Novel cytokinins: The predominant forms in mature buds of Pinus radiata

To elicit the roles of cytokinins in the regulation of maturation of Pinus radiata D. Don, the spectrum of endogenous cytokinins and their concentration in the mature buds were analysed using double-solvent extraction, column complex purification and separation, a novel immunoaffinity purification method, normal and reverse phase high-pressure liquid chromatography, enzymatic treatment, radioimmunoassay and electrospray MS/MS spectrometry. We have isolated two novel cytokinin glycosides whose proposed structures are isopentenyladenine-9-(glucopyranosyl riboside), dihydrozeatin-9-(glucopyranosyl riboside) and confirmed the presence of zeatin-9-(glucopyranosyl riboside). We have also found the presence of novel phosphorylated forms of these 3 cytokinin ribosyl-linked glycosides. Quantitative analyses revealed that the cytokinin ribosyl-linked glycosides predominate in P. radiata mature buds. Although cytokinin free base, riboside and nucleotide forms are also present, we could find no evidence of the traditional cytokinin O - or N-glucosides in the conifer buds. Thus, cytokinin metabolism in mature buds of P. radiata is very different from other species previously examined.     

Functional implications of structural differences between variants A and B of bovine beta-lactoglobulin

The structure of the trigonal crystal form of bovine beta-lactoglobulin variant B at pH 7.1 has been determined by X-ray diffraction methods at a resolution of 2.22 A and refined to values for R and Rfree of 0.239 and 0.286, respectively. By comparison with the structure of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 7.1, which was determined previously [Qin BY et al., 1998, Biochemistry 37:14014-14023], the structural consequences of the sequence differences D64G and V118A of variants A and B, respectively, have been investigated. Only minor differences in the core calyx structure occur. In the vicinity of the mutation site D64G on loop CD (residues 61-67), there are small changes in main-chain conformation, whereas the substitution V118A on beta-strand H is unaccompanied by changes in the surrounding structure, thereby creating a void volume and weakened hydrophobic interactions with a consequent loss of thermal stability relative to variant A. A conformational difference is found for the loop EF, implicated in the pH-dependent conformational change known as the Tanford transition, but it is not clear whether this reflects differences intrinsic to the variants in solution or differences in crystallization.     

A divalent major histocompatibility complex/IgG1 fusion protein induces antigen-specific T cell activation in vitro and in vivo

Activation of antigen-specific T cell clones in vivo might be possible by generating soluble MHC molecules; however, such molecules do not induce effective T cell responses unless cross-linked. As a first step in generating a soluble MHC molecule that could function as an antigen-specific immunostimulant, the extracellular domains of the murine H-2Kb MHC class I molecule were fused to the constant domains of a murine IgG1 heavy chain, resulting in a divalent molecule with both a TCR-reactive and an Fc receptor (FcR)-reactive moiety. The fusion protein can be loaded with peptide and can induce T cell activation in a peptide-specific, MHC-restricted manner following immobilization on plastic wells or following cross-linking by FcR+ spleen cells. The fusion protein induces partial T cell activation in vivo in a mouse transgenic for a TCR restricted to H-2Kb. This fusion protein molecule may be useful to study peptide-MHC interactions and may provide a strategy for boosting in vivo antigen-specific T cell responses, such as to viral or tumor antigens.     

Mutant vasopressin precursors that cause autosomal dominant neurohypophyseal diabetes insipidus retain dimerization and impair the secretion of wild-type proteins

Autosomal dominant familial neurohypophyseal diabetes insipidus is caused by mutations in the arginine vasopressin (AVP) gene. We demonstrated recently that mutant AVP precursors accumulate within the endoplasmic reticulum of neuronal cells, leading to cellular toxicity. In this study, the possibility that mutant AVP precursors interact with wild-type (WT) proteins to alter their processing and function was explored. WT and mutant precursors were epitope-tagged to allow them to be distinguished in transfected cells. An in vivo cross-linking reaction revealed homo- and heterodimer formation between WT and mutant precursors. Mutant precursors were also shown to impair intracellular trafficking of WT precursors from the endoplasmic reticulum to the Golgi apparatus. In addition to the cytotoxicity caused by mutant AVP precursors, the interaction between the WT and mutant precursors suggests that a dominant-negative mechanism may also contribute to the pathogenesis of familial neurohypophyseal diabetes insipidus.     

Spectroscopic Characterization of Two Soluble Transducers from the Archaeon Halobacterium salinarum

In the present study, structural aspects of the two soluble transducers, HtrX and HtrXI, from the archaeon H. salinarum have been examined using UV circular dichroism and steady-state fluorescence spectroscopies. Circular dichroism (CD) data indicate that both HtrX and HtrXI exhibit salt-dependent protein folding. Under low-ionic-strength conditions (0.2 M NaCl or KCl) the CD spectra of HtrXI is similar to that of the Gdn-HCl- or urea-denatured forms and is indicative of random coil structure. In contrast, the CD spectrum of HtrX under low-ionic-strength conditions contains roughly 85% -helical character, indicating a significant degree of folding. Addition of NaCl or KCl to solutions of HtrX or HtrXI results in CD features consistent with predominately -helical character (>95%) for both proteins. In addition, the transition points (i.e., ionic strengths at which the protein converts from random coil to -helical character) are quite distinct and dependent upon the type of salt present (i.e., either NaCl or KCl). Accessibility of tryptophan residues to the solvent was also examined for both HtrX and HtrXI in both folded and unfolded states using Kl quenching. The Stern–Volmer constants obtained suggest that the tryptophans (Trp35 in HtrX and both Trp47 and Trp74 in HtrXI) are partially exposed to the solvent, indicating that they are located near the surface of the protein in all three cases. Furthermore, fluorescence quenching with the single Trp mutants Trp74AIa and Trp47AIa of HtrXI indicates different environments for these two residues.     

Detection of Tryptophan to Tryptophan Energy Transfer in Proteins

Förster resonance energy transfer (FRET) studies usually involve observation of intensity or life-time changes in the donor or acceptor molecule and usually these donor and acceptor molecules differ (heterotransfer). The use of polarization to monitor FRET is far less common, although it was one of the first methods utilized. In 1960, Weber demonstrated that homotransfer between tryptophan molecules contributes to depolarization. He also discovered that the efficiency of homotransfer becomes much less effective upon excitation near the red-edge of the absorption. This “red-edge effect” was shown to be a general phenomenon of homotransfer. We have utilized Weber's red-edge effect to study tryptophan homotransfer in proteins. Specifically, we determined the polarization of the tryptophan fluorescence upon excitation at 295 nm and 310 nm (near the red-edge). Rotational diffusion leads to depolarization of the emission excited at either 295 nm or 310 nm, but homotransfer only contributes to depolarization upon excitation at 295 nm. Hence, the 310/295 polarization ratio gives an indication of tryptophan to tryptophan energy transfer. In single tryptophan systems, the 310/295 ratios are generally below 2 whereas in multi-tryptophan systems, the 310/295 ratios can be greater than 3.     

OGRe: a relational database for comparative analysis of mitochondrial genomes

Organellar Genome Retrieval (OGRe) is a relational database of complete mitochondrial genome sequences for over 250 Metazoan species. OGRe provides a resource for the comparative analysis of mitochondrial genomes at several levels. At the sequence level, OGRe allows the retrieval of any selected set of mitochondrial genes from any selected set of species. Species are classified using a taxonomic system that allows easy selection of related groups of species. Sequence alignments are also available for some species. At the level of individual nucleotides, the system contains information on base frequencies and codon usage frequencies that can be compared between organisms. At the level of whole genomes, OGRe provides several ways of visualizing information on gene order. Diagrams illustrating the genome arrangement can be generated for any selected set of species automatically from the information in the database. Searches can be done based on gene arrangement to find sets of species that have the same order as one another. Diagrams for pairwise comparison of species can be produced that show the positions of break-points in the gene order and use colour to highlight the sections of the genome that have moved. OGRe is available from http://www.bioinf.man.ac.uk/ogre.     

Thymocyte sensitivity and supramolecular activation cluster formation are developmentally regulated: a partial role for sialylation

TCR reactivity is tuned during thymic development. Immature thymocytes respond to low-affinity self-ligands resulting in positive selection. Following differentiation, T cells no longer respond to low-affinity ligands, but respond well to high-affinity (foreign) ligands. We show in this study that this response includes integrin activation, supramolecular activation cluster formation, Ca(2+) flux, and CD69 expression. Because glycosylation patterns are known to change during T cell development, we tested whether alterations in sialylation influence CD8 T cell sensitivity to low affinity TCR ligands. Using neuraminidase treatment or genetic deficiency in the ST3Gal-I sialyltransferase, we show that desialylation of mature CD8 T cells enhances their sensitivity to low-affinity ligands, although these treatments do not completely recapitulate the dynamic range of immature T cells. These studies identify sialylation as one of the factors that regulate CD8 T cell tuning during development.