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J Backer

Publications and source records attributed to J Backer.

12 recordsLinked to original sources
PubMed1994

Refolding and characterization of human recombinant heparin-binding neurite-promoting factor.

Heparin-binding neurite-promoting factor (HBNF) is a highly basic, cysteine-rich 136-residue protein, and a member of a new class of heparin-binding proteins. It exhibits a neurite-outgrowth promoting activity and its expression is both temporally and spacially regulated during fetal and postnatal development. A high interspecies sequence conservation suggests important, presently unknown, biological functions. HBNF is structurally and most likely functionally related to the product of a developmentally regulated gene, MK (midkine). To elucidate biological roles of these proteins, recombinant forms of the proteins were produced. Expression of human recombinant HBNF and MK in Escherichia coli lead to the formation of insoluble aggregated protein that accounted for about 25% of the total cellular protein. Homogeneous, monomeric forms of each protein were recovered from inclusion bodies by reduction with dithiothreitol and solubilization in 8 M urea. Refolding of the reduced and denatured protein occurred upon dialysis at pH 7.4. Human recombinant (hr) HBNF and hrMK prepared in this manner were further purified by heparin affinity chromatography. Chromatographic evidence demonstrates that refolding and concomitant disulfide bond formation in hrHBNF proceeds in high yield with minimal formation of stable nonnative disulfides. Studies on the redox status of the 10 cysteine residues of bovine brain HBNF and the refolded recombinant protein indicate that all cysteines are engaged in disulfide bond formation. The disulfide arrangements for the recombinant protein were found to be identical to those in the native protein isolated from bovine brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Two steps of insulin receptor internalization depend on different domains of the beta-subunit.

The internalization of signaling receptors such as the insulin receptor is a complex, multi-step process. The aim of the present work was to determine the various steps in internalization of the insulin receptor and to establish which receptor domains are implicated in each of these by the use of receptors possessing in vitro mutations. We find that kinase activation and autophosphorylation of all three regulatory tyrosines 1146, 1150, and 1151, but not tyrosines 1316 and 1322 in the COOH-terminal domain, are required for the ligand-specific stage of the internalization process; i.e., the surface redistribution of the receptor from microvilli where initial binding occurs to the nonvillous domain of the cell. Early intracellular steps in insulin signal transduction involving the activation of phosphatidylinositol 3'-kinase are not required for this redistribution. The second step of internalization consists in the anchoring of the receptors in clathrin-coated pits. In contrast to the first ligand specific step, this step is common to many receptors including those for transport proteins and occurs in the absence of kinase activation and receptor autophosphorylation, but requires a juxta-membrane cytoplasmic segment of the beta-subunit of the receptor including a NPXY sequence. Thus, there are two independent mechanisms controlling insulin receptor internalization which depend on different domains of the beta-subunit.

Amino Acid Sequence↗
PubMed1992

Anti-viral activity of human recombinant heparin-binding proteins HBNF and MK.

Herpes simplex viruses bind to cell surface heparan sulfate proteoglycans, as a first step of viral infection. We report here that two recombinant heparin-binding proteins HBNF and MK inhibit infectivity of human herpes simplex viruses types 1 and 2 and human cytomegalovirus. Carboxymethylated HBNF and MK, which retain affinity for heparin-Sepharose, do not exhibit anti-viral activities. Arguments are presented that anti-viral effects of HBNF and MK are due to the competition for the specific binding to the cell surface heparan sulfate proteoglycans.

Alkylation↗
PubMed1991

Purification and partial sequence analysis of pp185, the major cellular substrate of the insulin receptor tyrosine kinase.

Insulin stimulates the tyrosine phosphorylation of a 185-kDa putative cytosolic substrate protein (pp185) in diverse cell types. After intravenous insulin infusion into the live intact rat, pp185 and the 95-kDa insulin receptor beta-subunit were the major proteins that tyrosine phosphorylated in liver, skeletal muscle, and adipose tissue. Both proteins were maximally phosphorylated within 30 s, and both increased in phosphotyrosine content in parallel with increasing insulin dose. However, pp185 tyrosine phosphorylation was transient, with almost complete dephosphorylation within 2-3 min despite continued insulin stimulation. To identify pp185 directly, we purified pp185 from insulin-stimulated rat liver, using a denaturation-based extraction procedure that blocks endogenous protein phosphatases and thus allows a high yield, single step isolation of phosphotyrosyl proteins by anti-phosphotyrosine antibody immunoaffinity absorption. From 50 rat livers, 50-100 pmol of pp185 was isolated. Edman degradation of seven internal tryptic peptide fragments of pp185 yielded novel amino acid sequences, indicating that pp185 is a new protein. Antipeptide antibodies were raised which specifically recognize a single, 185-kDa insulin-stimulated phosphotyrosyl protein in liver, skeletal muscle, adipose tissue, and several cultured cell lines. These results indicate that pp185 is expressed in a variety of insulin-responsive tissues, is the major protein rapidly tyrosine phosphorylated under physiological conditions in the intact animal, and also provide a route for cloning the pp185 gene and elucidating the function of pp185 in insulin signal transduction.

Amino Acid Sequence↗
PubMed1991

Mutations in the juxtamembrane region of the insulin receptor impair activation of phosphatidylinositol 3-kinase by insulin.

CHO/IRF960/T962 cells express a mutant human insulin receptor in which Tyr960 and Ser962 in the juxtamembrane region of the receptor's beta-subunit are replaced by Phe and Thr, respectively. The mutant insulin receptor undergoes autophosphorylation normally in response to insulin; however, insulin fails to stimulate thymidine incorporation into DNA, glycogen synthesis, and tyrosyl phosphorylation of an endogenous substrate pp185 in these cells. Another putative substrate of the insulin receptor tyrosine kinase is phosphatidylinositol 3-kinase (Ptdlns 3-kinase). We have previously shown that Ptdlns 3-kinase activity in Chinese hamster ovary cells expressing the wild-type human insulin receptor (CHO/IR) increases in both antiphosphotyrosine [anti-Tyr(P)] immunoprecipitates and intact cells in response to insulin. In the present study a new technique (detection of the 85-kDa subunit of Ptdlns 3-kinase using [32P]phosphorylated polyoma virus middle T-antigen as probe) is used to monitor the Ptdlns 3-kinase protein. The 85-kDa subunit of Ptdlns 3-kinase is precipitated by anti-Tyr(P) antibodies from insulin-stimulated CHO/IR cells, but markedly less protein is precipitated from CHO/IRF960/T962 cells. The amount of Ptdlns 3-kinase activity in the immunoprecipitates was also reduced in the CHO/IRF960/T962 cells compared to CHO/IR cells. In intact CHO/IRF960/T962 cells, insulin failed to stimulate phosphate incorporation into one of the products of activated Ptdlns 3-kinase, phosphatidylinositol-3,4-bisphosphate [Ptdlns(3,4)P2], whereas it caused a 12-fold increase in CHO/IR cells. In contrast, phosphate incorporation into another product, phosphatidylinositol trisphosphate [PtdlnsP3], was only partially depressed in the CHO/IRF960/T962 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Temperature sensitive pet mutants in yeast Saccharomyces cerevisiae that lose mitochondrial RNA.

This is a description of a new class of temperature sensitive pet mutants in Saccharomyces cereviase that lose all or part of their mitochondrial RNA at the restrictive temperature. These mutants fall into 8 different complementation groups, mna1 to mna8, and 2 different classes based on their phenotype. Class I mutations, mna1-1 through mna5-1, cause complete or partial loss of mitochondrial RNA at the restrictive temperature. The mutation, mna1-1, is especially interesting since it causes a loss of both mitochondrial DNA and RNA when the mutant is grown on a fermentable carbon source at the restrictive temperature. However, when this mutant is grown at the permissive temperature on a non-fermentable carbon source then shifted to the restrictive temperature, only the mitochondrial RNA is lost. This indicates that the primary cause for the pet phenotype is due to the loss of mitochondrial RNA and not DNA. Class II mutations, mna6-1 through mna8-1, cause complete loss of the 14S rRNA after growth at the restrictive temperature in a fermentable carbon source. This loss appears to be specific for the 14S rRNA, since all other transcripts probed by Northern analysis are normal.

DNA, Mitochondrial↗
PubMed1985

Repair properties in yeast mitochondrial DNA mutators.

After ethylmethanesulfonate mutagenesis of the strain Saccharomyces cerevisiae D273-10B, out of 100,000 survivors, 1,000 were selected for their high production of petite mutants at 36 degrees C. Among these 1,000 mutators, 5 also showed an increased frequency of spontaneous point mutations measured at 25 degrees C. Further analysis revealed that in all mutators, except 2, petite accumulation proceeded at 25 degrees C as well as 36 degrees C. In these 2 mutants, the production of petite mutants was much higher at 36 degrees C than at 35 degrees C. In one of them, however, the mutator and the thermosensitive petite phenotypes were due to mutations in two unlinked nuclear genes. In the other mutants, both traits were the result of a mutation in a single nuclear gene. The mutators fell into three complementation groups (tpm1, tpm2, mup1). No complementation was observed between tpm1 mutants and the gam4 mutant previously described by Foury and Goffeau (1979). From the latter and the present works, only four complementation groups (gam1, gam2, gam4 or tpm1, mup1) have been identified and it is likely that the number of genes controlling specifically the spontaneous mutability of the mtDNA is low. The mutators exhibited a variety of responses to damaging agents such as UV light and ethidium bromide; especially in a representative mutant from the complementation group tpm1, the induction of rho- mutants was sensitive to UV light and resistant to ethidium bromide.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Repair↗
PubMed1983

Molecular mechanisms of tumor promotion and multistage carcinogenesis.

Carcinogenesis is a multistep process resulting from a complex interaction between multiple factors, both environmental and exogenous. In contract to initiating agents that act by damaging cellular DNA, the primary targets of the phorbol ester tumor promoters are membrane-associated receptors. We have proposed a stereochemical model to explain the interaction of these amphiphilic molecules, and of teleocidin and aplysiatoxin, with this receptor system. The model is consistent with evidence that a complex between protein kinase C and phospholipid is the actual receptor for these compounds. Recent data we have obtained with a compound present in tung oil, 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate (HHPA), and twelve of its congener's (provided by Y. Ito et al.) are also consistent with our stereochemical model. We have studied phorbol ester receptors in a wide variety of tissue culture cell types. Our data, together with other findings, provide evidence for considerable receptor heterogeneity and this may relate to the pleiotropic effects of these compounds. We have found a case of "masked" receptors in a rat liver cell line and shown that it is due to a cell-associated esterase. Normal human melanocyte cultures contain phorbol ester receptors and this is of particular interest since these cells actually require these or related compounds for optimal growth (in collaboration with M. Eisinger). The receptor studies provide clues to how tumor promoters can, via inductive mechanisms, produce alterations in the structure and function of cell membranes. It is not known, however, how in the multistep carcinogenic process promoters enhance the eventual outgrowth of permanently altered tumor cells. We have found that TPA and teleocidin produce a marked enhancement of transformation of C3H 10T1/2 cells induced by transfection with h-ras human bladder cancer oncogene. These and other results are discussed in terms of the role of alterations in cellular oncogenes and transcriptional enhancer sequences during multistage carcinogenesis.

Adenosine Triphosphate↗
PubMedDate not supplied

A proposed framework for teaching and evaluating critical thinking in nursing.

Critical thinking is now an explicit National League for Nursing outcome requirement for nursing education programs, and all nursing faculty currently are expected to teach and evaluate critical thinking. However, most of the related literature is theoretical and not on the practical level that might be directly usable by faculty. This article presents a beginning framework developed by a faculty task force at the Indiana University School of Nursing for teaching and evaluating critical thinking. The conceptual definition of critical thinking underlying the framework reflects the efforts of several hundred experts from various disciplines, is maximally inclusive, and lends itself well to operationalization in the nursing context. The task force made two assumptions for this project: first, that the principles of rational thinking are essentially the same at every educational level, although the expertise expected of students at different educational levels certainly differs; and, second, that at this time a focus on operational as opposed to theoretical definitions for the six identified components of critical thinking (interpretation, analysis, evaluation, inference, explanation, and self-regulation) would be particularly useful for faculty and student understanding.

Curriculum↗