Showing papers by "National University of Cuyo published in 2001"

A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation

Abstract: Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic portions and intracellular organelles in a membrane vacuole called the autophagosome. These vesicles fuse with lysosomes and the sequestered material is degraded. Owing to the complexity of the autophagic pathway and to its inaccessibility to external probes, little is known about the molecular mechanisms that regulate autophagy in higher eukaryotic cells. We used the autofluorescent drug monodansylcadaverine (MDC), a specific autophagolysosome marker to analyze at the molecular level the machinery involved in the autophagic process. We have developed a morphological and biochemical assay to study authophagy in living cells based on the incorporation of MDC. With this assay we observed that the accumulation of MDC was specifically induced by amino acid deprivation and was inhibited by 3-methlyadenine, a classical inhibitor of the autophagic pathway. Additionally, wortmannin, an inhibitor of PI3-kinases that blocks autophagy at an early stage, inhibited the accumulation of MDC in autophagic vacuoles. We also found that treatment of the cells with N-ethylmaleimide (NEM), an agent known to inhibit several vesicular transport events, completely blocked the incorporation of MDC, suggesting that an NEM-sensitive protein is required for the formation of autophagic vacuoles. Conversely, vinblastine, a microtubule depolymerizing agent that induces the accumulation of autophagic vacuoles by preventing their degradation, increased the accumulation of MDC and altered the distribution and size of the autophagic vacuoles. Our results indicate that in the presence of vinblastine very large MDC-vacuoles accumulated mainly under starvation conditions, indicating that the expansion of autophagosomes is upregulated by amino acid deprivation. Furthermore, these MDC-vacuoles were labeled with LC3, one of the mammalian homologues of the yeast protein Apg8/Aut7 that plays an important role in autophagosome formation.

Rab22a affects the morphology and function of the endocytic pathway.

Abstract: Soon after endocytosis, internalized material is sorted along different pathways in a process that requires the coordinated activity of several Rab proteins. Although abundant information is available about the subcellular distribution and function of some of the endocytosis-specific Rabs (e.g. Rab5 and Rab4), very little is known about some other members of this family of proteins. To unveil some of the properties of Rab22a, one of the less studied endosome-associated small GTPases, we have expressed the protein tagged with the green fluorescent protein in CHO cells. The results indicate that Rab22a associates with early and late endosomes (labeled by a 5 minute rhodamine-transferrin uptake and the cation-independent mannose 6-phosphate receptor, respectively) but not with lysosomes (labeled by 1 hour rhodamine horseradish peroxidase uptake followed by 1 hour chase). Overexpression of the protein causes a prominent morphological enlargement of the early and late endosomes. Two mutants were generated by site-directed mutagenesis, a negative mutant (Rab22aS19N, with reduced affinity for GTP) and a constitutively active mutant (Rab22aQ64L, with reduced endogenous GTPase activity). The distribution of the negative mutant was mostly cytosolic, whereas the positive mutant associated with early and late endosomes and, interestingly also with lysosomes and autophagosomes (labeled with monodansylcadaverine). Cells expressing Rab22a wild type and Rab22aS19N displayed decreased endocytosis of a fluid phase marker. Conversely, overexpression of Rab22aQ64L, which strongly affects the morphology of endosomes, did not inhibit bulk endocytosis. Our results show that Rab22a has a unique distribution along the endocytic pathway that is not shared by any other Rab protein, and that it strongly affects the morphology and function of endosomes.

Experimental evidence of stochastic resonance without tuning due to non-Gaussian noises.

Abstract: In order to test theoretical predictions, we have studied the phenomenon of stochastic resonance in an electronic experimental system driven by white non-Gaussian noise. In agreement with the theoretical predictions our main findings are an enhancement of the sensibility of the system together with a remarkable widening of the response (robustness). This implies that even a single resonant unit can reach a marked reduction in the need for noise tuning.

Ultrastructural and morphometric study of the sertoli cell of the viscacha (Lagostomus maximus maximus) during the annual reproductive cycle

Abstract: Changes in the morphology of viscacha Sertoli cells were studied during the annual reproductive cycle. Sertoli cells exhibited marked nuclear and cytoplasmic changes. Seasonal variation in nuclear size and shape, chromatin texture, and nucleolus characteristics was observed. The seasonal patterns of the volume densities of the endoplasmic reticulum (ER), mitochondria, Golgi complex, dense bodies and lipid inclusions were distinct. Morphometric analysis revealed that the Golgi complex is the organelle most sensitive to seasonal change. It declined drastically in the regressed testes and its recovery was slow. The ER and mitochondria exhibited seasonal variations in their pattern and content, that was minimal during winter. In contrast, an accumulation of lipid and dense bodies, such as primary and secondary lysosomes, accompanied the spermatogenic arrest. The volume densities of both organelles were maximum during the restoration of spermatogenesis. The length and organization of the inter-Sertoli junctions also changed with the reproductive cycle. The Sertoli cell number per tubular cross section decreased significantly during the testicular regression, coincident with the presence of Sertoli cells with marked signs of involution. The degree of regression and recovery exhibited by the viscacha Sertoli cells was closely related to that shown by the associated germ cells. Therefore, seasonal endocrine fluctuations and local factors could be involved in the regulation of the morphological and functional characteristics of the viscacha Sertoli cells. These hormonal fluctuations are synchronized by the photoperiod through the pineal gland and its hormone, melatonin. Anat Rec 262:176–185, 2001. © 2001 Wiley-Liss, Inc.

Magnetic relaxation in bulk and film manganite compounds

Abstract: We have investigated the time dependence of the magnetic and transport properties of ${\mathrm{La}}_{0.6}{\mathrm{Sr}}_{0.4}{\mathrm{MnO}}_{3}$ films and bulk samples. A significant magnetic aftereffect has been observed in all the samples through magnetization and resistivity measurements. The relaxation effects can be described by the addition of a logarithmic to an exponential contribution. The weight of each contribution is strongly temperature dependent, with a relaxation that is predominantly logarithmic at low temperatures $(Tl30 \mathrm{K})$ and exponential at high temperatures $(Tg100 \mathrm{K}).$ As the film thickness is decreased the magnetic relaxation becomes more important and the magnetic viscosity of the system increases. These results point out the important role of structural defects in the magnetic relaxation of the samples.

Stability of the Feasible Set Mapping in Convex Semi-Infinite Programming

Abstract: In this paper we approach the stability analysis of the feasible set mapping in convex semi-infinite programming for an arbitrary index set. More precisely, we establish its closedness and study the semicontinuity, in the sense of Berge, of this multivalued mapping- A certain metric is proposed in order to measure the distance between nominal and perturbed problems. Since we do not require any structure to the index set, our results cover the ordinary convex programming problem.

Detection of the mouse acrosome reaction by acid phosphatase. Comparison with chlortetracycline and electron microscopy.

Abstract: The sperm acrosome is a uniquely regulated secretory vesicle containing several hydrolase enzymes, including acid phosphatase (AP). The exocytotic event that releases these enzymes, the acrosome reaction, is required for fertilization in mammals. Different methods have been described in the scientific literature for detection of the acrosome reaction: double and triple stains, fluorescent-lectin stains, monoclonal antibodies against acrosomal antigens (immunodetection techniques), Coomassie blue, differential interference contrast or phase contrast, flow cytometry, and chlortetracycline (CTC). In contrast, only 1 method to detect AP released by live and reacted sperm has been described in the literature thus far. In this work we compare 2 classical methods, CTC and transmission electron microscopy (TEM), with the assay of AP released from the acrosome. AP released during the acrosome reaction was measured in the culture medium. Enzyme remaining in nonreacted sperm cells was released by Triton X-100 treatment. This enzyme-based methodology shows an increase of AP in the culture media after the acrosome reaction and a corresponding decrease in the detergent-releasable enzyme. The AP assay thus permits the detection of the mouse acrosome reaction and compares well with the CTC and TEM methods. This method is performed on the whole sperm population and so avoids the observer error that is inherent in light microscopic methods.

Recruitment of coat-protein-complex proteins on to phagosomal membranes is regulated by a brefeldin A-sensitive ADP-ribosylation factor

Abstract: Particle internalization in macrophages is followed by a complex maturation process. We have previously observed that proteins bound to phagocytosed particles are sorted from phagosomes into a heterogeneous population of vesicles that fuse with endosomes. However, the mechanism and the protein machinery involved in the formation of these phagosome-derived vesicles are largely unknown. It has been shown that vesicles coated with coat protein complex type I (COPI) have a role in both secretion and endocytosis. To address the possibility that COPI proteins might participate in the formation of phagosome-derived vesicles we studied the recruitment of beta-COP to highly purified phagosomes. The binding of beta-COP to phagosomal membranes was regulated by nucleotides and inhibited by brefeldin A (BFA). An ADP-ribosylation factor 1 (ARF1) mutant defective in GTP hydrolysis supported the binding of beta-COP to phagosomes independently of added nucleotide. AlF(4) and Gbetagamma subunits, agents known to modulate heterotrimeric G-protein activity, were tested in the beta-COP binding assay. AlF(4) increased beta-COP association, whereas binding was inhibited by the addition of Gbetagamma subunits. Our results suggest that COP proteins are recruited to phagosomal membranes by a mechanism that involves heterotrimeric GTP-binding proteins and a BFA-sensitive ARF. In addition, our findings indicate that COPI proteins are involved in the recycling of components from phagosomes to the cell surface.

Functional regional distribution of histamine receptors in the rat hippocampus: modulation of learning of an active avoidance response.

Abstract: The possible functional regional distribution of the histamine sensitive sites controlling the behavioral performance of learning of an active avoidance response in the hippocampal structure was studied. Adult male rats were stereotaxically implanted with microinjection cannula into three different regions of the hippocampus: the ventral, the dorsal posterior and the dorsal anterior hippocampus. Different groups of rats were microinjected into these subregions with either saline, 9, 45 or 90 nmol of histamine. Five min later all groups were subjected to learning of a conditioned response to an ultrasonic tone warning an electric shock to the feet of the animals. Three sessions of 8 trials were necessary in order to control animals learn the task. Results show that histamine microinjections into the ventral hippocampus were able to significantly inhibit the natural decrease of the latency to escape during trials, impairing the efficiency of learning. Histamine administration into the dorsal posterior hippocampus was slightly effective in prolonging latency and impairing learning, while injections into the dorsal anterior region of the hippocampus had no effect. Results give a further support to the idea that histamine sensitive neurons in the ventral hippocampus are modulating the memory and learning mechanisms in the rat. In addition, evidence has been found suggesting a regional distribution of histamine sensitive neurons controlling learning of avoidance responses along the septo-temporal axis of the rat hippocampus.

Sencillo dispositivo para determinar la posición del centro de masa de un sistema

Abstract: Con una regla de madera modificada y algunos otros elementos de bajo costo se puede armar un equipo para determinar el centro de masa de un sistema Los resultados presentan un excelente acuerdo con los obtenidos teoricamente El equipo permite mostrar que la posicion de este punto es independiente del sistema de referencia elegido y, ademas, revisar conceptos basicos de mecanica

Population screening of F508del (DeltaF508), the most frequent mutation in the CFTR gene associated with cystic fibrosis in Argentina.

Abstract: Cystic fibrosis (CF) is the most common autosomal recessive disease in the Caucasian population. The disease can be caused by one of the more than 900 different mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. However, the deletion of the phe508-codon is the most prevalent mutation observed. Our aim was to perform a screening for this mutation (ΔF508, or F508del) in the population of Mendoza, Argentina. For the screening, 1,000 blood samples were obtained from CF asymptomatic individuals and combined into 100 pools each containing 10 different blood samples. Pools containing at least one F508del carrier were detected by heteroduplex formation during the PCR amplification of exon 10. The PCR was designed to introduce a recognition site for a restriction enzyme that confirmed the presence of the deletion F508del in the positive pools. The results with this simple method indicate a frequency of carriers in the Mendoza population of 2.1% (1.3%-3.2, 95% confidence limits). The observed frequency of carriers is similar to that reported for European populations. Hum Mutat 18:167, 2001. © 2001 Wiley-Liss, Inc.

Estudios culturales, transdisciplinariedad e interdisciplinariedad (¿hegemonismo en las ciencias sociales latinoamericanas?).

Abstract: Los cultural studies surgidos en su momento en Inglaterra y posteriormente trasladados a los Estados Unidos, han ganado fuerte presencia en las ciencias sociales latinoamericanas en las dos ultimas decadas, a partir de su version local, expresada en autores como NGarcia Canclini, ROrtiz o Jesus MBarbero Su postura en favor de un abandono de los limites tradicionales entre las disciplinas sociales, les ha abierto las puertas a la vez en los mas variados campos de las ciencias sociales: comunicologia, antropologia, literatura y arte en general, incluso filosofia (a traves del tema modernidad/posmodernidad) La raigambre en los estudios culturales sajones no aparece explicitamente y los origenes en el campo del marxismo han sido abandonados, derivando hacia posiciones diversas en cuanto a la aceptacion o rechazo de manifestaciones de la actual sociedad de mercado globalizada (caso del consumo) Se hace necesario establecer una reflexion acerca de la validez y las limitaciones que pueda evaluarse en esta linea de investigacion, sus bases epistemologicas e ideologicas, y especialmente sus pretensiones interdisciplinarias

Perfil del docente y crisis cultural contemporanea

Abstract: This article aims to portray the most decisive challenges put forward by Argentina’s Federal Law of Education concerning the training and the action of teachers. With this in mind, special attention will be paid to the social-cultural environment of these policies, which more than an external context are indeed the solid ground that makes possible the apprehension of the educational process and the school system.

Threshold Reaction Cross Sections of Nickel Isotopes, Averaged Over a 235U Fission Neutron Spectrum

Abstract: We have measured, for nickel isotopes, several threshold reaction cross sections averaged over a 235U fission neutron spectrum. The measured reactions, and corresponding averaged cross sections fou...

Permeability barrier in the mantle epithelium lining the testis in the apple-snail Pomacea canaliculata (Gastropoda: Ampullariidae).

Abstract: Intercellular junctions are studied in the epithelium lining the testis of the freshwater snailPomacea canaliculata by conventional staining and lanthanum tracer techniques. The junctional complex consists of belt desmosomes and septate junctions. Septate junctions are of the pleated-sheet type and they are constantly associated with mitochondria. Gap and tight junctions appear to be absent. These septate junctions seem to be the structural correlate of an epithelial permeability barrier that separate the testis from the extrapallial space where the shell elements are deposited. These junctions may contribute to a functional barrier in the male gonad of Pomacea canaliculata. The results indicate that freshwater prosobranchs have junctional structures very close to those found in other molluscs.