Segregation of non‐p.R132H mutations in IDH1 in distinct molecular subtypes of glioma

Mutations in the gene encoding the isocitrate dehydrogenase 1 gene (IDH1) occur at a high frequency (up to 80%) in many different subtypes of glioma. In this study, we have screened for IDH1 mutations in a cohort of 496 gliomas. IDH1 mutations were most frequently observed in low grade gliomas with c.395G>A (p.R132H) representing >90% of all IDH1 mutations. Interestingly, non‐p.R132H mutations segregate in distinct histological and molecular subtypes of glioma. Histologically, they occur sporadically in classic oligodendrogliomas and at significantly higher frequency in other grade II and III gliomas. Genetically, non‐p.R132H mutations occur in tumors with TP53 mutation, are virtually absent in tumors with loss of heterozygosity on 1p and 19q and accumulate in distinct (gene‐expression profiling based) intrinsic molecular subtypes. The IDH1 mutation type does not affect patient survival. Our results were validated on an independent sample cohort, indicating that the IDH1 mutation spectrum may aid glioma subtype classification. Functional differences between p.R132H and non‐p.R132H mutated IDH1 may explain the segregation in distinct glioma subtypes. © 2010 Wiley‐Liss, Inc.     

抗血管生成因子Angiostatin与Endostatin作用下肿瘤血管生成的二维数值模拟

目的数值模拟抗血管生成因子Angiostatin和Endostatin对肿瘤血管生成的影响。方法建立肿瘤内外血管生成的二维离散数学模型。模型耦合两种抗血管生成因子Angiostatin和Endostatin的抑制效应,数值模拟在促血管生成因子诱导下肿瘤微血管网生成,讨论血管生成抑制因子的影响。结果抗血管生成因子Angiostatin对肿瘤内外血管网络生成的速度和成熟度有抑制作用。抗血管生成因子Angiostatin和Endostatin耦合作用时,在肿瘤血管生成的早期有明显的抑制效应;在肿瘤血管生成的中后期,它们可以降低肿瘤血管化程度。结论本文模型能够较好的模拟抗血管生成因子Angiostatin和Endostatin对内皮细胞迁移和增殖的抑制作用。     

Preserved contribution of nitric oxide to baseline vascular tone in deconditioned human skeletal muscle

Deconditioning is a risk factor for cardiovascular disease. Exercise reduces this risk, possibly by improving the vascular endothelial nitric oxide (NO) pathway. The effect of deconditioning on the NO pathway is largely unknown. This study was designed to assess baseline NO availability in the leg vascular bed after extreme, long-term deconditioning (spinal cord-injured individuals, SCI) as well as after moderate, short-term deconditioning (4 weeks of unilateral lower limb suspension, ULLS). For this purpose, seven SCI were compared with seven matched controls. Additionally, seven healthy subjects were studied pre- and post-ULLS. Leg blood flow was measured by venous occlusion plethysmography at baseline and during infusion of 5 incremental dosages of N ^G-monomethyl- l -arginine ( l -NMMA) into the femoral artery. Sodium nitroprusside (SNP) was infused to test vascular responsiveness to NO. Baseline leg vascular resistance tended to be higher in SCI compared with controls (37 ± 4 versus 31 ± 2 arbitrary units (AU), P = 0.06). Deconditioning altered neither the vasoconstrictor response to l -NMMA (increase in resistance in SCI versus controls: 102 ± 33% versus 69 ± 9%; pre- versus post-ULLS: 95 ± 18% versus 119 ± 15%), nor the vascular responsiveness to NO. In conclusion, two human in vivo models of deconditioning show a preserved baseline NO availability in the leg skeletal muscle vascular bed.     

Autosomal glycogenosis of liver and muscle due to phosphorylase kinase deficiency is caused by mutations in the phosphorylase kinase beta subunit (PHKB)

Glycogen storage disease due to phosphorylase kinase deficiency occurs in several variants that differ in mode of inheritance and tissue- specificity. This heterogeneity is suspected to be largely due to mutations affecting different subunits and isoforms of phosphorylase kinase. The gene of the ubiquitously expressed beta subunit, PHKB, was a candidate for involvement in autosomally transmitted phosphorylase kinase deficiency of liver and muscle. To identify such mutations, the complete PHKB coding sequence was amplified by RT-PCR of RNA isolated from blood samples of patients and analyzed by direct sequencing of PCR products. The characterization of mutations was complemented by PCR of genomic DNA. In one female and four male patients, we identified five independent nonsense mutations (Y418ter; R428ter; Y974H+E975ter; Q656ter in two cases), one single-base insertion in codon N421, one splice-site mutation affecting exon 31, and a large deletion involving the loss of exon 8. Although these severe translation-disrupting mutations occur in constitutively expressed sequences of the only known beta subunit gene of phosphorylase kinase, PHKB, they are associated with a surprisingly mild clinical phenotype, affecting virtually only the liver, and relatively high residual enzyme activity of approximately 10%.      

Delivery of a hammerhead ribozyme specifically down-regulates the production of fibrillin-1 by cultured dermal fibroblasts

The hammerhead ribozyme is a small catalytic RNA molecule. Potential hammerhead ribozymes that possess a catalytic domain and flanking sequence complementary to a target mRNA can cleave in trans at a putative cleavage site within the target molecule. We have investigated the potential of hammerhead ribozymes to down-regulate the product of the fibrillin-1 gene (FBN1). Fibrillin is a 347 kDa glycoprotein that is a major constituent of the elastin-associated microfibrils. Mutations in the FBN1 gene are responsible for Marfan syndrome (MFS), a common systemic disorder of the connective tissue. Many FBN1 mutations responsible for MFS appear to act in a dominant-negative fashion, raising the possibility that reduction of the amount of product from the mutant FBN1 allele might be a valid therapeutic approach for MFS. A trans-acting hammerhead ribozyme (FBN1-RZ1) targeted to the 5' end of the human FBN1 mRNA has been designed and synthesized, and shown to cleave its target efficiently in vitro. FBN1-RZ1 cleavage is magnesium dependent and efficient at both 37 and 50 degrees C. Delivery of the FBN1-RZ1 ribozyme into cultured dermal fibroblasts, by receptor- mediated endocytosis of a ribozyme-transferrin-polylysine complex, specifically reduces both cellular FBN1 mRNA and the deposition of fibrillin in the extracellular matrix. These results suggest that the use of hammerhead ribozymes is a valid approach to the study of fibrillin gene expression and possibly to the development of a therapeutic approach to MFS.      

The effect of bed rest and an exercise countermeasure on leg venous function

This study was performed to assess the effect of resistive vibration exercise during bed rest deconditioning on venous vascular dimension and function, as measured with ultrasound in the popliteal vein. Sixteen men were assigned to bed rest (BR-Ctrl) or bed rest with resistive vibration exercise (BR-RVE). Before and at 25 and 52 days of bed rest, popliteal vein diameter was measured at increasing cuff pressures. Venous capacitance and compliance were calculated from the pressure–volume curve. After 52 days of bed rest, BR-Ctrl showed no change in baseline popliteal vein diameter or compliance, while venous capacitance decreased. Resistive vibration exercise had no effect on the response in venous diameter, capacitance or compliance to 52 days of bed rest. The decline in venous capacitance due to long-term bed rest is not effectively counteracted by resistive vibration exercise, indicating that an alternative factor during bed rest deconditioning is responsible for venous changes.     

Value and cost of follow-up after adjuvant treatment of patients with Dukes' C colonic cancer

BACKGROUND: The clinical value and costs of different diagnostic tools used to identify potentially curable recurrent disease in patients treated adjuvantly for curatively resected Dukes' C colonic cancer were examined. METHODS: The study group comprised 496 patients treated with chemotherapy over a 1-year interval. Follow-up consisted of interim history, physical examination, liver ultrasonography or computed tomography (CT), measurement of carcinoembryonic antigen (CEA) levels, chest radiography and colonoscopy. RESULTS: Two hundred and thirteen patients had recurrent disease (median follow-up 43 months). Forty-two patients with recurrence (20 per cent) were treated with curative intent (median survival 38 months; 5-year survival rate 40 per cent). Recurrence was identified by liver ultrasonography or CT (n = 14), evaluation of symptoms (n = 12), colonoscopy (n = 8), CEA measurement (n = 3), chest radiography (n = 2), physical examination (n = 1) and other modalities in two patients. The mean cost of diagnostic procedures per curative resected recurrence for patients amenable to salvage surgery was US$9011. Of all treatable recurrences, 12 of 42 were identified by evaluation of symptoms only. Ultrasonography and colonoscopy identified 22 recurrences at a cost of US$11 790 per patient, while routine follow-up by CEA measurement, chest radiography and physical examination identified a further six at a cost of US$19 850 per patient. CONCLUSION: Potentially curable recurrences were detected primarily by liver imaging and colonoscopy. The yield of CEA measurement, chest radiography and physical examination was relatively low; such methods were expensive and should not be recommended in the routine follow-up of these patients.     

A processive versus a distributive mechanism of action correlates with differences in ability of normal and xeroderma pigmentosum group A endonucleases to incise damaged nucleosomal DNA

A DNA endonuclease, isolated from the nuclei of normal human and xeroderma pigmentosum complementation group A (XPA) cells, which recognizes predominately pyrimidine dimers, was examined for the mechanism by which it locates sites of damage on UVC-irradiated DNA. In reaction mixtures with low ionic strengths (i.e. lacking KCl), the normal and XPA endonuclease locate sites of UV damage on both naked and reconstituted nucleosomal DNA by different mechanisms. On both of these substrates, the normal endonuclease acts by a processive mechanism, meaning that it binds non-specifically to DNA and scans the DNA for sites of damage, whereas the XPA endonuclease acts by a distributive one, meaning that it randomly locates sites of damage on DNA. However, while both the normal and XPA endonucleases can incise UVC irradiated naked DNA, they differ in ability to incise damaged nucleosomal DNA. The normal endonuclease showed increased activity on UVC treated nucleosomal DNA compared with naked DNA, whereas the XPA endonuclease showed decreased activity on the damaged nucleosomal substrate. Since a processive mechanism of action is sensitive to the ionic strength of the micro-environment, the KCl concentration of the reaction was increased. At 70 mM KCI, the normal endonuclease switched to a distributive mechanism of action and its ability to incise damaged nucleosomal DNA also decreased. These studies show that there is a correlation between the ability of these endonucleases to act by a processive mechanism and their ability to incise damaged nucleosomal DNA; the normal endonuclease, which acts processively, can incise damaged nucleosomal DNA, whereas the XPA endonuclease, which acts distributively, is defective in ability to incise this substrate.      

Plasma antioxidant vitamins, chronic hepatitis B virus infection and urinary aflatoxin B1-DNA adducts in healthy males

Epidemiological evidence indicates that aflatoxin B1 (AFB1) intake is associated with an increased risk of hepatocellular carcinoma (HCC). The hepatocarcinogenesis is initiated by covalent binding of AFB1 to cellular DNA. To determine whether nutritional factors and hormonal status may influence the binding of AFB1 to hepatic DNA, a cross- sectional study was performed on a total of 42 male asymptomatic hepatitis B surface antigen (HBsAg) carriers and 43 male non-carriers in a cohort study on the multistage development of HCC in Taiwan. The major AFB1-DNA adduct in vivo, AFB1-N7-guanine, was measured by high- performance liquid chromatography in urine. Urinary AFB1-N7-guanine was detectable in 40% of the subjects. HBsAg carriers had a higher detection rate of urinary AFB1-DNA adducts than non-carriers and the difference was statistically significant after multivariate adjustment. After taking into account the total AFB1 urinary metabolite level, chronic HBsAg carrier status, and other potential confounders, plasma levels of cholesterol, alpha-tocopherol, and alpha- and beta-carotene were positively associated with the detection rate of the AFB1-DNA adducts in a dose-dependent manner, whereas plasma lycopene level was inversely related to the presence of the adducts in urine. The association of urinary AFB1-DNA adducts with the plasma levels of cholesterol, alpha-tocopherol, lycopene, and alpha- and beta-carotene was observed at both low and high exposure levels of AFB1. There was a synergistic interaction of plasma alpha-tocopherol with alpha- and beta- carotene on the adduct levels. No association with the adducts was found for plasma levels of retinol and testosterone. This study demonstrated different associations of antioxidant vitamins with AFB1- DNA adduct formation. The data consistent with our previous finding in cultured woodchuck hepatocytes that alpha-tocopherol and beta-carotene enhanced AFB1-DNA adduct formation suggest that prospective investigation of the relationship between plasma micronutrients and risk of AFB1-related HCC is warranted.