In vitro susceptibilities of clinical yeast isolates to three antifungal agents determined by the microdilution method

A comparative evaluation of the in vitro susceptibilities of 597 clinical yeast isolates to amphotericin B, fluconazole, and 5-fluorocytosine (5FC) was conducted. The broth macrodilution reference method of the National Committee for Clinical Laboratory Standards (NCCLS, M27-P) was adapted to the microdilution method. Microdilution endpoints for amphotericin B were scored as the lowest concentration in which a score of 0 (complete absence of growth) was observed and for 5FC and fluconazole as the lowest concentration in which a score of 2 (prominent decrease in turbidity; MIC-2) was observed compared to the growth control. The MIC values were read after 24 and 48 h incubation. A broad range of MIC values was observed with each antifungal agent. Amphotericin B was very active (MIC₉₀1.0 µg/ml) against all of the yeast isolates with the exception ofC. lusitaniae (MIC₉₀2.0 µg/ml). Fluconazole was most active againstC. parapsilosis (MIC₉₀ of 1.0 µg/ml) and least active againstC. krusei (MIC₉₀ of 32 µg/ml). 5FC was most active againstC. albicans, C. parapsilosis, C. tropicalis, andT. glabrata (MIC₉₀1.0 µg/ml) and was least active againstC. krusei andC. lusitaniae (MIC₉₀16 µg/ml). These data indicate that the microdilution method, performed in accordance with M27-P, provides a means of testing larger numbers of yeast isolates against an array of antifungal agents and allows this to be accomplished in a reproducible and standardized manner. Given these results, it appears that the microdilution method may be a useful alternative to the macrodilution reference method for susceptibility testing of yeasts.     

光皮桦组织培养离体再生研究

采用正交试验等设计,系统开展了光皮桦组织培养高效再生体系研究。结果表明:光皮桦茎段外植体最佳诱导培养基及激素组合为MS+0.50mg.L-1 6-BA+0.10mg.L-1 TDZ+30mg.L-1蔗糖+5.50g.L-1琼脂,丛生芽最佳生根培养基为1/2MS+1mg.L-1 IBA+20g.L-1蔗糖+5.50g.L-1琼脂;丛生芽在最佳生根培养基上培养15d后获健壮生根苗,移栽成活率达90%。该实验结果为光皮桦的优良品种快繁以及遗传转化体系建立奠定了良好的基础。     

粤紫萁离体再生技术研究

粤紫萁(Osmunda mildei)为新近形成的自然杂交种,其分布区狭窄、种群个体数量少、有性繁殖能力极低.该文以孢子为外植体,以MS、3/4MS、1/2MS和1/4MS为基本培养基,探讨不同生长调节剂对粤紫萁孢子萌发、原叶体增殖和孢子体形成的影响.结果表明:粤紫萁孢子中极少的可育孢子在植物生长调节剂的作用下,能够萌...     

In vitro chromosome doubling of nineZantedeschia cultivars

Tetraploid plants have been produced from nineZantedeschia cultivars usingin vitro colchicine treatment. Rapidly-multiplying shoot cultures were treated on a medium containing 0.05% colchicine for 1, 2 or 4 days to induce chromosome doubling. Following the treatment, most shoots were killed but the surviving shoots were multiplied for several subcultures. These shoots were then rootedin vitro and transferred to a greenhouse. Plants were screened 2 months later by measuring stomatal length, and 110 out of 565 plants were selected as putative tetraploids with a stomatal length significantly greater than in diploid control plants. Chromosome counts were carried out on root tips from 44 plants and confirmed that 38 were tetraploids, 2 were chimeras (predominantly tetraploid with a few octoploid cells), and 4 were diploids. Stomatal length has been rechecked in mature tetraploid plants of the cultivar Black Magic, demonstrating that stomatal length is a good indicator of ploidy level inZantedeschia. This study has shown that multiplying colchicine-treated shootsin vitro for several subcultures prior to transfer to soil produced very few chimeras. The stomatal length measurements are non-destructive and allow the rapid screening of a population for tetraploids.     

pNEgr-mIL-12真核表达载体的体外和体内生物学活性检测

肿瘤的基因-放射治疗是近年来发展起来的新技术.分别于体外和体内检测含辐射敏感启动子和mIL-12基因的真核表达载体（pNEgr-mIL-12）的生物学活性.体外经酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)法检测转染pNEgr-mIL-12重组质粒的COS-7和B16细胞可经辐射诱导mIL-12 p70表达,于1.5～2.0 Gy照射后表达增高最明显,COS-7细胞于照后4 h 达峰值,B16细胞的表达水平随照射后时间延长而逐渐增高;pNEgr-mIL-12重组质粒联合电离辐射治疗小鼠移植肿瘤,单次或多次注射pNEgr-mIL-12重组质粒,联合局部照射能够抑制小鼠移植肿瘤生长,与单纯照射组比较肿瘤生长速度减慢,瘤重降低,尤以多次给予质粒治疗组效果明显.为进一步探讨最佳治疗方案及临床肿瘤病人的基因放射治疗提供了初步依据.     

In vitro production of solasodine from Solanum trilobatum

Sucrose concentration in the culture medium affected chlorophyll content, trichome development and amount of solasodine in regenerated plantlets of Solanum trilobatum. High chlorophyll content and glandular trichomes were observed in the plants grown on Murashige and Skoog basal medium supplemented with 131.85 mM sucrose. The solasodine was quantified using reverse phase high performance liquid chromatography. The plantlets cultivated on this medium yielded 35.97 mg g⁻¹ (d.m.) solasodine whereas the field plants used as control yielded only 2.32 mg g⁻¹ (d.m.) of solasodine.     

大肠杆菌碱性磷酸酶的体外定向进化研究

大肠杆菌碱性磷酸酶（E.coli alkaline phosphatase, EAP, EC 3.1.3.1）是一个非特异性二聚体磷酸单酯酶. 采用易错聚合酶链反应(error prone PCR)的方法,在原有高活力突变株的基础上,对EAP远离活性中心催化三联体的区域进行定向进化,经两轮error prone PCR,获得催化活力较亲本D101S突变株提高3倍、较野生型酶提高35倍的进化酶4-186,并对该酶的催化动力学特征进行了分析. 进化酶基因的DNA测序表明4-186含两个有义氨基酸置换：K167R和S374C,二者既不位于底物结合位点,也不位于酶的金属离子结合位点.     

In vitro biohydrogenation of four dietary fats

This study was designed to determine in vitro rates of biohydrogenation of dietary unsaturated fatty acids by a mixed population of rumen microbes. The four dietary fats [Alifet High-Energy^® (AHE), Alifet-Repro^® (AR), Megalac^® (MG), and Energy Booster^® (EB)] differ in method of preparation, fatty acid composition, or both of these factors. Dietary fats (20 mg) were incubated with 4 mL strained rumen fluid diluted with 16 mL of medium, 0.8 mL of reducing solution buffer, and 200 mg of a synthetic diet (370 g cellulose, 370 g starch, and 160 g casein per kg DM) at 37 °C. Total contents were collected after 0, 6, 12, 24, or 36 h and change in fatty acid content determined. Disappearance of oleic acid was minimal (0.05–0.20) in AR and MG but moderate (about 0.60) in AHE and EB after 36 h of incubation. Rate of biohydrogenation of linoleic and linolenic acids from AR were similar (0.025 ± 0.009 h⁻¹) and 0.65 of these fatty acids remained intact after 36 h. Rate of biohydrogenation of linoleic acid was four times greater than for oleic acid (0.040 ± 0.013 h⁻¹ versus 0.009 ± 0.002 h⁻¹) in MG. Thus, 0.65 of the linoleic acid but only 0.20 of the oleic acid had disappeared from MG after 36 h. Trans-11 and trans-12 were the predominant trans-isomers in AHE and AR cultures whereas trans-9 and trans-10 were the predominant trans-isomers in EB and MG cultures. None of the dietary fats contained conjugated linoleic acid (CLA) but CLA was present in the incubation inoculum. The amount of CLA decreased with time but this was not affected by source of dietary fat. Most (0.90–0.95) of the long-chain fatty acids eicosapentaenoic (EPA) and docosahexaenoic (DHA) in AR remained after 36 h of incubation. Results demonstrate that biohydrogenation varied among fatty acids and among source of dietary fat and indicate that AR can be used to increase post-ruminal supply of linolenic, EPA and DHA.     

Correlation between broth microdilution and disk diffusion methods for antifungal susceptibility testing of caspofungin, voriconazole, amphotericin B, itraconazole and fluconazole against Candida glabrata

Candida glabrata is one of the most frequent organisms isolated from superficial and invasive fungal infections, after Candida albicans. This organism also exhibits intrinsically low susceptibility to azole antifungals and treatment often fails. The microdilution method is not very practical for use in routine susceptibility testing in the clinical laboratory, thus necessitating the use of other methods. In this study, we compared the in vitro activity of five antifungal agents in three different groups (echinocandin, polyene and azole) against 50 C. glabrata isolates by broth microdilution and disk diffusion methods recommended by Clinical Laboratory Standards Institute CLSI M27-A3 and CLSI M44-A, respectively. All the isolates were susceptible to amphotericin B (100%) and 98% of the isolates were susceptible to caspofungin by the broth microdilution method. Within the azole group drugs, voriconazole was the most active followed by fluconazole and itraconazole in vitro. The highest rate of resistance was obtained against itraconazole with a high number of isolates defined as susceptible-dose dependent or resistant. Although the disk diffusion method is easy to use in clinical laboratories, it shows very poor agreement with the reference method for fluconazole and itraconazole against C. glabrata (8% and 14%, respectively).     

In vitro pathogenicity assay for the ergot fungus Claviceps purpurea

The pathogenic development of the biotrophic ergot fungus Claviceps purpurea is strictly limited to the ovary of grasses. Early colonization stages occur within a defined spatio-temporal course of events, including the directed growth to the vascular tissue for nutrient supply. To characterize mutant strains with putative defects in pathogenicity, the close observation of the infection pathway is therefore indispensable. Here, we describe the establishment of a new pathogenicity assay, based on the in vitro cultivation of isolated rye ovaries. The pathogenic development of a wild-type strain of C. purpurea was compared with the infection of mature rye flowers on whole plants. Up to the sixth day post inoculation, the route of infection within the isolated ovaries was maintained and temporally equal to that seen in mature flowers. Therefore, the in vitro pathogenicity assay is an effective alternative to the whole-plant infection tests, and suitable for detailed infection studies and screening high numbers of mutants for defects in early pathogenesis.     

Use of spectrophotometric reading for in vitro antifungal susceptibility testing of Aspergillus spp

A comparative study of visual and spectrophotometric MIC endpoint determinations for antifungal susceptibility testing of Aspergillus species was performed. A broth microdilution method adapted from the National Committee for Clinical Laboratory Standards (NCCLS) was used for susceptibility testing of 180 clinical isolates of Aspergillus species against amphotericin B and itraconazole. MICs were determined visually and spectrophotometrically at 490 nm after 24, 48, and 72 h of incubation, and MIC pairs were compared. The agreement between the two methods was 99% for amphotericin B and ranged from 95 to 98% for itraconazole. It is concluded that spectrophotometric MIC endpoint determination is a valuable alternative to the visual reference method for susceptibility testing of Aspergillus species.     

In vitro effects of propolis on Giardia duodenalis trophozoites

In order to improve the current chemotherapy of Giardia infection, potential antigiardial agents have been screened, including natural products. Propolis, a resinous hive product collected by bees, has attracted attention as a useful and popular substance with several therapeutic activities. The present study was carried out aiming to evaluate the in vitro effects of an ethanolic extract of propolis on the growth and adherence of Giardia duodenalis trophozoites. Propolis inhibited the growth of trophozoites and the level of inhibition varied according to the extract concentration and incubation times. The highest reduction of parasite growth was observed in cultures exposed to 125, 250 and 500 microg/ml of propolis, in all incubation periods (24, 48, 72 and 96 h). Growth reduction by 50% was observed in 125 microg/ml propolis-treated cultures, while the concentrations of 250 and 500 microg/ml were able to inhibit growth by more than 60%. Propolis also inhibited parasite adherence and all assayed propolis concentrations promoted the detachment of trophozoites. Light microscope observations revealed changes of the pear-shaped aspect of the cell and reduction of flagellar beating frequency in the great part of the trophozoites. Our results hold the perspective for the utilization of propolis as an antigiardial agent.     

In vitro organogenesis of Citrus volkameriana and Citrus aurantium

In vitro organogenesis of Citrus volkameriana and C. aurantium was studied considering three explant types: epicotyl segment, internodal segment, and hypocotyl segment with attached cotyledon fragment. The explants were cultured in medium according to Grosser and Gmitter (EME) supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg dm^{− 3} 6-benzyl-aminopurine (BAP), incubated firstly in darkness for 4 weeks, and then transferred to 16-h photoperiod for 2 weeks. Comparing epicotyl and internodal segments, a higher percentage of responsive explants and a higher number of shoots per explant were obtained with epicotyl segments, regardless of the BAP concentration. For C. volkameriana the highest percentage of responsive epicotyl segments (42 %) was obtained in EME with 1.0 mg dm⁻³ BAP, while for C. aurantium (59 %) in EME with 0.5 mg dm⁻³ BAP. The organogenesis efficiency was the best with the use of the hypocotyl segment with attached cotyledon fragment (77 % for C. volkameriana and to 75 % for C. aurantium). With this explant the morphogenesis occurred only in the hypocotyl region. The in vitro organogenesis was characterized by histological analyses showing that the morphogenic process started in the cambium region near the explant cut end.     

In vitro cloning of Azadirachta indica from root explants

In vitro cultures of Azadirachta indica A. Juss. were raised by first culturing the root segments on modified Murashige and Skoog (MS) medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 9.84 μM N⁶-(2-isopentenyl) adenine (2iP), 5.71 μM indole-3-acetic acid (IAA), 81.43 μM adenine hemisulphate and 2.27 μM putrescine for 2 d followed by their transfer to the same medium except containing one-tenth of the initially used concentrations of BAP, 2iP and IAA. The regenerated shoots sustained proliferation in the basal medium supplemented with 1.11 μM BAP, 1.43 μM IAA and 135.72 μM adenine hemisulphate. The isolated shoots were rooted to produce plantlets in the presence of 2.46 μM indole-3-butyric acid (IBA). The plantlets showed uniform luxuriant growth under field conditions. True-to-type nature of the field-grown root-regenerated plants was ascertained by random amplified polymorphic DNA (RAPD) analysis.     

In vitro antifungal and anti-elastase activity of some aliphatic aldehydes from Olea europaea L. fruit

Olea europaea preparations are traditionally employed in a variety of troubles, including skin infections. Olive extracts and some of their pure compounds have shown antimicrobial activity in vitro. The present study deals with the antifungal activity of some aliphatic aldehydes from olive fruit [hexanal, nonanal, (E)-2-hexenal, (E)-2-heptenal, (E)-2-octenal, (E)-2-nonenal] against Tricophyton mentagrophytes (6 strains), Microsporum canis (1 strains) and Candida spp. (7 strains). The capability of these substances to inhibit elastase, a virulence factor essential for the dermatophytes colonization, and their cytotoxicity on cultures of reconstructed human epidermis, are also described. Aldehydes tested, inhibited the growth of T. mentagrophytes and M. canis in the range of concentration between <1.9 and 125 microg/ml; the unsaturated aldehydes showed the most broad spectrum of activity in that inhibited all strains tested. None of the aldehydes exhibited activity against Candida spp. strains. (E)-2-octenal and (E)-2-nonenal inhibited the elastase activity in a concentration-dependent manner; the anti-elastase activity suggests an additional target of the antimicrobial activity of these compounds. Aldehydes were devoid of cytotoxicity on cultures of human reconstructed epidermis. The antifungal activity of the aldehydes from olive fruit here reported, substantiates the use of olive and olive oil in skin diseases and suggests that these natural compounds could be useful agents in the topical treatment of fungal cutaneous infections.     

广藿香不同外植体离体培养的研究

以广藿香叶片、带节茎、不带节茎及根尖为材料进行离体培养,对影响离体再生的因素进行了研究。结果表明:BA有利于广藿香外植体出芽,浓度以0.1～0.5mg/L效果较好;不同外植体的出芽能力有较大差异,其中以叶片和带节茎出芽能力较强,出芽率均达100%;外植体在培养基上的接种方式对出芽也有一定影响,带节茎以形态学下端垂直插入,可以缩短出芽时间及增加单个外植体出芽的数量。无根苗生根以MT+IBA0.2mg/L培养基为好,苗的生长较为健壮。     

以转录因子AP-1为靶的decoy核酸体内外抗肿瘤研究

合成了双链寡聚核苷酸——decoy核酸,其与靶转录因子AP-1有高亲和性,可进入细胞作为decoy顺式元件,通过抑制特异的转录因子和调控区域的结合,调控基因转录而改变基因的表达.在体内外抗肿瘤试验中, decoy核酸有显著抑制肿瘤细胞增殖的作用,可以成为潜在性的肿瘤基因治疗药物.     

Chelating agent inhibition of Trypanosoma cruzi epimastigotes In vitro

A number of chelating agents and some of their derivatives are as effective as, or superior to, benznidazole, the compound currently in clinical use, in the suppression of the reproduction of epimastigotes of Trypanosoma cruzi, the protozoa that causes Chagas' disease. All compounds were examined at a culture concentration of 5 μg/mL. The most effective compounds included N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine, sodium diethylamine-N-carbodithioate, piperidine-N-carbodithioate and several of its analogs, a number of other carbodithioates with two nonpolar groups on the nitrogen, and tetraethylthiuram disulfide, a prodrug of sodium diethylamine-N-carbodithioate and widely used in the treatment of alcoholism. The introduction of additional ionic or nonionic polar groups on the chelating molecule generally results in a loss of tyrpanocidal activity. Common commercially available chelating agents which exhibited no activity included -penicillamine, meso-2,3-dimercaptosuccinic acid, and triethylenetetramine tetrahydrochloride. Dose-response data on the culture indicated that some of these compounds exhibited inhibition of Trypanosoma cruzi epimastigotes at concentrations as low as 0.625 μg/mL. It is proposed that the mechanism of action of these compounds is based on their ability to interface with the essential metal metabolism at intracellular sites of the epimastigote involving iron, copper, or zinc. The results also indicate that a certain degree of hydrophobicity may be necessary for the groups attached to the literal metal-bonding structure if the compounds are to successfully inhibit the epimastigotes of Trypanosoma cruzi. The development of antiprotozoal drugs which are chelating agents specifically designed to selectively disrupt the essential metal metabolism of Trypanosoma cruzi should furnish a new generation of drugs which can be used in the treatment of Chagas' disease.