Ecological Analysis of the Fish Distribution in Green Creek, A Spring-Fed Stream in Northern Ohio

Author Institution: Department of Zoology, The Ohio State UniversityThe distribution of fishes in Green Creek, a spring-fed tributary of Sandusky Bay in northern Ohio, was studied from June 1976 through June 1977. A total of 31 species representing 22 genera and 10 families was taken at 8 stations along the length of the creek. Two cold water springs interrupt the longitudinal succession of fishes and cause the stream to be divided into 4 divisions, each with its own ecological characteristics and fauna: I. Upland warm water tributaries with Catostomous commersoni and cyprinids dominant. II. Cold water trout stream produced by stocking Salmo gairdneri near the spring; Coitus bairdi is an abundant native species in this division. III. Marl substrate of low gradient with Catostomous commersoni, cyprinids, darters, and Coitus bairdi. IV. Estuary of Lake Erie with typical lake fishes. The stations with the highest macroinvertebrate biomass also had the highest fish biomass. Various physical and chemical measurements were made, and it was determined that the marl deposits of the springs were not conducive to fish productivity. The distribution of fishes in Green Creek does not follow the classical pattern of longitudinal zonation but is determined by the unusual physical and chemical parameters induced by the springs

Expression levels of GM3 synthase is transcriptionally regulated in HL60 cells differentiated in monocytoid lineage by phorbol esters

INTRODUCTION: During bi-directional differentiation of human myelogenous leukemia cell line HL-60 into monocytoid and granulocytoid lineages, ganglioside GM3 and neolacto series gangliosides (NeuAc-nLCs) are expressed in differentiation direction-specific manner (1). That is, GM3 markedly increases during monocytic differentiation of HL-60 cells induced by 12-O-tetradecanoyilphorbol-13-acetate (TPA), whereas NeuAc-nLCs noticeably increase in granulocytic differentiation induced by all-trans retinoic acid (RA). These observations suggest that the accumulation of specific gangliosides on the cell membrane plays an important role as a trigger in differentiation induction and as determinant of the differentiation direction in human hematopoietic cell lines (1). It is known that two key upstream glycosyltransferases, Lc3Cer synthase and GM3 synthase, play a critical role regulating the glycosphingolipid biosynthesis in HL-60 cells during bi-directional differentiation (2), but the mechanisms controlling expression and activity levels of these enzymes have not yet been elucidated. In this study our attention is directed to investigate the regulation of GM3 synthase activity. MATERIALS AND METHODS: HL-60 cells were grown in RPMI 1640 complete medium at 37\ub0C in a humidified atmosphere enriched with 5% CO2. Granulocytoid differentiation of HL-60 cells was induced by treatment with 1 mM RA for 48 hours; macrophage-like cell differentiation was produced by 4 nM TPA addition to the culture medium for the same period of time. Acidic and neutral glycolipid profiles of control, RA- and TPA-treated cells were quali-quantitatively analysed by HP-TLC and digital scanning of the plates. GM3 synthase activities were determined in control, RA- and TPA-treated cells by an in vitro radioactive assay using 50 mg and 100 mg of the microsomal enriched protein fraction as enzyme source. mRNA expression levels of GM3 synthase gene was determined by RT-PCR. The house-keeping gene encoding for hypoxantine phospho-ribosyl transferase (HPRT) was used as internal standard for quantitative evaluation of the RT-PCR products. RESULTS: After 48 hours RA treatment, a 30% granulocytic differentiation degree of HL-60 cells was evaluated by conventional cytoplasmic/nuclear histochemical staining of the cells. On the contrary, quite 80% of TPA-treated cells showed evident macrophage-like adherent ability and prominent pseudopods, phenotipic markers of differentiation. Indeed, no modification in the glycosphingolipid profiles, in the enzyme activities and in mRNA expression levels of the crucial glycosyltransferases (Lc3Cer synthase and GM3 synthase) were observed in RA-treated cells. On the other hand, in TPA-treated cells there is a sensible increase in ganglioside GM3 content, accompanied by a consistent up-regulation of GM3 synthase activity with respect to undifferentiated and to RA-treated cells. Through quantitative RT-PCR experiments performed on total RNA from undifferentiated, RA- and TPA-treated HL-60 cells, we demonstrate the strict correlation between GM3 synthase activity and its mRNA level: the GM3 synthase transcript is present in equal amount in either undifferentiated and RA-treated cells, but it is dramatically increased (quite 3 times) in TPA-treated cells. These results first give support to a regulation mechanism at the transcriptional level for this enzyme. REFERENCES (1) H. Nojiri et al., Characteristic expression of glycosphingolipid profiles in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60. Blood 64, 2:534-541 (1984); (2) M. Nakamura et al., Total metabolic flow of glycosphingolipid biosynthesis is regulated by UDP-GlcNAc:Lactosylceramide beta-1,3-N-Acetylglucosaminyltransferase and CMP-NeuAc:Lactosylceramide alfa-2,3sialyltransferase in human hematopoietic cell line HL-60 during differentiation. J. Biol. Chem. 267, 33: 23507-23541 (1992)

ISOLATION AND CHARACTERIZATION OF THE GM3 SYNTHASE cDNA FROM HUMAN PLACENTA

It is known that gangliosides have various important biological functions, and their functions as well as their biosynthesis are currently clarified (1, 2). In vertebrates, almost all the ganglio-series gangliosides are synthesized from a common precursor, ganglioside GM3, which has the simplest structure among the major gangliosides. GM3 itself is known to participate in induction of differentiation, modulation of proliferation, signal transduction and integrin-mediated cell adhesion. GM3 synthase (EC 2.4.99.9, ST3Gal V) is the enzyme involved in the last step of GM3 biosynthesis: it catalyses the transfer of a sialic acid moiety from CMP-sialic acid onto lactosylceramide, forming an a2-3 linkage. Whereas GM3 is ubiquitously distributed in the plasma membranes of all eukaryotic cells, GM3 synthase results expressed in a tissue specific manner, especially in brain, placenta, muscle and testis (3). Many important issues, such as human cDNA identification and characterization, genomic structure and regulation of gene expression, are still open. To isolate the coding sequence of the gene of GM3 synthase from human placenta we used the 5\u2019- and 3\u2019-Rapid Amplification of cDNA Ends technology (SMART RACE cDNA Amplification Kit, Clontech) using, as specific primers, oligonucleotides derived from the human GM3 synthase cDNA sequence from differentiated HL60 cells (3). The different PCR products were cloned into the pCR2.1 vector (TA Cloning Kit, InVitrogen) and the nucleotide sequence was determined. A cDNA, showing high sequence homology with that encoding the human GM3 synthase from TPA-differentiated HL60 cells (3), has been successfully isolated and cloned from human placenta. The major difference between these two cDNAs is in the 5\u2019-end, according to the existence of different promoter regions, responsible for tissue-specific expression of the gene. Furthermore, the cDNA from the human placenta contains, upstream and in frame with the ATG indicated as translation initiation site for the GM3 synthase of HL60 cells, another ATG codon inserted in a sequence compatible with Kozak\u2019s rule, suggesting that the protein of the human placenta has an additional portion in NH2-terminus. The complete coding region of the human placenta cDNA is going to be cloned in an expression vector, under the control of the CMV promoter, in order to evaluate its activity. On the other hand, in vitro translation experiments are going to be carried out to define the first start codon. 1) Hakomori S.I. (2000): Glycoconj. J. 17, 627-647 2) Kolter T. et al. (2002): J.Biol.Chem. 277, 25859-25862 3) Ishii A. et al. (1998): J.B.C. 273, 31652-3165

CLONING OF THE GM3 SYNTHASE cDNA FROM HUMAN PLACENTA AND GENOMIC ORGANISATION OF THE GENE

INTRODUCTION: Gangliosides are a large family of sialic acid-containing glycosphingolipids that play important roles in a large variety of biological processes. Both their functions and their biosynthetic pathways are currently clarified (1, 2). In vertebrates, almost all the ganglio-series gangliosides are synthesized from a common precursor, ganglioside GM3, which has the simplest structure among the major ganglioside. GM3 itself is known to participate in induction of differentiation, modulation of proliferation, signal transduction and integrin-mediated cell adhesion. GM3 synthase (EC 2.4.99.9, ST3Gal V) is the enzyme involved in the last step of GM3 biosynthesis: it catalyses the transfer of a sialic acid moiety from CMP-sialic acid onto lactosylceramide, forming an a2-3 linkage. Whereas GM3 is ubiquitously distributed in the plasma membranes of all eukaryotic cells, GM3 synthase results expressed in a tissue-specific manner, especially in brain, placenta, muscle and testis (3). Although its cDNA has been cloned from some mouse (4, 5) and human tissues (3, 6), studies on the genomic structure (7, 8) and on its transcriptional regulation (8, 9) provides contrasting results. MATERIALS AND METHODS: To isolate the complete coding sequence of the gene of GM3 synthase from human placenta we used the 5\u2019- and 3\u2019-Rapid Amplification of cDNA Ends technology (SMART RACE cDNA Amplification Kit, Clontech) using, as specific primers, oligonucleotides derived from the human GM3 synthase cDNA sequence from differentiated HL60 cells (3). The identity of some amplified DNA fragments was confirmed by Southern blot analysis (Gene ImagesTM AlkPhos DirectTM labelling and detection system, Amersham Pharmacia Biotech). The different PCR products were cloned into the pCR2.1 vector (TA Cloning Kit, InVitrogen) and the nucleotide sequence was determined (\u201cProgetto Camilla\u201d, M-Medical). The genomic structure of the human GM3 synthase gene has been determined through a human genome BLAST homology search of the public database (GenBank) using the GM3 synthase cDNA from human placenta as the query sequence. RESULTS: A cDNA, consisting of 2149 bp and showing high sequence homology with those encoding the human GM3 synthase from other human tissues (3, 6), has been successfully isolated and cloned from human placenta. Notwithstanding our approach, our cDNA has not the poli(A) tail. Between our cDNA and the other published ones, the major difference is in the 5\u2019-end, according to the existence of different promoter regions, responsible for tissue-specific expression of the gene. Furthermore, the cDNA from the human placenta contains, upstream and in frame with the ATG indicated as translation initiation site for the GM3 synthase of HL60 cells, another ATG codon inserted in a sequence compatible with Kozak\u2019s rule, suggesting that the protein of the human placenta could have an additional portion in NH2-terminus. The complete and partial coding regions of the human placenta cDNA are going to be cloned in an expression vector, under the control of the CMV promoter, in order to evaluate their GM3 synthase activity. The results of the human genome BLAST homology search of the public database using the GM3 synthase cDNA from human placenta as the query sequence showed that the gene consists of seven exons which span over 28.5 kb, with exons ranging in size up to 1242 bp. All exon-intron boundaries follows the GT-AG rule (10). 1) Hakomori S.I. (2000) Glycoconj. J. 17, 627-647 2) Kolter T. et al. (2002) J. Biol. Chem. 277, 25859-25862 3) Ishii A. et al. (1998) J. Biol. Chem. 273, 31652-31655 4) Kono M. et al. (1998) Biochem. Biophys. Res. Comm. 253, 170-175 5) Fukumoto S. et al. (1999) J. Biol. Chem. 274, 9271-9276 6) Kapitonov D. et al.(1999) Glycoconj J. 16, 337-350 7) Kim K.W. et al. (2001) Gene 273, 163-171 8) Kim S.W. et al. (2002) ) Biochim. Biophys. Acta 1578, 84-89 9) Zeng G. et al. (2003) Biochim. Biophys. Acta 1625, 30-35 10) Breathnach R. and Chambon P. (1981) Annu. Rev. Biochem. 50, 349-38

Experimental evidence of planar channeling in a periodically bent crystal

The usage of a Crystalline Undulator (CU) has been identified as a promising solution for generating powerful and monochromatic $\gamma$-rays. A CU was fabricated at SSL through the grooving method, i.e., by the manufacturing of a series of periodical grooves on the major surfaces of a crystal. The CU was extensively characterized both morphologically via optical interferometry at SSL and structurally via X-ray diffraction at ESRF. Then, it was finally tested for channeling with a 400 GeV/c proton beam at CERN. The experimental results were compared to Monte Carlo simulations. Evidence of planar channeling in the CU was firmly observed. Finally, the emission spectrum of the positron beam interacting with the CU was simulated for possible usage in currently existing facilities

Towards a Geological Information System: the CARGeo System and the Regione Lombardia Geological Database.

In the framework of the national mapping program «CARGNew Italian Geological Map at 1:50.000 scale», Regione Lombardia is generating a detailed map (1:10.000 scale) of its territory. Surveying criteria have been carefully defined in order to produce homogeneous geological maps: geological survey has been performed at the 1:10.000 scale, and data have been stored in a GIS-oriented database. The detailed survey scale improved the geological knowledge: the new maps represent an important tool for territorial planning requirements of public administrations and engineering geologists (e.g. in hydrogeological and seismic risk evaluation). Field geologists performed data input in the geological data base by alternating field campaigns and data input throughout the year, taking advantage of periods when field activities are slackened (i.e. according to climate conditions). In this way, data entry is nearly synchronous with data collection, and field data become quickly accessible. Data entry by the field geologists on one side slows down the field activity, however, it guarantees a precise digitalization of geometric data and a correct attribute assignment, allowing to optimize working time. To allow the data entry to non-GIS-specialized users, we developed an ArcView®-VisualBasic®-MSAccess® application, enabling the simultaneous acquisition of geometric and alphanumeric data. Data base management and cartographic production are performed with ArcInfo®, through specific procedures which, after data reorganization and control (both alphanumeric and geometric), lead to the final cartographic output at different scales. The 1:10.000 geological database is migrated in the ArcSDE structure and prepared for data view, query and download (www.cartografia.regione.lombardia.it/cargweb) using ESRI (ArcIMS) tools. From the 1:10.000 geological database we derived the database for the 1:50.000 CARG maps by both automatic and manual generalization according to the CARG-APAT standards. During the different phases of the project, several problems arose, due to both project organization and data storage system (from data collection in the field to elaboration and digitalization, and, in case, to final publication). – Data collection: the survey activity was divided between «bedrock » and «quaternary» specialists. The double survey provided a high-quality geological description of the territory, but slowed the generation of the data-flow. Based on this experience, the last assigned areas are surveyed by a single geologist, under the supervision of quaternary and bedrock experts. – Users feedback: geologists are normally used to draw their maps on paper; learning how to produce electronic maps can be difficult, and the software tools have to be studied very carefully and present user friendly interfaces. Nevertheless, in our experience, a training period has to be planned, and geologists have to be supported by a GIS expert, who can understand their needs and modify the software accordingly. – System architecture: the ArcView®-VisualBasic®-MSAccess® (Windows platform) – ArcInfo® (UNIX platform) environment, revealed problems in the client-server stability of an earlier version; some unsolved troubles remain, mainly related to the network architecture. The presence in the CARG-Regione Lombardia crew of consultant geologists, experienced and trained in collection, analysis and data entry in the final database, accelerated the critical phases of: – Data base derivation from Regione Lombardia dataset to CARG-APAT standard

GM3 IN THE REGULATION OF THE EXPRESSION AND ACTIVATION OF ErbB-2/EGFR HETERODIMERS

Gangliosides are a large and heterogeneous family of sialic acid containing glycosphingolipids, ubiquitous components of all eukaryotic cell membranes. They can partially segregate, together with cholesterol and specific signaling transduction proteins, such as receptor tyrosine-kinases, into unique more or less stable clusters or microdomains, indicated as \u201cglycolipid-enriched domains\u201d, \u201clipid rafts\u201d, \u201ccaveolae\u201d, contributing to the membrane structure, organization and function (1). Gangliosides are well-characterized modulators of receptor tyrosine-kinase (RTKs) phophorylation and dimerization (2). Our interest is particularly directed to study the relationship between gangliosides and two members of the tyrosine kinase ErbB receptor family: the epidermal growth factor receptor, EGFR or ErbB-1, and the structurally related protein ErbB-2. Unlike EGFR, ErbB-2 is a ligandless receptor: it can be activated by heterodimerization and cross-phosphorylation by other ligand-activated ErbB receptors (3,4). Our previous experimental evidence supports the functional relationship between ErbB-2 and gangliosides, in particular GM3 (5,6). In the present study, using the HC11 mouse mammary epithelial cell line, we investigated the ErbB-2 activation state and its tendency to form stable molecular complexes with EGFR and with ganglioside GM3, before and after EGF stimulation, by co-immunoprecipitation experiments with anti-ErbB-2 antibody and Western blot analyses. As HC11 cells express different ganglioside species, the exclusive association of GM3 with ErbB-2 and EGFR was ascertained by high performance-thin layer chromatography (HP-TLC) and TLC-immunostaining analyses of gangliosides extracted from the immunoprecipitates. Results display that in EGF-stimulated HC11 cells stable and tyrosine-phosphorylated ErbB2/EGFR dimers are formed and that GM3 is the unique ganglioside tightly associated to ErbB-2/EGFR dimers and to EGFR monomers, but not to ErbB2 monomers. In cells not stimulated with EGF a spontaneous but unproductive dimerization of ErbB2 and EGFR takes place and no ganglioside is found associated to the receptors. These observations indicate that the modulation of ErbB2 activation by GM3 may be mediated by EGFR, but that EGF stimulation is indispensable. After ganglioside depletion by [D]-PDMP, phosphorylated EGFR monomers are observed both before and after EGF stimulation, whereas ErbB-2 is present in the monomeric and unphosphorylated state even after EGF stimulation, suggesting that GM3 might have a bivalent key role in modulating the activation of ErbB-2 and EGFR. References 1. Fivaz, M., Abrami, L., Van Der Goot. F.G. Trends Cell Biol. 9(6), 212-213 (1999); 2. Bremer, E.G., Current topics in membranes 40, 387-411(1999); 3. Qian, X., LeVea, C.M., Freeman, J.K., Dougall, W.C., Greene, M.I., Proc. Natl. Acad. Sci. USA 91, 1500-1504 (1994); 4. Gulliford, T.J., Huang, G.C., Ouyang, X., Epstein, R.J., Oncogene 15, 2219-2223 (1997); 5. Sottocornola E., Berra B., and Colombo I., Biochim. Biophys. Acta-Molecolar and cell Biology of Lipids, 1635, 55-66 (2003); 6. Sottocornola E., Misasi R., Mattei V., Ciarlo L., Gradini R., Garofalo T., Berra B., Colombo I., and Sorice M., FEBS J. 273, 1821-1830 (2006)