A novel monoantennary complex-type sugar chain found in octopus rhodopsin: occurrence of the Gal{beta}1->4Fuc group linked to the proximal N-acetylglucosaniine residue of the trimannosyl core

The N-linked sugar chains were liberated as oligosaccha-ridesfrom octopus rhodopsin by hydrazinolysis. Most of the oligosaccharideswere neutral, and separated into two major components by columnchromatography using immobilized lectins and Bio-Gel P-4. Structuralanalysis of the one major component by sequential exoglycosidasedigestion, chemical fragmentation in combination with meth-ylationanalysis revealed that it is a nonasaccharide; Man16(Gaiβ13GlcNAcβ12Man13)Manβ14GlcNAcβ14(Galβ14Fuc16)GlcNAcThis structure is quite unique in that a novel galactosylatedfucose residue is attached to the reducing terminal N-acetyl-glucosamineresidue. galactosylated Fuc N-linked sugar chain novel structure octopus rhodopsin     

Immunostimulatory activities of monoglycosylated α-d-pyranosylceramides

We compared the immunostimulatory effects of chemically synthesized α-galactosylceramides (α-GalCers), α-glucosylceramides (α-GluCers), 6″-monoglycosylated α-GalCer and 6″- or 4″-monoglycosylated α-GluCer and made the following observations: (1) the length of the fatty acid side chain in the ceramide portions greatly affects the immunostimulatory effects of α-GalCers and α-GluCers; (2) the configuration of the 4″-hydroxyl group of the inner pyranose moiety plays an important role in the immunostimulatory effects of monoglycosylated α- -pyranosylceramides; (3) the free 4″-hydroxyl group of the inner pyranose of monoglycosylated α- -pyranosylceramides plays a more important role in their immunostimulatory effects than the free 6″-hydroxyl group.     

Possible Involvement of Phosphorylation of Occludin in Tight Junction Formation

Occludin is an integral membrane protein localizing at tight junctions in epithelial and endothelial cells. Occludin from confluent culture MDCK I cells resolved as several (>10) bands between 62 and 82 kD in SDS-PAGE, of which two or three bands of the lowest M_r were predominant. Among these bands, the lower predominant bands were essentially extracted with 1% NP-40, whereas the other higher M_r bands were selectively recovered in the NP-40–insoluble fraction. Alkaline phosphatase treatment converged these bands of occludin both in NP-40–soluble and -insoluble fractions into the lowest M_r band, and phosphoamino acid analyses identified phosphoserine (and phosphothreonine weakly) in the higher M_r bands of occludin. These findings indicated that phosphorylation causes an upward shift of occludin bands and that highly phosphorylated occludin resists NP-40 extraction. When cells were grown in low Ca medium, almost all occludin was NP-40 soluble. Switching from low to normal Ca medium increased the amount of NP-40–insoluble occludin within 10 min, followed by gradual upward shift of bands. This insolubilization and the band shift correlated temporally with tight junction formation detected by immunofluorescence microscopy. Furthermore, we found that the anti–chicken occludin mAb, Oc-3, did not recognize the predominant lower M_r bands of occludin (non- or less phosphorylated form) but was specific to the higher M_r bands (phosphorylated form) on immunoblotting. Immunofluorescence microscopy revealed that this mAb mainly stained the tight junction proper of intestinal epithelial cells, whereas other anti-occludin mAbs, which can recognize the predominant lower M_r bands, labeled their basolateral membranes (and the cytoplasm) as well as tight junctions. Therefore, we conclude that non- or less phosphorylated occludin is distributed on the basolateral membranes and that highly phosphorylated occludin is selectively concentrated at tight juctions as the NP-40–insoluble form. These findings suggest that the phosphorylation of occludin is a key step in tight junction assembly.     

Relations Between the Extracellular Concentrations of Choline and Acetylcholine in Rat Striatum

Abstract: Changes in extracellular levels of acetylcholine (ACh) and choline (Ch) in the striatum of rats were examined by in vivo microdialysis after intraperitoneal injections of drugs. A dopamine D₂ antagonist, sulpiride (20 mg/kg), and a muscarinic antagonist, atropine (3.5 mg/kg), increased ACh levels and decreased Ch levels. On the contrary, the D₂ agonist (±)-2-( N -phenylethyl- N -propyl)amino-5-hydroxytetralin (N-434; 5 mg/kg) and an anesthetic, pentobarbital (50 mg/kg), decreased ACh levels and increased Ch levels. Perfusion of 10 µ M hemicholinium-3 (HC-3), a Ch uptake inhibitor, through the striatum induced a complete inhibition of ACh release and increased Ch levels in all drug-treated groups. The degree of relative increase in the level of Ch induced by HC-3 differed among the drug-pretreated groups; compared with the control group, the relative increase was larger in the sulpiride- and atropine-treated groups and smaller in the N-434 and pentobarbital-treated groups. Thus, we demonstrated reciprocal relations between extracellular concentrations of Ch and ACh after treatments by drugs. The data suggest that in the striatum, which is rich in cholinergic innervation, the extracellular Ch concentration is to a large extent determined by activity of the cholinergic transmission reflected in high-affinity choline uptake.     

PHYLOGENYAND HISTORICAL ECOLOGY OF THE DESMARESTIACEAE (PHAEOPHYCEAE) SUPPORT A SOUTHERN HEMISPHERE ORIGIN1

Phylogenetic relationships in the Desmarestiales (Phaeophyceae) were inferred among the monotypic Arthrocladia (Arthrocladiaceae) and 27 isolates from Desmarestiaceae, representing 17 taxa of Desmarestia and the monotypic Antarctic genera Himantothallus and Phaeurus. Phaeurus and Arthrocladia were used as outgroups. Parsimony analyses of nuclear ribosomal DNA internal transcribed spacer (ITS1 and ITS2) sequences, in which gaps were both included and excluded, yielded well-resolved trees with a consistent general branching pattern. A parallel analysis of nine morphological and life-history characters and three ecological characters yielded a similar tree but provided little resolution in the terminal clades. The position of the monotypic Arthrocladia villosa within the Desmarestiales is consistent with monophyly for the order, but its position as the most primitive desmarestialean is not resolvable from the molecular data set. The basal position of Phaeurus, the Antarctic Desmarestia species, and Himantothallus is consistent with the hypothesis of a Southern Hemisphere origin for the family Desmarestiaceae. The more recent Northern Hemisphere “aculeata” clade evolved from an Antarctic ancestor. A “D. aculeata-like” species was ancestral to a lineage characterized by annual sporophytes with high sulfuric acid content, which radiated into many species, widely distributed in both hemispheres. Mapping of morphological and ecological characters onto the molecular tree confirm the informativeness of sulfuric acid-containing vacuoles and unilocular sporangial types. There is good congruence between phylogenetic tree topology and temperature impints in relation to biogeographic distribution, supporting the theory that temperature tolerance is a conservative trait.     

Sequences of E/NS1 Gene Junction from Four Dengue-2 Viruses of Northeastern Thailand and Their Evolutionary Relationships with Other Dengue-2 Viruses

We determined the 240-nueleotide sequences of the E/NS1 gene junction of four dengue-2 viruses by the primer extension dideoxy chain termination method. These viruses were isolated from dengue patients with different clinical severities in Nakhon Phanom, Northeastern Thailand in 1993. The results were compared with the 52 published dengue-2 sequences of the same gene region. Sequence divergence of four new isolates varied from 4.17% to 5.42% compared with dengue-2 prototype New Guinea C strain whereas it varied from 5.42% to 6.67% and from 6.67% to 7.09% when compared with Jamaica 1409 strain and PR159/S1 strain, respectively. All nucleotide substitutions were found at the 3rd position of the codons which were silent mutations. All 56 isolates studied were classified into five genotypic groups by constructing the dendrogram. The results indicated that four new isolates from Northeastern Thailand belong to genotype II of dengue virus serotype 2, and were most closely related to prototype New Guinea C strain. We also observed the variation in nucleotide and amino acid sequences among clusters of isolates (Thailand-1980, Malaysia-1989 and Thailand-1993) which were obtained from the dengue patients with different clinical severities. The significance of these genetic differences have been discussed in terms of the possible correlation between genetic variability and virulence.     

RAD51 homologues in Xenopus laevis: two distinct genes are highly expressed in ovary and testis

The RAD51 gene is a eukaryotic counterpart of the Escherichia coli recA gene which is involved in genetic recombination. Two distinct Xenopus laevis RAD51 cDNA clones (XRAD51.1 and XRAD51.2) were isolated from an oocyte cDNA library using the human RAD51 cDNA (HsRAD51) as a probe. Sequence analysis revealed that 98.2% of the amino-acid residues were identical between XRAD51.1 and XRAD51.2, and that both were 95% identical to HsRAD51. Both of the XRAD51 genes were expressed at a higher level in ovary and testis than in other somatic tissues, suggesting their involvement in meiotic recombination. The expression of XRAD51.1 was about eightfold in excess of that of XRAD51.2 in all of the tissues examined. Analysis of the rates of synonymous substitution in the coding sequences of the two XRAD51 suggests that these two genes diverged about 50 million years ago. The structural similarities of the XRAD51 proteins to RecA in E. coli and Rad51 in yeasts or vertebrates are discussed.     

Localization ofd-amino acid oxidase mRNA in the mouse kidney and the effect of testosterone treatment

d-Amino acid oxidase (DAO), which catalyzes oxidative deamination ofd-amino acids, is known to be highly expressed in the kidney. This study was designed to examine the localization of DAO mRNA in the mouse kidney using in situ hybridization histochemistry (ISH). For comparison, ISH for mRNA of ornithine decarboxylase (ODC), which is also highly expressed in the mouse kidney, was simultaneously performed. Adult, male mice which received 1 mg of testosterone propionate or vehicle injection, were sacrificed 14 h after injection and their kidneys were removed and processed for ISH. Hybridization signals for both mRNAs were exclusively located over the epithelial cells of the proximal tubule in the vehicle-treated animals. Signals for the DAO mRNA were observed at nearly the same hybridization intensity throughout the proximal tubule, whereas hybridization signals for the ODC mRNA were observed exclusively in the pars convoluta. Following testosterone treatment, ODC mRNA in the pars convoluta was expressed with a stronger intensity than that in the vehicle-injected animals. ODC mRNA was also expressed in the pars recta with a weaker intensity than in the pars convoluta. On the other hand, DAO mRNA expression was little affected by testosterone treatment. These results indicate that, although both genes are possibly expressed in the same cells, the expression of these genes is regulated by different mechanisms.     

Localization of immuno-analogues of erythrocyte protein 4.1 and spectrin in epidermis of psoriasis vulgaris

The presence and localization of immuno-analogues of human erythrocyte protein 4.1 and spectrin were examined in the epidermis of psoriasis vulgaris. Immunoblot analysis with antibodies against human erythrocyte protein 4.1 revealed that psoriatic epidermis contains a 4.1-like protein of 80 kDa, and also minor immunoreactive polypeptides, including a 45-kDa polypeptide. The 45-kDa band was not detected in non-lesional epidermis. Lesional epidermis of psoriasis contains spectrin-like proteins of 240 kDa. Analysis with immunofluorescence microscopy revealed that 4.1-like proteins were detected mainly in the cytoplasm of the suprabasal cells in lesional epidermis and in the peripheral cytoplasm of the basal cells in non-lesional epidermis. On the other hand, spectrin-like proteins were localized to the peripheral cytoplasm of basal keratinocytes in both lesional and non-lesional psoriatic epidermis. The present results indicate that proteins related to protein 4.1 and spectrin are consistently detected within epidermal cells of psoriasis, a chronic skin disease characterized by epidermal hyperplasia; the expression and distribution of protein 4.1 in lesional epidermis of psoriasis differs from that in non-lesional epidermis. These membrane skeletal proteins may be of significance in the hyperproliferative epidermis of psoriasis.     

Application of bacterial magnetic particles as novel DNA carriers for ballistic transformation of a marine cyanobacterium

Summary Bacterial magnetite particles (BMPs) of 50 to 100nm diam were used as DNA carriers for the ballistic transformation of the marine cyanobacteriumSynechococcus. BMPs were bombarded into the cyanobacterial cells at several bombardment velocities using a particle gun. Successful transformation and gene expression were confirmed by Southern hybridization and CAT assay, respectively. The BMPs were also observed in the cyanobacterial cells by transmission electron microscopy. These results suggested that BMPs can be used as carriers for introducing DNA into bacterial cells.