Snx3 Regulates Recycling of the Transferrin Receptor and Iron Assimilation

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Highlights? Snx3 is highly expressed in vertebrate hematopoietic tissues ? Silencing of Snx3 results in anemia and hemoglobin defects in vertebrates ? Snx3 and Vps35 physically interact with Tfrc ? Snx3 is required for endosomal recycling of Tf-Tfrc complex     

Weight-Bearing Locomotion in the Developing Opossum,Monodelphis domestica following Spinal Transection: Remodeling of Neuronal Circuits Caudal to Lesion

Complete spinal transection in the mature nervous system is typically followed by minimal axonal repair, extensive motor paralysis and loss of sensory functions caudal to the injury. In contrast, the immature nervous system has greater capacity for repair, a phenomenon sometimes called the infant lesion effect. This study investigates spinal injuries early in development using the marsupial opossum Monodelphis domestica whose young are born very immature, allowing access to developmental stages only accessible in utero in eutherian mammals. Spinal cords of Monodelphis pups were completely transected in the lower thoracic region, T10, on postnatal-day (P)7 or P28 and the animals grew to adulthood. In P7-injured animals regrown supraspinal and propriospinal axons through the injury site were demonstrated using retrograde axonal labelling. These animals recovered near-normal coordinated overground locomotion, but with altered gait characteristics including foot placement phase lags. In P28-injured animals no axonal regrowth through the injury site could be demonstrated yet they were able to perform weight-supporting hindlimb stepping overground and on the treadmill. When placed in an environment of reduced sensory feedback (swimming) P7-injured animals swam using their hindlimbs, suggesting that the axons that grew across the lesion made functional connections; P28-injured animals swam using their forelimbs only, suggesting that their overground hindlimb movements were reflex-dependent and thus likely to be generated locally in the lumbar spinal cord. Modifications to propriospinal circuitry in P7- and P28-injured opossums were demonstrated by changes in the number of fluorescently labelled neurons detected in the lumbar cord following tracer studies and changes in the balance of excitatory, inhibitory and neuromodulatory neurotransmitter receptors’ gene expression shown by qRT-PCR. These results are discussed in the context of studies indicating that although following injury the isolated segment of the spinal cord retains some capability of rhythmic movement the mechanisms involved in weight-bearing locomotion are distinct.     

Cretaceous and Eocene fossils of the rare extant genus Synneuron Lundstrom (Diptera: Canthyloscledidae): evidence of a true Pangean clade

The first two fossil species of the canthyloscelid genus Synneuron are described based on compression wings. Synneuron eomontana sp. nov. is described from the Middle Eocene Coal Creek Member of the Kishenehn Formation, in the USA, and Synneuron jelli sp. nov. is described from the Lower Cretaceous Koonwarra Fossil Bed of the Korumburra Group, in Australia. The wings are illustrated and compared to the extant species of the genus, to species of the three other recent genera of Canthyloscelidae and to an anisopodid. A phylogenetic analysis of the relationships between the species of Synneuron was performed. The Eocene fossil S. eomontana appears as sister of the pair of recent Holarctic species of the genus, while the Australian Cretaceous species S. jelli is sister of the clade with the species of Synneuron of the northern hemisphere. The sister group of Synneuron is the canthyloscelid clade (Hyperoscelis + Canthyloscelis), for which a middle Jurassic fossil is known. At the early Cretaceous, Gondwana was already separated from Laurasia and the disjunction between the species of Synneuron in Australia and the northern hemisphere clade of the genus suggest a true pangeic origin for the genus. The biology of the canthyloscelid larvae is shaped by its trophic specialization—xylosaprophagous. This suggests that the transition from the Pangean Jurassic gymnosperm-dominated forests to the late Cretaceous angiosperm-dominated forests may be related to the low recent diversity of Synneuron or of the canthyloscelids in the world—and maybe to the extinction of the genus in the southern hemisphere. This major turnover of the vegetation type along the Cretaceous may be also somehow related to the complete extinction of other groups of flies strictly associated with gymnosperms, as may be the case of the lower brachyceran family Zhangsolvidae. This speculation needs additional corroboration from other groups, that will become available with the combination of systematics, paleontology and biogeographical information of different early Cretaceous clades.     

p53 and Translation Attenuation Regulate Distinct Cell Cycle Checkpoints during Endoplasmic Reticulum (ER) Stress

Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G₁ phase, although recent studies have identified a novel ER stress G₂ checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G₂ delay involving CHK1 and a later induction of G₁ arrest associated both with the induction of p53 target genes and loss of cyclin D1. We show that substitution of p53/47 for p53 impairs the ER stress G₁ checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G₂ progression prior to ultimate G₁ arrest.     

Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.     

Simultaneous Surface Display and Secretion of Proteins from Mammalian Cells Facilitate Efficient in Vitro Selection and Maturation of Antibodies

A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.     

Scientific knowledge is possible with small-sample classification

A typical small-sample biomarker classification paper discriminates between types of pathology based on, say, 30,000 genes and a small labeled sample of less than 100 points. Some classification rule is used to design the classifier from this data, but we are given no good reason or conditions under which this algorithm should perform well. An error estimation rule is used to estimate the classification error on the population using the same data, but once again we are given no good reason or conditions under which this error estimator should produce a good estimate, and thus we do not know how well the classifier should be expected to perform. In fact, virtually, in all such papers the error estimate is expected to be highly inaccurate. In short, we are given no justification for any claims.Given the ubiquity of vacuous small-sample classification papers in the literature, one could easily conclude that scientific knowledge is impossible in small-sample settings. It is not that thousands of papers overtly claim that scientific knowledge is impossible in regard to their content; rather, it is that they utilize methods that preclude scientific knowledge. In this paper, we argue to the contrary that scientific knowledge in small-sample classification is possible provided there is sufficient prior knowledge. A natural way to proceed, discussed herein, is via a paradigm for pattern recognition in which we incorporate prior knowledge in the whole classification procedure (classifier design and error estimation), optimize each step of the procedure given available information, and obtain theoretical measures of performance for both classifiers and error estimators, the latter being the critical epistemological issue. In sum, we can achieve scientific validation for a proposed small-sample classifier and its error estimate.     

Carvedilol Prevents Ovariectomy-Induced Myocardial Contractile Dysfunction in Female Rat

Carvedilol has beneficial effects on cardiac function in patients with heart failure but its effect on ovariectomy-induced myocardial contractile dysfunction remains unclear. Estrogen deficiency induces myocardial contractile dysfunction and increases cardiovascular disease risk in postmenopausal women. Our aim was to investigate whether carvedilol, a beta receptor blocker, would prevent ovariectomy-induced myocardial contractile dysfunction. Female rats (8 weeks old) that underwent bilateral ovariectomy were randomly assigned to receive daily treatment with carvedilol (OVX+CAR, 20 mg/kg), placebo (OVX) and SHAM for 58 days. Left ventricle papillary muscle was mounted for isometric tension recordings. The inotropic response to Ca²⁺ (0.62 to 3.75 mM) and isoproterenol (Iso 10⁻⁸ to 10⁻² M) were assessed. Expression of calcium handling proteins was measured by western blot analysis. Carvedilol treatment in the OVX animals: prevented weight gain and slight hypertrophy, restored the reduced positive inotropic responses to Ca²⁺ and isoproterenol, prevented the reduction in SERCA2a expression, abolished the increase in superoxide anion production, normalized the increase in p22^phox expression, and decreased serum angiotensin converting enzyme (ACE) activity. This study demonstrated that myocardial contractile dysfunction and SERCA2a down regulation were prevented by carvedilol treatment. Superoxide anion production and NADPH oxidase seem to be involved in this response.     

Proteomic and microarray analyses of the Dictyostelium Zak1-GSK-3 signaling pathway reveal a role in early development

GskA, the Dictyostelium GSK-3 orthologue, is modified and activated by the dual-specificity tyrosine kinase Zak1, and the two kinases form part of a signaling pathway that responds to extracellular cyclic AMP. We identify potential cellular effectors for the two kinases by analyzing the corresponding null mutants. There are proteins and mRNAs that are altered in abundance in only one or the other of the two mutants, indicating that each kinase has some unique functions. However, proteomic and microarray analyses identified a number of proteins and genes, respectively, that are similarly misregulated in both mutant strains. The positive correlation between the array data and the proteomic data is consistent with the Zak1-GskA signaling pathway's functioning by directly or indirectly regulating gene expression. The discoidin 1 genes are positively regulated by the pathway, while the abundance of the H5 protein is negatively regulated. Two of the targets, H5 and discoidin 1, are well-characterized markers for early development, indicating that the Zak1-GskA pathway plays a role in development earlier than previously observed.