Analysis of telomerase activity and detection of its catalytic subunit,hTERT

The discovery of the enzyme telomerase and its subunits has led to major advances in understanding the mechanisms of cellular proliferation, immortalization, aging, and neoplastic transformation. The expression of telomerase in more than 85% of tumors provides an excellent tool for the diagnosis, prognosis, and treatment of cancer. However, the techniques employed in its detection appear to play a significant role in the interpretation of the results. The telomeric repeat amplification protocol (TRAP assay) has been the standard assay in the detection of telomerase activity and many variations of this technique have been reported. Recent advances in the development of the TRAP assay and the incorporation of techniques that provide a quantitative and qualitative estimate of telomerase activity are assessed in this review. In addition to histological and cytological examination of tissues, distribution patterns of the catalytic subunit of telomerase, hTERT, are frequently used in the prognosis of tumors. The methods involved in the detection of hTERT as a biomarker of cellular transformation are also analyzed.     

Immortalisation of human ovarian surface epithelium with telomerase and temperature-sensitive SV40 large T antigen

Epithelial ovarian cancer is the most common form of gynaecological malignancy. This lethal disease is thought to arise in ovarian surface epithelial (OSE) cells. The biology of these cells is not well understood, due to the limited amount of tissue that can be obtained from a single biopsy and their limited life span in culture. To overcome these problems, we have conditionally immortalised OSE cells with the catalytic subunit of telomerase (hTERT) and a temperature-sensitive form of SV40 Large T antigen (tsT). We have maintained these cells (designated OSE-C2) in culture for more than 100 population doublings after introduction of the immortalising genes. Early passage OSE-C2 cells have a near-tetraploid karyotype and exhibit a dual mesenchymal-epithelial phenotype, with consistent expression of vimentin and variable expression of cytokeratins and type III collagen, and absence of E cadherin expression. OSE-C2 cells proliferate steadily at the permissive temperature of 33 degrees C, but fail to increase in number at the nonpermissive temperature of 39 degrees C. Serum-deprived OSE-C2 cells are stimulated to grow at 33 degrees C by EGF, whereas they are growth inhibited at 33 degrees C by TGFbeta in the presence or the absence of serum. When temperature shifted to the nonpermissive temperature, OSE-C2 cells modulate to a more mesenchymal phenotype, and a proportion of the cells undergo senescence and/or apoptosis. Moreover, at the nonpermissive temperature, the levels of p53 and SV40 Large T antigen diminish, whilst the level of p21 increases, whereas the level of p16 and telomerase activity is unchanged. This experimental system shows that expression of telomerase alone only allows limited proliferative potential of OSE cells; expression of tsT is necessary to maintain these cells in culture for longer periods, perhaps by its ability to inactivate components of the p53/Rb pathway. OSE-C2 cells may be useful in studying the physiology and differentiation of human OSE cells and provide insight into the poorly understood earliest stages of epithelial ovarian cancer.     

Divalent hammerhead ribozyme targeting template region of human telomerase RNA has potent cleavage activity,but less inhibitory activity on telomerase

The template region of human telomerase RNA is a crucial area for regulating telomerase activity and would be a good target for ribozymes. In fact, potent telomerase inhibitory activity of the ribozyme targeting the GUC sequence of the 5(') end of this region (36-ribosome) has been well demonstrated. To search for a more potent ribozyme, we designed a divalent ribozyme to cleave both the phosphodiester bonds following the GUC and the 23 nucleotides downstream of GUA. An in vitro cleavage study showed that this divalent ribozyme cleaved telomerase RNA more efficiently than the 36-ribozyme or the 59-ribozyme to target the GUA. When this ribozyme was introduced into the carcinoma cells, its inhibitory effect on telomerase activity was less than that of the 36-ribozyme. The 59-ribozyme showed minimum activity on telomerase. This implies that, although the divalent ribozyme possesses a potent cleavage activity on hTR in vitro, the 36-ribozyme is most potent to suppress telomerase activity.     

Spatial differences in bacterioplankton composition along the Catalan coast (NW Mediterranean) assessed by molecular fingerprinting

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments was used to compare surface bacterioplankton assemblages along the Catalan coast (NW Mediterranean). Samples from three coastal stations were compared with samples taken inside the Barcelona harbour and open sea samples taken during a cruise. The bacterial assemblage of each sample showed a characteristic and reproducible DGGE fingerprint. Between 17 and 35 bands were detected in each sample, and about 40% of the bands accounted for more than 80% of the band intensity in each sample. The presence of bands as well as their relative intensity was used to compare bacterial assemblages. Clear differences between the harbour samples and the coastal samples were evident during all periods. Marked temporal changes in the bacterial assemblages were detectable for the coastal sites, suggesting seasonal succession of coastal bacterioplankton. During each season, two stations presented a very similar bacterial composition (Barcelona and Masnou) whereas bacterial assemblages in Blanes were slightly different. These differences were consistent with the different hydrography of the area. Diversity indices calculated from DGGE fingerprints were relatively similar for all samples analysed, even though harbour samples were expected to present lower diversity values.     

Do coral-reef fish faunas have a distinctive taxonomic structure?

Do the highly diverse fish faunas that associate with coral reefs have distinguishing taxonomic and ecological characteristics, as proposed by Choat and Bellwood (1991) and Bellwood (1996)? Does a 50?my old (Eocene) fossil fish fauna from Italy represent a coral-reef fish assemblage that provides unique information about the evolution of such assemblages, as claimed by Bellwood (1996)? I compared the structure of the reef fish faunas of adjacent tropical regions rich and poor in coral reefs, in both America and Polynesia, and found that they exhibit no substantive differences in relative species richness among families of typical “coral-reef” fishes. While coral-rich regions have larger reef fish faunas, a variety of factors probably contribute to such differences. Thus coral-reef fish faunas may lack a distinctive taxonomic structure. A similar comparative approach would be useful for assessing whether assemblages of fishes on coral reefs have distinctive ecological characteristics. Based on patterns of habitat use by modern tropical shorefishes, the Italian Eocene fauna includes few definite reef fishes, and may well consist primarily of non-reef fishes preserved in a non-reef habitat. Until we know more about the environment in which those fossils were preserved, that fauna can contribute little to understanding how coral reef fish assemblages have evolved.     

用硅藻群集指数(DAIpo)和河流污染指数(RPId)评价珠江广州河段的水质状况

用硅藻计于1990年采集珠江广州河段的9个采样点的附着硅藻群落．共得硅藻24个属,170个分类单位,其中,适腐性种8个,广腐性种84个,仍腐性种78个．群落相似性聚类分析表明,位于前、后航道中部的员村（st3）,新造（st8）,沙浴村（si10）三点的硅藻群落结构是为相似,向上,下游相似性逐渐减小．用硅藻群集指数（DAIpo）和河流污染指数（RPId）对各采样断面的水质进行分析表明：大盛（st12）和莲花山（st5）两点受污染最轻,属β-寡污带,黄沙点（st2）污染最严重,属α-中污带．对照点鸦岗（St1）也受到一定的污染,属α-寡污带,其它点的污染程度在α-寡污带与β-中污带之间．DAIpo值与BODS存在着显著的负相关（r＝0．86）．珠江广州河段的前、后航道的污染程度相差不大（RPId=56．42,56.26）,均属中度污染．     

The effects of erythropoietin signaling on telomerase regulation in non-erythroid malignant and non-malignant cells

Treatment with erythropoietin (EPO) in several cancers is associated with decreased survival due to cancer progression. Due to the major importance of telomerase in cancer biology we hypothesized that some of these effects may be mediated through EPO effect on telomerase. For this aim we explored the possible effects of EPO on telomerase regulation, cell migration and chemosensitivity in non-erythroid malignant and non-malignant cells.     

Long-term changes in a benthic assemblage associated with artificial reefs

The aim of the study was to evaluate the long-term development of a hard bottom benthic assemblage over a period of 20 years in an area off the mouth of a large river. The artificial reef of Fregene was selected because benthic assemblage data were available for the period 1981–1992. This artificial reef is located in the mid Tyrrhenian Sea, 5 nautical miles north of the two mouths of the Tevere River (Latium, Italy) and 1.5 nautical miles offshore from Fregene (Rome, Italy). The artificial reef was deployed in March 1981 for fisheries enhancement in 10–14 m of water on a sandy-silty seabed. The Tevere River carries suspended materials and a heavy load of organics since it transports Rome’s effluent, resulting in the eutrophic state of area waters. Benthic sampling was conducted in 2001 by SCUBA diving; two standard surfaces of 400 cm² were scraped from the vertical walls of the same uppermost block in four different periods. All organisms were identified and counted. The methodology used is the same as that adopted in the previous periods, so that the 2001 data could be compared with past collected data. The benthic assemblage was analysed by cluster analysis using the Bray-Curtis index and clustered using the group average clustering algorithm. The SIMPER procedure was used to identify those taxa that characterize each station group identified by cluster analysis. Changes in benthic assemblages and hydrological trends of the Tevere River were investigated using the cumulative sum series method. The 20-year development of the benthic community, starting from the new substratum, is composed of different phases characterised by different benthic assemblages. In particular five different phases were distinguished: 1. Pioneer species recruitment (May 1981–June 1981); 2. Mytilus galloprovincialis (mussel) dominance (August 1981–November 1983); 3. M. galloprovincialis regression (July 1984–October 1985); 4. M. galloprovincialis absence (91–92); 5. Bryozoans bioconstruction dominance (2001). The dynamic succession of the observed benthic assemblages exhibited a good relation with the Tevere River flow. The Tevere River flow, and the subsequent sedimentation process, seems to have strongly influenced the benthic assemblage succession of the Fregene artificial reef. Guest editors: G. Relini & J. Ryland Biodiversity in Enclosed Seas and Artificial Marine Habitats     

新疆塔里木拜城地区早白垩世舒善河组孢粉组合

基于新疆塔里木盆地北部拜城卡普沙良河剖面下白垩统舒善河组发现的29属48种孢子花粉的研究,建立舒善河组自下而上的4个孢粉亚组合,分别为Classopollis-Cycadopites-Lygodioisporites-Biretisporites(CCLB)亚组合,Classopollis-Dicheiropollis-Schizaeoisporites-Klukisporites(CDSK)亚组合,Classopollis-Dicheiropollis-Par-visaccites-Lygodiumsporites(CDPL)亚组合和Classopollis-Dicheiropollis-Pinuspollenites-Impardecispora(CDPI)亚组合。舒善河组孢粉植物群以松柏类掌鳞杉科植物占优势以及真蕨类海金沙科的繁盛为主要特征。该孢粉植物群与欧洲、亚洲、美洲和澳大利亚早白垩世尼欧克姆期孢粉植物群可以对比。根据孢粉学证据,舒善河组的时代属于尼欧克姆期或欧特里夫期至巴列姆期。