bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phosphorylation of glycolytic and gluconeogenic enzymes by the insulin receptor kinase
Authors: Sale Elizabeth M. , White Morris F. , Kahn C. Ronald 
Published: December 2004

AbstractVarious glycolytic and gluconeogenic enzymes were tested as substrates for the insulin receptor kinase. Phosphofructokinase and phosphoglycerate mutase were found to be the best substrates. Phosphorylation of these enzymes was rapid, stimulated 2‐ to 6‐fold by 10−7 M insulin and occurred exclusively on tyrosine residues. Enolase, fructose 1,6‐bisphosphatase, lactate dehydrogenases in decreasing order, were also subject to insulin‐stimulated phosphorylation but to a smaller extent than that for phpsphofructokinase or phosphoglycerate mutase.The phosphorylation of phosphofructokinase was studied most extensively since phosphofructokinase is known to catalyze a rate‐limiting step in glycolosis. The apparent Km of the insulin receptor for phosphofructokinase was 0.1 μM, which is within the physiologic range of concentration of this enzyme in most cells. Tyrosine phosphorylation of phosphofructokinase paralleled autophosphorylation of the β‐subunit of the insulin receptor with respect to time course, insulin dose response (half maximal effect between 10−9 and 10−8 M insulin), and cation requirement (Mn2+ > Mg2+ > > Ca2+). Further study will be required to determine whether the tyrosine phosphorylation of phosphofructokinase plays a role in insulin‐stimulated increases in glycolytic flux.
Site‐specific mutagenesis of cDNA clones expressing a poliovirus proteinase
Authors: Semler Bert L. , Johnson Victoria H. , Dewalt Patricia Gillis , Ypma‐Wong Mary Frances 
Published: December 2004

AbstractThe cleavage of poliovirus precursor polypeptides occurs at specific amino acid pairs that are recognized by viral proteinases. Most of the polio‐specific cleavages occur at glutamine‐glyeine (Q‐G) pairs that are recognized by the viral‐encoded proteinase 3C (formerly called P3–7c). In order to carry out a defined molecular genetic study of the enzymatic activity of protein 3C, we have made cDNA clones of the poliovirus genome. The cDNA region corresponding to protein 3C was inserted into an inducible bacterial expression vector. This recombinant plasmid (called pIN‐III‐C3–7c) utilizes the bacterial lipoprotein promoter to direct the synthesis of a precursor polypeptide that contains the amino acid sequence of protein 3C as well as the amino‐ and carboxy‐terminal Q‐G cleavage signals. These signals have been previously shown to allow autocatalytic production of protein 3C in bacteria transformed with plasmid pIN‐III‐C3–7c. We have taken advantage of the autocatalytic cleavage of 3C in a bacterial expression system to study the effects of site‐specific mutagenesis on its proteolytic activity. One mutation that we have introduced into the cDNA region encoding 3C is a single amino acid insertion near the carboxy‐terminal Q‐G cleavage site. The mutant recombinant plasmid (designated pIN‐III‐C3‐μ10) directs the synthesis of a bacterial‐polio precursor polypeptide that is like the wild‐type construct (pIN‐III‐C3–7c). However, unlike the wild‐type precursor, the mutant precursor cannot undergo autocatalytic cleavage to generate the mature proteinase 3C. Rather, the precursor is able to carry out cleavage at the amino‐terminal Q‐G site but not at the carboxy‐terminal site. Thus, we have generated an altered poliovirus proteinase that is still able to carry out at least part of its cleavage activities but is unable to be a suitable substrate for self‐cleavage at its carboxy‐terminal Q‐G pair.
Role of glandular kallikreins as growth factor processing enzymes: Structural and evolutionary considerations
Authors: Isackson Paul J. , Dunbar Joan C. , Bradshaw Ralph A. 
Published: December 2004

AbstractHormones and growth factors are generally released from larger precursors by limited proteolysis. The causative agents remain poorly defined with respect to location and properties. One subset of proteases, the glandular kallikreins, have been implicated in a few cases, in part because of their specific association with mature forms of some hormones. However, limited distribution and low copy number in some species cast doubt on this hypothesis, and they may well play other physiological functions that remain to be elucidated.
Growth factors, oncogenes, and multistage carcinogenesis
Authors: Weinstein I. Bernard 
Published: December 2004

AbstractThis paper presents evidence that the full repertoire of cellular genes involved in the carcinogenic process is several times larger than that of the known list of proto‐oncogenes. Furthermore, this repertoire includes genes whose normal function is related to growth stimulation, as well as genes whose normal function is to inhibit growth or induce terminal differentiation. Multistage carcinogenesis probably results from a complex series of changes in both categories of genes. Despite this complexity, carcinogenesis can be conceived in terms of disturbances in biochemical functions that normally control the expression or function of growth factors, receptors, and pathways of signal transduction. Several protein kinases play a central role in the process of signal transduction. Our laboratory has recently isolated cDNA clones for the enzyme protein kinase C (PKC). These clones should be useful for clarifying the role of PKC in growth control and tumor promotion. Finally, the existence of genes whose normal function is to inhibit cell growth provides a rationale for new strategies of cancer prevention and treatment.
Myc family of cellular oncogenes
Authors: DePinho Ronald , Mitsock Lisa , Hatton Kimi , Ferrier Pierre , Zimmerman Kathy , Legouy Edith , Tesfaye Abeba , Collum Robert , Yancopoulos George , Nisen Perry , Kriz Ronald , Alt Frederick 
Published: December 2004

AbstractThe myc family of cellular oncogenes contains three well‐defined members: c‐myc, N‐myc and L‐myc. Additional structural and functional evidence now suggests that other myc‐family oncogenes exist. The overall structure and organization of the c‐, N‐. and L‐myc genes and transcripts are very similar. Each gene contains three exons: encoding a long 5′ untranslated leader and a long 3′ untranslated region. The proteins encoded by these myc genes share several stretches of significant homology. The conservation of sequences at the carboxy‐terminus of the L‐myc protein suggests that it is also a DNA‐binding, nuclear‐associated protein. Each myc gene will cooperate with an activated Ha‐ras oncogene to cause transformation of primary rat embryo fibroblasts. Characteristics of several new myc‐family members are described.
Detergents inhibit exocytosis in PC 12 cells: Evidence for an effect on ion fluxes
Authors: Tapparelli Carlo , Grob Marianne , Burger Max M. 
Published: December 2004

AbstractMembrane events in exocytosis were studied by examining the effect of different detergents on the K+‐stimulated release of noradrenaline in the secretory cell line PC 12. The nonionic detergent Triton X‐100 and the cationic detergent cetyltrimethylammonium bromide (CTAB) inhibit the noradrenaline release evoked by 55 mM K+ by 50% at very low concentrations (30 μM and 10 μM, respectively). These values are tenfold lower than the critical micellar concentrations (CMC). No such effect was seen with the anionic detergent sodium dodecyl sulphate (NaDodSO4).The inhibitory effect of 30 μM Triton X‐100 is reversible, and the recovery from inhibition correlates with the loss of detergent from the cells as demonstrated by binding studies using [3H]Triton X‐100. The possible relationship between this inhibition of secretion and the structural properties of the detergent was investigated. The inhibition in the presence of purified Triton X‐100 subfractions turned out to be a function of the length odemonstrated by binding studies using [3H]Triton X‐100. The possible relationship between this inhibition of secretion and the structural properties of the detergent was investigated. The inhibition in the presence of purified Triton X‐100 subfractions turned out to be a function of the length of the oligometric ethyleneglycol chain (C6 to C26). The maximal effect was observed for Triton X‐100 molecules having a chain length of 16 carbon atoms, which can penetrate just half of the lipid bilayer of the membrane. Additionally, the phase transition at 13–14°C observed in an Arrhenius plot of noradrenaline release in stimulated cells was abolished.In the presence of 30 μM Triton X‐100, 22Na+ uptake, 86Rb+ release, and 45Ca2+ uptake were reduced by 50–60%. These data suggest that the site of action of Triton X‐100 is at the level of altering the movement of ions in PC 12 cells during the stimulatory phase of secretion.
Induction of granuloma‐dependent angiotensin‐converting enzyme and eosinophil chemotactic factor in the skin of athymic nude mice
Authors: Kanazawa Kazunori , Higuchi Mitsunari , Okamoto Mitsuse , Fukuyama Kimie , Epstein William L. 
Published: December 2004

AbstractActivities of angiotensin‐converting enzyme (ACE), other proteinases, and eosinophil chemotactic factor (ECF‐G) are known to be elevated in hepatic hypersensitivity granulomas of thymus intact (nu/+) mice after Schistosoma mansoni infection. The enzyme activities also increase, but to a lesser degree in hepatic granulomas of athymic nude (nu/nu) mice, and ECF‐G is not detectable. In this study isolated hepatic granulomas from nu/+ mice were grafted into the skin of uninfected nu/nu mice, and changes in those cellular functions were determined to examine whether the newly formed granulomas by recipient nu/nu cells acquire the functional activities as well as the histological appearance of nu/+ granulomas. ACE and ECF‐G rapidly disappeared from grafted sites during the first 5 days, corresponding to loss of nu/+ cells from the graft. Reduction in activities of arylsulfatases, lysozyme, and acid phosphatase also occurred, but to a lesser extent. Recovery of ACE and ECF‐G activities to the levels seen in nu/+ hepatic granulomas was observed by 14 days after grafting when nu/nu cells had accumulated in the grafts and formed new granulomas. Other enzymes increased to approximately half the levels seen in grafted donor granulomas. Circulating eosinophilia also increased. The findings indicate that nu/nu cells that accumulated in the skin grafts not only morphologically mimicked nu/+ type granulomas but also demonstrated nu/+ levels of cellular function. Analysis of skin granulomas developing in nu/+ mice after grafting of nu/+ hepatic granulomas showed the similar histology and enzymatic changes, whereas the skin sites inoculated with purified schistosome eggs alone caused neither significant histological changes nor elevation of ACE activity.
Identification and quantification of actin isoforms in vertebrate cells and tissues
Authors: Otey C. A. , Kalnoski M. H. , Bulinski J. C. 
Published: December 2004

AbstractThe cytoskeletal protein actin exists in vertebrates as six different isoforms, which are difficult to identify conclusively because of a high degree (>90%) of overall sequence homology. We have used IEF immunoblotting in combination with a panel of isoform‐specific and ‐selective antibodies to analyze the actin isoform composition of nine tissues from adult rat. In three nonmuscle tissues (lung, spleen, and testis), we detected a previously unreported isoform that we identified as smooth muscle α. The IEF immunoblot technique was also used to quantify the proportions of the isoforms expressed in these nine rat tissues.
Preparation of a monoclonal antibody to a melanoma growth‐stimulatory activity released into serum‐free culture medium by Hs0294 malignant melanoma cells
Authors: Lawson David H. , Thomas H. Greg , Roy Robert G. B. , Gordon David S. , Chawla Rajender K. , Nixon Daniel W. , Richmond Ann 
Published: December 2004

AbstractAutostimulatory growth factors may contribute to the ability of malignant cells to escape normal growth controls. We have previously shown that Hs0294 human malignant melanoma cells release into culture medium an acid‐soluble, heat‐stable, trypsin‐sensitive, autostimulatory monolayer mitogen which can be purified from acetic acid extracts of conditioned medium by gel filtration, reverse‐phase high‐performance liquid chromatography, and preparative electrophoresis. The majority of this melanoma growth‐stimulatory activity (MGSA) resides in a 16‐Kd moiety though bioactivity is also associated with 24–26 and < 14‐Kd forms of MGSA (Richmond and Thomas: J Cell Physiol 129:375, 1986). In order to further characterize this growth factor, monoclonal antibodies were prepared against a partially purified preparation of the autostimulatory melanoma mitogen. Monoclonal antibody clones were selected based on supernate inhibition of 3H‐thymidine incorporation in serum‐free Hs0294 melanoma cultures. One of these, termed FB2AH7, slows, but does not completely block, the growth of Hs0294 cells in scrum‐free medium in a dose‐dependent manner. This antibody does not slow the growth of normal rat kidney fibroblasts, which neither produce nor require this mitogen, in either serum‐free medium or medium containing 0.8% calf scrum. This monoclonal antibody also blocks the mitogenic effects of partially purified preparations of this melanoma growth stimulatory activity (MGSA) on both Hs0294 cells and normal rat kidney fibroblasts. The FB2AH7 antibody has been demonstrated to bind MGSA by Western blot and by immunoprecipitation procedures. Western blot analysis of reverse‐phase high‐performance liquid chromatography purified growth factor demonstrated that FB2AH7 antibody binds to the 16‐Kd and ∼ 13–14‐Kd forms of MGSA. FB2AH7 antibody can be used in immunoprecipitation experiments to bind the ∼ 13–16‐Kd forms of MGSA. The specificity of the binding of FB2AH7 antibody for MGSA but not other growth factors has been demonstrated in a modified dot blot assay. These data thus support the hypothesis that MGSA is an autostimulatory melanoma mitogen distinct from other growth factors.
Phosphorylation and dephosphorylation of soluble proteins in human eosinophils
Authors: Ramesh Kashi S. , Rocklin Ross E. , Pincus Stephanie H. 
Published: December 2004

AbstractThe effect of phorbol 12‐myristate 13‐acetate (PMA), calcium ionophore (A23187), opsonized zymosan (OZ), and N‐formylmethionyl‐leucyl‐phenylalanine (f‐Met‐Leu‐Phe) on protein phosphorylation was examined in purified eosinophils (eos) isolated from human peripheral blood. Eos were prelabeled with [32P]orthophosphate, stimulated with several activating agents for varying periods of time. The soluble proteins were then analyzed by one‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and autoradiography. In resting eos, there was phosphorylation of endogenous soluble proteins with molecular weights of 12, 16, 21, 40, and 66 kilodaltons (kDa). PMA, a potent activator of oxidative metabolism, induced phosphorylation of 19‐, 40‐, and 67‐kDa proteins. A23187, a strong degranulating stimulus, caused phosphorylation of 40‐, 53‐, and 67‐kDa proteins. OZ, a relatively weak stimulus for eos function, caused phosphorylation of 30–34‐, 59‐, 67‐, and 93‐kDa proteins. In addition, all the above stimuli caused a time‐dependent dephosphorylation of 21‐kDa protein. In contrast, f‐Met‐Leu‐Phe caused neither phosphorylatiop of new proteins nor dephosphorylation of preexisting eos proteins. These findings demonstrate that selected stimuli affect phosphorylation of soluble eos protein. These results also suggest that phosphorylation of specific proteins in eos is an intermediary step in external stimulus‐induced cell activation, which may involve many different cell functions.
Vaccinia virus and the EGF receptor: A portal for infectivity?
Authors: Marsh Y. Vivienne , Eppstein Deborah A. 
Published: December 2004

AbstractWe previously demonstrated that occupancy of the epidermal growth factor (EGF) receptor reduced the ability of vaccinia virus to infect L cells [Eppstein et al: Nature 318:663, 1985]. This result suggested that vaccinia virus was utilizing the EGF receptor as one pathway to infect cells. We have studied this system further, and now find that antibodies to the EGF receptor also reduce the ability of vaccinia virus to infect cells productively. Inclusion of both EGF and antibodies to the EGF receptor did not cause inhibition over that obtained by EGF alone, providing another line of evidence that the antiviral effects on vaccinia virus were at the level of the EGF receptor. The antiviral effects of EGF or synthetic peptides corresponding to the third disulfide loop of TGF‐α or the vaccinia virus growth factor were specific to vaccinia virus and did not inhibit replication of herpes simplex virus type 2 or vesicular stomatitis virus. The inhibitory effects on replication of vaccinia virus were obtained when EGF (but not insulin or growth hormone) was present prior to, but not after, productive viral adsorption. These results provided further evidence that the antivaccinia viral effects of EGF were at the level of initial receptor occupancy. As interferon (IFN) treatment has been shown to interfere with the action of some growth factors, including EGF, we examined the effects of IFN treatment of cells on the antivaccinia viral activity of EGF. Our results show that the antivaccinia effect of IFN‐β either interfered with or partially coalesced with the inhibitory effects of EGF. The former interpretation is consistent with the report that IFN treatment results in a decrease both in the apparent number and affinity of cell‐surface receptors for EGF [Zoon et al: Proc Natl Acad Sci USA 83:8226, 1986].
Growth regulation of human breast carcinoma occurs through regulated growth factor secretion
Authors: Lippman Marc E. , Dickson Robert B. , Gelmann Edward P. , Rosen Neal , Knabbe Cornelius , Bates Susan , Bronzert Diane , Huff Karen , Kasid Attan 
Published: December 2004

AbstractWe describe studies on human breast cancer in which it is shown that specific growth factors (IGF‐I, TGFα, PDGF) are secreted by human breast cancer cells and likely to be involved in tumor growth and progression. These activities are regulated by estradiol in hormone‐dependent breast cancer and secreted constitutively by hormone‐independent cells. These growth factor activities can induce the growth of hormone‐dependent cells in vivo in athymic nude mice. Hormone‐dependent breast cancer cells also secrete TGFβ, a growth‐inhibitory substance, when treated with antiestrogens. TGFβ functions as a negative autocrine growth regulator and is responsible for some of the growth‐inhibitory effects of antiestrogens.
Cloning and characterization of the human PIM‐1 gene: A putative oncogene related to the protein kinases
Authors: Meeker Timothy C. , Nagarajan Lalitha , ar‐Rushdi Abbas , Croce Carlo M. 
Published: December 2004

AbstractThe mouse PIM‐l gene has been implicated in the evolution of retrovirus‐associated mouse lymphomas. We have initiated a study of the human PIM‐1 gene because of its potential importance as a human oncogene. We have isolated genomic and cDNA clones for this gene and characterized this locus in detail. The predicted PIM‐1 protein is 313 amino acids in length. It has homology to a number of the protein kinases but does not have a transmembrane region. The amino acid corresponding to tyrosine‐416 of pp60v‐src is a tyrosine (position 198), which is consistent with the hypothesis that PIM‐1 is a tyrosine kinase rather than a serine threonine kinase. The PIM‐1 gene was found to have six exons and five introns derived from 5 kb of genomic DNA. The site of transcription initiation was localized by S1 nuclease protection studies which indicated that the mature PIM‐1 mRNA was approximately 2.7 kb in length. The promotor of this gene had no TATA or CAAT box but did have multiple GC boxes (CCGCCC) that might bind the Spl protein. The PIM‐1 gene was expressed in myeloid and B lymphoid cell lines, but not in T lymphoid and nonhemopoietic lines. This initial characterization of PIM‐1 will allow us to define its role in normal and malignant hematolymphoid differentiation.
Analysis of pp60c‐src tyrosine kinase activity and phosphotyrosyl phosphatase activity in human colon carcinoma and normal human colon mucosal cells
Authors: DeSeau Virginia , Rosen Neal , Bolen Joseph B. 
Published: December 2004

AbstractWe have compared the level of phosphotyrosyl phosphatase activity in lysates from normal human colon mucosal cells and human colon carcinoma cells and analyzed the effect of incubating these cells with sodium orthovanadate, an inhibitor of phosphotyrosyl phosphatase activity, on the relative abundance of acid‐stable phosphotyrosine and on in vitro protein kinase activity of pp60c‐src. Additionally, we compared the effect of lysing these cells in buffer containing only nonionic detergents with RIPA buffer, which contains both sodium dodecyl sulfate and deoxycholate, on the in vitro kinase activity of pp60c‐src. Our results show that the level of detectable phosphotyrosyl phosphatase activity in lysates derived from normal colon cells and colon carcinoma cells is very similar. Additionally, the abundance of acid‐stable phosphotyrosine in these cells cultured in the absence or presence of vanadate is not significantly different. However, incubation of these cells with vanadate significantly stimulates the activity of pp60c‐src derived from the normal colon cells in immune‐complex kinase assays, while having no detectable effect on the activity of pp60c‐src from the colon tumor cells. The in vitro protein kinase activity of pp60c‐src derived from RIPA buffer lysates of colon carcinoma cells was found to be elevated five‐ to sevenfold when compared with pp60c‐src from these same cells lysed in buffer containing only Nonidet‐P 40 as a detergent. The type of lysis buffer did not effect the activity of pp60c‐src from normal colon mucosal cells. These results provide additional evidence that the activity of pp60c‐src may be regulated differently in colon carcinoma and normal colon mucosal cells.
Antibody to the phosphomannosyl receptor inhibits recycling of receptor in fibroblasts
Authors: Nolan Catherine M. , Creek Kim E. , Grubb Jeffrey H. , Sly William S. 
Published: December 2004

AbstractThe 215‐kd phosphomannosyl receptor is involved in the transport of newly synthesized acid hydrolases to lysosomes and also mediates the pinocytosis of lysosomal enzymes by fibroblasts in culture. Recycling of receptors to the sorting sites is an integral part of both these processes. In this report, we describe the inhibition in human fibroblasts of both functions of the phosphomannosyl receptor by a rabbit. Antiserum to the bovine liver receptor. This inhibition cannot be completely accounted for by inhibition of ligand‐receptor interaction. Rather the antibody appears to cross‐link receptors and cause a removal of receptors from the sorting sites (plasma membrane and Golgi apparatus) and their accumulation in a compartment from which they do not recycle. Removal of receptors from the recycling pool by antibody is irreversible, and return of receptors requires synthesis of new protein. Degradation of “trapped receptors” is enhanced (t½ = 7.5 hr), but much more gradual than their removal from the functional receptor pool (t:½ = 30min).