Synthesis of Caffeoyl-Prolyl-Histidyl-Xaa Derivatives and Evaluation of Their Activities and Stability upon Long-Term Storage.

Antioxidants play a critical role in the treatment of degenerative diseases and delaying the aging of dermal tissue. Caffeic acid (CA) is a representative example of the antioxidants found in plants. However, CA is unsuitable for long-term storage because of its poor stability under ambient conditions. Caffeoyl-Pro-His-NH2 (CA-Pro-His-NH2, CA-PH) exhibits the highest antioxidant activity, free radical scavenging and lipid peroxidation inhibition activity among the histidine-containing CA-conjugated dipeptides reported to date. The addition of short peptides to CA, such as Pro-His, is assumed to synergistically enhance its antioxidative activity. In this study, several caffeoyl-prolyl-histidyl-Xaa-NH2 derivatives were synthesized and their antioxidative activities evaluated. CA-Pro-His-Asn-NH2 showed enhanced antioxidative activity and higher structural stability than CA-PH, even after long-term storage. CA-Pro-His-Asn-NH2 was stable for 3 months, its stability being evaluated by observing the changes in its NMR spectra. Moreover, the solid-phase synthetic strategy used to prepare these CA-Pro-His-Xaa-NH2 derivatives was optimized for large-scale production. We envision that CA-Pro-His-Xaa-NH2 derivatives can be used as potent dermal therapeutic agents and useful cosmetic ingredients.

Anti-inflammatory activity of palmitoylethanolamide ameliorates osteoarthritis induced by monosodium iodoacetate in Sprague-Dawley rats.

Novel treatment strategies are urgently required for osteoarthritis (OA). Palmitoylethanolamide (PEA) is a naturally occurring fatty acid amide with analgesic and anti-inflammatory effects. We aimed to examine its effect on OA and elucidate the molecular mechanism of actions in monosodium iodoacetate (MIA)-induced OA Sprague-Dawley rats. The experimental animals were divided into normal control group (injected with saline + treated with phosphate-buffered saline (PBS), NOR), control group (injected with MIA + treated with PBS, CON), 50 or 100 mg/kg body weight (BW)/day PEA-treated group (injected with MIA + treated with 50 or 100 mg of PEA/kg BW/day, PEA50 or PEA100), and positive control group (injected with MIA + treated with 6 mg of diclofenac/kg BW/day, DiC). The changes in blood parameters, body parameters, gene expression of inflammatory mediators and cytokines, knee thickness, and joint tissue were observed. Oral administration of PEA had no adverse effects on the BW, liver, or kidneys. PEA reduced knee joint swelling and cartilage degradation in MIA-induced OA rats. The serum levels of leukotriene B4, nitric oxide, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and prostaglandin E2 considerably reduced in the PEA100 group compared with those in the CON group. In the synovia of knee joints, the mRNA expression of iNOS, 5-Lox, Cox-2, Il-1ß, Tnf-α, and Mmp-2, -3, -9, and -13 apparently increased with MIA administration. Meanwhile, Timp-1 mRNA expression apparently decreased in the CON group but increased to the normal level with PEA treatment. Thus, PEA can be an effective therapeutic agent for OA.

Caffeoyl-Prolyl-Histidine Amide Inhibits Fyn and Alleviates Atopic Dermatitis-Like Phenotypes via Suppression of NF-κB Activation.

Caffeic acid (CA) is produced from a variety of plants and has diverse biological functions, including anti-inflammation activity. It has been recently demonstrated that caffeoyl-prolyl-histidine amide (CA-PH), which is CA conjugated with proline-histidine dipeptide, relieves atopic dermatitis (AD)-like phenotypes in mouse. In this study, we investigated the molecular mechanism underlying CA-PH-mediated alleviation of AD-like phenotypes using cell line and AD mouse models. We confirmed that CA-PH suppresses AD-like phenotypes, such as increased epidermal thickening, infiltration of mast cells, and dysregulated gene expression of cytokines. CA-PH suppressed up-regulation of cytokine expression through inhibition of nuclear translocation of NF-κB. Using a CA-PH affinity pull-down assay, we found that CA-PH binds to Fyn. In silico molecular docking and enzyme kinetic studies revealed that CA-PH binds to the ATP binding site and inhibits Fyn competitively with ATP. CA-PH further suppressed spleen tyrosine kinase (SYK)/inhibitor of nuclear factor kappa B kinase (IKK)/inhibitor of nuclear factor kappa B (IκB) signaling, which is required for nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. In addition, chronic application of CA-PH, in contrast with that of glucocorticoids, did not induce up-regulation of regulated in development and DNA damage response 1 (REDD1), reduction of mammalian target of rapamycin (mTOR) signaling, or skin atrophy. Thus, our study suggests that CA-PH treatment may help to reduce skin inflammation via down-regulation of NF-κB activation, and Fyn may be a new therapeutic target of inflammatory skin diseases, such as AD.

Predictive factors for pregnancy during the first four intrauterine insemination cycles using gonadotropin.

PURPOSE: Although a variety of factors have been reported as affecting pregnancy rates after intrauterine insemination (IUI), there have been conflicting results on prognostic factors. This study aimed to determine predictive factors for pregnancy in patients undergoing the first four IUI cycles. METHODS: A total of 348 IUI cycles using clomiphene citrate or letrozole combined with gonadotropin, or gonadotropin only were analyzed. Baseline clinical characteristics, variables related to ovulation induction and sperm parameters were compared between pregnant (n = 54) and non-pregnant groups (n = 294). Logistic regression analysis was performed to identify factors that could predict a pregnancy. RESULTS: The overall clinical pregnancy rate was 15.5% (54/348) per cycle and 30.0% (54/180) per couple. During the first four IUI cycles, logistic regression analysis revealed that woman who were 39 years or older (OR: 0.263, 95% CI: 0.076-0.906, p = 0.034), longer duration of infertility (OR: 0.967, 95% CI: 0.942-0.993, p = 0.012), endometriosis (versus unexplained infertility; OR: 0.177, 95% CI: 0.040-0.775, p = 0.022) and endometrial thickness below 7 mm (OR: 0.114, 95% CI: 0.015-0.862, p = 0.035) were unfavorable factors to predict clinical pregnancy. CONCLUSIONS: Women with old age, longer duration of infertility, the presence of endometriosis or thin endometrium in the preovulatory phase may have unfavorable outcomes during the first four IUI cycles.

Kisspeptin, leptin, and retinol-binding protein 4 in women with polycystic ovary syndrome.

OBJECTIVES: To compare plasma kisspeptin, serum leptin, and serum retinol-binding protein 4 (RBP4) levels in women with and without polycystic ovary syndrome (PCOS) and to correlate these among each other and with clinical, hormonal, and metabolic parameters. METHODS: Ninety women, including 54 women with PCOS and 36 without PCOS, participated in this study. For all patients, history and physical examinations were performed and blood samples were collected between days 3 and 8 of a spontaneous bleeding episode in the PCOS group and during normal menses of controls. Plasma kisspeptin, serum leptin, and serum RBP4 levels were measured using specific commercial assays. RESULTS: Kisspeptin, leptin, and RBP4 levels were significantly higher in the PCOS group than in controls. Kisspeptin and RBP4 levels were significantly higher among obese PCOS patients than controls. Leptin levels were higher among obese PCOS patients than non-obese PCOS patients or controls. Kisspeptin and leptin levels of PCOS patients were significantly correlated with RBP4 levels. When only obese PCOS patients were analyzed, kisspeptin levels correlated with only the free androgen index. CONCLUSIONS: These findings suggest that kisspeptin, leptin, and RBP4 are associated with metabolic disturbances in women with PCOS.

Agaricus bisporus Extract Exerts an Anti-Obesity Effect in High-Fat Diet-Induced Obese C57BL/6N Mice by Inhibiting Pancreatic Lipase-Mediated Fat Absorption.

Agaricus bisporus is well known as a source of polysaccharides that could improve human health. The objective of this study was to explore the anti-obesity effect of A. bisporus extract (ABE), abundant in polysaccharides, and its underlying mechanism. Pancreatic lipase inhibitory activity in vitro was determined after treatment with ABE and chitosan. Treatment with ABE and chitosan significantly decreased pancreatic lipase activity. Five-week-old male SD rats were randomly divided into three groups for acute feeding with vehicle, ABE at 80 mg/kg body weight (BW)/day, and ABE at 160 mg/kg BW/day. ABE dose-dependently increased plasma lipid clearance in an oral lipid tolerance test. Five-week-old male C57BL/6N mice were fed a control diet (CD), a high-fat diet (HFD), an HFD with ABE at 80 mg/kg BW/day, ABE at 160 mg/kg BW/day, or chitosan at 160 mg/kg BW/day for eight weeks. HFD-fed mice showed significant increases in body weight, fat mass, white adipose tissue, average lipid droplet size, and serum levels of glucose, triglyceride, ALT, and AST compared to those in the CD group. However, ABE or chitosan administration ameliorated these increases. ABE or chitosan significantly reduced dietary efficiency and increased fecal excretion levels of lipids, triglycerides, and total cholesterol. These in vitro and in vivo findings suggest that ABE might act as an anti-obesity agent by inhibiting pancreatic lipase-mediated lipid absorption, at least in part.

Kaempferia parviflora rhizome extract exerts anti-obesity effect in high-fat diet-induced obese C57BL/6N mice.

Kaempferia parviflora (KP) rhizome, also called black ginger, has been used as a herbal medicine for many centuries. This current study was aimed at exploring whether KP rhizome extract (KPE) had anti-obesity effects and the mechanism involved. Five-week-old C57BL/6N male mice were allocated into five groups for 8-week feeding with control diet (CD), high-fat diet (HFD), HFD + 150 mg/kg body weight (BW)/day KPE (HFD+K150), HFD + 300 mg/kg BW/day KPE (HFD+K300), and HFD + 600 mg/kg BW/day KPE (HFD+K600). KPE decreased BW, body fat mass, adipose tissue weight, adipocyte size, and serum levels of glucose, triglycerides, cholesterol, insulin, and leptin in HFD-induced obese C57BL/6N mice. KPE inhibited adipogenesis by decreasing CCAAT/enhancer binding protein α, peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein-1c, acetyl-CoA carboxylase 1, ATP-citrate lyase, and fatty acid synthase mRNA expression. KPE improved lipolysis by increasing carnitine palmitoyl transferase 1 and hormone-sensitive lipase mRNA expression. These results suggest that KPE may have inhibited HFD-induced obesity by regulating several pathways involved in decreasing adipogenesis and enhancing lipolysis. Thus, the results suggest that KPE (or KP) may be applicable as an anti-obesity agent.

Urinary vitamin D-binding protein is elevated in patients with endometriosis.

BACKGROUND: Recently, proteomic technologies have demonstrated that several proteins are differently expressed in various body fluids of patients with endometriosis compared with those without this condition. The aim of this study was to investigate proteins secreted in urine of patients with endometriosis using proteomic techniques in order to identify potential markers for the clinical diagnosis of endometriosis. METHODS: Urine samples were collected from women undergoing laparoscopy for different indications including pelvic masses, pelvic pain, suspicious endometriosis, infertility and diagnostic evaluation. Proteomic techniques and mass spectrometry were used to identify proteins secreted in the urine of the patients with and without endometriosis and quantification of identified protein was performed using western blot and specific commercial sandwich enzyme-linked immunosorbent assays (ELISA). RESULTS: Twenty-two protein spots were differentially expressed in the urine of patients with and without endometriosis, one of which was identified as urinary vitamin D-binding protein (VDBP). ELISA quantification of urinary VDBP corrected for creatinine expression (VDBP-Cr) revealed that urinary VDBP-Cr was significantly greater in patients with endometriosis than in those without (111.96 ± 74.59 versus 69.90 ± 43.76 ng/mg Cr, P = 0.001). VDBP-Cr had limited value as a diagnostic marker for endometriosis (Sensitivity 58%, Specificity 76%). When combined with serum CA-125 levels (the product of serum CA-125 and urinary VDBP-Cr), it did not significantly increase the diagnostic power of serum CA-125 alone. CONCLUSIONS: Urinary VDBP levels are elevated in patients with endometriosis. They have limited value as a potential diagnostic biomarker for endometriosis but suggest it would be worthwhile to investigate other urinary proteins for this purpose.

Expression of vascular endothelial growth factor (VEGF) and its soluble receptor-1 in endometriosis.

The aim of this study is to evaluate the expression of vascular endothelial growth factor (VEGF) and its soluble receptor (sFlt-1) in peritoneal fluid (PF), peritoneal endometriotic lesions and eutopic endometrial tissues of patients with endometriosis. Peritoneal fluid, peritoneal endometriotic lesions and eutopic endometrial samples from patients with endometriosis, and peritoneal fluid, peritoneal tissue and endometrial samples from patients without endometriosis were obtained during an operative laparoscopy. The mean PF concentrations of VEGF and sFlt-1 were significantly higher in patients with endometriosis than in the controls. In the peritoneal tissue, the expressions of VEGF and sFlt-1 were significantly higher, where the expression of sFlt-1 in endometrium was significantly lower in patients with endometriosis. These findings indicate that not only abnormal expressions of angiogenic factors, but also aberrant expressions of antiangiogenic factors in the peritoneal and endometrial environment seem to be involved in the pathogenesis of endometriosis.

Changes in serum levels of cartilage oligomeric matrix protein after estrogen and alendronate therapy in postmenopausal women.

BACKGROUND: Cartilage oligomeric matrix protein (COMP) is a biomarker for joint destruction and its serum levels are used for assessing therapeutic efficacy. This study aims to compare changes in serum COMP levels in postmenopausal women with osteopenia/osteoporosis receiving estrogen and alendronate. METHODS: A total of 62 postmenopausal women diagnosed with osteopenia or osteoporosis were treated with either estrogen (17ß-estradiol 1 mg, n = 30) or bisphosphonate (alendronate 5 mg, n = 32) for 6 months. The controls were healthy postmenopausal women (n = 30). Serum COMP and osteocalcin levels were measured at baseline and after 6 months of treatment. RESULTS: Estrogen decreased levels of COMP at 6 months compared to baseline levels (­8.35 ± 19.38%), whereas the bisphosphonate and control groups resulted in no significant changes (­5.50 ± 18.69 and ­1.49 ± 25.34%, respectively). Concentrations of osteocalcin decreased significantly in both treatment groups (estrogen ­25.60 ± 24.42% and alendronate ­13.76 ± 23.89%, respectively). There was a significant positive correlation between changes after 6 months in COMP and osteocalcin (R = 0.48, p = 0.002). CONCLUSIONS: Postmenopausal women treated with estrogen showed significantly decreased levels of COMP after 6 months. Estrogen might provide a further treatment modality in the prevention of joint destruction.

Pre-treatment diagnosis of endometrial cancer through a combination of CA125 and multiplication of neutrophil and monocyte.

AIM: This study aimed to investigate the clinical value of pre-treatment leukocyte differential counts and the prediction of endometrial cancer using leukocyte markers. MATERIAL AND METHODS: Medical records of 238 women with pathologically confirmed endometrial cancer between March 2000 and June 2009 at two Korean hospitals were reviewed and compared to 596 healthy people visiting the Health Promotion Center in Gangnam Severance Hospital. For all study subjects, leukocyte differential counts and CA125 levels in serum obtained prior to operation were recorded. Multiplication of neutrophil and monocyte (MNM) was determined by multiplying neutrophil and monocyte counts then dividing by 10000. Differences between endometrial cancer patients and healthy controls were compared. The sensitivity and specificity for each marker as well as the combined use of CA125 and other leukocyte markers were assessed using receiver operating characteristic curves. RESULTS: Mean white blood cell (WBC) counts were 6676 (6440-6913) cells/µL in endometrial cancer patients compared to 5663 (5542-5784) cells/µL in healthy controls (P<0.001). The area under curve (AUC) for CA125 was 0.689 with a sensitivity of 49.13% and specificity of 83.1% using an optimal cut-off value of 18.7U/mL. The AUC for MNM was 0.696 with a sensitivity of 62.9% and specificity of 69.1%. The combination of CA125 and MNM showed a higher AUC of 0.760 than use of CA125 or MNM alone. CONCLUSION: The combination of MNM and CA125 is a simple and cost-effective method for predicting endometrial cancer.

Gynostemma pentaphyllum extract and its active component gypenoside L improve the exercise performance of treadmill-trained mice.

BACKGROUND/OBJECTIVES: The effectiveness of natural compounds in improving athletic ability has attracted attention in both sports and research. Gynostemma pentaphyllum (Thunb.) leaves are used to make traditional herbal medicines in Asia. The active components of G. pentaphyllum, dammarane saponins, or gypenosides, possess a range of biological activities. On the other hand, the anti-fatigue effects from G. pentaphyllum extract (GPE) and its effective compound, gypenoside L (GL), remain to be determined. MATERIALS/METHODS: This study examined the effects of GPE on fatigue and exercise performance in ICR mice. GPE was administered orally to mice for 6 weeks, with or without treadmill training. The biochemical analysis in serum, glycogen content, mRNA, and protein expressions of the liver and muscle were analyzed. RESULTS: The ExGPE (exercise with 300 mg/kg body weight/day of GPE) mice decreased the fat mass percentage significantly compared to the ExC mice, while the ExGPE showed the greatest lean mass percentage compared to the ExC group. The administration of GPE improved the exercise endurance and capacity in treadmill-trained mice, increased glucose and triglycerides, and decreased the serum creatine kinase and lactate levels after intensive exercise. The muscle glycogen levels were higher in the ExGPE group than the ExC group. GPE increased the level of mitochondrial biogenesis by enhancing the phosphorylation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) protein and the mRNA expression of nuclear respiratory factor 1, mitochondrial DNA, peroxisome proliferator-activated receptor-δ, superoxide dismutase 2, and by decreasing the lactate dehydrogenase B level in the soleus muscle (SOL). GPE also improved PGC-1α activation in the SOL significantly through AMPK/p38 phosphorylation. CONCLUSIONS: These results showed that GPE supplementation enhances exercise performance and has anti-fatigue activity. In addition, the underlying molecular mechanism was elucidated. Therefore, GPE is a promising candidate for developing functional foods and enhancing the exercise capacity and anti-fatigue activity.

Gynostemma pentaphyllum extract and Gypenoside L enhance skeletal muscle differentiation and mitochondrial metabolism by activating the PGC-1α pathway in C2C12 myotubes.

BACKGROUND/OBJECTIVES: Peroxisome proliferator-activated receptor-gamma co-activator-1α (PGC-1α) has a central role in regulating muscle differentiation and mitochondrial metabolism. PGC-1α stimulates muscle growth and muscle fiber remodeling, concomitantly regulating lactate and lipid metabolism and promoting oxidative metabolism. Gynostemma pentaphyllum (Thumb.) has been widely employed as a traditional herbal medicine and possesses antioxidant, anti-obesity, anti-inflammatory, hypolipemic, hypoglycemic, and anticancer properties. We investigated whether G. pentaphyllum extract (GPE) and its active compound, gypenoside L (GL), affect muscle differentiation and mitochondrial metabolism via activation of the PGC-1α pathway in murine C2C12 myoblast cells. MATERIALS/METHODS: C2C12 cells were treated with GPE and GL, and quantitative reverse transcription polymerase chain reaction and western blot were used to analyze the mRNA and protein expression levels. Myh1 was determined using immunocytochemistry. Mitochondrial reactive oxygen species generation was measured using the 2'7'-dichlorofluorescein diacetate assay. RESULTS: GPE and GL promoted the differentiation of myoblasts into myotubes and elevated mRNA and protein expression levels of Myh1 (type IIx). GPE and GL also significantly increased the mRNA expression levels of the PGC-1α gene (Ppargc1a), lactate metabolism-regulatory genes (Esrra and Mct1), adipocyte-browning gene fibronectin type III domain-containing 5 gene (Fndc5), glycogen synthase gene (Gys), and lipid metabolism gene carnitine palmitoyltransferase 1b gene (Cpt1b). Moreover, GPE and GL induced the phosphorylation of AMP-activated protein kinase, p38, sirtuin1, and deacetylated PGC-1α. We also observed that treatment with GPE and GL significantly stimulated the expression of genes associated with the anti-oxidative stress response, such as Ucp2, Ucp3, Nrf2, and Sod2. CONCLUSIONS: The results indicated that GPE and GL enhance exercise performance by promoting myotube differentiation and mitochondrial metabolism through the upregulation of PGC-1α in C2C12 skeletal muscle.

Protein and lipid MALDI profiles classify breast cancers according to the intrinsic subtype.

BACKGROUND: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been demonstrated to be useful for molecular profiling of common solid tumors. Using recently developed MALDI matrices for lipid profiling, we evaluated whether direct tissue MALDI MS analysis on proteins and lipids may classify human breast cancer samples according to the intrinsic subtype. METHODS: Thirty-four pairs of frozen, resected breast cancer and adjacent normal tissue samples were analyzed using histology-directed, MALDI MS analysis. Sinapinic acid and 2,5-dihydroxybenzoic acid/α-cyano-4-hydroxycinnamic acid were manually deposited on areas of each tissue section enriched in epithelial cells to identify lipid profiles, and mass spectra were acquired using a MALDI-time of flight instrument. RESULTS: Protein and lipid profiles distinguish cancer from adjacent normal tissue samples with the median prediction accuracy of 94.1%. Luminal, HER2+, and triple-negative tumors demonstrated different protein and lipid profiles, as evidenced by permutation P values less than 0.01 for 0.632+ bootstrap cross-validated misclassification rates with all classifiers tested. Discriminatory proteins and lipids were useful for classifying tumors according to the intrinsic subtype with median prediction accuracies of 80.0-81.3% in random test sets. CONCLUSIONS: Protein and lipid profiles accurately distinguish tumor from adjacent normal tissue and classify breast cancers according to the intrinsic subtype.

Histology-directed matrix-assisted laser desorption/ionization analysis reveals tissue origin and p53 status of primary liver cancers.

To date, protein profiles for hepatocellular carcinomas and cholangiocarcinomas have not been systematically evaluated and compared with each other in an unbiased way. Thirty-six hepatocellular carcinomas and adjacent normal tissue samples were analyzed using histology-directed, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Four cholangiocarcinomas and adjacent normal tissue samples were also evaluated. Tissue samples were sectioned at 10 µm, with 1-3 sections thaw-mounted on a conductive indium tin oxide-coated glass slide. Sinapinic acid was manually deposited on areas of each tissue section enriched by epithelial cells, either tumor or normal, and mass spectra were acquired using a MALDI-time of flight instrument. According to class prediction analysis, average prediction accuracy in test sets (composed of 18 hepatocellular carcinoma-normal pairs) ranged from 93.0 to 95.8%. Cholangiocarcinomas and hepatocellular carcinomas had different protein profiles, as evidenced by average prediction accuracy of >95% in the test set for all classifiers. Permutation P-values for 0.632 + bootstrap cross validated misclassification rates (at feature selection P < 0.001) were less than 0.05 for predicting p53 immunostaining status. We conclude that MALDI MS profiles may be useful in assisting with the diagnosis and the differential diagnosis of primary liver cancers.

Aqueous extract of Petasites japonicus leaves promotes osteoblast differentiation via up-regulation of Runx2 and Osterix in MC3T3-E1 cells.

BACKGROUND/OBJECTIVES: Petasites japonicus Maxim (P. japonicus) has been used as an edible and medicinal plant and contains many bioactive compounds. The purpose of this study is to investigate the effect of P. japonicus on osteogenesis. MATERIALS/METHODS: The leaves and stems of P. japonicus were separated and extracted with hot water or ethanol, respectively. The total phenolic compound and total polyphenol contents of each extract were measured, and alkaline phosphatase (ALP) activity of each extract was evaluated to determine their effect on bone metabolism. To investigate the effect on osteoblast differentiation of the aqueous extract of P. japonicus leaves (AL), which produced the highest ALP activity among the tested extracts, collagen content was measured using the Sirius Red staining method, mineralization using the Alizarin Red S staining method, and osteocalcin production through enzyme-linked immunosorbent assay analysis. Also, real-time reverse transcription polymerase chain reaction was performed to investigate the mRNA expression levels of Runt-related transcriptional factor 2 (Runx2) and Osterix. RESULTS: Among the 4 P. japonicus extracts, AL had the highest values in all of the following measures: total phenolic compounds, total polyphenols, and ALP activity, which is a major biomarker of osteoblast differentiation. The AL-treated MC3T3-E1 cells showed significant increases in induced osteoblast differentiation, collagen synthesis, mineralization, and osteocalcin production. In addition, mRNA expressions of Runx2 and Osterix, transcription factors that regulate osteoblast differentiation, were significantly increased. CONCLUSIONS: These results suggest that AL can regulate osteoblasts differentiation, at least in part through Runx2 and Osterix. Therefore, it is highly likely that P. japonicus will be useful as an alternate therapeutic for the prevention and treatment of osteoporosis.

Effects of LNG-IUS on nerve growth factor and its receptors expression in patients with adenomyosis.

The levonorgestrel-releasing intrauterine system (LNG-IUS) is effective in the treatment of dysmenorrhea associated with adenomyosis. However, the mechanism of pain relief of LNG-IUS in patients with adenomyosis is unclear. We aimed to investigate the effects of LNG-IUS on the expression of nerve growth factor (NGF) and its receptors, NGFR p75 and TrkA in patients with adenomyosis. Endometrial and myometrial tissues were prepared from 17 LNG-IUS-treated patients and 15 hormonally untreated patients who had undergone hysterectomies for adenomyosis. Immunohistochemistry with antibodies against NGF, NGFR p75, and TrkA, was performed. The expression of NGF, NGFR p75, and TrkA in endometrium and myometrium of LNG-IUS-treated patients was significantly decreased compared to those of hormonally untreated patients. Our findings may indicate that the suppression of NGF and its receptors by LNG-IUS is another possible mechanism of relieving pain in patients with adenomyosis.

Expression and possible role of non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) in the human endometrium and endometriosis.

BACKGROUND: Non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) is involved in cellular processes such as inflammation, apoptosis and tumorigenesis. However, little is known about the expression and function of NAG-1 in the endometrium. This study aimed to evaluate the expression of NAG-1 in the endometrium and in the absence or presence of endometriosis and to investigate the effect of celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, on NAG-1 mRNA levels and apoptosis in human endometrial stromal cells (HESCs). METHODS: Eutopic endometrial samples were obtained during surgery from 40 patients with, and 40 patients without, endometriosis. Real-time PCR was used to quantify NAG-1 mRNA levels and immunohistochemistry was used to localize NAG-1 protein in the endometrium. To investigate the effects of celecoxib, HESCs were isolated and cultured with different concentrations of celecoxib or with 100 µM celecoxib at different times. Apoptosis was assessed by flow cytometry. RESULTS: NAG-1 mRNA levels and immunoreactivity showed cyclical changes through the menstrual cycle, increasing during the late secretory and menstrual phases. NAG-1 mRNA and protein levels were significantly lower in patients with endometriosis, compared with the control group. Celecoxib induced NAG-1 mRNA levels and apoptosis in cultured HESCs, with the effects dependent on drug concentrations and duration of treatment. Celecoxib treatment had no effect on prostaglandin E(2) levels in the culture supernatants. CONCLUSIONS: NAG-1 may be important in maintaining homeostasis in the normal endometrium and alterations in NAG-1 expression may be associated with the establishment of endometriosis. NAG-1 might be a therapeutic target for endometriosis.

Characterization of human monoclonal autoantibody Fab fragments against oxidized LDL.

Oxidized low-density lipoprotein (oxLDL) is a key autoantigen in atherosclerosis. The genetic structures and pathogenic roles of autoantibodies against this protein remain to be established. In this study, we cloned several monoclonal IgG autoantibody Fab fragments specific for oxLDL from peripheral blood lymphocytes of atherosclerosis patients, using phage display technology. The sequences of their variable regions were determined at the cDNA level. The closest germline counterparts for the heavy chains belonged to the V(H)3 or V(H)1 family. The sequences and lengths of complementarity-determining regions (CDR)3-V(H) were diverse, and frequent mutations of positively charged amino acids (particularly arginine) over entire V(H) and V(L) sequences were observed. It is proposed that anti-oxLDL autoantibody formation is driven by antigens. Among the Fabs, P2-8 and P3-175 bound to both MDA-LDL and Cu-oxLDL, and inhibited the uptake of oxLDL by macrophages, suggesting the epitope(s) recognized by the Fabs is a part of ligands on oxLDL that is involved in uptake by macrophage scavenger receptor. These human autoantibody Fabs require detailed investigation to ascertain their potential as agents for clinical applications.

The Prognostic Value of Individual Adhesion Scores from the Revised American Fertility Society Classification System for Recurrent Endometriosis.

PURPOSE: This study aimed to evaluate the prognostic value of each component of the revised American Fertility Society (rAFS) classification system for the first recurrence of endometriosis after conservative laparoscopy. MATERIALS AND METHODS: As this was a retrospective cohort study, data were collected by reviewing medical records. A total of 379 women ages 18 to 49 years were included. Women who underwent conservative laparoscopy with histologic confirmation of endometriosis at Gangnam Severance Hospital between March 2003 and May 2010 were included. Individual components of the rAFS classification system as well as preoperative serum CA-125 levels were retrospectively analyzed to assess their prognostic values for recurrence of endometriosis. RESULTS: Of 379 patients, 80 (21.2%) were found to have recurrence of endometriosis. The median duration of follow-up was 19.0 months, and the mean age at the time of surgery was 31.8±6.7 years. In endometriosis of advanced stage, younger age at the time of surgery, bilateral ovarian cysts at the time of diagnosis, a rAFS ovarian adhesion score >24, and complete cul-de-sac obliteration were independent risk factors of poor outcomes, and a rAFS ovarian adhesion score >24 had the highest risk of recurrence [hazard ratio=2.948 (95% CI: 1.116-7.789), p=0.029]. CONCLUSION: Our results suggest that of the rAFS adnexal adhesion scores, the ovarian adhesion score rather than the tubal adhesion score was associated with a significantly increased risk of recurrent endometriosis. The preoperative serum CA-125 level may be also a significant prognostic factor for recurrence, as known. However, it seemed to only have borderline significance in affecting recurrence in the current study.