4,585 research outputs found
Host cell-specific folding of the neuronal nicotinic acetylcholine receptor α7 and α8 subunits
The α7 and α8 nicotinic acetylcholine receptor (nAChR) subunits when expressed in Xenopus oocytes, form functional homo-oligomeric ion channels which are potently inhibited by the nicotinic antagonist α-bungarotoxin (αBTX). However, expression of the rat α7 and chick α8 subunits in several mammalian cell lines demonstrates that the folding of α7 and α8 into a conformation able to bind αBTX or conformation- sensitive antibodies, is critically dependent on the host cell type. In all cell lines, production of α7 (~58 kDa) or α8 (~57 kDa) subunit protein was verified by metabolic labelling and immunoprecipitation using subunit-specific antibodies which recognise linear epitopes. In contrast, for both α7 and α8 subunits, antibodies which recognise conformation-dependent extracellular epitopes fail to detect subunit protein in cell lines which fail to produce nicotinic radioligand binding, implying that these subunits are misfolded. The α7 subunit was expressed in a panel of nine mammalian cell lines from neuronal (N1E-115, NCB-20, Neuro2A, NG108-15, PC12, SH-SY5Y) and non-neuronal (HEK293, CHO, COS-7) origins. However, elevated levels of αBTX-binding could be detected in only two (PC12 and SH-SY5Y) of the nine cell lines examined. The α8 subunit was expressed in three cell lines (HEK293, GH4C1, SH-SY5Y) and readily formed αBTX-binding sites in a polyclonal SH-SY5Y-α8 cell line and when transiently expressed in GH4C1 cells. Saturation binding revealed that α8 nAChRs expressed in transfected GH4C1-α8 cells bind epibatidine with high affinity (KD=0.24 ± 0.4 nM) although no specific αBTX or epibatidine binding could be detected in transfected HEK293-α8 cells. HEK293 cells which fail to correctly fold the α7 and α8 nAChR subunits form functional cell surface rat muscle nAChRs and homo-oligomeric 5-HT3 receptors. In addition, chimaeric subunits encoding the N-terminal region of α7 or α8 and the C-terminal domain of the 5-HT3 receptor were expressed very efficiently in all cell lines examined. These results implicate the C-terminal domain of α7 and α8 in the cell-type specific folding and further chimeras need to be generated to more precisely determine which region mediates this effect
Phylogenetic analysis of ferlin genes reveals ancient eukaryotic origins
<p>Abstract</p> <p>Background</p> <p>The ferlin gene family possesses a rare and identifying feature consisting of multiple tandem C2 domains and a C-terminal transmembrane domain. Much currently remains unknown about the fundamental function of this gene family, however, mutations in its two most well-characterised members, dysferlin and otoferlin, have been implicated in human disease. The availability of genome sequences from a wide range of species makes it possible to explore the evolution of the ferlin family, providing contextual insight into characteristic features that define the ferlin gene family in its present form in humans.</p> <p>Results</p> <p>Ferlin genes were detected from all species of representative phyla, with two ferlin subgroups partitioned within the ferlin phylogenetic tree based on the presence or absence of a DysF domain. Invertebrates generally possessed two ferlin genes (one with DysF and one without), with six ferlin genes in most vertebrates (three DysF, three non-DysF). Expansion of the ferlin gene family is evident between the divergence of lamprey (jawless vertebrates) and shark (cartilaginous fish). Common to almost all ferlins is an N-terminal C2-FerI-C2 sandwich, a FerB motif, and two C-terminal C2 domains (C2E and C2F) adjacent to the transmembrane domain. Preservation of these structural elements throughout eukaryotic evolution suggests a fundamental role of these motifs for ferlin function. In contrast, DysF, C2DE, and FerA are optional, giving rise to subtle differences in domain topologies of ferlin genes. Despite conservation of multiple C2 domains in all ferlins, the C-terminal C2 domains (C2E and C2F) displayed higher sequence conservation and greater conservation of putative calcium binding residues across paralogs and orthologs. Interestingly, the two most studied non-mammalian ferlins (Fer-1 and Misfire) in model organisms <it>C. elegans </it>and <it>D. melanogaster</it>, present as outgroups in the phylogenetic analysis, with results suggesting reproduction-related divergence and specialization of species-specific functions within their genus.</p> <p>Conclusions</p> <p>Our phylogenetic studies provide evolutionary insight into the ferlin gene family. We highlight the existence of ferlin-like proteins throughout eukaryotic evolution, from unicellular phytoplankton and apicomplexan parasites, through to humans. We characterise the preservation of ferlin structural motifs, not only of C2 domains, but also the more poorly characterised ferlin-specific motifs representing the DysF, FerA and FerB domains. Our data suggest an ancient role of ferlin proteins, with lessons from vertebrate biology and human disease suggesting a role relating to vesicle fusion and plasma membrane specialization.</p
Generation of Phaseolus vulgaris ESTs and investigation of their regulation upon Uromyces appendiculatus infection
<p>Abstract</p> <p>Background</p> <p><it>Phaseolus vulgaris </it>(common bean) is the second most important legume crop in the world after soybean. Consequently, yield losses due to fungal infection, like <it>Uromyces appendiculatus </it>(bean rust), have strong consequences. Several resistant genes were identified that confer resistance to bean rust infection. However, the downstream genes and mechanisms involved in bean resistance to infection are poorly characterized.</p> <p>Results</p> <p>A subtractive bean cDNA library composed of 10,581 unisequences was constructed and enriched in sequences regulated by either bean rust race 41, a virulent strain, or race 49, an avirulent strain on cultivar Early Gallatin carrying the resistance gene <it>Ur-4</it>. The construction of this library allowed the identification of 6,202 new bean ESTs, significantly adding to the available sequences for this plant. Regulation of selected bean genes in response to bean rust infection was confirmed by qRT-PCR. Plant gene expression was similar for both race 41 and 49 during the first 48 hours of the infection process but varied significantly at the later time points (72–96 hours after inoculation) mainly due to the presence of the <it>Avr4 </it>gene in the race 49 leading to a hypersensitive response in the bean plants. A biphasic pattern of gene expression was observed for several genes regulated in response to fungal infection.</p> <p>Conclusion</p> <p>The enrichment of the public database with over 6,000 bean ESTs significantly adds to the genomic resources available for this important crop plant. The analysis of these genes in response to bean rust infection provides a foundation for further studies of the mechanism of fungal disease resistance. The expression pattern of 90 bean genes upon rust infection shares several features with other legumes infected by biotrophic fungi. This finding suggests that the <it>P. vulgaris</it>-<it>U. appendiculatus </it>pathosystem could serve as a model to explore legume-rust interaction.</p
Case report: Adult-onset limb girdle muscular dystrophy in sibling pair due to novel homozygous LAMA2 missense variant
Recessive pathogenic variants in the laminin subunit alpha 2 (LAMA2) gene cause a spectrum of disease ranging from severe congenital muscular dystrophy to later-onset limb girdle muscular dystrophy (LGMDR23). The phenotype of LGMDR23 is characterized by slowly progressive proximal limb weakness, contractures, raised creatine kinase, and sometimes distinctive cerebral white matter changes and/or epilepsy. We present two siblings, born to consanguineous parents, who developed adult-onset LGMDR23 associated with typical cerebral white matter changes and who both later developed dementia. The male proband also had epilepsy and upper motor neuron signs when he presented at age 72. Merosin immunohistochemistry and Western blot on muscle biopsies taken from both subjects was normal. Whole exome sequencing revealed a previously unreported homozygous missense variant in LAMA2 [Chr6(GRCh38):g.129297734G>A; NM_000426.3:c.2906G>A; p.(Cys969Tyr)] in the proband. The same homozygous LAMA2 variant was confirmed by Sanger sequencing in the proband's affected sister. These findings expand the genotypic and phenotypic spectrum of LGMDR23
Evidence for a dominant-negative effect in ACTA1 nemaline myopathy caused by abnormal folding, aggregation and altered polymerization of mutant actin isoforms
We have studied a cohort of nemaline myopathy (NM) patients with mutations in the muscle α-skeletal actin gene (ACTA1). Immunoblot analysis of patient muscle demonstrates increased γ-filamin, myotilin, desmin and α-actinin in many NM patients, consistent with accumulation of Z line-derived nemaline bodies. We demonstrate that nebulin can appear abnormal secondary to a primary defect in actin, and show by isoelectric focusing that mutant actin isoforms are present within insoluble actin filaments isolated from muscle from two ACTA1 NM patients. Transfection of C2C12 myoblasts with mutant actinEGFP constructs resulted in abnormal cytoplasmic and intranuclear actin aggregates. Intranuclear aggregates were observed with V163L-, V163M- and R183G-actinEGFP constructs, and modeling shows these residues to be adjacent to the nuclear export signal of actin. V163L and V163M actin mutants are known to cause intranuclear rod myopathy, however, intranuclear bodies were not reported in patient R183G. Transfection studies in C2C12 myoblasts showed significant alterations in the ability of V136L and R183G actin mutants to polymerize and contribute to insoluble actin filaments. Thus, we provide direct evidence for a dominant-negative effect of mutant actin in NM. In vitro studies suggest that abnormal folding, altered polymerization and aggregation of mutant actin isoforms are common properties of NM ACTA1 mutants. Some of these effects are mutation-specific, and likely result in variations in the severity of muscle weakness seen in individual patients. A combination of these effects contributes to the common pathological hallmarks of NM, namely intranuclear and cytoplasmic rod formation, accumulation of thin filaments and myofibrillar disorganizatio
Anthracycline-Induced Cardiotoxicity: Cardiac Monitoring by Continuous Wave-Doppler Ultrasound Cardiac Output Monitoring and Correlation to Echocardiography
Background: Anthracyclines are agents with a well-known cardiotoxicity. The study sought to evaluate the hemodynamic response to an anthracycline using real-time continuous-wave (CW)-Doppler ultrasound cardiac output monitoring (USCOM) and echocardiography in combination with serum biomarkers. Methods: 50 patients (26 male, 24 female, median age 59 years) suffering from various types of cancer received an anthracycline-based regimen. Patients' responses were measured at different time points (T0 prior to infusion, T1 6 h post infusion, T2 after 1 day, T3 after 7 days, and T4 after 3 months) with CW-Doppler ultrasound (T0-T4) and echocardiography (T1, T4) for hemodynamic parameters such as stroke volume (SV; SVUSCOM ml) and ejection fraction (EF; EFechocardiography%) and with NT-pro-BNP and hs-Troponin T (T0-T4). Results: During the 3-month observation period, the relative decrease in the EF determined by echocardiography was -2.1% (Delta T0-T4, T0 71 +/- 7.8%, T4 69.5 +/- 7%, p = 0.04), whereas the decrease in SV observed using CW-Doppler was -6.5% (Delta T0-T4, T0 54 +/- 19.2 ml, T4 50.5 +/- 20.6 ml, p = 0.14). The kinetics for serum biomarkers were inversely correlated. Conclusions: Combining real-time CW-Doppler USCOM and serum biomarkers is feasible for monitoring the immediate and chronic hemodynamic changes during an anthracycline-based regimen; the results obtained were comparable to those from echocardiography
Amino Alkynylisoquinoline and Alkynylnaphthyridine Compounds Potently Inhibit Acute Myeloid Leukemia Proliferation in Mice
B
ackground: Acute myeloid leukemia (AML) remains one of the most lethal, rarely cured cancers, despite decades of active development of AML therapeutics. Currently, the 5-year survival of AML patients is about 30% and for elderly patients, the rate drops to b10%. About 30% of AML patients harbor an activating mutation in the tyrosine kinase domain (TKD) of Fms-Like Tyrosine kinase 3 (FLT3) or a FLT3 internal tandem duplication (FLT3-ITD). In- hibitors of FLT3, such as Rydapt that was recently approved by the FDA, have shown good initial response but pa- tients often relapse due to secondary mutations in the FLT3 TKD, like D835Y and F691 L mutations.
Methods: Alkynyl aminoisoquinoline and naphthyridine compounds were synthesized via Sonogashira coupling. The compounds were evaluated for their in vitro and in vivo effects on leukemia growth. Findings: The compounds inhibited FLT3 kinase activity at low nanomolar concentrations. The lead compound, HSN431, also inhibited Src kinase activity. The compounds potently inhibited the viability of MV4–11 and MOLM-14 AML cells with IC50 values b1 nM. Furthermore, the viability of drug-resistant AML cells harboring the D835Y and F691 L mutations were potently inhibited. In vivo efficacy studies in mice demonstrated that the compounds could drastically reduce AML proliferation in mice.
Interpretation: Compounds that inhibit FLT3 and downstream targets like Src (for example HSN431) are good leads for development as anti-AML agents
Ketosis Suppression and Ageing (KetoSAge): The Effects of Suppressing Ketosis in Long Term Keto-Adapted Non-Athletic Females
Most studies on ketosis have focused on short-term effects, male athletes, or weight loss. Hereby, we studied the effects of short-term ketosis suppression in healthy women on long-standing ketosis. Ten lean (BMI 20.5 ± 1.4), metabolically healthy, pre-menopausal women (age 32.3 ± 8.9) maintaining nutritional ketosis (NK) for > 1 year (3.9 years ± 2.3) underwent three 21-day phases: nutritional ketosis (NK; P1), suppressed ketosis (SuK; P2), and returned to NK (P3). Adherence to each phase was confirmed with daily capillary D-beta-hydroxybutyrate (BHB) tests (P1 = 1.9 ± 0.7; P2 = 0.1 ± 0.1; and P3 = 1.9 ± 0.6 mmol/L). Ageing biomarkers and anthropometrics were evaluated at the end of each phase. Ketosis suppression significantly increased: insulin, 1.78-fold from 33.60 (± 8.63) to 59.80 (± 14.69) mmol/L (p = 0.0002); IGF1, 1.83-fold from 149.30 (± 32.96) to 273.40 (± 85.66) µg/L (p = 0.0045); glucose, 1.17-fold from 78.6 (± 9.5) to 92.2 (± 10.6) mg/dL (p = 0.0088); respiratory quotient (RQ), 1.09-fold 0.66 (± 0.05) to 0.72 (± 0.06; p = 0.0427); and PAI-1, 13.34 (± 6.85) to 16.69 (± 6.26) ng/mL (p = 0.0428). VEGF, EGF, and monocyte chemotactic protein also significantly increased, indicating a pro-inflammatory shift. Sustained ketosis showed no adverse health effects, and may mitigate hyperinsulinemia without impairing metabolic flexibility in metabolically healthy women
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