Structural insights into the distinct substrate preferences of two bacterial epoxide hydrolases.

Epoxide hydrolases (EHs), which catalyze the transformation of epoxides to diols, are present in many eukaryotic and prokaryotic organisms. They have recently drawn considerable attention from organic chemists owing to their application in the semisynthesis of enantiospecific diol compounds. Here, we report the crystal structures of BoEH from Bosea sp. PAMC 26642 and CaEH from Caballeronia sordidicola PAMC 26510 at 1.95 and 2.43 Å resolution, respectively. Structural analysis showed that the overall structures of BoEH and CaEH commonly possess typical α/ß hydrolase fold with the same ring-opening residues (Tyr-Tyr) and conserved catalytic triad residues (Asp-Asp-His). However, the two enzymes were found to have significantly different sequence compositions in the cap domain region, which is involved in the formation of the substrate-binding site in both enzymes. Enzyme activity assay results showed that BoEH had the strongest activity toward the linear aliphatic substrates, whereas CaEH had a higher preference for aromatic- and cycloaliphatic substrates. Computational docking simulations and tunnel identification revealed important residues with different substrate-binding preferences. Collectively, structure comparison studies, together with ligand docking simulation results, suggested that the differences in substrate-binding site residues were highly correlated with substrate specificity.

Reversible intracellular acidification and depletion of NTPs provide a potential physiological origin for centuries of dormancy in an Antarctic freshwater copepod.

A great diversity of crustacean zooplankton found in inland and coastal waters produce embryos that settle into bottom sediments to form an egg bank. Embryos from these banks can remain dormant for centuries, creating a reservoir of genetic diversity. A large body of literature describes the ecological and evolutionary importance of zooplankton egg banks. However, literature on the physiological traits behind dormancy in crustacean zooplankton are limited. Most data on the physiology of dormancy comes from research on one species of anostracan, the brine shrimp, Artemia franciscana. Anoxia-induced dormancy in this species is facilitated by a profound and reversible acidification of the intracellular space. This acidification is accompanied by a reversible depletion of adenosine triphosphate (ATP). The present study demonstrates that acidification of the intracellular space also occurs in concert with a depletion of nucleoside triphosphates (NTPs) in the Antarctic copepod, Boeckella poppei. Like A. franciscana, the depletion of NTPs and acidification are rapidly reversed during aerobic recovery in B. poppei. These data provide the first comparative evidence that extreme dormancy under anoxia in crustacean zooplankton is associated with intracellular acidification and an ability to recover from the depletion of ATP.

Complete Genome Sequence of the Antibiotic-Resistant Pseudomonas fluorescens Strain Ant01, from the Rhizosphere of Antarctic Moss.

Pseudomonas fluorescens Ant01 was isolated as an antibiotic-resistant strain from the rhizosphere of a moss from Barton Peninsula, King George Island, Antarctica. The assembled genome size is 6,249,144 bp, with 5,616 protein-coding genes, 69 tRNA genes, and 19 rRNA genes.

Comparative structural insight into the unidirectional catalysis of ornithine carbamoyltransferases from Psychrobacter sp. PAMC 21119.

Ornithine carbamoyltransferases (OTCs) are involved in the arginine deiminase (ADI) pathway and in arginine biosynthesis. Two OTCs in a pair are named catalytic OTC (cOTC) and anabolic OTC (aOTC). The cOTC is responsible for catalyzing the third step of the ADI pathway to catabolize citrulline into carbamoyl phosphate (CP), as well as ornithine, and displays CP cooperativity. In contrast, aOTC catalyzes the biosynthesis of citrulline from CP and ornithine in vivo and is thus involved in arginine biosynthesis. Structural and biochemical analyses were employed to investigate the CP cooperativity and unidirectional function of two sequentially similar OTCs (32.4% identity) named Ps_cOTC and Ps_aOTC from Psychrobacter sp. PAMC 21119. Comparison of the trimeric structure of these two OTCs indicated that the 80s loop of Ps_cOTC has a unique conformation that may influence cooperativity by connecting the CP binding site and the center of the trimer. The corresponding 80s loop region of in Ps_aOTC was neither close to the CP binding site nor connected to the trimer center. In addition, results from the thermal shift assay indicate that each OTC prefers the substrate for the unidirectional process. The active site exhibited a blocked binding site for CP in the Ps_cOTC structure, whereas residues at the active site in Ps_aOTC established a binding site to facilitate CP binding. Our data provide novel insights into the unidirectional catalysis of OTCs and cooperativity, which are distinguishable features of two metabolically specialized proteins.

Importance of rigidity of ice-binding protein (FfIBP) for hyperthermal hysteresis activity and microbial survival.

Ice-binding proteins (IBPs) are well-characterized proteins responsible for the cold-adaptation mechanisms. Despite extensive structural and biological investigation of IBPs and antifreeze proteins, only a few studies have considered the relationship between protein stabilization and thermal hysteresis (TH) activity as well as the implication of hyperactivity. Here, we investigated the important role of the head capping region in stabilization and the hyper-TH activity of FfIBP using molecular dynamics simulation. Data comparison revealed that residues on the ice-binding site of the hyperactive FfIBP are immobilized, which could be correlated with TH activity. Further comparison analysis indicated the disulfide bond in the head region is mainly involved in protein stabilization and is crucial for hyper-TH activity. This finding could also be generalized to known hyperactive IBPs. Furthermore, in mimicking the physiological conditions, bacteria with membrane-anchored FfIBP formed brine pockets in a TH activity-dependent manner. Cells with a higher number of TH-active IBPs showed an increased number of brine pockets, which may be beneficial for short- and long-term survival in cold environments by reducing the salt concentration. The newly identified conditions for hyper-TH activity and their implications on bacterial survival provide insights into novel mechanistic aspects of cold adaptation in polar microorganisms.

Crystal structure of a MarR family protein from the psychrophilic bacterium Paenisporosarcina sp. TG-14 in complex with a lipid-like molecule.

MarR family proteins regulate the transcription of multiple antibiotic-resistance genes and are widely found in bacteria and archaea. Recently, a new MarR family gene was identified by genome analysis of the psychrophilic bacterium Paenisporosarcina sp. TG-14, which was isolated from sediment-laden basal ice in Antarctica. In this study, the crystal structure of the MarR protein from Paenisporosarcina sp. TG-14 (PaMarR) was determined at 1.6 Šresolution. In the crystal structure, a novel lipid-type compound (palmitic acid) was found in a deep cavity, which was assumed to be an effector-binding site. Comparative structural analysis of homologous MarR family proteins from a mesophile and a hyperthermophile showed that the DNA-binding domain of PaMarR exhibited relatively high mobility, with a disordered region between the ß1 and ß2 strands. In addition, structural comparison with other homologous complex structures suggests that this structure constitutes a conformer transformed by palmitic acid. Biochemical analysis also demonstrated that PaMarR binds to cognate DNA, where PaMarR is known to recognize two putative binding sites depending on its molar concentration, indicating that PaMarR binds to its cognate DNA in a stoichiometric manner. The present study provides structural information on the cold-adaptive MarR protein with an aliphatic compound as its putative effector, extending the scope of MarR family protein research.

Whole genome survey and microsatellite motif identification of Artemia franciscana.

Artemia is an industrially important genus used in aquaculture as a nutritious diet for fish and as an aquatic model organism for toxicity tests. However, despite the significance of Artemia, genomic research remains incomplete and knowledge on its genomic characteristics is insufficient. In particular, Artemia franciscana of North America has been widely used in fisheries of other continents, resulting in invasion of native species. Therefore, studies on population genetics and molecular marker development as well as morphological analyses are required to investigate its population structure and to discriminate closely related species. Here, we used the Illumina Hi-Seq platform to estimate the genomic characteristics of A. franciscana through genome survey sequencing (GSS). Further, simple sequence repeat (SSR) loci were identified for microsatellite marker development. The predicted genome size was ∼867 Mb using K-mer (a sequence of k characters in a string) analysis (K = 17), and heterozygosity and duplication rates were 0.655 and 0.809%, respectively. A total of 421467 SSRs were identified from the genome survey assembly, most of which were dinucleotide motifs with a frequency of 77.22%. The present study will be a useful basis in genomic and genetic research for A. franciscana.

The ultrastructure of resurrection: Post-diapause development in an Antarctic freshwater copepod.

The copepod, Boeckella poppei, is broadly distributed in Antarctic and subantarctic maritime lakes threatened by climate change and anthropogenic chemicals. Unfortunately, comparatively little is known about freshwater zooplankton in lakes influenced by the Southern Ocean. In order to predict the impact of climate change and chemicals on freshwater species like B. poppei, it is necessary to understand the nature of their most resilient life stages. Embryos of B. poppei survive up to two centuries in a resilient dormant state, but no published studies evaluate the encapsulating wall that protects theses embryos or their development after dormancy. This study fills that knowledge gap by using microscopy to examine development and the encapsulating wall in B. poppei embryos from Antarctica. The encapsulating wall of B. poppei is comprised of three layers that appear to be conserved among crustacean zooplankton, but emergence and hatching are uniquely delayed until the nauplius is fully formed in this species. Diapause embryos in Antarctic sediments appear to be in a partially syncytial mid-gastrula stage. The number of nuclei quadruples between the end of diapause and hatching. Approximately 75% of yolk platelets are completely consumed during the same time period. However, some yolk platelets are left completely intact at the time of hatching. Preservation of complete yolk platelets suggests an all-or-none biochemical process for activating yolk consumption that is inactivated during dormancy to preserve yolk for post-dormancy development. The implications of these and additional ultrastructural features are discussed in the context of anthropogenic influence and the natural environment.

The protective effect of Leucosporidium-derived ice-binding protein (LeIBP) on bovine oocytes and embryos during vitrification.

Ice-binding proteins (IBPs) facilitate organism survival under extreme conditions by inhibiting thermal hysteresis and ice recrystallization. IBPs have been widely used as cryoprotectants to cryopreserve mammalian gametes and embryos. In the present study, we evaluated the protective effects of an Arctic yeast, Leucosporidium sp. AY30 derived ice-binding protein (LeIBP), on the vitrification of bovine metaphase II (MII) oocytes and embryos. When oocytes and embryos were frozen using the two-step vitrification method, the survival rate was significantly increased in the presence of LeIBP. The LeIBP supplementation decreased the levels of intracellular reactive oxygen species (ROS) and enhanced mitochondrial functions in the vitrified-warmed oocytes. Furthermore, LeIBP improved the developmental potential and suppressed apoptosis of the embryos derived from vitrified-warmed oocytes. Collectively, these data indicate that LeIBP can be used as a promising cryoprotectant to prevent cryoinjury during vitrification in bovine oocytes.

Structural and sequence comparisons of bacterial enoyl-CoA isomerase and enoyl-CoA hydratase.

Crystal structures of enoyl-coenzyme A (CoA) isomerase from Bosea sp. PAMC 26642 (BoECI) and enoyl-CoA hydratase from Hymenobacter sp. PAMC 26628 (HyECH) were determined at 2.35 and 2.70 Å resolution, respectively. BoECI and HyECH are members of the crotonase superfamily and are enzymes known to be involved in fatty acid degradation. Structurally, these enzymes are highly similar except for the orientation of their C-terminal helix domain. Analytical ultracentrifugation was performed to determine the oligomerization states of BoECI and HyECH revealing they exist as trimers in solution. However, their putative ligand-binding sites and active site residue compositions are dissimilar. Comparative sequence and structural analysis revealed that the active site of BoECI had one glutamate residue (Glu135), this site is occupied by an aspartate in some ECIs, and the active sites of HyECH had two highly conserved glutamate residues (Glu118 and Glu138). Moreover, HyECH possesses a salt bridge interaction between Glu98 and Arg152 near the active site. This interaction may allow the catalytic Glu118 residue to have a specific conformation for the ECH enzyme reaction. This salt bridge interaction is highly conserved in known bacterial ECH structures and ECI enzymes do not have this type of interaction. Collectively, our comparative sequential and structural studies have provided useful information to distinguish and classify two similar bacterial crotonase superfamily enzymes.

Crystal structure of a transcription factor, GerE (PaGerE), from spore-forming bacterium Paenisporosarcina sp. TG-14.

In cold and harsh environments such as glaciers and sediments in ice cores, microbes can survive by forming spores. Spores are composed of a thick coat protein, which protects against external factors such as heat-shock, high salinity, and nutrient deficiency. GerE is a key transcription factor involved in spore coat protein expression in the mother cell during sporulation. GerE regulates transcription during the late sporulation stage by directly binding to the promoter of cotB gene. Here, we report the crystal structure of PaGerE at 2.09 Šresolution from Paenisporosarcina sp. TG-14, which was isolated from the Taylor glacier. The PaGerE structure is composed of four α-helices and adopts a helix-turn-helix architecture with 68 amino acid residues. Based on our DNA binding analysis, the PaGerE binds to the promoter region of CotB to affect protein expression. Additionally, our structural comparison studies suggest that DNA binding by PaGerE causes a conformational change in the α4-helix region, which may strongly induce dimerization of PaGerE.

Antarctic blackfin icefish genome reveals adaptations to extreme environments.

Icefishes (suborder Notothenioidei; family Channichthyidae) are the only vertebrates that lack functional haemoglobin genes and red blood cells. Here, we report a high-quality genome assembly and linkage map for the Antarctic blackfin icefish Chaenocephalus aceratus, highlighting evolved genomic features for its unique physiology. Phylogenomic analysis revealed that Antarctic fish of the teleost suborder Notothenioidei, including icefishes, diverged from the stickleback lineage about 77 million years ago and subsequently evolved cold-adapted phenotypes as the Southern Ocean cooled to sub-zero temperatures. Our results show that genes involved in protection from ice damage, including genes encoding antifreeze glycoprotein and zona pellucida proteins, are highly expanded in the icefish genome. Furthermore, genes that encode enzymes that help to control cellular redox state, including members of the sod3 and nqo1 gene families, are expanded, probably as evolutionary adaptations to the relatively high concentration of oxygen dissolved in cold Antarctic waters. In contrast, some crucial regulators of circadian homeostasis (cry and per genes) are absent from the icefish genome, suggesting compromised control of biological rhythms in the polar light environment. The availability of the icefish genome sequence will accelerate our understanding of adaptation to extreme Antarctic environments.

Embryos of an Antarctic zooplankton require anoxia for dormancy, are permeable to lipophilic chemicals, and reside in sediments containing PCBs.

Zooplankton in Antarctic maritime lakes face challenges imposed by anthropogenic chemicals. Studies on temperate species suggest that lipophilic chemicals will accumulate in dormant embryos of Antarctic zooplankton and decrease hatching success, thereby threatening centuries of accumulated genetic diversity that would increase population resilience in the face of climate change. We evaluated the potential for lakes to act as sinks for legacy pollutants in the maritime Antarctic by testing sediments for polychlorinated biphenyls (PCBs) previously identified in soil, flora and fauna of lake catchments. Direct tests of embryo permeability to chemicals are confounded by potential adhesion of chemicals to the embryo surface and limited biomass available. Therefore, in order to assess the potential for lipophilic chemicals to penetrate and passively accumulate in dormant embryos of Antarctic lacustrine zooplankton, we evaluated the effect of anoxia on post-diapause development in the calanoid copepod, Boeckella poppei, and then used chemical anoxia induced by rotenone as a reporter for permeability of these embryos to moderately lipophilic chemicals. The data presented demonstrate that embryos of B. poppei from Antarctic lake sediments will passively accumulate moderately lipophilic chemicals while lying dormant in anoxic sediments. Implications for legacy POPs in sediments of Antarctic maritime lakes are discussed.

Crystal structure of dihydrodipicolinate reductase (PaDHDPR) from Paenisporosarcina sp. TG-14: structural basis for NADPH preference as a cofactor.

Dihydrodipicolinate reductase (DHDPR) is a key enzyme in the diaminopimelate- and lysine-synthesis pathways that reduces DHDP to tetrahydrodipicolinate. Although DHDPR uses both NADPH and NADH as a cofactor, the structural basis for cofactor specificity and preference remains unclear. Here, we report that Paenisporosarcina sp. TG-14 PaDHDPR has a strong preference for NADPH over NADH, as determined by isothermal titration calorimetry and enzymatic activity assays. We determined the crystal structures of PaDHDPR alone, with its competitive inhibitor (dipicolinate), and the ternary complex of the enzyme with dipicolinate and NADPH, with results showing that only the ternary complex had a fully closed conformation and suggesting that binding of both substrate and nucleotide cofactor is required for enzymatic activity. Moreover, NADPH binding induced local conformational changes in the N-terminal long loop (residues 34-59) of PaDHDPR, as the His35 and Lys36 residues in this loop interacted with the 2'-phosphate group of NADPH, possibly accounting for the strong preference of PaDHDPR for NADPH. Mutation of these residues revealed reduced NADPH binding and enzymatic activity, confirming their importance in NADPH binding. These findings provide insight into the mechanism of action and cofactor selectivity of this important bacterial enzyme.

Anticancer activities of ethanol extract from the Antarctic freshwater microalga, Botryidiopsidaceae sp.

BACKGROUND: Cancer is a leading cause of human death around the world and occurs through the highly complex coordination of multiple cellular pathways. Recent studies have revealed that microalgal extracts exhibit considerable pharmaceutical activities, including those against various cancer cells. Thus, microalgae are promising candidates as novel cancer therapeutic drugs. In this study, we evaluated the biological functions of the ethanolic extract of the Antarctic freshwater microalga, Bo tryidiopsidaceae sp., such as its antioxidant, anti-proliferative, apoptotic and anti-invasive properties. METHODS: To estimate antioxidant capacity of ethanol extract of Bo tryidiopsidaceae sp. (ETBO), free radical 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays were used. The anti-proliferative activity of ETBO was assessed in several cancer cell lines (A375, Hs578T and HeLa) and non-tumorigenic keratinocyte cells (HaCaT), using MTT assay. In addition, Annexin V binding was performed to detect ETBO-induced apoptotic cells, and the expression levels of apoptosis-regulating proteins, caspase-3, p53, and Bcl-2, were determined by western blot. Boyden chamber assays were used to determine anti-migratory and anti-invasive properties of ETBO. RESULTS: ETBO exhibited antioxidant activity and concentration-dependent anticancer activities, such as anti-proliferation and pro-apoptotic activities against cancer cells. Furthermore, the expression of the apoptosis-inducing proteins, p53 and caspase-3, significantly increased in response to ETBO, whereas the expression of the anti-apoptotic protein, Bcl-2, decreased. These data imply that ETBO induces apoptosis by caspase activation through the modulation of pro-apoptotic and anti-apoptotic gene, p53 and Bcl-2, respectively. In addition, ETBO significantly inhibited migration and invasion of cervical cancer cells in a concentration-dependent manner. CONCLUSION: In this study, ETBO exhibited considerable anticancer activities, such as inhibition of proliferation, invasion, and migration, as well as induction of apoptosis. These data suggest that ETBO is a promising therapeutic agent in cancer therapy and drug discovery.

Efficacy and Safety of Radiofrequency Ablation for Focal Hepatic Lesions Adjacent to Gallbladder: Reconfiguration of the Ablation Zone through Probe Relocation and Ablation Time Reduction.

PURPOSE: To evaluate the safety and efficacy of radiofrequency (RF) ablation for treatment of focal hepatic lesions adjacent to the gallbladder with electrode relocation and ablation time reduction. MATERIALS AND METHODS: Thirty-nine patients who underwent RF ablation for focal hepatic lesions adjacent to the gallbladder (≤ 10 mm) were evaluated retrospectively from January 2011 to December 2014 (30 men and 9 women; age range, 51-85 y; mean age, 65 y). Of 36 patients with hepatocellular carcinoma, 3 had a second treatment for recurrence (mean tumor size, 15 mm ± 6). Patients were divided into 2 subgroups based on lesion distance from the gallbladder: nonabutting (> 5 mm; n = 19) and abutting (≤ 5 mm; n = 20). Electrodes were inserted parallel to the gallbladder through the center of a tumor in the nonabutting group and through the center of the expected ablation zone between a 5-mm safety zone on the liver side and the gallbladder in the abutting group. Ablation time was decreased in proportion to the transverse diameter of the expected ablation zone. RESULTS: Technical success and technical effectiveness rates were 89.7% and 97.4%, respectively, with no significant differences between groups (P = 1.00). Local tumor progression was observed in 3 patients (1 in the nonabutting group and 2 in the abutting group; P = 1.00). There were no major complications. The gallbladder was thickened in 10 patients, with no significant difference between groups (P = .72). Biloma occurred in 1 patient in the nonabutting group. CONCLUSIONS: RF ablation with electrode relocation and reduction of ablation time can be a safe and effective treatment for focal hepatic lesions adjacent to the gallbladder.

Crystal structure and functional characterization of an isoaspartyl dipeptidase (CpsIadA) from Colwellia psychrerythraea strain 34H.

Isoaspartyl dipeptidase (IadA) is an enzyme that catalyzes the hydrolysis of an isoaspartyl dipeptide-like moiety, which can be inappropriately formed in proteins, between the ß-carboxyl group side chain of Asp and the amino group of the following amino acid. Here, we have determined the structures of an isoaspartyl dipeptidase (CpsIadA) from Colwellia psychrerythraea, both ligand-free and that complexed with ß-isoaspartyl lysine, at 1.85-Å and 2.33-Å resolution, respectively. In both structures, CpsIadA formed an octamer with two Zn ions in the active site. A structural comparison with Escherichia coli isoaspartyl dipeptidase (EcoIadA) revealed a major difference in the structure of the active site. For metal ion coordination, CpsIadA has a Glu166 residue in the active site, whereas EcoIadA has a post-translationally carbamylated-lysine 162 residue. Site-directed mutagenesis studies confirmed that the Glu166 residue is critical for CpsIadA enzymatic activity. This residue substitution from lysine to glutamate induces the protrusion of the ß12-α8 loop into the active site to compensate for the loss of length of the side chain. In addition, the α3-ß9 loop of CpsIadA adopts a different conformation compared to EcoIadA, which induces a change in the structure of the substrate-binding pocket. Despite CpsIadA having a different active-site residue composition and substrate-binding pocket, there is only a slight difference in CpsIadA substrate specificity compared with EcoIadA. Comparative sequence analysis classified IadA-containing bacteria and archaea into two groups based on the active-site residue composition, with Type I IadAs having a glutamate residue and Type II IadAs having a carbamylated-lysine residue. CpsIadA has maximal activity at pH 8-8.5 and 45°C, and was completely inactivated at 60°C. Despite being isolated from a psychrophilic bacteria, CpsIadA is thermostable probably owing to its octameric structure. This is the first conclusive description of the structure and properties of a Type I IadA.

Comprehensive Expression Profiling and Functional Network Analysis of Porphyra-334, One Mycosporine-Like Amino Acid (MAA), in Human Keratinocyte Exposed with UV-radiation.

Mycosporine-like amino acids (MAAs) have been highlighted as pharmacologically active secondary compounds to protect cells from harmful UV-radiation by absorbing its energy. Previous studies have mostly focused on characterizing their physiological properties such as antioxidant activity and osmotic regulation. However, molecular mechanisms underlying their UV-protective capability have not yet been revealed. In the present study, we investigated the expression profiling of porphyra-334-modulated genes or microRNA (miRNAs) in response to UV-exposure and their functional networks, using cDNA and miRNAs microarray. Based on our data, we showed that porphyra-334-regulated genes play essential roles in UV-affected biological processes such as Wnt (Wingless/integrase-1) and Notch pathways which exhibit antagonistic relationship in various biological processes; the UV-repressed genes were in the Wnt signaling pathway, while the activated genes were in the Notch signaling. In addition, porphyra-334-regulated miRNAs can target many genes related with UV-mediated biological processes such as apoptosis, cell proliferation and translational elongation. Notably, we observed that functional roles of the target genes for up-regulated miRNAs are inversely correlated with those for down-regulated miRNAs; the former genes promote apoptosis and translational elongation, whereas the latter function as inhibitors in these processes. Taken together, these data suggest that porphyra-334 protects cells from harmful UV radiation through the comprehensive modulation of expression patterns of genes involved in UV-mediated biological processes, and that provide a new insight to understand its functional molecular networks.

Porphyra-334, a mycosporine-like amino acid, attenuates UV-induced apoptosis in HaCaT cells.

The main aim of the current research was to study the effect of porphyra-334, one of mycosporine-like amino acids (MAAs), well known as UV-absorbing compounds, on UVinduced apoptosis in human immortalized keratinocyte (HaCaT) cells. Due to their UV-screening capacity and ability to prevent UV-induced DNA damage, MAAs have recently attracted considerable attention in both industry and research in pharmacology. Herein, human HaCaT cells were used to determine the biological activities of porphyra- 334 by various in vitro assays, including proliferation, apoptosis and Western blot assays. The proliferation rate of UV-irradiated HaCaT cells was significantly decreased compared to the control group. Pretreatment with porphyra- 334 markedly attenuated the inhibitory effect of UV and induced a dramatic decrease in the apoptotic rate. Expression of active caspase-3 protein was increased in response to UV irradiation, while caspase-3 levels were similar between cells treated with porphyra-334 and the non-irradiated control group. Taken together, our data suggest that porphyra-334 inhibits UV-induced apoptosis in HaCaT cells through attenuation of the caspase pathway.