Pyrrolizidine alkaloid profiles of the Senecio cineraria group (Asteraceae)

Alkaloid profiles of five Senecio species (Asteraceae), including S. ambiguus subsp. ambiguus, S. ambiguus subsp. nebrodensis, S. gibbosus subsp. bicolor, S. gibbosus subsp. gibbosus, and S. gibbosus subsp. cineraria, were studied. Eleven pyrrolizidine alkaloids were identified and their content was evaluated by GLC-MS and GLC analysis. Otosenine and florosenine were found to be the major alkaloids in all studied species. It is interesting that only S. ambiguus subsp. nebrodensis was characterized by a high content of the alkaloids jacobine, jacoline, jaconine, and jacozine.     

PKC α-mediated CREB activation is oxygen and age-dependent in rat myocardial tissue

Both hypoxia and aging affect the morphology and the function of rat myocardial tissue. Moreover the heart tries to counteract the impaired function by activating specific signalling cascades. Here we report the involvement of CREB protein in “in vivo” response to hypoxic challenge and during aging in rat hearts. CREB is activated in parallel to HIF-1α nuclear translocation in the young after hypoxia exposure followed by reoxygenation, while this kind of response is not so dramatic in the old, neither in terms of CREB activation, neither in terms of HIF-1α expression and translocation, suggesting in the old the existence of an impaired oxygen-sensing mechanism or an adaptation of the cells to hypoxia. Moreover in the young a PKC α/Erk pathway seems to be involved in the activation of HIF-1α along with CREB, suggesting an attempt of the young to counteract the damage evoked by hypoxia, while in the old a PKC α/p38 MAPK/CREB pathway could determine the occurrence of both aging and aged cell hypoxia response.     

NTPDase and 5'-nucleotidase activities in physiological and disease conditions: new perspectives for human health

Extracellular nucleotides and nucleosides act as signaling molecules involved in a wide spectrum of biological effects. Their levels are controlled by a complex cell surface-located group of enzymes called ectonucleotidases. There are four major families of ectonucleotidases, nucleoside triphosphate diphosphohydrolases (NTPDases/CD39), ectonucleotide pyrophosphatase/phosphodiesterases (E-NPPs), alkaline phosphatases and ecto-5'-nucleotidase. In the last few years, substantial progress has been made toward the molecular identification of members of the ectonucleotidase families and their enzyme structures and functions. In this review, there is an emphasis on the involvement of NTPDase and 5'-nucleotidase activities in disease processes in several tissues and cell types. Brief background information is given about the general characteristics of these enzymes, followed by a discussion of their roles in thromboregulatory events in diabetes, hypertension, hypercholesterolemia and cancer, as well as in pathological conditions where platelets are less responsive, such as in chronic renal failure. In addition, immunomodulation and cell-cell interactions involving these enzymes are considered, as well as ATP and ADP hydrolysis under different clinical conditions related with alterations in the immune system, such as acute lymphoblastic leukemia (ALL), B-chronic lymphocytic leukemia (B-CLL) and infections associated with human immunodeficiency virus (HIV). Finally, changes in ATP, ADP and AMP hydrolysis induced by inborn errors of metabolism, seizures and epilepsy are discussed in order to highlight the importance of these enzymes in the control of neuronal activity in pathological conditions. Despite advances made toward understanding the molecular structure of ectonucleotidases, much more investigation will be necessary to entirely grasp their role in physiological and pathological conditions.     

Identifying the genetic basis of ecologically and biotechnologically useful functions of the bacterium Burkholderia vietnamiensis

Signature-tagged mutagenesis (STM) was used to identify genetic determinants of fitness associated with two key ecological processes mediated by bacteria. Burkholderia vietnamiensis strain G4 was used as a model bacterium to investigate: phenol degradation as a model of bioremediation, and pea rhizosphere colonization as a prerequisite to biological control and phytoremediation. A total of 1900 mutants were screened and 196 putative fitness mutants identified; the genetic basis of 137 of these mutations was determined by correlation to the G4 genome. The phenol-STM screen was more successful at identifying phenol degradation mutations (83 mutants; 4.4% hit rate) than a conventional agar-based phenol screen (49 mutants, 5319 screened, 0.92% hit rate). The combination of both screens completely defined the components of the TOM pathway in strain G4 and also identified novel accessory genes not previously implicated in phenol utilization. The rhizosphere-STM screen identified 113 mutants (5.9% hit rate); 107 had reduced tag signals indicative of poor rhizosphere colonization (Rhiz-), while six mutants produced high hybridization signals suggesting increased rhizosphere competence (Rhiz+). Competition assays confirmed that 69% of Rhiz- mutants tested (24/35) were severely compromised in their rhizosphere fitness. Seventy Rhiz- mutations mapped to genes with the following putative functions: amino acid biosynthesis (25; 36%), general metabolism (18; 26%), hypothetical (9; 13%), regulatory genes (4; 5.7%), transport and stress (2 each; 2.8% respectively). One of the most interesting discoveries mediated by the rhizosphere-STM screen was the identification of three Rhiz+ mutants inactivated within a single virulence-associated autotransporter adhesin gene; this mutation consistently produced a hyper-colonization phenotype suggesting a highly novel role for this surface adhesin during plant interactions. Our study has shown that STM can be successfully applied to ecologically important microbial interactions, defining the underlying genetic systems important for biotechnological fitness of environmental bacteria such those from the Burkholderia cepacia complex.     

Congenital myasthenic syndromes: spotlight on genetic defects of neuromuscular transmission

The neuromuscular junction (NMJ) is a complex structure that efficiently communicates the electrical impulse from the motor neuron to the skeletal muscle to induce muscle contraction. Genetic and autoimmune disorders known to compromise neuromuscular transmission are providing further insights into the complexities of NMJ function. Congenital myasthenic syndromes (CMSs) are a genetically and phenotypically heterogeneous group of rare hereditary disorders affecting neuromuscular transmission. The understanding of the molecular basis of the different types of CMSs has evolved rapidly in recent years. Mutations were first identified in the subunits of the nicotinic acetylcholine receptor (AChR), but now mutations in ten different genes - encoding post-, pre- or synaptic proteins - are known to cause CMSs. Pathogenic mechanisms leading to an impaired neuromuscular transmission modify AChRs or endplate structure or lead to decreased acetylcholine synthesis and release. However, the genetic background of many CMS forms is still unresolved. A precise molecular classification of CMS type is of paramount importance for the diagnosis, counselling and therapy of a patient, as different drugs may be beneficial or deleterious depending on the molecular background of the particular CMS.     

p53 gain-of-function cancer mutants induce genetic instability by inactivating ATM

Tp53 is the most commonly mutated tumour-suppressor gene in human cancers. In addition to the loss of tumour-suppression function, some missense mutants gain novel oncogenic activities. To elucidate the nature of the gain of function, we introduced the most common p53 cancer mutations (R248W and R273H) independently into the humanized p53 knock-in (HUPKI) allele in mice. Tumour-suppressor functions of p53 are abolished in p53-mutant mice. Several lines of evidence further indicate gain-of-function of p53 mutants in promoting tumorigenesis. p53(R248W) mice rapidly succumb to certain types of cancers not commonly observed in p53(-/-) mice. Interchromosomal translocations, a type of genetic instability rarely observed in p53(-/-) cells, are readily detectable in p53-mutant pre-tumor thymocytes. Although normal in p53(-/-) mouse cells, the G(2)-M checkpoint is impaired in p53-mutant cells after DNA damage. These acquired oncogenic properties of mutant p53 could be explained by the findings that these p53 mutants interact with the nuclease Mre11 and suppress the binding of the Mre11-Rad50-NBS1 (MRN) complex to DNA double-stranded breaks (DSBs), leading to impaired Ataxia-telangiectasia mutated (ATM) activation. Therefore, p53 gain-of-function mutants promote tumorigenesis by a novel mechanism involving active disruption of critical DNA damage-response pathways.     

Wnt/β-catenin signaling is required for development of the exocrine pancreas

Background  

β-catenin is an essential mediator of canonical Wnt signaling and a central component of the cadherin-catenin epithelial adhesion complex. Dysregulation of β-catenin expression has been described in pancreatic neoplasia. Newly published studies have suggested that β-catenin is critical for normal pancreatic development although these reports reached somewhat different conclusions. In addition, the molecular mechanisms by which loss of β-catenin affects pancreas development are not well understood. The goals of this study then were; 1] to further investigate the role of β-catenin in pancreatic development using a conditional knockout approach and 2] to identify possible mechanisms by which loss of β-catenin disrupts pancreatic development. A Pdx1-cre mouse line was used to delete a floxed β-catenin allele specifically in the developing pancreas, and embryonic pancreata were studied by immunohistochemistry and microarray analysis.     

Selective alteration at the growth-hormone- releasing-hormone nerve terminals during aging in GHRH-green fluorescent protein mice

Growth hormone (GH) secretion decreases spontaneously during lifespan, and the resulting GH deficiency participates in aging-related morbidity. This deficiency appears to involve a defect in the activity of hypothalamic GH-releasing hormone (GHRH) neurons. Here, we investigated this hypothesis, as well as the underlying mechanisms, in identified GHRH neurons from adult ( approximately 13 weeks old) and aged ( approximately 100 weeks old) transgenic GHRH-green fluorescent protein mice, using morphological, biochemical and electrophysiological methods. Surprisingly, the spontaneous action potential frequency was similar in adult and aged GHRH neurons studied in brain slices. This was explained by a lack of change in the intrinsic excitability, and simultaneous increases in both stimulatory glutamatergic- and inhibitory GABAergic-synaptic currents of aged GHRH neurons. Aging did not decrease GHRH and enhanced green fluorescent protein contents, GHRH neuronal number or GHRH-fibre distribution, but we found a striking enlargement of GHRH-positive axons, suggesting neuropeptide accumulation. Unlike in adults, autophagic vacuoles were evident in aged GHRH-axonal profiles using electron microscopy. Thus, GHRH neurons are involved in aging of the GH axis. Aging had a subtle effect at the nerve terminal level in GHRH neurons, contrasting with the view that neuronal aging is accompanied by more widespread damage.     

Adaptation or selection? Old issues and new stakes in the postwar debates over bacterial drug resistance

The 1940s and 1950s were marked by intense debates over the origin of drug resistance in microbes. Bacteriologists had traditionally invoked the notions of 'training' and 'adaptation' to account for the ability of microbes to acquire new traits. As the field of bacterial genetics emerged, however, its participants rejected 'Lamarckian' views of microbial heredity, and offered statistical evidence that drug resistance resulted from the selection of random resistant mutants. Antibiotic resistance became a key issue among those disputing physiological (usually termed 'adaptationist') vs. genetic (mutation and selection) explanations of variation in bacteria. Postwar developments connected with the Lysenko affair gave this debate a new political valence. Proponents of the neo-Darwinian synthesis weighed in with support for the genetic theory. However, certain features of drug resistance seemed inexplicable by mutation and selection, particularly the phenomenon of 'multiple resistance'--the emergence of resistance in a single strain against several unrelated antibiotics. In the late 1950s, Tsutomu Watanabe and his collaborators solved this puzzle by determining that resistance could be conferred by cytoplasmic resistance factors rather than chromosomal mutation. These R factors could carry resistance to many antibiotics and seemed able to promote their own dissemination in bacterial populations. In the end, the vindication of the genetic view of drug resistance was accompanied by a recasting of the 'gene' to include extrachromosomal hereditary units carried on viruses and plasmids.     

Leishmanicidal activity of a supercritical fluid fraction obtained from Tabernaemontana catharinensis

The branches and leaves of Tabernaemontana catharinensis were extracted with supercritical fluid using a mixture of CO(2) plus ethanol (SFE), and the indole alkaloid enriched fraction (AF3) was selected for anti-Leishmania activity studies. We found that AF3 exhibits a potent effect against intracellular amastigotes of Leishmania amazonensis, a causative agent of New World cutaneous leishmaniasis. AF3 inhibits Leishmania survival in a dose-dependent manner, and reached 88% inhibition of amastigote growth at 100 microg/mL. The anti-parasite effect was independent of nitric oxide (NO), since AF3 was able to inhibit NO production induced by IFN-gamma plus LPS. In addition, AF3 inhibited TGF-beta production, which could have facilitated AF3-mediated parasite killing. The AF3 fraction obtained from SFE was nontoxic for host macrophages, as assessed by plasma membrane integrity and mitochondrial activity. We conclude that SFE is an efficient method for obtaining bioactive indole alkaloids from plant extracts. Importantly, this method preserved the alkaloid properties associated with inhibition of Leishmania growth in macrophages without toxicity to host cells.