Azelastine and suplatast shorten the distribution half-life of IgE in rats

We aim to clarify whether suplatast and azelastine (anti-allergic drugs) can shorten the half-life of imnunoglobulin E (IgE) in the circulating blood. Thirty Wistar rats were divided into six groups. Distilled water or anti-allergic drugs were given orally for 6 days after the first sensitization. Two milligrams of monoclonal dinitrophenyl (DNP)-specific rat IgE was administered to the rats, which had been given suplatast or azelastine orally. The level of DNP-specific rat IgE in the serum was estimated by IgE-capture enzyme-linked immunosorbent assay, and the turnover of IgE was analyzed from its pharmacokinetic parameters. The elimination half-life of rat IgE was about 12 h irrespective of the sensitized state. The intercompartmental rate constants (Kct and Ktc) in the suplatast-administered or azelastine-administered group were larger than those of the distilled water-administered group under non-sensitized conditions. These findings suggested that the anti-allergic drugs used in the present study facilitated the excretion of IgE from the circulation in rats.     

Role of the Rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway and its connection to calcineurin function

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.     

The XPC-HR23B complex displays high affinity and specificity for damaged DNA in a true-equilibrium fluorescence assay

The XPC-HR23B complex is a prime candidate for the initial damage recognition step during global genome nucleotide excision repair. A specific interaction between the XPC-HR23B complex and various types of damaged DNA substrates has been demonstrated in recent work by electrophoretic mobility shift assays or immunoprecipitation. Although these studies allowed the estimation of relative binding affinities for the different types of lesions, the presence of large amounts of competitor DNA or the need for glutaraldehyde fixation prevented the quantification of equilibrium constants. We have performed a quantitative study on the binding of XPC to damaged DNA using fluorescence anisotropy measurements. The XPC-HR23B complex binds with high affinity (K(D) approximately 1-3 nM) to fluorescent 36 bp DNA fragments containing a single cisplatin 1,3-intrastrand adduct or a six-nucleotide mispaired region. From stoichiometric titration experiments, it is concluded that approximately 70% of the XPC-HR23B preparation is active in DNA binding. Binding experiments employing fluorescent probes with a single defined photoproduct reveal a 30-fold preference of XPC for 6,4-photoproducts as compared to a cyclobutane dimer. Competition experiments with undamaged and damaged plasmid DNA indicate that the XPC-HR23B complex discriminates between damaged and undamaged sites with high specificity. The specificity factor is between 100 and 3000, depending on the number of nonspecific sites considered in the calculations. Upon addition of XPA to the XPC binding reaction mixtures, it was not possible to detect cooperative ternary complex formation on the platinated 36 bp probe.     

A new technique to co-localise membrane proteins with Homer/vesl

The minimal requirements were defined as necessary for cluster formation of the group 1 metabotropic glutamate receptor (mGluR), which is regulated by the Homer/vesl family of scaffolding proteins [Curr. Opin. Neurobiol. 10 (2000) 370]. Cluster formation of G-protein-coupled receptors (GPCRs) plays a fundamental role in signal transduction, particularly at the neuronal synapse. To understand the interaction of mGluR with PSD-Zip45, a Homer/vesl family member, we designed a series of chimeric receptor proteins, consisting of C-terminal mGluR1alpha sequences that were fused to endothelin B receptors (ET(B)Rs). In vitro and in vivo studies revealed that an extended 20 amino acid long C-terminal mGluR1alpha peptide, including the proline-rich core motif PPXXF, is sufficient to induce clustering of chimeric ET(B)R/mGluR1alpha receptors by PSD-Zip45. This result is especially important because it constitutes the basis for a new approach to form two-dimensional crystals of membrane proteins in situ, which may render unstable membrane proteins amenable to electron crystallographic structure determination.     

Marine Bacterial Sulfated Fucoglucuronomannan (SFGM) Lyase Digests Brown Algal SFGM into Trisaccharides

Abstract Three kinds of trisaccharides were prepared by digesting fucoidan from the brown alga Kjellmaniella crassifolia, with the extracellular enzymes of the marine bacterium Fucobacter marina. Their structures were determined as Δ^4,5GlcpUA1-2(L-Fucp(3-O-sulfate)α1-3)D-Manp, Δ^4,5GlcpUA1-2(L-Fucp(3-O-sulfate)α1-3)D-Manp(6-O-sulfate), and Δ^4,5GlcpUA1-2(L-Fucp(2,4-O-disulfate)α1-3)D-Manp(6-O-sulfate), which indicated the existence of a novel polysaccharide in the fucoidan and a novel glycosidase in the extracellular enzymes. In order to determine the complete structure of the polysaccharide and the reaction mechanism of the glycosidase, the fucoidan was partially hydrolyzed to obtain glucuronomannan, which is the putative backbone of the polysaccharide, and its sugar sequence was determined as (-4-D-GlcpUAβ1-2D-Manpα1-)_n, which disclosed that the main structure of the polysaccharide is (-4-D-GlcpUAβ1-2(L-Fucp(3-O-sulfate)α1-3)D-Manpα1-)_n. Consequently, the glycosidase was deduced to be an endo-α-D-mannosidase that eliminatively cleaves the α-D-mannosyl linkage between D-Manp and D-GlcpUA residues in the polysaccharide and produces the above trisaccharides. The novel polysaccharide and glycosidase were tentatively named as sulfated fucoglucuronomannan (SFGM) and SFGM lyase, respectively.     

A longitudinal study on hand and wrist skeletal maturation in chimpanzees ( Pan troglodytes), with emphasis on growth in linear dimensions

Skeletal maturation in the chimpanzee hand and wrist (the RUS system; radius, ulna, and short bones) was studied both longitudinally and cross-sectionally. Maturity states were evaluated in each of the 13 bones of the RUS system based on the TW2 method (Tanner and Whitehouse method), and the RUS score was calculated by the summation of scores for these bones. Individual variation was examined by means of residual curves and pseudo-velocity curves of RUS score and anterior trunk length (ATL). Norms of the age change pattern in RUS skeletal maturation and the growth of ATL were determined for each sex, and the relationships among ATL growth and skeletal and reproductive maturation were examined. We found a fairly good relationship between ATL growth and RUS skeletal maturation. Comparison of growth and development between humans and chimpanzees showed that growth characteristics are coupled with each other at puberty in male chimpanzees and in both sexes of humans. Although nutritional condition influenced ATL growth in infancy, it had no effect on the RUS maturational process. Social relationships appeared to influence both ATL growth and RUS maturation. Analyses on relationships between RUS skeletal maturation, ATL growth, and reproductive maturation, showed that RUS skeletal maturation is a good indicator of "physiological age".     

Thymine-rich single-stranded DNA activates Mcm4/6/7 helicase on Y-fork and bubble-like substrates

The presence of multiple clusters of runs of asymmetric adenine or thymine is a feature commonly found in eukaryotic replication origins. Here we report that the helicase and ATPase activities of the mammalian Mcm4/6/7 complex are activated specifically by thymine stretches. The Mcm helicase is specifically activated by a synthetic bubble structure which mimics an activated replication origin, as well as by a Y-fork structure, provided that a single-stranded DNA region of sufficient length is present in the unwound segment or 3' tail, respectively, and that it carries clusters of thymines. Sequences derived from the human lamin B2 origin can serve as a potent activator for the Mcm helicase, and substitution of its thymine clusters with guanine leads to loss of this activation. At the fork, Mcm displays marked processivity, expected for a replicative helicase. These findings lead us to propose that selective activation by stretches of thymine sequences of a fraction of Mcm helicases loaded onto chromatin may be the determinant for selection of initiation sites on mammalian genomes.