白囊耙齿菌对茜素红脱色条件优化及其毒性变化检测

白囊耙齿菌Irpex lacteus是分离自倒木上的一株可以分泌漆酶和锰过氧化物酶的白腐真菌。利用I. lacteus对固体条件下的活性黑、活性红、结晶紫、茜素红和孔雀石绿进行脱色能力的检测,通过单因素和正交试验优化I. lacteus对茜素红的脱色条件,并以3种作物发芽率为指标测定茜素红被I. lacteus脱色前后的毒性变化。结果显示,I. lacteus对5种染料均可脱色,其中对茜素红染料的脱色更为彻底;单因素和正交试验优化I. lacteus对茜素红的脱色条件为：pH 7.0、葡萄糖10.0g/L、硫酸铵0.66g/L、接种量2片（Φ=8.0mm）、100.0mL三角瓶装液20.0mL,优化条件下I. lacteus对茜素红脱色10d时的脱色率为88.26%,与未优化前的脱色率相比提高了60.50%;茜素红染料被I. lacteus脱色前后毒性大小排序为：染料原液>染料脱色后>PDB培养基处理,表明茜素红染料存在一定的毒性,I. lacteus脱色茜素红后可以使其毒性减弱。通过本研究,为I. lacteus在茜素红等染料废水脱色以及降低染料废水毒性方面的应用奠定基础。     

A Note on the Purification of Alcian Blue

Alcian blue 8GX is a copper phthalocyanin dye that shows a high degree of specificity for polyanionic substances such as hyaluronic acid, sialic acid and the chondroitin sulfates. This dye has proved useful for both histochemical and electrophoretic staining of these substances. The Biological Stain Commission has recently begun to certify Alcian blue (Schenk 1981). Commercially available lots contain approximately 50% dye. The remaining constituents have been identified as primarily boric acid, as well as sulfates and dextrins (Scott 1972, Horobin and Goldstein 1972). Horobin and Goldstein (1972) have pointed out that these contaminants may adversely affect staining in the critical electrolyte concentration procedure. Scott (1972), while not ascribing any adverse effects to the presence of boric acid, recommends its removal by differential precipitation with acetone. In this procedure one part of a 2-5% aqueous solution of the dye is added to 5-10 parts of acetone. The precipitated dye is approximately 80% pure. While this method is relatively simple, it does have several drawbacks. Low concentrations of Alcian blue (i.e., 2%) must be used to obtain purities near 80%. Thus a minimum of 250 ml of acetone is needed to purify 1 gram of dye. Furthermore, Horobin and Goldstein (1972) have reported that contamination by dextrin or unknown organic substances (detergent?) interferes with precipitation of the dye enough to make purification by Scott's method impossible. When difficulty in the precipitation of Alcian blue by Scott's method was encountered, the following simple method for the purification of the dye was developed.     

Decolorization of textile indigo dye by ligninolytic fungi

The indigo dye is extensively used by textile industries and is considered a recalcitrant substance, which causes environmental concern. Chemical products used on textile processing, which affect the environment through effluents, can be voluminous, colored and varied. Vat textile dyes, like indigo, are often used and dye mainly cellulosic fibers of cotton. Decolorization of this dye in liquid medium was tested with ligninolytic basidiomycete fungi from Brazil. Decolorization started in a few hours and after 4 days the removal of dye by Phellinus gilvus culture was in 100%, by Pleurotus sajor-caju 94%, by Pycnoporus sanguineus 91% and by Phanerochaete chrysosporium 75%. No color decrease was observed in a sterile control. Thin layer chromatography of fungi culture extracts revealed only one unknown metabolite of Rf=0.60, as a result of dye degradation.     

Horseradish peroxidase-catalyzed oxidative coupling of 3-methyl 2-benzothiazolinone hydrazone and methoxyphenols

The reaction between o-, m-, and p-methoxyphenols and 3-methyl-2-benzothiazolinone hydrazone (MBTH) is studied in the presence of horseradish peroxidase (HRP) and H₂O₂ as oxidative agent. The findings indicate that enzyme (H₂O₂ oxidoreductase; EC 1.11.1.7) catalyzes an oxidative coupling reaction between MBTH and phenols which produces azo dye compounds. On the basis of kinetic parameters and optimum pH values, a mechanism in which both MBTH and phenols seem to be activated by the HRP for achieving the oxidative coupling is proposed. Furthermore, in the current study, we have evaluated the possibility that these azo dyes may be useful in the measurement of peroxidase activity. The method is based on the observed increase in the absorbance at 502 nm (8,355 cm^{−1 −1} of extinction molar coefficient) due to the formation of a red azo dye compound resulting from the peroxidase-catalyzed oxidative coupling of MBTH and o-methoxyphenol (guaiacol). Using this assay system, HRP can be determined in picomolar levels by a fixed time method.     

Laboratory Hints from the Literature

DYES AND THEIR BIOLOGICAL USES Burckhalter, J. H., Jones, E. M., Holcomb, W. F., and Sweet, L. A. N-substituted 2-methoxy-6-chloro-9-aminoacridines. J. Amer. Chem. Soc., 65, 2012. 1943.

Galat, Alexander. New processes for sulfanilamide. Ind. and Eng. Chem., Ind. Ed., 36, 192. 1944.

Kumler, W. D., and Daniels, T. C. The relation between chemical structure and bacteriostatic activity of sulfanilamide type compounds. J. Amer. Chem. Soc., 65, 2190. 1943.

Kwartler, C. E., and Lucas, Philip. The preparation of sutfanil-amidoindazoles. J. Amer. Chem. Soc., 65, 1804. 1943.

Mueller, A. C., and Hamilton, C. S. The synthesis of 1-substituted aminobenzo(f)quinolines. J. Amer. Chem. Soc., 65, 1017. 1943.

Popkin, A. H. Derivatives of biphenylsulfonamides. I. Preparation of p-(o-aminopnenyl)-benzenesulfonamide. J. Amer. Chem. Soc., 65, 2043. 1943.

Popkin, A. H., and Perretta, Gertrude M. Derivatives of biphenyl-sulfonamide. II. Derivatives of p-(o-aminophenyl)-benzenesulfonamide. J. Amer. Chem. Soc., 65, 2046. 1943.

Shreve, R. N., and Bennett, R. B. Studies in azo dyes. I. Preparation and bacteriostatic properties of azo derivatives of 2,6-diaminopvridine. J. Amer. Chem. Soc., 65, 2241. 1943.

Shreve, R. N., and Bennett, R. B. Studies in azo dyes. II. Preparation and bacteriostatic properties of azo derivatives of 8-quinolino. J. Amer. Chem. Soc., 65, 2243. 1943.

Siebenmann, C., and Schnitzer, R. J. Chemotherapeutic study of p-nitrobenzoyl- and related compounds. J. Amer. Chem. Soc., 65, 2127 1943.

ANIMAL MICROTECHNIC Barrett, A. M. A method for staining sections of bone marrow. J. Path & Bact., 56, 133-5. 1944.

Barrett, A. M. On the removal of formaldehyde-produced precipitate from sections. J. Path. & Bact., 56, 135-6. 1944.

Chang, Min-Chueh. Disintegration of epidymal spermatozoa by application of ice to the scrotal testis. J. Exp. Biol., 20, 16-22. 1943.

Ercoli, N., and Lewis, M. N. The age factor in response of bone tissue to alizarin dyes and the mechanism of dye fixation. Anat. Rec., 87, 67-76. 1943.

Hess, Manfred, and Hollander, Franklin. Permanent metachromatic staining of gastric mucus smears. J. Lab. and Clin. Med., 29, 321-3. 1944.

Miller, John A. A new method of staining nervous tissue. Ohio J. of Sci., 44, 31-5. 1944.     

Chemometric Methods to Quantify 1D and 2D NMR Spectral Differences Among Similar Protein Therapeutics

NMR spectroscopy is an emerging analytical tool for measuring complex drug product qualities, e.g., protein higher order structure (HOS) or heparin chemical composition. Most drug NMR spectra have been visually analyzed; however, NMR spectra are inherently quantitative and multivariate and thus suitable for chemometric analysis. Therefore, quantitative measurements derived from chemometric comparisons between spectra could be a key step in establishing acceptance criteria for a new generic drug or a new batch after manufacture change. To measure the capability of chemometric methods to differentiate comparator NMR spectra, we calculated inter-spectra difference metrics on 1D/2D spectra of two insulin drugs, Humulin R® and Novolin R®, from different manufacturers. Both insulin drugs have an identical drug substance but differ in formulation. Chemometric methods (i.e., principal component analysis (PCA), 3-way Tucker3 or graph invariant (GI)) were performed to calculate Mahalanobis distance (D _M) between the two brands (inter-brand) and distance ratio (D _R) among the different lots (intra-brand). The PCA on 1D inter-brand spectral comparison yielded a D _M value of 213. In comparing 2D spectra, the Tucker3 analysis yielded the highest differentiability value (D _M = 305) in the comparisons made followed by PCA (D _M = 255) then the GI method (D _M = 40). In conclusion, drug quality comparisons among different lots might benefit from PCA on 1D spectra for rapidly comparing many samples, while higher resolution but more time-consuming 2D-NMR-data-based comparisons using Tucker3 analysis or PCA provide a greater level of assurance for drug structural similarity evaluation between drug brands.     

撕裂蜡孔菌在开放体系中对甲基橙染料的静态脱色研究

为了评价撕裂蜡孔菌处理偶氮染料的应用潜力,用性能稳定的甲基橙染料为材料,采用批次试验在开放性体系中研究了染料初始浓度、菌丝生物量、温度、pH等因素对该菌脱色能力的影响,运用菌丝体反接、染液光谱扫描、菌丝体显微观察等方法探讨了菌丝体脱色的可能机制,利用植物萌发试验进行了染料和脱色后溶液的毒性测试。结果表明,撕裂蜡孔菌在开放的静止体系中能够对甲基橙高效脱色,其最适脱色温度为35℃,最佳脱色pH值在6左右。菌丝对甲基橙的脱色表现在吸附和产酶降解两个方面,脱色过程中染料对菌丝体本身的影响较少。植物毒性分析显示撕裂蜡孔菌脱色48h后的产物对植物的毒性比甲基橙本身更强,若要彻底降解可能需要较长时间。本研究可为染料脱色工艺提供新的菌种。     

Effects of fixation on routine,special and immunohistochemical stains: introduction to a symposium for the Biological Stain Commission

Orcein was first proposed as an elastic fiber stain by Taenzer in 1890, and has proved very useful for the purpose. It is an important constituent of the author's “Quad” stain. Unfortunately not all orcein samples have proved equally satisfactory, whether they are derived from the lichens from which the dye was originally prepared or have been manufactured by a synthetic process. At the present time several brands are available, and two of the brands of the synthetic product investigated have not proved to be the same thing; one of the latter proves only fair as an elastin stain, the other is one of the best samples the author has ever tried.     

真核表达猪白细胞介素17及其生物活性研究

目的:为真核表达猪白细胞介素17(IL-17),研究产物在细胞培养下的免疫生物活性。方法:通过PCR扩增出猪IL-17基因并插入到真核表达载体p VAX1,然后转染到IPEC-J2细胞、Ha Ca T细胞和L02细胞中。在转染后第24、48和72h收集细胞,第48h收集上清液。收集细胞通过实时荧光定量PCR检测相关免疫基因的表达水平,收集上清液通过抑菌试验检测相关抗菌肽的生物活性。结果:采用p VAX1载体构建了表达猪IL-17的重组质粒,转染到细胞中。证实IL-17基因能诱导抗菌肽基因(RegⅢ、S100A8和BD2)的表达,显著上调JAK-STAT信号通路基因(JAK1、STAT1和STAT3)和细胞因子基因(IL-6、IL-12和TNF-α)的表达。此外,细胞上清液能够在不同程度上抑制大肠杆菌及金黄色葡萄球菌的增殖。结论:成功将猪IL-17基因真核表达,其表达产物能诱导效应细胞表达多种细胞因子,产生多种抗菌肽,具有抑菌能力;这为进一步研发猪IL-17作为抗菌免疫分子制剂奠定了初步基础。     

Effects of arbuscular mycorrhizal fungi on calorific value and contents of carbon and ash in Robinia pseudoacacia

该试验以根内球囊霉(Glomus intraradices)和地表球囊霉(G. versiforme)为接种剂, 研究了丛枝菌根真菌对刺槐(Robinia pseudoacacia)生物量、热值、含碳量、灰分、能量积累和碳素积累的影响。结果表明, 接种根内球囊霉和地表球囊霉对提高刺槐生物量、热值、能量积累和碳素积累都起到了重要作用。接种根内球囊霉和地表球囊霉后刺槐的总生物量比对照分别增加了89.61%和91.34%, 能量积累分别比对照增加102.20%和94.19%, 碳素积累分别比对照增加93.30%和77.21%; 同时发现刺槐的能量和碳主要分布在根系和叶, 而茎中能量和碳所占的比例较小。接种根内球囊霉提高了刺槐的干重热值, 其根、茎、叶的干重热值分别比对照增加7.72%、8.94%和8.41%; 接种地表球囊霉也显著(p < 0.05)提高了刺槐的干重热值, 但其效果低于根内球囊霉。接种根内球囊霉显著(p < 0.05)提高了刺槐根的含碳量, 对茎和叶的含碳量影响不明显。接种根内球囊霉和地表球囊霉都显著(p < 0.05)提高了刺槐茎和叶的去灰分热值。     

Orcein and Elastic Fibers

Orcein was first proposed as an elastic fiber stain by Taenzer in 1890, and has proved very useful for the purpose. It is an important constituent of the author's “Quad” stain. Unfortunately not all orcein samples have proved equally satisfactory, whether they are derived from the lichens from which the dye was originally prepared or have been manufactured by a synthetic process. At the present time several brands are available, and two of the brands of the synthetic product investigated have not proved to be the same thing; one of the latter proves only fair as an elastin stain, the other is one of the best samples the author has ever tried.     

Enzymatic reduction and oxidation of fibre-bound azo-dyes

A new customer and environmental friendly method of hair bound dye decolouration was developed. Biotransformation of the azo-dyes Flame Orange and Ruby Red was studied using different oxidoreductases. The pathways of azo dye conversion by these enzymes were investigated and the intermediates and metabolites were identified and characterised using UV–vis spectroscopy, high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Laccase from Pycnoporus cinnabarinus, manganese peroxidase (MnP) from Nematoloma frowardii and the novel Agrocybe aegerita peroxidase (AaP) were found to use a similar mechanism to convert azo dyes. They N-demethylated the dyes and concomitantly polymerized them to some extent. On the other hand the mechanism for cleavage of the azo bond by azo-reductases of Bacillus cereus and B. subtilis was based on reduction of the azo bond at the expense of NAD(P)H.     

Water Relations of Beetroot Seed Germination: I. Microbial Factors, with Special Reference to Laboratory Germination

Laboratory test results obtained with two seed lots of the cultivarDetroit Globe have shown: (1) 8 ml of water in a 9-cm diameterpetri-dish are excessive for good germination of 25 two-seededclusters, either natural or ‘rubbed’, which germinatewell in 3 ml of water. (2) Some of this depression in germinationis due to a ‘direct’ effect of the excessive water-supply.(3) But the benefit from either surface sterilization or theuse of aureomycin, a bacteriostatic antibiotic, indicates thatsome of the depression of germination in the presence of excesswater is the result of bacterial colonization of the seed clusters.The excess water effect can also be mitigated by preliminary‘rubbing’ of the seed clusters, presumably by removalof some of the bacterial inoculum, provided that the rubbingprocess itself does not injure the seeds. (4) There is goodevidence that the bacteria depress the germination of the seedsby competing with them for oxygen. (5) The effect of the bacterialpopulation on germination in excess water can be aggravatedby preliminary washing of the seed clusters. This is probablydue to a delicate equilibrium between the bacteria and at leastpart of the fungal population of the seeds which when retainedmay produce an antibacterial substance; but the water-solublephenolic acids contained in the seed clusters may also be involved:at high concentrations these inhibit germination, but they mayalso depress bacterial activity, even at lower than germination-inhibitingconcentrations. (6) Fungicides aggravate the depression of germinationon over-wet paper in the laboratory, but are of value in thefield. (7) There is evidence that different limiting factorsmay operate on different seed lots and care must be taken notto generalize from results obtained from a limited number ofseed samples.     

Quinacrine Mustard—a Selective Fluorescent Stain for the Y Chromosome in Human Tissues for Routine Cytogenetic Screening

A simple sensitive and specific cytochemical method for detection of the Y chromosome in man (George 1970, Pearson et al. 1970) is based on staining by alkylating fluorochromes. The dye binds strongly to the heterochromatic regions of the Y chromosome, thus causing these regions to fluoresce very brightly under ultraviolet (uv) light. The Y chromosome could be identified easily at metaphase and also as a highly fluorescent body in interphase cells. Two bodies were seen by Pearson et at. in a subject having the XYY anomaly. In prophase cells, it appears as a fluorescing chromatin thread. Its presence has been demonstrable only in males; hence the usefulness of this technique for rapid cytogenetic screening is obvious. The constancy and reproducibility of the technique seems to rule out the possibility of unreliability caused by nonspecific binding of the dye. The present report gives techniques applied to tissues usually studied in cytogenetic screening     

刺芹侧耳产漆酶条件优化及对偶氮染料甲基橙的脱色

漆酶是一种含铜的单电子多酚氧化酶,能够催化氧化各种酚类及多种染料,在处理染料废水方面具有巨大的潜力。刺芹侧耳Pleurotus eryngii具有较强的产漆酶能力,但漆酶产量在较大程度上受环境条件限制。本文研究了氮源含量、pH、温度、金属离子等环境条件对刺芹侧耳产漆酶能力的影响,优化了其产漆酶条件,并用其粗酶液对典型偶氮类染料甲基橙进行脱色,结果表明,在氮源0.5%（W/W）、pH 5.5、温度28℃、添加5.0mmol/L Mg ²⁺的培养条件下,刺芹侧耳产漆酶能力最强,培养6d时,漆酶酶活可达78.0U/L。用优化培养的刺芹侧耳粗酶液对偶氮染料甲基橙进行脱色,28h后脱色率可达90%,脱色反应为准一级动力学反应,甲基橙并未完全矿化,而是生成小分子中间产物。     

银耳菌糠黑色素的理化性质及抗氧化和抑菌活性

银耳菌糠中存在一种银耳的伴生菌——炭团菌(俗称香灰菌),其菌丝生长到一定阶段后会产生大量的黑色素,具有广泛的应用价值。本研究从银耳菌糠中提取黑色素,研究其理化性质、抗氧化活性及抑菌作用。通过紫外-可见光谱、傅里叶红外光谱对提取的黑色素进行鉴定,表明银耳菌糠黑色素具有黑色素的典型特征。通过对银耳菌糠黑色素理化性质的研究,表明银耳菌糠黑色素是一种趋于黑色并略带红色和黄色的粉末;该黑色素易溶于碱性溶液;具有较好的热稳定性和光稳定性,其稳定性受氧化剂和还原剂的影响较小,受Ca²⁺、Cu²⁺的影响较明显。通过总抗氧化能力(FRAP法)、自由基清除能力检测银耳菌糠黑色素的抗氧化活性,研究表明黑色素具有较高的抗氧化活性,羟自由基、ABTS自由基清除的EC₅₀值分别为0.429 mg/mL和0.016 mg/mL。本研究还检测了黑色素对革兰氏阳性、革兰氏阴性细菌的抑菌能力,结果表明该黑色素在浓度为3.2 mg/mL时对供试菌株的抑菌率超过90%,且对革兰氏阳性菌会较敏感。本研究为银耳菌糠的有效利用及其黑色素产品的开发提供了理论基础,具有较高的经济价值。     

Thermo-alkali-stable catalases from newly isolated Bacillus sp. for the treatment and recycling of textile bleaching effluents

Three thermoalkaliphilic bacteria, which were grown at pH 9.3–10 and 60–65 °C were isolated out of a textile wastewater drain. The unknown micro-organisms were identified as thermoalkaliphilic Bacillus sp. Growth conditions were studied and catalase activities and stabilities compared. Catalases from Bacillus SF showed high stabilities at 60 °C and pH 9 (t_1/2=38 h) and thus this strain was chosen for further investigations, such as electron microscopy, immobilization of catalase and hydrogen peroxide degradation studies. Degradation of hydrogen peroxide with an immobilized catalase from Bacillus SF enabled the reuse of the water for the dyeing process. In contrast, application of the free enzyme for treatment of bleaching effluents, caused interaction between the denaturated protein and the dye, resulting in reduced dye uptake, and a higher color difference of 1.3 ΔE* of dyed fabrics compared to 0.9 ΔE* when using the immobilized enzyme.     

网隙裂粉韧革菌不同生长时期转录组分析

网隙裂粉韧革菌Amylostereum areolatum是松树蜂Sirex noctilio携带并传播的共生真菌,与松树蜂之间存在严格的互利共生关系,其正常生长发育是松树蜂完成生活史的关键因子之一。为研究该共生菌生长发育的相关机制,我们对共生菌菌丝最大生长速率前期（7d）和后期（12d）样本进行转录组测序,在转录水平上分析差异表达基因在共生菌生长发育中的功能。结果显示,两个不同生长时期共有差异基因2 425个,其中在共生菌最大生长速率前期样本中上调的基因有946个,下调的有1 479个。Nr注释和GO功能富集分析结果表明,共生菌最大生长速率前期的差异表达基因主要与碳水化合物代谢、蛋白质合成以及水解酶活性相关。Pathway富集分析表明,差异表达基因显著富集在糖酵解/糖异生代谢通路上,并且这些基因在共生菌最大生长速率前期的样本中显著上调,表明其可能在网隙裂粉韧革菌的生长发育中发挥重要作用。通过分析两个不同生长时期共生菌基因的表达情况,挖掘参与共生菌生长发育的关键基因,旨在为探索松树蜂与其共生菌的互利共生机制提供理论基础。     

Staining Rickettsiae in Yolk-Sac Cultures

Rickettsiae in yolk sacs are not stained well by the Macchiavello technique, and experiments were undertaken to understand the mechanisms involved. It was found that the citric acid destaining step was not effective and that most of the basic fuchsin was lost from the rickettsiae during the application of methylene blue, another basic dye. A staining technique was then evolved with carbol basic fuchsin in pH 7.45 phosphate buffer (0.4% dye, 0.4% phenol, 0.07 M buffer), followed directly by 0.8% aqueous malachite green oxalate. This technique worked well for R. mooseri, R. prowazeki, R. rickettsii, R. akari, and R. buretii, but for R. tsutsugumushi a modification was needed, whereby 4% aqueous Fe(NO₃)₃·9H₂O was used as destaining solution, and 0.5% aqueous fast green as the counter-stain.     

瓜类细菌性果斑病菌拮抗细菌的筛选及其抑菌作用

从土壤中筛选出抑制哈密瓜细菌性果斑病的拮抗菌株,明确其分类地位,测定其生长条件及防病效果。采用梯度稀释法及平板对峙法进行菌株的分离及筛选,综合形态学观察、生理生化测定以及分子鉴定等多方面对菌株进行鉴定,确定生防菌种属,并测定其最适生长条件,利用平板对峙法测定其抑菌作用,并分别采用针刺接种法、叶面喷施法对生防菌的离体叶片和盆栽防病试验进行测定。本研究从土样中共分离出196株细菌,经对峙筛选发现有20株细菌具有抑菌效果,其中编号为131、791的两株细菌对哈密瓜细菌性果斑病病原菌具有较好的平板拮抗效果,抑菌圈直径分别为（19.03±0.13）mm,（17.55±0.29）mm。经鉴定菌株131为沙福芽孢杆菌（Bacillus safensis）,菌株791为贝莱斯芽孢杆菌（B. velezensis）。菌株131的最适生长条件为NB培养基,培养18 h,温度37℃,pH 7,接种量为2%。菌株791的最适生长条件为KB培养基,培养18 h,温度37℃,pH 6,接种量为1%。菌株131和791对哈密瓜细菌性果斑病的离体叶片防效分别为83.33%和87.53%,室内盆栽防效分别为69.86%和77.99%,同时也对多种病原真菌具有抑制作用。菌株131和791均为对哈密瓜细菌性果斑病具有较好防效的芽孢杆菌（Bacillus）,对瓜类作物细菌性果斑病的防治提供了理论补充,奠定了果斑病害生物防治的基础。