Comparative validation of c-kit exon 11 mutation analysis on cytology samples and corresponding surgical resections of gastrointestinal stromal tumours

Objective:  Studies have shown that c-kit mutation analysis of gastrointestinal stromal tumours (GISTs) obtained by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) can be routinely performed. We validated c-kit exon 11 mutational analysis on cell block material obtained from fine needle aspiration cytology (FNAC) for diagnostic purposes and compared it with the same analysis in formalin-fixed paraffin-embedded full sections of the corresponding resection specimens.
Methods:  c-kit mutation analysis was done on cell block material obtained from ten cases encountered in our department from 1999 to 2008 on which FNAC was attempted pre-operatively. The findings were compared with analysis on full paraffin section of the corresponding resected tumours in seven cases where patients opted for resection. c-kit exon 11 was examined via bidirectional nucleic acid sequencing.
Results:  Our results showed 100% concordance for the presence and type of exon 11 mutation in the resected and aspirated tumours in all seven cases. These mutations had diagnostic value when compared with other neoplasms that are part of the cytomorphological differential diagnosis, such as leiomyosarcoma or gastric adenocarcinomas.
Conclusion:  Molecular cytopathology is a powerful tool that can complement morphology and immunohistochemical assessment of cytological material in routine practice for the diagnosis and prognostication of GISTs. We briefly discuss the advantages and limitations of the fine needle method of obtaining tissue for the diagnosis and prognostication of GISTs, and its current therapeutic strategies.     

Combined MRI–PET dissects dynamic changes in plant structures and functions

Unravelling the factors determining the allocation of carbon to various plant organs is one of the great challenges of modern plant biology. Studying allocation under close to natural conditions requires non-invasive methods, which are now becoming available for measuring plants on a par with those developed for humans. By combining magnetic resonance imaging (MRI) and positron emission tomography (PET), we investigated three contrasting root/shoot systems growing in sand or soil, with respect to their structures, transport routes and the translocation dynamics of recently fixed photoassimilates labelled with the short-lived radioactive carbon isotope ¹¹C. Storage organs of sugar beet ( Beta vulgaris ) and radish plants ( Raphanus sativus ) were assessed using MRI, providing images of the internal structures of the organs with high spatial resolution, and while species-specific transport sectoralities, properties of assimilate allocation and unloading characteristics were measured using PET. Growth and carbon allocation within complex root systems were monitored in maize plants ( Zea mays ), and the results may be used to identify factors affecting root growth in natural substrates or in competition with roots of other plants. MRI–PET co-registration opens the door for non-invasive analysis of plant structures and transport processes that may change in response to genomic, developmental or environmental challenges. It is our aim to make the methods applicable for quantitative analyses of plant traits in phenotyping as well as in understanding the dynamics of key processes that are essential to plant performance.     

Productive infection of Piscirickettsia salmonis in macrophages and monocyte‐like cells from rainbow trout,a possible survival strategy

Piscirickettsia salmonis is the etiologic agent of the salmonid rickettsial septicemia (SRS), an endemic disease which causes significant losses in salmon production. This intracellular bacterium is normally cultured in salmonid epithelial cell lines inducing characteristic cytopathic effects (CPEs). In this study we demonstrate that P. salmonis is able to infect, survive, replicate, and propagate in the macrophages/monocytes cell line RTS11 derived from rainbow trout spleen, without inducing the characteristic CPEs and the host cells showing the same expression levels as non‐infected control cell. On the other hand, bacteria were capable of expressing specific proteins within infected cells. Infected macrophages cease proliferation and a fraction of them detached from the plate, transform to non‐adhesive, monocyte‐like cells with proliferative activity. Productive infection of P. salmonis into salmonid macrophage/monocyte cells in culture provides an excellent model for the study of host–pathogen interactions, almost unknown in the case of P. salmonis. Our results suggest that the infection of cells from the salmonid innate immune system without inducing an important cell death response should lead to the persistence of the bacteria and consequently their dissemination to other tissues, favoring the evasion of the first line of defense against pathogens. J. Cell. Biochem. 108: 631–637, 2009. © 2009 Wiley‐Liss, Inc.     

微乳体系中11β-羟基甲羟孕酮的C1,2生物脱氢

为改善过程传质,提高甾类药物中间体11β-羟基甲羟孕酮C1,2生物脱氢转化率,采用简单节杆菌Arthrobacter simplex UR016菌株在Tween-80/乙醇/食油/水构成的微乳体系中进行生物脱氢,并考察了微乳体系组成、转化温度、投料浓度对脱氢反应的影响。结果表明:以菌体培养液作为水相,食油作为油相构建微乳体系,食油最适加量为10g/L,表面活性剂Tween-80加量为4g/L;底物经醇溶后水析投料,乙醇最适加量为发酵液体积的7%(V/V);最适转化温度为33oC;当底物浓度为4g/L时,在构建的微乳体系中转化46h,脱氢转化率达88.6%,与水相转化工艺相比提高了66.2%。在该体系中疏水性11β-羟基甲羟孕酮底物得到了有效的增溶和扩散,生物脱氢转化率明显提高。     

A novel NADP-dependent dehydrogenase activity for 7α/β- and 11β-hydroxysteroids in human liver nuclei: A third 11β-hydroxysteroid dehydrogenase

Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP⁺ or NAD⁺, but not NADPH and NADH, as pyridine nucleotide cofactor with K_m values of 12 ± 2 and 390 ± 2 μM, compared to the K_m for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The K_m for corticosterone in the NADP⁺-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The K_cat values for nuclear activity with NADP⁺ was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2.     

Taxol biosynthesis: Identification and characterization of two acetyl CoA:taxoid-O-acetyl transferases that divert pathway flux away from Taxol production

Two cDNAs encoding taxoid-O-acetyl transferases (TAX 9 and TAX 14) were obtained from a previously isolated family of Taxus acyl/aroyl transferase cDNA clones. The recombinant enzymes catalyze the acetylation of taxadien-5α,13α-diacetoxy-9α,10β-diol to generate taxadien-5α,10β,13α-tri-acetoxy-9α-ol and taxadien-5α,9α,13α-triacetoxy-10β-ol, respectively, both of which then serve as substrates for a final acetylation step to yield taxusin, a prominent side-route metabolite of Taxus. Neither enzyme acetylate the 5α- or the 13α-hydroxyls of taxoid polyols, indicating that prior acylations is required for efficient peracetylation to taxusin. Both enzymes were kinetically characterized, and the regioselectivity of acetylation was shown to vary with pH. Sequence comparison with other taxoid acyl transferases confirmed that primary structure of this enzyme type reveals little about function in taxoid metabolism. Unlike previously identified acetyl transferases involved in Taxol production, these two enzymes appear to act exclusively on partially acetylated taxoid polyols to divert the Taxol pathway to side-route metabolites.     

Coexpression of CYP11B2 or CYP11B1 with adrenodoxin and adrenodoxin reductase for assessing the potency and selectivity of aldosterone synthase inhibitors

Excessive production of aldosterone has been implicated in the pathogenesis of hypertension and heart failure. One approach to ameliorate the deleterious effects of aldosterone is to suppress its biosynthesis. The enzyme aldosterone synthase (CYP11B2) is responsible for the final step of aldosterone synthesis. It requires electron transfer from the adrenodoxin/adrenodoxin reductase system to catalyze the production of aldosterone. A stable cell line simultaneously overexpressing recombinant human CYP11B2 as well as human adrenodoxin and adrenodoxin reductase was established to help maximize the enzyme activity. The homogenate of these cells was used to develop an in vitro CYP11B2 assay using 11-deoxycorticosterone as a substrate. By the same strategy, another stable cell line simultaneously overexpressing human 11β-hydroxylase (CYP11B1), an enzyme responsible for the final step of cortisol biosynthesis, and the two electron transfer proteins was also established, and an in vitro CYP11B1 assay using 11-deoxycortisol as a substrate was likewise developed to assess the selectivity of CYP11B2 inhibitors. FAD286, a reference CYP11B2 inhibitor, inhibited CYP11B2 and CYP11B1 activities with IC₅₀ values of 1.6 ± 0.1 and 9.9 ± 0.9 nM (mean ± SEM, n = 3–6), respectively. Kinetics studies revealed that the compound inhibited the activity of both enzymes competitively with respective K_i values of 0.8 ± 0.04 and 2.2 ± 0.2 nM (n = 3–4). These assays can be used for assessing the potency and selectivity of CYP11B2 inhibitors for the treatment of hypertension and heart failure.     

Apoptosis signal-regulating kinase 1 regulates the expression of caspase-11

Caspase-11 is an inducible caspase involved in the regulation of cell death and inflammation. In the present study, we examined whether apoptosis signal-regulating kinase 1 (Ask1)-mediated signaling pathway is involved in the expression of caspase-11 induced by lipopolysaccharide (LPS). We found that the induction of caspase-11 was suppressed by the inhibitors of NADPH oxidase (Nox) or knockdown of Nox4 that acts downstream of toll-like receptor 4 and generates Ask1-activating reactive oxygen species. Overexpression of dominant negative tumor necrosis factor receptor associate factor 6 also suppressed the induction of caspase-11. Importantly, knockdown or dominant negative form of Ask1 suppressed the induction of caspase-11 following LPS stimulation. Taken together, our results show that Ask1 regulates the expression of caspase-11 following LPS stimulation.