Second-generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakiness

BACKGROUND: The tetracycline-regulatable system is one of the most valuable tools for controlling gene expression. In its current form, however, the system is less than ideal for in vivo or gene therapy uses due to difficulties in set-up procedures, high basal leakiness, and unpredictable delivery and efficiency. METHODS: To address these issues, we have devised a second generation of tetracycline-regulated promoters (TREs). The second-generation TRE (SG-TRE) contains a shortened cytomegalovirus (CMV) minimal promoter together with eight tet operator sequences positioned in an optimized manner upstream of the TATA box. This construct displays far greater reduction in basal leakiness than maximal transgene expression. Conversely, maximal transgene expression is increased to a greater degree than basal leakiness by post-translational stabilization with bovine growth hormone poly A. RESULTS: In transient studies, the SG-TRE displays over 100 000-fold regulation efficiency in HeLa cells at 1:1 ratio of transactivator to reporter plasmid in the Tet-Off system. This novel promoter achieves a regulation efficiency 500- to 1000-fold higher than that of the original TRE (P(hCMV*-1)) in HeLa cells by displaying undetectable levels of basal leakiness without compromised maximal expression. In other cell lines, the SG-TRE proves to be more efficient than the original P(hCMV*-1) in a cell-dependent manner. Furthermore, the SG-TRE preserves its enhanced regulation efficiency and its reduced basal leakiness in the context of a single positive feedback regulatory vector that presents ease of delivery of the system for use in vivo. Finally, in vivo, the biological function of granulocyte-macrophage colony stimulating factor is tightly regulated in the context of SG-TRE delivered via adeno-associated viruses.     

Management of a right aberrant subclavian artery during complex hybrid stent-graft procedures: a rare and complex issue

A 76-year-old man with an ascending arch and proximal descending aortic aneurysm underwent a complex aortic replacement through a sternotomy with ligation of a right aberrant subclavian artery (RASA) distal to the right vertebral artery. The second-stage procedure was performed with a stent-graft deployed within the elephant trunk. At 6- and 12-month follow-up, the RASA was opacified by the patent right vertebral artery. Under ultrasound guidance, the patient's RASA stump was occluded by coils. Management of an RASA during complex hybrid stent-graft procedures is discussed.     

First nationwide survey of prevalence of overweight, underweight, and abdominal obesity in Iranian adults

Objective: The goal was to estimate the prevalence of overweight, obesity, underweight, and abdominal obesity among the adult population of Iran. Research Methods and Procedures: A nationwide cross‐sectional survey was conducted from December 2004 to February 2005. The selection was conducted by stratified probability cluster sampling through household family members in Iran. Weight, height, and waist circumference (WC) of 89,404 men and women 15 to 65 years of age (mean, 39.2 years) were measured. The criteria for underweight, normal‐weight, overweight, and Class I, II, and III obesity were BMI <18.5, 18.5 to 24.9, 25 to 29.9, 30 to 34.9, 35 to 39.9, and ≥40 (kg/m²), respectively. Abdominal obesity was defined as WC ≥102 cm in men and ≥88 cm in women. Results: The age‐adjusted means for BMI and WC were 24.6 kg/m² in men and 26.5 kg/m² in women and 86.6 cm in men and 89.6 cm in women, respectively. The age‐adjusted prevalence of overweight or obesity (BMI ≥25) was 42.8% in men and 57.0% in women; 11.1% of men and 25.2% of women were obese (BMI ≥30), while 6.3% of men and 5.2% of women were underweight. Age, low physical activity, low educational attainment, marriage, and residence in urban areas were strongly associated with obesity. Abdominal obesity was more common among women than men (54.5% vs. 12.9%) and greater with older age. Discussion: Excess body weight appears to be common in Iran. More women than men present with overweight and abdominal obesity. Prevention and treatment strategies are urgently needed to address the health burden of obesity.     

New synergistic co-culture of Corylus avellana cells and Epicoccum nigrum for paclitaxel production

Journal of Industrial Microbiology & Biotechnology - Paclitaxel is a main impressive chemotherapeutic agent with unique mode of action and broad-spectrum activity against cancers. Hazel...     

Fine-resolution mapping by haplotype evaluation: the examples of PFIC1 and BRIC

Loci for two inherited liver diseases, benign recurrent intrahepatic cholestasis (BRIC) and progressive familial intrahepatic cholestasis type 1 (PFIC1), have previously been mapped to 18q21 by a search for shared haplotypes in patients in two isolated populations. This paper describes the use of further haplotype evaluation with a larger sample of patients for both disorders, drawn from several different populations. Our assessment places both loci in the same interval of less than 1 cM and has led to the discovery of the PFIC1/BRIC gene, FIC1; this discovery permits retrospective examination of the general utility of haplotype evaluation and highlights possible caveats regarding this method of genetic mapping. Received: 21 September 1998 / Accepted: 29 December 1998     

Pravastatin reverses the membrane cholesterol reorganization induced by myocardial infarction within lipid rafts in CD14(+)/CD16(-) circulating monocytes

Large numbers of monocytes are recruited in the infarcted myocardium. Their cell membranes contain cholesterol-rich microdomains called lipids rafts, which participate in numerous signaling cascades. In addition to its cholesterol-lowering effect, pravastatin has several pleiotropic effects and is widely used as secondary prevention treatment after myocardial infarction (MI). The aim of this study was to investigate the effects of pravastatin on the organization of cholesterol within monocyte membrane rafts from patients who had suffered myocardial infarction. Monocytes from healthy donors and acute MI patients were cultured with or without 4μM pravastatin. Lipid rafts were extracted by Lubrol WX, caveolae and flat rafts were separated using a modified sucrose gradient. Cholesterol level and caveolin-1 expression in lipid rafts were determined. In healthy donors, cholesterol was concentrated in flat rafts (63±3 vs 13±1%, p<0.001). While monocytes from MI patients presented similar cholesterol distribution in both caveolae and flat rafts. Cholesterol distribution was higher in flat rafts in healthy donors, compared to MI patients (63±3 vs 41±2%, p<0.001), with less distribution in caveolae (13±1 vs 34±2%, p<0.001). Pravastatin reversed the cholesterol distribution in MI patients cells between flat rafts (41±2 vs 66±3%, p<0.001) and caveolae (34±2 vs 18±1%, p<0.001). In conclusion, MI redistributes cholesterol from flat rafts to caveolae indicating monocyte membrane reorganization. In vitro pravastatin treatment restored basal conditions in MI monocytes, suggesting another effect of statins.     

Quantitative proteomic analysis reveals induction of premature senescence in human umbilical vein endothelial cells exposed to chronic low‐dose rate gamma radiation

Chronic low‐dose ionizing radiation induces cardiovascular disease in human populations but the mechanism is largely unknown. We suggested that chronic radiation exposure may induce endothelial cell senescence that is associated with vascular damage in vivo. We investigated whether chronic radiation exposure is causing a change in the onset of senescence in endothelial cells in vitro. Indeed, when exposed to continuous low‐dose rate gamma radiation (4.1 mGy/h), primary human umbilical vein endothelial cells (HUVECs) initiated senescence much earlier than the nonirradiated control cells. We investigated the changes in the protein expression of HUVECs before and during the onset of radiation‐induced senescence. Cellular proteins were quantified using isotope‐coded protein label technology after 1, 3, and 6 weeks of radiation exposure. Several senescence‐related biological pathways were influenced by radiation, including cytoskeletal organization, cell–cell communication and adhesion, and inflammation. Immunoblot analysis showed an activation of the p53/p21 pathway corresponding to the progressing senescence. Our data suggest that chronic radiation‐induced DNA damage and oxidative stress result in induction of p53/p21 pathway that inhibits the replicative potential of HUVECs and leads to premature senescence. This study contributes to the understanding of the increased risk of cardiovascular diseases seen in populations exposed to chronic low‐dose irradiation.