Molecular systematics and paleobiogeography of the South American sigmodontine rodents [published erratum appears in Mol Biol Evol 1998 Feb;15(2):224]

The murid rodent subfamily Sigmodontinae contains 79 genera which are distributed throughout the New World. The time of arrival of the first sigmodontines in South America and the estimated divergence time(s) of the different lineages of South American sigmodontines have been controversial due to the lack of a good fossil record and the immense number of extant species. The "early-arrival hypothesis" states that the sigmodontines must have arrived in South America no later than the early Miocene, at least 20 MYA, in order to account for their vast present-day diversity, whereas the "late-arrival hypothesis" includes the sigmodontines as part of the Plio-Pleistocene Great American Interchange, which occurred approximately 3.5 MYA. The phylogenetic relationships among 33 of these genera were reconstructed using mitochondrial DNA (mtDNA) sequence data from the ND3, ND4L, arginine tRNA, and ND4 genes, which we show to be evolving at the same rate. A molecular clock was calibrated for these genes using published fossil dates, and the genetic distances were estimated from the DNA sequences in this study. The molecular clock was used to estimate the dates of the South American sigmodontine origin and the main sigmodontine radiation in order to evaluate the "early-" and "late-arrival" scenarios. We estimate the time of the sigmodontine invasion of South America as between approximately 5 and 9 MYA, supporting neither of the scenarios but suggesting two possible models in which the invading lineage was either (1) ancestral to the oryzomyines, akodonts, and phyllotines or (2) ancestral to the akodonts and phyllotines and accompanied by the oryzomyines. The sigmodontine invasion of South America provides an example of the advantage afforded to a lineage by the fortuitous invasion of a previously unexploited habitat, in this case an entire continent.      

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to “ribosomal stress” with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis.     

Inactivation of PEMT2 in hepatocytes initiated by DENA in fasted/refed rats

Phosphatidylethanolamine N-methyltransferase (PEMT) is the enzyme that converts phosphatidylethanolamine (PE) into phosphatidylcholine. We have previously shown that PEMT suppressed hepatoma growth by triggering apoptosis. We investigate whether PEMT controlled cell death and cell proliferation triggered by fasting/refeeding and whether it is a marker of early preneoplastic lesions. The induction of programmed cell death and suppression of cell proliferation by fasting were associated with enhanced PEMT expression and activity, and with a decrease in CTP:phosphocholine cytidylyltransferase expression. Refeeding returned the liver growth and expression of CTP:phosphocholine cytidylyltransferase to control levels, while the expression of PEMT decreased to below control values. After DENA administration, PEMT protein, evaluated by Western blotting, slightly increased, but it remained below control levels. The treatment with 20 mg/kg DENA to refed rats induced the appearance of initiated hepatocytes that were negative for PEMT expression. Present findings indicate that PEMT is a novel tumour marker for early liver preneoplastic lesions.     

Characterization of a novel mutation in the cardiac ryanodine receptor that results in catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease that manifests as syncope or sudden death during high adrenergic tone in the absence of structural heart defects. It is primarily caused by mutations in the cardiac ryanodine receptor (RyR2). The mechanism by which these mutations cause arrhythmia remains controversial, with discrepant findings related to the role of the RyR2 binding protein FKBP12.6. The purpose of this study was to characterize a novel RyR2 mutation identified in a kindred with clinically diagnosed CPVT.Single-strand conformational polymorphism analysis and direct DNA sequencing were used to screen the RyR2 gene for mutations. Site-directed mutagenesis was employed to introduce the mutation into the mouse RyR2 cDNA. The impact of the mutation on the interaction between RyR2 and a 12.6 kDa FK506 binding protein (FKBP12.6) was determined by immunoprecipitation and immunoblotting and its effect on RyR2 function was characterized by single cell Ca²⁺ imaging and [³H]ryanodine binding.A novel CPVT mutation, E189D, was identified. The E189D mutation does not alter the affinity of the channel for FKBP12.6, but it increases the propensity for store-overload-induced Ca²⁺ release (SOICR). Furthermore, the E189D mutation enhances the basal channel activity of RyR2 and its sensitivity to activation by caffeine.The E189D RyR2 mutation is causative for CPVT and functionally increases the propensity for SOICR without altering the affinity for FKBP12.6. These observations strengthen the notion that enhanced SOICR, but not altered FKBP12.6 binding, is a common mechanism by which RyR2 mutations cause arrhythmias.Key words: arrhythmia, calcium, death sudden, genetics, ion channels     

Mining cancer biology through bioinformatic analysis of proteomic data

ABSTRACT

Introduction: Discovery proteomics for cancer research generates complex datasets of diagnostic, prognostic, and therapeutic significance in human cancer. With the advent of high-resolution mass spectrometers, able to identify thousands of proteins in complex biological samples, only the application of bioinformatics can lead to the interpretation of data which can be relevant for cancer research.

Areas covered: Here, we give an overview of the current bioinformatic tools used in cancer proteomics. Moreover, we describe their applications in cancer proteomics studies of cell lines, serum, and tissues, highlighting recent results and critically evaluating their outcomes.

Expert opinion: The use of bioinformatic tools is a fundamental step in order to manage the large amount of proteins (from hundreds to thousands) that can be identified and quantified in a cancer biological samples by proteomics. To handle this challenge and obtain useful data for translational medicine, it is important the combined use of different bioinformatic tools. Moreover, a particular attention to the global experimental design, and the integration of multidisciplinary skills are essential for best setting of tool parameters and best interpretation of bioinformatics output.     

Spatial distribution of Ca(2+) signals during repetitive depolarizing stimuli in adrenal chromaffin cells

Exocytosis in adrenal chromaffin cells is strongly influenced by the pattern of stimulation. To understand the dynamic and spatial properties of the underlying Ca(2+) signal, we used pulsed laser Ca(2+) imaging to capture Ca(2+) gradients during stimulation by single and repetitive depolarizing stimuli. Short single pulses (10-100 ms) lead to the development of submembrane Ca(2+) gradients, as previously described (F. D. Marengo and J. R. Monck, 2000, Biophysical Journal, 79:1800-1820). Repetitive stimulation with trains of multiple pulses (50 ms each, 2Hz) produce a pattern of intracellular Ca(2+) increase that progressively changes from the typical Ca(2+) gradient seen after a single pulse to a Ca(2+) increase throughout the cell that peaks at values 3-4 times higher than the maximum values obtained at the end of single pulses. After seven or more pulses, the fluorescence increase was typically larger in the interior of the cell than in the submembrane region. The pattern of Ca(2+) gradient was not modified by inhibitors of Ca(2+)-induced Ca(2+) release (ryanodine), inhibitors of IP(3)-induced Ca(2+) release (xestospongin), or treatments designed to deplete intracellular Ca(2+) stores (thapsigargin). However, we found that the large fluorescence increase in the cell interior spatially colocalized with the nucleus. These results can be simulated using mathematical models of Ca(2+) redistribution in which the nucleus takes up Ca(2+) by active or passive transport mechanisms. These results show that chromaffin cells can respond to depolarizing stimuli with different dynamic Ca(2+) signals in the submembrane space, the cytosol, and the nucleus.     

Development of a classification and ranking method for the identification of possible biomarkers in two-dimensional gel-electrophoresis based on principal component analysis and variable selection procedures

The identification of biomarkers is one of the leading research areas in proteomics. When biomarkers have to be searched for in spot volume datasets produced by 2D gel-electrophoresis, problems may arise related to the large number of spots present in each map and the small number of samples available in each class (control/pathological). In such cases multivariate methods are usually exploited together with variable selection procedures, to provide a set of possible biomarkers: they are however usually aimed to the selection of the smallest set of variables (spots) providing the best performances in prediction. This approach seems not to be suitable for the identification of potential biomarkers since in this case all the possible candidate biomarkers have to be identified to provide a general picture of the "pathological state": in this case exhaustivity has to be preferred to provide a complete understanding of the mechanisms underlying the pathology. We propose here a ranking and classification method, "Ranking-PCA", based on Principal Component Analysis and variable selection in forward search: the method selects one variable at a time as the one providing the best separation of the two classes investigated in the space given by the relevant PCs. The method was applied to an artificial dataset and a real case-study: Ranking-PCA exhaustively identified the potential biomarkers and provided reliable and robust results.     

A Mycobacterium leprae Hsp65 mutant as a candidate for mitigating lupus aggravation in mice

Hsp60 is an abundant and highly conserved family of intracellular molecules. Increased levels of this family of proteins have been observed in the extracellular compartment in chronic inflammation. Administration of M. leprae Hsp65 [WT] in [NZBxNZW]F(1) mice accelerates the Systemic Lupus Erythematosus [SLE] progression whereas the point mutated K(409)A Hsp65 protein delays the disease. Here, the biological effects of M. leprae Hsp65 Leader pep and K(409)A pep synthetic peptides, which cover residues 352-371, are presented. Peptides had immunomodulatory effects similar to that observed with their respective proteins on survival and the combined administration of K(409)A+Leader pep or K(409)A pep+WT showed that the mutant forms were able to inhibit the deleterious effect of WT on mortality, indicating the neutralizing potential of the mutant molecules in SLE progression. Molecular modeling showed that replacing Lysine by Alanine affects the electrostatic potential of the 352-371 region. The number of interactions observed for WT is much higher than for Hsp65 K(409)A and mouse Hsp60. The immunomodulatory effects of the point-mutated protein and peptide occurred regardless of the catalytic activity. These findings may be related to the lack of effect on survival when F(1) mice were inoculated with Hsp60 or K(409)A pep. Our findings indicate the use of point-mutated Hsp65 molecules, such as the K(409)A protein and its corresponding peptide, that may minimize or delay the onset of SLE, representing a new approach to the treatment of autoimmune diseases.     

PKCδ sensitizes neuroblastoma cells to L-buthionine-sulfoximine and etoposide inducing reactive oxygen species overproduction and DNA damage

Neuroblastoma is a type of pediatric cancer. The sensitivity of neuroblastoma (NB) cancer cells to chemotherapy and radiation is inhibited by the presence of antioxidants, such as glutathione (GSH), which is crucial in counteracting the endogenous production of reactive oxygen species (ROS). We have previously demonstrated that cells depleted of GSH undergo apoptosis via oxidative stress and Protein kinase C (PKC) δ activation. In the present study, we transfected PKCδ in NB cells resistant to oxidative death induced by L-buthionine-S,R-sulfoximine (BSO), a GSH-depleting agent. Cell responses, in terms of ROS production, apoptosis and DNA damage were evaluated. Moreover, PKCδ activation was monitored by analyzing the phosphorylation status of threonine 505 residue, carrying out PKC activity assay and investigating the subcellular localization of the kinase. The cell responses obtained in BSO-resistant cells were also compared with those obtained in BSO-sensitive cells subjected to the same experimental protocol. Our results demonstrate, for the first time, that PKCδ induces DNA oxidation and ROS overproduction leading to apoptosis of BSO-resistant NB cells and potentiates the cytotoxic effects induced by BSO in sensitive cells. Moreover, PKCδ overexpression enhances the sensitivity of NB cells to etoposide, a well-characterised drug, commonly used in neuroblastoma therapy. Altogether our data provide evidence of a pro-oxidant role of PKCδ that might be exploited to design new therapeutic strategies aimed at selective killing of cancer cells and overcoming drug resistance. However, it becomes evident that a more detailed understanding of ROS-mediated signaling in cancer cells is necessary for the development of redox-modulated therapeutic approaches.     

A review of biology,fisheries and population structure of Dentex dentex (Sparidae)

The common dentex Dentex dentex (Linnaeus, 1758) is an iconic marine coastal fish in the Mediterranean Sea. It is a demersal sparid fish (0–200 m), that grows to a maximum length of 100 cm and a weight of 13 kg, with a relatively long life span (more than 20 years). As a high trophic level predator, it holds a key position in coastal marine food webs. The common dentex is of great economic importance for both artisanal (small-scale coastal fisheries) and recreational fishing. Despite its economic and ecological importance, scientific data on this species in its natural environment are still very scant. The global commercial catch of common dentex has fluctuated over the last 60 years on an interannual time scale, and has declined significantly since the 1990s. There are few data regarding fishing effort and total catch from recreational fishing for common dentex, but it appears that this species is particularly targeted by this activity. The common dentex is now classified as “vulnerable” in the Red List of Threatened Species in the Mediterranean Sea. This review summarizes the current literature on D. dentex in regard to biology, ecology, parasitology, population structure, commercial and recreational fishing, and management regulations. Future research directions to fill gaps in current knowledge are suggested.