Microanalysis of glycosaminoglycan-derived oligosaccharides labeled with a fluorophore 2-aminobenzamide by high-performance liquid chromatography: application to disaccharide composition analysis and exosequencing of oligosaccharides

A series of disaccharides derived from chondroitin sulfate and heparin/heparan sulfate were derivatized at their reducing ends with a fluorophore 2-aminobenzamide to develop a sensitive microanalytical method for glycosaminoglycans. The resulting labeled compounds derived from chondroitin sulfate or heparin/heparan sulfate were well-separated and quantified by HPLC equipped with a fluorescence detector. The detection limit was a low picomole level. This method was applied to the analysis of the disaccharide composition of tetra- and hexasaccharides derived from chondroitin sulfate and heparin/heparan sulfate as well as these glycosaminoglycan polysaccharides. The method was also successfully applied to the exosequencing of chondrohexasaccharides, where the fluorophore-labeled oligosaccharides were degraded exolytically from the nonreducing ends using bacterial eliminases. The resultant labeled fragments were identified by HPLC.     

Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer

Fetal liver, the major site of hematopoiesis during embryonic development, acquires additional various metabolic functions near birth. Although liver development has been characterized biologically as consisting of several distinct steps, the molecular events accompanying this process are just beginning to be characterized. In this study, we have established a novel culture system of fetal murine hepatocytes and investigated factors required for development of hepatocytes. We found that oncostatin M (OSM), an interleukin-6 family cytokine, in combination with glucocorticoid, induced maturation of hepatocytes as evidenced by morphological changes that closely resemble more differentiated hepatocytes, expression of hepatic differentiation markers and intracellular glycogen accumulation. Consistent with these in vitro observations, livers from mice deficient for gp130, an OSM receptor subunit, display defects in maturation of hepatocytes. Interestingly, OSM is expressed in CD45(+) hematopoietic cells in the developing liver, whereas the OSM receptor is expressed predominantly in hepatocytes. These results suggest a paracrine mechanism of hepatogenesis; blood cells, transiently expanding in the fetal liver, produce OSM to promote development of hepatocytes in vivo.     

Sulfated sialic acid-polymers inhibit the cytotoxic action of bee and snake venom

Colominic acid is an 2,8-linked sialic acid polymer produced by Escherichia coli. We found that synthetic sulfated-colominic acids (SC) remarkably inhibited the cytotoxicity of bee and snake venom toward mouse fibroblast cells, but colominic acids showed no inhibition themselves, indicating the important role of sulfate groups in the inhibitory activity of SC. Other sulfated carbohydrates such as chondroitin sulfates, heparin and heparan sulfate showed no inhibition. SC also exhibited potent inhibition of melittin, a highly basic peptide, which is a major cytotoxic component of bee venom. SC did not inhibit phospholipase A₂ activity in bee venom. This suggests that the inhibition of bee and snake venom by SC is due to inhibition of melittin and cardiotoxin, which is a cytolytic peptide in snake venom, respectively. SC with a higher sulfur content and a larger molecular mass showed more potent activity. The interaction between SC and melittin basically seems an ionic one, however, the conformation of SC is also likely important. For the binding of SC to melittin leading loss of its cytotoxic activity, the sulfate groups of SC must be properly arranged to interact with lysine and arginine residues of melittin molecules, which play an important role in the cytolytic activity. A higher molecular mass of SC substituted with more sulfate groups is required for more obvious inhibition of the cytotoxic activity.     

Effects of solvents interacting favorably with hydrophilic segments of the membrane surface of phosphatidylcholine on their gel-phase membranes in water

We have investigated the effects of two kinds of solvents forming the lamellar liquid-crystalline (L(alpha)) phase in phosphatidylcholine (PC) membranes in neat condition, such as formamide and 1,3-propanediol, on phase behaviors of multilamellar vesicle (MLV) of DPPC (DPPC-MLV). These solvents induced the interdigitated gel (L(beta)I) phase in DPPC-MLV in excess water above their critical concentrations. Solubility measurement indicates that these solvents interact favorably with the hydrophilic segment of the PC membrane but interact unfavorably with the alkyl chains. Based on these results, we propose the mechanism of the induction of the L(beta)I phase by these solvents.     

Ligation errors in DNA computing

DNA computing is a novel method of computing proposed by Adleman (1994), in which the data is encoded in the sequences of oligonucleotides. Massively parallel reactions between oligonucleotides are expected to make it possible to solve huge problems. In this study, reliability of the ligation process employed in the DNA computing is tested by estimating the error rate at which wrong oligonucleotides are ligated. Ligation of wrong oligonucleotides would result in a wrong answer in the DNA computing. The dependence of the error rate on the number of mismatches between oligonucleotides and on the combination of bases is investigated.     

Rho guanine nucleotide exchange factor xNET1 implicated in gastrulation movements during Xenopus development

During Xenopus development, embryonic cells dramatically change their shape and position. Rho family small GTPases, such as RhoA, Rac, and Cdc42, play important roles in this process. These GTPases are generally activated by guanine nucleotide exchange factors (GEFs); however, the roles of RhoGEFs in Xenopus development have not yet been elucidated. We therefore searched for RhoGEF genes in our Xenopus EST database, and we identified several genes expressed during embryogenesis. Among them, we focused on one gene, designated xNET1. It is similar to mammalian NET1, a RhoA-specific GEF. An in vitro binding assay revealed that xNET1 bound to RhoA, but not to Rac or Cdc42. In addition, transient expression of xNET1 activated endogenous RhoA. These results indicated that xNET1 is a GEF for RhoA. Epitope-tagged xNET1 was localized mainly to the nucleus, and the localization was regulated by nuclear localization signals in the N-terminal region of xNET1. Overexpression of either wild-type or a mutant form of xNET1 severely inhibited gastrulation movements. We demonstrated that xNET1 was co-immunoprecipitated with the Dishevelled protein, which is an essential signaling component in the non-canonical Wnt pathway. This pathway has been shown to activate RhoA and regulate gastrulation movements. We propose that xNET1 or a similar RhoGEF may mediate Dishevelled signaling to RhoA in the Wnt pathway.     

Intergeneric conjugal transfer of plasmid DNA from<Emphasis Type="Italic"> Escherichia coli</Emphasis> to<Emphasis Type="Italic"> Kitasatospora setae</Emphasis>, a bafilomycin B<Subscript>1</Subscript> producer

An effective transformation procedure for Kitasatospora setae was established based on transconjugation from Escherichia coli ET12567 (pUZ8002) using a C31-derived integration vector, pSET152, containing oriT and attP fragments. While no transconjugation was observed under the standard transconjugation conditions for Streptomyces species, sufficient transconjugation (>1×10^-6) was achieved on ISP4 medium containing 30 mM MgCl₂ using a 25- to 125-fold excess of E. coli donor cells. In addition, the sequence and location of the chromosomal integration site attB of K. setae was identified for the first time in genera of non-Streptomyces actinomycetes. K. setae contains a single C31 attB site. Similar to the case of Streptomyces species, the attB site of K. setae is present within an ORF encoding a pirin-homolog, but the K. setae-attB sequence deviates slightly from the consensus sequence of Streptomyces attB sequences.     

Doublecortin microtubule affinity is regulated by a balance of kinase and phosphatase activity at the leading edge of migrating neurons

Doublecortin (Dcx) is a microtubule-associated protein that is mutated in X-linked lissencephaly (X-LIS), a neuronal migration disorder associated with epilepsy and mental retardation. Although Dcx can bind ubiquitously to microtubules in nonneuronal cells, Dcx is highly enriched in the leading processes of migrating neurons and the growth cone region of differentiating neurons. We present evidence that Dcx/microtubule interactions are negatively controlled by Protein Kinase A (PKA) and the MARK/PAR-1 family of protein kinases. In addition to a consensus MARK site, we identified a serine within a novel sequence that is crucial for the PKA- and MARK-dependent regulation of Dcx's microtubule binding activity in vitro. This serine is mutated in two families affected by X-LIS. Immunostaining neurons with an antibody that recognizes phosphorylated substrates of MARK supports the conclusion that Dcx localization and function are regulated at the leading edge of migrating cells by a balance of kinase and phosphatase activity.     

Diurnal and seasonal changes in the mate-locating behavior of the satyrine butterfly <Emphasis Type="Italic">Lethe diana</Emphasis>

The time schedule of mate-locating behavior in the male satyrine butterfly Lethe diana was studied. Males displayed territorial behavior only in the afternoon, but it was not known whether they attempted to locate mates in the morning. In order for males of most butterfly species to mate with a virgin female, it would be most effective to initiate mate-locating behavior in the morning, when synchronous eclosions occur. Males of L.diana flew patrols in the morning and displayed territorial behavior in the afternoon. The cost of territorial behavior is probably less than that of patrolling, as territorial flights are shorter than those when patrolling. The temporal switching of mate-locating behavior is theoretically favored, since the reproductive value of females decreases later in the day, when males perform the less costly mate-locating behavior. Thus, the diurnal switching of male mate-locating behavior may be an adaptive strategy in response to the rapid decline in the reproductive value of females over the course of a day.     

Absolute stereochemistry of acremolactone A, a novel herbicidal epoxydihydropyranyl gamma-lactone from Acremonium roseum I4267

Acremolactone A was chemically degraded to the bicyclic hemiacetal gamma-lactone and an epoxycyclohexenol, and their stereochemistry was determined by spectroscopic methods. These observations and data from NOE experiments on acremolactone A led to the configurational assignment of all asymmetric carbons in acremolactone A, enabling its stereostructure to be established.