Mutant of Escherichia coli Tolerant to the Lysis Induced by Cephalexin

2,3-Diaminopropionate ammonia-lyase (DAPAL), which catalyzes α,β-elimination of 2,3-diaminopropionate regardless of its stereochemistry, was purified from Salmonella typhimurium. We cloned the Escherichia coli ygeX gene encoding a putative DAPAL and purified the gene product to homogeneity. The protein obtained contained pyridoxal 5′-phosphate and was composed of two identical subunits with a calculated molecular weight of 43,327. It catalyzed the α,β-elimination of both D- and L-2,3-diaminopropionate. The results confirmed that ygeX encoded DAPAL. The enzyme acted on D-serine, but its catalytic efficiency was only 0.5% that with D-2,3-diaminopropionate. The enzymologic properties of E. coli DAPAL resembled those of Salmonella DAPAL, except that L-serine, D- and L-β-Cl-alanine were inert as substrates of the enzyme from E. coli. DAPAL had significant sequence similarity with the catalytic domain of L-threonine dehydratase, which is a member of the fold-type II group of pyridoxal phosphate enzymes, together with D-serine dehydratase and mammalian serine racemase.     

聚乙二醇和金属离子诱导脂质体与细胞的融合

用荧光菜振能量转移技术检测ＰＥＧ和金属离子诱导脂质体和细胞的融合,发现有ＴＥＧ参与诱导时,虽然Ｃａ＾２＋对膜融合的促进作用仍专一地依赖于ＰＳ的存在,但其对ＰＳ的依赖性降低;Ｍｎ＾２＋促进含ＰＳ和ＰＥ的脂质体与细胞的融合,而Ｍｇ＾２＋无作用。以ＰＣ：ＣＬ：Ｃｈｏｌ为０．５：０．５：１的脂质体包埋天花粉蛋白,经ＰＥＧ诱导与骨髓瘤细胞ＳＰ２０融合,提高了天花粉蛋白对骨髓瘤细胞的杀伤力。     

Characterization of Cell Lysis in Pseudomonas putida Induced upon Expression of Heterologous Killing Genes

Active biological containment systems are based on the controlled expression of killing genes. These systems are of interest for the Pseudomonadaceae because of the potential applications of these microbes as bioremediation agents and biopesticides. The physiological effects that lead to cell death upon the induction of expression of two different heterologous killing genes in nonpathogenic Pseudomonas putida KT2440 derivatives have been analyzed. P. putida CMC4 and CMC12 carry in their chromosomes a fusion of the P_A1-04/03 promoter to the Escherichia coli gef gene and the X174 lysis gene E, respectively. Expression of the killing genes is controlled by the LacI protein, whose expression is initiated from the XylS-dependent Pm promoter. Under induced conditions, killing of P. putida CMC12 cells mediated by X174 lysis protein E was faster than that observed for P. putida CMC4, for which the Gef protein was the killing agent. In both cases, cell death occurred as a result of impaired respiration, altered membrane permeability, and the release of some cytoplasmic contents to the extracellular medium.     

Lysis of Yeast Cell Walls Induced by 2-Deoxyglucose at Their Sites of Glucan Synthesis

Six sites of 2-deoxyglucose (2DG)–induced lysis on three yeasts (Schizosaccharomyces pombe, Pichia farinosa, and Saccharomyces cerevisiae) coincided with the regions of growth of their glucan layers. Identification of the glucan layer as the site of lysis suggests a mechanism of attack by 2DG or by its derivatives. It is proposed that the glucan layer grows by addition of glucose into internal breaks of polysaccharide molecules. 2DG inhibited resynthesis (insertion of glucose) of the broken glycosidic linkage.     

pH降低和短杆菌肽D诱发的PE脂质体融合

 ｐＨ降低和短杆菌肽Ｄ诱发的ＰＥ脂质体融合王卓智,聂松青,林克椿（北京医科大学生物物理教研室,北京１０００８３）生物膜的融合是其在执行生理功能时的一种重要现象,如精子与卵子的融合,吞噬细胞形成吞噬体,出胞和入胞,以及病毒感染细胞等,都经历膜融合过程,此...     

Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

Cell lysis and molecular delivery in confluent monolayers of PtK₂ cells are achieved by the delivery of 6 ns, λ = 532 nm laser pulses via a 40×, 0.8 NA microscope objective. With increasing distance from the point of laser focus we find regions of (a) immediate cell lysis; (b) necrotic cells that detach during the fluorescence assays; (c) permeabilized cells sufficient to facilitate the uptake of small (3 kDa) FITC‐conjugated Dextran molecules in viable cells; and (d) unaffected, viable cells. The spatial extent of cell lysis, cell detachment, and molecular delivery increased with laser pulse energy. Hydrodynamic analysis from time‐resolved imaging studies reveal that the maximum wall shear stress associated with the pulsed laser microbeam‐induced cavitation bubble expansion governs the location and spatial extent of each of these regions independent of laser pulse energy. Specifically, cells exposed to maximum wall shear stresses τ_{w, max} > 190 ± 20 kPa are immediately lysed while cells exposed to τ_{w, max} > 18 ± 2 kPa are necrotic and subsequently detach. Cells exposed to τ_{w, max} in the range 8–18 kPa are viable and successfully optoporated with 3 kDa Dextran molecules. Cells exposed to τ_{w, max} < 8 ± 1 kPa remain viable without molecular delivery. These findings provide the first direct correlation between pulsed laser microbeam‐induced shear stresses and subsequent cellular outcome. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Enzymatic Kinetic Change of Ascorbate Oxidase Loaded into Liposomes Induced by Microwave Fields Exposure

Abstract

Enzymatic kinetic of enzyme Ascorbate Oxidase (AAO) loaded into liposomes has been studied during microwave (MW) fields irradiation at temperature of 25°C. DPPC:Chol (7:3 mole ratio) unilamellar vesicles of an average diameter of 110 nm were used. At the working frequency of 2.45 GHz, MW exposure at 2.8 mW/g and 5.6 mW/g Specific Absorption Rate (SAR) were investigated. At both SARs above cited, the free enzyme in solution did not show any effects induced by MW exposure. On the other hand, the enzyme loaded into liposomes exhibits a significant decrease of 13% (p<0.005, n=42) in the reaction velocity induced by MW exposure at 5.6 mW/g in comparison to control. At SAR of 2.8 mW/g no significant decrease was obtained.     

Single Particle Fluorescence Burst Analysis of Epsin Induced Membrane Fission

Vital cellular processes, from cell growth to synaptic transmission, rely on membrane-bounded carriers and vesicles to transport molecular cargo to and from specific intracellular compartments throughout the cell. Compartment-specific proteins are required for the final step, membrane fission, which releases the transport carrier from the intracellular compartment. The role of fission proteins, especially at intracellular locations and in non-neuronal cells, while informed by the dynamin-1 paradigm, remains to be resolved. In this study, we introduce a highly sensitive approach for the identification and analysis of membrane fission machinery, called burst analysis spectroscopy (BAS). BAS is a single particle, free-solution approach, well suited for quantitative measurements of membrane dynamics. Here, we use BAS to analyze membrane fission induced by the potent, fission-active ENTH domain of epsin. Using this method, we obtained temperature-dependent, time-resolved measurements of liposome size and concentration changes, even at sub-micromolar concentration of the epsin ENTH domain. We also uncovered, at 37°C, fission activity for the full-length epsin protein, supporting the argument that the membrane-fission activity observed with the ENTH domain represents a native function of the full-length epsin protein.     

Immunomagnetic purification of Colletotrichum lindemuthianum appressoria

We developed a method to purify appressoria of the bean anthracnose fungus Colletotrichum lindemuthianum for biochemical analysis of the cell surface and to compare appressoria with other fungal structures. We used immunomagnetic separation after incubation of infected bean leaf homogenates with a monoclonal antibody that binds strongly to the appressoria. Preparations with a purity of >90% could be obtained. Examination of the purified appressoria by transmission electron microscopy showed that most had lost their cytoplasm. However, the plasma membrane was retained, suggesting that there is some form of attachment of this membrane to the cell wall. The purified appressoria can be used for studies of their cell surface, and we have shown that there are clear differences in the glycoprotein constituents of cell walls of appressoria compared with mycelium.     

Immunomagnetic Purification of Colletotrichum lindemuthianum Appressoria

We developed a method to purify appressoria of the bean anthracnose fungus Colletotrichum lindemuthianum for biochemical analysis of the cell surface and to compare appressoria with other fungal structures. We used immunomagnetic separation after incubation of infected bean leaf homogenates with a monoclonal antibody that binds strongly to the appressoria. Preparations with a purity of >90% could be obtained. Examination of the purified appressoria by transmission electron microscopy showed that most had lost their cytoplasm. However, the plasma membrane was retained, suggesting that there is some form of attachment of this membrane to the cell wall. The purified appressoria can be used for studies of their cell surface, and we have shown that there are clear differences in the glycoprotein constituents of cell walls of appressoria compared with mycelium.     

Lysis of Micrococcus radiodurans

Two sulfur containing peptides have been isolated from a cationic peptide-rich fraction of dried Lentinus edodes by Chromatographic technic. They have been identified as γ-glutamylcystine and N,N-bis-³-glutamylcystinylglycine based on chemical, Chromatographic and spectroscopic properties.     

Immunomagnetic enrichment of interstitial cells of Cajal

Disruptions of networks of interstitial cells of Cajal (ICC), gastrointestinal pacemakers and mediators of neurotransmission, can lead to disordered phasic contractions and peristalsis by reducing and uncoupling electrical slow waves. However, detailed analysis of the ICC network behavior has been hampered by their scarcity, limited accessibility in intact tissues, and contamination with other cell types in culture. Our goal was to develop a simple technique to purify ICC from murine gastrointestinal muscles for functional studies. We identified ICC in live small intestinal muscles or primary cell cultures by Kit immunoreactivity using fluorescent antibodies. Because this technique also labels resident macrophages nonspecifically, parallel studies were performed in which nonfluorescent Kit antibodies and macrophages labeled with fluorescent dextran were used for subtractive analysis of ICC. In both groups, Kit-positive cells were tagged with superparamagnetic antibodies and sorted on magnetic columns. Efficacy was assessed by flow cytometry. ICC enrichment from primary cultures and freshly dissociated tissues was approximately 63-fold and approximately 8-fold, respectively. Unlike the cells derived directly from tissues, cells sorted from cultures frequently yielded extensive, nearly homogenous ICC networks on reseeding. Monitoring oscillations in mitochondrial Ca(2+) or membrane potential by imaging revealed spontaneous rhythmicity in these networks. Cells that did not bind to the columns yielded cultures that were depleted of ICC and dominated by smooth muscle cells. In conclusion, immunomagnetic sorting of primary cultures of ICC results in relatively homogenous, functional ICC networks. This technique is less suitable for obtaining ICC from freshly dispersed cells.     

Particle Simulation of Oxidation Induced Band 3 Clustering in Human Erythrocytes

Oxidative stress mediated clustering of membrane protein band 3 plays an essential role in the clearance of damaged and aged red blood cells (RBCs) from the circulation. While a number of previous experimental studies have observed changes in band 3 distribution after oxidative treatment, the details of how these clusters are formed and how their properties change under different conditions have remained poorly understood. To address these issues, a framework that enables the simultaneous monitoring of the temporal and spatial changes following oxidation is needed. In this study, we established a novel simulation strategy that incorporates deterministic and stochastic reactions with particle reaction-diffusion processes, to model band 3 cluster formation at single molecule resolution. By integrating a kinetic model of RBC antioxidant metabolism with a model of band 3 diffusion, we developed a model that reproduces the time-dependent changes of glutathione and clustered band 3 levels, as well as band 3 distribution during oxidative treatment, observed in prior studies. We predicted that cluster formation is largely dependent on fast reverse reaction rates, strong affinity between clustering molecules, and irreversible hemichrome binding. We further predicted that under repeated oxidative perturbations, clusters tended to progressively grow and shift towards an irreversible state. Application of our model to simulate oxidation in RBCs with cytoskeletal deficiency also suggested that oxidation leads to more enhanced clustering compared to healthy RBCs. Taken together, our model enables the prediction of band 3 spatio-temporal profiles under various situations, thus providing valuable insights to potentially aid understanding mechanisms for removing senescent and premature RBCs.     

Uptake of Archaeobacterial Liposomes and Conventional Liposomes by Phagocytic Cells

Abstract

Liposomes in the 200 nm size range were prepared from the ether lipids extracted from various Archaeobacteria (coined archaeosomes), and from conventional lipids. The entrapment of peroxidase or carboxyfluorescein was used to compare the in vitro uptake of various liposomes by murine peritoneal macrophages, J774A.1 macrophages and several non phagocytic cell lines. While liposomes composed of ester lipids dipalmitoylphosphatidylcholine, or dimyristoylphosphatidylcholine: dimyristoylphosphatidylglycerol: cholesterol (1.8:0.2:1.5, molar ratio) were taken up by macrophage species, the uptake of archaeosomes was 3 to 53 times greater. Uptake by non phagocytic HEp-2, HeLa, and EJ/28 cells was considerably less. Evidence from time-course studies using cytochalasins B+D, sub-optimal temperature or formaldehyde treatments of macrophages, indicated that the archaeosomes lost structural integrity following internalization within the J774A. 1 phagocytic cells. No cytotoxicity was observed in viability or growth assays with J774A. 1 cells, using high doses of three representative types of archaeosomes and one type of conventional-liposome. Therefore, archaeosomes may be well suited to applications where phagocytic cells are a target site.     

Lysis of erythrocytes byTrichomonas vaginalis

The in vitro hemolytic activity of 4 isolates ofTrichomonas vaginalis was investigated. Repetitive hemolysis assays of any one isolate showed cyclical fluctuations in hemolytic activity, varying over 24 hr of continuous culture. Maximal hemolytic activity was detected using trichomonads in the lag phase of the growth cycle. Investigations showed that hemolysis was a contact-dependent phenomenon and microscopic investigation of samples showed a significant correlation between hemolysis and attachment of erythrocytes to the trichomonad surface. Quantitative data from cytoadherence assays using [⁵¹Cr]-labeled erythrocytes were consistent with these observations. It is suggested that hemolytic activity is dependent upon adherence of red blood cells to the surface ofT. vaginalis.     

Immunomagnetic isolation of cells for serological BoLA typing

This paper describes a totally new immunomagnetic (IM) technique adapted to serological BoLA typing. The basic technique has recently been developed by Vartdal et al. (1986) for serological HLA typing. The main advantage is that bovine mononuclear cells (e.g. T-cells and possibly their subsets, B-cells and monocytes) can be quickly and specifically isolated with high yield and viability from whole blood in a one-step procedure. This is achieved by magnetic separation of rosettes formed between the cells and superparamagnetic monosized polystyrene microspheres (Dynabeads TM) coated with cross-species reactive monoclonal antibodies (MAbs) specific for various human T-cell antigens or for HLA class II monomorphic epitopes. The cells are isolated within 5 min after a 5-min incubation at 4 degrees C. Magnetic separation of rosettes with a strong cobalt-samarium magnet eliminates all the laborious centrifugation steps necessary with conventional procedures. The isolated cells, still attached to the particles, are available for microcytotoxic assay. This is carried out within 55 min, including a two-step application of alloantiserum and complement and addition of acridine orange/ethidium bromide for the staining of viable (green) and dead (red) cells. The high viability of isolated cells gives a very low background kill compared with the conventional cytotoxic assay. The IM typing technique is also superior in sensitivity to the conventional technique as standardized for the international BoLA comparison test. The IM technique is likely to have its greatest impact on class II typing; class II positive cells being separated very efficiently. Polymorphic HLA class II MAbs detected likely polymorphic BoLA class II epitopes.     

Immunomagnetic isolation of Xanthomonas campestris pv. pelargonii

Immunomagnetic fishing was developed as an improved procedure for increasing the bacterial target to non-target recovery ratio in suspensions containing mixtures of target and non-target organisms. A cell suspension containing the target Xanthomonas campestris pv. pelargonii and non-target organisms, is treated with rabbit polyclonal antiserum against X.c. pv. pelargonii and incubated for 1 h. The suspension is then mixed with paramagnetic iron oxide particles coated with goat anti-rabbit antibodies (immunomagnetic particles). After incubation, the polished surface of a 14 mm diameter neodymium supermagnet is placed at the air-water interace and the magnetic particles are attracted to the magnet. After all visible magnetic particles have attached to the bottom of the magnet, the magnet is dipped in sterile buffer to remove non-target organisms. The magnet with attached magnetic particles is rubbed evenly over an agar surface to dislodge the particles and attached bacteria. Conventional immunomagnetic isolation (immunomagnetic attraction) and immunomagnetic fishing were compared, for the recovery of the target organism in geranium leaf washings spiked with X.c. pv. pelargonii. With immunomagnetic attraction and immunomagnetic fishing, bacterial non-target organisms were reduced to 11.4 and 1.5% of the initial population, respectively, whereas the target was only reduced to 63.7 and 53.8%.