We examined the impact of purified bacterially synthesized GST-MDA-7 (IL-24) and ionizing radiation on the proliferation and survival of nonestablished human glioblastoma multiforme (GBM) cells. Glioma cell types expressing mutated PTEN and p53 molecules, activated ERBB1VIII, overexpressing wild type ERBB1 or without receptor overexpression were selected. In MTT assays, GST-MDA-7 caused a dose-dependent reduction in the proliferation of nonestablished glioma cells; however only at higher concentrations did GST-MDA-7 reduce cell viability. The anti-proliferative and cytotoxic effects of GST-MDA-7 were enhanced by radiation in a greater than additive fashion that correlated with JNK1/2/3 activation. The reduction in cell growth and enhancement in cell killing by the combination of GST-MDA-7 and radiation were blocked by an ROS scavenger, N-acetyl cysteine (NAC), a JNK1/2/3 inhibitor SP600125, a pan-caspase inhibitor (zVAD) and by an inhibitor of caspase 9 (LEHD), but not by an inhibitor of caspase 8 (IETD). Low concentrations of either GST-MDA-7 or radiation reduced clonogenic survival, however colony formation ability was significantly further decreased when the two treatments were combined, which was also blocked by inhibition of caspase 9 function. In general agreement with activation of the intrinsic caspase pathway, cell death correlated with reduced BCL-XL expression and with increased levels of the pro-apoptotic proteins BAD and BAX. Inhibition of caspase 9 after combination treatment blunted neither JNK1/2/3 activation nor the enhanced expression of BAD and BAX, but did block caspase 3 cleavage, reduced expression of BCL-XL and inhibition of ERK1/2 activity. In contrast, incubation with NAC blocked JNK1/2/3 activation and cell killing, but not the increases in BAD and BAX expression. These findings argue that after combination treatment JNK1/2/3 activation is a primary pro-apoptotic event and loss of BCL-XL expression and ERK1/2 activity are secondary caspase-dependent processes. This data also argues that GST-MDA-7 induces two parallel pro-apoptotic pathways via ROS-dependent and -independent mechanisms. Infection of primary human astrocytes with a recombinant adenovirus to express MDA-7, Ad.mda-7, but not infection with either Ad.cmv or Ad.mda-7 SP -lacking MDA-7 secretion, resulted in the suppression of GBM cell colony formation in soft agar overlay assays, an effect that was enhanced in a greater than additive fashion by radiation. Collectively, our findings demonstrate that MDA-7 reduces proliferation and enhances the radiosensitivity of nonestablished human GBM cells in vitro, and when grown in 3 dimensions, and that sensitization occurs independently of basal EGFR/ERK1/2/AKT activity or the functions of PTEN and p53.

ABSTRACT. Objective. In preterm infants, the rapid and accurate determination of the presence of a hemodynamically significant patent ductus arteriosus (PDA) is extremely important, but this is often difficult. Plasma B-type natriuretic peptide (BNP) measurement has been reported to be a helpful aid in the diagnosis of hemodynamically significant PDA in preterm infants. The aim of our study was to investigate the usefulness of a rapid BNP assay as a diagnostic marker of symptomatic PDA (sPDA) in preterm infants.Methods. Sixty-six preterm infants, ranging from 25 to 34 gestational weeks of age, underwent clinical and echocardiographic examinations for PDA every other day from the third day of life until the disappearance of ductal flow. Blood samples were collected and plasma BNP concentrations were measured simultaneously using a commercial kit, (Triage BNP test kit; Biosite Diagnositics, San Diego, CA). When >2 clinically significant features of PDA were noted, and a large ductal flow was confirmed by color Doppler echocardiography, sPDA was diagnosed and treated with indomethacin.Results. On the third day after birth, the mean BNP concentration in the sPDA group (n ‫؍‬ 23) was significantly higher than in the control group (n ‫؍‬ 43) (2896 ؎ 1627 vs 208 ؎ 313 pg/mL). Seventeen infants (74%) in the sPDA group became asymptomatic after an initial course of indomethacin and their BNP levels concomitantly decreased. Moreover, BNP concentrations were significantly correlated with the magnitudes of the ductal shunt, such as the ratio of left atrial to aortic root diameter and the diastolic flow velocity of the left pulmonary artery (r ‫؍‬ 0.726 and 0.877). The area under the receiver operator characteristic curve for the detection of sPDA was high: 0.997 (95% confidence interval: 0.991-1.004). The best cutoff of BNP concentration for the diagnosis of sPDA was determined to be 1110 pg/mL (sensitivity: 100%; specificity: 95.3%).Conclusion. In preterm infants, the circulating BNP levels correlated well with the clinical and echocardiographic assessments of PDA. Although not a stand-alone test, the rapid BNP assay provides valuable information for the detection of infants with sPDA that require treatment. Moreover, serial BNP measurements may be of value in determining the clinical course of PDA in preterm infants. ABBREVIATIONS. PDA, patent ductus arteriosus; hsPDA; hemodynamically significant patent ductus arteriosus; BNP, B-type natriuretic peptide; sPDA, symptomatic patent ductus arteriosus; LA/AO, a ratio of left atrium to the aortic root diameter; DFLPA, diastolic flow velocity of the left pulmonary artery; ROC, receiver operator characteristic; asPDA, asymptomatic patent ductus arteriosus. R apidly and accurately determining the indications of therapeutic closure of a hemodynamically significant patent ductus arteriosus (hsPDA) in preterm infants is extremely important. [1][2][3] The currently used tools, such as the clinical findings, which include heart failure and the typical echocardiographic features of hsPD...

All exons of the human thyroid peroxidase gene were cloned from phage and cosmid libraries and sequenced, including 2599 base pairs of upstream DNA. The gene contains 17 exons and covers at least 150 kilobase pairs of chromosome 2. The transcription start site was identified by both S1 mapping and primer extension; a typical TATA box was found 25 bases upstream of the putative start site. A comparison of the gene structures of thyroid peroxidase and a granulocyte protein, myeloperoxidase, revealed that the positions of the 3rd through 11th exon-intron junctions in thyroid peroxidase coincide exactly with those of the 2nd through 11th exon-intron junctions in myeloperoxidase except the 7th myeloperoxidase junction, that does not have any counterpart in thyroid peroxidase. The amino acid codon separation pattern in each junction is well conserved between both enzymes. Four exons, unique to thyroid peroxidase, are located at the 3' end of the gene (exons 13-16), each of which encompasses a different protein module. Three of these modules, representing exons 13, 14, and 15, bear significant similarities to C4b-beta 2 glycoprotein, the EGF-LDL receptor, and a typical transmembrane domain, respectively. The genes coding for these modules were probably fused to an ancestral peroxidase gene to generate the present thyroid peroxidase gene. The data suggest that intron loss, and/or insertion, and exon shuffling have played important roles in the evolution of the thyroid peroxidase gene.

Objective To investigate the effect of high glucose and spent peritoneal dialysate on the transforming growth factor-β1 (TGFβ1) synthesis of cultured human peritoneal mesothelial cells (HPMCs) and to examine the effect of costimulation with high glucose or spent dialysate, and cytokines, interleukin-1β (IL-1β), and tumor necrosis factor-α (TNFα) on TGFβ1 synthesis of HPMCs. Design HPMCs were exposed to different concentrations of glucose (30, 60, and 90 mmol/L) or spent peritoneal dialysate for 48 hours in the absence or presence of IL-1β (1 ng/mL) and TNFα (1 ng/mL). TGFβ1 mRNA expression was assessed by Northern blot analysis and TGFβ1 protein release by Western blot analysis and enzymelinked immunosorbent assay (ELISA). Results Exposure of HPMCs to high glucose conditions (30, 60, and 90 mmol/L of D-glucose) induced 2.3-, 3.6-, and 4.0-fold increases in TGFβ1 mRNA expression of HPMC with enhanced TGFβ1 protein synthesis and secretion into the media, whereas there were no significant changes in TGFβ1 synthesis with equimolar concentrations of D-mannitol. Incubation with spent dialysate also significantly increased TGFβ1 mRNA expression and protein secretion compared to control media ( p < 0.05). Stimulation with IL-1β (1 ng/mL) or TNFα (1 ng/mL) resulted in a significant increase in TGFβ1 mRNA expression after 48 hours: 2.7 and 2.1 times the control level, respectively. However, TNFα-induced increase in TGFβ1 mRNA expression was not translated into TGFβ1 protein secretion, while IL-1β stimulation induced a significant increase in TGFβ1 protein secretion as well as TGFβ1 mRNA expression. Combined stimulation by high glucose or spent dialysate, together with IL-1β or TNFα, showed a greater increase in TGFβ1 mRNA expression and protein secretion compared to stimulation by high glucose or spent dialysate alone. Conclusion Our results clearly show that high glucose solution and spent dialysate themselves might be sufficient to stimulate the production of TGFβ1 by peritoneal mesothelial cells. In peritoneal dialysis patients, this state of chronic induction of TGFβ1 is further exacerbated in the presence of peritonitis because of the stimulatory effect of proinflammatory cytokines, resulting in augmented TGFβ1 synthesis, thus promoting peritoneal fibrosis.

ABSTRACT:We investigated whether genetic polymorphisms of vascular endothelial growth factor (VEGF) and transforming growth factor-␤1 (TGF-␤1), potential candidate genes in the pathogenesis of urinary tract infection (UTI) and vesicoureteral reflux (VUR), are associated with the susceptibility to UTI and VUR, and renal scarring. We recruited 89 controls and 86 UTI and 58 VUR children. The UTI group was subdivided into two groups according to renal scarring. Two polymorphisms of VEGF and three of TGF-␤1 genes were investigated by using PCR-restriction fragment length polymorphism analysis. In both UTI and VUR groups, there was an increase in frequency of the VEGF -460 CC (control, 4.3; UTI, 15.9; VUR, 17.8%; p Ͻ 0.05), TGF-␤1 -509 CC (control, 8.7; UTI, 34.6; VUR, 35.1%; p Ͻ 0.001), and TGF-␤1 -800 GG genotypes (control, 19.1; UTI, 40.5; VUR, 40.4%; p Ͻ 0.05). An increase in the TGF-␤1 ϩ869 CC (scar-positive, 35.4; scar-negative, 10.3%; p Ͻ 0.05) and a decrease in the ϩ869 TC genotype (scar-positive, 29.2; scarnegative, 55.2%; p Ͻ 0.05) were observed in the scar-positive subjects. There were no differences in ϩ405 VEGF genotype frequencies. The VEGF T-460C and the TGF-␤1 C-509T, G-800A, and T869C polymorphisms could be genetic markers of the process of UTI and VUR. (Pediatr Res 62: 183-187, 2007)