Seed vigour studies in corn, soybean and tomato in response to fish protein hydrolysates and consequences on phenolic-linked responses

Seed priming with fish protein hydrolysates (FPH) has been studied for the enhancement of seed vigour of corn, soybean and tomato. The influence of FPH at 2.5 mL/L and 5.0 mL/L on traditional agronomic parameters for seed vigour (germination percentage, seedling weight, seedling height) and potential new vigour-associated parameters (phenolic content, antioxidant activity, guaiacol peroxidase (GuPX) activity, chlorophyll content) was investigated. FPH treatment preferentially stimulated seedling vigour in the following order: soybean>tomato>corn. For soybean, FPH at 2.5 mL/L and 5 mL/L improved the majority of the seed vigour parameters (seedling weight and height, phenolic content, antioxidant activity and chlorophyll content, and lignification-associated GuPX activity). Similarly, for tomato, FPH at 2.5 mL/L stimulated seedling weight and height, GuPX activity and chlorophyll content. However, FPH did not stimulate corn seed vigour. Our results suggest an ability of proline precursor-rich FPH to improve of plant growth and development (e.g., seed vigour) in phenolic-rich plant species through modulation of phenolic and chlorophyll metabolisms.     

水解鱼蛋白对大菱鲆幼鱼消化率的影响

设计5组等氮等脂等能的饲料, 在室内流水系统进行68d的养殖实验, 探讨高植物蛋白饲料中添加不同分子量水解鱼蛋白对大菱鲆Scophthalmus maximus L.（16.050.03） g幼鱼消化能力的影响。分别在高植物蛋白饲料中添加5.4%超滤水解鱼蛋白（UF）、5.5%未经超滤水解鱼蛋白（FPH）、5.5%超滤截留水解鱼蛋白（RF）, 其均占饲料蛋白的10%, 以及不添加水解蛋白（PP）, 以上各组鱼粉含量均为18%, 对照组（FM）鱼粉含量为67.5%。研究结果表明, FM组的大菱鲆特定生长率与UF、FPH及PP组无显著差异（P0.05）; 饲料效率、蛋白质效率和蛋白质沉积率在FM组与UF组无显著差异（P0.05）, 但显著高于FPH、RF及PP组（P0.05）; 饲料干物质和蛋白质消化率在UF、FPH及RF组显著高于PP组（P0.05）, 但显著低于FM组（P0.05）, 在UF组显著高于FPH和RF组（P0.05）; 不同处理对饲料氨基酸和牛磺酸的消化率均产生显著影响（P0.05）, 趋势为FM组最高, 其次为UF组, PP组最低; 半胱氨酸和牛磺酸的消化率在添加水解鱼蛋白的3组（UF、FPH和RF组）和不添加水解鱼蛋白的2组（FM和PP组）呈相反的趋势。上述结果表明, 在高植物蛋白饲料中添加低分子量水解鱼蛋白（UF）, 大菱鲆幼鱼的生长和饲料利用有升高的趋势, 但UF、FPH以及RF都显著提高了大菱鲆对饲料干物质、蛋白质和氨基酸的消化率, 且UF效果优于FPH和RF。此外, 添加不同分子量水解鱼蛋白都降低了牛磺酸的消化率。       

Use of fish protein hydrolysate in milk replacers

Fish protein hydrolysate (FPH) has been used as the sole source of protein in milk replacers for lambs in several experiments. In comparison with casein, diets containing FPH gave slightly inferior growth rates during the first 2 weeks of a 5-week rearing period. The lower growth rate was generally compensated for in the following 3 weeks so that overall growth rate and food utilization were similar with milk replacers containing casein or FPH. Diets based on FPH, lard and hydrolysed starch were found to give results similar to a milk replacer based on casein, butterfat and lactose. The use of fat fish to provide both the source of fat and protein has received some attention and needs to be further investigated.     

In-vitro Production of Meiosis Inducing Substance (MIS) by Isolated Amphibian (Rana pipiens) Follicle Cells*

Previous studies indicated that pituitary hormone induced oocyte maturation in preovulatory amphibian ovarian follicles is mediated by somatic elements of the follicle. In this study procedures were developed for isolating and culturing follicle cells and their ability to produce meiosis inducing substance (MIS) was assessed. Defolliculated oocytes surrounded by a single layer of follicle cells but not denuded oocytes matured in response to frog pituitary hormone (FPH) stimulation. Cultured follicle cells secreted MIS following stimulation with FPH. The amount of MIS activity produced was related to the number of follicle cells cultured and the dose of FPH utilized. Radioimmunoassay (RIA) analysis of medium from follicle cell cultures demonstrated that FPH stimulated steroid (progesterone) secretion from these cells. Addition of cAMP to follicle cell cultures enhanced FPH stimulated steroid production. The results indicate that follicle cells retain FPH responsiveness when uncoupled from the immature oocyte and exhibit both MIS and steroid secretory functions.     

Effects of different dietary levels of fish protein hydrolysates on growth, digestive enzymes, gut microbiota, and resistance to Vibrio anguillarum in European sea bass (Dicentrarchus labrax) larvae

Two fish protein hydrolysates (FPH) were incorporated into four diets prepared for start-feeding sea bass larvae, at two different levels (10% and 19% of total ingredients): a commercial FPH, CPSP, in which the molecular mass of the main fraction of soluble peptides (51%) was between 500-2500 Da, and an experimental FPH obtained by acidic silage of sardine offal, SH, with a main portion of soluble peptides (54%) ranging from 200 to 500 Da. The diet with 10% of the commercial FPH gave the best results in terms of growth, survival and intestinal development, as evaluated by the early activity of digestive enzymes in the brush border membrane (alkaline phosphatase and aminopeptidase N). This was related to the low level of Vibrio spp. counted in the larvae of group C10. The high dose of FPH, especially in the experimental preparation rich in short peptides, seemed to favour the dominance of Vibrio sp. TYH3, which behaved opportunistically. The effect of the experimental FPH was ambiguous, since early larvae challenged with Vibrio anguillarum were more resistant to the pathogen, especially at high FPH dose (group S19). This might be due either to direct antagonism between V. anguillarum and Vibrio sp. TYH3, or to the stimulation of the immune response in the larvae. These results indicate that different molecular weight fractions and concentrations of feed-soluble peptides may affect the growth performance and immunological status of sea bass larvae. Consequently, a low dose of commercial FPH seems advisable, both for larval development and for the bacterial environment, although further research is required to determine and characterize peptide fractions that may have a beneficial effect on growth and immune response, and to determine their optimal inclusion levels in diets for sea bass larvae.     

鱼溶浆、酶解鱼溶浆和酶解鱼浆完全替代鱼粉对黄颡鱼生长的影响

为了探究鱼溶浆、酶解鱼溶浆和酶解鱼浆完全替代鱼粉后对黄颡鱼(Pelteobagrus fulvidraco)生长的影响, 在黄颡鱼实用型日粮配方模式下, 以28%的日本级蒸汽鱼粉为对照(FM), 按照FM组鱼粉蛋白质量的15%、30%和45%三个梯度, 分别添加鱼溶浆(SW15、SW30和SW45)、酶解鱼溶浆(SWH15、SWH30和SWH45)、酶解鱼浆(FPH15、FPH30和FPH45)完全替代鱼粉, 设计共10组等氮等能日粮, 每组设定3个重复, 在池塘网箱中养殖初始体重(18.68±0.10) g /尾黄颡鱼60d。结果显示: (1)以FM为对照, SW的3个试验组黄颡鱼的SGR降低9.89%—20.33%(P<0.05), FCR增加18.79%—44.85%(P<0.05); SWH试验组中SWH15和SWH45的SGR相对FM组分别降低8.79%和14.84%, FCR相对FM组增加16.97%和27.88%, 差异显著(P<0.05); FPH试验组中FPH15和FPH45的SGR相对FM组降低13.18%和15.38%, FCR相对FM增加30.30%和29.70%, 差异显著(P<0.05); (2)SWH30和FPH30组的SGR、FCR与FM组无显著差异(P>0.05); (3)相对于SW, 同等添加量SWH的SGR增加4.40%—9.39%, FCR降低9.81%—15.31%(P<0.05); (4)与FM组的FIFO值相比, SW30、SWH30和FPH30的FIFO值仅为0.67、0.61和0.60, 降低67.67%—70.15%(P<0.05)。结果表明: 在黄颡鱼日粮中, 8.5%添加量的酶解鱼溶浆(干物质)、8.2%添加量的酶解鱼浆(干物质)可以完全替代鱼粉(28%), 而相应的FIFO值分别比鱼粉组降低了69.95%和70.15%; 酶解鱼溶浆对黄颡鱼的生长效果优于鱼溶浆。日粮中过高或过低添加量的鱼溶浆、酶解鱼溶浆和酶解鱼浆都会导致黄颡鱼的生长速度和饲料效率下降。     

饲料中不同水平鱼蛋白水解物对军曹鱼稚鱼生长及体组成的影响

设计4种等能等氮试验饲料,对照组以鱼粉为单一蛋白源,处理组分别以鱼蛋白水解物替代鱼粉蛋白的17%、34%和51%,军曹鱼(Rachycentron canadum)稚鱼的初始重为(3.79±0.22)g,实验在室内流水养殖系统中进行6周的饲养试验,每种饲料随机3个重复。试验结果显示不同水平鱼蛋白水解物对试验鱼的存活率没有显著影响(P>0.05),除了以鱼蛋白水解物替代鱼粉蛋白51%组外,其他各组的增重率(WG)和饲料摄入量(FI)都是随着饲料中鱼蛋白水解物替代鱼粉蛋白量的增加而增加。试验结果表明,在军曹鱼稚鱼饲料中用鱼蛋白水解物替代34%的鱼粉蛋白对于军曹鱼稚鱼的生长和饲料摄入量具有一定的促进作用。军曹鱼稚鱼血浆中胆固醇含量随着饲料中鱼蛋白水解物添加水平的升高而升高,而血浆中的总蛋白含量则无此规律,各处理组血浆中的甘油三酯无显著差异(P0.05)。       

饲料中添加水解鱼蛋白对半滑舌鳎稚鱼生长及生理生化指标的影响

试验研究了饲料中添加不同水平(0%、20%、40%和60%)的水解鱼蛋白(Fish potein hdrolysate,FPH)对半滑舌鳎(Cynoglossus semilaevis Günther)稚鱼生长及生理生化指标的影响。28d饲养实验结束后,对试验鱼生长指标、鱼体消化酶活性、碱性磷酸酶(AKP)及肠道发育情况进行分析。结果显示:实验鱼的存活率为64.44%—78.88%,其中20%与40%添加组存活率显著高于其他两组(P0.05);实验鱼的特定生长率(SGR)随饲料中FPH添加水平升高呈下降趋势,20%添加组特定生长率最高,显著高于其他3组(P0.05),且60%添加组与对照组(FM)之间无显著差异(P0.05);各试验组鱼体消化酶(脂肪酶、淀粉酶、胃蛋白酶和胰蛋白酶)的比活力在14d与28d时的强弱关系不一致。鱼体碱性磷酸酶比活力大小依次为:20%组40%组对照组60%组,各组间差异均显著(P0.05);半滑舌鳎肠道微绒毛长度和黏膜厚度均以20%添加组为最优,显著优于其他3组(P0.05)。以上结果表明,在实验条件下,饲料中FPH的添加水平为20%时,能提高半滑舌鳎稚鱼的存活率及生长性能,并增强其对营养物质的吸收能力。     

Protein synthesis involvement in regulating pituitary-induced progesterone levels in ovarian follicles of Rana pipiens

Involvement of protein synthesis in frog pituitary homogenate (FPH)-induced progesterone production and/or accumulation in ovarian follicles was investigated. In amphibians, cycloheximide (C), an inhibitor of protein synthesis, inhibits progesterone and FPH-induced germinal vesicle breakdown (GVBD). However, the site and mechanisms of action of cycloheximide within ovarian follicles have not been elucidated. Intrafollicular progesterone produced by FPH is considered to mediate oocyte maturation; thus, cycloheximide may interfere with production and/or action of progesterone. Simultaneous treatment of FPH-stimulated follicles with cycloheximide inhibited FPH-induced progesterone accumulation (measured by RIA) and the accompanying-GVBD in a dose-dependent fashion. Inhibitory effects of cycloheximide on either FPH-induced progesterone production or GVBD were not reversed when follicles were washed and returned to fresh medium devoid of FPH and cycloheximide. However, subsequent restimulation of washed follicles with FPH resulted in increased progesterone levels and oocyte maturation. The extent of reversibility, in terms of GVBD and progesterone production, after FPH restimulation varied between animals. Pretreatment of follicles with cycloheximide for 6 hours, without FPH, had little or no effect on progesterone production when follicles were washed and treated with FPH. Delayed addition of cycloheximide to follicles following FPH stimulation blocked further progesterone accumulation as indicated by measurement of intrafollicular progesterone at the time of cycloheximide addition and at the end of the incubation period. The results indicate that cycloheximide rapidly inhibits progesterone production and that continuous protein synthesis is required for progesterone accumulation. Furthermore, protein synthesis does not appear to be required for progesterone metabolism since intrafollicular progesterone declined with prolonged culture even in the presence of cycloheximide. The nature of protein(s) involved in follicular progesterone production remains to be elucidated. FPH mediation of oocyte maturation within ovarian follicles appears to depend upon protein synthesis in somatic follicle cells, which is required for progesterone production, and in the oocyte, to mediate the response to the steroid trigger.     

Performance of calves fed on milk replacers containing fish protein hydrolysate

Milk substitutes for calves are based upon dried skim milk and added fats of vegetable or animal origin. Many attempts have been made to replace part of the skim milk in these feeds by cheaper sources of protein.A milk product coagulates in the calf's abomasum and has a high digestibility. Milk replacers based on fish protein hydrolysate (FPH) do not coagulate in the abomasum, but have a high apparent digestibility coefficient (0.92) and the amino acid balance is similar to that of skim milk.Calves were fed on milk replacers prepared from FPH made from various materials. In general, FPH prepared from white fish (cod, blue whiting, white fish offal) was satisfactory although calf performance until weaning was poorer than for calves fed on milk replacers with dried skim milk (SMP). Some ways are suggested by which the quality of the product (FPH) could be maintained. FPH preparations from fatty fish, like sprats and mackerel, were also used. An antioxidant was added in the preparation of sprats and mackerel because of the unsaturated nature of fish fats. Milk replacers containing sprats prepared without an antioxidant and mackerel (31% body fat) with antioxidant were unsatisfactory and further development work is required on FPH materials from fatty fish.FPH from white fish can be dried without any appreciable loss in calf performance. The daily gain to weaning was 0.25 and 0.29 kg per day for calves fed on the dried and undried material, respectively. On the basis of present knowledge it is suggested that FPH can replace one third of skim milk in milk replacers for early weaned calves; the proportion recommended may rapidly increase with further technical development of the product.     

Enhancement of seed vigour following insecticide and phenolic elicitor treatment

Thiamethoxam (CGA 293'343) is a novel broad-spectrum neonicotinoid insecticide. It is commercially used as a seed treatment under the trademark Cruiser (CRZ). Although many reports detail its insecticidal, plant-protecting properties, there are minimal reports concerning the effect on seed germination activities which can be key control points of seedling vigour. In this report, we investigated the effect of CRZ, fish protein hydrolysates (FPH; a known elicitor of pentose-phosphate pathway) and the combination of CRZ and FPH (CF) on seed vigour of pea, soybean and corn. Seed vigour was investigated by estimating germination percentage, shoot height, shoot weight, total soluble phenolic content, antioxidant content, G6PDH (glucose-6-phosphate dehydrogenase) activity, and GPX (guaiacol peroxidase) activity. Addition of FPH to CRZ (CF) seemed to have a slightly positive effect on seed vigour, especially, CF and FPH treatment for corn and FPH treatment for pea, suggesting that pre-sowing treatments may cause positive/negative effects on seed vigour, depending on the concentration of treatments. Further research will be needed to determine their effects and the optimal concentration for seed priming.     

Functional,bioactive and antioxidative properties of hydrolysates obtained from cod (Gadus morhua) backbones

Fish protein hydrolysates (FPH) have good and well documented functional properties. Peptides obtained from various fish protein hydrolysates have also shown bioactive and antioxidative activities.The aim of this study was to evaluate how storage and preparation of cod (Gadus morhua) backbones influence the yield, functionality, bioactivity (CGRP and gastrin/CCK related molecules) and antioxidative properties of fish protein hydrolysates. A series of hydrolysis trials have been carried out using backbones from cod that were initially fresh or frozen and further hydrolysed for different times (10, 25, 45 and 60 min). Use of fresh raw material significantly increased yield of dry FPH, gave lighter and less yellow powders with better emulsification properties. Longer time of hydrolysis gave higher FPH yield, increased degree of hydrolysis and decreased water holding capacity of the powders. Among the hydrolysis times tested, 25 and 45 min hydrolysis demonstrated the best emulsification properties.FPH have potential to enhance product stability by preventing oxidative deterioration. The DPPH scavenging activity showed that antioxidative activity of hydrolysates could be due to the ability to scavenge lipid radicals. The ability of hydrolysates to inhibit iron induced lipid oxidation was not influenced by time of hydrolysis.This work also shows that it is possible to obtain bioactive molecules from cod backbones by protein hydrolysis. The content of bioactive peptides (gastrin/CCK- and CGRP-like peptides) could make the cod hydrolysates useful for incorporation in functional foods.     

Biosynthesis of protease by Pseudomonas aeruginosa MN7 grown on fish substrate

Fish powders and fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were prepared and tested as growth media for alkaline protease production by Pseudomonas aeruginosa MN7. Cultivated in fish substrate as carbon source, the strain exhibited a slightly greater protease production (about 7800 U ml^–1) than that obtained with commercial peptones (about 7222 U ml^–1). Furthermore, P. aeruginosa MN7 produced the same amount of protease when cultivated in medium containing only fish substrate or that containing all ingredients, indicating that the strain can obtain its carbon and nitrogen requirements directly from whole fish proteins. Moreover, it was found that extensive hydrolysis of fish proteins did not increase protease formation. Protease production in media containing only FPH prepared by Alcalase was about 70% of those obtained with MN7 protease digest of fish protein or with meat-fish powder. These results indicate that sardinella substrates are an excellent carbon and nitrogen source for the growth of P. aeruginosa MN7 and the production of protease.     

Antihyperlipidemic and antioxidant effects of feather protein hydrolysate in high‐fat diet‐fed mice

The hyperlipidemia is a serious health problem that increases the risk of many complications including cardiovascular disease. This study aims to evaluate the possible antihyperlipidemic effects of the feather protein hydrolysate (FPH) in a mice fed with a high‐fat diet (HFD)‐fed mice during 5 weeks. The FPH administration improved dose‐dependent lipid profile, as well as the liver and renal dysfunction indices in hyperlipidemic mice. The FPH also restored the antioxidant status in liver, kidney, and heart by lowering the lipid peroxidation and enhancing the antioxidant enzymes (catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase [SOD]). Moreover, the histological studies proved that FPH administration prevents hepatic steatosis, glomerular hyperfiltration risk, and cardiac muscle hypertrophy. Accordingly, the FPH is a promising novel medicinal ingredient for possible use in the hyperlipidemic treatment and related complications.     

Gain-of-Function Mutation of KIT Ligand on Melanin Synthesis Causes Familial Progressive Hyperpigmentation

Familial progressive hyperpigmentation (FPH) is an autosomal-dominantly inherited disorder characterized by hyperpigmented patches in the skin, present in early infancy and increasing in size and number with age. The genetic basis for FPH remains unknown. In this study, a six-generation Chinese family with FPH was subjected to a genome-wide scan for linkage analysis. Two-point linkage analysis mapped the locus for FPH at chromosome 12q21.31-q23.1, with a maximum two-point LOD score of 4.35 (Ø = 0.00) at D12S81. Haplotype analysis confined the locus within an interval of 9.09 cM, flanked by the markers D12S1667 and D12S2081. Mutation profiling of positional candidate genes detected a heterozygous transversion (c. 107A→G) in exon 2 of the KIT ligand (KITLG) gene, predicted to result in the substitution of a serine residue for an asparagine residue at codon 36 (p.N→S). This mutant “G” allele cosegregated perfectly with affected, but not with unaffected, members of the FPH family. Function analysis of the soluble form of sKITLG revealed that mutant sKITLGN36S increased the content of the melanin by 109% compared with the wild-type sKITLG in human A375 melanoma cells. Consistent with this result, the tyrosinase activity was significantly increased by mutant sKITLGN36S compared to wild-type control. To our knowledge, these data provided the first genetic evidence that the FPH disease is caused by the KITLGN36S mutation, which has a gain-of-function effect on the melanin synthesis and opens a new avenue for exploration of the genetic mechanism of FPH.     

Cholesterol mediation of progesterone production and oocyte maturation in cultured amphibian (Rana pipiens) ovarian follicles

Treatment of isolated amphibian ovarian follicles with frog pituitary homogenate (FPH) increases follicular progesterone levels, which, in turn, initiate oocyte maturation. Recent studies have demonstrated that follicular progesterone production requires concomitant protein synthesis at some stage preceding pregnenolone formation. Experiments were carried out to determine whether cholesterol metabolism plays a role in mediating these biochemical and physiological processes. Aminoglutethimide (AGI, and inhibitor of P450 side-chain cleavage enzyme) inhibited FPH-induced intrafollicular progesterone accumulation and oocyte maturation (or germinal vesicle breakdown, GVBD) in a dose-dependent manner. Follicular progesterone accumulation and GVBD were both stimulated, in the absence of FPH, after addition of 25-OH-cholesterol, but not cholesterol, to the culture medium. Higher levels of progesterone were present in defolliculated oocytes as compared to intact ovarian follicles after incubation with 25-OH-cholesterol. The results indicate that the surface epithelium and theca layer in the follicle wall retard 25-OH-cholesterol access to steroid-producing follicle cells. AGI blocked 25-OH-cholesterol-induced accumulation of progesterone and GVBD in defolliculated oocytes, suggesting that 25-OH-cholesterol does not directly induce GVBD and is metabolized by the follicle cells. The capacity of follicles to accumulate progesterone following preincubation with FPH or 25-OH-cholesterol along with AGI was compared. Intrafollicular levels of progesterone increased after AGI- and 25-OH-cholesterol-treated follicles were washed. In contrast, progesterone levels decreased in follicles pretreated with AGI and FPH after washing. The results indicate that considerable 25-OH-cholesterol, but not endogenous cholesterol (FPH stimulation), remains available for steroidogenesis after removal of AGI. A significant, but incomplete, inhibition of progesterone accumulation occurred when follicles were incubated in the presence of 25-OH-cholesterol and cycloheximide. This partial blockage produced by the protein synthesis inhibitor indicates that some basal protein synthesis is required for progesterone accumulation from exogenous 25-OH-cholesterol. We conclude that intracellular cholesterol stores in the follicle wall are utilized to mediate FPH induction of progesterone accumulation and oocyte maturation in amphibian follicles.     

Role of cAMP in modulating intrafollicular progesterone levels and oocyte maturation in amphibians (Rana pipiens)

The role of cAMP in regulating follicular progesterone levels and oocyte maturation was investigated following in vitro culture of amphibian (Rana pipiens) ovarian follicles. Intrafollicular levels of cAMP were manipulated with the use of a stimulator of cAMP synthesis (forskolin) or by exogenous addition of cAMP alone or either of these in combination with an inhibitor of cAMP catabolism (3-isobutyl-1-methyl xanthine, IBMX). Follicular progesterone content was determined by RIA and oocyte maturation was assessed cytologically. In the presence of increasing doses of forskolin (0-3 microM), cAMP (0-3 mM), or dibutyryl cAMP (dbcAMP, 0-2.5 mM) increasing but low levels of progesterone were detected. Increasing doses of IBMX (0-0.09 mM) alone had no significant effect on follicular steroid content. Exogenous cAMP, dbcAMP, or IBMX (0.09 mM) suppressed hormone-induced oocyte maturation. Simultaneous exposure of follicles to increasing doses of both forskolin (0-3 microM) and IBMX (0-0.09 mM) markedly increased intrafollicular progesterone levels to those produced by frog pituitary homogenate (FPH). A marked increase in progesterone levels also occurred when follicles were exposed to exogenous cAMP (3 mM) and IBMX (0.09 mM). These results indicate that exogenous cAMP is incorporated by follicle cells and that forskolin effects are mediated through cAMP. Changes in follicular progesterone levels (increase and decrease) over time following FPH or cAMP manipulation (cAMP + IBMX or forskolin + IBMX) were essentially identical. In contrast to cAMP, cGMP was inactive in inhibiting hormone induced GVBD or stimulating follicular progesterone accumulation. Elevation of follicular and medium levels of progesterone resulting from FPH or cAMP stimulation required the presence of the somatic follicular cells. The decrease in follicular progesterone levels with prolonged culture was not associated with a corresponding increase in progesterone levels in the medium. The decrease in follicular progesterone levels appears to reflect steroid catabolism rather than loss of steroid to the culture medium. The results suggest that the level of intracellular cAMP in the follicle cells is modulated by the relative activity of the adenylate cyclase system and phosphodiesterase and that FPH can affect both components. Thus, intracellular levels of cAMP play a key role in regulating follicular progesterone levels and FPH action on the follicle cells. The steroidogenic capacity of follicle cells can be manipulated independently of FPH stimulation.     

Induction of ovulation and oocyte maturation of amphibian (Rana dybowskii) ovarian follicles by protein kinase C activation in vitro.

We previously reported that protein kinase C (PKC) activation induced meiotic maturation (germinal vesicle breakdown, GVBD) of Rana dybowskii follicular oocytes cultured in vitro without hormone treatment. The experiments reported here were carried out to establish whether ovarian follicles ovulated in response to PKC activation during culture. A phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was used for PKC activation. TPA addition (10 microM) to cultured ovarian fragments induced ovulation and maturation of the oocytes similar to that seen following addition of frog pituitary homogenate (FPH, 0.05 pituitary/ml) or progesterone (0.5 microgram/ml). Such changes were not observed when ovarian fragments were treated with inactive phorbol ester. The time course of TPA-induced ovulation was similar to that produced by FPH-stimulated ovulation. Both TPA- and FPH-stimulated ovulation and maturation were blocked by treatment with cycloheximide, forskolin (an adenylate cyclase stimulator), and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7; a PKC inactivator). FPH treatment markedly increased progesterone levels in the medium during ovarian fragment culture whereas TPA treatment failed to elevate progesterone levels. Thus, TPA treatment mimics FPH and progesterone in inducing ovulation and meiotic maturation in cultured amphibian ovarian fragments. The data strongly suggest that PKC plays an important role in regulating ovulation as well as in modulating amphibian oocyte maturation during follicular differentiation.     

Intrinsic properties of muscle satellite cells are changed in response to long-term selection of mice for different growth traits

Satellite cell cultures were derived from mice selected long-term over 70 generations for body weight (DU-6, growth), carcass protein amount (DU-6P, protein) and an index combining body weight and endurance treadmill performance (DU-6+LB, growth + fitness) at 42 days of age and from an unselected control line (DU-Ks). They were grown under identical environmental conditions to examine intrinsic cellular differences in proliferation, protein metabolism and responsiveness to growth factors. Growth kinetics (DNA and protein amounts) were determined over a 12-day period. During exponential growth, all growth-selected cultures grew faster than the control culture: (DU-6+LB=DU-6P)>DU-6>DU-Ks. The differences in DNA and protein levels were maintained until day 8. DU-Ks cultures reached similar levels as the growth (DU-6) and protein (DU-6P) cultures in terms of DNA at day 12 of cultivation. Thus, the cultures from the growth and protein lines, but not from the growth + fitness line, exhibited larger protein:DNA ratios (cell size) than the control cultures. Cell cultures from the selected lines were more responsive to serum and epidermal growth factor in terms of [(3)H] thymidine incorporation into DNA, whereas no stimulation by insulin or insulin-like growth factor-I was detectable in cultures from selected lines or controls. During differentiation, protein metabolism in cultures from selected lines was characterised by higher rates of protein synthesis (PS) and degradation (PD), as measured by [(3)H] phenylalanine incorporation or release, respectively, than in control cells. The ratios of the relative differences from the control in PS and PD were only >1.0 in the growth and protein lines. In conclusion, long-term selection for growth therefore modifies the intrinsic capability of satellite cells for proliferation and protein metabolism, with changes being dependent on the selection trait.     

Changes in community composition during dilution cultures of marine bacterioplankton as assessed by flow cytometric and molecular biological techniques

Dilution cultures are a common technique for measuring the growth of bacterioplankton communities. In this study, the taxonomic composition of marine bacterioplankton dilution cultures was followed in water samples from Plymouth Sound and the English Channel (UK). Bacterial abundances as well as protein and DNA content were closely monitored by flow cytometry. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments and fluorescence in situ hybridization (FISH) were applied directly to the water samples and to cells sorted from the dilution cultures based on their protein and DNA content. As expected, a rapid activation of bacteria occurred. However, molecular techniques showed that the community developed in the dilution culture within 1 day was significantly different from that in the original water samples. Whereas in the original samples, cells detectable by FISH were dominated by members of the C ytophaga / Flavobacterium (CF) cluster, in dilution cultures, gamma-proteobacteria accounted for the majority of cells detected, followed by alpha-proteobacteria. An actively growing and an apparently non-growing population with average cellular protein contents of 24 and 4.5 fg respectively, were sorted by flow cytometry. FISH indicated mostly gamma- (64%) and alpha-proteobacteria (33%) in the first active fraction and 78% members of the CF cluster in the second fraction. Sequencing of DGGE bands confirmed the FISH assignments of the latter two groups. The data presented clearly show that even relatively short-term dilution experiments do not measure in situ growth, but rather growth patterns of an enrichment. Furthermore, it was demonstrated that the combination of flow cytometric analysis and sorting combined with FISH and DGGE analysis presented a fairly rapid method of analysing the taxonomic composition of marine bacterioplankton.