bluuebunny/crossref_metadata_2025_split huggingface dataset:
The frequency of bone marrow cells that bind erythropoietin
Authors: Weiss Tania L. , Kung Charles K. H. , Goldwasser Eugene , Meier Paul 
Published: December 2004

AbstractThe frequencies of rat and mouse bone marrow cells capable of binding erythropoietin were studied by both direct fluorescence and indirect immunofluorescence. We found that between 1–2% of the cells bound erythropoietin, that the binding was specific, and that the number of cells that bound erythropoietin was in part, a function of the erythropoietic state of the donor animal. A statistical method for evaluating the data obtained is included.
Cellular responses elicited by insulin mimickers in cells lacking detectable plasma membrane insulin receptors
Authors: Hofmann Cecilia , Crettaz Marco , Bruns Pamela , Hessel Patricia , Hadawi Gina 
Published: December 2004

AbstractMadin‐Darby canine kidney (MDCK) cells were previously shown to have few or no plasma membrane insulin binding sites [Hofmann et al: J Biol Chem 258:11774, 1983]. Accordingly, neither insulin‐stimulated incorporation of [14C]glucose into glycogen, nor insulin‐induced uptake of radiolabeled α‐aminoisobutyrate ([3H]AIB) could be demonstrated. To probe for receptors, MDCK cultures were surface‐labeled with Na125I or were labeled with [35S]methionine. When solubilized cells were immunoprecipitated with sera containing antibodies to the insulin receptor, and immunoprecipilates were analyzed on SDS‐gel electrophoresis, no evidence for insulin receptor components was found. Also, when intact MDCK cells were incubated first with serum containing antibodies to the insulin receptor and then with 125I‐protein A, no radiolabeling of insulin receptors occurred. Various agents reported to have insulin‐like activity were tested on MDCK cells. The insulino‐mimetic lectins concanavalin A and wheat germ agglutinin as well as hydrogen peroxide enhanced incorporation of [14C]glucose into glycogen and induced stimulated [13H]AIB uptake, whereas trypsin, vanadate, and serum containing antibodies to the insulin receptor were without effects. Altogether, these results showed that MDCK cells had few or no insulin receptors and were correspondingly insulin‐insensitive, However, since insulin‐associated responses could be elicited by some insulin mimickers, the post‐receptor limb of response in MDCK cells was apparently intact.
Increased turnover of surface insulin receptors in fibroblastic cultures from genetically diabetic (DB/DB) mice
Authors: Kadle Ranjana , Raizada Mohan K. , Fellows Robert E. 
Published: December 2004

AbstractThe turnover of surface insulin receptors in fibroblastic cultures from genetically diabetic (db/db) mice and nondiabetic (m/m) littermates has been determined by combining a heavy isotope density shift technique with cross‐linking of insulin to surface receptors. Our results indicate that the surface insulin receptors turn over faster in diabetic cells than in nondiabetic cells. In addition, fewer receptors are incorporated into the plasma membrane per hour in diabetic cells than in nondiabetic cells. It is possible to propose a model to account for the altered expression of surface insulin receptors in diabetic cells on the basis of abnormalities of receptor incorporation and turnover.
The cell attachment determinant in fibronectin
Authors: Pierschbacher M. D. , Hayman E. G. , Ruoslahti E. 
Published: December 2004

AbstractFibronectin possesses a domain that interacts with cell surfaces. The ability of fibronectin to promote cell attachment can be duplicated with a short amino acid sequence, glycyl‐L‐arginyl‐glycyl‐L‐aspartyl‐L‐serine, taken from that domain. The tripeptide Arg‐Gly‐Asp appears to be irreplaceable for maintenance of the activity of this peptide, wheareas the serine residue can be replaced with some, but apparently not all, possible residues. This recognition sequence, or a closely related sequence, is present in a number of proteins other than fibronectin that interact with cells. These proteins include collagens, fibrinogen, thrombin, a bacterial surface protein, and two viral proteins, as well as discoidin‐I, a protein implicated in the aggregation of Dictyostelium discoideum. A similar sequence is also repeated in some, but not all, fibronectin molecules, making it possible that some fibronectin molecules have more than a single cell attachment site. Synthetic peptides constructed from sequences taken from several of these other proteins have also been shown to promote cell attachment. The tripeptide sequence may, therefore, constitute an ancient cellular recognition mechanism common to many proteins.
Characterization of a membrane‐associated glycoprotein complex implicated in cell adhesion to fibronectin
Authors: Hasegawa Takayuki , Hasegawa Etsuko , Chen Wen‐Tien , Yamada Kenneth M. 
Published: December 2004

AbstractWe have characterized a 140‐kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band 1), 135,000 (band 2), and 120,000 (band 3). In two‐dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band 1), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two‐dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co‐migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.
Interaction between Raf and Myc oncogenes in transformation in vivo and in vitro
Authors: Cleveland J. L. , Jansen H. W. , Bister K. , Fredrickson T. N. , Morse H. C. , Ihle J. N. , Rapp U. R. 
Published: December 2004

Abstract3611 MSV, a raf‐oncogene‐transducing murine retrovirus, induces fibrosarcomas and erythroid hyperplasia in newborn mice after a latency of 4–8 wk. In contrast, new recombinant murine retroviruses carrying the myc oncogene (J‐3, J‐5 construct viruses) do not induce tumors before > 9 wk. A combination of both oncogenes in an infectious murine retrovirus (J‐2) induces hematopoietic neoplasms in addition to less prominent fibrosarcomas and pancreatic adenocarcinoma 1–3 wk after inoculation. The hematologic neoplasms consist of immunoblastic lymphomas of T and B cell lineage and erythroblastosis. If animals were inoculated with a variant of the J‐3 virus, which induces altered foci in cultures of NIH 3T3 cells, carcinoma developed in the pancreas with a 2–6 mo latency. In parallel to the synergistic action of both oncogenes on hematopoietic cells in vivo, we find that raf‐oncogene‐induced transformation of bone marrow cells in culture is enhanced by the addition of myc, which by itself does not transform these cells when grown in standard media. We conclude that concomitant expression of raf and myc oncogenes in hematopoietic and epithelial cells alters their respective transforming activities. The contribution of v‐myc in this synergism was examined by use of a series of recombinant murine retroviruses capable of expressing the avian v‐myc to study the effect of altered myc expression on hematopoietic/lymphoid cells. With either interleukin 3‐ or interleukin 2‐dependent cell lines, introduction of the recombinant viruses abrogated the requirement for IL 3 or IL 2 for growth, and associated with this was the suppression of c‐myc expression. The findings suggest that myc is a component in the signal transduction pathway for IL 3 and IL 2 and support an autoregulatory mechanism of c‐myc expression. In contrast to v‐myc, expression of v‐raf primary lymphoid hematopoietic cells has an immortilizing function without abrogating the requirement for IL 3 for growth. This suggests that v‐raf and v‐myc affect different components of growth regulation, as, for example, commitment (v‐myc ) and cell cycle progression (v‐raf).
The present status of erythrocyte spectrin structure: The 106‐residue repetitive structure is a basic feature of an entire class of proteins
Authors: Speicher David W. 
Published: December 2004

AbstractSpectrin, the major component of the erythroid membrane skeleton, is a long, asymmetrical rodlike protein that interacts with several other proteins to form a two‐dimensional membrane skeleton. Progress in several laboratories over the past few years including substantial partial peptide and nucleotide sequence determination has greatly enhanced our knowledge of the structrual properties of this large molecule (heterodimer = 465,000 daltons). The alpha and beta subunits are homologous with approximately 30% identity. They are aligned in an anti‐parallel side‐to‐side orientation with the amino‐ and carboxy‐termini near opposite physical ends of the molecule. The predominant structural feature elucidated from sequencing this large molecule is the nearly universal occurrence in both subunits of a single type of repetitive structure. The periodicity of this homologous structure is exactly 106 amino acid residues. As many as 36 homologous, but non‐identical, repeats exist and comprise more than 90% of the mass of the heterodimer. Each of these repetitive units is folded into a triple‐stranded structure that is highly helical. Peptide maps, antibody cross‐reactivity, peptide sequence analysis, and more recently nucleic acid sequences have defined several major properties of the erythroid molecule and related proteins in other tissues. Tissue‐specific spectrins have the same 106‐residue repetitive structure and show sequence homology to erythroid spectrin.
Demonstration of a relationship between talin and P235, a major substrate of the calcium‐dependent protease in platelets
Authors: Beckerle Mary C. , O'Halloran Theresa , Burridge Keith 
Published: December 2004

AbstractTalin is a 225,000‐Dalton protein we have purified from smooth muscle. In chick embryo fibroblasts talin is found in adhesion plaques (focal contacts), areas where the cell is closely opposed to the substratum. In comparison with other cytoskeletal proteins, we found talin to be unusually susceptible to proteolysis and have identified a 190,000‐Dalton proteolytic fragment of talin in the immunoblots of many tissues. These observations raised the possibility that the cleavage of talin to this fragment has physiological relevance. One system that we have investigated in which significant proteolysis occurs is platelets. During platelet activation several high‐molecular‐weight proteins are cleaved to lower‐molecular‐weight forms. Here we demonstrate that talin is closely related to one of these platelet high‐molecular‐weight proteins, P235. The purification of talin is comparable to that developed for P235, and the two proteins have similar biophysical properties. In addition, antibodies raised against chicken gizzard talin recognize P235 in purified form as well as in crude platelet extracts. The platelet protein also resembles smooth‐muscle talin in its susceptibility to endogenous proteolysis: P235 is rapidly cleaved to a 190‐200kD polypeptide by a calcium‐activated protease found in platelet extracts. Moreover, partial proteolysis of P235 and talin with chymotrypsin, elastase, or trypsin also generates remarkably similar one‐dimensional peptide maps. Because of their similar biophysical properties, immunological crossreactivity, and similar one‐dimensional partial peptide maps, we conclude that P235 is the platelet form of talin.
Isolation and characterization of nuclear lamina from ehrlich ascites tumor cells
Authors: Krachmarov Chavdar , Tasheva Bistra , Markov Dimitar , Hancock Ronald , Dessev George 
Published: December 2004

AbstractWe have developed a simple and rapid method for isolation of purified nuclear lamina from Ehrlich ascites tumor cells. The procedure employs chromatin structures prepared from whole cells at low ionic strength and is carried out under conditions that minimize the formation of artifactual protein‐DNA complexes. When the isolation is performed in the presence of EDTA, nuclear lamina without distinct pore complexes is obtained. In the absence of EDTA, intact pore complexes and a large amount of vimentin 100 A filaments are seen associated with nuclear lamina. The main nuclear lamina proteins are characterized using gel electrophoresis, immunoblotting, and two‐dimensional peptide mapping. An extensive structural homology is found between lamin A and lamin C. whose peptide maps differ by only one major spot, whereas lamin B has apparently unrelated pattern.
Ligand–receptor interactions: Detailed evaluation of occupancy‐dependent affinity
Authors: Faguet Guy B. 
Published: December 2004

AbstractThe exact nature of the curvilinearity of Scatchard plots derived from hormonal and nonhormonal binding systems has not been definitively resolved. Such plots are compatible with heterogeneous receptors with different but fixed affinities and with negatively interacting binding sites resulting in occupancy‐dependent affinity. In the current study we examined in detail the effect of receptor occupancy by the ligand on receptor affinity under a variety of experimental conditions. We chose the human lymphocyte–leukoagglutinin (LPHA) system, which closely mimics the IM9‐insulin model. Reliable estimates of total binding capacity (728 ng/106 cells) essential to our report were calculated from a wide database by the least‐squares model. At occupancies ≥ 0.085, receptors are associated with low and fixed affinity (1.5 × 106M−1), whereas at occupancies ⩽ 0.085, affinity is high and fixed (1.8 × 108M−1) or high but variable (1 × 107M−1 to 1.5 × 106M−1) depending on whether the binding is assumed to be noncooperative or cooperative, respectively. Calculation of receptor–ligand complex dissociation velocity over a wide range of occupancies (0.01–0.40) suggested that occupancy exerts an inversely proportional effect on affinity that is rapid and sustained. Cell activation (DNA synthesis) is initiated at receptor occupancy of ≅ 0.004 and is magnified as ligand binding to high affinity receptors increases up to ≅ 0.07 occupancy (functional sites), beyond which point further binding (to low affinity sites) becomes increasingly ineffective and cytotoxic (redundant sites). These findings suggest that occupancy influences affinity as postulated by the hypothesis of negative cooperativity. Through this effect occupancy may play a significant role in regulating ligand‐induced cell responses.
Actions of calcitonin, parathyroid hormone, and prostaglandin E2 on cyclic AMP formation in chicken and rat osteoclasts
Authors: Nicholson G. C. , Livesey S. A. , Moseley J. M. , Martin T. J. 
Published: December 2004

AbstractThe effects of calcitonin, parathyroid hormone, and prostaglandin E2 on cyclic AMP production were studied in osteoclast‐rich cultures derived from medullary bone of laying hens and from the long bones of newborn rats. Cyclic AMP was assayed biochemically in replicate cultures, and furthermore, changes in cytoplasmic fluorescence were sought by indirect immunofluorescence with rabbit anti‐cyclic AMP and FITC‐labelled goat anti‐rabbit IgG. Treatment of rat osteo‐clasts with calcitonin increased cyclic AMP formation as measured biochemically, and this was confirmed by the immunofluorescence method. No such increase took place in chick osteoclasts. Prostaglandin E2 increased cyclic AMP production in both rat and chick osteoclasts as determined by both methods. Since the immunofluorescence method failed to detect a response to parathyroid hormone either in chick or rat osteoclasts, its variable biochemical effects were concluded to be due to actions on contaminating osteoblasts in the cultures. Thus it has been possible with a combined biochemical and immunocytochemical approach to define the cyclic AMP responses to the calcium‐regulating hormones in rat and chick osteoclasts. The failure of calcitonin to increase cyclic AMP in chick osteoclasts identifies a need to investigate the nature of calcitonin action on avian osteoclasts, which may contribute to understanding of its actions on mammalian cells.
Sponge aggregation factor: In situ localization by fluorescent monoclonal antibody techniques
Authors: Gramzow Monika , Dorn August , Steffen Renate , Müller Werner E. G. 
Published: December 2004

AbstractThe aggregation factor (AF) from sponges mediates a heterophilic interaction of homologous cells. Applying electron microscopical means, we succeeded only very rarely in identifying the 90 S AF particle in tissue sections from Geodia cydonium. By means of a fluorescent antibody technique, we have now localized the cell binding domain of the AF in situ. Previous studies in this laboratory have led to the identification of the 47‐kDa cell binding protein of the AF, using the monoclonal antibody (mab) 5D2‐D11 [Gramzow M, Bachmann M, Zahn RK, Uhlenbruck G, Dorn A, Müller WEG, J Cell Biol, 102:1344–1349, 1986]. This mab and mab 7D5, directed against a 92‐kDa protein in the AF complex, were chosen for the fluorescent studies. By using mab 5D2‐D11, the plasma membranes of cells from different regions in the sponge could be brightly stained. However, mab 7D5 reacted only very weakly with the sponge surfaces. By applying the immuno‐blotting technique it was furthermore demonstrated that the cell binding protein is present both in the associated form with AF complex and in a free state. Moreover, it was established that the 47‐kDa binding protein is not present in (1) homologous glycoconjugates, (2) lectin, or (3) collagen; these components are known to be involved in cell‐matrix interaction.
Role of guanine nucleotide regulatory protein in polyphosphoinositide degradation and activation of phagocytic leukocytes by chemoattractants
Authors: Verghese Margrith W. , Smith Charles D. , Snyderman Ralph 
Published: December 2004

AbstractLeukocyte activation by Chemoattractants provides an important model to study the biochemical mechanisms of stimulus‐response coupling in these cells. Well‐defined chemotactic factors induce readily quantifiable responses in phagocytic leukocytes. These include directed migration and the production and release of toxic substances including oxygen radicals and lysosomal enzymes. The development of radiolabeled synthetic oligopeptides with potent chemotactic activity allowed the demonstration of chemoattractant receptors on polymorphonuclear leukocytes (PMNs) as well as macrophages. In membrane preparations from these cells, these receptors exist in high‐ and low‐affinity states which are regulated by guanosine di‐ and triphosphates. This suggested that chemoattractant receptors interact with guanine nucleotide regulatory proteins (N or G proteins). Although Chemoattractants elicit a rapid but transient increase in intracellular cAMP levels, they neither stimulate nor inhibit membrane‐bound adenylate cyclase, suggesting a novel role for N proteins in certain receptor‐transduction mechanisms. Stimulation of phagocytes by Chemoattractants is also associated with a rapid increase in cytosolic Ca2+ concentrations ([Ca2+]i) which appears to result from the production of inositol 1,4,5‐triphosphate (IP3) as a consequence of the diesteric cleavage of phosphatidylinositol 4,5‐bisphosphate (PIP2). Treatment of phagocytes with pertussis toxin (PT), which ADP‐ribosylates and thereby inactivates certain N proteins, abolishes the cells' responsiveness to chemoattractants. More direct evidence for a role of a PT‐sensitive N protein in leukocyte activation was provided by the demonstration that chemoattractants stimulate the hydrolysis of PIPi in PMN membranes only in the presence of GTP. This receptor‐mediated hydrolysis of PIPi is not observed in plasma membranes prepared from PT‐treated PMNs. Therefore, these studies suggest that occupancy of chemoattractant receptors activates a PT‐sensitive N protein. The activated N protein shifts the Ca2+ requirement for phospholipase C activity from supraphysiological levels to ambient cytosolic Ca2+ concentrations. Cleavage of PIP2 results in the formation of the second messenger molecules, IP3 and 1,2‐diacylglycerol, which can initiate cellular activation. These messengers also seem to activate responses which feed back to attenuate receptor stimulation of phospholipase.
Transforming growth factor‐α attenuates the acquisition of aromatase activity by cultured rat granulosa cells
Authors: Adashi Eli Y. , Resnick Carol E. , Twardzik Daniel R. 
Published: December 2004

AbstractThe effect of transforming growth factor‐α (TGFα) on granulosa cell differentiation, as assessed by the acquisition of aromatase activity, was evaluated in vitro by using a primary culture of rat granulosa cells. Harvested from immature, diethylstilbestrol‐treated rats, granulosa cells were cultured under serum‐free conditions for 72 hr in the presence of saturating concentrations (10−7M) of aromatase substrate androstenedione with or without the specified experimental agents. Basal aromatase activity, as assessed by the generation of radioimmunoassayable estrogen was negligible, remaining unaffected by treatment with TGFα; (10 ng/ml) by itself. Whereas treatment with follicle‐stimulating hormone (FSH) resulted in a substantial increase in the extent of aromatization, concurrent treatment with TGFα: (10 ng/ml) resulted in significant (P<0.05), yet reversible inhibition (78 ± 5.6%) of FSH action. Significantly, this effect of TGFα could not be accounted for by a decrease in cellular viability or plating efficiency nor by a decrease in the number of cells or their DNA content. Although independent of the FSH dose employed, the TGFα effect proved dose‐ and time‐dependent, with an apparent median inhibitory dose (EC50) of 0.33 ± 0.04 ng/ml, and a minimal time requirement of 48 hr. Capable of substantial inhibition of the forskolin‐stimulated accumulation of extracellular adenosine 3′, 5′ cyclic monophosphate (cAMP) and estrogen, TGFα had a measurable albeit limited effect on N6, 2‐′O‐Dibutyryladensine 3′:5′‐cyclic monophosphate‐supported estrogen production. Relative potency comparison revealed epidermal growth factor (EGF; EC50 = 0.24 ± 0.03ng/ml) and TGFα to be virtually equipotent as regards the attenuation of FSH‐stimulated estrogen biosynthesis. Taken together, our findings indicate that TGFα, like EGF, acting at subnanomolar concentrations, is capable of attenuating the FSH‐stimulated (but not basal) accumulation of estrogen. This effect of TGFα proved time‐ and dose‐dependent, involving virtually complete neutralization of FSH action at site(s) both proximal and distal to cAMP generation. As such, these findings provide yet another example of the remarkable qualitative and quantitative similarities between EGF and TGFα, thereby reaffirming the prospect that ligands of the EGF/TGFα receptor may play a modulatory role in the course of granulosa cell ontogeny.