bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human Interleukin‐3 inhibits the binding of granulocyte‐macrophage colony‐stimulating factor and interleukin‐5 to basophils and strongly enhances their functional activity
Authors: Lopez Angel F. , Eglinton Jeffrey M. , Lyons A. Bruce , Tapley Peter M. , To Luen B. , Park Linda S. , Clark Steven C. , Vadas Mathew A. 
Published: February 2005

AbstractThe human T cell‐derived cytokines interleukin (IL)‐3, granulocyte‐macrophage colony‐stimulating factor (CM‐CSF), and IL‐5 were examined for their ability to bind specifically to human basophils and to regulate their function. Scatchard analysis of equilibrium binding studies showed that IL‐3 and GM‐CSF, bound to basophils with apparent dissociation constants (KD) = 8 × 10−11M and 3.9 × 10−11M, respectively. Specificity studies under conditions that prevent receptor internalization showed that the binding of IL‐3, GM‐CSF, and IL‐5 was not inhibited by tumor necrosis factor (TNF)‐α, IL‐1β, interferon (IFN)‐γ, or G‐CSF. However, receptors for IL‐3, GM‐CSF, and IL‐5 interacted with each other on the basophil membrane, showing a unique spectrum of cross‐reactivity, with IL‐3 competing for GM‐CSF and IL‐5 binding, whereas GM‐CSF and IL‐5 showed little or no competition for IL‐3 binding. In order to relate the binding properties of these cytokines to function, they were tested for their ability to influence basophil histamine release in an IgE/anti‐IgE‐dependent system. We found a hierarchy in the stimulation of basophil with the order of potency being IL‐3 >GM‐CSF >IL‐5. In addition, IL‐3 stimulated larger amounts of histamine release than GM‐CSF or IL‐5. The observation that IL‐3 interacts with receptors for GM‐CSF and IL‐5 may have a bearing on its stronger functional effects and suggests a major role for IL‐3 in the pathogenesis of hypersensitivity syndromes.
Altered expression of keratin and vimentin in human retinal pigment epithelial cells in vivo and in vitro
Authors: Hunt Richard C. , Davis Alberta A. 
Published: February 2005

AbstractActively proliferating human retinal pigment epithelial (RPE) cells grown in tissue culture possess keratin‐containing intermediate filaments that react with a combination of AE1 and AE3 anti‐keratin monoclonal antibodies. Antibody reactivity is lost, however, from RPE cells as the cell population ceases to proliferate when it approaches confluence and attains morphological characteristics more similar to those in vivo. In contrast, clone 8.13 anti‐keratin antibody stains all cells in the culture at all stages of the growth cycle and cell densities. These findings were reflected in vivo using retinal pigment epithelium taken directly from the eye. Normal non‐proliferating RPE cells bound 8.13 antibody to cytoskeletal structures, as judged by indirect immunofluorescence, but did not bind AE1/AE3 antibodies. However, proliferating dedifferentiated RPE cells from the vitreous humor of patients with proliferative vitreoretinopathy possess filaments that bind both AE1/AE3 and 8.13 antibodies. Thus it appears that structures detected by AE1/AE3 antibodies only occur in actively growing RPE cells in vitro and in vivo. Keratins produced by RPE cells were identified using Western blotting. Species with molecular masses of 54 (keratin 7), 52 (keratin 8), 42 (keratin 18), and 40 (keratin 19) kiloDaltons were the most abundant in proliferating cultured cells, but cells isolated directly from the eye were found to lack keratin 7 and 19. Keratin 19 was, however, observed in proliferating RPE cells from some patients with proliferative vitreoretinopathy. The latter findings explain the differential staining observed with AE1/AE3 antibodies in cells in culture and isolated directly from the eye since these antibodies interact primarily with keratin 19 which is absent from non‐proliferating RPE cells. In contrast to the presence of keratin‐containing intermediate filaments in human RPE cells in vivo, there are apparently no detectable vimentin‐containing cytoskeletal structures. However, all RPE cells cultured in vitro develop filaments composed of vimentin which persist in cells that have reached confluence.
Stimulation of plasma membrane and matrix vesicle enzyme activity by transforming growth factor‐β in osteosarcoma cell cultures
Authors: Bonewald L. F. , Schwartz Z. , Swain L. D. , Ramirez V. , Poser J. , Boyan B. D. 
Published: February 2005

AbstractTransforming growth factor‐β (TGFβ) serves an important role in extracellular matrix formation by stimulating the production of numerous extracellular matrix proteins by connective tissue cells and by osteoblasts or bone‐forming cells. TGFβ has been shown to stimulate alkaline phosphatase (ALPase) activity in the rat osteoblast‐like osteosarcoma cell line ROS 17/2.8. Previous studies have shown that this enzyme is elevated during calcification of bone and that it is enriched in matrix vesicles, an extracellular organelle associated with initial hydroxyapatite formation. To test the hypothesis that TGFβ plays a role in regulating mineral deposition in the matrix, the effects of TGFβ on ALPase and phospholipase A2, two enzymes associated with mineralization, were examined. ROS 17/2.8 cells were cultured at high and low density with recombinant human TGFβ (0.1–10 ng/ml) to examine the influence of cell maturation on response to TGFβ. Maximal stimulation of ALPase activity in the low density cultures was seen at 5 ng/ml; in high‐density cultures, there was further stimulation at 10 ng/ml. There was a dose‐dependent increase in ALPase activity seen in the matrix vesicles and plasma membranes in both types of cultures. Matrix vesicle ALPase exhibited a greater response to factor than did the plasma membrane enzyme. However, in low density cultures, the two membrane fractions exhibited a parallel response with greatest activity consistently in the matrix vesicles. There was a dose‐dependent increase in phospholipase A2‐specific activity in the plasma membranes and matrix vesicles of both high‐ and low‐density cultures. In agreement with previous studies, TGFβ inhibited cellular proliferation 50%. The results show that addition of TGFβ stimulates the activity of enzymes associated with calcification. The effect of TGFβ is dependent on the stage of maturation of the cell. This study indicates that TGFβ may play an important role in induced bone formation, calcification, and fracture repair in addition to its role in promoting chondrogenesis.
Inhibition of gap‐junctional intercellular communication and enhanced binding of fibronectin‐coated latex beads by stimulation of DNA synthesis in quiescent 3T3‐L1 cells
Authors: Shiba Yoshiki , Sasaki Yasuto , Kanno Yoshinobu 
Published: February 2005

AbstractTo clarify the modulation of intercellular communication via gap junctions, associated with the growth induction of quiescent 3T3‐L1 cells, we investigated ‐the gap‐junctional intercellular communication in growth‐stimulated cells that were able to bind fibronectin‐coated beads. When quiescent 3T3‐L1 cells were incubated with fibronectin‐coated beads for the first 2 h after the addition of calf serum, 24.0% of the cells bound and phagocytosed beads. Among the cells with bound beads, the percentage of the cells labeled concurrently with bromodeox‐yuridine was 63.7% when examined 13 h after the addition of calf serum. Transient reduction of dye‐coupling, measured with Lucifer Yellow CH, was observed only in the cells with bound beads 2 h after addition of calf serum, but it was not observed in the cells without bound beads. When the quiescent cells were incubated with fibronectin‐coated beads for 2 h from 4–6 h after the addition of calf serum, the percentage of cells with bound beads increased to 53.1 %, but the decrease in dye‐coupling among the cells with bound beads was slight. These results suggest that the induction of cell growth causes a transient reduction in gap‐junctional intercellular communication in 3T3‐L1 cells with bound fibronec‐tin‐coated beads.
Glutamine regulates glutamine synthetase expression in skeletal muscle cells in culture
Authors: Feng Bo , Shiber Susan K. , Max Stephen R. 
Published: February 2005

AbstractMetabolite control of glutamine synthetase expression (by glutamine) was studied in L6 skeletal muscle cells. Depletion of glutamine from the culture medium for 24 hours resulted in a 3–4‐fold increase in glutamine synthetase activity. This effect was blocked by cycloheximide but not by actinomycin D. Addition of glutamine to L6 cells maintained in glutamine‐free medium caused a rapid return of glutamine synthetase activity to the control level. As reported, dexamethasone caused a striking increase in the glutamine synthetase mRNA level. In contrast, neither depletion nor addition of glutamine caused a change in the glutamine synthetase mRNA level. Therefore, regulation of glutamine synthetase by glutamine is exerted at a post‐transcriptional level.
Characterization of flat revertants isolated from cells transformed by Abelson murine leukemia virus (Ab‐MuLV)
Authors: Oka Takashi , Yutsudo Masuo , Inoue Hirokazu , Higuchi Fujito , Hakura Akira 
Published: February 2005

AbstractTransformed Fisher rat fibroblast cell lines by Abelson murine leukemia virus frequently revert to the normal phenotype in usual culture conditions. Molecular Biological analysis of thre revertant clones isolated from the transformants showed that their morphological reversions were due to inactivation of the v‐abl oncogene at multiple steps including transcription, translation or v‐abl protein kinase activity itself without any change in structural gene expression of helper virus. These findings suggest the existence of a specific mechanism(s) for elimination of the v‐abl oncogene by segregation, mutation, or gene rearrangement in these cells.
Prostaglandins do not mediate the actions of cholera toxin on pancreatic acini or gastric chief cells from the guinea pig
Authors: Raufman Jean‐Pierre , Singh Latika , Zakai M. David 
Published: February 2005

AbstractRecent reports suggest that prostaglandins, rather than cAMP, play a major role in mediating cholera toxin‐induced water and electrolyte secretion from rabbit intestinal loops. We examined the role of prostaglandins in mediating toxin‐induced pancreatic and gastric exocrine secretion. In these tissues, indomethacin, a potent inhibitor of prostaglandin synthesis, did not alter the stimulatory effects of cholera toxin on increases in cellular cAMP or enzyme secretion. Moreover, the addition of cholera toxin did not alter prostaglandin E2 release from either tissue. In contrast to their effects in rabbit intestinal loops, prostaglandins do not regulate cholera toxin‐induced enzyme secretion from the guinea pig pancreas or stomach.
Chondrocytes inhibit endothelial sprout formation in vitro: Evidence for involvement of a transforming growth factor‐beta
Authors: Pepper M. S. , Montesano R. , Vassalli J.‐D. , Orci L. 
Published: February 2005

AbstractUsing a quantitative in vitro model of spontaneous endothelial sprout formation, we have attempted to define physiological inhibitors of angiogenesis from hyaline cartilage, a tissue whose antiangiogenic properties have been well described. The model consists of embedding bovine microvascular endothelial cell aggregates into fibrin or collagen gels, which results in the formation of radially growing sprouts. When chondrocytes derived from the permanent cartilagenous region of the chick embryo sternum are cocultured with the endothelial cell aggregates, sprout formation is markedly inhibited. Addition of anti‐TGF‐β antibodies to the cocultures significantly reduces the inhibitory effect of chondrocytes on sprout formation. Chondrocyte‐conditioned medium or exogenously added TGF‐β1 have a similar albeit transient inhibitory effect. Depletion of TGF‐β from chondrocyte conditioned medium with anti‐TGF‐β antibodies and solid‐phase protein‐A significantly decreases the inhibition of sprout formation. These results demonstrate that a chondrocyte‐derived TGF‐β‐ like molecule inhibits capillary sprout formation in vitro and suggest that the antiangiogenic properties of cartilage may at least in part, be mediated by TGF‐β.
Epidermal growth factor‐induced hydrolysis of phosphatidylcholine by phospholipase D and phospholipase C in human dermal fibroblasts
Authors: Fisher Gary J. , Henderson Patricia A. , Voorhees John J. , Baldassare Joseph J. 
Published: February 2005

AbstractThe enzymatic pathways for formation of 1,2‐diradylglyceride in response to epidermal growth factor in human dermal fibroblasts have been investigated. 1,2‐Diradylglyceride mass was elevated 2‐fold within one minute of addition of EGF. Maximal accumulation (4‐fold) occurred at 5 minutes. Since both diacyl and ether‐linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether‐linked diglycerides following stimulation of fibroblasts with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2‐diradylglyceride was diacyl, the remaining 20% being ether‐linked. Addition of EGF caused accumulation of 1,2‐diacylglyceride with out alteration in the level of ether‐linked diglyceride. Thus, the observed induction of 1,2‐diradylglyceride by EGF was due exclusively to increased formation of 1,2‐diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2‐fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF‐induced phospholipase D activity, cells were labelled with exogenous [3H]1‐0‐hexadecyl, 2‐acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in 15 seconds after addition of EGF and was maximal (3‐fold) at 30 seconds. Consistent with the presence of EGF‐induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D‐catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2‐diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D‐catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2‐diglyceride. Thus, the induction of 1,2‐diacylglycerol by EGF may occur primarily via phospholipase C‐catalyzed hydrolysis of phosphatidylcholine.
Neuraminidase‐induced thrombocytopenia in mice: Effects on thrombopoiesis
Authors: Stenberg Paula E. , Levin Jack , Baker Georgiann , Mok Yuen , Corash Laurence 
Published: February 2005

AbstractPrevious studies to examine the effects of thrombocytopenia on thrombopoiesis have generally utilized immune‐mediated platelet depletion. We have developed a nonimmune model to exclude the possibility that adverse immune‐mediated effects have been misinterpreted as the physiological response to stimulation of thrombopoiesis. Thrombopoiesis was examined in mice after induction of thrombocytopenia with a single injection of the nonimmunologic agent neuraminidase (Ndase). Utilizing electron microscopy, we examined platelets and megakaryocytes (MK) obtained 8, 12, 24, 48, 72, 96, and 120 hr after administration of Ndase. Eight to 48 hr after induction of acute, severe throm‐bocytopenia (mean platelet count < 50,000/μl), the medians of the platelet sectional area distributions, as measured morphometrically, were significantly greater than the median platelet sectional area of pooled controls. The maximum median value for platelet sectional area was observed at 24 hr. The largest platelets in these samples contained more profiles of endoplasmic reticulum and Golgi cisternae, and a lower concentration of surface‐connected canalicular system, as compared with normal platelets. By 72 hr post‐injection of Ndase, virtually all platelets exhibited normal size and organelle complement. Mean platelet volumes, determined by electrical impedance analysis, paralleled the serial changes in platelet sectional areas. MK frequency and ploidy, measured by two‐color fluorescence activated flow cytometry, were unchanged 12 and 24 hr following Ndase. At 48 hr, total MK frequency increased significantly (P >0.01) from 0.11% to 0.17%, and MK ploidy distribution shifted with a reduction in 16N MK (P >0.005) and an increase in 32N MK (P >0.01). MK ploidy was maximally altered from normal at 72 hr with increased 32N MK frequency (32.0%, P <0.001) and increased 64N MK frequency (2.4%, P >0.005). Morphologic and morphometric examination of MK at all time points did not reveal detectable changes from normal in cytoplasmic appearance or size, respectively. Therefore, we have demonstrated marked alterations of morphology and size of platelets, and of MK ploidy, using this nonimmunologic model. These studies further support our previous observations that megakaryocyte ploidy and platelet volume are independently regulated in response to depletion of the circulating platelet mass, and they show that these changes are not dependent upon the mechanism of thrombocytopenia.
Plasminogen activators and inhibitors in the neuromuscular system: III. The serpin protease nexin I is synthesized by muscle and localized at neuromuscular synapses
Authors: Festoff B. W. , Rao J. S. , Hantaï D. 
Published: February 2005

AbstractRecent studies suggest that the nature of events leading to the formation, maintenance, and elimination of synapses may be regulated by cascade‐type, locally expressed proteases and protease inhibitors acting on adhesive extracellular matrix components. We have identified a molecule in conditioned medium of murine skeletal muscle cells that in molecular weight, target protease inhibition, heparin‐binding and cross‐reactivity with authenic antisera is similar to the human serine proteinase inhibitor, protease nexin I. Protease nexin I is a 43–50 kDa glycoprotein of the serpin superfamily (arg‐serpin class). Purified anti‐protease nexin I antibody (anti‐47 kDa) stains adult mouse skeletal muscle in discrete foci that precisely superimpose on synaptic neuromuscular junctions. Protease nexin I appears in patches on surfaces of cultured mouse skeletal myotubes, but not on myoblasts. These patches co‐localize with acetylcholine receptor clusters and acetylcholinesterase staining during cellular maturation in culture. Evidence that protease nexin I is a synaptic, extracellular antigen is particularly intriguing since it has been shown to be identical, in structure and activity, with a factor released by glial cells, called glia‐derived nexin that stimulates mouse neuroblastoma cell neurite outgrowth and inhibits granule cell migration Protease nexin I inhibits both tumor cell and myoblast plasminogen activator‐mediated destruction of extracellular matrix. Thus, such observations as presented in this report provide further evidence for involvement of cascade proteolytic systems, and their post‐translational regulation by specific serpins, in the remodeling that occurs in synapse formation and elimination.
Expression of bone matrix proteins associated with mineralized tissue formation by adult rat bone marrow cells in vitro: Inductive effects of dexamethasone on the osteoblastic phenotype
Authors: Kasugai Shohei , Todescan Reynaldo , Nagata Toshihiko , Yao Kam‐Ling , Butler William T. , Sodek Jaro 
Published: February 2005

AbstractThe nature and tissue distribution of non‐collagenous bone proteins synthesized by adult rat bone marrow cells, induced to differentiate in the presence of dexamethasone (DEX) and β‐glycerophosphate (β‐GP), was studied in vitro to determine the potential role of these proteins in bone formation. Northern hybridization analysis revealed a strong induction of bone sialoprotein (BSP) and osteocalcin in DEX‐treated cultures, whereas the constitutive expression of secreted phosphoprotein I (SPP‐1), type I collagen, SPARC, and alkaline phos‐phatase was stirnulated 6‐, 5‐, 3‐, and 2.5‐ fold, respectively. Metabolic labeling of proteins showed that the sialoproteins (SPP‐1 and BSP) were mostly secreted into the culture medium in the non‐mineralizing (‐β‐GP) cultures, but were the predominant non‐collagenous proteins associated with the hydroxyapatite of the bone nodules in mineralizing cultures (+β‐GP). Extraction of the tissue matrix with 4 M GuHCI and digestion of the demineralized tissue matrix with bacterial collagenase revealed that some BSP was also associated non‐covalently and covalently with the collagenous matrix. SPP‐1 was present in two distinct, 44 kDa and 55 kDa, forms in the conditioned medium of all cultures and was preferentially associated with the hydroxyapatite in the mineralizing cultures. In comparison, SPARC was abundant in culture media but could not be detected in de‐mineralizing extracts of the mineralized tissue. Radiolabeling with [35SO4] demonstrated that both SPP‐1 and BSP synthesized by bone cells are sulfated, and that a 35 kDa protein and some proteoglycan were covalently associated with the collagenous matrix in +DEX cultures. Labeling with [32PO4] was essentially confined to the sialoproteins; the 44 kDa SPP‐1 incorporating significantly more [32PO4] than the 55 kDa SPP‐1 and the BSP. These studies demonstrate that BSP and osteocalcin are only expressed in differentiated osteoblasts and that most of the major non‐collagenous bone proteins associate with the bone mineral. However, some novel proteins together with some of the BSP are associated with the collagenous matrix where they can influence hydroxyapatite formation.
The half‐lives of platelet‐derived growth factor A‐ and B‐chain mRNAS are similar in endothelial cells and unaffected by heparin‐binding growth factor‐1 or cycloheximide
Authors: Gay Cyril G. , Winkles Jeffrey A. 
Published: February 2005

AbstractPlatelet‐derived growth factor (PDGF) is mitogenic and chemotactic for vascular smooth muscle cells cultured in vitro, and, thus, may play a role in the smooth muscle cell proliferation and migration that occurs during atherosclerotic lesion development. Two related PDCF polypeptides, designated as the A and B chains, form functionally active PDGF‐AA, AB, or BB dinners. The PDGF A‐ and B‐chain genes are both transcribed in human umbilical vein endothelial (HUVE) cells and their expression is regulated by cytokines, growth factors, endotoxin, and phorbol ester. We reported previously that the angiogenic polypeptide heparin‐binding growth factor (HBGF)‐I induces PDGF A‐chain gene expression, but does not affect PDGF B‐chain gene expression. In this study, we determined whether mRNA stabilization contributed to this induction by measuring the half‐life of PDGF A‐chain mRNA in quiescent, HBGF‐1‐stimulated, and proliferating HUVE cells. PDGF A‐chain mRNA levels increase when quiescent HUVE cells are treated with the protein synthesis inhibitor cycloheximide; therefore, the effect of cycloheximide on PDGF A‐chain mRNA decay was also investigated. The half‐life of PDGF A‐chain transcripts in quiescent cells was ∼2.4 h and neither HBGF‐1 nor cycloheximide significantly altered this decay rate. We also estimated the half‐life of PDGF B‐chain mRNA under the three different growth conditions and in the absence or presence of cycloheximide. The half‐life in quiescent cells was ∼1.8 h and was unaffected by HBGF‐1 or protein synthesis inhibition. Therefore, the PDGF mRNAs have similar decay rates in HUVE cells, even though the 3′ untranslated region of B‐chain transcripts, but not A‐chain transcripts, contains AU‐rich sequence motifs postulated to confer rapid turnover in vivo.