Requirement of the Jak2 tyrosine kinase in Bcr -Abl oncogenic transformation

Philadelphia chromosome (Ph)-positive chronic myeloid leukemia is caused by a clonal myeloproliferative expansion of malignant primitive hematopoietic progenitor cells. The Ph results from the reciprocal translocation of the ends of chromosome 9 and 22, which generate Bcr-Abl fusion proteins. The Bcr-Abl proteins possess a constitutively activated Abl tyrosine kinase, which is the driving force responsible for causing leukemia. The activated Bcr-Abl tyrosine kinase stimulates multiple signal transduction pathway affecting growth, differentiation and survival of cells. It is known that the Bcr-Abl tyrosine kinase activates several signaling proteins including Stat5, which is a member of the Jak/Stat pathway that is activated by cytokines that control the growth and differentiation of normal hematopoietic cells. Our laboratory was the first one to report that Jak2 tyrosine kinase is activated in a human Bcr-Abl positive hematopoietic cell line. In this thesis, we further investigated the activation of Jak2 by Bcr-Abl. We found that Jak2 is activated not only in cultured Bcr-abl positive cell lines but also in blood cells from CML blast crisis patients. We also demonstrated that SH2 domain of Bcr-Abl is required for efficient activation Jak2. We further showed that Jak2 binds to the C-terminal domain of Bcr-Abl; tyrosine residue 1007, which is critical for Jak2 activation, is phosphorylated by Bcr-Abl. We searched downstream targets of Jak2 in Bcr-Abl positive cells. We treated Bcr-Abl positive cells with a Jak2 kinase inhibitor AG490 and found that c-Myc protein expression is inhibited by AG490. We further demonstrated that Jak2 inhibitor AG490 not only inhibit C-MYC transcription but also protect c-Myc protein from proteasome-dependent degradation. We also showed that AG490 did not affect Bcr-Abl kinase activity and Stat5 activation and its downstream target Bcl-xL expression. AG490 also induced apoptosis of Bcr-Abl positive cells, similar to Bcr-Abl kinase inhibitor STI571 (also termed Gliveec, a very effective drug for CML), but unlike STI571 the apoptosis effects induced by AG490 can not be rescued by IL-3 containing WEHI conditioned medium. We further established several Bcr-Abl positive clones that express a kinase-inactive Jak2 and found that these clones had reduced tumor formation in nude mice assays. Taken together, these results establish that Jak2 is activated in Bcr-Abl positive CML cells and it is required for c-Myc induction and the oncogenic effects of Bcr-Abl. Furthermore, Jak2 and Stat5 are two independent targets of Bcr-Abl. ^

Phosphorus components and barite in untreated and in carbonate-free sediments of ODP Hole 199-1221C

We determined changes in equatorial Pacific phosphorus (µmol P/g) and barite (BaSO4; wt%) concentrations at high resolution (2 cm) across the Paleocene/Eocene (P/E) boundary in sediments from Ocean Drilling Program (ODP) Leg 199 Site 1221 (153.40 to 154.80 meters below seafloor [mbsf]). Oxide-associated, authigenic, and organic P sequentially extracted from bulk sediment were used to distinguish reactive P from detrital P. We separated barite from bulk sediment and compared its morphology with that of modern unaltered biogenic barite to check for diagenesis. On a CaCO3-free basis, reactive P concentrations are relatively constant and high (323 µmol P/g or ~1 wt%). Barite concentrations range from 0.05 to 5.6 wt%, calculated on a CaCO3-free basis, and show significant variability over this time interval. Shipboard measurements of P and Ba in bulk sediments are systematically lower (by ~25%) than shore-based concentrations and likely indicate problems with shipboard standard calibrations. The presence of Mn oxides and the size, crystal morphology, and sulfur isotopes of barite imply deposition in sulfate-rich pore fluids. Relatively constant reactive P, organic C, and biogenic silica concentrations calculated on a CaCO3-free basis indicate generally little variation in organic C, reactive P, and biogenic opal burial across the P/E boundary, whereas variable barite concentrations indicate significant changes in export productivity. Low barite Ba/reactive P ratios before and immediately after the Benthic Extinction Event (BEE) may indicate efficient nutrient burial, and, if nutrient burial and organic C burial are linked, high relative organic C burial that could temporarily drawdown CO2 at this site. This interpretation requires postdepositional oxidation of organic C because organic C to reactive P ratios are low throughout the section. After the BEE, higher barite Ba/reactive P ratios combined with higher barite Ba concentrations may imply that higher export productivity was coupled with unchanged reactive P burial, indicating efficient nutrient and possibly also organic C recycling in the water column. If the nutrient recycling is decoupled from organic C, the high export production could be indicative of drawdown of CO2. However, the observation that organic C burial is not high where barite burial is high may imply that either C sequestration was restricted to the deep ocean and thus occurred only on timescales of the deep ocean mixing or that postdepositional oxidation (burn down) of organic matter affected the sediments. The decoupling of barite and opal may result from low opal preservation or production that is not diatom based.

Antibody C219 recognizes an α-helical epitope on P-glycoprotein

The ABC transporter, P-glycoprotein, is an integral membrane protein that mediates the ATP-driven efflux of drugs from multidrug-resistant cancer and HIV-infected cells. Anti-P-glycoprotein antibody C219 binds to both of the ATP-binding regions of P-glycoprotein and has been shown to inhibit its ATPase activity and drug binding capacity. C219 has been widely used in a clinical setting as a tumor marker, but recent observations of cross-reactivity with other proteins, including the c-erbB2 protein in breast cancer cells, impose potential limitations in detecting P-glycoprotein. We have determined the crystal structure at a resolution of 2.4 Å of the variable fragment of C219 in complex with an epitope peptide derived from the nucleotide binding domain of P-glycoprotein. The 14-residue peptide adopts an amphipathic α-helical conformation, a secondary structure not previously observed in structures of antibody–peptide complexes. Together with available biochemical data, the crystal structure of the C219-peptide complex indicates the molecular basis of the cross-reactivity of C219 with non-multidrug resistance-associated proteins. Alignment of the C219 epitope with the recent crystal structure of the ATP-binding subunit of histidine permease suggests a structural basis for the inhibition of the ATP and drug binding capacity of P-glycoprotein by C219. The results provide a rationale for the development of C219 mutants with improved specificity and affinity that could be useful in antibody-based P-glycoprotein detection and therapy in multidrug resistant cancers.

Role of Tyrosine Phosphorylation in Ligand-induced Regulation of Transcytosis of the Polymeric Ig Receptor

The polymeric Ig receptor (pIgR) transcytoses its ligand, dimeric IgA (dIgA), from the basolateral to the apical surface of epithelial cells. Although the pIgR is constitutively transcytosed in the absence of ligand, binding of dIgA stimulates transcytosis of the pIgR. We recently reported that dIgA binding to the pIgR induces translocation of protein kinase C, production of inositol triphosphate, and elevation of intracellular free calcium. We now report that dIgA binding causes rapid, transient tyrosine phosphorylation of several proteins, including phosphatidyl inositol-specific phospholipase C-γl. Protein tyrosine kinase inhibitors or deletion of the last 30 amino acids of pIgR cytoplasmic tail prevents IgA-stimulated protein tyrosine kinase activation, tyrosine phosphorylation of phospholipase C-γl, production of inositol triphosphate, and the stimulation of transcytosis by dIgA. Analysis of pIgR deletion mutants reveals that the same discrete portion of the cytoplasmic domain, residues 727–736 (but not the Tyr734), controls both the ability of pIgR to cause dIgA-induced tyrosine phosphorylation of the phospholipase C-γl and to undergo dIgA-stimulated transcytosis. In addition, dIgA transcytosis can be strongly stimulated by mimicking phospholipase C-γl activation. In combination with our previous results, we conclude that the protein tyrosine kinase(s) and phospholipase C-γl that are activated upon dIgA binding to the pIgR control dIgA-stimulated pIgR transcytosis.

Cardiolipin is a normal component of human plasma lipoproteins

Anticardiolipin (anti-CL) antibodies, diagnostic for antiphospholipid antibody syndrome, are associated with increased risks of venous and arterial thrombosis. Because CL selectively enhances activated protein C/protein S-dependent anticoagulant activities in purified systems and because CL is not known to be a normal plasma component, we searched for CL in plasma. Plasma lipid extracts [chloroform/methanol (2:1, vol/vol)] were subjected to analyses by using TLC, analytical HPLC, and MS. A plasma lipid component was purified that was indistinguishable from reference CL (M:1448). When CL in 40 fasting plasma lipid extracts (20 males, 20 females) was quantitated by using HPLC, CL (mean ± SD) was 14.9 ± 3.7 μg/ml (range 9.1 to 24.2) and CL was not correlated with phosphatidylserine (3.8 ± 1.7 μg/ml), phosphatidylethanolamine (64 ± 20 μg/ml), or choline-containing phospholipid (1,580 ± 280 μg/ml). Based on studies of fasting blood donors, CL (≥94%) was recovered in very low density, low density, and high density lipoproteins (11 ± 5.3%, 67 ± 11.0%, and 17 ± 10%, respectively), showing that the majority of plasma CL (67%) is in low density lipoprotein. Analysis of relative phospholipid contents of lipoproteins indicated that high density lipoprotein is selectively enriched in CL and phosphatidylethanolamine. These results shows that CL is a normal plasma component and suggest that the epitopes of antiphospholipid antibodies could include CL or oxidized CL in lipoproteins or in complexes with plasma proteins (e.g., β2-glycoprotein I, prothrombin, protein C, or protein S) or with platelet or endothelial surface proteins.

Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia

Many pathogens causing diarrhea do so by modulating ion transport in the gut. Respiratory pathogens are similarly associated with disturbances of fluid balance in the respiratory tract, although it is not known whether they too act by altering epithelial ion transport. Here we show that influenza virus A/PR/8/34 inhibits the amiloride-sensitive Na+ current across mouse tracheal epithelium with a half-time of about 60 min. We further show that the inhibitory effect of the influenza virus is caused by the binding of viral hemagglutinin to a cell-surface receptor, which then activates phospholipase C and protein kinase C. Given the importance of epithelial Na+ channels in controlling the amount of fluid in the respiratory tract, we suggest that down-regulation of Na+ channels induced by influenza virus may play a role in the fluid transport abnormalities that are associated with influenza infections.

Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis

Signal transducer and activator of transcription (STAT) proteins perform key roles in mediating signaling by cytokines and growth factors, including platelet-derived growth factor (PDGF). In addition, Src family kinases activate STAT signaling and are required for PDGF-induced mitogenesis in normal cells. One STAT family member, Stat3, has been shown to have an essential role in cell transformation by the Src oncoprotein. However, the mechanisms by which STAT-signaling pathways contribute to mitogenesis and transformation are not fully defined. We show here that disruption of Stat3 signaling by using dominant-negative Stat3β protein in NIH 3T3 fibroblasts suppresses c-Myc expression concomitant with inhibition of v-Src-induced transformation. Ectopic expression of c-Myc is able to partially reverse this inhibition, suggesting that c-Myc is a downstream effector of Stat3 signaling in v-Src transformation. Furthermore, c-myc gene knockout fibroblasts are refractory to transformation by v-Src, consistent with a requirement for c-Myc protein in v-Src transformation. In normal NIH 3T3 cells, disruption of Stat3 signaling with dominant-negative Stat3β protein inhibits PDGF-induced mitogenesis in a manner that is reversed by ectopic c-Myc expression. Moreover, inhibition of Src family kinases with the pharmacologic agent, SU6656, blocks Stat3 activation by PDGF. These findings, combined together, delineate the signaling pathway, PDGF → Src → Stat3 → Myc, that is important in normal PDGF-induced mitogenesis and subverted in Src transformation.

Alpha-lactalbumin affects the acceptor specificity of Lymnaea stagnalis albumen gland UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalactosaminyltransferase: synthesis of GalNAc beta 1-->4Glc.

The N,N'-diacetyllactosediamine (lacdiNAc) pathway of complex-type oligosaccharide synthesis is controlled by a UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalac-tesaminyltransferase (beta 4-GalNAcT) that acts analogously to the common UDP-Gal:GlcNAc beta-R beta 1-->4-galactosyltransferase (beta 4-GalT). LacdiNAc-based chains particularly occur in invertebrates and cognate beta 4-GalNAcTs have been identified in the snail Lymnaea stagnalis, in two schistosomal species, and in several lepldopteran insect cell lines. Because of the similarity in reactions catalyzed by both enzymes, we investigated whether L. stagnalis albumen gland beta 4-GalNAcT would share with mammalian beta 4-GalT the property of interacting with alpha-lactalbumin (alpha-LA), a protein that only occurs in the lactating mammary gland, to form a complex in which the specificity of the enzyme is changed. It was found that, under conditions where beta 4-GalT forms the lactose synthase complex with alpha-LA, the snail beta 4-GalNAcT was induced by this protein to act on Glc with a > 100-fold increased efficiency, resulting in the formation of the lactose analog GalNAc beta 1-->4Glc. This forms the second example of a glycosyltransferase, the specificity of which can be altered by a modifier protein. So far, however, no protein fraction could be isolated from L. stagnalis that could likewise interact with the beta 4-GalNAcT. Neither had lysozyme c, a protein that is homologous to alpha-LA, an effect on the specificity of the enzyme. These results raise the question of how the capability to interact with alpha-LA has been conserved in the snail enzyme during evolution without any apparent selective pressure. They also suggest that snail beta 4-GalNAcT and mammalian beta 4-GalT show similarity at a molecular level and allows the identification of the beta 4-GalNAcT as a candidate member of the beta 4-GalT family.

Contrasting roles for Myc and Mad proteins in cellular growth and differentiation.

The positive effects of Myc on cellular growth and gene expression are antagonized by activities of another member of the Myc superfamily, Mad. Characterization of the mouse homolog of human mad on the structural level revealed that domains shown previously to be required in the human protein for anti-Myc repression, sequence-specific DNA-binding activity, and dimerization with its partner Max are highly conserved. Conservation is also evident on the biological level in that both human and mouse mad can antagonize the ability of c-myc to cooperate with ras in the malignant transformation of cultured cells. An analysis of c-myc and mad gene expression in the developing mouse showed contrasting patterns with respect to tissue distribution and developmental stage. Regional differences in expression were more striking on the cellular level, particularly in the mouse and human gastrointestinal system, wherein c-Myc protein was readily detected in immature proliferating cells at the base of the colonic crypts, while Mad protein distribution was restricted to the postmitotic differentiated cells in the apex of the crypts. An increasing gradient of Mad was also evident in the more differentiated subcorneal layers of the stratified squamous epithelium of the skin. Together, these observations support the view that both downregulation of Myc and accumulation of Mad may be necessary for progression of precursor cells to a growth-arrested, terminally differentiated state.

Tumorigenic activity of the BCR-ABL oncogenes is mediated by BCL2.

BCR-ABL is a chimeric oncogene generated by translocation of sequences from the c-abl protein-tyrosine kinase gene on chromosome 9 into the BCR gene on chromosome 22. Alternative chimeric proteins, p210BCR-ABL and p190BCR-ABL, are produced that are characteristic of chronic myelogenous leukemia and acute lymphoblastic leukemia, respectively. Their role in the etiology of human leukemia remains to be defined. Transformed murine hematopoietic cells can be used as a model of BCR-ABL function since these cells can be made growth factor independent and tumorigenic by the action of the BCR-ABL oncogene. We show that the BCR-ABL oncogenes prevent apoptotic death in these cells by inducing a Bcl-2 expression pathway. Furthermore, BCR-ABL-expressing cells revert to factor dependence and nontumorigenicity after Bcl-2 expression is suppressed. These results help to explain the ability of BCR-ABL oncogenes to synergize with c-myc in cell transformation.

Clonagem molecular, expressão, purificação e caracterização estrutural da endoglucanase de Trichoderma harzianum visando o desenvolvimento de coquetéis enzimáticos para a produção de etanol lignocelulósico

O aumento na demanda mundial por energia, a perspectiva de encolhimento dos recursos energéticos e a preocupação global com a questão ambiental, despertaram o interesse por fontes alternativas de energia. A biomassa lignocelulósica é abundante e de baixo custo, com potencial para complementar a produção em larga escala de combustíveis. A degradação das moléculas constituintes da parede celular à açúcares fermentescíveis e então à etanol, ocorre através da hidrólise enzimática da biomassa. Contudo, a utilização de enzimas para esse fim encontra-se em estágio exploratório e representa um gargalo na implementação de tecnologias de etanol 2G em escala industrial, desencadeando a busca de celulases bioquimicamente mais ativas, estáveis e economicamente viáveis. O presente trabalho visou a caracterização da endoglucanase I do fungo Trichoderma harzianum, e para isso foi realizada expressão, ensaios bioquímicos e biofísicos do domínio catalítico (ThCel7B-CCD) e da proteína inteira (ThCel7B-full). A enzima exibiu um perfil acidofílico, com atividade ótima em pH 3,0 a 55°C. A proteína também se mostrou capaz de hidrolisar uma variedade de substratos, sendo a maior atividade hidrolítica em β-glucano (75 U mg-1). Ao analisar a estabilidade térmica medida a 55°C em pH 5, a atividade residual manteve-se intacta por mais de 2 meses. Outra característica relevante foi o elevado grau de sinergismo entre ThCel7B e ThCel7A. Análises de microscopia eletrônica de flocos de aveia submetidas à hidrólise com ThCel7B evidenciaram os efeitos de degradação do substrato em relação às amostras controle. O conjunto desses resultados, além de importante para a compreensão do mecanismo molecular de ThCel7B e de outras endoglucanases da família GH7, também revelou uma enzima de interesse biotecnológicos uma vez que o comportamento ácido e sua estabilidade térmica são características relevantes para aplicações industriais sob condições extremamente ácidas.

Functional consequences of sequence alterations in the ATM gene

The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is a high molecular weight, protein (similar to350 kDa) containing a C-terminal protein kinase domain and a number of other putative domains not yet functionally defined. The majority of ATM gene mutations in A-T patients are truncating, resulting in prematurely terminated products that are highly unstable. Missense mutations within the kinase domain and elsewhere in the molecule alter the stability of the protein and lead to loss of protein kinase activity. Only rarely are patients observed with two missense mutations and this gives rise to a milder disease phenotype. Evidence for a dominant interfering effect on normal ATM kinase activity has been reported in cell lines transfected with missense mutant ATM and in cell lines from some A-T heterozygotes. The dominant negative effect of mutant ATM is manifested by an enhancement of cellular radiosensitivity and may be responsible for the cancer predisposition observed in carriers of ATM missense mutations. In this review, we explore the domain structure of the ATM molecule, sites of interaction with other proteins and the consequences of specific amino acid changes on function. (C) 2003 Elsevier B.V. All rights reserved.

Intracellar signalling in the HGT-1 gastric cell line and in rat isolated parietal cells

The work presented in this thesis was undertaken to increase understanding of the intracellular mechanisms regulating acid secretion by gastric parietal cells. Investigation of the effects of protein kinase C on secretory activity induced by a variety of agents was a major objective. A further aim was to establish the sites at which epidermal growth factor (EGF) acts to stimulate prostaglandin E2 (PGE2) production and to inhibit acid secretion. These investigations were carried out by using the HGT-1 human gastric cancer cell line and freshly isolated rat parietal cells. In HGT-1 cells, the cyclic AMP response to histamine and to truncated glucagon-like peptide 1 (TGLP-1) was reduced when protein kinase C was activated by 12-0-tetradecanoylphorbol 13-acetate (TPA). Receptor-binding studies and experiments in which cyclic AMP production in HGT-1 cells was stimulated by gastric inhibitory polypeptide, cholera toxin and forskolin suggested that the effect of TPA was mediated by uncoupling of the histamine H2 receptor from the guanine nucleotide regulatory protein Gs, possibly by phosphorylation of the receptor. An involvement of protein kinase C α in this effect was suggested because an antibody to this isoform specifically prevented the inhibitory effects of TPA on histamine-stimulated adenylate cyclase activity in a membrane fraction prepared from HGT-1 cells. Carbachol-stimulated secretory activity in parietal cells was specifically inhibited by Ro 31-8220, a bisindolylmaleimide inhibitor of protein kinase C. Thus protein kinase C may play a role in the activation of the secretory response to carbachol. In parietal cells prelabelled with [3H]-arachidonic acid or [3H]myristic acid, EGF did not affect [3H]-fatty acid or [3H] - diacylglycerol content. No evidence for effects of EGF on phosphatidylinositol glycan-specific phospholipase C, phospholipase A2 or on low Km cyclic AMP phosphodiesterase activities were found.

Catching transcriptional regulation by thermostatistical modeling

Gene expression is frequently regulated by multiple transcription factors (TFs). Thermostatistical methods allow for a quantitative description of interactions between TFs, RNA polymerase and DNA, and their impact on the transcription rates. We illustrate three different scales of the thermostatistical approach: the microscale of TF molecules, the mesoscale of promoter energy levels and the macroscale of transcriptionally active and inactive cells in a cell population. We demonstrate versatility of combinatorial transcriptional activation by exemplifying logic functions, such as AND and OR gates. We discuss a metric for cell-to-cell transcriptional activation variability known as Fermi entropy. Suitability of thermostatistical modeling is illustrated by describing the experimental data on transcriptional induction of NF?B and the c-Fos protein.

Suppression of the Insulin Receptors in Adult Schistosoma japonicum Impacts on Parasite Growth and Development: Further Evidence of Vaccine Potential

To further investigate the importance of insulin signaling in the growth, development, sexual maturation and egg production of adult schistosomes, we have focused attention on the insulin receptors (SjIRs) of Schistosoma japonicum, which we have previously cloned and partially characterised. We now show, by Biolayer Interferometry, that human insulin can bind the L1 subdomain (insulin binding domain) of recombinant (r)SjIR1 and rSjIR2 (designated SjLD1 and SjLD2) produced using the Drosophila S2 protein expression system. We have then used RNA interference (RNAi) to knock down the expression of the SjIRs in adult S. japonicum in vitro and show that, in addition to their reduced transcription, the transcript levels of other important downstream genes within the insulin pathway, associated with glucose metabolism and schistosome fecundity, were also impacted substantially. Further, a significant decrease in glucose uptake was observed in the SjIR-knockdown worms compared with luciferase controls. In vaccine/challenge experiments, we found that rSjLD1 and rSjLD2 depressed female growth, intestinal granuloma density and faecal egg production in S. japonicum in mice presented with a low dose challenge infection. These data re-emphasize the potential of the SjIRs as veterinary transmission blocking vaccine candidates against zoonotic schistosomiasis japonica in China and the Philippines.