Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-mhz (1)h and (13)c NMR analysis.

We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen,Campylobacter jejuni OH4384, which has been associated with Guillain-Barre syndrome. We first cloned a gene encoding an α-2,3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a β-1,4-N-acetylgalactosaminyl-transferase (cgtA), a β-1,3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked α-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of3J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two relatedC. jejuni strains that express shorter ganglioside mimics in their LOS

Simulation of Organ Patterning on the Floral Meristem Using a Polar Auxin Transport Model

An intriguing phenomenon in plant development is the timing and positioning of lateral organ initiation, which is a fundamental aspect of plant architecture. Although important progress has been made in elucidating the role of auxin transport in the vegetative shoot to explain the phyllotaxis of leaf formation in a spiral fashion, a model study of the role of auxin transport in whorled organ patterning in the expanding floral meristem is not available yet. We present an initial simulation approach to study the mechanisms that are expected to play an important role. Starting point is a confocal imaging study of Arabidopsis floral meristems at consecutive time points during flower development. These images reveal auxin accumulation patterns at the positions of the organs, which strongly suggests that the role of auxin in the floral meristem is similar to the role it plays in the shoot apical meristem. This is the basis for a simulation study of auxin transport through a growing floral meristem, which may answer the question whether auxin transport can in itself be responsible for the typical whorled floral pattern. We combined a cellular growth model for the meristem with a polar auxin transport model. The model predicts that sepals are initiated by auxin maxima arising early during meristem outgrowth. These form a pre-pattern relative to which a series of smaller auxin maxima are positioned, which partially overlap with the anlagen of petals, stamens, and carpels. We adjusted the model parameters corresponding to properties of floral mutants and found that the model predictions agree with the observed mutant patterns. The predicted timing of the primordia outgrowth and the timing and positioning of the sepal primordia show remarkable similarities with a developing flower in nature

Synthesis of the human insulin gene. Part II. Further improvements in the modified phosphotriester method and the synthesis of seventeen deoxyribooligonucleotide fragments constituting human insulin chains B and mini-CDNA.

The purification of protected deoxyribooligonucleotides containing phosphotriester internucleotidic linkages has been improved by developing a deactivated silica gel chromatographic technique. The efficiency of this technique as applied in the modified phosphotriester approach has been demonstrated in the rapid synthesis of seventeen pure fragments constituting the sequence of human insulin B and mini-C DNA. The sequence of each oligomer was confirmed by the two-dimensional mobility shift method of fingerprinting

Modyfikacja chemiczna surowca skrobiowo-białkowego w celu uzyskania półproduktu do przerobu termoplastycznego

This article presents the results of tests which were carried out on a starch-protein biomaterial produced from wheat. The research was performed in order to assess optimal conditions for performing the chemical modification (acetylation and oxidation reactions) and its influence on the physico-chemical properties of the new biomaterial. Modifying agents were acetic acid anhydride with the presence of NaOH and K2CO3 activators as well as hydrogen peroxide, whose effects were catalysed with Cu2+ ions. Modified polymers with a degrees of substitution from 0.38 to 2.45 and oxidation from 1.0 to 35% were obtained in the process. The polymers obtained were characterized with various levels of starch degradation, minimal when the acetylation reaction activated with the K2CO3 method was used, moderate to high when the acetylation reaction was activated by NaOH, and maximal starch degradation was observed when oxidation was catalysed with Cu2+ ions. The modified polymers showed minor thermal granulation abilities and did not achieve thermoplastic abilities. Evaluation of the modification effects was carried out with absorption spectroscopy in infra-red radiation with the Fourier transformation (FTIR) technique, DSC, scanning electron microscopy and determining the boundary viscosity value.Poszukiwanie nowych materiałów przyjaznych środowisku skłania do przeanalizowania możliwości wykorzystania powszechnie występujących i prostych w pozyskaniu surowców roślinnych. Jednym z takich surowców jest materiał skrobiowo-białkowy. W artykule przedstawiono badania, którymi objęto biomateriał skrobiowo-białkowy wytwarzany w warunkach przemysłowych z pszenicy. Wykonane badania miały na celu określenie optymalnych warunków prowadzenia modyfikacji chemicznej oraz jej wpływu na właściwości fizykochemiczne nowego biomateriału. Badania zostały ukierunkowane pod kątem przydatności uzyskanego modyfikatu do wytwarzania materiałów termoplastycznych. Modyfikacja chemiczna obejmowała acetylację i utlenianie skrobi. Jako czynniki modyfikujące wykorzystano bezwodniki kwasu octowego w obecności aktywatorów NaOH i K2CO3 oraz nadtlenek wodoru stosowany w obecności katalizatora CuSO4. W wyniku przeprowadzonych badań uzyskano zmodyfikowane chemicznie produkty o szerokim zakresie stopni podstawienia (od 0,38 do 2,45 - w badaniach wstępnych 2,99) oraz utlenienia (od 1,0 do 35%). Charakteryzowały się one różnym stopniem degradacji skrobi, od niewielkiej w reakcji acetylacji bezwodnikiem octowym w obecności aktywatora K2CO3 do drastycznej, w reakcji utleniania nadtlenkiem wodoru katalizowanej CuSO4. Zmodyfikowane biopolimery wykazywały niewielkie zdolności do krystalizacji termicznej, nie uzyskały jednak właściwości termoplastycznych. Do oceny efektów modyfikacji wykorzystywano techniki spektroskopii absorpcyjnej w podczerwieni z transformacją Fouriera (FTIR), różnicowej kalorymetrii skaningowej (DSC), elektronowej mikroskopii skaningowej do oceny stopnia zdegradowania skrobi posłużono się wynikami badań granicznej liczby lepkościowej

Crystal structure of T4-lysozyme generated from synthetic coding DNA expressed in Escherichia coli

Authors affiliated with NRC Division of Biological Sciences at time of publishingPeer reviewed: YesNRC publication: Ye