Protein Tyrosine Phosphatase PTP1B Is Involved in Hippocampal Synapse Formation and Learning

ER-bound PTP1B is expressed in hippocampal neurons, and accumulates among neurite contacts. PTP1B dephosphorylates ß-catenin in N-cadherin complexes ensuring cell-cell adhesion. Here we show that endogenous PTP1B, as well as expressed GFP-PTP1B, are present in dendritic spines of hippocampal neurons in culture. GFP-PTP1B overexpression does not affect filopodial density or length. In contrast, impairment of PTP1B function or genetic PTP1B-deficiency leads to increased filopodia-like dendritic spines and a reduction in mushroom-like spines, while spine density is unaffected. These morphological alterations are accompanied by a disorganization of pre- and post-synapses, as judged by decreased clustering of synapsin-1 and PSD-95, and suggest a dynamic synaptic phenotype. Notably, levels of ß-catenin-Tyr-654 phosphorylation increased ∼5-fold in the hippocampus of adult PTP1B^−/− (KO) mice compared to wild type (WT) mice and this was accompanied by a reduction in the amount of ß-catenin associated with N-cadherin. To determine whether PTP1B-deficiency alters learning and memory, we generated mice lacking PTP1B in the hippocampus and cortex (PTP1B^fl/fl–Emx1-Cre). PTP1B^fl/fl–Emx1-Cre mice displayed improved performance in the Barnes maze (decreased time to find and enter target hole), utilized a more efficient strategy (cued), and had better recall compared to WT controls. Our results implicate PTP1B in structural plasticity within the hippocampus, likely through modulation of N-cadherin function by ensuring dephosphorylation of ß-catenin on Tyr-654. Disruption of hippocampal PTP1B function or expression leads to elongation of dendritic filopodia and improved learning and memory, demonstrating an exciting novel role for this phosphatase.     

Chlamydia isopodii sp. n., an obligate intracellular parasite of Porcellio scaber

In an ultrastructural study of the hepatopancreas of Porcellio scaber, an obligate intracellular parasite, Chlamydia, was noted in the epithelial cells. Although the infection was found to extend the entire length of the hepatopancreas, it was most extensive in the glandular region. Indirect immunofluorescence testing revealed no cross-reactivity with either lymphogranuloma venereum or psittacosis antisera.     

Unconjugated bilirubin inhibits VCAM-1-mediated transendothelial leukocyte migration

During lymphocyte migration, engagement of VCAM-1 stimulates the generation of endothelial cell-derived reactive oxygen species (ROS) and activation of matrix metalloproteinases, facilitating endothelial retraction. Because bilirubin is a potent antioxidant, we examined the hypothesis that this bile pigment inhibits VCAM-1-dependent cellular events. The migration of isolated murine splenic lymphocytes across monolayers of murine endothelial cell lines (which constitutively express VCAM-1) is significantly inhibited by physiological concentrations of bilirubin, in the absence of an effect on lymphocyte adhesion. Bilirubin administration also suppresses VCAM-1-stimulated ROS generation and reduces endothelial cell matrix metalloproteinase activity. In a murine asthma model characterized by VCAM-1-dependent airway inflammation, treatment of C57BL6/J mice with i.p. bilirubin decreases the total leukocyte count in the lung parenchyma and lavage fluid, through specific inhibition of eosinophil and lymphocyte infiltration. Blood eosinophil counts were increased in bilirubin-treated animals, while VCAM-1 expression in the capillary endothelium and cytokine levels in both lung lavage and supernatants from cultured lymph node lymphocytes were unchanged, suggesting that bilirubin inhibits leukocyte migration. Conclusion: bilirubin blocks VCAM-1-dependent lymphocyte migration in vitro and ameliorates VCAM-1-mediated airway inflammation in vivo, apparently through the suppression of cellular ROS production. These findings support a potential role for bilirubin as an endogenous immunomodulatory agent.     

RXR isoforms and endogenous retinoids in the fiddler crab, Uca pugilator

The pleiotropic effects of circulating ecdysteroids in the adult fiddler crab, Uca pugilator, during molting, regeneration, and reproduction are mediated by a limited number of receptor proteins. We hypothesize that hormonal effects in vivo may be the result of complex interactions between at least two receptor heterodimer conformations that differentially respond to multiple ecdysteroid/retinoid signals. Two splicing variants of the fiddler crab retinoid-X-receptor (UpRXR) differ from one another by the addition of a 33 amino acid insert in the ligand-binding domain. We show here that the ecdysteroid receptor in the fiddler crab (UpEcR) behaves differently depending upon the UpRXR isoform with which it is partnered. The two UpRXR variant partners for UpEcR confer slightly different responses in the binding of Ponasterone A (PA)—a naturally occurring ecdysteroid in the blood of Uca. UpRXR can bind 9-cis retinoic acid (9cRA) as well as terpenoids. 9cRA and the naturally occurring terpenoid, methyl farnesoate, influence the binding of PA to UpEcR/UpRXR dimers. Endogenous retinoids are found in the blastema of regenerating limbs of Uca and they (plus blood-borne terpenoids) may add additional levels of differential response by target tissues. Thus, the two sets of heterodimers tested here may represent different dynamic complexes whose properties are defined by the specific heterodimeric subunits involved and the specific ligands available.     

Slow heating of barley aleurone layers to heat-shock temperature preserves heat-shock-sensitive cellular properties

In barley (Hordeum vulgare L. cv. Himalaya) aleurone layers, heat shock causes the selective suppression of α-amylase synthesis by destabilizing this secretory protein's mRNA. The lamellar stacks of the endoplasmic reticulum (ER), which serve as the site of α-amylase mRNA translation, are dissociated by heat shock, suggesting that heat-shock-induced changes in ER may be important in selectively targeting α-amylase mRNAs for destabilization. We have found that samples maintained at heat-shock temperature (40°C) for 18 h recover the ability to synthesize α-amylase and that the ER membranes in these samples contain membrane phospholipids with enhanced levels of fatty acid saturation. This present study investigated whether gradual warming to 40°C over 3-6 h (ramping) would preserve α-amylase synthesis by permitting ER membrane phospholipid retailoring during the gradual temperature increase. Analyses by sodium dodecyl-sulfate polyacrylamide gel electrophoresis revealed that α-amylase synthesis was markedly increased in ramped samples. Furthermore, northern hybridization analyses and transmission electron microscopy showed that these samples had increased α-amylase mRNA levels and stacks of ER lamellae, respectively. Gas chromatographic analyses of ER membrane phospholipids indicated that the fatty acids of ramped samples were more saturated than their heat-shocked counterparts. These data indicate that heat-induced increases in aleurone ER membrane phospholipid fatty acid saturation may be important in maintaining secretory protein expression at normally nonpermissive heat-shock temperatures.     

Structure-based analysis of VDAC1: N-terminus location, translocation, channel gating and association with anti-apoptotic proteins

Structural studies place the VDAC1 (voltage-dependent anion channel 1) N-terminal region within the channel pore. Biochemical and functional studies, however, reveal that the N-terminal domain is cytoplasmically exposed. In the present study, the location and translocation of the VDAC1 N-terminal domain, and its role in voltage-gating and as a target for anti-apoptotic proteins, were addressed. Site-directed mutagenesis and cysteine residue substitution, together with a thiol-specific cross-linker, served to show that the VDAC1 N-terminal region exists in a dynamic equilibrium, located within the pore or exposed outside the β-barrel. Using a single cysteine-residue-bearing VDAC1, we demonstrate that the N-terminal region lies inside the pore. However, the same region can be exposed outside the pore, where it dimerizes with the N-terminal domain of a second VDAC1 molecule. When the N-terminal region α-helix structure was perturbed, intra-molecular cross-linking was abolished and dimerization was enhanced. This mutant also displays reduced voltage-gating and reduced binding to hexokinase, but not to the anti-apoptotic proteins Bcl-2 and Bcl-xL. Replacing glycine residues in the N-terminal domain GRS (glycine-rich sequence) yielded less intra-molecular cross-linked product but more dimerization, suggesting that GRS provides the flexibility needed for N-terminal translocation from the internal pore to the channel face. N-terminal mobility may thus contribute to channel gating and interaction with anti-apoptotic proteins.     

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.