Characterisation of the gene encoding a protective antigen from Babesia microti identified it as eta subunit of chaperonin containing T-complex protein 1.

Passive immunisations with a monoclonal antibody termed 1-5H showed a partial but significant inhibition of parasitaemia against Babesia microti challenge infection. By immunoscreening with 1-5H, a clone (termed p58 gene) was obtained from a cDNA expression library of B. microti and the complete nucleotide sequence was determined. A protein homology search showed significant amino acid identities to the η subunit of the chaperonin containing T-complex protein 1 (CCT) of human (59%), mouse (58%) and Plasmodium falciparum (62%). Genomic analyses indicated that the p58 gene is present as a single copy gene and contains a total of approximately 400-bp introns in the genome of B. microti. The mAb 1-5H recognised a 58-kDa protein of B. microti and was found to cross-react with a 60-kDa protein of Babesia rodhaini. These results suggest the possibility that the p58 protein is the CCT η subunit of B. microti and functions as a chaperonin.     

Babesia microti: Molecular and antigenic characterizations of a novel 94-kDa protein (BmP94)

A novel gene, BmP94, encoding 94-kDa protein of Babesia microti was identified by immunoscreening of the cDNA expression library. The full-length of BmP94 was expressed in Escherichia coli (rBmP94), which resulted in insoluble form with low yield, and the truncated hydrophilic C-terminus region of the gene was expressed as a soluble protein (rBmP94/CT) with improved productivity. Antiserum raised against rBmP94/CT recognized the 94-kDa native protein in the parasite extract by Western blot analysis. Next, an ELISA using rBmP94/CT was evaluated for diagnostic use, and it demonstrated high sensitivity and specificity when tested with the sera from mice experimentally infected with B. microti and closely related parasites. Moreover, the immunoprotective property of rBmP94/CT as a subunit vaccine was evaluated in BALB/c mice against a B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmP94 could be a promising candidate for diagnostic use for human babesiosis.     

Cloning and characterization of an mRNA encoding a novel G protein α-subunit abundant in mantle and gill of pearl oyster Pinctada fucata

Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel G_sα (pfG_sα) from the pearl oyster was isolated. The G_sα cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus (Octopus vulgaris) G_sα. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfG_sα has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster G_sα mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfG_sα in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfG_sα that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the G_sα in MC3T3-E1 cells. These results indicated that G_sα may be involved in regulation of physiological function, particularly in biological biomineralization.     

Identification and characterization of a novel secreted antigen 1 of Babesia microti and evaluation of its potential use in enzyme-linked immunosorbent assay and immunochromatographic test

Here, we identified a novel secreted antigen designated as Babesia microti secreted antigen 1 (BmSA1) by immunoscreening a B. microti cDNA expression library using the sera from hamsters immunized with plasma, putatively containing secreted antigens, from B. microti-infected hamsters. Antibodies raised in mice immunized with recombinant BmSA1 (rBmSA1) recognized a native 33-kDa parasite protein. An enzyme-linked immunosorbent assay (ELISA) of rBmSA1 detected specific antibodies as early as 6 and 4 days post-infection in sera from a hamster experimentally infected with B. microti Gray strain (US type) and a mouse experimentally infected with B. microti Munich strain (rodent isolate), respectively. Moreover, a rapid immunochromatographic test (ICT) using rBmSA1 detected specific antibodies in a hamster experimentally infected with B. microti from day 6 to at least day 270 post-infection, which was quite consistent with the results of the ELISA. In addition, analysis of the sera involved in the first case of human babesiosis in Japan (Kobe type) showed that specific antibodies were detectable in the patient and the positive donor by ELISA using rBmSA1, and the ICT result was identical to the ELISA data. Taken together, these results indicated that BmSA1 could be a promising and universal target for developing both ELISA and ICT for the serodiagnosis of human babesiosis and for an epidemiological survey of its rodent reservoir.     

High-Yield Expression and Purification of Human Interferon α-1 in Pichia pastoris

For several years, interferon α-1, also known as interferon α-D, has been studied for treatment of various viral diseases, such as hepatic fibrosis caused by hepatitis B, herpes simplex virus keratitis, and bovine respiratory diseases in calves. Currently, recombinant human interferon α-D (rHuIFNαD) is expressed intracellularly in Escherichia coli or secreted by Bacillus subtilis and Saccharomyces cerevisiae. In this report, we describe the process of obtaining a relatively high-yield secretion of biologically active recombinant rHuIFNαD using the Pichia pastoris system. The process produced as high as 0.7 mg of purified protein per 20 ml of shake culture of rHuIFNαD with better bioactivity than the commercially available rHuIFNαD molecule produced in E. coli.     

Affinity chromatography of α-amylase from Bacillus licheniformis

An affinity chromatographic method with a novel eluant from Bacillus licheniformis is described. α-amylase was bound to starch, starch-celite, starch-Sepharose columns and the bound α-amylase was rapidly eluted with 2% (w/v) white dextrin. The binding capacity of α-amylase to starch column is 380 μmol/g of starch. The purified enzyme showed a single polypeptide on SDS-polyacrylamide gel electrophoresis with a molecular weight of 58 kD. The specificity of purified enzyme was confirmed by immunodiffusion, immunoelectrophoresis. Single radial immunodiffusion and western blotting studies analyzed the synthesis of enzyme at different time points.     

Characterization of a root-specific β-thioglucoside glucohydrolase gene in Carica papaya and its recombinant protein expressed in Pichia pastoris

Plant β-thioglucoside glucohydrolases (TGGs or myrosinases) are a young class of enzymes in the glycosyl hydrolase family 1 and have a narrow distribution. TGG genes have mainly been cloned from crucifers, while TGGs in other species have received little attention. The TGG gene CpTGG2 and its recombinant protein from papaya were characterized in this paper. This is the first plant TGG gene without unusual intron splicing borders, as present in all other available TGG genes. Phylogenetic analysis indicated that plant myrosinases are divided into two major lineages. CpTGG2 is located in the lineage constituted by AtTGG4–6 from Arabidopsis thaliana, while the rest of myrosinases (including MA, MB and MC subfamilies) are grouped into another lineage. RT-PCR analysis indicated that CpTGG2 was specifically expressed in the root. The recombinant CpTGG2 expressed in yeast had a subunit mass of 70 kDa, and had low basal TGG activity without addition of ascorbate. Low concentrations of ascorbate stimulated CpTGG2 activity, while high concentrations were inhibitory. CpTGG2 was active in broad pH and temperature ranges, similar to AtTGG4 and AtTGG5. The apparent K_m and V_max were 2.24 mM and 24.3 μmol min⁻¹ mg⁻¹ when sinigrin was the substrate. The calculated k_cat/K_m value was 1.3 × 10⁴ S⁻¹ M⁻¹_. Our results reshaped and expanded the myrosinase family structure and provided clues to the evolution of myrosinase genes.     

Conversion of α-methyltropate to optically active α-phenylpropionate by tropate-degrading Rhodococcus sp. KU1314

Microorganisms which can assimilate tropate were screened from soil. Among them, we found a microorganism which has an ability to convert α-methyltropate to optically active α-phenylpropionate, and it was identified as Rhodococcus sp. KU1314. Substrate specificity of the microorganism has been studied. When the aryl group was phenyl, 4-methoxyphenyl and 2-naphthyl, the substrate gave optically active α-propionate in good yields. To estimate the reaction mechanism, some compounds considered to be the intermediates were subjected to the reaction. Both enantiomers of α-methyltropate were converted to (R)-α-phenylpropionate with almost the same enantiomeric excess (68 and 72% from R-and S-enantiomers, respectively) and yield (605 and 48% from R-and S-enantiomers, respectively).     

Cell wall α-1,3-glucans from a biocontrol isolate of Rhizoctonia: immunocytolocalization and relationship with α-glucanase activity from potato sprouts

The interface between plants and pathogens plays an important role in their interaction. Studies of fungal cell walls are scarce and previous results show the existence of α-1,3-glucans in addition to ß-glucans. In addition, α-1,3-glucans are not present in plant cell walls, and α-glucanase activity in plants has not been described before. In a previous work, we purified and characterized an α-1,3-glucan from a binucleated, non-pathogenic Rhizoctonia isolate, which induces plant defence responses. Therefore, in order to study the architecture of the fungal cell wall, and the accessibility and localization of the α-glucan elicitor, we prepared an antibody against the α-1,3-glucan and analysed its localization by TEM. Immunolocalization showed the presence of the α-1,3-glucan in the intercellular spaces and along the cell walls, mainly on the inner layers. This result, and the presence of the α-1,3-glucan in the liquid culture medium in which binucleated non-pathogenic Rhizoctonia was grown, confirmed that the α-glucan had been secreted. The α-1,3-glucan was also immunocytolocalized on potato sprouts tissue elicited with the glucan; gold particles were observed in vacuoles and close to the plasmalemma. In addition, α-glucanase activity in potato sprouts was detected using cell wall glucans from the pathogenic isolate R. solani AG-3 as substrates; whereas, when cell wall glucans from non-pathogenic isolates were used, no α-glucanase activity was detected. Our results suggest that the presence of α-1,3-glucans could be associated with the formation and integrity of the cell wall and also with plant–fungi interactions. This is the first report to describe α-glucanolytic activity in plants.     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

Cerebroside and β-carboline alkaloids from the ascidian Ascidia longistriata

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Highlights? Ascidia longistriata was collected for secondary metabolites investigation. ? This is the first report of compounds 1-3 from ascidians of the genus Ascidia. ? Compound 2 might be derived from an associated organism.     

The Gα4 G protein subunit interacts with the MAP kinase ERK2 using a D-motif that regulates developmental morphogenesis in Dictyostelium

G protein Gα subunits contribute to the specificity of different signal transduction pathways in Dictyostelium discoideum but Gα subunit-effector interactions have not been previously identified. The requirement of the Dictyostelium Gα4 subunit for MAP kinase (MAPK) activation and the identification of a putative MAPK docking site (D-motif) in this subunit suggested a possible interaction between the Gα4 subunit and MAPKs. In vivo association of the Gα4 subunit and ERK2 was demonstrated by pull-down and co-immunoprecipitation assays. Alteration of the D-motif reduced Gα4 subunit-ERK2 interactions but only slightly altered MAPK activation in response to folate. Expression of the Gα4 subunit with the altered D-motif in gα4⁻cells allowed for slug formation but not the morphogenesis associated with culmination. Expression of this mutant Gα4 subunit was sufficient to rescue chemotactic movement to folate. Alteration of the D-motif also reduced the aggregation defect associated with constitutively active Gα4 subunits. These results suggest Gα4 subunit-MAPK interactions are necessary for developmental morphogenesis but not for chemotaxis to folate.     

AtBS14a and AtBS14b, two Bet1/Sft1-like SNAREs from Arabidopsis thaliana that complement mutations in the yeast SFT1 gene

SNAREs are membrane-associated proteins that play a central role in vesicle targeting and intra-cellular membrane fusion reactions in eukaryotic cells. Here we describe the identification of AtBS14a and AtBS14b, putative SNAREs from Arabidopsis thaliana that share 60% amino acid sequence identity. Both AtBS14a and BS14b are dosage suppressors of the temperature-sensitive growth defect in sft1-1 cells and over-expression of either AtBS14a or AtBS14b can support the growth of sft1Δ cells but not bet1Δ cells. These data together with structure–function and biochemical studies presented herein suggest that AtBS14a and AtBS14b share properties that are consistent with them being members of the Bet1/Sft1 SNARE protein family.     

Studies on construction of a recombinant Eimeria tenella SO7 gene expressing Escherichia coli and its protective efficacy against homologous infection

Eimeria spp. are the causative agents of coccidiosis, a major disease affecting the poultry industry. A recombinant non-antibiotic Escherichia coli that expresses the Eimeria tenella SO7 gene was constructed and its protective efficacy against homologous infection in chickens was determined. The three-day-old chickens were orally immunized with the recombinant non-antibiotic SO7 gene expressing E. coli and boosted two weeks later. Four weeks after the second immunization, the chickens were challenged with 5 × 10⁴ homologous sporulated oocysts. The protective effects of the recombinant non-antibiotic E. coli were determined by measuring body weight change, mortality, histopathology, lesion scores, oocyst counts, the specific antibody response and the frequency of CD4⁺ and CD8⁺ lymphocytes in peripheral blood. The results showed that immunization with SO7 expressing E. coli resulted in significantly improved body weight gain, reduced lesion scores and oocyst shedding in immunized chickens compared to controls. Furthermore, administration of recombinant SO7 expressing E. coli leads to a significant increase in serum antibody, CD4⁺ and CD8⁺ T cells in peripheral blood of chickens. These results, therefore, suggest that the recombinant non-antibiotic E. coli that expresses the SO7 gene is able to effectively stimulate host protective immunity as evidenced by the induction of development of both humoral and cell-mediated immune responses against homologous challenge in chickens.     

Molecular characterization of rgp2, a gene encoding a small GTP-binding protein from rice

Summary We previously reported the isolation of rgp1, a gene from rice, which encodes a ras-related GTP-binding protein, and subsequently showed that the gene induces specific morphological changes in transgenic tobacco plants. Here, we report the isolation and characterization of an rgp1 homologue, rgp2, from rice. The deduced rgp2 protein sequence shows 53% identity with the rice rgp1 protein, but 63% identity with both the marine ray ora3 protein, which is closely associated with synaptic vesicles of neuronal tissue, and the mammalian rab11 protein. Conservation of particular amino acid sequence motifs places rgp2 in the rab/ypt subfamily, which has been implicated in vesicular transport. Northern blot analysis of rgp1 and rgp2 suggests that both genes show relatively high, but differential, levels of expression in leaves, stems and panicles, but low levels in roots. In addition, whereas rgp1 shows maximal expression at a particular stage of plantlet growth, rgp2 is constitutively expressed during the same period. Southern blot analysis suggests that, in addition to rgp1 and rgp2, several other homologues exist in rice and these may constitute a small multigene family.