Biomedical subjects

A Gazit

Publications and source records attributed to A Gazit.

At least 73 records · Page 4Linked to original sources

PubMed1995

Identification of sequences in the long terminal repeat of the lymphoproliferative disease virus required for efficient transcription.

We analyzed the long terminal repeat (LTR) of the lymphoproliferative disease virus of turkeys for sequences that influence its promoter activity by using the chloramphenicol acetyltransferase assay. A series of LTR deletion mutants and recombinants between LTR and simian virus 40 regulatory sequences were used for these studies. Through transfection experiments, we identified a negative regulatory element residing at the 5' end of the U3. The two imperfect direct repeats (DRs) located at nt - 170 to - 125 upstream of the RNA transcription site were identified as enhancer elements which could stimulate transcription of a heterologous promoter in an orientation independent manner. Specific interaction of nuclear factors with the DRs element was identified. The two DRs contain cArg motifs which are suggested to play a role in tissue specific expression of several cellular genes.

Alpharetrovirus↗

PubMed1995

Tyrosine kinase inhibition: an approach to drug development.

Protein tyrosine kinases (PTKs) regulate cell proliferation, cell differentiation, and signaling processes in the cells of the immune system. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases can lead to inflammatory responses and to diseases such as cancer, atherosclerosis, and psoriasis. Thus, inhibitors that block the activity of tyrosine kinases and the signaling pathways they activate may provide a useful basis for drug development. This article summarizes recent progress in the development of PTK inhibitors and demonstrates their potential use in the treatment of disease.

Animals↗

PubMed1995

Tyrphostin induces non-apoptotic programmed cell death in colon tumor cells.

The programmed cell death inducing effect of the EGF receptor tyrosine kinase inhibitor alpha-cyano-3,4-dihydroxycinnamthioamide (AG213) was investigated in vitro on HT-29 human colon tumor. AG213 at concentrations between 45 to 450 microM blocks the proliferation of HT-29 cells. Morphological findings suggest that the selective tyrosine kinase inhibitor AG213 induces Clarke III type (non-lysosomal vesiculate cytoplasmic) programmed cell death; unlike ATP analog non-selective tyrosine kinase inhibitors like Genistein which were found to induce apoptosis. Cycloheximide and Actinomycin-D reduced the effect of AG213 pointing to the fact that protein and RNA synthesis are also needed for this form of cell death. Acid phosphatase activity was found in the Golgi and in the newly formed intracytoplasmic vacuoles 3 hours after AG213 treatment which disappeared by 6 hours. The induction of Clarke III cell death by tyrosine kinase inhibitors may open a new modality to selective killing of tumor cells.

Acid Phosphatase↗

PubMed1995

Tyrphostins suppress the growth of psoriatic keratinocytes.

Tyrosine kinase inhibitors of the tyrphostin family which block EGF receptor kinase are reported to arrest the growth of psoriatic keratinocytes in vitro. Three tyrphostins with the potency ratio AG555 >> AG18 >> AG814 were found to arrest growth with no adverse cytotoxic effects. The potency ratio to inhibit keratinocyte proliferation follows their potency to inhibit EGF receptor kinase activity in vitro. These compounds represent novel leads for the therapy of psoriasis.

Benzylidene Compounds↗

PubMed1994

Biological activity and intracellular location of the Tat protein of equine infectious anemia virus.

The Tat protein of equine infectious anemia virus (EIAV) was synthesized in Escherichia coli using the inducible expression plasmid, pET16b, which contains a His.Tag leader, thus allowing for rapid and efficient enrichment of the histidine-tagged protein by metal affinity chromatography. Yields of up to 20 mg of Tat were obtained from 10(11) bacterial cells. The recombinant Tat protein was shown to potently trans-activate the EIAV long terminal repeat (LTR) following its introduction into canine cells by 'scrape loading'. The EIAV Tat protein was found to localize predominantly within the cytoplasm, in contrast to HIV-1 Tat. The availability of large amounts of purified functional EIAV Tat protein should greatly facilitate detailed structure-function analyses.

Amino Acid Sequence↗

PubMed1994

Selective platelet-derived growth factor receptor kinase blockers reverse sis-transformation.

A novel class of tyrosine kinase blockers represented by the tyrphostins AG1295 and AG1296 is described. These compounds inhibit selectively the platelet-derived growth factor (PDGF) receptor kinase and the PDGF-dependent DNA synthesis in Swiss 3T3 cells and in porcine aorta endothelial cells with 50% inhibitory concentrations below 5 and 1 microM, respectively. The PDGF receptor blockers have not effect on epidermal growth factor receptor autophosphorylation; weak effects on DNA synthesis stimulated by insulin, by epidermal growth factor, or by a combination of both; and over an order of magnitude weaker blocking effect on fibroblast growth factor-dependent DNA synthesis. AG1296 potently inhibits signaling of human PDGF alpha- and beta-receptors as well as of the related stem cell factor receptor (c-Kit) but has no effect on autophosphorylation of the vascular endothelial growth factor receptor KDR or on DNA synthesis induced by vascular endothelial growth factor in porcine aortic endothelial cells. Treatment by AG1296 reverses the transformed phenotype of sis-transfected NIH 3T3 cells but has no effect on src-transformed NIH 3T3 cells or on the activity of the kinase p60c-src(F527) immunoprecipitated from these cells. These potent and selective compounds represent leads for the development of novel agents to combat tumors driven by PDGF or to inhibit PDGF action in other diseases in which PDGF plays a key role, such as restenosis.

3T3 Cells↗

PubMed1994

Genome organization of a biologically active molecular clone of the lymphoproliferative disease virus of turkeys.

The lymphoproliferative disease retrovirus (LPDV) induces an acute, horizontally transmitted disease of turkeys that is often fatal. Although LPDV cannot be grown in cultured cells, it was possible to isolate molecular clones of biologically active integrated proviral genomes from spleens of infected turkeys. Based upon molecular hybridization and nucleotide sequence comparisons of its pol gene, LPDV was shown to represent a distinct group of avian retroviruses most closely related to avian sarcoma-leukemia viruses. Here we report the complete nucleotide sequence of the LPDV genome as well as amino acid sequence analysis of its gag gene products. The genetic organization of LPDV is characteristic of members of the oncovirus subfamily. Further sequence comparisons of the gag gene confirmed that LPDV is most closely related to Rous sarcoma virus (RSV). However, the gag, pro, and pol open reading frames (ORFs) were in different translational phases so that the expression of their mature gene products would require the double frame-shifting mechanism utilized by simian retroviruses, mouse mammary tumor virus, and human T-cell leukemia virus. In contrast, the RSV proteinase is synthesized as part of the gag precursor. The LPDV gag gene differs from that of RSV as well as from all other retroviruses in that it encodes a unique 31,000-Da (p31) protein, located between the MA and the CA coding sequences. FOur short ORFs of unknown function were present, Whether the putative products of these ORFs account for the acute nature of LPDV-induced disease remains to be determined.

Amino Acid Sequence↗

PubMed1994

Characterization of cDNAs species encoding the Tat protein of caprine arthritis encephalitis virus.

Two distinct species of caprine arthritis encephalitis virus (CAEV) tat cDNAs were isolated early after infection of a Himalayan tahr cell line. Sequence analyses predicted that one cDNA (pCEV/e1) represented a polycistronic transcript that encodes Tat and Rev as well as an N-terminally truncated transmembrane protein and a protein, designated X, whose function is unknown; whereas the other cDNA (pCEV/f1) encodes Tat and the env gene products. pCEV/e1 trans-activated a CAEV LTR-chloramphenicol acetyltransferase reporter gene in goat synovial membrane cells. This activity was shown to be encoded by the Tat open reading frame by analysis of a deletion mutant. Because the pCAEV/f1 insert was unstable in plasmid form, its Tat activity could not be convincingly demonstrated. The target sequences for Tat within the CAEV LTR were localized to the U3 region which, when placed in either orientation upstream of heterologous promoters, was able to confer responsiveness to Tat.

Amino Acid Sequence↗

PubMed1994

Trifluoroethanol stabilizes a helix-turn-helix motif in equine infectious-anemia-virus trans-activator protein.

The solution structure of the 75-amino-acid trans-activator (Tat) protein of the equine infectious-anemia virus in trifluoroethanol-containing solution was determined by two-dimensional and three-dimensional nuclear magnetic resonance spectroscopy, resulting in a total of 838 nuclear-Over-hauser-enhancement distance restraints, and restrained molecular-dynamics simulations. In contrast to the recently determined structure of this protein in trifluoroethanol-free pH 6.3 solution, the hydrophobic core and the adjacent basic RNA-binding region of the protein showed well-defined alpha-helical secondary structure in trifluoroethanol-containing solution. The helical regions comprise those parts of the molecule whose helix-forming tendencies were noted earlier in trifluoroethanol-free solution. Two helices (Gln38-Arg43 and Asp48-Ala64) are connected by a tight type-II turn centered at the strictly conserved Gly46 leading to a helix-turn-helix motif in the core and basic region of the protein. A third helix (Thr9-Asn13) is located in the less well defined N-terminal part of the protein. These observations may support the notion that the protein adopts a helical structure in the RNA-binding region on complex formation. Although the secondary-structure elements become better defined in trifluoroethanol-containing solution, the opposite is true for the hydrophobically stabilized tertiary structure. This adds a caveat to studies of protein structures in trifluoroethanol-containing solution in general.

Amino Acid Sequence↗

PubMed1994

Inhibition of topoisomerase I activity by tyrphostin derivatives, protein tyrosine kinase blockers: mechanism of action.

DNA topoisomerase I (topo I) is a member of a group of essential nuclear enzymes which control and modify the topological state of DNA and is recognized as the target for anticancer drugs. During the course of the catalytic activity of topo I, a covalent bond is formed between a tyrosine group at the active site of the enzyme and a 3' phosphate group along the DNA backbone. This chemical reaction resembles the protein kinase-mediated tyrosine phosphorylation process. We assumed, therefore, that tyrphostins, potent and selective blockers of protein tyrosine kinases, might affect topo I activity. We found that of three derivatives of tyrphostins (AG-555, AG-18, and AG-213) that inhibited topo I activity in an in vitro assay, AG-555 was the most active. Examination of the mechanism by which these compounds act as topo I inhibitors revealed that AG-555 blocked the binding of this enzyme to the DNA due to its interaction with the topo I enzyme. We showed that its mode of action differed from that observed for camptothecin, a known topo I inhibitor. However, AG-555 did not affect the activity of other major DNA binding enzymes (i.e., DNA ligase, DNA polymerase I, and reverse transcriptase). This study suggests that tyrphostins may serve as a new class of topo I inhibitors, and these results also present additional explanations for their antiproliferative effect.

3T3 Cells↗

PubMed1994

Inhibition of DNA topoisomerases I and II and induction of apoptosis by erbstatin and tyrphostin derivatives.

Inhibitors of protein tyrosine kinases (PTK) and DNA topoisomerases are potential antitumour agents. Drugs which bind to the ATP site of PTK, such as genistein, are common inhibitors to both types of enzymes. Eleven erbstatin and tyrphostin derivatives, which inhibit epidermal growth factor receptor PTK activity by competing with both the peptide substrate and ATP were tested for their capacity to inhibit DNA topoisomerases I and II. Erbstatin, two synthetic derivatives with a modified side chain and the tyrphostin AG 786 inhibited both topoisomerases in the same range of concentrations (20-50 microM). The tyrphostin AG 213 inhibited only topoisomerase II. In this series, absence of PTK inhibitory effect was correlated with the absence of DNA topoisomerase inhibition, while the detection of PTK inhibition may or may not be associated with DNA topoisomerase inhibition. In contrast to genistein, none of these molecules induced the stabilization of the topoisomerase-DNA cleavable complex, either in vitro or in vivo. Alcaline elution analysis revealed that erbstatin did not induce the formation of protein associated DNA strand breaks. However, an extensive degradation of the cellular DNA was observed which was shown to result from an internucleosomal fragmentation. Furthermore, typical morphological modifications associated with apoptosis were observed in the erbstatin treated cells by electron microscopy. These data indicate that erbstatin induces an apoptotic cell death.

Amino Acid Sequence↗

PubMed1994

Prevention of lipopolysaccharide-induced lethal toxicity by tyrosine kinase inhibitors.

Septic shock results from excessive stimulation of the host immune system, especially macrophages, by lipopolysaccharide (LPS), or endotoxin, which resides on the outer membrane of bacteria. Protein tyrosine kinase inhibitors of the tyrphostin AG 126 family protect mice against LPS-induced lethal toxicity. The protection correlates with the ability of these agents to block LPS-induced production of tumor necrosis factor alpha (TNF-alpha) and nitric oxide in macrophages as well as LPS-induced production of TNF-alpha in vivo. Furthermore, this inhibitory effect correlated with the potency of AG 126 to block LPS-induced tyrosine phosphorylation of a p42MAPK protein substrate in the murine macrophage.

Animals↗

PubMed1994

Platelet-derived growth factor isoforms prevent cell death during starvation of AKR-2B fibroblasts.

Confluent AKR-2B fibroblasts rapidly desintegrate upon removal of serum until a final density of approximately 50% of the initial value was reached after 12 h. This density remained unchanged for at least 48 h. Platelet-derived growth factor (PDGF)-BB stimulated more than 95% of these cells to divide. PDGF-AB or -BB added immediately after serum removal caused complete survival of the cells, but did not stimulate cell division as demonstrated by two-dimensional DNA flow cytometry. PDGF-AA was less effective leading to approximately 75% of the initial cell density. This effect could be augmented by the addition of ocadaic acid, a potent phosphatase inhibitor, suggesting that protein phosphorylation plays a role in this process. By using tyrphostin AG807 it was demonstrated that the signaling mechanism for survival requires receptor tyrosine autophosphorylation.

Animals↗

PubMed1994

Diagnostic test for lymphoproliferative disease virus infection of turkeys, using the polymerase chain reaction.

Lymphoproliferative disease virus, a type-C retrovirus, is the etiologic agent of a naturally acquired lymphoproliferative disorder in turkeys. The disease is characterized by rapid induction of lymphoproliferative lesions with morphologic and histopathologic features resembling those of reticuloendotheliosis. Owing to lack of overt clinical manifestations, early detection of lymphoproliferative disease virus is essential for preventing the rapid horizontal spread of the disease that can decimate flocks. We describe development of a simple, rapid, sensitive, and highly specific assay, using the polymerase chain reaction, capable of providing differential diagnosis of the disease soon after infection.

Animals↗

PubMed1994

Tyrphostin induced growth inhibition: correlation with effect on p210bcr-abl autokinase activity in K562 chronic myelogenous leukemia.

We have examined a series of tyrosine kinase inhibitors structurally related to erbstatin (tyrphostins) for inhibition of p210bcr-abl autokinase activity in vitro and for growth inhibition of chronic myelogenous leukemia (CML)K562 cells. Of the tyrphostins with IC50 for growth < 50 microM, AG814, AG946, AG952, AG896, AG953, AG956 and AG957 (structurally related to lavendustin A and piceatannol) completely inhibited p210bcr-abl kinase activity in an immune complex kinase assay. Another group of tyrphostins (AG807, AG568, AG763, AG1076, AG490, AG1318, AG556, AG1319, AG555 and AG1111) inhibits growth of K562 cells but not p210bcr-abl tyrosine kinase activity. Of the compounds which inhibit growth and p210bcr-abl tyrosine kinase activity, AG957 inhibits DNA synthesis as early as 2 h (60% inhibition at 20 microM of AG957), a time and concentration of drug where RNA and protein synthesis were not affected. AG957 inhibits p210bcr-abl tyrosine phosphorylation in living cells by 1 h without an inhibition of total protein phosphorylation. Growth inhibition by AG957 was reversible after 4 h of exposure, but irreversible after 24 h. AG957 can be considered as an important lead structure for the development of anti-bcr-abl tyrosine kinase antagonists. These data also raise the possibility that bcr-abl kinase activity is directly linked to maintenance of DNA synthesis in Philadelphia chromosome positive (Ph+) CML cells.

Adenosine Triphosphate↗

PubMed1994

Kinetics of inhibition by tyrphostins of the tyrosine kinase activity of the epidermal growth factor receptor and analysis by a new computer program.

The kinetics of inhibition of the epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase (TK) activity by erbstatin, tyrphostins, and lavendustin derivatives were studied in a system that employs poly(Glu6Ala3Tyr) (GAT) and ATP as substrates, after preactivation with EGF. All data were analyzed for computer best-fit curves by a program that was written for this purpose and is available upon request to those interested. The inhibition kinetics followed a sequential, Bi-Bi, rapid equilibrium, random mechanism, the mechanism of the EGFR-TK. Erbstatin and a few tyrphostins that contain a 3,4-dihydroxy-(cis)-cinnamonitrile [1-(3',4'-dihydroxyphenyl)-2-nitriloethene] group were found to be pure competitive inhibitors with respect to both substrates of the kinase reaction, i.e., GAT and ATP. Two tyrphostins, each containing an additional dihydroxyphenyl group in the alpha-position, were found to be pure competitive inhibitors with respect to GAT and noncompetitive (or mixed-competitive) inhibitors with respect to ATP. A lavendustin derivative with a 2,5-dihydroxyphenyl ring and a lavendustin derivative with a 3,4-dihydroyphenyl ring were also found to be competitive inhibitors with respect to both ATP and GAT. Various possible modes of binding at the EGFR-TK active center for the tyrphostins studied are proposed and the significance of the present findings, as well as the interpretations of computer analyses of kinetic data, is discussed.

Catechols↗

PubMed1993

Equine infectious anemia virus Tat is a predominantly helical protein.

Nuclear magnetic resonance (NMR) spectroscopy revealed features of the secondary structure of the equine infectious anemia virus (EIAV) Tat protein in solution. We could show that this protein, which is required in the replication cycle of lentiviruses, forms a predominantly helical structure in trifluoroethanol/water (40% by vol.) solution. In particular, the basic RNA-binding region and the adjacent core domain, which are highly conserved among lentiviral Tat proteins, show helix-type secondary structure under these conditions. Our observations, in concert with recent biochemical data from other laboratories, suggest that the core sequence region and the basic sequence region form interdependent structural domains, both possibly necessary for correct RNA binding.

Amino Acid Sequence↗

PubMed1993

Tyrphostin-induced inhibition of p210bcr-abl tyrosine kinase activity induces K562 to differentiate.

We report on the potency of two Tyrphostin tyrosine kinase blockers, AG 1112 and AG 568, to inhibit p210bcr-abl tyrosine kinase activity in K562 cells, concomitant with the induction of erythroid differentiation. AG 568 and especially AG 1112 represent a specific group of nontoxic protein tyrosine kinase blockers among more than 1,400 tested. These compounds possess therapeutic potential for purging Philadelphia chromosome-positive cells in preparation for autologous bone marrow transplantation in chronic myelogenous leukemia.

3T3 Cells↗