STUDY ON THE PROLIFERATIVE STATE OF HAEMOPOIETIC STEM CELLS (CFU)

Hydroxyurea was used to study the proliferation rate of haemopoietic stem cells (CFUJ in normal mice, after irradiation or transplantation into irradiated recipients. It was demonstrated that the proliferation rate of endogenous CFU_S (endo-CFU,) and exogenous CFU_S (exo-CFU_s) are identical. After irradiation (650 R) the surviving endo-CFU_s begin to proliferate immediately. By contrast exo-CFU, transplanted into the irradiated recipient mouse (850 R), begin to proliferate only after about 30 hr. However, injection of isoproterenol (which stimulates adenyl cyclase) or dibutyryl cyclic adenosine 3′,5′-monophosphate shortly after marrow cell graft, triggers the transplanted CFU_S into cell cycle as shown by an almost immediately increased sensitivity to hydroxyurea. Isoproterenol is capable of inducing DNA synthesis also in stem cells of normal mice but it takes about 20 hr before CFU, become to be increasingly sensitive to hydroxyurea.     

Nvbeta-actin and NvGAPDH as normalization factors for gene expression analysis in limb regenerates and cultured blastema cells of the adult newt, Notophthalmus viridescens

The red-spotted newt has the ability to fully regenerate complex structures by creating a pool of dedifferentiated cells that arise in response to tissue injury. An understanding of the mechanisms involved in the regenerative ability of the newt is limited by a lack of characterized assays. This deficiency includes the cloning and validation of housekeeping genes for normalizing gene expression data. We describe the cloning, characterization and real-time quantitative PCR evaluation of the normalization potential of the newt homologues of cytoplasmic beta-actin and GAPDH during newt limb regeneration and within the blastemal B1H1 cell line. Nvbeta-actin demonstrates a heterogeneous expression during limb regeneration and may be associated with differentiation state. The level of Nvbeta-actin expression in B1H1 cultures under conditions of myogenesis and serum resupplementation varies with the treatment. NvGAPDH is ubiquitously expressed during limb regeneration and within B1H1 cultures and does not demonstrate overall variations in expression levels. Thus, NvGAPDH is a more appropriate normalization factor in gene expression analyses during limb regeneration and treatments of B1H1 cultures.     

Impairment of enzymatic antioxidant defenses is associated with bilirubin-induced neuronal cell death in the cerebellum of Ugt1 KO mice

Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced encephalopathy and eventually death by kernicterus. Despite extensive studies, the molecular and cellular mechanisms of bilirubin toxicity are still poorly defined. To fill this gap, we investigated the molecular processes underlying neuronal injury in a mouse model of severe neonatal jaundice, which develops hyperbilirubinemia as a consequence of a null mutation in the Ugt1 gene. These mutant mice show cerebellar abnormalities and hypoplasia, neuronal cell death and die shortly after birth because of bilirubin neurotoxicity. To identify protein changes associated with bilirubin-induced cell death, we performed proteomic analysis of cerebella from Ugt1 mutant and wild-type mice. Proteomic data pointed-out to oxidoreductase activities or antioxidant processes as important intracellular mechanisms altered during bilirubin-induced neurotoxicity. In particular, they revealed that down-representation of DJ-1, superoxide dismutase, peroxiredoxins 2 and 6 was associated with hyperbilirubinemia in the cerebellum of mutant mice. Interestingly, the reduction in protein levels seems to result from post-translational mechanisms because we did not detect significant quantitative differences in the corresponding mRNAs. We also observed an increase in neuro-specific enolase 2 both in the cerebellum and in the serum of mutant mice, supporting its potential use as a biomarker of bilirubin-induced neurological damage. In conclusion, our data show that different protective mechanisms fail to contrast oxidative burst in bilirubin-affected brain regions, ultimately leading to neurodegeneration.Unconjugated bilirubin (UCB) is the metabolic product of heme degradation in mammals. The enzymes heme oxygenase 1 and 2 (HO-1, HO-2) catalyze the degradation of heme into biliverdin, which is subsequently reduced to bilirubin by biliverdin reductase. In the liver, UCB is conjugated to glucuronic acid by UDP-glucuronosil transferase (UGT1a1), rendering it water soluble and excretable in the bile. In neonates, the late induction of Ugt1a1 gene expression may result in a limited capacity to conjugate bilirubin. The imbalance between bilirubin production and its elimination result in neonatal hyperbilirubinemia. Moderate jaundice is present in a high proportion of babies and is considered beneficial because of the antioxidant and cytoprotective properties of UCB.¹ However, excessive hyperbilirubinemia in newborns may produce acute bilirubin encephalopathy (BE), bilirubin-induced neurological disorders (BINDs) and eventually death by kernicterus.² BIND is characterized by a wide array of neurological deficits, including irreversible abnormalities in motor, sensitive and cognitive functions, because of UCB accumulation in the cerebellum, hippocampus and basal ganglia. Infants affected by null mutations in the Ugt1 gene develop Crigler–Najjar syndrome type I (CNSI).³ CNSI patients present high UCB plasma levels and are particularly exposed to BE and BIND if untreated. Current therapy initially consists in intensive phototherapy (PT) but liver transplantation is required later in life because of a reduction in PT efficiency.^{4, 5}Despite extensive investigations in animal models and in vitro tissue culture cells, the basic mechanisms of hyperbilirubinemia neurotoxicity have not been fully clarified yet.^{6, 7} UCB affects a large number of cellular functions and neurological damage appears to be the result of their concerted disruption rather than misregulation of a single pathway. The reported observations range from energy metabolism,⁸ cell proliferation,⁹ DNA and protein synthesis,¹⁰ receptor functionality,¹¹ to neurotransmitter uptake and release.¹² Our group and others reported that UCB is associated with an increased oxidative stress condition in cell culture.^{13, 14} In vitro studies showed that exposure to UCB decreases neuronal and glia viability.^{15, 16} In primary cultures, it has been observed that UCB permeabilizes mitochondrial membranes, resulting in mitochondrial swelling, release of cytochrome c into the cytosol, caspase-3 activation, Bax translocation and cell death by apoptosis.^{14, 17} Bilirubin also decreases NGF signaling to AKT and ERKs, interfering with prosurvival signaling pathways.¹⁸ However, most of the studies leading to potential mechanisms of bilirubin neurotoxicity were performed on monotypic cultures with artificial dosing of bilirubin, lacking the complexity of the interactions between different cell types, as it occurs in vivo.The aim of this study was to get a deeper unbiased insight into the molecular processes underlying bilirubin-induced neurodegeneration in vivo by using a mouse model of neonatal hyperbilirubinemia that shows early lethality because of bilirubin-induced neurological damage.¹⁹ Previous experiments conducted by our lab showed that the cerebellum of the Ugt1a^−/− mouse is the most vulnerable region of the brain to bilirubin toxicity.^{19, 20} Cerebellar susceptibility to bilirubin resulted in important alterations of its architecture, being the external germinal layer (EGL) and the Purkinje cell layer (PCL) the most affected regions, associated with an increase of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells.^{19, 20}In this study, we performed a differential proteomic analysis of the cerebellum from Ugt1 mutant and wild-type (WT) mice followed by validation of candidate genes both at protein and RNA levels. Our data point to an impairment of enzymatic antioxidant processes as important intracellular mechanisms involved in the onset of bilirubin-induced neurotoxicity in vivo.     

Genetic diversity patterns in Curcuma reflect differences in genome size

The relationships between genome size and the systematic and evolutionary patterns in vascular plants are equivocal, although a close relationship between genome size and evolutionary patterns has been previously reported. However, several studies have also revealed the dynamic nature of genome size evolution and its considerable ‘ups’ and ‘downs’. Thus, in this study, the phylogenetic relationships among three previously revealed genome size groups and among species of the highly polyploid genus Curcuma were evaluated using AFLP. Our results suggest two main lineages within Indian Curcuma reflecting evolution of genome size. The first one includes hexaploids and higher polyploids of the previously recognized genome size group I, and the second one includes mainly hexaploids of genome size groups II and III. Within genome size group I, relationships among species seem to be influenced by reticulate evolution and higher polyploids are likely to be of allopolyploid origin. Reproductive systems in Indian Curcuma vary considerably among ploidy levels and these differences considerably affect morphological and genetic variation. In general, clonally reproducing species are expected to exhibit low genotypic diversity, but, at the same time, species of allopolyploid origin are expected to maintain higher levels of heterozygosity compared with their progenitors. We investigated intra‐populational genetic variability in Curcuma spp. to evaluate whether mode of reproduction or ploidy represent the main factor influencing the degree of genetic diversity. We found that hexaploid species exhibited significantly higher genetic diversity than higher polyploids (9x, 15x). Our results suggest that this genetic diversity pattern is largely influenced by the mode of reproduction, as higher polyploids reproduce exclusively vegetatively, whereas hexaploids reproduce mainly sexually. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 165 , 388–401.     

Acute toxicity and adverse effects of aqueous and ethanol extracts of Parkia biglobosa pods on biochemical parameters of Clarias gariepinus

The acute toxicity of the aqueous and ethanol extracts of Parkia biglobosa pods against Clarias gariepinus was investigated under laboratory conditions. Agitated behaviours and respiratory distress were also observed during the exposure period. The adverse effects on biochemical parameters were assessed using semi-static bioassays for 28 days. The ethanol extract of P. biglobosa pods was found to be more acutely toxic with a 96 h LC₅₀ value of 13.96 mg l^?1 than the aqueous extracts, with a 96 h LC₅₀ value of 19.95 mg l^?1 against C. gariepinus. Both extracts induced agitated behaviours and respiratory distress in exposed organisms. The activities of superoxide dismutase (SOD), catalase (CAT) and the concentration of malondialdehyde (MDA) were significantly lower (p < 0.05) in groups of organisms exposed to extracts of P. biglobosa when compared with the control group after 14 days. The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were also significantly (p < 0.05) lower compared with activities of the enzymes in the control group after 28 days. The current study has shown that the introduction of P. biglobosa pods into aquatic ecosystems is acutely toxic to fish and would possibly be to other non-target aquatic organisms especially invertebrates.