Development of a set of expression vectors in Hansenula polymorpha

Four expression vectors based on formate dehydrogenase promoter (FMDp) and methanol oxidase promoter (MOXp) from Hansenula polymorpha were developed to express heterologous genes in Hansenula polymorpha. A secretion signal sequence of the mating factor-alpha from Saccharomyces cerevisiae was inserted in the secretory expression plasmids for efficient secretion. A modified green fluorescent protein (mGFP5) was used as the marker of expression for the first time in H. polymorpha NCYC495 (leu 1.1) to determine the expression ability of these plasmids. The mGFP5 thus expressed retained its biochemical and physiological properties, such as accumulation inside cells and efficient secretion into the culture media. These results indicated that the four integrative vectors are useful expression systems which could be directly applied for production of heterologous proteins of interests in H. polymorpha.     

Epoxide hydrolase-catalyzed resolution of ethyl 3-phenylglycidate using whole cells of Pseudomonas sp.

A Pseudomonas sp. was isolated with enantioselective epoxide hydrolase activity to ethyl 3-phenylglycidate. Cells grown on sucrose and suspended in 10% (v/v) dimethyl formamide as co-solvent produced (2R,3S) ethyl 3-phenylglycidate with 95% ee and 26% yield in 12 h from 0.2% (w/v) of the racemate.     

Multiplex polymerase chain reaction/membrane hybridization assay for detection of genetically modified organisms

To improve detection efficiency and result accuracy, four screening primer pairs, four identifying primer pairs, one common primer pair and corresponding probes were designed for the development of multiplex polymerase chain reaction/membrane hybridization assay (MPCR-MHA) for detection of the foreign genes insert in genetically modified organisms (GMOs). After detecting condition and parameter were optimized and determined, MPCR reactions were developed for amplifying several target genes simultaneously in one tube. Primers were labeled with biotin at the 5'-end; biotinylated MPCR products were detected by hybridization to the oligonucleotide probes immobilized on a membrane with subsequent colorimetric detection to confirm hybridization. The testing of screening primers can judge whether the sample contains GMOs, and that of identifying primers can further judge what kinds of trait genes are contained in the sample. We detected nine soybean samples, six maize samples, seven potato samples and two rice samples by the MPCR-MHA method; at the same time we also detected them with single PCR-MHA method. The results between two methods have good consistency.     

Haemolymph parameters of Pacific white shrimp (Litopenaeus vannamei) infected with Taura syndrome virus

Pacific white shrimp (Litopenaeus vannamei) were injected with Taura syndrome virus (TSV) to assess shrimp immune responses and survival. TSV-infected shrimp suffered high mortality, but mock-infected and untreated shrimp experienced no mortality. Moribund shrimp were a pale, reddish colour and were lethargic and soft-shelled. Their haemolymph was clear red and coagulated poorly. In TSV-infected shrimp, the total haemocyte count (THC), hyalinocyte and granulocyte counts, and total plasma protein decreased significantly to 21%, 24%, 17% and 56% of untreated control values, respectively. Haemocyanin decreased to 67%, and clottable proteins to 80% of control values (P< 0.01). Copper and calcium ions, haemocytic transglutaminase (TGase) activity and plasma growth inhibitory activity against Vibrio harveyi also decreased significantly. Generation of intrahaemocytic superoxide anion, O(-2), in TSV-infected shrimp was significantly greater (P< 0.05) than in both control groups, no matter whether glucan stimulated or unstimulated. But the relative increase of intrahaemocytic O(-2) generation in TSV-infected shrimp response to glucan stimulation was lower in both controls. Plasma phenoloxidase (PO) activity increased significantly in TSV-infected shrimp. The plasma bacterial agglutinin titre against E. coli and V. harveyi, growth inhibition of E. coli and the concentration of magnesium ions in TSV-infected shrimp did not change significantly.In conclusion, ten of thirteen haemolymph parameters changed significantly during the host-TSV interaction. These parameters might be valuable references of shrimp health status.     

Description of a marine peritrichous ciliate, Pseudovorticella sinensis n. sp. (Ciliophora, Peritrichia) from China

A new marine peritrich ciliate, Pseudovorticella sinensis n. sp. was isolated from a shrimp-farming pond in the littoral area of Qingdao, China. The morphology, infraciliature, and silverline system were studied based on living and silver-impregnated specimens. This species is characterized by (1) an elongated bell-shaped body that measures 50-60 x 35-45 microm in vivo, (2) one large, ventrally located contractile vacuole, and (3) a pellicle covered by a layer of transparent, cortical vesicles. The number of transverse silverlines from the peristomial area to the aboral ciliary wreath is 26-32, and from the aboral ciliary wreath to the scopula is 12-15. The stalk measures about 160-250 microm long x 5-6 microm wide. The spasmoneme has one row of conspicuous thecoplasmic granules, which are about 0.8 microm in diameter.     

Autoimmune islet destruction in spontaneous type 1 diabetes is not beta-cell exclusive

Pancreatic islets of Langerhans are enveloped by peri-islet Schwann cells (pSC), which express glial fibrillary acidic protein (GFAP) and S100beta. pSC-autoreactive T- and B-cell responses arise in 3- to 4-week-old diabetes-prone non-obese diabetic (NOD) mice, followed by progressive pSC destruction before detectable beta-cell death. Humans with probable prediabetes generate similar autoreactivities, and autoantibodies in islet-cell autoantibody (lCA) -positive sera co-localize to pSC. Moreover, GFAP-specific NOD T-cell lines transferred pathogenic peri-insulitis to NOD/severe combined immunodeficient (NOD/SCID) mice, and immunotherapy with GFAP or S100beta prevented diabetes. pSC survived in rat insulin promoter Iymphocytic choriomeningitis virus (rip-LCMV) glycoprotein/CD8+ T-cell receptor(gp) double-transgenic mice with virus-induced diabetes, suggesting that pSC death is not an obligate consequence of local inflammation and beta-cell destruction. However, pSC were deleted in spontaneously diabetic NOD mice carrying the CD8+/8.3 T-cell receptor transgene, a T cell receptor commonly expressed in earliest islet infiltrates. Autoimmune targeting of pancreatic nervous system tissue elements seems to be an integral, early part of natural type 1 diabetes.     

Engineering tolerance and accumulation of lead and cadmium in transgenic plants

We have studied the utility of the yeast protein YCF1, which detoxifies cadmium by transporting it into vacuoles, for the remediation of lead and cadmium contamination. We found that the yeast YCF1-deletion mutant DTY167 was hypersensitive to Pb(II) as compared with wild-type yeast. DTY167 cells overexpressing YCF1 were more resistant to Pb(II) and Cd(II) than were wild-type cells, and accumulated more lead and cadmium. Analysis of transgenic Arabidopsis thaliana plants overexpressing YCF1 showed that YCF1 is functionally active and that the plants have enhanced tolerance of Pb(II) and Cd(II) and accumulated greater amounts of these metals. These results suggest that transgenic plants expressing YCF1 may be useful for phytoremediation of lead and cadmium.     

Characterization of Cryptocaryon irritans,a parasite isolated from marine fishes in Taiwan

The ciliated protozoan parasite Cryptocaryon irritans infecting marine fishes in Taiwan is described. Developmental characteristics and sequences of the ribosomal DNA regions such as part of 18 S, the entire first internal transcribed spacer, and part of 5.8 S of various Taiwan isolates of C. irritans were investigated. A total of 5 isolates was obtained from different fish-host species and localities, the majority from cultured fish species. C. irritans from Taiwan is able to shift its developmental characteristics, i.e. from non-adherent to adherent tomonts, from individualistic to aggregate-forming tomonts, from infection of the gills only to infection of the gills and body. Thus, it is not possible to classify strains of C. irritans on the basis of these parameters. Premature tomonts that developed from dead fishes were able to produce theronts that could infect fish host. Isolates from Pingtung and the USA had identical nucleotide sequences while an isolate from Malaysia was identical to an Israel isolate. Percentage variation among pairs of Taiwan isolates showed a higher degree of variation than isolate sequences listed in GenBank. Sequence analysis revealed highly aberrant isolates in Taiwan, and a phylogenetic tree distinguished a marine and a low-salinity variant. C. irritans from marine fishes in Taiwan, therefore, display some characteristics not previously reported. Since manipulation of salinity in brackishwater ponds and marine cage sites is not feasible, there is a need to develop new strategies for the control and prevention of cryptocaryoniasis.     

The unfolding intermediate state of calf intestinal alkaline phosphatase during denaturation in guanidine solutions

The equilibrium unfolding of calf intestinal alkaline phosphatase in guanidinium chloride (GdmCl) solutions was studied by following the fluorescence and ultraviolet difference spectra. At low concentrations of GdmCl (< 1.6 M), the fluorescence intensity decreased with a slight red shift of the emission maximum from 332 nm to 344 nm. An unfolding intermediate state was observed at a broad concentration range of GdmCl as a denaturant (between 1.6 and 2.6 M). This intermediate was characterized by increased fluorescence emission intensity, ultraviolet difference absorption at 236 nm and 260 nm, as well as increased binding to the protein and red shift of the fluorescence probe 1-anilinonaphthalene-8-sulfonic acid.