中国多变根毛霉感染性皮肤接合菌病13例回顾性分析

目的总结我国自1991年至今报道的所有多变根毛霉感染所致皮肤接合菌病的临床资料,探讨其流行病学特点、临床特征和治疗。方法文献搜索并回顾分析我国13例多变根毛霉原发性皮肤感染的临床资料。结果 13例皮肤多变根毛霉病,平均发病年龄为34±17(5～65)岁,男6例,女7例,病程5个月～16 a不等,感染报道多集中于长江流域;临床多表现为局部红斑、丘疹和/或结节,可缓慢扩展形成浸润性斑块伴溃疡坏死;发病前有明确皮肤损伤病史6例,无明确诱因7例。皮损多发生于暴露部位,面部10例,上肢3例。所有病例均经真菌学和病理学诊断确诊。治疗主要为两性霉素B和唑类抗真菌药物,其中8例治愈,2例疗效不佳,3例失随访。结论多变根毛霉皮肤接合菌病是一种重要的毁容性感染病,多与皮肤屏障损伤有关,提高对本病的认识有利于开展早期诊断和及时治疗,治疗首选两性霉素B。     

一种引致人体原发性皮肤毛霉病的非高温根毛霉属真菌

由石家庄中国人民解放军白求恩国际和平医院皮肤科李成龙大夫提供的一株人体皮肤病菌经我们研究鉴定为毛霉目(Mucorales)毛霉科(Mucoraceae)根毛霉属(Rhizomucor)的一个种,并定名为多变根毛霉新种(Rhizomucor variabilis Zheng & G.-q.Chen sp. nov.)。据李大夫介绍,这株菌是从一名居住在江苏农村到该医院看病的女病人的手上病部分离的。这个病人没有一般真菌病病人所患有的其他疾病如糖尿病、白血病等等。她也没有患有其他毛霉病,因此她的皮肤毛霉病是原发性的而不是继发性的。我们查阅文献结果,国内由根毛霉属真菌引致的毛霉病过去仅有过一次肺部感染的报道;国外则有过较多次数的由根毛霉引致的人体毛霉病,主要为肺部疾病并可引致继发性的皮肤病,尚未见有由根毛霉属引起的原发性皮肤毛霉病的报道。无论国内、外引起人体毛霉病的根毛霉均为微小根毛霉[Rhizomucor pusillus(Lindt) Schipper,包括Mucor pusillus Lindt,Mucor parasiticus Lucet & Costanin等异名]一种。因此,本病例为我国第二例由根毛霉弓l起的人体毛霉病及第一例由根毛霉引起的人体皮肤毛霉病,同时又是全世界第一例由根毛霉属除微小根毛霉以外的另外一个种引起的人体毛霉病,很可能还是全世界第一例由根毛霉引起的人体原发性皮肤毛霉病。多变根毛霉与根毛霉属内所有过去已报道过的种都有显著差异。它的最适生长温度为24-30℃,最低9℃,最高38℃;其他种均为高温真菌,它们的最高生长温度可达55℃或更高。形态方面,多变根毛霉也与属内其他已知种明显不同。它的菌落高达4-8 mm并呈鲜明的浅黄色;其他已知种菌落低矮,除奈尼塔尔根毛霉(Rhizomucor nainitalensis Joshi)外全部为深暗灰色,奈尼塔尔根毛霉菌落色泽虽然较浅,但为浅灰或灰黄色,与多变根毛霉的鲜明黄色不同。它的假根异常发达并可从菌体的各个部位如菌丝、匍匐丝、孢子枝、孢子囊、囊轴上长出;其他已知种的假根一般都不发达且从未见有从孢子枝、孢子囊、囊轴等处长出的描述。它的孢子枝的分枝常常长于主枝;其他已知种则分枝长度一般不超过主枝。它的囊轴形状多变:球形、近球形、扁球形、卵形、椭圆形、梨形等等,两边对称或不对称,溢缩或不绕缩,纵向深裂或不作纵向深裂;其他已知种的囊轴形状通常为倒卵形至梨形的规则形状。它的囊领明显;其他已知种的囊领均很不明显至缺如。它的孢囊孢子形状和大小变化都较大,卵形、椭圆形、矩圆形、近球形、近三角形或其他各种不规则形状,长度范围2.5-16.5 μm;其他已知种除上面已经提到过的奈尼塔尔根毛霉外,它们的孢囊孢子形状仅限于卵形、椭圆形、近球形等较规则的形状,长度范围总是在3-6 μm范围内,奈尼塔尔根毛霉的孢囊孢子形状虽然多变,但其决度亦在3-6 μm的范围内。此外,多变根毛霉的孢子囊、囊轴、孢囊孢子等各种构造均较大;其他已知种则较小,其中肿梗根毛霉[Rhizomucor tauricus (Milko & Schkurenko) Schipper]虽亦较大,但除此之外与多变根毛霉迥异。多变根毛霉未见有接合孢子,将我们保存的全部微小根毛霉菌株与它分别配对时,或将我们的几对别的属的(+)(-)测试菌株与它分别配对时,均未见形成接合孢子或有任何反应。     

泊沙康唑对临床来源接合菌的体外抗菌活性研究

目的研究泊沙康唑对临床来源的接合菌体外抗菌活性。方法对临床来源的43株接合菌采用ITS区序列分析进行准确鉴定,采用E-test纸片法研究泊沙康唑对43株菌的体外药物敏感性,观察其MIC值。结果经ITS序列分析鉴定,43株临床来源接合菌中最为常见的是小孢根霉和不规则毛霉(各12株),其次为米根霉(10株),ramosa(3株)。其他菌种分别为ornata、总状共头霉、微小根毛霉、卷曲毛霉、印度毛霉和冻土毛霉各1株。E-test纸片法测得泊沙康唑对毛霉属、根霉属和Lichtheimia spp.的MIC值范围分别为0.19～>32μg/mL、0.19～3μg/mL、0.002～0.38μg/mL,对总状共头霉和微小根毛霉的MIC值分别为1μg/mL和0.19μg/mL。结论泊沙康唑对大部分接合菌有效,不同种属MIC值范围不同,毛霉属真菌的MIC值差异较大。     

裴氏着色霉致着色芽生菌病1例并文献复习

报道1例裴氏着色霉致左手背及腕部着色芽生菌病,通过文献复习统计并分析该病在我国的流行病学及临床特点.患者男,48岁,左手背部红斑、疣状增生伴瘙痒10 a.皮损组织病理检查示真皮中下层有炎细胞及多核巨细胞,PAS染色见硬壳小体.经真菌培养和分子鉴定为裴氏着色霉.经伊曲康唑600 mg每日口服,治疗3个月后好转.     

利用Sed1p锚定蛋白在毕赤酵母表面展示米黑根毛霉脂肪酶及其应用

通过PCR扩增米黑根毛霉脂肪酶基因,在米黑根毛霉脂肪酶N端加入Flag标签。将米黑根毛霉脂肪酶基因与酿酒酵母细胞壁蛋白Sed1p基因的N端融合构建质粒pPIC9K-Flag-RML-Sed1,转化毕赤酵母GS115获得重组菌GS115/pPIC9K-Flag-RML-Sed1。重组菌经过甲醇诱导表达后,显微镜免疫荧光分析与流式细胞仪检测结果均证实米黑根毛霉脂肪酶已经成功展示在毕赤酵母上。该重组菌水解活力达到169.6U/g(Dry cell weight),在非水相中催化脂肪酸甲酯的合成,72h后脂肪酸甲酯的产率达82.36%。     

中国新记录巴克斯霉属Backusella的两个分类单元(英文)

最近在中国十个省市发现了一个新记录属——巴克斯霉属Backusella,包括一个种的两个变种,即卷曲巴克斯霉原变种B.circina var.circina和卷曲巴克斯霉改级光孢变种B. circina var. lamprospora stat. nov. ≡ B. lamprospora。文章对这两个变种提供了形态描述和线条图,还提供了属内区分所有可以承认的分类群的检索表。把这两个分类群从种级降为变种级的理由亦有所讨论。ITS rDNA序列分析表明,卷曲巴克斯霉是一个多系类群,与放射毛霉属Actinomucor、毛霉属Mucor和根毛霉属Rhizomucor有着密切的谱系关系。这一结果暗示目前的卷曲巴克斯霉种的概念较大,可称为广义卷曲巴克斯霉B.circina s.l.;如果仅仅依靠分子数据,很有可能给出错误的分类学鉴定结果。     

微小根毛霉感染机理研究

本文在首次报告二例肺结核、糖尿病患者合并肺微小根毛霉的基础上,进一步探讨免疫功能受损与微小根毛霉关系。本文采用不同剂量＾６０Ｃｏ－γ全身辐照小鼠,然后以不同途径注射同剂量的微小根毛霉的孢囊孢子,观察动物的感染情况和感染后的真菌检出率,结果发现各种辐射剂量均在辐射后７－１４日感染菌的检出率最高;各种脏器感染菌的检出率以脾脏最高;淋巴结最低;其他脏器的感染菌检出率也有不同程度的差异。     

微小根毛霉感染机理研究

本文在首次报告二例肺结核、糖尿病患者合并感染肺微小根毛霉的基础上,进一步探讨免疫功能受损与微小根毛霉感染关系。本文采用不同剂量~(60)Co-γ全身辐照小鼠,然后以不同途径注射同剂量的微小根毛霉的孢囊孢子,观察动物的感染情况和感染后的真菌检出率,结果发现各种辐射剂量均在辐射后7-14日感染菌的检出率最高;各种脏器感染菌的检出率以脾脏最高(66.7—81.8％);淋巴结最低(0.0—25.0％);其他脏器的感染菌检出率也有不同程度的差异。     

又一种引致人体原发性皮肤毛霉病的非商温根毛霉属真菌

一年多以前,我们报道了一种引致人类原发性皮肤毛霉病的病原真菌Rm6并定名为多变根毛霉((Rhizomucor variabilis Zheng & G.-q. Chen)。最近我们又发现了它的另外一个变种Rm7。它是北京医科大学第一医院的王端礼教授分离的,原编菌号4873。Rm7的病人和Rm6的情况很相近。她也是一个女病人,来自农村,患的同样是原发性皮肤病而没有患有其他的毛霉病或其他疾病,她的病也没有传染给她的家人。不同的是,她来自我国北部的河北省而不是南部的江苏省;她的病史更长,达16年之久;她的病部在脸上而不是在手上,并造成进食困难。Rm7和Rm6的培养物相似,也是亮黄色,但比较低矮。它与Rm6一样是非高温真菌,最适、最高、最低温度同样分别是24-30, 38,和9℃形态上的亲缘性也是显而易见的:两者都有匍匐菌丝和较发达的假根;孢子枝也是假轴分枝;孢子囊壁也是缓慢消解;有很不规则形状的囊轴;有明显的囊领;都有大量厚垣孢子。Rm7与Rm6的主要区别在于:(1)假根只从匍匐菌丝和气生菌丝上长出而不从孢子枝、孢子囊或囊轴等各处长出;(2)孢子枝可连续分枝达7次之多;(3)孢子枝在分枝处往往有一个隔膜;(4)往往在10天以上的培养物中才出现不规则形状的囊轴;(5)从未见有囊托;(6)孢囊孢子形状较规则,较短而不超过11 μm;(7)孢子囊和囊轴均较小,直径相应不超过70及30 μm.因此,我们将Rm7鉴定为Rm6的一个变种,并定名为较规则多变根毛霉新变种(Rhizomucor variabilis var. regularior Zheng & G.-q. Chen var.nov.)。这样,在根毛霉属内可以承认的共计5种6分类群。     

伊曲康唑联合特比萘芬治愈着色芽生菌病1例

报告1例由卡氏枝孢瓶霉引起的着色芽生菌病。患者男性,43岁,因右上臂局限性红色斑块性皮损5 a就诊。皮损组织病理为慢性炎性肉芽肿改变,可见硬壳细胞。真菌培养鉴定为卡氏枝孢瓶霉。给予伊曲康唑(200 mg/d)和特比萘芬(250 mg/d)联合治疗12周痊愈。     

Diagnostic Alternatives Used by Veterinary Surgeons for the Recording of Udder Diseases in the Norwegian Health Card System for Cattle

A questionnaire, in which 7 cases of udder disease were described, was distributed to 890 veterinarians in Norway. They were requested to classify the cases according to the diagnostic alternatives listed in the Norwegian Health Card System for Cattle (NHCSC). The NHCSC recordings are used for progeny testing of bulls, for disease monitoring, and for research purposes. The aim of this study was to evaluate the quality of the recordings for udder diseases. The questionnaire was answered by 633 veterinary surgeons. Four cases of clinical mastitis (CM) with abnormal secretion as well as other clinical signs of inflammation were correctly classified as CM by almost 100% of the veterinary surgeons. A cow in the final stage of lactation, showing no clinical signs except for clots in the milk, was considered not to be a clinical case by more than 25% of the veterinary surgeons. A typical case of subclinical mastitis (SM) was reported as SM by 83% of the veterinary surgeons, and as CM by 16%. A subclinical case with a recent history of clots in the secretion was classified as SM by 66% of the veterinary surgeons, but almost 40% either reported CM as their sole diagnosis or considered the case to be CM in combination with SM. Of the clinical cases, those exhibiting marked local signs of inflammation and a systemic reaction were correctly classified as acute clinical mastitis (ACM) by 96%–98% of the veterinary surgeons. In the NHCSC, the diagnostic alternatives for cases of CM are ACM and chronic clinical mastitis (CCM). One case, for which the diagnosis subacute clinical mastitis was appropriate according to standard definitions, was classified as CCM by 66%), and as ACM by 6%. Based on the information given in the questionnaire, the diagnosis for 2 of the clinical cases could have been either ACM or CCM, and for both cases each of these 2 alternatives was reported by more than 43% of the veterinary surgeons. A teat lesion, which was present together with ACM in one cow, was reported by 91% of the veterinary surgeons.     

Effect of capping agent on the particle size of CdSe nanoparticles

CdSe nanoparticles were synthesized by green route and chemical route methods. In the green route method the samples were capped by starch and in the chemical route method the samples were capped by mercaptoacetic acid (MAA). The samples were characterized by powder X‐ ray diffraction (XRD) and transmission electron microscopy (TEM). Both the samples showed zinc blend structure. The optical absorption spectra and Fourier transform infrared (FTIR) spectra were also studied. A blue shift was seen in the absorption spectra as compared with the bulk as well as the sample capped by starch. TEM images showed agglomeration for the starch‐capped sample as compared with the MAA‐capped sample. The particle size for the sample capped by MAA was found to be less as compared with the starch‐capped sample. A blue shift in the photoluminescence (PL) spectra was also recorded for the samples prepared by the chemical route as compared with the sample prepared by the green route as well as the bulk. The PL peak shifted towards the red side and increase in the peak intensity occurred with the change in the excitation wavelength. Change in PL intensity was observed with different pH at 685 nm. Copyright © 2016 John Wiley & Sons, Ltd.     

Purification and characterization of UDP-D-galactose 4-epimerase from the red alga Galdieria sulphuraria

UDP-D-galactose 4-epimerase of the unicellular red alga Galdieria sulphuraria has been purified to apparent electrophoretic homogeneity by chromatography on DEAE-Fractogel, hydroxylapatite and by affinity chromatography on Dyematrex Orange. The holoenzyme is a homodimer with an apparent molecular mass of 83 and 76 kDa as determined by gelfiltration and by sucrose gradient centrifugation, respectively. The size of the subunits was 42 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 4-epimerase from G. sulphuraria does not require external NAD for activity, unlike the enzyme from some other organisms, and inhibition by NADH was not observed. The apparent K_m for UDP-D-galactose was 64 μ M . The pH optimum was at 8 and the apparent equilibrium constant for UDP-Glc/UDP-Gal was 3.5. The enzyme in crude as well as in purified samples was unusually stable and was not inactivated even on incubation at 46°C for several hours.     

金黄色葡萄球菌肠毒素A的基因克隆、表达及活性试验

利用PCR从金黄色葡萄球菌标准株（Staphylococcus aureus, ATCC13565）中克隆了金黄色葡萄球菌肠毒素A(SEA)的基因,序列测定结果与报道完全一致。构建了表达载体pETSEA并获得高效表达,重组蛋白（rSEA）在37℃诱导时以包涵体形式存在,降低温度则出现可溶表达,可溶性rSEA占总rSEA的55%。可溶性rSEA经Ni²⁺亲和层析纯化,达电泳纯。通过同源模建对rSEA对SEA进行结构比较,结果表明尽管rSEA比野生型SEA多了9个氨基酸但其结构并没有明显的变化。单核细胞增殖试验进一步证明了该结论：将rSEA与SEA同外周血单个核细胞共同培养,两者均能有效地促进其增殖。将rSEA与体内激活的脾细胞共培养,则能增强脾细胞的体外抑瘤活性。     

Fluorescence immunolocalization of bound atrazine residues in plant tissue

Bound atrazine was detected inElodea canadensis by an improved immunohistochemical fluorescence procedure using anti-triazine antibodies from rabbits, biotin-labelled anti-rabbit immunoglobulin G and streptavidin-phycoerythrin conjugate. Whereas no labelling was found in control plants grown in charcoal-filtered, atrazine-free water, the labelling of plants obtained from their natural habitat and grown in tap water was sometimes nearly as high as in samples loaded with atrazine. The efficiency of the immunofluorescence procedure was compared using several antisera obtained by immunizing with different hapten conjugates and purified by various purification methods. The best results were observed with the atrazine analogue ametryn sulfoxide, which was coupled to bovine serum albumin for immunization and to Sepharose for immunoaffinity chromatography. The procedure described in this paper may serve as a general tool for detecting bound pesticide residues in plant material. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

A sulfone group-labeled TEM-DNA probe: comparison with a 32P-labeled probe in dot-hybridization

A non-radioactive DNA probe for the TEM-type beta-lactamase gene was obtained by using the 'Chemiprobe' system. It was used along with a 32P-labeled TEM probe to screen for TEM beta-lactamase gene in 107 bacterial isolates representing 7 Gram-negative genera and previously classified as TEM-positive or negative. The DNA to be tested was extracted from these bacterial isolates by the Birnboim-Doly method and, after blotting into charged nylon membranes, it was submitted to hybridization with either the TEM 'Chemiprobe' or the 32P-TEM probe. The TEM 'Chemiprobe' could detect as few as 25 pg specific DNA if it was used at a concentration of 5 ng per cm2 of membrane. The results obtained by both probes were concordant in 93.5% of the entire sample. The TEM 'Chemiprobe' was specific since only one false positive was observed. Furthermore, it appeared at least as sensitive as the 32P-labeled TEM probe. As the dot-hybridization with the sulfone-labeled probe was sensitive, simple and easy to perform, it will be useful for large-scale screening in clinical laboratory.     

Successful infection of the common marmoset (Callithrix jacchus) with human varicella-zoster virus.

The common marmoset, Callithrix jacchus, can be infected with human varicella-zoster virus (VZV), both wild-type strain KMcC and attenuated vaccine strain Oka/Merck. Infection was accomplished with either whole-cell-associated or cell extract VZV by combined oral-nasal-conjunctival application and was characterized by substantial and persistent anti-VZV antibody responses. The infectivity of VZV for marmosets was destroyed by treatment of inocula with heat or UV light. Diluted inocula with as few as 40 PFU/ml were infectious for marmosets. The lungs were demonstrated to be a major site of viral replication; both the presence of viral antigens and signs of pneumonia were demonstrated in lung tissues. Four serial passages of VZV KMcC were carried out in C. jacchus by a process of in vitro isolation and culturing of VZV from infected lung tissue and reapplication of the cultured isolates to fresh animals. The isolated viruses were identified as VZV both serologically and by restriction endonuclease analyses. The C. jacchus infectivity model should prove useful for determining the efficacy of subunit and live recombinant VZV vaccines as well as for the study of zoster.     

Peroxidatic activity in heart effluent: a biochemical parameter for the assessment of experimental ischemia-reperfusion injury

Peroxidatic activity in heart effluent was defined as a new biochemical parameter for the experimental study of myocardial ischemia. The peroxidatic reaction was determined by dot blot analysis with 3,3'-diaminobenzidine as hydrogen donor. After ischemia, the level of peroxidatic activity in heart effluent was 2-3 times higher than before. The effects in experimental modulation of ischemia, such as nicorandil or aprikalim protection, and the reversibility of protection by glibenclamide, could accurately be noted using the level of peroxidatic activity in heart effluent as a biochemical parameter. The results were in good agreement with those obtained for other enzymes used as biochemical parameters in experimental heart ischemia-reperfusion studies.     

Proteases in germinating finger millet (Eleusine coracana) seeds

Proteolytic activity was estimated in germinated finger millet seedlings using the endogenous trypsin/amylase inhibitor as substrate and also with haemoglobin and albumin as substrates. The maximal proteolytic activity was observed on the third day of germination. With the inhibitor as substrate, the proteolytic activity was maximal at pH 2.5. The protease that acted on the inhibitor required sulphydryl groups for maximal activity and was suppressed by diazoacetyl norleucine methyl ester and Pepstatin. The protease that acted on haemoglobin with optimum pH of 5.0, was more stable on storage, did not depend on sulphydryl groups for activity and was unaffected by reagents that react with carboxyl groups.     

Novel heterobifunctionalized polystyrene-polyethylene glycol resin for simultaneous preparation of free and immobilized peptides and biological activity detected by confocal microscopy

Summary Fast and convenient binding assays using synthetic peptides are of utmost and increasing importance, especially in the search for lead structures or in the field of diagnostics. A polymeric support suitable for solid-phase peptide synthesis was functionalized with two different anchor groups. The interior part of the aminomethylated polystyrene-1%-divinylbenzene resin beads, comprising about 98% of the total loading capacity, was modified by the acid-labile ADPV anchor whereas the 2% outer surface of the polymer was covalently coated with a PEG 10 000 derivative which renders the resin surface hydrophilic and biocompatible. The novel resin was characterized by introducing marker amino acids and by infrared spectroscopy. Employing this bifunctionalized resin for peptide synthesis, free as well as polymer-bound peptides were obtained which were tested for recognition by antibody. The resin-bound peptides proved to be suitable for ELISA and fluorescence assays, as shown by confocal laser microscopic investigations. Peptides from the interior part were obtained in high yield and purity as analyzed by HPLC, electrospray mass spectrometry and Edman degradation.