Slowed conduction and thin myelination of peripheral nerves associated with mutant rho Guanine-nucleotide exchange factor 10

Slowed nerve-conduction velocities (NCVs) are a biological endophenotype in the majority of the hereditary motor and sensory neuropathies (HMSN). Here, we identified a family with autosomal dominant segregation of slowed NCVs without the clinical phenotype of HMSN. Peripheral-nerve biopsy showed predominantly thinly myelinated axons. We identified a locus at 8p23 and a Thr109Ile mutation in ARHGEF10, encoding a guanine-nucleotide exchange factor (GEF) for the Rho family of GTPase proteins (RhoGTPases). Rho GEFs are implicated in neural morphogenesis and connectivity and regulate the activity of small RhoGTPases by catalyzing the exchange of bound GDP by GTP. Expression analysis of ARHGEF10, by use of its mouse orthologue Gef10, showed that it is highly expressed in the peripheral nervous system. Our data support a role for ARHGEF10 in developmental myelination of peripheral nerves.     

Identification and partial characterization of two inducible gelatin-cleavage activities localized to the sea urchin extraembryonic matrix,the hyaline layer

We have identified two inducible, gelatin-cleaving activities in the sea urchin extraembryonic matrix, the hyaline layer. Isolated hyaline layers, incubated in the presence of benzamidine, were devoid of gelatin-cleavage activities with apparent molecular mass less then 80k. However, when layers were incubated for 9-11 h in the absence of benzamidine, gelatin-cleavage activities, with apparent molecular mass 40- and 50k, were detected. Induction required the presence of NaCl and CaCl(2) at concentrations similar to those found in seawater and readdition of the reversible serine protease inhibitor benzamidine prevented induction. Both gelatin-cleaving activities were activated by calcium at a concentration similar to the calcium concentration found in seawater. Magnesium, also a major cationic species present in seawater, could not replace calcium as the activating ion. In addition, magnesium could not compete with calcium for binding to the gelatinases. Both cleavage activities showed substrate specificity and each failed to cleave bovine serum albumin, bovine hemoglobin or casein. Cleavage activity towards gelatin was inhibited by benzamidine and aminoethyl benzenesulfonyl fluoride, indicating that both activities belonged to the serine class of proteases. The induced 40-kDa activity displayed similar properties to those of a comigrating, gelatin-cleaving activity present in 69-h-old embryos.     

Differential binding of ligands to the apolipoprotein E receptor 2

Apolipoprotein E receptor 2 (apoER2) is an important participant in the Reelin signaling pathway that directs cell positioning during embryogenesis. ApoER2 is a cell surface molecule that elicits intracellular signal transduction through binding of Reelin. The structural requirements for Reelin binding to apoER2 and the receptor domains involved in this process are unclear at present. Using a series of receptor mutants, we characterized the interaction of apoER2 with Reelin and compared this interaction to that of apoER2 with the receptor-associated protein (RAP), an apoER2 ligand that does not induce signaling. By surface plasmon resonance we demonstrate that apoER2 exhibits 6-fold higher affinity for Reelin than the very low density lipoprotein receptor (VLDLR), which also functions as a Reelin receptor (K(D) 0.2 nM versus K(D) 1.2 nM). Acidic amino acid residues in complement-type repeat domains 1 and 3 of apoER2 are required for Reelin binding. The same regions of the receptor are also bound by RAP with a 25-fold lower affinity (K(D) 5 nM). Whereas RAP binds to apoER2 with a 1:1 stoichiometry, experimental evidence suggests that Reelin associates with two or more receptor molecules simultaneously to achieve high-affinity interaction. This finding indicates that aggregation of apoER2 by multivalent ligands such as Reelin may be the structural basis for signal transduction.     

Binding of Zn-chlorin to a synthetic four-helix bundle peptide through histidine ligation

We have used a two histidine-containing synthetic peptide (Sharp et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 10465-10470) as a scaffold to bind Zn(II) chlorin e6 (ZnCe6) through histidine ligation. Protocols for the preparation and purification of the peptide using an Escherichia coli expression system are presented. Size-exclusion chromatography and circular dichroism measurements indicate that the peptide self-assembles into a four-helix bundle protein. Two variants of the peptide lacking either one or both of the histidine residues were used to demonstrate the stoichiometry of ZnCe6 binding. Comparison of the titration profiles determined by UV-vis spectroscopy for the purified one- and two-histidine peptides suggests that the two-histidine peptide can bind two ZnCe6. The binding stoichiometry of ZnCe6 was verified by gel chromatography and native gel electrophoresis using the peptide variant lacking histidine residues as the control. Like many other chlorophyll analogue molecules, ZnCe6 can be photooxidized. The light-induced electron transfer between the ZnCe6-peptide complex and the added phenyl-p-benzoquinone was measured using time-resolved EPR spectroscopy and shown to be faster and have a higher yield than the electron transfer between unbound ZnCe6 and quinone. The implications of constructing a ZnCe6-peptide complex in terms of artificial photosynthesis are discussed.     

Effects of in vitro production on horse embryo morphology,cytoskeletal characteristics,and blastocyst capsule formation

Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was evaluated with light microscopy, and multiphoton scanning confocal microscopy was used to examine the distribution of microfilaments (AlexaFluor-Phalloidin stained) and the rate of apoptosis (cells with fragmented or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei). To examine the influence of culture on capsule formation, conceptuses were stained with a monoclonal antibody specific for capsular glycoproteins (OC-1). The blastocyst rate was higher for zygotes transferred to a sheep's oviduct (16%) than for those cultured in SOF (6.3%). Day 7 IVP embryos were small and compact with relatively few cells, little or no blastocoele, and an indistinct inner cell mass. IVP embryos had high percentages of apoptotic cells (10% versus 0.3% for in vivo embryos) and irregularly distributed microfilaments. Although they secreted capsular glycoproteins, the latter did not form a normal capsule but instead permeated into the zona pellucida or remained in patches on the trophectodermal surface. These results demonstrate that the initial layer of capsule is composed of OC-1-reactive glycoproteins and that embryo development ex vivo is retarded and aberrant, with capsule formation failing as a result of failed glycoprotein aggregation.     

Explicit water near the catalytic I helix Thr in the predicted solution structure of CYP2A4

The solution structure of mouse cytochrome P450 2A4 (CYP2A4), a monooxygenase of deoxysteroids, was obtained using homology modeling and molecular dynamics. The solvent-equilibrated CYP2A4 preserves the essential features of CYP450s. A comparison of the models CYP2A4 and CYP2A4 with testosterone bound CYP2A4/T illustrates the changes induced by the binding of the substrate. Experimental evidence links four amino acid residues to the catalytic activity, substrate specificity, and regioselectivity of this enzyme. Three of the four amino acids are found within contact distance of the testosterone substrate, and therefore may control the binding of the substrate through direct interaction. Remarkably, a water complex previously observed in x-ray crystal structure forms near the bulge in the central I helix that contains a conserved Thr. The properties of the I helix are computed in the context of the presence or absence of ligand.     

Mechanisms for nucleosome mobilization

Nucleosomes are the ubiquitous and fundamental packaging for eukaryotic genomes, and are the substrate for many processes in the nucleus. Nucleosomes are not static entities but can readily be moved by thermal energy and ATP-dependent chromatin remodeling complexes in a process known as sliding or shifting. We summarize from a mechanical perspective the twist defect and bulge diffusion mechanisms proposed as the most likely pathway for nucleosome mobilization. We then consider the elastic properties of DNA and how this affects the potential for each mechanism, concentrating on kinetic aspects of twist diffusion and possible planar bulge sizes and summarize the experimental evidence reflecting on each. Either, or both, mechanisms could occur, and careful experimentation focusing on their uniquely distinguishing features will be required to determine their relative contributions to chromatin dynamics.     

Micro-assembly of functionalized particulate monolayer on C18-derivatized SiO2 surfaces

This work describes a simple approach to immobilize functionalized colloidal microstructures onto a C(18)-coated SiO(2) substrate via specific or non-specific bio-mediated interactions. Biotinylated bovine serum albumin pre-adsorbed onto a C(18) surface was used to mediate the surface assembly of streptavidin-coated microbeads (2.8 microm), while a bare C(18) surface was used to immobilize anti-Listeria antibody-coated microbeads (2.8 microm) through hydrophobic interactions. For a C(18) surface pre-adsorbed with bovine serum albumin, hydrophobic polystyrene microbeads (0.8 microm) and positively charged dimethylamino microbeads (0.8 microm) were allowed to self-assemble onto the surface. A monolayer with high surface coverage was observed for both polystyrene and dimethylamino microbeads. The adsorption characteristics of Escherichia coli and Listeria monocytogenes on these microbead-based surfaces were studied using fluorescence microscopy. Both streptavidin microbeads pre-adsorbed with biotinylated anti-Listeria antibody and anti-Listeria antibody-coated microbeads showed specific capture of L. monocytogenes, while polystyrene and dimethylamino microbeads captured both E. coli and L. monocytogenes non-specifically. The preparation of microbead-based surfaces for the construction of microfluidic devices for separation, detection, or analysis of specific biological species is discussed.     

Pressure treatment of tailspike aggregates rapidly produces on-pathway folding intermediates

Protein folding and aggregation are in direct competition in living systems, yet measuring the two pathways simultaneously has rarely been accomplished. In order to identify the mechanism of high-pressure dissociation of aggregates, we compared the simultaneous on- and off-pathway behavior following dilution of freshly denatured P22 tailspike protein. Tailspike assembly at 100 microg/mL was monitored at four temperatures using a combination of size-exclusion chromatography and native polyacrylamide gel electrophoresis (PAGE) and folding and aggregation rates and yields were determined. As temperature increased, the yield of native trimeric tailspike decreased from 26.1 +/- 1.3 microg/mL at 20 degrees C to 0 microg/mL at 37 degrees C. Pressure treatment dissociated 60% of the trapped aggregates created at 37 degrees C and yielded 19.8 +/- 1.1 microg/mL of native trimer following depressurization and incubation at 20 degrees C. The rate of refolding of "freshly denatured" tailspike was compared to that following pressure treatment. The trimer formation rate increased by a factor of roughly five, and the aggregate rate decreased by a factor of three, following pressure treatment. Circular dichroism and high-pressure intrinsic tryptophan fluorescence measurements support the model that a structured intermediate is formed in a rapid manner under high pressure from a pressure-sensitive aggregate population.     

Characterization of an acetylated heteroxylan from Eucalyptus globulus Labill

A heteroxylan was isolated from Eucalyptus globulus wood by extraction of peracetic acid delignified holocellulose with dimethyl sulfoxide. Besides (1-->4)-linked beta-D-xylopyranosyl units of the backbone and short side chains of terminal (1-->2)-linked 4-O-methyl-alpha-D-glucuronosyl residues (MeGlcA) in a 1:10 molar ratio, this hemicellulose contained galactosyl and glucosyl units attached at O-2 of MeGlcA originating from rhamnoarabinogalactan and glucan backbones, respectively. About 30% of MeGlcA units were branched at O-2. The O-acetyl-(4-O-methylglucurono)xylan showed an acetylation degree of 0.61, as determined by 1H NMR spectroscopy, and a weight-average molecular weight (M(w)) of about 36 kDa (P=1.05) as revealed from size-exclusion chromatography (SEC) analysis. About half of the beta-D-xylopyranosyl units of the backbone were found as acetylated moieties at O-3 (34 mol%), O-2 (15 mol%) or O-2,3 (6 mol%). Practically, all beta-D-xylopyranosyl units linked at O-2 with MeGlcA residues were 3-O-acetylated (10 mol%).