Studies on Aspergillus Fumigatus; Properties of Intracellular Proteinase

Mycelial filtrates from Aspergillus fumigatus (AF) hydrolyzed protein substrate buffered at various pH values. Using casein as substrate there were distinct activity optima at pH 2.9, pH 6.2, and pH 10, with maximum activity at pH 6.2. Using haemoglobin as substrate there were activity optima at pH 3.6, pH. 4.6, and pH 10, with the biggest activity peak at pH 4.6.The pH stability at 4°G of the caseinase activity at pH 6.2 and pH 10 was strongest at pH 4, common to both, whereas the caseinase activity at pH 2.9 showed maximum pH stability at pH 6—7.The casein hydrolyzing activity at pH 2.9, pH 6.2, and pH 10 showed different optimum incubation temperatures and irregular heat inactivation.Normal rabbit serum inhibited the caseinase activity at pH 2.9 and pH 6.2 to some extent. The caseinase activity at pH 10 was almost completely inhibited. Antiserum against mycelial filtrate showed no definite inhibition beyond that exerted by normal serum.Following electrophoresis of antiserum, the presence of specific neutralizing antibodies against the casein precipitating enzyme of mycelial filtrate from AF could be established. Investigations of 14 AF strains showed immunological uniformity with respect to the casein precipitating enzyme.     

Immunological characterization of protective antigens prepared by alkaline treatment of whole cells and from the culture filtrate of Erysipelothrix rhusiopathiae.

Culture filtrate and alkaline-extracted antigens from whole cells of an attenuated strain of Erysipelothrix rhusiopathiae (strain Koganei: serovar 1a) were fractionated with ammonium sulfate; both induced protective immunity in mice. Sephadex G-200 gel filtration revealed three protein fractions in the alkaline-extracted antigen and four protein fractions in the culture filtrate antigen. A fraction in the alkaline extract (NaOH P-2) and in the culture filtrate (CF P-2) induced protection in mice against challenge with a different serovar strain (strain Agata: serovar 5). Anti-NaOH P-2 and anti-CF P-2 mouse sera were protective against different serovars. Glycoprotein fraction derived from CF P-2 antigen by affinity chromatography with Con A-Sepharose 4B did not show protective activity. Western blotting between the antisera (anti-NaOH P-2, Anti-CF P-2 and anti-Koganei strain) and the antigens (NaOH P-2, and sonicated antigens of Agata, Fujisawa and Koganei strains) showed strong recognition of the same bands at 62, 42 and 41 kDa.     

Studies of Aspergillus fumigatus; casein precipitating and proteolytic effects of mycelial filtrate

Schäffer (1900) and Butkewitch (1903) seem to have been the first to focus attention on the proteolytic activity of micro-fungi. The occurrence and properties of proteolytic enzymes from various fungus species were subsequently studied by several authors. Literature in this field was reviewed by e.g. Ito (1950), Gorbach & Koch (1955), Koch & Dedic (1957), Hagihara (1960), Davies (1963) and Roper & Fennell (1965).In connection with investigation of the proteolytic activity of several fungus species, a gelatin hydrolyzing effect of Aspergillus fumigatus (AF) was reported to be exerted by living organisms in pure culture (Jensen 1931), extracted mycelial material (Maxwell 1950, Dingle & Solomons 1952), and by fluid culture medium in which AF was cultured (Dion 1950a, b, Dingle & Solomons). Ay res & Tobie (1943) demonstrated moderate casein hydrolyzing activity in extracted mycelial material from 4 AF strains, and Amatayakul (1955) observed low fibrinolytic activity in 1 strain.Proteolytic activity measured by breakdown of gelatin and casein was demonstrated by Jonsson & Martin (1964) in culture medium in which AF had been cultured. Three activity optima were observed at pH values around 3, 6.5 and 10. A subsequent study indicated that the activity in neutral and alkaline environment was caused by the same enzyme (Martin & Jönsson 1965).By means of electrophoretic separation combined with reactions for enzyme characterization, Tran Van Ky et al. (1966) demonstrated proteolytic activity in mycelial extracts of 21-day AF cultures. A casein precipitating enzyme (CP enzyme) was demonstrated by Sandvik (1967) in the fluid phase of frozen and thawed skimmilk agar cultures from e.g. AF.In addition to haemolysin and toxin (Rutqvist 1965, 1968, Rutqvist & Persson 1966), mycelial filtrates of AF have proved to contain a proteolytic enzyme. An account is given in the following of a study of this enzyme with respect to (1) casein precipitating ability, (2) casein and gelatin hydrolyzing effect, and (3) relation to toxin and haemolysin.     

家蚕γ-谷氨酰环化转移酶的纯化及其性质的研究

首次从家蚕(Bombyx mori L.)蛹体脂肪织织及真皮细胞分离得到催化丫-L-谷氨酰-2-L-氨基丁酸反应生成吡咯烷酮羧酸和2-L-氨基丁酸的Y-谷氨酰环化转移酶。并采用硫酸铵沉淀、葡聚糖G-75柱层析、DEAE-纤维素DE_(32)柱层析和羟基磷灰石柱层析等步骤,将该酶提纯了1,285倍。高度纯化后的酶,对Y-L-谷氨酰-2-L-氨基丁酸具有最适pH7.2-7.4,最适酶反应温度为47℃,米氏常数(Km)为0.04M。在分离纯化过程中未发现蚕体内有该酶的同功酶存在,纯化后的酶溶解于pH8.0、0.01M的Tris—HCl缓冲掖中,-30℃经过一个月没有发现明显的失活现象,但在4℃中一个月后丧失活性76％。     

Antigens of Goat Spermatozoa that Recognize Homologous Zona Pellucida

Goat sperm surface proteins obtained from purified plasma membrane (PPM) vesicles (purity of membrane checked by marker enzymes and transmission electron microscopy) were size fractionated on an fast protein liquid chromatography (FPLC) gel filtration column. All the seven surface proteins (129, 100, 46, 28, 27, 18 and 10 kDa) obtained were further fractionated and purified on high-efficiency gel filtration (GFC-HPLC) as well as ion exchange (DEAE-HPLC) columns. Antibodies were generated against the PPM and the protein fractions. Such resolved and purified surface antigens were tested by Dot Blot Immunoassay and homologous in vitro sperm-zona binding assays. It was revealed that the binding of goat spermatozoa to homologous zona pellucida was inhibited by antisera raised against the five lower molecular weight surface antigens. Further, the components of FPLC-AIII (46 kDa; A represents antigenic protein) and IV (28 kDa) were most promising as the antibodies against these fractions inhibited sperm binding to zona pellucida even at a dilution of 1 : 1000 as tested by the sperm-zona binding assays.     

Bovine placental protease specificity toward muscle connective tissue proteins

Enzymes currently used to tenderize meat are not substrate-specific, resulting in extensive myofibrillar protein degradation that often produces an undesirable texture. Bovine placental metalloproteases, which selectively hydrolyze connective tissue proteins while leaving myofibrillar proteins intact, may tenderize meat without causing texture problems. Therefore, our objective was to extract and crudely purify bovine metalloproteases from bovine placenta for possible use as tenderizers in meat systems. Enzymes were extracted from homogenized tissue and purified by ammonium sulfate precipitation. Samples were collected before (crude enzyme) and after gel filtration on a Sephadex G-100 column. Spectrophotometric analysis identified one major peak (filtered enzyme). Gelatin, casein, and type I acid-soluble collagen zymography were used to determine substrate specificity. Beef myofibrillar proteins were incubated with crude and filtered enzyme fractions, enzymes quenched, and substrate degradation visualized using SDS-PAGE. Active gelatinases and collagenases exhibiting molecular weights of 57 to 65 kDa were detected on zymograms. Banding patterns from crude enzyme indicated two enzymes with both gelatinase and collagenase activity and a third enzyme with gelatinase activity only. Banding patterns from filtered enzyme indicated two enzymes with both gelatinase and collagenase activity. Proteolytic activity was not detected with casein, actin, or myosin heavy-chain substrates. Due to specificity for collagen and gelatin, these enzymes may be capable of improving the tenderness of certain cuts relatively high in connective tissue, while avoiding myofibrillar protein hydrolysis.     

鸡肠道抗菌肽的提取及活性研究

取新鲜鸡肠黏膜超声波破碎,乙酸浸提,经Sephadex G-50层析后,收集具有抑菌活性的组分进行超滤离心,抑菌实验表明滤过物具有抑菌活性.滤过物经SDS-PAGE分析为一个条带,分子量约为6.078KD,得到电泳纯的鸡肠道抗微生物肽.该肽具有一定的耐热性,但经90℃以上热处理后活性下降较大;pH值对其活性影响较大,在pH6～8时活性最强.     

Gel filtration of lignosulphonic acids and peptide-precipitating abilities of the separated fractions

Lignosulphonic acids in dialysed sulphite spent liquor and purified lignosulphonic acids were subjected to gel chromatography on Sephadex G-75, G-100 and G-200 and the fractions tested for peptide-precipitating ability. About 56 % of the total lignosulphonic acids in the dialysed sulphite spent liquor had estimated molecular weights above 90000 and about 72 % above 44000. About 94 % of the purified lignosulphonic acids had molecular weights above 90000 and the remaining 6 % had above 36000. The major peptide-precipitating activity of the lignosulphonic acids was due to fractions with molecular weights in excess of 90000. The percentage of peptides in the peptide-lignosulphonic acid precipitates was found to be 80–90. The molecular weights of the peptides used were found to have an upper limit of about 20000. The lower limit for molecular weights of lignosulphonic acid-precipitating peptides is estimated to be below 6000.Keyword: gel filtration, lignosulphonic acids, molecular weight, peptide-precipitating ability     

Methods of Preparing Supplementing Fractions from Fresh Unheated Bovine Sera for Use In Modified Direct Complement-Fixation Tests

Two methods of preparing partially purified, relatively stable supplementing factor from fresh bovine serum are described: gel filtration through Sephadex G-25, and anion exchange chromatography on a diethylaminoethyl (DEAE) cellulose column. In both procedures, an active, reconstituted precipitate prepared by dialysis of fresh unheated normal serum in the cold for 18 hours against phosphate buffer pH 6.2, 0.02 M, serves as the starting material.

The Sephadex G-25 column is equilibrated with acetate buffer pH 5.4, 0.2 M. The most actively-supplementing material appears in the eluates in which the pH has risen to 7.5 or higher. For the DEAE cellulose chromatography a gradient system is used: initial phosphate buffer 0.03 M, pH 8.0, limiting buffer Na H₂PO₄, 0.3 M. The greater part of the supplementing activity is eluated between pH 5.6 and 6.0, although some of the earlier fractions are also reactive. Pooled active eluates stored in the frozen state for nine months or longer maintained their supplementing titre in modified complement-fixation tests of two bacterial antigen-bovine antibody systems.

     

野桑蚕多酚氧化酶的部分生化特性分析

基于开发专一的酶抑制剂控制野桑蚕危害桑园的目的,采用硫酸铵分级沉淀及Sephadex G-100凝胶过滤等方法,纯化了野桑蚕(Bom byx mandarina)多酚氧化酶,纯化倍数为57.14倍。该酶对焦性没食子酸、邻苯二酚和L-多巴的米氏常数(Km)值分别为3.39、2.06和3.17 mmol/L,在pH 7.0、37℃时活性最高。利用硫脲、抗坏血酸等多种氧化酶抑制剂对该酶活性的抑制结果表明,所用抑制剂对其均有不同程度的抑制作用。此外,该酶对乙二胺四乙酸(EDTA)和金属离子比较敏感。     

The detection of IgM and IgG antibodies against Babesia bigemina in bovine sera using semi-defined antigens in enzyme immunoassays

Soluble extracts prepared from Babesia bigemina merozoites were tested for antigenicity in class-specific enzyme immunoassays currently being evaluated for the differential serodiagnosis of bovine babesiosis. Intact merozoites were harvested from erythrocytes from an experimentally-infected calf by controlled hypotonic lysis and differential ultra-centrifugation. The merozoites were disrupted by ultrasonication and a crude soluble extract obtained by ultracentrifugation. Fractionation of the crude extract on calibrated Sephadex G-200 columns consistently produced 4 fractions with molecular weights of 600, 40, 15 and 5 k (k = 10(3) daltons). Only the 600 and 15 k fractions proved to be antigenic when reacted against bovine immune sera. These fractions were incorporated into IgM- and IgG-specific enzyme immunoassays and used to determine the kinetics of the host-antibody responses to infection. The use of semi-defined antigens allowed assay standardization and good reproducibility of the results. A calf infected with a cryopreserved stabilate of B. bigemina originating from adult Boophilus microplus ticks developed a mild transient fever from 6-4 days post-infection (d.p.i.) and low parasitaemia levels from 7-16 d.p.i. IgG-antibodies first appeared at 7 d.p.i., peaked in intensity at 12 d.p.i. and then persisted at these levels until the end of the test period at 49 d.p.i. IgM-antibodies appeared at 7 d.p.i., peaked in intensity from 12-22 d.p.i., but then declined to low levels by 28 d.p.i. The importance of this transitory IgM-antibody response in the serodiagnosis of acute B. bigemina infections remains to be determined in clinical and field situations.     

A protective antigen for Turkeys purified from a type 1 strain of Pasteurella multocida

A protective antigen was purified from a saline extract of a Type 1 strain of Pasteurella multocida by chromatographic methods, and its chemical and immunological ccharacteristics were studied. Three protein peaks were obtained from crude extract by gel filtration with Sephadex G-200. A bacteria-specific antigen was detected only in the first peak fraction, which, after passing through an immunoadsorbent column to remove any components originating from the growth medium, was adsorbed onto DEAE-cellulose followed by elution with a gradient of NaCl. From the first peak fraction of the gel filtration, 4 protein peaks were obtained, the second and third peaks being the major ones. Carbohydrate/protein ratios of the peak fractions varied from 0.06 to 1.0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that 2 proteins of molecular weights 44 000 and 25 000 were present in all the fractions. The 4 DEAE-cellulose fractions (DP-1 to DP-4) contained a single antigenically identical material, and induced protective immunity in turkeys against challenge exposure. The second peak fraction from DEAE-cellulose (DP-2) protected turkeys when subcutaneously injected as 2 doses of 10 μg protein with a 14-day interval between doses. The DP-2 fraction induced antibodies in rabbits which formed a single precipitin line against the crude extract. The purified antigen (DP-2) from a Type 1 strain was antigenically distinct from a similar antigen purified from a Type 3 strain; there was no significant cross protection in turkeys between the 2 antigens. These results indicate that protective antigens purified from soluble extracts of a Type 1 or Type 3 strain possess similar physicochemical properties, but that they are immunologically distinct from each other.     

禽源多杀性巴氏杆菌(A:1)荚膜保护性抗原提纯及单克隆抗体细胞系建立

用Sep(?)adex G-200分之筛层析、DEAE-cellulose离子交换层析相结合,将禽源多杀性巴氏杆菌(A:1)荚膜抗原粗提物(CE)分为P1.P2两种成分,并经免疫保护试验证明,荚膜中含有保护性蛋白成份P1。P2分子量为129 100,且至少含有分子量分别为46700、42600和39800的3种蛋白亚基。用CE免疫BALB/c小鼠,建立了抗P1的单克隆抗体细胞系1B1和2B7,它们只与多杀性巴氏杆菌反应,而不与其他细菌交叉。     

Effects of filtration of semen doses from subfertile boars through neuter Sephadex columns.

This study was designed to develop a method of improving the quality of sperm obtained from subfertile Piétrain boars. Seminal doses were filtered through neuter Sephadex columns (G-25 Medium, G-50 Fine, G-50 Medium and G-75, length 10 +/- 0.5 cm, flow rate 1 ml/20 s). Doses were prepared by pooling 10 ml semen samples collected from 58 asthenoteratospermic boars and diluted the sperm-cell rich fraction 1 : 6 in Betsville thawing solution extender. Sperm quality was determined before and after the filtering process. Sperm morphology and motility were assessed using the computer program SCA 2002 production, and sperm vitality was evaluated by fluorescence multistaining. ORT and HRT tests were used to determine the osmotic resistance of spermatozoa, and metabolic performance was assessed by measuring l-lactate production. Results indicate that the filtration process rendered increased proportions of mature spermatozoa and of viable spermatozoa with an intact acrosome, nucleus and mitochondrial sheath. Sperm filtration led to decreased percentages of spermatozoa with proximal and distal droplets and of agglutinated spermatozoa, along with slightly diminished ORT values. HRT scores and L-lactate production were unaffected. Our findings indicate that filtering through a Sephadex column improves the sperm morphology and vitality of seminal doses obtained from subfertile boars, but produces no functional changes in the spermatozoa. All four column types yielded similar results.     

Retinol transport system in cattle and purification of retinol-binding protein from bovine serum

Retinol transport system in cattle was investigated, followed by the purification and characterization of bovine serum retinol-binding protein (RBP). Gel filtration of serum from cow produced two retinol peaks, peak 1 and 2. The major, peak 1 having higher molecular weight corresponded to the retinol peak from human serum which consisted of RBP and prealbumin (PA). The peak 2 which was not presented in the human serum had lower molecular weight (about 20,000). In the presence of 3.0 M urea, the peak 1 was almost disappeared and peak 2 was increased. On the other hand, in the serum from calf, major retinol peak was corresponded to the peak 2 from cow. These results suggested that, in cow, retinol was transported by the complex of RBP and another protein, presumably PA, but in calf, mainly by RBP alone. Purification of bovine RBP was carried out by using four chromatographic steps as follows; 1. DEAE-cellulose (pH 6.0), 2. Sephadex G-100 (using the buffer containing 3.0 M urea), 3. DEAE-cellulose (pH 8.3), 4. Sephadex G-100. From 1,100 ml of serum, 14.1 mg of bovine RBP was finally obtained and the overall recovery was estimated to be about 32%. Its molecular weight, ultraviolet absorption and fluorescence spectra, electrophoretic mobility, and amino acid composition were similar to those of other species.     

神经生长因子的提取,纯化及鉴定

经凝胶过滤层析－离子交换层析－凝胶过滤层析分离方法将蛇毒中的神经生长因子提取并纯化。得到的神经生长因子经ＳＤＳ－聚丙烯酰胺凝胶电泳测得其分子量为１７０００，由２３１个氨基酸组成。在２２０．６ｎｍ处有１个特征吸收峰。最低活性单位为１０ｎｇ／ｍＬ。     

The Effect of Unheated Bovine Serum on the Complement-Fixing Activity of Heat-Inactivated Bovine Antiserum with Homologous Antigen. IV. Chromatographic Studies

Unheated, non-dialyzed, normal bovine sera were fractionated by column chromatography on the cross-linked dextran, Sephadex G-25, and the fraction tested for “supplementing” properties, that is for complement-fixation augmenting activities when added to mixtures of heated bovine antiserum and homologous antigen. Supplementing activity was shown by precipitated fractions from earlier eluates with pH values below 7.2 and also by both supernatant and precipitated fractions of the later eluates with pH values from 7.6 to 8.1. The possibility is briefly discussed that certain alkaline protein substances of relatively lower molecular weight may be involved in the supplementing activities of the later fractions. Heating at 56°C. for 30 min. destroyed the supplementing activity of each of these fractions.

Some of the supplementing fractions proved to be anti-complementary, others were not or only slightly so. First component of complement, C¹1, was detected in the precipitated fractions of certain of the earlier eluates with pH values below 6.5; second component of complement, C¹2, was found exclusively in supernatant fractions of earlier eluates with pH values less than 6.2. Conglutinin was not separated from C¹1 by this method.

     

An evaluation of the mononuclear cells derived from bovine mammary gland dry secretions using leukocyte antigen specific monoclonal antibodies, light scattering properties and non-specific esterase staining.

The distribution of mononuclear cells isolated from the bovine mammary gland during the nonlactating (dry) period was examined using monoclonal antibodies against leukocyte cell surface antigens, cellular light scattering properties, and the presence of nonspecific esterase. Most of the mononuclear cells isolated during the dry period were lymphocytes. T cells predominated until about 1 week prior to parturition. During the week prior to calving, the percentage of B cells increased until it approximated T cells. The ratio of CD4:CD8 cells was 2-3:1 for mammary gland T cells. This was similar to the ratio found in peripheral blood. At dry-off, about 12% of mammary mononuclear cells were macrophages. The macrophage percentage increased (to about 30%) at mid-dry and remained at this levels until parturition. PMN's were isolated with the mononuclear cells during the first 2 weeks dry and the week prior to calving. Three methods were used to identify mammary macrophages. Esterase staining (as an enzymatic method), forward angle/90 degrees light scatter (based on size and internal complexity), and MHC class II/forward angle light scatter (based on size and surface markers) were compared. Each method yielded similar specificity for macrophage identification. Non-adherent cell fractions, obtained by passage of the cells over Sephadex G-10 columns, were enriched in CD4 positive T cells, somewhat depleted of B cells, and depleted of macrophages and PMN's. Cells eluted from G-10 columns, with lidocaine, were mostly lymphocytes, but reflected the cells loaded onto the column.     

The bovine immune response to Brucella abortus I. A water soluble antigen precipitated by sera of some naturally infected cattle.

Selected sera from cattle naturally infected with Brucella abortus precipitate water soluble antigens extracted by sonication from B. abortus. One of these antigens resembles antigen E (Baughn and Freeman) as it is excluded from Sephadex G-200 gels, migrates anodally when electrophoresed at pH 8.6, resists heating at 100 degrees C for ten minutes and appears to be susceptible to papain digestion. Precipitins specific for this antigen remained in sera from which all detectable Brucella agglutinating antibody had been removed by adsorption with live or heat killed B. abortus. The antigen has been extracted from smooth and rough strains of B abortus. Precipitins specific for this antigen have been detected in antisera produced against Brucella canis.     

Isolation and partial characterization of excretory/secretory antigens of Gastrothylax crumenifer

The present study was carried out to identify the excretory/secretory (E/S) antigens of the rumen infecting digenetic trematode Gastrothylax crumenifer that may be useful for the immunodiagnosis of rumen amphistomosis particularly during the pre-monsoon season during which this rumen parasite stops shedding eggs. The in vitro released E/S proteins were purified on a Sephadex G-200 column. The gel filtration profile revealed three distinct fractions F1-F3 where F1 and F3 appeared as sharp peaks while the F2 fraction was dispersed. The antibody titre against each of the purified E/S fractions was determined by ELISA using anti-whole E/S polyclonal antibodies raised in rabbit. Among the three fractions, the antibody titre against F1 was highest (1:12,800) whereas IgG titre was very low (1:50) for fraction F2 and F3 (1:100). Of the total polypeptides resolved on gradient SDS-PAGE, only a few antigenic polypeptides were detected in each fraction with hyperimmune anti-serum as revealed by Western Blot analysis. However, a 33 kDa antigen detected in each fraction appeared to be immunodominant which could be exploited for the diagnosis of the pouched amphistome.