bluuebunny/crossref_metadata_2025 huggingface dataset:
Vascular endothelial cells in cell‐mediated immunity: Adoptive transfer with in vitro conditioned cells is genetically restricted at the endothelial cell barrier
Authors: Standage Blayne A. , Vetto R. Mark , Jones Rich , Burger Denis R.
Published: December 2004

Delayed‐type hypersensitivity (DTH) is a cell‐mediated immune response that can be adoptively transferred in rats when greater than 2 × 108 cells from peritoneal exudate, lymph nodes, or spleen are used. We have shown that by using an in vitro conditioning step with antigen, transfer can be subsequently carried out with as few as 2 × 107 spleen cells. The magnitude of DTH was reflected in ear swelling after intradermal injection of antigen [tuberculin or keyhole limpet hemocyanin (KLH)] and confirmed histologically. The transfer was antigen specific, requiring the sensitizing antigen in both the in vitro conditioning step and in the ear test challenge. Adoptive transfer with conditioned cells was genetically restricted by alleles of the RT‐1 region [major histocompatibility complex (MHC) of the rat]. Brown Norway strain (n haplotype) immune cells would not transfer DTH to Lewis (1 haplotype), ACI (a haplotype), or Buffalo (b haplotype) rats, whereas each strain would transfer DTH to syngeneic recipients. Moreover, this pattern of restriction held for all strains when tested in reciprocal fashion. In additional experiments, F1 to parental bone marrow chimeras were constructed so that bone‐marrow‐derived cells and non‐bone‐marrow‐derived cells were of different RT‐1 haplotypes. When these chimeras were used as recipients, transfer of DTH was only observed when immune donor cells and recipient non‐bone‐marrow‐derived cells were syngeneic. These results point to the critical role of non‐bone‐marrow‐derived cells (endothelial cells) in the DTH reaction.
Design and characterization of peptides with amphiphilic β‐strand structures
Authors: Osterman David G. , Kaiser E. T.
Published: December 2004

To extend our studies on peptides and proteins with amphiphilic secondary structures, a series of peptides designed to form amphiphilic β‐strand structures was designed, synthesized, and characterized by circular dichroism and infrared spectroscopy. Amphiphilic β‐strand conformations may be likely to appear in a variety of surface‐active proteins, including apolipoprotein B and fibronectin. In a β‐strand conformation, the synthetic peptides will possess a hydrophobia face‐composed of valine side chains and a hydrophilic face composed of alternating acidic (glutamic acid) and basic (ornithine or lysine) residues. The peptides studied had a variety of chain lengths (5, 9, and 13 residues), and had the amino groups either free or protected with the trifluoroacetyl group. While the peptides did not possess a high potential for β‐sheet formation based on the Chou Fasman parameters, they possessed significant β‐sheet content, with up to 90% β‐sheet calculated for the 13‐residue protected peptide. The driving force for β‐sheet formation is the potential amphiphilicity of this conformation. The β‐strand conformation of the 13‐residue unprotected peptide was stable in 50% trifluoroethanol, 6 M guanidine hydrochloride, and octanol. The peptides are strongly sell‐associating in water, which would reduce the unfavorable contacts of the hydrophobic residues with water. It is clear that small peptides can be designed to form stable β‐strand conformations.
Cellular redistribution of β‐adrenergic receptors in a human astrocytoma cell line: A comparison with the epidermal growth factor receptor in murine fibroblasts
Authors: Wakshull E. , Hertel C. , O'Keefe E. J. , Perkins J. P.
Published: December 2004

The redistribution of β‐adrenergic receptors (β‐AR) during agonist‐induced desensitization has been compared to the process of receptor‐mediated endocytosis of epidermal growth factor (EGF) in human astrocytoma cells (1321N1). [125I]EGF exhibited saturable binding to high affinity (KD = 1–2 nM) receptor sites on intact 1321Nl cells. [125I]EGF was found to internalize rapidly using an acid wash technique to remove surface bound hormone. Sucrose density gradient fractionation following exposure to EGF revealed a redistribution of EGF binding sites from high density (heavy peak) to low density (light peak) regions of the gradient. The light peak binding probably represents EGF in internalized vesicles formed during endocytosis. Low temperature (4 °C) or the presence of the lectin concanavalin A (con A) inhibited this ligand‐induced movement of EGF receptors. When cells were incubated simultaneously with EGF and the β‐AR agonist isoproterenol, both receptors were found to co‐migrate in the low density regions of sucrose gradients. No evidence of heterologous ligand‐induced receptor endocytosis was found. These results suggest that the EGF receptors and β‐AR are processed in parallel by 1321N1 cells.
Wound fluid angiogenesis factor stimulates the directed migration of capillary endothelial cells
Authors: Banda Michael J. , Dwyer Karyn S. , Beckmann Alice
Published: December 2004

During wound healing, new capillaries grow into the wound site. An angiogenesis factor isolated from wound fluid stimulates the movement of capillary endothelial cells in a filter migration assay. Experiments were carried out to determine whether the movement seen in the assay was chemokinetic or chemotactic. Capillary endothelial cells were plated onto a collagen‐coated coverslip and inverted over a visualization apparatus. Cells exposed to a constant concentration of wound fluid angiogenesis factor (WAF) were more mobile than cells not exposed to WAF, and this movement was chemokinetic. When exposed to a gradient of WAF, the cells translocated toward the higher concentration; this directional movement was chemotactic. Cells in a gradient of WAF morphologically aligned with the gradient. These data support the idea that wound healing angiogenesis is regulated by the chemotaxis of capillary endothelial cells.
Antibodies specific for the carboxy‐terminal region of the major surface glycoprotein of simian rotavirus (SA11) and human rotavirus (Wa)
Authors: Streckert Hans‐Jürgen , Grunert Barbara , Werchau Hermann
Published: December 2004

Antibodies specific for the major outer capsid protein (VP7) of the simian rotavirus SA11 were obtained by immunization of rabbits with a synthetic peptide, Ser‐Ala‐Ala‐Phe‐Tyr‐Tyr‐Arg‐Val, corresponding to the eight carboxy‐terminal amino acids of the viral protein predicted from the nucleotide sequence of the gene segment 9 of the SA11 genome. As the carboxy‐terminal region of the VP7 of human rotavirus Wa has an identical sequence, cross‐reactivity of the raised antibodies was observed with this strain.
Treatment of a fatal transplantable erythroleukemia by procedures that lower endogenous erythropoietin
Authors: Hossain Azhar , Kim Jung‐Kon , Hankins W. David
Published: December 2004

The in vitro growth of primary erythroleukemia cells has been examined in the presence and absence of the hormone erythropoietin (EPO). Although these leukemic cells had previously been considered to be hormone‐independent, addition of EPO was found to be essential for maximum growth in culture. Erythroid colonies that grew in the presence of EPO were leukemogenic when returned to mice. Influence of EPO on the in vivo growth of leukemic cells was indicated by our findings that (1) administration of the hormone caused a more severe leukemia and rapid death, and (2) transfusion of red blood cells, which lowers endogenous EPO, led to decreased spleen size and increased survival of leukemic mice. We suggest from our results that hormone‐associated therapy might be efficacious in the treatment of this and, perhaps, other leukemias.
Human restriction fragment length polymorphisms and cancer risk assessment
Authors: Krontiris Theodore G. , DiMartino Nancy A. , Colb Mark , Mitcheson H. David , Parkinson David R.
Published: December 2004

The polymorphic restriction fragments of the human Ha‐ras locus, produced by the variable tandem repetition (VTR) of a short consensus sequence, fall into three classes based on allelic frequencies. Alleles of the “rare” class (individual frequencies < 0.5%) have been detected only in white blood cell and tumor DNA of cancer patients. This phenomenon is independent of ethnic origin. No significant association of rare alleles with cancer patients has been demonstrated at an independent tandem repeat locus, VTR4.1. The results suggest that the Ha‐ras restriction fragment length polymorphism is useful in cancer risk assessment.
Properties of catalase activity in vegetative and sporulating cells of yeast Saccharomyces cerevisiae
Authors: Ota Akihiro
Published: December 2004

Properties of catalase activities have been examined in the intact cells of early stationary phase and cells 3 hr after transfer to sporulation medium in Saccharomyces cerevisiae. The catalase activities of the two cells had a broad optimal pH from 6 to 8. Catalase activity in the intact cells increased throughout a 4‐hr period of the observation following transfer to sporulation medium. Almost all the catalase activity in vegetative cells was lost by the treatment at 60°C for 10 min. Catalase activities of both cells were inhibited by KCN, NaN3, o‐phenanthroline, and PCMB. The catalase activity of the vegetative cells was slightly more inhibited and inactivated than that of the sporulating cells by the inhibitors and by the treatment with HCl or NaOH.
The thyroid “microsomal” antigen is an epitope on the thyrotropin receptor
Authors: Islam Nazrul , Tuppal Raj , Hawe Beverley S. , Briones‐Urbina Rosario , Farid Nadir R.
Published: December 2004

Antimicrosomal antibodies are present in the sera of most patients with autoimmune thyroiditis, and Graves' disease. It has, in general, been difficult to separate antimicrosomal activity from that directed against the thyrotropin (TSH) receptor in Graves' IgG preparations. The “microsomal” antigen has been localized to the endoplasmic reticulum and microfollicular aspect of thyrocytes; its structure is however unknown. In an attempt to identify the thyroid microsomal antigen, we studied the interaction of Hashimoto's IgG with high microsomal antibody titre and negative for thyroglobulin with purified thyroid plasma and light microsomal membranes. We allowed Hashimoto's, Graves', and control IgGs to bind to protein blots of thyroid plasma membranes resolved on SDS‐PAGE under nonreducing conditions. All seven Hashimoto's IgG at a concentration of 2 mg/ml interacted with an M ∼︁ 197,000 polypeptide corresponding to the TSH holoreceptor. By contrast to Graves' IgG (which were positive at 1 mg/ml), however, this binding was not blocked by pretreatment of the protein blots with TSH. Normal IgGs showed no binding at concentrations of up to 2 mg/ml.

Both Hashimoto's and Graves' IgG interacted with TSH‐affinity column‐purified receptor preparations.

Two of the Hashimoto's IgGs induced adenylate cyclase activation in thyroid plasma membranes, three inhibited TSH‐stimulated enzyme activation, and two were without effect. Two classes of autoantibodies, other than TSH receptor directed, were encountered; one class raised to antigens common to all seven patients and another class unique to individual patients, eg, Mr 210,000 and Mr 20,000 polypeptides.

We propose that the TSH receptor has multiple epitopes (functional domains), and the one to which antimicrosomal antibody bind is likely to be spatially separated from that with which Graves' IgG and TSH interact. Differences in affinity or number of sites allows for the demonstration of Graves' IgG against a background of antimicrosomal antibody.
Number of receptor sites from Scatchard and Klotz graphs: Complementary approaches
Authors: Faguet Guy B.
Published: December 2004

Estimates of number of receptor sites and evaluation of the complexity of the binding process require collection of a spectrum of binding measurements and selection of a theoretical model to fit the experimental data. The appropriateness of the measurements and of the model can be visually judged on graphic displays of the model‐data fitting curves in Scatchard and semilogarithmic coordinates. This approach is helpful for detecting the two types of errors most frequently found in reports of binding studies: (1) underestimating the number of binding sites, and (2) failure to recognize the complexity of the binding process. While the former is readily recognizable on semilogarithmic but not on Scatchard plots of the model fitting the data, the latter might not be apparent on either plot. Collection of extensive measurements over a wide range of ligand concentrations with graphic display of the model‐data fitting curves in Scatchard and semilogarithmic coordinates should be used to recognize and prevent both errors.
Phenotypic characterization of Ewing sarcoma cell lines with monoclonal antibodies
Authors: Lipinski Marc , Braham Karim , Philip Irène , Wiels Joëlle , Philip Thierry , Dellagi Koussay , Goridis Christo , Lenoir Gilbert M. , Tursz Thomas
Published: December 2004

The histogenesis of Ewing sarcoma, the second most frequent bone tumor in humans, remains controversial. Four Ewing cell lines were analyzed by immunological methods. A panel of antibodies directed to T, B, and myelomonocytic markers gave negative results. Surface antigens recognized on Ewing cells were found to be related to the neuroectoderm lineage. Ganglioside GD2, a marker of neuroectodermal tissues and tumors, was present on all lines. These were also stained by the mouse monoclonal antibody HNK‐1, which detects a carbohydrate epitope present on several glycoconjugates of the nervous system, including two glycoproteins, the myelin‐associated glycoprotein and the neural cell‐adhesion molecule (N‐CAM), and an acidic glycolipid of the peripheral nervous system. The P61 monoclonal antibody, which reacts with a peptide moiety of N‐CAM, and a rabbit antiserum, raised to purified mouse N‐CAM and not recognizing the HNK‐1‐defined epitope, were also reactive. By contrast, all antibodies specific for hematopoietic cell surface antigens were totally negative. Besides these antigenic features, Ewing sarcoma cells are characterized by a specific t(11:22)(q24;q12) translocation also observed in neuroepithelioma, a neuroectodermal tumor, suggesting a possible evolutionary related origin. The recent finding that the human N‐CAM gene is located at the vicinity of the breakpoint on chromosome 11 indicates that it might be involved in genetic rearrangements occurring in this region.
Association of tyrosine protein kinase activity with mitochondria in human fibroblasts
Authors: Piedimonte Giuseppe , Silvotti Lucia , Borghetti Angelo F. , Chamaret Solange , Montagnier Luc
Published: December 2004

A tyrosine protein kinase activity has been detected in the mitochondrial fraction purified from human fibroblasts. By enzymatic and sedimentation analysis this activity appeared to be localized in the mitochondrial outer membrane. Mitochondrial tyrosine phosphorylation was strictly dependent on the presence of Mn2+ ions. An inverse relationship between cell proliferation and mitochondrial protein phosphorylation on tyrosine residues has been found: a marked increase in the mitochondrial tyrosine kinase activity occurred when a significant reduction in the growth rate followed serum step‐down. In mitochondria purified from resting cells, a protein band with apparent molecular weight of 50 kd appeared to be phosphorylated on tyrosine.
Formation of protease nexin‐thrombin complexes on the platelet surface
Authors: Gronke Robert S. , Curry Thomas K. , Baker Joffre B.
Published: December 2004

We have recently described a platelet factor that is similar to the fibroblast thrombin inhibitor protease nexin I (PNI) [12]. The present manuscript shows that this platelet form of PN (PNp) does not complex [125I]‐thrombin that has been blocked at its active site, consistent with the conclusion that it is a thrombin inhibitor. When platelets are incubated with [125I]‐thrombin, PNp‐[125MI]‐thrombin complexers accumulate both in the medium and on the platelet surface. In the case of fibroblasts, PNI‐[125I]‐thrombin Complexes that form in solution bind to the cells as a consequence of a receptor‐mediated clearance process [Low et al, Proc Natl Acad Sci USA 78:2340, 1981]. We show here that the PNp‐[125I]‐thrombin complexes that accumulate in platelet‐binding incubation medium do not bind to platelets. Thus, the platelet‐associated complexes must form by [125I]‐thrombin binding to PNp that is associated with the platelet surface. Pretreatment of platelets with heparin markedly increases the number of PNp‐[125I]‐thrombin complexes that form on platelets. The basis for this increase in unclear. This effect seems incompatible with a heparinlike factor acting as the surface binding site for PNp.
Growth‐promoting effects of esterolytically inactive thrombin on macrophages
Authors: Bar‐Shavit Rachel , Kahn Arnold J. , Mann Kenneth G. , Wilner George D.
Published: December 2004

It has been recognized for many years that α‐thrombin, like other better known mitogens (eg, PDGF, EOF, etc) is capable of initiating proliferation in quiescent cells belonging to the fibroblast family. However, unlike these other peptides, thrombin is a serine protease whose function as a growth stimulator for fibroblasts is intimately linked to its estefolytic activity. Thus, while native α‐thrombin is capable of evoking DNA synthesis in GoG1‐arrested cells, neither enzymatically inactive thrombin (eg, iPR2P‐α‐thrombin) nor partially degraded thrombin (eg, γ‐thrombin) shares in this capability. Data from our laboratory have shown that thrombin is chemotactic for peripheral blood monocytes and for cells belonging to the monocyte/macrophage family and that this activity is not dependent upon thrombin's enzymatic properties. Our recent findings demonstrate that thrombin also serves as a growth factor for these cells, and this mitogenic capability is independent of esterolytic function and resides in the same region of the molecule as that responsible for chemotaxis. Additionally, by means of techniques such as computer modeling and peptide synthesis, we have now been able to delineate a distinct mitogenic subsite within this chemotactic thrombin sequence. Thus, the sequence in the thrombin B chain that mediates chemotaxis represents a true cell interactive exosite additionally capable of stimulating growth and possibly other biological functions in cells of macrophage/monocyte lineage.
Transforming growth factors and control of neoplastic cell growth
Authors: Keski‐Oja Jorma , Leof Edward B. , Lyons Russette M. , Coffey Robert J. , Moses Harold L.
Published: December 2004

Transforming growth factors (TGFs) are peptides that affect the growth and phenotypic of cultured cells and bring about in nonmalignant fibroblastic cells phenotypic properties that resemble those of malignant cells. Two types of TGFs have been well characterized. One of these, TGFβ, is related to epidermal growth factor (EGF) and binds to the EGF receptor, whereas the other. TGFβ, is not structurally or functionally related to TGFβ or EGF and mediates its effects via distinct receptors.

TGFβ is produced by a variety of normal and malignant cells. Depending upon the assay system employed, TGFβ has both growth‐inhibitory and growth‐stimulating properties. Many of the mitogenic effects of TGFβ are probably an indirect result of the activation of certain growth factor genes in the target cell. The ubiquitous nature of the TGFβ receptor and the production of TGFβ in a latent form by most cultured cells suggests that the differing cellular responses to TGFβ are regulated either by events involved in the activation of the factor or by postreceptor mechanisms. The combined effects of TGFβ with other growth factors or inhibitors evidently play a central role in the control of normal and malignant cellular growth as well as in cell differentiation and morphogenesis. Since transforming growth factor as a concept has partially proven misleading and insufficient, there is a need to find a new nomenclature for these regulators of cellular growth and differentiation.
The role of ATP in the cytostructure of the hepatocytes
Authors: Katz Joseph , Wals P. A.
Published: December 2004

We have previously described the preparation of hepatocytes from which the plasm membrane was removed by digitonin treatment. Such “nude” cells were found to be very stable in sucrose media containing above 50 mM NaCl or KC1, but they disintegrate near instantly in salt‐free media, liberating nuclei, mitochondria, and other organelles. We show here that disintegration occurs at a physiologic pH and in the presence of oxygen. Disintegration was blocked by rotenone, oligomycin, KCN, and carboxyatractyloside, establishing that oxidative phosphorylation and ATP generation is essential for disintegration to occur. The addition of ATP, GTP, ITP, or ADP (but not AMP) in the presence of the inhibitors, induced breakdown.

Taxol, an inhibitor of tubulin depolymerization and phalloidin, a drug that stabilizes actin fibers, prevented disintegration in salt‐free media. The effect of these drugs was counteracted by the addition of ATP.

Our results show that two conditions are essential to induce the disintegration of the nude cell: media of low ionic strength, and ATP generation. The ATP effect is likely to be of physiological significance, suggesting role for ATP generation in affecting polymerization of cytoskeletal elements.
Hyaluronate‐binding protein of simian virus 40‐transformed 3T3 cells: Membrane distribution and reconstitution into lipid vesicles
Authors: Chi‐Rosso Gloria , Toole Bryan P.
Published: December 2004

Hyaluronate‐binding protein (HASP) has been extracted in detergent from the membranes of simian virus 40‐transformed 3T3 (SV‐3T3) cells (Underhill et al, J Biol Chem 258:8086–8091, 1983). When SV‐3T3 cells were treated with trypsin prior to isolation and dissolution of the membranes, no hyaluronate‐binding activity could be detected. This indicates that all of the detectable HABP of SV‐3T3 cells is located on the external surface of the plasma membrane rather than on internal membranes, which would be inaccessible to the trypsin. The detergent‐extracted HABP from SV‐3T3 membranes was reconstituted into the membrane of lipid vesicles, which were formed by addition of exogenous phosphatidylcholine and cholic acid to the extracts followed by removal of detergent by dialysis against 0.02 M Tris pH 8.0 in the presence of protease inhibitors. Reconstitution was assessed by sedimentation in a discontinuous sucrose gradient and by gel filtration on Sepharose 4B in the presence and absence of detergent. The characteristics of binding of hyaluronate to the reconstituted HABP were then compared with those studied previously for the original membrane‐bound HABP and the detergent‐extracted HABP (Underhill et al, J Biol Chem 258:8086–8091, 1983). It was observed previously that binding of hyaluronate to HABP in the cell membranes was of higher affinity and specificity than to HABP in the detergent extracts of these membranes. It was found here that reconstitution of the extracted HABP into the membranes of lipid vesicles led to restoration of affinity of binding to the level observed in the original cell membranes. However, whereas chondroitin sulfate does not compete significantly for binding of hyaluronate to cell membrane‐bound HABP, partial competition was observed for the reconstituted HABP as well as for detergent‐extracted HABP. Thus, it is concluded that the high affinity of binding of hyaluronate to the plasma membrane of SV‐3T3 cells is in part dependent on insertion of the HABP in the membrane, but that other interactions, not duplicated in our reconstitution experiments, must be necessary for the specificity of the HABP.
Autostimulatory mechanisms in myeloid leukemogenesis
Authors: Schrader J. W. , Leslie K. B. , Ziltener H. J. , Schrader S.
Published: December 2004

WEHI‐274 is a monocytic leukemia that arose in a BALB/c mouse infected with Abelson murine leukemia virus. A series of subclones were derived from early passages of this tumor. Three subsets of these leukemogenic subclones were identified. Two subsets demonstrated autostimulatory patterns of growth. This was due to the ectopic production of the T‐cell lymphokine the panspecific hemopoietin IL‐3 in one case and of the T‐cell lymphokine granulocyte‐macro‐phage colony‐stimulating factor (GM‐CSF) in the other. The third type of subclone did not secrete any autostimulatory growth factor. In the subclone producing IL‐3, one copy of IL‐3 gene was rearranged and abnormal IL‐3 RNA transcripts were present in the nucleus. Subclones producing GM‐CSF also contained abnormal GM‐CSF RNA transcripts, although no rearrangement of the GM‐CSF gene was detected. All three sets of subclones shared a common rearrangement of one c‐myb oncogene, suggesting that they share a common ancestor. These results suggest that initiation or progression of leukemogenic behavior in this abnormal clone occurred in three different ways, two of which involved autostimulation by the ectopic activation of T‐cell lymphokine genes.
Inhibition of epidermal growth factor‐stimulated DNA synthesis by a bovine sialoglycopeptide inhibitor occurs at an intracellular level
Authors: Bascom Charles C. , Sharifi Behrooz G. , Johnson Terry C.
Published: December 2004

The control of cell proliferation involves the complex interaction between growth factors and growth inhibitors. We have examined this interaction with the mitogen epidermal growth factor (EOF) and a recently purified 18 kD, pI 3, sialoglycopeptide that reversibly inhibits cellular metabolism of a variety of cells. The sialogly‐copeptide was a very potent inhibitor of EOF action; 0.22 nM of the inhibitor completely blocked the mitogenic effect of 1.60 nM of EGF. The sialoglycopeptide, however, did not affect the binding of EGF to 3T3 cells. Neither the mixed affinities (0.11–1.9 nM) of binding nor the total number of receptors (50,000 receptors/cell) for EGF were altered by the addition of the Sialoglycopeptide. In addition, competitive binding experiments demonstrated the specificity of inhibitor binding to 3T3 cells and also showed that EGF and the Sialoglycopeptide did not share the same receptor, suggesting that the inhibitor blocked EGF action at a postreceptor, intracellular event in the signal cascade. We further demonstrated that the Sialoglycopeptide had to be added within 2.5 hr after EGF to block effectively the stimulation of DNA synthesis by the growth factor, suggesting that the inhibitor blocked EGF stimulation at a relatively early step in the signal transduction mechanism.
Comparison of basement membrane matrix degradation by purified proteases and by metastatic tumor cells
Authors: Starkey Jean R. , Stanford David R. , Magnuson James A. , Hamner Steve , Robertson Nancy P. , Gasic Gabriel J.
Published: December 2004

We have examined the nature of biochemical degradation of an isolated basement membrane matrix (bovine lens capsule) using different methodologies. The first strategy was quantitation of the release of surface‐bound 125I and a second the documentation by SDS‐PAGE of the appearance of putative cleavage products and the loss of high‐molecular‐weight components from the matrix. Basement membrane matrix bands resolved on SDS‐PAGE were identified by their protease sensitivities as well as by Western immunoblots using monoclonal antibodies developed for this study. Radioiodinated components were found predominantly at positions on the gel equivalent to 160–200 kd and 400 kd proteins. Since these labeled moieties were sensitive to bacterial collagenase digestion and stained with anticollagen type IV antibodies, they were determined to represent various configurations of collagen type IV. Several other lower‐molecular‐weight bands also stained with the anticollagen IV antibodies. Monoclonal antibodies reactive with laminin exhibited a complex staining pattern on the gels, which included the expected 200 and 400 kd components. We confirmed that lens capsule basement membrane contained only a single heparan sulfate glycosaminoglycan species, and tumor cell‐induced glycosaminoglycan degradation within the basement membrane matrix was detected using cellulose acetate electrophoresis.

Distinctive putative cleavage products were resolved on SDS‐PAGE gels from matrices subjected to digestion by a variety of purified proteases as well as by metastatic tumor cells or their conditioned media. Tumor cells of different histio‐types produced different characteristic cleavage patterns, suggestive of the existence of several pathways of matrix degradation. Overall, primary tumor cells exhibited a greater degradative activity towards the basement membrane matrix than did long‐term tissue culture‐passaged cells. The same tumor cell line could exhibit considerably different patterns of both protein and glycosaminoglycan degradation depending on recent culture history. The relevance of these biochemical studies to the pathogenesis of malignant neoplasms is shown by: (1) the evaluation of degradative activities of B16 tumor cell populations exhibiting enhanced lung‐colonizing phenotypes, and (2) the ability of a known antimetastatic moiety with antiproteasc activity (Haementeria leech species salivary gland extract) to protect matrix components from degradation by tumor cell‐conditioned medium.
Steroid hormone antagonists at the receptor level: A role for the heat‐shock protein MW 90,000 (hsp 90)
Authors: Baulieu Etienne‐Emile
Published: December 2004

Antisteroid hormones compete for hormone binding at the receptor level and prevent the hormonal response. A new concept is proposed for explaining the antiglucocorticosteroid activity of RU 486 in the chick oviduct system. It is based on the ability of the antisteroid to stabilize the hetero‐oligomeric 8S‐form of the glucocorticosteroid receptor (GR), which involves the interaction of the 94k‐receptor and heat‐shock protein MW 90,000 (hsp 90). It is proposed that hsp 90 caps the DNA binding site of the receptor, and this prevents it from binding to the DNA of hormone regulatory elements (HRE) and increasing transcription of regulated genes. This paper reviews other antiglucocorticosteroid and antiestrogen systems with reference to this hypothesis and also describes a four‐step analysis of the molecular mechanism of antisteroid hormone action at the receptor level.
Ecto‐cyclic AMP‐receptor in goat epididymal intact spermatozoa and its change in activity during forward motility
Authors: Dey Chinmoy S. , Majumder Gopal C.
Published: December 2004

Goat epididymal intact spermatozoa have been shown to possess on the external surface specific receptors that bind with high affinity to exogenous [8‐3H]cyclic AMP. The ecto‐cyclic AMP‐receptor activity was not due to contamination of broken or “leaky” cells, if any. The binding reaction of [3H]cyclic AMP with the receptors was extremely rapid. Uptake of the labeled cyclic AMP to the sperm cytosolic fraction was undetectable. There was little leakage of cyclic AMP‐receptors from intact spermatozoa during the binding assays. The binding reaction was proportional to cell concentration, specific and saturable at 250 nM cyclic AMP. The binding of the labelled cyclic nucleotide was nearly completely displaced at saturating concentrations (2.5 μM) of the unlabelled nucleotide. The ecto‐receptors showed high specificity for binding to cyclic AMP. The Kd of the binding sites was approximately 1.7 × 10‐8 M. The binding interaction was highly sensitive to treatment with proteolytic enzymes: trypsin, chymotrypsin, or pronase (125 μg/ml). Sonication caused a nearly 450% increase of the ecto‐receptor activity. The specific activity of the ecto‐cyclic AMP‐receptor was approximately twofold higher in the vigorously forwardly motile spermatozoa than in the “composite” cells, suggesting that the ecto‐receptors may have a role in modulating flagellar motility.
Role of receptor occupancy in the transition from responsive to unresponsive states in cultured breast tumor cells
Authors: Darbre Philippa D. , King Roger J. B.
Published: December 2004

Progression from a steroid sensitive to insensitive state is characteristic of breast tumors, but little is known about the molecular mechanisms involved. Changes in steroid receptor can be associated with the progression. This paper reviews the cell culture data pertaining to loss of response and concludes that loss of receptor is a consequence rather than a cause of insensitivity. This view is based on evidence that loss of all response parameters occurs despite the presence of fully functional receptors as determined by transfection experiments. The postreceptor defect appears to be at the level of the hormone response element of the responsive genes arid may involve DNA methylation. The implications of the model for human breast cancer biology are discussed.
Identification and characterization of tyrosine kinase activity associated with mitochondrial outer membrane in sarcoma 180 cells
Authors: Piedimonte Giuseppe , Chamaret Solange , Dauguet Charles , Borghetti Angelo F. , Montagnier Luc
Published: December 2004

Tyrosine protein kinase activity has been detected in the mitochondrial fraction purified from sarcoma 180 tumor cells. Following hypotonic disruption of mitochondria, tyrosine kinase activity appeared to cosediment with monoamine oxidase, marker enzyme of mitochondrial outer membrane; meanwhile, serine and threonine kinases were found to be associated with the inner membrane and matrix of mitochondria. Mitochondrial tyrosine kinase(s) showed thermosensitivity and Mn 2+ dependence, useful properties for its characterization and separation from tyrosine kinases associated with other particulate fractions and from serine and threonine kinases associated with mitochondria. Following in vitro incubation of mitochondria with labelled ATP as substrate and analysis by PAGE, a complex pattern of phosphotyrosine containing proteins with a major band of 50–55 kilo‐daltons resulted.
Cytogenetic diversity in primary human tumors
Authors: Wolman Sandra R. , Camuto Patricia M. , Perle Mary Ann
Published: December 2004

Cytogenetic patterns from primary short‐term culture of breast cancer, renal carcinoma, and tumors of the central nervous system are presented to illustrate the range of karyotypic diversity of human solid tumors as well as their biologic differences in culture systems that support their growth. These studies have illustrated several major issues. (1) Results vary with the tissue of origin: primary cultures from breast are almost uniformly diploid, while renal tumors are near‐diploid, mosaic, and show clonal aberrations; and CNS tumors are heterogeneous: some diploid, some near‐diploid and some highly aneuploid. (2) Results after short‐term culture are selective, representing subpopulations from the heterogeneous cells that are detected on direct analysis of fresh tumors by cytogenetics or flow cytometry (FCM). It is not yet clear whether prognosis depends on the dominant population of the primary tumor or alternatively should be influenced by detection of small aneuploid subpopulations. (3) Evidence from all three tumor types supports the interpretation that cytogenetically normal diploid cells constitute part of some tumor populations, and may be better adapted to routine growth in culture than aneuploid subpopulations from the same primary tumors. These cells may also compose a major portion of the viable population of tumors in vivo and, therefore, could represent a useful model for studies of tumorigenesis and therapeutic regimens.
Isolation and visualization of Met‐72‐positive, metastatic variants present in B16 melanoma tumor masses
Authors: Parratto Nanette P. , Kimura Arthur K.
Published: December 2004

Metastatic variants of the B16 melanoma displaying high experimental metastatic potential have been shown to express high levels of a 72,000‐dalton glycoprotein (Met‐72) on their cell surface (Kimura AK, Xiang J: J Nat Can Inst 76:1247–1253, 1986). Monoclonal antibodies (MoAb) directed against the Met‐72 determinant have been used in this study as immunohistochemical reagents on preparations of fresh B16 melanoma tumors and their metastases. These immunohistochemical analyses have utilized frozen sections, impression smears, and cytospin preparations of fresh tumors harvested at various time points during tumor growth, to view the presence and location of Met‐72‐positive metastatic variants within tumor masses. Biotinylated anti‐Met‐72 MoAbs were reacted with freshly dissociated tumor cells from a B16 melanoma ovarian metastasis. These cells were then reacted with fluorescein isothiocyanate (FITC)‐streptavidin and analyzed by flow cytometry. A discrete population of positively staining cells was detected and isolated by cell sorting techniques. Met‐72‐positivc cells were then cloned and reanalyzed after several weeks of in vitro expansion and found to have high experimental metastatic potential to ovaries. Frozen sections of subcutaneous tumors and their metastases were analyzed by immunoperoxidase techniques. A consistent finding in these studies has been that the few tumor cells which showed high intensity of Met‐72 staining were positioned perivascularly and at the invading front of B16 melanoma tumors.
Analysis of cell surface glycoprotein changes related to hematopoietic differentiation
Authors: Reading Christopher L. , Hickey Catherine M. , Yong Weiben
Published: December 2004

A high‐resolution technique has been used to study differentiation‐related and leukemia‐associated glycoproteins. Cells are labeled with the membrane‐impermeable probe sulfo‐N‐hydroxysuccinimidyl‐biotin. Nonionic detergent extracts are subjected to affinity chromatography on a number of immobilized lectins and after polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS‐PAGE) and western transfer, the biotin‐labeled glycoproteins are visualized by using avidin‐horseradish peroxidase and 4‐chloronaphthol. With the aid of the lectins concanavalin A, Dolichos biflours agglutinin, Lens culinaris hemagglutinin, peanut agglutinin, pokeweed mitogen, Ricinus communus agglutinin I, soybean agglutinin, Ulex europeus agglutinin I (UEA), and wheat germ agglutinin, each purifies different glycoprotein subsets from the same cell type. Mature cells of distinct hematopoietic lineages differ considerably in their cell surface glycoprotein patterns.

This technique was used to analyze the glycoproteins of human leukemia cells before and after the induction of differentiation. K562 cells differentiated along different lineages after treatment with phorbol 12‐myristate 13‐acetate, sodium butyrate, dimethyl sulfoxide, or hemin. Limited specific alterations were observed with a number of lectins when K562 erythroleukemia cells were induced to differentiate. Among these, a number of bands were identified that were either lost or appeared after induction of differentiation with all four agents. In contrast, the glycoproteins bound by UEA were drastically diminished after induction of differentiation, and the remaining UEA‐bound glycoproteins bore little resemblance to those of the cells before treatment. This high‐resolution technique may be useful as a general method for the examination of cell surface glycoprotein differences. Once specific glycoprotein alterations are detected, lectin affinity chromatography and SDS‐PAGE allow purification of antigens for the production of monoclonal antibodies.
Intracellular localization and degradation of diphtheria toxin
Authors: Fedde Kenton N. , Sly William S.
Published: December 2004

The internalization of surface‐bound diphtheria toxin (DT) in BS‐C‐1 cells correlated with its appearance in intracellular endosomal vesicles; essentially no toxin appeared within secondary lysosomal vesicles. In contrast, internalized epidermal growth factor (EGF) was localized within both endosomal and lysosomal vesicles. Upon prein‐cubation of cells with leupeptin, a lysosomal protease inhibitor, a threefold increase in the accumulation of EGF into lysosomes was observed. Under identical conditions, essentially all of the diphtheria toxin remained within endosomes (less than 2% of the intracellular diphtheria toxin accumulated in the lysosomaJ fraction), indicating that the inability to detect diphtheria toxin in lysosomes was not due to its rapid turnover within this vesicle. Following internalization of EGF or DT, up to 40% of the lgand appeared in the medium as TCA‐soluble radioactivity. EGF degradation was partially leupeptin‐sensitive and markedly NH4Cl‐sensitive, indicating lysosomal degradation. In contrast, DT A‐fragment degradation was resistant to these inhibitors, while B‐fragment showed only partial sensitivity. These data suggest that the bulk of endocytosed diphtheria toxin is localized within endosomes and degraded by a pathway essentially independent of lysosomes.
Membrane association of the hyaluronate stimulatory factor from LX‐1 human lung carcinoma cells
Authors: Knudson Warren , Toole Bryan P.
Published: December 2004

LX‐1 human lung carcinoma cells interact with human fibroblasts in culture to cause an increase in hyaluronate production (Knudson et al: Proceedings of the National Academy of Sciences of the United States of America 81:6767, 1984). It is shown here that a similar increase in hyaluronate production also occurs when membranes derived from LX‐1 cells, or detergent extracts thereof, are added to cultures of the human fibroblasts. However, no stimulation occurs when membranes or extracts from fibroblasts are added to cultures of the LX‐1 cells. The hyaluronate stimulatory factor present in the detergent extracts is a heat‐ and trypsin‐sensitive protein, requires more than 12 h for its action on fibroblasts, causes an elevation in hyaluronate synthetase activity in membranes derived from the fibroblasts, and can be reconstituted into artificial lipid vesicles. Thus, it is concluded that the stimulatory factor is a membrane‐bound protein present on the surface of the LX‐1 cells and that it interacts with fibroblasts to induce increased hyaluronate synthesis.
Distinct mechanisms of interferon‐gamma and tumor necrosis factor‐alpha action in oncogene‐transformed mouse fibroblasts
Authors: Seliger Barbara , Killian Marion , Pfizenmaier Klaus
Published: December 2004

The potential mechanisms of interferon (IFN)‐gamma and tumor necrosis factor (TNF)‐alpha action on tumor cells have been investigated in a model of mouse fibroblasts transformed by distinct retroviral vectors carrying the v‐mos, c‐myc, and v‐Ha‐ras oncogene, respectively. Treatment with both cytokines not only caused growth inhibition of v‐mos‐ and c‐myc‐transformants, but also a reversion of transformation‐induced suppression of major histocompatibility complex (MHC) class I antigen expression in all transformed cell lines. The phenotypical reversion of transformants was preceded by a selective modulation of LTR‐controlled oncogene expression. TNF‐alpha primarily affected stability of oncogene‐specific RNAs without influencing the activity of retroviral promoters. In contrast, IFN‐gamma was effective at the transcriptional level, apparently due to inhibition of LTR activity as revealed from reduced CAT activity in IFN‐gamma‐treated LTR‐CAT transformants. This IFN‐gamma‐mediated down‐regulation of retroviral promoter activity seemed to be selective for Moloney‐virus‐derived promoters, since the activity of other viral and cellular promoters was not suppressed by IFN‐gamma.
Studies on the human retinoblastoma susceptibility gene
Authors: Lee Wen‐Hwa , Bookstein Robert , Lee Eva Y‐H. P.
Published: December 2004

The retinoblastoma susceptibility (RB) gene is unique among other cloned cancer genes because its causal role in a human cancer, retinoblastoma, was established by classical genetic methods before its isolation. Earlier hypotheses and experimental data suggested that inactivation of a gene in chromosome band 13q14 resulted in retinoblastoma formation. A gene in this region was identified as the RB gene on the basis of mutations found specifically in retinoblastoma tumors; however, its proposed biological activity in suppressing neoplasia has yet to be demonstrated. The RB gene product was identified as a nuclear phosphoprotein of 110 kD associated with DNA binding activity, suggesting that the RB protein may regulate other genes. Probes for the RB gene and gene product will be useful for genetic diagnosis of retinoblastoma susceptibility in affected families; for direct detection of mutant RB alleles; and, potentially, for genetic diagnosis of susceptibility to osteosarcoma and other tumors tentatively linked to RB‐gene dysfunction. Continued study of the RB gene should yield further insight into mechanisms of oncogenesis, development, and gene regulation.
Evidence for autocrine activation of a tyrosine kinase in a human gastric carcinoma cell line
Authors: Giordano Silvia , Renzo Maria Flavia Di , Narsimhan Radha P. , Tamagnone Luca , Gerbaudo Elena V. , Chiadó‐Piat Loredana , Comoglio Paolo M.
Published: December 2004

Phosphotyrosine (P‐Tyr) antibodies have been used to identify the phosphorylated forms of growth factor receptors and oncogene‐coded tyrosine kinases. Western blot analysis of a gastric carcinoma cell line with P‐Tyr antibodies revealed a tyrosine‐phosphorylated protein of Mr 145,000 (P145). In addition, in vitro phosphorylation with (γ‐32P)ATP of P‐Tyr immunoprecipitates of the same cells resulted in labelling of this protein on tyrosine. P145 appears to be a transmembrane glycoprotein, with features suggestive of a growth factor receptor. However, the in vivo or in vitro addition of known growth factors did not affect P145 tyrosine phosphorylation. We now report that P145 is rapidly dephosphorylated in vivo when cells are exposed to low pH, a condition known to dissociate ligands from their receptors. The addition of serum‐free medium, conditioned by the gastric carcinoma cells, fully restores the tyrosine phosphorylation lost with acid treatment. These data suggest that the activity responsible for P145 phosphorylation on tyrosine, whether intrinsic to P145 itself or due to an associated kinase, is stimulated by a factor secreted by the tumor cells themselves.
Selective expression of mRNA encoding platelet‐derived growth factor B chain following transfection of foreign genes into cell lines derived from baby hamster kidney
Authors: Sakariassen Kjell S. , Powell Jerry S. , Raines Elaine W. , Ross Russell
Published: December 2004

The genes for platelet‐derived growth factor (PDGF) A and PDGF B chains are expressed in a variety of biological situations. Active PDGF consists of two distinct but homologous polypeptide chains, PDGF A and PDGF B, which are found as heterodimers or homodimers. We report a novel situation in which there is selective expression of mRNA encoding PDGF B in cell lines derived from baby hamster kidney (BHK) following transfection with various gene/cDNA constructs and following growth selection with methotrexate. The process of transfection itself, and not expression of the proteins encoded by the transfected genes/cDNAs (hormones, enzymes, and structural proteins), induces expression of PDGF B. No PDGF B mRNA is detectable in control cell lines. Low levels of mRNA encoding PDGF A are constitutively present and are not changed by transfection and/or growth selection. PDGF‐like activity is present in the medium whenever PDGF B mRNA is detected. The composition of the secreted PDGF dimer cannot be established from our data, but quantitative analysis of mRNA suggests that the PDGF is a B‐B dimer. However, the data show that transcription of the PDGF A and PDGF B genes in BHK cells is regulated independently, similar to that reported for some human tumor cells. Furthermore, the selective expression of PDGF B in response to the introduction of foreign genes and to growth selection may be an important aspect of the reaction of these cells to nonoptimal growth conditions, allowing survival and growth of the cells that express PDGF B.