Protein Expression Analysis in Reversible Photobleached Cells of Scenedesmus vacuolatus after High Temperature Stress.

We have analyzed protein expression in the bleached small vegetative cells of synchronous Scenedesmus vacuolatus to investigate how unicellular algae lived through stress. These cells were subjected to heat treatment (46.5 °C for 1h in dark condition) and then cultured under continuous illumination for 24 h. Flow cytometry analysis of the chlorophyll autofluorescence intensity of S. vacuolatus cells indicated that heat-treated cells were completely bleached within 24 h of light cultivation. Transmission electron microscopy (TEM) images showed that bleached cells maintained thylakoid membrane structure, but with lower contrast. The bleached cells regained green color after 72 h, along with a recovery in contrast, which indicated a return of photosynthetic ability. Two-dimensional gel electrophoresis (2DE) showed that the protein expression patterns were very difference between control and bleached cells. ATP synthase subunits and glutamine synthetase were down-regulated among the many differences, while some of phototransduction, stress response proteins were up-regulated in bleached cells, elucidating bleached cells can undergo changes in their biochemical activity, and activate some stress response proteins to survive the heat stress and then revive. In addition, small heat shock proteins (HSPs), but not HSP40 and HSP70 family proteins, protected the bleaching cells.

Molecular identification for epigallocatechin-3-gallate-mediated antioxidant intervention on the H2O2-induced oxidative stress in H9c2 rat cardiomyoblasts.

BACKGROUND: Epigallocatechin-3-gallate (EGCG) has been documented for its beneficial effects protecting oxidative stress to cardiac cells. Previously, we have shown the EGCG-mediated cardiac protection by attenuating reactive oxygen species and cytosolic Ca2+ in cardiac cells during oxidative stress and myocardial ischemia. Here, we aimed to seek a deeper elucidation of the molecular anti-oxidative capabilities of EGCG in an H2O2-induced oxidative stress model of myocardial ischemia injury using H9c2 rat cardiomyoblasts. RESULTS: Proteomics analysis was used to determine the differential expression of proteins in H9c2 cells cultured in the conditions of control, 400 µM H2O2 exposure for 30 min with and/or without 10 to 20 µM EGCG pre-treatment. In this model, eight proteins associated with energy metabolism, mitochondrial electron transfer, redox regulation, signal transduction, and RNA binding were identified to take part in EGCG-ameliorating H2O2-induced injury in H9c2 cells. H2O2 exposure increased oxidative stress evidenced by increases in reactive oxygen species and cytosolic Ca2+ overload, increases in glycolytic protein, α-enolase, decreases in antioxidant protein, peroxiredoxin-4, as well as decreases in mitochondrial proteins, including aldehyde dehydrogenase-2, ornithine aminotransferase, and succinate dehydrogenase ubiquinone flavoprotein subunit. All of these effects were reversed by EGCG pre-treatment. In addition, EGCG attenuated the H2O2-induced increases of Type II inositol 3, 4-bisphosphate 4-phosphatase and relieved its subsequent inhibition of the downstream signalling for Akt and glycogen synthase kinase-3ß (GSK-3ß)/cyclin D1 in H9c2 cells. Pre-treatment with EGCG or GSK-3ß inhibitor (SB 216763) significantly improved the H2O2-induced suppression on cell viability, phosphorylation of pAkt (S473) and pGSK-3ß (S9), and level of cyclin D1 in cells. CONCLUSIONS: Collectively, these findings suggest that EGCG blunts the H2O2-induced oxidative effect on the Akt activity through the modulation of PIP3 synthesis leading to the subsequent inactivation of GSK-3ß mediated cardiac cell injury.

Association of genetic variants in senataxin and Alzheimer's disease in a Chinese Han population in Taiwan.

Development of Alzheimer's disease (AD) is characterized by progressive neuronal death and a decline in learning and memory. Mutations in human senataxin (SETX), an ortholog yeast protein of Sen1, have been identified to cause the syndrome of ataxia with oculomotor apraxia type 2 (AOA2) and juvenile amyotrophic lateral sclerosis (ALS4), two types of progressive motor neuron degeneration. However, the relationship between the SETX gene, which is involved in the regulation of RNA processing and DNA repair, and the predisposition for AD remains unclear. In this research, potential association of polymorphisms in the SETX gene with AD was investigated. A case-control study of a Chinese Han population in Taiwan was performed. Three single-nucleotide polymorphisms (SNPs), 3455T>G (rs3739922), 3576T>G (rs1185193) and 7759A>G (rs1056899) were studied. The experimental data showed that upon genotyping of the exonic polymorphism in the SETX gene, the T allele appeared at a lower rate than the G allele at position 3455 in AD patients compared with normal groups (P < 0.05, odds ratio (OR), 0.59, 95% confidence interval (CI), 0.40-0.89). Subjects with the GA genotype at position 7759 have higher incidences of AD development than with the AA genotype (P < 0.05, OR, 6.45, 95% CI, 1.24 to 33.70). Our results also showed that with six haplotypes (Hts) observed from the analyzed polymorphisms, distributions of the Ht4-GAA and Ht5-GCA haplotypes appeared to be significant 'risk' haplotypes between AD patients and controls (both P < 0.05, OR, 8.44, 95% CI, 1.07-66.60). These observations suggest that genetic variations in the SETX gene may contribute to AD pathogenesis in the Taiwanese Han population.

8-Alkylcoumarins from the fruits of Cnidium monnieri protect against hydrogen peroxide induced oxidative stress damage.

Three new 8-alkylcoumarins, 7-O-methylphellodenol-B (1), 7-methoxy-8-(3-methyl- 2,3-epoxy-1-oxobutyl)chromen-2-one (2), and 3'-O-methylvaginol (3), together with seven known compounds (4-10) were isolated from the fruits of Cnidium monnieri. Their structures were determined by detailed analysis of spectroscopic data and comparison with the data of known analogues. All the isolates were evaluated the cytoprotective activity by MTS cell proliferation assay and the results showed that all the three new 8-alkylcoumarins exhibited cytoprotective effect on Neuro-2a neuroblastoma cells injured by hydrogen peroxide.

The IgE gene in primates exhibits extraordinary evolutionary diversity.

Membrane-bound IgE (mIgE) on B lymphocytes is essential for IgE production. Earlier studies showed that the ε chain of mIgE (mε) on human B cells has a "long" isoform, with an extra "CεmX" domain of 52 amino acid (aa) residues between the CH4 domain and the membrane-anchor segment, as compared to the conventional "short" isoform. Because CεmX provides an antigenic site for targeting IgE-expressing B cells to down-regulate IgE production in patients with allergy, analysis of CεmX in various animals is of great interest. Hence, we analyzed the ε Ig gene, in particular, its membrane exon regions encoding the membrane anchor peptide segment and CεmX domain, of 26 species of the order Primates and 12 species of seven non-Primate orders using data obtained experimentally or retrieved from GenBank. Our analyses reveal the unexpected finding that the genes of three extant tarsier species do not contain the membrane exons for mIgE. Another striking finding is that early evolved Strepsirhini primates such as lemurs and lorises do not have gene segments for the long isoform, whereas New World monkeys such as marmosets and squirrel monkeys allow the transcription of only the long isoform. In Old World monkeys and apes, including humans, the ε gene allows the transcription of both isoforms. This work thus reveals the dramatic differences in the gene segment encoding the mε C terminal region among the four major primate lineages: the Strepsirhini primates, the tarsiers, New World monkeys, and Old World monkeys and apes/humans.

Constructing gene regulatory networks for long term photosynthetic light acclimation in Arabidopsis thaliana.

BACKGROUND: Photosynthetic light acclimation is an important process that allows plants to optimize the efficiency of photosynthesis, which is the core technology for green energy. However, currently little is known about the molecular mechanisms behind the regulation of the photosynthetic light acclimation response. In this study, a systematic method is proposed to investigate this mechanism by constructing gene regulatory networks from microarray data of Arabidopsis thaliana. METHODS: The potential TF-gene regulatory pairs of photosynthetic light acclimation have been obtained by data mining of literature and databases. Following the identification of these potential TF-gene pairs, they have been refined using Pearson's correlation, allowing the construction of a rough gene regulatory network. This rough gene regulatory network is then pruned using time series microarray data of Arabidopsis thaliana via the maximum likelihood system identification method and Akaike's system order detection method to approach the real gene regulatory network of photosynthetic light acclimation. RESULTS: By comparing the gene regulatory networks under the PSI-to-PSII light shift and the PSII-to-PSI light shift, it is possible to identify important transcription factors for the different light shift conditions. Furthermore, the robustness of the gene network, in particular the hubs and weak linkage points, are also discussed under the different light conditions to gain further insight into the mechanisms of photosynthesis. CONCLUSIONS: This study investigates the molecular mechanisms of photosynthetic light acclimation for Arabidopsis thaliana from the physiological level. This has been achieved through the construction of gene regulatory networks from the limited data sources and literature via an efficient computation method. If more experimental data for whole-genome ChIP-chip data and microarray data with multiple sampling points becomes available in the future, the proposed method will be improved on by constructing the whole-genome gene regulatory network. These advances will greatly improve our understanding of the mechanisms of the photosynthetic system.

Plant Growth Absorption Spectrum Mimicking Light Sources.

Plant factories have attracted increasing attention because they can produce fresh fruits and vegetables free from pesticides in all weather. However, the emission spectra from current light sources significantly mismatch the spectra absorbed by plants. We demonstrate a concept of using multiple broad-band as well as narrow-band solid-state lighting technologies to design plant-growth light sources. Take an organic light-emitting diode (OLED), for example; the resulting light source shows an 84% resemblance with the photosynthetic action spectrum as a twin-peak blue dye and a diffused mono-peak red dye are employed. This OLED can also show a greater than 90% resemblance as an additional deeper red emitter is added. For a typical LED, the resemblance can be improved to 91% if two additional blue and red LEDs are incorporated. The approach may facilitate either an ideal use of the energy applied for plant growth and/or the design of better light sources for growing different plants.

Photosynthetic plasticity of Phalaenopsis in response to different light environments.

The ability of a plant to dynamically acclimate to different light environments is, in general, genetically determined. Phalaenopsis amabilis is a CAM orchid with heavy self-shading. The aim of this study was to find out how the photosynthetic capacity of its mature lower leaves acclimates to the low light environment, and whether it possessed a potential for reacclimation following transfer of lower leaves to higher irradiance. We found that the photosynthetic performance of the leaves of Phalaenopsis was flexibly and reversibly adjusted to growth irradiance, making it possible to improve the light environment of the plant by increasing light exposure of lower leaves and bring about a higher photosynthetic production. We have tested the effectiveness of a simple setup using mirrors to augment light from the side and thus enhanced the irradiance in the shaded area of the plant. Both photosynthesis and starch contents of leaves as well as the number of flowers per plant increased greatly.

Allelic alterations at the STR markers in the buccal tissue cells of oral cancer patients and the oral epithelial cells of healthy betel quid-chewers: an evaluation of forensic applicability.

Although cancerous specimens are usually not used in forensic DNA typing, they might be forcibly employed under certain instances. On the other hand, though the oral epithelial samples have been applied to forensic identification, the great popularity of betel quid (BQ)-chewing in Taiwan, which is known to be a risk factor leading to an oral cancer, makes this application questionable. The DNA stability of nine short tandem repeat (STR) markers (the AmpFlSTR kit) was first investigated and then used to evaluate the forensic appropriateness of the oral samples of both healthy BQ-chewers and the archived clinical specimens from oral cancer patients. The analyses were performed on buccal samples from 100 BQ-chewers and 100 oral cancer patients, as well as their paired peripheral blood samples, and a group of 100 non-BQ-chewers were used for the control. In the group of 100 oral cancer patients, two types of DNA instability were found. They were major allelic imbalance, and allelic alterations including the expansion, the contraction and the un-classified type (i.e. can not be confirmed as the expansion or the contraction). The overall percentage of the cancerous subjects demonstrating DNA instability was 33% (five patients possessing both types of DNA instability). Both types of DNA instability showed a tendency of increasing with the severity of the pathological stage of oral cancer. Forty-four occurrences of major allelic imbalance were found from 21 cancer patients. The statistical result revealed that there was no significant difference in the allelic imbalanced occurrence among the nine STR loci. Allelic alterations were found in 17 patients, within which 12 individuals had the expansion, five had the contraction, and three were the un-classified type. Further, among these 17 patients, three were found to acquire multiple allelic alterations at multiple loci. In the group of 100 unrelated healthy BQ-chewers, two loci with major allelic imbalance were detected. However, the two imbalanced alleles were virtually half lost, and could still be recognized as heterozygous alleles. The statistical results of ANOVA, chi(2), and Scheffe tests indicated that the means of allelic imbalance at the nine STR loci of the oral cancerous group revealed a significant difference from those in the control group. Our results suggest that oral cancer tissues cannot be used as references for forensic purposes using the PCR-based STR systems, whereas the oral swabs from healthy BQ-chewers can be employed, but should be done with caution.

Isolation and sequencing a genomic DNA encoding for phenylalanine ammonia-lyase from Phalaenopsis.

The enzyme L-phenylalanine ammonia-lyase is universally present in higher plants and it catalyzes the first committed reaction for a central pathway that generates hundreds of different phenylpropanoid metabolites including anthocyanin, the main pigments in flowers. In this study, we have successfully isolated and analyzed a phenylalanine ammonia-lyase gene from Phalaenopsis. The gene spans 3265bp and consists of two exons and one intron and encodes a polypeptide of 703 amino acid residues.

Evaluation of the DNA stability of forensic markers used in betel-quid chewers' oral swab samples and oral cancerous specimens: implications for forensic application.

Chewed betel-quid (BQ) residues are often considered vital biological evidence at crime scenes, since the human DNA extracted from the residues is actually from buccal epithelial cells and can be associated with suspects. BQ-chewing is also a risk factor for oral diseases and/or cancers. Archived medical oral-specimens can be used to identify specific individuals under adverse conditions, although STR markers are known to be unstable in various tumor tissues. This study evaluates the DNA stability of forensic marker systems in BQ-chewers' oral epithelial cells, and in archived clinical specimens of oral cancer patients. The genotypes of oral and paired peripheral blood samples in 200 subjects were compared, using the commercialized typing systems of HLA-DQA1, PM (including LDLR, GYPA, HBGG, D7S8, and GC loci), and AmpFlSTR markers (including 9 STR loci and the Amelogenin gene). The 100 healthy BQ-chewers had consistent oral swab and paired blood sample genotypes analyzed withboth DQA1/PM and STR marker systems. In the 100 oral cancer patients, one discordant result at D7S8 was found in the 600DQA1/PM-marker loci, and 25 allelic alterations with expansion or contraction were detected in the 900 STR loci. The findings herein suggest that when cancerous specimens were tested, the HLA-DQA1/PM system with point polymorphism appears more reliable than the STR system with length polymorphism. Our results also indicate that healthy BQ-chewers' oral cotton swabs containing buccal epithelial cells are useful for forensic purposes using the HLA-DQA1, PM, and STR marker systems.

The reversible degeneration of heat-treated Scenedesmus vacuolatus under continuous light cultivation conditions.

To find out how microalgae cope with heat stress, the small vegetative cells of a synchronous Scenedesmus vacuolatus culture were subjected to heat treatment and then cultured under continuous illumination. The heat-treated cells were found first to enter a degenerative intermediate stage with low cellular activities almost right after the start of the cultivation, which was then followed by a revival. The changes in physiological activities and morphology of the treated cells throughout the whole period of regeneration were explored. The variations in cellular DNA content and protein composition were also investigated. Stressed cells at the end of the degeneration stage were completely bleached and were also characterized by condensed but undegraded chromatin, partially disintegrated chloroplasts but with the thylakoid membrane system retained, partially operating mitochondria, intact plasma membranes, and a dramatically changed profile of cellular proteins. All of our data indicate they were still alive but in a different physiological state than the control cells. Recovery started with regeneration of mitochondrial cristae and redispersion of chromatins. These were followed by regreening and resuscitation of chloroplasts, which often started from one part of a thylakoid membrane system and then spread out. This study provided a unicellular model for studying how plant cells react to a period of stress and recover.

NMR assignments of a hypothetical pseudo-knotted protein HP0242 from Helicobacter pylori.

Many knotted proteins have been discovered recently, but the folding process of which remains elusive. HP0242 is a hypothetical protein from Helicobacter pylori, which is a model system for studying the folding pathway of a knotted protein. In this study, we report the (1)H, (13)C, and (15)N chemical shift assignments of HP0242. The results will enable us to further investigate HP0242 by NMR experiments.

Association of glutathione S-transferase P1 (GSTP1) polymorphism with Tourette syndrome in Taiwanese patients.

The etiology of Tourette syndrome (TS) is multifactorial. TS vulnerability may be associated with genetic and environmental factors. From the genetic point of view, TS is heterogeneous. Previous studies showed that some single-nucleotide polymorphisms (SNPs) of the glutathione-S-transferase P1 (GSTP1) gene can affect cellular proliferation and apoptotic activity and TS is a neurodevelopmental disorder. We guessed that there was a relationship between TS and genetic variants of the GSTP1 gene. Therefore, in this study, we aimed to test the hypothesis that GSTP1 SNPs were associated with TS. We performed a case-control study. One hundred twenty-one TS children and 105 normal children were included in the study. Polymerase chain reaction was used to identify the GSTP1 gene polymorphism at position rs6591256 (A/G, promoter polymorphism) in TS patients and normal children. The polymorphism at position rs6591256 in the GSTP1 gene revealed significant differences in the allele (p=0.0135) and genotype (p=0.0159) distributions between the TS patients and the control group. The A allele was present at a higher frequency than the G allele in the TS patients compared with the control group (odds ratio [OR]=1.91, 95% confidence interval [CI]: 1.14-3.21). The AA genotype was associated with susceptibility to TS with an OR of 2.38 for the AA versus AG genotype (95% CI: 1.29-4.41). These findings suggest that variants in the GSTP1 gene may play a role in susceptibility to TS.

Characterization of the decline and recovery of heat-treated Scenedesmus vacuolatus.

BACKGROUND: To find out how algal cells cope with and recover from heat stress, the small vegetative cells of the synchronous Scenedesmus vacuolatus culture were subjected to a heat pretreatment (46.5°C for 1 h) followed by dark recultivation. The changes in physiological activities and morphology of Scenedesmus cells were continuously monitored throughout the course of decline and recovery. RESULTS: It was found that the heat treatment, though completely inhibited photosynthesis, did not kill Scenedesmus cells. These cells, during dark recultivation, could make a fast repair and regained the ability of proliferation. We suggest that they entered a 'stand-by' state, which was characterized by condensed chromatin, partially functional but morphologically altered chloroplasts, disappeared vacuoles, slightly shrunk protoplast and intact plasma membranes. These stressed cells, on the surface, seemingly were undergoing some kind of disintegration, could readily and quickly return to normal cells upon illumination. Cell death occurred only after a long period of darkness (>48 h). CONCLUSIONS: Our results suggest that the recovery of algal cells from stress damage may actually proceed in two steps. The middle "stand-by' stage normally is gone through too rapidly to be detected unless cells are kept in the dark.

DISINTEGRATION OF THE CELLS OF SIPHONOUS GREEN ALGA CODIUM EDULE (BRYOPSIDALES, CHLOROPHYTA) UNDER MILD HEAT STRESS(1).

The siphonous green alga Codium edule P. C. Silva (Bryopsidales, Chlorophyta) has the highest covering ratio among the macroalgae on the coral reef of Nanwan Bay in southern Taiwan, but its population in the subtidal region drastically decreases from July to September each year. The objective of this study was to determine whether the high temperature of summer could be the basis for this population decrease. Chlorophyll fluorescence measurements revealed that when the algae were incubated at 35°C (a temperature that can be reached in southern Taiwan during the summer), their photosynthetic activities were almost completely inhibited after about 8 h. The circadian rhythm of photosynthesis was disrupted at a temperature as low as 32°C. TEM studies showed that 4 h incubation at 35°C induced a decrease in turgidity accompanied by vacuole shrinkage and plasmolysis. The marked disintegrative changes, including damage to organelles, such as chloroplasts and nuclei, occurred after about 8 h, at which time central vacuoles collapsed and the cell interior was then filled with numerous small vesicles. Our results suggested that the rise in seawater temperature during the summer could be one of the major causes of the massive death of C. edule in the field.

A simple and highly efficient cloning method that employs PCR to directly create a fusion between insert and vector.

We have developed a very simple procedure for gene cloning using compound primers. This approach permits the insertion of a DNA fragment in a single step using a PCR-based cloning protocol, which employs annealing rather than ligation to create the recombinant in the plasmid. This method has been tested successfully to clone a Phalaenopsis gene coding for P450. A potential application of this protocol for constructing a cDNA library is also proposed.

A possible origin of the middle phase of polyphasic chlorophyll fluorescence transient.

The rise of the chlorophyll fluorescence of a whole leaf as induced by high-intensity actinic light comprises three distinct phases, and is termed the O-J-I-P polyphasic rise. The initial rise (the O-J phase) was found to be the most sensitive to light intensity, being slower and smaller with decreasing irradiation. The leaf was also found to be transparent for chlorophyll fluorescence to a considerable extent, so that the fluorescence originating from deep inside the sample could still be detected. In contrast, the actinic light used to induce fluorescence was strongly absorbed by chlorophylls, so that a steep light gradient was created along the light path. The fluorescence transient of a leaf, thus, was always a mixture of the fluorescence from the surface of the sample as well as that from the inside of the sample, whose O-J phase is slower as it is induced by a weaker actinic light. We have provided evidence suggesting that, in an intact leaf, the middle phase of the measured polyphasic fluorescence transient (the J-I phase) might actually reflect the initial rise of the transient coming from the abaxial layer of the leaf. Moreover, if the polyphasic fluorescence transient is used as an analytical tool for accessing information on the photosynthetic activities of leaves, the factors of concentration and thickness of the sample must be taken into account. To obtain the 'true' fluorescence transient of a sample, both the chlorophyll concentration and thickness of the sample must be kept as low as possible.

Cloning and expression of a putative cytochrome P450 gene that influences the colour of Phalaenopsis flowers.

Anthocyanins are responsible for reds through blues in flowers. Blue and violet flowers generally contain derivatives of delphinidin, whereas red and pink flowers contain derivatives of cyanidin or pelargonidin. Differences in hydroxylation patterns of these three major classes of anthocyanidins are controlled by the cytochrome P450 enzymes. Flavonoid-3',5'-hydroxylase, a member of the cytochrome P450 family, is the key enzyme in the synthesis of 3',5'-hydroxylated anthocyanins, generally required for blue or purple flowers. Here we report on the isolation of a cDNA clone of a putative flavonoid-3',5'-hydroxylase gene from Phalaenopsis that was then cloned into a plant expression vector. Transient transformation was achieved by particle bombardment of Phalaenopsis petals. The transgenic petals changed from pink to magenta, indicating that the product of the putative flavonoid-3',5'-hydroxylase gene influences anthocyanin pigment synthesis.