Genotype-phenotype correlations in valosin-containing protein disease: a retrospective muticentre study.

BACKGROUND: Valosin-containing protein (VCP) disease, caused by mutations in the VCP gene, results in myopathy, Paget's disease of bone (PBD) and frontotemporal dementia (FTD). Natural history and genotype-phenotype correlation data are limited. This study characterises patients with mutations in VCP gene and investigates genotype-phenotype correlations. METHODS: Descriptive retrospective international study collecting clinical and genetic data of patients with mutations in the VCP gene. RESULTS: Two hundred and fifty-five patients (70.0% males) were included in the study. Mean age was 56.8±9.6 years and mean age of onset 45.6±9.3 years. Mean diagnostic delay was 7.7±6 years. Symmetric lower limb weakness was reported in 50% at onset progressing to generalised muscle weakness. Other common symptoms were ventilatory insufficiency 40.3%, PDB 28.2%, dysautonomia 21.4% and FTD 14.3%. Fifty-seven genetic variants were identified, 18 of these no previously reported. c.464G>A (p.Arg155His) was the most frequent variant, identified in the 28%. Full time wheelchair users accounted for 19.1% with a median time from disease onset to been wheelchair user of 8.5 years. Variant c.463C>T (p.Arg155Cys) showed an earlier onset (37.8±7.6 year) and a higher frequency of axial and upper limb weakness, scapular winging and cognitive impairment. Forced vital capacity (FVC) below 50% was as risk factor for being full-time wheelchair user, while FVC <70% and being a full-time wheelchair user were associated with death. CONCLUSION: This study expands the knowledge on the phenotypic presentation, natural history, genotype-phenotype correlations and risk factors for disease progression of VCP disease and is useful to improve the care provided to patient with this complex disease.

Extracellular Vesicles Inhibit the Response of Pancreatic Ductal Adenocarcinoma Cells to Gemcitabine and TRAIL Treatment.

Pancreatic ductal adenocarcinoma remains an aggressive cancer with a low 5-year survival rate. Although gemcitabine has been a standard treatment for advanced pancreatic cancer, patients often develop resistance to this therapeutic. We have previously shown that treating pancreatic cancer cells in vitro with a combination of gemcitabine and the cytokine TRAIL significantly reduced both cell viability and survival. The data presented here demonstrate that this response to treatment is inhibited when cells are incubated with a conditioned medium derived from untreated cells. We show that this inhibition is specifically mediated by extracellular vesicles present in the conditioned medium, as seen by a significant decrease in apoptosis. Additionally, we further demonstrate that this effect can be reversed in the presence of GW4869, an inhibitor of exosome biogenesis and release. These results show that pancreatic cancer cell-derived extracellular vesicles can confer resistance to treatment with gemcitabine and TRAIL. The implications of these findings suggest that removal of EVs during treatment can improve the response of cells to gemcitabine and TRAIL treatment in vitro.

microRNA-21 Regulates Stemness in Pancreatic Ductal Adenocarcinoma Cells.

Pancreatic ductal adenocarcinoma (PDAC) is the most common and aggressive type of pancreatic cancer (PCa) with a low survival rate. microRNAs (miRs) are endogenous, non-coding RNAs that moderate numerous biological processes. miRs have been associated with the chemoresistance and metastasis of PDAC and the presence of a subpopulation of highly plastic "stem"-like cells within the tumor, known as cancer stem cells (CSCs). In this study, we investigated the role of miR-21, which is highly expressed in Panc-1 and MiaPaCa-2 PDAC cells in association with CSCs. Following miR-21 knockouts (KO) from both MiaPaCa-2 and Panc-1 cell lines, reversed expressions of epithelial-mesenchymal transition (EMT) and CSCs markers were observed. The expression patterns of key CSC markers, including CD44, CD133, CX-C chemokine receptor type 4 (CXCR4), and aldehyde dehydrogenase-1 (ALDH1), were changed depending on miR-21 status. miR-21 (KO) suppressed cellular invasion of Panc-1 and MiaPaCa-2 cells, as well as the cellular proliferation of MiaPaCa-2 cells. Our data suggest that miR-21 is involved in the stemness of PDAC cells, may play roles in mesenchymal transition, and that miR-21 poses as a novel, functional biomarker for PDAC aggressiveness.

Purpose-driven improvisation during organisational shocks: case narrative of three critical organisations and Typhoon Haiyan.

Disasters often produce elements of shock that may render pre-established plans for action limited or otherwise inapplicable to the current situation. Improvisation is considered to be a common response among organisations that operate in such turbulent environments. Despite the prevalence of such plans for action, existing research is reticent with regard to the processes concerning how improvisation relates to pre-established organisational goals. This study explores how improvisation can be employed by organisations to attain specific objectives, amidst the emergent nature of the initiative. Using case study narratives of three small organisations that were critical to the response after Typhoon Haiyan struck Leyte, Philippines, in November 2013, the paper demonstrates that improvisation can be absorbed as a conscious mechanism that can aid the attainment of pre-established goals. Furthermore, it conceptualises the buffering effect of improvisation to elucidate the process via which it purposefully directs an organisation to preserve and fulfil its aims.

WDR11-mediated Hedgehog signalling defects underlie a new ciliopathy related to Kallmann syndrome.

WDR11 has been implicated in congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS), human developmental genetic disorders defined by delayed puberty and infertility. However, WDR11's role in development is poorly understood. Here, we report that WDR11 modulates the Hedgehog (Hh) signalling pathway and is essential for ciliogenesis. Disruption of WDR11 expression in mouse and zebrafish results in phenotypic characteristics associated with defective Hh signalling, accompanied by dysgenesis of ciliated tissues. Wdr11-null mice also exhibit early-onset obesity. We find that WDR11 shuttles from the cilium to the nucleus in response to Hh signalling. WDR11 regulates the proteolytic processing of GLI3 and cooperates with the transcription factor EMX1 in the induction of downstream Hh pathway gene expression and gonadotrophin-releasing hormone production. The CHH/KS-associated human mutations result in loss of function of WDR11. Treatment with the Hh agonist purmorphamine partially rescues the WDR11 haploinsufficiency phenotypes. Our study reveals a novel class of ciliopathy caused by WDR11 mutations and suggests that CHH/KS may be a part of the human ciliopathy spectrum.

Store-operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCß1 to induce TRPC1 channel activation in vascular smooth muscle cells.

KEY POINTS: Depletion of Ca2+ stores activates store-operated channels (SOCs), which mediate Ca2+ entry pathways that regulate cellular processes such as contraction, proliferation and gene expression. In vascular smooth muscle cells (VSMCs), stimulation of SOCs composed of canonical transient receptor potential channel 1 (TRPC1) proteins requires G protein α q subunit (Gαq)/phospholipase C (PLC)ß1/protein kinase C (PKC) activity. We studied the role of stromal interaction molecule 1 (STIM1) in coupling store depletion to this activation pathway using patch clamp recording, GFP-PLCδ1-PH imaging and co-localization techniques. Store-operated TRPC1 channel and PLCß1 activities were inhibited by STIM1 short hairpin RNA (shRNA) and absent in TRPC1-/- cells, and store-operated PKC phosphorylation of TRPC1 was inhibited by STIM1 shRNA. Store depletion induced interactions between STIM1 and TRPC1, Gαq and PLCß1, which required STIM1 and TRPC1. Similar effects were produced with noradrenaline. These findings identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, where store-operated STIM1-TRPC1 interactions stimulate Gαq/PLCß1/PKC activity to induce channel gating. ABSTRACT: In vascular smooth muscle cells (VSMCs), stimulation of canonical transient receptor potential channel 1 (TRPC1) protein-based store-operated channels (SOCs) mediates Ca2+ entry pathways that regulate contractility, proliferation and migration. It is therefore important to understand how these channels are activated. Studies have shown that stimulation of TRPC1-based SOCs requires G protein α q subunit (Gαq)/phospholipase C (PLC)ß1 activities and protein kinase C (PKC) phosphorylation, although it is unclear how store depletion stimulates this gating pathway. The present study examines this issue by focusing on the role of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum Ca2+ sensor. Store-operated TRPC1 channel activity was inhibited by TRPC1 and STIM1 antibodies and STIM1 short hairpin RNA (shRNA) in wild-type VSMCs, and was absent in TRPC1-/- VSMCs. Store-operated PKC phosphorylation of TRPC1 was reduced by knockdown of STIM1. Moreover, store-operated PLCß1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH was reduced by STIM1 shRNA and absent in TRPC1-/- cells. Immunocytochemistry, co-immunoprecipitation and proximity ligation assays revealed that store depletion activated STIM1 translocation from within the cell to the plasma membrane (PM) where it formed STIM1-TRPC1 complexes, which then associated with Gαq and PLCß1. Noradrenaline also evoked TRPC1 channel activity and associations between TRPC1, STIM1, Gαq and PLCß1, which were inhibited by STIM1 knockdown. Effects of N-terminal and C-terminal STIM1 antibodies on TRPC1-based SOCs and STIM1 staining suggest that channel activation may involve insertion of STIM1 into the PM. The findings of the present study identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, in which store-operated STIM1-TRPC1 interactions stimulate PLCß1 activity to induce PKC phosphorylation of TRPC1 and channel gating.

Store depletion induces Gαq-mediated PLCß1 activity to stimulate TRPC1 channels in vascular smooth muscle cells.

Depletion of sarcoplasmic reticulum (SR) Ca(2+) stores activates store-operated channels (SOCs) composed of canonical transient receptor potential (TRPC) 1 proteins in vascular smooth muscle cells (VSMCs), which contribute to important cellular functions. We have previously shown that PKC is obligatory for activation of TRPC1 SOCs in VSMCs, and the present study investigates if the classic phosphoinositol signaling pathway involving Gαq-mediated PLC activity is responsible for driving PKC-dependent channel gating. The G-protein inhibitor GDP-ß-S, anti-Gαq antibodies, the PLC inhibitor U73122, and the PKC inhibitor GF109203X all inhibited activation of TRPC1 SOCs, and U73122 and GF109203X also reduced store-operated PKC-dependent phosphorylation of TRPC1 proteins. Three distinct SR Ca(2+) store-depleting agents, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, cyclopiazonic acid, and N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamineed, induced translocations of the fluorescent biosensor GFP-PLCδ1-PH from the cell membrane to the cytosol, which were inhibited by U73122. Knockdown of PLCß1 with small hairpin RNA reduced both store-operated PLC activity and stimulation of TRPC1 SOCs. Immunoprecipitation studies and proximity ligation assays revealed that store depletion induced interactions between TRPC1 and Gαq, and TRPC1 and PLCß1. We propose a novel activation mechanism for TRPC1 SOCs in VSMCs, in which store depletion induces formation of TRPC1-Gαq-PLCß1 complexes that lead to PKC stimulation and channel gating.

Differential weakness of finger extensor muscles: A clinical pattern of multifocal motor neuropathy.

INTRODUCTION: Several studies have suggested that differential weakness in muscles supplied by the same motor nerve supports the diagnosis of multifocal motor neuropathy (MMN). METHODS: We describe the clinical, electrophysiological, neuroimaging, and laboratory findings of patients with a lower motor syndrome whose clinical presentation included differential finger extension weakness that we have seen in our neuromuscular clinic. RESULTS: We identified 3 patients with hand weakness and 1 patient with asymmetric weakness of the upper extremity. Conduction blocks (CBs) were identified in 1 patient. Anti-GM1 immunoglobulin M antibodies were detected in 2 of the 3 patients tested. Only 1 patient responded to intravenous immunoglobulin (IVIg). Rituximab was administered in another patient, but we did not detect a response. CONCLUSIONS: We suggest that differential finger extension weakness is a feature that may be seen in MMN, even in the absence of CB or response to IVIg. Muscle Nerve 55: 433-437, 2017.

TRPM7 regulates proliferation and polarisation of macrophages.

Ion channels play pivotal roles in regulating important functions of macrophages, such as cytokine and chemokine production, migration, proliferation, phagocytosis and others. In this study, we have identified the transient receptor potential cation channel, subfamily M, member 7 (TRPM7) for the first time in macrophages. TRPM7 activity is differentially regulated in macrophages, i.e. current density in TRPM7 is significantly larger in anti-inflammatory M2-type macrophages than in untreated and in pro-inflammatory M1-type macrophages, whereas mRNA levels of TRPM7 remain unchanged upon cell polarisation. The specific TRPM7 inhibitors NS8593 and FTY720 abolish proliferation of macrophages induced by interleukin-4 (IL-4) and macrophage colony-stimulating factor (M-CSF), respectively, whereas proliferation arrest was not accompanied by induction of apoptosis or necrosis in macrophages. Furthermore, NS8593 and FTY720 prevented polarisation of macrophages towards the anti-inflammatory M2 phenotype. Inhibition of TRPM7 reduced IL-4-induced upregulation of arginase-1 (Arg1) mRNA levels and Arg1 activity, and abolished the inhibitory effects of IL-4 or M-CSF on LPS-induced TNF-α production by macrophages. In summary, our data suggest a main role of TRPM7 in the regulation of macrophage proliferation and polarisation.

Actin in transcription and transcription regulation.

Recent research has provided convincing evidence that actin plays several important roles in gene transcription. First, actin can bind transcription factors and determine their subcellular localization. Second, actin is a component of chromatin remodeling complexes involved in transcriptional activation. Third, actin binds directly to the RNA polymerases I, II and III, and is required for their full transcriptional activity. Fourth, actin associates with nascent mRNPs and participates in the recruitment of histone modifiers to transcribed genes. We do not know yet whether these functions are general, or restricted to certain subsets of genes.

Motor unit placement in a realistic muscle cross section model: performance of a new algorithm and effects of muscle architecture on surface EMG power spectral components.

Objetive.Surface electromyogram (sEMG) characteristics are strongly dependent on the spatial localization of motor units (MU). Thus, the definition of the muscle architecture is a crucial step in the simulation of sEMG. Muscles cross section or even the shape of the MU territories is not a regular geometric figure. Moreover, MU may have different fibre densities, and its distribution may not be random but follow a spatial regionalization by type. Despite these prior conditions, the method for MU placement in a muscle model should be able to achieve constant muscle fibre density, which is a generally accepted property of any muscle.Approach.An algorithm was developed that meets the previously established requirements for the distribution of MU in a muscle model. The algorithm considers the muscle cross section and MU territories as bitmaps and encodes the muscle fibre density distribution by pixel brightness. This allowed the use of digital image processing techniques to optimally distribute the MU. The impact of the different muscle architectures on sEMG was investigated using one of the most accepted models of motor unit pool recruitment combined with a model of MU action potential simulation. element-citationitalicMain results.The algorithm produced a completely regionalized architecture with constant muscle fibre density in a muscle with both an irregular cross-section and elliptic MU territory.Significance.In addition to the improvement in the realism of the muscle simulations, the algorithm allowed the investigation of the influence of the spatial distribution of MU on the sEMG power spectrum, thus helping to explain the existing inability to detect changes in motor unit recruitment strategies through the spectral analysis of interference patterns.

Inhibiting CDK4/6 in pancreatic ductal adenocarcinoma via microRNA-21.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with a 5-year survival rate of 5-10 %. The high mortality rate is due to the asymptomatic progression of clinical features in metastatic stages of the disease, which renders standard therapeutic options futile. PDAC is characterised by alterations in several genes that drive carcinogenesis and limit therapeutic response. The two most common genetic aberrations in PDAC are the mutational activation of KRAS and loss of the tumour suppressor CDK inhibitor 2A (CDKN2A), which culminate the activation of the cyclin-dependent kinase 4 and 6 (CDK4/6), that promote G1 cell cycle progression. Therapeutic strategies focusing on the CDK4/6 inhibitors such as palbociclib (PD-0332991) may potentially improve outcomes in this malignancy. MicroRNAs (miRs/miRNAs) are small endogenous non-coding RNA molecules associated with cellular proliferation, invasion, apoptosis, and cell cycle. Primarily, miR-21 promotes cell proliferation and a higher proportion of PDAC cells in the S phase, while knockdown of miR-21 has been linked to cell cycle arrest at the G2/M phase and inhibition of cell proliferation. In this study, using a CRISPR/Cas9 loss-of-function screen, we individually silenced the expression of miR-21 in two PDAC cell lines and in combination with PD-0332991 treatment, we examined the synergetic mechanisms of CDK4/6 inhibitors and miR-21 knockouts (KOs) on cell survival and death. This combination reduced cell proliferation, cell viability, increased apoptosis and G1 arrest in vitro. We further analysed the mitochondrial respiration and glycolysis of PDAC cells; then assessed the protein content of these cells and revealed numerous Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with PD-0332991 treatment and miR-21 knocking out. Our results demonstrate that combined targeting of CDK4/6 and silencing of miR-21 represents a novel therapeutic strategy in PDAC.

MAL and ternary complex factor use different mechanisms to contact a common surface on the serum response factor DNA-binding domain.

The transcription factor serum response factor (SRF) interacts with its cofactor, MAL/MKL1, a member of the myocardin-related transcription factor (MRTF) family, through its DNA-binding domain. We define a seven-residue sequence within the conserved MAL B1 region essential and sufficient for complex formation. The neighboring Q-box sequence facilitates this interaction. The B1 and Q-box regions also have antagonistic effects on MAL nuclear import, but the residues involved are largely distinct. Both MAL and the ternary complex factor (TCF) family of SRF cofactors interact with a hydrophobic groove and pocket on the SRF DNA-binding domain. Unlike the TCFs, however, interaction of MAL with SRF is impaired by SRF alphaI-helix mutations that reduce DNA bending in the SRF-DNA complex. A clustered SRF alphaI-helix mutation strongly impairs MAL-SRF complex formation but does not affect DNA distortion in the MAL-SRF complex. MAL-SRF complex formation is facilitated by DNA binding. DNase I footprinting indicates that in the SRF-MAL complex MAL directly contacts DNA. These contacts, which flank the DNA sequences protected from DNase I by SRF, are required for effective MAL-SRF complex formation in gel mobility shift assays. We propose a model of MAL-SRF complex formation in which MAL interacts with SRF by the addition of a beta-strand to the SRF DNA-binding domain beta-sheet region, while SRF-induced DNA bending facilitates MAL-DNA contact.

Lambert-Eaton Myasthenic syndrome: early diagnosis is key.

Lambert-Eaton myasthenic syndrome (LEMS) is an uncommon disorder of neuromuscular transmission with distinctive pathophysiological, clinical, electrophysiological and laboratory features. There are two forms of LEMS. The paraneoplastic (P-LEMS) form is associated with a malignant tumor that is most frequently a small cell lung carcinoma (SCLC), and the autoimmune (A-LEMS) form is often related to other dysimmune diseases. Approximately 90% of LEMS patients present antibodies against presynaptic membrane P/Q-type voltage-gated calcium channels (VGCC). These antibodies are directly implicated in the pathophysiology of the disorder, provoke reduced acetylcholine (ACh) at the nerve terminal and consequently lead to muscle weakness. LEMS is clinically characterized by proximal muscle weakness, autonomic dysfunction and areflexia. In clinically suspected cases, diagnoses are confirmed by serological and electrodiagnostic tests. The detection of P/Q-type VGCC antibodies is supportive when there is clinical suspicion but should be carefully interpreted in the absence of characteristic clinical or electrodiagnostic features. Typical electrodiagnostic findings (ie, reduced compound motor action potentials (CMAPs), significant decrements in the responses to low frequency stimulation and incremental responses after brief exercise or high-frequency stimulation) reflect the existence of a presynaptic transmission defect and are key confirmatory criteria. Diagnosis requires a high level of awareness and necessitates the initiation of a prompt screening and surveillance process to detect and treat malignant tumors. In clinically affected patients without cancer and after cancer treatment, symptomatic treatment with 3,4-diaminopyridine or immunosuppressive agents can significantly improve neurologic symptoms and the quality of life. We present a detailed review of LEMS with special emphasis on the pathophysiological mechanisms, clinical manifestation and diagnostic procedure.

HIF-mediated Suppression of DEPTOR Confers Resistance to mTOR Kinase Inhibition in Renal Cancer.

Mechanistic target of rapamycin (mTOR) is a fundamental regulator of cell growth, proliferation, and metabolism. mTOR is activated in renal cancer and accelerates tumor progression. Here, we report that the mTOR inhibitor, DEP domain-containing mTOR-interacting protein (DEPTOR), is strikingly suppressed in clear cell renal cell carcinoma (ccRCC) tumors and cell lines. We demonstrate that DEPTOR is repressed by both hypoxia-inducible factors, HIF-1 and HIF-2, which occurs through activation of the HIF-target gene and transcriptional repressor, BHLHe40/DEC1/Stra13. Restoration of DEPTOR- and CRISPR/Cas9-mediated knockout experiments demonstrate that DEPTOR is growth inhibitory in ccRCC. Furthermore, loss of DEPTOR confers resistance to second-generation mTOR kinase inhibitors through deregulated mTORC1 feedback to IRS-2/PI3K/Akt. This work reveals a hitherto unknown mechanism of resistance to mTOR kinase targeted therapy that is mediated by HIF-dependent reprograming of mTOR/DEPTOR networks and suggests that restoration of DEPTOR in ccRCC will confer sensitivity to mTOR kinase therapeutics.

Myoglobinopathy is an adult-onset autosomal dominant myopathy with characteristic sarcoplasmic inclusions.

Myoglobin, encoded by MB, is a small cytoplasmic globular hemoprotein highly expressed in cardiac myocytes and oxidative skeletal myofibers. Myoglobin binds O2, facilitates its intracellular transport and serves as a controller of nitric oxide and reactive oxygen species. Here, we identify a recurrent c.292C>T (p.His98Tyr) substitution in MB in fourteen members of six European families suffering from an autosomal dominant progressive myopathy with highly characteristic sarcoplasmic inclusions in skeletal and cardiac muscle. Myoglobinopathy manifests in adulthood with proximal and axial weakness that progresses to involve distal muscles and causes respiratory and cardiac failure. Biochemical characterization reveals that the mutant myoglobin has altered O2 binding, exhibits a faster heme dissociation rate and has a lower reduction potential compared to wild-type myoglobin. Preliminary studies show that mutant myoglobin may result in elevated superoxide levels at the cellular level. These data define a recognizable muscle disease associated with MB mutation.

Motor unit number index (MUNIX) derivation from the relationship between the area and power of surface electromyogram: a computer simulation and clinical study.

OBJECTIVE: The motor unit number index (MUNIX) is a technique based on the surface electromyogram (sEMG) that is gaining acceptance as a method for monitoring motor neuron loss, because it is reliable and produces less discomfort than other electrodiagnostic techniques having the same intended purpose. MUNIX assumes that the relationship between the area of sEMG obtained at increasing levels of muscle activation and the values of a variable called 'ideal case motor unit count' (ICMUC), defined as the product of the ratio between area and power of the compound muscle action potential (CMAP) by that of the sEMG, is described by a decreasing power function. Nevertheless, the reason for this comportment is unknown. The objective of this work is to investigate if the definition of MUNIX could derive from more basic properties of the sEMG. APPROACH: The CMAP and sEMG epochs obtained at different levels of muscle activation from (1) the abductor pollicis brevis (APB) muscle of persons with and without a carpal tunnel syndrome (CTS) and (2) from a computer model of sEMG generation previously published were analysed. MAIN RESULTS: MUNIX reflects the power relationship existing between the area and power of a sEMG. The exponent of this function was smaller in patients with motor CTS than in the rest of the subjects. The analysis of the relationship between the area and power of a sEMG could aid in distinguishing a MUNIX reduction due to a motoneuron loss from that due to a loss of muscle fibre. SIGNIFICANCE: MUNIX is derived from the relationship between the area and power of a sEMG. This relationship changes when there is a loss of motor units (MUs), which partially explains the diagnostic sensibility of MUNIX. Although the reasons for this change are unknown, it could reflect an increase in the proportion of MUs of great amplitude.

HEL/UAP56 binds cotranscriptionally to the Balbiani ring pre-mRNA in an intron-independent manner and accompanies the BR mRNP to the nuclear pore.

The splicing factor UAP56/HEL/Sub2p is essential for mRNA export. It has been proposed that UAP56/HEL/Sub2p interacts with the pre-mRNA during splicing and recruits the export factor Aly/REF/Yra1 (reviewed in ) to the spliced mRNA. However, UAP56/HEL/Sub2p also participates in the transport of intronless mRNAs, and thus its role in export is not necessarily coupled to splicing. Here, we characterize the HEL protein of Chironomus tentans and we analyze in situ the interaction of HEL with a natural export substrate, the Balbiani ring pre-messenger ribonucleoprotein (BR pre-mRNP, reviewed in ). Using immunoelectron microscopy, we show that HEL binds to the BR pre-mRNP cotranscriptionally and that incorporation of HEL into the pre-mRNP is independent of the location of introns along the BR pre-mRNA. We also show that HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation to the cytoplasm. Aly/REF is also released from the BR mRNP during translocation but after dissociation of HEL. In summary, we have shown that binding of HEL to the BR pre-mRNA occurs independently of splicing, and we have established the point in the export pathway at which HEL and Aly/REF interact with the mRNP.

Phenotypic patterns of desminopathy associated with three novel mutations in the desmin gene.

Desminopathy represents a subgroup of myofibrillar myopathies caused by mutations in the desmin gene. Three novel disease-associated mutations in the desmin gene were identified in unrelated Spanish families affected by cardioskeletal myopathy. A selective pattern of muscle involvement, which differed from that observed in myofibrillar myopathy resulting from mutations in the myotilin gene, was observed in each of the three families with novel mutations and each of three desminopathy patients with known desmin mutations. Prominent joint retractions at the ankles and characteristic nasal speech were observed early in the course of illness. These findings suggest that muscle imaging in combination with routine clinical and pathological examination may be helpful in distinguishing desminopathy from other forms of myofibrillar myopathy and ordering appropriate molecular investigations.