bluuebunny/crossref_metadata_2025 huggingface dataset:
α‐tocopherol, an exogenous factor of adult hippocampal neurogenesis regulation
Authors: Cecchini Tiziana , Ciaroni Sandra , Ferri Paola , Ambrogini Patrizia , Cuppini Riccardo , Santi Spartaco , Del Grande Paolo
Published: July 2003

In previous work, we found that adult hippocampal neurogenesis in rat is affected by vitamin E deficiency. Because vitamin E deficiency is a complex condition involving numerous biological systems, it is possible that its effect on postnatal new neuron production could be mediated by unknown changes in different factors that in turn play a role in this process. To clarify if vitamin E plays a direct role in regulating hippocampal neurogenesis, we studied the neurogenesis in adult control rats and in adult rats under supplementation with α‐tocopherol, the most important compound of vitamin E. The α‐tocopherol level in control and supplemented rats was monitored. Qualitative and quantitative analysis of cell proliferation and death was carried out and expression of immature neuron markers PSA‐NCAM, TUC 4, and DCX was investigated in hippocampus dentate gyrus. α‐Tocopherol levels increased significantly in both plasma and brain after supplementation. Cell proliferation was inhibited in α‐tocopherol‐supplemented rats, the number of dying cells was reduced, and the number of cells expressing the immature neuron markers was increased. The results obtained confirm and extend the idea that vitamin E is an exogenous factor playing a direct role in regulation of different steps of adult hippocampal neurogenesis. Some hypotheses about the possible mechanisms underlying the complex action of α‐tocopherol, related to its antioxidant and molecule‐specific non‐antioxidant properties, are proposed and discussed. © 2003 Wiley‐Liss, Inc.
Phase Separations in Water–Salt Solutions of Polyelectrolyte Complexes Formed by RNA and Polycations: Comparison with DNA Complexes
Authors: Wahlund Per‐Olof , Izumrudov Vladimir A. , Gustavsson Per‐Erik , Larsson Per‐Olof , Galaev Igor Yu.
Published: July 2003

Formation of insoluble polyelectrolyte complexes (PECs) between RNA and polycations was followed by measuring the residual RNA absorbance in the solution after separation of the precipitate. The polycations studied were poly(N,N′‐dimethyldiallylammonium) chloride (pendant type) and 2,5‐ionene bromide (integral type) with quaternary amino groups in every monomer unit. The data obtained were compared with the results of analogous studies of DNA‐containing PECs. This study is a part of a project aimed at the specific separation of plasmid DNA from RNA, a major problem in the preparative isolation of plasmid DNA. We thus deliberately chose a heterogenous RNA sample as it represents the RNA present in a real cell extract. In contrast to the exhaustive precipitation of DNA observed at certain φ values, a significant part of RNA was nonprecipitated at any φ = [+]/[−], that is, at any ratio of positively charged quaternary amino groups and negatively charged phosphate groups. The addition of sodium chloride increased the nonprecipitated fraction of RNA. DNA, on the other hand, was completely precipitated by both polycations at φ > 0.7. The less effective precipitation of RNA was probably due to the presence of a considerable fraction of short‐chained molecules, incapable of forming a sufficient cooperative system of salt bonds with the polycation. This assumption was supported by a separate experiment, in which the precipitation behavior of RNA fractions of different molecular masses was investigated. The same tendency, while less pronounced, was also ascertained for PECs formed by polycations with DNA fractions of different molecular masses. The possibility of using the revealed differences between DNA and RNA behavior for effective precipitation procedure useful in bioseparation is discussed. The difference in the precipitation behavior of nucleic acids of different molecular masses means there is a possibility for developing an enzymatic assay for DNAase and RNAase activity.

The relative residual absorbance of RNA (closed symbols) and DNA (open symbols) remaining in solution after precipitation with 2,5‐ionene bromide as a function of the charge ratio, [+]/[−], determined at different concentrations of sodium chloride (M).

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The relative residual absorbance of RNA (closed symbols) and DNA (open symbols) remaining in solution after precipitation with 2,5‐ionene bromide as a function of the charge ratio, [+]/[−], determined at different concentrations of sodium chloride (M).
Cationic Graft Copolymers as Carriers for Delivery of Antisense‐Oligonucleotides
Authors: Dautzenberg Herbert , Koňák Čestmír , Reschel Tomáš , Zintchenko Arkadi , Ulbrich Karel
Published: July 2003

Self‐assembling systems based on ionic complexes of DNA fragments (36 base pairs), bcl‐2 antisense oligonucleotides (octadecamer), oligophosphates (25 phosphate groups) or acrylic oligomers (18 groups of phosphonic acid) with poly(L‐lysine) (PLL) ($\overline M _{\rm w}$ = 130 000 and 88 000) grafted with short poly[N‐(2‐hydroxypropyl)methacrylamide] (PHPMA) chains ($\overline M _{\rm n}$ = 4 300 or 8 600) were studied by static and dynamic light scattering methods as systems suitable for gene therapy applications. The graft copolymers (GPLLs) with shorter PHPMA grafts ($\overline M _{\rm n}$ = 4 300) provide polyelectrolyte complexes (PECs) with smaller$\overline M _{\rm w}$andRHthan the corresponding GPLLs with longer grafts ($\overline M _{\rm n}$ = 8 600) and the same content of PLL. The lowest aggregation number of 2 was observed for PECs prepared from the GPLL with short grafts and 40 wt.‐% of PLL. The complexes of oligonucleotides and DNA fragments with GPLLs showed quite similar behavior to that with oligophosphates and acrylic oligomer. The complexes prepared from GPLLs containing 40 wt.‐% of PLL and at excess of oligophosphate were stable for at least 48 h under physiological conditions (0.15MNaCl) and in bovine serum albumin solutions (1 mg · mL−1). Additionally, polyanion exchange reactions of the PECs in contact with poly(styrenesulfonate) and DNA were studied in 0.15MNaCl solutions. The oligophosphates in complexes were at least partially substituted with high‐molecular‐weight polyanions. The structure of the initial PECs dominated the PEC structure after the exchange reaction.

The dependence of the molecular weight$\overline M _{\rm w}$(a) and the hydrodynamic radiusRH(b) of complexes of the oligophosphate (OPP) and four graft copolymers (GPLLi,i = 0–3) on the mixing ratioX.

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The dependence of the molecular weight$\overline M _{\rm w}$(a) and the hydrodynamic radiusRH(b) of complexes of the oligophosphate (OPP) and four graft copolymers (GPLLi,i = 0–3) on the mixing ratioX.
Loss of CD59 expression in breast tumours correlates with poor survival
Authors: Madjd Z , Pinder SE , Paish C , Ellis IO , Carmichael J , Durrant LG
Published: July 2003

CD59 (protectin), a phosphatidylinositol‐anchored glycoprotein, is a member of the cell membrane‐bound complement regulatory proteins that inhibits the formation of the terminal membrane attack complex (MAC) of complement. In this study, the expression of CD59 was evaluated in 520 breast carcinomas from patients with a mean follow‐up of 87 months. This expression was correlated with clinicopathological features and patient survival. Marked variation in the intensity of CD59 expression, which correlated with histological grade and Nottingham prognostic index (NPI), was found, with higher expression of CD59 found more often in well and moderately differentiated tumours and those of good prognosis (NPI ≤ 3.4). In contrast, high grade and poor prognosis (NPI > 5.4) carcinomas significantly demonstrated lack of CD59 expression (p < 0.001). Moreover, it was found that the percentage of CD59‐positive cells correlated significantly with patient survival, ie patients with a high percentage of positive cells (>50%) had a better overall survival (p = 0.006). A correlation was also found between the percentage of CD59‐positive cells and tumour type and also the development of distant metastases. No association was found between either the intensity or the percentage of cells expressing CD59 and vascular invasion, lymph node stage, tumour size, patient age or menopausal status. In multivariate analysis, CD59 percentage positivity was of independent prognostic significance with grade and lymph node stage. These findings indicate that loss of CD59 may offer a selective advantage for breast cancers, resulting in more aggressive tumours and conferring a poor prognosis for patients. Copyright © 2003 John Wiley & Sons, Ltd.
Quantitative analysis of colorectal tissue microarrays by immunofluorescence and in situ hybridization
Authors: Jubb AM , Landon TH , Burwick J , Pham TQ , Frantz GD , Cairns B , Quirke P , Peale FV , Hillan KJ
Published: July 2003

The accuracy and reliability of in situ studies may be compromised by qualitative interpretations. Quantitation imposes a greater degree of objectivity, is more reproducible, and facilitates the clarity of definitions. The aim of this study was to validate the utility of laser imaging systems for the in situ quantitative analysis of gene expression in tissue microarrays. Immunofluorescence was employed to quantify the expression of the tumour suppressor p53, a marker of proliferation (Ki67), an endothelial cell marker (CD31), and the mismatch repair proteins human Mut L homologue 1 and human Mut S homologue 2 in an arrayed series of colorectal tissues (n = 110). Quantitative data on this panel of antigens were compared objectively with qualitative scoring of immunohistochemical chromogen deposition. In addition, the expression of vascular endothelial growth factor (VEGF)‐A, placental growth factor, hepatocyte growth factor, and c‐Met mRNA was quantified by phosphor image analysis of in situ hybridization reactions. The quantified data on p53, Ki67, and CD31 expression were significantly associated with the pathologist's score (p ≤ 0.001). While hepatocyte growth factor and placental growth factor were not up‐regulated, c‐Met expression was increased up to 2.5‐fold and the median VEGF‐A expression was elevated 4‐fold (p = 0.003) in this series of colorectal tumours. Laser imaging systems are therefore feasible for high‐throughput, quantitative profiling of tissue microarrays. Copyright © 2003 John Wiley & Sons, Ltd.
Expression of p53, pRB, and p16 in lung tumours: a validation study on tissue microarrays
Authors: Leversha MA , Fielding P , Watson S , Gosney JR , Field JK
Published: July 2003

Tissue microarrays have been created from 326 lung tumours, including 173 squamous cell carcinomas (SCCs) and 132 adenocarcinomas (ADs). In order to evaluate the usefulness of this microarray series, the expression of p53, p16, and Rb proteins was compared by immunohistochemistry on both the tissue microarrays and the corresponding whole sections for all 326 tumours. The presence of replicate punches improved both the yield and the concordance of data relative to the whole section results, so that the consensus score from the replicates agreed with the whole section result in more than 90% of informative tumours. The large number of tumours in this series also allowed significant differences in protein expression patterns to be detected between SCC and AD, the major subtypes of non‐small cell lung carcinoma (NSCLC). SCC had higher levels of p53 staining (67% vs 52% in AD) and substantially increased p16 loss (SCC 75%, AD 53%) combined with greater retention of pRB expression (SCC 86% vs 67% in AD). The strong inverse correlation between p16 and pRB seen in SCC was essentially absent in AD. This study represents the largest single immunohistochemical survey of protein expression for p53, p16, and RB in NSCLCs. Copyright © 2003 John Wiley & Sons, Ltd.
Raman spectroscopy, a potential tool for the objective identification and classification of neoplasia in Barrett's oesophagus
Authors: Kendall Catherine , Stone Nicholas , Shepherd Neil , Geboes Karel , Warren Bryan , Bennett Robert , Barr Hugh
Published: July 2003

Histopathology remains the gold standard technique for the diagnosis of intraepithelial neoplasia (dysplasia) in Barrett's oesophagus, but it is highly subjective and relies on blind biopsy targeting. The aim of this study was to evaluate Raman spectroscopy, a rapid, non‐invasive, molecular, specific analytical technique, for the objective identification and classification of Barrett's neoplasia in vitro. A secondary objective was to demonstrate the need for a rigorous gold standard in the development of new diagnostic techniques. Forty‐four patients with a mean age of 69 years (range 34–89 years) undergoing surveillance for Barrett's oesophagus were included in the study. Three consultant pathologists independently assessed snap‐frozen oesophageal biopsy specimens. Raman spectra were measured on 87 histopathologically homogeneous samples. Spectral classification models were developed using multivariate analysis for the prediction of pathology. Histopathology and Raman classification results were compared. Raman spectral prediction with a consensus pathology classification model gave sensitivities between 73% and 100% and specificities of 90–100%. A high level of agreement (κ = 0.89) was demonstrated between the three‐subset biopsy targeting model and consensus pathology opinion. This compares favourably with the agreement measured between an independent pathologist and the consensus pathology opinion for the same spectra (κ = 0.76). Raman spectroscopy appears to provide a highly sensitive and specific technique for the identification and classification of neoplasia in Barrett's oesophagus. Copyright © 2003 John Wiley & Sons, Ltd.
Expression of nitric oxide synthase isoforms in human liver cirrhosis
Authors: Mohammed Nasser A , Abd El‐Aleem Seham , Appleton Ian , Maklouf Madiha M , Said Mahmoud , McMahon Raymond FT
Published: July 2003

Several mediators of systemic vasodilatation in liver cirrhosis have been reported. Among these is nitric oxide (NO), which has been proposed as one of the main mediators. In this study, sera and liver biopsies were analysed from 15 patients with clinically and pathologically diagnosed liver cirrhosis. In addition, sera from seven and liver biopsies from three healthy controls were used. Serum levels of nitrite (the end product of NO) were measured using the Griess reaction and the expression of the inducible nitric oxide synthase (iNOS) and constitutive nitric oxide synthase (ecNOS) proteins was investigated using immunohistochemistry. This study shows that serum nitrite levels (94 ± 9.8 µmol/l) in cirrhotic patients were significantly (p < 0.05) increased in comparison with the controls (36.6 ± 11.03 µmol/l). iNOS was completely absent from the control group but was highly expressed in the livers from the cirrhotic group. iNOS was seen mainly in the inflammatory cells infiltrating the portal tracts, blood monocyte‐like cells, hepatocytes, sinusoidal cells, and endothelial cells. However, expression of ecNOS was only seen in the vascular endothelial cells of both the control and the cirrhotic groups, but was much higher in the latter. It is therefore clear that NO is augmented in cirrhotic patients and it is mainly produced by induction of iNOS. Moreover, NO up‐regulation is dependent on the inflammatory stage of liver cirrhosis. ecNOS production could be a normal chronic adaptation mechanism of the endothelium to the chronically increased splanchnic blood flow secondary to portal hypertension. In the near future, the appropriate inhibition of NO activity by using NOS‐active agents may provide a novel strategy for the treatment of patients with liver cirrhosis. Copyright © 2003 John Wiley & Sons, Ltd.
Loss of PDCD4 expression in human lung cancer correlates with tumour progression and prognosis
Authors: Chen Yuan , Knösel Thomas , Kristiansen Glen , Pietas Agnieszka , Garber Mitchell E , Matsuhashi Sachiko , Ozaki Iwata , Petersen Iver
Published: July 2003

The programmed cell death 4 gene (PDCD4), a newly identified transformation suppressor, was analysed in lung tumour cell lines and primary lung carcinomas. Reduced PDCD4 mRNA expression was observed in two immortalized lung cell lines and 18 cancer cell lines by northern blot analysis. In the survey of primary lung tumours, PDCD4 cDNA was poorly represented in 47 lung tumours compared with normal lung tissue by cDNA microarray analysis and this poor representation was significantly associated with high‐grade (G3) adenocarcinomas (p = 0.012). Immunohistochemical analysis of 124 primary carcinomas comprising all subtypes demonstrated that PDCD4 protein expression was widely lost in tumour samples (83%) and was negatively related to poor prognosis (p = 0.013). The loss of PDCD4 expression correlated with higher grade and disease stage (p = 0.045 and 0.034, respectively), but not tumour size and nodal status. Similarly to the cDNA data, lack of PDCD4 expression was significantly linked to tumour grade in adenocarcinoma (n = 59, p = 0.048), while in squamous cell carcinoma (n = 58), no relationship between PDCD4 expression and clinicopathological parameters was established. These data suggest that the loss of PDCD4 expression is a prognostic factor in lung cancer and may correlate with tumour progression. Copyright © 2003 John Wiley & Sons, Ltd.
High expression of MDR1, MRP1, and MRP3 in the hepatic progenitor cell compartment and hepatocytes in severe human liver disease
Authors: Ros Jenny E , Libbrecht Louis , Geuken Mariska , Jansen Peter LM , Roskams Tania AD
Published: July 2003

An increase in bile ductular structures is observed in diverse human liver diseases. These structures harbour the progenitor cell compartment of the liver. Since ATP‐binding cassette (ABC) transporters may have a cytoprotective role in liver disease, an immunohistochemical study was performed on human liver specimens from patients with primary biliary cirrhosis (PBC), chronic hepatitis C virus (HCV) infection, submassive cell necrosis, and normal liver. The expression of MDR1, MDR3, BSEP, MRP1, MRP2, and MRP3 was determined using specific antibodies. Dilution series were constructed to determine the critical staining level in order to estimate the factor of up‐regulation. In normal liver, hepatocytes showed canalicular staining for MDR3, BSEP, and MRP2. MDR1 stained the canalicular membrane of hepatocytes as well as that of cholangiocytes. MRP3 showed low immunoreactivity of bile duct epithelial cells and centrilobular hepatocytes only. Normal liver showed no immunoreactivity for MRP1. In diseased liver, the expression of MDR3, BSEP, and MRP2 was relatively stable. In PBC, HCV, and submassive necrosis, the expression levels of MDR1, MRP1, and MRP3 were increased. The strongest immunoreactivity was seen after submassive necrosis, where remaining islands of hepatocytes showed strong canalicular staining for MDR1 and MRP3. Regenerating bile ductules at the interface of portal tracts and necrotic areas stained intensely for MDR1, MRP1, and MRP3. In conclusion, MDR1, MRP1, and MRP3 are up‐regulated in hepatocytes in severe human liver disease. Strong MDR1, MRP1, and MRP3 reactivity is seen in regenerating human bile ductules. Copyright © 2003 John Wiley & Sons, Ltd.
Ets 1 is expressed in capillary blood vessels but not in lymphatics
Authors: Wernert Nicolas , Okuducu Ali‐Fuat , Pepper Michael S
Published: July 2003

Little is known about the mechanisms that regulate lymphangiogenesis and, in particular, about regulation at the transcriptional level. To determine whether parallels exist in the mechanisms of angiogenesis and lymphangiogenesis, the expression of the Ets 1 transcription factor (which has previously been shown to be involved in angiogenesis) was examined in two transgenic mouse lines, namely RipVEGF‐C mice (which develop lymphatic capillaries ectopically around islets of Langerhans) and Rip1Tag2 mice (which develop insulinomas that are not lymphangiogenic). Crossing the two lines results in double transgenic mice that develop lymphatics around insulinomas, which in turn promotes metastasis to regional lymph nodes. By immunohistochemistry, it was found that, in contrast to blood vessels, lymphatic vessels in wild‐type and transgenic mice did not express Ets 1. Immunohistochemical staining for matrix metalloproteinase (MMP)‐2 and MMP‐9 as well as membrane type 1‐MMP (MT1‐MMP/MMP‐14), all of which are encoded by known or potential Ets 1 target genes, showed the same cellular distribution as Ets 1. These findings suggest that the transcriptional regulatory mechanisms of lymphangiogenesis differ from those involved in angiogenesis. Furthermore, if proteases are involved in lymphangiogenesis, these observations suggest that they are different from those considered to be important for blood vessel formation. Copyright © 2003 John Wiley & Sons, Ltd.
Nuclear localization of the metastasis‐related protein S100A4 correlates with tumour stage in colorectal cancer
Authors: Flatmark Kjersti , Pedersen Kjetil Boye , Nesland Jahn M , Rasmussen Heidi , Aamodt Geir , Mikalsen Svein‐Ole , Bjørnland Kristin , Fodstad Øystein , Mælandsmo Gunhild M
Published: July 2003

A large number of experimental studies have linked the S100A4 gene product to the metastatic phenotype of cancer cells and clinical evidence indicates a correlation between S100A4 expression and poor prognosis in several cancer types. The aim of the present study was to analyse the expression of the S100A4 protein in colorectal cancer. Paraffin‐embedded samples from 277 colorectal cancer patients were immunostained with anti‐S100A4 antibody. Cytoplasmic staining was observed in 178 of 277 samples (64%), whereas, unexpectedly, nuclear expression of S100A4 was found in 88 of 277 of the samples (32%). This novel finding was confirmed by western blot analysis of nuclear fractions isolated from frozen tumour tissue. Statistical analysis revealed a significant correlation between nuclear expression of S100A4 and tumour stage at diagnosis, while there was no such correlation between cytoplasmic staining and tumour stage. The nuclear localization of S100A4 in colorectal cancer and its relationship to tumour stage suggest that this protein may be involved in gene regulatory pathways of relevance to the metastatic phenotype of cancer cells. Copyright © 2003 John Wiley & Sons, Ltd.
Immunoreactivity for cyclin D3 is frequently detectable in high‐grade primary gastric lymphomas in the absence of the t(6;14)(p21.1;q32.3) chromosomal translocation
Authors: Pruneri Giancarlo , Fabris Sonia , Fasani Roberta , Del Curto Barbara , Capella Carlo , Pozzi Barbara , Motta Teresio , Andreola Salvatore , Ferreri Andres JM , Ponzoni Maurilio , Viale Giuseppe , Neri Antonino
Published: July 2003

Cyclin D3 plays a pivotal role in controlling the physiological progression from the G1 to the S phase of the cell cycle. Recent data suggest that cyclin D3 may be deregulated in extranodal non‐Hodgkin's lymphomas (NHLs) as a consequence of the t(6;14)(p21.1;q32.3) translocation. The present study investigated for the first time by dual‐colour fluorescence in situ hybridization (FISH) on interphase nuclei and immunohistochemistry the prevalence of the t(6;14) translocation and cyclin D3 immunoreactivity (IR) in a series of 29 stage I–IIE primary gastric NHLs (PGLs). No case showed the t(6;14) translocation. However, in five (17.2%) cases (two extranodal marginal zone lymphomas of MALT type, LGM; one diffuse large‐cell lymphoma with a MALT component, DLCLM; and two diffuse large‐cell lymphomas without a MALT component, DLCL), three to four cyclin D3 signals were detected by FISH. Co‐hybridization with probes specific for the centromeric region and long arm of chromosome 6 indicated trisomy in one case (DLCL), whereas in the remaining four cases the pattern was highly suggestive of the presence of an isochromosome 6p. One (12.5%) case of LGM, six (75%) cases of DLCLM, and seven (53.8%) cases of DLCL (p = 0.0378) were immunoreactive for cyclin D3. Cyclin D3 IR was detected in two (40%) of the five cases with extra cyclin D3 signals and in 12 of the remaining 24 cases (50%, p = 1.000). These results suggest that the t(6;14) may represent a rare event in the pathogenesis of PGL and that cyclin D3 deregulation is most likely the result of epigenetic mechanisms. Copyright © 2003 John Wiley & Sons, Ltd.
Typical genomic imbalances in primary MALT lymphoma of the orbit
Authors: Matteucci Caterina , Galieni Piero , Leoncini Lorenzo , Lazzi Stefano , Lauria Francesco , Polito Ennio , Martelli Massimo F , Mecucci Cristina
Published: July 2003

Primary orbital non‐Hodgkin lymphoma is a mucosa‐associated lymphoid tissue (MALT)‐type extranodal marginal zone lymphoma. Little information is available on its genome as conventional cytogenetics is limited by scarce biopsy material, while fluorescence in situ hybridization (FISH) explores only selected regions. Comparative genomic hybridization (CGH) performs full genomic analysis and is applicable to different sources of DNA, such as fresh and frozen cells, as well as paraffin‐embedded tissues. In this study, CGH was used to analyse primary MALT lymphoma of the orbit. Aneuploidy was identified in six of the ten cases studied. Gains (19) were more frequent than losses (5). The most frequent duplications involved chromosome 3 (common region at 3q24‐qter), as expected in marginal zone lymphoma, and chromosome 6 (common region at 6p21.1–21.3), which is typical of an orbital location. Other chromosome gains were found at 1p, 7, 8q, 9q, 12, 13, 17, 18, 19, 22, and X. Losses were located at 1q, 6q, 9q, 11q, and 13q. Two cases showed isolated duplications of chromosome 6p or 9q. Isolated imbalances were found only in tumours affecting the conjunctiva. Complex aneuploidies were observed in lymphoma of the retro‐orbital tissue. In summary, CGH in orbital MALT lymphoma provided new insights into typical genomic imbalances and underlying pathogenetic mechanisms. Copyright © 2003 John Wiley & Sons, Ltd.
Association of ets‐related transcriptional factor E1AF expression with tumour progression and overexpression of MMP‐1 and matrilysin in human colorectal cancer
Authors: Horiuchi Shina , Yamamoto Hiroyuki , Min Yongfen , Adachi Yasushi , Itoh Fumio , Imai Kohzoh
Published: July 2003

Expression of E1AF/PEA3 (ETV4), an ets family transcription factor, has been implicated in the invasive potential of several cancer cell lines through induction of matrix metalloproteinase (MMP) expression. The aim of this study was to examine E1AF mRNA expression and to determine whether it is correlated with progression of, and/or MMP expression in, human colorectal cancer. Using the semi‐quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR), 100 colorectal cancer tissues were analysed for E1AF mRNA expression. Expression of ER81 (ETV1) and ERM (ETV5), the other two members of the PEA3 subfamily, and Ets‐1 and Ets‐2 was also analysed. The results were correlated with clinicopathological characteristics and MMP expression. Immunohistochemical analysis and an in vitro invasion assay were also performed. E1AF mRNA expression was detected in 62% of the 100 colorectal cancer tissues, but was undetectable or only faintly detected in adjacent non‐tumour tissues. E1AF mRNA was detected in all of the ten liver metastases from colorectal cancers. E1AF expression correlated significantly with depth of invasion, lymphatic and venous invasion, lymph node and distant metastasis, advance in pathological tumour–node–metastasis stage, and recurrence. Patients with E1AF‐positive tumours had significantly shorter overall and disease‐free survival periods than did those with E1AF‐negative tumours (p < 0.0001 and p < 0.0001, respectively). E1AF expression retained its significant predictive value for overall and disease‐free survival in multivariate analysis that included conventional clinicopathological factors (p = 0.0066 and p = 0.0109, respectively). Among the MMPs analysed, expression of MMP‐1 and matrilysin correlated significantly with E1AF expression. In contrast, expression of ER81 and ERM did not correlate with clinicopathological characteristics or the expression of these MMPs. Immunohistochemical expression of E1AF was predominantly observed at the invasive front, where the expression of MMP‐1 and matrilysin and nuclear β‐catenin expression were often co‐localized. Antisense E1AF‐transfected HT‐29 colon cancer cells expressed reduced levels of MMP‐1 and matrilysin and were less invasive in vitro than neo‐transfected HT‐29 cells. The results of this study suggest that E1AF, the expression of which is closely correlated with the expression of MMP‐1 and matrilysin, plays a key role in the progression of colorectal cancer. Copyright © 2003 John Wiley & Sons, Ltd.
Survivin gene expression in early‐stage non‐small cell lung cancer
Authors: Falleni Monica , Pellegrini Caterina , Marchetti Antonio , Oprandi Barbara , Buttitta Fiamma , Barassi Fabio , Santambrogio Luigi , Coggi Guido , Bosari Silvano
Published: July 2003

Survivin is an inhibitor of apoptosis protein, overexpressed in most human malignancies and implicated in mitosis regulation and preservation of cell viability. In order to investigate the prevalence and clinical significance of survivin in early‐stage non‐small cell lung carcinoma (NSCLC), survivin mRNA levels and protein expression were evaluated, using quantitative real‐time RT‐PCR and immunohistochemistry, respectively, in a series of 83 patients with stage I (IA and IB) surgically resected NSCLC. Detectable survivin mRNA levels could be demonstrated in all non‐neoplastic lung tissue samples and in the tumours analysed. Survivin mRNA levels were elevated in 80 carcinomas (96%) compared to normal lung (p = 0.008). Among all tumours, survivin transcripts were present at a higher level in squamous cell carcinomas (p = 0.0022). Cytoplasmic and nuclear immunoreactivity was found in 70% and 80% of tumours, respectively and both were present in 54%. Cytoplasmic immunoreactivity correlated with tumour stage (p = 0.019). Survivin expression levels did not correlate with patient survival. In one specimen, cytoplasmic and focal nuclear immunostaining was observed in dysplastic bronchial squamous metaplasia. These results document that survivin overexpression is almost always present in early‐stage NSCLC, suggesting that this protein may play a role in lung tumourigenesis. This ubiquitous expression makes survivin an appealing new target for novel therapies in lung cancer. In addition, this study also documents that survivin overexpression could be exploited for diagnostic purposes and that quantitative real‐time RT‐PCR can be a useful tool for evaluating survivin activation in NSCLC. Copyright © 2003 John Wiley & Sons, Ltd.
The wt1‐heterozygous mouse; a model to study the development of glomerular sclerosis
Authors: Menke Aswin L , IJpenberg Annemieke , Fleming Stewart , Ross Allyson , Medine Claire N , Patek Charles E , Spraggon Lee , Hughes Jeremy , Clarke Alan R , Hastie Nicholas D
Published: July 2003

In the present study, it is shown that mice heterozygous for wt1 develop glomerular sclerosis and the nature and time course of events leading to the glomerular scarring are determined. Wt1‐heterozygous (wt1het) mice and their wild‐type littermates were closely monitored from birth and plasma levels of urea, creatinine, and albumin were compared with histological data and clinical features. One of the first indications of nephropathy in the wt1het mouse was the development of proteinuria, accompanied by progressive elevation of the plasma levels of urea and creatinine. Subsequently, the mice developed albuminuria, which correlated with thickening of the glomerular basement membrane and fusion of the podocyte foot processes. Glomerulosclerosis was a relatively late event, accompanied by severe albuminuria and loss of WT1, nephrin, CD2AP, and α‐actinin‐4. Copyright © 2003 John Wiley & Sons, Ltd.
β‐catenin abnormalities and associated insulin‐like growth factor overexpression are important in phyllodes tumours and fibroadenomas of the breast
Authors: Sawyer Elinor J , Hanby Andrew M , Poulsom Richard , Jeffery Rosemary , Gillett Cheryl E , Ellis Ian O , Ellis Paul , Tomlinson Ian PM
Published: July 2003

The aim of this study was to assess the expression of IGF‐I and IGF‐II in phyllodes tumours and fibroadenomas and to see if there is any correlation between nuclear β‐catenin expression and IGF‐I and IGF‐II expression in these tumours. In a previous study, it has been shown that Wnt signalling is important in the pathogenesis of phyllodes tumours of the breast. Epithelial Wnt5a overexpression and stromal Wnt2 overexpression were associated with abnormal, nuclear localization of β‐catenin in the stromal cells of these tumours. However, not all tumours with β‐catenin accumulation showed Wnt overexpression. One other possible cause of β‐catenin accumulation is overexpression of insulin‐like growth factors (IGFs), as both IGF‐I and IGF‐II have been shown to stabilize β‐catenin. In this study, 30 fibroadenomas of the breast were assessed for β‐catenin expression using immunohistochemistry and the results were compared with previous data from 119 phyllodes tumours. In situ hybridization was used to assess IGF‐I and IGF‐II expression in 23 phyllodes tumours and 16 fibroadenomas. Nineteen phyllodes tumours (83%) showed widespread overexpression of IGF‐II and 5/23 (22%) showed widespread overexpression of IGF‐I. IGF‐I expression correlated with nuclear β‐catenin staining in phyllodes tumours. Malignant phyllodes tumours showed no or little IGF‐I expression. There was a degree of nuclear β‐catenin expression in the stroma (weak in 33%, moderate in 27%, and strong in 40%) in all fibroadenomas and nuclear β‐catenin staining correlated with IGF‐I overexpression. Extensive IGF‐II overexpression was also found in the majority of fibroadenomas (12/16). These results support the hypothesis that IGF‐I and IGF‐II overexpression may be important in the pathogenesis of fibroepithelial neoplasms of the breast and that IGF‐I overexpression is likely to be contributing to the nuclear β‐catenin localization observed in the tumours. Copyright © 2003 John Wiley & Sons, Ltd.
Measurement of telomere length on tissue sections using quantitative fluorescence in situ hybridization (Q‐FISH)
Authors: Ferlicot Sophie , Youssef Nelly , Feneux Danielle , Delhommeau François , Paradis Valérie , Bedossa Pierre
Published: July 2003

Loss of telomere repeat sequences occurs after each cell division and telomere shortening has been implicated in cellular senescence. The measurement of telomere length might therefore assess the lifespan of a cell. The aim of this study was to set up and validate a technique enabling the assessment of telomere length on tissue sections. Quantitative fluorescence in situ hybridization (Q‐FISH) with telomeric probes was performed on smears and sections from cell preparations or human tissues. The mean fluorescence intensity of telomere spots (FI/spot) was automatically quantified by image analysis. Telomeric restriction fragment (TRF) length was assessed by Southern blotting. There was a positive significant correlation between telomere length, as assessed by Q‐FISH, and TRF length determined by Southern blotting in corresponding samples (p < 0.01, r = 0.6 for tissue and p < 0.01, r = 0.79 for cells). FI/spot was higher on smears than on sections, but pairwise comparison showed a significant correlation both for cells and for tissues (r = 0.77, p < 0.001 for cells and p ≤ 0.01, r = 0.64 for tissue). Finally, since telomere length is expected to shorten with age, FI/spot was assessed in liver samples according to the age of patients: a negative correlation was demonstrated (r = 0.76, p < 0.01). Inter‐assay variation was 7% for Q‐FISH performed on tissue sections and 12% on touch preparations. This study shows that Q‐FISH can be performed with confidence on fixed frozen tissue sections in order to assess telomere length. It is an easy, accurate, and reproducible in situ method for assessing telomeres in the context of cell type and tissue architecture. Copyright © 2003 John Wiley & Sons, Ltd.
Lymphatics and vascular channels; the differences behind similarity
Authors: Jones Pamela F
Published: July 2003

Although sharing many features and functions, there is a mounting body of evidence to suggest that there are subtle differences between lymphatics and vascular channels. New data suggest that Ets‐1 and Ets‐1‐target MMP genes are differentially expressed in lymphatic and vascular channels. Since it is generally accepted that the Ets family of transcription factors play a significant role in the regulation of the expression of angiogenic factors, the finding that Ets‐1 is apparently not involved in lymphangiogenesis may highlight one aspect where angiogenesis and lymphangiogenesis are differently regulated. Copyright © 2003 John Wiley & Sons, Ltd.
Tissue‐based stem cells: ABC transporter proteins take centre stage
Authors: Alison Malcolm R
Published: July 2003

In adult tissues, with the notable exception of the haematopoietic system, the identity of somatic stem cells has often been inferred based on no more evidence than their location. Functional data (clonogenicity) to confirm such an appellation has been absent due to the inability to find appropriate cellular markers with which to isolate live stem cells. Flow cytometry has isolated haematopoietic stem cells based on their ability to exclude Hoechst dye. Named the side population (SP), this phenotype may be a universal marker of stem cells in adult tissues. Copyright © 2003 John Wiley & Sons, Ltd.
Digital Holography for Instantaneous Spray Diagnostics on a Plane
Authors: Burke Jan , Hess Cecil F. , Kebbel Volker
Published: July 2003

We present a measurement technique that is capable of simultaneously determining sizes and positions of multiple transparent droplets in a plane from scattered light features. The technique is largely independent of particle intensity and mutual obscuration. Reflected and refracted light from the droplets in a pulsed laser sheet is recorded holographically to yield the smallest possible probe volume and the largest possible number density. Larger droplets are best analyzed at the image plane; in this case, the droplets appear as two spots (glare points), whose separation is proportional to the droplet diameter. Smaller droplets are easier to analyze at an out‐of‐focus plane, where their images appear as fringe patterns whose spatial frequency is related to droplet size. Photographic techniques allow only one of these planes to be chosen and are therefore not suitable for multidisperse sprays. Optical holography allows to analyze arbitrary depths, but often suffers from low sensitivity and long analysis times. With digital holography, the spray images are captured digitally by a CCD camera and reconstructed numerically; as in optical holography, the particle reconstruction plane can be freely chosen a posteriori to optimize the measurement. We discuss the issues raised by the transition from holographic film to a CCD sensor as the recording medium, and demonstrate the capabilities of the digital technique.
Thermal and metabolic factors affecting bioaccumulation of triazine herbicides by rainbow trout (Oncorhynchus mykiss)
Authors: Tarja Nakari , Kirsti Erkomaa , Marja Luotola , Kari Ennola
Published: July 2003

The bioaccumulation of the triazine herbicides terbutryn and terbuthylazine in rainbow trout was investigated at water temperatures of 4°C, 10°C, and 17°C. The synergetic effect of water temperature and herbicide on fish liver detoxification metabolism was also studied by measuring liver ethoxyresorufin‐O‐deethylase (EROD) and UDP‐glucuronosyltransferase (UDPGT) activities. The bioaccumulation of terbutryn increased significantly with increasing water temperature. However, temperature seemed to have no effect on the bioaccumulation of terbuthylazine. Both chemicals decreased the measured enzyme activities. Also, temperature effects were significant. In all tests the highest liver EROD activities were measured in fish exposed to a water temperature of 4°C, and the activities decreased with increasing water temperature. The thermal effects on fish liver UDPGT activity differed between the herbicides. The highest activities at a water temperature of 4°C were measured in fish exposed to terbutryn, whereas fish exposed to terbuthylazine had the highest liver UDPGT activities at 17°C. Measuring liver detoxification metabolism together with bioaccumulation increases the understanding of bioaccumulation of chemicals in different environmental conditions. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 219–226, 2003.
Cylindrospermopsin‐induced protein synthesis inhibition and its dissociation from acute toxicity in mouse hepatocytes
Authors: Froscio Suzanne M. , Humpage Andrew R. , Burcham Philip C. , Falconer Ian R.
Published: July 2003

The toxicology of the cyanobacterial alkaloid cylindrospermopsin (CYN), a potent inhibitor of protein synthesis, appears complex and is not well understood. In exposed mice the liver is the main target for the toxic effects of CYN. In this study primary mouse hepatocyte cultures were used to investigate the mechanisms involved in CYN toxicity. The results show that 1–5 μM CYN caused significant concentration‐dependent cytotoxicity (52%–82% cell death) at 18 h. Protein synthesis inhibition was a sensitive, early indicator of cellular responses to CYN. Following removal of the toxin, the inhibition of protein synthesis could not be reversed, showing behavior similar to that of the irreversible inhibitor emetine. In contrast to the LDH leakage, protein synthesis was maximally inhibited by 0.5 μM CYN. No protein synthesis occurred over 4–18 h at or above this concentration. Inhibition of cytochrome P450 (CYP450) activity with 50 μM proadifen or 50 μM ketoconazole diminished the toxicity of CYN but not the effects on protein synthesis. These findings imply a dissociation of the two events and implicate the involvement of CYP450‐derived metabolites in the toxicity process, but not in the impairment of protein synthesis. Thus, the total abolition of protein synthesis may exaggerate the metabolite effects but cannot be considered a primary cause of cell death in hepatocytes over an acute time frame. In cell types deficient in CYP450 enzymes, protein synthesis inhibition may play a more crucial role in the development of cytotoxicity. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 243–251, 2003.
Acute toxicity and bioaccumulation of arsenic in tilapia (Oreochromis mossambicus) from a blackfoot disease area in Taiwan
Authors: Liao Chung‐Min , Chen Bo‐Ching , Singh Sher , Lin Ming‐Chao , Liu Chen‐Wuing , Han Bor‐Cheng
Published: July 2003

The general objective of our work was to determine the acute toxicity and bioaccumulation of arsenic (As) in tilapia (Oreochromis mossambicus) from the blackfoot disease (BFD) area in Taiwan. The average concentration of As in pond water ranged from 17.8 to 49 μg L−1. Acute toxicity tests showed that the As concentration that caused toxicity to tilapia ranged from 69 060 μg As L−1, in the 24‐h toxicity test, to 28 680 μg As L−1, in the 96‐h toxicity test. We measured As concentrations in various tissues of tilapia to identify the affinities of tissues for As. Significant correlations were found among the As concentrations in all tissues. The highest bioconcentration factor (BCF) was found in the intestine (maximum value: 2270). The order of BCFs was: intestine > stomach > liver ≃ gill > muscle. Arsenic concentrations in all tissues were allometric, negatively correlating with fish body weight [r2 = 0.63 ± 0.045 (mean ± SE), p < 0.05]. Our results also revealed that As concentrations in muscle tissue were positively correlated with As accumulation in the viscera (r2 = 0.85, p < 0.05). Significantly higher concentrations of As were obtained in the viscera of tilapia [12.65 ± 10.17 μg g−1 dry wt (mean ± SD)] than in the muscle tissue (3.55 ± 0.42 μg g−1 dry wt). Our results suggest that a simple way of reducing the health risk associated with consuming tilapia is to trim and cook the fish properly, that is, removing the viscera of tilapia can greatly reduce the amount of As ingested and consequently reduce the health risks. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 252–259, 2003.
Biochemical and histopathological effects of glyphosate herbicide on Nile tilapia (Oreochromis niloticus)
Authors: Jiraungkoorskul W. , Upatham E. S. , Kruatrachue M. , Sahaphong S. , Vichasri‐Grams S. , Pokethitiyook P.
Published: July 2003

In Oreochromis niloticus that had been exposed for 3 months to sublethal concentrations (5 and 15 ppm) of the commercial glyphosate herbicide (C3H8NO5P) Roundup, the organs exhibited varying degrees of histopathological change. In the gills filament cell proliferation, lamellar cell hyperplasia, lamellar fusion, epithelial lifting, and aneurysm were observed. In the liver there were vacuolation of hepatocytes and nuclear pyknosis. Kidney lesions consisted of dilation of Bowman's space and accumulation of hyaline droplets in the tubular epithelial cells. The structural damages could be correlated to the significant increase (p ≤ 0.05) in aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activities in the second and third months of exposure. The results indicated that long‐term exposure to glyphosate at sublethal concentrations had adverse effects on the histopathological and biochemical alterations of the fish. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 260–267, 2003.
Effect of chronic exposure to lead on estrogen action in the prepubertal rat uterus
Authors: Tchernitchin Nina N. , Clavero Andrea , Mena Miguel A. , Unda Cristina , Villagra Rebeca , Cumsille Miguel , Tchernitchin Andrei N.
Published: July 2003

Lead is a widely spread environmental pollutant known to affect both male and female reproductive systems in humans and in experimental animals. The present study investigated the effect of a chronic exposure to lead on different parameters of estrogen stimulation in the uteri of prepubertal rats. Chronic exposure to lead enhanced some parameters of estrogen stimulation, inhibited other estrogenic responses, while the remainder were unaltered. Estrogen‐induced uterine eosinophilia (24 h), the proportion of uterine eosinophils in the mesometrium (6 h), and luminal epithelial hypertrophy and RNA content (24 h) appeared to be enhanced by lead exposure, compared to lead‐unexposed control animals. Eosinophilia in the endometrium (6 h), the proportion of uterine eosinophils in the endometrium (6 and 24 h), edema in superficial and deep endometria (6 h), luminal epithelial hypertrophy (6 h), and mitotic response (cell proliferation) in all uterine cell types were inhibited by lead exposure, whereas circular myometrial hypertrophy was not significantly modified. The effects of lead exposure on responses to estrogen found in this study showed some differences with those previously reported for acute or subacute exposure to lead. The results revealed an interaction with the different mechanisms of estrogen action in the uterus at various levels, suggesting that some uterine cell types are more sensitive to lead than others. The relevance of the results for lead‐induced infertility is discussed in this article, and possible mechanisms of action are proposed. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 268–277, 2003.