Tailored ?-Cyclodextrin Blocks the Translocation Pores of Binary Exotoxins from C. Botulinum and C. Perfringens and Protects Cells from Intoxication

Background

Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin are binary exotoxins, which ADP-ribosylate actin in the cytosol of mammalian cells and thereby destroy the cytoskeleton. C2 and iota toxin consists of two individual proteins, an enzymatic active (A-) component and a separate receptor binding and translocation (B-) component. The latter forms a complex with the A-component on the surface of target cells and after receptor-mediated endocytosis, it mediates the translocation of the A-component from acidified endosomal vesicles into the cytosol. To this end, the B-components form heptameric pores in endosomal membranes, which serve as translocation channels for the A-components.

Methodology/Principal Findings

Here we demonstrate that a 7-fold symmetrical positively charged ß-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-ß-cyclodextrin, protects cultured cells from intoxication with C2 and iota toxins in a concentration-dependent manner starting at low micromolar concentrations. We discovered that the compound inhibited the pH-dependent membrane translocation of the A-components of both toxins in intact cells. Consistently, the compound strongly blocked transmembrane channels formed by the B-components of C2 and iota toxin in planar lipid bilayers in vitro. With C2 toxin, we consecutively ruled out all other possible inhibitory mechanisms showing that the compound did not interfere with the binding of the toxin to the cells or with the enzyme activity of the A-component.

Conclusions/Significance

The described ß-cyclodextrin derivative was previously identified as one of the most potent inhibitors of the binary lethal toxin of Bacillus anthracis both in vitro and in vivo, implying that it might represent a broad-spectrum inhibitor of binary pore-forming exotoxins from pathogenic bacteria.     

Two new Cladophialophora species,C. tumbae sp. nov. and C. tumulicola sp. nov., and chaetothyrialean fungi from biodeteriorated samples in the Takamatsuzuka and Kitora Tumuli

During dismantling and relocation of the Takamatsuzuka Tumulus stone chamber, many Cladophialophora and chaetothyrialean black fungi, such as Exophiala and Phialophora, were isolated from samples taken from the joints between the stone walls. However, inside the stone chamber of the Kitora Tumulus, after intermittent UV irradiation in 2009, these black fungi were also isolated from samples taken from the stone walls. Molecular phylogenetic analyses based on only nrLSU and the concatenated (nrLSU D1/D2 + ITS) sequences revealed that the 35 Takamatsuzuka and Kitora Tumuli isolates of Cladophialophora and the chaetothyrialean black fungi were divergent. Two new species of Cladophialophora are described herein: C. tumulicola from the viscous gels and various substrates on the stone walls of the Takamatsuzuka and Kitora Tumuli and C. tumbae from black substances on the plastic cover over the “thief hole,” soil and plaster pieces between the stone walls, and the exterior of the Takamatsuzuka Tumulus chamber. Also, molecular phylogenetic placements for the remaining eight Takamatsuzuka and Kitora Tumuli isolates of chaetothyrialean black fungi have been determined or suggested.     

Extracellular α Galactosidase (E.C. 3.2.1.22) from Aspergillus Ficuum NRRL 3135 Purification and Characterization

Abstract

Extracellular α-galactosidase, a glycoprotein from the extracellular culture fluid of Aspergillus ficuum grown on glucose and raffinose in a batch culture system, was purified to homogeneity in five steps by inn exchange and hydrophobic Interaction chromatography. The molecular mass of the enzyme was 70.8 Kd by SDS polyacrylamide gel electrophoresis and 74.1 Kd by gel permeation HPLC. On the basis of a molecular mass of 70.7 Kd, the molar extinction coefficient of the enzyme at 279 nm was estimated to be 6.1 × 10⁴ M^?1 cm^?1. The purified enzyme was remarkably stable at 0°C. It had a broad temperature optimum and maximum catalytic activity was at 60°C. It retained 33% of its activity after 10 min. at 65°C. It had a pH optimum of 6.0. It retained 62% of its activity after 12 hours at pH 2.3. The K_ms for p-nitrophenyl-α-D-galactopyranoside, o-nitrophenyl-α-D-galactopyranoside and m-nitrophenyl-α-D-galactopyranoside are: 1462, 839 and 718 μ. The enzyme was competitively inhibited by mercury (19.8 μ), silver (21.5μM), copper (0.48 mM), zinc (0.11 mM), galactose (64.0 mM) and fructose (60.3 mM). It was inhibited non-competitively by glucose (83.2 mM) and uncompetitively by mannose (6.7 mM).     

C.P.S.—Its Strengths and Weaknesses

The following, a supplement to the annual report of the Board of Trustees of California Physicians'' Service that was published in the March 1961 issue of California Medicine, was delivered at the 1961 Annual Session of the House of Delegates by Dr. John G. Morrison, chairman of the board.     

Interspecific Transition Among Caragana microphylla,C. davazamcii and C. korshinskii Along Geographic Gradient. Ⅰ. Ecological and RAPD Evidence

The former plant population survey has shown that three genetically-related species, Caragana microphylla Lam., C. davazamcii Sancz. and C. korshinskii Kom., form a geographical replacement series in Nei Mongol Plateau. The present study on population distribution, taxonomy, morphology, development and genetic structure demonstrated that the geographical distribution of these three species was successive and in gradual change, thus forming a geographical cline which extended from the east to the west of Nei Mongol Plateau. With an analysis of climate change over time, it was considered that the formation of this geographical cline was a result of plant adaptation to its natural environment.     

Phenotypic and genotypic analysis of clarithromycin-resistant Helicobacter pylori from Bogotá D.C., Colombia

Resistance of Helicobacter pylori to clarithromycin is the most common cause of treatment failure in patients with H. pylori infections. This study describes the MICs and the presence of 23S rRNA mutations of H. pylori isolates from Bogotá, D.C., Colombia. H. pylori were isolated from gastric biopsies from patients with functional dyspepsia. Clarithromycin susceptibility was investigated by agar dilution and strains were considered resistant if the MIC was ≥1 μg/ml. DNA sequences of the 23S rRNA gene of strains resistant and sensitive to clarithromycin were determined to identify specific point mutations. Clarithromycin resistance was present in 13.6% of patients by agar dilution. The A2143G, A2142G and A2142C mutations were found in 90.5, 7.1, and 2.4% of H. pylori strains with resistance genotype.The resistant phenotype was associated with 23S rRNA resistance genotype in 85.7% of isolates. The point mutations in 23S rRNA were well correlated with MICs values for clarithromycin.     

Cholinesterases from the marine mussels Mytilus galloprovincialis Lmk. and M. edulis L. and from the freshwater bivalve Corbicula fluminea Müller

In order to improve the molecular basis for the use of bivalve cholinesterases as a reliable biomarker for aquatic pollution, the polymorphism and characterization of these enzymes in Mytilus edulis, Mytilus galloprovincialis and Corbicula fluminea were investigated. All results are consistent with the presence of only one pharmacological form of cholinesterase in each species. The molecular masses were 180 kDa for the two marine mussels and 240 kDa for C. fluminea. The cholinesterases are anchored to the membrane by a glycosyl inositol phosphate like the G^a form (type I) described in vertebrates. Surprisingly, these cholinesterases were poorly inhibited by organophosphorous compounds compared to enzymes from other sources. This suggests that these bivalves could be used as a biomarker for acute rather than chronic contaminations by anticholinesterase insecticides.     

Interspecific Transition Among Caragana microphylla,C. davazamcii and C. korshinskii Along Geographic Gradient. Ⅱ. Characteristics of Photosynthesis and Water Metabolism

The characteristics of photosynthesis and water metabolism of Caragana microphylla Lam., C. davazamcii Sancz. and C. korshinskii Kom. populations in different sites (117.6o-105.7o E, 44.6o-38.8o N) were studied. (1) From the east to the west, the responses of the three species to photosynthetically available radiation (PAR) in net photosynthesis rate increased, the relative humidity of the air which corresponded to the occurrence of maximum photosynthesis rate decreased, and the corresponding air temperature increased. Along the same gradient, the before-noon superiority of the photosynthesis became evident, and the photosynthesis rate and the light use efficiency (LUE ) increased, while the transpiration rate decreased, thus the water use efficiency (WUE ) increased notably, and the leaf water content decreased gradually. From the east to the west, the plants took a water-saving strategy step by step with higher photosynthesis rate and lower transpiration rate. These physiological changes in the plants were adaptable to the conditions of light, temperature and humidity in the habitat of the plants, and might be the biological foundation for the geographical transition among C. microphylla , C. davazamcii and C. korshinskii. (2) The adaptation of photosynthetic system of C. microphylla , C. davazamcii and C. korshinskii to PAR, air humidity and temperature exhibited the interspecific continuity, which was consistent with the environmental gradient. In different species and different sites, the diurnal changes of net photosynthesis rate, the daily cumulative value of net photosynthesis, the diurnal changes of transpiration rate, the daily cumulative value of transpiration, the water use efficiency and the diurnal changes of leaf water content varied with longitudinal descent (from the east to the west). The characteristics of photosynthesis and water metabolism indicated that the geographical transition among C. microphylla , C. davazamcii and C. korshinskii was in gradual change, and these three species formed a geographical cline.     

Seed structure in Crocus sativus L. ×, C. cartwrightianus Herb., C. thomasii Ten., and C. hadriaticus Herb. at SEM

Crocus sativus L. (Iridaceae) is a sterile triploid geophyte widely cultivated for the production of the spice saffron and only reproduced by means of corms. Extensive research has identified Crocus cartwrightianus Herb. as being a probable progenitor of C. sativus. However, other diploid Crocus species of the same C. sativus group, such as C. thomasii Ten. and C. hadriaticus Herb., have been considered as possible progenitors of saffron. Of the characteristics for distinguishing critical genera, species and intraspecific taxa of angiosperms, the most widely adopted have been seed organisation and patterns of spermoderma microstructure detected at SEM. The aim of this study is to use SEM to analyse the seeds of C. sativus ×, a cross obtained by C. sativus with pollen of C. cartwrightianus Herb. and the seeds of allopollinated C. cartwrightianus, C. thomasii Ten., and C. hadriaticus Herb. Results indicate that the seed surface microstructure of C. sativus × is very similar to that of C. cartwrightianus while being different from those of C. thomasii and C. hadriaticus.     

Production and properties of ∝-mannosidase from aspergillus sp.

Summary Aspergillus sp NCIM 508 produced 22 U/L of extracellular -mannosidase activity in a medium containing 8 % brewer's yeast cells. The optimum period and pH range for maximum production of the enzyme were 7 days and 4.0–6.0, respectively. The optimum pH and temperature for enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable for 24 h at 28°C, in the pH range 6.0–7.0. The enzyme retained 100 and 65 % of its original activity after heating for 15 min at 45 and 55°C, respectively. The Km and V_max for p-nitrophenyl--D- mannoside (PNPM) were 71M and 7.5 × 10^–2 moles/min/mg, respectively. The enzyme was strongly inhibited by 1 mM Hg⁺⁺ and Cu⁺⁺ and partially by Co.⁺⁺ (NCL Communication No.; 5780)     

The amino acid sequence of cytochrome c′ from Alcaligenes sp. N.C.I.B. 11015

The amino acid sequence of the cytochrome c' from Alcaligenes sp. N.C.I.B. 11015 (Iwasaki's ;Pseudomonas denitrificans') has been determined. This organism is the only non-photosynthetic bacterium in which the protein has been found. The protein consists of a single polypeptide chain of 127 residues, with a single haem covalently attached to two cysteines. Unlike normal cytochromes c, the haem attachment site is very close to the C-terminus. The amino acid sequence around the haem attachment site is very similar to that of Chromatium vinosum D cytochrome c'. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50022 at the British Library (Lending Division), (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.     

Purification and Properties of L-Methionine γ-Lyase from Aeromonassp.

Four types of β-xylosidases from a concentrated culture filtrate of Pénicillium wortmanni IFO 7237, designated as xylosidase-1, -2, -3, and -4 were purified to homogeneity on SDS polyacrylamide gel electrophoresis by an alcohol precipitation, DEAE-Sephadex A-25 ion exchange chromatography, and isoelectric focusing. The molecular weights of xylosidase-1, -2, -3, and -4 were estimated to be 110,000, 195,000, 210,000, and 180,000 respectively and their isoelectric points to be 3.7, 4.28, 4.6, and 4.8. The pH optima of β-xylosidase activities were from 3 to 4.5. The optimum temperature for enzyme activities was from 55°C to 65°C. On the enzymic hydrolysis of phenyl ß-d- xyloside, the reaction product of each enzyme was found to be β-d-xylose with retention of configuration. All the four ß-xylosidases were free of α-xylosidase and ß-glucosidase activities. All the enzyme activities of four β-xylosidases were strongly inhibited by Hg²⁺ and N- bromosuccinimide. With respect to the hydrolysis patterns and HPLC analysis of hydrolyzates from xylooligosaccharides, xylosidase-2 was totally different from other three as a distinct enzyme. Xylosidase-1 was also in a separate group although xylosidase-3 and -4 showed closely related action patterns as a different group.