Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

ABL-MYC is a recombinant retrovirus that constitutively expresses the v-abl and c-myc oncogenes. When used to infect immunized mice this virus rapidly and efficiently induces plasmacytomas of which an unusually high percentage secrete antigen (Ag)-specific monoclonal antibodies. These findings suggested that ABL-MYC targets Ag-stimulated B cells for transformation and that infection of lymphoid cells in vitro might be a useful, alternative method for generating monoclonal, Ag-specific plasmacytomas (ASPCTs). Therefore, we used helper virus-free ABL-MYC to infect suspensions of cells from spleens and other lymphoid organs from mice that had been immunized with a variety of Ags and transplanted them into naive mice. The results show that ABL-MYC preferentially transforms splenocytes that are Ag-reactive. They also demonstrate that ASPCTs can be produced by in vitro infection of cell suspensions from the spleen, lymph nodes and Peyer's patches of mice that had been immunized intraperitoneally with sheep red blood cells, Escherichia coli core RNA polymerase or Epstein-Barr virus gp340 protein or immunized orally with live Giardia lamblia parasites. The ASPCTs usually consisted of one to three colnes, secreted antibodies that were quantitatively and qualitatively similar to those obtained from hybridomas, and could continue to secrete Ag-reactive antibody over eight transplant generations.
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PMID:The ABL-MYC retrovirus generates antigen-specific plasmacytomas by in vitro infection of activated B lymphocytes from spleen and other murine lymphoid organs. 889 Aug 96

This report describes a precise molecular analysis of a rare case of Philadelphia chromosome (Ph) positive acute myeloid leukemia (AML) (FAB classification M2). Phenotypic markers were positive for cells of the myeloid lineage, but negative for B cell and T cell lineage. The leukemic cells carried a Philadelphia chromosome. Major breakpoint cluster region (M-BCR) rearrangement was detected by the Southern blot analysis. Reverse transcriptase polymerase chain reaction analysis revealed the presence of b3a2 BCR/ABL mRNA transcripts. The patient achieved complete remission by conventional remission induction therapy for acute myeloid leukemia. M-BCR rearrangement could not be detected during complete remission. After hematological remission of an 8-month duration, the patient relapsed and died of respiratory distress due to pneumonia. Our case indicate Ph-positive AML with M-BCR rearrangement actually exists. Ph-positive AML carries either M-BCR rearrangement expressing the P210 BCR-ABL or minor breakpoint cluster region (m-BCR) rearrangement producing the P190 BCR-ABL. Therefore, additional other factor (s) apart from the Ph chromosome must be responsible for the acute malignant transformation.
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PMID:Molecular analysis of a case of Philadelphia chromosome-positive acute myeloid leukemia. 906 90

For the great majority of patients with chronic myeloid leukaemia (CML), the Philadelphia (Ph) chromosome is a specific marker of the malignant clone. The standard method to assess the quality of remission in these patients is cytogenetic analysis of bone marrow derived metaphases. However, the molecular definition of the t(9;22) and its consequences has enabled other tests to be developed that can specifically detect CML cells. Fluorescence in situ hybridization (FISH) analyses chromosomes to detect either the juxtaposition of BCR and ABL sequences or the disruption of these genes; Southern blotting analyses genomic DNA to determine whether the BCR gene is rearranged; reverse-transcriptase polymerase chain reaction (RT-PCR) analyses RNA to determine the presence or absence of BCR-ABL transcripts; Western blotting analyses cell lysates to determine the presence or absence of BCR-ABL protein. Each of these techniques has particular advantages and pitfalls but in general they may be used to replace or at least to reduce the frequency of conventional cytogenetic analysis. Partly because of economic factors and the lack of standardization or effective quality control, these assays are still largely restricted to research laboratories. The sensitivity with which residual leukaemia can be detected suggests that FISH, Southern blotting and Western blotting are likely to be most useful in assessing patient response to interferon-alpha or other forms of treatment that typically induce partial remission. RT-PCR is by far the most sensitive assay and is probably most appropriate for monitoring patients who are in complete remission.
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PMID:Assessing residual leukaemia. 937 71

Philadelphia (Ph) chromosome-positive leukemias, with the bcr-abl gene translocation, have a dismal prognosis. The identification of Ph-positive patients is vitally important because only aggressive therapeutic approaches, such as allogeneic bone marrow transplantation, may result in long-term disease-free survival. Routine diagnostic methods, such as Southern blot analysis and cytogenetics, may lead to false-negative results. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis is considered the most sensitive tool for the detection of the bcr-abl translocation, and it is widely used alone or in combination with karyotyping or Southern blot analysis to identify Ph-positive cases. In this study, we used fluorescence in situ hybridization (FISH) with BCR and ABL double-color probes for detecting Ph-positive leukemias. The FISH results were compared with the results of cytogenetic and RT-PCR analyses in 75 patients with leukemia or other myeloproliferative syndromes (chronic myeloid leukemia, 30; acute lymphoblastic leukemia, 24; acute myelogenous leukemia, 6; essential (hemorrhagic) thrombocythemia, 12; chronic myelomonocytic leukemia, 2; and polycythemia vera, 1). FISH analysis proved to be simple, extremely reliable and sensitive; bcr-abl fusion detection was successful in the presence of all types of molecular junctions i.e., (b2a2, b3a2, and e1a2). Furthermore, a Ph-positive case that proved fusion negative by RT-PCR was identified as positive by FISH. The sensitivity of RT-PCR and FISH related to Ph-positive cases were 97% and 100%, respectively. Regarding specificity, in 4 (5%) of 75 patients, RT-PCR provided false-positive results. Cross-contamination was identified because a new specimen was harvested and reanalyzed when FISH, cytogenetics, and RT-PCR results were contradictory. We believe FISH is an optimal diagnostic method to detect bcr-abl translocation that can be used alone or to validate the results of RT-PCR analysis.
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PMID:A comparative analysis of FISH, RT-PCR, and cytogenetics for the diagnosis of bcr-abl-positive leukemias. 942 14

We previously demonstrated that the macrophage cell lines RAW 264.7 and WEHI-3 exhibit distinct patterns of gene expression in response to IFN-gamma. This difference is controlled at the transcriptional level and results from a specific inability of the less mature WEHI-3 cells to utilize either the IFN-stimulated response element or the gamma-activated sequence DNA regulatory element in response to stimulation with IFN-gamma, while other aspects of IFN-gamma gene induction remain intact. In the work described here, we examined the components of the IFN-gamma signal transduction pathway in RAW 264.7 and WEHI-3 cells to determine whether differences in pathway components or activity exist in WEHI-3 cells that could give rise to this difference in transcriptional response. Reverse transcriptase-PCR (RT-PCR) and flow cytometric analyses indicated that the levels of IFN-gamma receptor mRNA accumulation and protein expression are comparable for RAW 264.7 and WEHI-3 cells. RT-PCR and immunoblot analyses revealed that the principal components of this signaling pathway, including JAK1, JAK2, and STAT1, are present in both RAW 264.7 and WEHI-3 cells. However, analysis of STAT1 DNA-binding activity by electrophoretic mobility shift assay and of STAT1 phosphorylation by immunoblot revealed that this DNA-binding factor is active in RAW 264.7, but not in WEHI-3, cells after IFN-gamma stimulation. These results demonstrate that the components of the IFN-gamma signal transduction pathway are intact in WEHI-3 cells, but stimulation of these cells by IFN-gamma does not result in STAT1 activation.
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PMID:Analysis of the IFN-gamma-signaling pathway in macrophages at different stages of maturation. 957 37

Ribozymes have been shown to be potent inhibitors of gene expression and viral function. Effects of ribozyme-mediated repression to target gene in living cells are correlated with the amounts of expression and stabilities of ribozyme molecules. In our previous study, it was demonstrated that a minimized hammerhead ribozyme, minizyme, with high activity forms a dimeric structure with a common stem II. We constructed dimeric minizymes that could cleave the BCR-ABL chimeric (b2a2) mRNA which had been difficult target for conventional hammerhead ribozymes without damaging the normal ABL mRNA. In order to achieve high expression of these dimeric minizymes in vivo for the treatment of CML, we embedded the dimeric minizyme portion downstream of a tRNA(Val) promoter sequence which could be recognized by RNA polymerase III. We determined cleavage activities of tRNA-embedded dimeric minizymes and compared the activities between tRNA-embedded hammerhead ribozyme and tRNA-embedded dimeric minizymes. All tRNA-embedded dimeric minizymes tested were capable of cleavage the target substrate. The activity of the tRNA-embedded dimeric minizyme targeted at BCR-ABL mRNA was almost the same as that of the naked dimeric minizymes. Interestingly, the cleavage activity of tRNA-embedded dimeric minizymes was higher than that of tRNA-embedded conventional hammerhead ribozyme.
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PMID:Activities of tRNA-embedded dimeric minizymes. 958 22

The HSPDE4A gene spans 50 kb, consists of at least 17 exons and is orientated 5'-3', telomere to centromere. It is located at chromosome 19p13.2, being 350 kb proximal to the gene encoding TYK2 and 850 kb distal to the gene encoding the low-density lipoprotein receptor. Its structure is consistent with the production of active 'long' and 'short' isoenzymes as the result of alternative mRNA splicing at two splice junctions. Identified is the single alternatively spliced 5' exon encoding the unique N-terminal region of the long isoenzyme HSPDE4A4B (pde46). The upstream conserved regions, UCR1 and UCR2, which form characteristic domains of PDE4 long forms are each encoded by three exons. The PDE4A-subfamily-specific linker region LR1, which joins UCR1 and UCR2, is encoded by two exons, whereas LR2, which joins UCR2 to the catalytic unit, is encoded by a single exon. Identification of exons encoding an enzymically inactive product of this gene, HSPDE4A8A (2el), indicates that this is an authentic gene product. The 5' exon encoding the unique N-terminal region of the human homologue of the rodent isoform RNPDE4A1A (RD1) was located, and the splice junction used to produce this short PDE4A isoform shown to occur at a different position from that seen in both the rat PDE4B and PDE4D genes. Reverse transcriptase PCR analysis indicates that RD1 homologues are conserved across species, having a conserved membrane-targeting region and a hypervariable LR2 region. Human RD1 was expressed transiently in COS-7 cells and detected as an 83 kDa species primarily associated with the high-speed membrane fraction. Human RD1 exhibited a Km for cAMP of about 3 microM, an IC50 value for inhibition by the PDE4-selective inhibitor rolipram of about 0.3 microM and was considerably more thermostable than rat RD1. Human RD1 was generated as a mature 80 kDa species in an in vitro transcription-translation system and shown to be capable of binding to membranes. Knowledge of the gene structure and the associated sequence information should facilitate analysis of the involvement of PDE4A in hereditary disorders that may result from alterations in enzyme expression, activity, regulation and intracellular targeting and serve as a resource for determining authenticity of cloned PDE4A species.
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PMID:Identification and characterization of the human homologue of the short PDE4A cAMP-specific phosphodiesterase RD1 (PDE4A1) by analysis of the human HSPDE4A gene locus located at chromosome 19p13.2. 967 30

The FER locus in the mouse encodes two tyrosine kinases, p94fer and p51ferT. While p94fer accumulates in the cytoplasm and nucleus of most mammalian cells the expression of p51ferT is restricted to the nucleus of meiotic primary spermatocytes. The cellular function of the FER kinases is not understood, nor has a substrate for these enzymes been characterized. To identify putative substrates of p94fer and p51ferT, the two enzymes were used as 'baits' in the yeast two-hybrid screening system. cDNAs encoding the mouse TATA element modulatory factor (TMF) were repeatedly isolated in this assay. TMF was previously shown to bind the TATA element in RNA polymerase II promoters and impaired their functioning in a cell free transcription system. Both p94fer and p51ferT phosphorylated the TMF protein in in vitro and in vivo kinase assays. Sequential deletions showed that the carboxy-terminal region of TMF was essential for phosphorylation. In situ hybridization analysis revealed the preferential accumulation of TMF transcripts in meiotic spermatogenic and oogenic cells. p94fer and p51ferT may thus modulate the suppressive activity of TMF during cellular growth and in defined differentiation processes.
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PMID:Tyrosine phosphorylation of the TATA element modulatory factor by the FER nuclear tyrosine kinases. 974 51

A 40-year-old man had chronic myeloid leukemia (CML) and an apparently normal karyotype. Fluorescence in situ hybridization with a BCR/ABL1-S probe, which is formatted to display a BCR/ABL fusion signal on chromosome 22, gave a positive fusion signal on a chromosome 9. Therefore this patient has a BCR/ABL fusion gene on chromosome 9. The BCR/ABL1-D probe, formatted to display a fluorescent signal for both the reciprocal products of a 9/22 rearrangement, gave a positive fusion signal on the derivatives 9 and 22. These findings favor either a cryptic reciprocal exchange between BCR and ABL loci or the reversal of a Philadelphia translocation. An insertion of BCR next to ABL is ruled out. The reverse-transcriptase polymerase chain reaction provided molecular evidence that a typical CML chimeric product resulting from a fusion of BCR exon 2 with C-ABL exon II, a2b2, is present.
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PMID:A Philadelphia-negative chronic myeloid leukemia with a BCR/ABL fusion gene on chromosome 9. 980 34

Severe congenital neutropenia (SCN) or Kostmann's syndrome is characterized by a stop in differentiation of myeloid progenitor cells at the myelocytic or promyelocytic stage. The pathophysiology of SCN is still unclear. We previously showed that the tyrosine kinase JAK2 is phosphorylated and activated in neutrophils from patients with severe congential neutropenia. We investigated the role of tyrosine phosphatases in this disease. Expression of the SH2 domain-containing tyrosine phosphatases SHP-1 and SHP-2 was analyzed in myeloid cells from patients with SCN in comparison to healthy donors. We investigated tyrosine phosphatase expression in myeloid cells at the protein level by Western blot analysis using polyclonal antisera against SHP-1 and SHP-2. Whereas SHP-1 and SHP-2 were hardly detectable in neutrophils from healthy donors, neutrophils from patients with SCN revealed high amounts of these two proteins in Western blot analyses. Reverse transcriptase-polymerase chain reaction and Northern blot analyses demonstrated no dramatic differences of SHP-1 mRNA in neutrophils from congenital neutropenia patients as compared to healthy donors. SHP-2 mRNA was hardly detectable in the neutrophils from patients and in normal neutrophils. Increased expression of SHP protein correlated with elevated activity of both SHP-1 and SHP-2 in neutrophils of patients with SCN. Taken together, these data indicate differential regulation for SHP-1 and SHP-2 at the protein level in neutrophils from SCN patients in comparison to healthy donors. We suggest that overexpression of SHP-1 and SHP-2 protein in neutrophils and not in mononuclear cells from patients with SCN might be related to the disease, e.g., by defective dephosphorylation of proteins involved in intracellular signaling pathways.
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PMID:SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 are dramatically increased at the protein level in neutrophils from patients with severe congenital neutropenia (Kostmann's syndrome). 1037 93


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