Immunomodulatory, Antioxidant, Anticancer, and Pharmacokinetic Activity of Ulvan, a Seaweed-Derived Sulfated Polysaccharide: An Updated Comprehensive Review.

Cancer is one of the most worldwide spread diseases and causes maximum death. Treatment of cancer depends on the host immune system and the type of drugs. The inefficiency of conventional cancer treatments as a result of drug resistance, nontargeted delivery, and chemotherapy-related negative side effects has caused bioactive phytochemicals to come into focus. As a result, recent years have seen an increase in research into screening and identifying natural compounds with anticancer properties. Recent studies on the isolation and use of polysaccharides derived from various marine algal species have revealed a variety of biological activities, including antioxidant and anticancer properties. Ulvan is a polysaccharide derived from various green seaweeds of the Ulva species in the family Ulvaceae. It has been demonstrated to have potent anticancer and anti-inflammatory properties through the modulation of antioxidants. It is vital to understand the mechanisms underlying the biotherapeutic activities of Ulvan in cancer and its role in immunomodulation. In this context, we reviewed the anticancer effects of ulvan based on its apoptotic effects and immunomodulatory activity. Additionally, we also focused on its pharmacokinetic studies in this review. Ulvan is the most conceivable candidate for use as a cancer therapeutic agent and could be used to boost immunity. Moreover, it may be established as an anticancer drug once its mechanisms of action are understood. Due to its high food and nutritive values, it can be used as a possible dietary supplement for cancer patients in the near future. This review may provide fresh perspectives on the potential novel role of ulvan, reveal a brand-new cancer-prevention strategy, and improve human health.

Antioxidant and chemotherapeutic efficacies of seaweed-derived phlorotannins in cancer treatment: A review regarding novel anticancer drugs.

The ineffectiveness of traditional cancer therapies due to drug resistance, nontargeted delivery, and chemotherapy-associated adverse side effects has shifted attention to bioactive phytochemicals. Consequently, research efforts toward screening and identification of natural compounds with anticancer properties have increased in recent years. Marine seaweed-derived bioactive compounds, such as polyphenolic compounds, have exhibited anticancer properties. Phlorotannins (PTs), a major group of seaweed-derived polyphenolic compounds, have emerged as powerful chemopreventive and chemoprotective compounds, regulating apoptotic cell death pathways both in vitro and in vivo. In this context, this review focuses on the anticancer activity of polyphenols isolated from brown algae, with a special reference to PTs. Furthermore, we highlight the antioxidant effects of PTs and discuss how they can impact cell survival and tumor development and progression. Moreover, we discussed the potential therapeutic application of PTs as anticancer agents, having molecular mechanisms involving oxidative stress reduction. We have also discussed patents or patent applications that apply PTs as major components of antioxidant and antitumor products. With this review, researcher may gain new insights into the potential novel role of PTs, as well as uncover a novel cancer-prevention mechanism and improve human health.

Unveiling the genomic structures and evolutionary events of the saxitoxin biosynthetic gene sxtA in the marine toxic dinoflagellate Alexandrium.

Marine dinoflagellates Alexandriumare known to produce saxitoxin (STX) and cause paralytic shellfish poisoning (PSP) which can result in mortality in human. SxtA is considered a core gene for the biosynthesis of STX. However, its gene coding structure and evolutionary history have yet to be fully elucidated. Here, we determined the full-length sequences of sxtA cDNA and genomic coding regions from two toxic dinoflagellates, Alexandrium catenella (LIMS-PS-2645 and LIMS-PS-2647) andA. pacificum (LMBE-C4), characterised their domain structures, and resolved evolutionary events. The sxtA gene was encoded on the genome without introns, and was identical in length (4002 bp) between two A. catenella strains, but their sequences differed from A. pacificum (5031 bp). SxtA consists of four domains, sxtA1, sxtA2, sxtA3, and sxtA4; however, A. pacificum has an extra domain TauD near sxtA1. Each domain had >64.4% GC content, with the highest being 71.6% in sxtA3. Molecular divergence was found to be significantly higher in sxtA4 than in the other domains. Phylogenetic trees of sxtA and separate domains showed that bacteria diverged earliest, followed by non-toxic, toxic cyanobacteria, toxic dinoflagellates. While sxtA domains in Alexandrium were similar to the PKS-like structure with the conserved sxtA1, sxtA2, and sxtA3. PKS_KS may be replaced by sxtA4 in toxic Alexandrium. These suggest that sxtA in Alexandrium may have evolved by acquiring specific domains, whose modification and complexity markedly affect toxin biosynthesis.

Phytoplankton Toxins and Their Potential Therapeutic Applications: A Journey toward the Quest for Potent Pharmaceuticals.

Phytoplankton are prominent organisms that contain numerous bioactive substances and secondary metabolites, including toxins, which can be valuable to pharmaceutical, nutraceutical, and biotechnological industries. Studies on toxins produced by phytoplankton such as cyanobacteria, diatoms, and dinoflagellates have become more prevalent in recent years and have sparked much interest in this field of research. Because of their richness and complexity, they have great potential as medicinal remedies and biological exploratory probes. Unfortunately, such toxins are still at the preclinical and clinical stages of development. Phytoplankton toxins are harmful to other organisms and are hazardous to animals and human health. However, they may be effective as therapeutic pharmacological agents for numerous disorders, including dyslipidemia, obesity, cancer, diabetes, and hypertension. In this review, we have focused on the properties of different toxins produced by phytoplankton, as well as their beneficial effects and potential biomedical applications. The anticancer properties exhibited by phytoplankton toxins are mainly attributed to their apoptotic effects. As a result, phytoplankton toxins are a promising strategy for avoiding postponement or cancer treatment. Moreover, they also displayed promising applications in other ailments and diseases such as Alzheimer's disease, diabetes, AIDS, fungal, bacterial, schizophrenia, inflammation, allergy, osteoporosis, asthma, and pain. Preclinical and clinical applications of phytoplankton toxins, as well as future directions of their enhanced nano-formulations for improved clinical efficacy, have also been reviewed.

Algal Phlorotannins as Novel Antibacterial Agents with Reference to the Antioxidant Modulation: Current Advances and Future Directions.

The increasing drug resistance of infectious microorganisms is considered a primary concern of global health care. The screening and identification of natural compounds with antibacterial properties have gained immense popularity in recent times. It has previously been shown that several bioactive compounds derived from marine algae exhibit antibacterial activity. Similarly, polyphenolic compounds are generally known to possess promising antibacterial capacity, among other capacities. Phlorotannins (PTs), an important group of algae-derived polyphenolic compounds, have been considered potent antibacterial agents both as single drug entities and in combination with commercially available antibacterial drugs. In this context, this article reviews the antibacterial properties of polyphenols in brown algae, with particular reference to PTs. Cell death through various molecular modes of action and the specific inhibition of biofilm formation by PTs were the key discussion of this review. The synergy between drugs was also discussed in light of the potential use of PTs as adjuvants in the pharmacological antibacterial treatment.

Molecular cloning and oxidative-stress responses of a novel Phi class glutathione S-transferase (GSTF) gene in the freshwater algae Closterium ehrenbergii.

Glutathione S-transferases (GSTs) belong to a family of enzymes involved in diverse biological processes, including detoxification and protection against oxidative damage. Here, we determined the full-length sequence (915 bp) of a novel Phi class cytosolic glutathione S-transferase (GSTF) gene from the green algae Closterium ehrenbergii. We examined the gene structure and expression patterns in response to metals and endocrine disrupting chemicals (EDCs). It was significantly upregulated by metals, but responded differently to EDCs. The highest up-regulation of CeGSTF was registered under 0.1 mg/L CuCl2 and 0.01 mg/L CuSO4 treatments. In a 72-h course experiment with treatment of 0.1 mg/L CuCl2 , CeGSTF was dramatically induced at 6 h, and then gradually decreased with increasing exposure time. This was consistent with the increase in both GST activity and ROS production in copper-treated cells. These results suggest that CeGSTF may be involved in detoxification mechanisms associated with oxidative stress in green algae.

Identification of a Metacaspase Gene in the Bloom-Forming Dinoflagellate Prorocentrum minimum and its Putative Function Involved in Programmed Cell Death.

Programmed cell death (PCD) in dinoflagellates has been introduced as a new concept that facilitates the demise of harmful algal blooms. Metacaspases (MCAs) play a role in PCD, but their function in dinoflagellates is unclear. Here, we cloned a novel MCA gene (PmMCA) from the harmful dinoflagellate Prorocentrum minimum and examined its molecular characteristics and gene expression during cell death. The gene was encoded in the nuclear genome with two introns. The putative protein contained 288 amino acids and three conserved MCA signature motifs. Phylogenetic analysis showed that PmMCA may have the same ancestor as other dinoflagellates. PmMCA expression and cell apoptosis were significantly induced under copper exposure, considerably affecting cell growth. These results suggest that PmMCA could be involved in PCD triggered by copper stress.

Low Temperature and Cold Stress Significantly Increase Saxitoxins (STXs) and Expression of STX Biosynthesis Genes sxtA4 and sxtG in the Dinoflagellate Alexandrium catenella.

Toxic dinoflagellate Alexandrium spp. produce saxitoxins (STXs), whose biosynthesis pathway is affected by temperature. However, the link between the regulation of the relevant genes and STXs' accumulation and temperature is insufficiently understood. In the present study, we evaluated the effects of temperature on cellular STXs and the expression of two core STX biosynthesis genes (sxtA4 and sxtG) in the toxic dinoflagellate Alexandrium catenella Alex03 isolated from Korean waters. We analyzed the growth rate, toxin profiles, and gene responses in cells exposed to different temperatures, including long-term adaptation (12, 16, and 20 °C) and cold and heat stresses. Temperature significantly affected the growth of A. catenella, with optimal growth (0.49 division/day) at 16 °C and the largest cell size (30.5 µm) at 12 °C. High concentration of STXs eq were detected in cells cultured at 16 °C (86.3 fmol/cell) and exposed to cold stress at 20→12 °C (96.6 fmol/cell) compared to those at 20 °C and exposed to heat stress. Quantitative real-time PCR (qRT-PCR) revealed significant gene expression changes of sxtA4 in cells cultured at 16 °C (1.8-fold) and cold shock at 20→16 °C (9.9-fold). In addition, sxtG was significantly induced in cells exposed to cold shocks (20→16 °C; 19.5-fold) and heat stress (12→20 °C; 25.6-fold). Principal component analysis (PCA) revealed that low temperature (12 and 16 °C) and cold stress were positively related with STXs' production and gene expression levels. These results suggest that temperature may affect the toxicity and regulation of STX biosynthesis genes in dinoflagellates.

Chloroacetanilides inhibit photosynthesis and disrupt the thylakoid membranes of the dinoflagellate Prorocentrum minimum as revealed with metazachlor treatment.

The chloroacetanilides are among the most commonly used herbicides worldwide, which contaminate aquatic environments and affect aquatic phototrophs. Their sub-lethal toxicity has been evaluated using freshwater algae; however, the modes of cellular toxicity and levels of toxicity to marine organisms are not fully understood. In the present study, we assessed the cellular and molecular effects of chloroacetanilides on marine phototrophs using the dinoflagellate Prorocentrum minimum and the herbicide metazachlor (MZC). The MZC treatment led to a considerable reduction in cell number and pigment, and the EC50 of MZC was calculated to be 0.647 mg/L. The photosynthetic parameters, Fv/Fm and chlorophyll fluorescence significantly decreased with MZC exposure time in a dose-dependent manner. In addition, MZC significantly induced photosynthesis genes, including PmpsbA, PmpsaA, and PmatpB, and the antioxidant PmGST, but not PmKatG. These findings were well matched to reactive oxygen species (ROS) production in MZC-treated cells. Interestingly, we observed inflated vacuoles, undivided chloroplasts, and breakdown of thylakoid membranes in MZC-treated cells. These results support the hypothesis that MZC severely damages chloroplasts, resulting in dysfunction of the dinoflagellate photosynthesis and possibly marine phototrophs in the environment.

Transcriptome survey and toxin measurements reveal evolutionary modification and loss of saxitoxin biosynthesis genes in the dinoflagellates Amphidinium carterae and Prorocentrum micans.

In the present study, we characterized the potential toxin genes for polyketide synthase (PKS) and saxitoxin (STX) biosynthesis using the transcriptomes of two non-STX producing dinoflagellates Amphidinium carterae and Prorocentrum micans. RNA sequencing revealed 94 and 166 PKS contigs in A. carterae and P. micans, respectively. We first detected type III PKS, which was closely related to bacteria. In addition, dozens of homologs of 20 STX biosynthesis genes were identified. Interestingly, the core STX-synthesizing genes sxtA and sxtB were only found in P. micans, whereas sxtD was detected in A. carterae alone. Bioinformatic analysis showed that the first two core genes (sxtA and sxtG) had a low sequence similarity (37.0-67.6%) and different domain organization compared to those of other toxigenic dinoflagellates, such as Alexandrium pacificum. These might result in the breakdown of the initial reactions in STX production and ultimately the loss of the ability to synthesize the toxins in both dinoflagellates. Our findings suggest that toxin-related PKS and sxt genes are commonly found in non-STX producing dinoflagellates. In addition to their involvement in the synthesis of toxins, our result indicates that genes may also have other molecular metabolic functions.

Molecular characterization and expression analysis of copper-zinc superoxide dismutases from the freshwater alga Closterium ehrenbergii under metal stress.

Superoxide dismutase (SOD) acts as the first line of defense against reactive oxygen species (ROS) within cells. In the present study, we determined two novel CuZnSOD genes (designated as CeCSD1 and CeCSD2) from the toxicity-testing freshwater algae Closterium ehrenbergii and examined their structural features, phylogenetic relationships, and gene expression under exposure to different metals. Putative CeCSD1 (204 aa, 20.6 kDa) and CeCSD2 (155 aa, 15.3 kDa) proteins had conserved CuZnSOD family motifs and metal (Cu, Zn) binding sites, but different N-terminus structures, that is, CeCSD1 has a signal peptide to chloroplasts. Phylogenetic analysis of each protein revealed that C. ehrenbergii was well clustered with other green algae and plants. Real-time PCR results showed that the gene expression obviously increased with heavy metal exposure. In addition, excess copper considerably increased the SOD activity and ROS generation but decreased the photosynthetic efficiency in treated cells. These results suggest that CeCSDs are involved in the antioxidant defense system and can be regarded as potential biomarkers for monitoring metal contaminants in aquatic environments.

The herbicide alachlor severely affects photosystem function and photosynthetic gene expression in the marine dinoflagellate Prorocentrum minimum.

Alachlor is one of the most widely used herbicides and can remain in agricultural soils and wastewater. The toxicity of alachlor to marine life has been rarely studied; therefore, we evaluated the physiological and transcriptional responses in the marine dinoflagellate Prorocentrum minimum. The herbicide led to considerable decreases in P. minimum cell numbers and pigment contents. The EC50 was determined to be 0.373 mg/L. Photosynthesis efficiency and chlorophyll autofluorescence dramatically decreased with increasing alachlor dose and exposure time. Real-time PCR analysis showed that the photosynthesis-related genes PmpsbA, PmatpB, and PmrbcL were induced the most by alachlor; the transcriptional level of each gene varied with time. PmrbcL expression increased after 30 min of alachlor treatment, whereas PmatpB and PmpsbA increased after 24 h. The PmpsbA expression level was highest (5.0 times compared to control) after 6 h of alachlor treatment. There was no significant change in PmpsaA expression with varying treatment time or concentration. Additionally, there was no notable change in the expression of antioxidant genes PmGST and PmKatG, or in ROS accumulation. These suggest that alachlor may affect microalgal photosystem function, with little oxidative stress, causing severe physiological damage to the cells, and even cell death.

Differential transcriptional responses of carotenoid biosynthesis genes in the marine green alga Tetraselmis suecica exposed to redox and non-redox active metals.

The green microalga, Tetraselmis suecica, is commonly used in scientific, industrial, and aquacultural purposes because of its high stress tolerance and ease of culture in wide spectrums of environments. We hypothesized that carotenoids help to protect Tetraselmis cells from environmental stress by regulating genes in biosynthetic pathways. Here, we determined three major carotenogenic genes, phytoene synthase (PSY), phytoene desaturase (PDS), and ß-lycopene cyclase (LCY-B) in T. suecica, and examined the physiological parameters and gene expression responses when exposed to redox-active metals and non-redox-active metals. Phylogenetic analyses of each gene indicated that T. suecica clustered well with other green algae. Real-time PCR analysis showed that TsPSY, TsPDS, and TsLCY-B genes greatly responded to the redox-active metals in CuSO4 followed by CuCl2, but not to the non-redox-active metals. The redox-active metals strongly affected the physiology of the cells, as determined by cell counting, reactive oxygen species (ROS) imaging, and photosynthetic efficiency. This suggests that carotenoids protect the cells from oxidative damage caused by metals, thereby contributing to cell survival under various stress conditions.

Molecular cloning and oxidative-stress responses of a novel manganese superoxide dismutase (MnSOD) gene in the dinoflagellate Prorocentrum minimum.

Dinoflagellate algae are microeukaryotes that have distinct genomes and gene regulation systems, making them an interesting model for studying protist evolution and genomics. In the present study, we discovered a novel manganese superoxide dismutase (PmMnSOD) gene from the marine dinoflagellate Prorocentrum minimum, examined its molecular characteristics, and evaluated its transcriptional responses to the oxidative stress-inducing contaminants, CuSO4 and NaOCl. Its cDNA was 1238 bp and contained a dinoflagellate spliced leader sequence, a 906 bp open reading frame (301 amino acids), and a poly (A) tail. The gene was coded on the nuclear genome with one 174 bp intron; signal peptide analysis showed that it might be localized to the mitochondria. Real-time PCR analysis revealed an increase in gene expression of MnSOD and SOD activity when P. minimum cells were separately exposed to CuSO4 and NaOCl. In addition, both contaminants considerably decreased chlorophyll autofluorescence, and increased intracellular reactive oxygen species. These results suggest that dinoflagellate MnSOD may be involved in protecting cells against oxidative damage.

Small heat shock protein genes of the green algae Closterium ehrenbergii: Cloning and differential expression under heat and heavy metal stresses.

The freshwater green algae Closterium ehrenbergii has been considered as a model for eco-toxicological assessment in aquatic systems. Heat shock proteins (HSPs) are a class of highly conserved proteins produced in all living organisms, which participate in environmental stress responses. In the present study, we determined the cDNA sequences of small heat shock protein 10 (sHSP10) and sHSP17.1 from C. ehrenbergii, and examined the physiological changes and transcriptional responses of the genes after exposure to thermal shock and toxicants treatments. The open reading frame (ORF) of CeHSP10 was 300 bp long, encoding 99 amino acid (aa) residues (10.53 kDa) with a GroES chaperonin conserved site of 22 aa. The CeHSP17.1 had a 468 bp ORF, encoding 155 aa with a conserved C-terminal α-crystallin domain. For heat stress, cells presented pigment loss and possible chloroplast damage, with an up-regulation in the expression of both sHSP10 and sHSP17.1 genes. As for the heavy metal stressors, an increase in the production of reactive oxygen species was registered in a dose dependent manner, with a significant up-regulation of both sHSP10 and sHSP17.1 genes. These results suggest that sHSP genes in C. ehrenbergii may play a role in responses to stress environments, and they could be used as an early detection parameter as biomarker genes in molecular toxicity assessments.

A Review of the Biological Activities of Microalgal Carotenoids and Their Potential Use in Healthcare and Cosmetic Industries.

Carotenoids are natural pigments that play pivotal roles in many physiological functions. The characteristics of carotenoids, their effects on health, and the cosmetic benefits of their usage have been under investigation for a long time; however, most reviews on this subject focus on carotenoids obtained from several microalgae, vegetables, fruits, and higher plants. Recently, microalgae have received much attention due to their abilities in producing novel bioactive metabolites, including a wide range of different carotenoids that can provide for health and cosmetic benefits. The main objectives of this review are to provide an updated view of recent work on the health and cosmetic benefits associated with carotenoid use, as well as to provide a list of microalgae that produce different types of carotenoids. This review could provide new insights to researchers on the potential role of carotenoids in improving human health.

Transcriptomic profiles reveal the genome-wide responses of the harmful dinoflagellate Cochlodinium polykrikoides when exposed to the algicide copper sulfate.

BACKGROUND: Harmful algal blooms (HABs) caused by the dinoflagellate Cochlodinium polykrikoides lead to severe environmental impacts in oceans worldwide followed by huge economic losses. Algicide agent copper sulfate (CuSO4) is regard as an economical and effective agent for HABs mitigation; its biochemical and physiological effects were revealed in C. polykrikoides. However, molecular mechanisms of CuSO4 effect on the C. polykrikoides, even other HAB species, have not been investigated. The present study investigated the transcriptional response of C. polykrikoides against CuSO4 treatments, with the aim of providing certain molecular mechanism of CuSO4 effect on the C. polykrikoides blooms. RESULTS: RNA-seq generated 173 million reads, which were further assembled to 191,212 contigs. 43.3 %, 33.9 %, and 15.6 % of contigs were annotated with NCBI NR, GO, and KEGG database, respectively. Transcriptomic analysis revealed 20.6 % differential expressed contigs, which grouped into 8 clusters according to K-means clustering analysis, responding to CuSO4; 848 contigs were up-regulated and 746 contigs were down-regulated more than 2-fold changes from 12 h to 48 h exposure. KEGG pathway analysis of eukaryotic homologous genes revealed the differentially expressed genes (DEGs) were involved in diverse pathway; amongst, the genes involved in the translation, spliceosome, and/or signal transduction genes were highly regulated. Most of photosystem related genes were down-regulated and most of mitochondria related genes were up-regulated. In addition, the genes involved in the copper ion binding or transporting and antioxidant systems were identified. Measurement of chlorophyll fluorescence showed that photosynthesis was significantly inhibited by CuSO4 exposure. CONCLUSIONS: This study reported the first transcriptome of the C. polykrikoides. The widely differential expressed photosystem genes suggested photosynthetic machinery were severely affected, and may further contribute to the cell death. Furthermore, gene translation and transcription processes may be disrupted, inhibiting cell growth and proliferation, and possibly accelerating cell death. However, antioxidant systems resistant to CuSO4 caused stress; mitochondrion may compensate for photosynthesis efficiency decreasing caused energy deficiency. In addition, various signal transduction pathways may be involved in the CuSO4 induced regulation network in the C. polykrikoides. These data provide the potential transcriptomic mechanism to explain the algicide CuSO4 effect on the harmful dinoflagellate C. polykrikoides.

Physiological and biochemical responses of the freshwater green algae Closterium ehrenbergii to the common disinfectant chlorine.

Chlorine (Cl2) is widely used as a disinfectant in water treatment plants and for cleaning swimming pools; it is finally discharged into aquatic environments, possibly causing damage to the non-target organisms in the receiving water bodies. Present study evaluated the effects of the biocide Cl2 to the green alga Closterium ehrenbergii (C. ehrenbergii). Growth rate, chlorophyll a levels, carotenoids, chlorophyll autofluorescence, and antioxidant enzymes were monitored up to 72-h after Cl2 exposure. C. ehrenbergii showed dose-dependent decrease in growth rate and cell division after exposure to Cl2. By using cell counts, the median effective concentration (EC50)-72-h was calculated to be 0.071mgL(-1). Cl2 significantly decreased the pigment levels and chlorophyll autofluorescence intensity, indicating that the photosystem was damaged in C. ehrenbergii. In addition, it increased the production of reactive oxygen species (ROS) in the cells. This stressor significantly increased the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase, and glutathione, and affected the physiology of the cells. These results indicate that Cl2 induces oxidative stress in the cellular metabolic process and leads to physiological and biochemical damages in the green algae. Cl2 discharged in industrial effluents and from water treatment plants may cause harmful effects to the C. ehrenbergii a common freshwater microalgae and other non-target organisms.

Implications of High Molecular Divergence of Nuclear rRNA and Phylogenetic Structure for the Dinoflagellate Prorocentrum (Dinophyceae, Prorocentrales).

The small and large nuclear subunit molecular phylogeny of the genus Prorocentrum demonstrated that the species are dichotomized into two clades. These two clades were significantly different (one-factor ANOVA, p < 0.01) with patterns compatible for both small and large subunit Bayesian phylogenetic trees, and for a larger taxon sampled dinoflagellate phylogeny. Evaluation of the molecular divergence levels showed that intraspecies genetic variations were significantly low (t-test, p < 0.05), than those for interspecies variations (> 2.9% and > 26.8% dissimilarity in the small and large subunit [D1/D2], respectively). Based on the calculated molecular divergence, the genus comprises two genetically distinct groups that should be considered as two separate genera, thereby setting the pace for major systematic changes for the genus Prorocentrum sensu Dodge. Moreover, the information presented in this study would be useful for improving species identification, detection of novel clades from environmental samples.

Molecular characterisation and expression analysis of a novel calreticulin (CRT) gene in the dinoflagellate Prorocentrum minimum.

Calreticulin is a multifunctional Ca(2+)-binding protein that has been well characterised in mammalian cells. Here, we characterised a novel calreticulin (CRT2) gene in the dinoflagellate Prorocentrum minimum, which codes for a calcium binding protein and examined its expression pattern following the addition of calcium and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). PmCRT2 is encoded in the nuclear genome of P. minimum without introns. The full length cDNA of PmCRT2 was found to be 1,493 base pairs (bp) in length, which ranges from the dinoflagellate spliced leader sequence to the poly (A) tail and contains a 1,173-bp open reading frame, a 70-bp 5'-untranslated region (UTR), and a 207-bp 3'-UTR. On the basis of in silico analyses that revealed the distinct domain architectures of PmCRT2, we classified this protein under the calreticulin family. PmCRT2 gene expression was up-regulated in the presence of excess calcium in a dose-dependent manner; however, PmCRT2 expression was down-regulated by the addition of EGTA. These results clearly indicate that PmCRT2 plays a vital role in calcium regulation and this may be involved in the stress response of P. minimum.