Enzymatic characteristics of a recombinant neutral protease I (rNpI) from Aspergillus oryzae expressed in Pichia pastoris.

A truncated neutral protease I (NpI) from Aspergillus oryzae 3.042 was expressed in Pichia pastoris with a high enzyme yield of 43101 U/mL. Its optimum pH was about 8.0, and it was stable in the pH range of 5.0-9.0. Its optimum temperature was about 55 °C and retained >90% activity at 50 °C for 120 min. Recombinant NpI (rNpI) was inhibited by Cu(2+) and EDTA. Eight cleavage sites of rNpI in oxidized insulin B-chain were determined by mass spectrometry, and five of them had high hydrophobic amino acid affinity, which makes it efficient in producing antihypertensive peptide IPP from ß-casein and a potential debittering agent. The high degree of hydrolysis (DH) of rNpI to soybean protein (8.8%) and peanut protein (11.1%) compared to papain and alcalase makes it a good candidate in the processing of oil industry byproducts. The mutagenesis of H(429), H(433), and E(453) in the deduced zinc-binding motif confirmed rNpI as a gluzincin. All of these results show the great potential of rNpI to be used in the protein hydrolysis industry.

Molecular cloning, recombinant expression and antibacterial activity analysis of hepcidin from Simensis crocodile (Crocodylus siamensis).

Hepcidin, a cysteine-rich cationic antibacterial peptide, plays an important role in human defense against pathogen infection. However, its role in reptile immune response and whether it is involved in antibacterial immune have not yet been proven. In order to study the antibacterial activity of Crocodylus siamensis hepcidin (Cshepc), a common reptile which lives in topic region of Southeast Asia, a cDNA sequence of Cshepc was cloned, which included an open reading frame (ORF) of 300 bp encoding a 99 amino acid preprohepcidin. Cshepc has eight cysteines formed four conserved disulfide bridges, similarly to that of human's. Sequence analysis showed that Cshepc mature peptide was more conserved than that of preprohepcidin. Tissue expression analysis indicated that Cshepc transcripts were highly expressed in the liver, muscle and heart of C. siamensis. Recombinant expressed hepcidin could significantly inhibit the growth of the Gram-negative bacteria Escherichia coli and Aeromonas sobria as well as the Gram-positive bacterium Staphylococcus aureus, and Bacillus subtilis in vitro, suggesting that Cshepc, like human hepcidin could play a role in the antibacterial function in hosts innate immune response.

Roles of L5-7 loop in the structure and chaperone function of SsHSP14.1.

The small heat shock protein SsHSP14.1 from the hyper-thermophilic archeaon, Sulfolobus solfataricus (S. solfataricus) was able to protect proteins from thermal aggregation and prevent enzymes from heat induced inactivation. According to the 3D (dimensional) structural model of SsHSP14.1 developed by us before, the region L5-7 (ß5-ß7, 68-82 residues) plays an important role for the oligomerization of SsHSP14.1 and its chaperone function. Here, to validate the findings, an in-depth investigation was conducted of both the wild type SsHSP14.1 and its deletion mutant DEL75-79. With E. coli proteins and bromelain as substrate, the deletion mutant DEL75-79 can protect them from thermo-aggregating as effective as the wild protein. Interestingly, unlike the wild protein, DEL75-79 was unable to prevent bromelain and EcoRI from thermo-inactivating. Results of size exclusion HPLC showed that the oligomerization state was changed in mutant protein. This was in accordance with the changed structure and lower hydrophobicity of DEL75-79. These outcomes proved that the L5-7 loop did play a role for the oligomerizing SsHSP14.1, and that the residues 75-79 were indispensable for its function of prevent enzymes from thermo-inactivating.

A novel protein isolated from the serum of rabbitfish (Siganus oramin) is lethal to Cryptocaryon irritans.

The susceptibility of eight marine fish species cultured in South China were tested for infection by the parasitic ciliate, Cryptocaryon irritans, via a challenge examination and an immobilization assay. All species of fish (representing six different families) that we investigated were infected by C. irritans except the rabbitfish (Siganus oramin), which displayed resistance to C. irritans infection. The infection intensity of rabbitfish (0.92+/-0.97, p<0.05) was significantly lower while the immobilization titres of rabbitfish serum were significantly higher (44.51+/-22.98, p<0.05) than the other seven species of fish. Additionally, the serum of the rabbitfish presented a strong killing effect to C. irritans in vitro. Light microscopy, scanning electron microscopy and fluorescence microscopy confirmed that rabbitfish serum could induce the theront cilia fall off, rupture of the cell membrane because of the swell and rupture of the macronucleus. Rabbitfish serum could also induce the rupture of the trophont membrane and content efflux. Herein a novel antiparasitic protein (APP) was isolated and purified from the serum of rabbitfish (S. oramin) by using a series of salting-out, cation exchange chromatography and two step of reversed phase high performance liquid chromatography (RP-HPLC). Analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that APP was a homogenous polymeric protein with an N-terminal amino acid sequence of SSVEKNLAACLRDND. Its monomeric molecular mass, determined by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometer (MALDI-TOF-TOF-MS), was found to be 61,739.87 Da. Results of homology analyses indicated that this protein was a newly discovered functional protein in the rabbitfish serum. Laser confocal fluorescence microscopy conformed that the action site of the APP was mainly on the cell membrane and nucleus of theront, which agreed with the results of light microscopy, fluorescence microscopy and scanning electron microscopy. These findings suggest that this protein may contribute considerably to the innate host defence mechanism to combat microbes of the rabbitfish.

Characterization and expression of an actin-depolymerizing factor from Eimeria tenella.

Actin-depolymerizing factor (ADF) plays an important role in remodeling the actin cytoskeleton which contributes much to the invasion of host cells by the apicomplexan parasite. The gene encoding for Eimeria tenella ADF with one intron was cloned and identified by the E. tenella genome raw sequence data ( http://www.sanger.ac.uk/projects/E.tenella/ ). The deduced polypeptide sequence was only composed of 118 amino acids (13.14 kDa) without signal peptide and nuclear localization sequence. The amino acid sequence was most similar to the ADF of Toxoplasma gondii, 69.1%. Compared the putative three-dimensional structures between E. tenella and yeast, the actin filament binding sites on the segment from the alpha4-helices to the C-terminal were mostly missed in E. tenella. Real-time RT-PCR and dot blot both revealed that ADF expression was relatively stronger in the sporozoites and merozoites than in sporulated and unsporulated oocysts in both mRNA and protein levels. Northern blot analysis suggested that there was only one form of ADF transcripts in all different life stages of E. tenella. Actin-binding experiment showed that the recombinant fusion ADF protein could bind with actin, which indicated that ADF probably plays an important role in the invasion host of E. tenella.

Optimization of fermentation media for nitrite oxidizing bacteria using sequential statistical design.

The sequential statistical experimental design (Plackett-Burman, factorial, response surface and steepest ascent experiment) was applied to optimize the culture medium of nitrite oxidizing bacteria for improving the nitrite oxidizing rate. Estimated optimum medium composition of the nitrite oxidizing rate was as follows: NaHCO3, 1.86gl(-1); NaNO2, 2.04gl(-1); Na2CO3, 0.2gl(-1); NaCl, 0.2gl(-1); KH2PO4, 0.1gl(-1); MgSO4 x7H2O, 0.1gl(-1); and FeSO4 x 7H2O, 0.01gl(-1). The nitrite oxidizing rate was increased by 48.0% and reached a maximum at 859.5+/-8.4mgNO2-N/gMLSS.d as compared to 580.7+/-25.8mgNO2-N/gMLSS x d. In the field trial, 50L of nitrite oxidizing bacteria concentrate (1.99gVSS/L) with 850mgNO2-N/gMLSS x d were added to 0.6ha of the aquaculture water. Nitrite level in all treated ponds remained very low compared to the steady increase observed in all of the control ponds during 7 days.

Comparative studies on the immunogenicity of theronts, tomonts and trophonts of Cryptocaryon irritans in grouper.

Cryptocaryon irritans is a ciliated obligate parasite of marine fish, causing white spot disease in tropical and sub-tropical regions. Recent studies have shown that fish can mount immune responses against C. irritans and acquire protection. The present study compared the protective immunity in grouper (Epinephelus coioides) against C. irritans induced by theronts, trophonts and tomonts via intraperitoneal (i.p.) injection and tentatively identified an immobilization antigen. Specific antibody titres of immunized fish serum were determined by enzyme-linked immunosorbent assay and immobilization assays at weeks 0, 1, 2, 4, 6 and 8 post-immunization, and immunized grouper were challenged with theronts to detect the survival percentage of fish. Fish immunized with theronts produced higher levels of serum antibody against C. irritans than fish immunized with trophonts or tomonts. A significantly higher level of immune protection was achieved in fish immunized with theronts but not in fish immunized with tomonts or trophonts. Western blot analysis using polyclonal immobilization antibodies of rabbit immunized with theront revealed an immunoreactive band of the protective (immobilization) antigens of C. irritans, the size of which is approximately 34 kDa. The sera of mice immunized with the protein could immobilize theronts of C. irritans. These results demonstrated that the theront stage of C. irritans elicited stronger protective immunity than the trophont and tomont stages in grouper. The tentative identification of protective antigen provides the solid foundation for the development of a defined vaccine against C. irritans.

The evaluation for generic-level monophyly of Ancyrocephalinae (Monogenea, Dactylogyridae) using ribosomal DNA sequence data.

To overcome the limited regional and taxonomic coverage in previous studies of the Ancyrocephalinae (Monogenea, Dactylogyridae), we present further findings on the molecular systematics and phylogeny of a broader selection of specimens, aiming to build a molecular phylogeny of the Ancyrocephalinae, and to assess the monophyly of each available genus, using D1-D2 domain of LSU rDNA and the combined LSU and partial sequence of SSU rDNA data sets. Our studies showed that 18 Haliotrema species were highly dispersive to form several clades with species from ten closely related genera. The host range of Euryhaliotrematoides species was not only restricted in butterfly fishes (Chaetodontidae), but widen to include the Lutjanidae. Euryhaliotrema was phylogenetically more closely related to Aliatrema than to Euryhaliotrematoides. Given that the species Haliotrema kurodai, Haliotrema spirotubiforum and Haliotrema anguiformis had a funnel-shaped base of the coiled male copulatory organ (MCO) lacking an accessory piece, and our molecular evidence, we proposed to transfer the three species to the Aliatrema as new combinations. We proposed to combine Bravohollisia and Caballeria into one genus, mainly based on molecular evidence and their similar MCO characters. Using SSU+ITS1 data set, Scutogyrus was robustly resolved as a polyphyletic group and its status should be questioned. Based on the present molecular evidence and their similar MCO characters, we proposed to combine Cichlidogyrus and Scutogyrus into one genus, Cichlidogyrus, by treating Scutogyrus as the synonym of Cichlidogyrus. Generally, the present study indicated that the vagina can make little contribution for understanding the generic-level monophyly, due to its high variability even among phylogenetically very closely related species, but it was useful for species determination. Given that phylogenetically closely related species from the same or closely related host species may have similar MCO characters but distinct haptoral characters, we consider that it is dangerous to erect a genus mainly based on different haptoral characters, particularly those separated from existing genera. The resultant phylogenetic reconstructions indicated that the radiation of some Haliotrema species has correlated with their hosts, because species from closely related fishes have correspondingly shown close relationships. Though the present study can not provide the comparison of host-parasite associations, phylogenetically closely related Haliotrema species which have similar morphological characters (both MCOs and haptors) but distinct/distantly related host species may indicate a speciation model of host-switching.

Protective immunity in grouper (Epinephelus coioides) following exposure to or injection with Cryptocaryon irritans.

The protective immunity of grouper (Epinephelus coioides) against Cryptocaryon irritans was determined after immunisation by surface exposure or intraperitoneal injection. Specific antibody titres of immunised fish serum and skin culture supernatant were determined by enzyme-linked immunosorbent assay and immobilisation assays. Specific antibody can be detected in some immunised fish at Week 1 and in all immunised fish at Week 2, and the peaks were between Weeks 4-6. Specific antibody was still evident in the serum and skin of immunised fish at Week 8, and provided good protection against challenge with C. irritans. These findings indicated that humoral and skin mucosal immunity play important roles in fish against C. irritans infection.

PCR-based identification and delineation of members within the Pseudorhabdosynochus lantauensis complex (Monogenea: Diplectanidae).

Molecular methods using genetic markers in the nuclear ribosomal DNA (rDNA) were established to identify and distinguish between two members within the Pseudorhabdosynochus lantauensis complex and two morphologically distinct congeners, Pseudorhabdosynochus epinepheli and Pseudorhabdosynochus coioidesis, from different marine fish species and various geographical origins. Supported by selective DNA sequencing, it was demonstrated that the polymerase chain reaction (PCR)-coupled single-strand conformation polymorphism analysis of the first internal transcribed spacer and restriction fragment length polymorphism analysis of a variable region (representing the D1-D3 domains) in the large subunit of rDNA achieved the identification and delineation of all four taxa examined. These PCR-based approaches provide useful complementary tools to traditional methods for the accurate identification of species within the genus Pseudorhabdosynochus (irrespective of developmental stage) and have major implications for studying the ecology, transmission, and population genetic structures of these and other related parasites and for the prevention and control of the diseases they cause.

Are Tritrichomonas foetus and Tritrichomonas suis synonyms?

Tritrichomonas suis, a tritrichomonad of pigs, and the related species Tritrichomonas foetus, a tritrichomonad of cattle, are morphologically identical. The taxonomic relationship between these two tritrichomonads has been questioned ever since they were established as distinct species in 1843 and 1928, respectively. Here, we compare the similarities of morphology, ultrastructure, distribution, host specificity, characteristics of in vitro cultivation, immunology, biochemistry and analysis of molecular data from published sources between these two species. All data indicate that these two tritrichomonad species are identical. Thus, we propose that T. foetus and T. suis are synonyms.

PCR-SSCP as a molecular tool for the identification of Benedeniinae (Monogenea: Capsalidae) from marine fish.

PCR-based single strand conformation polymorphism (SSCP) analysis was used to characterize monogenean specimens of the subfamily Benedeniinae of morphologically uncertain specific status from different marine fish species, using Neobenedenia melleni, N. girellae and Entobdella corona for comparison. The first internal transcribed spacer (ITS-1) and the 5' terminal variable region (D1-D3 domains) of the large subunit ribosomal DNA (lsrDNA) were amplified separately from individual monogeneans, and the amplicons were subjected to PCR-SSCP analyses, followed by direct sequencing. Both SSCP patterns and the ITS-1 sequences data allowed specimens representing Entobdella spp. from the host Taeniura melanospilos to be unequivocally distinguished from those representing Neobenedenia spp. and those representing Benedenia spp. Neobenedenia girellae, a morphologically controversial species, had identical SSCP banding pattern and ITS-1 sequence to that of N. melleni, supporting the proposal that N. girellae is a synonym of N. melleni. Neobenedenia spp. and Benedenia spp. had identical SSCP patterns and ITS-1 sequences. These findings and the PCR-SSCP approach taken should have implications for the accurate identification and assessment of taxonomic validity of other important monogenean groups of the marine fish.

Cloning of Etp28 Gene of Eimeria tenella (Guangdong Strain) Sporozoites and Its Expression in Baculovirus Expression System.

A refractile body antigen Etp28 Gene of Eimeria tenella (Guangdong Strain) sporozoites was cloned by PCR from the synthesized first strand cDNA. It has a high homology with the previously reported Etp28 gene of Merck Strain LS18. The gene was expressed through standard procedures in the modified Autographa californica nuclear polyhedrosis virus (AcMNPV-OCC(-)) expression system and large amount of heterologous fusion protein (GST-6xHis-Etp28) was obtained. The expression product was about 21.3% of the total cellular protein of an infected cell and corresponding to approximately 0.42 mg of recombinant protein per 10(6) cells. And the immunoprotective trial showed that this candidate for recombinant vaccine conferred partial protection against chicken coccidiosis.