Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori

Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm.

Phylogenetic analysis of human group C rotavirus circulating in Brazil reveals a potential unique NSP4 genetic variant and high similarity with Asian strains

Group C rotaviruses (RVC) cause gastroenteritis in humans and animals worldwide, and the evidence for a possible zoonotic role has been recently provided. To gain information on the genetic diversity and relationships between human and animal RVC, we sequenced the VP4, VP7, and NSP4 genes of 12, 19, and 15 human strains, respectively, detected in São Paulo state during historical (1988 and 1993) and recent (2007 and 2008) Brazilian rotavirus surveillance. All RVC strains analyzed in the present study grouped into human genotype (G4-P[2]-E2), and did not show any evidence of animal ancestry. Phylogenetic analysis showed that RVC samples detected in 1988 and 1993 clustered together with strains from distinct continents, indicating that historical RVC strains circulating in São Paulo were closely related to those strains circulating worldwide. All three genes (VP7, VP4 and NSP4) of São Paulo RVC strains isolated in 2007-2008 exhibited close phylogenetic relationship with human RVC strains isolated in China and Japan, suggesting that they are genetically linked, and that a gene flow could be occurring between this Asian countries and Brazil. We identified two distinct clusters in the NSP4 phylogenetic tree. One cluster formed exclusively by human Brazilian strains detected in 1997 and 2003-2004 in Rio de Janeiro, Bahia, and Rio Grande do Sul states (Subgroup II) previously described in a different study, that displayed low sequence identities to other human strains formerly published, and to the Brazilian RVC strains (Subgroup I) characterized in the present study. These data suggests the circulation of two genetic profiles of the NSP4 gene in Brazil. High sequence diversity in NSP4 gene was previously reported in Asia, and additional diversity in NSP4 RVC strains spreading in the world should be expected. More in-depth molecular and epidemiological analysis of human RVC throughout the world will be needed to understand their diversity and clarify their evolution, as well as to develop classifications schemes.

Isolation and characterization of avian metapneumovirus from chickens in Korea

Avian metapneumovirus (aMPV) causes upper respiratory tract infections in chickens and turkeys. Although the swollen head syndrome (SHS) associated with aMPV in chickens has been reported in Korea since 1992, this is the study isolating aMPV from chickens in this country. We examined 780 oropharyngeal swab or nasal turbinate samples collected from 130 chicken flocks to investigate the prevalence of aMPV and to isolate aMPV from chickens from 2004-2008. Twelve aMPV subtype A and 13 subtype B strains were detected from clinical samples by the aMPV subtype A and B multiplex real-time reverse transcription polymerase chain reaction (RRT-PCR). Partial sequence analysis of the G glycoprotein gene confirmed that the detected aMPVs belonged to subtypes A and B. Two aMPVs subtype A out of the 25 detected aMPVs were isolated by Vero cell passage. In animal experiments with an aMPV isolate, viral RNA was detected in nasal discharge, although no clinical signs of SHS were observed in chickens. In contrast to chickens, turkeys showed severe nasal discharge and a relatively higher titer of viral excretion than chickens. Here, we reveal the co-circulation of aMPV subtypes A and B, and isolate aMPVs from chicken flocks in Korea.

Glicoproteína del virus rábico: Estructura, inmunogenicidad y rol en la patogenia / Rabies vims glycoprotein: Structure, immunogenicity and pathogenic role

Rabies glycoprotein is the only exposed protein which is inserted in the viral lipidie envelope. This 65-67 kda protein is a N-glycosilated transmembrane protein forming trimers on the viral surface. It has been identified as the major pathogenicity determinant, playing a role in the budding, viral axonal transport during infection, apoptosis and immune evasion. It is also the major antigen responsible for the protective immune response and it is been used in commercial recombinant vaccines. Its structure, antigenicity and pathogenic role have been well studied, identifying main antigenic sites that have the responsibility for virulence, cellular receptors attachment and epitope acquisition.

La glicoproteína del virus rábico es la única proteína viral expuesta, encontrándose inserta en la envoltura lipídica. Esta molécula de 65-67 kda corresponde a una proteína trans-membrana N-glicosilada que se dispone en forma de trímeros en la superficie viral. Ha sido identificada como el mayor determinante de pato-genicidad, participando además en procesos de yemación, flujo axonal del virion durante la infección, apoptosis y evasión de la respuesta inmune. Es también el principal antígeno inductor de la respuesta inmune protectora siendo utilizado en vacunas recom-binantes comerciales. Su estructura, antigenicidad e implicancias en la patogenia han sido bien estudiadas identificándose los principales sitios antigénicos responsables de la patogenicidad, unión a receptores celulares y formación de epitopos.

Sequence analysis of the S1 glycoprotein gene of infectious bronchitis viruses: identification of a novel phylogenetic group in Korea

Twelve Korean infectious bronchitis viruses (IBVs) were isolated in the field from chickens suspected of being carriers of infectious bronchitis between 2001 and 2003. The S1 glycoprotein genes of these IBV isolates were amplified by reverse transcriptase-polymerase chain reaction (RTPCR) and analyzed by restriction fragment length polymorphism (RFLP) analysis. These Korean IBV isolates were classified into three groups according to their RFLP patterns obtained using the restriction enzyme HaeIII. Half of the twelve isolates were similar to the KM91 RFLP pattern, which is a common pattern in Korea. Three more isolates were related to the Arkansas strain pattern, but with some unique variations. The other three viruses showed variant RFLP patterns. For a comparison with the published sequences for non-Korean IBV strains, amplified PCR products from the twelve isolates were cloned and sequenced. The Korean IBV field isolates had 71.2-99.7% nucleotide sequence homology with each other and 45.9-80.7% nucleotide sequence homology with non-Korean IBV strains. With respect to the deduced amino acid sequence, the Korean IBV isolates had 71.5-99.3% similarity with each other and 44.9-80.3% similarity with non-Korean IBV strains. Phylogenetic tree analysis revealed that some of the IBV isolates appear to belong to a new group, different from the non-Korean IBV strains or from previously isolated Korean IBV strains. Specifically, the new Korean IBV isolates K10217-03, K3-3 and K1255-03 represented a separate group. These findings suggest that the Korean IBVs appear to be continuously evolving.

Glycobiology of Leishmania donovani.

Leishmania donovani, the causative organism of visceral leishmaniasis (VL) is one of the deadliest of the entire known Leishmania species. This protozoan parasite displays immense adaptability to survive under extremely harsh conditions. Cell surface glycoconjugates play a pivotal role in parasite virulence and infectivity. This review mainly highlights on the importance of these molecules and their reported roles with special emphasis on L. donovani sialobiology. The recently evolved information reported by our group regarding the identification and characterization of sialoglycans and their possible mode(s) of acquisition as also the detailed identification, characterization of anti-O-acetylated sialic acid (anti-OAcSA) antibodies and their emerging biological roles, notably as molecules that may aid in host defense against the pathogen has been vividly discussed in this review.

Comparative diagnostic potentiality of ELISA and dot-ELISA in prepatent diagnosis of experimental Fasciola gigantica infection in cattle.

A glycoprotein (27 kDa) was isolated from crude somatic antigen of Fasciola gigantica by two steps affinity chromatography and was used in early detection of experimental fasciolosis in cattle by indirect ELISA and in dot-ELISA formats. Although, anti-27 kDa antibodies could be detected after 3 weeks post infection (WPI) by dot - ELISA which was one week later than indirect ELISA. The test, dot-ELISA, was more convenient in field application. By the test (dot-ELISA) the infection could be equally detected in animals infected with 100, 200 and 300 metacercariae of F. gigantica with high sensitivity. Further, the antigen (27 kDa) was not found to react with goat sera infected with Paramphistomum epiclitum, which are giving strong reaction to homologous immature and mature fluke antigens of P. epiclitum.

A review of genetic and structural understanding of the role of myocilin in primary open angle glaucoma.

Primary open angle glaucoma (POAG) is the most common form of glaucoma and the second leading cause of blindness in the world. Discovery of the candidate gene MYOC (TIGR/MYOC) encoding the protein myocilin, believed to have a role in cytoskeletal function, might play a key role in understanding the pathogenesis of POAG. MYOC is expressed in many ocular tissues, including trabecular meshwork (TM), a specialised eye tissue essential in regulating intraocular pressure (IOP). Later it was shown to be the trabecular meshwork inducible-glucocorticoid response protein (TIGR). Mutations in MYOC have been identified as the cause of hereditary juvenile-onset open-angle glaucoma (JOAG). The unprocessed myocilin with signal peptide is a 55-kDa protein with 504 amino acids. Mature myocilin is known to form multimers. Wild type myocilin protein is normally secreted into the trabecular extracellular matrix (ECM) and there appears to interact with various ECM materials. It is believed that the deposition of high amounts of myocilin in trabecular ECM could affect aqueous outflow either by physical barrier and/or through cell-mediated process leading to elevation of IOP. The N-terminal region of the myocilin has sequence similarity to myosin (muscle protein) and the C-terminal of the protein has an olfactomedin-like domain. Structural and genetic studies of the MYOC gene and its protein product along with molecular modeling could lead to better understanding of the pathogenesis of POAG. This review highlights the current understanding of myocilin and the relevance of genetic and structural work.

Glándulas linguales de Blandin y Nuhn; estudio histoquímico de los glicoconjugados de las células serosas y mucosas / Blandin and Nuhn lingual glands; an histochemical study of serous and mucous sugars

Se emplearon las técnicas histoquímicas convencionales para mucinas (PAS, Alcian blue y Azul de toluidina) y la técnica de avidina-biotina para estudiar la unión de las lectinas PNA, UEA-1, RCA-1, ConA, DBA, SBA y WGA a los azúcares específicos en cortes histológicos de glándulas inguales anteriores de Blandin y Nuhn. Las células secretoras mucosas y serosas exhibieron diferentes grados de coloración dependiendo de la lectina y el tipo celular. Estos resultados proveen las bases para la comparación de posibles cambios en las enfermedades de las glándulas salivales menores

Carboxy terminal region of a chloroplast DNA polymerase accessory factor stimulates DNA polymerase activity.

p43, a glycoprotein of pea chloroplast (ct), acts as an accessory protein of pea chloroplast DNA polymerase. p43 binds to DNA, binds to ct-DNA polymerase and stimulates the ct-DNA polymerase activity. In the work presented here, the C-terminal domain of p43 (p22) has been overexpressed in E. coli. South Western analysis reveals that the recombinant p22 lacks in DNA binding activity. However, the recombinant p22 can form complex with the pea ct-DNA polymerase quite efficiently and stimulates the DNA polymerase activity to a greater extent than the native p43. Thus the DNA binding domain of p43 appears to be spatially separate from the domain responsible for the DNA polymerase accessory activity. The DNA binding domain is also highly O-glycosylated and loss of glycosylation of p43 leads to enhanced DNA binding as well as repression of ct-DNA polymerase activity. These findings allow us to propose a model to explain how glycosylation of p43 helps ct-DNA polymerase latch onto the DNA template for enhanced processivity. The predictive components of the model have been discussed.

Members of the Tc-85 protein family from Trypanosoma cruzi are adhesion proteins

In recent years, attention has been focused on the characterization of surface proteins from Trypanosoma cruzi in an attempt to understand the invasion mechanism of the parasite. Among the molecules described by different laboratories, we report an 85-KDa glycoprotein specific for the trypomastigote stage (Tc-85) which has been implicated in the invasion of host cells by the parasite. The hypothesis that different members of the Tc-85 protein family are involved in the adhesion of the parasite to the host is discussed.

Unique structural features of red kidney bean purple acid phosphatase.

Purple acid phosphatase from red kidney beans (Phaseolus vulgaris) has been purified to homogeneity and characterized. The enzyme is a homodimer of 60 kDa subunits each containing one atom of zinc and iron in the active site. Circular dichroism spectral studies on the purified enzyme reveals that a large portion of the peptide backbone is in the unordered and beta-turn conformation. A unique feature of the red kidney bean acid phosphatase, which we have found, is that one of the two cysteines of each subunit is involved in the formation of an inter-subunit disulphide. The thiol group of the other cysteine is not necessary for the activity of the enzyme. Western blot analysis with antibodies raised against kidney bean acid phosphatase could not recognize acid phosphatases from other sources except from potato. This paper emphasizes the fact that acid phosphatases are functionally, but not structurally, conserved enzymes.

Mechanism of action of an eukaryotic initiation factor-2 (eIF-2) associated 67 kDa glycoprotein (p67) and an eIF-2 kinase (dsI).

Mechanism of regulation of eIF-2 alpha-subunit phosphorylation by dsI and p67 was studied. The results are as follows: (1) At low dsI concentration, p67 protected equimolar concentration of eIF-2. (2) At high dsI concentration, dsI efficiently phosphorylated eIF-2 alpha-subunit even when equimolar concentrations of both p67 and eIF-2 were present. Significantly increased p67 concentration was necessary to protect eIF-2 alpha-subunit at high dsI concentration. (3) dsI was also phosphorylated as it phosphorylated eIF-2 alpha-subunit. p67 inhibited both eIF-2 alpha-subunit and dsI phosphorylation similarly. (4) Although the [32P]-labelled dsI formed during the reaction could be effectively chased upon subsequent addition of excess unlabelled eIF-2 and ATP, the [32P] labelled eIF-2 formed under identical conditions, retained most of the radioactivity. (5) dsI coimmunoprecipitated with three subunit eIF-2 and p67 inhibited this coimmunoprecipitation reaction. It has been proposed: Three subunit eIF-2 and free p67 are in equilibrium with eIF-2 bound to p67 and, eIF-2.p67 complex is resistant to dsI phosphorylation. Activated dsI is already phosphorylated. At high concentration, dsI(P) can bind to free three subunit eIF-2 and form eIF-2.dsI(P) complex. dsI(P) in this complex then transfers its phosphoryl residue to eIF-2 and forms eIF-2 alpha(P) in an irreversible reaction. In a subsequent reaction, unphosphorylated dsI is autophosphorylated using [gamma 32P]-ATP and the cycle continues. Inhibition of eIF-2 alpha-subunit phosphorylation by p67 blocks this phosphorylation cycle and consequent dsI phosphorylation.

Identification of endogenous soluble glycoprotein receptors of bovine brain grey matter 14 kDa beta-galactoside-binding lectin.

The 14 kDa beta-galactoside-binding lectin from bovine brain grey matter (BBL) covalently attached to caproic acid-Sepharose by the N-hydroxy succinimide procedure was used to isolate endogenous glycoprotein receptors of this lectin. BBL-Sepharose could sugar-specifically retain several endogenous soluble glycoproteins with subunit molecular mass (in kDA) 44, 51, 60, 123 and 186. BBL, conjugated with horse radish peroxidase, could sugar-specifically recognize several glycoprotein subunits with molecular mass (in kDA) 58, 87, and 117 and 186 on Western blots. The only protein from an extract of bovine brain grey matter, that retained on Sepharose-immobilized endogenous N-linked glycoproteins and subsequently eluted with beta-galactosides was BBL as confirmed by electrophoresis and agglutination inhibition measurement. N-linked glycoproteins from bovine heart and even from human placenta were also efficient receptors of BBL. These results suggest that 14 kDa beta-galactoside-binding lectin is the major protein, if not the only one, that sugar-specifically interacts with endogenous soluble glycoproteins in bovine brain grey matter.

Structural features of LPPG and related compounds isolated from Trypanosoma cruzi trypomastigotes

A hydrophobic fraction isolated from trypomastigotes of Trypanosoma cruzi is being characterized using immunological and chemical techniques. The lipopeptidophosphoglycan (LPPG) was identified in this fraction since it gave a positive reaction with anti-LPPG rabbit serum and had similar structural features such as the presence of ceramide as the lipid moiety, furanoic galactose, and a glycan moiety consistent with that obtained from an authentic sample of epimastigote LPPG, as judged by thin-layer chromatography. Furthermore, the hydrophobic fraction contained other glycolipids with different structural features. The lipid moiety of these compounds is alkylglycerol rather than a ceramide, the carbohydrate chain appears to be less complex than that in LPPG and no reactivity was observed towards an anti-LPPG serum

Interaction of pulmonary surfactant-associated protein (SP-A) with surfactant lipids.

A pulmonary surfactant-associated protein complex with components of 36, 32 and 28 kDa was isolated from human lung homogenates and reassembled with surfactant lipids prepared as small unilamellar liposomes. The role of divalent cations in the assembly of this recombinant lipoprotein complex was studied by monitoring the changes in turbidity, intrinsic tryptophanyl fluorescence and surface activity. The protein-facilitated lipid aggregation was promoted on addition of 5 to 20 mM Ca2+. Intrinsic fluorescence measurements on SP-A (28-36 kDa) indicated that the tryptophan side chains were in a relatively hydrophobic environment, that the wavelength of maximum fluorescence emission and also the relative fluorescence, were changed upon the binding of lipid. Tryptophanyl fluorescence of the lipoprotein assembly was quenched as indicated by a reduction in the effective Stern-Volmer constant. These results suggest that Ca2+ lipid-protein interactions are involved in the structure and function of extracellular lung surfactant assembly.

Isolation, purification and preliminary studies on fish eye lens glycoprotein.

Glycoprotein from the eye lens of fish, Mystus cavasius, was isolated by extraction with 1% Triton X-100 in saline. The crude extract which was found to be electrophoretically heterogeneous was fractionated on a DEAE-cellulose column. One of the fractions obtained in major amount was further resolved by column chromatography using Sephadex G-150 into two homogeneous fractions (GP-1 and GP-2]. GP-1 contained carbohydrates (11.2%) and protein (77.5%). The constituent sugars were D-glucose, D-mannose, D-galactosamine and N-acetyl neuraminic acid. The principal amino acids were aspartic acid, serine, glutamic acid, glycine and alanine. The proportions of these residues were determined.

Mechanism of host cell membrane modification by tumour: a model for paraneoplastic syndromes.

A not well-appreciated but clinically important aspect of malignant tumours is their effects on distantly located host cells. The effects, termed paraneoplastic syndromes, also pose an intriguing mechanistic problem: how do malignant cells influence properties of host cells not in contact with them? Erythrocytes from the circulation of rats bearing intraperitoneal Yoshida ascites sarcoma exhibit higher agglutinability with concanavalin A (Con A) than the cells from normal animals. Since the tumour and the red cells are not in contact, the enhanced agglutinability of the latter is a paraneoplastic effect. The mechanism by which the tumour brings about this effect is investigated as a model for paraneoplastic syndromes. The cell-free ascites fluid is able to impart high agglutinability on cells from normal animals in vitro. Also, when injected intraperitoneally in normal animals, the ascites fluid is able to enhance the agglutinability of erythrocytes in circulation. Apparently the tumour produces a substance(s) that appears in the ascites fluid and is able to diffuse into circulation, explaining the mechanism by which it can reach distant sites. From the cell-free ascites fluid three fractions have been isolated that are active in vitro. Of these, only one showed activity in vivo. From this fraction, a glycoprotein has been purified to homogeneity that confers maximal Con A-agglutinability on normal erythrocytes at 8 x 10(-7)M, at which concentration 6,400 molecules bind per cell. The protein has a molecular weight of 600 kDa in the native state and a pI of 5.35. It is made up of 4 identical subunits of Mr 170,000. It is detected in the plasma of tumour-bearing but not normal rats.(ABSTRACT TRUNCATED AT 250 WORDS)