Functional characterization and substrate specificity analysis of Δ6-desaturase from marine microalga <Emphasis Type="Italic">Isochrysis</Emphasis> sp.

Objectives

To express a Δ6-desaturase gene and produce gamma-linolenic acid (GLA) and stearidonic acid (SDA) in prokaryotic expression system (Escherichia coli), and analyze its substrate specificity in the omega-3 fatty acid biosynthetic pathway.

Results

Full-length ORF (1448 bp) of Δ6Des-Iso was isolated from Isochrysis sp. and characterized using multiple sequence alignment, phylogenetic analysis, transmembrane domain, and protein tertiary structure. Δ6Des-Iso is a front-end desaturase consisting of three conserved histidine domains and a cytochrome b₅ domain. Δ6Des-Iso was cloned and expressed in E. coli with the production of GLA and SDA. Recombinant E. coli utilized 27 and 8% of exogenously supplied alpha-linolenic acid (ALA) and linoleic acid (LA) to produce 6.3% of SDA and 2.3% of GLA, respectively, suggesting that isolated Δ6Des-Iso is specific to the omega-3 pathway.

Conclusion

For the first time production of GLA and SDA in a prokaryotic system was achieved.

     

Solution structure of copper ion-induced molecular aggregates of tyrosine melanin.

Melanin, the ubiquitous biological pigment, provides photoprotection by efficient filtration of light and also by its antioxidant behavior. In solutions of synthetic melanin, both optical and antioxidant behavior are affected by the aggregation states of melanin. We have utilized small-angle x-ray and neutron scattering to determine the molecular dimensions of synthetic tyrosine melanin in its unaggregated state in D(2)O and H(2)O to study the structure of melanin aggregates formed in the presence of copper ions at various copper-to-melanin molar ratios. In the absence of copper ions, or at low copper ion concentrations, tyrosine melanin is present in solution as a sheet-like particle with a mean thickness of 12.5 A and a lateral extent of approximately 54 A. At a copper-to-melanin molar ratio of 0.6, melanin aggregates to form long, rod-like structures with a radius of 32 A. At a higher copper ion concentration, with a copper-to-melanin ratio of 1.0, these rod-like structures further aggregate, forming sheet-like structures with a mean thickness of 51 A. A change in the charge of the ionizable groups induced by the addition of copper ions is proposed to account for part of the aggregation. The data also support a model for the copper-induced aggregation of melanin driven by pi stacking assisted by peripheral Cu(2+) complexation. The relationship between our results and a previous hypothesis for reduced cellular damage from bound-to-melanin redox metal ions is also discussed.     

Do effects of ultraviolet radiation on microbial films have indirect effects on larval attachment of the barnacle Balanus amphitrite?

We have examined the indirect effects of UV-A and UV-B on cypris attachment of the barnacle Balanus amphitrite Darwin through their effects on microbial films. Specifically, we tested the hypothesis that both UV-A and UV-B radiation can indirectly affect the larval attachment of barnacles by altering the microbial film bioactivity. Microbial films were developed from mid-intertidal region (∼1 m above Mean Low Water Level) for 6 days and subjected to ambient levels of ultraviolet radiation. Response of cyprids to untreated and UV-treated microbial films was investigated using double-dish still water choice bioassay. Results showed that both UV-A and UV-B caused a decrease in the percentage of respiring bacterial cells in microbial films and this effect increased with UV energy. With the same UV energy, UV-B caused a greater decrease in respiring bacterial cells than UV-A. However, despite strong UV radiation, the bioactivities of microbial films (i.e., stimulation of cypris attachment) remain unchanged. Results of this study suggest that increased UV radiation, which might occur due to ozone depletion, may not significantly affect the barnacle recruitment by means of affecting the inductive larval attachment cues of microbial films.     

N-terminal polybasic motifs are required for plasma membrane localization of Galpha(s) and Galpha(q)

Heterotrimeric G proteins typically localize at the cytoplasmic face of the plasma membrane where they interact with heptahelical receptors. For G protein alpha subunits, multiple membrane targeting signals, including myristoylation, palmitoylation, and interaction with betagamma subunits, facilitate membrane localization. Here we show that an additional membrane targeting signal, an N-terminal polybasic region, plays a key role in plasma membrane localization of non-myristoylated alpha subunits. Mutations of N-terminal basic residues in alpha(s) and alpha(q) caused defects in plasma membrane localization, as assessed through immunofluorescence microscopy and biochemical fractionations. In alpha(s), mutation of four basic residues to glutamine was sufficient to cause a defect, whereas in alpha(q) a defect in membrane localization was not observed unless nine basic residues were mutated to glutamine or if three basic residues were mutated to glutamic acid. betagamma co-expression only partially rescued the membrane localization defects; thus, the polybasic region is also important in the context of the heterotrimer. Introduction of a site for myristoylation into the polybasic mutants of alpha(s) and alpha(q) recovered strong plasma membrane localization, indicating that myristoylation and polybasic motifs may have complementary roles as membrane targeting signals. Loss of plasma membrane localization coincided with defects in palmitoylation. The polybasic mutants of alpha(s) and alpha(q) were still capable of assuming activated conformations and stimulating second messenger production, as demonstrated through GST-RGS4 interaction assays, cAMP assays, and inositol phosphate assays. Electrostatic interactions with membrane lipids have been found to be important in plasma membrane targeting of many proteins, and these results provide evidence that basic residues play a role in localization of G protein alpha subunits.     

Response of bacterioplankton community structures to hydrological conditions and anthropogenic pollution in contrasting subtropical environments

Bacterioplankton community structures under contrasting subtropical marine environments (Hong Kong waters) were analyzed using 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of predominant bands for samples collected bimonthly from 2004 to 2006 at five stations. Generally, bacterial abundance was significantly higher in the summer than in the winter. The general seasonal variations of the bacterial community structure, as indicated by cluster analysis of the DGGE pattern, were best correlated with temperature at most stations, except for the station close to a sewage discharge outfall, which was best explained by pollution-indicating parameters (e.g. biochemical oxygen demand). Anthropogenic pollutions appear to have affected the presence and the intensity of DGGE bands at the stations receiving discharge of primarily treated sewage. The relative abundance of major bacterial species, calculated by the relative intensity of DGGE bands after PCR amplification, also indicated the effects of hydrological or seasonal variations and sewage discharges. For the first time, a systematic molecular fingerprinting analysis of the bacterioplankton community composition was carried out along the environmental and pollution gradient in a subtropical marine environment, and it suggests that hydrological conditions and anthropogenic pollutions altered the total bacterial community as well as the dominant bacterial groups.     

Proteomic analysis during larval development and metamorphosis of the spionid polychaete Pseudopolydora vexillosa

Background  

While the larval-juvenile transition (metamorphosis) in the spionid polychaete Pseudopolydora vexillosa involves gradual morphological changes and does not require substantial development of juvenile organs, the opposite occurs in the barnacle Balanus amphitrite. We hypothesized that the proteome changes during metamorphosis in the spionids are less drastic than that in the barnacles. To test this, proteomes of pre-competent larvae, competent larvae (ready to metamorphose), and juveniles of P. vexillosa were compared using 2-dimensional gel electrophoresis (2-DE), and they were then compared to those of the barnacle.     

Effect of all-trans retinol on in vitro development of preimplantation buffalo embryos

Vitamin A is a well-known antioxidant and is essential for embryonic development, growth and differentiation. Oxidative stress is involved in the etiology of defective embryo development. The present study evaluated whether the presence of all-trans retinol (0, 1, 2, 5 and 10 μM) in maturation medium or embryo culture medium would enhance the developmental competence of preimplantation buffalo embryos in vitro. In experiment I, cumulus oocytes complex were matured with varying concentrations of all-trans retinol. Treatment with 5 μM all-trans retinol improved the blastocyst formation (P < 0.001) when compared with control and significant increase (P < 0.01) in total cell number was observed in 5 μM group when compared with control. Supplementation of all-trans retinol in embryo culture medium for the entire culture period under 5% O2 and 20% O2 was tested in experiments II and III, respectively. Supplementation of 10 μM all-trans retinol under 5% O2, significantly reduced blastocyst formation and cell numbers. Presence of 5 μM all-trans retinol under 20% O2 enhanced the frequency of blastocyst formation and total cell number (P < 0.001) when compared with control. DNA damage of individual embryos cultured under 20% oxygen concentration was measured by the comet assay. Supplementation of 5 μM all-trans retinol significantly reduced the comet tail (P < 0.001) when compared with control. Supplementation of all-trans retinol in embryo culture medium for first 72 h of the 8-day culture period under 5% O2 was tested in experiment IV. Addition of 5 μM all-trans retinol resulted in significant increase in blastocyst rate and total cell number (P < 0.001) when compared with control. Our results demonstrate that addition of all-trans retinol to maturation or embryo culture medium may enhance the developmental competence of buffalo embryos in vitro by enhancing blastocyst formation rate and total cell number.     

Quantitative analysis of oyster larval proteome provides new insights into the effects of multiple climate change stressors

The metamorphosis of planktonic larvae of the Pacific oyster (Crassostrea gigas) underpins their complex life‐history strategy by switching on the molecular machinery required for sessile life and building calcite shells. Metamorphosis becomes a survival bottleneck, which will be pressured by different anthropogenically induced climate change‐related variables. Therefore, it is important to understand how metamorphosing larvae interact with emerging climate change stressors. To predict how larvae might be affected in a future ocean, we examined changes in the proteome of metamorphosing larvae under multiple stressors: decreased pH (pH 7.4), increased temperature (30 °C), and reduced salinity (15 psu). Quantitative protein expression profiling using iTRAQ‐LC‐MS/MS identified more than 1300 proteins. Decreased pH had a negative effect on metamorphosis by down‐regulating several proteins involved in energy production, metabolism, and protein synthesis. However, warming switched on these down‐regulated pathways at pH 7.4. Under multiple stressors, cell signaling, energy production, growth, and developmental pathways were up‐regulated, although metamorphosis was still reduced. Despite the lack of lethal effects, significant physiological responses to both individual and interacting climate change related stressors were observed at proteome level. The metamorphosing larvae of the C. gigas population in the Yellow Sea appear to have adequate phenotypic plasticity at the proteome level to survive in future coastal oceans, but with developmental and physiological costs.     

Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC₅₀ values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H₂O₂) induced RBC haemolysis (IC₅₀ 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H₂O₂-induced damage; inhibited H₂O₂ induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations.