Novel structure of the conserved gram-negative lipopolysaccharide transport protein A and mutagenesis analysis

Lipopolysaccharide (LPS) transport protein A (LptA) is an essential periplasmic localized transport protein that has been implicated together with MsbA, LptB, and the Imp/RlpB complex in LPS transport from the inner membrane to the outer membrane, thereby contributing to building the cell envelope in Gram-negative bacteria and maintaining its integrity. Here we present the first crystal structures of processed Escherichia coli LptA in two crystal forms, one with two molecules in the asymmetric unit and the other with eight. In both crystal forms, severe anisotropic diffraction was corrected, which facilitated model building and structural refinement. The eight-molecule form of LptA is induced when LPS or Ra-LPS (a rough chemotype of LPS) is included during crystallization. The unique LptA structure represents a novel fold, consisting of 16 consecutive antiparallel β-strands, folded to resemble a slightly twisted β-jellyroll. Each LptA molecule interacts with an adjacent LptA molecule in a head-to-tail fashion to resemble long fibers. Site-directed mutagenesis of conserved residues located within a cluster that delineate the N-terminal β-strands of LptA does not impair the function of the protein, although their overexpression appears more detrimental to LPS transport compared with wild-type LptA. Moreover, altered expression of both wild-type and mutated proteins interfered with normal LPS transport as witnessed by the production of an anomalous form of LPS. Structural analysis suggests that head-to-tail stacking of LptA molecules could be destabilized by the mutation, thereby potentially contributing to impair LPS transport.     

Apolipoprotein C-II amyloid fibrils assemble via a reversible pathway that includes fibril breaking and rejoining

Alzheimer's and several other diseases are characterized by the misfolding and assembly of protein subunits into amyloid fibrils. Current models propose that amyloid fibril formation proceeds via the self-association of several monomers to form a nucleus, which then elongates by the addition of monomer to form mature fibrils. We have examined the concentration-dependent kinetics of apolipoprotein C-II amyloid fibril formation and correlated this with the final size distribution of the fibrils determined by sedimentation velocity experiments. In contrast to predictions of the nucleation-elongation model, the final size distribution of the fibrils was found to be relatively independent of the starting monomer concentration. To explain these results, we extended the nucleation-elongation model to include fibril breaking and rejoining as integral parts of the amyloid fibril assembly mechanism. The system was examined under conditions that affected the stability of the mature fibrils including the effect of dilution on the free pool of monomeric apolipoprotein C-II and the time-dependent recovery of fibril size following sonication. Antibody-labelling transmission electron microscopy studies provided direct evidence for spontaneous fibril breaking and rejoining. These studies establish the importance of breaking and rejoining in amyloid fibril formation and identify prospective new therapeutic targets in the assembly pathway.     

活体生物发光与荧光成像技术在干细胞研究中的应用

活体生物发光与荧光成像技术是近年发展起来的新兴技术,以其操作简便、灵敏度高、创伤性小在生命科学研究中有着较大的优势,目前已被广泛应用于基因标记、细胞凋亡、免疫细胞研究、肿瘤转移等诸多领域,尤其在新兴的干细胞研究方面更是发挥着不可替代的作用。综述了活体生物发光与荧光成像技术的原理、优势、应用范围及发展前景,特别对近年来该技术在胚胎干细胞的肝向分化、人造血干细胞重建小鼠造血系统、神经祖细胞治疗中枢神经系统肿瘤等方面的应用做了详细介绍。     

Optical imaging of nociception in primary somatosensory cortex of non-human primates

While the activation of primary somatosensory (SI) cortex during pain perception is consistently reported in functional imaging studies on normal subjects and chronic pain patients, the specific roles of SI, particularly the subregions within SI, in the processing of sensory aspects of pain are still largely unknown. Using optical imaging of intrinsic signal (OIS) and single unit electrophysiology, we studied cortical activation patterns within SI cortex (among Brodmann areas 3a, 3b and 1) and signal amplitude changes to various intensities of non-nociceptive, thermal nociceptive and mechanical nociceptive stimulation of individual distal finerpads in anesthetized squirrel monkeys. We have demonstrated that areas 3a and 1 are preferentially involved in the processing of nociceptive information while areas 3b and 1 are preferentially activated in the processing of non-nociceptive (touch) information. Nociceptive activations of individual fingerpad were organized topographically suggesting that nociceptive topographic map exits in areas 3a and 1. Signal amplitude was enhanced to increasing intensity of mechanical nociceptive stimuli in areas 3a, 3b and 1. Within area 1, nociceptive response co-localizes with the non-nociceptive response. Therefore, we hypothesize that nocicepitve information is area-specifically represented within SI cortex, in which nociceptive inputs are preferentially represented in areas 3a and 1 while non-nociceptive inputs are preferentially represented in areas 3b and 1.     

分子与细胞事件的光学可视化——解读2008年诺贝尔化学奖

绿色荧光蛋白（green fluorescent protein,GFP）及其突变体作为报告基因,已被广泛应用于基因表达调控、蛋白质空间定位、生物分子之间相互作用、转基因动物以及药物药效评价和作用机理研究等方面,极大地推动了现代生物学的发展．随着光电信息技术的不断进步,基于荧光报告基因的光学分子成像技术将在细胞、细胞网络、组织、器官和个体等不同层次实现分子与细胞事件的实时可视化,从而在重大疾病的早期诊断和药物研发中发挥重要作用．     

Anisakis simplex and Kudoa sp.: evaluation of specific antibodies in appendectomized patients

High prevalence and intensity of infection with anisakid larvae has been reported in commercially important fish in Spain. Likewise, Kudoa-infected fish have lately been detected in both fresh and frozen fish. In the present study the possible relation between appendectomy and specific antibodies to these fish parasites was investigated. One hundred and sixty patients were enrolled in this study. They were divided into two groups of eighty patients each and matched for sex and age: Group 1 (appendectomized) and Group 2 (control group). Total immunoglobulins (Ig’s), IgG, IgM, IgA and IgE against Anisakissimplex or Kudoa sp. antigens were analysed by ELISA. The mean values of the specific antibodies were lower in the appendectomy group, although significant differences were not observed in the case of IgG, IgA and IgE anti-A. simplex and IgE anti-Kudoa sp. In summary, appendectomy significantly decreased serum specific immunoglobulin levels against these food borne parasite antigens. This decrease was detectable from three months to three years post-appendectomy. It is necessary to study the influence of the surgical removal of other important parts of the GALT on these anti-parasite humoral immune responses.     

Effects of an optical brightener on the development, body weight and sex ratio of Spodoptera frugiperda (Lepidoptera: Noctuidae)

The incorporation of stilbene-derived optical brighteners into baculovirus formulations can substantially enhance virus infectivity and persistence of inoculum in the field. We evaluated the effect of the optical brightener Tinopal UNPA-GX (Sigma Chemical Co.) on the weight, development time, adult emergence and sex ratio of the principal pest of maize in the Americas, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae). Tinopal UNPA-GX was incorporated into semi-synthetic diet at a final concentration of 0.1 or 0.05% (w/v) and larvae of S. frugiperda were individually reared on the Tinopal-treated or an untreated control diet. In both Tinopal treatments, the weights of larvae and pupae were significantly reduced by up to 36 and 13%, respectively. The duration of larval development was increased by up to 30% compared to the controls. Adult emergence was high in all treatments (≈90%) and was not affected by the presence of Tinopal in the diet. Significant differences in the proportion of adult females were observed in the 0.05% Tinopal treatment but not in the control or 0.1% Tinopal treatments. By affecting the integrity of the peritrophic membrane and the rate of sloughing of intestinal epithelial cells, natural pest populations exposed to optical brightener residues from bioinsecticide applications are likely to suffer a reduction in growth rate and other correlates of insect fitness.     

Interplay of temperature,thermal‐stresses and strains in laser‐assisted modification of collagenous tissues: Speckle‐contrast and OCT‐based studies

Moderate heating of collagenous tissues such as cartilage and cornea by infrared laser irradiation can produce biologically nondestructive structural rearrangements and relaxation of internal stresses resulting in the tissue reshaping. The reshaping results and eventual changes in optical and biological properties of the tissue strongly depend on the laser‐irradiation regime. Here, a speckle‐contrast technique based on monochromatic illumination of the tissue in combination with strain mapping by means of optical coherence elastography (OCE) is applied to reveal the interplay between the temperature and thermal stress fields producing tissue modifications. The speckle‐based technique ensured en face visualization of cross correlation and contrast of speckle images, with evolving proportions between contributions of temperature increase and thermal‐stresses determined by temperature gradients. The speckle‐technique findings are corroborated by quantitative OCE‐based depth‐resolved imaging of irradiation‐induced strain‐evolution. The revealed relationships can be used for real‐time control of the reshaping procedures (e.g., for laser shaping of cartilaginous implants in otolaryngology and maxillofacial surgery) and optimization of the laser‐irradiation regimes to ensure the desired reshaping using lower and biologically safer temperatures. The figure of waterfall OCE‐image demonstrates how the strain‐rate maximum arising in the heating‐beam center gradually splits and drifts towards the zones of maximal thermal stresses located at the temperature‐profile slopes.