Yellowtail insulin-like growth factor 1: molecular cloning and response to various nutritional conditions.

Insulin-like growth factor 1 (IGF1) plays an important role in fish growth. This study investigated the IGF1 response to various nutritional conditions in yellowtail. First, we cloned 1,075 bp of yellowtail IGF1 cDNA, which codes for a protein of 185 amino acids (aa). This is composed of 44 aa for the signal peptide; 68 aa for the mature peptide comprising the B, C, A, and D domains; and 73 aa for the E domain. The mature yellowtail IGF1 showed high identity to IGF1 of other teleosts. Insulin-like growth factor 1 mRNA expression in the liver and white muscle was measured to observe the IGF1 response to various nutritional conditions, because the liver has the highest IGF1 expression and white muscle comprises the largest fraction of the fish body. Only white muscle IGF1 mRNA expression decreased significantly by 3 wk of fasting and recovered by refeeding. In subsequent feeding ratio (1%, 2%, and 3%/BW/d) experiments, significant correlations to growth were observed in white muscle IGF1 mRNA expression at 2- and 6-wk points and in hepatic IGF1 mRNA expression at 4 wk point. These data suggest that IGF1 expression both in hepatic and white muscle is important for somatic growth in yellowtail. Furthermore, white muscle IGF1 mRNA expression showed better responses to somatic growth and nutrition status in our two experiments than hepatic IGF1 mRNA expression.

Previtellogenic oocyte growth in salmon: relationships among body growth, plasma insulin-like growth factor-1, estradiol-17beta, follicle-stimulating hormone and expression of ovarian genes for insulin-like growth factors, steroidogenic-acute regulatory protein and receptors for gonadotropins, growth hormone, and somatolactin.

Body growth during critical periods is known to be an important factor in determining the age of maturity and fecundity in fish. However, the endocrine mechanisms controlling oogenesis in fish and the effects of growth on this process are poorly understood. In this study interactions between the growth and reproductive systems were examined by monitoring changes in various components of the FSH-ovary axis, plasma insulin-like growth factor 1 (Igf1), and ovarian gene expression in relation to body and previtellogenic oocyte growth in coho salmon. Samples were collected from females during two hypothesized critical periods when growth influences maturation in this species. Body growth during the fall-spring months was strongly related to the degree of oocyte development, with larger fish possessing more advanced oocytes than smaller, slower growing fish. The accumulation of cortical alveoli in the oocytes was associated with increases in plasma and pituitary FSH, plasma estradiol-17beta, and ovarian steroidogenic acute regulatory protein (star) gene expression, whereas ovarian transcripts for growth hormone receptor and somatolactin receptor decreased. As oocytes accumulated lipid droplets, a general increase occurred in plasma Igf1 and components of the FSH-ovary axis, including plasma FSH, estradiol-17beta, and ovarian mRNAs for gonadotropin receptors, star, igf1, and igf2. A consistent positive relationship between plasma Igf1, estradiol-17beta, and pituitary FSH during growth in the spring suggests that these factors are important links in the mechanism by which body growth influences the rate of oocyte development.

Metabolic hormones modulate the effect of growth hormone (GH) on insulin-like growth factor-I (IGF-I) mRNA level in primary culture of salmon hepatocytes.

Liver production of insulin-like growth factor-I (IGF-I) is a major point of control in the growth hormone (GH)/IGF axis, the endocrine system regulating body growth in fishes and other vertebrates. Pituitary GH stimulates hepatocyte production of IGF-I; however, in catabolic states, hepatocyte GH resistance results in decreases in liver IGF-I production. To investigate endocrine mechanisms leading to the development of hepatocyte GH resistance, we examined the regulation of IGF-I mRNA level by GH and metabolic hormones in primary culture of salmon hepatocytes. Cells were cultured in RPMI medium, and exposed to insulin (Ins, 10(-6) M), glucagon (Glu, 10(-6) M), triiodothyronine (T3, 10(-7) M), dexamethasone (Dex, 10(-6) M) and glucagon-like peptide (GLP, 10(-6) M), in the presence and absence of GH (5 x 10(-9) M). GH always increased IGF-I mRNA. None of the other hormones tested alone affected IGF-I mRNA. However, Dex, Ins and Glu reduced the response to GH. The response to GH was inhibited by Dex at concentrations of 10(-12) M and above, by Ins at 10(-9) M and above, and by Glu only at 10(-6) M. Inhibition of GH response by glucocorticoids is found in other vertebrates. Salmon hepatocytes were very sensitive to Dex, suggesting that glucocorticoids may play an important role in salmon growth regulation even in unstressed conditions. Inhibition of GH response by Ins is the opposite of what is found in mammals and chickens, suggesting that the role of Ins in growth regulation may differ between fishes and tetrapods. To examine mechanisms for modulation of GH sensitivity, we measured hepatocyte GH receptor (GHR) mRNA levels. Ins inhibited and Dex stimulated GHR mRNA, suggesting that different mechanisms mediate the inhibition of GH response by these hormones. This study shows that glucocorticoids, Ins, and Glu induce GH resistance in cultured salmon hepatocytes.

Salmon serum 22 kDa insulin-like growth factor-binding protein (IGFBP) is IGFBP-1.

Western ligand blotting of salmon serum typically reveals three insulin-like growth factor (IGF) binding proteins (IGFBPs) at 22, 28 and 41 kDa. Physiologic regulation of the 22 kDa IGFBP is similar to that of mammalian IGFBP-1; it is increased in catabolic states such as fasting and stress. On the other hand, its molecular mass on Western ligand blotting is closest to mammalian IGFBP-4. The conflict between physiology and molecular mass makes it difficult to determine the identity of the 22 kDa IGFBP. This study therefore aimed to identify the 22 kDa IGFBP from protein and cDNA sequences. The 22 kDa IGFBP was purified from chinook salmon serum by a combination of IGF-affinity chromatography and reverse-phase chromatography. The N-terminal aminoacid sequence of the purified protein was used to design degenerate primers. Degenerate PCR with liver template amplified a partial IGFBP cDNA, and full-length cDNA was obtained by 5'- and 3'-rapid amplification of cDNA ends (RACE). The 1915-bp cDNA clone encodes a 23.8 kDa IGFBP, and its N-terminal amino-acid sequence matched that of purified 22 kDa IGFBP. Sequence comparison with six human IGFBPs revealed that it is most similar to IGFBP-1 (40% identity and 55% similarity). These findings indicate that salmon 22 kDa IGFBP is IGFBP-1. Salmon IGFBP-1 mRNA is predominantly expressed in the liver, and its expression levels appear to reflect circulating levels. The 3'-untranslated region of salmon IGFBP-1 mRNA contains four repeats of the nucleotide sequence ATTTA, which is involved in selective mRNA degradation. In contrast, amino-acid sequence analysis revealed that salmon IGFBP-1 does not have an Arg-Gly-Asp (RGD) integrin recognition sequence nor a Pro, Glu, Ser and Thr (PEST)-rich domain (a segment involved in rapid turnover of protein), both of which are characteristic of mammalian IGFBP-1. These findings suggest that association with the cell surface and turnover rate may differ between salmon and mammalian IGFBP-1.

A quantitative real-time RT-PCR assay for salmon IGF-I mRNA, and its application in the study of GH regulation of IGF-I gene expression in primary culture of salmon hepatocytes.

The hormone insulin-like growth factor-I (IGF-I) regulates vertebrate growth. The liver produces most circulating IGF-I, under the control of pituitary growth hormone (GH) and nutritional status. To study the regulation of liver IGF-I production in salmon, we established a primary hepatocyte culture system and developed a TaqMan quantitative real-time RT-PCR assay for salmon IGF-I gene expression. A portion of the coho salmon acidic ribosomal phosphoprotein P0 (ARP) cDNA was sequenced for use as a reference gene. A systematic bias across the 96 well PCR plate was discovered in an initial IGF-I assay, which was corrected when the assay was redesigned. IGF-I mRNA levels measured with the validated assay correlated well with levels measured with an RNase protection assay, and were highest in liver compared with other tissues. We examined the time course of hepatocyte IGF-I gene expression over 48 h in culture, the response to a range of GH concentrations in hepatocytes from fed and fasted fish, and potential effects of variation in IGF-I in the medium. IGF-I gene expression decreased over time in culture in hepatocytes in plain medium, and in cells treated with 5 nM GH with or without a combination of metabolic hormones (1 microM insulin, 100 nM triiodothyronine, and 0.1 nM dexamethasone). GH stimulated IGF-I gene expression at all time points. In cells treated with GH plus metabolic hormones, IGF-I gene expression was intermediate between the controls and GH alone. Increasing concentrations of GH resulted in biphasic IGF-I gene expression response curves in cells from fed and fasted fish, with the threshold for stimulation from 0.5 to 2.5 nM GH, maximal response from 5 to 50 nM, and a reduced response at 500 nM. Medium IGF-I (5 nM) did not affect basal or GH stimulated IGF-I gene expression. This study shows that primary hepatocyte culture and the TaqMan IGF-I assay can be used to study the regulation of hepatic IGF-I gene expression in salmon, and provides the first evidence of a biphasic response to GH concentration in fish hepatocyte culture.

Development and validation of chemiluminescent immunoassay for vitellogenin in five salmonid species.

A highly sensitive and specific chemiluminescent immunoassay (CLIA) was developed for quantification of vitellogenin (Vg) in five salmonids. The CLIA for salmon Vg was performed using the two-site method, with anti-masu salmon beta'-component as primary antibody and chemiluminescent acridinium-labeled anti-rainbow trout lipovitellin F(ab)'(2) as the second antibody. Using cutthroat trout Vg as the standard, the working range of the CLIA was from 60 pg to 500 ng Vg/ml. Intra- and inter-assay coefficients of variation ranged from 3.04 to 6.67% and 3.23 to 5.86%, respectively. For the various salmonid species, serially diluted samples of serum from vitellogenic fish ran parallel to their purified Vg standard curve in the CLIA. In male cutthroat trout maturing during the 4 months before spawning, serum Vg levels ranged from 1.56 to 8000 ng/ml. High levels of Vg in some individuals may have resulted from temporary elevation of estradiol-17beta levels in the same fish during December or January (1-2 months before spawning). This is the first report on changes in serum Vg levels in maturing male trout using CLIA, the most sensitive assay for Vg yet developed.

Comparison of extraction methods and assay validation for salmon insulin-like growth factor-I using commercially available components.

Insulin-like growth factor-binding proteins (IGFBPs) may interfere with accurate measurement of plasma IGFs in radioimmunoassay (RIA). Although several simplified extraction methods for IGFs have been developed, these methods are not always validated for differing physiological states, developmental stages, and animal species. For teleost fish, neither the necessity of plasma extraction nor the validity of extraction methods for IGF RIA is widely established. We systematically examined the validity of acid-ethanol (AE) extraction, AE extraction followed by cryoprecipitation (AEC extraction), and SP-Sephadex extraction in RIA for salmon IGF-I using commercially available components (GroPep Pty Ltd). Displacement curves of plasma extracted by AE, AEC, and SP-Sephadex were parallel to those of the standard. Measured IGF-I levels in plasma from several developmental stages and under different physiological and experimental conditions were significantly increased by the extractions and comparable to those after acid-size exclusion chromatography (SEC). On Western ligand blotting using digoxigenin-labeled human IGF-I, the intensity of IGFBP bands remaining in plasma were reduced after extraction, although some IGFBPs remained. However, these residual IGFBPs did not interfere measurably with the RIA based on quantitative comparison of IGF-I levels with acid-SEC. We conclude that with this RIA extraction is necessary for measurement of salmon IGF-I in plasma since measured values were routinely lower in unextracted samples, and AE, AEC, and SP-Sephadex extractions are applicable to the IGF-I RIA using the commercially available components. Using the validated RIA for IGF-I, plasma IGF-I levels in nonmaturing and precociously maturing chinook salmon in spring were measured after AE extraction. During spring, nonmaturing and maturing fish fed and grew well, and plasma IGF-I level was significantly correlated with body weight in both fish. This result indicates that circulating IGF-I plays a key role in controlling growth in precociously maturing chinook salmon in spring as in nonmaturing fish.

Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12.

Chitinase A1 from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III-like domains, and a C-terminal chitin-binding domain (ChBD). In order to study the biochemical properties and structure of the ChBD, ChBD(ChiA1) was produced in Escherichia coli using a pET expression system and purified by chitin affinity column chromatography. Purified ChBD(ChiA1) specifically bound to various forms of insoluble chitin but not to other polysaccharides, including chitosan, cellulose, and starch. Interaction of soluble chitinous substrates with ChBD(ChiA1) was not detected by means of nuclear magnetic resonance and isothermal titration calorimetry. In addition, the presence of soluble substrates did not interfere with the binding of ChBD(ChiA1) to regenerated chitin. These observations suggest that ChBD(ChiA1) recognizes a structure which is present in insoluble or crystalline chitin but not in chito-oligosaccharides or in soluble derivatives of chitin. ChBD(ChiA1) exhibited binding activity over a wide range of pHs, and the binding activity was enhanced at pHs near its pI and by the presence of NaCl, suggesting that the binding of ChBD(ChiA1) is mediated mainly by hydrophobic interactions. Hydrolysis of beta-chitin microcrystals by intact chitinase A1 and by a deletion derivative lacking the ChBD suggested that the ChBD is not absolutely required for hydrolysis of beta-chitin microcrystals but greatly enhances the efficiency of degradation.

Lyotropic-salt-induced changes in monomer/dimer/tetramer association equilibrium of formyltransferase from the hyperthermophilic Methanopyrus kandleri in relation to the activity and thermostability of the enzyme.

Formyltransferase from Methanopyrus kandleri is composed of only one type of subunits of molecular mass 32 kDa. The enzyme requires the presence of lyotropic salts for activity and thermostability. We report here that the enzyme is in a monomer/dimer/tetramer association equilibrium, the association constant being affected by lyotropic salts. At 0.01 M K2HPO4/KH2PO4, pH 7.2, the enzyme (0.4 mg/ml) was mainly present in a monomeric form. Upon increase of the phosphate concentration, the concentration of the dimer increased up to a phosphate concentration of 0.6 M, then decrease at the expense of tetramer formation up to a phosphate concentration of 1.0 M. The specific activity at 4 C increased from <0.1 U/mg at 0.01 M, over 1.5 U/mg at 0.6 M to 3.6 U/mg at 1.0 M. Similar results were obtained with ammonium sulfate as lyotropic salt. The findings indicate that both oligomerization and activity increase with increasing salt concentrations, suggesting that there is a causal connection. To determine this, we exploited the observation that oligomer formation was not induced by the weak lyotropic salt NaCl up to a concentration of 1.5 M and that the dissociation of the dimer into the monomer at 4 degrees C proceeded very slowly (50% in approximately 6 h). This allowed us to study the effect of NaCl on the activity of the oligomers at NaCl concentrations not sufficient to induce oligomerization. At 4 degrees C, the activity of the oligomers increased from 0.3 U/mg at 0.25 M NaCl to 3.4 U/mg at 1.0 M NaCl. At these NaCl concentrations, the monomers were inactive. The findings indicate that oligomerization is a prerequisite for enzyme activity in the presence of NaCl. The salt-dependent induction of oligomerization was parallelled by an increase in thermostability; strong lyotropic salts conferred thermostability at much lower concentrations than the weak lyotropic NaCl.

Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride.

Enthalpy and heat capacity changes for the deprotonation of 18 buffers were calorimetrically determined in 0.1 M potassium chloride at temperatures ranging from 5 to 45 degrees C. The values of the dissociation constant were also determined by means of potentiometric titration. The enthalpy changes for the deprotonation of buffers, except for the phosphate and glycerol 2-phosphate buffers, were found to be characterized by a linear function of temperature. The enthalpy changes for the second dissociation of phosphate and glycerol 2-phosphate where divalent anion is formed on dissociation were fitted with the second order function of temperature rather than the first order. Temperature dependence of buffer pH calculated by using the enthalpy and heat capacity changes obtained was in good agreement with the temperature variation of the pH values actually measured in the temperature range between 0 and 50 degrees C for all the buffers studied. On the basis of the results obtained, a numeric table showing the temperature dependence of pK values for the 18 buffers is presented.

Immune complex transfer two-site chemiluminescent immunoassay for serum growth hormone in alevin chum salmon.

An immune complex transfer two-site chemiluminescent immunoassay (CLIA) for salmon growth hormone (GH) was developed to measure serum GH in alevin chum salmon (Oncorhynchus keta) using a chemiluminescent acridinium ester as a label. The immune complex transfer method dramatically reduced non-specifically bound of acridinium ester labelled antibody without a decrease in the specific binding. Consequently, we could detect lower levels of GH than achieved previously in a two-site CLIA for salmon GH. The detection limit of the assay was 7.8 fg/ml and the standard curve was linear up to 250 fg/ml. Coefficients of variation were 2.2-7.7% within-assay and 5.3-91% between-assay. We have developed a highly sensitive and reproducible GH method and applied it to measurement of GH in alevin chum salmon.

Isothermal titration calorimetric studies on the associations of putidaredoxin to NADH-putidaredoxin reductase and P450cam.

Putidaredoxin (Pdx), an iron-sulfur protein containing a 2Fe-2S cluster, serves as a physiological electron mediator from NADH-putidaredoxin reductase (PdR) to P450cam in the P450cam monooxygenation reaction cycle. Previous studies have revealed that the associations of Pdx with P450cam and PdR are not strongly dominated by electrostatic interactions, although such interactions stabilize most electron-transfer complexes [A.R. De Pascalis, I. Jelesarov, F. Ackermann, W.H. Koppenol, M. Hiroasawa, D.B. Knaff, H.R. Bosshard, Protein Sci. 2 (1993) 1126-1135]. In the present study, to elucidate the interactions dominating the specific associations in the electron-transfer reaction mediated by Pdx, the thermodynamic properties--entropy (delta S), enthalpy (delta H), and heat capacity changes (delta Cp)--for PdR/Pdx and P450cam/Pdx association reactions have been examined by isothermal titration calorimetry (ITC). Although the binding enthalpy change, delta Hbind, for the PdR/Pdx association is positive at 10 degrees C, it declines linearly with temperature in the range 10-22 degrees C and becomes negative above 11 degrees C. On the other hand, the binding entropy change, delta Sbind, is positive at all temperatures examined in this study, indicating that the association of Pdx to PdR is entropically driven. On the basis of the temperature dependence of delta Hbind, delta Cpbind for the association of Pdx to PdR was estimated as -1.24 kJ mol-1 K-1. This value is larger than those reported for other electron-transfer protein systems (e.g., -0.68 kJ mol-1 K-1 for ferredoxin/ferredoxin: NADP+ reductase), suggesting that the PdR/Pdx association may be dominated by hydrophobic rather than electrostatic components. For the P450cam/Pdx association, the negative delta Sbind and highly favorable delta Hbind were observed, behavior that stands in sharp contrast to the association reactions in other electron-transfer proteins. The energetics of the P450cam/Pdx association are similar to those of binding reaction of antibody to antigen in which van der Waals and hydrogen bonding interactions are dominant, resulting in high specificity in the association of Pdx with P450cam.

Development of an ELISA for chum salmon (Oncorhynchus keta) growth hormone.

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of low levels of serum growth hormone (GH) in chum salmon (Oncorhynchus keta). The antiserum to GH (a-rsGH) was obtained from a rabbit immunized with recombinant chum salmon GH. The noncompetitive ELISA was performed by a sandwich method using a-rsGH rabbit IgG as the first antibody, its biotinylated Fab' fragment as the second antibody, and the avidin-biotin reaction for signal amplification. This assay could be run in 3 days and routinely detected GH at concentrations as low as 0.5 ng/ml. The development of an ELISA for GH made possible quantification of serum GH levels. In this assay system, parallel dilution curves were obtained using purified chum salmon GH and GH's from several species of the genus Oncorhynchus.

Stress and social support in mental and physical health of Chinese students in Japan.

This study examined the influence of stress and social support on mental and physical health and happiness of 175 Chinese students enrolled in 13 Japanese universities. Needed support accounted for only 10% of the variance in reported stress, indicating that the relation between the two variables was not strong and they were generally independent. With greater scores on stress or needed support and lower scores on perceived or received support, depression and somatic complaints become more severe. The higher the scores on perceived or received support, the higher the reported happiness. Both perceived and received support showed a buffering effect on somatic complaints. Finally, stress and needed support had an interesting interaction, indicating that only among students reporting more stress did students who experienced greater need for support report more severe depression than those who experienced less need for support.

Chemiluminescent immunoassay (CLIA) for salmon growth hormone (GH).

A highly sensitive and specific chemiluminescent immunoassay (CLIA) was developed for quantification of growth hormone (GH) in salmonid species. The CLIA for salmon GH was performed using the sandwich method with anti-GH IgG as the first antibody and chemiluminescent acridinium ester-labelled specific anti-GH F(ab')2 as the second antibody. The measurable range of salmon GH in the CLIA was 39-1250 pg/mL using a short assay (1 day) protocol and 3.9-125 pg/mL in a longer (2-day) assay. The dilution curve in the CLIA of serum from masu salmon (Oncorhynchus masou) was parallel to the standard curve of recombinant chum salmon (Oncorhynchus keta) GH. Seasonal changes of serum GH levels were measured in 1 year-old masu salmon cultivated in a pond from March to November. Their serum GH levels increased during smoltification from March to April, achieved a maximum level of 21 ng/mL in August, and then declined gradually to 11 ng/mL in October.

The relationship between leadership and sociometric status among preschool children.

In the present study the relationship between leadership behavior and sociometric status among preschool children was examined. Leadership behaviors of 24 6-year-old Japanese children were observed during free play and scored on the basis of a 2-dimensional leadership scale (facilitation of play and consideration-evaluation of playmates; Fukada, Fukada, & Hicks, 1994). Participants were categorized into three sociometric-status groups. Children who had high sociometric status showed higher scores on both leadership dimensions than those with low sociometric status.

Transition of serum vitellogenin cycle in Sakhalin taimen (Hucho perryi).

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) and a single radial immunodiffusion (SRID) were developed for measurement of serum vitellogenin (Vg) levels in Sakhalin taimen (Hucho perryi). Regarding specificity for serum Vg, an antiserum raised against lipovitellin of taimen (a-Lv) was adequate for both assays. ELISA and SRID could detect Vg in serum at concentrations as low as 10 ng/ml and 25 micrograms/ml, respectively. In estrogen administration experiments, the level of serum Vg began clearly increasing within 12 to 24 hr after injection of immature females with estradiol-17 beta (E2). The appearance and levels of Vg in males treated with E2 were delayed and smaller, respectively, than for females. Vg levels varied throughout natural vitellogenesis from 0-4 micrograms/ml (3 years old) to approximately 30 mg/ml (5-6 years old). We observed an early transitory peak of serum Vg levels (primary reaction) at the time of early vitellogenesis and chronic high Vg levels (for 6-7 months) in winter period before ovulation. Changes of serum E2 levels were correlated with Vg levels. However, E2 levels decreased a month earlier than Vg levels near ovulation. It appears that the duration of vitellogenesis in taimen is considerably longer than that in other salmonids, lasting more than 2 years.