Structural study of the sugar chains of human platelet thrombospondin

The asparagine-linked sugar chains of human platelet thrombospondin were released as oligosaccharides by hydrazinolysis. About 12 mol of sugar chains was released from one thrombospondin molecule. This was converted to radioactive oligosaccharides by sodium borotritide reduction after N-acetylation, and separated into one neutral and four acidic fractions by paper electrophoresis. More than 90% of the oligosaccharides were recovered in the acidic fraction. The acidic oligosaccharides were mostly converted to neutral oligosaccharides by sialidase treatment, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were further fractionated by Bio-Gel P-4 column chromatography. Structural study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed that the thrombospondin contains mono-, bi-, tri-, and tetraantennary complex-type sugar chains in addition to a small amount of high-mannose type. Approximately 70% of the complex-type sugar chains was fucosylated at asparagine-linked N-acetylglucosamine residue and 19% of the biantennary complex-type sugar chains was bisected.     

Complete primary structure of the transacylase (E2b) subunit of the human branched chain alpha-keto acid dehydrogenase complex

We isolated from a placental cDNA library by immunoscreening a cDNA clone encoding the transacylase (E2b) precursor of the human branched chain alpha-keto acid dehydrogenase (BCKDH) complex. The cDNA insert consists of 2,649 base pairs with an open reading frame of 1,431 base pairs which can be translated into 477 amino acids and a 3'-untranslated region of 1,205 base pairs. The deduced amino acid sequence includes a leader peptide of 56 amino acid residues, a lipoyl-bearing domain, a E3-binding domain and an inner core domain. A mature human E2b subunit is likely to contain 421 amino acid residues with a calculated Mr 46,322. The nucleotide sequence of the open reading frame and the deduced amino acid sequence of the human E2b shows 91.6% and 92.0% homology with those of the bovine E2b subunit, respectively.     

Lobeline production by hairy root culture of Lobelia inflata L.

Hairy roots were obtained following inoculation of the stems of Lobelia inflata L. with Agrobacterium rhizogenes strain ATCC 15834. These hairy roots contained agropine and mannopine. In addition, lobeline was detected by HPLC and confirmed by mass spectrometry. Various media were tested for the growth of hairy roots as well as for the content of lobeline in hairy roots. The growth rate of hairy roots cultured in Nitsch and Nitsch's medium was approximately one third of those cultured in other media. The lobeline content of hairy roots (18–54 g/g dry weight) cultured in these media was the same order of magnitude compared with that of roots of L. inflata (24 g/g dry weight) cultivated in pots. The hairy roots cultured in Nitsch and Nitsch's medium were morphologically different from those cultured in other media.Abbreviations MS medium Murashige and Skoog's medium - 1/2 MS medium one-half strength of the standard Murashige and Skoog's medium - B5 medium Gamborg's B5 medium - NN medium Nitsch and Nitsch's medium - FW fresh weight - DW dry weight     

Epidermal growth factor (EGF)-induced morphological changes in the basement membrane of chick embryonic skin

Summary The effect of epidermal growth factor (EGF) on the basement membrane structure of chick embryonic skin cultured in a chemically defined medium (BGJb) containing 20 mM hydrocortisone, and EGF at 10, 50, or 100 ng/ml supplemented with 5% delipidized fetal calf serum, was examined by electron microscopy. During development of the epidermis in vitro, EGF (100 ng/ml) caused striking changes to occur in the basement membrane structure and in the keratinization process. The basement membrane frequently became discontinuous with many gaps apparent in section, and occasionally became folded following detachment from the basal surface of the epidermis and protruded into the underlying dermis. In the basal and intermediate cells of EGF-treated epidermis, tonofilament bundles were decreased in number, while desmosomes and hemidesmosomes revealed no significant changes in morphology.     

Digit development and embryonic weight in mice: analysis of sex-related time difference and mating period-related interlitter variability

The embryonic growth and digit formation in limb buds were more advanced in male embryos than in female embryos at a specific time (day 12.0) of midgestation. Furthermore, when the number of digits was compared between the sexes according to their body weight, male embryos were found to be more advanced than females in the differentiation of the digit in limb buds. This is the first demonstration of the presence of a time difference in digit development between the sexes of mouse embryos. In the short-period, morning-mating group, embryonic weights at day 12.0 were lower than those in the overnight-mating group. However, the digit development was not very much delayed in proportion to the difference in body weights, and some "catch-up" phenomena were observed in this group. Interlitter and intralitter variability in body weights of mouse embryos at day 12.0 was greater in the overnight-mating group than in the short-period-mating group. These findings suggest that, in embryonic stage-related teratological experiments in mice, a short-period-mating schedule is advised and that the incidence of developmental anomalies should be analyzed separately for male and female fetuses.     

The genotoxicity of a variety of aniline derivatives in a DNA repair test with primary cultured rat hepatocytes

The genotoxicity of a variety of aniline derivatives was examined by a DNA repair test with rat hepatocytes. Out of 37 aniline derivatives, 6 chemicals, i.e., 2,4,6-trimethylaniline (mesidine), 2,4-xylidine, 3,5-diaminobenzoic acid, 3,4-diaminochlorobenzene, 2-chloro-4-methylaniline and 4-chloro-N-methylaniline, elicited positive DNA repair responses. The results are in agreement with the bacterial mutagenicities with or without norharman of these compounds. Positive compounds of unknown carcinogenicity in the present assay, i.e., 3,5-diaminobenzoic acid, 2-chloro-4-methylaniline and 4-chloro-N-methylaniline are suspected of being potentially carcinogenic.     

Carbohydrate binding specificity of immobilized Allomyrina dichotoma lectin II

The carbohydrate binding specificity of Allomyrina dichotoma lectin II was investigated by analyzing the behavior of various complex type oligosaccharides and human milk oligosaccharides on an A. dichotoma lectin II-agarose column. Basically, the lectin interacts with the Gal beta 1----4GlcNAc group. Substitution of their terminal galactose residues by Neu5Ac alpha 2----6 will enhance their affinity to the lectin. By contraries, substitution at the C-2 or C-3 position of their terminal galactose with other sugars including sialic acid deprives their affinity to the lectin. With this characteristic, the immobilized lectin column can be used to separate complex type oligosaccharides with the Neu5Ac alpha 2----6Gal beta 1----4GlcNAc group from their isomeric oligosaccharides with the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc group, where Neu5Ac is N-acetylneuraminic acid.     

Genetic diversity among sublines originating from a single somatic hybrid cell of Duboisia hopwoodii + Nicotiana tabacum

Summary The genetic instability of an intertribal hybrid cell line, Duboisia hopwoodii + Nicotiana tabacum, obtained by mechanical isolation of a single hybrid cell was studied. Ten subclones of calli derived from this hybrid cell line were cultured for 3 years, and their genetic makeup clarified as to nuclear DNA content, chromosome constitution, and peroxidase isozymes. Nuclear DNA content differed in each subclone. In most subclones, mean DNA content was lower than the mean DNA content in the original hybrid cell line determined 1 year after fusion. This decrease in DNA content is partly attributable to the elimination of tobacco chromosomes that occurred in all subclones. The extent to which tobacco chromosomes were eliminated varied among the subclones — evidence that chromosome elimination occurred slowly. Peroxidase isozyme analysis indicated the loss of a tobacco-specific isozyme, thus confirming results obtained by chromosome analysis. Shoots regenerated from two hybrid subclones after 2 years were also heterogeneous in morphology and nuclear DNA content.     

Patterns of prey utilization in a web of orb-weaving spiderAraneus pinguis (Karsch)

The pattern of prey utilization of the orb-weaving spider Araneus pinguis was studies by comparing between arthropods restrained in the empty webs (spiders were removed) and those unattacked in the intact webs (spiders were not removed). The number of arthropods was larger in the empty webs than in the intact webs. In the empty webs, web area, mesh width, number of radii, and signal thread length were presumed to affect the number of arthropods left in a single web. As for the intact webs, web area, mesh width, and web-exposure time were important factors. In the empty webs, the density of arthropods decreased away from the hub. On the other hand, the density of unattacked arthropods in the intact webs was the same throughout the web. Arthropods in the empty webs were larger than those in the intact webs. The upper limit in size of unattacked arthropods increased along with the distance from the hub in the intact webs, but not in the empty webs. These results indicate increase in the minimum size of eaten arthropods increased in the former. This positive sizedistance relation may have resulted from the adaptive switching of spiders’ alternative foraging methods (i.e., the rapid attack at encounter and the later eating during web deconstruction) on the basis of the prey profitability.     

Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain.

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3′-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5′-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.