Inhibitory Effects of Anti-sense PTTG on Malignant Phenotype of Human Ovarian Carcinoma Cell Line SK-OV-3

To construct eukaryotic expression vector expressing full length anti-sense pituitary tumor transforming gene (PTTG) mRNA and observe its blocking effect on the potential invasion of human ovarian carcinoma cell line SK-OV-3. PCR primers containing designed enzyme cut sites were used for cloning full-length PTTG gene fragment, and the resulting PCR product was inserted into the eukaryotic vector pcDNA3. 1 in the antisense direction. The recombinant vector was then transfected into SK-OV-3 by Lipofectamine. The positive cell clone was screened by G418, PTTG and bFGF at protein level expression were detected by Western blot. The biological behavior change of transfection positive cells was observed by colony formation in soft agar assay. Our results showed that SK-OV-3 clones stably expressing full-length recombinant pcDNA3. 1-PTTGas were obtained. The expressions of PTTG and bFGF protein in transfected cells were decreased by 61.5% and 52.3%, respectively as compared with non-transfected ones. The number of colony formation was reduced significantly in transfected cells as compared with empty vector transfected and non-transfected cells. It is concluded that the recombinant vector pcDNA3. 1-PTTGas is a novel tool and provides an alternative anti-sense gene therapy targeted at PTTG in human carcinoma.     

Value of Quantitative Tissue Velocity Imaging in the Detection of Regional Myocardial Function in Dogs with Acute Subendocardial Ischemia

This study evaluated the application of quantitative tissue velocity imaging （QTVI） in assessing regional myocardial systolic and diastolic functions in dogs with acute subendocardial ischemia. Animal models of subendocardial ischemia were established by injecting microspheres （about 300 μm in diameter） into the proximal end of left circumflex coronary artery in 11 hybrid dogs through cannulation. Before and after embolization, two-dimensional echocardiography, QTVI and real-time myocardial contrast echocardiography （RT-MCE） via intravenous infusion of self-made microbubbles,were performed, respectively. The systolic segmental wall thickening and subendocardial myocardial longitudinal velocities of risk segments before and after embolization were compared by using paired t analysis. The regional myocardial video intensity versus contrast time could be fitted to an exponential function： y=A·（1-exp-β·t）, in which the product of A and β provides a measure of myocardial blood flow. RT-MCE showed that subendocardial normalized A·β was decreased markedly from 0.99±0.19 to 0.35±0.11 （P〈0.05） in 28 left ventricular （LV） myocardial segments after embolization, including 6 basal and 9 middle segments of lateral wall （LW）, 8 middle segments of posterior wall （PW） and 5 middle segments of inferior wall （IW）. However, there was no statistically significant difference in subepicardial layer before and after embolization. Accordingly, the ratio of A·β of subendocardial myocardium to subepicardial myocardium in these segments was significantly decreased from 1.10±0.10 to 0.31±0.07 （P〈0.05）. Although the systolic wall thickening did not change 5 min after the embolization in these ischemic segments （29%±3% vs 31%±5%, P〉0.05）, the longitudinal peak systolic velocities （Vs） and early-diastolic peak velocities （Ve） recorded by QTVI were declined significantly （P〈0.05）. Moreover, the subendocardial velocity curves during isovolumic relaxation predominant     

Longitudinal Nutritional Intakes in Italian Pregnant Women in Comparison with National Nutritional Guidelines

Background: Nutritional quality during pregnancy is crucial for mother and child health and their short/long-term outcomes. The aim of this study is to evaluate the adherence to nutritional recommendations in Italy during the three pregnancy trimesters in Normal Weight (NW) and Over Weight (OW) women. Methods: Data from a multicenter randomized controlled trial included 176 women (NW = 133; OW = 43) with healthy singleton pregnancies enrolled within 13 + 6 weeks of gestation. Dietary intake was assessed every trimester by a Food Frequency Questionnaire. Results: OW and NW had similar gestational weight gain. However, as Institute of Medicine (IOM) recommend lower gestational weight gain (GWG) for OW, they exceeded the suggested range. In both groups, caloric intake during the three trimesters never met recommendations. Protein intake in first and second trimester was higher than recommendations, as was sugars percentage. Dietary fiber intake was lower in OW. Polyunsaturated fatty acids, calcium, iron and folic acid requirements were never satisfied, while sodium intake exceeded recommendations. Conclusions: NW and OW women in Italy do not adhere to nutritional recommendations during pregnancy, with lower caloric intake, protein and sugars excess and inadequacies in micronutrients intake. Pregnant women in Italy should be provided with an adequate counseling and educational intervention as well as supplementation when indicated.     

Increased Expression of PI-3K in Asthmatic Rat T Lymphocytes

In order to explore the expression of PI-3K in T lymphocytes of asthmatic rats and the relationship between PI-3K and activation of T lymphocytes, 24 Wistar rats were randomly divided into 4 groups: normal control group, asthmatic one-week group, asthmatic two-week group and asth-matic four-week group. T cells were purified from blood of each rat and the expression of PI-3K was observed by immunocytochemical fluorescence staining, the semiquantitative fluorescence intensity was measured by HPIAS-2000 analytic software, and the expression of IL-4 in supernatants was de-tected by ELISA. The results showed that the fluorescence intensity of T lymphocytes in asthmatic groups was significantly higher than that in normal control (P<0.001), indicating that the expression of PI-3K in T lymphocytes of asthmatic rats was significantly higher than that in those of normal controls, and the difference between acute and chronic stage asthmatic groups was significant (P<0.05). The expression levels of IL-4 protein in supernatants of asthmatic T lymphocytes were sig-nificantly higher than those in the normal controls (P<0.05). There was a significant positive correla-tion between the expression of PI-3K in T lymphocytes and the IL-4 protein expression in super-natants (r=0.583, P<0.01). It was suggested that PI-3K signal pathway may participate in the proc-esses of activation and other cytological effects of asthmatic T lymphocytes, thus may play an impor-tant roles in the pathogenesis of asthma.     

Inhibitory activity of nuclear factor-κB potentiates cisplatin-induced apoptosis in A549 cells

Whether inhibiting the activity of nuclear factor （NF）-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated. The recombinant plasmid pcDNA3.1（＋）/IκBα expressing IκBα was constructed. The in vitro cultured A549 cells were transfected with pcDNA3.1 （＋）/IκBα alone, or pcDNA3.1（＋）/IκBα combined with cisplatin. The mitochondrial membrane potential （△ψm） was determined by rhodamine 123, the activity of caspase-3 was tested by colorimetric assay, and cell apoptosis was detected by flow cytometry with the annexin V/propidium iodide assay. The results showed that the activity of NF-κB in A549 cells was inhibited by transfecting pcDNA3.1（＋）/IκBα. Transfection of pcDNA3.1（＋）/IκBα alone did not promote apoptosis. Treatment of cisplatin alone had a little effect on cell apoptosis. Transfection of pcDNA3.1（＋）/IκBα combined with cisplatin treatment significantly induced apoptosis of A549 ceils. It was concluded that inhibiting the activity of NF-κB potentiated cisplatin-induced apoptosis of A549 cells.     

Impact of blood source and component manufacturing on neurotrophin content and in vitro cell wound healing

BackgroundWe evaluated neurotrophin (NF) levels and their impact on in vitro cell wound healing in eye drops from differently prepared blood sources (cord blood [CB], and peripheral blood [PB]) in the same donor, to avoid intrasubject biological variability.Materials and methodsTwenty healthy adult donor PB samples, and twenty CB samples acquired at the time of delivery were processed to obtain serum (S), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and S retrieved from PRP after activation with Ca-gluconate (PRP-R). The levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) were assessed with a Luminex xMAP (Luminex Corporation), and by using multikine kits from R&D system, and were statistically analysed in the eight different preparations. The impact of S, PRP, PPP, PRP-R from both sources on a cell line responding to NF supplementation (MIO-M1, UCL Institute of Ophthalmology, London, UK) was tested with a scratch wound assay, and analysed by IncuCyte S3 equipment.ResultsAll the preparations from CB showed higher NF levels, except for BDNF where no difference was found as compared to PB. PRP showed higher NF levels with respect to S, PPP and PRP-R in this decreasing order. Younger donors in PB contributed with higher NF levels. The scratch assay showed different cell migration results, with a complete wound closure only recorded with the supplementation of CB-S, and a progressive reduction by using PRP, PRP-R, and PPP from both sources.DiscussionProtocols of preparation and choice of blood source determine different NF levels in the final products. The therapeutic use of a natural neurotrophin pool from blood sources might have a clinical impact in several different settings. Efforts are needed to standardise the manufacturing and the product content in order to establish and modulate the posology of the final supplementation.     

Postoperative Liver Dysfunction and Future Remnant Liver: Where Is the Limit?

Background The future remnant liver (FRL) limit for safe major hepatectomy with low risk of postoperative liver failure has not yet been well defined. Methods Between April 2000 and September 2004, every patient scheduled for major hepatectomy in our institution underwent CT-volumetry of FRL. Patients with FRL <25% underwent portal vein embolization (PVE). Exclusion criteria were PVE, associated vascular resection and liver cirrhosis. The FRL was correlated with short-term results in patients with normal liver (group A) and those with impaired liver function secondary to neoadjuvant chemotherapy or cholestasis (bilirubin >2 mg/100 ml) (group B). Liver dysfunction was defined as both PT <50% and serum bilirubin level >5 mg/100 ml for three or more consecutive days. Results A total of 119 patients were analyzed, 72 in group A and 47 in group B. The FRL value was the only significant risk factor for postoperative liver dysfunction in the univariate and multivariate analysis (p = 0.009). The FRL did not correlate with postoperative mortality and morbidity. Bilirubin and prothrombin time (PT) on days 3 and 7 were significantly correlated to FRL in both groups. In group A, patients with postoperative liver dysfunction had a FRL<30% (3 versus 0; p = 0.005). According to receiving operator characteristic (ROC) curve analysis, a FRL value of 26.5% predicted postoperative liver dysfunction with 66.7% sensitivity, 97.1% specificity, 50% positive predictive value (PPV), and 98.5% negative predictive value (NPV). In group B, patients with postoperative liver dysfunction had a FRL <35% (4 versus 0; p = 0.027). According to ROC curve analysis, a FRL value of 31.05% predicted postoperative liver dysfunction with 75% sensitivity, 79.1% specificity, 25% PPV, and 97.1% NPV. Conclusions Hepatectomy can be considered safe when FRL is >26.5% in patients with healthy liver and >31% in patients with impaired liver function.