Voluntary locomotor activity mitigates oxidative damage associated with isolation stress in the prairie vole (Microtus ochrogaster)

Organismal performance directly depends on an individual''s ability to cope with a wide array of physiological challenges. For social animals, social isolation is a stressor that has been shown to increase oxidative stress. Another physiological challenge, routine locomotor activity, has been found to decrease oxidative stress levels. Because we currently do not have a good understanding of how diverse physiological systems like stress and locomotion interact to affect oxidative balance, we studied this interaction in the prairie vole (Microtus ochrogaster). Voles were either pair housed or isolated and within the isolation group, voles either had access to a moving wheel or a stationary wheel. We found that chronic periodic isolation caused increased levels of oxidative stress. However, within the vole group that was able to run voluntarily, longer durations of locomotor activity were associated with less oxidative stress. Our work suggests that individuals who demonstrate increased locomotor activity may be better able to cope with the social stressor of isolation.     

Effects of surface roughness on the coefficients of friction in model orthodontic systems

Orthodontists, like others (Engel, P.A. (1976) Impact Wear of Materials. Elsevier Scientific, New York.), often equate the smoothness of surfaces with the absence of friction. To investigate whether the surface roughness of opposing materials influence the coefficients of friction and ultimately the movement of teeth, arch wires were slid between contact flats to simulate orthodontic arch wire-bracket appliances. From laser specular reflectance measurements, the RMS surface roughness of these arch wires varied from 0.04 microns for stainless steel to 0.23 microns for nickel titanium. Using the same technique, the roughnesses of the contact flats varied from 0.03 microns for the 1 micron lapped stainless steel, to 0.26 microns for the as-received alumina. After each of the arch wire-contact flat couples was placed in a friction tester, fifteen normal forces were systemically applied at 34 degrees C. From plots of the static and kinetic frictional forces vs the normal forces, dry coefficients of friction was obtained that were greater than those reported in the dental literature. The all-stainless steel couples had lower kinetic coefficients (0.120-0.148) than the stainless steel-polycrystalline alumina couple (0.187). When pressed against the various flats, the beta-titanium arch wire (RMS = 0.14 microns) had the highest coefficients of friction (0.445-0.658), although the nickel titanium arch wire was the roughest (RMS = 0.23 microns). Scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX) verified that mass transfer of the beta-titanium arch wire occurred by adhesion onto the stainless steel flats or by abrasion from the sharply faceted polycrystalline alumina flats.     

The MDCK variety pack: choosing the right strain

The MDCK cell line provides a tractable model for studying protein trafficking, polarity and junctions (tight, adherens, desmosome and gap) in epithelial cells. However, there are many different strains of MDCK cells available, including the parental line, MDCK I, MDCK II, MDCK.1, MDCK.2, superdome and supertube, making it difficult for new researchers to decide which strain to use. Furthermore, there is often inadequate reporting of strain types and where cells were obtained from in the literature. This review aims to provide new researchers with a guide to the different MDCK strains and a directory of where they can be obtained. We also hope to encourage experienced researchers to report the stain and origin of their MDCK cells.     

Evidence of an increased incidence of day 3 parasitaemia in Suriname: an indicator of the emerging resistance of Plasmodium falciparum to artemether

The emerging resistance to artemisinin derivatives that has been reported in South-East Asia led us to assess the efficacy of artemether-lumefantrine as the first line therapy for uncomplicated Plasmodium falciparum infections in Suriname. This drug assessment was performed according to the recommendations of the World Health Organization in 2011. The decreasing number of malaria cases in Suriname, which are currently limited to migrating populations and gold miners, precludes any conclusions on artemether efficacy because adequate numbers of patients with 28-day follow-up data are difficult to obtain. Therefore, a comparison of day 3 parasitaemia in a 2011 study and in a 2005/2006 study was used to detect the emergence of resistance to artemether. The prevalence of day 3 parasitaemia was assessed in a study in 2011 and was compared to that in a study in 2005/2006. The same protocol was used in both studies and artemether-lumefantrine was the study drug. Of 48 evaluable patients in 2011, 15 (31%) still had parasitaemia on day 3 compared to one (2%) out of 45 evaluable patients in 2005/2006. Overall, 11 evaluable patients in the 2011 study who were followed up until day 28 had negative slides and similar findings were obtained in all 38 evaluable patients in the 2005/2006 study. The significantly increased incidence of parasite persistence on day 3 may be an indication of emerging resistance to artemether.     

Molecular Basis of Mucopolysaccharidosis Type IIIB in Emu (Dromaius novaehollandiae): An Avian Model of Sanfilippo Syndrome Type B<

Sanfilippo syndrome type B, or mucopolysaccharidosis (MPS) IIIB, is an autosomal recessive disease caused by a deficiency of lysosomal α-N-acetylglucosaminidase (NAGLU). In Dromaius novaehollandiae (emu), a progressive neurologic disease was recently discovered, which was characterized by NAGLU deficiency and heparan sulfate accumulation. To define the molecular basis, the sequences of the normal emu NAGLU cDNA and gene were determined by PCR-based approaches using primers for highly conserved regions of evolutionarily distant NAGLU homologues. It was observed that the emu NAGLU gene is structurally similar to that of human and mouse, but the introns are considerably shorter. The cDNA had an open reading frame (ORF) of 2259 bp. The deduced amino acid sequence is estimated to share 64% identity with human, 63% with mouse, 41% with Drosophila, 39% with tobacco, and 35% with the Caenorhabditis elegans enzyme. Three normal and two affected emus were studied for nucleotide sequence covering the entire coding region and exon–intron boundaries. Unlike the human gene, emu NAGLU appeared to be highly polymorphic: 19 variations were found in the coding region alone. The two affected emus were found to be homozygous for a 2-bp deletion, 1098-1099delGG, in exon 6. The resulting frameshift predicts a longer ORF of 2370 bp encoding a polypeptide with 37 additional amino acids and 387 altered amino acids. The availability of mutation screening in emus now permits early detection of MPS IIIB in breeding stocks and is an important step in characterizing this unique, naturally occurring avian model for the development of gene transfer studies.     

Phosphoinositide hydrolysis in mitogen-stimulated human peripheral-blood T lymphocytes.

Both phytohaemagglutinin and antibodies to the CD3 molecule induced proliferation and phosphoinositide hydrolysis in human peripheral-blood T lymphocytes, but the magnitude of the inositol phosphate response was small and the rate of accumulation slow [significant increases in Ins(1,4,5)P3 were observed only after 10 min]. Hence this response differs from the well-characterized Ins(1,4,5)P3 responses of many other systems. This slow response, its abrogation in Ca2+-depleted medium, the slow and maintained increase in Ca2+ as measured by Quin-2, and the ability of the Ca2+ ionophore A23187 to stimulate Ins(1,4,5)P3 accumulation all suggest that the increase in Ins(1,4,5)P3 occurs, at least in part, as a result of receptor-mediated Ca2+ influx in mitogen-stimulated T lymphocytes.     

Arabidopsis FAB1/PIKfyve Proteins Are Essential for Development of Viable Pollen

Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] is a phospholipid that has a role in controlling membrane trafficking events in yeast and animal cells. The function of this lipid in plants is unknown, although its synthesis has been shown to be up-regulated upon osmotic stress in plant cells. PtdIns(3,5)P₂ is synthesized by the PIKfyve/Fab1 family of proteins, with two orthologs, FAB1A and FAB1B, being present in Arabidopsis (Arabidopsis thaliana). In this study, we attempt to address the role of this lipid by analyzing the phenotypes of plants mutated in FAB1A and FAB1B. It was not possible to generate plants homozygous for mutations in both genes, although single mutants were isolated. Both homozygous single mutant plant lines exhibited a leaf curl phenotype that was more marked in FAB1B mutants. Genetic transmission analysis revealed that failure to generate double mutant lines was entirely due to inviability of pollen carrying mutant alleles of both FAB1A and FAB1B. This pollen displayed severe defects in vacuolar reorganization following the first mitotic division of development. The presence of abnormally large vacuoles in pollen at the tricellular stage resulted in the collapse of the majority of grains carrying both mutant alleles. This demonstrates a crucial role for PtdIns(3,5)P₂ in modulating the dynamics of vacuolar rearrangement essential for successful pollen development. Taken together, our results are consistent with PtdIns(3,5)P₂ production being central to cellular responses to changes in osmotic conditions.Phosphoinositides make up a minor fraction of total membrane lipids in all eukaryotic organisms. Their production is spatially restricted to the cytoplasmic leaflet of specific organellar membranes and temporally regulated by phosphatidylinositol (PtdIns) kinases and phosphatases. Three of the five hydoxyl groups of PtdIns can be phosphorylated, either singly or combinatorially, to produce seven different phosphoinositides. These different phosphoinositides can recruit and/or activate proteins with specific phosphoinositide-binding domains and have been implicated in the regulation of many important cellular functions, including membrane trafficking, cell growth, and cytoskeleton remodeling (Di Paolo and De Camilli, 2006).In animal cells, phosphorylation at the 3 position of PtdIns and its phosphorylated derivatives can be carried out by three different classes of PtdIns 3-kinase (classes I–III; Cantley, 2002). Plants and yeast only have class III PtdIns 3-kinases that are orthologs of the Saccharomyces cerevisiae protein Vps34p (Mueller-Roeber and Pical, 2002). Vps34p orthologs are thought to use PtdIns as their sole lipid substrate and produce PtdIns 3-phosphate (PtdIns3P). PtdIns3P is involved in endosomal/lysosomal protein sorting in eukaryotic cells in addition to cellular signaling events (Backer, 2008).In plants, PtdIns3P is essential for normal growth and development. Arabidopsis (Arabidopsis thaliana) plants carrying a VPS34 antisense construct have severe developmental defects (Welters et al., 1994). Furthermore, using pharmacological inhibitors of PtdIns3P production and analysis of transgenic plants defective in downstream signaling from PtdIns3P, it has been shown that this lipid has a role to play in many diverse physiological processes, such as root hair growth (Lee et al., 2008a). The phenotypes observed in studies of PtdIns3P function in plants are consistent with a role in endosomal and vacuolar trafficking in plants (Kim et al., 2001; Lee et al., 2008a), as in other eukaryotes. Recently, an attempt to generate vps34 homozygous mutant plant lines was unsuccessful due to failure of the mutant vps34 allele to transmit through the male germ line (Lee et al., 2008b).Importantly, PtdIns3P is the precursor to another phosphoinositide, PtdIns 3,5-bisphosphate [PtdIns(3,5)P₂], which also has vital roles in endosomal trafficking in eukaryotes (Dove et al., 2009). Thus, it is possible that some of the effects in plants attributed to PtdIns3P in previous studies may actually be due to an inability of cells to produce PtdIns(3,5)P₂. PtdIns(3,5)P₂ is produced by the PtdIns3P 5-kinases PIKfyve and Fab1p in animal and yeast cells, respectively. PIKfyve/Fab1p proteins have an N-terminal FYVE domain necessary for binding to PtdIns3P-containing membranes, a central Cpn60_TCP1 (for HSP chaperonin T complex 1) homology domain, and a C-terminal kinase domain. In Arabidopsis, there are a number of genes encoding putative Fab1p homologs, but only two of them, FAB1A (At4g33240) and FAB1B (At3g14270), encode proteins having FYVE domains at their N termini (Mueller-Roeber and Pical, 2002). It is likely that these proteins are PtdIns3P 5-kinases in Arabidopsis. Despite the importance of PtdIns(3,5)P₂ in yeast and animals, very little is known about its function in plants. However, it has been shown that hyperosmotic stress can induce the rapid synthesis of PtdIns(3,5)P₂ in cell suspension cultures from a number of plant species (Meijer and Munnik, 2003) and in pollen tubes from tobacco (Nicotiana tabacum; Zonia and Munnik, 2004). This production is consistent with a requirement for PtdIns(3,5)P₂ in vacuolar membrane reorganization, as water moves from the vacuole to the cytosol upon cells being placed under hyperosmotic stress. In animal cells, defective PtdIns(3,5)P₂ production leads to cytoplasmic vacuolation of endosome-derived membranes (Ikonomov et al., 2001; Jefferies et al., 2008). It seems that there is a general requirement in all eukaryotes for PtdIns(3,5)P₂ production in endomembrane remodeling. This remodeling could be mediated by proteins that bind to PtdIns(3,5)P₂. A number of candidates have been identified, including yeast Svp1p (Dove et al., 2004), its mammalian homolog WIP149 (Jeffries et al., 2004), CHMP3 (Whitley et al., 2003), and Ent3p (Friant et al., 2003).In this study, we aimed to further investigate the role of PtdIns(3,5)P₂ in plant physiology and the function of PIKfyve/Fab1p orthologs in Arabidopsis by generating mutant plant lines homozygous for T-DNA insertions in both FAB1A and FAB1B. We failed to generate double homozygous fab1a/fab1b knockout plants but observed subtle phenotypes in both fab1a and fab1b single homozygous plants. The data show that pollen with a fab1a/fab1b genotype has an abnormal vacuolar phenotype and does not contribute to the next generation. Our data are consistent with the hypothesis that the male gametophytic defect observed in vps34 mutant pollen (Lee et al., 2008b) is due to an inability of this pollen to generate PtdIns(3,5)P₂ and is not a direct result of the lack of PtdIns3P.