bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of amplified DNA, analyzed by pulsed field gradient gel electrophoresis
Authors: Borst P. , Van Der Bliek A. M. , Van Der Velde‐Koerts T. , Hes E.
Published: December 2004

Pulsed field gradient electrophoresis allows the separation of large DNA molecules up to 2,000 kilobases (kb) in length and has the potential to close the resolution gap between standard electrophoresis of DNA molecules (smaller than 50 kb) and standard cytogenetics (larger than 2,000 kb). We have analysed the amplified DNA in four cell lines containing double minute chromosomes (DMs) and two lines containing homogeneously staining regions. The cells were immobilized in agarose blocks, lysed, deproteinized, and the liberated DNA was digested in situ with various restriction endonucleases. Following electrophoretic separation by pulsed field gel electrophoresis, the DNA in the gel was analysed by Southern blotting with appropriate probes for the amplified DNA. We find that the DNA in intact DMs is larger than 1,500 kb. Our results are also compatible with the notion that the DNA in DMs is circular, but this remains to be proven. The amplified segment of wild‐type DNA covers more than 550 kb in all lines and possibly up to 2,500 kb in some. We confirm that the repeat unit is heterogeneous in some of the amplicons. In two cell lines, however, with low degrees of gene amplification, we find no evidence for heterogeneity of the repeats up to 750 (Y1‐DM) and 800 kb (3T6‐R50), respectively. We propose that amplicons start out long and homogeneous and that the heterogeneity in the repeat arises through truncation during further amplification events in which cells with shorter repeats have a selective advantage. Even if the repeats are heterogeneous, however, pulsed field gradient gels can be useful to establish linkage of genes over relatively short chromosomal distances (up to 1,000 kb). We discuss some of the promises and pitfalls of pulsed field gel electrophoresis in the analysis of amplified DNA.
Changes in surface glycopeptides after malignant transformation of rat liver cells and during the regression of hepatoma cells
Authors: Chaumeton Brigitte , Saunier Brigitte , Nato Farida , Goulut Chantal , Bourrillon Roland
Published: December 2004

Normal liver cells, Zajdela's hepatoma cells, and regressing hepatoma cells were metabolically labeled with either radioactive glucosamine or mannose. Glycopeptides obtained by exhaustive pronase digestion of these cells were compared after fractionation by gel filtration on Bio‐Gel P‐6.

Chemical analysis, affinity chromatography on immobilized lectins, alkaline treatment, and susceptibility toward endo‐β‐N‐acetylglucosaminidase and tunica‐mycin revealed dramatic changes in the glycopeptide patterns of transformed cells during the recovery of normal phenotype.

The most prominent feature was the presence on the surface of hepatoma cells of a large glycopeptide, which was absent from normal liver cells and disappeared almost completely during the regression of hepatoma cells. This large glycopeptide had a Mr of 70,000, contained essentially O‐glycosidically linked glycan chains, and did not result from a hypersialylation.

N‐glycosidically linked glycopeptides, high‐mannose, and complex‐type oligosaccharides were present in distinct proportions according to the differentiation state. Transformation of liver cells led to a reduction of high‐mannose type oligosaccharides and an increase in the degree of branching of complex‐type oligosaccharides. In addition, “bisected” glycopeptides were present only on hepatoma cells. The pattern of N‐linked glycopeptides of normal liver cells was recovered during the regression of hepatoma cells.

The origin of glycopeptide differences between normal and transformed cells and the evidence of a relation between carbohydrate changes, in particular the appearance of a large glycopeptide, and tumorigenicity are discussed.
Estrogen‐induced lysosomal proteases secreted by breast cancer cells: A role in carcinogenesis?
Authors: Rochefort Henri , Capony Françoise , Garcia Marcel , Cavaillès Vincent , Freiss Gilles , Chambon Monique , Morisset Muriel , Vignon Françoise
Published: December 2004

In an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis, metastatic human breast cancer cell lines (MCF7, ZR75‐1) were found to secrete a 52,000 dalton (52K) protein under estrogen stimulation. Following its purification to homogeneity, the 52K protein was identified as a secreted procathepsin‐D‐like aspartyl protease bearing man‐nose‐6‐phosphate signals. This precursor display an in vitro autocrine mitogenic activity on estrogen‐deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its autoactivation. The total protease (52K + 48K and 34K) was detected and assayed by monoclonal antibodies and was found to be highly concentrated in proliferative and cystic mastopathies. In breast cancer, its cytosolic concentration appears to be correlated more to tumor invasiveness than to hormone responsiveness. The mRNA of the 52K protease accumulates rapidly following estradiol treatment, as was shown by Northern blot analysis with cloned cDNA. The 52K cathepsin‐D‐like protease is the first example of a lysosomal protease induced by estrogens in cancer cells. Results obtained using different approaches suggest that two cysteinyl cathepsins are also related to cell transformation and invasiveness. It has been proposed that cathepsin‐B is involved in breast cancer and metastatic melanoma, and its regulation by estrogen has been shown in the rat uterus. Cathepsin‐L corresponds to the major excreted protein (MEP) whose synthesis and secretion are markedly increased by transformation of NIH 3T3 cells with Ki ras and are regulated by several growth factors. In addition to secreted autocrine growth factors and to other proteases (plasminogen activator, collagenase), lysosomal cathepsins may therefore play an important role in the process of tumor growth and invasion as long as their precursor is secreted abundantly.
Interactions between growth factor receptors and corresponding monoclonal antibodies in human tumors
Authors: Rodeck Ulrich , Herlyn Meenhard , Koprowski Hilary
Published: December 2004

Monoclonal antibodies (MAbs) to the human epidermal growth factor (EOF) receptor, the type I insulin‐like growth factor (IGF) receptor, and the nerve growth factor (NGF) receptor were used to study the growth regulation of malignant cells. Anti‐EGF receptor MAb 425 inhibited the growth of A 431 squamous carcinoma cells which express high numbers of EGF receptors on their surfaces. Growth inhibition induced by MAb 425 was accompanied by alterations of the cell‐cycle distribution of these cells, indicating the ability of a monoclonal antibody to act as a biologically active ligand. Growth stimulation of melanoma cells by EGF was unrelated to EGF receptor expression on the cell surface. Insulin‐and IGF‐I‐induced growth stimulation of melanoma cells was inhibited by MAb α‐3 which reacts with the type I IGF receptor. This result indicates that the type I IGF receptor mediated growth stimulation not only by IGF‐I but also by insulin. Normal melanocytes and cells of all stages of tumor progression expressed in tissue culture the receptor for NGF, but no effect on the growth of these cells has been observed.
Human teratocarcinoma stem cells: Glycolipid antigen expression and modulation during differentiation
Authors: Andrews Peter W.
Published: December 2004

Teratocarcinomas are germ cell tumors in which pluripotent stem cells, embryonal carcinoma (EC) cells, undergo differentiation along the pathways resembling those occurring during early embryogenesis. Human EC cell lines established in vitro provide a model for studying embryonic cellular differentiation in a way that is pertinent to early human development. The predominant glycolipid antigens expressd by EC cells of both humans and mice have globoseries core structures; in humans they are terminally modified to yield the monoclonal antibody‐defined stage‐specific embryonic antigens SSEA‐3 and SSEA‐4, and also globo‐ABH antigens; in the mouse terminal modification yields the Forssman antigen rather than SSEA‐3 and ‐4. These observations focus attention on the possible role of the P‐blood group system, which regulates synthesis of globoseries oligosaccharides, in the behavior of cells in the early embryo and in teratocarcinomas. Marked changes in the core structures of the cell surface glycolipids occur as the EC cells differentiate; thus globoseries structures rapidly diminish and are replaced by lactoseries and then by ganglioseries glycolipids. During differentiation of the NTERA‐2 line of pluripotent human EC cells into neurons and other cell types, the various subsets of differentiated cells that arise are distinguished by their differential expression of new glycolipid antigens, particularly ganglioside GT3 (recognized by antibody A2B5), and ganglioside 9‐0‐acetyl GD3 (recognized by antibody ME311). Neurons are found among the A2B5+/ME311− cells.
Melanoma growth stimulatory activity: Isolation from human melanoma tumors and characterization of tissue distribution
Authors: Richmond Ann , Thomas H. Greg
Published: December 2004

Melanoma growth stimulatory activity (MGSA) is an acid and heat stable, autostimulatory growth factor which was first isolated from culture medium conditioned by the Hs294T human melanoma cell line. In this report, we describe the purification of MGSA from acid ethanol extracts of Hs294T tumors grown in nude mice using a series of Bio‐Gel P30, reverse phase‐high performance liquid chromatography and heparin‐sepharose steps. This modified procedure provides a 10‐fold improved yield of MGSA over previously published procedures. Purified MGSA‐stimulated melanoma cell growth in both 3H‐thymidine and cell number assays over a concentration range of 0.06 to 6 ng/ml. The MGSA bioactivity was primarily associated with fractions which exhibited molecular weights of 16 and 13–14 Kd based upon sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Purified platelet‐derived growth factor (PDGF), insulin‐like growth factor (IGF‐1), transforming growth factor‐beta (TGFβ), and epidermal growth factor (EGF) in combination with TGFβ did not stimulate 3H‐thymidine incorporation in Hs294T cells under the conditions used for MGSA bioassay. Monoclonal antibody to MGSA was used to screen melanoma and benign nevus cultures as well as fixed sectioned tissue for MGSA. The majority of the melanoma cultures were MGSA positive, while most nevus cultures were MGSA negative. However, when fixed sectioned tissue was screened for MGSA immunoreactivity, melanoma tissue was MGSA positive and three‐fourths of the benign nevi were MGSA positive. In addition, epidermal keratinocytes and several tissues exhibiting proliferative disorders contained immunoreactive MGSA. These data suggest that MGSA may be a normal regulator of growth and that the microenvironment of the cell may regulate both production of MGSA and response to MGSA.
Effects of fibroblasts and endothelial cells on inactivation of target proteases by protease nexin‐1, heparin cofactor II, and C1‐inhibitor
Authors: Hiramoto Sheree A. , Cunningham Dennis D.
Published: December 2004

Previous studies have shown that glycosaminoglycans in the extracellular matrix accelerate the inactivation of target proteases by certain protease inhibitors. It has been suggested that the ability of the matrix of certain cells to accelerate some inhibitors but not others might reflect the site of action of the inhibitors. Previous studies showed that fibroblasts accelerate the inactivation of thrombin by protease nexin‐1, an inhibitor that appears to function at the surface of cells in extravascular tissues. The present experiments showed that endothelial cells also accelerate this reaction. The accelerative activity was accounted for by the extracellular matrix and was mostly due to heparan sulfate. Fibroblasts but not endothelial cells accelerated the inactivation of thrombin by heparin cofactor II, an abundant inhibitor in plasma. This is consistent with previous suggestions that heparin cofactor II inactivates thrombin when plasma is exposed to fibroblasts and smooth muscle cells. Neither fibroblasts nor endothelial cells accelerated the inactivation of C1s by plasma C1‐inhibitor.
Modulation of the epidermal growth factor receptor by brain‐derived growth factor in Swiss mouse 3T3 cells
Authors: Huang Shuan Shian , Lokeshwar Vinata B. , Huang Jung San
Published: December 2004

Incubation of Swiss mouse 3T3 cells at 37°C with bovine brain‐derived growth factor (BDGF) decreased the cell surface 125I‐EGF binding activity of these cells by 70–80%. This down‐modulation of the EOF receptor by BDGF was time, temperature, and dose dependent Scatchard plot analysis indicated that BDGF binding led to a selective decrease in the number of high‐affinity EGF receptors. The BDGF‐induced down‐modulation of the EGF receptor was completely blocked by protamine, a potent inhibitor of receptor binding and mitogenic activities of BDGF.

BDGF down‐modulated the EGF receptor in phorbol myristic acetate (PMA)‐pretreated cells, as well as in control cells. Furthermore, PMA‐pretreated cells responded mitogenically to BDGF, whereas PMA itself failed to stimulate the mitogenic response of PMA‐pretreated cells. This BDGF‐induced down‐modulation of the EGF receptor in PMA‐desensitized cells suggests that BDGF down‐regulates the EGF receptor by a mechanism distinct from that of PMA.

Incubation of cells with compounds which are known to inhibit pinocytosis blocked the down‐modulation induced either by BDGF or by platelet‐derived growth factor (PDGF) but had no effect on the PMA‐induced down‐modulation. Incubation of cells with inhibitors of receptor recycling enhanced the BDGF‐induced down‐modulation of the EGF receptor. These results suggest that BDGF and PDGF induce down‐modulation of the EGF receptor by increasing the internalization of cell surface high‐affinity receptors and that the internalization process may not be required for down‐modulation induced by PMA.
Mechanism of induction of class I major histocompatibility antigen expression by murine leukemia virus
Authors: Faller Douglas V. , Wilson Lise D. , Flyer David C.
Published: December 2004

Alterations in expression of major histocompatibility complex (MHC) antigens on tumor cells clearly correlate with the tumorgenicity and metastatic potential of those cells. These changes in the biological behavior of the tumor cells are presumably secondary to resulting changes in their susceptibility to immune recognition and destruction. Murine leukemia viruses (MuLV) exert regulatory effects on class I genes of the MHC locus. MuLV infection results in substantial increases in cell surface expression of all three class I MHC antigens. These viral effects on MHC antigen expression profoundly influence immune‐mediated interaction with the infected cells, as assessed by cytotoxic T lymphocyte recognition and killing. Control of class I MHC and beta‐2 microglobulin genes by MuLV takes place via a trans‐acting molecular mechanism. MuLV controls expression of widely separated endogenous cellular MHC genes, transfected xenogeneic class I MHC genes, and unintegrated chimeric genes consisting of fragments of class I MHC genes linked to a bacterial reporter gene. These findings indicate that MuLV exerts its effects on MHC expression via a trans mechanism. The MuLV‐responsive sequences on the MHC genes appear to lie within 1.2 kilobases upstream of the initiation codon for those genes.
Phosphorylation of ankyrin down‐regulates its cooperative interaction with spectrin and protein 3
Authors: Cianci Carol D. , Giorgi Mauro , Morrow Jon S.
Published: December 2004

Ankyrin mediates the primary attachment between beta spectrin and protein 3. Ankyrin and spectrin interact in a positively cooperative fashion such that ankyrin binding increases the extent of spectrin tetramer and oligomer formation (Giorgi and Morrow: submitted, 1988). This cooperative interaction is enhanced by the cytoplasmic domain of protein 3, which is prepared as a 45–4l‐kDa fragment generated by chymotryptic digestion of erythrocyte membranes. Using sensitive isotope‐ratio methods and non‐denaturing PAGE, we now demonstrate directly (1) the enhanced affinity of ankyrin for spectrin oligomers compared to spectrin dimers; (2) a selective stimulation of the affinity of ankyrin for spectrin oligomer by the 43‐kDa cytoplasmic domain of protein 3; and (3) a selective reduction in the affinity of ankyrin for spectrin tetramer and oligomer after its phosphorylation by the erythrocyte cAMP‐independent membrane kinase. The phosphorylation of ankyrin does not affect its binding to spectrin dimer. Ankyrin also enhances the rate of interconversion between dimer‐tetramer‐oligomer by 2–3‐fold at 30°C, and in the presence of the 43‐kDa fragment, ankyrin stimulates the rate of oligomer interconversions by nearly 40‐fold at this temperature. These results demonstrate a long‐range cooperative interaction between an integral membrane protein and the peripheral cytoskeleton and indicate that this linkage may be regulated by covalent protein phosphorylation. Such interactions may be of general importance in nonerythroid cells.
Caldesmon: A common actin‐linked regulatory protein in the smooth muscle and nonmuscle contractile system
Authors: Sobue Kenji , Kanda Keiko , Tanaka Toshihiko , Ueki Noboru
Published: December 2004

Caldesmon was originally purified from gizzard smooth muscle as a major calmo‐dulin‐binding protein which also interacts with actin filaments. It has an alternative binding ability to either calmodulin or actin filaments depending upon the concentration of Ca2+(“flip‐flop binding”). Two forms of caldesmon (Mr's in the range of 120–150 kDa and 70–80 kDa) have been demonstrated in a wide variety of smooth muscles and nonmuscle cells. Immunohistochemical studies suggest that caldesmon is colocalized with actin filaments in vivo. Considering its abundance, the Ca2+ ‐dependent flip‐flop binding ability to either calmodulin or actin filaments, and its intracellular localization, caldesmon is expected to be involved in contractile events. Recent results from our laboratory have led to the conclusion that caldesmon regulates the smooth muscle and nonmuscle actin‐myosin interaction and the smooth muscle actin‐high Mr actin‐binding protein (ABP or filamin) interactin in a flip‐flop manner. It might function in cell motility by regulating the contractile system.
Effect of transforming growth factor‐α on inositol phospholipid metabolism in human epidermoid carcinoma cells
Authors: Kato Miwako , Takenawa Tadaomi , Twardzik Daniel R.
Published: December 2004

Transforming growth factor‐α (TGF‐α) stimulates (in a dose‐dependent manner) the incorporation of [32P]Pi into phosphatidylinositol(PI), phosphatidylinositol 4‐phosphate (PIP), phosphatidylinositol 4,5‐bisphosphate (PIP2), and phosphatidic acid (PA) in the human epidermoid carcinoma cell line (A431). The effect of TGF‐α on the incorporation was found to be similar to that of EGF. On the other hand, a striking difference in the activation of diacylglycerol (DG) kinase activity was seen between TGF‐α and EGF. At least 100 times more TGF‐α was required to achieve maximal stimulation of DG kinase activity relative to EGF. These results suggest that the activation of DG kinase by TGF‐α may involve a mechanism independent from or subsequent to activation of the EGF receptor.
Structure and expression of Ick transcripts in human lymphoid cells
Authors: Perlmutter Roger M. , Marth Jamey D. , Lewis David B. , Peet Richard , Ziegler Steven F. , Wilson Christopher B.
Published: December 2004

The murine Ick gene encodes a membrane‐associated protein tyrosine kinase that has been implicated in lymphocyte oncogenesis. Here we report the structure of normal human Ick transcripts and the pattern of expression of these transcripts in developing thymus and in peripheral T cell subsets. The human Ick gene encodes a 509 amino acid polypeptide that is closely related to the murine Ickencoded protein throughout its length. Analysis of the deduced amino acid sequence of human p56Ick demonstrates that an amino‐terminal domain, widely divergent among the seven known src family members, has been conserved between murine and human p56Ick, and thus probably includes sequences crucial to the lymphocyte‐specific function of this molecule. Human Ick transcripts were detected in CD4+ and CD8+ T cells, in partially purified B cells, and in Epstein‐Barr virus‐immortalized B cell lines, but not in monocytes, granulocytes, or in nonhematopoietic cell types. Human Ick transcripts are readily detectable in fetal thymocytes at 70 days of gestation, but not at 57 days of gestation, indicating that Ick expression appears coordinately with the appearance of lymphoid cells in the developing thymus. Thus Ick gene expression is a marker for cells of the lymphocyte lineage in man. We conclude that the Ick gene probably participates in a signal transduction pathway uniquely present in lymphoid cells.
Cyclin mRNA and protein expression in recombinant interleukin 2‐stimulated cloned murine T lymphocytes
Authors: Shipman P. M. , Sabath D. E. , Fischer A. H. , Comber P. G. , Sullivan K. , Tan E. M. , Prystowsky M. B.
Published: December 2004

Expression of cyclin, a non‐histone nuclear protein, during recombinant interleukin 2 (rIL2)‐driven cell‐cycle progression of cloned T lymphocytes has been assessed. We found that expression of cyclin protein, as detected by immunofluorescence, is tightly associated with proliferation, and not merely S‐phase, of L2 cells stimulated with rIL2. Cyclin immunofluorescence was detected in all cell‐cycle phases (G1/S/G2/M, as detected by flow cytometry) of proliferating L2 cells. Accumulation of cyclin mRNA levels was induced as early as 1 h after stimulation, was maximal at 25–49 h, and remained elevated throughout stimulation, as detected by Northern blot analysis. A cDNA‐encoding murine cyclin was cloned from a cDNA library prepared from IL2‐stimulated cloned T cells. The sequence of the 5′ end of the murine cyclin cDNA was determined and found to be 88% and 82% similar to the sequences of cDNA clones encoding rat and human cyclin, respectively. The present studies demonstrate that cyclin protein and mRNA accumulation are highly regulated during IL2‐induced proliferation of a cloned T cell. These data provide a framework for addressing the molecular mechanisms regulating cyclin gene expression during cellular proliferation.
A polypeptide growth inhibitor isolated from lactating bovine mammary gland (MDGI) is a lipid‐carrying protein
Authors: Böhmer Frank‐D. , Mieth Maren , Reichmann Gunter , Taube Christel , Grosse Richard , Hollenberg Morley D.
Published: December 2004

Mammary‐derived growth inhibitor (MDGI), a polypeptide growth inhibitor isolated from lactating bovine mammary tissue, previously shown to have extensive sequence homology with fatty acid‐binding proteins, was demonstrated to meet the criteria of a fatty acid‐binding protein. The protein was found to bind [3H]palmitic acid in a saturable manner and to be complexed with endogeneous free fatty acids. [3H]palmitic acid, when bound to the protein, was more rapidly taken up by the target cells (human mammary carcinoma cells [MaTu]) than was free [3H]palmitic acid, suggesting a lipid carrier function for the inhibitor. It is suggested that the fatty acid‐binding properties of MDGI may relate to its ability to inhibit cell growth in vitro and to regulate other cellular functions.
Retroviruses expressing different levels of the normal epidermal growth factor receptor: Biological properties and new bioassay
Authors: Velu Thierry J. , Beguinot Laura , Vass William C. , Zhang Ke , Pastan Ira , Lowy Douglas R.
Published: December 2004

Two retroviral DNAs that encode the normal human epidermal growth factor (EGF) receptor hEGFR have been generated by inserting a hEGFR cDNA into two different retroviral vectors. One DNA (pCO11‐EGFR‐neo) also contained a linked selectable marker gene (neoR). The other (pCO12‐EGFR) only expresses hEGFR. When introduced into NIH3T3 cells, the two DNAs and the viruses derived from them induced a fully transformed phenotype, including focal transformation and growth in agar or low serum, but transformation depended entirely upon EGF being present in the growth medium. Compared with pCO11‐EGFR‐neo, pCO12‐EGFR induced EGF‐dependent transformation 2–5 times more efficiently and expressed higher numbers of receptors (4 × 105 vs. 1 × 105 EGF receptors per cell). The results indicate that transforming potential is directly related to the number of EGF receptors. In defined, serum‐free medium that contained only very low concentrations of insulin (0.6 μg/ml) and transferrin (0.6 μg/ml), hEGFR‐virus infected cells were able to grow with EGF as the only growth factor. Moreover, daily incubation of the cells with EGF for only 30 min was sufficient to induce growth. NR6 cells, which lack endogenous EGF receptors, were transformed as efficiently as NIH3T3 cells by the hEGFR virus. The dose‐dependent growth response to EGF of infected NR6 cells grown in serum‐free medium can be used as a highly sensitive bioassay for the quantitative assessment of EGF and transforming growth factor type α (TGFα). This bioassay is at least as sensitive as previously reported radioimmunoassays and can measure a much wider concentration range (10 pg–100 ng/ml). Uninfected NR6 cells or NR6 cells infected by helper virus alone can be used as controls for the EGF specificity of growth stimulation.
Does the lack of regression‐associated mRNA expression render a rat ventral prostate epithelial cell line androgen independent?
Authors: Montpetit Michael L. , Tenniswood Martin P.
Published: December 2004

A series of rapidly dividing epithelial (RDE) cell lines have been isolated from primary cultures of rat ventral prostate (RVP) epithelial cells. Unlike androgen‐dependent secretory epithelial cells, the RDE cells in culture do not express the androgen‐dependent secretory proteins, nor do they express the androgen‐repressed cell death sequences (TRPM‐2) found in the epithelial cells during prostatic regression. Screening of a cDNA clone library established from RDE cell mRNA has yielded a number of RDE cell‐specific sequences. One of these, RDE‐.25 is a 250‐base mRNA. The sequence of RDE‐.25 shows considerable homology with the rat growth hormone gene and two murine oncogene sequences. We believe that the absence of androgen‐repressed cell death sequence expression confers androgen independence for survival and growth, while the expression of RDE‐.25 may represent an autocrine growth stimulus which greatly increases the rate of cell division in these cells.
Phorbol diester enhances calcium ionophore A23187‐induced [3H]acetate incorporation into platelet‐activating factor in murine macrophages: Predominant incorporation into 1‐O‐acyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine
Authors: Wey Howard E.
Published: December 2004

Pretreatment of macrophages with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) has been shown to enhance the release of arachidonic acid from cell phospholipids in response to agonist stimulation. This study describes the ability of TPA to also alter calcium ionophore A23187‐induced incorporation of [3H]acetate into platelet activating factor (PAF). Cultured murine peritoneal macrophages were preincubated with [3H]acetate (25 μCi) and TPA (10 ng/ml) for 10 min, and subsequently incubated with 0.1 μM A23187 for 0.5–10 min. Buffer and cells were then extracted and PAF resolved by normal‐phase HPLC. Sequential exposure to TPA and A23187 resulted in a greatly enhanced incorporation (11,861 dpm/106 cells) of [3H]acetate into PAF compared to TPA alone, which did not significantly influence [3H]acetate incorporation into PAF, and 0.1 μM A23187, which induced minimal incorporation (688 dpm/106 cells). Macrophage‐produced [3H]PAF was resolved by HPLC, extracted, treated with phospholipase‐C, and acetylated to facilitate quantitation of 1‐O‐alkyl‐2‐acetyl‐GPC (PAF) from 1‐O‐acyl‐2‐acetyl‐GPC (acylPAF). A23187 alone (1 μM) produced 72% 1‐O‐acyl‐2‐[3H]acetyl‐GPC, and A23187 (0.1 μM) following TPA pretreatment produced 81% 1‐O‐acyl‐2‐[3H]acetyl‐GPC. Less than 2% of the radioactivity of acylPAF was in the acyl moiety. These data support a role for protein kinase C in modulating agonist‐induced PAF synthesis. The results also suggest that acetyltransferase of murine macrophages does not possess specificity for 1‐O‐alkyl‐2‐lyso‐GPC, and that availability of specific species of lyso‐phospholipid may determine the type of PAF produced.
Cascade of autophosphorylation in the β‐subunit of the insulin receptor
Authors: White Morris F. , Kahn C. Ronald
Published: December 2004

Insulin stimulated autophosphorylation of the β‐subunit of the insulin receptor purified from Fao hepatoma cells or purified from Chinese hamster ovary (CHO/HIRC) or Swiss 3T3 (3T3/HIRC) cells transfected with the wild‐type human insulin receptor cDNA. Autophosphorylation of the purified receptor occurred in at least two regions of the β‐subunit: the regulatory region containing Tyr‐1146, Tyr‐1150, and Tyr‐1151, and the C‐terminus containing Tyr‐1316 and Tyr‐1322. In the presence of antiphosphotyrosine antibody (α‐PY), autophosphorylation of the purified receptor was inhibited nearly 80% during insulin stimulation. Tryptic peptide mapping showed that α‐PY inhibited autophosphorylation of both tyrosyl residues in the C‐terminus and one tyrosyl residue in the regulatory region, either Tyr‐1150 or Tyr‐1151. Thus, a bis‐phosphorylated form of the regulatory region accumulated in the presence of α‐PY, which contained Tyr(P)‐1146 and either Tyr(P)‐1150 or 1151. In intact Fao, CHO/HIRC, and 3T3/HIRC cells, insulin stimulated tyrosyl phosphorylation of the β‐subunit of the insulin receptor. Tryptic peptide mapping indicated that the regulatory region of the β‐subunit was mainly (>80%) bis‐phosphorylated; however, all three tyrosyl residues of the regulatory region were phosphorylated in about 20% of the receptors. As the phosphotransferase was activated by tris‐phosphorylation but not bis‐phosphorylation of the regulatory region of the β‐subunit (White et al.: Journal of Biological Chemistry 263:2969–2980, 1988), the extent of autophosphorylation in the regulatory region may play an important regulatory role during signal transmission in the intact cell.
Sulfation of the tumor cell surface sialomucin of the 13762 rat mammary adenocarcinoma
Authors: Hull Steven R. , Carraway Kermit L.
Published: December 2004

ASGP‐1, the major cell surface sialomucin of the 13762 ascites rat mammary adenocarcinoma, is at least 0.5% of the total ascites cell protein and has sulfate on 20% of its O‐linked oligosaccharide chains. We have used this system to investigate the O‐glycosylation pathway in these cells and to determine the temporal relationship between sulfation and sialylation. The two major sulfated oligosaccharides (S‐1 and S‐2) were isolated as their oligosaccharitols by alkaline boro‐hydride elimination, anion exchange HPLC, and ion‐suppression HPLC. From structural analyses S‐1 is proposed to be a branched, sulfated trisaccharide −O4S‐GlcNAcβ1,6‐(Galβ1,3)‐GalNAc and S‐2 its sialylated derivative −O4S‐GlcNAcβ1,6‐(NeuAcα2,3‐Galβ1,3)‐GalNac. Pulse labeling with sulfate indicated that sulfation occurred primarily on a form of ASGP‐1 intermediate in size between immature and mature sialomucin. Pulse‐chase analyses showed that the intermediate could be chased into mature ASGP‐1. The concomitant conversion of S‐1 into S‐2 had a half‐time of less than 5 min. Monensin treatment of the tumor cells led to a 95% inhibition of sulfation with the accumulation of unsulfated trisaccharide GlcNAcβ1,6‐(Galβ1,3)‐GalNAc and sialylated derivative GlcNAcβ1,6‐(NeuAcα2,3‐Galβ1,3)‐GalNac. These data suggest that sulfation of ASGP‐1 is an intermediate synthetic step, which competes with β‐1,4‐galactosylation for the trisaccharide intermediate and thus occurs in the same compartment as β‐1,4‐galactosylation. Moreover, sulfation precedes sialylation, but the two are rapidly successive kinetic events in the oligosaccharide assembly of ASGP‐1.
Two‐dimensional protein profiles of cultured stromal and epithelial cells from hyperplastic human prostate
Authors: Sherwood Edward R. , Berg Lori A. , McEwan Robert N. , Pasciak Robert M. , Kozlowski James M. , Lee Chung
Published: December 2004

Studies were undertaken to compare and contrast the two‐dimensional protein profiles of epithelial and stromal cells from hyperplastic human prostate to establish the protein composition of the two major cellular components of the prostate. Epithelial and stromal cells were isolated from human prostate obtained from patients undergoing open prostatectomy for benign prostatic hyperplasia (BPH). Proteins, isolated from the two cell populations and separated by two‐dimensional (2D) electrophoresis, were analyzed by silver staining, fluorography of [35S]‐methionine‐labeled proteins, and immunoprotein blotting. Isolated prostatic epithelial cells, but not stromal cells, contained cytokeratin polypeptides 5, 6, 7, 8, 13, 14, 15, 16, 17, 18, and 19. Although vimentin could not be identified in silver stained 2D gels and fluorographs of cultured prostatic epithelial cells, a low level of immunoreactivity was noted following immunoblot analysis of epithelial cell proteins by the use of an anti‐vimentin polyclonal. Vimentin was prominently expressed in cultured prostatic stromal cells and could be identified on silver stained 2D gels, fluorographs, and immunoblots of stroma‐derived proteins. In addition, stromal marker proteins SM1, SM2, and SM3 were identified in 2D gels of stromal cells to distinguish them from epithelial cells. These studies demonstrate (1) the two‐dimensional protein profile and cytokeratin polypeptide composition of cultured epithelial cells from hyperplastic human prostate and (2) the 2D protein profile of cultured prostatic stromal cells and identification of specific stromal marker proteins.
Cytovillin and other microvillar proteins of human choriocarcinoma cells
Authors: Pakkanen Raimo , Vaheri Antti
Published: December 2004

Microvilli were isolated from cultured human JEG‐3 choriocarcinoma cells using a gentle shearing method. The protein components of the isolated microvilli were examined by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting. The major Mr 42,000 and Mr 100,000 polypeptide bands reacted with anti‐actin and anti‐α‐actinin antisera, respectively. Extraction of the isolated JEG‐3 microvilli with Triton X‐100 left an insoluble cytoskeletal residue containing mainly actin, α‐acinin, and polypeptides of Mr 200,000, 55,000 and 35,000. The Mr 35,000 polypeptide remained insoluble only at high concentrations of free Ca2+. Immunoblotting analysis of the JEG‐3 microvilli indicated that they were devoid of tropomyosin, although the total JEG‐3 protein lysates gave a strong positive reaction with anti‐tropomyosin, antiserum. The different subcellular localization of cytovillin and tropomyosin was also shown by indirect immunofluorescence microscopy. Cytovillin, an Mr 75,000 microvillus‐specific membrane protein of JEG‐3 cells, existed in an oligomeric form (dimer or trimer) as shown by gel filtration of Triton X‐100 solubilized microvillar proteins and by native polyacrylamide gel electrophoresis of purified cytovillin. Disulfide bridges were not involved in the aggregation, because the mobility of cytovillin was similar under reducing and nonreducing conditions in SDS‐PAGE. Cytovillin was shown to be closely related to ezrin, a minor component of chicken intestinal brush border microvilli.
Phenotypic effects of overexpression of PKCβ1 in rat liver epithelial cells
Authors: Hsieh Ling‐Ling , Hoshina Sadayori , Weinstein I. Bernard
Published: December 2004

We have used a previously described retroviral expression vector pMV7‐PKCβ1 to develop derivatives of two rat liver epithelial cell lines, K16 and K22, that stably express about tenfold‐higher PKC activity than control cells. Despite these high levels of PKC, these cells did not exhibit gross morphologic changes, anchorage‐independent growth, or tumorigenicity. K16PKC‐4 and K22PKC‐2, two lines with the highest PKC enzyme activity, were studied further in terms of several responses to the phorbol ester tumor promoter TPA. When treated with 100 ng/ml of TPA, the control K16MV7 and K22MV7 cells displayed a slight change in morphology, whereas the K16PKC‐4 and K22PKC‐2 cells displayed a marked change in morphology. Northern blot analyses demonstrated that TPA induced increased levels of fos, myc, phorbin, and ODC RNAs in control K16MV7 and K22MV7 cells, with maximum induction occurring at about 0.5, 1, 8, and 8 h, respectively. In K16PKC‐4 and K22PKC‐2 cells, TPA induction of phorbin and ODC RNAs was markedly enhanced, but this was not the case for myc and fos RNAs. In addition, the levels of myc RNA were constitutively higher in both K16PKC‐4 and K22PKC‐2 cells than in the control cells. Taken together, these results provide direct evidence that PKC plays a critical role in modulating the expression of myc, phorbin, and ODC RNAs. On the other hand, overexpression of PKCβ1 is not itself sufficient to cause cell transformation.
Drug‐induced alterations of tumor necrosis factor‐mediated cytotoxicity: Discrimination of early versus late stage action
Authors: Kull Frederick C. , Besterman Jeffery M.
Published: December 2004

The killing of L‐M cells by murine tumor necrosis factor (mTNF) was investigated by a combination of drug, antiserum neutralization, and kinetic studies. Kinetic studies with 125I‐mTNF showed that the bulk of association of ligand with L‐M cells peaked within 2 hr and the ligand was not degraded. Cell surface receptors were depleted (down regulated) by 6 hr when death commenced. The off‐rates of acid dissociabie (surface bound) and acid‐indissociable (internalized) compartments were found to be 9 min and 35 hr, respectively. Nevertheless, complete cell killing required the persistent presence of mTNF for up to 20 hr. This requirement was ablated by the concomitant addition of cycloheximide. Antiserum completely inhibited cytotoxicity when it was applied up to 4 hr after mTNF, but antiserum added I hr after mTNF was not neutralizing in the presence of cydohe.ximide. Thus, the inclusion of cycloheximide temporally dissociated early events (internalization and signal transduction) from lysis. Other drugs with and without cycloheximide were found to preferentially affect either early or later aspects of cell death. Phorbol myristate acetate and the ionophore A23187 were potent inhibitors of cytotoxicity and staurosporine was a potent enhancer. These agents were more effective when added 1 hr before mTNF and cycloheximide than when added 1 hr after and likely affected early events in the cytolytic program. In contrast, chloroquine and cAMP likely affect more terminal aspects of cytotoxicity. Dibutyrylcyclic AMP, cholera, and pertussis toxins enhanced cytotoxicity. They were equipotent when added either before or after mTNF regardless of the presence of cycloheximide. Likewise, chloroquine was an equipotent inhibitor when added either before or after mTNF regardless of the presence ofcycloheximide. Agents that primarily affect association events may be more likely to impinge on other TNF‐mediated activities than agents that primarily affect lysis.
Inhibition of epidermal growth factor receptor activity by retinoic acid in glioma cells
Authors: Steck Peter A. , Hadi Azra , Lotan Reuben , Yung W. K. Alfred
Published: December 2004

The growth inhibitory effects of exogenously added retinoic acid (RA) on various cultured human glioma cells was observed to be heterogenous, with an ID50 ranging from 10−7 M to no response. The protein tyrosine kinase activity of epidermal growth factor receptor (EGF‐receptor) appeared to parallel the cell's growth responsiveness to RA. Cells sensitive to RA‐induced growth inhibition exhibited a dose‐dependent decrease in EGF‐receptor activity, whereas RA‐resistant cells showed no alterations in EGF‐receptor protein tyrosine kinase activity or expression. The modulation of EGF‐receptor by RA was further examined with RA‐sensitive (LG) and ‐resistant (NG‐1 ) cell lines. Both cell lines were approximately equal in their ability to bind and internalize epidermal growth factor in the presence or absence of RA. Several independent assays suggested that the inhibition of EGF‐receptor activity was independent of protein kinase C modulation as mediated by phorbol myristate acetate. However, alterations in associated glycoconjugates of EGF‐receptor were observed among the sensitive cells but not the resistant cells. These results suggest RA‐induced growth inhibition in sensitive cells may arise, at least in part, through alterations in EGF‐receptor activity and structure.
Analysis of gene expression during adipogenesis in 3T3‐F442A preadipocytes: Insulin and dexamethasone control
Authors: Moustaid Naïma , Lasnier Françoise , Hainque Bernard , Quignard‐Boulange Annie , Pairault Jacques
Published: December 2004

In the present study, we have investigated dexamethasone and insulin regulation of the expression of adipose‐specific mRNA, namely, glycerophosphate dehydrogenase (G3PDH) and adipsin, at different stages of differentiation. During adipose conversion, insulin promotes an accumulation of G3PDH mRNA which is linked to cell differentiation; in fully differentiated cells, insulin is not required to maintain G3PDH gene expression. Differentiating cells in serum deprived medium already exhibit, at day I, a maximal amount of mRNA encoding for adipsin, which is tenfold decreased by 10 nM of insulin; insulin also exerts a negative effect on the abundance of adipsin mRNA in mature cells. This result indicates that adipsin appears to be a very early marker of adipose conversion, the gene expression of which is down‐regulated by the presence of insulin. Dexamethasone (DEX) decreases the G3PDH message at all stages of adipose conversion, while it promotes the accumulation of adipsin mRNA mainly in differentiating cells. In DEX‐treated adipocytes, the transcription efficiency of the G3PDH gene is not altered, and reduction to 50% of the message is due essentially to an approximately twofold decrease in its half‐life.
Characterization of the hamster DDT‐1 cell aFGF/HGBF‐I gene and cDNA and its modulation by steroids
Authors: Hall Jeffrey A. , Harris Marie A. , Malark Marlene , Mansson Per‐Erik , Zhou Hua , Harris Stephen E.
Published: December 2004

Syrian hamster DDT‐1 cells are derived from smooth muscle of the ductus deferens. DDT‐1 cell growth is increased by the addition of testosterone (T). Acidic fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF) also known as heparin binding growth factor I and II (HBGF‐I and HBGF‐II) can replace T in the stimulation of growth in these cells. This phenomenon is correlated with testosterone's ability to elevate aFGF/HBGF‐I mRNA. The increase steady‐state levels of aFGF/HBGF‐I mRNA were documented by northern blots and by in situ hybridization. Using a 520 bp human aFGF/HBGF‐I cDNA probe, a genomic clone with a 38 kb DNA insert was isolated from a cosmid library. By restriction enzyme analysis and southern hybridization, it was determined that there are three coding exons. DNA sequence analysis showed all of the coding region and 3′ noncoding sequences were on this clone. A 5′ noncoding exon not in the 38 kb insert is indicated, based on the cDNA sequences and genomic sequences of aFGF/HBGF‐I's from hamster DDT‐1 cells and several other species. The cDNA for hamster aFGF/HBGF‐I was isolated from a DDT‐1 lambda gt11 library and sequenced. Comparison of the coding region of aFGF/HBGF‐I from four species shows a >90% conservation of amino acid sequence.
Developmental changes in levels of growth hormone mRNA in zucker rats
Authors: Ahmad Iqbal , Steggles Alan W. , Carrillo Alberto J. , Finkelstein Judith A.
Published: December 2004

Levels of pituitary growth hormone (GH) messenger RNA (mRNA) were compared in groups of genetically obese (fa/fa) and lean (Fa/‐) littermate male Zucker rats at four different ages, 3, 5, 9, and 11 weeks, in order to determine the earliest age at which a difference between the two groups could be detected. No difference was seen in three‐week‐old animals. Five weeks of age was the earliest time at which the level of GH mRNA was significantly decreased in the obese rats; this decrease was present at all subsequent ages. Mean serum growth hormone levels were lower in obese animals at all ages, but the differences were not statistically significant because of the large individual variation associated with the pulsatile nature of GH release. The earliest occurrence of differences in GH mRNA level is later than some of the obesity associated abnormalities present in adipose tissue. The earliest time of the GH mRNA differences can be associated with the time when decreased protein deposition is initially seen in the obese rats. Because of this association, decreased GH mRNA may enhance the development of obesity.
Evans‐Elemente. Eine Entgegnung zu Masings Bemerkungen zur Theorie der Korrosion durch Lokalelemente
Authors: Wickert K. , Wiehr H.
Published: December 2004

Zusammenfassend läßt sich sagen, daß die Deutung der Evanschen Versuche erst dann mit unseren Ergebnissen übereinstimmt, wenn die Aktivitätshöhe der Elektrodenoberflächen berücksichtigt werden. Alle Formulierungen berücksichtigt werden. Alle Formulierungen allgemeinen Inhalts über die Evans‐Elemente führen zu Widersprüchen, wenn die Oberflächenaktivität nicht berücksichtigt wird. Da die Korrosion als ein Geschehen im atomaren Bereich aufzufassen ist, müssen auch die theoretischen Grundlagen der Korrosion aus diesem Gebiet entnommen werden. Das hat leider in einigen Fällen zu neuen Wortprägungen geführt, die aber unseres Erachtens hingenommen werden müssen.
Die Isolierung von Magnetblechen durch eine Glimmer‐Phosphatschicht
Authors: Wüstefeld A.
Published: December 2004

Entwicklung der Verfahren zur Isolierung von Magnetblechen (Isolierung durch Papier oder Lack) — Grundlagen, Vorteile und Durchführung des Micacid‐Isolierverfahrens — Eigenschaften micacidisolierter Magnetbleche.
Werkstoffe und Böden
Authors: Haase L. W.
Published: December 2004

Allgemeines chemisches Verhalten sowie spezielles Verhalten der Eisenmetalle (Reinsteisen, Flußstahl, Gußeisen), Nichteisenmetalle (Kupfer, Zink, Blei und deren Legierungen) und nichtmetallischen Baustoffe (Zement, Asbestzement, Beton, Ton, Steinzeug und Holz) in Erdböden.
Das Korrosionsverhalten von ferritischem und perlitischem Gußeisen mit Kugelgraphit. Eine kritische Betrachtung
Authors: Franke E.
Published: December 2004

Gefügeaufbau von „verformbarem Gußeisen”︁ — Korrosionsversuche mit Ni‐Mg‐ bzw. Mg‐behandeltem Gußeisen mit Kugelgraphit und mit handelsüblichem Gußeisen — Auswertung und Deutung der Versuchsergebnisse (Einfluß von Graphitmenge und ‐verteilungsform auf den Angriff in sauren, neutralen und alkalischen Mitteln) — Bodenkorrosion von „verformbarem Gußeisen”︁.
Experimentell bestimmte pH‐Werte in verdünnten NH3, CO2‐ und NaOH‐Lösungen. (Zum Gebrauch in Hochdruckkraftwerken)
Authors: Wickert K. , Wiehr H. , Jahr H.‐G.
Published: December 2004

pH‐Wert von Betriebswässern und Korrosion der Metalle — Versuchsaufgabe, ‐einrichtungen und ‐durchführung — NH3‐bzw. CO2‐bzw. NaOH‐Gehalt und pH‐Wert von Wässern — Grobe pH‐Schätzungen — Temperaturkoeffizient des pH‐Wertes — Ermittlung des Salzgehaltes ammoniakhaltiger Kondensate aus elektrischer Leitfähigkeit und pH‐Wert — Zusammenfassung.
Graphitfarben
Authors: Hadert H.
Published: December 2004

Graphitfarben

Bei der allgemeinen Bleiknappheit finden heute Rostschutzfarben aus Graphit wieder größere Beachtung. Es werden deshalb die Eigenschaften geeigneter Graphitsorten sowie deren Anwendung als Rostschutzfarbe allein oder in Verbindung mit Bleifarben geschildert.