The effects and comparative differences of neutrophil specific chemokines on neutrophil chemotaxis of the neonate

Neutrophil specific chemokines are potent chemoattractants for neutrophils. IL-8/CXCL8 is the most extensively studied member of this group, and its concentrations increase during inflammatory conditions of the newborn infant including sepsis and chronic lung disease. A significant amount of information exists on the effects of IL-8/CXCL8 on neutrophil chemotaxis of neonates, but little is known about the other neutrophil specific chemokines. The aim of this study was to determine the relative potency of the neutrophil specific chemokines on chemotaxis of neonatal neutrophils and to compare this effect with the effect on adult neutrophils. Neutrophils were isolated from cord blood or healthy adult donors and incubated in a Neuroprobe chemotaxis chamber. Chemokine concentrations ranging from 1-1000 ng/mL were used. Differences in chemotactic potency existed among the seven neutrophil specific chemokines. Specifically, at 100 ng/mL, the order was IL-8/CXCL8>GRO-alpha/CXCL1>GCP-2/CXCL6>NAP-2/CXCL7>ENA-78/CXCL5>GRO-gamma/CXCL2>GRO-beta/CXCL3. This pattern was observed for adult and neonatal neutrophils. We conclude that (1) neutrophils from cord blood exhibit the same pattern of potency for each ELR chemokine as neutrophils from adults, and (2) migration of neonatal neutrophils is significantly less than that of adults at every concentration examined except the lowest (1 ng/mL).     

The new higher level classification of eukaryotes with emphasis on the taxonomy of protists

This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional "kingdoms." The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.     

Interaction of RRF and EF-G from E. coli and T. thermophilus with ribosomes from both origins--insight into the mechanism of the ribosome recycling step

Ribosome recycling factor (RRF), elongation factor-G (EF-G), and ribosomes from Thermus thermophilus (tt-) and Escherichia coli (ec-) were used to study the disassembly mechanism of post-termination ribosomal complexes by these factors. With tt-RRF, ec-EF-G can release bound-tRNA from ec-model post-termination complexes. However, tt-RRF is not released by ec-EF-G from ec-ribosomes. This complex with tt-RRF and ec-ribosomes after the tRNA release by ec-EF-G is regarded as an intermediate of the disassembly reaction. Not only tt-RRF, but also mRNA, cannot be released from ec-ribosomes by tt-RRF and ec-EF-G. These data suggest that the release of RRF from ribosomes is coupled or closely related to the release of mRNA during disassembly of post-termination complexes. With tt-ribosomes, ec-EF-G cannot release ribosome-bound ec-RRF even though they are from the same species, showing that proper interaction of ec-RRF and ec-EF-G does not occur on tt-ribosomes. On the other hand, in contrast to a published report, tt-EF-G functions with ec-RRF to disassemble ec-post-termination complexes. In support of this finding, tt-EF-G translocates peptidyl tRNA on ec-ribosomes and catalyzes ec-ribosome-dependent GTPase, showing that tt-EF-G has in vitro translocation activity with ec-ribosomes. Since tt-EF-G with ec-RRF can release tRNA from ec-post-termination complexes, the data are consistent with the hypothesis that the release of tRNA by RRF and EF-G from post-termination complexes is a result of a translocation-like activity of EF-G on RRF.     

SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments

Footprinting is a powerful and widely used tool for characterizing the structure, thermodynamics, and kinetics of nucleic acid folding and ligand binding reactions. However, quantitative analysis of the gel images produced by footprinting experiments is tedious and time-consuming, due to the absence of informatics tools specifically designed for footprinting analysis. We have developed SAFA, a semi-automated footprinting analysis software package that achieves accurate gel quantification while reducing the time to analyze a gel from several hours to 15 min or less. The increase in analysis speed is achieved through a graphical user interface that implements a novel methodology for lane and band assignment, called "gel rectification," and an optimized band deconvolution algorithm. The SAFA software yields results that are consistent with published methodologies and reduces the investigator-dependent variability compared to less automated methods. These software developments simplify the analysis procedure for a footprinting gel and can therefore facilitate the use of quantitative footprinting techniques in nucleic acid laboratories that otherwise might not have considered their use. Further, the increased throughput provided by SAFA may allow a more comprehensive understanding of molecular interactions. The software and documentation are freely available for download at http://safa.stanford.edu.     

The ClpX chaperone modulates assembly of the tubulin-like protein FtsZ

Summary Assembly of the tubulin-like cytoskeletal protein FtsZ into a ring structure establishes the location of the nascent division site in prokaryotes. Factors that modulate FtsZ assembly are essential for ensuring the precise spatial and temporal regulation of cytokinesis. We have identified ClpX, the substrate recognition subunit of the ClpXP protease, as an inhibitor of FtsZ assembly in Bacillus subtilis. Genetic data indicate that ClpX but not ClpP inhibits FtsZ-ring formation in vivo. In vitro, ClpX inhibits FtsZ assembly in a ClpP-independent manner through a mechanism that does not require ATP hydrolysis. Together our data support a model in which ClpX helps maintain the cytoplasmic pool of unassembled FtsZ that is required for the dynamic nature of the cytokinetic ring. ClpX is conserved throughout bacteria and has been shown to interact directly with FtsZ in Escherichia coli. Thus, we speculate that ClpX functions as a general regulator of FtsZ assembly and cell division in a wide variety of bacteria.     

Recombinant shark natural antibodies to thyroglobulin

As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL.     

Pathogenic mutations and polymorphisms in the lipoprotein receptor-related protein 5 reveal a new biological pathway for the control of bone mass

PURPOSE OF REVIEW: This review summarizes recent findings concerning the genomic variations of the lipoprotein receptor-related protein 5 (LPR5) in relation to bone biology. RECENT FINDINGS: Mutations in the LRP5 gene causing high bone mass (HBM) and osteoporosis-pseudoglioma (OPPG) underscored the role of the Wnt-LRP5 canonical signaling on bone formation. Additional LRP5 activating mutations have been identified in a variety of sclerosing bone dysplasias, improving the diagnostic classification of these disorders. Association of polymorphisms in LRP5 with bone mineral density indicate that LRP5 genetic variation contribute to the risk of osteoporosis. Transgenic mice carrying the LRP5 HBM mutation have improved bone biomechanical properties, and the molecular mechanisms by which this mutation exerts its effects have been clarified. A number of KO mice have shown the complex effects of the Wnt-LRP5 pathway on bone mass and skeletal morphology. In vitro studies indicate that osteoblasts produce a variety of Wnts, the LRP5 co-receptor frizzled (Fzd), as well as LRP5 and Wnt inhibitors, i.e. dickkopf (Dkk1) and frizzled-related proteins (Sfrps), respectively, and delineate the role of these molecules in regulating the commitment of mesenchymal stem cells along the osteoblastic lineage. SUMMARY: Identification of pathogenic mutations and allelic variations in LRP5 has improved our understanding of the physiology of bone mass acquisition and the pathophysiology of several bone diseases, including osteoporosis. Understanding how complex interactions between agonistic and inhibitory factors in the Wnt-LRP5 canonical pathway influence osteoblast functions has the potential of providing new anabolic treatments for osteoporosis.