New record for Galba neotropica (d'Orbigny, 1835) in Argentina,with a detailed analysis of its morphology and molecular characteristics

We report for the first time the presence of Galba neotropica in Santa Fe province, Pampean Argentina. Until the present work, the identity of the intermediate host of Fasciola hepatica in this region, the most important livestock production area of Argentina, was unknown. This report extends the geographic distribution of G. neotropica and is the first to provide molecular and morphological information on this species in Argentina. During summer 2013, snails were collected on private livestock farms with high prevalence of fasciolosis in cattle. Specimens were identified by using the nuclear sequences of the internal transcribed spacers ITS-1 and ITS-2, the shell and the shape and size of the male reproductive organs. Molecularly, ITS-1 and ITS-2 nuclear sequences exhibit 100% nucleotide identity with G. neotropica from Lima, Peru. Morphologically, G. neotropica from Santa Fe province was indistinguishable from Galba viatrix and Galba cubensis, the other two cryptic species within this South American group of lymnaeids. The combination of molecular and morphological analyses is strongly recommended to identify lymnaeids at species level. The identity of the intermediate host and the subsequent knowledge of its susceptibility, behaviour, distribution, ecology and biology are important components in developing effective measures to control fasciolosis.     

PCR approach for detection of Fragile X syndrome and Huntington disease based on modified DNA: limits and utility

A group of mutations characterized by trinucleotide repeat expansion causes human diseases such as the Fragile X syndrome, Huntington disease (HD), and myotonic dystrophy. Methods based on PCR amplification of the CGG and CAG repeats region could facilitate the development of a rapid screening assay; unfortunately, amplification across CGG and CAG repeats can be inefficient and unreliable due to the G + C base composition. The utility of the PCR on modified DNA for amplification of the CGG and CAG repeats at the Fragile X syndrome and HD has been reported. In the present study, we analyzed the utility of PCR on modified DNA as a rapid screening method for diagnosis of patients with Fragile X syndrome and HD. A comparative analysis realized with 38 Fragile X and 29 HD patients showed that the molecular diagnosis by simple PCR on modified DNA has a sensitivity and specificity of 100% in Fragile X patients and 94.1% and 91.6% in HD patients. The results achieved from the statistical analysis allowed us to conclude that the amplification by simple PCR on modified DNA is a reliable and useful method for the molecular diagnosis of the Fragile X syndrome, but not for the HD.     

A message emerging from development: the repression of mitochondrial β-F1-ATPase expression in cancer

Mitochondrial research has experienced a considerable boost during the last decade because organelle malfunctioning is in the genesis and/or progression of a vast array of human pathologies including cancer. The renaissance of mitochondria in the cancer field has been promoted by two main facts: (1) the molecular and functional integration of mitochondrial bioenergetics with the execution of cell death and (2) the implementation of ¹⁸FDG-PET for imaging and staging of tumors in clinical practice. The latter, represents the bed-side translational development of the metabolic hallmark that describes the bioenergetic phenotype of most cancer cells as originally predicted at the beginning of previous century by Otto Warburg. In this minireview we will briefly summarize how the study of energy metabolism during liver development forced our encounter with Warburg’s postulates and prompted us to study the mechanisms that regulate the biogenesis of mitochondria in the cancer cell.     

Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis

Background  

Recombinant proteins expressed in Escherichia coli vectors are generally contaminated with endotoxin. In this study, we evaluated the ability of Polymyxin B to neutralize the effect of LPS present as contaminant on Schistosoma mansoni recombinant proteins produced in E. coli in inducing TNF-α and IL-10. Peripheral blood mononuclear cells from individuals chronically infected with S. mansoni were stimulated in vitro with recombinant Sm22.6, Sm14 and P24 antigens (10 μg/mL) in the presence of Polymyxin B (10 μg/mL).     

Nucleotide binding to human UMP-CMP kinase using fluorescent derivatives -- a screening based on affinity for the UMP-CMP binding site

Methylanthraniloyl derivatives of ATP and CDP were used in vitro as fluorescent probes for the donor-binding and acceptor-binding sites of human UMP-CMP kinase, a nucleoside salvage pathway kinase. Like all NMP kinases, UMP-CMP kinase binds the phosphodonor, usually ATP, and the NMP at different binding sites. The reaction results from an in-line phosphotransfer from the donor to the acceptor. The probe for the donor site was displaced by the bisubstrate analogs of the Ap5X series (where X = U, dT, A, G), indicating the broad specificity of the acceptor site. Both CMP and dCMP were competitors for the acceptor site probe. To find antimetabolites for antivirus and anticancer therapies, we have developed a method of screening acyclic phosphonate analogs that is based on the affinity of the acceptor-binding site of the human UMP-CMP kinase. Several uracil vinylphosphonate derivatives had affinities for human UMP-CMP kinase similar to those of dUMP and dCMP and better than that of cidofovir, an acyclic nucleoside phosphonate with a broad spectrum of antiviral activities. The uracil derivatives were inhibitors rather than substrates of human UMP-CMP kinase. Also, the 5-halogen-substituted analogs inhibited the human TMP kinase less efficiently. The broad specificity of the enzyme acceptor-binding site is in agreement with a large substrate-binding pocket, as shown by the 2.1 A crystal structure.     

Internalization of beta-amyloid peptide by primary neurons in the absence of apolipoprotein E

Extracellular accumulation of beta-amyloid peptide (Abeta) has been linked to the development of Alzheimer disease. The importance of intraneuronal Abeta has been recognized more recently. Although considerable evidence indicates that extracellular Abeta contributes to the intracellular pool of Abeta, the mechanisms involved in Abeta uptake by neurons are poorly understood. We examined the molecular mechanisms involved in Abeta-(1-42) internalization by primary neurons in the absence of apolipoprotein E. We demonstrated that Abeta-(1-42) is more efficiently internalized by axons than by cell bodies of sympathetic neurons, suggesting that Abeta-(1-42) uptake might be mediated by proteins enriched in the axons. Although the acetylcholine receptor alpha7nAChR, previously suggested to be involved in Abeta internalization, is enriched in axons, our results indicate that it does not mediate Abeta-(1-42) internalization. Moreover, receptors of the low density lipoprotein receptor family are not essential for Abeta-(1-42) uptake in the absence of apolipoprotein E because receptor-associated protein had no effect on Abeta uptake. By expressing the inactive dynamin mutant dynK44A and the clathrin hub we found that Abeta-(1-42) internalization is independent of clathrin but dependent on dynamin, which suggests an endocytic pathway involving caveolae/lipid rafts. Confocal microscopy studies showing that Abeta did not co-localize with the early endosome marker EEA1 further support a clathrin-independent mechanism. The lack of co-localization of Abeta with caveolin in intracellular vesicles and the normal uptake of Abeta by neurons that do not express caveolin indicate that Abeta does not require caveolin either. Instead partial co-localization of Abeta-(1-42) with cholera toxin subunit B and sensitivity to reduction of cellular cholesterol and sphingolipid levels suggest a caveolae-independent, raft-mediated mechanism. Understanding the molecular events involved in neuronal Abeta internalization might identify potential therapeutic targets for Alzheimer disease.     

Effects of Clomipramine Administration on Syrian Hamster Circadian System and Behavior

The aim of the present work is to discuss the available data on neonatal and adult antidepressant treatment in relation to animal models of depression and serotonergic modulation of the circadian system, with a particular emphasis on our own published and unpublished work on the effects of clomipramine (a serotonin reuptake inhibitor) on the Syrian hamster circadian behavior. Neonatal clomipramine treatment (15 mg/kg from postnatal days 8 to 21) significantly augmented the amplitude of the wheel running rhythm, as well as delayed its acrophase and increased the time to reentrain after a 6-h phase advance of the light-dark cycle. Neonatally clomipramine-treated hamsters had a shorter circadian period than saline-treated animals under constant light - but not under constant dark- conditions, exhibited decreased phase advances after light pulses applied at late subjective night and greater phase advances after i.p. administration of the 5-HT_1A-receptor agonist 8-OH-DPA at midday. These animals also exhibited more locomotor activity than controls, but did not display the typical circadian variation in anxiety-related behavior, as measured in a plus-maze paradigm. They also showed an increased 5-HIAA/5-HT ratio in hypothalamus and midbrain raphe, while 5-HT content was decreased in frontal cortex and anterior hypothalamic areas. Since drugs linked to the serotonergic system are able to modify the circadian system, we decided to test whether acute and chronic clomipramine administration in adulthood was able to change: a) the phase of free running activity rhythms; (b) light-induced phase shifts, and (c) hypothalamic 5-HT turnover. Acute clomipramine injection had a phase-dependent effect on the free running activity rhythm, with phase advances at CT 0-8 being significantly higher than at CT 8-16. Pretreatment with clomipramine inhibited phase advances in response to light pulses when applied at CT 19 while phase delays at CT 14 remained unaffected. This acute treatment also decreased 5-HT turnover in the SCN at both CTs. In contrast, chronic clomipramine administration potentiated light-induced phase advances, without changes in period, amplitude or central 5-HT turnover. Taken together, these data support the view that clomipramine, as other antidepressant drugs, can affect the expression of the circadian rhythmicity in Syrian hamsters, possibly through serotonergic mechanisms in the case of acute treatments, and more complex behavioral interaction in the case of neonatal and chronic treatments.