Associations among genotype, clinical phenotype, and intracellular localization of trafficking proteins in ARC syndrome

Arthrogryposisrenal dysfunctioncholestasis (ARC) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in vacuolar protein sorting 33 homologue B (VPS33B) and VPS33B interacting protein, apicalbasolateral polarity regulator (VIPAR). Cardinal features of ARC include congenital joint contractures, renal tubular dysfunction, cholestasis, severe failure to thrive, ichthyosis, and a defect in platelet alpha-granule biogenesis. Most patients with ARC do not survive past the first year of life. We report two patients presenting with a mild ARC phenotype, now 5.5 and 3.5 years old. Both patients were compound heterozygotes with the novel VPS33B donor splice-site mutation c.1225+5G>C in common. Immunoblotting and complementary DNA analysis suggest expression of a shorter VPS33B transcript, and cell-based assays show that c.1225+5G>C VPS33B mutant retains some ability to interact with VIPAR (and thus partial wild-type function). This study provides the first evidence of genotypephenotype correlation in ARC and suggests that VPS33B c.1225+5G>C mutation predicts a mild ARC phenotype. We have established an interactive online database for ARC (https://grenada.lumc.nl/LOVD2/ARC) comprising all known variants in VPS33B and VIPAR. Also included in the database are 15 novel pathogenic variants in VPS33B and five in VIPAR. Hum Mutat 33:16561664, 2012. (c) 2012 Wiley Periodicals, Inc.

T-3 Rapidly Increases SLC2A4 Gene Expression and GLUT4 Trafficking to the Plasma Membrane in Skeletal Muscle of Rat and Improves Glucose Homeostasis

Background: Glucose transporter 4 (GLUT4) is highly expressed in muscle and fat tissue, where triiodothyronine (T-3) induces solute carrier family 2 facilitated glucose transporter member 4 (SLC2A4) gene transcription. T-3 was also shown to rapidly increase glucose uptake in myocytes exposed to cycloheximide, indicating that it might act nongenomically to regulate GLUT4 availability. We tested this hypothesis by evaluating, in thyroidectomized rats (Tx rats), the acute and/or chronic T-3 effects on GLUT4 mRNA expression and polyadenylation, protein content, and trafficking to the plasma membrane (PM) in skeletal muscle, as well as on blood glucose disappearance rate (kITT) after insulin administration. Methods: Rats were surgically thyroidectomized and treated with T-3 (0.3 to 100 mu g/100 g body weight) from 10 minutes to 5 days, and killed thereafter. Sham-operated (SO) rats were used as controls. Total RNA was extracted from the skeletal muscles (soleus [SOL] and extensorum digitalis longus [EDL]) and subjected to Northern blotting analysis using rat GLUT4 cDNA probe. Total protein was extracted and subjected to specific centrifugations for subcellular fractionation, and PM as well as microsomal (M) fractions were subjected to Western blotting analysis, using anti-GLUT4 antiserum as a probe. GLUT4 mRNA polyadenylation was examined by a rapid amplification of cDNA ends-poly(A) test (RACE-PAT). Results: Thyroidectomy reduced skeletal muscle GLUT4 mRNA, mRNA poly(A) tail length, protein content, and trafficking to the PM, as well as the kITT. The acute T-3 treatment rapidly (30 minutes) increased all these parameters compared with Tx rats. The 5-day T-3 treatment increased GLUT4 mRNA and protein expression, and restored GLUT4 trafficking to the PM and kITT to SO values. Conclusions: The results presented here show for the first time that, in parallel to its transcriptional action on the SLC2A4 gene, T-3 exerts a rapid post-transcriptional effect on GLUT4 mRNA polyadenylation, which might increase transcript stability and translation efficiency, leading to the increased GLUT4 content and availability to skeletal muscle, as well as on GLUT4 translocation to the PM, improving the insulin sensitivity, as shown by the kITT.

Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics

Background: Proteinaceous toxins are observed across all levels of inter-organismal and intra-genomic conflicts. These include recently discovered prokaryotic polymorphic toxin systems implicated in intra-specific conflicts. They are characterized by a remarkable diversity of C-terminal toxin domains generated by recombination with standalone toxin-coding cassettes. Prior analysis revealed a striking diversity of nuclease and deaminase domains among the toxin modules. We systematically investigated polymorphic toxin systems using comparative genomics, sequence and structure analysis. Results: Polymorphic toxin systems are distributed across all major bacterial lineages and are delivered by at least eight distinct secretory systems. In addition to type-II, these include type-V, VI, VII (ESX), and the poorly characterized "Photorhabdus virulence cassettes (PVC)", PrsW-dependent and MuF phage-capsid-like systems. We present evidence that trafficking of these toxins is often accompanied by autoproteolytic processing catalyzed by HINT, ZU5, PrsW, caspase-like, papain-like, and a novel metallopeptidase associated with the PVC system. We identified over 150 distinct toxin domains in these systems. These span an extraordinary catalytic spectrum to include 23 distinct clades of peptidases, numerous previously unrecognized versions of nucleases and deaminases, ADP-ribosyltransferases, ADP ribosyl cyclases, RelA/SpoT-like nucleotidyltransferases, glycosyltranferases and other enzymes predicted to modify lipids and carbohydrates, and a pore-forming toxin domain. Several of these toxin domains are shared with host-directed effectors of pathogenic bacteria. Over 90 families of immunity proteins might neutralize anywhere between a single to at least 27 distinct types of toxin domains. In some organisms multiple tandem immunity genes or immunity protein domains are organized into polyimmunity loci or polyimmunity proteins. Gene-neighborhood-analysis of polymorphic toxin systems predicts the presence of novel trafficking-related components, and also the organizational logic that allows toxin diversification through recombination. Domain architecture and protein-length analysis revealed that these toxins might be deployed as secreted factors, through directed injection, or via inter-cellular contact facilitated by filamentous structures formed by RHS/YD, filamentous hemagglutinin and other repeats. Phyletic pattern and life-style analysis indicate that polymorphic toxins and polyimmunity loci participate in cooperative behavior and facultative 'cheating' in several ecosystems such as the human oral cavity and soil. Multiple domains from these systems have also been repeatedly transferred to eukaryotes and their viruses, such as the nucleo-cytoplasmic large DNA viruses. Conclusions: Along with a comprehensive inventory of toxins and immunity proteins, we present several testable predictions regarding active sites and catalytic mechanisms of toxins, their processing and trafficking and their role in intra-specific and inter-specific interactions between bacteria. These systems provide insights regarding the emergence of key systems at different points in eukaryotic evolution, such as ADP ribosylation, interaction of myosin VI with cargo proteins, mediation of apoptosis, hyphal heteroincompatibility, hedgehog signaling, arthropod toxins, cell-cell interaction molecules like teneurins and different signaling messengers.

Modeling the Emergence of Circadian Rhythms in a Clock Neuron Network

Circadian rhythms in pacemaker cells persist for weeks in constant darkness, while in other types of cells the molecular oscillations that underlie circadian rhythms damp rapidly under the same conditions. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms leading to damped or self-sustained oscillations remain largely unknown. There exist many mathematical models that reproduce the circadian rhythms in the case of a single cell of the Drosophila fly. However, not much is known about the mechanisms leading to coherent circadian oscillation in clock neuron networks. In this work we have implemented a model for a network of interacting clock neurons to describe the emergence (or damping) of circadian rhythms in Drosophila fly, in the absence of zeitgebers. Our model consists of an array of pacemakers that interact through the modulation of some parameters by a network feedback. The individual pacemakers are described by a well-known biochemical model for circadian oscillation, to which we have added degradation of PER protein by light and multiplicative noise. The network feedback is the PER protein level averaged over the whole network. In particular, we have investigated the effect of modulation of the parameters associated with (i) the control of net entrance of PER into the nucleus and (ii) the non-photic degradation of PER. Our results indicate that the modulation of PER entrance into the nucleus allows the synchronization of clock neurons, leading to coherent circadian oscillations under constant dark condition. On the other hand, the modulation of non-photic degradation cannot reset the phases of individual clocks subjected to intrinsic biochemical noise.

Motor Neuron Disease and Acquired Axonal Neuropathy Association in HIV Infection: Case Report and Update

Background: A possible viral etiology has been documented in the genesis of motor neuron disorders and acquired peripheral neuropathies, mainly due to the vulnerability of peripheral nerves and the anterior horn to certain viruses. In recent years, several reports show association of HIV infection with Amyotrophic Lateral Sclerosis Syndrome, Motor Neuron Diseases and peripheral neuropathies. Objective: To report a case of an association between Motor Neuron Disease and Acquired Axonal neuropathy in HIV infection, and describe the findings of neurological examination, cerebrospinal fluid, neuroimaging and electrophysiology. Methods: The patient underwent neurological examination. General medical examinations were performed, including, specific neuromuscular tests, analysis of cerebrospinal fluid, muscle biopsy and imaging studies. Results and Discussion: The initial clinical presentation of our case was marked by cramps and fasciculations with posterior distal paresis and atrophy in the left arm. We found electromyography tracings with deficits in the anterior horn of the spinal cord and peripheral nerves. Dysphagia and release of primitive reflexes were also identified. At the same time, the patient was informed to be HIV positive with high viral load. He received antiretroviral therapy, with load control but with no clinical remission. Conclusion: Motor Neuron disorders and peripheral neuropathy may occur in association with HIV infection. However, a causal relationship remains uncertain. It is noteworthy that the antiretroviral regimen may be implicated in some cases.

SCG postnatal remodelling - hypertrophy and neuron number stability - in Spix's Yellow-toothed Cavies (Galea spixii)

Whilst a fall in neuron numbers seems a common pattern during postnatal development, several authors have nonetheless reported an increase in neuron number, which may be associated with any one of a number of possible processes encapsulating either neurogenesis or late maturation and incomplete differentiation. Recent publications have thus added further fuel to the notion that a postnatal neurogenesis may indeed exist in sympathetic ganglia. In the light of these uncertainties surrounding the effects exerted by postnatal development on the number of superior cervical ganglion (SCG) neurons, we have used state-of-the-art design-based stereology to investigate the quantitative structure of SCG at four distinct timepoints after birth, viz., 1-3 days, 1 month, 12 months and 36 months. The main effects exerted by ageing on the SCG structure were: (i) a 77% increase in ganglion volume; (ii) stability in the total number of the whole population of SCG nerve cells (no change - either increase or decrease) during post-natal development; (iii) a higher proportion of uninucleate neurons to binucleate neurons only in newborn animals; (iv) a 130% increase in the volume of uninucleate cell bodies; and (v) the presence of BrdU positive neurons in animals at all ages. At the time of writing our results support the idea that neurogenesis takes place in the SCG of preas, albeit it warrants confirmation by further markers. We also hypothesise that a portfolio of other mechanisms: cell repair, maturation, differentiation and death may be equally intertwined and implicated in the numerical stability of SCG neurons during postnatal development. (C) 2011 ISDN. Published by Elsevier Ltd. All rights reserved.

A Java-based simulation environment for networks of simplified neuron models

ACR is supported by a research grant from CNPq.

Highlights of the XXI annual meeting of the Brazilian Society of Protozoology, the XXXII annual meeting on Basic Research in Chagas' disease & an international symposium on vesicle trafficking in parasitic Protozoa – 7 to 9 November 2005, Caxambu, Minas Gerais, Brazil

This report focuses on the 2005 Annual meeting held in Caxambu, Minas Gerais, Brazil that was convened and organized by the Brazilian Society of Protozoology http://www.sbpz.org.br/. This is an annual event and details of these meetings can be found on the Society's website. Within the space available it has been impossible to cover all the important and fascinating contributions and what is presented are our personal views of the meetings scientific highlights and new developments. The contents undoubtedly reflect each author's scientific interests and expertise. Fuller details of the round tables, seminars and posters can be consulted on line at http://www.sbpz.org.br/livroderesumos2005.php.

ANP impairs the dose-dependent stimulatory effect of ANG II or AVP on H+-ATPase subcellular vesicle trafficking

The effect of angiotensin II (ANG II) or arginine vasopressin (AVP) alone or plus atrial natriuretic peptide (ANP) on H+-ATPase subcellular vesicle trafficking was investigated in MDCK cells following intracellular pH (pHi) acidification by exposure to20 mMNH4Cl for 2 min in a Na+-free solution containing Schering 28080, conditions under which H+-AT-Pase is the only cell mechanism for pHi recovery. Using the acridine orange fluorescent probe (5mM) and confocal microscopy, the vesicle movement was quantified by determining, for each experimental group, the mean slope of the line indicating the changes in apical/basolateral fluorescence density ratio over time during the first 5.30 min of the pHi recovery period. Under the control conditions, the mean slope was 0.079 ± 0.0033 min-1 (14) and it increased significantly with ANG II [10-12 and 10-7 M, respectively to 0.322 ± 0.038 min-1 (13) and 0.578 ± 0.061 min-1 (12)] or AVP [10-12 and 10-6 M, respectively to 0.301 ± 0.018 min-1 (12) and 0.687 ± 0.049 min-1 (11)]. However, in presence of ANP (10-6 M, decreases cytosolic free calcium), dimethyl-BAPTA/AM (5 × 10-5 M, chelates intracellular calcium) or colchicine (10-5 M, 2-h preincubation; inhibits microtubule-dependent vesicular trafficking) alone or plus ANG II or AVP the mean slopes were similar to the control values, indicating that such agents blocked the stimulatory effect of ANG II or AVP on vesicle trafficking. The results suggest that the pathway responsible for the increase in cytosolic free calcium and the microtu-bule-dependent vesicular trafficking are involved in this hormonal stimulating effect. Whether cytosolic free calcium reduction represents an important direct mechanism for ANP impairs the dose-dependent stimulatory effect of ANG II or AVP on H+-ATPase subcellular vesicle trafficking, or is a side effect of other signaling pathways which will require additional studies.