Effects of Pb(â ¡) exposure on Chlorella protothecoides and Chlorella vulgaris growth, malondialdehyde, and photosynthesis-related gene transcription.

Greater exposure to Pb(Ⅱ) increases the likelihood of harmful effects in the environment. In this study, the aquatic unicellular alga Chlorella protothecoides (C. protothecoides) and Chlorella vulgaris (C. vulgaris) were chosen to assess the acute and chronic toxicity of Pb(Ⅱ) exposure. Results of the observations show dose-response relationships could be clearly observed between Pb(Ⅱ) concentration and percentage inhibition (PI). Exposure to Pb(Ⅱ) increased malondialdehyde (MDA) content by up to 4.22 times compared with the control, suggesting that there was some oxidative damage. ANOVA analysis shows that Pb(Ⅱ) decreased chlorophyll (chl) content, indicating marked concentration-dependent relationships, and the lowest levels of chl a, chl b, and total-chl were 14.53, 18.80, and 17.95% of the controls, respectively. A real-time PCR assay suggests the changes in transcript abundances of three photosynthetic-related genes. After 120 h exposure Pb(Ⅱ) reduced the transcript abundance of rbcL, psaB, and psbC, and the relative abundances of the three genes of C. protothecoides and C. vulgaris in response to Pb(Ⅱ) were 54.66-98.59, 51.68-95.59, 37.89-95.48, 36.04-94.94, 41.19-91.20, and 58.75-96.80% of those of the controls, respectively. As for 28 d treatments, the three genes displayed similar inhibitory trend. This research provides a basic understanding of Pb(Ⅱ) toxicity to aquatic organisms.

Acute and chronic toxic effects of bisphenol A on Chlorella pyrenoidosa and Scenedesmus obliquus.

The acute and chronic toxic effects of Bisphenol A (BPA) on Chlorella pyrenoidosa (C. pyrenoidosa) and Scenedesmus obliquus (S. obliquus) were not well understood. The indoor experiments were carried out to observe and analyze the BPA-induced changes. Results of the observations showed that in acute tests BPA could significantly inhibit the growth of both algae, whereas chronic exposure hardly displayed similar trend. Superoxide dismutase (SOD) and Catalase (CAT) activities of both algae were promoted in all the treatments. Chlorophyll a synthesis of the two algae exhibited similar inhibitory trend in short-term treatments, and in chronic tests C. pyrenoidosa hardly resulted in visible influence, whereas in contrast, dose-dependent inhibitory effects of S. obliquus could be clearly observed. The experimental results indicated that the growth and Chlorophyll a syntheses of S.obliquus were more sensitive in response to BPA than that of C. pyrenoidosa, whereas for SOD andCAT activities, C. pyrenoidosa was more susceptible. This research provides a basic understanding of BPA toxicity to aquatic organisms.

Effects of different glycerol feeding strategies on S-adenosyl-l-methionine biosynthesis by P(GAP)-driven Pichia pastoris overexpressing methionine adenosyltransferase.

Methionine adenosyltransferase (MAT) was overexpressed within Pichia pastoris employing the promoter of glyceraldehyde-3-phosphate dehydrogenase gene (P(GAP)), to biosynthesize S-adenosyl-l-methionine (SAM). Effects of five glycerol feeding tactics on MAT activity were first investigated. Strategies A-C were based on limited feeding correlated with dissolved oxygen (DO) at 50.0%, 25.0% and 0.0%, respectively. For strategies D and E, unlimited supplementation was executed by pulsed feeding mode. Gradual decline (2-0%) (w:v) of the residual glycerol level was shown between any two pulses in strategy D, while a nearly stable content (2%) throughout fed-batch cultivation with strategy E. With shifting strategies A-E in alphabetical order, gradual improvements of MAT activities were achieved, with the maximum of 9.05Ug(-1) dried biomass for strategy E, since the specific glycerol consumption rate (F(G)) ascended due to the elevated specific oxygen uptake rate (qO(2)). The success was ascribed to the enhancement of oxygen transfer rate (OTR), because 2% glycerol improved oxygen saturation content in broth (C*) and volumetric oxygen transfer coefficient (k(L)a). Strategy E also led to the highest values of ATP and biomass besides MAT. Consequently, the highest SAM yield and volumetric level were obtained at 0.058gg(-1) and 9.26gl(-1), respectively.

Real-time viable-cell mass monitoring in high-cell-density fed-batch glutathione fermentation by Saccharomyces cerevisiae T65 in industrial complex medium.

An on-line monitoring of viable-cell mass in high-cell-density fed-batch cultivations of Saccharomyces cerevisiae grown on an industrial complex medium was performed with an in situ capacitance probe fitted to a 50-l fermentor. Conventional off-line biomass determinations of several parameters, including dry cell weight (DCW), optical density at 600 nm wavelength (OD(600)), packed mycelial volume (PMV) and number of colony forming units (CFU), were performed throughout the bioprocess and then compared with on-line viable-cell concentrations measured using a capacitance probe. Capacitance versus viable biomass and all off-line biomass assay values were compared during glutathione fermentation in industrial complex culture media. As a result, the relationship between the number of colony forming units and capacitance with a correlation coefficient (R) of 0.995 was achieved. Simultaneously, compared with those determined by at-line indirect estimation methods including oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER), the specific growth rates estimated by on-line capacitance measurement could be more reliable during glutathione fermentation. Therefore, it is concluded that a capacitance probe is a practical tool for real-time viable biomass monitoring in high-cell-density fed-batch cultivation in a complex medium.

Mitochondrion-targeted photosensitizer enhances the photodynamic effect-induced mitochondrial dysfunction and apoptosis.

Recently, the mitochondrion has been considered as a novel pharmacological target for anticancer therapy due to its crucial role involved in arbitrating cell apoptosis. We have previously demonstrated that 488-nm laser irradiation induced a specific mitochondrial reactive oxygen species (mROS) formation and apoptotic death. In this study, we used a second generation of photosensitizers, the benzoporphyrin-derivative monoacid ring A (BPD-MA). We investigated specifically mechanisms at the mitochondrial level for BPD-MA coupled with 690-nm laser irradiation, the photodynamic effect (PDE) of BPD-MA, using conventional and laser scanning imaging microscopy in intact C6 glioma cells. We demonstrated BPD-MA localized mainly in the mitochondrial area. The phototoxicity induced by 1-10 J 690-nm laser irradiation was minor as compared to that induced by 488-nm laser irradiation. Unlike other mitochondrion-targeted photosensitizers, the dark toxicity induced by BPD-MA (0.05-5 mg/mL, effective doses used for the PDE) was relatively low. Nevertheless, the PDE of BPD-MA using 0.5 mg/mL coupled with 5J 690-nm irradiation induced profound and rapid (< 1 min) mitochondrial swelling, mROS formation, and severe plasma membrane blebbing as compared to that induced by 488-nm laser irradiation (< 10 min). Later, the PDE of BPD-MA resulted in positive propidium iodide cell-death stain and positive TUNEL apoptotic nuclear stain and DNA laddering. Finally, the PDT of BPD-MA also instantaneously promoted the mitochondrion to diminish its covalent binding with a mitochondrial marker, MitoTracker Green. We conclude that the PDT of BPD-MA targeted primarily and compellingly the mitochondrion to induce effective mitochondria-mediated apoptosis and thus may serve as a powerful photosensitizer for clinical cancer therapy.

Mitochondrial reactive oxygen species generation and calcium increase induced by visible light in astrocytes.

Mitochondria contain photosensitive chromophores that can be activated or inhibited by light in the visible range. Rather than utilizing light energy, however, mitochondrial electron transport oxidation-reduction reaction and energy coupling could be stimulated or damaged by visible light. Our previous work demonstrated that reactive oxygen species (ROS) were generated in cultured astrocytes after visible laser irradiation. With confocal fluorescence microscopy, we found that ROS were generated mostly from mitochondria. This mitochondrial ROS (mROS) formation plays a critical role in photoirradiation-induced phototoxicity and apoptosis. In this study, we measured changes of mitochondrial calcium level ([Ca(2+)](m)) in cultured astrocytes (RBA-1 cell line) irradiated with blue light and examined the association between mROS formation and [Ca(2+)](m) level changes. Changes of intracellular ROS and [Ca(2+)](m) were visualized using fluorescent probes 2',7'-dichlorodihydrofluorescein (DCF), and rhod-2. After exposure to visible light irradiation, RBA-1 astrocytes showed a rapid increase in ROS accumulation particularly in the mitochondrial area. Increase in [Ca(2+)](m) was also induced by photoirradiation. The levels of increase in DCF fluorescence intensity varied among different astrocytes. Some of the cells generated much higher levels of ROS than others. For those cells that had high ROS levels, mitochondrial Ca(2+) levels were also high. In cells that had mild ROS levels, mitochondrial Ca(2+) levels were only slightly increased. The rate of increase in DCF fluorescence seemed to be close to the rate of rhod-2 fluorescence increase. There is a positive and close correlation between mitochondrial ROS levels and mitochondrial Ca(2+) levels in astrocytes irradiated by visible light.

Isolation, characterization, and molecular cloning of the cDNA encoding a novel phytase from Aspergillus niger 113 and high expression in Pichia pastoris.

Phytases catalyze the release of phosphate from phytic acid. Phytase-producing microorganisms were selected by culturing the soil extracts on agar plates containing phytic acid. Two hundred colonies that exhibited potential phytase activity were selected for further study. The colony showing the highest phytase activity was identified as Aspergillus niger and designated strain 113. The phytase gene from A. niger 113 (phyI1) was isolated, cloned, and characterized. The nucleotide and deduced amino acid sequence identity between phyI1 and phyA from NRRL3135 were 90% and 98%, respectively. The identity between phyI1 and phyA from SK-57 was 89% and 96%. A synthetic phytase gene, phyI1s, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide that was designed and synthesized using P. pastoris biased codon. For the phytase expression and secretion, the construct was integrated into the genome of P. pastoris by homologous recombination. Over-expressing strains were selected and fermented. It was discovered that ~4.2 g phytase could be purified from one liter of culture fluid. The activity of the resulting phytase was 9.5 U/mg. Due to the heavy glycosylation, the expressed phytase varied in size (120, 95, 85, and 64 kDa), but could be deglycosylated to a homogeneous 64 kDa species. An enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0) and an optimum temperature of 60 degrees C.

[Influence of signal peptide sequences on the expression of heterogeneous proteins in Pichia pastoris].

Pichia pastoris has been developed to be a very efficient expression host for the heterogeneous proteins since its alcohol promoter was isolated and cloned, and its transformation technique was established. For further improving the secretion expression of heterogeneous proteins, in this research, the signal sequences were studied. At first, the Saccharomyces cerevisiae mating factor alpha prepro-leader sequence was synthesized using successive PCR and designated as MF4I. Then, ten different signal sequences were constructed by adding the N-terminal residues of Pichia pastoris Aox1 protein to the N-terminal of the MF4I. These ten signal sequences were used for directing phytase gene secretion in Pichia pastoris, the secretion of phytase were increased in Pichia pastoris strains containing new signal sequence. Among these strains, the phytase secretion was highest in strain contain signal sequence added with A, I, P three Aox1 N-terminal residues; the phytase secretion of Pichia pastoris was 90 mg/L in flake. The secretion was six-fold of that with original Saccharomyces cerevisiae mating factor alpha prepro-leader sequence. In addition, insert of ten residues E E A E A E A E P K can further increase the phytase secretion by 35%, the secretion reach 120 mg/L.

[High expression of a heat-stable phytase in Pichia pastoris].

It is difficult to obtain naturally occurring phytase having the required thermostability for application in animal feeding. The 1.3 kb thermal stable phytase gene (fphy) of Aspergillus fumigatus was synthesized by using a successive PCR method for the optimal expression in Pichia pastoris. Though the nucleotides of synthetic fphy share only 74% homology with the natural gene, the amino acid sequences coded by both genes were identical. After being cloned into the yeast expression vector (pPIC9) and inserted into the chromosome of Pichia pastoris by homologous recombination, phytase was expressed in the yeast and secreted from the cell. The strains for phytase over-expression were selected out by SDS-PAGE and enzyme analysis. After fermentation in 5 L fermention tank and induced by 0.5% methanol for 60 h, about 5.6 g purified phytase was obtained per liter culture fluid. The activity of phytase was 130 000 u per microlitre fluid. The thermostable phytase remained 40% active after exposure at 90 degrees for 80 min.

[Control effect of different covering patterns on indigenous nutrient release from sediment].

A laboratory simulation experiment was conducted to study the release of sediment phosphorous and nitrogen under the effects of coating the sediment with plastic, clinoptilolite, calcite, quartz sand, and calcium nitrate, aimed to provide scientific basis and technical support to control the sediment nutrient release under the background of water environment pollution by different concentrations of nitrogen and phosphorus. The control efficacy of test coating materials for sediment total phosphorous release was in the order of plastic > calcium nitrate > clinoptilolite > calcite > quartz sand, and that for sediment total nitrogen release was in the order of clinoptilolite > plastic > calcite > quartz sand > calcium nitrate. As for the release of sediment NO(3-)-N, the control efficacy of test coating materials was calcium nitrate > quartz sand > clinoptilolite > calcite > plastic coating; whereas for the release of sediment NH(4+)-N, the sequence was calcium nitrate > plastic coating > clinoptilolite > calcite > quartz sand. Water temperature had definite relativity to the sediment nutrient release. With the increase of water temperature, the concentrations of water total phosphorous and nitrogen and NO(3-)-N increased, while the concentration of water NH(4+)-N presented a declining trend.

Efficient extraction of intracellular reduced glutathione from fermentation broth of Saccharomyces cerevisiae by ethanol.

Reduced glutathione (GSH) from fermentation broth of Saccharomyces cerevisiae was extracted with ethanol without disruption of the cells. The effects of ethanol concentration, extraction temperature and extraction time were assessed by using 2(3) full factorial designs (FFD). Preliminary studies showed that ethanol concentration had the most influence on GSH yield by ethanol extraction, based on the first order regression coefficients derived using MINITAB software, and an optimal ethanol concentration (25%, v/v) was obtained. However, compared to the conventional extraction technique (hot water extraction), there was no significant advantage in yield of GSH from yeast cells using ethanol extraction under these optimized conditions. But ethanol extraction has several advantages, such as lower energy consumption and lower protein concentration of extraction broth, which may reduce the complexity and cost of the purification process. Hence, ethanol extraction which does not disrupt yeast cells could be an inexpensive, simple and efficient alternative to conventional extraction techniques in the GSH industry.

[Over-expression in Escherichia coli and characterization of apolipoprotein AI].

Apolipoprotein AI (apo AI), the major protein component of human high-density lipoprotein (HDL), is a single-chain polypeptide of 243 amino acids. Several epidemiological studies have shown that the plasma concentrations of HDL has the role of reverse cholesterol transport (RCT) and inversely correlated with the incidence of coronary artery disease. Because apo AI lacks post-translational modifications, it is convenient to express human apo AI in Escherichia coli expression system. However, there is a poor stability of the mRNA and the apo AI protein in E. coli, it is difficult to express mature apo AI in recombinant bacteria, moreover, even as a fusion protein, apo AI is still sensitive to degradation and can not be cleaved efficiently from the fusion tags. In contrast, proapolipoprotein AI (proapo AI, having an additional polypeptide containing the amino acids Arg-His-Phe-Trp-Gln-Gln at the amino-teminal of the mature protein) proved stable and undegraded in Escherichia coli, and therefore, in this research, an expression system of E. coli including a plasmid of P(R)P(L) tandem promoter was adapted to produce proapo AI. Furthermore, site-directed mutagenesis of the proapo AI cDNA was performed to generate a Clu8Asp mutation in the amino-terminal sequence of proapo AI which created an acid labile Asp-Pro peptide bond between amino acid 8 and 9, and permitted specific chemical cleavage to remove pro-peptide. After inducing with a shift of temperature, yields of recombinant proapo AI achieved about 40% of total cell protein and the recombinant proapo AI expressed proved as a form of inclusion body in cells, so protein need to renature. First of all, the protein was dissolved in buffer with denaturant, and renaturation was carried out on a hydrophobic interaction column (Phenyl Sepharose), ion-exchange chromatography and gel-filtration chromatography were then used to further purify the protein. The purified recombinant apo AI was detected by a set of tests including Western-blotting, Circular dichroism spectra and lipid-binding test, the results shown that recombinant apo AI has similar structural and lipid-binding properties identical to those of native plasma apo AI, which facilitates further research and application.

[Quantitative studies of the production phase in rHSA fermentation].

The model equations of the production phase of rHSA fermentation were derived on the base of both elemental balance and metabolic balance, then the unknown parameters of the model were estimated by multivariable optimization. The possible reasons of discrepancy of production rate between different period of fermentation were discussed. The model could preferably described the relations between different macroscopic reaction rates of the process and keys for the high-efficiency expression of HAS were deduced.