The Netrin-4/Laminin γ1/Neogenin-1 complex mediates migration in SK-N-SH neuroblastoma cells

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It arises during development of the sympathetic nervous system. Netrin-4 (NTN4), a laminin-related protein, has been proposed as a key factor to target NB metastasis, although there is controversy about its function. Here, we show that NTN4 is broadly expressed in tumor, stroma and blood vessels of NB patient samples. Furthermore, NTN4 was shown to act as a cell adhesion molecule required for the migration induced by Neogenin-1 (NEO1) in SK-N-SH neuroblastoma cells. Therefore, we propose that NTN4, by forming a ternary complex with Laminin γ1 (LMγ1) and NEO1, acts as an essential extracellular matrix component, which induces the migration of SK-N-SH cells.     

Distribution and antifungal susceptibility of Candida clinical isolations coming from six health care centers in the metropolitan area of Caracas, Venezuela (years 2003-2005)

The aim of this study was to investigate the frequency and antifungal susceptibility of Candida clinical isolations coming from patients with candidiasis in six health care centers of Caracas, Venezuela metropolitan area. The laboratory reports were retrospectively revised from January 2003 through August 2005. The isolated yeasts identification was carried out by conventional methods and antifungal susceptibility was evaluated by ATB-fungus (bioMérieux, France) and Etest (AB Biodisk, Solna, Sweden). One thousand nine hundred seventy seven (1.977) yeasts were studied and their susceptibility testing were carried out only in 1,414 of them. C. albicans was the most isolated yeast (46.7%) and none-albicans Candida-species represented more than half of the isolations (53.4%). All the isolated yeasts evaluated presented CMIs<1 microg/ml to anfotericina B and showed variable susceptibility percentages to fluconazole (91.5%), itraconazole (80%) and voriconazole (98.6%).     

Propensity of lignin to associate: light scattering photometry study with native lignins

Many studies of lignins in solution invoke association and aggregation phenomena to explain their solution behavior (e.g., reprecipitation onto pulp fibers, condensation, etc.). Following their colloidal (apparent) molecular weights in solution as a function of time allows us to explore observable dissociation phenomena. These measurements were carried out using multiple angle laser light scattering (MALLS) photometry in the static mode. The challenges and opportunities of measuring the specific refractive index increment (dn/dC) of lignin solutions and determining the kinetics of the dissociation process were thus investigated. Hardwood and softwood representative lignins were isolated, and method for their full dissolution in THF was further developed, which then lead to accurate dn/dC values being obtained as a function of time. When coupled to additional work using light scattering static measurements and Zimm plots for the same solutions, this effort offers insight into the aggregation and ensuing dissociative events that operate within the lignin macromolecules.     

Regulation of phosphoenolpyruvate carboxylase phosphorylation by metabolites and abscisic acid during the development and germination of barley seeds

During barley (Hordeum vulgare) seed development, phosphoenolpyruvate carboxylase (PEPC) activity increased and PEPC-specific antibodies revealed housekeeping (103-kD) and inducible (108-kD) subunits. Bacterial-type PEPC fragments were immunologically detected in denatured protein extracts from dry and imbibed conditions; however, on nondenaturing gels, the activity of the recently reported octameric PEPC (in castor [Ricinus communis] oil seeds) was not detected. The phosphorylation state of the PEPC, as judged by l-malate 50% inhibition of initial activity values, phosphoprotein chromatography, and immunodetection of the phosphorylated N terminus, was found to be high between 8 and 18 d postanthesis (DPA) and during imbibition. In contrast, the enzyme appeared to be in a low phosphorylation state from 20 DPA up to dry seed. The time course of 32/36-kD, Ca(2+)-independent PEPC kinase activity exhibited a substantial increase after 30 DPA that did not coincide with the PEPC phosphorylation profile. This kinase was found to be inhibited by l-malate and not by putative protein inhibitors, and the PEPC phosphorylation status correlated with high glucose-6-phosphate to malate ratios, thereby suggesting an in vivo metabolic control of the kinase. PEPC phosphorylation was also regulated by photosynthate supply at 11 DPA. In addition, when fed exogenously to imbibing seeds, abscisic acid significantly increased PEPC kinase activity. This was further enhanced by the cytosolic protein synthesis inhibitor cycloheximide but blocked by protease inhibitors, thereby suggesting that the phytohormone acts on the stability of the kinase. We propose that a similar abscisic acid-dependent effect may contribute to produce the increase in PEPC kinase activity during desiccation stages.     

Central Role of the Oxygen-dependent Degradation Domain of Drosophila HIFα/Sima in Oxygen-dependent Nuclear Export

The Drosophila HIFα homologue, Sima, is localized mainly in the cytoplasm in normoxia and accumulates in the nucleus upon hypoxic exposure. We have characterized the mechanism governing Sima oxygen-dependent subcellular localization and found that Sima shuttles continuously between the nucleus and the cytoplasm. We have previously shown that nuclear import depends on an atypical bipartite nuclear localization signal mapping next to the C-terminus of the protein. We show here that nuclear export is mediated in part by a CRM1-dependent nuclear export signal localized in the oxygen-dependent degradation domain (ODDD). CRM1-dependent nuclear export requires both oxygen-dependent hydroxylation of a specific prolyl residue (Pro850) in the ODDD, and the activity of the von Hippel Lindau tumor suppressor factor. At high oxygen tension rapid nuclear export of Sima occurs, whereas in hypoxia, Sima nuclear export is largely inhibited. HIFα/Sima nucleo-cytoplasmic localization is the result of a dynamic equilibrium between nuclear import and nuclear export, and nuclear export is modulated by oxygen tension.     

Changes in the Viral Distribution Pattern after the Appearance of the Novel Influenza A H1N1 (pH1N1) Virus in Influenza-Like Illness Patients in Peru

Background

We describe the temporal variation in viral agents detected in influenza like illness (ILI) patients before and after the appearance of the ongoing pandemic influenza A (H1N1) (pH1N1) in Peru between 4-January and 13-July 2009.

Methods

At the health centers, one oropharyngeal swab was obtained for viral isolation. From epidemiological week (EW) 1 to 18, at the US Naval Medical Research Center Detachment (NMRCD) in Lima, the specimens were inoculated into four cell lines for virus isolation. In addition, from EW 19 to 28, the specimens were also analyzed by real time-polymerase-chain-reaction (rRT-PCR).

Results

We enrolled 2,872 patients: 1,422 cases before the appearance of the pH1N1 virus, and 1,450 during the pandemic. Non-pH1N1 influenza A virus was the predominant viral strain circulating in Peru through (EW) 18, representing 57.8% of the confirmed cases; however, this predominance shifted to pH1N1 (51.5%) from EW 19–28. During this study period, most of pH1N1 cases were diagnosed in the capital city (Lima) followed by other cities including Cusco and Trujillo. In contrast, novel influenza cases were essentially absent in the tropical rain forest (jungle) cities during our study period. The city of Iquitos (Jungle) had the highest number of influenza B cases and only one pH1N1 case.

Conclusions

The viral distribution in Peru changed upon the introduction of the pH1N1 virus compared to previous months. Although influenza A viruses continue to be the predominant viral pathogen, the pH1N1 virus predominated over the other influenza A viruses.     

The role of alpha-glucosidase in germinating barley grains

The importance of α-glucosidase in the endosperm starch metabolism of barley (Hordeum vulgare) seedlings is poorly understood. The enzyme converts maltose to glucose (Glc), but in vitro studies indicate that it can also attack starch granules. To discover its role in vivo, we took complementary chemical-genetic and reverse-genetic approaches. We identified iminosugar inhibitors of a recombinant form of an α-glucosidase previously discovered in barley endosperm (ALPHA-GLUCOSIDASE97 [HvAGL97]), and applied four of them to germinating grains. All four decreased the Glc-to-maltose ratio in the endosperm 10 d after imbibition, implying inhibition of maltase activity. Three of the four inhibitors also reduced starch degradation and seedling growth, but the fourth did not affect these parameters. Inhibition of starch degradation was apparently not due to inhibition of amylases. Inhibition of seedling growth was primarily a direct effect of the inhibitors on roots and coleoptiles rather than an indirect effect of the inhibition of endosperm metabolism. It may reflect inhibition of glycoprotein-processing glucosidases in these organs. In transgenic seedlings carrying an RNA interference silencing cassette for HvAgl97, α-glucosidase activity was reduced by up to 50%. There was a large decrease in the Glc-to-maltose ratio in these lines but no effect on starch degradation or seedling growth. Our results suggest that the α-glucosidase HvAGL97 is the major endosperm enzyme catalyzing the conversion of maltose to Glc but is not required for starch degradation. However, the effects of three glucosidase inhibitors on starch degradation in the endosperm indicate the existence of unidentified glucosidase(s) required for this process.     

Relationship between Tobacco,cagA and vacA i1 Virulence Factors and Bacterial Load in Patients Infected by Helicobacter pylori

MethodsBiopsies of the gastric corpus and antrum from 155 consecutive patients in whom there was clinical suspicion of infection by H. pylori were processed. In 106 patients H. pylori infection was detected. Molecular methods were used to quantify the number of microorganisms and presence of cagA and vacA i1 genes. A standardized questionnaire was used to obtain patients’ clinical data and lifestyle variables, including tobacco and alcohol consumption. Adjusted Odds Ratios (OR_adjusted) were estimated by unconditional logistic regression.ResultscagA was significantly associated with active-smoking at endoscope: OR_adjusted 4.52. Evidence of association was found for vacA i1 (OR_adjusted 3.15). Bacterial load was higher in active-smokers, although these differences did not yield statistical significance (median of 262.2 versus 79.4 copies of H. pylori per cell).ConclusionsThe association between smoking and a higher risk of being infected by a virulent bacterial population and with higher bacterial load, support a complex interaction between H. pylori infection and environmental factors.     

The paracellular channel for water secretion in the upper segment of the Malpighian Tubule of Rhodnius prolixus

Lumen to bath J ₁₂/C ₁ and bath to lumen J ₂₁/ C ₂ fluxes per unit concentration of 19 probes with diameters (d ^m) ranging from 3.0–30.0 Å (water, urea, erythritol, mannitol, sucrose, raffinose and 13 dextrans with d ^m 9.1–30.0 Å) were measured during volume secretion (J _v ) in the upper segment of the Malpighian Tubule of Rhodnius by perfusing lumen and bath with ¹⁴C or ³H-labeled probes. J _net=(J ₁₂/C ₁ – J ₂₁/C ₂) was studied as a function of J _v · J _v was varied by using different concentrations of 5-hydroxy tryptamine. J _net for ³H-water was not different from J _v We found: (i) A strong correlation between J _net and J _v for 8 probes d ^m =3.0–11.8 Å (group a probes), indicating that the convective component of J _net is more important than its diffusive component and than unstirred layers effects which are negligible. Therefore group a probes are solvent dragged as they cross the epithelium, (ii) There is no correlation between J _net and J _v for 11 probes with d ^m=11.8–30 Å (group b). Therefore these probes must cross the epithelium by diffusion and not by solvent drag, (iii) In a plot of J _net/J _v vs. d ^m group a probes show a steep linear relation with a slope = –0.111, while for group b probes the slope is –0.002. Thus there is a break between groups a and b in this plot. We tried to fit the data with models for restricted diffusion and convention through cylindrical or parallel slit pathways. We conclude that (i) group a probes are dragged by water through an 11.0 Å-wide slit, (ii) Most of J _v must follow an extracellular noncytosolic pathway, (iii) Group b probes must diffuse through a 42 Å-wide slit, (iv) A cylindrical pathway does not fit the data.E.G. is a Visiting Scientist at IVIC. It is a pleasure to thank Drs. A.E. Hill and Bruria Shachar-Hill for their suggestion of the use of dextrans, their instruction and help with the dextran separation technique, and their extensive discussions. Dr. R. Apitz, Mr H. Rojas and Mrs. Fulvia Bartoli were most helpful with suggestions during the course of the experimental work. Mr. Jose Mora was fundamental help with the equipment. Mrs. Lelis Ochoa and Mr. Luis F. Alvarez helped with some of the drawings. This work was partially supported by CONICIT, Fundación Polar and CDCH of UCV. It is a pleasure to thank Dr. H. Passow and Dr. K.J. Ullrich at the Max Planck Institut für Biophysik (Frankfurt/Main) where this work was initiated.