text: [CANCER RESEARCH 44, 2991-2999, July 1984] Coexpression of Simple Epithelial Keratins and Vimentin by Human Mesothelium and Mesothelioma in Vivo and in Culture Paul J. LaRocca1 and James G. Rheinwald2 Division of Cell Growth and Regulation, Dana-Farber Cancer Institute and Department of Physiology and Biophysics, Harvard Medical School, Boston, Massachusetts 02115 ABSTRACT wanted to determine whether vimentin is present in the cells of the intact mesothelium also, or whether it is only acquired after We have determined the intermediate filament proteins present cell detachment or cultivation, as has been suggested by others in normal and malignant mesothelium in vivo. Pure sheets of (12, 21, 32, 39). The set of keratin proteins synthesized by normal lung mesothelium were obtained by transfer to agar- normal human peritoneal mesothelial cells in culture is different coated slides or by gentle scraping and cytocentrifugation. Cy- from that of stratified squamous epithelial and many glandular toplasmic filament networks in the mesothelium were labeled via epithelial cell types (22, 27, 38, 43). We wished to determine indirect immunofluorescence both by anti-Mr 40,000 keratin and whether mesotheliomas express any keratins different from anti-vimentin antisera. Two-dimensional gel electrophoresis of those of normal mesothelium, inasmuch as squamous cell car the Triton:high-salt-insoluble proteins of normal lung mesothel cinomas often synthesize keratins inappropriate for their cell type ium disclosed the presence of vimentin and all but the largest of origin (23, 43, 44). We report here that normal mesothelial (M, 55,000) of the four simple epithelial keratins synthesized by cells in the intact tissue coexpress keratins and vimentin, that mesothelial cells in culture. Samples of three peritoneal and three the largest keratin M, 55,000 appears to be induced by conditions pleural mesotheliomas were found to contain either all four simple of culture, and that mesotheliomas express vimentin and the epithelial keratins or all but the M, 55,000 keratin. Notably, none same set of keratins as does normal mesothelium. of the keratins characteristic of stratified and many glandular epithelia and their malignant forms was present in these meso-, MATERIALS AND METHODS theliomas. Two mesothelioma samples from which the tumor cells could be obtained free of other cell types were found to Preparation of Mesothelial Tissues. Sheets of normal lung mesothe contain vimentin as well as simple epithelial keratins and to lium were obtained free of underlying connective tissue by a modification synthesize these same proteins during short-term culture. None of the imprinting method (42). A molten solution of 1% agarose (Sea of the mesotheliomas placed in culture grew progressively in Kem) in water was applied to glass slides and dried overnight. The medium optimal for the growth of normal mesothelial cells. These surface of freshly excised human fetal lung was rinsed in isotonic PBS3 data demonstrate that malignant mesothelial cells preserve the and exposed to the air. Just as the mesothelium began to appear dry, an agarose-coated slide was pressed against the lung for several sec normal pattern of intermediate filament protein synthesis, includ onds. When the slide was pulled away, parts of the mesothelial cell layer ing coexpression of simple epithelial keratins and vimentin, and adhered to the agar and were peeled off. Slides were examined under suggest the use of this characteristic as an aid in the identification an inverted microscope with phase contrast optics to identify areas of cells of mesothelial origin. containing large sheets of mesothelium. These were rinsed with PBS to remove any blood cells. Slides were then either briefly air-dried and immersed in cold (-20°) methanol to fix for immunofluorescence micros INTRODUCTION copy, or the agarose and cell material were scraped into Triton:high-salt The mesothelium is the simple squamous epithelium that lines extraction buffer and stored frozen for future electrophoretic analysis. the pleural, pericardial, and peritoneal cavities and covers the Tissues to be used as electrophoresis samples were collected on slides outer surfaces of the organs contained therein (6, 7). Unlike most that had been coated with electrophoretically purified agarose in order to reduce the levels of contaminants which would be stained in the other epithelia, the mesothelium is of embryonic mesodermal subsequent silver staining procedure. As an alternate method for obtain origin. Perhaps because of this the malignant form, mesothe ing small sheets and clusters of mesothelial cells for ¡mmunofluores- lioma, can adopt either sarcomatous or carcinomatous forms, cence, the lung surface was gently scraped with a rubber policeman into often making differential diagnosis difficult (9, 18, 37). PBS. The cell suspension was then diluted 1:1 with serum and applied This laboratory has recently determined the cell type-specific to a glass slide by spinning in a cytocentrifuge. The slides were then air requirements of normal, diploid human mesothelial cells for rapid dried and fixed in cold methanol. For these experiments, lungs were growth in long-term, serial culture (4). Mesothelial cells are obtained from 18 to 22-week-old human fetuses or from adult rats. different from most other normal epithelial cell types in that they Surgical specimens of solid peritoneal and pleural mesotheliomas (kindly provided by Dr. J. M. Corson, Brigham and Women's Hospital) express in culture high levels of 2 different types of intermediate filament proteins: keratins and vimentin (42,44). Because normal were collected within 1 h after resection and minced into 1 cu mm pieces. Some fragments from each tumor were placed in culture, and some were mesothelial cells that detach from the mesothelium and float in frozen in Triton:high-salt buffer until extracted for electrophoresis. Me ascites fluid also contain both vimentin and keratins (4), we sothelioma cells were also obtained from a pleural effusion of mesothe lioma (MS-7). This effusion was found by cytopathological evaluation to 1 Recipient of National Research Service Awards T32 CA09361 and consist of >90% mesothelioma cells. The cells were concentrated by IF32AG05303 ¡n partial support of this investigation. 1 Recipient of grants from the National Cancer Institute and the National Institute on Aging. 3The abbreviation used is: PBS, phosphate-buffered saline (137 mm NaCI:3 HIM Received July 25,1983; accepted March 30,1984. KCI:16 mw HajrtPQt:2muKHJPO*, pH 7.3). JULY 1984 2991 P. J. La/tocca and J. G. Rheinwald centrifugation from 200 ml of effusion. One fourth of the cell pellet was of pure tumor cells were placed into culture in the growth medium extracted for electrophoretic analysis, and the rest was used to initiate described above. cultures. Keratin Nomenclature. Keratins 40KM, 44KM, 52KM and 55KM, the "mesothelial keratins" described in Wu ef a/. (43), are keratins 19,18, 8, Frozen ampuls of cells concentrated from the ascites fluid of a patient and 7, respectively, independently identified as "simple epithelial keratins" with an ovarian surface epithelial neoplasia were thawed and applied to slides by cytocentrifugation for examination by immunofluorescence. This by Moll ef a/. (22). was the same ascites fluid from which the normal mesothelial strain LP- 9 have been derived (4, 43). Indirect Immunofluorescence Microscopy. Anti-M, 40,000 keratin RESULTS antiserum, raised in rabbits, is specific for this particular keratin (43, 44). Anti-stratum corneum keratins antiserum, raised in rabbits, reacts M, 40,000 Keratin Immunofluorescence of Mesothelia. Nor strongly with epidermal keratins and cross-reacts to various degrees mal mesothelium was transferred from the outer surface of a with all the "mesothelial" or "simple epithelial" keratins (43, 44). An anti- fetal human lung to an agarose-coated slide by an imprinting human fibroblast vimentin antiserum was raised in rabbits. An anti- technique described by Whitaker et al. (42). Cell shape, nuclear vimentin antiserum raised in guinea pigs was kindly provided by Dr. position, and intermediate filament content was examined by Werner Franke, German Cancer Research Center, Heidelberg, West phase-contrast microscopy (Fig. 1a) and indirect immunofluor Germany. Mouse monoclonal antibody OC125, which recognizes a sur escence with anti-40KM keratin. Favorable areas of the trans face antigen of ovarian carcinoma cells (1), was obtained from Dr. Robert Bast of this institute. Rhodamine- and fluorescein-conjugated secondary ferred mesothelium were found to contain this keratin in a fibrous antibodies were obtained from Miles and Cappel Laboratories. The network (Fig. 1b) similar in appearance to that of cultured epithe procedures used for indirect immunofluorescence microscopy and pho lial cells. Heterogeneity of staining presumably resulted from tography has been described (4,44). variability in the amount of cytoskeleton transferred to the slide. Cell Labeling, Extraction, and Electrophoresis. Methods were sim In these preparations, the apical portion of the plasma membrane ilar to those previously described (4, 43, 44). Proteins synthesized by adheres to the agarose, and the cells apparently fracture such cultured cells were labeled by a 4-hr incubation with [^SJmethionine (50 that variably amounts of the cytoplasmic contents are removed ßd/ml) (specific activity, ~800 Ci/mmol, MEN Chemicals) in otherwise methionine-free minimal Eagle's medium (Flow Laboratories) supple to the slide, even though the nucleus (or its outline) can be seen in each transferred cell (Fig. 1a). mented with 20% dialyzed fetal calf serum. Labeled cultured cells, agarose slide-collected mesothelium, and minced tumor tissue were Small clusters to large sheets of mesothelial cells could also rinsed with PBS, Dounce homogenized in Triton:high-salt extraction be dislodged by scraping the lung surface with a rubber police buffer (20 mw Tris-HCI, pH 7.4:0.6 M KCI:1% Triton X-100:1 ITIMphenyl- man and depositing the PBS:serum suspension of these cells on methylsulfonylfluoride) at 0°, and then sonicated for 45 sec. Lysates a slide with the aid of a cytocentrifuge (Fig. 1c). Clusters of easily were then centrifuged at 10,000 x g for 45 min at 4°.The insoluble identified mesothelial cells stained positively for the 40KM keratin pellets (containing the keratins and vimentin) were then dissolved in and also revealed details of the cytoplasmic filament network O'Farrell 2-dimensional lysis buffer. The proteins were separated in the (Fig. 1d). This was consistent with the results of our preliminary first dimension by isoelectric focusing (29) and in the second dimension examination of paraffin sections of mesothelium by immunoper- by sodium dodecyl sulfate:polyacrylamide gel electrophoresis (10% ac- oxidase staining (44). The mesothelium was also stained strongly rylamide) (20). High-purity agarose (Sea Kern) was used to hold the by anti-total stratum corneum keratins, an antiserum which isoelectric focusing tube gel in place at the top of the sodium dodecyl cross-reacts with most of the keratins (43, 44) (data not shown), sulfate:polyacrylamide slab gel. The possible presence of basic (isoelec as had been reported previously (5, 36). tric points 7 to 8) keratins was examined by separating the proteins by nonequilibrium pH gradient electrophoresis for 3.5 hr in the first dimen The M, 40,000 keratin was also detected by immunofluores sion, according to the method of O'Farrell ef al. (30). Labeled proteins cence in secondary cultures of mesotheliomas MS-7 (Fig. 1ft) were detected by scintillation fluor-enhanced (Enhance; MEN Chemicals) and MS-10 (data not shown). The mesothelioma cells had a autoradiography. morphology in culture (Fig. 1g) distinct from that of normal Protein Detection in Gels by Silver Staining. An ultrasensitive silver mesothelial cells (Fig. 1e). MS-7 and MS-10 could not grow method (25) was used with the following modifications. After fixation in progressively in the culture medium permissive for long-term 10% glutaraldehyde, the gel was rinsed for at least 48 hr to more growth of normal pleural and peritoneal mesothelial cells and completely remove glutaraldehyde and consequently reduce the back ceased dividing in secondary culture. We initiated 5 other cultures ground staining. Ethanolamine (5 ¿J/ml)was added to the citric acid of solid pleural and peritoneal mesotheliomas, but the only cells fixative to prevent the formaldehyde from overdeveloping the silver (10). Wet gels were then photographed over a light box with Kodak Technical that grew had morphologies and growth requirements of either Pan film (ASA 50), which was developed in a 1:36 dilution of HC-110 for normal mesothelial cells or of normal connective tissue fibro- 8 min. Gels were placed in a solution of 0.02% sodium carbonate and blasts and were not tumorigenic in nude mice. Apparently the stored in sealed plastic bags for future examination. mesothelioma cells present in those 5 tumor specimens could Cell Culture Methods. A population of normal, diploid, human pleural not grow at all under the conditions we used. mesothelial cells (strain HPM-2) was cultured from pleural effusion fluid Detection of Vimentin in Normal Mesothelial Cells by Im withdrawn from an adult female. The normal, diploid, human peritoneal munofluorescence. This laboratory previously had reported that mesothelial strain LP-9 has been described previously (4, 43). LP-9 and normal mesothelial cells present among the cells in an ascites HPM-2 were grown as described (4), in either M199 or M199/ fluid specimen, which had been allowed to attach to a coverslip, MCDB202:15% fetal calf serum (Sterile Systems):hydrocortisone (0.4 contained both keratin and vimentin (4). We wished to confirm Mg/ml; Calbiochem):epidermal growth factor (10 ng/ml; Collaborative Research). In order to maximize keratin synthesis (Fig. 3, b to d), cells this coexpression in ascites fluid mesothelial cells that never had were grown to confluence during one passage in the absence of epider been exposed, even briefly, to culture conditions, and also to mal growth factor. determine whether the cells of intact mesothelium contain vimen Cells from a pleural effusion mesothelioma (MS-7) and from a solid tin. We applied human ascites fluid cells and intact rat lung peritoneal mesothelioma (MS-10) which could be dislodged as clusters mesothelium (dislodged by scraping with a rubber policeman) to 2992 CANCER RESEARCH VOL. 44 Intermediate Filament Proteins of Mesothelium and Mesothelioma Table 1 Keratin types and vimentin coexpression in normal and malignant mesothelia Keratins (40KM/19) (44KH/18) (46K>,) (52IW8) (55KM/7) Vimentin Normal mesothelium8 Pleural v6 +° + - ++ + Pleural (HPM-2) c + ++/-++ + Peritoneal (LP-9) c + + +/- ++ + Malignant mesothelium Pleural (MS-2) Pleural (MS-11) Pleural (MS-7) Peritoneal(MS-4) Peritoneal (MS-5) Peritoneal(MS-10) " Data summarized from Fig. 3 and Refs. 4 and 43. v, in vivo',c, in culture. 0 +, moderate amounts; ++, relatively high amounts; +/-, variable amounts among experiments; -, none present. " Data summarized from Figs. 3 and 4. e Amount in mesotheliomacells indeterminate because of connective tissue present in tumor specimen. ' Relative amounts unknown due to partial proteolysis. slides by cytocentrifugation and double labeled them with anti- synthesized these same proteins in secondary culture (Fig. 3, e vimentin and anti-keratin antisera. As shown in Fig. 2, a to c, all and g). MS-10 contained vimentin, keratins 40KM and 44KM, a mesothelial cells in rat lung mesothelium coexpressed vimentin small amount of keratin 52KM, and what appeared to be proteo- and keratin. Consistent with the previous report from this labo lytic digestion products of 52KM (Fig. 3f). Cultured MS-10 cells ratory (4), some of the ascites fluid cells coexpressed vimentin synthesized vimentin and all 4 simple epithelial keratins (Fig. 3h). and keratin (Fig. 2, d to f). Vimentin-positive, keratin-negative Thus, malignant mesothelial cells may also increase their expres cells in the ascites fluid were assumed to be macrophages (3, sion of 55KM in culture. 11); these cells attached and spread under culture conditions Samples of 3 other solid peritoneal mesotheliomas and 4 other but did not divide (4). The keratin-positive, vimentin-negative pleural mesotheliomas were extracted and analyzed by 2-dimen cells were presumed to be neoplastic cells of ovarian surface sional gel electrophoresis. Three samples gave no interpretable epithelial origin because cells in the sample which stained with spots due to failures of isoelectric focusing or to paucity of the ovarian surface-specific monoclonal antibody OC125 (1) were mesothelioma cells in a predominantly fibrous tumor. Four sam vimentin negative (Fig. 2, g to /) and keratin positive (data not ples could be scored (Fig. 4). Keratins 40KM, 44KM, and 52KM shown). Thus, keratin and vimentin coexpression is a normal were present in all samples, with keratin 55KM present in all but characteristic of mesothelial cells and is retained by cells that one sample (Fig. 4o). One tumor (Fig. 4d) also contained a become detached from the mesothelium. protein migrating in the position of keratin 46KM. Vimentin was Electrophoretic Analyses of Mesothelial and Mesothelioma detected in 3 of the samples, but since these contained connec Cytoskeletal Proteins. The Triton:high-salt-insoluble protein tive tissue, the vimentin content of the tumor cells themselves fraction extracted from normal and malignant mesothelial sam was indeterminate. ples were analyzed by 2-dimensional gel electrophoresis to Proteolysis of vimentin (14, 28) and of keratin 52KM (35) was determine precisely the intermediate filament proteins present extensive in some tumor extracts (Fig. 3f; Fig. 4, a, b, and d), (Table 1). Fetal human pleural mesothelium collected by stripping precluding quantitative estimation of these proteins when it with an agar-coated slide, precluding contamination with under occurred. Evaluation of gels made from extracts of small tissue lying connective tissue, contained vimentin, keratins 40KM and samples was also complicated because the silver staining 44Km, and, in greatest abundance, keratin 52KM (Fig. 3a). Keratin method used to detect very low levels of protein also stained 55KM, synthesized by adult peritoneal mesothelial cells in culture minor impurities in the buffers and in the agarose used to (4, 43) (see Fig. 3, c and d) was not detectable. Normal pleural immobilize the tube gel during the second dimension of electro mesothelial cells (HPM-2) synthesized vimentin and all 4 simple phoresis. These impurities were most common in the M, 56,000 epithelial keratins in culture, negating the possibility that cells of to 70,000 range (eg., cf. Fig. 3 c and d, and see Fig. 3 a and Fig. pleural origin are unable to synthesize keratin 55KM. A rapidly 4 b, c, and d). Despite the staining background, no proteins turning over, basic M, 46,000 keratin which is expressed at migrating at the position of any known keratin (22,43) other than variable levels by mesothelial cells growing in culture (4, 43) (see the simple epithelial keratins were identified in any of the me Fig. 3cO,also was not detected in the in vivo mesothelium. sothelioma extracts. A relatively pure cell suspension of pleural mesothelioma (MS- 7) and clusters of cells teased from a solid peritoneal mesothe DISCUSSION lioma (MS-10) were extracted directly and their intermediate filament proteins compared with those synthesized by cells from We previously had identified vimentin in the normal mesothelial these same tumors after 1 to 2 weeks in culture. MS-7 contained cells present in ascites fluid and rising to higher levels in these vimentin and keratins 40KM, 44KM, and 52KM in vivo and also cells during serial culture (4). Nevertheless, it was necessary to JULY 1984 2993 P. J. LaRocca and J. G. Rheinwald examine mesothelial cells in situ, since it has been proposed that medium permissive for rapid, long-term growth of normal me vimentin may become expressed as a consequence of loss of sothelial cells. This suggests that mesothelioma cells have sig cell-cell contact after detachment from an epithelial sheet (21, nificantly altered growth requirements which are difficult to sat 32). It is also known that some epithelial cell types (12, 39) but isfy in culture. We suspect that this is the reason that we have not others (4, 34) abnormally synthesize vimentin under certain been unable to find any published reports of mesothelioma cell conditions of culture. lines. Normal mesothelial cells are anchorage independent, form We have found by indirect immunofluorescence microscopy of ing large colonies in semisolid medium.4 Thus, there is no basis scraped mesothelium and by 2-dimensional gel electrophoretic at present for culturing mesothelioma cells or selectively identi analysis of pure, agar slide-transfer preparations of mesothelium fying them. that this tissue contains both keratins and vimentin in vivo. In addition to the coexpression of keratins and vimentin in Previous reports of an absence of detectable vimentin in cross- mesothelioma, the specific subset of keratins expressed by sections of chick (36) and mammalian (33) mesothelium by mesotheliomas could be used to aid in the differential diagnosis immunofluorescence microscopy may have been due to the of tumors in the pleural and peritoneal cavities. Tumors of relatively low vimentin content in mesothelial cells compared to stratified epithelial and mammary epithelial origin contain 2 or the high levels present in fibroblasts and endothelial cells and more keratins from a set of more basic (M, 58,000 and 56,000) because the mesothelium is very thin and easily damaged during and more acidic (M, 52,000, 50,000, and 46,000) keratins (ker handling and fixation. Mesothelial cells may express keratins and atins 5 and 6 and keratins 13, 14, and 17, respectively) (22-24, vimentin because they are derived from the embryonic meso- 26, 44). Carcinomas of the colon, stomach, and ovary would not derm (this subject is reviewed in Refs. 9, 18, and 37), inasmuch be distinguishable from mesothelioma by the keratin pattern as most other cell types of mesodermal origin, including mes- alone because these tumors also are restricted to simple epithe enchyme-derived cells and cells of some hematopoietic lineages, lial keratins (22-24). Nevertheless, mesotheliomas are carci- express vimentin (2, 8, 12, 13, 15, 31, 36). noembryonic antigen negative or very weakly positive (5,19, 40, The types of keratins in normal mesothelial tissue are restricted 41 ), while adenocarcinomas of the colon are strongly carcinoem- to the simple epithelial keratins, as we had predicted from the bryonic antigen-positive (17). Ovarian epithelial neoplasms can previous results of this laboratory on peritoneal mesothelial cells be recognized by a specific antibody against a surface antigen in culture (4, 43). Samples of human pleural mesothelium con (1). Our results indicate that vimentin is likely to be present in tained the 40 KM, 44 KM, and 52 KM simple epithelial keratins, mesotheliomas, in contrast to its absence in tumors of all other but very little or no keratin 55 KM. This lack of keratin 55 KMwas epithelial cell types (31). Vimentin has also been found recently not simply a regional difference between pleural and peritoneal in several renal carcinomas (16) which originate from another mesothelial cells, however, because pleural mesothelial cells mesoderm-derived epithelial cell type. The data we have pre expressed the keratin 55 KM in culture. We do not know the sented here suggest that coexpression of simple epithelial ker mechanism of this specific regulation of keratin 55 KMbut it could atins and vimentin is a useful marker for distinguishing normal be the result of very rapid cell division or the abnormal culture and malignant cells of mesothelial origin from those of most other substratum. We had found previously that synthesis and content cell types. of all 4 keratins are controlled coordinately in mesothelial cells in culture as a function of growth rate (4, 34). ACKNOWLEDGMENTS Solid and effusion mesotheliomas and 2 short-term cultured We are very grateful to Dr. J. M. Corson, Brigham and Women's Hospital, for mesothelioma populations contained 3 or 4 of the simple epithe providing mesothelioma samples and pathology reports and for helpful comments on the manuscript. Susan Rehwoldt and Terese O'Connell provided excellent lial keratins and vimentin. Six of 9 surgical samples were suc cessfully analyzed by 2-dimensional gel electrophoretic separa technical assistance with cell culture and preparation of vimentin antiserum. We thank Dr. Oswaldo Alberti, Brigham and Women's Hospital, for providing fetal tion and silver staining. Qualitative determination of the inter human lung samples, Dr. Werner Franke, German Cancer Research Institute, Heidelberg, West Germany, for his gift of guinea pig anti-vimentin, and Dr. Robert mediate filament proteins was possible by this method, but Bast of this institute for a sample of OC125 antibody. 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Cell Bid., 92: of specific keratin subsets and vimentin in normal human epithelial cells—a 299-312,1982. function of cell type and condition of growth during serial culture. In: Cancer 16. Herman, C., Moesker, 0., Kant, A., Huysmans, A., Vooijs, G. P., and Ramaek- Cells 1/The Transformed Phenotype, pp. 217-228. Cold Spring Harbor, NY: ers, F. C. S. Is renal cell (Grawitz) tumor a carcinosarcoma? Evidence from Cold Spring Harbor Laboratory, 1984. analysis of intermediate filament types. Virchows Arch. B Cell Pathd., 44: 73- 35. Schiller, D. L., and Franke, W. W. Limited proteolysis of cytokeratin A by an 83, 1983. endogenous protease: removal of positively charged terminal sequences. Cell 17. Heyderman, E., and Neville, A. M. A shorter immunoperoxidase technique for Biol. Int. Rep., 7: 3,1983. the demonstration of carcinoembryonic antigen and other cell products. J. Clin. 36. Schmid, E. A., Tappscott, S., Bennet, G. S., Croop, J., Fellini, S. A., Hoitzer, Pathol. (Lond.), 30: 138-140, 1977. H., and Franke, W. W. Differential location of different types of intermediate- 18. Klemperer, P., and Rabin, C. B. Primary neoplasms of the pleura. Arch. Pathol., sized filaments in various tissues of the chicken embryo. Differentiation, 75: 77:385-412,1931. 27-40,1979. 19. Kwee, W. S., Veldhuizen, R. W., Golding, R. P., Mullink, H., Stan, J., Donner, 37. Stout, A. P., and Murray, M. R. Localized pleural mesothelioma. Arch. Pathol., R., and Boon, M. E. Histologie distinction between malignant mesothelioma, 34:951-962, 1942. benign lesion and carcinoma metastasis. Evaluation of the application of 38. Tseng, S. C. G., Jarvinen, M. J., Nelson, W. G., Huang, J.-W., Woodcock- morphometry combined with histochemistry and immunostaining. Virchows Mitchell, J., and Sun, T.-T. Correlation of specific keratins with different types Arch. A Pathol. Anat. Histol., 397: 287-299, 1982. of epithelial differentiation: monoclonal antibody studies. Cell, 30: 361-372, 20. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head 1982. of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970. 39. Virtanen, I., Lehto, V. P., Lehtonen, E., Vartio, T., Kurki, P., Wager, O., Small, 21. Lane, E. B., Hogan, B. L. M., Kurkinen, M., and Garrels, J. I. Coexpression of J. V., Dahl, D., and Badley, R. A. Expression of intermediate filaments in vimentin and cytokeratins in parietal endoderm cells of early mouse embryo. cultured cells. J. Cell Sci., 50: 45-63,1981. Nature (Lond.), 303: 701-704,1983. 40. Walts, A. A., Said, J. W., and Banks-Schlegel, S. Keratin and carcinoembryonic 22. Moll, R., Franke, W. W., Schiller, D. L., Geiger, B., and Krepier, R. The catalog antigen in exfoliated mesothelial and malignant cells: an immunoperoxidase of human cytokeratins: patterns of expression in normal epithelia. Cell, 37: study. Am. J. Clin. Pathol., 80: 671-676, 1983. 11-24,1982. 41. Wang, N. S., Huang, S. N., and Gold, P. Absence of carcinoembryonic antigen- 23. Moll, R., Krepier, R., and Franke, W. W. Complex cytokeratin polypeptide like material in mesothelioma: an immunohistochemical differentiation from patterns observed in certain human carcinomas. Differentiation 23: 256-269, other cancers. Cancer (Phila.), 44: 937-943,1979. 1983. 42. Whitaker, D., Papadimitriou, J. M., and Walters, M. N.-l. The mesothelium: 24. Moll, R., Levy, R., Czemobilsky, B., Hohlweg-Majert, P., Dallenback-Hellweg, techniques for investigating the origin, nature and behaviour of mesothelial G., and Franke, W. W. Cytokeratins of normal epithelia and some neoplasms cells. J. Pathol., 732: 263-271, 1980. of the female genital tract. Lab. Invest., 49: 599-610, 1983. 43. Wu, Y.>J., Parker, L. M., Binder, N. E., Beckett, M. A., Sinard, J. H., Griffiths, 25. Morrisey, J. H. Silver stain for proteins in polyacrylamide gels: a modified C. T., and Rheinwald, J. G. The mesothelial keratins: a new family of cyto- procedure with enhanced uniform sensitivity. Anal. Biochem., 777: 307-310, skeletal proteins identified in cultured mesothelial cells and nonkeratinizing 1981. epithelia. Cell, 37: 693-703, 1982. 26. Nelson, W. G., Battifora, H., Santana, H., and Sun, T-T. Specific keratins as 44. Wu, Y.-J. and Rheinwald, J. G. A new small (40kd) keratin filament protein molecular markers of neoplasms with a stratified epithelial origin. Cancer Res., made by some cultured human squamous cell carcinomas. Cell, 25: 627-635, 44: 1600-1603, 1984. 1981. JULY 1984 2995 ••>> ' ^v^ , . >-v% ; f, ^'. •#M , 1¿v / ?'.' -"i * •»il ä • r4 ' f<4V Fig. 1. Morphology and M, 40,000 keratin immunofluoresœnce of human mesothelium in vivo and of mesothelioma cells in culture, a, fetal lung mesothelium transferred to an agarose-coated slide; b, immunofluorescence of same field as a; c, scraped and cytocentrifuged fetal lung mesothelium; cf, immunofluorescence of same field as c; e, normal peritoneal mesothelial strain LP-9 in secondary culture; I, ¡mmunofluorescence of a different LP-9 culture; g, pleural mesothelioma strain MS-7 in secondary culture; h, immunofluorescence of a different culture of MS-7. a, c, e, and g are phase contrast; b, d, f, and h are anti-M, 40,000 keratin immunofluorescence. Bar in a is 50 urn and is the same magnification for 6; bar in c is 50 »mand is the same magnification for d; bar in e is 200 urn and is the same magnification for g; bar in f is 50 ^m and is the same magnification for h. 2996 Intermediate Filament Proteins of Mesothelium and Mesothelioma Fig. 2. Vimentin and keratin immunofluorescence of intact lung mesothelium and of mesothelial cells In ascites fluid, a to c, intact mesothelium scraped from rat lung. Double labeling with guinea pig antivimentin and fluorescein (b) and with rabbit anti-M, 40,000 keratin and rhodamine (c). Small, retractile cells in a are erythrocytes. d to f. human ascites fluid cells from patient with ovarian epithelial neoplasia, double labeled with guinea pig antivimentin and fluorescein (e) and with rabbit anti-stratum comeum keratins and rhodamine (f). g to i, human ascites fluid cells from the same patient, double labeled with rabbit antivimentin and rhodamine (h) and with mouse monoclonal anti-ovarian carcinoma cell surface and fluorescein (/). Bar in a is 50 pm and is the same magnification for b to i. In d, arrows indicate mesothelial cells. In d and g, arrowheads indicate neoplastic ovarian epithelial cells. Vimentin-positive, keratin-negative cells in d to t are presumably macrophages. JULY 1984 2997 bt t a ':v'<ïr--.'.¡'.'i •.-, • .:••?'•;."•'"' v 'T^ v C b b b' ^ 1a 2998 CANCER RESEARCH VOL. 44 Intermediate Filament Proteins of Mesothelium and Mesothelioma K —y » a^~ b c- Fig. 4. Two-dimensional gel electrophoretic separation of mesothelioma cytoskeletal proteins. Triton:high-salt-insoluble proteins of solid tumor samples separated horizontally by charge (loll, basic; right, acidic) and vertically by sodium dodecyl sulfate:polyacrylamide (10%) gel electrophoresis. Gels a and c were focused isoelectrically between pH 6.5 and 5.0. Gels b and d were separated by nonequilibrium pH gradient gel electrophoresis to permit detection of more basic proteins, a, peritoneal mesothelioma MS-4; £>, pleural mesothelioma MS-2; c, peritoneal mesothelioma MS-5; d, pleural mesothelioma MS-11. All gels are silver stained. Lower case letters and arrows within each frame, position of keratins, vimentin, and actin, as in the legend of Fig. 3; arrowheads, proteolytic breakdown products of vimentin and the 52K« keratin. * in frame a, prominent species of size and charge different from that of any known keratin, v, vimentin. Fig. 3. Two-dimensional gel electrophoretic separation of the cytoskeletal proteins of normal human mesothelium and mesotheliomas. Triton:high-salt-insolubte protein fractions of tissues, tumors, and cultured cells separated horizontally by their charge (left, basic; right, acidic) and vertically by sodium dodecyl sulfate;polyacrylamide (10%) gel electrophoresis. Gels a, c, e, and g were isoelectrically focused between pH 6.5 and 5.0 in the horizontal dimension. Gels b, d, f, and h were separated by nonequilibrium pH gradient gel electrophoresis in the horizontal dimension in order to permit detection of more basic proteins, a, human fetal lung mesothelium collected by slide transfer (spots marked with * are agarose contaminants detected by silver staining); b, cultured adult lung mesothelial strain HPM-2; c and d. cultured adult peritoneal mesothelial cell strain LP-9; e, pleural effusion mesothelioma MS-7; f, peritoneal mesothelioma MS-10; g, secondary culture of MS-7; h, primary culture of MS- 10. Proteins in a, d, and e were revealed by silver staining, in frame f by Coomassie blue staining, and in b, c, g, and h by autoradiography of [^Slmethionine-proteins. Lower case letters within each frame, positions of keratins and vimentin: a, 40«M/19;b, 44KM/18; c, 52K«/8;d, 55KM/7; e, 46KM. Arrows, actin. c' is presumed to be a proteolytic digestion product of keratin 52KM/8. v, vimentin. JULY 1984 2999


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What does HRAN stand for?
What does HRAN mean? This page is about the various possible meanings of the acronym, abbreviation, shorthand or slang term: HRAN.
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HRAN | Hispanic Radio Amateur Network | ||||
HRAN | Hospital Regional da Asa Norte | ||||
HRAN | High radio access network | ||||
HRAN | Human Rights for All Now | ||||
HRAN | Himalayan Rescue Association Nepal | ||||
HRAN | Human Rights Agenda Network |
What does HRAN mean?
- Hraň
- Hraň is a village and municipality in the Trebišov District in the Košice Region of south-eastern Slovakia.
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I have been planing a build on a Pontiac Torpedo (though I am in no position to build it just yet). One of the modifications I would like to do is to put the transaxle from a first generation Tempest in the back. This would improve weight distribution. As I would have to shorten the axles I figure I could put a pair of universal joints on it providing fully independent suspension, as in the '65 Corvair. Thing is, the manual transmissions on Tempests where only 3 or 4 speeds, which I do not think is enough. An overdrive kit could double this. I am not entirely sure if the gearbox and axle segments can be separated without screwing up the suspension mounts. Thus I think it might be necessary to put the overdrive unit between the engine and the transaxle. Would this create any problems?
I am as big a Pontiac fan as anyone but, I think this is, generally, a bad idea. They have poor susp geometry, hard to find parts, bearings, etc. Plus, they are weak and more. For sure, if you put enough money and energy into anything you can make it good but, you may wish to choose another direction for a rear end and trans. Best to you whatever you choose to do. Steve
I am assuming by Pontiac "Torpedo" you mean the fastback 2 doors sedn/coupes of '41 and later. If that is correct, I fully agree with Applekrate on all counts. However, if you are determined to do this, I don't see why it is necessary to seperate the trans/final drive units. Seems to me you'll have to do something different than the original 'sagging driveshaft' anyway, as it will be too short. So why not place the overdrive unit just ahead of the driveshaft/transaxle connecting point. Something like a Gear Vendors Overdrive that can be mounted "inline". Further, why not use the far superior Corvair '65/'69 IRS with the Pontiac transaxle? All of this requires A LOT of facbrication, with dubious outcome......but I'm just offering suggestions in case you can't be persuaded otherwise. Basically, as Applekrate indicated, you are pairing components from two completely different categories (weight/size) of vehicles. If you are open to re-thinking this, consider using either Jaguar or Corvette IRS, either of which is readily available, rugged, reduces unsprung weight, better brakes, etc, etc. The overdrive comes with the 2004R or 700R4 or 4L80 or T-5 etc. Ray
I want everything on the car to be either taken from a Pontiac car or new build. And yes, I do know that a Tempest transaxle is just a slightly modified Corvair transaxle. I am fully aware that these modifications will take a great deal of time, effort and money. If I wanted something cheap, quick and easy I would get a Mini Cooper. Mr. Applekrate said the tranaxles from a Tempest are "weak." Could you please clarify this? I understand that the torque converters on the automatics tend to balloon and burst with relatively little power. Would the manual transaxle be capable of handling a ~300 HP engine.
While I applaud your attempt to keep it all-Pontiac, think about the configuration of the Tempest transaxle. Any OD unit would need to go between the trans output shaft and the differential gears. That's pretty tough to do in the stock configuration. While this violates your self-imposed requirement, consider a C5 Corvette transaxle. It uses a T56 or 4L60E as the basis for the trans.
The OD unit could be located behind the engine and ahead of the transaxle unit.........it can be anywhere between the engine and the wheels to be effective. Ray
The trans was basically a standard Saginaw unit. If there is an overdrive version of that trans maybe it could be used? While I no longer remember the particulars, the '63 transaxle was stronger than the '61 - '62. For a heavy car, with 300HP, that is driven hard, the transaxle would be a weak link. In a light car and/or driven normally the transaxle would be adequate but not overbuilt. Since you would need to do something about the solid driveshaft anyway, replace it with a "regular" driveshaft and a custom trans input shaft. That has been done before. Because the driveshaft is on the input side of the trans it doesn't need to be as big/heavy as a "regular" driveshaft. I know you want to keep this all Pontiac, but there are a number of alternatives out there that would be a better option(and in the end cheaper) than the Tempest. I've never understood the idea that it's ok to mix and match parts that were made 50 years apart so long as they were made by the same manufacturer, but not ok to use an equally unrelated part made by a different manufacturer. I know that's another discussion, that some are just hung up on the concept, and that it's your car, so do as you wish.


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Are the memory optimizations going to be in 3.15? The one done by curaga.
Phoronix: Radeon DRM Kernel Driver Gets More Changes With Linux 3.15
A Radeon pull request for drm-next changes was sent in this morning for ultimately landing with the Linux 3.15 kernel...
Are the memory optimizations going to be in 3.15? The one done by curaga.
There was no news in his topic. Did he actually implement anything?
He's still working on the trainer: https://github.com/clbr/hotbos
No, I blew away all your money on hookers, blow and bitcoin. No refunds.
I found a bug, and the fix has gone into 3.14 and several stable versions. Some of the work is currently on dri-devel, looking to be for 3.16. For the rest, it'll take a few weeks to implement and benchmark.


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Bears, Tim Jennings agree to terms
CHICAGO -- The Bears took care of some in-house business before the official start of NFL free agency by re-signing veteran cornerback Tim Jennings to a two-year deal.
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Jennings was set to be an unrestricted free agent after starting 28 games for the Bears the past two seasons.
Signed by the Bears prior to the 2010 regular season, Jennings began as a reserve, but overtook Zack Bowman for the starting cornerback spot opposite Charles Tillman in Week 4.
Although Jennings managed just three interceptions the past two seasons, he did develop the reputation as a solid run-stopping cornerback, registering 153 tackles, six tackles for a loss and two forced fumbles.
The Bears still might choose to try and upgrade at cornerback, either in free agency or the draft, but with fellow corners Bowman and Corey Graham also about to hit free agency, bringing back one of the three made sense. Jennings also could play the nickel position if necessary.
Jeff Dickerson covers the Bears for ESPNChicago.com and ESPN 1000.
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Statements about State Finances
The Associated Press
— Georgia
Rhode Island Center for Freedom and Prosperity
"Common welfare programs give income of $43,330 or $20.83 an hour."
— Rhode Island
Greta Van Susteren
New Jersey has had "seven credit downgrades" since Chris Christie became governor.
— PunditFact
Allan Fung
Gina Raimondo "paid 70 million in taxpayer dollars to millionaire hedge fund managers."
— Rhode Island


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Arizona Cardinals
Updated Dec 31, 2012 - 3:36 pm
Cardinals Press Conference Video
GM Rod Graves and head coach Ken Whisenhunt have been fired by the Arizona Cardinals.
Watch team president Michael Bidwill's press conference here.
Watch live streaming video from nflcardinals at livestream.com
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Cardinals Audio
- Matt Maiocco- CSNBayArea.com 49ers beat writer - Tuesday March 3Matt Maiocco explains why the San Francisco 49ers have interest in Darnell Dockett.
- Dave Pasch- ESPN play-by-play announcer - Monday March 2Dave Pasch believes that Steve Keim doesn't want Adrian Peterson to get away again.
- Darren Urban, AZCardinals.com - Friday February 27Darren Urban discusses Darnell Dockett's release from the Arizona Cardinals.
- Daniel Jeremiah- NFL Network analyst - Friday February 27Daniel Jeremiah had a near death experience involving Steve Keim and Bruce Arians.
- Bickley Blast - Thursday February 26Adrian Peterson and Dez Bryant may have more in common than we thought.


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Away: LA Dodgers [58-49 SU] Home: Chi. Cubs [49-59 SU]
Game Time: 4:05 PM Stadium: Wrigley Field
Recent Meetings: 08/01/13 - LA 6 vs. CHC 4
Team Leaders: LA Dodgers: Home Runs: N/A Batting Avg: N/A Stolen Bases: N/A
Chi. Cubs: Home Runs: N/A Batting Avg: N/A Stolen Bases: N/A
More: Live Odds In-Depth Matchup Information Consensus Betting Information
No Kemp, Hanley, or Crawford for the Dodgers tonight. Travis Wood has had a very solid year and has been pitching better than Ryu.
The Dodgers ML is dropping even though everyone is on the dodgers. I'm taking the Chicago Cubs


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Archives :: Chem Ideas
Displaying 321 - 340 of 879 stories.
March 26, 2012
Topping the headlines last week, share buybacks and dividends are increasing as chemical companies continue to run up large cash balances. Over 60% of cash flow from operations was...http://www.chemweek.com/chem_ideas/Vincent-Valk/Chemical-Finance-Digest-Mar-19-23_42313.html March 23, 2012
The rapid transformation of the North American petrochemicals outlook will dominate discussions at key industry conferences over the next few weeks, including the American Fuel and...http://www.chemweek.com/chem_ideas/Rob-Westervelt/Petchem-Revival_42254.html March 23, 2012
This Week on CW: Growth in the chemical industry in China is slowing, but still strong, and higher than in developed economies. Most analysts say the country can avoid a...http://www.chemweek.com/chem_ideas/Vincent-Valk/Chemical-Industry-Weekly-News-Round-Up-Mar-23_42276.html March 23, 2012
Many Western pharmaceutical companies – and indeed pharma fine chemical contract manufacturers - have a tough road to tread. As Aslam Malik, president of Ampac Fine Chemicals...http://www.chemweek.com/chem_ideas/Alex-Scott/Fine-Chemicals-Why-High-Tech-Manufacturing-Really-is-the-Place-to-Be_42251.html March 20, 2012
Topping the headlines last week, Wacker Chemie reported higher volumes and sales for 2011, but lower Ebitda and net income. The company also expects a further Ebitda decline for this...http://www.chemweek.com/chem_ideas/Vincent-Valk/Chemical-Finance-Digest-Mar-12-16_42128.html March 20, 2012
AMPAC FINE CHEMICALS: RELYING ON TECHNOLOGY CAPABILITY TO DRIVE GROWTH Alex Scott Ampac Fine Chemicals (AFC; Rancho Cordova, CA), the wholly owned pharmaceutical fine chemicals...http://www.chemweek.com/chem_ideas/Guest-Author/PharmaFine-Chemicals-Roundup-March-20_42121.html March 19, 2012
Opportunities on a scale not seen in decades are emerging for chemical makers, David Weidman, chairman and CEO of Celanese told attendees in accepting the Society of the Chemical Industry (SCI) American Section's Chemical Industry Medal in New York last week....http://www.chemweek.com/chem_ideas/Rob-Westervelt/New-Realities_42073.html March 16, 2012
This Week on CW: Shell Chemical says it is evaluating a site in Pennsylvania for a new, world-scale ethane cracker that will mostly serve the U.S. northeast. The company has...http://www.chemweek.com/chem_ideas/Vincent-Valk/Chemical-Industry-Weekly-News-Round-Up-Mar-16_42093.html March 16, 2012
This week, BASF has made yet another advance in the lithium battery materials sector through its licensing in of lithium iron phosphate (LFP) battery materials from LiFePO4+C...http://www.chemweek.com/chem_ideas/Alex-Scott/Chemical-Industry-Weekly-Innovation-News-Round-up-March-16_42084.html March 14, 2012
by Hannu Vornamo, Secretary General, Helsinki Chemicals Forum The international community has been looking to enhance chemicals safety on a continuous basis for a long while now...http://www.chemweek.com/chem_ideas/Guest-Author/Helsinki-Chemicals-Forum-to-Focus-on-Internationalization-of-Reach_41981.html March 13, 2012
U.S. Bill Would Fund FDA Pharma Inspections By Kara Sissell Several U.S. lawmakers have introduced a bill that would result in more resources for FDA officials to conduct inspection of...http://www.chemweek.com/chem_ideas/Guest-Author/PharmaFine-Chemicals-Roundup-March-13_41963.html March 13, 2012
by Girish Malhotra, president of Epcot International (Pepper Pike, OH) During my career I have been involved in process simplification and cost reduction of different chemistries...http://www.chemweek.com/chem_ideas/Girish-Malhotra/Neglected-Tropical-Disease-Drugs-What-are-they-Telling-Us-About-Innovation_41922.html March 9, 2012
No single major innovation or technology development stand out this week - but there are some fascinating developments across a range of fronts. In plant biotechnology, Bayer...http://www.chemweek.com/chem_ideas/Alex-Scott/Chemical-Industry-Weekly-Innovation-News-Round-up-March-9_41900.html March 5, 2012
By Nadim Chaudhry, CEO of Green Power Conferences We polled more than 100 leaders in the industry for their thoughts and predictions about the biofuels and biochemicals...http://www.chemweek.com/chem_ideas/Guest-Author/Survey-Future-Investments-in-Biofuels-will-be-in-Asia_41789.html March 2, 2012
This Week on CW: Some late earnings trickled in this week, and the news was mixed, though leaning negative. DSM's fourth-quarter profits fell 43% due to flagging economic...http://www.chemweek.com/chem_ideas/Vincent-Valk/Chemical-Industry-Weekly-News-Round-Up-March-2_41783.html March 2, 2012
So called megatrends associated with earth's growing human population have been in evidence this week: In the agchems field the adoption of GM crops by area grew 8% in 2011 to 160...http://www.chemweek.com/chem_ideas/Alex-Scott/Chemical-Industry-Weekly-Innovation-News-Round-up-March-2_41772.html February 28, 2012
Topping the headlines last week, BASF reported a small increase in fourth-quarter net income, as the company was hit by economic weakness in Europe. Net income totaled €1.13...http://www.chemweek.com/chem_ideas/Vincent-Valk/Chemical-Finance-Digest-Feb-20-24_41659.html February 28, 2012
by Andy Talkington and Jim Aslaksen, Senior Client Partners and co-leaders of Korn/Ferry International's Global Chemicals Practice Within a few years, a large wave of...http://www.chemweek.com/chem_ideas/Guest-Author/Senior-level-Retirements-Set-to-Take-their-Toll-on-the-Chemical-Industry_41640.html February 28, 2012
PHARMA FINE CHEMICAL SUPPLY CHAIN UNDER THREAT Alex Scott Industry experts, including Tom Beil, v.p./quality and regulatory affairs at Sigma-Aldrich Fine Chemicals (SAFC), say the fine...http://www.chemweek.com/chem_ideas/Guest-Author/PharmaFine-Chemicals-Roundup-February-28_41638.html February 24, 2012
This Week on CW: BASF's fourth-quarter earnings were adversely impacted by economic weakness, but the company still showed profitability and forecasts growth for this...http://www.chemweek.com/chem_ideas/Vincent-Valk/Chemical-Industry-Weekly-News-Round-Up-Feb-24_41588.html |
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Design Glossy Black FinishAudio-
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Save the music you have collected and play it at work, in your car, or at home on your CD player. Record sound files from analog or digital sources, clean-up the audio, organize the tunes into playlists, and write a standard Redbook audio CD that can be played in any CD player.
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Kopin Corporation To Announce Second-Quarter 2012 Financial Results On Tuesday, August 7
Kopin Corporation (NASDAQ: KOPN) plans to announce its second-quarter 2012 financial results before the market opens on Tuesday, August 7, 2012 and host a conference call for investors and analysts that day at 9:00 a.m. ET.
On the call, President and Chief Executive Officer Dr. John C.C. Fan and Richard Sneider, the Company's Treasurer and Chief Financial Officer, will discuss Kopin's financial results, strategy and business outlook.
To participate, please dial (877) 407-5790 (U.S. and Canada) or (201) 689-8328 (International). The call will also be available as a live and archived audio webcast on the "Investors" section of the Kopin website, www.kopin.com.
About Kopin
Kopin Corporation is developing Golden-i®, a proprietary voice-activated, cloud computing, wireless, hands-free technology with microdisplay capabilities for use in headset computers. Kopin's unique HBTs (heterojunction bipolar transistors), which help to enhance battery life, talk time and signal clarity, have been integrated into billions of wireless handsets as well as into WiFi, VoIP and high-speed Internet data transmission systems. Kopin's proprietary technologies are protected by more than 200 global patents and patents pending. For more information, please visit Kopin's website at www.kopin.com and www.mygoldeni.com. Kopin, CyberDisplay, Golden-i and The NanoSemiconductor Company are trademarks of Kopin Corporation. Kopin – The NanoSemiconductor Company™Check Out Our Best Services for Investors
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S.V.A.T. Astatine is the study association for Advanced Technology. We are there for our students and are run by our students.
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Still looking for a suit to wear or lacking dance moves? Come to the board room for more info on the other events that will be held in preparation of the prom.


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Poor Canada is all I can say. It really is not that bad, but as you say, maybe after all the excitement of seeing your mum and the bus journey maybe it is not all that great.
I hope the next installment may be a little brighter for the sake of Canada.
Trip Start
Jun 01, 2008
Trip End
The next day was spent entirely on a train, from 5:15 am to 9pm, i was knackered when i got to Toronto and it was raining and miserable... and so it began.
Now i'll keep it brief but basically i was not a fan of Toronto, well it was OK, there were some really cool little areas and stuff and it wasn't really Toronto's fault.
From the moment i left America and my mum in NY i was generally miserable, as i said i'll keep it brief but i felt chronically homesick, or should i say bus- sick and wanted more than anything to go back to California or back out on the road. I was sad, so i slept A LOT. Despite the weird morocan man who would lay awake staring at me over his bed until i yelled at him and a nice aussie guy in my room threatened to 'kick his fucking head in' for me. Which was nice!
Yes so bus sick, slept a lot, wandered the streets by day, hung out in a lot of book shops, trailed around kensington market and queen street (oh yes there's one here too) and checked out the cool shops and cafes.
I did go up the CN tower because well you have to don't you! It was ok, although i was disappointed in the 'glass floor' which i in my mind had decided was the whole floor, but no, just a little section, still though cool to stand on, jump up and down on and look through!
As i say i spent most of my time watching TV and sleeping and drinking which was fine really. Probably what i needed, it was quite good fun andd gave me time to drink a lot of wine and pine aimlessly for california my bus and eventually good old Britain, i write recovered fully and back on form but tragically for toronto it took the brunt of me being grumpy. But alas, these things happen!!


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Blog Archives
Project Logicality: Argumentation's Basic Assumptions
This is the second re-release in my Project Logicality series, and it was posted in its original form in April of 2011. I've corrected and re-written this and reposted it here, hopefully clearer and to the point. May all your arguments be rational and all your disputes be resolved. ~ Troythulu
We persuade others through our arguments, to get them to accept the statements and claims we make as likely true of their own free choice, justified on the basis of the reasons we give rather than prove them absolutely. Argumentation contributes to healthy discussion and debate, to let those so arguing find common ground, and to make easier a willingness to compromise.
People argue daily, though seldom with skill, and in my view, argumentation as a well-honed tool of a functional democratic republic is needed more than ever with the increasing decay of social discourse, political polarization and interpersonal conflicts that ever more are seen as irreconcilable.
In this post, I'll describe the basic assumptions and basic conditions that go into any attempt at constructive argument, and before I do, I'll note as before that good argument is intellectual in force, not coercive or deceptive. It is an ethical means of influencing others, limiting their freedom of action without imposing on their freedom of will.
First, argument is carried out under conditions of uncertainty: We generally don't argue about things we think certain, though that doesn't prevent us from talking about them.
We argue about things because we think it important enough to convince others of them, and things may well turn out to be otherwise. If things were absolutely self-evident, they would be so to all, and there would be no need to convince anyone of them.
These differences may be implied and apparent to an analyst, concealed in the context of an argument, or explicit, obvious to an audience. Bear in mind that even the concept of certainty can depend on the audience addressed and the assumptions they bring to the table as to what it means.
Second, Argumentation must consider the needs of an audience. people argue about things that matter to them, attempting to resolve what they think are conflicting positions that cannot simply be settled by any non-argumentative means; appealing to common knowledge, or widely-shared empirical methods; things they consider to be non-trivial, matters important enough to need resolution.
This is not to pander to their biases, or to say that one claim is just as good as any other, it's just that in being ethical, we must consider what is likely to persuade a given audience as if they were exercising their critical judgment on the merits of the arguments we give, and the soundness of the justifications we offer for our claims.
The audience is the final judge of whether an argument is strong or weak, justified or not, assenting to it if it is strong or justified, rejecting it if not.
Third, argumentation is both adversarial and cooperative. we make choices in arguments, choices in what arguments to select, and how to arrange and present them, based upon the audience we are addressing.
The adversarial components of argumentation help the rigor of the discussion; they help us avoid hasty generalizations; they reduce omission of important details. Skilled arguers seek first to find common ground which is itself the bedrock upon which they can meaningfully discuss their disagreement. Ultimately, these enhance our confidence of the outcome, a confidence pending better arguments to be made in future.
Fourth, argumentation involves restrained partisanship. It requires a cooperative effort between arguer and audience, despite the contentiousness often associated with everyday argument.
Arguers must share a common system of terms, assumptions, and meanings. This allows resolution of the dispute, and is needed to permit any meaningful argument at all.
Fifth, and finally, argument involves elements of risk. This is the risk of losing the argument, the risk of being shown wrong, the risk of having to alter one's views and position, and in either case the emotional disruption of wounding one's self-esteem or losing face with others.
But the cooperative aspect of argument means that in willingly accepting these risks, each arguer is respecting the rights and personhood of the other, and in so doing, claiming that same privilege of respect from the other for him or herself.
I think that these are good situational benchmarks, and are the optimal conditions, I would argue even necessary conditions, under which can be made any serious attempt to argue constructively, for the purpose of reaching the best possible conclusions given the means at hand.
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Sunday Evening Commentarium for 2013/05/26
I'm involved in a new writing project for this blog, and this post you're reading now is an endeavor of mine to publish at least one original piece of material each week. I'll be posting on things that annoy me, things that interest me, and things that make me go WTF??
This edition will be all those in one.
At the local game shop I went to this weekend, during a talk with friends, whose opinions on certain matters are quite divergent from mine, I was chided for giving an argument that undermined itself, allegedly for being qualified in probabilistic terms, rather than couched in certain ones.
Personally, I think that's very strange, and even occult, that an argument about contingent matters of fact could be undermined by an admission that one could be wrong.
That sounds to me more like intellectual honesty…
What my friend sees as a weakness in argument, a lack of certitude, is being misapplied to the wrong type of argument. While he may have been thinking "deduction," I was thinking "induction." And inductive argument is not logically certain, either from the form or the content of an argument. Inductive argument is strong or weak instead of strictly valid or invalid.
After all, that's what most scientific reasoning is like — concerning the more or less probable rather than the absolute and certain — at least in my experience concerning all the scientists I've read and have chatted with online. And I don't think that that's unique to just those I've come across.
Rhetorically, I suppose, confidence, or rather, its appearance, is an advantage. We are more often apt to lend more credence than we should to the statements of those who seem sure of themselves.
But what I know about skeptical thinking tells me that the appearance of confidence is far from a reliable indicator of competence, and may in fact show the opposite. Bluster does not equal brains.
Not just those I disagree with either, but anyone without a proven record of expertise in their field.
What undermines an argument for me is the use of language and buzzwords attempting to appeal to ideals, values, beliefs and moral judgements — emotive things — one may not share with the speaker, especially when there is no concern shown for the audience spoken to or about, the use of not just loaded language, but truly inflammatory loaded language as if to rally the troops and dismiss the enemy in one shot.
This is what I see on most political websites, especially on propaganda outlets of the far right and far left that see a lot of traffic, of which many such site owners seem inordinately proud, perhaps thinking that popularity equals accuracy, or seeking to convey that impression to their more uncritical readers.
Unfortunately for far too many people, otherwise sensible, this works. But only those already inclined to agree with the speakers or writer's views to begin with. There's that whole thing about confirmation bias, with a slant toward tribalism leading to political polarization and a shutdown of rational discussion through useless media shouting matches.
Add to that an ignorance of the basic concepts and principles of cogent argumentation, and you have the sad situation this country is in right now, where no one wants to talk or listen to each other. It's a climate of toxicity, dangerous to a functioning democracy and a powerful tool of political corruption.
Despots like it when their subjects can't or won't get together and talk with each other. The opposite often leads to revolutions, or fair elections, which simply will not do to oligarchs who wish to keep their ill-gotten power and gerrymandered votes.
So if there were one rhetorical fallacy that really stands out, one that I see the most of in online and real-time discussions of politics, or religious apologetics, hands down the most obvious would be be loaded language, seconded by false dilemmas, quote-mining and poisoning the well.
It's a good idea not to assume without reason that your audience knows certain things, but don't treat them like gullible idiots either — never cynically assume stupidity, and don't talk down to them — underestimation is a weakness, as many a failed politician has learned when the voting process has not been improperly influenced in his favor.
The greatest sin in any argument is to ignore or insult the audience one writes or speaks to, and writing in a deliberately inflammatory manner may win one converts among those who already agree, but while useful in that regard, it just puts off everyone else, especially those outside of the rhetorical echo chamber who can see it — or hear it — for what it is.
Sunday Evening Commentarium is a regular installment posted at 6:00 PM Eastern Time each Sunday, on a question or matter bringing itself to my attention during the previous week.
Charlie Chaplin – Let Us All Unite!
Nov 27, 2012 via melodysheep
mp3: http://melodysheep.bandcamp.com/album/remixes-for-the-soul //
Charlie Chaplin's 1940 anti-fascist speech from The Great Dictator, as a song. Subscribe for more like this!
Sources used:
The Great Dictator: https://www.youtube.com/watch?v=jH3zh2XHGVE&hd=1
WWII in HD: https://www.youtube.com/watch?v=XqMlo51U_KQ
The Greatest Speech Ever Made: http://www.youtube.com/watch?v=WibmcsEGLKo
I'm sorry, but I don't want to be an emperor, that's not my business. I should like to help everyone if possible – Jew, gentile, black man, white. We all want to help one another, human beings are like that. In this world there is room for everyone, and the good earth is rich, and can provide for everyone. But we have lost the way.
Greed has poisoned men's souls
Has barricaded the world with hate
We think too much and feel too little
More than machinery, we need humanity
More than cleverness, we need kindness
Without these qualities life would be violent
And all would be lost
Do not despair
The hate of men will pass
And dictators die
And the power they took from the people
Will return to the people
Let us all unite!
Let us fight for a new world
To do away with greed
Now let us fight to free the world
To fulfill that promise
Let us all unite!
Soldiers, don't give yourselves to brutes
Men who despise you and slave you
Tell you what to do, what to think and what to feel
Who drill you, diet you, treat you like cattle
Don't give yourselves to these unnatural men
Machine men with machine minds and machine hearts
You are not machines
You are men
You the people have the power
To make this life free and beautiful
Let us use that power
To make this life a wonderful adventure
Post Election Day Thoughts…
Now that the dust is clear and I've no need to worry for the next couple of years or so for my country's political future, I'm actually just a wee bit disappointed…
You see, I was rooting for Galvatron as the next world leader, as he's got a number of good policies and sound business practices at his disposal — and lots of loyal servants scared sh*tless of him as well.
First, he takes no guff from anyone — none of that nonsense of assassination attempts and coups by ambitious lieutenants, as he simply converts into a tank, cannon, or dragon (or whatever, depending on the version,) and destroys them.
Think Starscream from the 1985 Transformers animated feature (Oh, crap — I'm showing my age…) and you get the idea — Discipline, discipline, discipline — and this underlies the core of his philosophy which is…
Order. Law. Total, complete order and control over everything (Wow! Absolutist control-freak much, Galvatron? — too bad you lost the election; I was hoping to help you betray Unicron and regain your autonomy — maybe in 2016 or so…), everywhere, and this speaks of deeply ingrained personal issues, though, but never mind, wink, wink.
Second, Galvatron's got an frankness you don't see in most politicians — he tells you what he's going to do, and then does it, and that kind of honesty is very appealing to those of us rendered cynical about the American political process.
Third, and finally, his determination to get the job done when it needs to be, regardless of obstacles. Who wouldn't want a leader who uses his immense power to conquer — er, I mean, lead — the world into a place and new age of change, into a brighter, shinier, more gleaming metal future where we can have universal access to all the spare parts we need to keep ourselves functional, and of course, useful to the Predacon cause.
I think I need to stop now — really. So there.


text:
Red carpet row escalates
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Isaiah Washington's sorry biggoted ass should be fired. Where is the outrage from the press, if the situation was reversed they would never get their panties out of the crack of their bums.
I am very offended and feel he should be punished. I don't understand how2 white men were punished so severely about jewish and the n word but washington is just getting a slap on the wrist for the f word. shame
I am not a biggoted person in any way shape or form. Being African American my parents taught me to always be tolerant even when others weren't. To further this, my sister who I love dearly is gay.With that being said I don't think that Washington using the F word to deffend himself against reports that he did is in any way a slur against the gay community. I think that Giuliano's claim that Washington's comments put gays in harms way is just riddicioulous! Just as Michael Richards use of the N word did not put blacks in harms way. It was very offensive, but no one was endangered by it.
This must be Prats on display day on Pink news.Now we have another Prat, works in show business, and not aware (Like hell!), that show business is successful because of gay people!
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