Influence of Hydrocortisone on Chick Embryo Retina Development

Treatment of chick embryos in ovo with hydrocortisone-21-phosphate (a single dose of 150 micrograms) caused a marked reduction of retinal thymidine kinase activity 24 h later. The inhibitory effect was highest (65-70%) in 8-10-day-old embryos and declined with age, disappearing after day 15. It was accompanied by a reduction in thickness of the retinal layers. Adrenocorticotropic hormone (ACTH) treatment (10 micrograms daily for 2 days) also produced an age-dependent inhibitory effect on retinal thymidine kinase, whereas treatment with a single dose of 200 micrograms of metopirone, a compound that prevents the 11 beta-hydroxylation of steroid molecules in the adrenal glands, impeded the decrease in thymidine kinase activity that normally occurs in chick embryo retina after day 9 of development. In addition, metopirone prevented the inhibition exerted by ACTH on thymidine kinase activity but had no effect on the action of hydrocortisone.     

Biochemical Aspects of Chick Embryo Retina Development: The Effects of Glucocorticoids

In chick embryo retina during development, DNA synthesis and the activities of DNA polymerase, thymidine kinase, thymidylate synthetase, and ornithine decarboxylase (ODC) declined in parallel from day 7 to 12. The administration in ovo of hydrocortisone reduced significantly, particularly at 8-10 days of incubation, both DNA synthesis and the four enzyme activities tested. The effect was dose dependent, reaching the maximum with 50-100 nmol of hydrocortisone, 8-16 h after treatment. The highest inhibition was found for ODC activity (70%), followed by thymidine kinase activity (62%) and DNA synthesis (45%), whereas activities of DNA polymerase and thymidylate synthetase were reduced only by 30%. The inhibitory effect was exerted by all the glucocorticoids tested, with dexamethasone and hydrocortisone being the most efficacious. The results support the view that glucocorticoids reduce the proliferative events in chick embryo retina, particularly at 8-10 days of embryonic life.     

Inhibitory effect of D-glucosamine on glycolysis in bovine retina.

1. The effects of glucosamine concentration on the size of the lactate pool, on the levels of ATP, ADP, AMP and on the radioactivity incorporation from [1-14-C] glucosamine into lactate, N-acetylglucosamine and glucosamine-6-P were studied using whole bovine retinas. 2. The radioactive lactate, evaluated in relation to glucosamine molarity, after a modest initial increase, diminishes significantly. On the contrary the N-acetyl [1-14-C] glucosamine, the [1-14-C] glucosamine-6-P and, consequently, also the [1-14-C] glucosamine-6-P/[-14-C] lactate ratio increase with glucosamine molarity. 3. The retinal content of ATP shows a modest increment after incubation with low concentrations of D-glucosamine (0.5--2.0 mM) and a remarkable fall at higher concentrations. 4. Using retinal homogenates D-glucosamine clearly lowers the lactate production from glucose, glucose-6-P and fructose-1, 6-P2. 5. D-Glucosamine acts as an inhibitor of retinal glyceraldehyde-3-P dehydrogenase and lactate dehydrogenase by decreasing the initial velocity of these reactions. 6. It is concluded that D-glucosamine causes a reduction in the lactate production, by inhibiting two enzymes of the glycolytic pathway: glyceraldehyde-3-P dehydrogenase and lactate dehydrogenase. The fall in the adenine nucleotides content is a consequence of a dephosphorylation of ATP for the phosphorylation of glucosamine without concomitant resynthesis of ATP "via glycolysis".     

Paleoecological and paleobiogeographic considerations of Ordovician graptolites from the Cordillera Oriental,Jujuy Province,Argentina

We analyse new paleoecological data from South American graptolite records to understand the patterns that influence the space-tempo distribution of the group. A cluster analysis including taxa from the Baltograptus cf. B. deflexus and Didymograptellus bifidus Zones is carried out to evaluate the paleogeographic relationships between north-western Argentina and the other regions in the world. Three different biofacies were recognised in the Acoite Formation. The first biofacies takes place in the lower part of the unit, corresponding to pelitic levels (Tetragraptus phyllograptoides and Tetragraptus akzharensis Zones), and it is represented by pandemic forms. The other two biofacies belong to the endemic or neritic shallow water environments, and develop in deposits corresponding to the middle part of the Acoite Formation (bottom of the Baltograptus cf. B. deflexus Zone) in the Sierra de Aguilar, and in the upper half of the unit exposed in the Los Colorados area (D. bifidus Zone), respectively. The statistical analysis highlight the paleobiogeographic relationship between north-western Argentina and south-western China during the middle-upper Floian (Baltograptus cf. B. deflexus and D. bifidus Zones), and it supports the hypothesis that during the Early Ordovician north-western Argentina was located in the middle to high latitudes, included in the cold water faunal realm.     

Genetic and molecular characterization of the human Osteosarcoma 3AB‐OS cancer stem cell line: A possible model for studying osteosarcoma origin and stemness

Finding new treatments targeting cancer stem cells (CSCs) within a tumor seems to be critical to halt cancer and improve patient survival. Osteosarcoma is an aggressive tumor affecting adolescents, for which there is no second‐line chemotherapy. Uncovering new molecular mechanisms underlying the development of osteosarcoma and origin of CSCs is crucial to identify new possible therapeutic strategies. Here, we aimed to characterize genetically and molecularly the human osteosarcoma 3AB‐OS CSC line, previously selected from MG63 cells and which proved to have both in vitro and in vivo features of CSCs. Classic cytogenetic studies demonstrated that 3AB‐OS cells have hypertriploid karyotype with 71–82 chromosomes. By comparing 3AB‐OS CSCs to the parental cells, array CGH, Affymetrix microarray, and TaqMan® Human MicroRNA array analyses identified 49 copy number variations (CNV), 3,512 dysregulated genes and 189 differentially expressed miRNAs. Some of the chromosomal abnormalities and mRNA/miRNA expression profiles appeared to be congruent with those reported in human osteosarcomas. Bioinformatic analyses selected 196 genes and 46 anticorrelated miRNAs involved in carcinogenesis and stemness. For the first time, a predictive network is also described for two miRNA family (let‐7/98 and miR‐29a,b,c) and their anticorrelated mRNAs (MSTN, CCND2, Lin28B, MEST, HMGA2, and GHR), which may represent new biomarkers for osteosarcoma and may pave the way for the identification of new potential therapeutic targets. J. Cell. Physiol. 228: 1189–1201, 2013. © 2012 Wiley Periodicals, Inc.     

Apoptosis meets proteasome, an invaluable therapeutic target of anticancer drugs

This report reviews the current status of extensive efforts directed towards the interpretation of crosstalk between apoptosis and proteasome to understanding the molecular mechanism of anticancer agents targeting proteasome, with particular focus on MG132 and PS-341. The discovery that all cancer cells have retained the apoptotic death program has offered to the researchers new biochemical targets to design anticancer drugs. Moreover, the demonstration that proteasome inhibition induces apoptosis and sensitizes cancer cells to traditional tumoricidal agents has proposed the proteasome as an attractive target for development of new anticancer drugs. Since then, a number of both naturally occurring and synthetic inhibitors of the proteasome have been identified. The best characterized and most widely used inhibitors of the proteasome are the peptide aldehydes; among these MG132, due to its broad spectrum of action, low cost and rapid reversibility of action, still remains the first choice to study proteasome function in cell and tissue cultures. Recently, a very potent new class of selective and reversible proteasome inhibitors which contains an inhibitory boronate group has been described. PS-341 represent the first of this promising class of agents that could have application in cancer therapy and it is the only that has progressed to clinical trials.     

Effects of early dexamethasone therapy on pulmonary fibrogenic mediators and respiratory mechanics in preterm infants

Corticosteroid administration may prevent chronic lung disease in premature newborns, perhaps by modulating the synthesis of various cytokines, including those involved in fibrogenic processes. This study analyses the levels of three fibrogenic cytokines, namely vascular endothelial growth factor, transforming growth factor-beta 1 and basic fibroblast growth factor in tracheobronchial aspirate fluids collected from 20 premature newborns randomly assigned to the early dexamethasone group or to the control group. Furthermore, pulmonary function tests were performed on days 0 and 2 following the start of therapy. The results show that early corticosteroid administration reduces transforming growth factor-beta 1 and basic fibroblast growth factor levels, and abolishes the spontaneous vascular endothelial growth factor increase observed in untreated patients, concomitantly with significant improvement of dynamic lung compliance and shorter duration of the intubation period in the treated group of patients. Significant correlations were observed between the levels of transforming growth factor-beta 1 and vascular endothelial growth factor, indicating that the production of both these cytokines is coordinated. Finally, transforming growth factor-beta 1 ratios (day 2/day 0), representing early variations of the cytokine levels, were significantly different between treated and untreated subjects and correlated with the dynamic lung compliance ratios and the extubation day, suggesting that the downmodulation of some fibrogenic mediators may be involved in the mode of action of dexamethasone.     

Effects of dexamethasone on human synovial fibroblast-like cells, from osteoarthritic joints, in culture.

The effect of Dexamethasone (DEX) on cell division and macromolecular synthesis was investigated in a line (McCoy cells, A 9) of synovial fibroblast-like cells derived from human osteoarthritic joints. DEX markedly reduced the proliferation of McCoy cells in a time and dose-dependent manner. The maximal inhibition (45%) was found at 500 nM DEX 24 h after incubation and was accompanied by the appearance of giant macrophage-like cells. After DEX treatment cells showed increased content of DNA, proteins and RNA together with the reduction of [3H]-thymidine incorporation into the TCA-precipitable fraction.     

Modeling human osteosarcoma in mice through 3AB-OS cancer stem cell xenografts

Osteosarcoma is the second leading cause of cancer‐related death for children and young adults. In this study, we have subcutaneously injected—with and without matrigel—athymic mice (Fox1nu/nu) with human osteosarcoma 3AB‐OS pluripotent cancer stem cells (CSCs), which we previously isolated from human osteosarcoma MG63 cells. Engrafted 3AB‐OS cells were highly tumorigenic and matrigel greatly accelerated both tumor engraftment and growth rate. 3AB‐OS CSC xenografts lacked crucial regulators of beta‐catenin levels (E‐cadherin, APC, and GSK‐3beta), and crucial factors to restrain proliferation, resulting therefore in a strong proliferation potential. During the first weeks of engraftment 3AB‐OS‐derived tumors expressed high levels of pAKT, beta1‐integrin and pFAK, nuclear beta‐catenin, c‐Myc, cyclin D2, along with high levels of hyperphosphorylated‐inactive pRb and anti‐apoptotic proteins such as Bcl‐2 and XIAP, and matrigel increased the expression of proliferative markers. Thereafter 3AB‐OS tumor xenografts obtained with matrigel co‐injection showed decreased proliferative potential and AKT levels, and undetectable hyperphosphorylated pRb, whereas beta1‐integrin and pFAK levels still increased. Engrafted tumor cells also showed multilineage commitment with matrigel particularly favoring the mesenchymal lineage. Concomitantly, many blood vessels and muscle fibers appeared in the tumor mass. Our findings suggest that matrigel might regulate 3AB‐OS cell behavior providing adequate cues for transducing proliferation and differentiation signals triggered by pAKT, beta1‐integrin, and pFAK and addressed by pRb protein. Our results provide for the first time a mouse model that recapitulates in vivo crucial features of human osteosarcoma CSCs that could be used to test and predict the efficacy in vivo of novel therapeutic treatments. J. Cell. Biochem. 113: 3380–3392, 2012. © 2012 Wiley Periodicals, Inc.