Deficiency of Type I Platelet-Activating Factor-Acetylhydrolase Catalytic Subunits Causes an Increase in Body Weight.

Type I platelet-activating factor-acetylhydrolase (PAF-AH) forms a complex consisting of two catalytic subunits (α1 and/or α2) with a regulatory subunit (ß). Although this protein was discovered as an enzyme that degrades an acetyl ester linked at the sn-2 position of platelet-activating factor (PAF), its physiological function remains unknown. In this study, to examine whether knockout mice lacking the catalytic subunits of this enzyme showed a different phenotype from that of wild-type mice, we measured and compared the body weights of knockout mice and control mice. The body weights of knockout mice were significantly increased compared to those of the control mice during 6 to 20 weeks from birth. Food intake was also significantly increased in knockout mice compared with control mice during these periods. Since a decrease in testis weight was reported in the knockout mice, we expected a decrease in testosterone levels. We measured and compared the amounts of testosterone in the serum and testis of knockout and control mice using liquid chromatography-tandem mass spectrometry, and found that testosterone levels in both the serum and testis were significantly decreased in the knockout mice compared with the control mice. These results suggest that a deficiency of type I PAF-AH catalytic subunits causes an increase in body weight, in part, due to reduced testosterone levels in male mice.

Transcriptional Regulation of Acyl-CoA:Glycerol-sn-3-Phosphate Acyltransferases.

Acyl-CoA:glycerol-sn-3-phosphate acyltransferase (GPAT) is an enzyme responsible for the rate-limiting step in the synthesis of glycerophospholipids and triacylglycerol (TAG). The enzymes of mammalian species are classified into four isoforms; GPAT1 and GPAT2 are localized in the mitochondrial outer membrane, whereas GPAT3 and GPAT4 are localized in the endoplasmic reticulum membrane. The activity of each enzyme expressed is associated with physiological and pathological functions. The transcriptional regulation is well known, particularly in GPAT1. GPAT1 mRNA expression is mainly regulated by the binding of the transcriptional factor SREBP-1c to the specific element (the sterol regulatory element) flanking the GPAT1 promoter. The TAG level is controlled by the insulin-induced transcriptional expression of GPAT1, which occupies most of the GPAT activity in the liver. The transcriptional regulation of the other three GPAT isoforms remains undetermined in detail. It is predicted that retinoic acid serves as a transcription factor in the GPAT2 promoter. PPARγ (peroxisome proliferator-activated receptor γ) increases the mRNA expression of GPAT3, which is associated with TAG synthesis in adipose tissues. Although GPAT has been considered to be a key enzyme in the production of TAG, unexpected functions have recently been reported, particularly in GPAT2. It is likely that GPAT2 is associated with tumorigenesis and normal spermatogenesis. In this review, the physiological and pathophysiological roles of the four GPAT isoforms are described, alongside the transcriptional regulation of these enzymes.

Non-alcoholic steatohepatitis-associated hepatic fibrosis and hepatocellular carcinoma in a combined mouse model of genetic modification and dietary challenge.

AIM: Experimental models of non-alcoholic steatohepatitis (NASH) are still required for understanding the pathophysiology of this disease. This study aimed to examine whether disease progression is accelerated by combining dyslipidemic genetic modification and dietary challenges and develop NASH-associated hepatic fibrosis, cirrhosis, and carcinoma in a short period. METHODS: Low-density lipoprotein receptor knockout mice were fed a modified choline-deficient amino acid-defined diet, including 1 w/w% cholesterol and 41 kcal% fat, and was comprehensively profiled over 1 year. RESULTS: Microvesicular and macrovesicular steatosis in the liver was observed from the first week after starting the modified choline-deficient amino acid-defined diet. Macrovesicular steatosis was exacerbated with time and was observed in almost all hepatocytes at week 8, but slightly decreased at week 16. Infiltration of macrophages and neutrophils, and upregulation of hepatic inflammatory cytokines such as tumor necrosis factor-α and interleukin-1ß were also observed from week 1. Plasma hepatic transaminase activities were increased at week 1, reached a peak at week 4, and gradually decreased thereafter. In parallel with increases in hepatic gene expression of collagen-I, the hepatic fibrosis area expanded after week 4 and massively spread all over the liver by week 8. Hepatocellular hyperplasia was observed from week 24. Hepatocellular adenoma and carcinoma were observed from week 31 and 39, respectively. CONCLUSION: These results suggest that, in a rodent NASH model with the combination of genetic modification and dietary challenges, hepatic steatosis, inflammatory cell infiltration and hepatic injury, hepatic fibrosis, hepatocellular hyperplasia, adenoma, and carcinoma can be developed in a relatively short period.

Pharmacological evaluation of pioglitazone and candesartan cilexetil in a novel mouse model of non-alcoholic steatohepatitis, modified choline-deficient, amino acid-defined diet fed low-density lipoprotein receptor knockout mice.

AIM: Low-density lipoprotein receptor knockout (LDLR-KO) mice fed a modified choline-deficient and amino acid-defined (mCDAA) diet show non-alcoholic steatohepatitis (NASH)-like pathophysiology. In order to pharmacologically benchmark this model, effects of pioglitazone (a thiazolidinedione) and candesartan cilexetil (an angiotensin II type 1 receptor blocker) on steatosis and liver fibrosis were examined. METHODS: Pioglitazone (10 mg/kg) and candesartan cilexetil (3 mg/kg) were given orally once daily to LDLR-KO mice under mCDAA diet for 7 weeks. Blood biochemistry and hepatic histology were assessed, and hepatic gene expression levels and triglyceride content were measured. RESULTS: Pioglitazone suppressed hepatic COL1A1 gene expression by 43% and attenuated hepatic fibrosis areas by 49%. Pioglitazone also decreased plasma alanine aminotransferase levels, liver weight, hepatic triglyceride content, and hepatic expression of other fibrosis-related genes such as TGFB1, SPP1, TIMP1, and IL6. Candesartan cilexetil suppressed hepatic COL1A1 gene expression by 33%, whereas the other end-points including hepatic fibrosis areas were not affected. CONCLUSIONS: Pioglitazone showed anti-fibrotic effects accompanied by improving hepatic transaminase activity and hepatic lipid accumulation, but the effect of candesartan cilexetil was only limited, unlike previous reports for angiotensin II type 1 receptor blockers. As the pharmacological effects of pioglitazone in the current animal model are similar to those reported in patients with NASH, this model may represent some aspects of the pathophysiology of NASH. Further profiling using other agents or mechanisms that have been tested in the clinic will better clarify the utility of the animal model.

Involvement of Leukotriene B4 Released from Keratinocytes in Itch-associated Response to Intradermal Interleukin-31 in Mice.

A recent study suggests that interleukin-31 (IL-31) exerts its effect via indirect mechanisms rather than through direct stimulation of cutaneous nerves. However, the underlying peripheral mechanisms of IL-31-induced itch in the skin remain unclear. Therefore, the present study investigated the peripheral mechanisms underlying IL-31-induced itch in mice. IL-31-induced itch-related response was inhibited by anti-allergic drugs (tranilast and azelastine), but not by an H1 histamine receptor antagonist (terfenadine). Furthermore, a 5-lipoxygenase inhibitor (zileuton), but not a cyclooxygenase inhibitor (indomethacin), and a leuko-triene B4 (LTB4) receptor antagonist (CMHVA) attenuated the action of IL-31. IL-31 receptor-immunoreactivity was observed in the epidermis and primary sensory neurones. IL-31 receptor mRNA was expressed in mouse keratinocytes and dorsal root ganglia neurones. IL-31 increased the production of LTB4 in mouse keratinocytes. These results suggest that IL-31 elicits itch not only through direct action on primary sensory neurones, but also by inducing LTB4 production in keratinocytes.

Association of serum copper levels and glycemic control in patients with type 2 diabetes.

It has been suggested that copper ion is involved in the pathogenesis of various diseases. The aim of this study is to examine the association of serum copper levels and glycemic control in patients with type 2 diabetes. We recruited a total of 132 patients with type 2 diabetes, and measured their serum copper levels by atomic absorption spectrometry. Serum copper levels were positively correlated with HbA1c levels (r=0.176, p=0 .044). In addition, after 3-month glycemic control, we evaluated whether the improvement of glycemic control influenced serum copper levels. As hemoglobin A1c (HbA1c) levels were decreased (from 8.7% to 6.8%, p<0.001), copper levels tended to be decreased (from 105.7 µg/dL to 101.8 µg/dL, p=0.069). In conclusion, it is likely that serum copper levels are associated with glycemic control in patients with type 2 diabetes.

TAK-875, an orally available G protein-coupled receptor 40/free fatty acid receptor 1 agonist, enhances glucose-dependent insulin secretion and improves both postprandial and fasting hyperglycemia in type 2 diabetic rats.

G protein-coupled receptor 40/free fatty acid receptor 1 (GPR40/FFA(1)) is highly expressed in pancreatic ß cells and mediates free fatty acid-induced insulin secretion. This study examined the pharmacological effects and potential for avoidance of lipotoxicity of [(3S)-6-({2',6'-dimethyl-4'-[3-(methylsulfonyl)propoxy]biphenyl-3-yl}meth-oxy)-2,3-dihydro-1-benzofuran-3-yl]acetic acid hemi-hydrate) (TAK-875), a novel, orally available, selective GPR40 agonist. Insulinoma cell lines and primary rat islets were used to assess the effects of TAK-875 in vitro. The in vivo effects of TAK-875 on postprandial hyperglycemia, fasting hyperglycemia, and normoglycemia were examined in type 2 diabetic and normal rats. In rat insulinoma INS-1 833/15 cells, TAK-875 increased intracellular inositol monophosphate and calcium concentration, consistent with activation of the Gqα signaling pathway. The insulinotropic action of TAK-875 (10 µM) in INS-1 833/15 and primary rat islets was glucose-dependent. Prolonged exposure of cytokine-sensitive INS-1 832/13 to TAK-875 for 72 h at pharmacologically active concentrations did not alter glucose-stimulated insulin secretion, insulin content, or caspase 3/7 activity, whereas prolonged exposure to palmitic or oleic acid impaired ß cell function and survival. In an oral glucose tolerance test in type 2 diabetic N-STZ-1.5 rats, TAK-875 (1-10 mg/kg p.o.) showed a clear improvement in glucose tolerance and augmented insulin secretion. In addition, TAK-875 (10 mg/kg, p.o.) significantly augmented plasma insulin levels and reduced fasting hyperglycemia in male Zucker diabetic fatty rats, whereas in fasted normal Sprague-Dawley rats, TAK-875 neither enhanced insulin secretion nor caused hypoglycemia even at 30 mg/kg. TAK-875 enhances glucose-dependent insulin secretion and improves both postprandial and fasting hyperglycemia with a low risk of hypoglycemia and no evidence of ß cell toxicity.

Mycobacterium leprae promotes triacylglycerol de novo synthesis through induction of GPAT3 expression in human premonocytic THP-1 cells.

Mycobacterium leprae (M. leprae) is the etiological agent of leprosy, and the skin lesions of lepromatous leprosy are filled with numerous foamy or xanthomatous histiocytes that are parasitized by M. leprae. Lipids are an important nutrient for the intracellular survival of M. leprae. In this study, we attempted to determine the intracellular lipid composition and underlying mechanisms for changes in host cell lipid metabolism induced by M. leprae infection. Using high-performance thin-layer chromatography (HPTLC), we demonstrated specific induction of triacylglycerol (TAG) production in human macrophage THP-1 cells following M. leprae infection. We then used [14C] stearic acid tracing to show incorporation of this newly synthesized host cell TAG into M. leprae. In parallel with TAG accumulation, expression of host glycerol-3-phosphate acyltransferase 3 (GPAT3), a key enzyme in de novo TAG synthesis, was significantly increased in M. leprae-infected cells. CRISPR/Cas9 genome editing of GPAT3 in THP-1 cells (GPAT3 KO) dramatically reduced accumulation of TAG following M. leprae infection, intracellular mycobacterial load, and bacteria viability. These results together suggest that M. leprae induces host GPAT3 expression to facilitate TAG accumulation within macrophages to maintain a suitable environment that is crucial for intracellular survival of these bacilli.

Simultaneous Detection of Six Different Types of Pesticides by an Immunosensor Based on Surface Plasmon Resonance.

Six pesticides, azoxystrobin, boscalid, chlorfenapyr, imazalil, isoxathion, and nitenpyram, were simultaneously detected by using a surface plasmon resonance (SPR) immunosensor. The working ranges were 3.5 - 19 ng/mL for azoxystrobin, 4.5 - 50 ng/mL for boscalid, 2.5 - 25 ng/mL for chlorfenapyr, 5.5 - 50 ng/mL for imazalil, 3.5 - 50 ng/mL for isoxathion, and 8.5 - 110 ng/mL for nitenpyram. They showed adequate recovery results in tomato samples: 104 - 116% for azoxystrobin, 94 - 101% for boscalid, 90 - 112% for chlorfenapyr, 96 - 106% for imazalil, 107 - 119% for isoxathion, and 104 - 109% for nitenpyram. The correlation coefficient with liquid chromatography (HPLC or LC-MS/MS) using vegetable samples also agreed well: 0.91 - 0.99 as R2 without strong bias, except for nitenpyram for which the SPR immunosensor sensitivity was too low. The SPR immunosensor will have high applicability for pesticide residue analyses in vegetable samples.

Development of a direct competitive enzyme-linked immunosorbent assay for determination of the fungicide mepanipyrim and its metabolite.

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for determination of anilinopyrimidine fungicide mepanipyrim in vegetables. Two derivatives of mepanipyrim and mepanipyrim propanol type metabolite which carried carboxy acid were synthesized and conjugated with keyhole limpet hemocyanin. BALB/c mice were immunized to prepare anti-mepanipyrim monoclonal antibodies (MoAbs) by obtained conjugates. The dc-ELISAs based on the prepared MoAbs, MPP107 and MPP204, showed working ranges between 0.12 and 1.8 ng/mL with mepanipyrim for MPP107, 0.12 and 2.4 ng/mL with mepanipyrim for MPP204, and 0.2 ng/mL and 5.7 ng/mL with the mepanipyrim propanol type for MPP204. The dc-ELISAs showed the sufficient sensitivity to determine the mepanipyrim residues for the MRLs of 1-15 mg/kg among the majority of vegetables and fruits in Japan. Recovery and/or correlation results from HPLC suggested that the dc-ELISAs would be applicable to the residue analysis of mepanipyrim and its propanol type in vegetables.

Expression of a novel 90-kDa protein, Lsd90, involved in the metabolism of very long-chain fatty acid-containing phospholipids in a mitosis-defective fission yeast mutant.

The fission yeast lsd1/fas2 strain carries a temperature-sensitive mutation of the fatty-acid-synthase alpha-subunit, exhibiting an aberrant mitosis lsd phenotype, with accumulation of very-long-chain fatty-acid-containing phospholipid (VLCFA-PL). A novel 90-kDa protein, Lsd90 (SPBC16E9.16c), was found to be newly expressed in small particle-like structures in lsd1/fas2 cells under restrictive conditions. Two mismatches leading to a double frame shift were found between the sequences of the lsd90(+) gene registered in the genomic database and the sequences determined experimentally at the amino acid, cDNA and genomic DNA levels. Unexpectedly, overexpression and disruption of the lsd90(+) gene in either lsd1/fas2 or wild-type cells did not affect either cell growth or expression of the lsd phenotype. The amounts of VLCFA-PL that accumulated in lsd90-overexpressing lsd1/fas2 cells were significantly lower than those in lsd1/fas2 cells, suggesting the involvement of Lsd90 in the metabolism of VLCFA-PL.

MafA is a key regulator of glucose-stimulated insulin secretion.

MafA is a transcription factor that binds to the promoter in the insulin gene and has been postulated to regulate insulin transcription in response to serum glucose levels, but there is no current in vivo evidence to support this hypothesis. To analyze the role of MafA in insulin transcription and glucose homeostasis in vivo, we generated MafA-deficient mice. Here we report that MafA mutant mice display intolerance to glucose and develop diabetes mellitus. Detailed analyses revealed that glucose-, arginine-, or KCl-stimulated insulin secretion from pancreatic beta cells is severely impaired, although insulin content per se is not significantly affected. MafA-deficient mice also display age-dependent pancreatic islet abnormalities. Further analysis revealed that insulin 1, insulin 2, Pdx1, Beta2, and Glut-2 transcripts are diminished in MafA-deficient mice. These results show that MafA is a key regulator of glucose-stimulated insulin secretion in vivo.

Development of an Immunosensor Based on Surface Plasmon Resonance for Simultaneous Residue Analysis of Three Pesticides -Boscalid, Clothianidin, and Nitenpyram- in Vegetables.

A simultaneous immunosensor based on surface plasmon resonance (SPR) was developed for determination of 3 pesticides -boscalid, clothianidin and nitenpyram- instead of the direct competitive enzyme-linked immunosorbent assays (dcELISAs) widely used as individual determination methods. Carboxy groups that introduced compounds to their pesticides were designed, and conjugates of them and bovine serum albumin were immobilized onto separate channels of the same sensor chip. When a mixture of 3 monoclonal antibodies reacted to each pesticide, and 3 pesticides were injected into the SPR immunosensor, each channel showed specific reactivity at 15 - 93 ng mL-1 for boscalid, 6.7 - 27 ng mL-1 for clothianidin, and 7.3 - 62 ng mL-1 for nitenpyram. Recovery tests using vegetables spiked with a mixture of 3 pesticides showed good results: 75 - 90%, 88 - 104%, and 72 - 105%, respectively, with a high correlation to results of the dcELISAs. The SPR immunosensor would be useful for the determination of pesticide residues in vegetables.

Suppression of MafA-dependent transcription by transforming growth factor-beta signaling.

MafA is a basic leucine zipper (b-Zip) type transcription factor that binds to the insulin promoter and regulates insulin transcription synergistically with Pdx-1 and NeuroD. Transforming growth factor-beta (TGF-beta) signaling has been reported to regulate activity of b-Zip transcription factor such as ATF-2 and acts as an important regulator of insulin gene transcription and pancreatic beta cell maintenance. To investigate the relationship between MafA-dependent transcriptional activation and TGF-beta signaling, we examined the effects of TGF-beta signal on MafA-dependent transactivation of the rat insulin II gene promoter (RIPII-251) and a synthetic MafA-dependent promoter. MafA-dependent activation of the reporters was inhibited in the presence of Smad2/Smad4 or Smad3/Smad4 and a constitutively active TGF-beta type I receptor and this inhibition was dependent upon the presence of MafA. Co-immunoprecipitation analyses revealed that MafA physically interacts with Smad2 or Smad3. These results suggest that MafA-dependent transcriptional activation is negatively regulated by TGF-beta signaling.

Amelioration of sexual behavior and motor activity deficits in a castrated rodent model with a selective androgen receptor modulator SARM-2f.

Sarcopenia and cachexia present characteristic features of a decrease in skeletal muscle mass and strength, anorexia, and lack of motivation. Treatments for these diseases have not yet been established, although selective androgen receptor modulators (SARMs) are considered as therapeutic targets. We previously reported that a novel SARM compound, SARM-2f, exhibits anabolic effect on muscles, with less stimulatory effect on prostate weight compared with testosterone, in rat Hershberger assays and cancer cachexia models. In this study, we studied the mechanism of action for SARM-2f selectivity and also assessed whether the muscle increase by this compound might lead to improvement of muscle function and physical activity. First, we examined the tissue distribution of SARM-2f. Tissue concentration was 1.2-, 1.6-, and 1.9-fold as high as the plasma concentration in the levator ani muscle, brain, and prostate, respectively. This result showed that the tissue-selective pharmacological effect did not depend on SARM-2f concentration in the tissues. The ability of SARM-2f to influence androgen receptor (AR)-mediated transcriptional activation was examined by reporter assays using human normal prostate epithelial cells (PrEC) and skeletal muscle cells (SKMC). SARM-2f exerted higher activity against AR in SKMC than in PrEC. Mammalian two hybrid assays showed different co-factor recruitment patterns between SARM-2f and dihydrotestosterone. Next, we studied the effect of SARM-2f on motivation and physical functions such as sexual behavior and motor activities in castrated rat or mouse models. SARM-2f restored the sexual behavior that was lost by castration in male rats. SARM-2f also increased voluntary running distance and locomotor activities. These results suggest that tissue-specific AR regulation by SARM-2f, but not tissue distribution, might account for its tissue specific androgenic effect, and that the muscle mass increase by SARM-2f leads to improvement of physical function. Together, these findings suggest that SARM-2f might represent an effective treatment for sarcopenia and cachexia.

Plasma platelet activating factor-acetylhydrolase (PAF-AH).

The platelet-activating factor-acetylhydrolase (PAF-AH) is an enzyme which catalyzes the hydrolysis of acetyl ester at the sn-2 position of PAF. The family of PAF-AHs consists of two intracellular isoforms (Ib and II), and one secreted isoform (plasma). These PAF-AHs show different biochemical characteristics and molecular structures. Plasma PAF-AH and intracellular isoform, II degrade not only PAF but also oxidatively fragmented phospholipids with potent biological activities. Among these PAF-AHs, plasma PAF-AH has been the target of many clinical studies in inflammatory diseases, such as asthma, sepsis, and vascular diseases, because the plasma PAF-AH activity in the patients with these diseases is altered when compared with normal individuals. Finding a genetic deficiency in the plasma PAF-AH opened the gate in elucidating the protecting role of this enzyme in inflammatory diseases. The most common loss-of-function mutation, V279F, is found in more than 30% of Japanese subjects (4% homozygous, 27% heterozygous). This single nucleotide polymorphism in plasma PAF-AH and the resulting enzymatic deficiency is thought to be a genetic risk factor in various inflammatory diseases in Japanese subjects. Administration of recombinant plasma PAF-AH or transfer of the plasma PAF-AH gene improves pathology in animal models. Therefore, substitution of plasma PAF-AH would be an effective in the treatment of the patients with the inflammatory diseases and a novel clinical approach. In addition, the detection of polymorphisms in the plasma PAF-AH gene and abnormalities in enzyme activity would be beneficial in the diagnosis of the inflammatory diseases.

Red blood cells highly express type I platelet-activating factor-acetylhydrolase (PAF-AH) which consists of the alpha1/alpha2 complex.

Although red blood cells account for about 30% of total PAF-AH activity found in the blood, the physiological function of this enzyme is unknown. To understand the role and regulatory mechanism of this enzyme, we purified it from easily obtainable pig red blood cells. PAF-AH activity was mainly found in the soluble fraction of the red blood cells. Two peaks of enzyme activity appeared with increasing concentration of imidazole on column chromatography on nickel-nitroacetic acid (Ni-NTA) resin. We called these peaks of small and large enzyme activities fractions X and Y, respectively, and then further purified the enzymes by sequential chromatofocusing on Mono P and gel filtration on TSK G-3000. In the final preparation from fraction Y, two proteins bands corresponding to 26 kDa and 28 kDa were related to enzyme activity. Determination of the partial amino acid sequences of the proteins of 26 kDa and 28 kDa revealed that these proteins were identical to alpha(1) and alpha(2), respectively, both of which are catalytic subunits of Type I intracellular PAF-AH. On Western analysis, the 26 kDa and 28 kDa protein bands cross-reacted with specific monoclonal antibodies to alpha(1) and alpha(2), respectively. Since the apparent molecular weight of the natural enzyme was estimated to be about 60 kDa, the enzyme activity in fraction Y was thought to be that of a heterodimer consisting of alpha(1) and alpha(2). On the other hand, the enzyme activity in fraction X was thought to be that of a homodimer consisting of alpha(2). Other blood cells such as polymorphonuclear leukocytes and platelets only contained the alpha(2)/alpha(2) homodimer. It has been reported that the alpha(1)/alpha(2) heterodimer is poorly expressed in adult animals except for in the spermatogonium. Taken altogether, these results suggest that high expression of the alpha(1)/alpha(2) heterodimer is important for the physiological function of mature red blood cells.

Analysis of the Fungicide Boscalid in Horticultural Crops Using an Enzyme-Linked Immunosorbent Assay and an Immunosensor Based on Surface Plasmon Resonance.

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and an immunosensor based on surface plasmon resonance (SPR-sensor) were developed for fungicide boscalid determination in horticultural crops. To produce antiboscalid monoclonal antibodies (MoAb BSC7 and MoAb BSC72) for these assays, a hapten of boscalid was synthesized and conjugated to keyhole limpet hemocyanin for Balb/c mouse immunization. The working range of the dc-ELISA was 0.8-16 ng/mL with MoAb BSC7 and 2.5-120 ng/mL with MoAb BSC72, and that of the SPR-sensor was 17-80 ng/mL with MoAb BSC7. The dc-ELISA and SPR-sensor were compared for their sensitivity in determining boscalid residues at the maximum residue limit of 1-40 mg/kg for horticultural crops in Japan. Recovery of the spiked boscalid was 85-109% by the SPR-sensor and 100-124% by the dc-ELISA. On real tomato samples, the results obtained by both of these immunoassays correlated well with the results obtained by high-performance liquid chromatography.

Nuclear factor-kappa B is required for tumor necrosis factor-alpha-induced manganese superoxide dismutase expression in human endometrial stromal cells.

We recently found that manganese superoxide dismutase (Mn-SOD) is up-regulated by TNF alpha at the transcription level in human endometrial stromal cells (ESC) and that TNF alpha-induced Mn-SOD expression is mediated by protein kinase C (PKC)-dependent phosphorylation. This study was undertaken to investigate whether nuclear factor-kappa B (NF-kappa B), a transcription factor, is involved in Mn-SOD induction by TNF alpha or PKC in human ESC. Electrophoretic mobility shift assay revealed that TNF alpha (1 ng/ml) and phorbol 12-myristate 13-acetate (TPA; 0.4 micro M), PKC activator, caused marked increases in nuclear NF-kappa B DNA binding activity. Secondly, ESC were incubated with MG132 (proteasome inhibitor) or SN50 (inhibitor of translocation of NF-kappa B into the nucleus) in the presence of TNF alpha or TPA. TNF alpha and TPA significantly increased Mn-SOD activities and Mn-SOD mRNA levels, and those effects were completely inhibited by MG132 and SN50. TNF alpha alone caused no effect on cell viability, but in the presence of MG132, TNF alpha significantly decreased cell viability. This inhibitory effect of MG132 was blocked by simultaneous addition of N-acetyl-L-cysteine, an antioxidant. In conclusion, the present study showed the involvement of NF-kappa B in Mn-SOD induction by TNF alpha or PKC in human ESC. This phenomenon could be a self-defense system of ESC against TNF alpha-mediated oxidative stress.

Role of transient hyperprolactinemia in the late follicular phase of the gonadotropin-stimulated cycle.

Background: Serum prolactin (PRL) concentration is known to transiently increase in rats; however, its change is obscure and the role of it is also unclear in women. We studied the relationship between estradiol (E2) and PRL production and the role of transient hyperprolactinemia in the late follicular phase of the gonadotropin-stimulated cycle. Methods: (1) Serum E2 and PRL concentrations were measured on an early follicular day and immediately before a human chorionic gonadotropin (hCG) injection in 60 patients with normoprolactinemia. Twelve of the 60 patients also received a gonadotropin injection with bromocriptine, and serum hormone levels were compared with those without bromocriptine. (2) Preovulatory serum E2 and PRL concentrations were compared between the natural and clomiphene treatment cycles in 14 hormonally normal women. (3) Changes of serum PRL concentrations were measured before and after E2 loading in five premature ovarian failure (POF) patients. (4) The E2 production by granulosa cells in the presence of PRL was measured. Results and Conclusion: Serum E2 and PRL concentrations were significantly increased by the gonadotropin injection. Bromocriptine treatment completely inhibited the PRL increase, but further increased serum E2 concentration on the late follicular day. The E2 loading increased serum PRL levels in POF patients. The clomiphene treatment increased serum E2 but decreased PRL concentrations. Prolactin significantly decreased E2 production by granulosa cells. A feedback loop may exist between E2 and PRL to control the excess E2 production induced by gonadotropin injection. (Reprod Med Biol 2002; 1: 69-74).