The 10 kDa protein of Taenia solium metacestodes shows genus specific antigenicity

Genus specific antigenicity of the 10 kDa protein in cyst fluid (CF) of Taenia solium metacestodes was demonstrated by comparative immunoblot analysis. When CFs from taeniid metacestodes of T. saginata, T. solium, T. taeniaeformis and T. crassiceps were probed with specific monoclonal antibody (mAb) raised against 150 kDa protein of T. solium metacestodes, specific antibody reactions were observed in 7 and 10 kDa proteins of T. solium and in 7/8 kDa of T. saginata, T. taeniaeformis and T. crassiceps. The mAb did not react with any protein in hydatid fluid of Echinococcus granulosus and E. multilocularis. This result revealed that the 10 kDa peptide of T. solium metacestodes and its equivalent proteins of different Taenia metacestodes are genus specific antigens that are shared among different Taenia species.     

Immunoblot findings of calcareous corpuscles binding proteins in cyst fluid of Taenia solium metacestodes

After collecting calcareous corpuscles from plerocercoid of Spirometra mansoni (sparganum), we evaluated the antigenic values of calcareous corpuscles binding proteins obtained from the cyst fluid of Taenia solium metacestodes. Immunoblot analysis revealed that cysticercosis patient sera strongly recognized 10 and 95 kDa calcareous corpuscles binding proteins. This result demonstrated that calcareous corpuscles are bound with major secretory antigenic proteins, which is possibly involved in the secretory pathways of the 10 and 95 kDa proteins presenting in the cyst fluid of T. solium metacestodes.     

Uptake and secretion of host proteins by Taenia crassiceps metacestodes

Although the presence of intact host proteins in the cyst fluid of cyclophyllidean metacestodes has been well documented, the underlying reason for protein uptake is poorly understood. To investigate this discrepancy, both the cyst fluid (CF) and excreted/secreted (E/S) proteins were collected in vitro from Taenia crassiceps metacestodes 16 wk postinfection in Balb/cJ female mice. The CF and E/S were subsequently immunoblotted using rabbit anti-mouse whole serum antibodies as a probe. The results show that whole host proteins were not only internalized by metacestodes but also secreted as well. The predominant secreted host protein was 66 kDa and was confirmed to be mouse serum albumin. The amount of secreted albumin decreased daily, whereas the concentration of albumin in the cyst fluid remained consistent. This suggests that the secretion of albumin is a coordinated function rather than a random event. It is probable that albumin cycling may be an evolved mechanism providing multiple benefits for the larvae, including osmoregulation and protection from innate immune responses.     

Usefulness of 8 kDa protein of Fasciola hepatica in diagnosis of fascioliasis

This study was designed to detect and evaluate an antigenicity of low molecular weight proteins of Fasciola hepatica in fascioliasis. Low molecular weight protein of F. hepatica was purified by ammonium sulfate precipitation and Sephacryl S-100 HR gel filtration. The protein obtained was estimated to be 8 kDa on 7.5-15% gradient sodium dodecyl sulfate gel electrophoresis. Immunoblotting studies showed that the 8 kDa protein reacted with human fascioliasis sera, but not other trematodiasis sera. This result suggests that the 8 kDa protein of F. hepatica is one of diagnostic antigens in human fascioliasis without cross-reaction with other human trematodiasis.     

Characterization of partially purified 8 kDa antigenic protein of Clonorchis sinensis

The 8 kDa antigenic protein of Clonorchis sinensis was partially purified by ammonium sulfate precipitation and subsequently by a column chromatographic steps. The purified protein was separated into 7 and 8 kDa protein bands through SDS-tricine gel electrophoresis, while the protein was found to migrate to a 8 kDa band in 7.5-15% SDS-PAGE. The molecular weight of the antigen was estimated to be 110 kDa by Superose 6 HR 10/30 gel filtration. The purified antigen strongly reacted with the human sera of clonorchiasis. The hyperimmune sera of BALB/c mice immunized against the 8 kDa protein were reacted with both the crude extract and the excretory-secretory product of adult worms, but not with the metacercarial extract. Immunohistochemical staining demonstrated that the protein was distributed to the tegument and subtegumental cells and also to the seminal receptacle. The present findings suggest that the 8 kDa protein is a partition of the multicomplex protein originating from various organs of adult C. sinensis, and that it is composed of several 7 and 8 kDa proteins.     

Measurement of 150 kDa protein of Taenia solium metacestodes by antibody-sandwich ELISA in cerebrospinal fluid of neurocysticercosis patients.

An antigenic protein in cystic fluid of Taenia solium metacestodes (CF) of 150 kDa was measured by antibody-sandwich ELISA in serum and cerebrospinal fluid (CSF) of neurocysticercosis patients. Capture antibodies were rabbit antisera against CF (RACF) and a monoclonal antibody (MAb) against 150 kDa protein in CF. Lower limit of antibody-sandwich ELISA was 8 ng/ml of the protein. Except CF, no tested helminths extracts reacted. Levels of the protein in 351 sera from 255 patients (55 surgery confirmed and 202 antibody and CT/MRI confirmed) were below sensitivity of the assay. Of 276 CSF from 212 patients, 31 samples (11.2%) showed positive findings. This assay, therefore, was not sensitive enough to be a diagnostic. Instead, the 150 kDa protein appeared in CSF in such situations as in 2 days after praziquantel treatment, or as in a patient infected with a racemose cysticercus with degenerated cyst wall. Of cases whose follow-up CSF were assayed, 2 cases showed that the protein appeared intermittently. These results suggest strongly that appearance of free 150 kDa protein is associated with cyst wall rupture. In CSF which contained the 150 kDa protein over 61 ng/ml, the protein was recognized in SDS-PAGE before and after immunoprecipitation.     

Taenia saginata: Production and characterization of monoclonal antibodies against Taenia saginata metacestode antigens

Cysticercosis is a major cause of economic loss in bovine production due to meat condemnation. Chemotherapy is being used in Brazilian cattle and a diagnostic test to improve the treatment program is desired. We produced monoclonal antibodies against crude (TAEB) and cyst fluid (TAEF) Taenia saginata metacestode antigens using immunized BALB/c mice. After cell fusion, 10 TAEB and nine TAEF hybrids were selected and cloned resulting in 18 IgG₁ and 32 IgM TAEB clones, and 9 IgG₁ and 9 IgM TAEF clones. Ascites was produced and Western blot testing was performed resulting in reactivity to protein fractions of low molecular weight (<18 kDa), 43, 55, 66 and 100 kDa. The indirect immunofluorescence test, with one monoclonal antibody against crude and one against cyst fluid antigens, recognized antigenic fractions of both the scolex and the bladder wall of metacestodes from naturally infected bovine.     

Component proteins in cystic fluid of Taenia solium metacestodes collected surgically from neurocysticercosis patients.

Surgically collected cystic fluid of Taenia solium metacestodes from patients of intracranial cystic lesion were compared in their protein composition with those from naturally infected pigs in Cheju Do, Korea and Ecuador. In non-denaturing discontinuous-polyacrylamide gel electrophoresis (disc-PAGE), no discernible differences were recognized in banding patterns between the cystic fluids from Cheju Do and Ecuador, and between the cystic fluids from pigs and human lesions except wider bands that corresponded to human albumin and gamma-globulin (in 4 of 9 patients). In reducing SDS-PAGE, bands in the cystic fluid from Ecuador showed the same banding pattern with that from Cheju Do but two bands of 21 and 17 kDa were stained darker. Cystic fluids from patients revealed the same protein compositions of the major protein bands of 94, 64, 15, 10 and 7 kDa as in the cystic fluid of pig origin, but human albumin (66 kDa), heavy and light chains of gamma globulin (55 and 22.5 kDa) were contaminated in 4 of 9 cystic fluids. Human CSF proteins seem to have been contaminated during cystic fluid collection. In any cystic fluid from patients, the major protein component was 150 kDa which was subdivided into 15, 10 and 7 kDa in reducing SDS-PAGE.     

Partial characterization of a 29 kDa cysteine protease purified from Taenia solium metacestodes

A 29 kDa cysteine protease of Taenia solium metacestodes was purified by Mono Q anion-exchanger and Superose 6 HR gel filtration chromatography. The enzyme was effectively inhibited by cysteine protease inhibitors, such as iodoacetic acid (IAA) and trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) while inhibitors acting on serine- or metallo-proteases did not affect the enzyme activity. The purified enzyme degraded human immunoglobulin G (IgG), collagen and bovine serum albumin (BSA), but human IgG was more susceptible for proteolysis by the enzyme. To define the precise biological roles of the enzyme, more detailed biochemical and functional studies would be required.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Partial characterization of a 17 kDa protein of Clonorchis sinensis

A 17 kDa protein from Clonorchis sinensis adults was purified by a procedure including Sephacryl S-200 HR gel filtration and Q-Sepharose anion exchange chromatography. The protein was proved to be a cysteine protease as it showed hydrolytic activity toward Cbz-Phe-Arg-AMC in the presence of dithiothreitol and was inhibited by specific inhibitors such as iodoacetic acid or trans epoxy-succinly-L-leucyl-amido(4-guanidino) butane. The polyclonal antibody raised against the protein reacted to 17 kDa proteins of trematodes such as Paragonimus westermani, Fasciola hepatica, Opisthorchis viverrini, Gymnophalloides seoi, and Metagonimus yokogawai. The antibody recognized the 17 kDa and 16 kDa cysteine proteases purified from C. sinensis, P. westermani, and G. seoi as well. These results suggest that the 17 kDa protein may be a cysteine protease commonly present in trematodes.     

Degradations of human immunoglobulins and hemoglobin by a 60 kDa cysteine proteinase of Trichomonas vaginalis

The present study was undertaken to investigate the role of cysteine proteinase of Trichomonas vaginalis in escaping from host defense mechanism. A cysteine proteinase of T. vaginalis was purified by affinity chromatography and gel filtration. Optimum pH for the purified proteinase activity was 6.0. The proteinase was inhibited by cysteine and serine proteinase inhibitors such as E-64, NEM, IAA, leupeptin, TPCK and TLCK, and also by Hg²⁺, but not affected by serine-, metallo-, and aspartic proteinase inhibitors such as PMSF, EDTA and pepstatin A. However, it was activated by the cysteine proteinase activator, DTT. The molecular weight of a purified proteinase was 62 kDa on gel filtration and 60 kDa on SDS-PAGE. Interestingly, the purified proteinase was able to degrade serum IgA, secretory IgA, and serum IgG in time- and dose-dependent manners. In addition, the enzyme also degraded hemoglobin in a dose-dependent manner. These results suggest that the acidic cysteine proteinase of T. vaginalis may play a dual role for parasite survival in conferring escape from host humoral defense by degradation of immunoglobulins, and in supplying nutrients to parasites by degradation of hemoglobin.     

Purification and characterization of an extracellular exochitinase,beta-N-acetylhexosaminidase,from the fungal mycoparasite Stachybotrys elegans

The mycoparasite Stachybotrys elegans produces two exo- and one endo-acting chitinases when grown on chitin. We purified to homogeneity one of the exo-acting chitinases, beta-N-acetylhexosaminidase and partially characterized its physical and biochemical properties. The native enzyme has a molecular mass of 120 kDa when determined by gel filtration and 68 kDa by sodium dodecyl sulfate - polyacrylamide gel electrophoresis indicating that the native protein probably occurs as a dimer in solution. The purified beta-N-acetylhexosaminidase is most active at pH 5.0 and 40 degrees C and hydrolyzes the p-nitrophenyl-N-acetyl-beta-D-glucosaminide with apparent Km of 84.6 microM. Polyclonal antibodies raised against the 68-kDa beta-N-acetylhexosaminidase (NAG-68) indicated that the antibody is highly specific and recognizes the protein in crude filtrate preparation. This suggests that the protein is a not a proteolytic product of another protein. Western blot analysis showed that the activity of NAG-68 was induced when S. elegans was grown on purified cell wall fragments of its host, Rhizoctonia solani, as well as during antagonistic interaction of the mycoparasite and host when both were grown on synthetic medium with or without supplemental carbon source.     

A novel inhibitor protein of N-myristoyltransferase from Escherichia coli

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal of the glycine residue of various eukaryotic and viral proteins of diverse functions. Earlier, we have demonstrated that NMT activity is elevated in colon and gall bladder cancer. Attenuation of NMT activity may prove a novel therapeutic protocol for cancer. We report here a novel inhibitor protein of NMT being expressed in Escherichia coli cells containing the human NMT gene on increasing the incubation period from 5 to 24h. The inhibitor protein was purified by SP-Sepharose column chromatography, heat treatment, ammonium sulfate precipitation, and Superose 12 HR/30 FPLC column chromatography. The inhibitor protein had an apparent molecular mass of 10kDa by gel filtration. It inhibited human NMT in a concentration-dependent manner with 50% inhibition at 640+/-4.68nM. The inhibitor protein showed no direct interaction with myristoyl-CoA and demonstrated no demyristoylase or protease activity. Therefore, we conclude that the inhibitor protein acts directly on NMT.     

Evidence of a novel allergenic protein Narcin in the bulbs of Narcissus tazetta

Several plant-derived allergens have been identified which result in the formation of immunoglobulin E antibodies. Primarily, these allergens belong to the protein families including seed storage proteins, structural proteins and pathogenesis-related proteins. Several allergens are also reported from flower bulbs which cause contact dermatitis. Such symptoms are highly common with the bulb growers handling different species of Narcissus. Narcissus toxicity is also reported if the bulbs are consumed accidentally. The present study aimed to characterize the protein from the bulbs of Narcissus tazetta responsible for its allergenic response. A 13 kDa novel allergenic protein, Narcin was isolated from the bulbs of Narcissus tazetta. The protein was extracted using ammonium sulfate fractionation. The protein was further purified by anion exchange chromatography followed by gel filtration chromatography. The N-terminal sequence of the first 15 amino-acid residues was determined using Edman degradation. The allergenicity of the protein was measured by cytokine production using flow cytometry in peripheral blood mononuclear cells. Further estimation of total IgE was performed by ELISA method. This novel protein was found to induce pro-inflammatory cytokines and thus induce allergy by elevating total IgE level. The novel protein, Narcin isolated from Narcissus tazetta was found to exhibit allergenic properties.     

Further characterization of the light breast cyst fluid protein, GCDFP-15

A light protein of breast cyst fluid from women with gross cystic disease, termed GCDFP-15 in the literature, has been investigated. This light protein was purified by preparative electrophoresis on sodium dodecyl sulfate polyacrylamide gel. Its isoelectric point has been determined as 3.75 and its molecular weight has been estimated at 17 400. The light protein was a glycoprotein containing about 163 amino acid residues; the glucidic fraction corresponded to 11% of the molecular weight. The N-terminal amino acid was blocked and the C-terminal amino acid was determined as valine. Antisera raised against this light protein have proved to be specific. In the literature, there is evidence suggesting that apocrine secretion is of prime importance in conditioning the biochemical composition of breast cyst fluid. Further information is needed to substantiate the hypothesis that in gross cystic disease the apocrine epithelium itself or some of its functional aspects are associated with the risk of neoplasia. The physicochemical characterization of the breast cyst fluid protein can contribute to the study of its biosynthesis and provide a better understanding of the physiopathology of gross cystic disease and its relationship to breast carcinoma.     

Human dehydroepiandrosterone sulfotransferase: purification and characterization of a recombinant protein

Dehydroepiandrosterone sulfate is the most abundant sulfated steroid transformed in human tissues and serves as a precursor for steroid hormones. Recombinant human dehydroepiandrosterone sulfotransferase (DHEA-ST) expressed in glutathione sulfotransferase fusion form in E. coli was purified using glutathione sepharose 4B affinity adsorption chromatography, a Factor Xa cleavage step, and Q-sepharose fast flow column chromatography. The homogeneous preparation had an activity toward dehydroepiandrosterone (DHEA) of 150+/-40 nmol/min per mg of protein under the assay conditions at an overall yield of 38.4%. The recombinant human DHEA-ST was shown to have a subunit mass of 34 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, while having a molecular mass of 67.2 kDa by Superose-12 gel filtration. Our results indicate that the active recombinant enzyme expressed in E. coli is a homodimer.Biochemical properties for purified DHEA-ST were studied using DHEA as a substrate. The optimum pH ranged from pH 7 to 8, and the optimum temperature 40-45 degrees C. Ninety percent of basal DHEA-ST activity remained even after the enzyme was treated at 45 degrees C for 15 min. The 50% inactivation concentration of NaCl for DHEA-ST activity was determined to be around 500 mM. The K(m) value for DHEA was 1.9+/-0.3 microM and V(max)=190+/-18 nmol/min per mg of protein at 37 degrees C, pH 7.5.     

Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis

A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.     

Purification and characterization of mannitol dehydrogenase and identification of the corresponding cDNA from the head blight fungus,Gibberella zeae (Fusarium graminearum)

The mannitol-2-dehydrogenase (MtDH) from Gibberella zeae was purified and the corresponding cDNA identified. Purification of MtDH was accomplished using a combination of ammonium sulfate fractionation, anion exchange and dye-ligand chromatography. Final purification was achieved following electroelution from a native gel. Molecular mass determination based on SDS-PAGE indicated that the denatured protein was 29 kDa. Native protein mass was determined to be 110 kDa using gel permeation chromatography, indicating a tetrameric form. The pH optima for mannitol oxidation and fructose reductase activities were 9.0, and 7.0, respectively. Activity with sorbitol as the substrate was 21% of activity with mannitol. Kinetic parameters were determined by direct-linear plots of enzyme activity vs. substrate concentrations. Fructose concentrations above 600 mM and NADPH concentrations above 0.3 mM caused substrate inhibition. Comparisons of predicted amino acid sequences of several fungal MtDHs indicated high conservation within the phyla. A possible role for MtDH in generation of turgor pressure for forcible ascospore discharge is discussed.     

Heterogeneity of streptococcus group A cell wall protein components

Cell wall surface proteins of group A streptococcus (M 29) were isolated by mild chemical extraction with 1 M hydroxylamine pH 6.0 (37 degrees C). The proteins were purified by ammonium sulfate fractionation, gel filtration on Sephadex G-150 and ion-exchange chromatography on DEAE-Trisacryl M. Using two independent methods (disc electrophoresis in 7.5% PAAG pH 8.9 and high pressure gel filtration), it was shown that after chromatography on Sephadex G-150 the original protein fraction contains up to 8 protein components, while SDS-PAAG electrophoresis performed according to Laemmli revealed up to 25 protein components in the same fraction. During SDS-PAAG electrophoresis six protein fractions performed after ion-exchange chromatography were resolved into 40 protein components whose molecular masses vary from 13 to 80 kDa. Possible reasons for the heterogeneity of surface proteins of group A streptococcus cell wall are discussed.