Potential roles of hydroxybenzoate paralytic shellfish toxins in modulating toxin biokinetics in scallops.

Paralytic shellfish toxins (PSTs) produced by some marine dinoflagellates can cause severe human intoxication via vectors like bivalves. Toxic dinoflagellate Gymnodinium catenatum produce a novel group of hydroxybenzoate PSTs named GC toxins, but their biokinetics in bivalves haven't been well examined. In this experiment, we analyzed PSTs in bay scallops Argopecten irradians exposed to G. catenatum (strain MEL11) to determine their accumulation, elimination, anatomical distribution, and biotransformation. To our surprise, up to 30% of the PSTs were accumulated in the adductor muscle of scallops at the end of the experiment, and the toxicity of adductor muscle exceeded the regulatory limit of 800 µg STXeq/kg in only 6 days. High concentration of toxins in the adductor muscle are likely linked to the rapid transfer of GC toxins from viscera to other tissues. Moreover, most GC toxins in scallops were found rapidly transformed to decarbamoyl toxins through enzyme-mediated hydrolysis, which was further supported by the in vitro incubation experiments. Our study demonstrates that GC toxins actively participate in toxin distribution and transformation in scallops, which may increase the risks of food poisoning associated with the consumption of scallop adductor muscle. ENVIRONMENTAL IMPLICATION: The negative impacts of harmful algal blooms (HABs) have become a global environmental concern under the joint effects of cultural eutrophication and climate change. Our study, targeted on the biokinetics of paralytic shellfish toxins in scallops exposed to Gymnodinium catenatum producing unique GC toxins, aims to elucidate potential risks of seafood poisoning associated with GC toxins. The findings of this study will help us to understand the roles of GC toxins in seafood poisoning, and to develop effective management strategies against toxic algal blooms and phycotoxins.

Marked shifts of harmful algal blooms in the Bohai Sea linked with combined impacts of environmental changes.

The Bohai Sea, a semi-enclosed inland sea in China and an important mariculture region, has experienced extensive harmful algal blooms (HABs) and their negative impacts for several decades. To investigate the changes of HABs and their potential drivers over time and space, a dataset of 230 HAB events (1952-2017), along with corresponding environmental data (1990-2017) was compiled. The frequency of HAB events in the Bohai Sea has increased over time but plateaued in the last decade, and our analysis showed that history of HABs in the Bohai Sea could be categorized into three periods based on their frequency, scale, and HAB-forming species. The seasonal window of HAB events has started earlier and lasted longer, and the main hotspot has moved from Bohai Bay to coastal waters of Qinhuangdao over time. There were marked shifts in the representative HAB-forming microalgae, from dinoflagellates in the first period (before 2000) to haptophytes in the second period (2000-2009), and pelagophytes in the third period (2009 onwards). These community changes are accompanied by a trend toward diversification of HAB-forming microalgae, decrease in cell-size, and increase in negative impacts. Statistical analyses indicate that long-term changes in HABs in the Bohai Sea are linked with the combined effects of climate change, eutrophication and mariculture development. The results of the present study require to refine future monitoring programs, develop adaptive management strategies and predictive models for HABs in the Bohai Sea.

An investigation on bloom dynamics of Alexandrium catenella and A. pacificum and toxin accumulation in shellfish along the coast of Qinhuangdao, China.

The dinoflagellate genus Alexandrium comprises most of the toxic bloom-forming species producing paralytic shellfish toxins (PSTs) in the sea. Recently, repeated paralytic shellfish poisoning episodes have been recorded in Qinhuangdao located at the west coast of the Bohai Sea. To elucidate the relationship between toxic Alexandrium blooms and the poisoning episodes, a year-round investigation was carried out in this region from July 2020 to July 2021. Two qPCR assays were used to detect A. catenella and A. pacificum, and LC-MS/MS was applied to analyze PSTs in phytoplankton and shellfish samples. The blooms of A. catenella and A. pacificum were found in April and July, respectively, and PST content in three bivalves exhibited notable increase following the bloom of A. catenella. The results revealed bloom dynamics of the two toxic Alexandrium species in the Bohai Sea for the first time, and further confirmed A. catenella as the causative agent of poisoning episodes.

Toxin production of dinoflagellate Gymnodinium catenatum isolated from the East China Sea.

Dinoflagellate Gymnodinium catenatum is an important producer of paralytic shellfish toxins (PSTs), including a novel group of hydroxybenzoate derivatives named GC toxins. In the East China Sea, G. catenatum has been considered as the causative agent for several paralytic shellfish poisoning (PSP) episodes, yet the knowledge on their toxin production was still quite limited. In this study, toxins produced by a strain of G. catenatum (MEL11) isolated from the East China Sea were determined, using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Changes of toxin profile in the stain MEL11 in response to nutrient and temperature variations were also examined. A total of 11 PST components dominated by hydroxybenzoate analogs and N-sulfocarbamoyl toxins were detected, which was different from other G. catenatum strains previously established in the East China Sea in the presence of GC5 and the lack of dcGTX2&3. Cellular toxin composition and content of the strain had no apparent change within a range of temperature from 20°C to 26°C. In contrast, nutrient limitation and nitrogen source had notable impacts on toxin production. The molar percentage of GC toxins decreased remarkably at the stationary growth phase under nutrient-deprived conditions of both nitrogen (N) and phosphorus (P). The replacement of nitrate with ammonium as the source of N significantly promoted PST production by G. catenatum. The study revealed the potential diversity of toxin profiles of G. catenatum in the East China Sea, and highlighted the effects of nutrients on production of GC toxins by G. catenatum.

Green Tides in the Yellow Sea Promoted the Proliferation of Pelagophyte Aureococcus anophagefferens.

Harmful algal blooms formed by fast-growing, ephemeral macroalgae have expanded worldwide, yet there is limited knowledge of their potential ecological consequences. Here, we select intense green tides formed by Ulva prolifera in the Yellow Sea, China, to examine the ecological consequences of these blooms. Using 28-isofucosterol in the surface sediment as a biomarker of green algae, we identified the settlement region of massive floating green algae in the area southeast of the Shandong Peninsula in the southern Yellow Sea. The responses of the phytoplankton assemblage from the deep chlorophyll-a maximum layer were then resolved using high-throughput sequencing. We found striking changes in the phytoplankton community in the settlement region after an intensive green tide in 2016, characterized by a remarkable increase in the abundance of the pelagophyte Aureococcus anophagefferens, the causative species of ecosystem disruptive brown tides. Our study strongly suggests that the occurrence of massive macroalgal blooms may promote blooms of specific groups of microalgae through alteration of the marine environment.

Pigment Characterization of the Giant-Colony-Forming Haptophyte Phaeocystis globosa in the Beibu Gulf Reveals Blooms of Different Origins.

The giant-colony-forming haptophyte Phaeocystis globosa has caused several large-scale blooms in the Beibu Gulf since 2011, but the distribution and dynamics of the blooms remained largely unknown. In this study, colonies of P. globosa, as well as membrane-concentrated phytoplankton samples, were collected during eight cruises in the Beibu Gulf from September 2016 to August 2017. Pigments were analyzed by high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD). The pigment 19'-hexanoyloxyfucoxanthin (hex-fuco), generally considered a diagnostic pigment for Phaeocystis, was not detected in P. globosa colonies in the Beibu Gulf, whereas 19'-butanoyloxyfucoxanthin (but-fuco) was found in all colony samples. Moreover, but-fuco in membrane-concentrated phytoplankton samples exhibited a similar distribution pattern to that of P. globosa colonies, suggesting that but-fuco provided the diagnostic pigment for bloom-forming P. globosa in the Beibu Gulf. Based on the distribution of but-fuco in different water masses in the region prior to the formation of intensive blooms, it is suggested that P. globosa blooms in the Beibu Gulf could originate from two different sources. IMPORTANCE Phaeocystis globosa has formed intensive blooms in the South China Sea and even around the world, causing huge social economic losses and environmental damage. However, little is known about the formation mechanism and dynamics of P. globosa blooms. 19'-Hexanoyloxyfucoxanthin (hex-fuco) is often used as the pigment proxy to estimate Phaeocystis biomass, while this is challenged by the giant-colony-forming P. globosa in the Beibu Gulf, which contains only 19'-butanoyloxyfucoxanthin (but-fuco) but not hex-fuco. Using but-fuco as a diagnostic pigment, we traced two different origins of P. globosa blooms in the Beibu Gulf. This study clarifies the development process of P. globosa blooms in the Beibu Gulf, which provides a basis for the early monitoring and prevention of the blooms.

The dinoflagellate Alexandrium catenella producing only carbamate toxins may account for the seafood poisonings in Qinhuangdao, China.

An outbreak of paralytic shellfish poisoning, recorded in April 2016 in Qinhuangdao China, was suspected to be caused by a toxic species in genus Alexandrium. Shortly after the poisoning outbreak, shellfish and net-concentrated phytoplankton samples were collected from the Bohai Sea, and analysed using high performance liquid chromatography coupled with fluorescence detection. Paralytic shellfish toxins (PSTs) were detected in both phytoplankton and shellfish samples, with similar toxin profiles dominated by carbamate toxins. High throughput sequencing data for phytoplankton samples collected previously in the coastal waters of Qinhuangdao were then analysed, and 8 operational taxonomic units (OTUs) were assigned to Alexandrium affine, A. andersonii/A. ostenfeldii, A. catenella, A. fraterculus, A. hiranoi/A. pseudogonyaulax, A. margalefii, A. pacificum and A. pohangense, among which A. catenella, A. pacificum and A. ostenfeldii could be potential producers of PSTs. During a cruise in 2019, three isolates of Alexandrium were established by cyst germination, and identified as A. catenella based on the sequences of the 28S ribosomal RNA gene (28S rDNA) D1-D2 region. Interestingly, all the three strains had the same toxin profile consisting of gonyautoxins 1, 3, 4 (GTX1, 3, 4) and neosaxitoxin (NEO). The toxin profile is similar to those of phytoplankton samples collected previously in the coastal waters of Qinhuangdao, but remarkably different from the general toxin profile of A. catenella dominated by N-sulfocarbamoyl toxins C1-2 in the Bohai Sea and the Yellow Sea. The results suggest that A. catenella is most likely to be the causative species of the poisoning outbreak in Qinhuangdao. As far as we know, this is the first report of A. catenella in the Bohai Sea producing PSTs dominated by high potent gonyautoxins GTX1-4. Occurrence of the highly toxic A. catenella will increase the risk of paralytic shellfish poisoning, which necessitates in-depth mechanism studies and increasing monitoring efforts.

Increasing dominance of dinoflagellate red tides in the coastal waters of Yellow Sea, China.

The Yellow Sea (YS) has been subjected to harmful algal blooms (HABs) for several decades. In this study, we compiled and analyzed a dataset of 165 red tides from 1972 to 2017 and a dataset of green tides from 2008 to 2017 in the YS. The most notable feature of red tides in the YS is the increasing dominance of dinoflagellate red tides in terms of frequency, scale, seasonal distribution, spatial coverage, and red tide causative species. The increasing dominance of dinoflagellate red tides is closely related to eutrophication and the development of the mariculture industry in the YS. However, the dinoflagellate red tides in the northern Yellow Sea (NYS) and the southern Yellow Sea (SYS) have different features. The apparent changes in red tides in the SYS in terms of frequency and seasonal patterns might have been caused by recurrent large-scale green tides in the last decade.

Biokinetics and biotransformation of paralytic shellfish toxins in different tissues of Yesso scallops, Patinopecten yessoensis.

Paralytic shellfish toxins (PSTs) are a group of natural toxic substances often found in marine bivalves. Accumulation, anatomical distribution, biotransformation and depuration of PSTs in different tissues of bivalves, however, are still not very well understood. In this study, we investigated biokinetics and biotransformation of PSTs in six different tissues, namely gill, mantle, gonad, adductor muscle, kidney, and digestive gland, in Yesso scallops Patinopecten yessoensis exposed to a toxic strain of dinoflagellate Alexandrium pacificum. High daily accumulation rate (DAR) was recorded at the beginning stage of the experiment. Most of the PSTs in toxic algae ingested by scallops were retained and the toxicity level of PSTs in scallops exceeded the regulatory limit within 5 days. At the late stage of the experiment, however, DAR decreased obviously due to the removal of PSTs. Fitting results of the biokinetics model indicated that the amount of PSTs transferred from digestive gland to mantle, adductor muscle, gonad, kidney, and gill in a decreasing order, and adductor muscle, kidney, and gonad had higher removal rate than gill and mantle. Toxin profile in digestive gland was dominated by N-sulfocarbamoyl toxins 1/2 (C1/2), closely resembled that of the toxic algae. In contrast, toxin components in kidney were dominated by high-potency neosaxitoxin (NEO) and saxitoxin (STX), suggesting that the kidney be a major organ for transformation of PSTs.

Distribution of Alexandrium pacificum cysts in the area adjacent to the Changjiang River estuary, China.

The coastal waters adjacent to the Changjiang River estuary (CRE) are characterized by nutrient pollution and recurrent harmful algal blooms. In this study, resting cysts of Alexandrium pacificum Litaker and A. catenella (Whedon & Kof.) Balech, two major species within the A. tamarense species complex in Chinese coastal waters, were studied using sediment samples collected from the area adjacent to the CRE in May 2014 and December 2015. Cysts were detected with two real-time quantitative PCR assays, as well as the primuline-staining method. Only cysts of A. pacificum were found in the study area, which mainly distributed in the mud depositional zone near the CRE. A low-abundance region of the cysts present in spring is in accordance with the intrusive pathway of the Nearshore Kuroshio Branch Current (NKBC), suggesting that A. pacificum blooms could be regulated by seasonal intrusion of NKBC.

Resting cysts of Alexandrium catenella and A. pacificum (Dinophyceae) in the Bohai and Yellow Seas, China: Abundance, distribution and implications for toxic algal blooms.

The Alexandrium tamarense species complex consists of 5 closely related species that are important bloom-forming dinoflagellates with a complex life cycle. The formation of resting cyst is a key strategy to resist harsh environmental conditions. In this study, the resting cysts of two major bloom-forming species of the A. tamarense species complex in China, A. catenella (Whedon & Kof.) Balech (previously A. fundyense, or A. tamarense species complex Group I) and A. pacificum Litaker (A. tamarense species complex Group IV), were studied in surface sediment collected from the Bohai Sea (BS) and Yellow Sea (YS) during two cruises conducted in 2012 and 2015. Cyst abundance of the A. tamarense species complex was first quantified by the primuline-staining method, and cysts of the two species were subsequently determined using two real-time quantitative PCR (qPCR) assays. Results showed that resting cysts of the A. tamarense species complex were more abundant in the YS than the BS (mean of 480 and 33 cysts g dry weight, DW-1 of sediment, respectively). Cysts were mainly found in the central portion of the northern YS, the area SE (southeast) of the Shandong peninsula, and the area near the Subei Shoal in the southern YS, where surface sediment had a high percentage of clay and silt (particle size < 63 µm) content. The maximum cyst abundance recorded was 3090 cysts g DW-1 of sediment in 2012 and 3448 cysts g DW-1 in 2015, respectively. Cysts were mainly composed of A. catenella in the YS and the BS, while those of A. pacificum were only detected occasionally at some sampling sites in the YS. Highly abundant resting cysts in surface sediment of the YS may serve as "seed banks" for recurrent toxic blooms of A. catenella and the associated shellfish contamination by paralytic shellfish toxins in the YS.

Spatiotemporal variation of paralytic shellfish toxins in the sea area adjacent to the Changjiang River estuary.

The Changjiang (Yangtze River) River estuary (CRE) and its adjacent coastal waters is a notable region for nutrient pollution, which results in severe problems of coastal eutrophication and harmful algal blooms (HABs). The occurrence of HABs, particularly those of dinoflagellate Alexandrium spp. capable of producing paralytic shellfish toxins (PSTs), has an increasing risk of contaminating seafood and poisoning human-beings. The investigation of PSTs, however, is often hampered by the relatively low abundance of Alexandrium spp. present in seawater. In this study, a monitoring strategy of PSTs using net-concentrated phytoplankton from a large volume of seawater was employed to examine spatiotemporal variations of PSTs in the CRE and its adjacent waters every month from February to September in 2015. Toxins in concentrated phytoplankton samples were analyzed using high-performance liquid chromatography coupled with a fluorescence detector (HPLC-FLD). The results showed that PSTs could be detected in phytoplankton samples during the sampling stage in the CRE and its adjacent waters. Toxin content increased gradually from February to May, reached the peak in June, and then decreased rapidly from July to September. The maximum value of PST content was 215 nmol m-3 in June. Low-potency toxins N-sulfocarbamoyl toxins 1/2 (C1/2) were the most dominant components of PST in phytoplankton samples from February to June in 2015, while high-potency gonyautoxin 4 (GTX4) became the dominant component from July to September. Toxins were mainly detected from three regions, the sea area north to the CRE, the sea area east to the CRE, and sea area near Zhoushan Island south to the CRE. Based on the results of this study, it can be inferred that the three regions around the CRE in May and June is of high risk for PST contamination and seafood poisoning.

Resolving phytoplankton taxa based on high-throughput sequencing during brown tides in the Bohai Sea, China.

Large-scale blooms formed by pico-sized phytoplankton, which strongly inhibited feeding activity and growth of cultured scallops, have been recorded along the coast of Qinhuangdao in the Bohai Sea since 2009. Based on pigment profiles and clone library analysis of phytoplankton samples during the blooms, the major bloom-forming species was primarily identified as pelagophyte Aureococcus anophagefferens Hargraves et Sieburth, the causative species of intensive brown tides in the United States and South Africa. Due to the indistinct morphological features of the bloom-forming microalgae and limited knowledge on the composition of phytoplankton communities, there were still disputes concerning the causative species of the blooms. In this study, the method of high-throughput sequencing targeted 18S rDNA V4 region was used to study the composition of pico- and nano-sized phytoplankton communities in 2013 and 2014. A total of 18 groups of eukaryotic microalgae at the class level and more than 2000 operational taxonomic units (OTUs) were identified in phytoplankton samples collected from the brown-tide zone in the Qinhuangdao coastal waters. For both years, A. anophagefferens was the most dominant species during the bloom period and its maximum relative abundance exceeded 60 percent. Along with other evidence, the results further confirm that A. anophagefferens is the major causative species of the pico-sized phytoplankton blooms in the Bohai Sea. The outbreak of brown tide exhibited a strong inter-annual variation between 2013 and 2014, and an increasing dominance of dinoflagellates could be observed in the Qinhuangdao coastal waters.

Contamination status of lipophilic marine toxins in shellfish samples from the Bohai Sea, China.

Lipophilic marine toxins in shellfish pose significant threats to the health of seafood consumers. To assess the contamination status of shellfish by lipophilic marine toxins in the Bohai Sea, nine species of shellfish periodically collected from five representative aquaculture zones throughout a year were analyzed with a method of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Lipophilic marine toxins, including okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), yessotoxin (YTX), homo-yessotoxin (homo-YTX), azaspiracids (AZA2 and AZA3), gymnodimine (GYM), and 13-desmethyl spirolide C (13-DesMe-C), were detected in more than 95 percent of the shellfish samples. Toxins PTX2, YTX, 13-DesMe-C and GYM were predominant components detected in shellfish samples. Scallops, clams and mussels accumulated much higher level of lipophilic marine toxins compared to oysters. Toxin content in shellfish samples collected from different sampling locations showed site-specific seasonal variation patterns. High level of toxins was found during the stages from December to February and June to July in Hangu, while from March to April and August to September in Laishan. Some toxic algae, including Dinophysis acuminata, D. fortii, Prorocentrum lima, Gonyaulax spinifera and Lingulodinium polyedrum, were identified as potential origins of lipophilic marine toxins in the Bohai Sea. The results will offer a sound basis for monitoring marine toxins and protecting the health of seafood consumers.

Cadmium: Toxic effects on placental and embryonic development.

Cadmium is a non-essential trace metal that has strong teratogenic and mutagenic effects in living organisms. The content is more highly enriched in women than in men and can enter the embryo through the placenta and destroy the placenta's morphological structure, resulting in fetal growth restriction. In this report, we review published data linking pregnancy exposure to cadmium to placenta and fetal growth and development toxicity and summarize the related mechanisms. An understanding of how cadmium exposure contributes to placental and fetal development is necessary for the development of prevention and control strategies for fetal development defects caused by cadmium exposure during pregnancy.

Genetic evidence in tracking the origin of Ulva prolifera blooms in the Yellow Sea, China.

Recurrent green tides have been recorded in the Yellow Sea for 11 consecutive years. The origin of floating green algae in the Yellow Sea, however, remains a subject of debate. Previous studies suggest that the major bloom-forming green alga Ulva prolifera represent a unique ecotype different from other attached populations of U. prolifera in China. In this study, 97 green algal samples collected during the 2012 green-tide event and from other locations along the coastline of China were analyzed. Based on the sequences of nuclear ribosomal RNA gene (rDNA) internal transcribed spacer (ITS) region and 5S rDNA spacer region, the green alga U. prolifera in the samples were identified. The intraspecific genetic diversity within U. prolifera was then examined using sequences of 5S rDNA spacer and a marker of sequence characterized amplified region (SCAR) highly specific for bloom-forming U. prolifera in the Yellow Sea. The screening results for SCAR marker demonstrated that U. prolifera attached to aquaculture rafts in Subei Shoal belong to the same ecotype of the bloom-forming U. prolifera in the Yellow Sea. These findings offer genetic evidence that aquaculture rafts in Subei Shoal are a major source of floating green algae in the Yellow Sea.

Neuroprotective effects of epoxyeicosatrienoic acids.

Eicosatrienoic acids (EETs) are a class of intermediates produced during arachidonic acid metabolism mediated by cytochrome P450 epoxygenases that exert multiple physiological effects on the nervous system. EETs promote three metabolic processes, including esterification, hydrolysis and degradation or extension. EETs are hydrolyzed by soluble epoxide hydrolase (sEH) to form corresponding diols, thereby reducing their biological activity. Strategies regulating sEH expression or activity affect EET hydrolysis and alter relative cell concentrations, thus influencing EET function. This article summarizes the metabolic pathway of eicosatrienoic acid in organisms and highlights its neuroprotective effects on the central nervous system, which include regulating neuronal excitability, increasing cerebral blood flow, inhibiting neuronal apoptosis, reducing neuroinflammation, mitigating brain injury and promoting recovery of neurological function in subjects with nervous system diseases.

Analysis of sterols in selected bloom-forming algae in China.

Sterols, a group of stable lipid compounds, are often used as biomarkers in marine biogeochemical studies to indicate sources of organic matter. In this study, sterols in 13 species of major bloom-forming algae in China, which belong to Dinophyceae, Bacillariophyceae, Ulvophyceae, and Pelagophyceae, were analyzed with gas chromatography-mass spectrometry (GC-MS) to test their feasibility in representing different types of harmful algal blooms (HABs). It was found that (24Z)-stigmasta-5,24-dien-3ß-ol (28-isofucosterol) was a major sterol component in green-tide forming macroalga Ulva prolifera. In bloom-forming dinoflagellates Alexandrium spp., Prorocentrum micans and Scrippsiella trochoidea, (22E)-4α,23-dimethyl-5α-ergost-22-en-3ß-ol (dinosterol) was detected in addition to cholest-5-en-3ß-ol (cholesterol), (22E)-ergosta-5,22-dien-3ß-ol, (22E)-stigmasta-5,22-dien-3ß-ol and other minor sterol components. In brown-tide forming pelagophyte Aureococcus anophagefferens, (24E)-24-propylcholesta-5,24-dien-3ß-ol ((24E)-24-propylidenecholesterol) and (24Z)-24-propylcholesta-5,24-dien-3ß-ol ((24Z)-24-propylidenecholesterol) were detected together with cholesterol, (22E)-stigmasta-5,22-dien-3ß-ol, stigmast-5-en-3ß-ol and campest-5-en-3ß-ol. Among the selected bloom-forming diatoms, Chaetoceros sp. and Pseudo-nitzschia spp. only produced cholesterol, while Cylindrotheca closterium produced solely (22E)-ergosta-5,22-dien-3ß-ol. Sterol content in four bloom-forming algal species correlates well with their biomass or abundance. It's proposed that 28-isofucosterol could serve as a promising biomarker for green algae in green-tide studies. Dinosterol and (24Z)-24-propylidenecholesterol can be used as potential biomarkers to represent bloom-forming dinoflagellates and pelagophytes, while (22E)-ergosta-5,22-dien-3ß-ol is not a good indicator for diatoms.

14,15-EET Suppresses Neuronal Apoptosis in Ischemia-Reperfusion Through the Mitochondrial Pathway.

Neuronal apoptosis mediated by the mitochondrial apoptosis pathway is an important pathological process in cerebral ischemia-reperfusion injury. 14,15-EET, an intermediate metabolite of arachidonic acid, can promote cell survival during ischemia/reperfusion. However, whether the mitochondrial apoptotic pathway is involved this survival mechanism is not fully understood. In this study, we observed that infarct size in ischemia-reperfusion injury was reduced in sEH gene knockout mice. In addition, Caspase 3 activation, cytochrome C release and AIF nuclear translocation were also inhibited. In this study, 14,15-EET pretreatment reduced neuronal apoptosis in the oxygen-glucose deprivation and re-oxygenation group in vitro. The mitochondrial apoptosis pathway was also inhibited, as evidenced by AIF translocation from the mitochondria to nucleus and the reduction in the expressions of cleaved-caspase 3 and cytochrome C in the cytoplasm. 14,15-EET could reduce neuronal apoptosis through upregulation of the ratio of Bcl-2 (anti-apoptotic protein) to Bax (apoptosis protein) and inhibition of Bax aggregation onto mitochondria. PI3K/AKT pathway is also probably involved in the reduction of neuronal apoptosis by EET. Our study suggests that 14,15-EET could suppress neuronal apoptosis and reduce infarct volume through the mitochondrial apoptotic pathway. Furthermore, the PI3K/AKT pathway also appears to be involved in the neuroprotection against ischemia-reperfusion by 14,15-EET.

Prokaryotic expression and polyclonal antibody preparation of a novel Rab-like protein mRabL5.

Rab GTPases, which belong to the Ras superfamily, represent a group of small molecular weight GTP binding proteins that are involved in various steps along the exocytic and endocytic pathways. We first identified mRabL5 (GenBank Accession No. NP_080349), a novel Mus musculus Rab-like protein, present as a Golgi-associated protein. Here we presented the results of the cloning, prokaryotic expression, purification, and polyclonal antibody production of the novel Rab-like protein. In order to obtain a specific antibody against mRabL5, we prepared two GST fusion proteins, full-length mRabL5 GST fusion protein and mRabL5 C terminus GST fusion protein, to immunize rabbits. Western blot analysis showed that both antibodies prepared against full length of mRabL5 and its C terminus, respectively, can recognize mRabL5 protein. Immunofluorescence of mRabL5 in NIH3T3 cells using the two antibodies showed its perinuclear clustering distribution pattern. The polyclonal antibodies preparation against mRabL5 provided a good tool for us to study the functional involvement of mRabL5.