Nucleotide sequence of the DdeI restriction-modification system and characterization of the methylase protein.

The DdeI restriction-modification system was previously cloned and has been maintained in E. coli on two separate and compatible plasmids (1). The nucleotide sequence of the endonuclease and methylase genes has now been determined; it predicts proteins of 240 amino acids, Mr = 27,808, and 415 amino acids, Mr = 47,081, respectively. Inspection of the DNA sequence shows that the 3' end of the methylase gene had been deleted during cloning. The clone containing the complete methylase gene was made and compared to that containing the truncated gene; only clones containing the truncated form support the endonuclease gene in E. coli. Bal-31 deletion studies show that methylase expression in the Dde clones is also dependent upon orientation of the gene with respect to pBR322. The truncated and complete forms of the methylase protein were purified and compared; the truncated form appears to be more stable and active in vitro. Finally, comparison of the deduced amino acid sequence of M. DdeI with that of other known cytosine methylases shows significant regions of homology.     

Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts.

The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.     

Parameters of Male and Female Recombination Influenced by the T-007 Second Chromosome in DROSOPHILA MELANOGASTER

The T-007 second chromosome line of Drosophila melanogaster, previously shown to contain genetic elements responsible for male recombination induction, appears to affect several parameters of recombination in females. In T-007 heterozygous females, the distribution of recombination (but not the total frequency) is changed from that observed in control females; relative increases are observed in the more proximal regions of the second, third and X chromosomes, while relative decreases are observed more distally. These changes are paralleled by altered coefficient of coincidence values and in an increased nondisjunction frequency of second chromosomes. The distribution of recombination in females is strikingly similar to that observed in males as measured along the second and third chromosomes, and the frequency of nondisjunction of the X and Y chromosomes is increased in T-007 heterozygous males. Based upon these results and responses to the effect of structurally rearranged heterologues (the "interchromosomal effect"), it is suggested that T-007 affects the preconditions for meiotic exchange in females. It is not yet known if elements responsible for these effects are the same elements responsible for the numerous other traits associated with the T-007 second chromosome.     

Determination of Wolbachia genome size by pulsed-field gel electrophoresis

Genome sizes of six different Wolbachia strains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases. Enzymes SmaI, ApaI, AscI, and FseI cleaved the studied Wolbachia strains at a small number of sites and were used for the determination of the genome sizes of wMelPop, wMel, and wMelCS (each 1.36 Mb), wRi (1.66 Mb), wBma (1.1 Mb), and wDim (0.95 Mb). The Wolbachia genomes studied were all much smaller than the genomes of free-living bacteria such as Escherichia coli (4.7 Mb), as is typical for obligate intracellular bacteria. There was considerable genome size variability among Wolbachia strains, especially between the more parasitic A group Wolbachia infections of insects and the mutualistic C and D group infections of nematodes. The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined. They also indicated that the Wolbachia genome is circular.     

556例多发伤患者的急诊救治临床分析

目的探讨多发伤患者的救治策略。方法回顾分析我科2000年1月至2008年5月急诊抢救的556例多发伤患者的临床资料。结果 16例患者经抢救无效死亡,死亡率2.88%;其余患者均经紧急抢救及行必要实验室检查,病情稳定,好转率达97.12%。平均抢救时间为(1.37±1.05)h。结论强化多发伤的急诊科早期救治,树立创伤急救"黄金1 h"观念,是提高多发伤患者生存率及降低死亡率的关键。     

Sporogenesis in Eusporangiate Ferns: II. Polyplastidic Meiosis in Ophioglossum (Ophioglossales)

Ophioglossum petiolatum . Unlike Angiopteris (Marattiales), which is monoplastidic, Ophioglossum undergoes polyplastidic meiosis like members of the fern-seed plant clade. The meiotic spindle is distinctly multipolar in origin and is consolidated into a bipolar spindle that is variously twisted and curved to accommodate the large number of chromosomes. Although a phragmoplast forms after first meiosis, no wall is deposited. Instead, an organelle band consisting of intermingled plastids and mitochondria is formed in the equatorial region between the dyad domains. Following second meiosis, a complex of phragmoplasts forms among sister and non-sister nuclei. Cell plates are deposited first between sister nuclei and then in the region of the organelle band resulting in a tetrad of spores each with a equal allotment of organelles. Received 30 January 2001/ Accepted in revised form 24 April 2001     

Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.     

Wolbachia Lipoprotein Stimulates Innate and Adaptive Immunity through Toll-like Receptors 2 and 6 to Induce Disease Manifestations of Filariasis

Wolbachia endosymbiotic bacteria have been implicated in the inflammatory pathogenesis of filariasis. Inflammation induced by Brugia malayi female worm extract (BMFE) is dependent on Toll-like receptors 2 and 6 (TLR2/6) with only a partial requirement for TLR1. Removal of Wolbachia, lipids, or proteins eliminates all inflammatory activity. Wolbachia bacteria contain the lipoprotein biosynthesis genes Ltg and LspA but not Lnt, suggesting Wolbachia proteins cannot be triacylated, accounting for recognition by TLR2/6. Lipoprotein databases revealed 3–11 potential lipoproteins from Wolbachia. Peptidoglycan-associated lipoprotein (PAL) and Type IV secretion system-VirB6 were consistently predicted, and B. malayi Wolbachia PAL (wBmPAL) was selected for functional characterization. Diacylated 20-mer peptides of wBmPAL (Diacyl Wolbachia lipopeptide (Diacyl WoLP)) showed a near identical TLR2/6 and TLR2/1 usage compared with BMFE and bound directly to TLR2. Diacyl WoLP induced systemic tumor necrosis factor-α and neutrophil-mediated keratitis in mice. Diacyl WoLP activated monocytes induce up-regulation of gp38 on human lymphatic endothelial cells and induced dendritic cell maturation and activation. Dendritic cells primed with BMFE generated a non-polarized Th1/Th2 CD4⁺ T cell profile, whereas priming with Wolbachia depleted extracts (following tetracycline treatment; BMFEtet) polarized to a Th2 profile that could be reversed by reconstitution with Diacyl WoLP. BMFE generated IgG1 and IgG2c antibody responses, whereas BMFEtet or inoculation of TLR2 or MyD88^−/− mice produced defective IgG2c responses. Thus, in addition to innate inflammatory activation, Wolbachia lipoproteins drive interferon-γ-dependent CD4⁺ T cell polarization and antibody switching.Human filariasis is a major neglected tropical disease. More than 150 million individuals are infected with the filarial worms responsible for lymphatic filariasis (LF)⁴ (Wuchereria bancrofti and Brugia malayi) and onchocerciasis (Onchocerca volvulus). Over 40 million suffer from disfiguring and incapacitating disease with an estimated 1.5 billion people at risk of infection, ranking filariasis as one of the major causes of global morbidity (1).A feature of filarial pathogenesis is a host inflammatory response provoked by the death of larvae and adult stages within parasitized tissues (2). All causative agents of LF and O. volvulus harbor an intracellular symbiotic bacterium, Wolbachia, and are reliant on this endosymbiont for embryogenesis, growth, and survival (3). Previous studies have determined that the inflammatory potential of B. malayi and O. volvulus is dependent on the presence of Wolbachia. For example, Wolbachia-containing filarial extracts induce activation and tolerance in murine macrophages (4, 5), activate human monocytes (6), and activate human and murine neutrophils (7, 8). In addition, O. volvulus and B. malayi extracts containing Wolbachia stimulate neutrophil recruitment to the corneal stroma and development of corneal haze in a murine model of ocular onchocerciasis, in contrast with an aposymbiotic filaria (9). Moreover, isolated Wolbachia from filaria or from insect cells can replicate these effects (8, 10). The activation of neutrophils results in further neutrophil recruitment leading to the disruption of normal corneal clarity and development of stromal haze (11).Activation and subsequent desensitization of macrophages by Wolbachia molecules has been shown to be dependent on TLR2 and the adaptor molecule MyD88 (5, 10). Further studies have established that Wolbachia-induced inflammation is dependent on TLR2 and TLR6 recognition and signaling through the MyD88/Mal pathway and are independent of TRIF and TRAM (12). However, Wolbachia ligands for TLR2/TLR6 have not been characterized. To address this, we used the TLR receptor recognition profile to identify TLR2/6 ligands in the Wolbachia genome. In this study, we demonstrate that Wolbachia-derived diacyl-lipoproteins are candidate stimulatory molecules required for TLR2/6 ligation and production of pro-inflammatory cytokine and chemokine responses. Furthermore, we show that a synthetic Wolbachia lipopeptide (Diacyl WoLP) induces TLR2/6-dependent corneal inflammation, and TLR2-dependent TNFα responses in filarial disease models and up-regulates surface markers of human lymphatic endothelium. Diacyl WoLP also induced activation and maturation of dendritic cells and generated type 1 CD4⁺ T cell and antibody responses to filarial antigens.