Sperm-activating proteins from unfertilized eggs of the Pacific herring, Clupea pallasii

Ripe unfertilized eggs of the Pacific herring, Clupea pallasii , release sperm-activating proteins into seawater at the time of fertilization. Five species of herring sperm-activating proteins (HSAP) with different pl values (4.8, 4.9, 5.0, 5.1 and 5.4) were purified from the egg-conditioned medium by gel filtration and isoelectric focusing. Molecular mass of the HSAP (pl = 5.1), the major species of the five HSAP, was determined to be 8.1 kDa by mass spectrometry. Molecular weights of all of the HSAP were estimated to be 7700 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The first 20 amino acid sequences from N-terminal ends of three HSAP (pl = 4.9, 5.0 and 5.1) were almost identical, suggesting that the HSAP have similar structures.     

Development of microsatellite loci in Pacific herring (Clupea pallasi)

Fifteen polymorphic di‐, tri‐ and tetranucleotide microsatellite markers were developed in Pacific herring (Clupea pallasi) to aid in the delineation of population structure of herring in British Columbia. Twelve of the microsatellite loci were analysed in over 4000 wild herring. Expected heterozygosities ranged 0.73–0.95, and only two loci significantly deviated from Hardy–Weinberg equilibrium.     

Two egg-derived molecules in sperm motility initiation and fertilization in the Pacific herring (Clupea pallasi)

Sperm of the Pacific herring are immotile at spawning. Two egg-derived molecules are capable of initiating sperm motility. One is herring sperm activating protein(s) (HSAPs) and the other is sperm motility initiation factor (SMIF). These two motility initiators differ in their location and association with the chorion, and in thier isoelectric points and molecular weights. In this study we have investigated the roles of these two inducers with respect to motility and fertilization. Using computer analysis of sperm motility, we found that HSAPs, as well as the C-terminal HSAPs peptide, elicit a linear motility pattern, while SMIF induced a highly circular and asymmetric pattern. HSAPs induced a two-fold increase in intracellular calcium, whereas SMIF induced a four-fold increase of motility initiation. SMIF-exposed sperm, preloaded with BAPTA-AM, showed a more linear motility and this motility trajectory decreased with their fertilizing capability. The difference in intracellular calcium levels between HSAPs and SMIF is consistent with the observed linear and circular motility. In the absence of SMIF, HSAPs do not support fertilization. Fertilization is rescued in these experiments if SMIF is reintroduced. We propose that diffusible HSAPs are not essential for fertilization, but enhance sperm-egg collisions via linear motility. SMIF, which is bound to the micropylar region of the chorion, is required for fertilization and induces circular motility that is a prerequisite for sperm to enter the micropylar canal and fertilize the egg.     

放线菌代谢物抑制脯酰氨内酞酶活性及培养条件的初步研究

以脯酰氨内酞酶为靶酶,从供试菌中筛选出一株放线菌A45发酵液乙酸乙酯提取物对该酶有较强的抑制活性,并对该活性的部分进行酶抑制动力学实验,结果表明乙酸乙酯提取物对该酶抑制率达到54.2%,且其活性和浓度呈量效关系,其IC50为60.5μg/mL,动力学表现为非竞争抑制类型,抑制常数Ki=119.1μg/mL;以对该酶的抑制率为指标,进行该菌株液体培养条件和培养时间的初步研究,结果最适培养时间为6d,最佳培养基配方（g/L）为可溶性淀粉30 g,酵母浸粉2g,MgSO.47H2O 0.4g,CaCO30.3g,K2HPO40.4g。     

Development and characterization of a cell line from Pacific herring, Clupea harengus pallasi, sensitive to both naphthalene cytotoxicity and infection by viral hemorrhagic septicemia virus

A cell line, PHL, has been successfully established from newly hatched herring larvae. The cells are maintained in growth medium consisting of Leibovitz's L-15 supplemented with 15% fetal bovine serum (FBS), and have been cryopreserved and maintain viability after thawing. These cells retain a diploid karotype after 65 population doublings. PHL are susceptible to infection by the North American strain of viral hemorrhagic septicemia (VHS) virus, and are sensitive to the cytotoxic effects of naphthalene, a common environmental contaminant. Naphthalene is a component of crude and refined oil, and may be found in the marine environment following acute events such as oil spills. In addition, chronic sources of naphthalene contamination include offshore drilling and petroleum contamination from areas such as docks and marinas that have creosote-treated docks and pilings and also receive constant small inputs of petroleum products. This cell line should be useful for investigations of the toxicity of naphthalene and other petroleum components to juvenile herring. In addition, studies of the VHS virus will be facilitated by the availability of a susceptible cell line from an alternative species.     

Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: implications for Celiac Sprue therapy

Celiac Sprue is a multi-factorial disease characterized by an inflammatory response to ingested wheat gluten and similar proteins in rye and barley. Proline-rich gluten peptides from wheat, rye, and barley are relatively resistant to gastrointestinal digestion, and therefore persist in the intestinal lumen to elicit immunopathology in genetically susceptible individuals. In this study, we characterize the in vitro gluten detoxifying properties of a therapeutically promising prolyl endopeptidase from Myxococcus xanthus (MX PEP), and describe the development of a prototypical enteric-coated capsule containing a pharmacologically useful dose of this enzyme. A high-cell density fed-batch fermentation process was developed for overproduction of recombinant MX PEP in E. coli, yielding 0.25-0.4 g/L purified protein. A simple, scalable purification and lyophilization procedure was established that yields >95% pure, highly active and stable enzyme as a dry powder. The dry powder was blended with excipients and encapsulated in a hard gelatin capsule. The resulting capsule was enteric coated using Eudragit L30-D55 polymer coat, which provided sufficient resistance to gastric conditions (> 1 h in 0.01 M HCl, pH 2 with pepsin) and rapid release under duodenal conditions (15-30 min release in pH 6.0 in the presence of trypsin and chymotrypsin). In conjunction with pancreatic enzymes, MX PEP breaks down whole gluten into a product mixture that is virtually indistinguishable from that generated by the Flavobacterium meningosepticum (FM) PEP as judged by chromatographic assays. Competitive studies involving selected immunogenic peptides mixed with whole gluten reveal that both PEPs have a wide range of substrate specificity. Our results support further in vitro and in vivo evaluation of the MX PEP capsule as an oral therapeutic agent for Celiac Sprue patients.     

Comparison of fatty acid profiles of spawning and non-spawning Pacific herring, Clupea harengus pallasi

Crude lipid and fatty acid composition from liver, intestine, roe, milt and flesh of spawning and non-spawning Pacific herring (Clupea harengus pallasi) were examined to determine the relative effects of spawning on the nutritional value of herring. Depletion of lipid due to spawning condition was significant (P < 0.01) in all organ tissues and flesh of spawning herring. The lipid content ranged from an average of 1.9 to 3.4% (wet weight basis) in different organ tissues of spawning herring, to 10.5 to 16% in non-spawning fish. The fatty acid profile exhibited many differences in the relative distribution of individual fatty acids among organ tissues and between the two fish groups. Oleic acid (C18:1n-9), a major monounsaturated fatty acid (MUFA) found in all tissue lipids, decreased significantly (P < 0.01) in spawning fish. The two monoenes, C20:1n-9 and C22:1n-11, occurred at high concentrations in the flesh but at only minor proportion in the digestive organs and gonads. Spawning herring also had significantly (P < 0.01) higher polyunsaturated fatty acids (PUFA) content in the organ tissues, particularly in the milt and ovary, with docosahexaenoic acid (C22:6n-3, DHA) having the greatest proportion. Among the n-6 fatty acids, only C18:2n-6 and C20:4n-6 occurred at notable amounts and were present in higher proportions in spawning fish. We concluded that although relatively higher n-3 fatty acid content was found in the organ lipids of spawning herring, they are not an energy-dense prey food source due to the fact that both flesh and gonads contain a very low amount of lipid.     

Hatch-period-dependent early growth and survival of Pacific herring Clupea pallasii in Miyako Bay, Japan

Considerable interannual variation in the abundance of larval and juvenile Pacific herring Clupea pallasii was detected in Miyako Bay, on the Pacific coast of northern Japan; abundances were high in 2001 and 2003 and low in 2000 and 2002. Hatch dates and growth rates for larval and juvenile survivors were estimated through otolith analysis. Water temperature and food availability were monitored on the spawning and nursery grounds in the inner part of the bay. The number of spawning females caught in nets set around the spawning ground was recorded during each spawning season (January to May) in 2000–2003. No correlation was found between the number of spawning females and the abundance of larvae and juveniles on the spawning and nursery grounds. The hatch dates of surviving larvae and juveniles were concentrated at the end of the spawning season in 2001 and in the middle of the season in 2003. The larvae experienced relatively high prey concentrations during the first-feeding period in 2001 but low concentrations in 2003. Survival of larvae during the first-feeding period may be a function of prey concentration as well as water temperature. In 2003, low water temperature would reduce starvation mortality during the first-feeding period. In contrast, unfavourable feeding conditions with higher temperatures during the first-feeding period seemed to result in low larval survival in 2000 and 2002. The 2001 larvae grew faster than those in 2003 because of the late hatch dates and the higher ambient temperatures that resulted. Temperature might be a major factor controlling growth rates of C. pallasii larvae in Miyako Bay.     

烟曲霉脯氨酰内肽酶在巴斯德毕赤酵母中的分泌表达与重组酶性质

【目的】研究烟曲霉脯氨酰内肽酶cDNA基因的异源表达及重组酶性质。【方法】以烟曲霉CICIM F0044总RNA为模板,反转录合成cDNA;再以cDNA为模板,通过PCR扩增去除自身信号肽的脯氨酰内肽酶基因,构建表达载体pPIC9K-PEP;电转化酵母宿主菌Pichia pastoris GS115,获得重组菌PEP-09;纯化并分析重组酶性质。【结果】重组菌摇瓶发酵酶活力最高可达647.3 U/L。表达产物纯化后的分子量为63 kD左右。重组酶最适反应温度为65°C,有较好的温度稳定性,在55°C保温8 h能保留90%以上的酶活力。该酶最适pH为5.5,在pH 3.0 9.0范围内有很好稳定性,在pH 6.0 8.0的缓冲液中37°C保温10 d酶活没有明显变化。【结论】烟曲霉脯氨酰内肽酶cDNA基因在巴斯德毕赤酵母中实现了分泌表达,重组酶活性稳定,有一定的应用潜力。     

Association of plasma IgM with body size, histopathologic changes, and plasma chemistries in adult Pacific herring Clupea pallasi

Pacific herring Clupea pallasi immunoglobulin is an IgM-like molecule comprised of heavy and light chains with molecular weights of 79 and 25 to 27 kD, respectively. Purified immunoglobulin was used to generate highly specific polyclonal antibodies for development of a sandwich ELISA. The ELISA was used to quantify total plasma IgM in 602 Pacific herring captured in Prince William Sound and Sitka Sound, Alaska, USA. Plasma IgM concentrations ranged from 0.13 to 5.32 mg ml-1. Using multiple stepwise regression analysis, plasma IgM was highly correlated (p < or = 0.01) with body length, Ichthyophonus hoferi infection, plasma albumin, plasma cholesterol, liver macrophage aggregates, and focal skin reddening. I. hoferi was the only organism significantly associated with plasma IgM. Gender, site, and season (spring vs fall) did not contribute to significant differences in plasma IgM. This study contributes to the understanding of the interaction of body size, plasma chemistries, and pathological changes upon circulating immunoglobulins in fish.     

Combined effects of physicochemical variables (pH and salinity) on sperm motility: characterization of sperm motility in European sea bass Dicentrarchus labrax

Gamete activation in fish is an important step in terms of artificial fertilization of oocytes, cryopreservation studies and other experimental manipulations. Salinity and pH differences in activation media affect to sperm motility and fertilizing ability. These experiments were therefore designed to investigate the combined effects of pH (range 5.0–9.0) and salinity (20, 30, 37, and 45‰) of activation media on sperm motility of European sea bass Dicentrarchus labrax. The best results were obtained at salinity 37‰ and a pH of 9.0. Our results also demonstrated that non-progressive motility at salinity 45‰ was observed in the range of 5.0–9.0 pH. In conclusion, spermatozoa can be motile at a wide range of pH and salinity values although the percent of motile spermatozoa and motility duration are negatively affected by low pH values.     

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/10⁶ cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN₂) at 5 × 10^–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH Thyrotropin-Releasing Hormone or Thyroliberin - His-Pro-DKP Histidyl-ProlineDiketopiperazine - TRH-OH acid TRH or deamidated TRH - LH-RH Luteinizing Hormone-Releasing Hormone - Z-Gly-Pro-CHN₂ N-benzyloxycarboxyl-Gly-Pro-diazomethylketone - PGP Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-) - PE Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26)     

Characterization of secretory proteins from cultured cauda epididymal cells that significantly sustain bovine sperm motility in vitro

Epididymis provides a safe environment in which stored-spermatozoa could survive for days before ejaculation. In vitro studies suggested that epididymal proteins seem to be implicated in sperm survival during coincubation with cultured epididymal cells. This study was basically designed to confirm if secretory proteins from bovine epididymal cell cultures provide sperm protection against rapid loss of sperm motility in vitro. Bovine spermatozoa were incubated in conditioned media (CM), which were prepared from cultured cauda epididymal cell (CEC). Motion parameters were recorded using a computer-assisted sperm analyzer. Sperm-free protein extracts from CM were fractionated by ultrafiltration through a 10-kDa cut off membrane. A significantly positive effect on sperm motility was observed when spermatozoa were incubated in CM (54 +/- 4%) and CM > 10 kDa (57 +/- 4%) compared to CM < 10-kDa fraction (30 +/- 3%) or fresh media (34 +/- 3%), after a 6-hr incubation period. This beneficial effect on sperm motility was abolished when the CM > 10-kDa fraction was heat-treated at 100 degrees C for 10 min. The CM > 10 kDa fraction provides factors that remained active even though spermatozoa were washed twice after a 2-hr preincubation period. To identify potential beneficial factors, bovine spermatozoa were incubated with radiolabeled proteins obtained using (35)S-methionine in culture medium. SDS-PAGE analysis of proteins extracted from CM-preincubated spermatozoa revealed the presence of a 42-kDa protein strongly associated to the sperm surface. This 42-kDa spot was trypsin-digested and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as a protein homologue to a 35-kDa bovine estrogen-sulfotransferase. This protein can play a role in epididymal biology and sperm function. Taken together, these results suggest that specific epididymal proteins can be implicated in the sperm protection in vitro, and can be characterized in our cell culture system.     

Spatially structured interactions between a migratory pelagic predator, the Norwegian spring-spawning herring Clupea harengus L., and its zooplankton prey

Spring-spawning herring Clupea harengus was patchily distributed over large parts of the Norwegian Sea in May 1995–2005, during the early phase of the annual feeding migration. Overall, herring tended to be found in areas with intermediate biomasses of zooplankton prey, intermediate water temperatures and relatively high salinities. Herring had more food in their stomachs in areas of relatively low water temperature and high herring abundance. Hydrographical conditions revealed that herring was feeding mainly within Atlantic water masses, and more intensely in western and northern regions of the Norwegian Sea. Zooplankton biomass was patchily distributed, and was generally higher towards the western parts of the Norwegian Sea. Here, zooplankton biomass in year _{i +1} was also negatively associated with herring spawning stock biomass in year _i , while there was no evidence for such an association in the eastern region; indicating that herring may have a geographically structured 'top–down' effect on the recruitment of its zooplankton prey. The fact that herring was not typically associated with the areas containing the greatest zooplankton biomasses may reflect that the fish had not yet reached the most profitable feeding grounds or alternatively that herring was depleting zooplankton biomass.     

Interannual and regional variations in body length, weight and energy content of age-0 Pacific herring from Prince William Sound, Alaska

Age-0 Pacific herring were surveyed in October of 4 years in a large northern Gulf of Alaska estuary, to determine the range of variations in length, weight and whole body energy content (WBEC). These parameters reflect their preparedness for surviving their first winter's fast. During the surveys there were distinct regional and interannual variations in all three parameters for individual groups of herring in Prince William Sound. Likewise, with each collection there was typically a large range of size and WBEC values. The average standard length was (±S.D.) 80±13 mm (range=40–118), the mean whole body wet weight was 5·7±3·0 g (range=0·7–29·2) and the average WBEC of all age-0 herring captured, regardless of year or site (n=1471), was 5·4± 1·0 kJ g⁻¹ wet weight (range=2·4–9·4). The large range of WBEC and size indicates that age-0 herring at different capture sites were not all equally prepared for surviving their first winter.     

Purification and characterization of an endopeptidase that has an important role in the carboxyl terminal processing of antihypertensive peptides in Lactobacillus helveticus CM4

AIMS: To purify and characterize a peptidase that can catalyse C-terminal processing of antihypertensive peptide from Lactobacillus helveticus CM4. METHODS AND RESULTS: An endopeptidase which seems to process the carboxyl terminal end of two antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, was purified from Lactobacillus helveticus CM4 by four stages of column chromatography, using synthetic pro-peptide as a substrate. The molecular weight of the purified enzyme was estimated to be 67,000 by SEPHACRYL S-200 and 70,000 by SDS-PAGE analysis. The purified enzyme generated: (i) Val-Pro-Pro from Val-Pro-Pro-Phe-Leu and Val-Pro-Pro-Phe-Leu-Gln-Pro, and (ii) Ile-Pro-Pro from Ile-Pro-Pro-Leu-Thr and Ile-Pro-Pro-Leu-Thr-Gln-Thr, but theses peptides could not be generated from Val-Pro-Pro-Phe, Val-Pro-Pro-Phe-Leu-Gln, Ile-Pro-Pro-Leu and Ile-Pro-Pro-Leu-Thr-Gln. Part of the amino terminal sequence of the purified enzyme had homology to a previously reported pepO gene product. CONCLUSION: These results suggest that the purified endopeptidase isolated in this study have an important role in the carboxyl terminal processing of two antihypertensive peptides in Lact. helveticus CM4.     

Sperm motility in the horseshoe crab. III. Isolation and characterization of a sperm motility initiating peptide

Sperm motility in Limulus is initiated by a sperm motility initiating factor (SMI) that emanates from Limulus eggs. This report describes the partial purification of SMI (greater than 230-fold purification with respect to protein content) with 40% recovery. SMI appears to be a hydrophobic peptide of 500–2,000 MW. Although probably not purified to homogeneity, SMI is estimated to be active at a concentration of less than 0.2 μM.     

Effects of alternative prey on predation intensity from herring Clupea harengus and sandeel Ammodytes marinus on capelin Mallotus villosus larvae in the Barents Sea

Stomach contents from lesser sandeel Ammodytes marinus and herring Clupea harengus caught at one and three stations, respectively, were analysed to investigate predation intensity on capelin Mallotus villosus larvae. Most capelin larvae were found in the anterior sections of the stomachs close to the oesophagus, indicating that rapid digestion rates of larvae reduced the abundance in the posterior sections. The anterior sections of the stomachs had alternating layers of either copepods or capelin larvae and krill. This shows that the individual predators switched between feeding on either copepods or on krill and capelin larvae. A total of 549 capelin larvae was found in 440 fish stomachs. Capelin larvae were found in 20% of the sandeel stomachs, and 24, 34 and 62% of the stomachs from the three herring stations, respectively. Many of the predator stomachs contained more than five larvae, and up to 28 larvae were found in one herring stomach. The herring appeared to prey selectively on the largest capelin larvae, and the number of larvae per stomach was much higher in stomachs where krill had recently been eaten than where copepods were dominant. The predation intensity measured in this study is much higher than reported in earlier studies, and it is suggested that the predators were feeding using a searching image, and that the concentrations of alternative prey (copepods and krill) varied around a switching threshold.     

An analysis of allozyme variation in herring <Emphasis Type="Italic">Clupea pallasii</Emphasis> from the White and Barents Seas

An analysis of genetic variation is made according to four allozyme loci of reproductive (spawning) groups of oligovertebrate herring Clupea pallasii collected in various bays of the White Sea and the south-eastern part of the Barents Sea in 1995–2002. The temporal stability of genetic characteristics during several years is shown. The analysis of genetic variation revealed a significant difference between herring from the south-eastern part of the Barents Sea and spring-spawning and summer-spawning herring from inner bays of the White Sea. The analysis of geographic variation of genetic characteristics of spawning aggregations revealed the change in frequencies of alleles of loci LDH-2* and MDH-4* from the north-east to the south-west along the coast.     

Occurrence and survival of the larval nematode Anisakis sp. in the flesh of fresh, frozen, brined, and smoked pacific herring, Clupea harengus pallasi

Two hundred and twenty Pacific herring (Clupea harengus pallasi) from Yaquina Bay, Oregon, were divided into five groups to test the effects of various methods of handling and processing (fresh, frozen, brined, cold smoked, and cold smoked-gibbed) on the occurrence and survival of Anisakis larvae in the flesh. Viscera and flesh were digested separately using a pepsin-HC1 technique, and counts were taken of Anisakis larvae. Parasite loads were statistically largest in the flesh of frozen, brined, and smoked fish. Smoked fish harbored the greatest proportions of larvae in the flesh. Human consumption of brined or cold smoked Pacific herring represents a potential public health hazard.