Amphipathic Helical Cationic Antimicrobial Peptides Promote Rapid Formation of Crystalline States in the Presence of Phosphatidylglycerol: Lipid Clustering in Anionic Membranes

Five AHCAPs exhibiting a broad-spectrum of antimicrobial activity, were examined with regard to their action in lipid mixtures with two anionic lipids, PG and CL. We find that all of the peptides studied were capable of promoting the formation of crystalline phases of DMPG in mixtures of DMPG and CL, without prior incubation at low temperatures. This property is indicative of the ability of these peptides to cluster CL away from DMPG. In contrast, the well studied antimicrobial cationic peptide magainin 2 does not cluster anionic lipids. We ascribe the lower anionic lipid clustering ability of magainin to its low density of positive charges compared with the five other AHCAPs used in this work. The peptide MSI-1254 was particularly potent in segregating these two anionic lipids. Consequently, clusters enriched in DMPG appear in a lipid mixture with CL. These can rapidly form higher temperature crystalline phases because of the increased permeability of the bilayer caused by the AHCAPs. The polyaminoacids, poly-L-Lysine and poly-l-arginine are also very effective in causing this segregation. Thus, the clustering of anionic lipids by AHCAPs is not confined only to mixtures of anionic with zwitterionic lipids, but it extends to mixtures containing different anionic headgroups. The resulting effects, however, have different consequences to the biological activity. This finding broadens the scope for which an AHCAP agent will cluster lipids in a membrane.     

The comet assay for DNA damage and repair

The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.     

Quantitative determination of curcuminoids in Curcuma rhizomes and rapid differentiation of Curcuma domestica Val. and Curcuma xanthorrhiza Roxb. by capillary electrophoresis

The three major curcuminoids, curcumin, demethoxycurcumin and bis-demethoxycurcumin, from Curcuma domestica Val. (Curcuma longa L.) and Curcuma xanthorrhiza Roxb. (Zingiberaceae) were fully separated and quantified in less than 5 min using a capillary zone electrophoresis method with standard fused-silica capillaries and photodiode array detection. An electrolyte solution of 20 mM phosphate, 50 mM sodium hydroxide and 14 mM beta-cyclodextrin was found to be appropriate. Quantification was performed with 3,4-dimethoxy-trans-cinnamic acid as internal standard, and the limit of detection was 0.01 mg/mL. Extraction, stabilisation during sample storage and quantification procedures were optimised and carried out with drugs and commercial curry powder from different provenances. The results were compared with the photometric method of the monograph Curcumae xanthorrhizae rhizoma of the European Pharmacopoeia.     

Differentiation-related proteins of the broad bean rust fungus Uromyces viciae-fabae,as revealed by high resolution two-dimensional polyacrylamide gel electrophoresis

On artificial polyethylene membranes providing a thigmotropic signal, uredospores of the broad bean rust fungus Uromyces viciae-fabae differentiated a series of infection structures which in nature are necessary to invade the host tissue through the stomata. Within 24 h germ tubes, appressoria, substomatal vesicles, infection hyphae and haustorial mother cells were developed successively. Alterations in protein metabolism during infection structure differentiation of this obligate plant pathogen were analyzed in the absence of the host plant by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver staining. The norm pattern representing the 2-DE protein patterns of the whole developmental sequence of infection structures of U. viciae-fabae showed 733 spots. During infection structure differentiation 55 proteins were newly formed, altered in quantity, or disappeared. Major alterations in the protein pattern occurred during uredospore germination and when infection hyphae were formed. Uredospore germination was characterized by a decrease of acidic proteins and an increase mainly of proteins with isoelectric points ranging from weakly acidic to basic.Abbreviations 2-DE two-dimensional polyacrylamide gel electrophoresis - DAPI 4,6-diamino-phenylindol - kDa kilo Dalton - pl isoelectric point - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

凝胶过滤与镍柱亲和纯化金葡菌a-溶血素重组蛋白的生物学特性比较

以在宿主菌株BL21(DE3)中成功表达的重组金黄色葡萄球菌a-溶血素蛋白为研究对象, 分析比较通过凝胶过滤层析(Gel filtration chromatography)和镍柱亲和层析纯化试剂盒(Ni-NTA spin columns)纯化所得到的重组蛋白的蛋白含量和生物特性方面的差异。SDS-PAGE分析检测纯化产物, Bradford法测定蛋白含量, 兔红细胞测定半数溶血效价。结果显示这2种方法得到的纯化产物在53 kD处均呈现单一清晰带, 达电泳级纯度。与此同时, 凝胶过滤对目的蛋白的纯化率为14.04%, 蛋白含量为0.337 mg/mL, 溶血活性为1519 HU/mg; 镍柱亲和层析的纯化率为17.5%, 蛋白含量为0.35 mg/mL, 溶血活性为1463 HU/mg。由此可见, 凝胶过滤得到的纯化产物在蛋白含量和蛋白活性方面丝毫不亚于镍柱亲和层析纯化试剂盒。     

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci

Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.     

Two uncommon phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.)

Phospholipase D (PLD) has been detected in seedlings of Papaver somniferum L. cv. Lazúr (Papaveraceae). Purification of the enzyme revealed the existence of two forms of PLD (named as PLD-A and PLD-B). The two enzymes strongly differ in their catalytic properties. The pH optima were found at pH 8.0 for PLD-A and at pH 5.5 for PLD-B. While both enzymes show hydrolytic activity toward phosphatidylcholine (PC) and phosphatidyl-p-nitrophenol (PpNP), PLD-B only was able to catalyze the exchange of choline in PC by glycerol. Both enzymes were activated by Ca²⁺ ions with an optimum concentration of 10 mM. In contrast to PLDs from other plants, PLD-B was still more activated by Zn²⁺ ions with an optimum concentration of 5 mM. The apparent molecular masses of PLD-A and PLD-B, derived from sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), were estimated to be 116.4 and 114.1 kDa. N-terminal protein sequencing indicated N-terminal blockage in both cases. The isoelectric points were found to be 8.7 for PLD-A and 6.7 for PLD-B. Both enzymes were shown to be N-linked glycoproteins. This paper is the first report on PLD in poppy and indicates some important differences of the two enzyme forms to other PLDs known so far.     

Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells

Summary Furosemide-binding proteins were isolated from cholate-solubilized membranes of Ehrlich ascites tumor cells by affinity chromatography, using furosemide as ligand. Solubilized proteins retarded by the affinity material were eluted by furosemide. In reducing and denaturing gels, the major proteins eluted by furosemide were 100 and 45 kDa. In nonreducing, nondenaturing gels, homodimers of both polypeptides were found, whereas no oligomeric proteins containing both polypeptides were seen. It is concluded that the furosemide gel binds two distinct dimeric proteins. The isolated proteins were reconstituted into phospholipid vesicles and the K⁺ transport activity of these vesicles was assayed by measurement of⁸⁶Rb⁺ uptake against a large opposing K⁺ gradient. The reconstituted system was found to contain a K⁺ transporting protein, which is sensitive to Ba²⁺ like the K⁺ channel previously demonstrated to be activated in intact cells after cell swelling.     

Enterococcus faecalis infection in root canals – host‐derived or exogenous source?

Aims: Enterococcus faecalis is associated with a significant number of refractory endodontic infections. Previous studies report a prevalence of Ent. faecalis ranging from 24% up to 77% in teeth with failed endodontic treatment. The origin of the micro‐organism remains unclear, as enterococci do not belong to the normal oral microflora. The aim of this study was to determine whether these enterococci were of endogenous or exogenous origin. Methods and Results: Fifty consecutive patients with apical periodontitis in need of endodontic orthograde re‐treatment were included. Samples were collected from root canals, saliva and faeces and subjected to microbiological culturing. The genetic relationship between Ent. faecalis from root canals and isolates from the different host sources was determined using pulsed‐field gel electrophoresis. In 16% (8/50) of the patients, enterococci were collected from the root canal samples. The genetic analysis showed that the isolates from the root canals were not related to those from the normal gastrointestinal microflora. None of these patients had enterococci in their saliva samples. Conclusions: Endodontic infections with Ent. faecalis are probably not derived from the patient’s own normal microflora, which indicates that these infections ent. faecalis are of exogenous origin. Significance and Impact of the Study: This is the first study to genetically compare endodontic infectious Ent. faecalis isolates with isolates from the hosts’ own normal microflora.     

Chemiluminescent immunoassay of thyroxine enhanced by microchip electrophoresis

A homogeneous chemiluminescent immunoassay of thyroxine (T4) enhanced by microchip electrophoresis separation has been developed. The method deployed the competitive immunoreaction of T4 and horseradish peroxidase (HRP)-labeled T4 (HRP-T4) with anti-T4 mouse monoclonal antibody (Ab). HRP-T4 and the HRP-T4-Ab complex were separated and quantified by using microchip electrophoresis (MCE) with chemiluminescence (CL) detection. Highly sensitive CL detection was achieved by means of HPR-catalyzed luminol-H₂O₂ reaction. Due to the effective MCE separation, the CL analytical signal was less prone to sample matrix interference. Under the selected assay conditions, the MCE separation was accomplished within 60 s. The linear range for T4 was 5-250 nM with a detection limit of 2.2 nM (signal/noise ratio = 3). The current method was successfully applied for the quantification of T4 in human serum samples. It was demonstrated that the current MCE-CL-enhanced competitive immunoassay was quick, sensitive, and highly selective. It may serve as a tool for clinical analysis of T4 to assist in the diagnosis of thyroid gland functions.