Airway patterning: A paradigm for restricted signalling.

Intercellular signalling is limited by the range of cell responsiveness, often mediated by repressors. A recently identified repressor, Sprouty, inhibits MAP kinase signalling in flies, mice and humans and has a conserved function in patterning the airways of these divergent species.     

Vertebrate development: Et in Arkadia

The similarities in organiser formation in Xenopus and mouse embryos have remained elusive. Recent evidence suggests a common mechanism, in which an intracellular protein, Arkadia, is required for formation of the mouse organiser and potentiates the effects of the signalling protein Nodal.     

Orientation of commissural axons in vitro in response to a floor plate-derived chemoattractant

Developing axons are guided to their targets by molecular cues in their local environment. Some cues are short-range, deriving from cells along axonal pathways. There is also increasing evidence for longer-range guidance cues, in the form of gradients of diffusible chemoattractant molecules, which originate from restricted populations of target cells. The guidance of developing commissural axons within the spinal cord depends on one of their intermediate cellular targets, the floor plate. We have shown previously that floor plate cells secrete a diffusible factor(s) that can alter the direction of commissural axon growth in vitro. Here we show that the factor is an effective chemoattractant for commissural axons. It can diffuse considerable distances through a collagen gel matrix and through dorsal and ventral neural epithelium in vitro to reorient the growth of virtually all commissural axons. The orientation of axons occurs in the absence of detectable effects on the survival of commissural neurons or on the rate of commissural axon extension. The regionally restricted expression of the factor suggests that it is present in the embryonic spinal cord in a gradient with its high point at the floor plate. These observations support the idea that the guidance of commissural axons to the ventral midline of the spinal cord results in part from the secretion of a chemoattractant by the floor plate.     

Successful immunotherapy with matrix metalloproteinase-derived peptides in adjuvant arthritis depends on the timing of peptide administration

We have recently found that matrix metalloproteinases (MMPs) are targets for T-cell and B-cell reactivity in experimental arthritis. In the present article, we investigate whether modulation of MMP-specific T-cell responses could influence the course of adjuvant arthritis (AA). Lewis rats were treated nasally with MMP peptides prior to or after AA induction. Administration of the MMP-10 or the MMP-16 peptide prior to AA induction reduced the arthritic symptoms. In contrast, administration of the MMP-10 peptide after AA induction aggravated the arthritic symptoms. The present study shows the possible usefulness of MMP peptides for immunotherapy. However, a clear understanding of proper timing of peptide administration is crucial for the development of such therapies.     

NMR structure and functional characterization of a human cancer-related nucleoside triphosphatase

A screen of the human cancer genome anatomy project (CGAP) database was performed to search for new proteins involved in tumorigenesis. The resulting hits were further screened for recombinant expression, solubility and protein aggregation, which led to the identification of the previously unknown human cancer-related (HCR) protein encoded by the mRNA NM_032324 as a target for structure determination by NMR. The three-dimensional structure of the protein in its complex with ATPgammaS forms a three-layered alpha/beta sandwich, with a central nine-stranded beta-sheet surrounded by five alpha-helices. Sequence and three-dimensional structure comparisons with AAA+ ATPases revealed the presence of Walker A (GPPGVGKT) and Walker B (VCVIDEIG) motifs. Using 1D (31)P-NMR spectroscopy and a coupled enzymatic assay for the determination of inorganic phosphate, we showed that the purified recombinant protein is active as a non-specific nucleoside triphosphatase, with k(cat)=7.6x10(-3) s(-1). The structural basis for the enzymatic activity of HCR-NTPase was further characterized by site-directed mutagenesis of the Walker B motif, which further contributes to making the HCR-NTPase an attractive new target for further biochemical characterization in the context of its presumed role in human tumorigenesis.     

Tensile strength of bone fixation of hydroxyapatite coated Schanz screws of the Heidelberg External Fixation System (HEFS)--comparative torque measurements in clinical use and in cadaver tibia]

It is claimed in the literature that hydroxyapatite(HA)-coated screws of external fixators have superior fixation strength in bone, which is postulated to lead to a substantial decrease in loosening and infection rates. We report on a study of the maximum torque values developed while inserting and removing 30 HA-coated Schanz screws of 8 Heidelberg external fixation systems applied to the tibia to correct leg length differences and axial deformities. The infection rate was determined in accordance with defined criteria, and was found to be about 20% for the HA-coated screws. Screws without infection showed an extraction torque above insertion torque, screws with infection an extraction torque below. A significant correlation (p = 0.05) was seen between infection and decrease in fixation strength (quotient: loosening torque/tightening torque). To exclude the impact of such biological processes as osteointegration and bone remodelling, the clinical results were compared with the torques measured for coated and uncoated Schanz screws in a human cadaveric tibia. A significantly higher fixation strength in bone was found for HA-coated screws in comparison with uncoated screws (p = 0.002). These data warrant a clinical study directly comparing HA-coated and uncoated Schanz screws.     

Granule-bound starch synthase: structure, function, and phylogenetic utility

Interest in the use of low-copy nuclear genes for phylogenetic analyses of plants has grown rapidly, because highly repetitive genes such as those commonly used are limited in number. Furthermore, because low- copy genes are subject to different evolutionary processes than are plastid genes or highly repetitive nuclear markers, they provide a valuable source of independent phylogenetic evidence. The gene for granule-bound starch synthase (GBSSI or waxy) exists in a single copy in nearly all plants examined so far. Our study of GBSSI had three parts: (1) Amino acid sequences were compared across a broad taxonomic range, including grasses, four dicotyledons, and the microbial homologs of GBSSI. Inferred structural information was used to aid in the alignment of these very divergent sequences. The informed alignments highlight amino acids that are conserved across all sequences, and demonstrate that structural motifs can be highly conserved in spite of marked divergence in amino acid sequence. (2) Maximum-likelihood (ML) analyses were used to examine exon sequence evolution throughout grasses. Differences in probabilities among substitution types and marked among-site rate variation contributed to the observed pattern of variation. Of the parameters examined in our set of likelihood models, the inclusion of among-site rate variation following a gamma distribution caused the greatest improvement in likelihood score. (3) We performed cladistic parsimony analyses of GBSSI sequences throughout grasses, within tribes, and within genera to examine the phylogenetic utility of the gene. Introns provide useful information among very closely related species, but quickly become difficult to align among more divergent taxa. Exons are variable enough to provide extensive resolution within the family, but with low bootstrap support. The combined results of amino acid sequence comparisons, maximum-likelihood analyses, and phylogenetic studies underscore factors that might affect phylogenetic reconstruction. In this case, accommodation of the variable rate of evolution among sites might be the first step in maximizing the phylogenetic utility of GBSSI.      

Effects of medium viscosity on kinetics of the enzymatic reaction catalyzed by bacterial RNase

Effects of medium viscosity on kinetic parameters of poly(U) hydrolysis catalyzed by RNase from Bac. intermedius 7P (binase) were studied in solutions of sucrose (4-50 wt. %) and glycerol (35-62 wt. %) in Tris--sodium acetate buffer (pH 7.5) at 25 degreesC. The rate constant of reaction kcat was practically unchanged over a wide range of viscosities (1-15 cP for sucrose and 2.5-3 cP for glycerol). In glycerol solutions, kcat slightly increased with viscosity increase from 4 to 10 cP. Addition of NaCl to the buffer medium resulted in an inhibitory effect of Na+ on kcat, prevented by 50% sucrose or 60% glycerol. It is concluded that binase-catalyzed poly(U) cleavage occurs through a "tense"-substrate mechanism, similarly to reactions catalyzed by alpha-chymotrypsin, trypsin, and laccase.