N4WBP5, a potential target for ubiquitination by the Nedd4 family of proteins, is a novel golgi-associated protein

Nedd4 belongs to a family of ubiquitin-protein ligases that is characterized by 2-4 WW domains, a carboxyl-terminal Hect ((h) under bar omologous to (E) under bar6-AP (C) under bar arboxyl (t) under bar erminus)-domain and in most cases an amino-terminal C2 domain. We had previously identified a series of proteins that associates with the WW domains of Nedd4. In this paper, we demonstrate that one of the Nedd4-binding proteins, N4WBP5, belongs to a small group of evolutionarily conserved proteins with three transmembrane domains. N4WBP5 binds Nedd4 WW domains via the two PPXY motifs present in the amino terminus of the protein. In addition to Nedd4, N4WBP5 can interact with the WW domains of a number of Nedd4 family members and is ubiquitinated. Endogenous N4WBP5 localizes to the Golgi complex. Ectopic expression of the protein disrupts the structure of the Golgi, suggesting that N4WBP5 forms part of a family of integral Golgi membrane proteins. Based on previous observations in yeast, we propose that N4WBP5 may act as an adaptor for Nedd4-like proteins and their putative targets to control ubiquitin-dependent protein sorting and trafficking.

Elucidating the molecular mechanisms that underlie the target control of motoneuron death

Approximately half of the motoneurons generated during normal embryonic development undergo programmed cell death. Most of this death occurs during the time when synaptic connections are being formed between motoneurons and their target, skeletal muscle. Subsequent muscle activity stemming from this connection helps determine the final number of surviving motoneurons. These observations have given rise to the idea that motoneuron survival is dependent upon access to muscle derived trophic factors, presumably through intact neuromuscular synapses. However, it is not yet understood how the muscle regulates the supply of such trophic factors, or if there are additional mechanisms operating to control the fate of the innervating motoneuron. Recent observations have highlighted target independent mechanisms that also operate to support the survival of motoneurons, such as early trophic-independent periods of motoneuron death, trophic factors derived from Schwann cells and selection of motoneurons during pathfinding. Here we review recent investigations into motoneuron cell death when the molecular signalling between motoneurons and muscle has been genetically disrupted. From these studies, we suggest that in addition to trophic factors from muscle and/or Schwann cells, specific adhesive interactions between motoneurons and muscle are needed to regulate motoneuron survival. Such interactions, along with intact synaptic basal lamina, may help to regulate the supply and presentation of trophic factors to motoneurons.

The VCAM-1 gene that encodes the vascular cell adhesion molecule is a target of the Sry-related high mobility group box gene, Sox18

VCAM-1 (vascular cell adhesion molecule-1) and Sox18 are involved in vascular development. VCAM-1 is an important adhesion molecule that is expressed on endothelial cells and has a critical role in endothelial activation, inflammation, lymphatic pathophysiology, and atherogenesis. The Sry-related high mobility group box factor Sox18 has previously been implicated in endothelial pathologies. Mutations in human and mouse Sox18 leads to hypotrichosis and lymphedema. Furthermore, both Sox18 and VCAM-1 have very similar spatio-temporal patterns of expression, which is suggestive of crosstalk. We use biochemical techniques, cell culture systems, and the ragged opossum (RaOP) mouse model with a naturally occurring mutation in Sox18 to demonstrate that VCAM-1 is an important target of Sox18. Transfection, site-specific mutagenesis, and gel shift analyses demonstrated that Sox18 directly targeted and trans-activated VCAM-1 expression. Importantly, the naturally occurring Sox18 mutant attenuates the expression and activation of VCAM-1 in vitro. Furthermore, in vivo quantitation of VCAM-1 mRNA levels in wild type and RaOP mice demonstrates that RaOP animals show a dramatic and significant reduction in VCAM-1 mRNA expression in lung, skin, and skeletal muscle. Our observation that the VCAM-1 gene is an important target of SOX18 provides the first molecular insights into the vascular abnormalities in the mouse mutant ragged and the human hypotrichosis-lymphedematelangiectasia disorder.

The receptor tyrosine kinase regulator sprouty1 is a target of the tumor suppressor WT1 and important for kidney development

WT1 encodes a transcription factor involved in kidney development and tumorigenesis. Using representational difference analysis, we identified a new set of WT1 targets, including a homologue of the Drosophila receptor tyrosine kinase regulator, sprouty. Sprouty1 was up-regulated in cell lines expressing wild-type but not mutant WT1. WT1 bound to the endogenous sprouty1 promoter in vivo and directly regulated sprouty1 through an early growth response gene-1 binding site. Expression of Sprouty1 and WT1 overlapped in the developing metanephric mesenchyme, and Sprouty1, like WT1, plays a key role in the early steps of glomerulus formation. Disruption of Sprouty1 expression in embryonic kidney explants by antisense oligonucleotides reduced condensation of the metanephric mesenchyme, leading to a decreased number of glomeruli. In addition, sprouty1 was expressed in the ureteric tree and antisense-treated ureteric trees had cystic lumens. Therefore, sprouty1 represents a physiologically relevant target gene of WT1 during kidney development.

Winding patterns for actively shielded shim coils with asymmetric target-fields

A method is presented for calculating the currents and winding patterns required to design independent zonal and tesseral shim coils for magnetic resonance imaging. Both actively shielded and unshielded configurations are considered, and the region of interest can be located asymmetrically with respect to the coil's length. Streamline, target-field and Fourier-series methods are utilized. The desired target-field is specified at two cylindrical radii, on and inside a circular conducting cylinder of length 2L and radius a. The specification is over some asymmetric portion pL < z < qL of the coil's length (-1 < p < q < 1). Arbitrary functions are used in the outer sections, -L < z < pL and qL < z < L, to ensure continuity of the magnetic field across the entire length of the coil. The entire field is then periodically extended as a half-range cosine Fourier series about either end of the coil. The resultant Fourier coefficients are then substituted into the Fourier-series expressions for the internal and external magnetic fields, and current densities and stream functions on both the primary coil and shield. A contour plot of the stream function directly gives the required coil winding patterns. Spherical harmonic analysis and shielding analysis on field calculations from a ZX shim coil indicate that example designs and theory are well matched.

A novel target-field method for magnetic resonance shim coils: III. Shielded zonal and tesseral coils

This paper continues the development of a new approach for the design of shim and gradient coils, used in magnetic resonance imaging (MRI) applications. A cylindrical primary coil of radius a and length 2L is placed inside a co-axial shield cylinder of radius b. An active shielding strategy is used to create a desired target field at an arbitrarily specified (cylindrical) location within the primary coil, and to annul the field at a certain radius outside the shield. The form of the interior target field may be chosen arbitrarily by the designer, although zonal and tesseral harmonics are typically used in MRI applications. The method presented here designs coil windings on both the primary and shielding cylinders, to produce fields that conform to the specified interior target field and the annulled field exterior to the shield. An additional feature of the method presented here is that the target field inside the primary coil is matched at two different radii, to improve overall accuracy. The method is illustrated by designing several shielded shim coils, for creating higher order tesseral fields located asymmetrically within the coil. The simpler case of pure zonal fields is discussed separately and applied to the design of some higher order shielded coils.