure with cyclosporine A (200-800 ng/ml) in vitro. In vitro mRNA for B1 receptors was significantly higher using therapeutic (200-400 ng/ml) concentration of cyclosporine A, whereas expression of B2 receptor and kallistatin increased after exceeding therapeutic concentration (400-800 ng/ml).Thus, treatment of UC with cyclosporine A seems to be safe in relation to tissue kallikrein-kinin system profile.

Caco-2 Cells↗

PubMed2005

[Detection of RbAp46 expression in bone marrow cells of leukemia patients by real-time quantitative RT-PCR].

OBJECTIVE: To investigate retinoblastoma (Rb) associated protein 46 (RbAp46) gene expression levels in bone marrow (BM) cells of leukemia patients. METHODS: Real-time quantitative reverse polymerase chain reaction (QRT-PCR) method was used for detecting RbAp46 expression levels in BM cells of 140 patients with acute leukemia (AL), 13 with chronic myelogenous leukemia in chronic phase (CML-CP), 7 with CML in blast crisis (CML-BC) and 32 with non-leukemic disorders. RESULTS: The M-Estimators of RbAp46 were higher in 98 newly diagnosed ALs and 5 relapsed ALs than in 28 ALs in complete remission (CR) and 32 non-leukemic controls (178.23 and 213.65 vs 85.89 and 88.08, respectively). No statistic difference was found between the CR group and control group, or between the newly diagnosed group and relapsed group. The M-Estimators of RbAp46 in patients with CML-CP was 58.27, similar to that in control, but much lower than that in CML-BC (173.24). Among 98 newly diagnosed ALs, the M-Estimators of RbAp46 in M(3) and M(4) were the lowest in all of the subtypes. Furthermore, the RbAp46 expression levels were not correlated with the expression of the fusion genes of bcr/abl, PML-RARalpha, and multidrug resistant gene (mdr1), but were positively correlated with Wilms' tumor gene (WT1) expression levels and negatively with AML1/ETO fusion gene expression. CONCLUSION: RbAp46 expression levels in ALs and CML-BC were strikingly higher than that in non-leukemias and CML-CP, and might participate in leukemogenesis.

Adolescent↗

PubMed2006

Gene expression profile of bovine bone marrow mesenchymal stem cell during spontaneous chondrogenic differentiation in pellet culture system.

Bovine bone marrow mesenchymal stem cells (MSCs) cultured in condensate culture, spontaneous and independent for any external biostimulants, undergo chondrogenic differentiation. In the present study, the bovine MSC chondrogenesis pathway was studied by analyzing stage-specific gene expression using quantitative "Real Time" reverse transcriptase polymerase chain reaction (qRT-PCR). Results showed that bovine MSCs underwent complete chondrogenesis; the initial stage was characterized by expression. of sox9 messenger ribonucleic acid (mRNA), followed by high transcription of chondrocyte specific genes, collagen type II and IX, biglycan and cartilage oligomeric matrix protein, and the final prehypertrophic and/or hypertrophic stage was distinguished by increased expression of collagen type X. From day 7 to day 14 of differentiation increased mRNA expression of the transforming growth factors beta1 and beta2, basic fibroblast growth factor (FGF 2), bone morphogenic protein 6 (BMP 6), insulin-like growth factors 1, parathyroid hormone related peptide and indian hedgehog (Ihh) were detected. These results suggest that these well know chondrogenic growth factors may play a role in bovine chondrogenesis in autocrine and/or paracrine manner. On day 21 of the culture, FGF 2, BMP 6 and Ihh were highly expressed, compared to cells cultured in monolayer manner, which suggests a possible function in maintaining the terminal stage of differentiation. This data extends our knowledge about the unusual species-specific bovine MSC chondrogenesis, allowing us to define the phenotype of the differentiated cells. Furthermore, this study contributes to our in understanding of known chondrogenic-growth factors in autocrine and/or paracrine manner playing a role in the spontaneous differentiation.

Animals↗

PubMedDate not supplied

Study of housekeeping gene expression in human keratinocytes using OLISA, a long-oligonucleotide microarray and q RT-PCR.

In recent years, applications of microarray platforms have been extended to different areas of research including cosmetic and pharmaceutical. Although microarray technology is still improving its sensitivity and flexibility, researchers often turn toward quantitative RT-PCR for data validation. Assessment of messenger RNA quantity by these methods is based on comparison with internal standard genes, mainly housekeeping genes, so called because their synthesis occurs normally at a constant level. However, numerous studies showed that expression of these genes could vary in given situations. Here, we report results on four housekeeping genes (GAPDH, beta-2 microglobulin, S40 and S26 ribosomal sub-units) with constant expression levels established on OLISA microarray using different keratinocyte cultures. Moreover, qRT-PCR validation demonstrates that S26 ribosomal is a good housekeeping gene on keratinocytes and skin studies. Our data indicate that S26 gene can be routinely used to standardize results to investigate differentially expressed genes in a healthy human skin.

Cells, Cultured↗

PubMed2005

[Quantitative assessment of mRNA TGF-beta1 in liver tissue in connection with serum mean daily level of TGF-1 in chronic hepatitis B patient].

Monitoring of fibrosis process with the use of histopathologic studies on liver's bioptates is limited, so it is attempted to find reliable, obtained with less invasive methods, sensitive and reflecting fibrosis dynamics markers of this process. The aim of the study was simultaneously to assess liver's expression as well as circadian and mean daily TGF-betal concentration in serum of patients with chronic hepatitis type B in comparison to control group. Studies were performed on 50 patients (9 women, 41 men) aged 45.9 +/- 2.3 years with chronic hepatitis type B. Control group consisted of 20 patients (mean age 38.6 +/- 3.7 years), in which so called minimal changes without fibrosis were observed in histophatologic bioptate of liver. Blood for studies was collected every 4 houres during the day. Serum concentration of TGF-betal was assessed with the use of ELISA method, and expression of mRNA TGF-betal in liver with QRT-PCR method. No significant difference between circadian as well as mean daily serum TGF-betal concentration beetwen control group and the group with chronic hepatitis type B was shown. Increased expression of mRNA, TGF-betal in bioptate of liver of patients with chronic hepatitis type B in comparison to control group was noted. In "minimal changes" control group presence of significant positive correlation between expression of mRNA TGF-beta1 in liver and concentration of this cytokine in serum was shown, in the group of patients with chronic hepatitis B this connection was not noted.

Circadian Rhythm↗

PubMed2006

The impact of hyperthermic chemotherapy on human gastric cancer cell lines: preliminary results.

In this preliminary study, we evaluated the impact of hyperthermia (HT) and hyperthermic chemotherapy (HTCT) on six human gastric cancer cell lines and explored the mechanisms of cell-killing effect under HTCT. Treatment conditions were categorized into 4 modes: i) normothermic control (NT), ii) HT, iii) normothermic chemotherapy (NTCT) and iv) HTCT. According to the data of MTT and LM observations, isolated HT only temporarily inhibited cell proliferation and had no cell-killing effect on gastric cancer cell lines employed in our study except for SNU-1. Combining with HT enhanced the cytotoxicity of CDDP in all gastric cell lines and the concentration inhibiting cell proliferation and inducing cell death of HTCT was much lower than that of NTCT. There was a synergistic effect of HT and chemotherapy on inhibiting proliferation in each cell line in a certain range of CDDP concentration. The data of TEM and FCM proved that HTCT induced cell death with two modes - apoptosis and necrosis, and apoptosis was the major type. Microarray illustrated that, under HTCT, a total of 58 gene expressions were regulated according to the filtering criteria, including 10 extra genes with an expression change below the threshold or even unchanged when treated with either HT or CDDP alone. Five of these 10 genes were verified by QRT-PCR. These genes may include the target genes for the enhancing effect of HT on chemotherapy and their effects should be further validated by functional analysis.

Antineoplastic Agents↗

PubMed2006

Transpupillary thermotherapy-induced modification of angiogenesis- and coagulation-related gene expression in the rat posterior fundus.

PURPOSE: To study gene expression changes in the rat retina and choroid following transpupillary thermotherapy (TTT) and to identify molecular mechanisms that may enhance treatment of choroidal neovascularization, complicating age-related macular degeneration. METHODS: One fundus of Brown Norway rats was treated with an 810 nm diode laser while the contralateral fundus received no treatment. The mRNA was extracted and processed for cDNA microarray analysis. Genes with increased expression were validated by semiquantitative reverse transcription polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR). RESULTS: Of the 14,815 cDNA elements on the array, 12 genes were up-regulated in TTT treated eyes. Upregulation of eight of these 12 genes could be verified by semiquantitative RT-PCR. The eight verified genes were EPCR, IL-1beta, MCP-1, TSP-1, Fgl, Asns, MT-2, and NMDMC, which included 4 angiogenesis- and coagulation-related genes. CONCLUSIONS: This study demonstrates upregulation of angiogenesis- and coagulation-related genes following TTT. The response profile and its temporal relationships provide insight into the molecular mechanisms that lead to vascular occlusion and antiangiogenesis induced by TTT.

Animals↗

PubMed2025

Studying the Role of HOX Genes in Thrombocyte Development.

In our laboratory, we study thrombopoiesis and hemostasis using zebrafish as a model organism to unravel the mechanisms of differentiation and development of thrombocytes. We have shown in our earlier work that thrombocytes are functional equivalents of platelets and have transcriptional machinery similar to megakaryocytes. We recently found evidence that hox genes play a role in their development. We used piggyback gene knockdown and thrombocyte quantification assays to understand the influence of these ancient developmental regulators on thrombopoiesis. In this chapter, we describe methods used to discover these hox genes.

Animals↗

PubMed2026

Integrated metabolomics, transcriptional, and physicochemical analysis reveals key metabolites and genes associated with somatic embryogenesis in Phyllostachys pubescens.

Phyllostachys pubescens (Moso bamboo) is a significant perennial crop species that provides valuable nutritional and industrial uses, as well as carbon sequestration. Due to its remarkable growth rate, bamboo offers an ideal system for studying organogenesis, particularly in monocots. Somatic embryogenesis (SE) serves as a useful technique for crop breeding and improvement. SE in moso bamboo (Phyllostachys pubescens) remains challenging due to limited knowledge of its transcriptional and metabolomic reprogramming. To address this, we optimized callus initiation (MS + 18.1 µM 2,4-D + 8.5 µM picloram), callus proliferation (MS + 12.5 µM 2,4-D + 8.5 µM picloram), and somatic embryogenesis (MS + 1.1 µM 2,4-D + 3.3 µM metatopolin), using nodal segments as explants. UHPLC-Q-TOF-MS-based metabolite profiling revealed distinct biochemical trajectories across developmental stages of P. pubescens. NEC (non-embryogenic callus) was enriched in flavonoids, alkaloids, and saponins, while in-vitro shoots showed flavonoids and glycosides enrichment, and ex-vitro shoots showed high accumulation of glycosides and terpenoids. In contrast, EC (embryogenic callus) showed elevated levels of fatty acid derivatives (α-ESA, 26-Methyl Nigranoate), phytoalexins (Wyerone acid), sesquiterpene (Alpha-santalal, Beta-guaiene), flavonoid glycosides, and plant hormones (Cis-Zeatin, Gibberellin A45), indicating a metabolically active state supporting somatic embryogenesis. Similarly, genes and transcription factors controlling cell differentiation and embryogenesis were upregulated during SE. This study provides a comprehensive resource to facilitate future genomic and genetic investigations aimed at deciphering the molecular basis of organogenesis and advancing research on somatic embryogenesis in bamboo.

Plant Somatic Embryogenesis Techniques↗

PubMed2025

Genome-wide identification, characterization, and expression pattern analysis of the glyoxalase gene family in Phyllostachys pubescens during abiotic stresses.

BACKGROUND: The glyoxalase pathway comprising of three enzymes i.e., glyoxalase I (GLYI), glyoxalase II (GLYII), and glyoxalase III (GLYIII), which play vital role in mitigating abiotic stresses by detoxifying the stress induced cytotoxic metabolite methylglyoxal (MG). Phyllostachys pubescens an ecologically and economically important forest species, plays vital roles in carbon sequestration and climate change mitigation. A genome-wide study was conducted to identify and characterize GLYI, GLYII, and unique DJ-1/GLYIII gene candidates in P. pubescens. The identified members were evaluated based on phylogenetic analysis, gene structure, chromosomal distribution, gene duplication, presence of conserved domain(s) and cis regulatory region. RESULTS: A total of 19 GLYI, 18 GLYII, and 15 GLYIII members were identified, each featuring characteristic domains: glyoxalase, metallo-β-lactamase, and DJ-1/PfpI, respectively. The presence of different cis-elements in the promoter region of the glyoxalase genes gives insights into their role and regulation under hormonal response, developmental processes and stress adaptation. Besides this, stress responsive transcription factors binding sites also dominated the promoter regions of glyoxalase genes. Expression analysis of various glyoxalase genes demonstrated significant variability under different stress conditions, underscoring their potential roles in stress modulation. Significant upregulation of all of the PhGLYI, PhGLYII, and PhGLYIII were observed under cold, drought, heavy metal and salinity stress suggesting their involvement in oxidative stress management, osmotic regulation and remodelling cellular redox homeostasis. Among the glyoxalase genes, PhGLYI-15, PhGLYII-9, and PhGLYIII-3 showed consistent upregulation under various abiotic stresses. CONCLUSIONS: Our findings reveal that glyoxalase genes crucially contribute towards the improvement of cellular osmotic potential in moso bamboo under different abiotic stresses. This study enhances our understanding of glyoxalase genes' evolution and functional roles in plants and opens new avenues for developing stress resilient crop varieties for sustainable agriculture.

Lactoylglutathione Lyase↗