Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

Benzyl-N-acetyl-α-d-galactosaminide inhibits the sialylation and the secretion of mucins by a mucin secreting HT-29 cell subpopulation

We have analysed the mucins synthesized by the HT-29 MTX cell subpopulation, derived from the HT-29 human colon carcinoma cells through a selective pressure with methotrexate (Lesuffleuret al., 1990,Cancer Res 50: 6334–43), in the presence of benzyl-N-acetyl--galactosaminide (GalNAc-O-benzyl), which is a potential competitive inhibitor of the 1,3-galactosyltransferase that synthesizes the T-antigen. The main observation was a 13-fold decrease in the sialic acid content of mucins after 24 h of exposure to 5mm GalNAc-O-benzyl. This effect was accompanied by an increased reactivity of these mucins to peanut lectin, testifying to the higher amount of T-antigen. The second observation was a decrease in the secretion of the mucins by GalNAc-O-benzyl treated cells. The decrease in mucin sialyation was achieved through thein situ -galactosylation of GalNAc-O-benzyl into Gal1–3GalNAc-O-benzyl, which acts as a competitive substrate of Gal1–3GalNAc 2,3-sialyltransferase, as shown by the intracellular accumulation of NeuAc2–3Gal1–3GalNAc-O-benzyl in treated cells.Abbreviations BSM bovine submaxillary mucin - MTX methotrexate - PBS sodium phosphate 10mm, NaCl 0.15m, pH 7.4 buffer - pNp p-nitrophenol - TBS Tris/HCl 10mm, NaCl 0.15m, pH 7.4 buffer Enzymes: CMP-NeuAc: Gal1–3/4GlcNAc 2,3-sialyltransferase, ST3(N), EC 2.4.99.6; CMP-NeuAc: Gal1–4GlcNAc 2,6-sialyltransferase, ST6(N), EC 2.4.99.1; CMP-NeuAc: Gal1–3GalNAc 2,3-sialyltransferase, ST3(O), EC 2.4.99.4; CMP-NeuAc: R-GalNAc1-O-Ser 2,6-sialyltransferase, ST6(O)-I, EC 2.4.99.3; CMP-NeuAc: NeuAc2–3Gal1–3GalNAc 2,6-sialyltransferase, ST6(O)-II, EC 2.4.99.7; UDP-GlcNAc: Gal1–3GalNAc-R·(GlcNAc to GalNAc) 1,6-N-acetylglucosaminyltransferase, EC 2.4.1.102; UDP-GlcNAc: GalNAc-R 1,3-N-acetylglucosaminyltransferase, EC 2.4.1.147; UDP-Gal: GalNAc-R 1,3-galactosyltransferase, EC 2.4.1.122.     

Studies on aneuploids of Petunia

Summary The seven possible primary trisomics of Petunia (2 n= 14) located in the progenies of triploid, hypertriploid and hypotriploid plants were distinguished from one another and from diploid on the basis of cytological and morphological criteria. They were provisionally named as Oval, Semi, Slender, Pseudonormal, Arrow, Narrow and Giant. In three of the trisomics, the extra chromosome was identified for the first time at pachytene stage. Postpachytene studies revealed no precise relationship between the length of extra chromosome and the frequency of multiple association.     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Karyosystematics of thePhleum alpinum polyploid complex (Poaceae)

The karyotypes of three taxa from thePhleum alpinum group of sect.Phleum (P. alpinum subsp.rhaeticum, 2n = 14,P. commutatum, 2n = 28, and informally namedP. commutatum, 2n = 14) were investigated by Giemsa C-banding. The overall similarity of diploid genomes suggests thatP. alpinum subsp.rhaeticum andP. commutatum are closely related — their karyotypes vary only with respect to their average amounts of telomeric heterochromatin. TheP. commutatum genome contains less telomeric heterochromatin than the genome ofP. alpinum subsp.rhaeticum, but in theP. alpinum group as a whole almost fluent transition between low (1.5%) and high (25.5%) amounts of telomeric heterochromatin was observed among populations. In the karyotype of tetraploidP. commutatum, seven distinguishable chromosome types were observed. Each of them is represented at somatic metaphase by four chromosomes. C-band structure of karyotype and average amount of telomeric heterochromatin suggest that this taxon has originated from hybridization between two related diploid forms of theP. commutatum — P. alpinum complex.     

Dense-core vesicles in motor nerve terminals

Summary Motor nerve terminals on white and intermediate muscle fibers of the Atlantic hagfish (Myxine glutinosa, L.) contain translucent synpatic vesicles and about 1–2% dense-core vesicles. Terminals on red muscle fibers contain up to 40% dense-core vesicles with diameter 800–1100 Å. Examinations for formaldehyde-induced fluorescence indicate yellow fluorescence (5-HT ?) apparently corresponding with terminal axons on red muscle fibers in craniovelar muscles. Possibly red muscle fibers of Myxine receive monoaminergic innervation.The author is indebted to Dr. Finn Walvig, Biological station, University of Oslo, Drøbak, for supply of hagfishes.     

Subtle behavioral variation in wild chimpanzees, with special reference to Imanishi’s concept of kaluchua

Here we consider the concept of kaluchua (a word adopted from the English culture) in group-living animals developed by Imanishi in the 1950s. He distinguished it from bunka (the Japanese equivalent to the English culture) because he thought that bunka had strong connotations of noble and intellectual human-like activities. Although he did not rigidly define kaluchua, his original concept of kaluchua was much broader than bunka and represented non-hereditary, acquired behavior that was acknowledged socially. However, instead of social life, complex feeding skills have often formed the central topic in the current studies of animal culture. In order to provide evidence that more subtle behavioral variations exist among wild chimpanzee (Pan troglodytes) populations, we directly compared the behaviors of two well-habituated chimpanzee groups, at Bossou and Mahale. During a 2-month stay at Bossou, M.N. (the first author) saw several behavioral patterns that were absent or rare at Mahale. Two of them, mutual genital touch and heel tap were probably customary for mature females and for mature males, respectively. Index to palm and sputter are still open to question. These subtle patterns occurred more often than tool use during the study period, suggesting that rarity is not the main reason for their being ignored. Unlike tool use, some cultural behavioral patterns do not seem to require complex skills or intellectual processes, and sometimes it is hard to explain the existence of such behaviors only in terms of function.     

Floral sexual dimorphism and flower choice by pollinators in a nectarless monoecious vine Akebia quinata (Lardizabalaceae)

We investigated the pollination system and movement patterns of pollinators among flowers of the nectarless, monoecious vine Akebia quinata in natural populations and experimental floral arrays. Female flowers did not offer any rewards for pollinators and were larger than male flowers. Pollinators of A.quinata , such as small solitary bees and hoverflies, clearly discriminated between male and female flowers. Hoverflies always visited male flowers and rarely visited female flowers. In contrast, solitary bees tended to visit female flowers first when entering the array, but then switched to male flowers within the same foraging bout. This tendency disappeared when the sepal size of female flowers was experimentally reduced to the size of male flowers. Thus, observed non-randomness in flower choice by solitary bees may be caused by female>male sexual dimorphism and may increase the chance of cross-pollination and lower the probability of geitonogamous pollination in a single visit to a plant. Therefore, floral sexual dimorphism in A.quinata is considered to be an adaptation for deceptive pollination associated with discriminating pollinators.     

Anti-diabetic activity of <Emphasis Type="Italic">β</Emphasis>-glucans and their enzymatically hydrolyzed oligosaccharides from <Emphasis Type="Italic">Agaricus blazei</Emphasis>

-Glucans were prepared from Agaricus blazei Murill by repeated extraction with hot water. The average molecular weights of -glucans were 30–50 kDa by gel filtration chromatography. Oligosaccharides (AO), derived from hydrolyzing -glucans with an endo--(16)-glucanase from Bacillus megaterium, were mainly di- and tri-saccharides. Though -glucans and AO both showed anti-hyperglycemic, anti-hypertriglyceridemic, anti-hypercholesterolemic, and anti-arteriosclerotic activity indicating overall anti-diabetic activity in diabetic rats, AO had about twice the activity of -glucans with respect to anti-diabetic activity.     

Differential accumulation of four phaseolin glycoforms in transgenic tobacco

An intron-less phaseolin gene [15] was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical -phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (wt^i–) and mutant phaseolin glycoforms (dgly ₁, dgly ₂ and dgly _1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the dgly ₁ and dgly ₂ mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin dgly _1,2 gene. Additionally, the profile of 25–29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.Abbreviations bp base pair(s) - DAF days after flowering - GUS -glucuronidase - kb kilobase - kDa kilodalton     

An improved micropropagation system for Chrysanthemum based on Pluronic F-68-supplemented media

Supplementation of MS-based medium containing 4.4 M 6-benzyladenine and 2.7 M -naphthaleneacetic acid with 0.001–0.1% (w/v) of the non-ionic, co-polymer surfactant, Pluronic F-68, significantly (p<0.05) increased the mean fresh weight gain of cultured leaf explants of chrysanthemum (Dendranthema grandiflora) Tone Maid and Early Charm by a maximum of 74% and 34%, respectively. The percentage of individual explants giving adventive shoots was also stimulated by Pluronic F-68; for cv. Early Charm, 0.001% (w/v) Pluronic F-68 induced the maximum response, whereas for cv. Tone Maid, the maximum response occurred with 0.01% surfactant. Shoot regeneration from explants was also enhanced at these concentrations of surfactant, compared to explants cultured in the absence of Pluronic F-68.Abbreviations BA 6-benzyladenine - MS Murashige & Skoog 1962 - NAA -naphthaleneacetic acid     

Chromate reduction by Microbacterium liquefaciens immobilised in polyvinyl alcohol

A polyvinyl alcohol-based immobilisation technique has been utilised for entrapping the newly-isolated chromate-reducing bacterium, Microbacterium liquefaciens MP30. Three immobilisation methods were evaluated: PVA-nitrate, PVA-borate and PVA-alginate. Chromate reduction was studied in batch and continuous-flow bioreactors, where the beads maintained integrity during continuous operation. PVA-borate and PVA-alginate cell beads showed a higher rate and extent of chromate reduction than PVA-nitrate cell beads in batch experiments. With the former 100 M Cr(VI) was removed within 4 days, while only 40 M Cr(VI) was removed using the latter, and with no increase in Cr(VI) removal subsequently. Cell activity was maintained during immobilisation but the rate of Cr(VI) removal by immobilised cells was only half that of an equivalent mass of free cells. Using PVA-alginate cell beads in a continuous-flow system, chromate removal was maintained at 90–95% from a 50 M solution over 20 days without signs of bead breakdown.     

Nucleotide sequence and phylogenetic implication of the ATPase subunits β and ε encoded in the chloroplast genome of the brown alga Dictyota dichotoma

We have cloned and sequenced the genes atpB and atpE, coding for CF₁ subunits and , respectively, of the chloroplast genome of the brown alga Dictyota dichotoma. Although the coding site of atpE cannot be demonstrated by heterologous Southern hybridizations, a 417 bp reading frame 3 to atpB was identified as the gene atpE by sequence similarities with atpE genes from other sources. A maximum sequence identity of 30% is found between the predicted amino acid sequence of the Dictyota subunit and the corresponding cyanobacterial subunits. Including conserved amino acid replacements, the Dictyota subunit exhibits about 70% sequence similarity with the cyanobacterial and land plant subunits. As in cyanobacteria, the atpE gene does not overlap the preceding gene atpB. The deduced amino acid sequence of atpB is 74–79% identical to the corresponding cyanobacterial and chloroplast subunits. Entirely conserved are regions referred to as the catalytic and/or regulatory sites of ATP formation, including interacting regions between subunits and . A phylogram predicted from F₁/CF₁- subunits of eleven different organisms suggests a common evolutionary origin of plastids from chlorophytes and brown algae.     

Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca²⁺. To determine if Ca²⁺ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca²⁺, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca²⁺, was observed only in the perfusion solution containing Ca²⁺, indicating that myosin is responsible for the inhibitory effect of Ca²⁺ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca²⁺ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP adenosine 5-triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

The carotenoids of Flavobacterium strain R1560

The main carotenoid of Flavobacterium strain R1560 has been identified as (3R,3R)-zeaxanthin. Also present were small amounts of 15-cis-phytoene, phytofluene, -carotene (7,8,7,8-tetrahydro-, -carotene plus 7,8,11,12-tetrahydro-, -carotene), neurosporene, lycopene, -zeacarotene, -carotene, -carotene, -cryptoxanthin, rubixanthin, 3-hydroxy--zeacarotene and several apo-carotenals. Zeaxanthin production was inhibited by nicotine (10 mM), and lycopene and rubixanthin accumulated. The biosynthesis of zeaxanthin is discussed in terms of pathways and also of half-molecule reaction sequences. The presence of zeaxanthin may be a characteristic of a group of Flavobacterium species, and may thus be useful in the taxonomic classification of these organisms.     

Theβ-subunit of human chorionic gonadotropin containsN-glycosidic trisialo tri- and tri′-antennary carbohydrate chains

TheN-linked carbohydrate chains of the-subunit of highly purified urinary human chorionic gonadotropin have been re-investigated. The oligosaccharides were released enzymatically by peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F, and fractionated by a combination of FPLC and HPLC. As a result of the application of improved fractionation methods, apart from the earlier reported carbohydrate chains, also small amounts of trisialo tri- and tri-antennary oligosaccharides were found. The primary structures of the latter carbohydrate chains have been determined by 500-MHz¹H-NMR spectroscopy to beAbbreviations hCG human chorionic gonadotropin - hCG- -subunit - hCG- -subunit - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52) - endo-F endo--N-acetylglucosaminidase-F (E.C. 3.2.1.96) - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - CBB coomassie brilliant blue R 250 - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

Further cytogenetic analyses of the Croatian triploid shallot “Ljutika” (Allium cepa var.viviparum, Alliaceae) and its comparison with the Indian triploid “Pran”

Feulgen and silver-stained karyotypes and meiosis of two triploid viviparous onion forms (Allium cepa var.viviparum), the Croatian Ljutika and the Indian Pran, were comparatively analyzed. The results of chromosome measurements show that Ljutika and Pran are karyologically not identical, although significant similarities were found in the morphology of their chromosomes. Five geographically distant clones of Ljutika showed good agreement in the number and gross morphology of the chromosomes and in the number and position of NORs and interphase nucleoli. Heterotrivalents were predominant in meiosis of Ljutika but a relatively high frequency of higher multivalents together with univalents and bivalents were also observed. The relationship between Ljutika and Pran and their possible origin are discussed.     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

Plant regeneration from callus and explants of Sesbania spp.

Root, hypocotyl and cotyledon explants of Sesbania bispinosa, Sesbania cannabina, Sesbania formosa, and Sesbania sesban were cultured on Murashige and Skoog medium with benzyladenine (BA; 2.22, 4.44, 8.88 M) in combination with 2,4-dichlorophenoxyacetic acid (2,4-d; 2.26, 4.52, 9.05 M), indolebutyric acid (IBA; 0.25, 0.49, 4.92 M) or naphthaleneacetic acid (NAA; 2.69, 5.37, 10.74 M). Although all explant types developed some callus, callus occurred earliest and continued to grow fastest with hypocotyls. Media including 2.4-d or NAA gave the fastest growing callus. Callus was subcultured up to 10 times at 20-day intervals and retained a rapid growth rate. Shoots regenerated readily from both hypocotyls or cotyledons but not from roots. Shoot organogenesis was most frequent with IBA (0.25–4.92 M) in combination with BA (4.44–8.88 M) and did not occur with 2,4-d. With each species at least one medium induced shoot differentiation from more than 50 percent of the callus pieces. With one exception, media containing IBA that induced shoot organogenesis on explants also did so in callus, but media containing NAA, even when effective with explants, did not cause differentiation of callus. Shoots that differentiated were excised and cultured on MS medium without growth regulators or with IBA (2.46, 4.92, 9.84 M). Roots developed after 3–8 days on an appropriate rooting medium, often without IBA. Rooted plantlets were transplanted to pots in a greenhouse and developed into normal plants. Suitable media and protocols for initiating and subculturing callus and regenerating whole plants in vitro from callus and explants have thus been established for four species of Sesbania.