Spontaneous activity was unaffected by ration size in Nile tilapia and gibel carp

Two growth trials using a range of ration sizes from starvation to maximum feeding suggested that linear relationships existed between specific growth rate and ration size for Nile tilapia and givel carp, Continuous measurement of activity showed that activity level, in terms of distance swum per day, was not affected significantly by ration size in both Nile tilapia and gibel carp. (C) 2001 The Fisheries Society of the British Isles.

Locomotor activity rhythm in the Japanese eel Anguilla japonica elvers

Under artificial LD cycles (6, 12, 18 L), the elvers of Japanese eel, Anguilla japonica, showed a 24 h cycle of locomotor activity rhythm being most active at light transitions: the eels' activity rose to a primary peak after lights-off, followed by a quiescent period during which they buried into the shelters or lying motionlessly on sand for most of the time, and then reached a secondary peak before lights-on. Elvers could resynchronize their activity rhythm with a new photo cycle within 4 d. Moreover, their activity level at dark phase significantly increased as the light period was prolonged: higher activity levels during shorter dark period. However, the elvers did not display clearly the existence of a circadian rhythm under constant light or dark conditions. The timing of daily activity rhythm evidenced in the Japanese eels may occur through the action of the LD cycles with a weak participation of an endogenous circadian system. In all the LD cycles, over 99% of the activity occurred in the dark phase, indicating that the eels were always nocturnally active no matter what time of day it might be. Under 12 L conditions, the eels' activity level and the time outside sand were significantly elevated both at light and dark phases as temperature increased from 10 similar to 15 to 20 similar to 25 degrees C. The activity rhythm pattern (i.e., two peaks occurring around light transitions) did not apparently change among temperatures. However, in contrast with the primary activity peaks immediately after lights-off at 20 and 25 degrees C, the timing of the primary peaks at 10 and 15 degrees C showed a latency of a few hours following lights-off, indicating the inhibiting effect of low temperature on the eels' activity.

若尔盖湿地3种两栖类的种间竞争及畜牧业对其食性的影响

从空间、时间、食物3个方面研究了若尔盖湿地3种两栖类的种间竞争，运用生态位理论探讨了3种两栖类利用环境资源的能力以及它们之间的共存模式，研究了3种两栖类年周期食性，并考察了畜牧业对3种两栖类食性及食物竞争格局的影响；此外，还通过实验室研究对2种两栖类幼体的种间竞争策略进行了考察。主要结果如下： 1、两栖类的空间资源利用状况：在3种两栖类成体生态位宽度的比较中，岷山蟾蜍（Bufo minshanicus）成体在牧场性质(0.41)、牛粪数量(0.42)、植被盖度(0.20)、地表温度(0.50)、地表湿度(0.51) 5个维度上的生态位宽度最窄；而倭蛙（Nanorana pleskei）成体在牛粪数量(0.81)、植被高度(0.63)、植被盖度(0.47)、小水体距离(0.68) 4个维度上的生态位宽度最宽。在3种两栖类亚成体生态位宽度的比较中，岷山蟾蜍亚成体在牧场性质(0.66)、牛粪数量(0.58)、植被高度(0.64)、小水体距离(0.51)、地表湿度(0.79) 5个维度的上生态位宽度最宽；倭蛙亚成体在牧场性质(0.39)、牛粪数量(0.30)、地表温度(0.18)、地表湿度(0.33) 4个维度上的生态位宽度最窄。高原林蛙（Rana kukunoris）在地表温度（成体：0.62；亚成体：0.56）、地表湿度（成体：0.84；亚成体：0.60）两个维度上具有较大的生态位宽度值，而在小水体距离维度上（成体：0.27；亚成体：0.14）的生态位宽度值则很小。比较3种无尾两栖类在不同生长阶段（成体、亚成体）的生态位宽度，发现高原林蛙和倭蛙的亚成体对栖息环境的要求更高。3种两栖类空间资源利用的相似程度很高，高原林蛙与倭蛙之间的生态重叠度(0.87)较之它与岷山蟾蜍(0.81)的生态位重叠度更大。 2、两栖类的日活动节律：高原林蛙成体、亚成体、岷山蟾蜍亚成体活动的最低气温为0℃、2℃、8℃；岷山蟾蜍和高原林蛙亚成体出现的数量与气温成极显著的正相关（r=0.797, p<0.001；r=0.794, p<0.001），高原林蛙成体出现的数量与气温有一定相关性（r=0.456, p<0.05）；晴天时两栖类的活动性明显高于阴天（p<0.001）；多云转晴天气，高原林蛙和岷山蟾蜍亚成体出现两次日活动高峰，分别为中午12:30左右和下午15:30～16:30之间；多云天气，高原林蛙和岷山蟾蜍亚成体出现两次日活动高峰，分别为9:30～10:30之间和15:30～16:30之间。 3、两栖类的食物资源利用状况：春、秋两季，高原林蛙最主要的食物是蜉金龟科(Aphodiidae)昆虫，相对重要性指数(IRI)最高（春季：35.28%，秋季：28.57%），其次为昆虫的幼虫，以及双翅目的毛蚊科(Bibionidae)、蝇科(Muscidae)、丽蝇科(Calliphoridae)昆虫，秋季，蝗虫是高原林蛙食物组成中的重要部分；岷山蟾蜍最主要的食物是蚂蚁(IRI，春季：85.54%，秋季：49.70%)，其次为蜉金龟科、象甲科(Curculionidae)、步甲科(Carabidae)、粪金龟科(Geotrupidae) 等鞘翅目昆虫；倭蛙春季的最主要食物也是蜉金龟科昆虫(IRI，春季：13.41%)，其次为蚂蚁、毛蚊科昆虫、昆虫的幼虫以及狼蛛科(Lycosidae)。3种两栖类中，倭蛙的食性生态位宽度相对较宽（0.43），而岷山蟾蜍（0.09）和高原林蛙（0.22）的生态位宽度较窄，与春季相比，两栖类在秋季的食谱更宽。以利用食物种类为标准，春季高原林蛙与倭蛙的生态位重叠度(0.40)比它与岷山蟾蜍的生态位重叠度（0.33）更大。 4、畜牧业对两栖类食性及食物竞争格局的影响：以藏牦牛粪为食物或寄居场所的昆虫，如蜉金龟科、粪金龟科、毛蚊科、蝇科、丽蝇科昆虫和某些昆虫幼虫，是3种两栖类食物谱中最主要的组成部分，蜉金龟科昆虫在高原林蛙食谱中的比例更高，高原林蛙可能从畜牧业发展中获得更多的好处，使之在食物竞争方面处于优势地位。与无放牧样地相比，在有放牧样地的中，两栖类食谱中的蜉金龟科昆虫数量更多(有放牧：31.94%；无放牧：21.32%)、出现频率更高(有放牧：76.38%；无放牧：44%)。然而在不同样地上（有放牧/无放牧），两栖类的食物组成无显著性差异(P=0.188)，两栖类的数量(P=0.075)、肥满度(P=0.537)均没有显著差别。 5、两栖类幼体的竞争策略：实验室条件下，通过活动性水平，变态时的体重、增长率和完成变态所需时间考察自然条件下常同水塘分布的中华蟾蜍（Bufo gargarizans）和高原林蛙蝌蚪的竞争策略。结果表明：中华蟾蜍蝌蚪在不同食物资源条件下，所选择的生存策略可能不同，即食物资源充足时，增加活动性获取更多食物，食物资源有限时，降低活动性且提前完成变态；与中华蟾蜍蝌蚪相比，在食物资源有限时高原林蛙蝌蚪获取食物能力可能更强。 This paper presented the study of competition of three amphibians (Rana kukunoris, Nanorana pleskei, Bufo minshanicus) based on spatial, temporal and dietary scales in Zoige wetland. We measured coexistence patterns of three amphibians and analyzed their ability of exploiting resource. Effects of grazing on the diet composition and diet competition of amphibians were analyzed by their diet composition during spring and autumn. Furthermore, we examined the competitive ability of larval common frogs (Rana kukunoris)and common toads(Bufo gargarizans) in a laboratory experiment, and analyzed their competitive strategies respectively. The results were as follows: 1 .The status of using spatial resource Niche breadths of B. minshanicus adults on 5 dimensional axes including character of pasture(0.41), number of yaks dung(0.42), vegetation coverage(0.20), temperature (0.50)and humidity(0.51) of ground surface were narrower than adults of R. kukunoris and N. pleskei. Niche breadths of B. minshanicus subadults were broader than R.kukunoris subadults and N.pleskei subadults on 5 dimensional axes including character of pasture (0.66), number of yaks dung (0.58), vegetation height (0.64), distance to small waterbodies (0.51), humidity of ground surface (0.79). Niche breadths of N. pleskei subadults were the narrowest in three anurans subadults on 4 dimensional axes including character of pasture (0.39), number of yaks dung (0.30), temperature (0.18) and humidity (0.33) of ground surface, niche breadths of N. pleskei adults were the broadest in three anurans adults on 4 dimensional axes including number of yaks dung (0.81), vegetation height (0.63) and coverage(0.47), distance to small waterbodies(0.68).Comparatively, niche breadths of R. kukunoris were broader on the two microclimate factors including temperature(adults：0.62；subadults：0.56) and humidity (adults：0.84；subadults：0.60)of ground surface, but was narrow on distance to small waterbodies(adults：0.27；subadults：0.14). Strategies for using habitat resource of adults and subadults of the three species anuran were different. Generally, subadults of R. kukunoris and N. pleskei needs better habitat condition. It was quite similar that three anurans exploited spatial resource, Niche overlap between R. kukunoris and N. pleskei (0.87) was greater than that between R. kukunoris and B.minshanicus(0.81). 2.Daily activity rhythm R. kukunoris audlts were active when air temperatures were as low as 0℃, R. kukunoris subadults were active at 2℃, B.minshanicus subaudlts were active at 8℃. Positive correlation was found between activities of amphibians and air temperature, Subadults of R.kukunoris, (r=0.797, p<0.001), Subadults,of,B.minshanicus, (r=0.794, p<0.001), andbadults,of,R.kukunoris(r=0.456, p<0.05).Amphibians were more active during sunny days than cloudy days. In cloudy turning into sunny, R. kukunoris and B.minshanicus subadults had two active peak: at noon about 12:30 and 15:30～16:30 pm; in cloudy, R. kukunoris and B.minshanicus subadult had two active peak too : 9:30～10:30am,15:30～16:30pm. 3.Diet analysis Aphodiidae was the most commonly consumed food item by R. kukunoris based on index of relative importance (IRI) during spring (35.28%) and autumn (28.57%) in Zogie wetland. Besides Aphodiidae, larval insect, dipterans such as Bibionidae, Muscidae, Calliphoridae also were important food item for R. kukunoris, in autumn, locust was one of important food item for R. kukunoris. The most important food item for B.minshanicus during spring (IRI:85.54%) and autumn (IRI:49.70%) was ants, following, was coleopterans, such as Aphodiidae, dung beetle. Aphodiidae (IRI:13.41%) were the most important consumed food item by N. pleskei during spring too, following, was ants and Bibionidae. Dietary breadth of N. pleskei (0.43) were greater than R. kukunoris (0.22) and B. minshanicus (0.09). As a whole, Dietary breadth of amphibians during aurumn were greater than spring. Based on prey item, dietary overlap between R. kukunoris and N. pleskei (0.40) was greater than that between R. kukunoris and B.minshanicus (0.33) during spring. 4.Effects of grazing on the diet composition and diet competition of amphibians Amphibians are an important part of the pasture ecosystems as prey and predator. In Zogie wetland, major diet of amphibians was closely associated with dung of yaks, for example, Aphodiidae, Bibionidae, Muscidae, dung beetle. Dung of yaks was major diet and habitat of these insects. Proportion of Aphodiidae was higher in diet composition of R. kukunoris than N. pleskei and B.minshanicus, with development of pasturage, R. kukunoris may have a diet competitive advantage over N. pleskei and B.minshanicus. Number of Aphodiidae in diet composition of amphibians was higher in samples with grazing (31.94%) than in those without grazing (21.32%). Occurrence Frequency of Aphodiidae in diet composition of amphibians was higher in samples with grazing (76.38%) than in those without grazing (44%). However, There was not significantly different on diet composition (P=0.188), and number (P=0.075) and the relative fatness (P=0.537) of amphibians between grazing samples and without grazing. 5.Competitive strategies of amphibian larvae I examined the competitive ability of larval toads (Bufo gargarizans) and frogs (Rana kukunoris) which co-occur in the nature pond by activity level, the growth rate and mass at metamorphosis and larval period in a laboratory experiment. The results suggest: In laborary, B.gargarizans adapted himself to different food level by changing activity. At high food level, B. gargarizans increased activity to gain more diet. At low food level, B. gargarizans decreased activity and achieved early metamorphosis. When food resource was limit, R. kukunoris could gain more food than B. gargarizans.

太阳活动变化对中低纬电离层的影响

With the variations of solar activity, solar EUV and X-ray radiations change over different timescales (e.g., from solar cycle variation to solar flare burst). Since solar EUV and X-ray radiations are the primary energy sources for the ionosphere, theirs variations undoubtedly produce significant and complicated effects on the ionosphere. So the variations of solar activity significantly affect the ionosphere. It is essential for both ionospheric theory and applications to study solar activity effects on the ionosphere. The study about solar activity variations of the ionosphere is an important part of the ionospheric climatology. It can enhance the understanding for the basic processes in the ionosphere, ionospheric structure and its change, ionosphere/thermosphere coupling, and so on. As for applications, people need sufficient knowledges about solar activity variations of the ionosphere in order to improve ionospheric models so that more accurate forecast for the ionospheric environments can be made. Presently, the whole image about the modalities of ionospheric solar activity variations is still unknown, and related mechanisms still cannot be well understood. This paper is about the effects of the 11-year change in solar activity to the low- and mid-latitude ionosphere. We use multi-type ionospheric observations and model to investigate solar activity effects on the electron density and ionospheric spatial structure, and we focus on discussing some related mechanisms. The main works are as follows: Firstly, solar activity variations of ionospheric peak electron density (NmF2) around 1400 LT were investigated using ionosonde observations in the 120°E sector. The result shows that the variation trend of NmF2 with F107 depends on latitudes and seasons. There is obvious saturation trend in low latitudes in all seasons; while in middle latitudes, NmF2 increases linearly with F107 in winter but saturates with F107 at higher solar activity levels in the other seasons. We calculated the photochemical equilibrium electron density to discuss the effects induced by the changes of neutral atmosphere and dynamics processes on the solar activity variations of NmF2. We found that: (1) Seasonal variation of neutral atmosphere plays an important role in the seasonal difference of the solar activity variations of NmF2 in middle latitudes. (2) Less [O]/[N2] and higher neutral temperature are important for the saturation effect in summer, and the increase of vibrational excited N2 is also important for the saturation effect. (3) Dynamics processes can significantly weaken the increase of NmF2 when solar activity enhances, which is also a necessary factor for the saturation effect. Secondly, solar activity variations of nighttime NmF2 were investigated using ionosonde observations in the 120°E sector. The result shows that the variation trends of NmF2 with F107 in nighttime are different from that in daytime in some cases, and the nighttime variation trends depend on seasons. There is linear increase trend in equinox nighttime, and saturation trend in summer nighttime, while the increase rate of NmF2 with F107 increases when solar activity enhances in winter nighttime (we term it with “amplification trend”). We discussed the possible mechanisms which affect the solar activity variations of nighttime NmF2. The primary conclusions are as follows: (1) In the equatorial ionization anomaly (EIA) crest region, the plasma influx induced by the pre-reversal enhancement (PRE) results in the change of the variation trend between NmF2 and F107 from “saturation” to “linear” after sunset in equinoxes and winter; while the recombination process at the F2-peak is the primary factor that affects the variation trend of NmF2 with F107 in middle latitudes. (2) The recombination coefficient at the F2-peak height reaches its maximum at moderate solar activity level in winter nighttime, which induces NmF2 attenuates more quickly at moderate solar activity level. This is the main reason for the amplification trend. (3) The change of the recombination process at the F2-peak with solar activity depends on the increases of neutral parameters (temperature, density et al.) and the F2-peak height (hmF2). The seasonal differences in the changes of neutral atmosphere and hmF2 with solar activity are the primary reasons for the seasonal difference in the variation trend of nighttime NmF2 with F107. Finally, we investigated the solar activity dependence of the topside ionosphere in low latitudes using ROCSAT-1 satellite (at 600 km altitude) observations. The primary results and conclusions are as follows: (1) Latitudinal distribution of the plasma density is local time, seasonal, and solar activity dependent. In daytime, there is a plasma density peak at the dip equator. The peak is obviously enhanced at high solar activity level, and the strength of the peak strongly depends on seasons. While at sunset, two profound plasma density peaks (double-peak structure) are found in solar maximum equinox months. (2) Local time dependence of the latitudinal distribution is due to the local time variation of the equatorial dynamics processes. Double-peak structure is attributed to the fountain effect induced by strong PRE. Daytime peak enhances with solar activity since the plasma density increases with solar activity more strongly at the dip equator due to the equatorial vertical drift, and its seasonal dependence is mainly due to the seasonal variations of neutral density and the equatorial vertical drift. In the sunset sector, seasonal and solar activity dependences of the latitudinal distribution are related to the seasonal and solar activity variations of PRE. (3) The variation trend of the plasma density with solar activity shows local time, seasonal, and latitudinal differences. That is different from the changeless amplification trend at the DMSP altitude (840 km). Profound saturation effect is found in the dip equator region at equinox sunset. This saturation effect in the topside ionosphere is realated to the increase of PRE with solar activity. Solar activity variation trend of the topside plasma density was discussed quantitatively by Chapman-α function. The result shows that the effect induced by the change of the scale height is dominant at high altitudes; while the variation trend of ROCSAT-1 plasma density with solar activity is suggested to be related to the changes of the peak height, the scale height, and the peak electron density with solar activity.

Effects of age and dietary protein level on digestive enzyme activity and gene expression of Pelteobagrus fulvidraco larvae

The present research studied the effects of age and dietary protein level on pepsin, trypsin and amylase activity and their mRNA level in Petteobagrus fulvidraco larvae from 3 to 26 days after hatch (DAH). Three DAH larvae were fed three isoenergetic diets, containing 42.8% (CP 43), 47.3% (CP 47) and 52.8% (CP 53) crude protein. Live food (newly hatched Artemia, unenriched) was included as a control. The effects of age on enzyme activity and mRNA were as follows: pepsin and trypsin activity in all treatment groups showed a significant (P < 0.05) increase at the beginning and decrease later although the timing of decrease was not the same among treatment groups and between the digestive enzymes. Pepsin and trypsin mRNA level followed the pattern of their respective enzyme changes. Age significantly affected amylase activity (P < 0.05) while age had no effect on amylase mRNA during the experimental period. The four diets significantly (P < 0.05) affected activity and mRNA level of pepsin and trypsin. Diets did not affect amylase activity or mRNA level. These results suggest that the effects of age on pepsin and trypsin gene expressions are at the transcriptional level. Dietary protein level does affect pepsin and trypsin gene expression in the early life of P. fulvidraco. There were no transcriptional effects on amylase gene expression. (c) 2005 Elsevier B.V. All rights reserved.

A selenium-containing abzyme, the activity of which surpassed the level of native glutathione peroxidase

Using two different glutathione derivatives as hapten, we have prepared two abzymes, which display glutathione peroxidase (GPX) activity. Their GPX activities are 0.2 and 1.6 times that of natural GPX from rabbit liver, respectively. Selenium content analysis indicates that the activity difference between the two abzymes is possibly attributed to the conformation difference of the abzymes.

Anti-HIV-1 activity of trichobitacin, a novel ribosome-inactivating protein

AIM: To determine whether trichobitacin, a novel ribosome-inactivating protein purified from the root tubers of Trichosanthes kirilowii, possesses the anti-HIV activity. METHODS: The inhibition of syncytial cell formation induced by human immunodeficiency virus type 1 (HIV-1),was determined under microscope, reduction of HIV-1 p24 antigen expression level was measured by ELISA, and decrease in numbers of HIV-1 antigen positive cells in acutely and-chronically infected cultures were detected by indirect immunofluorescence assay. RESULTS: Trichobitacin Was-found to greatly suppress syncytial cell formation induced by HIV-1 and to markedly reduce both expression of HIV-1 p24 antigen and the number of HIV antigen positive cells in acutely but not chronically HIV-1 infected culture. The median inhibitory concentration (IC50) in inhibition of syncytial cell formation and HIV antigen positive cells were 5 mu g.L-1 (95 % confidence limits: 1.3 - 20 mu g.L-1) and 0.09 mg.L-1 (95 % confidence limits: 0.011 - 0.755 mg.L-1), respectively. CONCLUSION: Trichobitacin is a novel ribosome-inactivating protein with anti-HIV-l activity.

Effect of CO2 concentrations on the activity of photosynthetic CO2 fixation and extracelluar carbonic anhydrase in the marine diatom Skeletonema costatum

The growth and activity of photosynthetic CO2 uptake and extracellular carbonic anhydrase (CA(ext)) of the marine diatom Skeletonema costatum were investigated while cultured at different levels of CO2 in order to see its physiological response to different CO2 concentrations under either a low (30 mumol . m(-2) . s(-1)) or high (210 mumol . m(-2) . s(-1)) irradiance. The changes in CO2 concentrations (4-31 mumol/L) affected the growth and net photosynthesis to a greater extent under the low than under the high light regime. CAext was detected in the cells grown at 4 mumol/L CO2 but not at 31 and 12 mumol/L CO2, with its activity being about 2.5-fold higher at the high than at the low irradiance. Photosynthetic CO2 affinity (1/K-1/2(CO2)) of the cells decreased with increased CO2 concentrations in culture. The cells cultured under the high-light show significantly higher photosynthetic CO2 affinity than those grown at the low-light level. It is concluded that the regulations of CA(ext) activity and photosynthetic CO2 affinity are dependent not only on CO2 concentration but also on light availability, and that the development of higher CA(ext) activity and CO2 affinity under higher light level could sufficiently support the photosynthetic demand for CO2 even at low level of CO2.

Influence of intraperitoneal injection of rare earth compounds on the activities of antioxidant enzymes and the level of lipid peroxidation in mice livers

Following intraperitoneal injection of lanthanum and terbium chloride and their complexes of diethyltriaminopentagacetic acid (DTPA) to adult mice with a dose of 0.28 mmol/kg body weight/day for three days. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the content of lipid end product, malonaldehyde (MDA) in the mice livers have been assayed respectively. The results show that the activity of SOD was increased and the content of MDA was reduced for LaCl3 treated mice and the two targets were not changed for TbCl3, but the activity of GSH-Px was reduced markedly for both LaCl3 and TbCl3 while the above three targets were not changed for La-DTPA and Tb-DTPA complexes.

A thioredoxin with antioxidant activity identified from Eriocheir sinensis

Thioredoxin, with a redox-active disulfide/dithiol in the active site, is the major ubiquitous disulfide reductase responsible for maintaining proteins in their reduced state. In the present study, the cDNA encoding thioredoxin-1 (designated EsTrx1) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsTrx1 was of 641 bp, containing a 51 untranslated region (UTR) of 17 bp, a 3' UTR of 306 bp with a poly (A) tail, and an open reading frame (ORF) of 318 bp encoding a polypeptide of 105 amino acids. The high similarity of EsTrx1 with Trx1s from other animals indicated that EsTrx1 should be a new member of the Trx1 sub-family. Quantitative real-time PCR analysis revealed the presence of EsTrx1 transcripts in gill, gonad, hepato-pancreas, muscle, heart and haemocytes. The expression of EsTrx1 mRNA in haemocytes was up-regulated after Listonella anguillarum challenge, reached the maximum level at 6 h post-stimulation, and then dropped back to the original level gradually. In order to elucidate its biological functions, EsTrx1 was recombined and expressed in E. coli BL21 (DE3). The rEsTrx1 was demonstrated to possess the expected redox activity in enzymatic analysis, and to be more potent than GSH in antioxidant capacity. These results together indicated that EsTrx1 could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to bacterial challenge. (C) 2009 Elsevier Ltd. All rights reserved.

High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a similar to6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use. (C) 2004 Elsevier Inc. All rights reserved.

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: Gene cloning, expression and activity analysis

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549 bp full-length cDNA of EsproPO contained a 2040 bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5'-untranslated region (UTR) of 68 bp, and a long 3'-UTR of 1441 bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2 h, 12 h and 48 h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab. (c) 2007 Elsevier Ltd. All rights reserved.