A comparative study of bark lectins from three elderberry (Sambucus) species

Three elderberry lectins isolated from the bark of three different species of the genus Sambucus which are native to Europe (S. nigra), North America (S. canadensis), and Japan (S. sieboldiana) were studied comparatively with regard to their carbohydrate binding properties and some structural features. All three lectins contained two identical carbohydrate binding sites per molecule and showed a very high specificity for the Neu5Ac(alpha 2-6)-Gal/GalNAc sequence. However, relative affinities for various oligosaccharides were significantly different among them, suggesting differences in the detailed structure of the carbohydrate binding sites of these lectins. The three lectins were immunologically related, but not identical, and all were composed of hydrophobic and hydrophilic subunit regions, although the molecular sizes of these subunits were slightly different among the three lectins. N-terminal sequence analysis of the subunits of these lectins suggested that they have a very similar structure in this region but also indicated the occurrence of N-terminal processing such as the deletion of several amino acid residues at the N-termini for both hydrophobic and hydrophilic subunits of all three lectins. Tryptic peptide mapping of the three lectins showed a similar pattern for all of them but also showed the presence of some unique peptides for each lectin.     

ccl19 and ccl21 affect cell movements and differentiation in early Xenopus development

We characterized Xenopus laevis C-C motif chemokine ligand 19.L (ccl19.L) and C-C motif chemokine ligand 21.L (ccl21.L) during early Xenopus embryogenesis. The temporal and spatial expression patterns of ccl19.L and ccl21.L tended to show an inverse correlation, except that the expression level was higher in the dorsal side at the gastrula stage. For example, even at the dorsal sector of the gastrulae, ccl19.L was expressed in the axial region and ccl21.L was expressed in the paraxial region. Dorsal overexpression of ccl19.L and ccl21.L and knockdown of Ccl19.L and Ccl21.L inhibited gastrulation, but their functions were different in cell behaviors during morphogenesis. Observation of Keller sandwich explants revealed that overexpression of both ccl19.L and ccl21.L and knockdown of Ccl21.L inhibited the convergent extension movements, while knockdown of Ccl19.L did not. ccl19.L-overexpressing explants attracted cells at a distance and ccl21.L-overexpressing explants attracted neighboring cells. Ventral overexpression of ccl19.L and ccl21.L induced secondary axis-like structures and chrd.1 expression at the ventral side. Upregulation of chrd.1 was induced by ligand mRNAs through ccr7.S. Knockdown of Ccl19.L and Ccl21.L inhibited gastrulation and downregulated chrd.1 expression at the dorsal side. The collective findings indicate that ccl19.L and ccl21.L might play important roles in morphogenesis and dorsal–ventral patterning during early embryogenesis in Xenopus.     

XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1-TAK1 in the BMP signaling pathway.

Signals elicited by transforming growth factor-beta (TGF-beta) superfamily ligands are generated following the formation of heteromeric receptor complexes consisting of type I and type II receptors. TAK1, a member of the MAP kinase kinase kinase family, and its activator, TAB1, participate in the bone morphogenetic protein (BMP) signaling pathway involved in mesoderm induction and patterning in early Xenopus embryos. However, the events leading from receptor activation to TAK1 activation remain to be identified. A yeast interaction screen was used to search for proteins that function in the pathway linking the receptors and TAB1-TAK1. The human X-chromosome-linked inhibitor of apoptosis protein (XIAP) was isolated as a TAB1-binding protein. XIAP associated not only with TAB1 but also with the BMP receptors in mammalian cells. Injection of XIAP mRNA into dorsal blastomeres enhanced the ventralization of Xenopus embryos in a TAB1-TAK1-dependent manner. Furthermore, a truncated form of XIAP lacking the TAB1-binding domain partially blocked the expression of ventral mesodermal marker genes induced by a constitutively active BMP type I receptor. These results suggest that XIAP participates in the BMP signaling pathway as a positive regulator linking the BMP receptors and TAB1-TAK1.     

RHAMM is a centrosomal protein that interacts with dynein and maintains spindle pole stability

The receptor for hyaluronan-mediated motility (RHAMM), an acidic coiled coil protein, has previously been characterized as a cell surface receptor for hyaluronan, and a microtubule-associated intracellular hyaluronan binding protein. In this study, we demonstrate that a subset of cellular RHAMM localizes to the centrosome and functions in the maintenance of spindle integrity. We confirm a previous study showing that the amino terminus of RHAMM interacts with microtubules and further demonstrate that a separate carboxy-terminal domain is required for centrosomal targeting. This motif overlaps the defined hyaluronan binding domain and bears 72% identity to the dynein interaction domain of Xklp2. RHAMM antibodies coimmunprecipitate dynein IC from Xenopus and HeLa extracts. Deregulation of RHAMM expression inhibits mitotic progression and affects spindle architecture. Structure, localization, and function, along with phylogenetic analysis, suggests that RHAMM may be a new member of the TACC family. Thus, we demonstrate a novel centrosomal localization and mitotic spindle-stabilizing function for RHAMM. Moreover, we provide a potential mechanism for this function in that RHAMM may cross-link centrosomal microtubules, through a direct interaction with microtubules and an association with dynein.     

Identification of two Smad4 proteins in Xenopus. Their common and distinct properties

Smad family proteins have been identified as mediators of intracellular signal transduction by the transforming growth factor-beta (TGF-beta) superfamily. Each member of the pathway-restricted, receptor-activated Smad family cooperates and synergizes with Smad4, called co-Smad, to transduce the signals. Only Smad4 has been shown able to function as a common partner of the various pathway-restricted Smads in mammals. Here we have identified a novel Smad4-like molecule in Xenopus (XSmad4beta) as well as a Xenopus homolog of a well established Smad4 (XSmad4alpha). XSmad4beta is 70% identical to XSmad4alpha in amino acid sequence. Both of the Xenopus Smad4s can cooperate with Smad1 and Smad2, the pathway-restricted Smads specific for bone morphogenetic protein and TGF-beta, respectively. However, they show distinct properties in terms of their developmental expression patterns, subcellular localizations, and phosphorylation states. Moreover, XSmad4beta, but not XSmad4alpha, has the potent ability to induce ventralization when microinjected into the dorsal marginal region of the 4-cell stage of the embryos. These results suggest that the two Xenopus Smad4s have overlapping but distinct functions.     

Phosphatidylserine synthesis required for the maximal tryptophan transport activity in Saccharomyces cerevisiae

Saccharomyces cerevisiae cho1/pss mutants, which are severely impaired in phosphatidylserine (PS) synthesis, do not have detectable amounts of PS in their lipid fractions. Their derivatives with mutations that cause defects in tryptophan synthesis grew poorly in a medium containing 5 micrograms/ml of L-tryptophan, a concentration that met the requirements of tryptophanauxotrophic CHO1/PSS strains. The rates of tryptophan uptake of trp1 cho1/pss mutants were low at low tryptophan concentrations. This defect in the use of tryptophan was restored either by expression of CHO1/PSS or by introduction of a gene encoding tryptophan transporter, TAT1 or TAT2. These results indicate that PS synthesis is required for the maximal tryptophan-transporting activity of S. cerevisiae at low tryptophan concentrations.     

Primary structure of guinea-pig Hageman factor: sequence around the cleavage site differs from the human molecule.

The guinea-pig and human Hageman factors differ in their sensitivity to activation by particular bacterial proteinases. To understand this difference, the primary structure and cleavage site on activation of the guinea-pig molecule were determined and compared with the human molecule. By the use of a synthetic oligodeoxyribonucleotide probe which encoded a part of human Hageman factor cDNA, a cDNA clone was isolated from a lambda gt11 cDNA library of guinea-pig liver and sequenced. The cDNA clone was identified as that of guinea-pig Hageman factor by the complete identity of the deduced amino-acid sequence with the actual sequence of the amino-terminal portion of guinea-pig Hageman factor molecule and the active form. The cDNA included part of a leader sequence and the entire coding region of the Hageman factor molecule. Guinea-pig Hageman factor was composed of the same domain structures as the human counterpart with an overall 72% homology in the amino-acid sequence. However, the sequences around the cleavage site were surprisingly different; -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val359-(human) and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val346-(guinea-pig). The amino-acid substitutions around the cleavage site might explain the difference in sensitivity to activation between the human and guinea-pig molecules.     

Development of multidrug resistance type I Cmdr1 expression in chicken embryonic gonads

A primary role of P-glycoprotein (P-gp), encoded by the multidrug resistance type I gene, is to protect against naturally occurring xenotoxics. Recently, the preferential expression of chicken multidrug resistance type I (Cmdr1) was identified in the embryonic gonads during the early periods of development. Here we investigated the expression of Cmdr1 and P-gp in the gonads during embryogenesis, and compared to that in the ovarian follicles of domestic hens (Gallus gallus). As revealed by immunohistochemistry, P-gp was highly expressed in theca cells of mature follicles, whereas the expression was low in immature follicles. Immunohistochemical analysis showed that expression of Cmdr1-type P-gp was very low in embryonic gonads. Cmdr1 mRNA was undetectable in the gonads of 5-day embryos (E5) by RT-PCR, whereas Cmdr1 mRNA was significantly detectable in the developing gonads at E9 and E21. In the testicular tissues, germ cells were distributed along developing seminiferous cords as identified by a specific marker gene, whereas Cmdr1-type P-gp positive cells were observed evenly on testicular tissues. Collectively, it is concluded that Cmdr1 expression is initiated in the chicken ovary and testis after sexual differentiation, but expression of Cmdr1-type P-gp is very low through embryogenesis.