Isolation, purification, and partial characterization of an enterotoxin from extracts of Entamoeba histolytica trophozoites.

Soluble cell-free extracts of pathogenic Entamoeba histolytica, as well as serum-free minimal media in which trophozoites are incubated, contain substances that cause the rapid rounding up and detachment of tissue-cultured monolayers of mammalian cells (cytopathic activity) and induce fluid secretion in ligated intestinal loops of indomethacin-pretreated rats (enterotoxic activity). A semiquantitative assay for the determination of the cytopathic activity based on the rate of detachment of tissue-cultured baby hamster kidney cells was developed. Two peaks containing cytopathic activity were obtained upon gel filtration of the soluble extracts: peak I, with over 60% of the activity, emerged in the 30,000 to 50,000 molecular weight region, and peak II, containing the remaining activity, was in the 15,000 to 25,000 molecular weight region. The activity of peak I was found to be heat labile and inhibited by sialoglycoproteins such as fetuin and mucin (5 mg/ml), as well as by sialic acid. Protease inhibitors such as antitrypsin, pepstatin, phenylmethylsulfonyl fluoride, metaloprotease inhibitors, and bacitracin had no effect on the cytopathic activity. Marked inhibition of cytopathic activity was observed, however, with iodoacetamide and p-chloromercuribenzoate, which affect sulfhydryl groups. The toxic material in peak II was found to have ionophoric activity and was not inhibited by sialic acid-containing compounds. The materials from both peaks had enterotoxic activity in intestinal ligated loops. The active substance from peak I was further purified (200X) on an agarose-fetuin affinity column, yielding one major protein band with an apparent molecular weight of ca. 30,000 on sodium dodecyl sulfate. Amino acid analysis revealed that the protein was very poor in sulfur amino acids. The sialic acid-sensitive toxic activity was higher in known virulent strains such as HM-1:IMSS and could be markedly augmented after preincubation of the trophozoites with certain Escherichia coli strains.     

Augmentation of host defense by a unicellular green alga, Chlorella vulgaris, to Escherichia coli infection.

Protection against Escherichia coli inoculated intraperitoneally into mice was enhanced by intraperitoneal, intravenous, or subcutaneous administration of a water-soluble, high-molecular-weight fraction extracted from a dialyzed hot-water extract from a strain of Chlorella vulgaris (CVE-A). The enhancing effect was detected with doses over 2.0 mg/kg and when doses were administered 1, 4, or 7 days before the infection. The elimination of bacteria from the spleen of CVE-A-treated mice was increased, and this enhanced elimination may have been related to the acceleration of superoxide generation and chemokinesis in polymorphonuclear leucocytes by CVE-A treatment. A cyclophosphamide-induced decrease in protection against E. coli could be prevented by subcutaneous administration of CVE-A.     

Transient expression of firefly luciferase in protoplasts of the green alga Chlorella ellipsoidea

Summary We report here on the development of a transient expression system for Chlorella ellipsoidea using a heterologous gene, firefly luciferase. Cells of this unicellular green alga were converted to protoplasts and treated with plasmid pDO432, which bears luciferase under the control of the CaMV 35S promoter. This treatment resulted in detectable luciferase activity in cell extracts. Expression required Cellulysin treatment, active cell metabolism, and the addition of carrier DNA and polyethylene glycol. Linearization of the luciferase plasmid did not significantly alter the activity. A time course of expression showed that luciferase is made rapidly, within about 7 h after addition of DNA, but that the activity disappears over the course of a few days. These experiments represent an important first step in the development of a Chlorella transformation system.     

Characterization and partial purification of the Croatian national standard Dermatophagoides pteronyssinus allergen extract

Lyophilized Dermatophagoides pteronyssinus ( Der p ) allergen extract (AE) and partially purified Der p extract (PAE) Were Prepared and characterized. Partial purification of AE was performed by gel filtration on Sephadex G-1OO and Sephacryl S-300. Crossed immunoelectrophoresis (CIE) disclosed the same precipitating lines in AE and PAE preparations. The relative potencies of AE and PAE were determined and compared with the WHO International Standard for Der p by the RAST inhibition method. The potencies were 6.5 × 10⁵ IU and 1.5 × 10⁶ IU, respectively. Biologic standardization by quantitative skin testing was performed with AE (20 selected patients) and PAE (12 patients). Median C_h was calculated by linear regression analysis (log-log model). One ampoule of AE contained 65 300 BU and 1 ml (vial) of PAE contained 166000 BU. Der p AE could serve as a Croatian national standard for further production of Der p allergenic extracts.     

The allergens of dog II. Identification and partial purification of a major dander allergen

A dog hair and dander (DHD) extract was prepared from hair obtained from mixed breeds. By SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting, using sera from 32 dog-allergic subjects, a number of IgE radio-staining bands could be seen. In 78% of sera a protein of molecular weight (MW) of 21 000 daltons, designated Ag X, was found to bind IgE and in 34% it did so strongly. This allergen was isolated from DHD by size-exclusion and ion exchange chromatography. The final product was a single allergen of MW of 21 000 and an isoelectric point of approximately 5.2. An additional protein-staining band could still be seen of MW of 24 000 daltons. Using a serum which contained IgE antibodies only to Ag X, this allergen was found only in DHD extract and dog saliva and was absent from dog serum and urine. It was the same dog allergen that we [1] reported as Ag 8 using crossed radio-immunoelectrophoresis (CRIE) and that Blands et al. [2] and Løwenstein [3] described as Ag 13. We propose that this major dog allergen be given the title Can f I according to the new allergen nomenclature.     

重阳木花粉过敏原的分离、纯化和鉴定

目的对重阳木花粉变应原蛋白进行分离、纯化和鉴定。方法采用Coca s液提取重阳木花粉的粗提液,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离粗提液蛋白质组分,并测定其分子质量;收集过敏患者血清,用Western blot法鉴定其变应原成分;通过离子交换层析对重阳木花粉变应原进行初步纯化和免疫印迹鉴定。结果分离得到重阳木花粉18条蛋白带,其中分子质量为12和14 ku的是重阳木花粉的特异性变应原,通过离子交换柱层析方法纯化得到其相应的纯化蛋白。结论对重阳木花粉变应原进行了初步的分离、纯化和鉴定,为临床重阳木花粉过敏疾病的诊断和治疗奠定了基础。     

Studies of dialysates from Dermatophagoides farinae: partial purification and haptenic properties of dialysates from Dermatophagoides farinae.

Dialysates from Dermatophagoides farinae were partially purified. Fractionation on HPLC and anion exchange chromatography revealed that the dialysates consisted of 5 major fractions of glycoprotein whose apparent molecular sizes were 5.1, 4.1, 3.2, and less than 1.35 kD on HPLC. The apparent molecular size of two fractions was 5.3 and 2.9 kD on SDS-PAGE. They were basic glycoproteins which had a pI ranging from 7.46 to 8.71 on PAG-IEF. These fractions were allergenic in the RAST and ELISA inhibition tests but not in the skin prick test (SPT). Our results suggest that the dialysates from D. farinae have haptenic properties. The dialysates from D. farinae (low molecular weight) and its 5 fractions bound noncovalently to human serum albumin (HSA) at the free tyrosine residues of HSA. They proved to bind noncovalently to serum proteins and collagens. Once they bound to proteins, the conjugates became allergenic not only in the RAST and ELISA inhibition test but also in the SPT. Our results provide evidence that the dialysates from D. farinae have haptenic properties.     

Isolation, purification, and partial characterization of a lipopolysaccharide from Haemophilus pleuropneumoniae.

Lipopolysaccharide (LPS) from Haemophilus pleuropneumoniae 1536, serotype 2, was isolated and purified by a procedure designed to be equally satisfactory for both smooth- and rough-type LPS. The LPS yield was 53%. Analysis of the preparations revealed that protein, nucleic acid, and cellular phospholipid contamination was negligible (less than 0.1%). Analysis of the sugar content of the LPS by gas-liquid chromatography and colorimetric analysis revealed the presence of rhamnose, mannose, galactose, glucose, heptose, glucosamine, galactosamine, and 3-deoxy-D-manno-2-octulosonic acid. The heptose and glucose contents appeared to be unusually high. The fatty acids of the LPS consisted of a mixture of C14:0 and C16:0 in a ratio of about 4.5:1 (50% of the total) and 3-hydroxy C14:0. When used as a preparatory dose for the dermal Shwartzman reaction, as little as 10 micrograms of the LPS injected intradermally in rabbits produced reddening and swelling. After intravenous injection of a 100-micrograms LPS provoking dose, necrosis was observed at all intradermal injection sites. Limulus amebocyte lysate gelation was observed with an LPS concentration as low as 0.5 ng/ml. A typical biphasic fever response was noted in rabbits injected with as little as 0.25 ng of LPS per kg of body weight.     

Preparation,cryopreservation, and growth of cells prepared from the green turtle (Chelonia mydas)

Techniques are described for preparing, preserving, and growing cell cultures from 30 to 40-day old green turtle embryos (2.0–3.0 cm length) including cells derived from skeletal muscle, liver, heart, kidney, eye, lung, and brain. Acceptable growth of all cells occurred in all standard cell culture media tested, with optimum growth temperature near 30 °C. These cell cultures will be used in the study of sea turtle viral diseases including fibropapillomatosis, which is currently epidemic in some green turtle populations.     

Further characterization of a low-molecular weight allergen fragment isolated from the green pea.

A low molecular weight allergen fragment present in the pea dialysate fraction was purified by ion-exchange chromatography and gel filtration. The highly purified allergen fragment inhibits both antigen-indiced passive cutaneous anaphlaxix reactions in guinea-pigs sensitized with rabbit anti-pea extract sera and Prausnitz-Küstner reactions in non-allergic volunteers sensitized with the sera of patients sensitive to green peas. Preliminary analysis of the purified allergen fragment indicates that it is a glycoprotein with a molecular weight of 1800 +/- 250.     

Studies on the stability of diluted allergen extracts using the radioallergosorbent test (RAST)

Allergen extracts from birch pollen, timothy pollen and horse dandruff were investigated. The influence of storage time, volume of extract in the vial and various additives on the potency of diluted extracts was tested with the radioallergosorbent test (RAST). The potency of diluted pollen extracts was found to decrease more rapidly than the potency of diluted dandruff extracts. The differences in allergen activity seen in RAST between freshly diluted extracts and stored extract dilutions were roughly reproducible in skin tests and histamine release tests from passively sensitized chopped human lung. The higher allergen stability of the dandruff extracts could, at least in part, be explained by the higher protein content of these extracts. An adsorption of allergen to the glass wall was shown to be the most probable cause to the more rapid decrease of allergen activity seen in pollen extracts stored in vials only partly filled. With addition of 0.2% Tween 20 after storage the potency of the extracts became almost equivalent to that of corresponding dilutions stored in filled vials.     

兔抗人重组生长激素rhGH多克隆抗体的制备、纯化及鉴定

目的制备抗人生长激素(growth hormone,GH)的多克隆抗体并鉴定其性能。方法真核细胞表达质粒pcDNA3.0-GHcDNA免疫家兔,制备rhGH多克隆抗体;ELISA法检测抗体效价。亲和层析法纯化后,进行Western blot、免疫组织化学和激光共聚焦显微镜检测,鉴定抗体的特异性与适用范围。结果得到兔抗人重组人生长激素rhGH多克隆抗体,效价达1:40000。纯化后的抗体可特异识别超表达的hGH和人内源性GH,可用于免疫组织化学实验、Western blot及激光共聚焦。结论用质粒免疫家兔制备的抗GH抗体具有较高的效价以及特异性,为hGH的功能性研究提供了有力的支持。     

Purification and partial characterization of the allergen Ag-54 from Cladosporium herbarum

One important allergen, Ag-54, was extracted from Cladosporium herbarum and purified by a combination of diafiltration, gel filtrations and isoelectric focusing. The allergen, when examined by gel electrophoresis and high-performance liquid chromatography, was found to be essentially homogeneous with a molecular weight of about 20,000 daltons. Ag-54 is of glycoprotein nature. It contained 19.5% protein, calculated from its amino acid composition and 80.2% carbohydrate, quantified by methanolysis, trimethylsilyl derivatization and gas-liquid chromatography. The carbohydrate moiety was composed only of mannose, galactose and glucose in the ratio 1.0:0.6:1.3. Frequently occurring amino sugars like glucosamine and galactosamine could not be detected. Mass spectrometry of trimethylsilyl-derivatized methylglycosides demonstrated that sialic acid was not present.     

Isolation and purification of a major allergen from Parietaria officinalis pollen

A major allergen of Parietaria officinalis, a species responsible for a large number of respiratory allergies in Mediterranean areas, has been identified and characterized. This allergen (Pol) was found in the fraction which precipitates between 70 and 100% ammonium sulphate saturation. Pol showed a molecular weight of 15,000 daltons as determined by SDS-PAGE and HPLC. The pI of Pol was in the pH region 4-6, IEF showing four major bands. Two major bands were shown by CIE, CRIE and immunoblotting; major contaminants or aggregates were also revealed by the latter technique and by HPLC. Pol showed an allergic specific activity 2 times higher than the crude extract; moreover it was shown to be a major allergen since it inhibited 29 out of 30 sera from allergic patients sensitive to P. officinalis.