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Biomedical subjects

J M Bishop

Publications and source records attributed to J M Bishop.

At least 73 records · Page 4Linked to original sources
PubMed1993

Predisposition to neoplastic transformation caused by gene replacement of H-ras1.

Homologous recombination was used to introduce a nominally transforming mutation into an endogenous H-ras1 gene in Rat1 fibroblasts. Although both the mutant and the remaining normal allele were expressed equally, the heterozygous cells were not neoplastically transformed. Instead, spontaneously transformed cells arose from the heterozygotes at a low frequency, and the majority of these cells had amplified the mutant allele. Thus, the activated H-ras1 allele was not by itself dominant over the normal allele but predisposed cells to transformation by independent events, such as amplification of the mutant allele.

Alleles↗
PubMed1993

The human gene AHNAK encodes a large phosphoprotein located primarily in the nucleus.

AHNAK is a newly identified human gene notable for the exceptional size (c.a. 700 kD) and structure of its product, and for the repression of its expression in human neuroblastoma cells. Here we report the identification and partial characterization of the protein encoded by AHNAK. The protein is located principally (but not exclusively) in the nucleus and is phosphorylated on both serine and threonine. The abundance of the protein increases appreciably when cells withdraw from the division cycle, in response to either withdrawal of serum (fibroblasts) or differentiation (neuroblastoma cells). By contrast, the amount of phosphorylation appears to diminish in those settings. The considerable abundance and conjectured fibrous structure of AHNAK protein suggest a role in cytoarchitecture, but no function can yet be discerned.

Amino Acid Sequence↗
PubMed1993

Domains of E1A that bind p105Rb, p130, and p300 are required to block nerve growth factor-induced neurite growth in PC12 cells.

Nerve growth factor (NGF) causes PC12 cells to cease division and undergo sympathetic neuron-like differentiation, including neurite outgrowth. We have tested whether differentiation and division share overlapping control mechanisms in these cells. To do this, we have perturbed the activity of proteins known to participate in cell-cycle regulation by introducing the E1A oncogene or its mutant forms via microinjection into PC12 cells. The E1A protein binds to several putative cell cycle control proteins, including p105Rb (the product of the retinoblastoma susceptibility gene), as well as others of unknown function such as p130, p107, and p300. Similar to previous results, we find that wild-type E1A abrogates NGF-induced neurite extension. However, NGF does cause neurite outgrowth in the presence of E1A mutants known to have greatly reduced binding to either p105Rb and p130 or p300. Our experiments suggest that p105Rb, p130, and p300 may participate either in E1A-mediated inhibition of differentiation or in the NGF signal transduction pathway. We also report here that NGF affects phosphorylation of p105Rb, suggesting that Rb mediates at least some of NGF's effects. Our results raise the possibility that putative cell-cycle control proteins may participate not only in NGF-induced cessation of division but also in differentiation.

Adenovirus E1A Proteins↗
PubMed1993

Torso, a receptor tyrosine kinase required for embryonic pattern formation, shares substrates with the sevenless and EGF-R pathways in Drosophila.

The maternally expressed Drosophila gene torso (tor) is a receptor tyrosine kinase that, when activated, initiates a signal transduction cascade that is responsible for the proper differentiation of the terminal, nonsegmented regions of the embryo. l(1)pole hole, the Drosophila raf-1 serine-threonine kinase homolog, and corkscrew, a tyrosine phosphatase, have been shown previously to function in this signal transduction pathway. We have identified other products in this pathway by carrying out a mutagenesis screen for dominant suppressors of a tor gain-of-function allele. More than 40 mutations, some of which fall into seven complementation groups, have been characterized genetically. Two of these correspond to mutations in ras-1 and Son of sevenless (Sos), which also function in the sevenless and EGF receptor (Der) tyrosine kinase pathways. The phenotypes of several other Su(tor) mutations suggest that they also function in other receptor tyrosine kinase-activated pathways at different times during Drosophila development.

Alleles↗
PubMed1993

Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase.

We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid. This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade. Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor. This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.

3T3 Cells↗
PubMed1992

Directed establishment of rat brain cell lines with the phenotypic characteristics of type 1 astrocytes.

Interest in obtaining cell lines for use in studies on the development and biochemistry of the central nervous system has motivated efforts to establish cells from primary brain cultures by the use of oncogene-transfer techniques. In previous reports, cell lines derived from astrocytes in this way have had immature or abnormal phenotypes. We have explored the possibility of specifically "targeting" expression of exogenous oncogenes to differentiated astrocytes by using the promoter of the gene encoding glial fibrillary acidic protein, which is expressed almost exclusively in such cells. We report here that cell lines displaying the phenotypic characteristics of type 1 astrocytes can be established reproducibly in this manner. Given the heterogeneity of primary cultures, the availability of clonal cell lines displaying characteristics of type 1 astrocytes should greatly facilitate our understanding of the biology of these cells.

Animals↗
PubMed1992

A human gene (AHNAK) encoding an unusually large protein with a 1.2-microns polyionic rod structure.

We report here the identification and partial characterization of a human gene (designated AHNAK) that encodes an unusually large protein (approximately 700 kDa). AHNAK is expressed by means of a 17.5-kilobase mRNA in diverse cellular lineages but is typically repressed in cell lines derived from human neuroblastomas and in several other types of tumors. Unique-sequence domains at the two ends of the protein flank a large internal domain (approximately 4300 amino acids) composed of highly conserved repeated elements, most of which are 128 amino acids in length. The repeated elements in turn display a redundant motif, marked by the recurrence of proline at every seventh residue. Within these sequences, hydrophobic and hydrophilic residues alternate in a manner that is incompatible with a helical coiled-coil structure. Instead, we propose a structure resembling a beta-strand but with a periodicity of 2.33. The structure would engender a polyionic rod approximately 1.2 microns long. Preliminary evidence indicates that the protein resides predominantly within the nucleus, but no function has yet been discerned.

Adrenal Gland Neoplasms↗
PubMed1992

Specific association of the proto-oncogene product pp60c-src with an intracellular organelle, the PC12 synaptic vesicle.

The protein product of the proto-oncogene c-src is a membrane-associated tyrosine kinase of unknown function. Identification of pp60c-src target membranes may elucidate the function of the c-src protein. The available evidence indicates that pp60c-src associates with distinct membranes within single cell types and has different distributions in different cell types. Our experiments demonstrate targeting of pp60c-src to an isolatable and biochemically identified membrane fraction in the neuroendocrine cell line PC12. The c-src protein was found to be specifically associated with synaptic vesicles since: (a) the pp60c-src immunofluorescent pattern overlapped with a synaptic vesicle marker, synaptophysin; (b) a significant proportion (44%) of the pp60c-src from PC12 but not fibroblast postnuclear supernatants was recovered in a small vesicle fraction; (c) an anti-synaptophysin cytoplasmic domain antibody immunodepleted all of the pp60c-src vesicles in this fraction, and (d) pp60c-src copurified during a 100-fold purification of PC12 synaptic vesicles. These results suggest a role for the c-src protein in the regulation of synaptic vesicle function.

Animals↗
PubMed1992

Human protein-tyrosine kinase gene HCK: expression and structural analysis of the promoter region.

The vertebrate gene HCK encodes a protein-tyrosine kinase that is closely related to the product of the proto-oncogene SRC. HCK is expressed principally in monocytic and granulocytic hematopoietic cells, in coordination with differentiation of these cells. Here we report an initial description of the mechanisms by which expression of human HCK is controlled. Induction of the gene during differentiation was manifested by an increase in the steady-state levels of HCK RNA and protein product. The accumulation of RNA apparently resulted from modulation of transcription itself, since no change occurred in the stability of the transcripts. Transcription initiated at multiple sites, clustered c. 145 nucleotides upstream of the first intron of HCK. The sequence of 660 bp upstream of the major initiation site was determined, revealing candidate binding sites for Sp1 and AP-2 transcription factors, but neither TATA nor CAAT elements. Comparison to the same region of the mouse hck locus showed five small regions of similarity, only two of which were topographically analogous between the two sequences. It appears that expression of HCK is regulated primarily through control of transcription, but the mechanisms by which tissue-specific expression and increase of transcription during differentiation are achieved remain to be explored.

Base Sequence↗
PubMed1991

Premature termination of transcription from the P1 promoter of the mouse c-myc gene.

Modulation of transcriptional elongation within the c-myc gene is thought to play a major role in determining levels of c-myc mRNA in both normal and tumor cells. A discrete site of blockage to transcriptional elongation has previously been localized at the 3' end of exon 1 of the mouse and human c-myc genes. We here identify an additional site of transcriptional attenuation that is located between the P1 and P2 promoters of the c-myc gene and that mediates premature termination of transcripts initiating from the P1 promoter. A 95-nucleotide DNA fragment derived from this region prematurely terminated transcription when placed downstream from the promoter of the H-2Kbm1 gene and assayed by expression in Xenopus oocytes. We also show that the previously identified attenuation signal in exon 1 of the mouse c-myc gene can mediate premature termination of P1-initiated transcripts. Premature termination of P1-initiated transcripts presumably increases transcription from the downstream P2 promoter; aberrant regulation of this termination may explain the increased use of the P1 promoter that is characteristic of certain tumors in which myc is overexpressed.

Animals↗
PubMed1991

Dominant male sterility in mice caused by insertion of a transgene.

While examining a series of transgenic mouse lines carrying the HCK protooncogene, we encountered one line in which males hemizygous for the transgene were sterile. The sterile males mated normally but failed to impregnate females. Light and electron microscopy revealed that spermatogenesis proceeds normally until nuclear condensation, which occurs but gives rise to a variety of abnormally shaped nuclei. Expression of the transgene was not detectable. Thus, the insertion itself probably caused the abnormal phenotype by disrupting a gene (or genes) important in spermatogenesis. The mutation is genetically dominant, causing an abnormal phenotype even though the sterile mice carry an ostensibly normal counterpart of the disrupted locus. The mutant phenotype is completely penetrant only in some genetic backgrounds, suggesting a modifying influence from a second locus. Junctions between the inserted transgene and adjoining cellular DNA were cloned, allowing us to confirm the heterozygous nature of the genetic disruption and to detect and associated deletion. We have designated the mutation Lvs (lacking vigorous sperm) and presume that it may define a previously undescribed locus important in spermatogenesis.

Animals↗
PubMed1991

Identification of platelet membrane proteins that interact with amino-terminal peptides of pp60c-src.

Platelets contain exceptionally high levels of pp60c-src and, thus, provide a convenient system for investigating the physiological function of this protein-tyrosine kinase. We have employed chemical cross-linking of myristylated amino-terminal peptides of pp60c-src to platelet membranes in order to identify platelet membrane components that interact with pp60c-src to regulate or mediate its activity. We detected specific binding of radioiodinated peptides to platelet membrane proteins of 32, 50, 92, and 105 kDa. The 32-kDa protein may be related to the putative src receptor component recently identified in fibroblast membranes. The most reactive platelet protein, however, is the 50-kDa protein, which is either absent or nonreactive in fibroblast membranes. Binding of src peptides to this protein was saturable, and we estimate the presence of approximately 1 x 10(6) of the 50-kDa binding sites per platelet. The specificity of the peptide binding to the 50- and 32-kDa platelet proteins was analyzed by competition with different peptides. The binding sites displayed an absolute requirement for an N-myristoyl moiety and a strong preference for pp60c-src amino-terminal sequences. The identification of these src-interacting proteins may help to decipher the biochemical pathways in which platelet pp60c-src is involved.

3T3 Cells↗
PubMed1991

Isolation of Legionella maceachernii from an immunocompromised patient with severe underlying lung disease.

OBJECTIVE: To present the first case in Australia in which Legionella maceachernii has been documented as a cause of infection. CLINICAL FEATURES: A 71-year-old retired carpenter with underlying autoimmune disease and pulmonary fibrosis was admitted to hospital with increasing shortness of breath and fever, and pneumonia was diagnosed. INTERVENTION AND OUTCOME: He was treated with ampicillin then gentamicin and flucloxacillin and later imipenem but his condition deteriorated with progression of the pneumonia and abscess formation and he died 25 days after admission. Subsequently, L. maceachernii was isolated from three sputum samples collected before the patient died. CONCLUSION: There is a need for clinical awareness of the value of sputum culture in the diagnosis of infection due to Legionella spp. other than L. pneumophila.

Aged↗
PubMed1991

Nerve growth factor rapidly stimulates tyrosine phosphorylation of phospholipase C-gamma 1 by a kinase activity associated with the product of the trk protooncogene.

Nerve growth factor (NGF) promotes the survival and differentiation of specific populations of neurons. The molecular mechanisms by which cells respond to NGF are poorly understood, but two clues have emerged recently. First, NGF rapidly stimulates tyrosine phosphorylation of several unidentified proteins in the NGF-responsive pheochromocytoma cell line PC12 [Maher, P. (1988) Proc. Natl. Acad. Sci. USA 85, 6788-6791]. Second, the protein-tyrosine kinase encoded by the protooncogene trk (p140trk), a member of the receptor class of tyrosine kinases, becomes activated and phosphorylated on tyrosine after NGF treatment of PC12 cells [Kaplan, D. R., Martin-Zanca, D. & Parada, L. F. (1991) Nature (London) 350, 158-160]. We now report that NGF rapidly induces tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), and we present evidence that the responsible tyrosine kinase is either p140trk or a closely associated protein. Treatment of responsive cells with NGF elicited phosphorylation of PLC-gamma 1 on tyrosine and serine. PLC-gamma 1 immunoprecipitated from NGF-stimulated cells was phosphorylated in vitro by coprecipitating protein kinase activity, and the phosphorylations occurred principally on tyrosine. The responsible kinase could be depleted from cellular lysates by antibodies specific for p140trk. This procedure also depleted a 140-kDa protein that normally coprecipitated with PLC-gamma 1 and became phosphorylated on tyrosine in vivo in response to NGF. Analysis of tryptic peptides from PLC-gamma 1 indicated that the residues phosphorylated in vitro by p140trk-associated kinase activity were largely congruent with those phosphorylated in vivo after NGF treatment. Our findings identify PLC-gamma 1 as a likely substrate for the trk-encoded tyrosine kinase, and they provide a link between NGF-dependent activation of p140trk and the stimulation of intracellular second messenger pathways.

Adrenal Gland Neoplasms↗
PubMed1991

The MYC protein activates transcription of the alpha-prothymosin gene.

The proto-oncogene MYC encodes a nuclear protein whose biochemical and physiological functions remain uncertain. We used an estrogen-regulated version of the MYC protein to explore these functions. Activation of MYC in quiescent rat and mouse fibroblasts elicited re-entry into and progression through the cell cycle, bypassing early events that would follow stimulation of the cells with serum. Activation of MYC led to a rapid increase in transcription of the alpha-prothymosin gene, even in the absence of protein synthesis. We conclude that the product of MYC acts directly on transcription, in accord with inferences based on the structure of the MYC protein. The function of alpha-prothymosin is not known, but our results suggest that the protein may play a role in the proliferation of mammalian cells.

Actins↗
PubMed1991

Expression during Drosophila development of DER, a gene related to erbB-1 and neu: correlations with mutant phenotypes.

We used in situ hybridization to study expression of the DER gene during Drosophila development. DER encodes a transmembrane cell-surface receptor with a cytoplasmic protein-tyrosine kinase domain, and resembles the vertebrate genes that encode the EGF receptor and the neu protein. We examined most stages of development in the Drosophila life cycle and found a substantial correlation between DER expression and the phenotypes associated with a variety of mutant alleles. Of particular note were high levels of expression in the primordia of the mouth parts, which are the embryonic tissues most sensitive to reductions in DER activity; discrete expression in a subset of neural cells essential for construction of the axonal scaffold, a structure that is deformed in mutant embryos; uneven expression in the eye disc, the development of which is abnormal in both mild hypomorphs and hypermorphs; and expression in the follicular epithelial cells of the ovary, which are responsible for producing the eggshell of developing oocytes and do so aberrantly in the mildest hypomorphs. However, DER transcripts were also detected in a subset of tissues that have not been reported to be abnormal in mutant organisms. Our findings agree with and extend recently reported results for the DER protein, indicating that DER is regulated primarily at the level of transcription, in contrast to previous suggestions. We conclude that the phenotypes displayed by recessive mutants can be attributed to loss of DER function in the affected tissues.

Animals↗