bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and characterization of an activated form of the protein tyrosine kinase Lck from an Escherichia coli expression system
Authors: Flint Nicholas A. , Amrein Kurt E. , Jascur Thomas , Burn Paul 
Published: May 2005

AbstractThe lymphocyte‐specific, nonreceptor protein tyrosine kinase Lck has been purified from an Escherichia coli expression system using a monoclonal antibody column followed by dye‐affinity chromatography. Polyacrylamide gel electrophoretic analysis of purified protein revealed a single 56 kDa band, indicating that recombinant Lck was purified to near‐homogeneity. The purified enzyme displayed tyrosine kinase activity as measured by both autophosphorylation and phosphorylation of exogenous substrates. Biochemical properties including protein phosphorylation and kinetic characteristics of the enzyme have been assessed. Peptide map analysis revealed that bacterially expressed Lck is phosphorylated predominantly on the autophosphorylation site (tyrosine‐394), which is characteristic for activated protein tyrosine kinases. Indeed, we found that the recombinant enzyme is approximately fivefold more active than Lck from resting T cells, which is extensively phosphorylated at the regulatory carboxy‐terminal tyrosine residue (tyrosine‐505). Thus, we have overproduced recombinant human Lck in E. coli and developed a simple two‐step purification procedure which yields highly active enzyme. This will enable the identification and characterization of potential regulators and targets of Lck and thereby greatly facilitate studies which will clarify its role in T cell signal transduction. © 1994 Wiley‐Liss, Inc.
Comparative effects of hepatocyte growth factor and epidermal growth factor on motility, morphology, mitogenesis, and signal transduction of primary rat hepatocytes
Authors: Stolz Donna Beer , Michalopoulos George K. 
Published: May 2005

AbstractHepatocyte growth factor (HGF) and epidermal growth factor (EGF) are major hepatacyte mitogens, but HGF, also known as scatter factor (SF), has also been shown as a potent motogen for epithelial and endothelial cells. The mechanisms by which HGF is a stronger motogen compared to other mitogens are not understood. Here we report a comparative study of the effect of the two growth factors on cultured primary rat hepatocytes regarding their differential effects on morphology, mitogenicity, and motility as well as the phosphorylation of cytoskeletal‐associated proteins. Using three different motility assays, both HGF and EGF increased the motility of hepatocytes, but HGF consistently elicited a significantly greater motility response than EGF. Additionally, HGF induced a more flattened, highly spread morphology compared to EGF. To examine if HGF and EGF phosphorylated different cytoskeletal elements as signal transduction targets in view of the observed variation in morphology and motility, primary cultures of 32P‐loaded rat hepatocytes were stimulated by either HGF or EGF for up to 60 min. Both mitogens rapidly stimulated four isoforms of MAP kinase with similar kinetics and also rapidly facilitated the phosphorylation of cytoskeletal‐associated F‐actin. Two cytoskeletal‐associated proteins, however, were observed to undergo rapid phosphorylation by HGF and not EGF during the time points described. One protein of 28 kDa was observed to become phosphorylated fivefold over controls, while the EGF‐stimulated cells showed only a slight increase in the phosphorylation of this protein. Another protein with an apparent mwt of 42 kDa was phosphorylated 20‐fold at 1 min and remained phosphorylated over 50‐fold over control up to the 60 min time point. This protein was observed to become phosphorylated by EGF only after 10 min, and to a lesser extent (20‐fold). Taken together, the data suggest that HGF and EGF stimulate divergent as well as redundant signal transduction pathways in the hepatocyte cytoskeleton, and this may result in unique HGF‐ or EGF‐specific motility, morphology, and mitogenicity in hepatocytes. © 1994 Wiley‐Liss, Inc.
Induction of c‐fos and c‐jun mRNA at the M/G1 border is required for cell cycle progression
Authors: Cosenza Stephen C. , Yumet Gladys , Soprano Dianne Robert , Soprano Kenneth J. 
Published: May 2005

AbstractThe proto‐oncogenes c‐fos and c‐jun have been shown in numerous model systems to be induced within minutes of growth factor stimulation, during the G0/G1 transition. In this report we use the mitotic shake‐off procedure to generate a population of highly synchronized Swiss 3T3 cells. We show that both of these immediate‐early, competence genes are also induced during the M/G1 transition, immediately after completion of mitosis. While c‐fos mRNA levels drop to undetectable levels within 2 hr after division, c‐jun mRNA levels are maintained at a basal level which is ∼ 30% maximum throughout the remainder of G1. In order to access the functional significance of these patterns of c‐fos and c‐jun expression, antisense oligodeoxynucleotides specific to c‐fos or c‐jun were added to either actively growing Swiss 3T3 cells or mitotically synchronized cells, and their ability to inhibit DNA synthesis and cell division determined. Our results show that treatment of Swiss 3T3 cells with either c‐fos or c‐jun antisense oligodeoxynucleotides, while actively growing, during mitosis, or in early G1, results in a reduction in ability to enter S and subsequently divide. This was also true if Swiss 3T3 cells were treated during mid‐G1 with c‐jun antisense oligodeoxynucleotides. These results demonstrate that the regulation of G1 progression following mitosis is dependent upon the expression and function of the immediate‐early, competence proto‐oncogenes c‐fos and c‐jun. © 1994 Wiley‐Liss, Inc.
Overcoming obstacles to metastasis ‐ defenses against host defenses: Osteopontin (OPN) as a shield against attack by cytotoxic host cells
Authors: Denhardt David T. , Chambers Ann F. 
Published: May 2005

AbstractOsteopontin (OPN) serves both a cell attachment function and a cell signalling function via the αvβ3 integrin, in its cell attachment capacity it can promote attachment of both osteolasts to bone hydroxyapatite and various other cell types to basement membrane/extracellular matrix. In its cell signalling capacity it initiates a signal transduction cascade that includes changes in the intracellular calcium ion levels and the tyrosine phosophorylation status of several proteins including paxillin. Effects on gene expression include suppression of the induction of nitric oxide synthase by inflammatory mediators. OPN can also reduce cell oxidant and inhibit the killing of tumor cells by activated macrophages and endothelial cells. We hepothesize that those cancer cells that produce OPN at elevated levels can suppress the oxidative burst, inhibit NO production, and thus protect themselves from killing by specific host cell types.
Identification of the major sites of phosphorylation in IGF binding protein‐3
Authors: Hoeck Wolfgang G. , Mukku Venkat R. 
Published: May 2005

AbstractInsulin‐like growth factor binding protein‐3 (IGFBP‐3) is the major carrier of insulin‐like growth dactor I and II in the circulation. IGFBP‐3 is secreted by various tissues and cell lines as a glycosylated phosphoprotein. We have identified two major serine phosphorylation sites located at amino acids 111 and 113 of the human protein. These serine residues ngighboring amino acids potentially involved in defining a protrein kinase recognition sequence were mutated to alanine using PCR. Single and double point mutants were stably transfected into CHO‐cells and analyzed for their level of phosphorylation. Mutation of both serines reduced phosphorylation by 80% in the full‐length protein and completely abolished phosphorylation in a 17 kDa IGFBP‐3 fragment, derived from digestion with EndoProteinase Lys‐C. The 17kDa fragment containd serines 111 and 113. S111A/S113A, a double serine‐to‐alanine mutant at positions 111 and 113, showed a strongly reduced glycosylation pattern that appears to be the result of anino acid substitutions rather tha n lack of phosphrylation. Mutant S111A/S113A, despite being non‐phosphorylated and non‐glycosylated, is functionally similar to the wild‐type IGFBP‐3 in terms of IGF‐1 binding. These results enhance our non‐glycosylated, is functionally similar to the wild‐type IGFBP‐3 in terms of IGF‐I binding. These results enhance our understanding on the functional role of glycosylation and phosphorylation of IGFBP‐3.
Vitamin D receptor alleles and bone physiology
Authors: White C. P. , Morrison N. A. , Gardiner E. M. , Eisman J. A. 
Published: May 2005

AbstractThe vitamin D endocrine system is central to the control of bone and calcium homeostasis. The active hormonal aform of vitamin D, 1,25 dihydroxyvitamin D (calcitriol), the circulating level of which is tightly regulated, acts through a specific receptor to mediate its genomic actions on almost every aspect of calcium homeostasis. Because of its transactivation function, it possible that a small difference in vitamin D receptor level could be amplified into a biologically significant alteration in physiological setpoint. The recent finding that polymorphisms in the vitamin D receptor gene are predictive of bone density (morrison et al., Nature 367:284–287, 1994) is the first example of an allelic effect in such a homeostatically controlled system. This raises the possibility that such central operators may exist in other regulatory pathways, and could expllain a large part of the observed “ormal” population distribution that exists for all physiological paraameters.
Pleckstrin homology (PH) domains in signal transducton
Authors: Ingley Evan , Hemmings Brian A. 
Published: May 2005

AbstractA diverse array of molecules involved in signal transduction have recently been recognised as containing a new homology domain, the pleckstrin homology (PH) domain. These include kinases (both serine/threonine and tyrosine specific), all currently known mammalian phospholipase Cs, GTPases, GTPage‐activatng proteins, GTpace‐exchange factors, “adapter” proteins, cyotskeletal proteins, and kinase substrates. This has sparked a new surge of research into elucidating its sturcture and function. The NMR solution structure of the PH domains of β‐spectrin and pleckstrin (the N‐terminal domain) both display a core consisting of seven anti‐parallel β‐sheet strands. The carboxy terminus is folded into a long α‐helix. The molecule is electrostatically polarised and contains a pocket which may be involved in the inding of a ligand. The PH domain overall topological relatedness to the retinoid inding protein family of molecules would suggest a lipid ligand could bind to this pocket. the prime function of the PH domain still remains to be elucidated. However, it has been shown to be important in signal transduction, most probably by mediating protein‐protein interactions. An extended PH domain of the β‐adrenergic receptor kinase (βARK), as well as that of several other molecules, can bind to βγ subunits of the heterotrimeric G‐proteins. The possibility that the PH domain, which is found in so many signalling molecules, being generally inovolved in βγ binding site appear to be concomitant in βARK, detailed analysis indicates that the PH domain is not generally a βγ binding domain. Thus, the race is on to find the ligands of each PH domain and determine a common nature to their interaction.
Identification of a novel heparin binding domain in RHAMM and evidence that it modifies HA mediated locomotion of ras‐transformed cells
Authors: Yang Baihua , Hall Christine L. , Yang Bing Luo , Savani Rashmin C. , Turley Eva A. 
Published: May 2005

AbstractWe have previously reported that the hyaluronan (HA) receptor RHAMM (Receptor for Mediated Motility) [Turley et al., 1991] and that HA stimulation of the motility of ras‐transformed fibroblasts is mediated via its interaction with RHAMM. Here we show that RHAMM also contains binding sites for heparin (HP) anbd that interaction of HP with these sites can regulates the locomotion of ras‐transformed fibroblasts. At low concentrations (0.01 mg/ml), HP inhibited HA‐induced locomotion of ras‐transformed cells in a manner independent of RHAMM. At higher, but still physioligical concentrations (0.1 mg/ml), HP alone stimulated cell locomotion and this stimulation appeared to be RHAMM‐dependent as it was blocked by anti‐RHAMM antibodies. Other related glycosaminogolycans such as chondroitin sulfate and dermatin sulfate had no effect on cell motility. In ligand blotting assays, GST‐RHAMM fusion protein was shown to bind biotin‐labelled HP and this binding was displaceable with unabelled HP. In similar lignad binding analyses conducted with truncations of RHAMM fusion protein, the binding region was found to be localizeed in the same 35 amino acid segment of RHAMM that contains the two HA binding domains. Synthetic peptides corresponding to these HA binding domains were retained on and bound effectively to an HP‐Sepharose affinity column. Fusion protein generated by linkage of these peptides to the non‐HP binding amino terminus of RHAMM conferred HP binding capacity to the genetically engineered proteins. Conversely, deletion of the HA binding domains of RHAMM resulted in fusion proteins devoid of HP binding activity. The relative affinities of RHAMM for HA and HP, as determined by competition and transblot assays as well as quantification of binding at various salt concentrations, indicated that RHAMM had lower affinity for HP than that for HA. These results demonstrate the existence of new HP binding motif that has biological relevance to locomotion.
IFI 16 gene encodes a nuclear protein whose expression is induced by interferons in human myeloid leukaemia cell lines
Authors: Dawson Michelle J. , Trapani Joseph A. 
Published: May 2005

AbstractWe have characterized the induction of mRNA and protein products of the human IFI 16 gene in response to IFN‐γ, IFN‐α, and IFN‐β2 (IL‐6). We demonstrate that the IFI 16 gene product is a novel nucleoprotein expressed in association with the differentiation of myeloid precursor cell lines. In Northern blots, IFI 16 mRNA was increased ∼25‐fold above barely detectable levels in unstimulated promyelocytic HL‐60 cells, in response to IFN‐γ. Other myeloid cell lines, U937 and K562, also demonstrated a marked IFN‐γ‐inducibility of IFI 16 mRNA. However, all three cell lines were far less responsive to IFN‐α, and there was no response to IL‐6. By comparison, a panel of T and B cell lines demonstrated high constitutive expression of IFI 16 mRNA that was not regulated by these cytokines. Culture of HL‐60 cells in medium containing dimethylsulfoxide, retinoic acid, and 1,25 dihydroxyvitamin D3, agents that stimulate the differentiation of HL‐60 along myeloid pathways, also caused the induction of IFI 16 mRNA. To characterize the protein product of IFI 16, a monoclonal antibody was raised against a recombinant bacterial protein comprising the amino terminal 159 amino acids of IFI 16 fused to glutathione S‐transferase. The antibody, designated 1G7, was used in Western blotting to demonstrate the strong induction of a cluster of proteins of 85–95 kDa in the nuclear extracts of IFN‐γ‐treated HL‐60. The nuclear localization of IFI 16 antigen was confirmed by immunohistochemical staining of HL‐60 cells treated with IFN‐γ, dimethylsulfoxide, and retinoic acid. IFI 16 was also detected in the nuclei of monocytes, neutrophils, and lymphocytes in normal peripheral blood. Database comparisons of the IFI 16 amino acid sequence revealed 51% identity with the recently cloned myeloid cell nuclear differentiation antigen (MNDA), and extensive similarity to protein products of the Gene 200 cluster of IFN‐inducible genes, Ifi 202 and Ifi 204. The amino terminal domain of IFI 16 encodes a putative nuclear localization signal, 124PGAQKRKK, which is strongly conserved in MNDA and 204. Nuclear IFI 16 was able to bind double‐stranded DNA in vitro and exhibited a similar elution profile from DNA‐cellulose as previously observed for MNDA and 204. Therefore, IFI 16 and MNDA are members of a novel family of human DNA‐binding proteins whose expression is associated with myeloid cell differentiation induced by cytokines and chemical agents.
Temporal variation of c‐fos proto‐oncogene expression during osteoblast differentiation and osteogenesis in developing rat bone
Authors: Machwate M. , Jullienne A. , Moukhtar M. , Marie P. J. 
Published: May 2005

AbstractTo delineate the implication of c‐fos protooncogenic in the osteogenie process, we have investigated the temporal pattern of c‐fos mRNA expression in fetal and neonatal rat bone during intramembranous and endochondral bone formation. Northern blot analysis of mRNA extracted from calvaria and femur showed that expression of c‐fos, Histone H4, and osteocalcin mRNAs followed a temporal sequence during bone development. The levels of histone H4 mRNA, a marker of cell proliferation, were high at early stages of fetal development of calvaria and femur, and decreased until birth. In both the postnatal calvaria and femur, c‐fos mRNA levels increased transiently at birth and preceded a rise in osteocalcin transcripts, a marker of the mature osteoblast phenotype. The immunohistochemical analysis showed that c‐fos protein was expressed in osteoprogenitor cells in the perichondrium and periosteum, and not in mature osteoblasts which expressed markers of differentiated osteoblasts such as type‐I collagen, bone sialoprotein, and osteocalcin. Thus, the transient c‐fos proto‐oncogene expression during the postnatal life that precedes the osteocalcin expression may be involved in the transition from the precursor state to mature osteoblasts. These results suggest that c‐fos proto‐oncogene may play an important role in osteogenesis during rat postnatal life.
Proximal promoter binding protein contributes to developmental, tissue‐restricted expression of the rat osteocalcin gene
Authors: Heinrichs Arianne A. J. , Bortell Rita , Bourke Michael , Lian Jane B. , Stein Gary S. , Stein Janet L. 
Published: May 2005

AbstractOsteocalcin is a 6 kD tissue‐specific calcium binding protein associated with the bone extracellular matrix. The osteocalcin gene is developmentally expressed in postproliferative rat osteoblasts with regulation at least in part at the transcriptional level. Multiple, basal promoter and enhancer elements which control transcriptional activity in response to physiological mediators, including steroid hormones, have been identified in the modularly organized osteocalcin gene promoter. The osteocalcin box (OC box) is a highly conserved basal regulatory element residing between nucleotides −99 and −76 of the proximal promoter. We recently established by in vivo competition analysis that protein interactions at the CCAAT motif, which is the central core of the rat OC box, are required for support of basal transcription [Heinrichs et al. J Cell Biochem 53:240–250, 1993]. In this study, by the combined utilization of electrophoretic mobility shift analysis, UV cross linking, and DNA affinity chromatography, we have identified a protein that binds to the rat OC box. Results are presented that support involvement of the OC box‐binding protein in regulating selective expression of the osteocalcin gene during differentiation of the rat osteoblast phenotype and suggest that this protein is tissue restricted.
Humoral hypercalcemia of malignancy: Some enigmas on the clinical features
Authors: Ikeda Kyoji , Ogata Etsuro 
Published: May 2005

AbstractHumoral hypercalcemia of malignancy (HHM) is a common paraneoplastic syndrome mediated by tumor‐derived parathyroid hormone‐related peptide (PTHRP), which bears structural and functional similarities to PTH. Thus the clinical features of HHM are very similar to those of primary hyperparathyroidism (1° HPT), a prototype of humoral hypercalcemia caused by PTH. On the other hand, HHM syndrome differs from 1° HPT in several aspects, including serum 1,25(OH)2D levels, acid‐base balance, and bone remodeling process, the reason of which remains largely unknown. We approached these questions using a unique animal model of HHM, nude rats implanted with PTHRP‐overproducing human carcinomas. In this review we will summarize the results and discuss the implications in understanding the disease mechanism.
Tyrosine phosphorylation of phosphatase inhibitor 2
Authors: Williams John P. , Jo Hanjoong , Hunnicutt Ruthann E. , Brautigan David L. , McDonald Jay M. 
Published: May 2005

AbstractInhibitor 2 is a heat‐stable protein that complexes with the catalytic subunit of type‐1 protein phosphatase. The reversible phosphorylation of Thr 72 of the inhibitor in this complex has been shown to regulate phosphatase activity. Here we show that inhibitor 2 can also be phosphorylated on tyrosine residues. Inhibitor 2 was 32P‐labeled by the insulin receptor kinase in vitro, in the presence of polylysine. Phosphorylation of inhibitor 2 was accompanied by decreased electrophoretic mobility. Dephosphorylation of inhibitor 2 by tyrosine phosphatase 1B, restored normal electrophoretic mobility. Phosphotyrosine in inhibitor 2 was detected by immunoblotting with antiphosphotyrosine antibodies and phosphoamino acid analysis. In addition, following tryptic digestion, one predominant phosphopeptide was recovered at the anode. The ability of inhibitor 2 to inhibit type‐1 phosphatase activity was diminished with increasing phosphorylation up to a stoichiometry of 1 mole phosphate incorporated/mole of inhibitor 2, where inhibitory activity was completely lost. These data demonstrate that inhibitor 2 can be phosphorylated on tyrosine residues by the insulin receptor kinase, resulting in a molecule with decreased ability to inhibit type‐1 phosphatase activity.
Noncoordinate changes in the steady‐state mrna expressed from aldolase A and aldolase C genes during differentiation of chicken myoblasts
Authors: Meighan‐Mantha Rachel L. , Tolan Dean R. 
Published: May 2005

AbstractIn chickens, as in all vertebrates, tissue‐specific expression of aldolase isozymes A, B, and C is developmentally coordinated. These developmental transitions in aldolase expression have been studied most extensively by charting enzyme activity during normal and abnormal development of specific vertebrate tissues. Indeed, aldolase expression has been a key marker for normal differentiation and for retrodifferentiation during carcinogenesis. Aldolase expression during chicken myoblast differentiation offers a model for investigating the regulatory mechanisms of these developmental transitions at the level of gene expression. For these studies, cDNAs encoding the most isozyme‐specific regions of both chicken aldolase A and C were cloned. The chicken aldolase A cDNA represents the first report of this sequence. Aldolase steady‐state mRNA expression was measured during chicken myoblast differentiation in primary cultures using RNase protection assays with cRNA probes generated from these aldolase cDNA clones. Steady‐state mRNA for aldolase C, the predominant embryonic aldolase isozyme in chickens, did not significantly change throughout myoblast differentiation. In contrast, expression of steady‐state mRNA for aldolase A, the only aldolase isozyme found in adult skeletal muscle, was not detected until after myoblast fusion was approximately 50% completed. Aldolase A expression gradually increased throughout myoblast differentiation until approximately 48 h after fusion was completed when there was a dramatic increase. These results are contrasted with those of Turner et al. (1974) [Dev Biol 37:63–89] that showed a coordinated switch in isozyme activities between the embryonic aldolase C and the muscle‐specific aldolase A. This discordant expression indicates that the aldolase A and C genes may employ different regulatory mechanisms during myoblast differentiation.