GC Type Research Articles

Testing of analytical and purification methods for HTR helium coolant

The paper describes a method of determination of gaseous impurities (H2, O2, N2, CO, CH4, CO2) in high purity helium as well as the methods of gaseous impurities removal. A device used for the determination was the Master GC type gas chromatograph with a helium detection unit and a ShinCarbon ST column. A temperature program was used to improve the conditions of separation and to reach maximum response of the detector. The obtained limits of detection defined as three times the standard deviation of the background value of tested components are H2 0.3vppm, O2 0.4vppm, N2 0.2vppm, CO 2vppm, CH4 3vppm, CO2 2vppm. Analysis time is less than 20min. The developed method will be used for checking the purity of helium environment in the experimental loop to model experimental conditions in the high temperature gas cooled reactor (HTGR).Within the scope of the proposal of the helium purification system, the efficiency of removing selected impurities (CO, CO2, CH4) on adsorbents (based on zeolites and active carbon) was tested. A laboratory scale apparatus and an FTIR spectrometer were used for experiments.

Effect modification of air pollution on Urinary 8-Hydroxy-2'-Deoxyguanosine by genotypes: an application of the multiple testing procedure to identify significant SNP interactions

BackgroundAir pollution is associated with adverse human health, but mechanisms through which pollution exerts effects remain to be clarified. One suggested pathway is that pollution causes oxidative stress. If so, oxidative stress-related genotypes may modify the oxidative response defenses to pollution exposure.MethodsWe explored the potential pathway by examining whether an array of oxidative stress-related genes (twenty single nucleotide polymorphisms, SNPs in nine genes) modified associations of pollutants (organic carbon (OC), ozone and sulfate) with urinary 8-hydroxy-2-deoxygunosine (8-OHdG), a biomarker of oxidative stress among the 320 aging men. We used a Multiple Testing Procedure in R modified by our team to identify the significance of the candidate genes adjusting for a priori covariates.ResultsWe found that glutathione S-tranferase P1 (GSTP1, rs1799811), M1 and catalase (rs2284367) and group-specific component (GC, rs2282679, rs1155563) significantly or marginally significantly modified effects of OC and/or sulfate with larger effects among those carrying the wild type of GSTP1, catalase, non-wild type of GC and the non-null of GSTM1.ConclusionsPolymorphisms of oxidative stress-related genes modified effects of OC and/or sulfate on 8-OHdG, suggesting that effects of OC or sulfate on 8-OHdG and other endpoints may be through the oxidative stress pathway.

Soluble Human Interleukin-2 Receptor (SIL-2R) as a Potential Predictor for Central Nervous System Relapse In Patients with Diffuse Large B-Cell Lymphoma In Rituximab Era: A 4.4-Year Follow up Analysis.

AbstractAbstract 2814 Background:Central nervous system (CNS) relapse is considered an infrequent but severe, nearly fatal complication of diffuse large B-cell lymphoma (DLBCL) following initial chemotherapy. Intrathecal (IT) prophylaxis cannot be recommended for all DLBCL patients because of the low probability of CNS relapse. Rituximab (R) added to CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) has been reported as likely to reduce the frequency of CNS relapse in patients with DLBCL, however, the efficacy of rituximab on the prognosis of CNS relapse compared with those who relapsed at other sites and predictive factors for CNS relapse in patients treated with rituximab contained chemotherapy remain unclear. The main objective was to determine the high-risk patients who need IT prophylaxis and the outcomes of CNS relapse compared with relapse on other sites. Patients and Methods:Diffuse large B cell lymphoma patients treated with rituximab contained CHOP regimen with or without radiotherapy and IT prophylaxis at Cancer Institute Hospital of JFCR between October 2003 and December 2006 were analyzed retrospectively. CNS relapse was defined as leptomeningeal, brain parenchymal, or intradural involvement with lymphoma, based on radiological findings or the presence of malignant lymphoma cells in spinal fluid. Results:A total number of 137 patients were identified. IT prophylaxis was administered in 13 of 137 patients (9.48%) depending on the sites of lymphoma involvement, such as bone marrow involvement, testis, paranasal sinuses. 17 of 137 patients (12.4%) underwent radiotherapy after chemotherapy because of early stage or their residual disease. With a median follow-up period of 4.4 years, 9 patients had experienced CNS relapse (6.7%: 3.0–12.1, 95%CI) among 30 documented relapses, with 9 presenting with nodal relapse alone and 21 presenting with extranodal relapse including CNS. IT prophylaxis and the addition of radiotherapy did not affect the frequency of CNS relapse (P=0.6 and 0.26). Median time to CNS relapse was 20 months. Overall survival (OS) was significantly inferior in CNS relapse patients to other-sites relapse patients, (median OS, 61 months vs. did not occur; P =.042). Nevertheless, OS was not significantly different between patients with CNS relapse or at other sites. In univariate analysis, factors associated with CNS relapse (P < 0.05) included age over 65 years and serum levels of soluble IL-2 receptors (sIL-2R) ≧10 ULN (upper limit of normal) but not sex, PS ≧3, stage ≧4, B symptom, bulky mass, elevated LDH >2 ULN, elevated MIB1 index ≧90%, poor revised-international prognostic index (R-IPI), extranodal sites ≧2, or type of GC or non-GC. Multivariate Cox regression analysis identified increased serum levels of (sIL-2R) (P =0.037) as an independent predictive factor for CNS relapse (P=0.04, HR=7.02: 95%CI=1.87-26.22). Five of 9 CNS relapse patients were still alive with the combination treatment of whole brain irradiation, systemic chemotherapy (R- dexamethasone, cisplatin, cytarabine) and intrathecal chemotherapy. Conclusions:The incidence rate of CNS relapse in 137 DLBCL patients treated with R-CHOP, CEOP regimens may be lower than with CHOP in agreement with previous studies. Furthermore, rituximab may improve OS after CNS relapse. Ten times increased serum s-IL2R is a potential independent risk factor for CNS relapse and should be included in the IT prophylaxis indication in patients with DLBCL. Disclosures:No relevant conflicts of interest to declare.

RCHOP Therapy Overcome Gastric Diffuse Large B Cell Lymphoma without Surgery

AbstractAbstract 1760 BackgroundOther than MALT, gastric diffuse large B cell lymphoma is a very common disease in malignant lymphoma. However the treatment strategy is controversial. Although a high efficacy of rituximab plus CHOP (RCHOP) has been proven in diffuse large B cell lymphoma (DLBCL), the decision to operate is still usual among surgeons in limited stage of gastric DLBCL. Koch et al. described that CHOP14 with radiotherapy is very effective for aggressive gastric lymphoma without surgery. In this report, though, the histology was various and mixed with MALT and DLBCL. Studies about only gastric DLBCL were very few, but, in some small size studies about RCHOP to gastric DLBCL, a good prognosis has been shown. If chemotherapy with radiotherapy alone gave rise to a complete response to gastric lymphoma, the patients could avoid gastric resection. Therefore it is necessary to devise a strategy for gastric DLBCL treatment. Patients and MethodsWe retrospectively analyzed the patients who diagnosed with gastric DLBCL between November 2003 and October 2008. The cases with limited stage were treated with 3 cycles of RCHOP with radiotherapy and the cases with advanced stage were treated with 6 cycles of RCHOP without radiation. All cases were evaluated with PET/CT after treatment. Statistical analysis of progression free survival (PFS) and overall survival (OS) were calculated using Kaplan-Meier estimators. Comparison between categories was performed by means of log-rank test. We estimated age-adjusted IPI, GC or non GC subtype, and hemoglobin level (Hb<11.0 g/dl) as risk factors. Using Cox proportional hazards regression analysis, multivariate analysis was performed about these risk factors. ResultTotal 40 cases were analyzed in this study. Median age was 65.5 years old and males were 21 (52.5%). For age-adjusted IPI, low was 22, low-intermediate was 9, high intermediate was 3, and high was 6. 15 cases (27.5%) were limited stage and they all received 3 cycles of RCHOP with radiation. 25 cases (72.5%) were stage II or more, and they all were treated with 6 cycles of RCHOP. GC and non-GC types were 30 (75%) and 10 (25%), respectively. The average of observation duration was 1274 days. Four cases dropped out from these regimens because of adverse event. CR rate was 92.8% (37/40) and one case progressed and one case relapsed. Two year-OS and PFS rate were 95.2% and 91.8%, respectively. Main adverse events were grade 4 neutropenia47.5% and febrile neutropenia 17.5%. There was no case with gastric bleeding after chemotherapy. There was one case of SIADH induced by vincristine, one case of grade 3 of hepatotoxity, confusion, and one case of intestinal pneumonia, respectively. Secondary gastric cancer after radiotherapy did not occur. We performed univariate statistical analysis about aa-IPI, GC or non GC subtype, and Hb level, then, there was tendency of poor prognosis only in high risk of aa-IPI (p=0.0512). However there was no significant difference in multivariate analysis. DiscussionRCHOP with or without radiation therapy was a very effective regimen and well-tolerated for gastric DLBCL patients. The result of our study, which was a small sample size, showed a better result than previous reports about DLBCL with RCHOP. Although the cases with severe bleeding or deep ulcer were included in this study, bleeding stopped and severe ulcers improved after chemotherapy in all such cases, so surgical treatment was not necessary. There was no report about direct comparison of the therapy with or without surgical treatment in gastric DLBCL, although RCHOP with radiation could preserve the organ and keep quality of life after treatment. We should recommend RCHOP with or without radiation for gastric DLBCL as the first line treatment. Disclosures:No relevant conflicts of interest to declare.

サルコイドーシス患者のGm, Km, Gcタイプ

サルコイドーシス患者のGm, Km, Gcタイプ

Abstract 3735: Dasatinib induces dephosphorylation of tyrosine proteins and apoptosis in both B-cell lymphoma lines and primary B-cell Non-Hodgkin Lymphoma cells in vitro for a subgroup of patients with phospho-Src (Y416) expression

Abstract The Non-Hodgkin Lymphomas (NHLs) are a heterogeneous group of disease with differing clinical behavior and responses to treatment. Approximately 85% of NHLs belonging to the B-cell lineage. Src family kinases (SFKs) are the predominant tyrosine kinase expressed in B-cell lines and are important in regulating multiple signaling pathways. We attempted to correlate the efficacy of SFK inhibitors with the expression of p-SFKs in lymphoma cells, to examine p-Src (Y416) expression in NHL tissues, and study dasatinib-induced apoptosis in primary lymphoma cells. In screening for the effects of different kinases inhibitors on B-cell lymphoma lines, we observed that SFK inhibitors, PP2 and dasatinib, inhibited proliferation and induced apoptosis at 24 hrs (PP2: 31% at 10 μM; dasatinib: 39% at 100 nM) in Burkitt's lymphoma (BL) line Raji. Apoptosis was associated with cleavage of caspases-3 &amp; 8. SFK inhibitor-induced apoptosis in Raji cells correlated with high level expression of p-Src. A similar effect was observed in a follicular lymphoma (FL) line DOHH2 despite expression of BCL2 in both. Raji and DOHH2 cells. In contrast, dasatinib showed no apoptotic induction in B-cell lymphoma lines Ramos (BL line) &amp; Sud-HL4 [GC type diffuse large B-cell lymphoma (DLBCL) line] which have no detectable expression of p-Src. Dasatinib caused complete de-phosphorylation of tyrosine proteins (around 160 kDa, 110 kDa and 60 kDa) in Raji cells as early as 15 mins. We are currently trying to identify these proteins. IHC staining of B-cell NHLs by tissue microarray indicated 26/55 (47.3%) of NHL tissues are positive for p-Src, which including DLBCL, FL, BL, small lymphocytic lymphoma (SLL), mantle cell lymphoma, marginal zone lymphoma (MZL) and lymphoblastic lymphoma. Fresh lymphoma cells from 12 cases [DLBCL (3), FL (3), SLL (2), MZL (1), NK/T cell lymphoma (1) and two reactive cases] were studied for in vitro effects of dasatinib. 6 cases (3 FL, 2 DLBCL and one reactive case) had clear expression of p-Src and underwent dasatinib-induced apoptosis (100 nM, 24h) of B-cells gated by CD45+/CD19+ co-expression. De-phosphorylation of p-Src and cleavage of caspase-3 were observed in the two DLBCL cases. The other 6 cases had undetectable p-Src and showed no apoptotic induction upon expose to dasatinib. Purified B-cells from 3 healthy donors have no detectable p-Src and no dasatinib induced apoptosis. These data suggest that 1) rational application of molecularly targeted therapy for aggressive NHL may be possible by directly examining key signaling nodes promoting survival and proliferation. 2) dasatinib is a potential therapeutic candidate for a subgroup of aggressive NHL which express p-Src (Y416). 3) profiling patients’ lymphoma cells for phospho-SFK might be a strategy selection of such therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3735.

Quantification of Rat Spermatogenesis in Late Puberty after Neonatal Hormonal Manipulation

ABSTRACTIt has been known for many years that estrogens administration to experimental animals during the neonatal period or adulthood can impair sperm production and maturation. In earlier studies the negative effects of E were explained only as a result of suppression of gonadotropin secretion during the treatment. We aimed to assess GC development on day 35 (different GC types) in tandem with Sertoli cell support toward GCs (efficiency of spermatogenesis) by complex systems of quantitative criteria. We used experimental model for manipulation of neonatal hormonal environment by treatment with DES-10 μg, DES-1 μg, DES-0.1 μg or GnRHa. DES-10 greatly affected testis development and spermatogenesis in rat whereas GnRHa was quite less effective in producing negative impact. In contrast during the onset of puberty (d 18) both treatments exerted similar negative effect, and this time dependent response of the testis corresponds to different hormonal profiles. Quantitative evaluation of late pubertal spermatogenesis demonstrate that the most differentiated GC population- spermatides was the most sensitive to hormonal manipulation compared to spermatogonia (Sg) and spermatocytes (Sc). Different mechanisms are probably involved in mediation of the effect of E and gonadotropins and direct E action on GC differentiation is suggested. Our finding would elucidate our understanding about the hormonal regulation of different germ sell steps of spermatogenesis.

The Use of ABL and GUS as Control Genes for Normalizing Quantification of Fusion Transcripts by Real Time PCR: Our Experience of More Than 1000 Samples.

Abstract 4727 IntroductionReal-time quantitative PCR (RT-PCR) is a sensitive and accurate tool for monitoring leukemic patients by amplification of fusion gene (FG) transcripts.In order to correct variations in RNA quality and quantity and to calculate the sensitivity of each measurement, a control gene (CG) transcript should be amplified in parallel to the FG transcript.In our laboratory two CG are used: Abelson (ABL) and beta-glucuronidase (GUS).Clinical application for monitoring the treatment of chronic myelogenous leukemia (CML) patients with tyrosine kinase inhibitor makes the quantification of which CG to use very important into the clinical practice today. ObjectiveThe aim of this study was to assess the degree of agreement between the GC, compare the expression of GC (type and time of sampling and pathology), try to redefine the limits between the 4 groups of quality (very good, good, poor and very poor quality samples) and to focus on CML patients. Materials and methodsA total of 1087 samples (peripheral blood and bone marrow) were obtained from 475 patients from January 2005 to December 2007. This study includes patients with acute myeloid leukaemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukaemia (CML) and Hypereosinophilic syndrome (HES).We collected 366 samples of 41 CML patients between 2002 and March 2009 including 32 CML at diagnosis.RT-PCR analysis was performed using the EAC protocols. ResultsComparison between CML, AML, ALL and HES patients showed that ABL and GUS expression did not differ significantly (Bland and Altman method).ABL and GUS gene expressions did not differ significantly between CML patients at diagnosis and the others. ConclusionBased on the literature and our local experience of more than 1000 samples, we propose to use only ABL as CG in routine practice in onco-hematology particularly for CML patients at diagnosis. Disclosures:No relevant conflicts of interest to declare.

Magnifying Zoom Imaging and Spectral Estimation Technology for the Endoscopic Differential Diagnosis of Subtypes of Hyperplastic Colorectal Polyps

New histopathologic evidence demonstrated distinct subtypes of hyperplastic epithelial polyps. There is the goblet cell-type hyperplastic polyp (GC), the vesicular cell-type hyperplastic polyp (VC), and the serrated adenoma (SA). While the majority of hyperplastic polyps are not related to colorectal carcinogenesis, the serrated dysplastic polyps might progress to colorectal carcinoma. With high resolution and magnifying zoom imaging along with spectral estimation technology (FICE) it is possible a detailed in vivo examination of the superficial microstructure of colorectal lesions. Aim: To evaluate differences on the superficial microstructure (pits, microvessels) among colorectal hyperplastic polyps correlating their endoscopic aspects with the histopathologic findings. Methods: A total of 54 lesions (polyps and flat lesions) were evaluated with the Fujinon 4400 processor and 590 series colonoscope that allows FICE and magnifying zoom imaging of 100×. All macroscopic features were evaluated but the pit pattern (modified Kudo) and the capillary vessel patterns were the focus of the study. Mean size of the lesions was 5.4 +/- 1.2mm. According to histopathologic examination there were 8 polyps GC type, 36 polyps VC type and 10 lesions SA type. All GC polyps presented enlarged square-shaped pits and hipovascularity with rare capillaries on the surface. VC type polyps presented star shaped pits and the majority (28/36) exhibited a typical capillary pattern characterized by sparse, short and straight vessels with tiny ramifications. SA lesions showed a mixed pattern composed of linear and star-shaped enlarged pits. Half of the SA lesions were hypovascular and half showed numerous spiral and tortuous capillary vessels on the surface. Conclusion: Detailed endoscopic analysis of the pit and the capillary vessel patterns of colorectal polyps by magnifying zoom imaging and FICE helps the in vivo differential diagnosis between subtypes of colorectal hyperplastic polyps and serrated adenomas.

Age related Epstein Barr virus associated B cell lymphoproliferative disorder in Peru

19514 Background: Age related Epstein Barr virus (EBV) associated B cell lymphoproliferative disorder is a recent new entity described in Japanese patients with diffuse large B cell lymphoma (DLBCL) and infection with EBV. It usually affects elderly patients with poor survival. Methods: In order to establish the correlation of EBV with DLBCL, we investigated the EBV status by RNA- in situ hybridization (ISH) in newly diagnosed nodal DLBCL.Between January 2002 and December 2004, seventy-four newly diagnosed nodal DLBCL patients were included in the analysis. Clinical data were reviewed retrospectively and patient’s biopsies were analyzed for the presence of EBV encoded RNA (EBER) by ISH and the immunohistochemical expression of BCL6, CD10 and MUM-1/IRF4 by tissue microarray (TMA) technique. Results: In this cohort the mean age was 66.1 years old (range 23–84) at diagnostic; male/female ratio 39/35; 46 patients (65.7%) with advanced stage (III/IV); 53 cases (71.6%) were of the non Germinal Center DLBCL type (non GC). This group was associated with a higher IPI than the Germinal Center DLBCL type (GC). Also there was a trend that the non GC type had a poorer overall survival than the GC type (p=0.171). Eleven cases of the cohort (14.9%) were identified as EBER-positive and were called as Age related EBV associated B cell lymphoproliferative disorder. Most of the cases EBER (+) were of the non GC group (10/11), showing a trend between EBER+ and non-GC DLBCL (OR=0.21, p=0.11 fisher 2-tails). EBER positive cases were associated with an advanced age (> 60 years), poorer status performance, more advanced stage, higher IPI risk group and poorer outcome to initial treatment. Overall survival in the EBER positive cases and negative were 1.1 vs. 15.5 months respectively (p=0.001). At the multivariable level de EBER status is an independent variable compared with the International Prognostic Index (IPI) (p=0.001) with a 3.2-fold (95% CI, 1.5 - 7.2) risk for death for positive cases Conclusions: Age related EBV associated B cell lymphoproliferative disorder is frequent between DLBCL in Peru. It was related to more advanced age, poor status performance, non-GC group and very short overall survival in comparison with DLBCL. New treatment strategies should be defined in this subgroup of poor prognostic. No significant financial relationships to disclose.

Antitumor activity and tumor localization of liposomal glucocorticoids in B16 melanoma-bearing mice

Prednisolone disodium phosphate (PLP) encapsulated in long-circulating liposomes (LCL) (LCL-PLP) inhibited tumor growth by 80–90% after a single dose of 20 mg/kg, whereas PLP in the free form was completely ineffective at the same single dose. To generalize our findings with LCL-PLP, the antitumor activity and side effects of LCL containing synthetic glucocorticoids (LCL-GC) other than PLP were investigated. In addition to PLP, budesonide disodium phosphate, dexamethasone disodium phosphate, and methylprednisolone disodium phosphate were selected based on the difference in their potency to activate the human glucocorticoid receptor. The present study shows that the tumor localization of each GC is governed by the transport capacity of the LCL composed of dipalmitoylphosphatidylcholine, cholesterol, and polyethylene glycol 2000-distearoylphosphatidylethanolamine in a molar ratio of 1.85:1.0:0.15. The antitumor potency of the LCL-GC strongly depends on the potency of the type of GC encapsulated. LCL-encapsulated budesonide disodium phosphate (LCL-BUP) had the highest antitumor activity which is likely due to the much higher potency of BUP encapsulated in LCL versus the other three GC types. The high potency of LCL-BUP confers the risk for occurrence of strong side effects. However, at the dose of 3 mg/kg, LCL-BUP was highly efficacious without the occurrence of adverse effects.

BCL6 represses NFκB activity in diffuse large B‐cell lymphomas

BCL6 is a transcriptional repressor whose deregulated expression plays a key role in diffuse large B-cell lymphomas (DLBCLs). BCL6 expression characterizes one of the two main subtypes (GC type) of DLBCL, while the other (ABC type) is recognized by increased NFkappaB activation. The mechanistic basis of this distinction remains unclear and the BCL6 targets have been only partially explored. Here we describe how NFkappaB activity is increased after BCL6 silencing by shRNA in DLBCL cells, leading us to propose that BCL6 represses NFkappaB activity. We also demonstrate that this repression is brought about by a mechanism involving protein-protein interaction between BCL6 and NFkappaB members, both in vitro and in vivo. Analysis of a series of DLBCLs shows a negative correlation between the expression of NFkappaB target genes and BCL6. This combined approach using silenced cells and a series of human DLBCL samples leads us to a better understanding of the role of BCL6 as an NFkappaB regulator in B-cells.

Expression of liver-enriched nuclear factors and their isoforms in α-fetoprotein-producing gastric carcinoma cells

Alpha-fetoprotein (AFP)-producing gastric cancer (AFP-GC) is a highly malignant variant of adenocarcinoma with aberrant hepatocellular phenotype. A detailed understanding of the regulation of its liver phenotype is lacking. Liver-enriched nuclear factors (LENFs) are implicated in the transcriptional regulation of AFP in the fetal liver. To investigate the regulatory role of LENFs in AFP-GCs, the expression of LENFs including CCAAT/enhancer binding protein (C/EBP)-β, C/EBP-α, hepatocyte nuclear factor (HNF)-1α, HNF-1β and HNF-4α was investigated in 3 cell lines of AFP-GC and 7 cell lines of control GC. The liver activating protein (LAP), an activating isoform of C/EBP-β, was predominantly expressed in AFP-GCs, whereas the liver inhibitory protein (LIP), an inhibitory isoform of C/EBP-β, predominated in the control GCs. HNF-1α was relatively suppressed in AFP-GCs. HNF-4α was expressed in one of three AFP-GC cell lines. C/EBP-α and HNF-1β were expressed at the same levels in both cell types of GC. AFP-GCs expressed a set of hepatocyte-related proteins (e.g., transferrin and albumin) while they still retained the several glandular cell-related proteins (e.g., MUC2). The induction of LIP reduced transferrin expression and induced CEA expression in an AFP-GC line. Collecting these results, it was suggested that the contribution of LENFs, especially isoforms of C/EBP-β, is possibly important in phenotypic regulation of AFP-GCs.

A new type of GC–MS with advanced capabilities

We have combined the benefits of supersonic molecular beam interface and its related fly-through electron ionization (EI) ion source with the advanced features of the Varian 1200L gas chromatography–mass spectrometry (GC–MS) and mass spectrometry–mass spectrometry (MS–MS), resulting in a new and powerful GC–MS platform with record setting performance. Electron ionization of vibrationally cold molecules in the supersonic molecular beams (SMB) (cold EI) provided mass spectra with enhanced molecular ion, yet with good library search results and superior identification probabilities. We found that high GC column flow rates lower the elution temperature for any given compounds. This allows much larger molecules to elute at the maximum temperature of standard columns. We analyzed a mixture of heavy linear chain hydrocarbons all the way to C 84H 170 with a molecular weight of 1179.3 amu, using a 4 m 0.25 mm i.d. column and 32 ml/min He flow rate. Furthermore, we obtained a dominant molecular ion to all these compounds. The lower elution temperatures also greatly enhance the ability to analyze very thermally labile compounds such as carbamate pesticides. The experimental 1200 system is capable of triple quadrupole based MS–MS. We found that MS–MS on the molecular ion is much more effective than on fragment ions, and thus, the enhancement of the molecular ion directly improves the MS–MS sensitivity. Fast GC–MS analysis was also explored, based on very high column flow rate for fast splitless injections without affecting the sensitivity, and on the high system selectivity due to the combination of enhanced molecular ion and MS–MS. We demonstrate a few seconds long GC–MS–MS analysis of diazinon, spiked at 10 ng/g in a mixed fruit and vegetable extract. The feature of enhanced molecular ion provides significant enhancement in the detection sensitivity via SIM and RSIM on the molecular ion. While octafluoronaphthalene (OFN) detection limit of below 1 fg in SIM mode is shown, the harder is the compound analysis by standard GC–MS the greater is the gain in sensitivity by 1200-SMB. For example, the use of the 1200-SMB resulted in an improvement of over 50 times in the RSIM detection limit of cholesterol on its molecular ion at m/ z = 386, and decabromobiphenylether could be analyzed in SIM mode at 1 ppb with S/N > 50 (peak-to-peak noise).

Mprobe 2.0

DNA chips have proven to be effective tools in detecting gene expression levels. Compared with DNA chips using complementary DNA as probes, oligonucleotide microarrays using oligonucleotides as probes have attracted great attention because of their well known advantages. The design of gene-specific probes for each target is essential to the development of oligonucleotide microarrays. We have previously reported the development of a probe design software termed Mprobe 1.0. Here, we present a new version of this software, termed Mprobe 2.0. Several new features are included in Mprobe 2.0. Firstly, a paradox-based sequence database management system has been developed and integrated into the software, which consequently allows interoperability with sequences in GenBank, EMBL, and FASTA formats. Secondly, in contrast to setting a fixed threshold for the secondary structure of probes in Mprobe 1.0 and other related software, Mprobe 2.0 employs a different method. After parameters such as GC type, probe melting temperature and GC contents have been evaluated, candidate probes are sorted by the free energy from high to low value, followed by specificity analysis. Thirdly, Mprobe 2.0 provides users with substantial parameter options in the visual mode. Mprobe 2.0 possesses an easier interface for users to manage sequences annotated in different formats and design the optimal probes for oligonucleotide microarrays and other applications. The program is free for non-commercial users and can be downloaded from the web page http://www.biosun.org.cn/mprobe/ Wuju Li (wujuli@yahoo.com or liwj@nic.bmi.ac.cn).

Investigations of a new field in gas chromatography: Capillary columns with a super-thick layer of stationary liquid phase

Basic characteristics (efficiency, selectivity, non-equilibrium) of capillary columns with a super-thick layer of stationary liquid phase are investigated. In contrast to traditionally used capillary columns with standard stationary phase thickness of 0.1–0.5 um, some new variables are now established. Firstly, the values of relative retention depend on carrier gas linear velocity. Secondly, the asymmetry of chromatographic peaks increased in accordance with the increase in carrier gas velocity. Thirdly, it was theoretically and experimentally shown that dependence of the height equivalent to a theoretical plate (HETP) on carrier gas velocity is linear. The above noted variables are evidences that the new type of GC is realized under these conditions. The use of capillary columns with super-thick layer of stationary liquid phase is practical when the following problems have to be solved: (1) Separation of highly volatile substances; (2) Preliminary concentration of trace compounds from strong diluted samples; (3) Improvements in measurement and accuracy due to the advantages of splitless injection into wide bore columns with super-thick films. Solutions to some analytical tasks while using super-thick stationary liquid phase are shown: (1) Large volume injection into capillary column with sample transfer speed up to 100 μL min −1; (2) Isothermal splitless injection; (3) Separation of low boiling compounds; (4) Separation of polar substances (alcohols).

Identification of novel synthetic organic compounds with supersonic gas chromatography–mass spectrometry

Several novel synthetic organic compounds were successfully analyzed with a unique type of GC–MS titled Supersonic GC–MS following a failure in their analysis with standard GC–MS. Supersonic GC–MS is based on interfacing GC and MS with a supersonic molecular beam (SMB) and on electron ionization of sample compounds as vibrationally cold molecules while in the SMB, or by cluster chemical ionization. The analyses of novel synthetic organic compounds significantly benefited from the extended range of compounds amenable to analyses with the Supersonic GC–MS. The Supersonic GC–MS enabled the analysis of thermally labile compounds that usually degrade in the GC injector, column and/or ion source. Due to the high carrier gas flow rate at the injector liner and column these compounds eluted without degradation at significantly lower elution temperatures and the use of fly-through EI ion source eliminated any sample degradation at the ion source. The cold EI feature of providing trustworthy enhanced molecular ion ( M +), complemented by its optional further confirmation with cluster CI was highly valued by the synthetic organic chemists that were served by the Supersonic GC–MS. Furthermore, the provision of extended mass spectral structural, isomer and isotope information combined with short (a few minutes) GC–MS analysis times also proved beneficial for the analysis of unknown synthetic organic compounds. As a result, the synthetic organic chemists were provided with both qualitative and quantitative data on the composition of their synthetic mixture, and could better follow the path of their synthetic chemistry. Ten cases of such analyses are demonstrated in figures and discussed.

Prognostic significance of CD44 expression in diffuse large B cell lymphoma of activated and germinal centre B cell-like types: a tissue microarray analysis of 90 cases

Background: Gene expression profiling of diffuse large B cell lymphoma (DLBCL) revealed three disease types: germinal centre B cell-like (GC), activated B cell-like (ABC), and a “third” type. Expression of...

Identification of a guanylyl cyclase-activating protein-binding site within the catalytic domain of retinal guanylyl cyclase 1.

Regulation of cAMP and cGMP production is a fundamental step in a broad range of signal transduction systems, including phototransduction. To identify regions within photoreceptor guanylyl cyclase 1 (GC1) that interact with GC-activating proteins (GCAPs), we synthesized the intracellular fragment of GC1, residues 491-1110, as a set of 15 amino acid long, partially overlapping peptides on the surface of individual pins arranged in a microtiter plate format. This pin assay identified 8 peptides derived from different regions of the GC1 intracellular domain that bind GCAPs. Peptide variants containing these sequences were synthesized as free peptides and tested for their ability to inhibit GC1 stimulation by GCAPs. A free peptide,968GTFRMRHMPEVPVRIRIG, from the catalytic domain of GC1 was the strongest inhibitor of GCAP1/GCAP2-mediated activation. In native GC1, this polypeptide fragment is likely to form a loop between alpha-helix 3 and beta-strand 4. When this region in GC1 was replaced by the corresponding sequence of GCAP-insensitive GC type A, GCAPs did not stimulate the GC1 mutant. The corresponding loops in related adenylyl cyclase (AC) are involved in the activating and inhibiting interactions with Gs alpha and Gi alpha, respectively. Thus, despite interacting with different activating proteins, both AC and GC activity may be modulated through their respective regions within catalytic domains.

Nature and distribution of constitutive heterochromatin in fishes, genus Hypostomus (Loricariidae).

Some Hypostomus species were studied concerning the features of the karyotype structure and the constitutive heterochromatin. The karyotype of Hypostomus sp. F from the São Francisco river (Minas Gerais state, Brazil) is now described for the first time. A diversity in the diploid number, ranging from 2n = 68 to 2n = 80, as well as in the karyotype formulae, is evident in this fish group. Two types of heterochromatin, GC- and AT-rich, could be identified with the use of base-specific fluorochromes. In some species heterochromatic bands are mainly located on the centromeric and telomeric chromosomal regions, while in other species they are also observed at interstitial locations. Hypotheses concerning this heterochromatic distribution in Hypostomus karyotypes are discussed. A case of supernumerary heterochromatic segment and a centric fusion appear to be related with two variant karyotypic formulae observed among specimens from the Mogi-Guaçu and São Francisco rivers, respectively. The available data permit us to characterize a divergent karyotypic evolution among the Hypostomus species already analyzed, both at the macro- and microstructural levels, that is, their general karyotype organization and particular features related to chromosomal banding or staining, respectively.