Transcriptomic Study of Gracilaria Changii (Gracilariales, Rhodophyta) By Expressed Sequence Tags and Cdna Microarray Approach

Gracilaria is one of the most extensively harvested seaweeds throughout the world due to its economic importance as an agarophyte for global agar production. In this study, Gracilaria changii, an indigenous seaweed species in Malaysia known for its high quality agar was chosen for transcription profiling. A total of 990 expressed sequence tags (ESTs) consisting of 766 tentative unique genes (TUGs) have been generated. These TUGs comprise 643 TUSs (tentative unique singletons) and 123 TUCs (tentative unique contigs). The putative identity of TUGs was identified by using Basic Local Alignment Search Tool X (BLASTX) algorithm and classified according to the functional groups in Kyoto Encyclopedia of Genes and Genomes (KEGG). The result showed that 198 TUGs (25.85%) have significant matches to the annotated proteins and 81 TUGs (10.57%) have significant matches to the unknown proteins in the non-redundant protein database in the GenBank; whereas the remaining 487 (63.58%) TUGs had non-significant matches or no matches to sequence from other organisms. Similar to animals and plants, G. changii showed preference for purine residues at -3 position and guanidine at +4 position at the translational initiation signal. On the other hand, the 3' untranslated region of G. changii was found to have relatively less stringent polyadenylation process compared with plants and animals in producing mature mRNA. A cDNA microarray consisting of approximately 3,000 cDNA probes was constructed and used for the hybridization of cDNAs synthesized from G. changii samples cultured under conditions with and without light to understand the genetic acclimation of G. changii to light deprivation. The results suggested that genes related to photosynthesis, oxidative stress and sulfate metabolism were down-regulated during light deprivation. The cDNA microarray data were further verified by using real-time PCR. The results of the real-time PCR analysis of four genes encoding light-harvesting complex I polypeptide (DV962275.1), low molecular mass early light-inducible protein (DV964113.1), 14-3-3 protein (DV965610.1) and sonic hedgehog protein precursor (DV967367.1) supported the expression patterns demonstrated using cDNA microarray

Transcriptomic analysis of Gracilaria changii (Rhodophyta) in response to hyper- and hypo-osmotic stresses

Osmotic stress is one of the most significant natural abiotic stresses that occur in the intertidal zones. Seaweeds may physiologically acclimate to changing osmolarity by altering their transcriptome. Here, we investigated the transcriptomic changes of Gracilaria changii (B. M. Xia et I. A. Abbott) I. A. Abbott, J. Zhang et B. M. Xia in response to hyper- and hypoosmotic stresses using a cDNA microarray approach. Microarray analysis revealed that 199 and 200 genes from ∼3,300 genes examined were up- and down-regulated by >2-fold in seaweed samples treated at 50 parts per thousand (ppt) artificial seawater (ASW) compared with those at 30 ppt ASW, respectively. The number of genes that were up- and down-regulated by >2-fold in seaweed samples treated at 10 ppt ASW compared with those at 30 ppt ASW were 154 and 187, respectively. A majority of these genes were only differentially expressed under hyper- or hypoosmotic conditions, whereas 67 transcripts were affected by both stresses. The findings of this study have shed light on the expression profiles of many transcripts during the acclimation of G. changii to hyperosmotic and hypoosmotic conditions. This information may assist in the prioritization of genes to be examined in future studies

Molecular cloning and characterization of GDP-mannose-3′,5′-epimerase from Gracilaria changii

GDP-mannose-3′,5′-epimerase (GME) is an enzyme involved in the biosynthesis of GDP-l-galactose which is a building unit of agar and cell wall polysaccharides. GME catalyzes the formation of GDP-β-l-galactose and GDP-l-gulose from GDP-mannose. In this study, the gene and transcript encoding GME from the red alga Gracilaria changii (GcGME) were cloned. The structural gene sequence of GcGME is devoid of an intron. The cis-acting regulatory element involved in light response is the most abundant element at the 5′-flanking region of GcGME. The open reading frame of GcGME consists of 1,053 nucleotides with 351 amino acids. This cDNA was cloned into pET32a expression vector for recombinant protein production in Escherichia coli. High yield of soluble recombinant GcGME (55 kDa) was expressed upon isopropyl β-d-1-thiogalactopyranoside induction. The enzyme activity of recombinant GcGME was detected using thin layer chromatography and high-performance liquid chromatography. The transcript abundance of GcGME was the highest in G. changii and the lowest in Gracilaria salicornia corresponding to their agar contents. The characterization of GcGME from G. changii is important to facilitate the understanding of its role in agar production of this seaweed

Profiling the transcriptome of Gracilaria changii (Rhodophyta) in response to light deprivation

Light regulates photosynthesis, growth and reproduction, yield and properties of phycocolloids, and starch contents in seaweeds. Despite its importance as an environmental cue that regulates many developmental, physiological, and biochemical processes, the network of genes involved during light deprivation are obscure. In this study, we profiled the transcriptome of Gracilaria changii at two different irradiance levels using a cDNA microarray containing more than 3,000 cDNA probes. Microarray analysis revealed that 93 and 105 genes were up- and down-regulated more than 3-fold under light deprivation, respectively. However, only 50% of the transcripts have significant matches to the nonredundant peptide sequences in the database. The transcripts that accumulated under light deprivation include vanadium chloroperoxidase, thioredoxin, ferredoxin component, and reduced nicotinamide adenine dinucleotide dehydrogenase. Among the genes that were down-regulated under light deprivation were genes encoding light harvesting protein, light harvesting complex I, phycobilisome 7.8 kDa linker polypeptide, low molecular weight early light-inducible protein, and vanadium bromoperoxidase. Our findings also provided important clues to the functions of many unknown sequences that could not be annotated using sequence comparison

Impact of the putative cancer stem cell markers and growth factor receptor expression on the sensitivity of ovarian cancer cells to treatment with various forms of the HER inhibitors and cytotoxic drugs

Increased expression and activation of human epidermal growth factor receptor (EGFR) and HER-2 have been reported in numerous cancers. The aim of this study was to determine the sensitivity of a large panel of human ovarian cancer cell lines (OCCLs) to treatment with various forms of small molecule tyrosine kinase inhibitors (TKIs) and cytotoxic drugs. The aim was to see if there was any association between the protein expression of various biomarkers including three putative ovarian cancer stem cell (CSC) markers (CD24, CD44, CD117/c-Kit), P-glycoprotein (P-gp), and HER family members and response to treatment with these agents. The sensitivity of 10 ovarian tumour cell lines to the treatment with various forms of HER TKIs (gefitinib, erlotinib, lapatinib, sapitinib, afatinib, canertinib, neratinib), as well as other TKIs (dasatinib, imatinib, NVP-AEW541, crizotinib) and cytotoxic agents (paclitaxel, cisplatin and doxorubicin), as single agents or in combination, was determined by SRB assay. The effect on these agents on the cell cycle distribution, and downstream signaling molecules and tumour migration were determined using flow cytometry, western blotting, and the IncuCyte Clear View cell migration assay respectively. Of the HER inhibitors, the irreversible pan-TKIs (canertinib, neratinib and afatinib) were the most effective TKIs for inhibiting the growth of all ovarian cancer cells, and for blocking the phosphorylation of EGFR, HER-2, AKT and MAPK in SKOV3 cells. Interestingly, while the majority of cancer cells were highly sensitive to treatment with dasatinib, they were relatively resistant to treatment with imatinib (i.e., IC50 >10 µM). Of the cytotoxic agents, paclitaxel was the most effective for inhibiting the growth of OCCLs, and of various combinations of these drugs, only treatment with a combination of NVP-AEW541 and paclitaxel produced a synergistic or additive anti-proliferative effect in all three cell lines examined (i.e., SKOV3, Caov3, ES2). Finally, of the TKIs, only treatment with afatinib, neratinib and dasatinib were able to reduce the migration of HER-2 overexpressing SKOV3 cells. We did not find any significant association between the expression of putative ovarian CSC marker, HER family members, c-MET, ALK, and IGF-IR and the response to the irreversible HER TKIs. Our results support the need for further investigations of the therapeutic potential of these irreversible HER family blockers in ovarian cancer, and the therapeutic potential of dasatinib when used in combination with the inhibitors of the HER family members in ovarian cancer

The Interleukin-6 inflammation pathway from cholesterol to aging – Role of statins, bisphosphonates and plant polyphenols in aging and age-related diseases

We describe the inflammation pathway from Cholesterol to Aging. Interleukin 6 mediated inflammation is implicated in age-related disorders including Atherosclerosis, Peripheral Vascular Disease, Coronary Artery Disease, Osteoporosis, Type 2 Diabetes, Dementia and Alzheimer's disease and some forms of Arthritis and Cancer. Statins and Bisphosphonates inhibit Interleukin 6 mediated inflammation indirectly through regulation of endogenous cholesterol synthesis and isoprenoid depletion. Polyphenolic compounds found in plants, fruits and vegetables inhibit Interleukin 6 mediated inflammation by direct inhibition of the signal transduction pathway. Therapeutic targets for the control of all the above diseases should include inhibition of Interleukin-6 mediated inflammation