Ultrastructural and immunohistological characterization of the sirc corneal cell line

Summary A widely utilized rabbit corneal cell line, SIRC, was characterized ultrastructurally and immunohistologically. Although SIRC cells are often described as being of epithelial origin, important ultrastructural and antigenic characteristics indicate that these cells are fibroblastic and not epithelial. SIRC cells lack desmosomes, cytoplasmic filaments, and cytokeratin—structures that are characteristic of corneal epithelial cells. By contrast, the dendritic morphology, presence of vimentin, and the extensive dense accumulations of ribosomes and rough endoplasmic reticulum are consistent with a fibroblastic phenotype. Collectively, the morphology, ultrastructural features, and antigenic composition favor the hypothesis that SIRC cells are fibroblastic cells (keratocytes) and not corneal epithelial cells. This work supported in part by grant EY 07641 from the National Institutes of Health, Bethesda, MD, and an unrestricted grant from Research to Prevent Blindness, Inc., New York.     

Characterization of malignant mesenchymal cell line (UISO-RS-3) derived from a human rhabdomyosarcoma and inhibition by pharmacologic doses of estrogen

Summary A new tumor cell line has been established from a malignant pleural effusion in a 28-yr-old female patient with a primary alveolar rhabdomyosarcoma of the left buttock. The in vitro and in vivo growth characteristics, morphologic features, abnormal karyotype, and immunohistochemical staining pattern indicate that this cell line is comprised of primitive malignant mesenchymal cells derived from a human rhabdomyosarcoma. Receptor studies done on tumors grown in male athymic mice revealed a single class of high affinity saturable cytoplasmic estrogen receptor (Bmax 2.6 fm/mg cytosol protein, Kd 0.34 mM). Likewise, sucrose density gradient analysis demonstrated specific low-capacity, high-affinity estradiol binding predominately in the 8S region. Cell growth in monolayer culture and on soft agar in the presence of estradiol was inhibited by pharmacologic concentrations of estradiol in a dose-responsive manner compared with control. We describe a newly characterized malignant mesenchymal cell line derived from an alveolar rhabdomyosarcoma that is inhibited by pharmacologic doses of estradiol in vitro. These findings suggest further investigation into the mechanism(s) of this estrogen-induced inhibition in rhabdomyosarcomas.     

Gonadotropes in a novel rat pituitary tumor cell line,RC-4B/C. Establishment and partial characterization of the cell line

Summary An epithelial cell line (RC-4B/C) was established from a pituitary adenoma obtained from a 3-yr-old (ACI/fMai × F344/fMai)F1 male rat. Before Year 5 in vitro, RC-4B/C cells could not be viably recovered from cryogenic storage. Recovery of viable cells from cryogenic storage in Year 5 was associated with a more transformed phenotype, including the appearance of endogenous C-type rat retroviral particles. The ultrastructural appearance of the cells was similar to that of differentiated anterior pituitary cell; the cultured cells contained numerous, electron dense, secretory granules, Golgi complexes, and extended arrays of rough endoplasmic reticulum. Immunocytochemical study showed that all cell types present in the rat anterior pituitary gland were present in the cell line. The percentage of luteinizing hormone beta (LHβ) cells in the cell line was higher (19.9%) and that of growth hormone cells was lower (12.2%) than in normal male rat pituitary, whereas the cell line contained a comparable percentage of follicle stimulating hormone beta (FSHβ), prolactin (PRL), ACTH, and thyrotropin beta cells. Radioimmunoassay data demonstrated the PRL content of the cells was comparable to that of normal male rat pituitary gland, whereas the content of LH and FSH was 70- and 800-fold lower, respectively. Assay of specific receptor sites for gonadotropin releasing hormone (GnRH) using Scatchard plots of the data established the RC-4B/C cells contained GnRH receptor sites of the same affinity as in the pituitary gland, but of twofold lower capacity. These data suggest the RC-4B/C cell line warrants further study as a model for the induction and maintenance of the gonadotropic function of the pituitary gland. An abstract of portions of these results was presented at the 8th International Congress of Endocrinology, Kyoto, Japan, 1988. This work was supported in part by grants DK-17631 (E.H.L.), CA-24145 (W.G.B.), CA-31102 (H.G.B.), AG-01753 (D.E.H.) and HD-1778 (M.T.D.) from the National Institutes of Health, Bethesda, MD, and by a grant from the Association pour la Recherche sur le Cancer, France (M.J.). The NIH is not responsible for the contents of this publication nor do the contents necessarily represent the official views of that agency. Jolanta Polkowska was a recipient of a Foundation Simone et Cino del Duca grant.     

Diversity and phytogeography of vascular epiphytes in a tropical–subtropical transition island,Taiwan

We present the first checklist of vascular epiphytes in Taiwan, based on herbarium specimens, literature records, and field observations. Epiphyte phytogeography was analyzed using Takhtajan's modified division in floristic regions. We ascertain the presence of 336 species of vascular epiphytes (24 families, 105 genera) in Taiwan. Pteridophytes contribute most species (171 species), followed by orchids (120 species). Epiphytes contribute 8% to Taiwanese floristic diversity and epiphyte endemism is near 21.3%. The extensive mountain system is probably the most effective driver for epiphyte diversification and endemicity in Taiwan. Phytogeographically, Taiwanese epiphytes exhibit equal affinity to the Malesian region, southern China and Indo-China and Eastern Asiatic regions. However, some species have a disjunctive distribution between Taiwan and SW China and/or E Himalaya, presumably related to low habitat similarity with adjacent China and/or the legacy of Late Quaternary climate change. Vascular epiphyte distribution patterns corroborate the phytogeographical separation of the island of Lanyu from the main island of Taiwan along Kanto's Neo-Wallace Line.     

Strong relationships between vegetation and two perpendicular climate gradients high on a tropical mountain in Hawai‘i

Aim We compared vegetation patterns at high elevation on a tropical mountain with edaphic properties and position along climate gradients to examine this landscape’s potential sensitivity to climate change. Location Our study covers the cloud forest, the ecotone at the cloud forest’s upper limit, and the alpine grassland, on the north‐east corner of windward Haleakalā, Hawai‘i. The study area brackets the mean trade wind inversion (TWI), encompasses a perpendicular, east–west precipitation gradient and includes multiple edaphic contexts. Methods We collected vegetation structure and composition data in 134 plots from 1900 to 2400 m elevation, stratified east to west. We used classification trees to compare species assemblage groups with spatial (elevation, easting, aspect) and edaphic (substrate age, texture, degree slope) variables derived from a 10‐m digital elevation model and a digital geological map. Results The forest line was physiognomically sharp, and a Shipley–Keddy test showed that species distributional limits were aggregated there. Forest line elevation was not consistent, but dropped nearly 200 m from east to west. Indicator taxa for positions above or below the forest line varied from east to west. Hierarchical clustering identified species assemblage groups with significantly different composition that were distributed across the TWI and/or along east–west climate gradients. Classification trees showed that edaphic properties were not well associated with species assemblage groups, but position along two perpendicular climate gradients was. Compositional turnover was detected along both elevational and east–west gradients. Turnover of the cloud forest’s epiphytic community was particularly pronounced across east–west gradients. Lichen abundance was significantly higher at the drier end of the east–west moisture gradient, and bryophyte abundance was higher at the wetter end. Main conclusions Modern spatial patterns suggest that this landscape will respond to changes in moisture balance through changes in species assemblage and structure, especially at the ecotone. Furthermore, ecotone response to climate change may vary from east to west because of differences in species‐specific constraints or climatic context.     

The comparative morphology of pit organs in elasmobranchs

The pit organs of elasmobranchs (sharks, skates and rays) are free neuromasts of the mechanosensory lateral line system. Pit organs, however, appear to have some structural differences from the free neuromasts of bony fishes and amphibians. In this study, the morphology of pit organs was investigated by scanning electron microscopy in six shark and three ray species. In each species, pit organs contained typical lateral line hair cells with apical stereovilli of different lengths arranged in an “organ‐pipe” configuration. Supporting cells also bore numerous apical microvilli taller than those observed in other vertebrate lateral line organs. Pit organs were either covered by overlapping denticles, located in open grooves bordered by denticles, or in grooves without associated denticles. The possible functional implications of these morphological features, including modification of water flow and sensory filtering properties, are discussed. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

The γ-gliadin gene content of a derivative from a somatic hybrid between bread wheat and tall wheatgrass

A PCR-based strategy was applied to obtain the DNA sequence of γ-gliadin open reading frames present in line II-12, a derivative from a somatic hybrid between bread wheat (Triticum aestivum L.) cv. Jinan177 and tall wheatgrass (Lophopyrum ponticum, 10×). A total 50 analysable sequences were obtained, 18 from II-12 and 16 each from the parents. Amplicon length ranged from 720 to 936 bp, corresponding to a putative mature protein of 239–309 residues. The primary structure of these putative proteins comprised five domains, of which only two varied in length. Phylogenetic analyses showed that the mature γ-gliadin sequences fell into four major clades. Group 1 contained sequences shared between II-12 and L. ponticum, suggesting that some L. ponticum γ-gliadin genes are present in the introgression line. Group 3 has five Jinan177 and five II-12 sequences, indicating that II-12 also carries wheat versions of Gli-1. Group 2 and 4 comprised four and two II-12, three and one Jinan177 as well as one and four L. ponticum sequences, respectively. Fewer genes encoding coeliac disease epitopes were present in II-12 than in the wheat donor parent. Three II-12 γ-gliadins and one from the wheat parent contained an odd number of cysteine residues, and two of them had an additional cysteine residue at the amino end of domain V. The possible use of II-2 for improving quality of bread wheat is discussed.     

A simulation test of the resource‐averaging hypothesis of ecotone formation

Abstract. The pattern at an ecotone may indicate the processes that created that ecotone. Such patterns may in turn affect the responses of ecotones to environmental change. The resource averaging hypothesis suggests a process for the development of tree lines that should produce patterns that are modifications of patterns in soil resources. A computer simulation model that embodies the resource averaging hypothesis is used to generate tree‐line patterns. Different spatial patterns in the variation of soil resources are represented in the model. The patterns of tree line computed by the simulation closely correspond to the patterns of soil resources that were input. These patterns are compared to patterns recorded in the field and by aerial photography. For the patterns of soil resources observed at some alpine tree lines, the model cannot produce the kinds of patterns of vegetation observed. Resource averaging alone cannot be an explanation of such tree lines.     

Homogeneous assay for real-time and simultaneous detection of thymidine kinase 1 and deoxycytidine kinase activities

Measurement of thymidine kinase-1 (TK1) and deoxycytidine kinase (dCK) activity may be useful in cancer disease management. Therefore, a one-step homogeneous assay for real-time determination of TK1 and dCK was developed by combining enzyme complementation with fluorescent signal generation using primer extension and a quenched probe oligodeoxyribonucleotide system at 37 °C. Complementation, for producing dCTP and TTP from nucleoside substrates, was carried out by dTMP kinase and/or UMP/CMP kinase and nucleoside diphosphate kinase. dNTP was continuously incorporated into a fixed oligodeoxyribonucleotide primer, template, and probe system, and the fluorescent signal was generated by using the combined actions of primer extension and 5′ exonuclease activity of Thermophilus aquaticus (Taq) DNA polymerase for specific relief of fluorescent quenching. Fluorescence was captured at 1-min intervals using a real-time polymerase chain reaction (PCR) instrument. A horizontal threshold line, crossing all sample relative fluorescent units (RFU) values at the level of the RFU of the blank sample at the end of the assay (i.e., 90 min), was drawn, obtaining RFU measurement data in minutes for each sample. Duplex proof of principle was demonstrated by the independent determination of different amounts of dCK and TK1 in combination. R² values of 0.90 were demonstrated with Prolifigen TK-REA U/L reference values obtained from pathological canine and human serum samples.     

Targeted Integration of Single-Copy Transgenes in Drosophila melanogaster Tissue-Culture Cells Using Recombination-Mediated Cassette Exchange

Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the φC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ∼6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied.