Mechanism of oocyte maturation and ovulation and its application to seed production in the Japanese eel.

Reduction in eel resources and catches of glass eels as seedlings for aquaculture have been a serious concern in recent years in both Europe and East Asia. Thus, technical advancement to produce eel seeds for artificial cultivation is most desired. Fundamental information on oocyte maturation and ovulation and its application to artificial induction of sexual maturation are needed to produce good quality seeds of the Japanese eel. This review introduces hormonal mechanisms of cytoplasmic maturation (such as hydration, lipid coalescence, and clearing of the ooplasm) and the maturational competence (the ability to respond to maturation-inducing steroid) and nuclear maturation (germinal vesicle breakdown). In addition, previous and newly developed methods for induction of spawning have been described.

Development of a single-tube PCR-pyrosequencing method for the simultaneous and rapid detection of four variant alleles of CYP2C9 gene polymorphism.

BACKGROUND AND OBJECTIVE: CYP2C9 is a polymorphic enzyme that has been reported to metabolize several clinically useful drugs such as warfarin, phenytoin and non-steroidal anti-inflammatory drugs. We designed a rapid single-tube multiplex assay to detect four variant alleles of the CYP2C9 in a single polymerase chain reaction (PCR) and a single pyrosequencing reaction. METHODS: A multiplex PCR was designed to amplify two fragments simultaneously, one containing 430C>T (CYP2C9*2) polymorphism and other containing 1075A>C (CYP2C9*3), 1076T>C (CYP2C9*4) and 1080C>G (CYP2C9*5) polymorphisms. RESULTS: Four variants of the CYP2C9 gene could be simultaneously detected using only two varieties of pyrosequencing primers in a single-tube. The success rate for the four SNPs (*2, *3,*4 and *5) was high. Genotypes obtained by the multiplex reaction were 100% concordant with genotypes obtained using direct DNA sequencing (n = 96). The analysis time was halved, compared with existing simplex pyrosequencing. The system allowed high-throughput analysis of over 384 samples per hour. DISCUSSION: Our method reduces running cost and halves analysis time, compared to simplex pyrosequencing. Another advantage of this method is that it analyses and determines multiple bases around the polymorphic site thereby reducing the possibility of scoring a truncated PCR product.

Enhancement of ganglioside GM3 synthesis in okadaic-acid-treated granulosa cells.

Okadaic acid is a potent inhibitor of type-2A (PP2A) and type-1 (PP1) protein phosphatases and has been proved to be a valuable tool for studies on the protein phosphorylation. We have investigated the effects of okadaic acid on rat granulosa cells in order to determine whether the regulation of ganglioside synthesis involves protein phosphorylation via inhibition of PP2A and PP1. Granulosa cells expressed luteinizing hormone (LH) receptors, measured as the binding of 125I-deglycosylated human chorionic gonadotropin (hCG) to intact cells, and synthesized the gangliosides NeuAc alpha 2-->3Gal beta 1-->4Glc beta 1-->1Cer (GM3) and Gal beta 1-->3GalNAc beta 1-->4[NeuAc alpha 2-->3]Gal beta 1-->4Glc beta 1-->1Cer (GM1), demonstrated by metabolic labeling of glycosphingolipids with [3H]galactose, in response to follicle-stimulating hormone (FSH). When FSH-stimulated granulosa cells were treated with 10 nM okadaic acid for 15 h, down-regulation of LH receptors, dissociation of LH receptor-effector coupling and significant decreases of intracellular and extracellular 3',5'-cyclic adenosine monophosphate (cAMP) levels were observed. The okadaic acid-induced desensitization to gonadotropin in granulosa cells was accompanied by increased ganglioside synthesis. The amount of 3H-labeled ganglioside GM3, the major ganglioside (about 95% of the total) synthesized by mature granulosa cells, was enhanced in okadaic acid-desensitized cells (to 215% of the control value) and in those desensitized by hCG (by 354%), forskolin (by 190%) and 12-O-tetradecanoylphorbol 13-acetate (by 143%). The results of this study suggest that an increase in the phosphorylation state of cells is accompanied by enhancement of ganglioside synthesis.

Granulosa cell luteinizing hormone receptor expression is modulated by ganglioside-specific ligands.

The ganglioside GM1 (Gal beta 1-->3GalNAc beta 1-->4[NeuAc alpha 2-->3] Gal beta 1-->4Glc beta 1-->1Cer) was synthesized during granulosa cell development in vitro, and the effect of the interaction between cell-surface GM1 and its ligands on the luteinizing hormone (LH) receptor expression was investigated. GM1 synthesis, demonstrated by metabolic labeling of glycosphingolipids with [3H]galactose and binding studies using the 125I-B-subunit of cholera toxin, a specific ligand for GM1, was increased in follicle-stimulating hormone (FSH)-treated granulosa cells. When granulosa cells were cultured for 72 h in a medium containing the B-subunit of cholera toxin, FSH-induced LH-receptor contents determined by measuring the binding of 125I-deglycosylated human chorionic gonadotropin to intact cells, was augmented. The stimulatory effect of the B-subunit was dependent on the FSH concentration and culture duration. The augmentation was observed after culture for 48 h, and marked increases were evident after 72 h, which coincided with an increase of the 125I-B-subunit binding capacity. Scatchard analysis of the LH-receptor binding indicated that treatment with the B-subunit increased the number of LH-binding sites (6580 sites/cell after treatment with 20 ng/ml FSH; 11,290 sites/cell after FSH plus 100 ng/ml B-subunit), but did not alter the binding affinity. A specific antibody against GM1 mimicked the stimulatory effect of the B-subunit. The augmentation was not accompanied by granulosa cell proliferation. These findings suggest that binding of exogenous or possible endogenous ligands to cell-surface GM1 produces signals and modulates the cellular behavior during granulosa cell development.

Inhibitory effect of dibutyryl cyclic adenosine monophosphate on the induction of alkaline phosphatase in human fetal liver cell line.

When human fetal liver cells (HuL-1-317), cultured continuously in a serum-free medium, were incubated with a combination of prednisolone, butyrate and a hypertonic concentration of NaCl at 37 degrees C, alkaline phosphatase activity increased. However, the addition of dibutyryl adenosine cyclic monophosphate (Bt2cAMP) to these agents inhibited the increase in alkaline phosphatase activity in a dose-dependent manner: the inhibitory effect of Bt2cAMP was significant at 0.05 mM, but disappeared at 0.01 mM. Both cycloheximide and actinomycin D inhibited the increase in alkaline phosphatase activity with the combination described above. Western blotting showed that this enzyme activity increase was a consequence of greater biosynthesis of enzyme molecules in HuL-1-317 cells, and that Bt2cAMP regulated the synthesis of enzyme molecules. We conclude that the changes in alkaline phosphatase activity under various conditions are based on the changes in the number of enzyme molecules in HuL-1-317 cells.

Identification of protein disulfide isomerase and calreticulin as autoimmune antigens in LEC strain of rats.

Long Evans Cinnamon (LEC) rats, showing spontaneous hereditary hepatitis and hepatic carcinoma, were found to possess autoimmune antibodies to liver microsomal proteins, particularly to proteins with the molecular weight of 56kD and 55kD. The antibodies occurred in association with acute lethal hepatitis in the LEC rats in our previous study. Two-dimensional immunoblot analysis of the antigenic proteins revealed that the 56kDa and 55kDa proteins showed 4.2 and 4.0 pI values and were estimated to be protein disulfide isomerase (PDI) and calreticulin, respectively, from NH2-terminal amino acid sequence analysis. These proteins were further identified by immunoblot analyses using purified proteins and specific antibodies. PDI was a major autoimmune antigenic protein.

Effect of thyrotropin on cell orientation and follicle reconstruction in rotated suspension culture of hog thyroid cells.

Isolated porcine thyroid cells were cultured in suspension by rotation on a gyratory shaker. In the absence of TSH, the cells associated into small, globular, and hollow aggregates or pseudofollicles and had microvilli only at the outer surface of the pseudofollicles which faced the culture medium. These pseudofollicles were inactive in terms of thyroglobulin synthesis and iodination. In the presence of TSH, cell aggregates were enlarged to 1 mm in diameter. Inside the aggregates. Inside the aggregates, the cells were reorganized into numerous functional follicles in which microvilli were oriented towards the follicular lumen. In both the presence and absence of TSH, Golgi apparatuses were found in the cytoplasm near the cell periphery bearing microvilli. These findings indicate that the TSH effects on thyroid cell morphology are to arrange the polarity of follicular cells in a proper direction and to enlarge the aggregates they form.

Dualistic effects of thyroid hormone on a human hepatoma cell line: inhibition of thyroxine-binding globulin synthesis and stimulation of alpha 1-acid glycoprotein synthesis.

In order to examine the action of thyroid hormone on the secretory proteins of the liver, we investigated the effects of thyroid hormones on the synthesis of T4-binding globulin (TBG), alpha 1-acid glycoprotein (AGP), and albumin in a human hepatoblastoma cell line, Hep G2. Hep G2 cells grown to be confluent in medium with 10% fetal calf serum were further cultured in serum-free medium for 4 days, and followed by treatment with hormones for 2 days changing the medium every 24 h. On day 2 (the second 24 h of hormone treatment), about 30% of TBG accumulation was inhibited by 10(-12) M T3 and 50% was inhibited by 10(-8) M T3, although no change was observed on day 1 (the first 24 h of hormone treatment). This inhibitory effect of T3 closely resembled the effect of T3 on [35S]methionine-labeled TBG synthesis by the cells incubated for 3 h after 42 h of pretreatment. About 30-55% of the newly synthesized [35S]TBG immunoprecipitated with anti-TBG serum was inhibited by 10(-8) M T3. These results showed that thyroid hormone inhibited TBG synthesis in Hep G2 cells. On the other hand, T3 stimulated the accumulation of AGP in the media on day 1 (140% of control by 10(-8) M T3), and the effect increased drastically on day 2 (250% of control by 10(-8) M T3). No effect of T3 on albumin accumulation or total protein synthesis was seen. The concentrations of T4 which had significant effects on TBG and AGP accumulation were 10 and 10(3) times higher than those of T3, respectively. In conclusion, thyroid hormone has dualistic effects on the secretory proteins synthesized by a human hepatoblastoma cell line: physiological concentrations of thyroid hormones decrease the synthesis of TBG, but increase the synthesis of AGP.

Purification and characterization of a 3,5,3'-L-triiodothyronine-specific binding protein from bullfrog tadpole plasma: a homolog of mammalian transthyretin.

We analyzed the nature of the thyroid hormone-binding proteins in bullfrog plasma using N-bromoacetyl-[125I]T3 as an affinity labeling probe. Polyacrylamide gel electrophoresis under nondenaturing conditions of the bullfrog N-bromoacetyl-[125I]T3-labeled plasma proteins revealed two proteins with specific binding to T3. A labeled protein that migrated faster than albumin (T-T3BP) was detected only in plasma obtained from tadpoles at stages earlier than, but not at the end of, metamorphic climax. Another protein that migrated more slowly than albumin appeared in the plasma at the late climax stage and was present in the adult stage. To study the function of T-T3BP during metamorphosis, it was purified from tadpole plasma to the single protein. The molecular mass of this protein was estimated to be 56 kilodaltons by gel filtration, but only 16 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which indicates that the molecule comprised four identical subunits. The amino acid composition of T-T3BP and the amino acid sequence of its N-terminal portion were highly homologous with those of mammalian transthyretins. These molecular features indicate that T-T3BP is a homolog of mammalian transthyretins. However, in contrast to mammalian transthyretins, the affinity of bullfrog transthyretin for T3 was 360 times higher than that for T4. Scatchard analysis revealed that T-T3BP possessed a single class of T3-binding site, with a Kd of 0.67 nM at 0 C. These results suggest that bullfrog transthyretin may play an important role in transporting T3 in the blood during metamorphosis.

Actions of 3,5,3'-tri-iodothyronine on the synthesis and secretion of major plasma proteins by a human hepatoblastoma cell line (Hep G2).

We have elucidated the action of tri-iodothyronine (T3) on the synthesis and secretion of seven major plasma proteins in a human hepatoblastoma cell line, Hep G2, and established an in vitro experimental model of human liver cells for the study of the mechanism of the action of thyroid hormone. Hep G2 cells cultured in serum-free medium were treated with various concentrations of T3. During the first 24 h of T3 treatment, accumulation of alpha-fetoprotein in the medium was decreased in a dose-dependent manner (10(-11)-10(-8) M), and the inhibitory effect was enhanced during the second 24 h of T3 treatment. On the other hand, alpha 1-antitrypsin accumulation in the medium during the second 24 h of hormone treatment was decreased by T3 (10(-9)-10(-8) M), although no change in accumulation was observed during the first 24 h of T3 treatment. The newly synthesized [35S]Met-labelled alpha 1-acid glycoprotein was increased by T3 and reached 3.4-fold within 37 h of 10(-8) M T3 treatment. The stimulatory effect increased time-dependently (4.6-fold after 61 h). In contrast, the synthesis of alpha-fetoprotein was reduced to half of that of the control after T3 treatment for 37 h. Although the content of newly synthesized [35S]alpha 1-antitrypsin was not affected by 10(-8) M T3 treatment during 3 days of hormone treatment, the accumulation of alpha 1-antitrypsin in the medium decreased to 87%; in contrast, total cellular newly synthesized alpha 1-antitrypsin increased to 105-130% of that of the control.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel action of epidermal growth factor in rat granulosa cells: its potentiation of gonadotrophin action.

It is well known that epidermal growth factor (EGF) induces down-regulation of LH receptors and desensitization to gonadotrophin stimulation in gonadal cells, including granulosa cells. In a previous study we showed that EGF receptor levels in rat granulosa cells were increased up to fourfold after 96 h of culture with human GH in the presence of FSH, and the present study has evaluated the action of EGF on these cells. The induced EGF receptors were identical in size to the pre-existing receptors as assessed by affinity labelling with 125I-EGF. After 48 h in culture, various amounts of EGF (0.5-10 ng) were added and the cells were cultured for a further 48 h. The addition of EGF caused down-regulation of LH receptors in cells expressing high levels of EGF receptors. However, this down-regulation was less than that in control cells. After the cells were washed, cAMP synthesis in response to human chorionic gonadotrophin (hCG) increased by two to three times the control value and this increase was closely correlated with an increase in EGF receptor content. However, stimulation with cholera toxin or forskolin showed no such augmentation, indicating that it may not be due to quantitative alterations in G proteins and their effector systems. Induction of EGF potentiation required long-term exposure to EGF, for at least more than 24 h. In addition, progesterone synthesis was sensitive to stimulation with lower doses of hCG. These findings indicate that the activation of hGH-induced EGF receptors may potentiate gonadotrophin action in granulosa cells.

Uptake of 3,5,3'-L-tri-iodothyronine in human erythrocytes.

The mechanisms of 3,5,3'-L-tri-iodothyronine (T3) uptake into human erythrocytes were examined. Purified membranes of human erythrocytes were shown to have two classes of T3-binding sites with one being a high-affinity site (dissociation constant, 59.2 +/- 17.8 nmol/l; maximum binding capacity, 344.3 +/- 95.5 fmol/micrograms protein). Furthermore, it was shown that there were two pathways for T3 uptake in human erythrocytes; one was saturable, stereospecific (T3 much greater than thyroxine greater than 3,5,3'-D-tri-iodothyronine), energy-dependent and dominant at 15 degrees C; the other was not displaced by unlabelled T3 and was energy-independent but did not occur by passive diffusion. The former pathway which, it is suggested, is a receptor-mediated transport pathway, was inhibited by monodansylcadaverine, phloretin or oligomycin at 15 or 37 degrees C, but the latter pathway was not inhibited by these inhibitors. Our results strongly suggest that uptake of T3 by the energy-independent pathway became predominant over the energy-dependent pathway at 37 degrees C and accounted for 83% of total T3 uptake of human erythrocytes.

Co-ordinate actions of FSH and insulin-like growth factor-I on LH receptor expression in rat granulosa cells.

The actions of FSH and Insulin-like growth factor-I (IGF-I) were studied in cultured rat ovarian granulosa cells. Cells became differentiated and expressed LH receptors when they were incubated for 72 h with 200 micrograms FSH/l (high FSH) but not 20 micrograms FSH/l (low FSH). Treatment with high but not low FSH increased the release of both immunoreactive and bioactive IGF-I into the medium. A combination of low FSH and IGF-I reproduced the effect of high FSH on LH receptor expression. We then examined the critical time when low FSH and IGF-I exerted their effects. In the presence of continuous low FSH, IGF-I was capable of inducing LH receptor expression even when added 24 h after the addition of low FSH. However, when IGF-I was added at 36 h, LH receptor expression measured at 72 h was greatly reduced. In contrast to the action of IGF-I, continuous exposure to low FSH was required for LH receptor expression, and IGF-I had no effect when FSH was not included for the entire 72 h of culture. DNA synthesis as assessed by both [3H]thymidine incorporation and nuclear bromodeoxyuridine labelling was moderate at the beginning of culture and markedly reduced at 24 h both in the presence and absence of either high FSH or low FSH plus IGF-I. In the presence of either high FSH or a combination of low FSH plus IGF-I, DNA synthesis remained decreased for up to 72 h whereas it began to increase in the absence of either high FSH or a combination of low FSH plus IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Human growth hormone augmentation of epidermal growth factor binding sites on rat granulosa cells.

The effect of human GH (hGH) on the regulation of epidermal growth factor (EGF) receptor was investigated during differentiation of FSH-treated rat granulosa cells, which has been reported to be mediated by a cAMP-dependent mechanism. By measuring the binding of [125I]iodo-EGF to the intact cells, FSH was shown to cause increases in the number of EGF binding sites after culture for 72 h. When granulosa cells were cultured with hGH, the number of FSH-induced EGF binding sites was augmented, with a half-maximal effect at about 10 micrograms hGH/l and a maximal stimulatory concentration of 100 micrograms/l. The stimulatory effect of hGH was absolutely dependent on insulin which by itself showed stimulatory effects on EGF binding sites. Scatchard analysis of EGF binding sites indicated that treatment with hGH increased the number of EGF binding sites (17,200 sites/cell after treatment with FSH; 31,700 sites/cell after FSH plus hGH), but did not alter the binding affinity. The augmentation was observed after culturing for 48 h and increased progressively with time, reaching 280% of the level after FSH treatment by 120 h. Although progesterone synthesis was increased by hGH, the markers of cell differentiation such as cAMP synthesis and LH binding sites were suppressed, indicating hGH inhibition of the cAMP-mediated signal. The action of hGH on the EGF binding sites was not accompanied by cell proliferation. These findings indicate that hGH has a novel action on the regulation of rat granulosa cell EGF binding sites and that the granulosa cell may possess both cAMP-dependent and -independent mechanisms for expression of EGF binding sites.

Biphasic effects of exogenous ganglioside GM3 on follicle-stimulating hormone-dependent expression of luteinizing hormone receptor in cultured granulosa cells.

The major ganglioside NeuAc alpha 2-->3Gal beta 1-->4Glc beta 1-->1Cer (GM3) present in cultured rat granulosa cells was examined for potential function in the expression of luteinizing hormone (LH) receptor on the cell surface in response to follicle-stimulating hormone (FSH). Synthesis of GM3 was stimulated concentration-dependently by FSH, and the stimulation was enhanced synergistically by insulin, as revealed by metabolic labeling of glycosphingolipids with [3H]galactose. When granulosa cells were cultured in the media containing GM3 (0.2-20 microM), biphasic changes in FSH-dependent expression of LH receptor were observed, as measured by the binding of 125I-deglycosylated human choriogonadotropin to the intact cells. Exogenous GM3 suppressed expression of LH receptor in the cells treated with a low dose of FSH (20 ng/ml), which was characterized by a low GM3 level, to 30% of control at 10 microM, with a half-maximal inhibitory concentration of 8 microM. In contrast, in the cells treated with a high dose of FSH (100 ng/ml) and insulin, which was characterized by a high GM3 level, expression of LH receptor was enhanced by exogenous GM3, to 148% of control at 10 microM, with a half-maximal effective concentration of 2 microM. Exogenous GM3 produced concomitant changes in the levels of extracellular cAMP. These effects of exogenous GM3 were not accompanied by changes in granulosa cell proliferation. Exogenous GM3 also modulated the LH receptor expression by the synergistic action of 12-O-tetradecanoylphorbol 13-acetate with insulin, with no significant changes in cellular DNA contents, suggesting that exogenous GM3 does not modulate directly the action of FSH at its receptor sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor-promoting phorbol ester acts synergistically with insulin to induce lutropin receptor expression in rat granulosa cells.

Lutropin (LH) receptors in rat granulosa cells are expressed by activation of cAMP-dependent protein kinase in response to follitropin (FSH). In the present study, 12-O-tetradecanoylphorbol 13-acetate (TPA) could cause a dose-dependent expression of LH receptors in the presence of insulin, but not in the absence of insulin, as measured by binding of 125I-deglycosylated human choriogonadotropin (DGhCG). The synergistic action of TPA with insulin was achieved at 1 nM and 10 mIU/ml, respectively. The receptor expression induced by this synergistic action was accompanied by cAMP accumulation which was detected after a lag time of 6 h following exposure to TPA. However, a synthetic diacylglycerol and non-protein kinase C activating phorbol derivatives did not mimic the effect of TPA on the receptor expression. In addition, insulin modulated the inhibitory effect of TPA in FSH-induced LH receptor expression, indicating a peculiar action of insulin in the receptor expression. Indomethacin treatment led to a dose-dependent inhibition in the receptor expression in the cells treated with TPA plus insulin more than that in the cells with FSH plus insulin, suggesting that the synergistic action was dependent upon cyclooxygenase and/or phospholipase A2 activity. It was shown by Scatchard analysis of LH receptors and kinetic studies of hCG-stimulated cAMP formation that the synergistic action of TPA with insulin led to expression of functional LH receptors coupled with the adenylate cyclase system in cultured granulosa cells.

Basic fibroblast growth factor induces luteinizing hormone receptor expression in the presence of insulin-like growth factor-I in ovarian granulosa cells.

Effect of basic fibroblast growth factor (bFGF) on the expression of receptors for luteinizing hormone (LH), a marker of differentiation, was studied using estrogen-primed rat ovarian granulosa cells in primary culture. bFGF had no effect by itself but dose-dependently induced expression of functional LH receptors in the presence of insulin-like growth factor-I (IGF-I). The effect of a combination of bFGF and IGF-I was delayed in onset and the magnitude of the response was smaller when compared to the action of follicle-stimulating hormone (FSH). Scatchard analysis revealed that dissociation constant (Kd) and number of LH receptors induced by bFGF and IGF-I were 0.47 nM and 6.48 fmol/10(6) cells, respectively. Unlike FSH, bFGF plus IGF-I did not cause an immediate increase in cAMP release, however, considerable amount of cAMP release was observed in cells incubated for 72 h with bFGF plus IGF-I. Indomethacin, an inhibitor of cyclooxygenase, attenuated both LH receptor expression and cAMP release induced by bFGF plus IGF-I but had little effect on the action of FSH. Finally, a combination of bFGF and IGF-I increased production of prostaglandin E2 in granulosa cells. These results indicate that bFGF is capable of inducing LH receptor in the presence of IGF-I by a mechanism involving production of prostaglandin E2.

RNA SYNTHESIS IN CULTURED CHICK EMBRYO CELLS IN GROWING AND CONFLUENT PHASES.

RNA synthesis at the growing phase in monolayer cultures of chick embryo fibroblasts was compared with that at confluent phases by zonal sedimentation, base composition and hybridization experiments. The nuclei were isolated by treatment with Nonidet p-40. The ratio of RNA/DNA in isolated nuclei was higher at the growing phase than that of confluent. The rate of RNA synthesis was reduced in the cells at confluent phase to 15.1% of that at the growing phase. The sucrose density gradient sedimentation pattern of nuclear RNA was on the whole the same in both phases. According to the distribution of 14 C-uridine incorporated into nuclear RNA, 45S ribosomal precursor RNA was more distinct for the growing cell, while the radioactivities were found to be polydispersed, including the RNA which sedimented faster than 28S RNA in the cells at confluent phase. The base compositions and hybridization analyses indicated that ribosomal RNA was synthesized more actively in the growing cells. About 50% of newly synthesized RNA was ribosomal in the growing cells but 35% in the confluent. It was found that newly synthesized 18S and 28S ribosomal RNAs appeared in cytoplasm after 21 and 33 min lag periods respectively. These times were exactly same in both growing and confluent phases.

Alterations in the enzyme activity and protein contents of protein disulfide isomerase in rat tissues during fasting and refeeding.

Protein disulfide isomerase (PDI) is an enzyme that participates in the formation of disulfide bonds. It is also known to be the subunits of some enzymes and the membrane-associated thyroid hormone-binding protein. In this study, we measured the quantitative distribution of PDI protein in rat tissues and examined the relationship between protein level and enzyme activity in PDI during fasting and refeeding. Western blotting with specific anti-PDI antiserum detected the PDI protein band of 55 kd. Among several tissues, liver contained the largest amount of PDI protein, followed by kidney and fat, in which one-third to one-fourth of the hepatic PDI protein existed. The PDI protein band was also detected in heart and muscle. Fasting for 3 days decreased PDI protein levels in rat liver by 40%; control levels were recovered after 3 days of refeeding. The same change was observed in kidney. PDI activity, measured by the scrambled ribonuclease method, did not show the parallel alteration to PDI protein level in liver and kidney. Isomerase activity decreased to 50% of control values during fasting, but did not recover by refeeding. Thyroidal status did not affect either PDI protein level or isomerase activity. These findings show that fasting and refeeding affect PDI protein and enzyme activity, and that PDI protein level does not always reflect PDI activity.

Production of human protein disulfide isomerase by Bacillus brevis.

Human protein disulfide isomerase (PDI; EC 5.3.4.1) was expressed and secreted into the culture medium using Bacillus brevis as host and pNU200 which codes the promoter and signal sequence of major cell wall protein of B. brevis as vector. The accumulation of recombinant human PDI (rhPDI) reached about 5 mg l-1 in the late exponential phase of the bacterial growth. The purified rhPDI was found to be exactly processed at the carboxyl terminus of the signal sequence. It was as active as natural PDI derived from human placenta as determined by its ability to reactivate scrambled ribonuclease that was a fully oxidized mixture containing randomly formed disulfide bonds. The activity was significantly accelerated in the presence of dithiothreitol or a mixture of reduced and oxidized glutathione. These indicate that the characteristics of rhPDI are similar to those reported for mammalian PDI and that it can be used for refolding inactive proteins having incorrect disulfide bonds.