The DNA translocating ATPase of bacteriophage T4 packaging motor

In double-stranded DNA bacteriophages the viral DNA is translocated into an empty prohead shell by a powerful ATP-driven motor assembled at the unique portal vertex. Terminases consisting of two to three packaging-related ATPase sites are central to the packaging mechanism. But the nature of the key translocating ATPase, stoichiometry of packaging motor, and basic mechanism of DNA encapsidation are poorly understood. A defined phage T4 packaging system consisting of only two components, proheads and large terminase protein (gp17; 70 kDa), is constructed. Using the large expanded prohead, this system packages any linear double-stranded DNA, including the 171 kb T4 DNA. The small terminase protein, gp16 (18 kDa), is not only not required but also strongly inhibitory. An ATPase activity is stimulated when proheads, gp17, and DNA are actively engaged in the DNA packaging mode. No packaging ATPase was stimulated by the N-terminal gp17-ATPase mutants, K166G (Walker A), D255E (Walker B), E256Q (catalytic carboxylate), D255E-E256D and D255E-E256Q (Walker B and catalytic carboxylate), nor could these sponsor DNA encapsidation. Experiments with the two gp17 domains, N-terminal ATPase domain and C-terminal nuclease domain, suggest that terminase association with the prohead portal and communication between the domains are essential for ATPase stimulation. These data for the first time established an energetic linkage between packaging stimulation of N-terminal ATPase and DNA translocation. A core pathway for the assembly of functional DNA translocating motor is proposed. Since the catalytic motifs of the N-terminal ATPase are highly conserved among >200 large terminase sequences analyzed, these may represent common themes in phage and herpes viral DNA translocation.     

噬菌体Qβ病毒样颗粒的制备、纯化及鉴定

目的: 利用大肠杆菌原核表达系统制备噬菌体Qβ VLPs,验证其自组装能力,纯化后免疫动物以确定其免疫原性,同时制备兔多克隆抗体验证其在哺乳动物细胞中内化能力。方法: 合成pET-28-Qβ-CP质粒,利用大肠杆菌原核表达系统制备Qβ VLPs,通过蔗糖密度梯度离心纯化VLPs,将经凝胶层析柱Sephacryl S-400纯化的Qβ VLPs用透射电镜观察颗粒形态;纯化后的Qβ VLPs加入或不加入佐剂分别免疫新西兰兔,获得血清后用Protein G纯化得到兔多克隆抗体,通过Western blot确定制备的兔多克隆抗体特异性,并利用间接免疫荧光法对Qβ VLPs的细胞内化能力进行鉴定。结果: 制备并获得纯度较高的Qβ VLPs,通过透射电镜观察到大量直径约为28 nm的颗粒;Western blot结果表明制备的兔多克隆抗体能特异性识别Qβ VLPs,且在免疫实验中加入佐剂与不加入佐剂分别免疫动物,对抗体水平的影响不显著。间接免疫荧光法结果表明Qβ VLPs在哺乳动物细胞中具有内化能力。结论: 成功制备Qβ VLPs为后续研发以噬菌体Qβ VLPs为载体的相关疫苗奠定基础。     

Localization of vanadium-containing particles in the lungs of uranium/vanadium miners

Several geological formations of the Utah-Colorado mining region mined for uranium ore during and after World War II had been mined earlier for vanadium. Therefore, most miners and millers from that region were exposed to those metals’ ores or tailings at one time or another. Preliminary investigation to determine uranium and vanadium retained in the lungs of a former uranium miner and miller from this region, who died of lung cancer (mesothelioma), showed a high nonuniform distribution of vanadium. This observation led to the hypothesis that the vanadium content in the lungs could be associated with inhaled particles. Further examination of spectra of characteristic X-rays obtained by scanning particle-induced X-ray emission (microPIXE) of an autopsy sample of this lung indicated that vanadium was indeed present in localized sites within the 20-μm spatial resolution of the proton beam. This work points out that the microPIXE-RBS (Rutherford backscattering) test for vanadium can be used for site localization of inhaled particles retained in the lungs. Further studies are in progress to: (i) locate uranium-bearing particles in lung tissues of former uranium miners and millers; and (ii) evaluate the local doses of alpha radiation received from these particles.     

Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2

The high level of immunogenicity of peptides displayed in dense repetitive arrays on virus-like particles makes recombinant VLPs promising vaccine carriers. Here, we describe a platform for vaccine development based on the VLPs of RNA bacteriophage MS2. It serves for the engineered display of specific peptide sequences, but will also allow the construction of random peptide libraries from which specific binding activities can be recovered by affinity selection. Peptides representing the V3 loop of HIV gp120 and the ECL2 loop of the HIV coreceptor, CCR5, were inserted into a surface loop of MS2 coat protein. Both insertions disrupted coat VLP assembly, apparently by interfering with protein folding, but these defects were suppressed efficiently by genetically fusing coat protein's two identical polypeptides into a single-chain dimer. The resulting VLPs displayed the V3 and ECL2 peptides on their surfaces where they showed the potent immunogenicity that is the hallmark of VLP-displayed antigens. Experiments with random-sequence peptide libraries show the single-chain dimer to be highly tolerant of six, eight and ten amino acid insertions. MS2 VLPs support the display of a wide diversity of peptides in a highly immunogenic format, and they encapsidate the mRNAs that direct their synthesis, thus establishing the genotype/phenotype linkage necessary for recovery of affinity-selected sequences. The single-chain MS2 VLP therefore unites in a single structural platform the selective power of phage display with the high immunogenicity of VLPs.     

Development and application of thermo-sensitive immunomicrospheres for antibody purification

The latex particles composed of poly(styrene/N-isopropylacrylamide/glycidyl methacrylate) [P(St/NIPAM/GMA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] were prepared by emulsifier-free emulsion polymerization. These latex particles with submicrometer size showed the thermosensitivity originated from the thermo-sensitive nature of NIPAM. That is, the minimum NaCI concentration for flocculation of these latex particles [critical flocculation concentration (CFC)] decreased significantly with increasing temperature and reached constant values at above the critical temperature [critical flocculation temperature (CFT)]. At a certain NaCl concentration, the thermo-sensitive latex particles were flocculated by raising temperature, and conversely, the flocculated thermo-sensitive latex particles were completely dispersed by lowering temperature. Bovine serum albumin (BSA) was covalently immobilized onto the P(St/NIPAM/GMA) and P(St/NIPAM/MMA) latex particles with high efficiency. The BSA-immobilized P(St/NIPAM/GMA) and P(St/NIPAM/MAA) latex particles (immunomicrospheres) showed the similar dependencies of CFC on temperature to the bare latex particles. These thermo-sensitive immunomicrospheres were successfully used for the immunoaffinity purification of anti-BSA antibodies from antiserum. (c) 1994 John Wiley & Sons, Inc.     

Distinct Mutants of Retrograde Intraflagellar Transport (IFT) Share Similar Morphological and Molecular Defects

A microtubule-based transport of protein complexes, which is bidirectional and occurs between the space surrounding the basal bodies and the distal part of Chlamydomonas flagella, is referred to as intraflagellar transport (IFT). The IFT involves molecular motors and particles that consist of 17S protein complexes. To identify the function of different components of the IFT machinery, we isolated and characterized four temperature-sensitive (ts) mutants of flagellar assembly that represent the loci FLA15, FLA16, and FLA17. These mutants were selected among other ts mutants of flagellar assembly because they displayed a characteristic bulge of the flagellar membrane as a nonconditional phenotype. Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images. Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A. The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.     

Specific arrangements of membrane particles at sites of exo-endocytosis in the freeze-etched neurohypophysis

Summary Images have been obtained from freeze-etch replicas of neurohypophyses which are consistent with the view that orderly arranged aggregates of membrane particles occur in regions where fragments of membrane are being added to and taken away from the plasma membrane during secretion. Aggregates of particles included rosette-like and necklace-like patterns similar to those described by other authors at sites of exocytosis and endocytosis.Dedicated to Prof. W. Bargmann at the occasion of his 70th birthday. A short account has been presented at the 8th International Congress of Electron Microscopy (Dreifuss, Akert, Sandri and Moor, 1974).This work was supported by grants from the Swiss National Foundation for Scientific Research Nos. 3.257.74, 3.368.0.74, 3.774.72 and 3.045.73; the Hartmann-Müller Foundation for Medical Research in Zürich and the Dr. Eric Slack-Gyr Stiftung.     

Immunoregulatory role of intestinal surfactant-like particles during Salmonella typhimurium infection

Surfactants like particles (SLP) are secreted by Intestinal epithelium. These particles have the ability to lower surface tension of intestinal epithelial cells and contain small amounts of surfactant specific proteins A, B, and D. In the intestinal lumen they are known to function as lubricants and/or as a vehicle to deliver digestive enzymes to the luminal fluid. These particles have been found to have the ability in binding of uropathogenic E.coli. But their immunological function is not known. The present study was designed to assess the role of the SLP in the regulation of immune response during Salmonella (S) typhimurium infection using a rat an enteric model. The animals were divided in four different groups including control (PBS), rats fed fat diet (corn oil), rats fed fat diet followed with S. typhimurium infection and rats with S. typhimurium infection alone. The Peyer's patches (PP), intraepithelial (IE) and lamina propria (LP) mononuclear cells were isolated from the above-mentioned groups. These mononuclear cells were then incubated in presence of S. typhimurium lysate alone, SLP alone and S. typhimurium lysate and SLP together. T cell markers CD4 and CD8, cytokines mainly pro-inflammatory ones including IFN-gamma, TNF-alpha, IL-12 etc were studied under such conditions. In addition histological studies were also carried out under these conditions. We report in this study that SLP plays an important role in modulating the cytokine level during infection. The pro-inflammatory cytokines were found significantly reduced in SLP induced diet along with the infection group compared to the infection group alone. Histopathological studies revealed the breakdown of duodenal villi after infection while only broadening of villi was observed in rats given corn oil induced SLP along with infection. These results suggested an important immuno-modulatory role for SLP during Salmonella infection.     

形貌调控与镀层修饰结合制备口服疫苗载体*

口服疫苗因其具有接种方便、能产生黏膜免疫等优点而备受关注,但胃肠道屏障、酸性环境和蛋白酶等不利条件制约了口服疫苗免疫效果的发挥。为提升其免疫效果,将形貌调控与镀层修饰策略结合制备新型口服疫苗载体,具体为将溶剂蒸发法与快速膜乳化法结合制备聚乳酸-羟基乙酸共聚物(PLGA)杆状颗粒,并采用能够增强免疫反应的β-葡聚糖及具有更高降解pH的硫醇化修饰的羟丙基甲基纤维素苯二甲酸酯(T-HPMCP)对PLGA杆状颗粒镀层修饰。在制备PLGA杆状颗粒时,通过对外水相条件的摸索制备出了适合小肠上皮细胞摄取的长度在2~4 μm、宽度在1~2 μm的PLGA杆状颗粒。体外实验结果表明通过T-HPMCP修饰的疫苗载体在酸性环境下保持稳定有利于抗原活性保护,同时能够在pH≥7.4时分解而使抗原释放。细胞和动物实验结果表明其特殊的杆状形貌可实现较高的肠道上皮摄取速率及转运效率,并且β-葡聚糖的修饰能活化树突状细胞(DC),提升OVA特异性IgA和IgG抗体水平。综上,制备的镀层PLGA杆状颗粒作为口服疫苗载体可提升机体免疫应答,为口服疫苗的研究提供了新的材料和思路。     

Analysis of glycoproteins in human serum by means of glycospecific magnetic bead separation and LC-MALDI-TOF/TOF analysis with automated glycopeptide detection.

Comprehensive proteomic analyses require efficient and selective pre-fractionation to facilitate analysis of post-translationally modified peptides and proteins, and automated analysis workflows enabling the detection, identification, and structural characterization of the corresponding peptide modifications. Human serum contains a high number of glycoproteins, comprising several orders of magnitude in concentration. Thereby, isolation and subsequent identification of low-abundant glycoproteins from serum is a challenging task. selective capturing of glycopeptides and -proteins was attained by means of magnetic particles specifically functionalized with lectins or boronic acids that bind to various structural motifs. Human serum was incubated with differentially functionalized magnetic micro-particles (lectins or boronic acids), and isolated proteins were digested with trypsin. Subsequently, the resulting complex mixture of peptides and glycopeptides was subjected to LC-MALDI analysis and database searching. In parallel, a second magnetic bead capturing was performed on the peptide level to separate and analyze by LC-MALDI intact glycopeptides, both peptide sequence and glycan structure. Detection of glycopeptides was achieved by means of a software algorithm that allows extraction and characterization of potential glycopeptide candidates from large LC-MALDI-MS/MS data sets, based on N-glycopeptide-specific fragmentation patterns and characteristic fragment mass peaks, respectively. By means of fast and simple glycospecific capturing applied in conjunction with extensive LC-MALDI-MS/MS analysis and novel data analysis tools, a high number of low-abundant proteins were identified, comprising known or predicted glycosylation sites. According to the specific binding preferences of the different types of beads, complementary results were obtained from the experiments using either magnetic ConA-, LCA-, WGA-, and boronic acid beads, respectively.     

Alteration of negatively charged residues in the 89 to 99 domain of apoA-I affects lipid homeostasis and maturation of HDL

In this study, we investigated the role of positively and negatively charged amino acids within the 89-99 region of apolipoprotein A-I (apoA-I), which are highly conserved in mammals, on plasma lipid homeostasis and the biogenesis of HDL. We previously showed that deletion of the 89-99 region of apoA-I increased plasma cholesterol and phospholipids, but it did not affect plasma triglycerides. Functional studies using adenovirus-mediated gene transfer of two apoA-I mutants in apoA-I-deficient mice showed that apoA-I[D89A/E91A/E92A] increased plasma cholesterol and caused severe hypertriglyceridemia. HDL levels were reduced, and approximately 40% of the apoA-I was distributed in VLDL/IDL. The HDL consisted of mostly spherical and a few discoidal particles and contained preβ1 and α4-HDL subpopulations. The lipid, lipoprotein, and HDL profiles generated by the apoA-I[K94A/K96A] mutant were similar to those of wild-type (WT) apoA-I. Coexpression of apoA-I[D89A/E91A/E92A] and human lipoprotein lipase abolished hypertriglyceridemia, restored in part the α1,2,3,4 HDL subpopulations, and redistributed apoA-I in the HDL2/HDL3 regions, but it did not prevent the formation of discoidal HDL particles. Physicochemical studies showed that the apoA-I[D89A/E91A/E92A] mutant had reduced α-helical content and effective enthalpy of thermal denaturation, increased exposure of hydrophobic surfaces, and increased affinity for triglyceride-rich emulsions. We conclude that residues D89, E91, and E92 of apoA-I are important for plasma cholesterol and triglyceride homeostasis as well as for the maturation of HDL.     

The size distribution of primary biological aerosol particles in the multiphase atmosphere

Primary biological aerosol particles are a ubiquitouscomponent of the atmospheric aerosol and have greatimportance within the whole atmosphere. Besides theireffect on air hygiene (i.e. causing allergic diseases), theycontribute to cloud and rain development. They amountto almost 25% of the total number of aerosolparticles both in dry air and in cloudwater. They showno seasonal variation in concentration but incomposition.     

The use of coated paramagnetic particles as a physical label in a magneto-immunoassay

An ideal label for use in an immunoassay would require no further chemical or electromagnetic stimulation prior to its detection and would be free from interference from the sample matrix. Micron sized paramagnetic particles are able to perturb magnetic fields. This perturbation can be directly detected using a suitable electronic device and is independent of the sample matrix. In this study coated paramagnetic particles were used as a physical label in a non-competitive solid phase ‘sandwich’ assay for the detection of human transferrin. The transferrin acted as a ‘biological bridge’ allowing a dose dependant immobilization of the paramagnetic particles to a polyethylene terephthalate solid phase. Quantitation of the paramagnetic label was achieved using an electronic detection system allowing a linear dose response with a femtomolar detection limit (260 fmol).