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Colorectal cancer (CRC) is a leading cause of mortality worldwide. We conducted a genome-wide association study meta-analysis of 100,204 CRC cases and 154,587 controls of European and East Asian ancestry, identifying 205 independent risk associations, of which 50 were unreported. We performed integrative genomic, transcriptomic and methylomic analyses across large bowel mucosa and other tissues. Transcriptome- and methylome-wide association studies revealed an additional 53 risk associations. We identified 155 high confidence effector genes functionally linked to CRC risk, many of which had no previously established role in CRC. These have multiple different functions, and specifically indicate that variation in normal colorectal homeostasis, proliferation, cell adhesion, migration, immunity and microbial interactions determines CRC risk. Cross-tissue analyses indicated that over a third of effector genes most likely act outside the colonic mucosa. Our findings provide insights into colorectal oncogenesis, and highlight potential targets across tissues for new CRC treatment and chemoprevention strategies.

In the version of this article initially published, the author affiliations incorrectly listed “Candiolo Cancer Institute FPO-IRCCS, Candiolo (TO), Italy” as “Candiolo Cancer Institute, Candiolo, Italy.” The change has been made to the HTML and PDF versions of the article.

The adductor muscle of the pen shell Atrina pectinata (AMPS) is a popular protein‐enriched food item in Asian Pacific countries, and has only been marketed in the frozen condition, as a result browning and decreased sensory attributes occur. To overcome these problems, superheated steam roasting (at 270°C for 4 min) combined with the hot smoke (10 min) using a selective Oak sawdust was employed to develop a new AMPS product yielding high physicochemical properties during storage periods (0–13 days) especially at 10°C. The processed AMPS showed high sensory preferences because of good odor, color, and textural properties. It also significantly inhibited bacterial growth, volatile basic nitrogen, thiobarbituric acid‐reactive species, and pH changes, and eventually possessed higher nutritional composition with low trimethylamine N‐oxide level. Results indicate that saturated steam allows AMPS at good physicochemical conditions, whereas hot smoke‐derived aroma compounds prolong its shelf life through antioxidant and antimicrobial activities.

The antioxidant properties of the Pacific chub mackerel (Scomber japonicus) muscle protein hydrolysates prepared by enzymatic hydrolysis were investigated. After enzyme hydrolysis at 50°C for 60 min, more than 80% of the S. japonicus muscle protein was hydrolyzed. The highest 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity (71.69%) occurred in whole muscle protein hydrolysates treated at 50°C for 30 min with Protamex, and the highest 2,2ʹ-azino-bis(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS) radical-scavenging activity (95.39%) was observed in white muscle protein hydrolysates treated at 50°C for 30 min with Neutrase. The highest superoxide dismutase (SOD)-like activity (32.84%) was recorded in white muscle protein hydrolysates treated at 50°C for 120 min with Protamex. Changes in the molecular weight distribution of S. japonicus muscle proteins after enzymatic hydrolysis were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A robust and a convenient enzyme hydrolysis technique for obtaining S. japonicus muscle protein hydrolysates with useful biological activities, within a short time (

There is technology available for anti-thrombus with earthworms, but the procedure is complex and extracts protein with inferior purity. In order to develop a simplified process with a stronger purity of protease, we investigated the Lumbricus rubellus earthworm and Perinereis linea lugworm. We purified water extracts cut off at 10 kDa of molecular weight using ultrafiltration because proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. We purified EW1 (raw earthworm extract), EW2 (molecular weight (m.w) > 10 kDa of earthworm extract), and EW3 (m.w 10 kDa), and LW3 (m.w EW1 > EW3 > LW2 > LW1 > LW3. In particular, EW2 and LW2 showed a potent fibrinolytic effect in two different worm specimens. The protein content of each sample was detected as 2.34 (EW1), 3.03 (EW2), 2.80 (LW1), 3.71 (LW2) mg/ml respectively, and their molecular weights were measured using SDS-PAGE. The samples contained the following amounts of total fatty acids: EW1, 3.61%; EW2, 0.48%; LW1, 4.96%; and LW2, 0.23%.We developed a process to increase the thrombolytic effect with a higher purity protein. The study results demonstrate this procedure and provide basic data for developing an anti-thrombolytic agent. Keywords: Lumbricus rubellus, Perinereis linea, Fibrinolytic effect, Fatty acid

Secretory carrier membrane proteins (SCAMPs) are widely distributed integral membrane proteins implicated in membrane trafficking. Secretory carrier membrane protein 5 (SCAMP5) is expected to be involved in regulation of the immune response because it is expressed in a variety of immune tissues and promotes the secretion of cytokines in monocytes and macrophages. In this study, we performed an analysis of the molecular characteristics and phylogenetic of the SCAMP5 gene identified in Pagrus major (PmSCAMP5). In addition, we analysed PmSCAMP5 gene expression levels in the tissues of red sea bream infected with various pathogens [Edwardsiella piscicida (E. piscicida), Streptococcus iniae (S. iniae) and Red sea bream iridovirus (RSIV)], and we analysed PmSCAMP5 gene expression levels in the tissues of healthy red sea bream. This study was carried out to provide basic data on the non-specific immune system of the red sea bream. Keywords: Secretory carrier membrane protein 5, Red sea bream, Edwardsiella piscicida, Streptococcus iniae, Red sea bream iridovirus

This study investigated the anti-inflammatory effects of mixed extracts of Achyranthes japonica Nakai (AJ) and Aralia continentalis Kitagawa (AC) (ratios of 1:2, 1:3, 1:5, 2:1, 3:1 and 5:1) on RAW264.7 macrophages and evaluated the anti-inflammatory effects of the mixed extracts of AJ and AC by measuring IL-1β, IL-6, and TNFα using the ELISA kit assay. In particular, the formation of nitric oxide (NO) was found to decrease in the group treated with the combined extracts of AJ and AC at all ratios. In particular, extracts of ratio of 2:1 (AJ:AC) deceased the formation of NO level that is approximately 60% of the group treated with only lipopolysaccharide (LPS). Also, extracts of ratio of 2:1 (AJ:AC) reduced the production of IL-1β, IL-6, TNFα and PGE2 with statistical significance. Volunteers over the age of 50 who complain of discomfort in knee joints were selected as the experimental subjects. The subjects took daily administration of 2000 mg of the combined extracts of ratio of 2:1 (AJ:AC) for 12 weeks. A survey (VAS (Visual Analog Scale), WOMAC (Western Ontario and McMaster Universities Osteoarthritis Index)) was conducted after the 12 weeks of oral administration. The experimental group showed the change between each visit and baseline time compared with the control group. In the intention-to-treat (ITT) analysis, VAS score and WOMAC stiffness score decreased significantly. And the WOMAC total score and function score tended to decrease. In the per-protocol (PP) analysis, the WOMAC stiffness score was significantly decreased and the VAS and WOMAC total and function scores were decreased. There was no significant difference in all parameters of ITT and PP in radiological examinations.

Based on the particle swarm algorithm to establish a mathematical model, this article counts the air quality index, tobacco sales, the number of cancer hospitals and the number of health personnel from 2008 to 2019, and quickly calculates the morbidity and mortality of lung cancer over the years, and uses statistics The relative error between the value and the calculated value is the evaluation standard. The result proves that the relative error between the calculated value and the statistical value of the morbidity and mortality of lung cancer is less than 10%, indicating that the calculation result of the particle swarm algorithm has good confidence.

The frequent occurrence of adverse drug reactions in medical events has become the focus of increasing attention in various countries. The quality of adverse reaction reports is the basis for medical risk assessment and analysis. Only by obtaining high-quality and valuable information can scientific treatment be carried out. Analysis, however, at present, there is no quantitative analysis on the relationship between the factors affecting adverse drug reactions and the degree of adverse reactions. There are few reasonable mathematical models. This article adopts the method of deep learning to establish a “human-machine material law ring”. “The mathematical model between the five factors and the degree of adverse drug reactions in China analyzes adverse drug reactions in China, and provides effective guidance for reducing drug accidents and improving China’s adverse drug reaction monitoring system.

Bachelor thesis in nursing.

The adductor muscle of the pen shell Atrina pectinata (AMPS) is a popular protein‐enriched food item in Asian Pacific countries, and has only been marketed in the frozen condition, as a result browning and decreased sensory attributes occur. To overcome these problems, superheated steam roasting (at 270°C for 4 min) combined with the hot smoke (10 min) using a selective Oak sawdust was employed to develop a new AMPS product yielding high physicochemical properties during storage periods (0–13 days) especially at 10°C. The processed AMPS showed high sensory preferences because of good odor, color, and textural properties. It also significantly inhibited bacterial growth, volatile basic nitrogen, thiobarbituric acid‐reactive species, and pH changes, and eventually possessed higher nutritional composition with low trimethylamine N‐oxide level. Results indicate that saturated steam allows AMPS at good physicochemical conditions, whereas hot smoke‐derived aroma compounds prolong its shelf life through antioxidant and antimicrobial activities.

Arthrospira platensis (A. platensis) is an economically important microalgae because it has carbohydrates, lipids, proteins and a number of phytochemicals. It is also a valuable source used in the production of biodiesel and functional foods. In this study, A. platensis was exposed to electron beam irradation (240 kGy) and induced random mutagenesis for strain improvement. Several mutants were obtained, and the resulting mutant was designated as EB29. The growth rate and chlorophyll content of EB29 was similar to those of wild type. However, the lipid content of EB29 was increased seven-fold compared to that of wild type when comparing the nile red fluorescent intensity. Semi-quantitative analysis of EB29 using the calibration plot of standard lipid, triolein, represented , which increased 2 times compared to wild type (). When analyzing the fatty acid profile of EB29, polyunsaturated fatty acids (PUFAs), such as gamma-linolenic acid (GLA) in EB29 increased about six-fold. Moreover, fatty acids affecting the quality of biodiesel increased compared to that of wild type. Thus, electron beam could be used for the strain improvement of microalgae in order to accumulate PUFAs and alteration of fatty acid profile for biodiesel.

GABA는 포유류의 신경전달물질의 분비를 억제하며, 신경안정 작용, 스트레스 해소, 우울증 완화, 불면 등에 효과가 있는 것으로 알려져 있다. 곡류식품에 존재하는 GABA 함량은 일반미 1∼4 mg/100 g, 현미 4∼8 mg/100 g으로 알려져 있지만, 그 함량이 낮아 자연 식품으로부터 섭취하는 양으로는 생리활성을 기대하기 어렵다. 본 연구에서는 mono sodium glutamate (MSG)발효로 얻은 GABA 코팅 쌀이 신경안정제 뿐만 아니라 수면유도제로 쓰이는 melatonin과 serotonin의 생성 촉진 효과를 알아보았다. 그 결과 melatonin의 경우 GABA (25 mg/mL) 투여군은 3.515 ± 0.149 pg/mL, 일반 미 공급군의 경우 2.632 ± 0.188 pg/mL이며, GABA 코팅 쌀 공급군은 3.578 ± 0.158 pg/mL로 나타났다. Serotonin 함량 을 측정한 결과 GABA (25 mg/mL), 일반미 공급군은 각각 5.183 ± 0.142 ng/mL, 4.784 ± 0.108 ng/mL가 측정되었으며 GABA 코팅 쌀을 공급한 실험군은 5.918 ± 0.169 ng/mL의 serotonin이 생성되었다. GABA 코팅 쌀의 지속적인 섭취로 인해 melatonin과 serotonin은 유의적인 증가는 있었지만, 누적효과는 없었다. 기능성 식품으로 개발된 GABA 코팅 쌀을 지속적으로 섭취하면 일반미에서 섭취하여 얻을 수 있는 melatonin과 serotonin의 유도효과를 얻을 수 있고, GABA 25 mg/mL를 섭취하는 것과 비슷한 효과를 나타낼 수 있다고 사료된다.

The peptides of enzymatic hydrolysates from oyster were determined by inhibitory activity against angiotensin-converting enzyme. The ACE inhibitory activity of enzymatic oyster hydrolysates increases with hydrolysis time. Among enzymatic oyster hydrolysates, oyster hydrolysates incubated with Protamex showed the best ACE inhibitory activity after 10 h. Hydrolysates were filtered through a HiSep ultrafiltration membrane (M.W. cut-off 30 kDa, 10 kDa) to obtain the peptide fractions with ACE inhibition activity. These fractions were applied to an HPLC column (watchers 120 ODS-AP ()). Six active fractions were collected and the range of ACE inhibition was from 29.56 to 85.85%. Peptide was purified from fraction B, showing the highest ACE inhibitory activity, and its sequence was Leu-Gln-Pro. These results suggest that PEH may be beneficial for developing antihypertensive food and drug.

This research explored and identified the protein composition of rat kidneys after acupuncture at the Taixi acupoint (KI3). Twelve adult male Wistar rats were randomly divided into a control group (n = 6) and an acupuncture group (n = 6). Rats in the acupuncture group received electroacupuncture on the bilateral KI3 for seven days. The kidneys were perfused with ice-cold saline and all kidney proteins were isolated. After protein sample preparation, two-dimensional gel electrophoresis (2-DE) was performed. The interesting spots were analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). There were nine protein spots with three-fold up-regulation in the kidney after the acupuncture. NAD-dependent isocitrate dehydrogenase and quinone reductase, the proteins involved in energy metabolism, the reduction of endogenous quinones, chemoprotection, and electrophilic stress, were identified. The data indicated that acupuncture at the KI3 of the kidney meridian of the foot shaoyin was able to increase NAD-dependent isocitrate dehydrogenase and quinone reductase expression in the kidney, and supported the relationship between the kidney and KI3.

Random mutagenesis was performed on beta-agarase, AgaB, from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutants E99K, T307I and E99K-T307I were approx. 140, 190 and 200%, respectively, of wild type beta-agarase (661 U/mg) at 40 degrees C. All three mutant enzymes were stable up to 50 degrees C and E99K-T307I had the highest thermostability. The melting temperature (Tm) of E99K-T307I, determined by CD spectra, was increased by 5.2 degrees C over that of the wild-type enzyme (54.6 degrees C). Activities of both the wild-type and E99K-T307I enzymes, as well as their overall thermostabilities, increased in 1 mM CaCl2. The E99K-T307I enzyme was stable at 55 degrees C with 1 mM CaCl2, reaching 260% of the activity the wild-type enzyme held at 40 degrees C without CaCl2.

Bisphenol A (BPA), generally known as bisphenols, has been identified as a potential estrogenic substance. BPA must be conjugated to carrier protein and BSA was commonly used. 4,4-Bis(4-hydroxyphenyl) valeric acid (BHPVA) has a bisphenolic structure and a long carbon chain with a reactive carboxyl group on the end. In this study, BHPVA-BSA was used to produce polyclonal antibody against bisphenolic structure, and a modified competitive ELISA method for quantification of BPA was developed. This system was based on BHPVA-BSA for polyclonal antibody production against bisphenolic structure, and BHPVA-HRP for determination of BPA substituting detection antibody in competitive reaction. Recovery was assessed at 10 different concentrations (2-1000 ng/ml) of BHPVA, and the recovery range was from 96.3% to 107.2%. The variation was from 6.2% to 9.8% for intra assay and from 10.1% to 12.6% for inter assay. The quadratic was used to establish the curve regression. The range was found to be between 2 and 1000 ng/ml. This modified competitive ELISA method has proven to be a very useful tool for quantification of BPA without the unexpected interaction of BSA and anti-BSA polyclonal antibody.

The heat shock response is induced by environmental stress, pathophysiological state and non-stress conditions and wide spread from bacteria to human. Although translations of most proteins are stopped under a heat shock response, heat shock proteins (HSPs) are produced to protect cell from stress. When heat shock response is induced, conformation of HSF1 was changed from monomer to trimer and HSF1 specifically binds to DNA, which was called a heat shock element(HSE) within the promoter of the heat shock genes. Human HSF1(hHSF1) contains five cysteine(Cys) residues. A thiol group(R-SH) of Cys is a strong nucleophile, the most readily oxidized and nitrosylated in amino acid chain. This consideration suggests that Cys residues may regulate the change of conformation and the activity of hHSF1 through a redox-dependent thiol/disulfide exchange reaction. We want to construct role of five Cys residues of hHSF by redox reagents. According to two studies, Cys residues are related to trimer formation of hHSF1. In this study, we want to demonstrate the correlation between structural change and DNA-binding activity of HSF1 through forming disulfide bond and trimerization. In this results, we could deduce that DNA binding activity of DNA binding domain wasn't affected by redox for always expose outside to easily bind to DNA. DNA binding activity of wild-type HSF's DNA binding domain was affected by conformational change, as conformational structure change (trimerization) caused DNA binding domain.

A sandwich enzyme-linked immunosorbant assay (ELISA) was developed to quantitatively detect Bullfrog (Rana catesbeiana) vitellogenin (VTG) levels. This procedure involved a sandwich ELISA using monoclonal antibodies of BVmA1 and BVmA2 against Bullfrog-VTG, and BVmA1 conjugated to horseradish peroxidase as the detection antibody. The assay range was between 9.4 ng/ml and 1200 ng/ml and the recovery of the VTG added to Bullfrog control male serum was 92.0-108.8%. Male Bullfrog was induced by injection 17beta-estradiol (E2) for four weeks and Bullfrog-VTG levels were measured each week. Histological analysis was performed for investigating the correlation of the effect to male reproduction and Bullfrog-VTG level variation depending on E2 dose. After two weeks of E2 exposure, the induced Bullfrog-VTG level was significantly higher than Bullfrog control female (p0.05). After four weeks of E2 exposure, the rupture and fusion of seminiferous tubule in the testes of male Bullfrog were shown and provided direct evidence that the reproduction of male Bullfrog was affected by estrogenic compounds. Bullfrog-VTG bioassay, using the sandwich ELISA, could be a sensitive and useful tool for quantification of estrogenic principles in aquatic environments.

Department of Biology, Pusan National University, Pusan 609-735, Korea Received January 31, 2002) . Fucoidan ! , "#$ ! , "% ! , &' ! , virus ! ( !,- )*+ $ ./ 01 2 3. /$ 4 . 5 67 fucoidan- 89 :; !:; CD ?@AB > *+ fucoidanE G F. HIJ - KL :$ - M @NO ? . C P FGE - .QR ' fucoidan> FGE - R STQ fucoidanA *+> V$ U , > RPAE ssDNA W ,- X G Y) >[ Z7 \ ] ^ . 5 3 J_ 89J G= F J fucoidanE - `, *+ O A;a . : F fucoidan, b , replication protein A(RPA), SV40 DNA cSABSTRACT.Fucoidan is a kind of polysaccharides in brown seaweeds. For the past years have beenextensively studied due to their numerous biological activities : anticancer, anticoagulant, antithrombotic, anti-inflammatory and antiviral. In this study, we have extracted fucoidan from the Korean brown seaweeds andexamined it’s anticancer activities for employed SV40 DNA replication assay, RPA-ssDNA binding assay ofreplication protein A(RPA: known as human single-stranded DNA-binding protein essential for DNA rep-lication) and MCF7 cell growth inhibition assay. In addition to, we found that chemically sulfated fucoidan'santicancer activity is more higher than natural and desulfated fucoidan. It seem that fucoidan's sulfate groupaffect on DNA replication, cause of decrease RPA's DNA binding activity. These results suggests that sulfatedfucoidan from Korean brown seaweeds have anticancer activity.Key words: Sulfated fucoidan, anticancer, replication protein A(RPA), SV40 DNA replication

Received 10 October 2001, Accepted 13 November 2001Eukaryotic replication protein A (RPA) is a single-stranded(ss) DNA binding protein with multiple functionsin DNA replication, repair, and genetic recombination. The70-kDa subunit of eukaryotic RPA contains a conservedfour cysteine-type zinc-finger motif that has beenimplicated in the regulation of DNA replication and repair.Recently, we described a novel function for the zinc-fingermotif in the regulation of human RPA’s ssDNA bindingactivity through reduction-oxidation (redox). Here, weshow that yeast RPA’s ssDNA binding activity is regulatedby redox potential through its RPA32 and/or RPA14subunits. Yeast RPA requires a reducing agent, such asdithiothreitol (DTT), for its ssDNA binding activity. Also,under non-reducing conditions, its DNA binding activitydecreases 20 fold. In contrast, the RPA70 subunit does notrequire DTT for its DNA binding activity and is notaffected by the redox condition. These results suggest thatall three subunits are required for the regulation of RPA’sDNA binding activity through redox potential.Keywords: Replication protein A (RPA), RPA70, Single-stranded DNA binding protein, Redox

We conducted a study to determine prevalence of HPV types in oropharyngeal cancers in the United States and establish a prevaccine baseline for monitoring the impact of vaccination. HPV DNA was extracted from tumor tissue samples from patients in whom cancer was diagnosed during 1995–2005. The samples were obtained from cancer registries and Residual Tissue Repository Program sites in the United States. HPV was detected and typed by using PCR reverse line blot assays. Among 557 invasive oropharyngeal squamous cell carcinomas, 72% were positive for HPV and 62% for vaccine types HPV16 or 18. Prevalence of HPV-16/18 was lower in women (53%) than in men (66%), and lower in non-Hispanic Black patients (31%) than in other racial/ethnic groups (68%–80%). Results indicate that vaccines could prevent most oropharyngeal cancers in the United States, but their effect may vary by demographic variables.

In this study, the effects of the ursodeoxycholic acid (UDCA), and its derivatives, on DNA replication were examined using simian virus (SV40) DNA replication in vitro. We found that UDCA and its derivatives inhibited SV40 DNA replication, and predominantly inhibited the initiation stage of DNA replication. UDCA and its derivatives inhibited the DNA cleavage by topoisomerase I (topo I). Among them, HS-1183 significantly reduced the activity of topo I. UDCA, at 100 microM, significantly reduced polymerase alpha-primase (pol alpha-primase) activity, but HS-1030 and HS-1183 showed a weak inhibitory effect. The ssDNA binding activity of replication protein A (RPA) was little affected by UDCA and HS-1030, but was weakly inhibited by HS-1183. Based on their properties, we suggest that UDCA and its derivatives might inhibit some molecules that is required to establish replication forks during the initiation reaction and their cytotoxicity might be related to the inhibitory effect they have on this fundamental cellular process.

To obtain cytochrome labeled with a stable isotope, the conditions of cultivation and the composition of medium for DvMF were examined. The growth of DvMF was steady and reproducible under purging with and under pH control. DvMF was able to go on a defined medium without natural products. The composition of medium containing a small amount of l as sole nitrogen source was established. Then, uniformly labeled cytochrome was obtained during the culture of DvMF in a defined medium with Cl; it was confirmed by HMQC.

This study investigated the propagated sensation along meridians (PSM) produced respectively by acupuncture at a specific acupoint of right-side Quchi (LI11), a nonacupoint on meridian (control meridian point), and neither meridian nor acupoint (control point). All the stimulated points were on the right brachioradialis along the large intestine meridian of hand Yangming. Surface electromyography (sEMG) was used to reflect the activity of the brachioradialis along the large intestine meridian of hand Yangming. The PSM rate of LI11 (59.21%) and the control meridian point (53.95%) were significantly higher than the control point (38.16%) (P<0.05). After acupuncture, the brachioradialis sEMG amplitude was5.08±2.93 uV at LI11,3.08±1.18 uV at the control point, and2.77±1.36 uV at the control meridian point. The amplitude of LI11 was significantly higher than both the control meridian point and the control point (P<0.05). When the sEMG activity of brachioradialis returned to the stable base line, brachioradialis sEMG duration at LI11 (265±87.87 s) was significantly longer than that at the control meridian point (91.69±42.98 s) and the control point (83.31±32.76 s) (P<0.05). In conclusion, acupuncture activated PSM at all points but showed an acupoint specificity at LI11 and a meridian specificity at the control meridian point.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Recent studies have shown that extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) promotes adhesion, invasion and metastasis of malignant tumor cells. The aim of this study was to investigate the impact of EMMPRIN/CD147 expression on prognosis and its correlation with clinicopathological characteristics in patients with osteosarcoma. The expression of EMMPRIN/CD147 in 55 surgical specimens from patients with osteosarcoma at stage IIA or above, 15 non-tumor rib bone tissues, three human osteosarcoma cell lines (Saos-2, U-2OS and MG-63), the human osteoblast cell line HOB and the malignant melanoma cell line A375 were examined by immunohistochemistry, western blot analysis and ELISA, respectively. The potential association of the levels of EMMPRIN/CD147 expression in osteosarcoma specimens with the overall survival of patients was statistically analyzed. We found that the EMMPRIN/CD147 was expressed in 45 out of 55 osteosarcomas, with immunoreactivity primarily within the membrane and cytoplasm of tumor cells, but not in the non-tumor bone tissues. We also observed that EMMPRIN/CD147 was expressed in Saos-2, U-2OS, MG-63 and A375, but not in HOB cells. The levels of EMMPRIN/CD147 expression correlated positively with the pathological degree of osteosarcoma and negatively with the survival period of patients with osteosarcoma. The expression of EMMPRIN/CD147 is a potential factor in the development and prognosis of osteosarcoma and may be a novel therapeutic target of human osteosarcoma.

Ghrelin, a 28-amino acid brain-gut peptide expressed in periphery tissues and the central nervous system, has been demonstrated to increase insulin sensitivity in adipocytes. Recent data have indicated that insulin resistance exists in the brain and is related to Alzheimer's Disease (AD). The aim of this study was to investigate whether ghrelin increased high glucose-induced hippocampal neuron insulin sensitivity, and further modulated tau phosphorylation. Hippocampal neurons were cultured in concentrations of 25 mM and 75 mM glucose. The effect of ghrelin on hippocampal neuronal insulin sensitivity was detected by [(3)H]-2-deoxy-D-glucose uptake. The expression of Akt, glycogen synthase kinase-3beta (GSK-3beta) and tau phosphorylation was determined via Western blotting. Culturation in 75 mM glucose resulted in decreased neuronal glucose uptake and an increase in the level of tau phosphorylation at Ser 199. In neurons treated with ghrelin for 1 h, neuronal glucose uptake was increased and tau hyperphosphorylation was improved. Ghrelin activated Akt and GSK-3beta phosphorylation, whereas phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin eliminated ghrelin's effect on neuronal glucose uptake and tau phosphorylation. This study demonstrated that ghrelin increased insulin-stimulated neuronal glucose uptake in 25 mM or 75 mM glucose, raised insulin sensitivity, improved insulin resistance and decreased tau abnormal phosphorylation via the PI3-K/Akt-GSK pathway. Ghrelin is a potential new medicine in the treatment of AD.

Serum vitellogenin (VTG) contents of wild goldfish (Carassius auratus) were investigated as a sensitive biomarker for artificial estrogenic compounds in aquatic environments. Goldfish was sampled from a pristine area, a river situated 5 km downstream from a sewage treatment works (STW), and also from the Young-San River in Korea. The female yolk precursor protein VTG was not detected when gonadosomatic index (GSI) was less than 0.85%, while VTG levels of10 microg/ml were found in males whose GSI was less than 1.53%. In male goldfish sampled from STW and the Young-San River, the higher VTG corresponded to lower GSI. This study suggested a trend that gonad development was connected to VTG levels in both sexes, and the application of GSI and histological analysis provide an attractive possibility that it could be included in the panel of markers used for estrogenic activity investigation of aquatic environments.

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantitatively detect Carassius auratus vitellogenin (VTG) levels. The protein levels in fish plasma are useful aquatic biomarkers of estrogenic compounds. This procedure involved an ELISA using monoclonal antibodies of CVmA2 and CVmA7 against Carassius auratus VTG, and CVmA7 conjugated to horseradish peroxidase as the detection antibody. The assay range was between 1 and 401.5 ng/ml and the recovery of the VTG added to Carassius auratus plasma was 92.5-109%. An in vitro assay was performed to measure low levels of the VTG, using primary hepatocytes of Carassius auratus induced by 17-beta estradiol (E2). The detection limit was 1 ng/ml and 137 ng/ml at the maximum. Within each sex of wild Carassius auratus, VTG levels from the river next to sewage treatment works (STWs) were much higher than those from the feeding stream. The Carassius auratus VTG bioassay could be a sensitive and useful tool for quantification of estrogenic principles in aquatic environments.

The eukaryotic replication protein A (RPA) is a heterotrimeric protein complex. It consists of 70, 32, and 14 kDa subunits that are involved in DNA replication, repair, and genetic recombination. RPA is a 4-cysteine type zinc-finger protein. RPA's zinc-finger domain is not essential for DNA binding activity, but it is involved in the regulation of RPA's DNA binding activity through reduction-oxidation (redox). In this study, we show that yeast RPA's ssDNA binding activity is regulated by redox potential through its subcomplexes of 32 and 14 kDa subunits. In contrast, the subunits' complex, RPA70, formed a stable complex with ssDNA, even under non-reducing conditions. The addition of DTT and H202 had no effect on its DNA binding activity. In RPA70, since the addition of the subcomplexes of the 32 and 14 kDa subunits, it restored the modulating ssDNA binding activity to native RPA's DNA binding activity. These results suggest that the subcomplexes of the 32 and 14 kDa subunits may be involved in the modulating RPA's DNA binding activity through redox change. These studies, therefore, show the novel structure and function relationship of a multiprotein complex in that the role of a specific domain (or one subunit) is regulated by the other subunits.

Department of Chemistry, School of Natural Science, Pusan National University, Busan 609-735, Korea *E-mail: jaspark@pusan.ac.kr †Department of Biomedicinal Chemistry, and Institute of Functional Materials, Inje University, Kimhae 621-749, Korea Department of Biochemistry & Molecular Biology and the Walther Cancer Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA Received September 1, 2009, Accepted October 6, 2009

Eukaryotic replication protein A (RPA) is comprised of a three subunits (70, 32, 14 kDa; p70, p32, p14 respectively). RPA has been identified as a human protein necessary for SV40 DNA replication in vitro and acting as a singlestrand DNA (ssDNA) binding protein with multiple functions in DNA replication, repair, and genetic recombination. Recent studies suggest that RPA functions are regulated in response DNA damage and that its ssDNA binding activity may be involved in this regulatory event. In our previous study, we found that RPA’s ssDNA binding activity is regulated by redox through cysteins in the putative zinc finger domain of the p70 subunit. A number of DNAbinding proteins have been identified in which their DNA binding activity is regulated by redox, although the regulatory role of the zinc finger domain is not clear. RPA70 has an evolutionarily conserved 4 cystein in the zinc-finger domain at 473 through 503 amino acids. Even though previous studies showed that the zinc-finger domain does not participate in DNA binding directly, it is still possible that the zinc-finger domain regulates DNA binding activity. To verify this possibility, we built mutant RPA (ZFD4) by substitution four cysteins for alanine and checked the DNA binding activity. The ZFD4 formed a stable complex with the ssDNA, even under oxidized conditions, and the amounts of DTT had no effect on its binding property (Figure 1a). The ZFD4 also showed strong binding activity in the non-reducing condition without DTT. The addition of H2O2 did not have much of effect on RPA-DNA complex formation (Figure 1a), whereas the wild-type RPA had H2O2 dependent activity. 7-9 These results suggest that the zinc-finger domain could regulate the ssDNA binding activity of RPA and that the cystein residues of the p70 subunit are essential in this process. We examined the effect of phosphorylation on ZFD4’s in regulation of ssDNA binding activity of RPA in the various redox conditions. The results showed phosphorylation of ZFD4 does not affect its DNA binding activity and suggests that phosphorylation could occur at other sites of the RPA complex (Figure 1a). In an effort to understand RPA’s regulatory function, we examined the structural changes of hRPA by the ssDNA binding using protease sensitive digestions. We examined