Fibroblast growth factor 21 analogue LY2405319 lowers blood glucose in streptozotocin-induced insulin-deficient diabetic mice by restoring brown adipose tissue function.

AIM: To investigate the effects of LY2405319, an analogue of fibroblast growth factor 21 (FGF21), on glucose homeostasis in streptozotocin (STZ)-induced insulin-deficient mice (STZ mice). METHODS: Nine-week-old male C57BL/6J mice were administered a single intraperitoneal injection of STZ (150 mg/kg). One week later, after confirmation of hyperglycaemia, saline or LY2405319 (5 mg/kg) was injected subcutaneously daily for 4 weeks. Changes in glucose homeostasis, energy metabolism and brown adipose tissue (BAT) function were assessed. RESULTS: The STZ mice had elevated blood glucose and reduced plasma FGF21 levels, impaired glucose uptake in the BAT, and BAT mitochondria with absent or swollen cristae and fewer lipid vacuoles. LY2405319 significantly reduced blood glucose levels and this was associated with increased BAT glucose uptake and changes in gene expression and morphology, indicating improved mitochondrial lipid metabolism in the BAT. Importantly, the ability of LY2405319 to lower blood glucose in STZ mice was compromised after removing interscapular BAT. CONCLUSIONS: Our results show that LY2405319 reduces blood glucose levels in insulin-deficient diabetes by improving BAT metabolism. Additional studies investigating the therapeutic potential of FGF21 for the treatment of type 1 diabetes are warranted.

The impact of the National Denture Service on oral health-related quality of life among poor elders.

The objective of this study was to assess the effects of the Korean National Denture Service (NDS) for poor elderly people requiring dentures on oral health-related quality of life (OHRQOL). Data from follow-up studies were collected from 439 subjects at eight public health centres who answered every question of a questionnaire, and the OHRQOL was measured at the baseline and at 3-month follow-up after receiving the NDS according to the type of denture provision. The multivariate linear mixed model with a public health centre as a random effect for the score change of Oral Health Impact Profile (OHIP)-14K was carried out to confirm the factors related to the improvement in OHRQOL. The mean OHIP-14K was 28.60 at the baseline time points, and there was a decrease in the OHIP-14 scores to 21.14 ± 12.52 at the 3-month follow-up of the removable partial denture beneficiaries. The changes in OHIP-14K among complete denture beneficiaries were 21.53 ± 12.01 for previously dentate subjects and 22.54 ± 11.12 for edentate subjects. The multivariate linear mixed model of dentate subjects demonstrated that the improvement in the OHRQOL was associated with the number of remaining teeth, satisfaction with denture and self-reported oral health status after 3 months. In the case of the edentate model, satisfaction with denture was the only factor related to the improvement in OHRQOL. This study revealed considerable improvement in OHRQOL among poor elderly people after NDS. Satisfaction with provision of dentures was associated with improvement in the OHRQOL.

VDUP1 potentiates Ras-mediated angiogenesis via ROS production in endothelial cells.

Vitamin D3 up-regulated protein 1 (VDUP1) is a tumor suppressor of which expression is reduced in a variety of cancer cells, and enforced expression inhibits the tumor cell proliferation. It inhibits the activity of thioredoxin, thus contributing cellular ROS generation. Since ROS is a critical factor for angiogenesis, we investigated the role of VDUP1 in angiogenesis and endothelial proliferation. The expression of VDUP1 was upregulated by overexpression of an oncogene, Ras. Enforced expression of VDUP1 increases ROS production and proliferation of Ras-overexpressing endothelial cells. Overexpression of VDUP1 increases the resistance to the anchorage-dependent cell death and tube formation of the Ras-overexpressing endothelial cell. In addition, the removal of ROS by ROS scavenger attenuates the effect of VDUP1 on tube formation. These results suggest that VDUP1 is involved in Ras-mediated angiogenesis via ROS generation in endothelial cells.

Tripartite structure of Saccharomyces cerevisiae Dna2 helicase/endonuclease.

In order to gain insights into the structural basis of the multifunctional Dna2 enzyme involved in Okazaki fragment processing, we performed biochemical, biophysical and genetic studies to dissect the domain structure of Dna2. Proteolytic digestion of Dna2 using subtilisin produced a 127 kDa polypeptide that lacked the 45 kDa N-terminal region of Dna2. Further digestion generated two subtilisin-resistant core fragments of approximately equal size, 58 and 60 kDa. Surprisingly, digestion resulted in a significant (3- to 8-fold) increase in both ATPase and endonuclease activities compared to the intact enzyme. However, cells with a mutant DNA2 allele lacking the corresponding N-terminal region were severely impaired in growth, being unable to grow at 37 degrees C, indicating that the N-terminal region contains a domain critical for a cellular function(s) of Dna2. Analyses of the hydrodynamic properties of and in vivo complex formation by wild-type and/or mutant Dna2 lacking the N-terminal 45 kDa domain revealed that Dna2 is active as the monomer and thus the defect in the mutant Dna2 protein is not due to its inability to multimerize. In addition, we found that the N-terminal 45 kDa domain interacts physically with a central region located between the two catalytic domains. Our results suggest that the N-terminal 45 kDa domain of Dna2 plays a critical role in regulation of the enzymatic activities of Dna2 by serving as a site for intra- and intermolecular interactions essential for optimal function of Dna2 in Okazaki fragment processing. The possible mode of regulation of Dna2 is discussed based upon our recent finding that replication protein A interacts functionally and physically with Dna2 during Okazaki fragment processing.

Intracellular annexin A2 regulates NF-κB signaling by binding to the p50 subunit: implications for gemcitabine resistance in pancreatic cancer.

Annexin A2 (ANXA2) expression is highly upregulated in many types of cancer. Although cell surface localization of ANXA2 has been reported to have a critical role in the progression and metastasis of a variety of tumors, including pancreatic cancer, the biological role of intracellular ANXA2 is not fully understood. Herein the role of intracellular ANXA2 was investigated in a pancreatic cancer cell line. We first determined whether ANXA2 is involved in NF-κB signaling pathways. ANXA2 bound to the p50 subunit of NF-κB in a calcium-independent manner, and the ANXA2-p50 complex translocated into the nucleus. Furthermore, ANXA2 increased the transcriptional activity of NF-κB in both the resting and activated states and upregulated the transcription of several target genes downstream of NF-κB, including that encoding interleukin (IL)-6, which contributes to anti-apoptotic signaling. In Mia-Paca2 cells, we determined the effects of wild-type ANXA2 and an ANXA2 mutant, Y23A, which suppresses the cell surface localization, on upregulation of NF-κB transcriptional activity and secretion of IL-6. Both wild-type and Y23A ANXA2 induced anti-apoptotic effects in response to treatment with tumor necrosis factor-α or gemcitabine. Based on these results, we suggest that ANXA2 mediates resistance to gemcitabine by directly increasing the activity of NF-κB. Collectively, these data may provide additional information about the biological role of ANXA2 in pancreatic cancer and suggest that ANXA2 is a potential biomarker for the drug resistance phenotype and a candidate therapeutic target for the treatment of pancreatic cancer.

Dimeric beta-cyclodextrin-based supramolecular ligands and their copper(II) complexes as metalloenzyme models.

New supramolecular ligands possessing linear 13- and 15-membered pyridine diamidetriamine chelators between the primary sides of two beta-cyclodextrin cavities were synthesized, and characterized by MALDI-MS, NMR, IR and UV-Visible spectroscopy. Fluorescence and pH-metric titration were carried out in order to ascertain their behavior as bifunctional hosts for fluorescent guests and Cu(II) ion. The pKa value for the Cu(II) promoted deprotonation of amide ligands was determined to be 6.2 from pH-absorbance profile. Above pH 8.0, two deprotonated amides and three amino groups chelated Cu(II) ion, and yielded penta-coordinated Cu(II) complexes. The Cu(II) complexes catalyzed the hydrolysis of p-nitrophenyl acetate, adamantate and amino acids. Especially, the complex containing 13-membered chelator is an artificial metalloesterase with catalytic rate constant kcat = 3.8 x 10(-3) min-1 and Michaelis constant K(m) = 3.5 x 10(-4) M for the hydrolysis of p-nitrophenyl adamantate via metal-hydroxide mechanism.

Synthesis and antibacterial activities of 4-hydroxy-o-phenylphenol and 3,6-diallyl-4-hydroxy-o-phenylphenol against a cariogenic bacterium Streptococcus mutans OMZ 176.

For the purpose of survey of the antibacterial activity against a cariogenic bacterium Streptococcus mutans OMZ 176 with the introduction of hydroxyl and allyl groups to o-phenylphenol (Fig. 2, 1), 4-hydroxy-o-phenylphenol (2), and 3,6-diallyl-4-hydroxy-o-phenylphenol (4) were synthesized, successively. The synthesized compounds, 2 and 4 showed more potent antibacterial activity than the starting material, 1. The hydroxyl group was supposed to the essential element for the antibacterial activity and the introduction of allyl group to phenolic ring to be another element to increase the activity.

A cytotoxic constituent fromSophora flavescens.

A cytotoxic constituent was isolated by bioassay-guided procedure from the roots ofSophora flavescens Aiton (Leguminosae). The constituent was identified as sophoraflavanone G (I) by means of chemical methods and in comparsion with spectral data of standard compound. The ED(50) values of constituent I were 0.78, 1.57, 2.14 and 8.59 mug/ml against A549, HeLa, K562 and L1210 cell lines respectively. ConstituentI exhibited highly cytotoxic activities against A 549, K562 and HeLa cells, but showed a mild activity (ED(50) value, 5 mug/ml) against L1210 cells. Among the tested cell lines, A549 cells were the most sensitive to constituentI.

Antitumor activity of 2(S)-5,2',5'-trihydroxy-7,8-dimethoxyflavanone and its analogues.

In an effort to increase of the antitumor activity of 2(S)-5,2',5'-trihydroxy-7,8-dimethoxyflavanone isolated fromScutellaria indica, we synthesized its analogues,II, III, andIV. They showed potent cytotoxicityin vitro against cancer cell lines, L1210, K562 and A549. On the basis of ED(50) values against the cancer cell lines,III exhibited about 2-7 times stronger activity thanI against various cell lines. We tested the antitumor activity of the analogues against Sarcoma 180 cellsin vivo and evaluated the structure-activity relationship. The antitumor activity appeared to be related to the hydrogen bond between carbonyl group at C-4 and hydroxyl group at C-5, in contrast to cytotoxic action.

Antifungal activity of magnolol and honokiol.

Two neolignan compounds, magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl, 1) and honokiol (5,5'-diallyl-2,4'-dihydroxybiphenyl, 2), were isolated from the stem bark of Magnolia obovata and evaluated for antifungal activity against various human pathogenic fungi. Compound 1 and 2 showed significant inhibitory activities against Trichophyton mentagrophytes, Microsporium gypseum, Epidermophyton floccosum, Aspergillus niger, Cryptococcus neoformans, and Candida albicans with minimum inhibitory concentrations (MIC) in a range of 25-100 microg/ml. Therefore, compound 1 and 2 could be used as lead compounds for the development of novel antifungal agents.

Two new lignans from Lindera obtusiloba blume.

Two new furanolignans (3, 5), together with three known lignans (1, 2, 4), were isolated from the stem of Lindera obtusiloba (Lauraceae). The structures of the compounds were determined as actifolin (1), pluviatilol (2), 5,6-dihydroxymatairesinol (3), (+)-syringaresinol (4), and (+)-9'-O-trans-feruloyl-5,5'-dimethoxylariciresinol (5) on the basis of physicochemical and spectroscopic evidences. Compounds 1, 2, 3, and 5 showed cytotoxicity against a small panel of human tumor cell lines with ED50 values of 3.40 to approximately 19.27 microg/ml.

Hypocholesterolemic effect of naringin associated with hepatic cholesterol regulating enzyme changes in rats.

The effects of the citrus bioflavonoid naringin were tested by using it as a supplement in a high-cholesterol diet. Male rats were fed for 42 days with a 1% (wt/wt) high cholesterol diet either with or without naringin-supplementation (0.1%, wt/wt) to study the effect on plasma lipid levels, hepatic lipid contents, hepatic enzyme activity, and the excretion of fecal neutral sterols. Naringin did not significantly alter the levels of plasma triglycerides, however, the levels of plasma cholesterol (3.80 +/- 0.31 mmol/L vs. 2.61 +/- 0.30 mmol/L, mean +/- SE; p < 0.05) and hepatic cholesterol (70.3 +/- 4.3 mg/g vs. 54.3 +/- 3.8 mg/g, mean +/- SD; p < 0.05) were significantly lowered compared to those of the control. HMG-CoA reductase (2487.0 +/- 210.0 pmole/min/mg vs. 1879.0 +/- 236.0 pmole/min/mg, mean +/- SE; p < 0.05) and ACAT (806.0 +/- 105.0 pmole/min/mg vs. 643.0 +/- 80.0 pmole/min/mg, mean +/- SE; p < 0.05) activities were both substantially lower in the naringin-supplemented group than in the control. The naringin supplementation markedly decreased the excretion of fecal neutral sterols (204.7 +/- 28.5 mg/day) compared to the control (521.9 +/- 53.9 mg/day). The combination of the inhibited HMG-CoA reductase (-24.4%) and ACAT (-20.2%) activities as a result of naringin supplementation could account for the decrease of fecal neutral sterols.

Inguinal approach for the management of unilateral non-palpable testis: is diagnostic laparoscopy necessary?

OBJECTIVE: During the last decade laparoscopy has increasingly been advocated as the primary investigative procedure for the management of the non-palpable testis. We reviewed the medical records in a consecutive series of boys with non-palpable testis to examine the contribution of the initial inguinal approach in the management of unilateral non-palpable testis. MATERIALS AND METHODS: Among the 183 consecutive patients with cryptorchidism from 2003 to 2012, there were 21 patients with unilateral and three with bilateral non-palpable testes. All unilateral patients then underwent inguinal and scrotal exploration through an inguinal incision. For those patients with an intra-abdominal peeping testis, the gonad was placed into the scrotum after meticulous cranial mobilization of the spermatic cord. RESULTS: Patient age ranged from 11 months to 144 months (mean age: 23 months). Among the 21 unilateral cases, testicular absence or atrophy was confirmed in seven patients with a scrotal nubbin in six, and blind-ending vas and vessels at the external inguinal ring in one patient. Among the remaining 14 patients with sizeable testes, 12 testes were intra-abdominal peeping testes and two testicles were seen within the distal inguinal canal, which may be missed on physical examination owing to patient obesity. The intra-abdominal peeping testicle had the opened processus vaginalis entering the internal ring in which testicle was found. These were fixed into the scrotum successfully by cranial mobilization of spermatic vessel sometimes cutting the internal oblique muscle and by Prentiss and Fowler-Stephen's maneuver. Diagnostic laparoscopy was done on three patients with bilateral cases. CONCLUSIONS: Given the result that most of nubbins are within the scrotum and testes with intra-abdominal peeping testes are fixed down safely into the scrotum, the inguinal approach may suffice for the management of unilateral non-palpable testis. Laparoscopy should be reserved for patients with bilateral non-palpable undescended testes.

Antitumor activity of Ad-IU2, a prostate-specific replication-competent adenovirus encoding the apoptosis inducer, TRAIL.

In this study, we analyzed the preclinical utility and antitumor efficacy of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) delivered by Ad-IU2, a prostate-specific replication-competent adenovirus (PSRCA), against androgen-independent prostate cancer. Through transcriptional control of adenoviral early genes E1a, E1b and E4, as well as TRAIL by two bidirectional prostate-specific enhancing sequences (PSES), the expression of TRAIL and adenoviral replication was limited to prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA)-positive cells. Ad-IU2 induced fivefold greater apoptosis selectively in PSA/PSMA-positive CWR22rv and C4-2 cells than an oncolytic adenoviral control. Furthermore, prolonged infection with Ad-IU2 reversed TRAIL resistance in LNCaP cells. Ad-IU2 showed superior killing efficiency in PSA/PSMA-positive prostate cancer cells at doses five- to eight-fold lower than required by a PSRCA to produce a similar effect; however, this cytotoxic effect was not observed in non-prostatic cells. As an enhancement of its therapeutic efficacy, Ad-IU2 exerted a TRAIL-mediated bystander effect through direct cell-to-cell contact and soluble factors such as apoptotic bodies. In vivo, Ad-IU2 markedly suppressed the growth of subcutaneous androgen-independent CWR22rv xenografts compared with a PSRCA at 6 weeks after treatment (3.1- vs 17.1-fold growth of tumor). This study shows the potential clinical utility of a PSRCA armed with an apoptosis-inducing ligand.

Enhanced combined tumor-specific oncolysis and suicide gene therapy for prostate cancer using M6 promoter.

Enzyme pro-drug suicide gene therapy has been hindered by inefficient viral delivery and gene transduction. To further explore the potential of this approach, we have developed AdIU1, a prostate-restricted replicative adenovirus (PRRA) armed with the herpes simplex virus thymidine kinase (HSV-TK). In our previous Ad-OC-TK/ACV phase I clinical trial, we demonstrated safety and proof of principle with a tissue-specific promoter-based TK/pro-drug therapy using a replication-defective adenovirus for the treatment of prostate cancer metastases. In this study, we aimed to inhibit the growth of androgen-independent (AI), PSA/PSMA-positive prostate cancer cells by AdIU1. In vitro the viability of an AI- PSA/PSMA-expressing prostate cancer cell line, CWR22rv, was significantly inhibited by treatment with AdIU1 plus GCV (10 microg ml(-1)), compared with AdIU1 treatment alone and also cytotoxicity was observed following treatment with AdIU1 plus GCV only in PSA/PSMA-positive CWR22rv and C4-2 cells, but not in the PSA/PSMA-negative cell line, DU-145. In vivo assessment of AdIU1 plus GCV treatment revealed a stronger therapeutic effect against CWR22rv tumors in nude mice than treatment with AdIU1 alone, AdE4PSESE1a alone or in combination with GCV. Our results demonstrate the therapeutic potential of specific-oncolysis and suicide gene therapy for AI-PSA/PSMA-positive prostate cancer gene therapy.

Far upstream element-binding protein-1, a novel caspase substrate, acts as a cross-talker between apoptosis and the c-myc oncogene.

Far upstream element-binding protein-1 (FBP-1) binds to an upstream element of the c-myc promoter and regulates the c-myc mRNA level. Earlier, FBP-1 was identified as a candidate substrate of caspase-7. Here, we report that FBP-1 is cleaved by executor caspases, both in vitro and during apoptosis. Cleavage occurs at the caspase consensus site (DQPD(74)) located within the classical bipartite nuclear localization signal sequence. In cells subjected to apoptotic stimuli, the caspase-mediated cleavage of FBP-1 leads to its decreased presence in the nucleus, concomitant with the marked downregulation of c-Myc and its various target proteins. By contrast, cells transfected with a non-cleavable mutant of FBP-1 (D74A) maintain higher levels of c-Myc and are protected from apoptosis. On the basis of these results, we suggest that the oncogenic potential of c-Myc is 'switched off' after apoptosis induction as a consequence of the caspase-mediated cleavage of FBP-1.

A comparison of oral health related quality of life between complete and partial removable denture-wearing older adults in Korea.

The purpose of this study was to compare the oral health related quality of life (OHRQoL) between older Korean adults with complete and partial removable dentures using oral health impact profile (OHIP). A pool of 4250 Korean older adults was identified by random stratification by area from the sample cohort of Korean National Survey of Oral Health in the year of 2000. Out of the potential pool, 445 subjects with removable partial dentures (RPDs) or complete dentures (CDs) agreed to be interviewed by telephone. Each question in the survey was about how frequently each event was experienced during the past 12 months. To analyse the association between OHIP scores and removable denture status, bivariable analysis and multiple linear regression analysis were used. RPD users showed higher scores on eight items than CD users. They were 'breath stale', 'food catching', 'sensitive teeth', 'toothache', 'tense', 'unable to brush teeth', 'less tolerant to family members' and 'irritable with others'. RPD users might be associated with higher scores of functional limitation, physical pain and psychological discomfort than CD users. RPD users had slight tendency to be associated with higher OHIP-49 score than CD users, but it was not statistically significant. The findings of the present study confirm that there is no general OHRQoL difference between RPD and CD users. But in the items and subscales of OHIP, RPD users might experience more impaired OHRQoL than CD users.

Pharmacological effects of methanolic extract from the root of Scutellaria baicalensis and its flavonoids on human gingival fibroblast.

The methanolic extract from the root of Scutellaria baicalensis Georgi and its flavonoids, wogonin, baicalein, and baicalin were evaluated for anti-inflammatory action and the activatory effect on gingival fibroblasts. In LPS-induced production of IL-1 beta, three flavonoids at 1 microgram/ml expressed a significant (> 50%) inhibitory effect, similar to that of prednisolone. Moreover, the flavonoids inhibited IL-1 beta-induced synthesis of PGE2 and LTB4 considerably, although the effect of wogonin on LTB4 synthesis was marginal. In addition, three flavonoids exerted a moderate inhibition (33-36%) of collagenolytic activity, comparable to 40% inhibition by tetracycline. Meanwhile, the cellular activity of fibroblasts was augmented remarkably (40%) by baicalein (2) and slightly by baicalin (3) or wogonin (1). Consistent with the cellular activation, flavonoids enhanced the synthesis of both collagen and total protein in fibroblasts, in contrast to growth factors which increased only the synthesis of total protein. Although the effects of the methanolic extract resembled those of the flavonoids, the extract expressed a preferential effect on the synthesis of collagen and total protein.

Effect of the weak Ca(2+)-binding site of subtilisin J by site-directed mutagenesis on heat stability.

The functional role of the negatively charged amino acid residue in subtilisin J from Bacillus stearothermophilus has been investigated by site-directed mutagenesis. Glu-195 located at the weak Ca2+-binding site was replaced with Gln to examine the role of Glu-195 in the heat stability of subtilisin J. Mutant enzyme was expressed in Bacillus subtilis and was purified from the culture supernatant. When the mutant enzyme was expressed at 37 degrees C in the presence of 2mM calcium chloride, the pattern of enzyme production was quite different from that of wild-type. The purified Gln-195 mutant enzyme was analyzed with respect to optimal temperature, optimal pH, and heat stability. The mutation was found to decrease the heat stability but not catalytic efficiency (kcat/Km) and optimal pH. These results demonstrate the important role of the negatively charged side chains at the weak Ca(2+)-binding site in the heat stability of subtilisin.

RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes.

Extensive work on the maturation of lagging strands during the replication of simian virus 40 DNA suggests that the initiator RNA primers of Okazaki fragments are removed by the combined action of two nucleases, RNase HI and Fen1, before the Okazaki fragments join. Despite the well established in vitro roles of these two enzymes, genetic analyses in yeast revealed that null mutants of RNase HI and/or Fen1 are not lethal, suggesting that an additional enzymatic activity may be required for the removal of RNA. One such enzyme is the Saccharomyces cerevisiae Dna2 helicase/endonuclease, which is essential for cell viability and is well suited to removing RNA primers of Okazaki fragments. In addition, Dna2 interacts genetically and physically with several proteins involved in the elongation or maturation of Okazaki fragments. Here we show that the endonucleases Dna2 and Fen1 act sequentially to facilitate the complete removal of the primer RNA. The sequential action of these enzymes is governed by a single-stranded DNA-binding protein, replication protein-A (RPA). Our results demonstrate that the processing of Okazaki fragments in eukaryotes differs significantly from, and is more complicated than, that occurring in prokaryotes. We propose a novel biochemical mechanism for the maturation of eukaryotic Okazaki fragments.