Dual action of intracellularly released calcium on the quantal mediator secretion

The mice diaphragm muscle and microelectrode technique were used to check the influence of ryanodine (0.5 mcM) on spontaneous and evoked mediator release under conditions of potassium depolarization (8-16 mM [K+]ex or rhythmic (4-100 Hz) stimulation of motor nerve terminals. Weak tonic calcium loading (by muscle exposition to 8 mM [K+]ex) caused a two-fold frequency increase if miniature and plate potentials (MEPPs), which was returned to the basal level by subsequent application of ryanodine. This inhibitory effect of ryanodine was blocked by apamin (500 nM) a blocker of K+(Ca)-channels. A greater calcium load of terminals (in solution with 16 mM [K+]ex) caused a 15-fold increase of MEPPs frequency. Subsequent ryanodine application caused an additional 2-3-fold increase of MEPPs frequency. During rhythmic activity of motor synapses, ryanodine was able to decrease the amplitude of EPP by 60% at plateau phase at short low frequency (4 Hz) of discharges and to increase the amplitude of EPP by 60-150% at high frequency (70-100 Hz) of discharges. It is concluded that rynodine induced calcium release from intraterminal Ca2+-stores can influence dual: excitatory or inhibitory, action on spontaneous and evoked mediator release, due to different intraterminal calcium loads and regimen of synaptic activity.     

Protective effects of anti-ricin A-chain antibodies delivered intracellularly against ricin-induced cytotoxicity

AIM: To evaluate the ability of anti-ricin A-chain antibodies, delivered intracellularly, to protect against ricin-induced cytotoxicity in RAW264.7 cells.METHODS: Anti-deglycosylated ricin A-chain antibody and RAC18 anti-ricin A-chain monoclonal antibody were delivered intracellularly by encapsulating in liposomes or via conjugation with the cell-penetrating MTS-transport peptide. RAW264.7 cells were incubated with these antibodies either before or after ricin exposure. The changes in cytotoxicity were estimated by MTT assay. Co-localization of internalized antibody and ricin was evaluated by fluorescence microscopy.RESULTS: Internalized antibodies significantly increased cell viability either before or after ricin exposure compared to the unconjugated antibodies. Fluorescence microscopy confirmed the co-localization of internalized antibodies and ricin inside the cells.CONCLUSION: Intracellular delivery of antibodies to neutralize the ricin toxin after cellular uptake supports the potential use of cell-permeable antibodies for post-exposure treatment of ricin intoxication.     

Mycobacterium xenopi and drinking water biofilms

The ability of Mycobacterium xenopi to colonize an experimental drinking water distribution system (a Propella reactor) was investigated. M. xenopi was present in the biofilm within an hour following its introduction. After 9 weeks, it was always present in the outlet water (1 to 10 CFU 100 ml(-1)) and inside the biofilm (10(2) to 10(3) CFU cm(-2)). Biofilms may be considered reservoirs for the survival of M. xenopi.     

DNA fingerprinting of Mycobacterium xenopi strains

Mycobacterium xenopi is an environmental bacterium that occasionally causes disease in humans. A method is described for DNA fingerprinting strains of this species. Seven of 10 strains from humans were clearly distinguishable from each other using DNA fingerprinting. This method will enable the investigation of possible environmental sources and human spread of disease due to this species.     

Mycobacterium xenopi and Drinking Water Biofilms

The ability of Mycobacterium xenopi to colonize an experimental drinking water distribution system (a Propella reactor) was investigated. M. xenopi was present in the biofilm within an hour following its introduction. After 9 weeks, it was always present in the outlet water (1 to 10 CFU 100 ml⁻¹) and inside the biofilm (10² to 10³ CFU cm⁻²). Biofilms may be considered reservoirs for the survival of M. xenopi.     

Secondary fatty alcohols of Mycobacterium xenopi.

Secondary alcohols of Mycobacterium xenopi were studied by gas chromatography and gas chromatography-mass spectrometry. Mycobacterial cells were hydrolysed and the liberated alcohols separated by extraction and analysed both underivatized and as trimethylsilyl, methyl ether- and pentafluorobenzoyl derivatives. Seven straight-chain secondary alcohols containing from 18 to 24 carbon atoms and two branched-chain secondary alcohols with 21 and 23 carbon atoms were present in all of the studied strains.     

Modeling biocide action against biofilms

A phenomenological model of biocide action against microbial biofilms was derived. Processes incorporated in the model include bulk flow in and out of a well-mixed reactor, transport of dissolved species into the biofilm, substrate consumption by bacterial metabolism, bacterial growth, advection of cell mass within the biofilm, cell detachment from the biofilm, cell death, and biocide concentration-dependent disinfection. Simulations were performed to analyze the general behavior of the model and to perform preliminary sensitivity analysis to identify key input parameters. The model captured several general features of antimicrobial agent action against biofilms that have been observed widely by experimenters and practitioners. These included (1) rapid disinfection followed by biofilm regrowth, (2) slower detachment than disinfection, and (3) reduced susceptibility of microorganisms in biofilms. The results support the plausibility of a mechanism of biofilm resistance in which the biocide is neutralized by reaction with biofilm constituents, leading to a reduction in the bulk biocide concentration and, more significantly, biocide concentration gradients within the biofilm. Sensitivity experiments and analyses identified which input parameters influence key response variables. Each of three response variables was sensitive to each of the five input parameters, but they were most sensitive to the initial biofilm thickness and next most sensitive to the biocide disinfection rate coefficient. Statistical regression modeling produced simple equations for approximating the response variables for situations within the range of conditions covered by the sensitivity experiment. The model should be useful as a tool for studying alternative biocide control strategies. For example, the simulations suggested that a good interval between pulses of biocide is the time to minimum thickness. (c) 1996 John Wiley & Sons, Inc.     

Identification of Mycobacterium xenopi by gas chromatography

For the purposes of the following study we cultured 32 strains of Mycobacterium xenopi isolated from clinical specimens and several strains of other slowly growing mycobacteria. The cultures were grown in liquid medium and then analysed--after saponification, methylation, extraction with organic solvent and washing of the organic phase--using a highly sensitive manual gas-liquid chromatographic assay for the determination of secondary alcohol 2-OH-docosanol. The percentage of this compound was compared with that previously measured in strains of Mycobacterium xenopi grown on solid medium. The presence of this specific alcohol was always apparent, even though its quantity was lower than that obtained by growing mycobacteria on solid medium. The absence of interference peaks around the compound was checked by analyzing strains of other slowly growing mycobacteria in the same conditions.     

New Targets for Drug Discovery against Malaria

A mathematical model which predicts the intraerythrocytic stages of Plasmodium falciparum infection was developed using data from malaria-infected mice. Variables selected accounted for levels of healthy red blood cells, merozoite (Plasmodium asexual phase) infected red blood cells, gametocyte (Plasmodium sexual phase) infected red blood cells and a phenomenological variable which accounts for the mean activity of the immune system of the host. The model built was able to reproduce the behavior of three different scenarios of malaria. It predicts the later dynamics of malaria-infected humans well after the first peak of parasitemia, the qualitative response of malaria-infected monkeys to vaccination and the changes observed in malaria-infected mice when they are treated with antimalarial drugs. The mathematical model was used to identify new targets to be focused on drug design. Optimization methodologies were applied to identify five targets for minimizing the parasite load; four of the targets thus identified have never before been taken into account in drug design. The potential targets include: 1) increasing the death rate of the gametocytes, 2) decreasing the invasion rate of the red blood cells by the merozoites, 3) increasing the transformation of merozoites into gametocytes, 4) decreasing the activation of the immune system by the gametocytes, and finally 5) a combination of the previous target with decreasing the recycling rate of the red blood cells. The first target is already used in current therapies, whereas the remainders are proposals for potential new targets. Furthermore, the combined target (the simultaneous decrease of the activation of IS by gRBC and the decrease of the influence of IS on the recycling of hRBC) is interesting, since this combination does not affect the parasite directly. Thus, it is not expected to generate selective pressure on the parasites, which means that it would not produce resistance in Plasmodium.     

Protection by DMSO against cell death caused by intracellularly localized iodine-125, iodine-131 and polonium-210

The mechanisms by which DNA-incorporated radionuclides impart lethal damage to mammalian cells were investigated by examining the capacity of dimethyl sulfoxide (DMSO) to protect against lethal damage to Chinese hamster V79 cells caused by unbound tritium ((3)H(2)O), DNA-incorporated (125)I- and (131)I-iododeoxyuridine ((125)IdU, (131)IdU), and cytoplasmically localized (210)Po citrate. The radionuclides (3)H and (131)I emit low- and medium-energy beta particles, respectively, (125)I is a prolific Auger electron emitter, and (210)Po emits 5.3 MeV alpha particles. Cells were radiolabeled and maintained at 10.5 degrees C for 72 h in the presence of different concentrations of DMSO (5-12.5% v/v), and the surviving fraction compared to that of unlabeled controls was determined. DMSO afforded no protection against the lethal effects of the high-LET alpha particles emitted by (210)Po. Protection against lethal damage caused by unbound (3)H, (131)IdU and (125)IdU depended on the concentration of DMSO in the culture medium. Ten percent DMSO provided maximum protection in all cases. The dose modification factors obtained at 10% DMSO for (3)H(2)O, (131)IdU, (125)IdU and (210)Po citrate were 2.9 +/- 0.01, 2.3 +/- 0.5, 2.6 +/- 0.2 and 0.95 +/- 0.07, respectively. These results indicate that the toxicity of Auger electron and beta-particle emitters incorporated into the DNA of mammalian cells is largely radical-mediated and is therefore indirect in nature. This is also the case for the low-energy beta particles emitted by (3)H(2)O. In contrast, alpha particles impart lethal damage largely by direct effects. Finally, calculations of cellular absorbed doses indicate that beta-particle emitters are substantially more toxic when incorporated into the DNA of mammalian cells than when they are localized extracellularly.     

A new type of serine-containing glycopeptidolipid from Mycobacterium xenopi

An unknown immunogenic glycopeptidolipid, named GPL X-1, was isolated from Mycobacterium xenopi, which is a nontuberculous mycobacterium responsible for pulmonary and disseminated infectious diseases mainly occurring in immunocompromised patients. The glycopeptidolipid was purified until homogeneity, in the native form, by direct phase high performance liquid chromatography. A new route is proposed for the structural elucidation of its unusual lipopeptidic core. The presence of allothreonine (aThr), phenylalanine, and serine in the molecular ratio 1:1:2, respectively, was established by reverse phase high performance liquid chromatography analysis of the phenylthiocarbamyl amino acid derivatives. From the molecular mass (1828 Da) of the native glycopeptidolipid, determined by cesium ion liquid secondary ion mass spectrometry using the amphipathic triethylene glycol monobutyl ether matrix, it was deduced that the tetrapeptide was amidified by a dodecanoic acid. The complete structure, C12-Ser-Ser-Phe-aThr-OCH3, of the lipopeptidic core was established by pyrolysis electron impact-mass spectrometry of the native glycopeptidolipid. To date, this is the first example of a mycobacterial glycopeptidolipid with a C12-tetrapeptidic core containing serine. A novel approach, based on two dimensional 1H,1H correlated spectroscopy analysis of the native and peracetylated GPL X-1, was developed, allowing the structural determination of the monosaccharidic residues with their alkali-labile groups "in situ" on the whole complex molecule. 2-O-Acyl-alpha-L-Rhap, alpha-L-Rhap, 2,4-di-O-acyl-6-deoxy-alpha-L-Glcp, 2,3,4-tri-O-Me-alpha-L-Rhap, and 3-O-Me-6-deoxy-alpha-L-Talp were identified, where Me, Rhap, and Talp are methyl, rhamnopyranosyl, and talopyranosyl, respectively. The latter two were localized at the carbohydrate non-reducing ends, and the C-3's of the remaining monosaccharide residues were found involved in the interglycosidic linkage. The alpha anomeric configurations were inferred from the JC-1,H-1 heteronuclear coupling constants, and the L absolute configurations for all the monosaccharide residues were established by gas chromatography analysis of the trimethylsilyl (+/-)-2-butyl glycosides. Finally, by pyrolysis electron impact mass spectrometry of peracetylated GPL X-1, the following tetrasaccharide appendage structure was proposed: 2,3,4-tri-O-Me-L-Rhap(alpha 1----3)-2-O-lauryl-L-Rhap(alpha 1----3)-L-Rhap- (alpha 1----3)-2,4-di-O-(acetyl,lauryl)-6-deoxy-alpha-L-Glcp. Compared to the oligosaccharidic glycopeptidolipid structures, the particular features of the GPL X-1 tetrasaccharide structure arise from the presence of monosaccharide residues esterified by C12 fatty acids and from the absence of the basal disaccharide core, L-Rhap-(alpha 1----2)-6-deoxy-alpha-L-Talp.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vapour action of fungicides against powdery mildews

Certain fungicides prevented the growth of powdery mildews at distances extending several millimetres beyond the edge of localized deposits on leaves. Most of the materials also acted at a distance when separated from the leaf by glass cover-slips, indicating that the effect was due to the emission of vapour. In addition to sulphur and lime-sulphur, whose vapour action is already known, fungicides based on drazoxolon, oxythioquinox, binap-acryl, dinitro-octyl phenyl crotonates and a phthalimido-phosphonothionate were found to act in this way. Two systemic fungicides, griseofulvin and triamiphos, did not give a detectable vapour action. Tests were conducted mostly on open glasshouse benches. A draught from a fan shifted the zones of inhibition to one side of the deposits, but did not reduce their areas. Vapour effects were similar in extent on plants maintained at 18–25° C and at 27–32° C. Variation in the areas protected was studied in relation to the size and fungicide content of the deposits, to different powdery mildews, to time of incubation and to different types of formulation. Deposits applied to leaves by high- or low-volume sprays at concentrations used in the field gave significant protection at a distance. Vapour effects were demonstrated also on mildew conidia incubated on glass slides bearing a spot of fungicide, and on infected plants placed in beakers coated on the bottom with fungicide. Movement of fungicides in the gaseous state is discussed in relation to the control of foliage diseases in the field.     

Drug treatment and novel drug target against Cryptosporidium

Cryptosporidiosis emergence triggered the screening of many compounds for potential anti-cryptosporidial activity in which the majority were ineffective. The outbreak of cryptosporidiosis which occurred in Milwaukee in 1993 was not only the first significant emergence of Cryptosporidium spp. as a major human pathogen but also a huge waterborne outbreak thickening thousands of people from a major city in North America. Since then, outbreaks of cryptosporidiosis are regularly occurring throughout the world. New drugs against this parasite became consequently urgently needed. Among the most commonly used treatments against cryptosporidiosis are paromomycin, and azithromycin, which are partially effective. Nitazoxanide (NTZ)'s effectiveness was demonstrated in vitro, and in vivo using several animal models and finally in clinical trials. It significantly shortened the duration of diarrhea and decreased mortality in adults and in malnourished children. NTZ is not effective without an appropriate immune response. In AIDS patients, combination therapy restoring immunity along with antimicrobial treatment of Cryptosporidium infection is necessary. Recent investigations focused on the potential of molecular-based immunotherapy against this parasite. Others tested the effects of probiotic bacteria, but were unable to demonstrate eradication of C. parvum. New synthetic isoflavone derivatives demonstrated excellent activity against C. parvum in vitro and in a gerbil model of infection. Newly synthesized nitro- or non nitro- thiazolide compounds, derived from NTZ, have been recently shown to be at least as effective as NTZ against C. parvum in vitro development and are promising new therapeutic agents.     

抗新冠肺炎药物研究进展

新冠病毒引发的急性呼吸道传染病造成了全球大流行的新冠肺炎,严重危害世界公共卫生安全,迫切需要研发有效治疗新冠肺炎的药物。综述了疫情暴发初期抗新冠肺炎药物研发的进展,重点介绍“老药新用”、小分子及抗体创新药物研发和中药等。通过“老药新用”研究发现多个老药具有抑制新冠病毒复制作用,其中瑞德西韦、法匹拉韦、氯喹和羟氯喹等进入临床研究,尤其是瑞德西韦成为被美国FDA批准用于新冠肺炎治疗的首个药物。针对新冠病毒识别宿主细胞受体的S蛋白开展的抗体发现和靶向3CL蛋白酶及RNA依赖的RNA聚合酶等新冠病毒复制过程中的关键蛋白质开展小分子抑制剂发现是抗新冠肺炎创新药物研究中的主要方向。此外,中药在防治新冠肺炎中发挥了重要作用,金花清感颗粒、莲花清瘟胶囊、血必净注射液、双黄连口服液、清肺排毒汤、化湿败毒方、宣肺败毒方等都进入了新冠肺炎治疗的临床研究及应用。