Loss of Myotubularin Function Results in T-Tubule Disorganization in Zebrafish and Human Myotubular Myopathy

Myotubularin is a lipid phosphatase implicated in endosomal trafficking in vitro, but with an unknown function in vivo. Mutations in myotubularin cause myotubular myopathy, a devastating congenital myopathy with unclear pathogenesis and no current therapies. Myotubular myopathy was the first described of a growing list of conditions caused by mutations in proteins implicated in membrane trafficking. To advance the understanding of myotubularin function and disease pathogenesis, we have created a zebrafish model of myotubular myopathy using morpholino antisense technology. Zebrafish with reduced levels of myotubularin have significantly impaired motor function and obvious histopathologic changes in their muscle. These changes include abnormally shaped and positioned nuclei and myofiber hypotrophy. These findings are consistent with those observed in the human disease. We demonstrate for the first time that myotubularin functions to regulate PI3P levels in a vertebrate in vivo, and that homologous myotubularin-related proteins can functionally compensate for the loss of myotubularin. Finally, we identify abnormalities in the tubulo-reticular network in muscle from myotubularin zebrafish morphants and correlate these changes with abnormalities in T-tubule organization in biopsies from patients with myotubular myopathy. In all, we have generated a new model of myotubular myopathy and employed this model to uncover a novel function for myotubularin and a new pathomechanism for the human disease that may explain the weakness associated with the condition (defective excitation–contraction coupling). In addition, our findings of tubuloreticular abnormalities and defective excitation-contraction coupling mechanistically link myotubular myopathy with several other inherited muscle diseases, most notably those due to ryanodine receptor mutations. Based on our findings, we speculate that congenital myopathies, usually considered entities with similar clinical features but very disparate pathomechanisms, may at their root be disorders of calcium homeostasis.     

Ascaris lumbricoides pseudocoelomic body fluid induces a partially activated dendritic cell phenotype with Th2 promoting ability in vivo

Dendritic cells (DCs) matured with helminth-derived molecules that promote Th2 immune responses do not follow conventional definitions of DC maturation processes. While a number of models of DC maturation by Th2 stimuli are postulated, further studies are required if we are to clearly define DC maturation processes that lead to Th2 immune responses. In this study, we examine the interaction of Th2-inducing molecules from the parasitic helminth Ascaris lumbricoides with the maturation processes and function of DCs. Here we show that murine bone marrow-derived DCs are partially matured by A. lumbricoides pseudocoelomic body fluid (ABF) as characterised by the production of IL-6, IL-12p40 and macrophage inflammatory protein 2 (MIP-2) but no enhanced expression of cluster of differentiation (CD)-14, T-cell co-stimulatory markers CD80, CD86, CD40, OX40L and major histocompatibility complex class II was observed. Despite these phenotypic characteristics, ABF-stimulated DCs displayed the functional hallmarks of fully matured cells, enhancing DC phagocytosis and promoting Th2-type responses in skin-draining lymph node cells in vivo. ABF activated Th2-associated extracellular signal-regulated kinase-1 and nuclear factor-kB intracellular signalling pathways independently of toll-like receptor 4. Taken together, we believe this is the first paper to demonstrate A. lumbricoides murine DC-Th cell-driven responses shedding further light on DC maturation processes by helminth antigens.     

Urine-concentrating defects exacerbate with age in male offspring with a low-nephron endowment

Fetal uninephrectomy (uni-x) in male sheep at 100 days of gestation (term = 150 days) reduces overall nephron endowment without affecting birth weight. Offspring have a lower glomerular filtration rate (GFR) and elevated mean arterial pressure (MAP) at 6 mo of age. This study investigated whether this reduction in renal function was associated with impaired urine-concentrating ability at 6 mo of age and exacerbated with ageing (4 yr) and examined response to 1) nonpressor dose of exogenous arginine vasopressin (AVP; 0.2 μg·kg(-1)·h(-1) iv) and 2) 30 h of water deprivation. Basal MAP was higher in uni-x animals at both ages, and became further elevated with age compared with the sham group (elevation in MAP with age; sham: ~4 mmHg, uni-x: 9 mmHg, P(group × age) < 0.01). GFR declined with ageing in both groups with the decrease being greater with age in the uni-x group (further 26%, P(group × age) < 0.001). In response to AVP infusion, urine osmolality increased in both treatment groups; this response was significantly lower in the uni-x animals and became further reduced with ageing. Uni-x animals had reduced renal expression of vasopressin-2 receptor and aquaporin-2 at both ages (P < 0.01). The increase in plasma AVP levels in response to dehydration was similar between the treatment groups, suggesting the urine-concentrating defect was associated with these renal gene changes rather than defects in AVP secretion. Renal insufficiency due to a low-nephron endowment increases the risk of hypertension and chronic renal disease and may incur greater vulnerability to physiological challenges such as water deprivation as observed in the uni-x animals.     

Paralog-specific primers for the amplification of nuclear Loci in tetraploid barbels (barbus: cypriniformes)

Thirty paralog-specific primers were developed, following an intron-primed exon-crossing strategy, for S7 and growth hormone genes in Barbus (subgenera Barbus and Luciobarbus). We found that paralog-specific amplification requires the use of only one paralog-specific primer, allowing their simultaneous use with universal exon-primed intron-crossing primers of broad taxonomic applicability. This hybrid annealing strategy guarantees both specificity and generality of amplification reactions and represents a step forward in the amplification of duplicated nuclear loci in polyploid organisms and members of multigene families. Assays of several representative taxa identified high levels of segregating single nucleotide polymorphisms (SNPs) and nucleotide diversity within each of these subgenera. Additionally, several insertions-deletions (indels) that are diagnostic across species are found in intronic regions. Therefore, these primers provide a reliable source of valuable nuclear SNP and indel data for population and species level studies of barbels, such as applied conservation and basic evolutionary studies.     

SIGNIFICANT ROLE FOR HISTORICAL EFFECTS IN THE EVOLUTION OF REPRODUCTIVE ISOLATION: EVIDENCE FROM PATTERNS OF INTROGRESSION BETWEEN THE CYPRINID FISHES,LUXILUS CORNUTUS AND LUXILUS CHRYSOCEPHALUS

Samples of Luxilus cornutus, Luxilus chrysocephalus, and their hybrids were collected along hypothesized routes of dispersal from Pleistocene refugia to examine the significance of geographic variation in patterns of introgression between these species. Patterns of allozyme and mitochondrial DNA (mtDNA) variation were generally consistent with those from previous studies. Tests of Hardy-Weinberg equilibrium revealed significant deficiencies of heterozygotes in all samples, indicating some form of reproductive isolation. Mitochondrial DNAs of each species were not equally represented in F₁ hybrids; however, this bias was eliminated when the two largest samples were excluded from the analysis. Backcross hybrids exhibited biased mtDNA introgression, as samples from Lake Erie (eastern) and Lake Michigan (western) drainages showed significant excesses of mtDNAs from L. chrysocephalus and L. cornutus, respectively, relative to frequencies of diagnostic allozyme markers. The extent and direction of allozyme and mtDNA introgression was quantified by calculating isolation index values from morphologically “pure” individuals of each species from each locality. Analysis of variance of these measures identified limited introgression of allozyme variants with no geographic pattern, but significant differences in direction of mtDNA introgression between drainages (i.e., postglacial dispersal route). Association between patterns of mtDNA introgression and dispersal route across the latitudinal width of the contact zone is best explained by genetic divergence during past isolation of ancestral populations from these drainages. These results identify a significant role for historical effects in the evolution of reproductive isolation and the process of speciation.     

Adipocyte Dysfunction in a Mouse Model of Polycystic Ovary Syndrome (PCOS): Evidence of Adipocyte Hypertrophy and Tissue-Specific Inflammation

Clinical research shows an association between polycystic ovary syndrome (PCOS) and chronic inflammation, a pathological state thought to contribute to insulin resistance. The underlying pathways, however, have not been defined. The purpose of this study was to characterize the inflammatory state of a novel mouse model of PCOS. Female mice lacking leptin and insulin receptors in pro-opiomelanocortin neurons (IR/LepR^POMC mice) and littermate controls were evaluated for estrous cyclicity, ovarian and adipose tissue morphology, and body composition by QMR and CT scan. Tissue-specific macrophage infiltration and cytokine mRNA expression were measured, as well as circulating cytokine levels. Finally, glucose regulation during pregnancy was evaluated as a measure of risk for diabetes development. Forty-five percent of IR/LepR^POMC mice showed reduced or absent ovulation. IR/LepR^POMC mice also had increased fat mass and adipocyte hypertrophy. These traits accompanied elevations in macrophage accumulation and inflammatory cytokine production in perigonadal adipose tissue, liver, and ovary. These mice also exhibited gestational hyperglycemia as predicted. This report is the first to show the presence of inflammation in IR/LepR^POMC mice, which develop a PCOS-like phenotype. Thus, IR/LepR^POMC mice may serve as a new mouse model to clarify the involvement of adipose and liver tissue in the pathogenesis and etiology of PCOS, allowing more targeted research on the development of PCOS and potential therapeutic interventions.     

Comparative analysis of Dp427-deficient mdx tissues shows that the milder dystrophic phenotype of extraocular and toe muscle fibres is associated with a persistent expression of beta-dystroglycan

The cell biological hypothesis of Duchenne muscular dystrophy assumes that deficiency in the membrane cytoskeletal element dystrophin triggers a loss in surface glycoproteins, such as beta-dystroglycan, thereby rendering the sarcolemmal membrane more susceptible to micro-rupturing. Secondary changes in ion homeostasis, such as increased cytosolic Ca2+ levels and impaired luminal Ca2+ buffering, eventually lead to Ca2+-induced myonecrosis. However, individual muscle groups exhibit a graded pathological response during the natural time course of x-linked muscular dystrophy. The absence of the dystrophin isofom Dp427 does not necessarily result in a severe dystrophic phenotype in all muscle groups. In the dystrophic mdx animal model, extraocular and toe muscles are not as severely affected as limb muscles. Here, we show that the relative expression and sarcolemmal localization of the central trans-sarcolemmal linker of the dystrophin-glycoprotein complex, beta-dystroglycan, is preserved in mdx extraocular and toe fibres by means of two-dimensional immunoblotting and immunofluorescence microscopy. Thus, with respect to improving myology diagnostics, the relative expression levels of beta-dystroglycan appear to represent reliable markers for the severity of secondary changes in dystrophin-deficient fibres. Immunoblotting and enzyme assays revealed that mdx toe muscle fibres exhibit an increased expression and activity of the sarcoplasmic reticulum Ca2+-ATPase. Chemical crosslinking studies demonstrated impaired calsequestrin oligomerization in mdx gastrocnemius muscle indicating that abnormal calsequestrin clustering is involved in reduced Ca2+ buffering of the dystrophic sarcoplasmic reticulum. Previous studies have mostly attributed the sparing of certain mdx fibres to the special protective properties of small-diameter fibres. Our study suggests that the rescue of dystrophin-associated glycoproteins, and possibly the increased removal of cytosolic Ca2+ ions, might also play an important role in protecting muscle cells from necrotic changes.     

Phylogenetic relationships among superfamilies of Hymenoptera

The first comprehensive analysis of higher‐level phylogeny of the order Hymenoptera is presented. The analysis includes representatives of all extant superfamilies, scored for 392 morphological characters, and sequence data for four loci (18S, 28S, COI and EF‐1α). Including three outgroup taxa, 111 terminals were analyzed. Relationships within symphytans (sawflies) and Apocrita are mostly resolved. Well supported relationships include: Xyeloidea is monophyletic, Cephoidea is the sister group of Siricoidea + [Xiphydrioidea + (Orussoidea + Apocrita)]; Anaxyelidae is included in the Siricoidea, and together they are the sister group of Xiphydrioidea + (Orussoidea + Apocrita); Orussoidea is the sister group of Apocrita, Apocrita is monophyletic; Evanioidea is monophyletic; Aculeata is the sister group of Evanioidea; Proctotrupomorpha is monophyletic; Ichneumonoidea is the sister group of Proctotrupomorpha; Platygastroidea is sister group to Cynipoidea, and together they are sister group to the remaining Proctotrupomorpha; Proctotrupoidea s. str. is monophyletic; Mymarommatoidea is the sister group of Chalcidoidea; Mymarommatoidea + Chalcidoidea + Diaprioidea is monophyletic. Weakly supported relationships include: Stephanoidea is the sister group of the remaining Apocrita; Diaprioidea is monophyletic; Ceraphronoidea is the sister group of Megalyroidea, which together form the sister group of [Trigonaloidea (Aculeata + Evanioidea)]. Aside from paraphyly of Vespoidea within Aculeata, all currently recognized superfamilies are supported as monophyletic. The diapriid subfamily Ismarinae is raised to family status, Ismaridae stat. nov. © The Will Henning Society 2011.     

Caspase-mediated specific cleavage of BubR1 is a determinant of mitotic progression

The fidelity of chromosomal duplication is monitored by cell cycle checkpoints operational during mitosis. One such cell cycle delay is invoked by microtubule-targeting agents such as nocodazole or paclitaxel (Taxol) and is mediated by mitotic checkpoint proteins that include BubR1. Relatively little is known about the regulation of expression and stability of BubR1 (or other checkpoint proteins) and how these factors dictate the durability of the cell cycle delay. We report here that treatment of HeLa cells with spindle-disrupting agents resulted in caspase activation and precipitated the cleavage of BubR1. This mechanism ultimately leads to reduced levels of full-length protein, which are accompanied by abrogation of the mitotic block; the checkpoint abrogation is substantially accelerated by inhibition of de novo protein synthesis. In contrast, inhibition of caspase activity blocked BubR1 degradation and prolonged mitosis. To confirm a direct link between caspase activity and BubR1 protein expression, we identified by site-directed mutagenesis the specific caspase cleavage sites cleaved after exposure to paclitaxel. Surprisingly, BubR1 has two sites of cleavage: primarily at Asp607/Asp610 and secondarily at Asp576/Asp579. BubR1 mutated at both locations (BubR1Delta579Delta610) was resistant to paclitaxel-induced degradation. Expression of BubR1Delta579Delta610 augmented the mitotic delay induced by spindle disruption in transfected cells as well as in clones engineered to inducibly express the mutant protein upon exposure to doxycycline and ultimately led to increased aneuploidy. Underscoring the importance of these caspase cleavage sites, both tetrapeptide motifs are identified in the amino acid sequences of human, mouse, chicken, and Xenopus BubR1. These results are potentially the first to link the control of the stability of a key mitotic checkpoint protein to caspase activation, a regulatory pathway that may be involved in killing defective cells and that has been evolutionarily conserved.