Thermodynamic and kinetic studies on the mechanism of binding of methylumbelliferyl glycosides to jacalin.

The binding of Artocarpus integrifolia lectin (jacalin) to 4-methylumbelliferyl (Meumb)-glycosides, Gal alpha Meumb, Gal beta Meumb, GalNAc alpha Meumb, GalNAc beta-Meumb, and Gal beta 3GalNAc beta Meumb was examined by extrinsic fluorescence quenching titration and stopped flow spectrofluorimetry. The binding was characterized by 100% quenching of fluorescence of Meumb-glycosides. Their association constants range from 2.0 x 10(4) to 1.58 x 10(6) M-1 at 15 degrees C. Entropic contribution is the major stabilizing force for avid binding of Meumb-glycosides indicating the existence of a hydrophobic site that is complementary to their methylumbelliferyl group. The second order association rate constants for interaction of these sugars with lectin at 15 degrees C vary from 8.8 x 10(5) to 3.24 x 10(6) M-1 S-1, at pH 7.2. The first order dissociation rate constants range from 2.30 to 43.0 S-1 at 15 degrees C. Despite the differences in their association rate constants, the overall values of association constants for these saccharides are determined by their dissociation rate constants. The second order rate constant for the association of Meumb-glycosides follows a pattern consistent with the magnitude of the activation energies involved therin. Activation parameters for association of all ligands illustrate that the origin of the barrier between binding of jacalin to Meumb-glycosides is entropic, and the enthalpic contribution is small. A correlation between these parameters and the structure of the ligands on the association rates underscores the importance of steric factors in determining protein saccharide recognitions.     

Characterization of species-specific repeated DNA sequences from B. nigra

Summary The construction and characterization of two genome-specific recombinant DNA clones from B. nigra are described. Southern analysis showed that the two clones belong to a dispersed repeat family. They differ from each other in their length, distribution and sequence, though the average GC content is nearly the same (45%). These B genome-specific repeats have been used to analyse the phylogenetic relationships between cultivated and wild species of the family Brassicaceae.     

An ultrastructural study of the clitellar epithelium of the earthworm Metaphire posthuma (vail.)

The ultrastructure of clitellar epithelium of Metuphire posthuma revealed mainly three types of secretory cells. Most prominent among these are the large slender granular cells which contain a large number of secretory granules filling in the entire columncr region of the cell. The secretory granules are 2-4mu in diameter with a limiting membrane and containing numerous tiny vesicles in a matrix of varying electron density. Basolateral rough endoplasmic reticulum and extensive Golgi cisternae were seen interspersed with the secretory granules. The Golgi cisternae in these cells were quite prominent extending all around the secretory granules. The secretory granules of type 2 cells are spheroid bodies with motley appearance due to varying electron density of the matrix. The immature granules contain fibrillar material. Type 3 cells contained electron lucent membrane-bound mucous like secretory granules which are reticulated with filamentous materials. All the three cell types open to the exterior at the cuticular region which is characterised by the presence of numerous microvilli.     

Preoperative diagnosis of bilateral renal oncocytoma by needle aspiration cytology. A case report

The cytohistologic and ultrastructural findings in a case of bilateral renal oncocytoma diagnosed by needle aspiration cytology are presented. This case appears to be the first reported example in which the preoperative needle aspiration cytologic diagnosis of bilateral renal oncocytoma was made due to the presence of characteristic tumor cells that were seen singly or in small clusters with rounded polygonal shapes and abundant eosinophilic granular cytoplasm. The preoperative diagnosis of bilateral oncocytoma permitted a timely decision as to the appropriate management in this case.     

Evidence for autophosphorylation of hyaluronate binding protein and its enhanced phosphorylation in rat histiocytoma.

This report documents for the first time the in vitro autophosphorylation of purified 68 kDa hyaluronate binding protein in presence of [32P] ATP. The rate of phosphorylation is proportional to the concentration of protein and to the time of incubation up to 5 min. By both phosphoamino acid and western blot analysis with antiphosphotyrosine antibodies, we have confirmed that the phosphorylation occurs at tyrosine residues. Immunoprecipitation with anti HA binding protein antibody shows a 5 fold increase in the phosphorylation in macrophage histiocytoma compared to normal macrophage. Supplementing hyaluronate with hyaluronate binding protein in the medium is further shown to enhance total protein phosphorylation in rat histiocytoma.     

Nucleotide sequences of three different isoforms of beta-tubulin cDNA from Chinese hamster ovary cells.

The complete cDNA sequences of two clones encoding beta-tubulin isotypes and the partial sequence of a third isoform from Chinese hamster ovary cells have been determined. The deduced amino acid sequences of the three isoforms show extensive homology to each other as well as with other alpha and beta-tubulin sequences from various species. These results provide evidence for the expression of three different isoforms of beta-tubulin in Chinese hamster ovary cells.     

The HPLC profile of proteins of thermoprotection-acquired wheat (Triticum aestivum L.) seedlings

A pre-treatment of 40 °Cprovided thermoprotection to wheat seedlings against 43 °C, which was otherwise a lethal temperature. Due to temperature pretreatment, the rate of protein synthesis at 45 °C increased in both plumules and radicles. The HPLC profile of plumule and radicle proteins of thermoprotection-acquired seedlings was different from the plumules and radicles of non-treated seedlings.     

Activating mutations in the NH2- and COOH-terminal moieties of the Gs alpha subunit have dominant phenotypes and distinguishable kinetics of adenylyl cyclase stimulation.

The alpha subunit polypeptides of the G proteins Gs and Gi2 stimulate and inhibit adenylyl cyclase, respectively. The alpha s and alpha i2 subunits are 65% homologous in amino acid sequence but have highly conserved GDP/GTP binding domains. Previously, we mapped the functional adenylyl cyclase activation domain to a 122 amino acid region in the COOH-terminal moiety of the alpha s polypeptide (Osawa et al: Cell 63:697-706, 1990). The NH2-terminal half of the alpha s polypeptide encodes domains regulating beta gamma interactions and GDP dissociation. A series of chimeric cDNAs having different lengths of the NH2- or COOH-terminal coding sequence of alpha s substituted with the corresponding alpha i2 sequence were used to introduce multi-residue non-conserved mutations in different domains of the alpha s polypeptide. Mutation of either the amino- or carboxy-terminus results in an alpha s polypeptide which constitutively activates cAMP synthesis when expressed in Chinese hamster ovary cells. The activated alpha s polypeptides having mutations in either the NH2- or COOH-terminus demonstrate an enhanced rate of GTP gamma S activation of adenylyl cyclase. In membrane preparations from cells expressing the various alpha s mutants, COOH-terminal mutants, but not NH2-terminal alpha s mutants markedly enhance the maximal stimulation of adenylyl cyclase by GTP gamma S and fluoride ion. Neither mutation at the NH2- nor COOH-terminus had an effect on the GTPase activity of the alpha s polypeptides. Thus, mutation at NH2- and COOH-termini influence the rate of alpha s activation, but only the COOH-terminus appears to be involved in the regulation of the alpha s polypeptide activation domain that interacts with adenylyl cyclase.