The effect of latent heat of fusion on heat transfer in fluidized-bed coating of thin plates

This paper presents a numerical study of fluidized-bed coating on thin plates using an orthogonal collocation technique. Inclusion of the latent heat of fusion term in the boundary conditions of the mathematical model accounts for the fact that some polymer powders used in coating may be partially crystalline. Predictions of coating thickness on flat plates were made with actual polymers used in fluidized-bed coating. Reasonably good agreement between numerical predictions of the coating thickness and experimental coating data of Richart was obtained for steel panels preheated to 316 degreesC. A good agreement was also obtained between numerical predictions and our coating thickness data for nylon-11 and polyethylene powders. Predicted coating thickness for polyethylene powder on flat plates were obtained with values of heat transfer coefficient closer to those obtained from our experiments. (C) 2002 Elsevier Science B.V. All rights reserved.

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.

In vivo DNA electrotransfer

The use of electrotransfer for DNA delivery to prokaryotic cells, and eukaryotic cells in vitro, has been well known and widely used for many years. However, it is only recently that electric fields have been used to enhance DNA transfer to animal cells in vivo, and this is known as DNA electrotransfer or in vivo DNA electroporation. Some of the advantages of this method of somatic cell gene transfer are that it is a simple method that can be used to transfer almost any DNA construct to animal cells and tissues in vivo; multiple constructs can be co-transfected; it is equally applicable to dividing and nondividing cells; the DNA of interest does not need to be subeloned into a specific viral transfer vector and there is no need for the production of high titre viral stocks; and, as no viral genes are expressed there is less chance of an adverse immunologic reaction to vector sequences. The ease with which efficient in vivo gene transfer can be achieved with in vivo DNA electrotransfer is now allowing genetic analysis to be applied to a number of classic animal model systems where transgenic and embryonic stem cell techniques are not well developed, but for which a wealth of detailed descriptive embryological information is available, or surgical manipulation is much more feasible. As well as exciting applications in developmental biology, in vivo DNA electrotransfer is also being used to transfer genes to skeletal muscle and drive expression of therapeutically active proteins, and to examine exogenous gene and protein function in normal adult cells situated within the complex environment of a tissue and organ system in vivo. Thus, in effect providing the in vivo equivalent of the in vitro transient transfection assay. As the widespread use of in vivo electroporation has really only just begun, it is likely that the future will hold many more applications for this technology in basic research, biotechnology and clinical research areas.

Neuronal calcium-binding proteins and schizophrenia

Calcium-binding proteins (CBPs) such as calbindin, parvalbumin and calretinin are used as immunohistochemical markers for discrete neuronal subpopulations. They are particularly useful in identifying the various subpopulations of GABAergic interneurons that control output from prefrontal and cingulate cortices as well as from the hippocampus. The strategic role these interneurons play in regulating output from these three crucial brain regions has made them a focus for neuropathological investigation in schizophrenia. The number of pathological reports detailing subtle changes in these CBP-containing interneurons in patients with schizophrenia is rapidly growing. These proteins however are more than convenient neuronal markers. They confer survival advantages to neurons and can increase the neuron's ability to sustain firing. These properties may be important in the subtle pathophysiology of nondegenerative phenomena such as schizophrenia. The aim of this review is to introduce the reader to the functional properties of CBPs and to examine the emerging literature reporting alterations in these proteins in schizophrenia as well as draw some conclusions about the significance of these findings. (C) 2002 Elsevier Science B.V. All rights reserved.

GATA proteins identify a novel ventral interneuron subclass in the developing chick spinal cord

Members of the GATA transcription factor gene family have been implicated in a variety of developmental processes, including that of the vertebrate central nervous system. However, the role of GATA proteins in spinal cord development remains unresolved. In this study, we investigated the expression and function of two GATA proteins, GATA2 and GATA3, in the developing chick spinal cord. We show that both proteins are expressed by a distinct subpopulation of ventral interneurons that share the same dorsoventral position as CHX10-positive V2 interneurons. However, no coexpression is observed between the two GATA proteins and CHX10. By in vivo notochord grafting and cyclopamine treatment, we demonstrate that the spatially restricted pattern of GATA3 expression is regulated, at least in part, by the signaling molecule Sonic hedgehog. In addition, we further show that Sonic hedgehog induces GATA3 expression in a dose-dependent manner. Using in ovo electroporations, we also demonstrate that GATA2 is upstream of GATA3 in the same epigenetic cascade and that GATA3 is capable of inducing GATA2 expression in vivo. Furthermore, the ectopically expressed GATA proteins can repress differentiation of other ventral cell fates, but not the development of progenitor populations identified by PAX protein expression. Taken together, our findings strongly suggest an important role for GATA2 and GATA3 proteins in the establishment of a distinct ventral interneuron subpopulation in the developing chick spinal cord. (C) 2002 Elsevier Science (USA).

Circular proteins - no end in sight

Circular proteins are a recently discovered phenomenon. They presumably evolved to confer advantages over ancestral linear proteins while maintaining the intrinsic biological functions of those proteins. In general, these advantages include a reduced sensitivity to proteolytic cleavage and enhanced stability. In one remarkable family of circular proteins, the cyclotides, the cyclic backbone is additionally braced by a knotted arrangement of disulfide bonds that confers additional stability and topological complexity upon the family. This article describes the discovery, structure, function and biosynthesis of the currently known circular proteins. The discovery of naturally occurring circular proteins in the past few years has been complemented by new chemical and biochemical methods to make synthetic circular proteins; these are also briefly described.

Matching SOX: partner proteins and co-factors of the SOX family of transcriptional regulators

SOX transcription factors perform a remarkable variety of important roles in vertebrate development, either activating or repressing specific target genes through interaction with different partner proteins. Surprisingly, these interactions are often mediated by the conserved, DNA-binding HMG domain, raising questions as to how each factor's specificity is generated. We propose a model whereby non-HMG domains may influence partner protein selection and/or binding stability.

Prediction of Golgi Type II membrane proteins based on their transmembrane domains

Motivation: A major issue in cell biology today is how distinct intracellular regions of the cell, like the Golgi Apparatus, maintain their unique composition of proteins and lipids. The cell differentially separates Golgi resident proteins from proteins that move through the organelle to other subcellular destinations. We set out to determine if we could distinguish these two types of transmembrane proteins using computational approaches. Results: A new method has been developed to predict Golgi membrane proteins based on their transmembrane domains. To establish the prediction procedure, we took the hydrophobicity values and frequencies of different residues within the transmembrane domains into consideration. A simple linear discriminant function was developed with a small number of parameters derived from a dataset of Type II transmembrane proteins of known localization. This can discriminate between proteins destined for Golgi apparatus or other locations (post-Golgi) with a success rate of 89.3% or 85.2%, respectively on our redundancy-reduced data sets.

GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway

Endocytosis of cell-surface proteins via specific pathways is critical for their function. We show that multiple glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed to the recycling endosomal compartment but not to the Golgi via a nonclathrin, noncaveolae mediated pathway. GPI anchoring is a positive signal for internalization into rab5-independent tubular-vesicular endosomes also responsible for a major fraction of fluid-phase uptake; molecules merely lacking cytoplasmic extensions are not included. Unlike the internalization of detergent-resistant membrane (DRM)-associated interleukin 2 receptor, endocytosis of DRM-associated GPI-APs is unaffected by inhibition of RhoA or dynamin 2 activity. Inhibition of Rho family GTPase cdc42, but not Rac1, reduces fluid-phase uptake and redistributes GPI-APs to the clathrin-mediated pathway. These results describe a distinct constitutive pinocytic pathway, specifically regulated by cdc42.

Antiserum raised against the Epstein-Barr virus BARF0 protein reacts to HLA-DR beta chain

The role that Epstein-Barr virus plays in nasopharyngeal carcinoma and Burkitt's lymphoma has been under intense study for many years. With only a limited set of viral genes being expressed in these tumours it has been difficult to understand how the virus could cause/aid in the generation of the tumours. In 1997 a paper was published by Fries et al. [Fries et al. (1997) Identification of a novel protein encoded by the BamHI A region of the Epstein-Barr virus. J Virol 71: 2765-2771.] in which a rabbit serum was generated and used to identify protein products (RK-BARF0) encoded from the BamH1 A region of EBV. In this paper we have isolated these proteins from two-dimensional gels and identified them, using mass spectrometry, as components of HLA DR.

Molecular and functional analyses of Kunjin virus infectious cDNA clones demonstrate the essential roles for NS2A in virus assembly and for a nonconservative residue in NS3 in RNA replication

A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by similar to100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an similar to5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type lie residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.

Ras proteins: Different signals from different locations

Ras signalling has classically been thought to occur exclusively at the inner surface of a relatively uniform plasma membrane. Recent studies have shown that Ras proteins interact dynamically with specific microdomains of the plasma membrane as well as with other internal cell membranes. These different membrane microenvironments modulate Ras signal output and highlight the complex interplay between Ras location and function.

Direct visualization of Ras proteins in spatially distinct cell surface microdomains

Localization of signaling complexes to specific micro-domains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent micro-domain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.