Purification and identification of a clotting protein from the hemolymph of Chinese shrimp (Fenneropenaeus chinensis)

The clotting protein (CP) plays important and diverse roles in crustaceans, such as coagulation and lipid transportation. A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis (named as Fc-CP) with Q sepharose HP anion-exchange chromatography and phenyl sepharose HP hydrophobic interaction chromatography. Fc-CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca²⁺, suggesting that the clotting reaction is catalyzed by a Ca²⁺-dependent transglutaminase in shrimp hemocytes. The molecular mass of Fc-CP was 380 kDa under non-reducing conditions and 190 kDa under reducing conditions as was determined with SDS-PAGE. CP exists as disulfide-linked homodimers and oligomers. The N-terminal amino acid sequence of Fc-CP was identical to that of shrimps including Penaeus monodon, Farfantepenaeus paulensis and Litopenaeus vannamei; and similar to that of other decapods. The purified Fc-CP was digested with trypsin and verified on an ABI 4700 matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. Our results will aid to better understanding the coagulation mechanism of shrimp hemolymph.     

Purification and characterization of 2-haloacid dehalogenase from marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis

2-haloacid dehalogenases constitute a group of dehalogenases which are capable of dehalogenating the halogenated organic compounds. So far, the 2-haloacid dehalogenases have been found in many bacteria, but not in Paracoccus genus. In the present study, one enzyme 2-haloacid dehalogenase(designated as Deh99), induced by DL-2-chloropropionate(DL-2-CPA), was purified from the marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis. The enzyme of Deh99 was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography(Q-Sepharose HP), and Superdex 200 gel filtration chromatography. The molecular weight of Deh99 was estimated to be 25.0 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and 50.0 kDa natively by gel filtration chromatography. The enzyme of Deh99 stereospecifically dehalogenated L-2-CPA to produce D-lactate, with an apparent Michaelis-Menten constant(Km) value of 0.21 mmol L-1 for L-2-CPA. The optimal pH and temperature for Deh99 activity were 10.0 and 40℃, respectively. The enzyme of Deh99 acted on short-carbon-chain 2-haloacids, with the highest activity towards monochloroacetate. The activity of Deh99 was slightly affected by DTT and EDTA, but strongly inhibited by Cu2+ and Zn2+. The enzyme of Deh99 shows unique substrate specificity and inhibitor sensitivities compared to previously characterized 2-haloacid dehalogenases and is the reported one about purified 2-haloacid dehalogenase isolated from the bacteria of Paracoccus genus.     

Isolation and characterization of a marine bacterium producing protease from Chukchi Sea, Arctic

A Gram negative bacterium Ar/W/b/75°25＇N/1 producing extracellular alkaline protease was isolated from surface water of latitude 75°25＇N, and longitude 162°25＇W in Chukchi sea, Arctic. The strain can grow at the temperature range from 7℃ to 30℃, and grow better at 30(℃. It can not grow at 40℃. Keeping certain salinity concentration in medium is necessary for cell growth. It grows well in medium containing salinity concentration from 0. 5 % to 10 % sodium chloride. Glucose, sucrose and soluble starch can be utilized by the strain, among which glucose is the optimal carbon source. Peptone is the optimal organic nitrogen source for cell growth and protease producing, and ammonium nitrate is the optimal inorganic nitrogen source.About 75.7% of total protease of the strain are extracellular enzyme. Optimal temperature for proteolytic activity is at 40℃. Protease of the strain keeps stable below 40℃, and shows high proteolytic activity within the pH range from 7 to 11.     

Isolation,identification, and cytotoxicity of a new isobenzofuran derivative from marine Streptomyces sp. W007

A new isobenzofuran derivative( 1) was isolated from the marine Streptomyces sp. W007 and its structure was determined through extensive spectroscopic analyses, including 1D-NMR, 2D-NMR, and ESI-MS. The absolute configuration of compound 1 was determined by a combination of experimental analyses and comparison with reported data, including biogenetic reasoning, J-coupling analysis, NOESY, and 1 H- 1 HCOSY. Compound 1 exhibited no cytotoxicity against human cells of gastric cancer BGC-823, lung cancer A549, and breast cancer MCF7.     

Isolation and characterization of a fucoidan-degrading bacterium from Laminaria japonica

Fucoidan, a polysaccharide containing abundant fucose and sulfate ester group, was prepared from Laminaria japonica. In order to obtain fucoidan-degrading enzyme, bacteria capable of degrading fucoidan were screened from kelp. A bacterial strain named RC2-3 was obtained, which degraded fucoidan by the maximum extent of 54% ± 1.3%, the highest among all bacterial isolates. High-performance size exclusion chromatography(HPSEC) showed that the molecular weight of fucoidan was gradually reduced by RC2-3 with culturing time, suggesting the production of fucoidan-degrading enzyme by RC2-3. Phylogenetic analysis of partial 16S ribosomal RNA gene(16S rDNA) sequence showed that RC2-3 belonged to the family Flavobacteriaceae. However, it showed different physiological and biochemical characteristics from the known Flavobacteriaceae members producing fucoidan-degrading enzyme, thus RC2-3 was proposed to be a new member of this family.     

Inhibitory activity of an extract from a marine bacterium Halomonas sp. HSB07 against the red-tide microalga Gymnodinium sp. （Pyrrophyta）

In recent years, red tides occurred frequently in coastal areas worldwide. Various methods based on the use of clay, copper sulfate, and bacteria have been successful in controlling red tides to some extent. As a new defensive agent, marine microorganisms are important sources of compounds with potent inhibitory bioactivities against red-tide microalgae, such as Gymnodinium sp. （Pyrrophyta）. In this study, we isolated a marine bacterium, HSB07, from seawater collected from Hongsha Bay, Sanya, South China Sea. Based on its 16S rRNA gene sequence and biochemical characteristics, the isolated strain HSB07 was identified as a member of the genus Halomonas. A crude ethyl acetate extract of strain HSB07 showed moderate inhibition activity against Gymnodinium sp. in a bioactive prescreening experiment. The extract was further separated into fractions A, B, and C by silica gel column chromatography. Fractions B and C showed strong inhibition activities against Gymnodinium. This is the first report of inhibitory activity of secondary metabolites of a Halomonas bacterium against a red-tide-causing microalga.     

Isolation,identification, and cytotoxicity of a new isobenzofuran derivative from marine <Emphasis Type="Italic">Streptomyces</Emphasis> sp. W007

A new isobenzofuran derivative (1) was isolated from the marine Streptomyces sp. W007 and its structure was determined through extensive spectroscopic analyses, including 1D-NMR, 2D-NMR, and ESI-MS. The absolute configuration of compound 1 was determined by a combination of experimental analyses and comparison with reported data, including biogenetic reasoning, J-coupling analysis, NOESY, and ¹H-¹HCOSY. Compound 1 exhibited no cytotoxicity against human cells of gastric cancer BGC-823, lung cancer A549, and breast cancer MCF7.     

A new identification method for five species of oysters in genus Crassostrea from China based on high-resolution melting analysis

The high phenotypic plasticity in the shell of oysters presents a challenge during taxonomic and phylogenetie studies of these economically important bivalves. However, because DNA can exhibit marked differences among morphologically similar species, DNA barcoding offers a potential means for oyster identification. We analyzed the complete sequences of the cytochrome oxidase subunit I （COI） of five common Crassostrea species in China （including Hong Kong oyster C. hongkongensis, Jinjiang oyster C. ariakensis, Portuguese oyster C. angulata, Kumamoto oyster C. sikamea, and Pacific oyster C. gigas） and screened for distinct fragments. Using these distinct fragments on a high-resolution melting analysis platform, we developed an identification method that does not rely on species-specific PCR or fragment length polymorphism and is efficient, reliable, and easy to visualize. Using a single pair of primers （Oyster- COI-1）, we were able to successfully distinguish among the five oyster species. This new method provides a simple and powerful tool for the identification of oyster species.     

Antiangiogenic activity of low-temperature lysozyme from a marine bacterium <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

We extracted marine low-temperature lysozyme (MLTL), a novel lysozyme, from a marine microorganism through fermentation. Our previous study suggested that a low molecular weight (16 kDa) may exert anti-tumor activity through antiangiogenesis. In this study, we extracted a high weight (39 kDa) and investigated its antiangiogenic activity in vivo and in vitro. Using zebrafish embryos as an in vivo study model, we found that treatment with MLTL significantly inhibited the growth of subintestinal vessels (SIVs) in a dose-dependent manner and that 400 μg/ml MLTL was sufficient to block the growth of SIVs. An in vitro study conducted using human umbilical vein endothelial cells (HUVECs) revealed that MLTL suppressed the proliferation, migration and tube formation of HUVECs in a dose-dependent manner. Interestingly, assays by flow cytometry and DNA electrophoresis indicated that MLTL was able to induce apoptosis of HUVECs. Moreover, further study demonstrated that the disruption of intracellular Ca²⁺ homeostasis may play an important role in MLTL induced apoptosis of HUVECs. Taken together, the results of this study demonstrate for the first time that MLTL inhibits angiogenesis through its pleiotropic effects on vascular endothelial cells and induces apoptosis through regulation of cellular Ca²⁺ levels. The results of this study also revealed a possible mechanism underlying the antiangiogenic effect of MLTL and suggested that MLTL may be a promising new antiangiogenic agent for use in cancer therapy.     

Isolation and characteristics of one marine psychrotrophic cellulase-generating bacterium Ar/w/b/75°/10/5 from Chuckchi Sea,Arctic

Microorganisms living in polar zones play an important part as the potential source of organic activity materials with low temperature characteristics in the bio-technological applications. A psychrotrophic bacterium (strain Ar/w/b/75°/10/5) , producing cellulose at low temperatures during late-exponential and early-stationary phases of cell growth, was isolated from sea ice-covered surface water in Chuckchi Sea, Arctic. This bacterium, with rod cells, was Gram-negative, slightly halophilic. Colony growing on agar plate was in black. Optimum growth temperature was 15℃. No cell growth was observed at 351 or above. Optimum salt concentration for cell growth was between 2 and 3 % of sodium chloride in media. Maximal cellulase activity was detected at a temperature of 35℃ and pH8. Cellulase was irreversibly inactivated when incubated at 55℃ within 30 min. Enzyme can be kept stable at the temperature no higher than 25℃. Of special interest was that this bacterium produced various extracellular enzymes i     

A new genus and species of shrimp(Crustacea:Axiidea:Axiidae) from the Caroline Ridge,Northwest Pacific

A new genus and species of axiid shrimp,Carolinaxius kexuae gen.et sp.nov.is described and illustrated based on a single specimen collected from an unnamed seamount in the Caroline Ridge,Northwest Pacific.Although both chelipeds are mis sing,the specimen can be distinguished from other axiid genera by a combination of characteristics:narrowly triangular ro strum;median carina and lateral gastric carina each with one prominent tooth;submedian gastric carinae converging po steriorly,with teeth;cornea weakly pigmented,eyestalk with acute distome sial tooth on dorsal surface;male pleopod 1 two-articled;pleopod 2 with appendix interna and appendix masculina;pleopods 3-5 with appendix interna.The molecular phylogeny suggests the new genus is most closely related to another recently described genus living inside hexactinellid sponges on seamounts in the Indian Ocean,Montanaxius Dworschak,2016.However,it differs from Montanaxius in the shape of the rostrum,the arrangement of teeth on the carapace,and the shape of the eyestalk.Besides,the significant molecular differences support the two belonging to different genera.     

A review of Epipenaeon ingens Nobili, 1906 (Isopoda: Bopyridae) host species and documentation of a new host, Metapenaeopsis stridulans (Alcock, 1905) (Decapoda: Penaeidae)

We collected 3 596 Metapenaeopsis stridulans (Decapoda: Penaeidae) from the southeast coast of India between January and December 2007. Sixty three specimens (43 females and 18 males) were parasitized by the bopyrid isopod Epipenaeon ingens (Isopoda: Bopyridae). This is the first report of the occurrence of E. ingens in this host; therefore, it was considered as a new host record. The highest level of infestation (3.2%) occurred in October 2007, coincident with observations of gravid females (9). The total prevalence and presence of gravid females were 17.46% and 28%, respectively. Infestation caused a characteristic bulge of the branchial chamber, growth retardation, and degeneration of the sex organs, but had no effect on the host weight.     

A new record of a flathead fish (Teleostei: Platycephalidae) from China based on morphological characters and DNA barcoding

A new record of Platycephalus sp.1 (sensu Nakabo, 2002) was documented based on morphological characters and DNA barcoding. We collected 174 specimens of the genus Platycephalus from Chinese coastal waters of Dongying, Qingdao, Zhoushan, and Beihai. Samples were identified as Platycephalus sp.1 morphologically. The coloration, meristic traits, and morphometric measurements are consistent with previously published records. In brief, it is an orange-brown flathead fish with dark brown spots scattered on head and body, lateral line scales 83 to 99 with one or two spine-bearing anteriormost pored scale, no yellow blotch on the caudal fin. Cytochrome oxidase I subunit (COI) gene fragments were sequenced for phylogenetic analysis. The mean evolutionary distance within the species Platycephalus sp.1 was 0.1%. Net evolutionary distances between Platycephalus sp.1 and other species of Platycephalus ranged from 10.8% to 19.7%, which is much greater than the threshold for species delimitation. The COI sequence analysis strongly supports the validity of Platyceohalus sp.1 at genetic level.     

A NEW SPECIES OF RANINOIDES （CRUSTACEA ： DECAPODA ： RANINIDAE） FROM OFF THE SOUTHEASTERN COAST OF HAINAN ISLAND AND NANSHA ISLANDS

Ranlnoides longifions new species, off SE coast of Hainan Island, dorsal surface of carapace with an arched granular edge across carapace between the anterolateral teeth, median frontal tooth longest, second frontal teeth subquadrate.     

ON THE FAMILY LYONSIIDAE (MOLLUSCA:BIVALVIA), WITH DESCRIPTION OF A NEW GENUS AND SPECIES FROM THE CHINA SEAS

The present paper deals with 4 species of Family Lyonsiidae , collected from the China Seas : one new genus and new species, Sinolyonsia sinica gen . et sp. nov, one species, Lyonsia kawamurai recorded for the first time from the China Seas , and 2 species L . ventricosa Gould and Agriodesma navicula (Adams et Reeve) reported previously from the Yellow Sea by the author .     

Assessment and analysis of rockfall-caused tree injuries in a Turkish fir stand: A case study from Kastamonu-Turkey

Rockfalls can cause serious damage to people,property,facilities and transportation corridors.Furthermore,rockfalls are major hazards in mountain areas with negative impacts on individual trees and forested ecosystems.We conducted a study of rockfall events on 117 mapped(91% of total trees in the stand with1.3 m in height and5 cm diameter at breast height)Turkish fir trees(Abies bornmuelleriana Mattf.)in a stand within the Kayaarkasi-Topular Village,Inebolu district, Kastamonu province of Turkey.The study site of 0.35 Ha is located on the transition zone of frequently passing rockfall fragments(～40 cm in diameter) generally causing healable injuries.Parameters of trees and injuries were recorded and analysed as to injury number,height and size.Bivariate correlation analysis were used to investigate the relationships between:a)diameter at breast height and number of injuries per tree,b)diameter at breast height and total injury size,c)the number of injuries and total injury size and d)the number of injuries per tree and distance from the source of the rockfall area.Results indicate that the average height of injury,average number of injuries and average injury area to be 81.3 cm(STDEV:49.8),7.46(STDEV:4.4)and 628.6 cm2 (STDEV:678.2),respectively.In total 84% of all injuries were recorded within 160°sector at the upslope side of trees and callus tissue that had closed wounds was observed in 79.1% of all injuries. Furthermore 14.5% of injured trees had callus tissue in the process of closing wounds while 6.4% of injuries had not formed any callus tissue.The most common injury types were bark and wood injuries. Bivariate correlation analysis indicated strong relationships between diameter at breast height and the number of injuries(rs=0.524),injury number and total injury area(rs=0.653)and distance from rockfall area and injury numbers relations(rs=-0.518).A weaker relationship was found between diameter at breast height and total injury area(rs=0.363).These results indicate that bigger trees are more prone to rockfall injuries.As expected,trees further from the rockfall area seem to be less prone to rockfall injuries.From our results,it can be inferred that the protection of people and property can be increased through the maintanance of forest in areas immediately below areas prone to rockfall.This stand is still managed in selective forest management system.In order to protect the settlement at the deposition zone it has to manage for protective goals with uneven-aged and multilayered stand structure.