Structural analysis of the lipopolysaccharide from Neisseria meningitidis strain BZ157 galE: localisation of two phosphoethanolamine residues in the inner core oligosaccharide

The structure of the phase-variable lipopolysaccharide (LPS) from the group B Neisseria meningitidis strain BZ157 galE was elucidated. The structural basis for the LPS's variation in reactivity with a monoclonal antibody (MAb) B5 that has specificity for the presence of phosphoethanolamine (PEtn) at the 3-position of the distal heptose residue (HepII) was established. The structure of the O-deacylated LPS was deduced by a combination of monosaccharide analyses, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. These analyses revealed the presence of a novel inner core oligosaccharide (OS) structure in the MAb B5 reactive (B5+) LPS that contained two PEtn residues simultaneously substituting the 3- and 6-positions of the HepII residue. The determination of this structure has identified a further degree of variability within the inner core OS of meningococcal LPS that could contribute to the interaction of meningococcal strains with their host.     

Composition of the lipopolysaccharide from different capsular serotype strains of Haemophilus influenzae

Lipopolysaccharide (LPS) from all six serotype strains of Haemophilus influenzae was similar in composition. The oligosaccharide, of each LPS, was composed of glucose, galactose, heptose and 2-keto-3-deoxyoctonic acid. The lipid A was composed of glucosamine, phosphate and the fatty acids 14:0 and 3-OH 14:0. Each LPS also contained ethanolamine and ethanolamine phosphate, and the oligosaccharides from two strains additionally contained small amounts of glucosamine. Although the LPS was similar in composition, different serotypes had quantitative differences, especially in the galactose content, which correlated with the antigenic specificity of their homologous antisera and with their mobility on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A survey by SDS-PAGE showed that LPS from strains of the serotypes a, c and d was characteristically of lower Mr than the LPS from most (80%) serotype b strains.     

Identification of phosphorylated 3-deoxy-manno-octulosonic acid as a component of Haemophilus influenzae lipopolysaccharide.

A phosphorylated 3-deoxy-manno-octulosonic acid (KDO) was released from the lipopolysaccharide (LPS) of the deep rough mutant (Rb+169) of Haemophilus influenzae by acid hydrolysis. Both phosphorylated and dephosphorylated KDO, produced by treatment with alkaline phosphatase, were identified by gas chromatography-mass spectrometry after trimethylsilylation. This technique provides a rapid and reliable method for the identification of phosphorylated KDO in LPS.     

Structural analysis of lipopolysaccharides from Haemophilus influenzae serotype f. Structural diversity observed in three strains.

Structural elucidation of the lipopolysaccharide (LPS) from three serotype f Haemophilus influenzae clinical isolates RM6255, RM7290 and RM6252 has been achieved using NMR spectroscopy techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MSn on permethylated dephosphorylated OS. This is the first study to report structural details on LPS from serotype f strains. We found that the LPSs of all strains were highly heterogeneous mixtures of glycoforms expressing the common H. influenzae structural element l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-[beta-d-Glcp-(1-->4)]-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A with variable length of OS chains linked to each of the heptoses. The terminal heptose (HepIII) in RM6255 is substituted at the O-3 position by a beta-d-Glcp residue whereas HepIII in strains RM7290 and RM6252 is substituted at O-2 by the globoside unit (alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glc) or truncated versions thereof. The central heptose (HepII) is substituted by an alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp-(1-->4)-alpha-d-Glcp unit in RM7290 and RM6252 or truncated versions thereof. Strain RM6255 does not express galactose in its LPS and only shows a cellobiose unit elongating from HepII (beta-d-Glcp-(1-->4)-alpha-d-Glcp). ESI-MSn on dephosphorylated and permethylated OS provided information on the existence of additional minor isomeric glycoforms.     

电针介导的基因转移

基因转移是实现基因治疗的关键技术之一 ,目前尚缺少简便、易行、有效、安全的方法 .首次将我国传统的针刺技术与现代转基因技术结合起来 ,创建了一种电针转基因的方法 .应用针灸针携带外源基因 ,经皮针刺 ,进行直流电刺激 ,可实现有效的基因转移 .     

DNA methyltransferase homologue TRDMT1 in Plasmodium falciparum specifically methylates endogenous aspartic acid tRNA

In eukaryotes, cytosine methylation regulates diverse biological processes such as gene expression, development and maintenance of genomic integrity. However, cytosine methylation and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, here we investigated the presence of cytosine methylation in the nucleic acids of the protozoan Plasmodium falciparum. Interestingly, P. falciparum has TRDMT1, a conserved homologue of DNA methyltransferase DNMT2. However, we found that TRDMT1 did not methylate DNA, in vitro. We demonstrate that TRDMT1 methylates cytosine in the endogenous aspartic acid tRNA of P. falciparum. Through RNA bisulfite sequencing, we mapped the position of 5-methyl cytosine in aspartic acid tRNA and found methylation only at C38 position. P. falciparum proteome has significantly higher aspartic acid content and a higher proportion of proteins with poly aspartic acid repeats than other apicomplexan pathogenic protozoans. Proteins with such repeats are functionally important, with significant roles in host-pathogen interactions. Therefore, TRDMT1 mediated C38 methylation of aspartic acid tRNA might play a critical role by translational regulation of important proteins and modulate the pathogenicity of the malarial parasite.     

DNA Barcoding of Economically Important Mushrooms: A Case Study on Lethal Amanitas from China

Some species of the genus Amanita are economically important gourmet mushrooms, while others cause dramatic poisonings or even deaths every year in China and in many other countries. A DNA barcode is a short segment or a combination of short segments of DNA sequences that can distinguish species rapidly and accurately. To establish a standard DNA barcode for poisonous species of Amanita in China, three candidate markers, the large subunit nuclear ribosomal RNA (nLSU), the internal transcribed spacer (ITS), and the translation elongation factor 1 alpha (tef1 α) were tested using the eukaryotic general primers for their feasibility as barcodes to identify seven species of lethal fungi and two species of edible ones which can easily be confused with the lethal ones known from China. In addition, A.phalloides—a European and North American species closely related to one of the seven taxa, A.subjunquillea was also included. PCR amplification and sequencing success rate, intra and inter specific variation and rate of species identification were considered as main criteria for evaluation of the candidate DNA barcodes. Although the nLSU had high PCR and sequencing success rates (100% and 100% respectively), occasional overlapping occurred between the intra and inter specific variations. The PCR amplification and sequencing success rates of ITS were 100% and 85.7% respectively. ITS showed high sequence variation among species group and low variation within a given species. There was a relatively high PCR amplification and sequencing success rate for tef1 α (85.7% and 100% respectively), and its intra and inter specific variation was higher than that of ITS or nLSU. All three candidate markers showed hight species resolution. ITS and tef1 α had a more clearly defined barcode gap than nLSU. Our study showed that the tef1 α and nLSU can be proposed as supplementary barcodes for the genus Amanita, while ITS can be used as a primary barcode marker considering that the ITS region may become a universal barcode marker for the fungal kingdom.