The red alga Porphyridium as a host for molecular farming: Efficient production of immunologically active hepatitis C virus glycoprotein.

Microalgae are promising production platforms for the cost-effective production of recombinant proteins. We have recently established that the red alga Porphyridium purpureum provides superior transgene expression properties, due to the episomal maintenance of transformation vectors as multicopy plasmids in the nucleus. Here, we have explored the potential of Porphyridium to synthesize complex pharmaceutical proteins to high levels. Testing expression constructs for a candidate subunit vaccine against the hepatitis C virus (HCV), we show that the soluble HCV E2 glycoprotein can be produced in transgenic algal cultures to high levels. The antigen undergoes faithful posttranslational modification by N-glycosylation and is recognized by conformationally selective antibodies, suggesting that it adopts a proper antigenic conformation in the endoplasmic reticulum of red algal cells. We also report the experimental determination of the structure of the N-glycan moiety that is attached to glycosylated proteins in Porphyridium. Finally, we demonstrate the immunogenicity of the HCV antigen produced in red algae when administered by injection as pure protein or by feeding of algal biomass.

The structure of PSI-LHCI from Cyanidium caldarium provides evolutionary insights into conservation and diversity of red-lineage LHCs.

Light-harvesting complexes (LHCs) are diversified among photosynthetic organisms, and the structure of the photosystem I-LHC (PSI-LHCI) supercomplex has been shown to be variable depending on the species of organisms. However, the structural and evolutionary correlations of red-lineage LHCs are unknown. Here, we determined a 1.92-Å resolution cryoelectron microscopic structure of a PSI-LHCI supercomplex isolated from the red alga Cyanidium caldarium RK-1 (NIES-2137), which is an important taxon in the Cyanidiophyceae. We subsequently investigated the correlations of PSI-LHCIs from different organisms through structural comparisons and phylogenetic analysis. The PSI-LHCI structure obtained shows five LHCI subunits surrounding a PSI-monomer core. The five LHCIs are composed of two Lhcr1s, two Lhcr2s, and one Lhcr3. Phylogenetic analysis of LHCs bound to PSI in the red-lineage algae showed clear orthology of LHCs between C. caldarium and Cyanidioschyzon merolae, whereas no orthologous relationships were found between C. caldarium Lhcr1-3 and LHCs in other red-lineage PSI-LHCI structures. These findings provide evolutionary insights into conservation and diversity of red-lineage LHCs associated with PSI.

Simple prerequisite of presequence for mitochondrial protein import in the unicellular red alga Cyanidioschyzon merolae.

Mitochondrial biogenesis relies on hundreds of proteins that are derived from genes encoded in the nucleus. According to the characteristic properties of N-terminal targeting peptides (TPs) and multi-step authentication by the protein translocase called the TOM complex, nascent polypeptides satisfying the requirements are imported into mitochondria. However, it is unknown whether eukaryotic cells with a single mitochondrion per cell have a similar complexity of presequence requirements for mitochondrial protein import compared to other eukaryotes with multiple mitochondria. Based on putative mitochondrial TP sequences in the unicellular red alga Cyanidioschyzon merolae, we designed synthetic TPs and showed that functional TPs must have at least one basic residue and a specific amino acid composition, although their physicochemical properties are not strictly determined. Combined with the simple composition of the TOM complex in C. merolae, our results suggest that a regional positive charge in TPs is verified solely by TOM22 for mitochondrial protein import in C. merolae. The simple authentication mechanism indicates that the monomitochondrial C. merolae does not need to increase the cryptographic complexity of the lock-and-key mechanism for mitochondrial protein import.

Devosia rhodophyticola sp. nov. and Devosia algicola sp. nov., isolated from a marine red alga.

Two Gram-stain-negative, obligately aerobic, motile rod bacteria, designated as G2-5T and G20-9T, exhibiting catalase- and oxidase-positive activities, were isolated from the phycosphere of a Chondrus species, a marine red alga. Strain G2-5T exhibited optimal growth at 30 °C and pH 5.0-6.0 and in the presence of 0.5-1.0% NaCl. In contrast, strain G20-9T demonstrated optimal growth at 25 °C and pH 6.0 and in the presence of 0.5-1.5% NaCl. Both strains contained ubiquinone-10, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C18 : 0 and 11-methyl-C18 : 1 ω7c, and diphosphatidylglycerol and phosphatidylglycerol as the major respiratory isoprenoid quinone, cellular fatty acids and polar lipids, respectively. The genomic DNA G+C contents were 57.2 mol% for strain G2-5T and 57.5 mol% for strain G20-9T. Strains G2-5T and G20-9T exhibited 98.2 % 16S rRNA gene sequence similarity, along with 82.3 % average nucleotide identity (ANI) and 25.0 % digital DNA-DNA hybridization (dDDH) values, indicating that they represent different species. Phylogenetic analyses based on both 16S rRNA gene and genome sequences revealed that strains G2-5T and G20-9T formed distinct phylogenic lineages within the genus Devosia. Strains G2-5T and G20-9T were most closely related to Devosia limi DSM 17137T and Devosia beringensis S02T with 97.7 and 96.9 % 16S rRNA gene sequence similarities, respectively. The ANI and dDDH values between strains G2-5T and G20-9T and other Devosia species were lower than 73.9 and 19.2 %, respectively, suggesting that they constitute novel species within the genus Devosia. Based on their distinct phenotypic, chemotaxonomic, and molecular characteristics, strains G2-5T and G20-9T represent two novel species of the genus Devosia, for which the names Devosia rhodophyticola sp. nov. (G2-5T=KACC 22601T=JCM 35404T) and Devosia algicola sp. nov. (G20-9T=KACC 22650T=JCM 35405T) are proposed, respectively.

Octadecabacter algicola sp. nov. and Octadecabacter dasysiphoniae sp. nov., isolated from a marine red alga and emended description of the genus Octadecabacter.

Two Gram-stain-negative, strictly aerobic, catalase- and oxidase-positive and non-motile rod-shaped bacteria, strains D2-3T and G9-8T, were isolated from a marine red alga. Both strains contained ubiquinone-10 as the sole isoprenoid quinone. As the major cellular fatty acids (>5.0 %), D2-3T contained C16 : 0, 11-methyl-C18 : 1ω7c, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), whereas G9-8T contained C16 : 0, 11-methyl-C18 : 1ω7c, C12 : 1 3-OH, and summed feature 8. The DNA G+C contents of D2-3T and G9-8T were 54.4 % and 56.0 %, respectively. As the major polar lipids, phosphatidylglycerol, diphosphatidylglycerol and unidentified phospholipid, aminolipid and lipid were identified from both strains, and phosphatidylcholine was additionally detected from G9-8T only. The 16S rRNA gene sequence similarity of D2-3T and G9-8T was 98.5 % and their digital DNA-DNA hybridization (DDH) value was 19.1 %. Phylogenetic analyses based on 16S rRNA gene and genome sequences revealed that D2-3T and G9-8T formed respectively distinct phylogenetic lineages within the genus Octadecabacter. D2-3T and G9-8T were most closely related to Octadecabacter ascidiaceicola RA1-3T and Octadecabacter antarcticus 307T, with 98.9 % and 98.5 % 16S rRNA gene sequence similarities, respectively, and digital DDH values between D2-3T and O. ascidiaceicola and between G9-8T and O. antarcticus were 18.3 % and 19.5 %, respectively. Phenotypic, chemotaxonomic and molecular features support the hypothesis that D2-3T and G9-8T represent two novel species of the genus Octadecabacter, for which the names Octadecabacter algicola sp. nov. and Octadecabacter dasysiphoniae sp. nov. are proposed. The type strains of O. algicola and O. dasysiphoniae are D2-3T (=KACC 22493T =JCM 34969T) and G9-8T (=KACC 22488T =JCM 34973T), respectively.

Alkalicoccobacillus porphyridii sp. nov., isolated from a marine red alga, reclassification of Shouchella plakortidis and Shouchella gibsonii as Alkalicoccobacillus plakortidis comb. nov. and Alkalicoccobacillus gibsonii comb. nov., and emended description of the genus Alkalicoccobacillus Joshi et al. 2022.

A Gram-stain-positive alkali-tolerant and strictly aerobic bacterium, designated strain P16T, was isolated from a marine red alga, Porphyridium cruentum, in the Yellow Sea, Republic of Korea. Cells were motile rods with peritrichous flagella and exhibited catalase and oxidase activities. The optimal growth of strain P16T was observed to occur at 30 °C and pH 8.0 and in the presence of 2.0 % (w/v) NaCl. Menaquinone-7 was identified as the sole respiratory quinone. Strain P16T contained anteiso-C15 : 0, iso-C15 : 0, iso-C14 : 0 and iso-C16 : 0, and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine as major cellular fatty acids and polar lipids, respectively. The G+C content of strain P16T was 40.8 mol%. Strain P16T was most closely related to Shouchella plakortidis P203T, Shouchella gibsonii DSM 8722T and Alkalicoccobacillus murimartini LMG 21005T with 98.1, 98.1 and 98.0 % 16S rRNA gene sequence similarities, respectively. Phylogenetic analyses based on 16S rRNA gene and genome sequences revealed that strain P16T, S, plakortidis, S. gibsonii and A. murimartini formed a single phylogenetic lineage cluster, and genomic relatedness analyses showed that they are different species. Based on phylogenetic, phenotypic, chemotaxonomic and molecular features, strain P16T represents a novel species of the genus Alkalicoccobacillus, for which the name Alkalicoccobacillus porphyridii sp. nov. is proposed. The type strain is P16T (=KACC 19520T=JCM 32931T). In addition, S. plakortidis and S. gibsonii are reclassified as Alkalicoccobacillus plakortidis comb. nov. (type strain P203T=DSM 19153T=NCIMB 14288T) and Alkalicoccobacillus gibsonii comb. nov. (type strain PN-109T=ATCC 700164T=DSM 8722T=KCCM 41407T), respectively.

Characterisation of Bioactive Peptides from Red Alga Gracilariopsis chorda.

In this study, we studied the bioactive peptides produced by thermolysin hydrolysis of a water-soluble protein (WSP) from the red alga Gracilariopsis chorda, whose major components are phycobiliproteins and Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCo). The results showed that WSP hydrolysate exhibited significantly higher ACE inhibitory activity (92% inhibition) compared to DPP-IV inhibitory activity and DPPH scavenging activity. The phycobiliproteins and RuBisCo of G. chorda contain a high proportion of hydrophobic (31.0-46.5%) and aromatic (5.1-46.5%) amino acid residues, which was considered suitable for the formation of peptides with strong ACE inhibitory activity. Therefore, we searched for peptides with strong ACE inhibitory activity and identified two novel peptides (IDHY and LVVER). Then, their interaction with human ACE was evaluated by molecular docking, and IDHY was found to be a promising inhibitor. In silico analysis was then performed on the structural factors affecting ACE inhibitory peptide release, using the predicted 3D structures of phycobiliproteins and RuBisCo. The results showed that most of the ACE inhibitory peptides are located in the highly solvent accessible α-helix. Therefore, it was suggested that G. chorda is a good source of bioactive peptides, especially ACE-inhibitory peptides.

Two new halogenated metabolites from the red alga Laurencia sp.

Two new halogenated metabolites, laurenhalogens A (1) and B (2), along with four known ones (3-6), were isolated from the red alga Laurencia sp. The structures of 1 and 2 were determined by the means of UV, IR, MS, NMR and X-ray diffraction analysis. In addition, the antibacterial activities of 1-6 were also evaluated.

A new chlorobenzoate derivative from the red alga Solieria sp.

A new chlorobenzoate derivative, solieriate (1), together with six known compounds (2-7), were isolated from the red alga Solieria sp. The structures of 1-7 were determined by comprehensive spectroscopic methods and X-ray diffraction analysis. Compound 1 is the first example of halogenated derivative isolated from this genus. In addition, 1 exhibited moderate antibacterial activity on A. baumannii with MIC value of 64 µg/ml.

Functional characterization of a cellulose synthase, CtCESA1, from the marine red alga Calliarthron tuberculosum (Corallinales).

In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthases (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum using sequence similarity-based approaches, and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1, was closely related to other putative red algal CESA genes. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared with plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal CESAs. The relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are probably functional differences between the algal and land plant CESAs.

Aureibaculum algae sp. nov. isolated from the Pacific red alga Ahnfeltia tobuchiensis.

A Gram stain-negative, aerobic, rod-shaped, motile by gliding and yellow-orange-pigmented bacterium, designated strain 10Alg 115T, was isolated from the red alga Ahnfeltia tobuchiensis. The phylogenetic analysis based on 16S rRNA gene sequences placed the novel strain within the family Flavobacteriaceae, phylum Bacteroidetes. The nearest neighbor of the new isolate was Aureibaculum marinum KCTC 62204T with sequence similarity of 98.1%. The average nucleotide similarity and digital DNA-DNA hybridization values between the novel strain and Aureibaculum marinum KCTC 62204T were 80% and 22.3%, respectively. The prevalent fatty acids of strain 10Alg 115T were iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH, iso-C16:0 3-OH and C15:0. The polar lipid profile consisted of phosphatidylethanolamine, two unidentified aminolipids and two unidentified lipids. The DNA G + C content of the type strain calculated from the whole-genome sequence was 32.2 mol%. A combination of the genotypic and phenotypic data showed that the algal isolate represents a novel species of the of genus Aureibaculum, for which the name Aureibaculum algae sp. nov. is proposed. The type strain is 10Alg 115T (= KCTC 62086T = KMM 6764T).

Algicella marina gen. nov., sp. nov., a novel marine bacterium isolated from a Pacific red alga.

A novel Gram-staining negative, strictly aerobic, rod-shaped, and non-motile bacterium, designated strain 9Alg 56T, was isolated from the red alga Tichocarpus crinitus. The phylogenetic analysis based on 16S rRNA gene sequences placed the novel strain within the family Rhodobacteraceae, the order Rhodobacterales, the class Alphaproteobacteria, the phylum Pseudomonadota. The nearest neighbors of the new strain were Pontivivens insulae KCTC 42458T, Oceanibium sediminis KCTC 62076T, Halovulum dunhuangense YYQ-30T and Monaibacterium marinum C7T with 16S rRNA gene sequence similarity of 94.7, 94.4%, 93.1 and 92.7%, respectively. The AAI/ANI/dDDH values between 9Alg 56T and the five species of the closest genera (Pontivivens, Oceanibium, Halovulum, Monaibacterium, and 'Oceanomicrobium') were 58.63-63.91%/ 75.91-77.37%/ 19.3-20.4%. The prevalent fatty acids of strain 9Alg 56T were C18:1 ω7c, C18:0 and C14:0 3-OH. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylcholine, and two unidentified lipids. The DNA G+C content of strain 9Alg 56T was 61.5 mol%. A combination of the genotypic and phenotypic data showed that the algal isolate represents a novel genus and species, for which the name Algicella marina gen. nov., sp. nov. is proposed. The type strain is 9Alg 56T (= KCTC 72005T = KMM 6775T).

Characterization of Antioxidant Activity of Heated Mycosporine-like Amino Acids from Red Alga Dulse Palmaria palmata in Japan.

We recently demonstrated the monthly variation and antioxidant activity of mycosporine-like amino acids (MAAs) from red alga dulse in Japan. The antioxidant activity of MAAs in acidic conditions was low compared to that in neutral and alkali conditions, but we found strong antioxidant activity from the heated crude MAA fraction in acidic conditions. In this study, we identified and characterized the key compounds involved in the antioxidant activity of this fraction. We first isolated two MAAs, palythine, and porphyra-334, from the fraction and evaluated the activities of the two MAAs when heated. MAAs possess absorption maxima at around 330 nm, while the heated MAAs lost this absorption. The heated MAAs showed a high ABTS radical scavenging activity at pH 5.8-8.0. We then determined the structure of heated palythine via ESI-MS and NMR analyses and speculated about the putative antioxidant mechanism. Finally, a suitable production condition of the heated compounds was determined at 120 °C for 30 min at pH 8.0. We revealed compounds from red algae with antioxidant activities at a wide range of pH values, and this information will be useful for the functional processing of food.

A cotransformation system of the unicellular red alga Cyanidioschyzon merolae with blasticidin S deaminase and chloramphenicol acetyltransferase selectable markers.

BACKGROUND: The unicellular red alga Cyanidioschyzon merolae exhibits a very simple cellular and genomic architecture. In addition, procedures for genetic modifications, such as gene targeting by homologous recombination and inducible/repressible gene expression, have been developed. However, only two markers for selecting transformants, uracil synthase (URA) and chloramphenicol acetyltransferase (CAT), are available in this alga. Therefore, manipulation of two or more different chromosomal loci in the same strain in C. merolae is limited. RESULTS: This study developed a nuclear targeting and transformant selection system using an antibiotics blasticidin S (BS) and the BS deaminase (BSD) selectable marker by homologous recombination in C. merolae. In addition, this study has succeeded in simultaneously modifying two different chromosomal loci by a single-step cotransformation based on the combination of BSD and CAT selectable markers. A C. merolae strain that expresses mitochondrion-targeted mSCARLET (with the BSD marker) and mVENUS (with the CAT marker) from different chromosomal loci was generated with this procedure. CONCLUSIONS: The newly developed BSD selectable marker enables an additional genetic modification to the already generated C. merolae transformants based on the URA or CAT system. Furthermore, the cotransformation system facilitates multiple genetic modifications. These methods and the simple nature of the C. merolae cellular and genomic architecture will facilitate studies on several phenomena common to photosynthetic eukaryotes.

Photosynthesis of the Cyanidioschyzon merolae cells in blue, red, and white light.

Photosynthesis and respiration rates, pigment contents, CO2 compensation point, and carbonic anhydrase activity in Cyanidioschizon merolae cultivated in blue, red, and white light were measured. At the same light quality as during the growth, the photosynthesis of cells in blue light was significantly lowered, while under red light only slightly decreased as compared with white control. In white light, the quality of light during growth had no effect on the rate of photosynthesis at low O2 and high CO2 concentration, whereas their atmospheric level caused only slight decrease. Blue light reduced markedly photosynthesis rate of cells grown in white and red light, whereas the effect of red light was not so great. Only cells grown in the blue light showed increased respiration rate following the period of both the darkness and illumination. Cells grown in red light had the greatest amount of chlorophyll a, zeaxanthin, and ß-carotene, while those in blue light had more phycocyanin. The dependence on O2 concentration of the CO2 compensation point and the rate of photosynthesis indicate that this alga possessed photorespiration. Differences in the rate of photosynthesis at different light qualities are discussed in relation to the content of pigments and transferred light energy together with the possible influence of related processes. Our data showed that blue and red light regulate photosynthesis in C. merolae for adjusting its metabolism to unfavorable for photosynthesis light conditions.

Macrocyclic polyketides with siderophore mode of action from marine heterotrophic Shewanella algae: Prospective anti-infective leads attenuate drug-resistant pathogens.

AIMS: Biotechnological and chemical characterization of previously undescribed homologous siderophore-type macrocyclic polyketides from heterotrophic Shewanella algae Microbial Type Culture Collection (MTCC) 12715 affiliated with Rhodophycean macroalga Hypnea valentiae of marine origin, with significant anti-infective potential against drug-resistant pathogens. METHODS AND RESULTS: The heterotrophic bacterial strain in symbiotic association with intertidal macroalga H. valentiae was isolated to homogeneity in a culture-dependent method and screened for bioactivities by spot-over-lawn assay. The bacterial organic extract was purified and characterized by extensive chromatographic and spectroscopic methods, respectively, and was assessed for antibacterial activities with disc diffusion and microtube dilution methods. The macrocyclic polyketide compounds exhibited wide-spectrum of anti-infective potential against clinically significant vancomycin-resistant Enterococcus faecalis (VREfs), methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Klebsiella pneumonia with minimum inhibitory concentration of about 1-3 µg ml-1 , insomuch as the antibiotics chloramphenicol and ampicillin were active at ≥6·25 µg ml-1 . The studied compounds unveiled Fe3+ chelating activity, which designated that their prospective anti-infective activities against the pathogens could be due to their siderophore mechanism of action. In support of that, the bacterium exhibited siderophore production on bioassay involving the cast upon culture agar plate, and the presence of siderophore biosynthetic gene (≈1000 bp) (MF 981936) further corroborated the inference. In silico molecular modelling with penicillin-binding protein (PBP2a) coded by mecA genes of MRSA (docking score -11·68 to -12·69 kcal mol-1 ) verified their in vitro antibacterial activities. Putative biosynthetic pathway of macrocyclic polyketides through stepwise decarboxylative condensation initiated by malonate-acyl carrier protein further validated their structural and molecular attributes. CONCLUSIONS: The studied siderophore-type macrocyclic polyketides from S. algae MTCC 12715 with significant anti-infective potential could be considered as promising candidates for pharmaceutical and biotechnological applications, especially against emerging multidrug-resistant pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: This study exhibited the heterotrophic bacteria in association with intertidal macroalga as propitious biological resources to biosynthesize novel antibacterial agents.

Characterization of ACE Inhibitory Peptides Prepared from Pyropia pseudolinearis Protein.

More than 7000 red algae species have been classified. Although most of them are underused, they are a protein-rich marine resource. The hydrolysates of red algal proteins are good candidates for the inhibition of the angiotensin-I-converting enzyme (ACE). The ACE is one of the key factors for cardiovascular disease, and the inhibition of ACE activity is related to the prevention of high blood pressure. To better understand the relationship between the hydrolysates of red algal proteins and the inhibition of ACE activity, we attempted to identify novel ACE inhibitory peptides from Pyropia pseudolinearis. We prepared water soluble proteins (WSP) containing phycoerythrin, phycocyanin, allophycocyanin, and ribulose 1,5-bisphosphate carboxylase/oxygenase. In vitro analysis showed that the thermolysin hydrolysate of the WSP had high ACE inhibitory activity compared to that of WSP. We then identified 42 peptides in the hydrolysate by high-performance liquid chromatography and mass spectrometry. Among 42 peptides, 23 peptides were found in chloroplast proteins. We then synthesized the uncharacterized peptides ARY, YLR, and LRM and measured the ACE inhibitory activity. LRM showed a low IC50 value (0.15 µmol) compared to ARY and YLR (1.3 and 5.8 µmol). In silico analysis revealed that the LRM sequence was conserved in cpcA from Bangiales and Florideophyceae, indicating that the novel ACE inhibitory peptide LRM was highly conserved in red algae.

Induction of Apoptotic Cell Death in Human Leukemia U937 Cells by C18 Hydroxy Unsaturated Fatty Acid Isolated from Red Alga Tricleocarpa jejuensis.

Our previous studies have found that (±)-(E)-12-hydroxyoctadec-10-enoic acid (HOEA) isolated from the red alga Tricleocarpa jejuensis showed cytotoxic effects on various living organisms including harmful microalgae, Gram-positive bacteria, and mammalian tumor cells. Since natural products with apoptosis-inducing ability can be promising anti-cancer agents, in this study, we investigated the cytotoxic mechanism of HOEA on U937 cells focusing on apoptosis induction. HOEA showed much stronger cytotoxic and cytolytic effects on U937 cells than elaidic acid, which has similar structure but no 12-hydroxy group, suggesting that hydroxy group is important for the cytotoxicity of HOEA. HOEA induced apoptotic nuclear morphological changes, DNA fragmentation, and decrease in mitochondrial membrane potential. Furthermore, time-dependent increase in annexin V+/PI+ cell population in HOEA-treated U937 cells was detected. Among the apoptosis-related reagents, caspase-family inhibitor almost completely inhibited HOEA-induced DNA fragmentation. In the analyses using specific caspase-substrates, extremely high cleavage activity toward caspase-3/7/8 substrate was observed in HOEA-treated U937 cells, and weak activities of caspase-1 and -3 were detected. Analyses using specific caspase inhibitors suggested that caspase-3 and caspase-8 might be predominantly responsible for the cleavage activity. Activation of these caspases were also confirmed by western blotting in which significant levels of cleaved forms of caspase 3, caspase 8, and PARP were detected in HOEA-treated U937 cells. Our results suggest that HOEA is capable of inducing apoptosis in U937 cells in which caspase-3 and caspase-8 might play important roles. Since the cytotoxic effect of HOEA is not strictly specific to tumor cells, development of appropriate drug delivery system for selective tumor targeting is necessary for the clinical applications to reduce the possible side effects.

Probing the Anti-Cancer Potency of Sulfated Galactans on Cholangiocarcinoma Cells Using Synchrotron FTIR Microspectroscopy, Molecular Docking, and In Vitro Studies.

Sulfated galactans (SG) isolated from red alga Gracilaria fisheri have been reported to inhibit the growth of cholangiocarcinoma (CCA) cells, which was similar to the epidermal growth factor receptor (EGFR)-targeted drug, cetuximab. Herein, we studied the anti-cancer potency of SG compared to cetuximab. Biological studies demonstrated SG and cetuximab had similar inhibition mechanisms in CCA cells by down-regulating EGFR/ERK pathway, and the combined treatment induced a greater inhibition effect. The molecular docking study revealed that SG binds to the dimerization domain of EGFR, and this was confirmed by dimerization assay, which showed that SG inhibited ligand-induced EGFR dimer formation. Synchrotron FTIR microspectroscopy was employed to examine alterations in cellular macromolecules after drug treatment. The SR-FTIR-MS elicited similar spectral signatures of SG and cetuximab, pointing towards the bands of RNA/DNA, lipids, and amide I vibrations, which were inconsistent with the changes of signaling proteins in CCA cells after drug treatment. Thus, this study demonstrates the underlined anti-cancer mechanism of SG by interfering with EGFR dimerization. In addition, we reveal that FTIR signature spectra offer a useful tool for screening anti-cancer drugs' effect.

Occurrence of Cis-11,12-Methylene-Hexadecanoic Acid in the Red Alga Solieria pacifica (Yamada) Yoshida.

Fatty acids in marine algae have attracted the attention of natural chemists because of their biological activity. The fatty acid compositions of the Solieriaceae families (Rhodophyceae, Gaigartinales) provide interesting information that unusual cyclic fatty acids have been occasionally found. A survey was conducted to profile the characteristic fatty acid composition of the red alga Solieria pacifica (Yamada) Yoshida using gas chromatography-mass spectrometry (GC-MS), infrared spectroscopy (IR), and proton nuclear magnetic resonance spectroscopy (1H-NMR). In S. pacifica, two cyclopentyl fatty acids, 11-cyclopentylundecanoic acid (7.0%), and 13-cyclopentyltridecanoic acid (4.9%), and a cyclopropane fatty acid, cis-11,12-methylene-hexadecanoic acid (7.9%) contributed significantly to the overall fatty acid profile. In particular, this cyclopropane fatty acid has been primarily found in bacteria, rumen microorganisms or foods of animal origin, and has not previously been found in any other algae. In addition, this alga contains a significant amount of the monoenoic acid cis-11-hexadecenoic acid (9.0%). Therefore, cis-11,12-methylene-hexadecanoic acid in S. pacifica was likely produced by methylene addition to cis-11-hexadecenoic acid.