Luminal localization of blood-brain barrier sodium, potassium adenosine triphosphatase is dependent on fixation.

Cytochemical data in the literature reporting localization of sodium, potassium adenosine triphosphatase (Na(+), K(+)-ATPase) in the blood-brain barrier (BBB) have been contradictory. Whereas some studies showed the enzyme to be located exclusively on the abluminal endothelial plasma membrane, others demonstrated it on both the luminal and abluminal membranes. The influence of fixation on localization of the enzyme was not considered a critical factor, but our preliminary studies showed data to the contrary. We therefore quantitatively investigated the effect of commonly used fixatives on the localization pattern of the enzyme in adult rat cerebral microvessels. Fixation with 1%, 2%, and 4% formaldehyde allowed deposition of reaction product on both the luminal and abluminal plasma membranes. The luminal reaction was reduced with increasing concentration of formaldehyde. Glutaraldehyde at 0.1%, 0.25%, 0.5%, in combination with 2% formaldehyde, drastically inhibited the luminal reaction. The abluminal reaction was not significantly altered in all groups. These results show that luminal localization of BBB Na(+), K(+)-ATPase is strongly dependent on fixation. The lack of luminal localization, as reported in the literature, may have been the result of fixation. The currently accepted abluminal polarity of the enzyme should be viewed with caution.     

Modular TRAPP complexes regulate intracellular protein trafficking through multiple Ypt/Rab GTPases in Saccharomyces cerevisiae

Ypt/Rab are key regulators of intracellular trafficking in all eukaryotic cells. In yeast, Ypt1 is essential for endoplasmic reticulum (ER)-to-Golgi transport, whereas Ypt31/32 regulate Golgi-to-plasma membrane and endosome-to-Golgi transport. TRAPP is a multisubunit complex that acts as an activator of Ypt/Rab GTPases. Trs85 and Trs130 are two subunits specific for TRAPP III and TRAPP II, respectively. Whereas TRAPP III was shown to acts as a Ypt1 activator, it is still controversial whether TRAPP II acts as a Ypt1 or Ypt31/32 activator. Here, we use GFP-Snc1 as a tool to study transport in Ypt and TRAPP mutant cells. First, we show that expression of GFP-Snc1 in trs85Δ mutant cells results in temperature sensitivity. Second, we suggest that in ypt1ts and trs85Δ, but not in ypt31Δ/32ts and trs130ts mutant cells, GFP-Snc1 accumulates in the ER. Third, we show that overexpression of Ypt1, but not Ypt31/32, can suppress both the growth and GFP-Snc1 accumulation phenotypes of trs85Δ mutant cells. In contrast, overexpression of Ypt31, but not Ypt1, suppresses the growth and GFP-Snc1 transport phenotypes of trs130ts mutant cells. These results provide genetic support for functional grouping of Ypt1 with Trs85-containing TRAPP III and Ypt31/32 with Trs130-containing TRAPP II.     

An evaluation of information content as a metric for the inference of putative conserved noncoding regions in DNA sequences using a genetic algorithms approach

In previous work, we presented GAMI, an approach to motif inference that uses a genetic algorithms search. GAMI is designed specifically to find putative conserved regulatory motifs in noncoding regions of divergent species, and is designed to allow for analysis of long nucleotide sequences. In this work, we compare GAMI's performance when run with its original fitness function (a simple count of the number of matches) and when run with information content, as well as several variations on these metrics. Results indicate that information content does not identify highly conserved regions, and thus is not the appropriate metric for this task, while variations on information content as well as the original metric succeed in identifying putative conserved regions.     

Synthesis and Biological Evaluation of New Natural Phenolic (2E,4E,6E)‐Octa‐2,4,6‐trienoic Esters

In the present study the esterification of the OH groups of resveratrol, caffeic acid, ferulic acid, and β‐sitosterol with an antioxidant polyconjugated fatty acid, (2E,4E,6E)‐octa‐2,4,6‐trienoic acid, was achieved. As the selective esterification of OH groups of natural compounds can affect their biological activity, a selective esterification of resveratrol and caffeic acid was performed by an enzymatic approach. The new resulting compounds were characterized spectroscopically (FT‐IR, NMR mono, and bidimensional techniques); when necessary the experimental data were integrated by quantum chemical calculations. The antioxidant, anti‐inflammatory and proliferative activity was evaluated. The good results encourage the use of these molecules as antioxidant and/or anti‐inflammatory agents in dermocosmetic application.     

Mutagenicity of antiamebic and anthelmintic drugs in the Salmonella typhimurium microsomal test system

4 amebicides (chloroquine diphosphate, diiodohydroxyquin, iodochlorohydroxyquin and dehydroemetine) and 6 anthelmintics (bephenium hydroxynaphthoate, 4-hexylresorcinol, mebendazole, niclosamide, pyrantel pamoate and pyrvinium pamoate) were tested for mutagenicity in the Salmonella typhimurium microsomal test system. Frameshift mutations were induced by dehydroemetine and niclosamide following activation by microsomal enzymes, while pyrvinium pamoate induced both frameshift and base-pair substitution mutations with or without metabolic activation. The urine of mice treated with dehydroemetine or pyrvinium pamoate showed no mutagenic activity. However, urine obtained from mice treated with niclosamide was mutagenic in strains TA98 and TA1538. The fluctuation assay showed chloroquine diphosphate to be mutagenic in TA1537, a strain which detects frameshift mutations.     

Validation of a fish-based index of biotic integrity for streams and rivers of central Mexico

An existing version of a fish assemblage-basedindex of biotic integrity (IBI) for the streamsand rivers of west central Mexico was testedwith independent data to validate itsusefulness as a measure of ecosystem qualityand to determine the geographic area where itis effective. Fish assemblages from 63 uplandsites in 10 basins in central Mexico(Armería, Ameca, Coahuayana, Marabasco,Purificación, Grande de Morelia, Grande deSantiago, Lerma, Balsas and Pánuco) wereassessed using the metrics and scoring criteriafrom the existing IBI and then compared withindependent evaluations of habitat and waterquality. IBI scores were congruent withhabitat and water quality values in theArmería, Purificación and Marabascobasins, where the IBI was first developed, aswell as in the adjacent Ameca and Coahuayanabasins. We conclude that the IBI can be usedwithout modification to assess environmentalquality in non-coastal streams and riverswithin these five basins. Further data areneeded from the Grande de Morelia, Grande deSantiago and middle Lerma basins, but ourresults suggest that the existing IBI may alsobe effective here. However, the existing IBIdoes not consistently reflect habitat and waterquality conditions in the Balsas and Pánucobasins and must be modified before it can beapplied there. Necessary modifications in theBalsas basin appear to be small and relatedprimarily to changes in the scoring criteriafor metrics. However, in the Pánuco basinmore substantive changes in the nature of themetrics are required. Changes in the IBI forthese basins are proposed. The IBI is nowvalidated for use in river monitoring,conservation and restoration efforts in 5basins in west central Mexico and suggestionsfor its application in other basins areavailable here.     

Reproductive biology of the invasive species Pseudoxiphophorus bimaculatus and Poecilia sphenops in the Teuchitlán River,México

Reproductive biology of invasive species is not often studied relative to the invasion process, although it may provide an accurate indicator of the invasion stage. We evaluated the reproductive biology of the exotic fish species Pseudoxiphophorus bimaculatus and Poecilia sphenops in the Teuchitlán River, Jalisco, Mexico by fertility, size at first maturity, sex ratio, gonad maturity stage, gonadosomatic index, condition factor, size‐structure, and habitat. The reproductive variables were related to environmental characteristics using the non‐metric analysis of multidimensional scaling. A total of 1374 specimens of P. bimaculatus and 571 of P. sphenops were captured by seine netting and electrofishing. Maximum fertility of P. bimaculatus was 15.99 ± 2.27 embryonated eggs and embryos and, for P. sphenops, 31.26 ± 4.17. Females predominated among P. bimaculatus, while in P. sphenops the sex ratio was ~1:1. We found mature female and male of P. bimaculatus in degraded sites and juveniles in the springs. Poecilia sphenops reproduced along the river. The establishment of both invasive species in the Teuchitlán River is evidence that they share the reproductive habitat with native fish species, and tend to spread and colonize new areas.     

Trophic control changes with season and nutrient loading in lakes

Experiments have revealed much about top‐down and bottom‐up control in ecosystems, but manipulative experiments are limited in spatial and temporal scale. To obtain a more nuanced understanding of trophic control over large scales, we explored long‐term time‐series data from 13 globally distributed lakes and used empirical dynamic modelling to quantify interaction strengths between zooplankton and phytoplankton over time within and across lakes. Across all lakes, top‐down effects were associated with nutrients, switching from negative in mesotrophic lakes to positive in oligotrophic lakes. This result suggests that zooplankton nutrient recycling exceeds grazing pressure in nutrient‐limited systems. Within individual lakes, results were consistent with a ‘seasonal reset’ hypothesis in which top‐down and bottom‐up interactions varied seasonally and were both strongest at the beginning of the growing season. Thus, trophic control is not static, but varies with abiotic conditions – dynamics that only become evident when observing changes over large spatial and temporal scales.     

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (P_f) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the ‘PfFit’), to yield P_f.     

Desmoglein-2: a novel regulator of apoptosis in the intestinal epithelium

Intestinal epithelial intercellular junctions regulate barrier properties, and they have been linked to epithelial differentiation and programmed cell death (apoptosis). However, mechanisms regulating these processes are poorly defined. Desmosomes are critical elements of intercellular junctions; they are punctate structures made up of transmembrane desmosomal cadherins termed desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) that affiliate with the underlying intermediate filaments via linker proteins to provide mechanical strength to epithelia. In the present study, we generated an antibody, AH12.2, that recognizes Dsg2. We show that Dsg2 but not another desmosomal cadherin, Dsc2, is cleaved by cysteine proteases during the onset of intestinal epithelial cell (IEC) apoptosis. Small interfering RNA-mediated down-regulation of Dsg2 protected epithelial cells from apoptosis. Moreover, we report that a C-terminal fragment of Dsg2 regulates apoptosis and Dsg2 protein levels. Our studies highlight a novel mechanism by which Dsg2 regulates IEC apoptosis driven by cysteine proteases during physiological differentiation and inflammation.