The increase in cytosolic levels of protein kinase C precedes the growth factor requirements during the transition stem cells to differentiated progeny in the MCF-7 breast tumor cells.

The expression of regulatory genes within the context of a differentiation program can have profound long-term consequences in tissues with permanent renewing populations. The breast tumor cell line MCF-7 retains in culture some of the characteristics of a unidirectional differentiation pathway. We show that the cytosolic activity of the regulatory enzyme protein kinase C (PKC) precedes and continues the sequence of maturation in pre-differentiated subpopulations derived from a stem cell fraction. However, the activity declines in the most differentiated, post-mitotic fraction. These results indicate that PKC may be considered among the regulatory genes in MCF-7 cells that specify maturation of the stem cell progeny.     

Rapid chemiluminometric determination of gabapentin in pharmaceutical formulations exploiting pulsed‐flow analysis

In this study, a straightforward and automated pulsed flow‐based procedure was developed for the chemiluminometric determination of gabapentin [1‐(aminomethyl)cyclo‐hexaneacetic acid], a new generation antiepileptic drug, in different formulated dosage forms. The software‐controlled time‐based injection method capitalizes on the decrease of the background chemiluminescence (CL) readout of the luminol–hypochlorite reaction in the presence of gabapentin. In short, gabapentin works as a hypochlorite scavenger. The analytical procedure was implemented in a multi‐pumping flow network furnished with a suite of microdispensing solenoid‐actuated pumps. The diaphragm‐type micropumps might be configured to operate as fluid propellers, commutation units and metering injectors. A dynamic linear working range for gabapentin concentrations in the range 60–350 µmol/L was obtained, with an estimated detection limit of 40 µmol/L. The flow analyser handles about 41 injections/h and yields precise results (RSD < 2%). The miniaturized flow analyser thus has potential to be exploited for in‐line monitoring of drug manufacturing within the quality assurance framework of modern pharmaceutical companies. Copyright © 2008 John Wiley & Sons, Ltd.     

Infection with Usutu Virus Induces an Autophagic Response in Mammalian Cells

Usutu virus (USUV) is an African mosquito-borne flavivirus closely related to West Nile virus and Japanese encephalitis virus, which host range includes mainly mosquitoes and birds, although infections in humans have been also documented, thus warning about USUV as a potential health threat. Circulation of USUV in Africa was documented more than 50 years ago, but it was not until the last decade that it emerged in Europe causing episodes of avian mortality and some human severe cases. Since autophagy is a cellular pathway that can play important roles on different aspects of viral infections and pathogenesis, the possible implication of this pathway in USUV infection has been examined using Vero cells and two viral strains of different origin. USUV infection induced the unfolded protein response, revealed by the splicing of Xbp-1 mRNA. Infection with USUV also stimulated the autophagic process, which was demonstrated by an increase in the cytoplasmic aggregation of microtubule-associated protein 1 light chain 3 (LC3), a marker of autophagosome formation. In addition to this, an increase in the lipidated form of LC3, that is associated with autophagosome formation, was noticed following infection. Pharmacological modulation of the autophagic pathway with the inductor of autophagy rapamycin resulted in an increase in virus yield. On the other hand, treatment with 3-methyladenine or wortmannin, two distinct inhibitors of phosphatidylinositol 3-kinases involved in autophagy, resulted in a decrease in virus yield. These results indicate that USUV virus infection upregulates the cellular autophagic pathway and that drugs that target this pathway can modulate the infection of this virus, thus identifying a potential druggable pathway in USUV-infection.     

Growth and siderophore production of Xylella fastidiosa under iron-limited conditions

In this study, the production of siderophores by Xylella fastidiosa from the citrus bacteria isolate 31b9a5c (FAPESP – ONSA, Brazil) was investigated. The preliminary evidence supporting the existence of siderophore in X. fastidiosa was found during the evaluation of sequencing data generated in our lab using the BLAST-X tool, which indicated putative open reading frames (ORFs) associated with iron-binding proteins. In an iron-limited medium siderophores were detected in the supernatant of X. fastidiosa cultures. The endophytic bacterium Methylobacterium extorquens was also evaluated. Capillary electrophoresis was used to separate putative siderophores produced by X. fastidiosa. The bacterial culture supernatants of X. fastidiosa were identified negative for hydroxamate and catechol and positive for M. extorquens that secreted hydroxamate-type siderophores.     

The seeds of Campsiandra angustifolia (Fabaceae: Caesalpiniodeae) as a reflex of selective pressures on dispersal and establishment

We indirectly evaluated the selective pressures on dispersal and establishment of Campsiandra angustifolia, a common water-dispersed tree from the Peruvian Amazon, analyzing the variation in the relationship between the volume occupied by dispersal and establishment structures in a total of 535 seeds from 13 trees located at three different habitats. The seeds differed one order of magnitude in their total volume. However, independently of their size and the location of the maternal tree, the relationship between the volume occupied by dispersal and establishment structures was relatively constant (approximately 1) and showed a normal distribution with low skewness, indicating stabilizing selection. These results suggest that, in the habitats studied, dispersal and establishment processes may have similar importance to C. agustifolia. In species with seeds confined in pods, and therefore strongly space-limited, the relative volume of their seeds occupied by dispersal and establishment structures could be a better measure of the trade-off between these two processes than the variation in seed size.     

Morphological characterization of a human glioma cell l ine

A human malignant continuous cell line, named NG97, was recently established in our laboratory. This cell line has been serially subcultured over 100 times in standard culture media presenting no sign of cell senescence. The NG97 cell line has a doubling time of about 24 h. Immunocytochemical analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP) and S-100 protein, and negative for vimentin. Under phase-contrast microscope, cultures of NG97 showed cells with variable morphological features, such as small rounded cells, fusiform cells (fibroblastic-like cells), and dendritic-like cells. However, at confluence just small rounded and fusiform cells can be observed. At scanning electron microscopy (SEM) small rounded cells showed heterogeneous microextentions, including blebs and filopodia. Dendritic-like cells were flat and presented extensive prolongations, making several contacts with small rounded cells, while fusiform cells presented their surfaces dominated by microvilli.     

Molecular regulation of melanocyte senescence

Cell senescence is the loss of ability to divide after a finite number of divisions, seen in normal mammalian somatic cells and often disrupted in cancer cells. The three genes so far associated with familial melanoma susceptibility--INK4A, CDK4 and ARF, are all implicated in the molecular pathways controlling cell senescence. Here we review those pathways, both as generally studied in fibroblasts and epithelial cells, and as specifically analysed in melanocytes. Key molecular effectors in melanocyte senescence appear to include some in common with other cell types - telomere attrition and the p16/RB pathway, and one that is not commonly mentioned in this connection, the cAMP signalling pathway that also regulates melanocyte differentiation. These findings are discussed in relation to the role of cell senescence in the development and molecular genetics of melanoma and its precursor lesions.     

Genomic organization, chromosomal localization, alternative splicing, and isoforms of the human synaptosome-associated protein-23 gene implicated in vesicle-membrane fusion processes

Synaptosome-associated protein-23 (SNAP23) is a component of the cellular mechanism required for specific membrane fusion and targetting of intracellular vesicles. We have cloned the full-length human cDNA and the SNAP23 gene. The SNAP23 gene has eight exons, with the initiation codon located in exon 2, and maps to the human chromosome 15q21-22 region. The human SNAP23 gene can generate two types of message, the full-length message (SNAP23A) and a shorter message (SNAP23B). The latter is the result of alternative splicing where exon 5 is joined to exon 7 and the skipping of exon 6; it thus lacks a region that is required for non-specific binding to plasma membranes. The two isoforms, expressed as fusion proteins with glutathione-S-transferase, interact in vitro with human syntaxin 6, thus retaining the specific protein interaction required for membrane fusion. Alterations in the SNAP23 gene might be involved in neurological and other diseases with defects in vesicle-membrane fusion processes that map to 15q15-21.     

1-Oleoyl Lysophosphatidic Acid: A New Mediator of Emotional Behavior in Rats

The role of lysophosphatidic acid (LPA) in the control of emotional behavior remains to be determined. We analyzed the effects of the central administration of 1-oleoyl-LPA (LPA 18∶1) in rats tested for food consumption and anxiety-like and depression-like behaviors. For this purpose, the elevated plus-maze, open field, Y maze, forced swimming and food intake tests were performed. In addition, c-Fos expression in the dorsal periaqueductal gray matter (DPAG) was also determined. The results revealed that the administration of LPA 18∶1 reduced the time in the open arms of the elevated plus-maze and induced hypolocomotion in the open field, suggesting an anxiogenic-like phenotype. Interestingly, these effects were present following LPA 18∶1 infusion under conditions of novelty but not under habituation conditions. In the forced swimming test, the administration of LPA 18∶1 dose-dependently increased depression-like behavior, as evaluated according to immobility time. LPA treatment induced no effects on feeding. However, the immunohistochemical analysis revealed that LPA 18∶1 increased c-Fos expression in the DPAG. The abundant expression of the LPA₁ receptor, one of the main targets for LPA 18∶1, was detected in this brain area, which participates in the control of emotional behavior, using immunocytochemistry. These findings indicate that LPA is a relevant transmitter potentially involved in normal and pathological emotional responses, including anxiety and depression.