Real-time Quantitative Reverse Transcriptase-polymerase Chain Research Articles

Ontogeny of Cytochrome P450 Aromatase mRNA Expression in the Developing Sheep Brain

The intraneuronal conversion of testosterone to oestradiol constitutes a critical step in the development and sexual differentiation of the brain of many short gestation mammalian species and has been inferred to play a similar role in long gestation sheep. This conversion is catalysed by cytochrome P450 aromatase (CYP19), which is expressed in specific brain structures during foetal development. The present study was undertaken to examine the specific neuroanatomical distribution and relative expression of aromatase mRNA in the developing sheep hypothalamus. The foetal sheep is a highly tractable model system for localising the region-specific expression of aromatase in the brain during prenatal development that can help predict regions where oestrogen acts to shape neural development. Our results, obtained using real time quantitative reverse transcriptase-polymerase chain reaction, revealed that aromatase mRNA was expressed throughout mid to late gestation in the foetal preoptic area and amygdala. In the preoptic area, aromatase expression declined with advancing gestation, whereas, it increased in the amygdala. No sex differences were observed in either brain area. We next investigated the anatomical distribution of aromatase using in situ hybridisation histochemistry and found that the pattern of mRNA expression was largely established by midgestation. High expression was observed in the medial preoptic nucleus, bed nucleus of the stria terminalis and corticomedial amygdala. We also observed substantial expression in the dorsal striatum. These results extend our understanding of the developmental expression of aromatase in the foetal sheep brain and lend support to the view that it plays an essential role in sexual differentiation and maturation of the neuroendocrine, motor and reward control systems.

Assessment of Prognostic Circulating Tumor Cells in a Phase III Trial of Adjuvant Immunotherapy After Complete Resection of Stage IV Melanoma

To verify circulating tumor cell (CTC) prognostic utility in stage IV resected melanoma patients in a prospective international phase III clinical trial. Our studies of melanoma patients in phase II clinical trials demonstrated prognostic significance for CTCs in patients with AJCC stage IV melanoma. CTCs were assessed to determine prognostic utility in follow-up of disease-free stage IV patients pre- and during treatment. After complete metastasectomy, patients were prospectively enrolled in a randomized trial of adjuvant therapy with a whole-cell melanoma vaccine, Canvaxin, plus Bacille Calmette-Guerin (BCG) versus placebo plus BCG. Blood specimens obtained pretreatment (n = 244) and during treatment (n = 214) were evaluated by quantitative real-time reverse-transcriptase polymerase chain reaction (qPCR) for expression of MART-1, MAGE-A3, and PAX3 mRNA biomarkers. Univariate and multivariate Cox analyses examined CTC biomarker expression with respect to clinicopathological variables. CTC biomarker(s) (≥ 1) was detected in 54% of patients pretreatment and in 86% of patients over the first 3 months. With a median follow-up of 21.9 months, 71% of patients recurred and 48% expired. CTC levels were not associated with known prognostic factors or treatment arm. In multivariate analysis, pretreatment CTC (> 0 vs. 0 biomarker) status was significantly associated with disease-free survival (DFS; HR 1.64, P = 0.002) and overall survival (OS; HR 1.53, P = 0.028). Serial CTC (>0 vs. 0 biomarker) status was also significantly associated with DFS (HR 1.91, P = 0.02) and OS (HR 2.57, P = 0.012). CTC assessment can provide prognostic discrimination before and during adjuvant treatment for resected stage IV melanoma patients.

Correlation of Pfg377 ortholog gene expression of Plasmodium vivax and mosquito infection

To determine the expression of Pfg377 ortholog gene in Plasmodium vivax, and examine its correlation with mosquito infection. Seventy clinical blood samples positive for P. vivax by microscopy, were used for the mosquito infectivity assay. Infectivity to female Anopheles dirus was determined from oocyst counts. The transcripts of Pfg377 ortholog gene of P. vivax from blood samples infective and non-infective to mosquitoes were examined using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Of 70 P. vivax positive blood samples, 50 (71.4%) samples were mosquito-infective and 20 (28.6%) were not. In infective samples, the expression level of Pfg377 ortholog gene was significantly higher than in the non-infective group (P<0.05). In infective samples, the expression level of Pfg377 ortholog gene at ≥100 copies/ml of blood cut-off point correlated with ≥10 oocysts/mosquito cut-off point of average oocyst numbers and with ≥50% cut-off point of per cent infected mosquitoes (Pearson's chi-square correlation, P=0.014 and P=0.026, respectively). The cut-off point of the expression level of Pfg377 ortholog gene could be used to predict the infectiousness of P. vivax gametocytes leading to mosquito infection and parasite transmission in the field.

Bone Marrow of Multiorgan Donors Underutilized

The demand for human hematopoietic stem and progenitor cells (HSPCs) for transplantation is increasing. Thus, effective alternative sources of HSPCs are required. Consequently, we sought to expand the accessibility of hematopoietic cells for clinical purposes by the investigation of hematopoietic reconstitution after transplantation of human HSPCs harvested from the bone marrow (BM) of heparinized deceased organ donors (HDODs). For multipart research comparison, human BM HDODs-, healthy donor-derived, umbilical cord blood nuclear cells, or CD34(+) cells were transplanted into sublethally irradiated NOD/SCID mice. Twenty-eight days after transplantation nuclear cells were isolated from the murine BM, spleen, and peripheral blood and were used to quantitatively detect human CD45 antigen by quantitative real-time reverse transcriptase-polymerase chain reaction and flow cytometry. The clonogenic growth of human colony-forming units was also investigated. We found that umbilical cord blood-derived HSPCs showed the greatest transplantation potential in our in vivo model. Interestingly, the transplantation potential of HSPCs collected from the BM of HDODs was of the same quality as cells obtained from healthy BM donors. Based on these results, we conclude that HDODs are a strongly underappreciated source of HSPCs that are ready to use for clinical purposes.

Upregulated expression of ADAM12 is associated with progression of oral squamous cell carcinoma

ADAMs are a disintegrin and metalloproteinase family of membrane-associated metalloproteinases characterized by their multidomain structure, and have been reported to be associated with various malignant tumors. The aim of this study was to identify crucial members of the ADAM family in oral squamous cell carcinoma (OSCC), and to reveal their biological function and clinical significance. To clarify whether ADAM family genes are involved in OSCC, changes in the expression profile were investigated by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis and immunohistochemical analysis. Functional analysis was performed by comparing cellular proliferation of siADAM-transfected cell lines and parental cell lines. Real-time qRT-PCR analysis identified significantly upregulated expression of ADAM12 in OSCC-derived cell lines. This was validated in OSCC samples using real-time qRT-PCR and immuno-histochemical staining. ADAM12 expression was correlated with TNM classification; significantly greater expression of ADAM12 was observed in tumors with higher T classification and more advanced stages. Moreover, siADAM12-transfected cells showed both a suppressed proliferation rate and increased transforming growth factor (TGF)-β3 expression. Our data indicate that ADAM12 is overexpressed in OSCC and might accelerate cellular proliferation. Its function may be associated with TGF-β signaling. This study suggests that controlling the expression or activity of ADAM12 could be a useful strategy in the development of an effective cure for OSCC.

BIRC6/Apollon gene expression in childhood acute leukemia: impact on therapeutic response and prognosis

Although BIRC6/Apollon seems to play a critical role as an antiapoptotic regulator, its clinical relevance in acute leukemia remains largely elusive. Therefore, we aimed to investigate BIRC6 gene expression in childhood acute leukemia in relation to clinicopathological characteristics at presentation, therapeutic response, and prognosis. BIRC6 expression level was assessed in 75 children with acute leukemia; 30 patients with acute myeloblastic leukemia (AML) and 45 patients with acute lymphoblastic leukemia (ALL) using real-time quantitative reverse transcriptase-polymerase chain reaction. The median level of BIRC6 expression did not differ significantly between AML and ALL patients. BIRC6 expression level was higher in patients with AML and ALL with extramedullary involvement, white blood cell (WBC) count ≥ 10 × 10(9) /L, and unfavorable cytogenetics at diagnosis. BIRC6 gene expression was higher in patients with unfavorable response to therapy at day 14, those who developed relapse or died in both leukemic groups. The best cutoff value of BIRC6 to predict therapeutic response and disease outcome was determined. AML and ALL patients with BIRC6 overexpression had significantly shorter overall and disease free survivals. This is the first report to study BIRC6 gene in pediatric ALL. Our results suggested that BIRC6 gene expression could be considered as an adverse risk factor in childhood acute leukemia and, hence, could be used to guide therapeutic regimens.

Dickkopf-1 Expression Is a Novel Prognostic Marker for Gastric Cancer

Aim. To investigate the involvement of Dickkopf-1 expression in gastric cancer. Methods. Dickkopf-1 mRNA and protein expression were determined by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunohistochemistry in specimens of primary cancer and their adjacent noncancerous tissues in gastric cancer patients. Results. Dickkopf-1 mRNA and protein expression levels were both significantly upregulated in gastric cancer lesions compared with adjacent noncancerous tissues. Its positive expression was correlated with depth of invasion, vessel invasion, lymph node and distant metastasis, and TNM stage of tumors. Additionally, in stages I, II, and III gastric cancers, the 5- year survival rate of patients with a high expression of Dickkopf-1 was significantly lower than that in patients with low expression. In stage IV, Dickkopf-1 expression did not correlate with the 5-year survival rate. Further multivariate analysis suggested that the up-regulation of Dickkopf-1 was an independent prognostic indicator for gastric cancer. Conclusion. A subset of cases with gastric cancer revealed the up-regulation of Dickkopf-1, which was associated with a progressive pathological feature and an aggressive clinical course. Therefore, Dickkopf-1 expression may be predictor for poor prognosis in patients with gastric cancer. This is the first report describing the involvement of Dickkopf-1 in gastric cancer.

Significance of regenerating islet-derived type IV gene expression in gastroenterological cancers

The regenerating islet-derived members (Reg), a group of small secretory proteins, which are involved in cell proliferation or differentiation in digestive organs, are upregulated in several gastrointestinal cancers, functioning as trophic or antiapoptotic factors. Regenerating islet-derived type IV (RegIV), a member of the Reg gene family, has been reported to be overexpressed in gastroenterological cancers. RegIV overexpression in tumor cells has been associated with carcinogenesis, cell growth, survival and resistance to apoptosis. Cancer tissue expressing RegIV is generally associated with more malignant characteristics than that without such expression, and RegIV is considered a novel prognostic factor as well as diagnostic marker in some gastroenterological cancers. We previously investigated the expression levels of RegIV mRNA of 202 surgical colorectal cancer specimens with quantitative real-time reverse-transcriptase polymerase chain reaction and reported that a higher level of RegIV gene expression was a significant independent predictor of colorectal cancer. The biologic functions of RegIV protein in cancer tissue, associated with carcinogenesis, anti-apoptosis and invasiveness, are being elucidated by molecular investigations using transfection techniques or neutralizing antibodies of RegIV, and the feasibility of antibody therapy targeting RegIV is being assessed. These studies may lead to novel therapeutic strategies for gastroenterological cancers expressing RegIV. This review article summarizes the current information related to biological functions as well as clinical importance of RegIV gene to clarify the significance of RegIV expression in gastroenterological cancers.

Serum microRNA is a promising biomarker for osteogenesis imperfect

The purpose of our study was to screen preliminary differential expression bone-related microRNAs (miRNAs) in serum of patients with osteogenesis imperfacta and to clarify whether serum microRNA is a promising biomarker for osteogenesis imperfecta. geNorm and several other programes were performed to select suitable reference genes for quantitative detection of serum miRNAs from 6 candidate control genes. With geometric averaging of selected reference genes as a normalization factor, fluorescence-based quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to detect expression levels of more than 100 bone-related miRNAs obtained by means of miRanda, Targetscan and Pictar software calculations and reading the literature. Through analysis of expression stability and pairwise variations, all 6 candidate reference genes had a stable expression level in serum of 8 healthy controls and 8 patients with different characrteristics, and the optimal number of reference genes for normalization was 4 (snRNAU6, miR-92a, miR-16, and Let-7a). For further validation, the expression stability of 4 reference genes remained steady in serum of another 8 healthy controls and 16 patients with osteogenesis imperfecta (M < 1.5). When normalized using multiple control genes, 11 bone-related miRNAs showed differential expression in serum of 8 osteogenesis imperfecta patients compared with 8 healthy controls. In conclusion, we identified snRNAU6, miR-92a, miR-16, and Let-7a as an internal reference gene group for qRT-PCR normalization and screening results revealed that there existed many differential expression bone-related miRNAs in serum of patients with osteogenesis imperfecta compared with healthy controls, and that these miRNAs had potential to be biomarkers for serologic tests and diagnosis of osteogenesis imperfecta with analysis of bioinformation.

Effects of the antipsoriatic drug dithranol on E2A and caspase-9 gene expression in vitro

Although the precise causes of psoriasis remain to be elucidated, psoriasis has been known as a disorder in which factors in the immune system, enzymes and other biochemical substances that regulate skin cell division are functionally imbalanced, thereby resulting in rapid proliferation of keratinocytes and incomplete keratinization. The expression of candidate genes such as E2A and caspase-9, which have been recognized to play a critical role in cellular proliferation/differentiation and apoptosis, is of great interest. They may be therapeutically targeted by the antipsoriatic drug, dithranol. We examined the molecular effects of dithranol on the mRNA and protein expression levels of E2A and caspase-9 in the HaCaT keratinocyte cell line. The HaCaT cells were treated with 0-0.5 μg/mL dithranol for 30 min. After dithranol was washed out, the HaCaT cells were cultured for 2 h, and their total cellular RNA and proteins were isolated. Quantitative real-time reverse transcriptase-polymerase chain reaction and Western blot were performed to determine the mRNA and protein levels of these two genes. We found that dithranol treatment in the range of 0.25-0.5 μg/mL slightly upregulated the mRNA expression of E2A and caspase-9 approximately 1.5- and 1.2-fold, respectively. However, undetectable change and minor downregulation of the protein expression levels were observed for E2A and caspase-9, respectively. Consequently, these genes appear not to be viable therapeutic targets for dithranol.

Epithelial-mesenchymal transition is associated with increased invasiveness of side population cells from hepatoma SMMC-7721 cells

To study the in vitro invasiveness of side population (SP), cells purified from hepatoma SMMC-7721 cells and the expressions of epithelial-mesenchymal transition (EMT) markers of SP cells and non-SP (NSP) cells, fluorescence-activated cell sorting (FACS) was used to sort SP cells and NSP cells from SMMC-7721 cells. The in vitro invasiveness of both subpopulations was tested by Transwell chamber assay. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot were applied to evaluate the expressions of Vimentin, E-cadherin, N-cadherin and Snail in SP cells and NSP cells, respectively. In Transwell invasion assay, the number of SP cells (66.3 ± 7.4) penetrating the basilar membrane was significantly higher than that of NSP cells (27.5 ± 6.5) ( P < 0.01). The mRNA expressions of Vimentin, N-cadherin and Snail was significantly increased in SP cells (9.527-fold, 6.834fold and 8.173-fold, respectively) when compared with that in NSP cells ( P < 0.01). In contrast, the decrease of E-cadherin mRNA expression in SP cells (5.353-fold) was markedly higher than that in NSP cells ( P<0.01). The protein expressions of E-cadherin, Vimentin, N-cadherin and Snail in SP cells were markedly different from those in NSP cells (0.174 ± 0.014 and 0.935 ± 0.012; 1.117 ± 0.012 and 0.314±0.011; 0.975 ± 0.017 and 0.179 ± 0.013; 0.917 ± 0.014 and 0.202 ± 0.013; P < 0.01). The results suggest that SP cells from hepatoma SMMC-7721 cells are highly invasive in vitro which may be related to the changes in expressions of EMT markers.

Abnormal anandamide metabolism in celiac disease

The endocannabinoid system has been extensively investigated in experimental colitis and inflammatory bowel disease, but not in celiac disease, where only a single study showed increased levels of the major endocannabinoid anandamide in the atrophic mucosa. On this basis, we aimed to investigate anandamide metabolism in celiac disease by analyzing transcript levels (through quantitative real-time reverse transcriptase–polymerase chain reaction), protein concentration (through immunoblotting) and activity (through radioassays) of enzymes responsible for anandamide synthesis (N-acylphosphatidyl-ethanolamine specific phospholipase D, NAPE-PLD) and degradation (fatty acid amide hydrolase, FAAH) in the duodenal mucosa of untreated celiac patients, celiac patients on a gluten-free diet for at least 12 months and control subjects. Also, treated celiac biopsies cultured ex vivo with peptic–tryptic digest of gliadin were investigated. Our in vivo experiments showed that mucosal NAPE-PLD expression and activity are higher in untreated celiac patients than treated celiac patients and controls, with no significant difference between the latter two groups. In keeping with the in vivo data, the ex vivo activity of NAPE-PLD was significantly enhanced by incubation of peptic–tryptic digest of gliadin with treated celiac biopsies. On the contrary, in vivo mucosal FAAH expression and activity did not change in the three groups of patients, and accordingly, mucosal FAAH activity was not influenced by treatment with peptic–tryptic digest of gliadin. In conclusion, our findings provide a possible pathophysiological explanation for the increased anandamide concentration previously shown in active celiac mucosa.

Abnormal DNA methylation of ITGAL (CD11a) in CD4+ T cells from infants with biliary atresia

Recent evidence indicates that alterations to epigenetic DNA methylation patterns contribute to many autoimmune diseases. Biliary atresia (BA) is a virus-induced autoimmune disease characterized by impaired T cells, which may be due to aberrant DNA methylation. CD11a, a subunit of the β2-integrin LFA-1 (CD11a/CD18) with costimulatory functions, is overexpressed due to hypomethylation of its promoter regulatory elements in CD4+ T cells from patients with many autoimmune diseases. However, it is unknown whether aberrant expression and methylation of CD11a occur in T cells from infants with BA. We aimed to compare the CD11a expression level and the methylation status of the CD11a promoter region in CD4+ T cells from BA infants and healthy controls (HC). We used real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) to examine CD11a mRNA levels in CD4+ T cells from BA and HC infants. Bisulfite sequencing was used to determine the methylation status of the CD11a promoter and flanking regions in CD4+ T cells from BA and HC infants, and in CD4+ T cells with DNA methylation inhibitors. We found that CD11a expression is significantly decreased in BA CD4+ T cells (P=0.007). This was associated with hypermethylation of the CD11a promoter region in CD4+ T cells from infants with BA. Treatment with a DNA methylation inhibitor decreased CD11a promoter methylation and increased CD11a mRNA. Therefore, DNA hypermethylation at the CD11a locus contributes to the lowered expression of CD11a in BA CD4+ T cells.

Higher BMP receptor expression and BMP-2-induced osteogenic differentiation in tendon-derived stem cells compared with bone-marrow-derived mesenchymal stem cells

Surgical reattachment of tendon to bone often fails due to regeneration failure of the specialised tendon-bone junction (TBJ). The use of mesenchymal stem cells for TBJ regeneration has been reported with promising results. Tendon-derived stem cells (TDSCs) with high proliferative and multi-lineage differentiation potential have been isolated. As stem cells residing in tendons, TDSCs can be considered a new cell source for TBJ repair. Bone morphogenic protein 2 (BMP-2) is a potent osteogenic factor with roles in normal bone healing and pathological ectopic bone formation in soft tissues. The use of BMP-2 to promote TBJ repair has been well reported. This study aimed to compare TDSCs to the gold standard bone-marrow-derived mesenchymal stem cells (BMSCs) with respect to osteogenic response to BMP-2 in vitro. The clonogenicity and multi-differentiation potential of TDSCs and BMSCs were identified by colony-forming-unit assay, osteogenic, adipogenic and chondrogenic differentiation assays. Their osteogenic response to BMP-2 in vitro was examined by alkaline phosphatase (ALP) cytochemical staining, ALP activity assay and Alizarin red S staining of calcium nodule formation. Messenger RNA (mRNA) and BMP receptor (types IA, IB and II) protein expression were examined by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blotting. Our results showed that both TDSCs and BMSCs exhibited stem cell properties, including clonogenicity and multi-differentiation potential. TDSCs expressed higher mRNA and protein levels of BMP receptors IA, IB and II. They also exhibited higher osteogenic differentiation with and without BMP-2 stimulation compared with BMSCs. TDSCs with/without BMP-2 might be an attractive source for TBJ repair compared with BMSCs.

Multiparametric Flow Cytometry for Identification and Fluorescence Activated Cell Sorting of Five Distinct B-Cell Subpopulations in Normal Tonsil Tissue

The purpose of this study was to establish a procedure capable of isolating distinct B-cell subpopulations from human tonsils as a basis for subsequent molecular analyses. Overall, 5 distinct B-cell subpopulations were purified from fresh tonsils based on their fluorescence surface marker expression: naive B cells, centroblasts, centrocytes, memory B cells, and plasmablasts. The immunophenotypic identity of the subpopulations was verified by quantitative real-time reverse transcriptase-polymerase chain reaction using the proliferation marker MKI-67 and 6 B-cell-associated differentiation markers (BACH2, BCL6, PAX5, IRF4, PRDM1, and XBP1). Furthermore, within the centroblast compartment, large and small centroblasts could be distinguished and large centroblasts were shown to proliferate with a morphologic appearance of a "centroblast"-like cell but with lower gene expression of the germinal center markers BCL6 and BACH2 vs small centroblasts. This study has established a detailed and fast procedure for simultaneous sorting of up to 5 distinct maturation-associated B-cell subpopulations from human tonsils.

L-003 BETA1-ADRENERGIC RECEPTOR GENE METHYLATION AND CORRELATION WITH BLOOD PRESSURE IN SPONTANEOUSLY HYPERTENSIVE RATS

Background To investigate the role of DNA methylation in the control of expression of beta1-AR gene. Detection the expression of beta1-adrenergic receptor (beta1-AR) between different individuals in spontaneously hypertensive rats (SHR) whose genotype is consistent. Methods Measured forty spontaneously hypertensive rats (SHRs) blood pressure (BP) at 14:00-16:00 every day. Using gene sequencing to check SHR beta1-Adrenergic Receptor (beta1-AR) genotype; Real-time quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR), real-time reverse transcriptase-polymerase chain reaction (RT-PCR)to analyze myocardial mRNA expression of beta1-AR gene; methylation specific PCR (MSP) and bisulfite sequencing to detect methylated status in beta1-AR gene; protein expression of beta1-AR were identified by Western blot. Randomly divided them into 176–185mmHg, 186–195mmHg, 196–205mmHg three groups according their BP. Results There was no difference in any two groups in heart rate, body length, tail length, body weight, heart weight (P > 0.05). No mutation was detected in SHR beta1-AR gene sequence; MSP and bisulfite sequencing showed that SHR beta1-AR gene have DNA methylation. Expression of myocardial beta1-AR gene mRNA in methylation and non-methylation group are 27.8 ± 21.7, 110.2 ± 87.5 respectively (P < 0.05). Expression of myocardial beta1-AR gene in both group are 0.2 ± 0.1, 0.4 ± 0.1 respectively (P < 0.05). RT-PCR shows expression of myocardial beta1-AR mRNA are 0.21 ± 0.04, 0.27 ± 0.02, 0.33 ± 0.03 respectively, RQ-PCR shows expression of myocardial beta1-AR mRNA are 25.6 ± 6.4, 67.0 ± 15.5,149.3 ± 10.6 respectively;Expression of myocardial beta1-AR gene are 0.25 ± 0.08, 0.33 ± 0.09, 0.41 ± 0.06 respectively, demonstrates that different tissues of SHR cardiac beta1-AR expression was different, the difference was significant (P < 0.05). Conclusion Beta1-AR gene promoter methylatied in SHR myocardium, and influence the expression of beta1-AR gene. SHR beta1-AR expressed differently, the expression tended to increase with the increase of BP.

Megakaryocytopoiesis and Thrombopoiesis in Hematopoietic Stem Cells Exposed to Ionizing Radiation

Hematopoietic processes, especially megakaryocytopoiesis and thrombopoiesis, are highly sensitive to extracellular oxidative stresses such as ionizing radiation and chemotherapeutic agents. This study examined the terminal maturation of megakaryocytes and platelet production in hematopoietic stem/progenitor cells (HSPCs) exposed to ionizing radiation. Highly purified CD34(+) cells derived from human placental/umbilical cord blood were exposed to X rays (2 Gy, 150 kVp, 20 mA; 0.5-mm aluminum and 0.3-mm copper filters) at a dose rate of approximately 1 Gy/min and then cultured in a serum-free medium supplemented with thrombopoietin and interleukin-3. The number of cells generated from X-irradiated CD34(+) cells decreased with the time in culture. However, the fraction of CD34(+)Tie-2(+) and CD41(+)Tie-2(+) cells among the total cells generated from X-irradiated cells increased significantly in comparison to nonirradiated controls on day 7. In addition, the CD42a(+) particles, which appeared to be platelets, generated from the X-irradiated HSPCs appeared to be normal. Quantitative real-time reverse transcriptase-polymerase chain reaction analysis of the expression of various genes in cells harvested from the cultures showed that the early hematopoiesis-related genes FLI1, HOXB4 and Tie-2, the cytokine receptor genes KIT and IL3RA, and the oxidative stress-related genes HO1 and NQO1 were upregulated on day 7. These results suggest that normal terminal maturation of megakaryocytes and platelet production occur in residual HSPCs after exposure to ionizing radiation despite the adverse effect of radiation on proliferation and differentiation of HSPCs. Ionizing radiation may have the potential to promote both megakaryocytopoiesis and thrombopoiesis.

Oncofetal protein, IMP‐3, a potential marker for prediction of postoperative peritoneal dissemination in gastric adenocarcinoma

The aim of this study was to determine the expression of insulin-like growth factor-II messenger RNA (mRNA)-binding protein-3 (IMP-3) and its clinical significance in gastric cancers, as well the prognostic value of its expression in the peritoneal lavage fluid after surgery. IMP-3 expression was examined by immunohistochemistry in 96 primary gastric tumors. IMP-3 mRNA expression in peritoneal lavage fluid obtained at laparotomy was determine by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Positive staining for IMP-3 was observed in 74% (71/96) of the tumors. IMP-3 expression in gastric tumors correlated significantly with worst overall survival (OS) and recurrence-free survival. Multivariate analyses identified pathological N stage and IMP-3 expression as significant independent prognostic factors for disease-free survival. Eight (28%) of 36 peritoneal lavage samples were cytologically negative but positive for IMP-3 mRNA expression by RT-PCR. The OS of patients with IMP-3-positive peritoneal lavage was significantly worse than of those with negative expression. IMP-3 expression in primary gastric tumors was an independent poor prognostic factor. IMP-3 mRNA expression in peritoneal lavage fluid was a predictor of recurrence after surgery in gastric cancer and a marker of poor prognosis.

Developmental changes in the expression of Κv1 potassium channels in rat vestibular ganglion cells

The primary afferent neurons of the vestibular ganglion convey sensory information from hair cells in the semicircular canals and otolith organs to the vestibular nuclei, the adjacent brainstem and the cerebellum. The intrinsic firing properties of vestibular ganglion cells (VGCs) are heterogeneous and have been classified into phasic, intermediate and tonic firing types on the basis of their response to injected depolarizing currents. A previous study from our group showed that the proportion of phasic discharging VGCs decreased during the first postnatal weeks. Moreover, α-dendrotoxin (α-DTX), a Kv1 potassium channels antagonist, turned neuron phasic firing to tonic, thus suggesting that these channels play an important role in the developmental changes of VGCs firing patterns. Here, by using immunohistochemistry, Western blotting and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we explored the change in the expression of α-DTX-sensitive K+ channels, Kv1.1, Kv1.2 and Kv1.6 in rat VGCs during early postnatal periods. We showed that expression of Kv1.6 protein is down-regulated together with expression of Kv1.6 mRNA after postnatal day 7 in rat VGCs whereas expression of Kv1.1 and Kv1.2 proteins did not change during the same developmental period. Our results suggest that down-regulation of the Kv1.6 protein and mRNA may be associated with maturation of excitable properties of primary vestibular neurons.

Role of Neuropilin-1 and its expression in Egyptian Acute Myeloid and Acute Lymphoid Leukemia patients

Neuropilins are expressed in tumors vasculature and cells. Their expression is thought to be correlated with tumor angiogenesis and progression. In this study, we analyzed NRP-1 expression level in 40 acute leukemia patients [20 acute myeloid leukemia (AML) and 20 acute lymphoblastic leukemia (ALL)] and 10 healthy controls using Real-Time Quantitative Reverse-Transcriptase Polymerase Chain Reaction (RTQ-PCR) aiming to show Neuropilin-1 (NRP-1) gene expression pattern in acute leukemia patients and its role in disease severity and progression. NRP-1 was expressed in 80% and 95% of ALL and AML respectively with levels higher in patients than controls and in ALL than AML patients. NRP-1 levels were significantly correlated with blast percentage and complete remission. We conclude that NRP-1 is significantly associated with acute leukemia and that its level might serve as an indicator for disease severity and progression. NRP-1 signaling may represent a novel therapeutic approach for the treatment of acute leukemia subsets.