Processing and stability of type IIc sodium-dependent phosphate cotransporter mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria

Mutations in the apically located Na(+)-dependent phosphate (NaPi) cotransporter, SLC34A3 (NaPi-IIc), are a cause of hereditary hypophosphatemic rickets with hypercalciuria (HHRH). We have characterized the impact of several HHRH mutations on the processing and stability of human NaPi-IIc. Mutations S138F, G196R, R468W, R564C, and c.228delC in human NaPi-IIc significantly decreased the levels of NaPi cotransport activities in Xenopus oocytes. In S138F and R564C mutant proteins, this reduction is a result of a decrease in the V(max) for P(i), but not the K(m). G196R, R468W, and c.228delC mutants were not localized to oocyte membranes. In opossum kidney (OK) cells, cell surface labeling, microscopic confocal imaging, and pulse-chase experiments showed that G196R and R468W mutations resulted in an absence of cell surface expression owing to endoplasmic reticulum (ER) retention. G196R and R468W mutants could be partially stabilized by low temperature. In blue native-polyacrylamide gel electrophoresis analysis, G196R and R468W mutants were either denatured or present in an aggregation complex. In contrast, S138F and R564C mutants were trafficked to the cell surface, but more rapidly degraded than WT protein. The c.228delC mutant did not affect endogenous NaPi uptake in OK cells. Thus, G196R and R468W mutations cause ER retention, while S138F and R564C mutations stimulate degradation of human NaPi-IIc in renal epithelial cells. Together, these data suggest that the NaPi-IIc mutants in HHRH show defective processing and stability.     

Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose

The kinetics of cellulose hydrolysis have long been described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s(-1) at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.     

Muscleblind-like 1 knockout mice reveal novel splicing defects in the myotonic dystrophy brain

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTG(exp)) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSA(LR) transgenic mouse model which expresses a pathogenic range CTG(exp). In the present study, we addressed the possibility that MBNL1 sequestration by CUG(exp) RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1(ΔE3/ΔE3) knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1(ΔE3/ΔE3) knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1(ΔE3/ΔE3) brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUG(exp) RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain.     

Modulation of social interactions by immune stimulation in honey bee, <Emphasis Type="Italic">Apis mellifera</Emphasis>, workers

Background  

Immune response pathways have been relatively well-conserved across animal species, with similar systems in both mammals and invertebrates. Interestingly, honey bees have substantially reduced numbers of genes associated with immune function compared with solitary insect species. However, social species such as honey bees provide an excellent environment for pathogen or parasite transmission with controlled environmental conditions in the hive, high population densities, and frequent interactions. This suggests that honey bees may have developed complementary mechanisms, such as behavioral modifications, to deal with disease.     

Flying lemurs – The 'flying tree shrews'? Molecular cytogenetic evidence for a Scandentia-Dermoptera sister clade

Background  

Flying lemurs or Colugos (order Dermoptera) represent an ancient mammalian lineage that contains only two extant species. Although molecular evidence strongly supports that the orders Dermoptera, Scandentia, Lagomorpha, Rodentia and Primates form a superordinal clade called Supraprimates (or Euarchontoglires), the phylogenetic placement of Dermoptera within Supraprimates remains ambiguous.     

Lysophosphatidylcholine induces delayed myelination in the juvenile ventral hippocampus and behavioral alterations in adulthood

Maternal virus infection or maternal polyinosinic-polycytidilic acid injection confers behavioral alterations including deficit in prepulse inhibition on the offspring. We previously found delayed myelination specifically in the early postnatal hippocampus in the polyinosinic–polycytidilic acid-injection model. To test whether the transient delay in myelination in the juvenile hippocampus leads to abnormal behaviors after adolescence, we injected lysophosphatidylcholine, a potent demyelinating agent, into the ventral hippocampus of the 10-day-old rat. The lysophosphatidylcholine treatment yielded hypomyelination at postnatal day 16, but myelination reverted to normal level in the adult rat. Neuronal arrays and morphology were not disturbed in this model. We then performed a battery of behavioral tests on the lysophosphatidylcholine-treated and control PBS-injected rats. The lysophosphatidylcholine-treated rats showed deficit in prepulse inhibition, motor hyperactivity in response to methamphetamine and anxiety-related behaviors, all of which are typical behaviors observed in the maternal infection models. These findings suggest that the timing of myelination in the early postnatal hippocampus is crucial for the proper development of sensorimotor and emotional functions. The lysophosphatidylcholine-treated rat without a gross anatomical defect is useful as a model for psychotic disorders.     

Expression of ASIC2 in ciliated cells and stereociliated cells

Acid-sensing ion channel 2 (ASIC2) plays a role as a mechanorecptor and acid receptor in the peripheral and central nervous systems. However, several recent studies have suggested that ASIC2 is expressed in several organs, in addition to the nervous system. We have examined the expression and distribution of ASIC2 in rat ciliated cells (trachea and oviduct) and stereociliated cells (epididymis, Corti organ, and ampullary crest) by immunohistochemistry and transmission electron microscopy (TEM). Immunohistochemistry revealed that ASIC2 was expressed in both ciliated cells and stereociliated cells, but the localization differed between these cell types. In ciliated cells, ASIC2 was coexpressed with a cilial marker (acetylated tubulin). In stereociliated cells stained with a stereocilial marker (phalloidin), ASIC2 was observed in the cell body. Observation by TEM suggested that ASIC2 expression was present at the apical side of the cilial membrane in ciliated cells and at the apical side of the cell body in stereociliated cells. This study thus indicates that the proton receptor ASIC2 is expressed in both ciliated and stereociliated cells.     

Protocol for high-resolution separation of rodent myosin heavy chain isoforms in a mini-gel electrophoresis system

Skeletal muscle comprises several fiber types classified based on their contractile and metabolic properties. Skeletal muscle fiber types are classified according to their myosin heavy chain isoforms (MyHC I, IIa, IIx, and IIb). We attained good separation of MyHC isoforms in a mini-gel system by modifying a previously developed electrophoresis protocol. Increased glycerol and decreased cross-linking agent concentrations improved the separation of MyHC isoforms. Sample preparation with dithiothreitol and protease inhibitors produced clear MyHC band boundaries. This protocol included silver staining, with a linear range. The protocol provided high resolution and a highly accurate assay of rodent MyHC isoforms.     

Mechano-sensitive channels regulate the stomatal aperture in Vicia faba

In the bright fields, stomata of the plants are fully opened to raise the transpiration rate and CO₂ uptake required for photosynthesis. Stomatal opening is driven by the activation of plasma membrane H⁺-ATPase and K⁺_in channels, and the Ca²⁺-dependent inactivation and blockage of both components were supposed to be inevitable function to regulate the stomatal aperture. Although, it is still obscure how these activities are regulated at the open state. Application of an amphipathic membrane creator, trinitrophenol (TNP), instantly generates the convex curvature in the plasma membrane, which occurs in the phases of stomatal opening and closure. TNP surely activates mechanosensitive Ca²⁺-permeable channels and attenuates the promotion of stomatal opening, but does not inhibit and promote stomatal closure. These results suggest that activation of mechanosensitive Ca²⁺-permeable channels regulates the opening phase of stomata in plants.