bluuebunny/crossref_metadata_2025_split huggingface dataset:
Serum‐activated T51B rat liver cells transiently accumulate cyclic AMP‐dependent protein kinases on their surfaces during the G1 phase
Authors: Kleine Leonard P. , Whitfield James F. 
Published: February 2005

AbstractConfluent T51B rat liver epithelial cells promptly began accumulating cyclic AMP‐binding sites on their surfaces when they were stimulated from quiescence by serum growth factors in medium containing 1.8 mM Ca2+, but they began losing the accumulated binding sites shortly before initiating DNA replication. When the medium contained only 0.02 mM Ca2+, the cells still accumulated surface cyclic AMP‐binding sites, but they did not initiate DNA replication and tended to continue accumulating the binding sites. The cyclic AMP‐binding sites were eliminated completely by treating intact cells for 5 minutes with 0.005% trypsin (which did not damage the cells), and cyclic AMP caused them to be released from intact, undamaged cells into the medium. The binding sites also comigrated electrophoretically with purified regulatory subunits of type I cyclic AMP‐dependent protein kinase, and to a lesser extent the regulatory subunit of type II cyclic AMP‐dependent protein kinase. Therefore, it is likely that a transient accumulation of cyclic AMP‐dependent protein kinases on the outer surface of the plasma membrane is part of the T51B rat liver cell's prereplicate program.
Colony‐stimulating factor‐1 induces thromboplastin activity in murine macrophages and human monocytes
Authors: Lyberg T. , Stanley E. R. , Prydz H. 
Published: February 2005

AbstractHighly purified colony‐stimulating factor‐1 induced thromboplastin activity in murine macrophages and human monocytes in vitro. The activity increase was inhibited by cycloheximide and prevented by antibodies to CSF‐1.
Human iliac artery endothelial cells express both genes encoding the chains of platelet‐derived growth factor (PDGF) and synthesize PDGF‐like mitogen
Authors: Sitaras N. M. , Sariban E. , Pantazis P. , Zetter B. , Antoniades H. N. 
Published: February 2005

AbstractIn human umbilical vein and bovine aortic endothelial cells in culture c‐sis gene expression and secretion of platelet‐derived growth factor (PDGF) has been previously demonstrated. We now report the presence of PDGF‐1 and PDGF‐2/sis mRNA transcripts in primary cultures of human iliac artery endothelial cells (HIA‐EC). Concomitantly, these cells synthesize and secrete PDGF‐like proteins identified by direct immunoprecipitation with specific PDGF antiserum. The PDGF proteins secreted by HIA‐EC have molecular weights of 31 and 35 kd under nonreducing conditions. Upon reduction these proteins are converted to the monomeric 15‐ and 16‐kd forms. Conditioned media derived from HIA‐EC stimulated the incorporation of 3H‐thymidine by 3T3 cells and competed with 125I‐PDGF for its binding to 3T3 cell membrane receptors. The biologic activity was stable to heating at 100°C for 10 min and sensitive to reducing agents, properties similar to those of authentic PDGF. Production of PDGF‐like mitogen by the human arterial endothelial cells may play an important role in the paracrine modulation of arterial wall regeneration following vascular injury.
Generation of osteoclasts in cultures of rabbit bone marrow and spleen cells
Authors: Fuller K. , Chambers T. J. 
Published: February 2005

AbstractThe primary and specific function of the osteoclast is the resorption of bone. We have applied this criterion, and a monoclonal antibody that binds specifically to osteoclasts, to cultures of tissues that may contain osteoclastic precursors. Bone marrow and spleen cells were incubated for up to 4 weeks in the presence or absence of parathyroid hormone, interleukin 1, or 1,25(OH)2 vitamin D3, on plastic coverslips or slices of devitalised bone. Osteoclasts (as judged by the presence of resorption cavities and the appearance of monoclonal antibody‐positive cells) did not develop in cultures incubated without added hormones, nor in cultures containing parathyroid hormone or interleukin 1, but were regularly observed when bone marrow cells were incubated with 1,25(OH)2vitamin D3. Although multinucleate giant cells were common after incubation, especially in the presence 1,25(OH)2vitamin D3, monoclonal antibody bound not to these cells but to a minor and distinctive population of mononuclear cells and cells of low multinuclearity. We found no excavations and no monoclonal antibody‐positive cells after incubation of peritoneal macrophages with 1,25(OH)2D3. These results provide direct evidence of osteoclastic function arising in cultures of haemopoietic tissues.
Identification of an enhancer‐like element upstream from a cell cycle dependent human H4 histone gene
Authors: Helms S. R. , van Wijnen A. J. , Kroeger P. , Shiels A. , Stewart C. , Hirshman J. , Stein J. L. , Stein G. S. 
Published: February 2005

AbstractWe have identified a segment of DNA in the region 6,500 nucleotides upstream from a cell‐cycle‐dependent human H4 histone gene (pF0108A) which exhibits properties of an enhancer element. This distal element is not required for cap site initiation from the F0108A H4 histone gene. When the enhancer element is present in the genome as a stable integrated sequence, either in its natural upstream location or in a construct where the element is moved just upstream from the proximal promoter sequences, a 25‐fold increase in the level of human H4 histone RNAs is observed. This increased level of mRNA reflects an increase in the rate of transcription. The enhancer effect is also observed when the distal element is inserted in inverse orientation with respect to this gene. In addition, the far upstream element can increase expression of a prokaryotic chloramphenicol acetyl transferase (CAT) gene under control of the simian virus 40 (SV40) early promotor, indicating that the ability to influence transcription is not confined to the gene with which it is normally associated. The ability of the histone gene distal enhancer element to function in both mouse and human cells indicates that transacting regulatory factors encoded by either the human or murine genome are capable of mediating the functional properties of this element, further supporting the cross‐species compatibility of regulatory sequences and molecules that influence transcription of human histone genes.
Cell cycle changes in water properties in sea urchin eggs
Authors: Cameron I. L. , Cook K. R. , Edwards D. , Fullerton G. D. , Schatten G. , Schatten H. , Zimmerman A. M. , Zimmerman S. 
Published: February 2005

AbstractThis study concerned changes in the motional properties of cellular water during the first cell cycle of fertilized sea urchin eggs (Lytechinus variegatus). There was a significant decrease in proton NMR T1 relaxation time and in cytoplasmic ice crystal growth during mitosis and a significant increase in T1 time and cytoplasmic ice crystal size during cleavage. This was not caused by egg water content changes as reflected by egg volume measurements. Removal of both the fertilization membrane and the hyaline layer shortly after fertilization did not alter the pattern of T1 time changes at mitosis and cleavage as compared to whole eggs; thus, the pattern of T1 time changes was attributed to intracellular events. Treatment of fertilized eggs with cytochalasin B, an inhibitor of actin polymerization, did not block the fall in T1 time at mitosis, but did block cytokinesis and the increase in T1 time, which normally occurred at cleavage. A significant pattern of actin disassembly and reassembly at mitosis and cytokinesis was found by studies on the total amount of monomeric actin (G actin) using the DNase I assay. This led to the hypothesis that the observed changes in T1 time and ice crystal size during the first cell cycle were due to the depolymerization and polymerization of cytoplasmic actin. To test this, the effect of the in vitro polymerization of purified actin on the T1 time and on ice crystal growth was examined. It was concluded that changes in the T1 time and ice crystal growth upon polymerization of actin in vitro resembled the changes seen in vivo. These results suggest that changes in the motional properties of cytoplasmic water during the first cell cycle are due, at least in part, to the state of polymerization of cytoplasmic actin.
Covalent modification of the permeability pathways induced in the human erythrocyte membrane by the malarial parasite Plasmodium falciparum
Authors: Breuer W. V. , Kutner S. , Sylphen J. , Ginsburg H. , Cabantchik Z. I. 
Published: February 2005

AbstractThe intraerythrocytic malarial parasite Plasmodium falciparum induces permeation pathways in the plasma membrane of its host, the red blood cell. The pathways display porelike properties with selectivity toward anions and neutral molecules. They are shown here to be susceptible to chemical modification by 4,4′‐diisothiocyano‐2,2′‐dihydrostilbene disulfonic acid (H2DIDS), an amino‐reactive reagent which is impermeant to uninfected cells. At pH 7.4 the reagent affected transport only marginally while freely entering into infected cells and reacting with intracellular hemoglobin. On the other hand, at pH above 8.5, the compound blocked the pathways efficiently (IC50 = 50 μM, at 37°C for 10 min) as judged by four criteria: (1) selective lysis of infected erythrocytes in the presence of isotonic polyols; (2) uptake of [14C] sorbitol into infected cells; (3) uptake of the fluorescent anion Nbd‐taurine into infected cells under conditions in which the native anion transport system was inhibited; and (4) labeling of intracellular hemoglobin by the permeating reagent [3H]H2DIDS. The inhibitory effect was observed only with mature forms of parasitized cells, i.e., from the trophozoite stage and onward, while the pathways of immature ring forms were refractive. However, when the probe was incorporated into the interior of hemoglobin‐depleted resealed ghosts prepared from ring forms, it was found to inhibit the pore‐mediated transport. On the basis of these and other studies we postulate that the H2DIDS‐sensitive sites on the pathways are endofacial, thus requiring penetration of the probe (probably through the same pathway) for their inactivation. Labeling studies with the radiolabeled modifier implicate 120‐Kd, 63‐Kd, and/or 51‐Kd polypeptides as candidates for the pore components.
Infection of L6E9 myoblasts with Trypanosoma cruzi alters adenylate cyclase activity and guanine nucleotide binding proteins
Authors: Morris Stephen A. , Bilezikian John P. , Tanowitz Herbert , Wittner Murray 
Published: February 2005

AbstractWe studied the consequences of infection of L6E9 myoblasts with T. cruzi on the adenylate cyclase complex to test the hypothesis that infection alters the functional properties of the guanine nucleotide regulatory proteins, Ns and Ni. Stimulating activities of adenylate cyclase due to isoproterenol, isoproterenol plus Gpp(NH)p, or forskolin (activities mediated by Ns) are not altered by infection. However, inhibitory activites mediated by Ni [Gpp(NH)p, acetylcholine, and adenosine inhibition of forskolin‐dependent adenylate cyclase activity] are compromised by infection. The reduction in adenosine's inhibition of forskolin‐dependent adenylate cyclase activity is seen throughout the effective concentration range of adenosine. Pertussis toxin does not change basal or stimulated adenylate cyclase activity in infected cells compared with normal uninfected cells, nor does it alter the inhibiting action of adenosine. To evaluate the coupling proteins (Ns and Ni) involved in the stimulation and inhibition of adenylate cyclase more directly, cholera‐ and pertussis‐toxin‐dependent ADP ribosylation studies were performed. The incorporation of [32P]ADP ribose in the presence (specific) or absence (nonspecific) of the toxins was markedly decreased in membranes prepared from infected cells. However, in membranes prepared from infected or uninfected cells previously treated with pertussis toxin, there was a significant reduction in specific pertussis‐toxin‐dependent ADP ribosylation. The infection‐associated diminution in toxin‐dependent ADP ribosylation complements the impaired inhibition of adenylate cyclase data. Collectively, the data further substantiate an infection‐associated alteration in the adenylate cyclase complex, probably at the level of the guanine nucleotide binding proteins.
Rapid in vitro formation of smooth endoplasmic reticulum aggregates within peptide‐producing islet cells
Authors: Knodel Sandra , Meda Paolo , Orci Lelio 
Published: February 2005

AbstractWe report here that heptanol (3.5 mM) induces in vitro a rapid formation of smooth endoplasmic reticulum aggregates (SERA) within isolated islets of Langerhans. SERA appeared after only 15 min of exposure to the alkanol and increased in number during the first 30 min of incubation. At that time, SERA represented 2% and 6% of the volume of B‐ and non‐B‐cells, respectively. Removal of heptanol resulted in the rapid disappearance of SERA, whereas reintroduction of the alkanol rapidly induced these structures again. SERA formation was seen in different types of endocrine and nonendocrine islet cells. In the insulin‐producing B‐cells, SERA formation was not modified by conditions known to alter the secretory activity and the microtubular‐microfilament network or to inhibit protein synthesis. By contrast, SERA formation was inhibted by low temperature and by conditions depleting the energy sources of the cells. Similar observations were made in the presence of either octanol (1 mM) or nonanol (1 mM) but not of shorter chain alkanols, alkanes, oxidative derivates of either heptanol or octanol, and of other unrelated lipid soluble compounds. Incubations in the presence of long‐chain alkanols provide, therefore, a unique model to study in vitro the formation and disposal of smooth endoplasmic reticulum, as well as a system in which rapid membrane biogenesis is amenable to direct experimental testing.
Insulin and α2‐macroglobulin‐methylamine undergo endocytosis by different mechanisms in rat adipocytes: I. Comparison of cell surface events
Authors: Goldberg Ronald I. , Smith Robert M. , Jarett Leonard 
Published: February 2005

AbstractThis ultrastructural study compared the endocytosis of a peptide hormone, ferritin‐labeled insulin (Fm‐I) or gold‐labeled insulin (Au‐I), and a non‐hormonal ligand, gold‐labeled alpha‐2‐macroglobulin‐methylamine (Au‐α2MGMA), by rat adipocytes. Quantitative analysis of the cell surface showed that coated pits occupied 0.4% of the adipocyte surface. This was one fifth to one tenth of that which has been reported on fibroblasts and hepatocytes, cell types in which receptor‐mediated endocytosis has been extensively studied. In contrast, uncoated micropinocytotic invaginations were quite numerous and occupied 13.1% of the adipocyte cell surface. The frequency of microphinocytotic invaginations, 13.8 per μm2 of plasma membrane, was 7–12 times greater than has been reported on fibroblasts. Therefore, the ultrastructure of the endocytic apparatus on rat adipocytes was different from more commonly studied cell types. At 4°C, Au‐α2MGMA concentrated within coated pits to a density that was 52 times greater than that on the uncoated plasma membrane. Au‐α2MGMA was excluded from micropinocytotic invaginations by more than 93%; this exclusion was unrelated to the size of the Au‐α2MGMA particle. In contrast, at 4°C, Fm‐I did not concentrate within coated pits and occupied micropinocytotic invaginations in a random manner. At 37°C, coated pits accounted for all of the endocytosis of Au‐α2MGMA, proving that these structures were functional despite their atypically low density. In contrast, greater than 99% of the endocytosis of Fm‐I or Au‐I occurred through micropinocytotic invaginations. These results demonstrated for the first time by a comparative, quantitative, ultrastructural method that insulin and Au‐α2MGMA undergo endocytosis by dissimilar mechanisms on rat adipocytes. Dissimilarities in the endocytosis of insulin and Au‐α2MGMA may be related to the different biological roles of these two molecules.
Insulin and α2‐macroglobulin‐methylamine undergo endocytosis by different mechanisms in rat adipocytes: II. Comparison of intracellular events
Authors: Goldberg Ronald I. , Smith Robert M. , Jarett Leonard 
Published: February 2005

AbstractA previous ultrastructural study showed that gold‐labeled insulin (Au‐I) and the non‐hormonal ligand gold‐labeled alpha‐2‐macroglobulin‐methylamine (Au‐α2MGMA) underwent endocytosis by dissimilar cell surface structures on rat adipocytes. The present ultrastructural study compared the intracellular routes taken by these two ligands in adipocytes. Intracellular Au‐α2MGMA was initially found within apparent coated vesicles but Au‐I was not, consistent with the previous demonstration that Au‐α2MGMA underwent endocytosis by coated pits whereas Au‐I was internalized by uncoated micropinocytotic invaginations. Early in the endocytic pathway, the two ligands were segregated within separate small vesicles and tubulovesicles. Au‐α2MGMA was concentrated in a small number of these structures whereas Au‐I was sparsely distributed among a relatively large number. Subsequently, the two endocytic pathways converged as the ligands intermingled within pale multivesicular bodies and lysosome‐like structures. Au‐I was less efficiently transferred to lysosomes than Au‐α2MGMA since a greater proportion of intracellular Au‐I remained associated with small vesicles and tubulovesicles. This study indicates that early intracellular events in the endocytic pathways of insulin and α2MGMA are distinct. These findings are discussed in light of the fundamentally dissimilar biological roles of these two molecules and the possible involvement of the endocytic pathway in the insulin signaling mechanism.
Proteoglycan synthesis by primary chick skeletal muscle during in vitro myogenesis
Authors: Miller Raymond R. , Rao Jasti S. , Festoff Barry W. 
Published: February 2005

AbstractThe proteoglycans synthesized by primary chick skeletal muscle during in vitro myogenesis were compared with those of muscle‐specific fibroblasts. Cultures of skeletal muscle cells and muscle fibroblasts were separately labeled using [35S] sulfate as a precursor. The proteoglycans of the cell layer and medium were separately extracted and isolated by ion‐exchange chromatography on DEAE‐Sephacel followed by gel filtration chromatography on Sepharose CL‐2B. Two cell layer‐associated proteoglycans synthesized both by skeletal muscle cells and muscle fibroblasts were identified. The first, a high molecular weight proteoglycan, eluted from Sepharose CL‐2B with a Kav of 0.07 and contained exclusively chondroitin sulfate chains with an average molecular weight greater than 50,000. The second, a relatively smaller proteoglycan, eluted from Sepharose CL‐2B with a Kav of 0.61 and contained primarily heparan sulfate chains with an average molecular weight of 16,000. Two labeled proteoglycans were also found in the medium of both skeletal muscle and muscle fibroblasts. A high molecular weight proteoglycan was found with virtually identical properties to that of the high molecular weight chondroitin sulfate proteoglycan of the cell layer. A second, smaller proteoglycan had a similar monomer size (Kav of 0.63) to the cell layer heparan sulfate proteoglycan, but differed from it in that this molecule contained primarily chondroitin sulfate chains with an average molecular weight of 32,000. Studies on the distribution of these proteoglycans in muscle cells during in vitro myogenesis demonstrated that a parallel increase in the relative amounts of the smaller proteoglycans occurred in both the cell layer and medium compared to the large chondroitin sulfate proteoglycan in each compartment. In contrast, muscle‐derived fibroblasts displayed a constant ratio of the small proteoglycans of the cell layer and medium fractions, compared to the larger chondroitin sulfate proteoglycan of the respective fraction as a function of cell density. Our results support the concept that proteoglycan synthesis is under developmental regulation during skeletal myogenesis.
Growth of a human carcinoma (HEp3) in nude mice: Rapid and efficient metastasis
Authors: Ossowski L. , Russo H. , Gartner M. , Wilson E. L. 
Published: February 2005

AbstractOur aim was to identify conditions which would permit the development of spontaneous metastasis of a human tumor in nude mice in a rapid and predictable manner and to explore ways to quantitate metastasis. Using a human squamous carcinoma–HEp3–we determined that invasiveness and metastasis were influenced by the host. HEp3 cells grew very rapidly and without a significant lag period in Balb/c and NIH(S)‐II nude mice kept in aseptic conditions; a much longer lag period was observed in NIH‐Swiss mice kept in conventional conditions. The HEp3 tumor displayed a highly invasive behavior in N‐NIH(S)‐II mice, in which it invaded the body wall, gaining access to the peritoneal cavity. Microinvasion was noted in all strains of mice inoculated with HEp3 cells. To prolong survival of the mice until metastases became evident, primary tumors were excised when they weighed 1–2 gm. N‐NIH(S)‐II and Balb/c nude mice, maintained in germ‐free conditions, were most receptive to the development of metastases–lung metastases developed in 80% of these mice. Over 60% of all metastases were present within 4 weeks following the removal of the primary. Only 26% of tumor bearing NIH‐Swiss developed lung metastases. Lung metastases were observed in some mice in the absence of local microinvasion, local tumor recurrence, and regardless of the presence of lymph node involvement. They were also noted in mice from which primary tumors were not excised. We compared three methods of lung metastasis detection: histology, detection of tumor cells in the cultures of lung minces, and the measurement of the levels of human urokinase‐type plasminogen activator directly in the lysates of lungs. The detection of tumor cells in cultures of lung minces appeared to be the most sensitive of these methods and the determination of enzyme in lung lysates seemed to hold most promise for a quantitative approach. These data indicate that, the type of tumor, as well as the genetic background and the maintenance conditions of the host, have to be carefully selected to ensure the successful outcome of the particular tumor‐host interaction being studied. Adherence to these guidelines allowed us, in the case of the HEp3 tumor, to develop a rapid, predictable, and efficient model in which to study factors affecting metastasis of this human tumor.