Carboxyamidotriazole-orotate inhibits the growth of imatinib-resistant chronic myeloid leukaemia cells and modulates exosomes-stimulated angiogenesis

The Bcr/Abl kinase has been targeted for the treatment of chronic myelogenous leukaemia (CML) by imatinib mesylate. While imatinib has been extremely effective for chronic phase CML, blast crisis CML are often resistant. New therapeutic options are therefore needed for this fatal disease. Although more common in solid tumors, increased microvessel density was also reported in chronic myelogenous leukaemia and was associated with a significant increase of angiogenic factors, suggesting that vascularity in hematologic malignancies is a controlled process and may play a role in the leukaemogenic process thus representing an alternative therapeutic target. Carboxyamidotriazole-orotate (CTO) is the orotate salt form of carboxyamidotriazole (CAI), an orally bioavailable signal transduction inhibitor that in vitro has been shown to possess antileukaemic activities. CTO, which has a reduced toxicity, increased oral bioavailability and stronger efficacy when compared to the parental compound, was tested in this study for its ability to affect imatinib-resistant CML tumor growth in a xenograft model. The active cross talk between endothelial cells and leukemic cells in the bone marrow involving exosomes plays an important role in modulating the process of neovascularization in CML. We have thus investigated the effects of CTO on exosome-stimulated angiogenesis. Our results indicate that CTO may be effective in targeting both cancer cell growth and the tumor microenvironment, thus suggesting a potential therapeutic utility for CTO in leukaemia patients.     

Characterization of the protein ubiquitination response induced by Doxorubicin

Doxorubicin is commonly considered to exert its anti-tumor activity by triggering apoptosis of cancer cells through DNA damage. Recent reports have shown that Doxorubicin elicits a marked heat shock response, and that either inhibition or silencing of heat shock proteins enhance the Doxorubicin apoptotic effect in neuroblastoma cells. In order to investigate whether Doxorubicin may also act through protein modification, we performed a proteomic analysis of ubiquitinated proteins. Here we show that nanomolar Doxorubicin treatment of neuroblastoma cells caused: (a) dose-dependent over-ubiquitination of a specific set of proteins in the absence of measurable inhibition of proteasome, (b) protein ubiquination patterns similar to those with Bortezomib, a proteasome inhibitor, (c) depletion and loss of activity of ubiquitinated enzymes such as lactate dehydrogenase and α-enolase, and (d) a decrease in HSP27 solubility, probably a consequence of its binding to denatured proteins. These data strongly reinforce the hypothesis that Doxorubicin may also exert its effect by damaging proteins.     

Delayed development of interstitial cells of Cajal in the ileum of a human case of gastroschisis

The Interstitial Cells of Cajal (ICC) are responsible for rhythmic electrical activity. A paralytic ileus is present in gastroschisis (GS), a malformation due to a defective closure of the abdominal wall through which part of the intestine herniates during pregnancy. In experimental GS, ICC morphological immaturity was shown in the rat foetus at-term but it could not be demonstrated whether differentiation is accomplished post-natally. For this purpose we morphologically investigated ICC, as well as enteric neurons and smooth muscle cells, in a case of human GS at birth and 1 month later when peristaltic activity had initiated. A 36 weeks gestation female was born by c/section with prenatal diagnosis of GS and possible volvulus of the herniated intestine. At birth, the necrotic intestine was resected and both ileostomy and colostomy were performed. The intestine continuity was restored after 4 weeks. Intestinal specimens, taken during both operations at the level of the proximal stoma, were immunostained with c-kit, neuron-specific-enolase and alpha-smooth-muscle-actin antibodies and some processed for electron microscopy. ICC were present at the myenteric plexus only. At birth, these cells were rare and ultrastructurally immature; 1 month later, when partial enteral feeding was tolerated, they formed rows or groups and many of them were ultrastructurally differentiated. Neurons and smooth muscle cells, immature at birth, had developed after 1 month. Therefore, ICC differentiation, as well as that of neurons and smooth muscle cells, is delayed at birth and this might explain the paralytic ileus in GS. One month later, differentiation quickly proceeded at all cellular levels paralleling the increasing tolerance of enteral nutrition.     

Predicting anaerobic biogasification potential of ingestates and digestates of a full-scale biogas plant using chemical and biological parameters

The aim of this work was to develop simple and fast tests to predict anaerobic biogasification potential (ABP) of ingestates and digestates from a biogas plant. Forty-six samples of both ingestates and digestates were collected within an eight-month observation period and were analyzed in terms of biological and chemical parameters, namely, ABP test, oxygen demand in a 20-h respirometric test (OD20), total solids (TS), volatile solids (VS), total organic carbon (TOC), total Kjeldahl nitrogen (TKN), ammonia, cell solubles (CS), acid detergent fibers (ADF), lignin (ADL), cellulose, and hemicellulose. Considering both quantitative (VS and TOC) and qualitative aspects (OD20 and CS) of organic matter (OM), four models (linear regressions; 0.80相似文献   

Effect of salt and pH on the reductive half-reaction of Mycobacterium tuberculosis FprA with NADPH

Despite a number of studies, the formation of the Michaelis complexes between ferredoxin-NADP (+) reductases and NADP(H) eluded detailed investigations by rapid kinetic techniques because of their high formation rates. Moreover, the reversible nature of the reaction of hydride ion transfer between these enzymes and NADPH prevented the obtainment of reliable estimates of the rate constant of the hydride transfer step. Here we show that by working at a high salt concentration, the mechanism of the reaction with NADPH of FprA, a Mycobacterium tuberculosis homologue of adrenodoxin reductase, is greatly simplified, making it amenable to investigation by rapid reaction techniques. The approach presented herein allowed for the first time the observation of the formation of the Michaelis complex between an adrenodoxin reductase-like enzyme and NADPH, and the determination of the related rate constants for association and dissociation. Furthermore, the rate constant for the reaction of hydride ion transfer between NADPH and FAD could be unambiguously assessed. It is proposed that the approach described should be applicable to other ferredoxin reductase enzymes, providing a valuable experimental tool for the study of their kinetic properties.     

Role of α2‐macroglobulin in regulating amyloid β‐protein neurotoxicity: protective or detrimental factor?

alpha2-Macroglobulin (alpha2M) has been identified as a carrier protein for beta-amyloid (Abeta) decreasing fibril formation and affecting the neurotoxicity of this peptide. The alpha2-macroglobulin receptor/low density lipoprotein receptor related protein (LRP) is involved in the internalization and degradation of the alpha2M/Abeta complexes and its impairment has been reported to occur in Alzheimer's disease. Previous studies have shown alpha2M to determine an enhancement or a reduction of Abeta toxicity in different culture systems. In order to clarify the role of alpha2M in Abeta neurotoxicity, we challenged human neuroblastoma cell lines with activated alpha2M in combination with Abeta. Our results show that in neuroblastoma cells expressing high levels of LRP, the administration of activated alpha2M protects the cells from Abeta neurotoxicity. Conversely, when this receptor is not present alpha2M determines an increase in Abeta toxicity as evaluated by MTT and TUNEL assays. In LRP-negative cells transfected with the full-length human LRP, the addition of activated alpha2M resulted to be protective against Abeta-induced neurotoxicity. By means of recombinant proteins we ascribed the neurotoxic activity of alpha2M to its FP3 fragment which has been previously shown to bind and neutralize transforming growth factor-beta. These studies provide evidence for both a neuroprotective and neurotoxic role of alpha2M regulated by the expression of its receptor LRP.