Initial radiofrequency ablation failure for hepatocellular carcinoma: repeated radiofrequency ablation versus transarterial chemoembolisation.

AIM: To compare the long-term therapeutic outcomes of repeated radiofrequency ablation (RFA) with that of transarterial chemoembolisation (TACE) in patients with local tumour progression (LTP) after initial RFA treatment for hepatocellular carcinoma (HCC). MATERIALS AND METHODS: This retrospective study was approved by the institutional review board and the requirement for informed consent was waived. Between July 2006 and February 2012, 713 patients underwent RFA for single HCC as a first-line treatment. Fifty-eight patients who showed LTP as initial tumour recurrence post-RFA treatment were included. Patients were treated with either repeated RFA (n=33) or TACE (n=25). TACE was performed as an alternative therapeutic option when repeated RFA was not feasible based on the planning ultrasonography. Recurrence-free and overall survival rates were estimated using the Kaplan-Meier method. Prognostic factors for outcomes were evaluated using the Cox proportional hazards model. RESULTS: Both groups did not show significant differences in terms of baseline characteristics, with the exception being the proportion of subphrenic tumours (p=0.031). The RFA and TACE groups did not differ significantly in their 5-year recurrence-free and overall survival rates (17% versus 10.7% and 72.7% versus 51.9%, respectively, with all p-values >0.05). In addition, multivariate analyses revealed that type of treatment was not associated with recurrence-free or overall survival in patients with post-RFA LTP. CONCLUSION: TACE is an effective treatment, comparable to repeated RFA, in patients with LTP after initial RFA when repeated RFA is not feasible.

Influence of hepatitis B virus reactivation on the recurrence of HBV-related hepatocellular carcinoma after curative resection in patients with low viral load.

It is unclear whether the reactivation of hepatitis B virus (HBV) influences the prognosis of hepatocellular carcinoma (HCC) after resection in patients with chronic hepatitis B. The aim of this study was to identify the influence of HBV reactivation on the recurrence of hepatitis B-related HCC after curative resection in patients with low viral load (HBV DNA <2000 IU/mL). We retrospectively analysed a total of 130 patients who underwent curative resection for HBV-related early stage HCC (single nodule; <5 cm/two or three nodules; <3 cm) with pre-operative HBV DNA levels <2000 IU/mL with serial HBV DNA tests. The predictive factors including HBV reactivation for the recurrence of HBV-related HCC after curative resection were investigated. Fifty-three patients (41%) had HBV reactivation after resection among 130 patients. HBV reactivation was observed in 22 of 53 patients with undetectable baseline HBV DNA and in 31 of 77 patients with detectable baseline HBV DNA. Cumulative recurrence rates after resection at 1, 2 and 3 years were 17.0%, 23.3% and 31.4%, respectively. The multivariable analysis demonstrated that the risk factors for the recurrence were the presence of microvascular invasion (hazard ratio (HR) 2.62, P = 0.003), multinodularity (HR 4.61, P = 0.005), HBV reactivation after resection (HR 2.03, P = 0.032) and HBeAg positivity (HR 2.06, P = 0.044). HBV reactivation after curative resection is associated with the recurrence of HBV-related HCC in patients with low viral load.

Proton pump inhibitor use significantly increases the risk of spontaneous bacterial peritonitis in 1965 patients with cirrhosis and ascites: a propensity score matched cohort study.

BACKGROUND: The risk of spontaneous bacterial peritonitis (SBP) associated with proton pump inhibitor (PPI) use has been raised in cirrhotic patients with ascites. However, this is based on case-control studies, often with a small series. AIM: To determine whether PPI use increases the risk of SBP using a large cohort. METHODS: This retrospective cohort study included 1965 cirrhotic patients with ascites diagnosed between January 2005 and December 2009. The SBP incidence rate was compared between the PPI and non-PPI groups before and after propensity score matching to reduce the effect of selection bias and potential confounders. Multivariate analysis was conducted to confirm the association of PPI use with SBP. RESULTS: After excluding 411 patients, 1554 were analysed. Among them, 512 patients (32.9%) were included in the PPI group. The annual SBP incidence rate was higher in the PPI group than in the non-PPI group (10.6% and 5.8%, P = 0.002) before matching. Indications for PPI use and dose of PPI were similar between patients with and without SBP. In the propensity score matched cohort (402 pairs), the SBP incidence rate was also higher in the PPI group than in the non-PPI group (10.8% vs. 6.0%, P = 0.038). Multivariate analysis revealed that PPI use (Hazard ratio 1.396; 95% confidence interval, 1.057-1.843; P = 0.019) was the independent risk factor for SBP. CONCLUSIONS: Proton pump inhibitor use significantly increases the risk of spontaneous bacterial peritonitis in cirrhotic patients with ascites. Proton pump inhibitor use should be undertaken with greater caution and appropriately in patients with cirrhosis.

Percutaneous cryoablation of small hepatocellular carcinomas using a 17-gauge ultrathin probe.

AIM: To evaluate the feasibility and safety of percutaneous cryoablation (PCA) of small hepatocellular carcinomas (HCCs) using a 17 G ultrathin cryoprobe. MATERIALS AND METHODS: Twenty patients (male:female ratio14:6) with 20 HCCs, who were not surgical candidates, underwent ultrasound (US)-guided PCA for treatment of HCCs. Single HCCs less than 3cm in diameter were included in this study. Ablation was performed using a 17 G cryoprobe. The effectiveness was determined by the changes in alpha-foetoprotein level and degree of tumour necrosis on follow-up computed tomography (CT); complete response (100% necrosis), partial response (100%>necrosis≥30%), stable disease (any cases not qualifying for either partial response or progressive disease) and progressive disease (increase of at least 20% in diameter of viable tumour). Haemoglobin, white blood cell count (WBC), serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), and total bilirubin were compared before and after the procedure, and the technical feasibility, complications, clinical outcomes and survival of each patient were also evaluated. RESULTS: All procedures were technically successful. Each patient complained of negligible pain and there was no other procedure-related complication or mortality. The mean level of alpha-foetoprotein declined significantly from 53.2 to 20.4ng/ml 1 month after the procedure (p<0.05). At 1-month follow-up CT, there were 13 complete responses, four partial responses, three patients with stable disease, and no patients had progressive disease. Six of seven lesions that did not present with a complete response underwent further treatment. On long-term follow up (6-30 months; mean 20.7), a local recurrence was seen in one of 13 lesions (8%) with complete response revealed. Laboratory findings showed no significant changes except for the transient increase of SGOT and SGPT. CONCLUSION: US-guided PCA using a 17 G cryoprobe was feasible and safe for the treatment of HCC smaller than 3cm.

Decreased expression of p12 is associated with more advanced tumor invasion in human gastric cancer tissues.

BACKGROUND/AIMS: p12(DOC-1) is a well-known growth suppressor; however, its role in gastric carcinogenesis is still unclear. We investigated the expression of p12(DOC-1) in gastric cancer tissues and its possible correlation with p53 expression, and determined its clinical significance. METHODS: Immunohistochemical staining using the tissue array method was performed on 180 human gastric carcinomas. The clinicopathological features and prognostic significance were analyzed. RESULTS: Of the 180 tissue samples, p53 expression was positive in 85 (47.2%) and p12(DOC-1) expression was negative in 140 (77.8%). The negative expression of p12(DOC-1) was significantly associated with a more advanced depth of tumor invasion and stage (p < 0.05). No apparent correlation was found between p12(DOC-1) and p53 expressions. The 5-year survival rate of the p12(DOC-1)-positive cases (53.7%) was higher than that of the p12(DOC-1)-negative cases (39.3%); however, neither p12(DOC-1) nor p53 expression status had any statistically significant prognostic value. Multivariate analysis revealed that lymph node metastasis, distant metastasis, lymphatic invasion and perineural invasion were independent prognostic factors. CONCLUSIONS: This is the first report that suggests that p12(DOC-1) may be involved in the development and progression of gastric cancer. Further studies are required to clarify its exact role in the mechanism of gastric carcinogenesis.

Celecoxib induces hepatic stellate cell apoptosis through inhibition of Akt activation and suppresses hepatic fibrosis in rats.

BACKGROUND AND AIMS: Activated hepatic stellate cells (HSCs) but not quiescent HSCs express cyclo-oxygenase-2 (COX-2), suggesting that the COX-2/prostanoid pathway has an active role in hepatic fibrogenesis. However, the role of COX-2 inhibitors in hepatic fibrogenesis remains controversial. The aim of this study was to investigate the antifibrotic effects of celecoxib, a selective COX-2 inhibitor. METHODS: The effects of various COX inhibitors-that is, ibuprofen, celecoxib, NS-398 and DFU, were investigated in activated human HSCs. Then, the antifibrotic effect of celecoxib was evaluated in hepatic fibrosis developed by bile duct ligation (BDL) or peritoneal thioacetamide (TAA) injection in rats. RESULTS: Celecoxib, NS-398 and DFU inhibited platelet-derived growth facor (PDGF)-induced HSC proliferation; however, only celecoxib (> or =50 microM) induced HSC apoptosis. All COX inhibitors completely inhibited prostaglandin E(2) (PGE(2)) and PGI(2) production in HSCs. Separately, PGE(2) and PGI(2) induced cell proliferation and extracellular signal-regulated kinase (ERK) activation in HSCs. All COX inhibitors attenuated ERK activation, but only celecoxib significantly inhibited Akt activation in HSCs. Celecoxib-induced apoptosis was significantly attenuated in HSCs infected with adenovirus containing a constitutive active form of Akt (Ad5myrAkt). Celecoxib had no significant effect on PPARgamma (peroxisome proliferator-activated receptor gamma) expression in HSCs. Celecoxib inhibited type I collagen mRNA and protein production in HSCs. Oral administration of celecoxib (20 mg/kg/day) significantly decreased hepatic collagen deposition and alpha-SMA (alpha-smooth muscle actin) expression in BDL- and TAA-treated rats. Celecoxib treatment significantly decreased mRNA expression of COX-2, alpha-SMA, transforming growth factor beta1 (TGFbeta1) and collagen alpha1(I) in both models. CONCLUSIONS: Celecoxib shows a proapoptotic effect on HSCs through Akt inactivation and shows antifibrogenic effects in BDL- and TAA-treated rats, suggesting celecoxib as a novel antifibrotic agent of hepatic fibrosis.

Plasma protein profiles in early asthmatic responses to inhalation allergen challenge.

Although mediators, such as lipids, cytokines, and chemokines, are related to the appearance of an IPR, there has been no reliable indicator to predict conditions for the appearance of an IPR. In this study, we adopted a proteomic approach to investigate the pathogenesis at the level of the plasma proteins and to develop plasma markers to predict the appearance of an IPR following an inhalation challenge with Dermatophagoides pteronyssinus (D.p.). Sixteen mild asthmatics were recruited. Plasma was obtained before challenge and when a decline in forced expiratory volume in 1 s (FEV(1)) values greater than 20% from the phosphate-buffered saline value was achieved during D.p. allergen challenge (positive responders), or at 60 min after the highest concentration of D.p. allergen was inhaled (negative responders). After comparing normalized volumes of the spots in the two groups, differentially expressed spots were identified using intra-gel digestion and mass spectrometric analysis. Before D.p. antigen challenge, four spots of gamma fibrinogen and its isoforms were significantly decreased and two spots of complement C3 fragments were significantly increased in the positive responders compared to the negative responders. After D.p. antigen challenge, complement C3 fragment was persistently higher, while gamma fibrinogen was lower in the positive responders than in the negative responders. A validation study using Western blotting showed that gamma fibrinogen expression in the IPR-positive asthmatics was significantly decreased compared to the average of the IPR-negative asthmatic control group. These results indicate that alterations in the complement cascade and fibrinogen may predispose patients to the appearance of an immediate response to D.p. allergen challenge and may provide plasma markers to predict the appearance of an IPR.

High prevalence of significant histology in asymptomatic chronic hepatitis B patients with genotype C and high serum HBV DNA levels.

Current treatment guidelines suggest that antiviral therapy be considered for chronic hepatitis B (CHB) patients with high viral load if a biopsy shows significant liver disease despite alanine aminotransferase (ALT) levels two times or less than the upper limit of normal (ULN). We evaluated the histological findings in CHB patients with high viral load and persistently normal or slightly elevated serum ALT levels. Between January 2003 and June 2006, 105 consecutive treatment-naive patients with CHB who underwent ultrasonography-guided percutaneous liver biopsy, had detectable serum HBV DNA (>10(5) copies/mL) in a direct hybridization assay and normal or slightly elevated serum ALT levels (≤2 × ULN) for at least 12 months were included in a prospective study. Histological assessment was based on the METAVIR scoring system. Significant histology was defined as fibrosis stage ≥F2 or necroinflammation grade ≥A2. Among the 105 CHB patients with high viral load and persistently normal or slightly elevated serum ALT levels for at least 12 months, significant fibrosis (F2-F4 fibrosis) was observed in 63 patients (60.0%) and the actual significant histology was found in 65 patients (61.9%). On multivariate analysis, serum ALT levels and age at which they entered the study were independent factors associated with significant histology. Odds ratios for significant histology increased progressively according to serum ALT levels and age. In conclusion, a large proportion of CHB patients with genotype C, high viral load and ALT ≤2 × ULN had significant liver disease on liver biopsy and should be considered for antiviral therapy.

Diagnostic application of serum proteomic patterns in gastric cancer patients by ProteinChip surface-enhanced laser desorption/ionization time-of-flight mass spectrometry.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is one of thecurrently used techniques to identify biomarkers for cancers. This study was planned to establish a system to accurately distinguish gastric cancer patients by using SELDI-TOF-MS. A total of 100 serum samples obtained from 60 individuals with gastric cancer and 40 healthy individuals were screened. Protein expression profiles were expressed on CM10 ProteinChip arrays and analyzed. Peak intensities were analyzed with the Biomarker Wizard software to identify peaks showing significantly different intensities between normal and cancer groups. Classification analysis and construction of decision trees were done with the Biomarker Pattern software 5.0. Seventeen protein peaks showed significant differences between the two groups. The decision tree which gave the highest discrimination included four peaks at mass 5,919, 8,583, 10,286, and 13,758 as splitters. The sensitivity and specificity for classification of the decision tree were 96.7% (58/60) and 97.5% (39/40), respectively. When the protein biomarker pattern was tested on a blinded test set, it yielded a sensitivity of 93.3% (28/30) and a specificity of 90% (18/20). These results suggest that serum protein profiling by the SELDI system may distinguish gastric cancer patients from healthy controls with relatively high sensitivity and specificity.

Phenotypic and genetic antibiogram of methicillin-resistant staphylococci isolated from bovine mastitis in Korea.

Staphylococcus aureus belongs to the group of major contagious mastitis pathogens, whereas the coagulase-negative staphylococci (CNS) are also capable of causing opportunistic bovine mastitis. Many of these strains are resistant to penicillin or ampicillin because of the long-term use of beta-lactam antibiotics in agricultural and healthcare settings. Based on the simple and highly specific coagulase genotyping by PCR-RFLP used for discriminating among Staph. aureus strains, the relationship between phenotypic antibiogram and the polymorphism of coagulase gene was determined in this study. The staphylococci strains (835 Staph. aureus and 763 CNS) were isolated from 3,047 bovine mastitic milk samples from 153 dairy farms in 8 provinces from 1997 to 2004 in the Republic of Korea. Twenty-one (2.5%) Staph. aureus and 19 (2.4%) CNS strains were resistant to methicillin [oxacillin minimum inhibitory concentration (MIC) > or = 4 microg/mL]. The mecA gene was also found in 13 methicillin-resistant Staph. aureus (MRSA) and 12 methicillin-resistant CNS (MRCNS) isolates with a significantly higher detection rate of the mecA gene in MRSA with high MIC (> or = 16 microg/mL) compared with those with MIC < or = 8 microg/mL. Methicillin-resistant Staph. aureus and MRCNS were also more resistant to other antibiotics (ampicillin, cephalothin, kanamycin, and gentamicin) than methicillin-susceptible staphylococci. Among 10 different coa PCR-RFLP patterns (A to J) in 706 Staph. aureus strains, the main types were A (26.9%), B (17.0%), G (10.5%), and H (15.4%), with the frequent observation of the A and H types (6 and 10 isolates) in MRSA. This study indicates that major epidemic Staph. aureus clones may be spread between different dairy farms, and the profile of coa genotype can be applied for epidemiological investigations and control of bovine mastitis, particularly one caused by MRSA with specific prevalent coa types.

Expression of hMLH1 is inactivated in the gastric adenomas with enhanced microsatellite instability.

Microsatellite instability (MSI) and frameshift mutations in the genes containing coding nucleotide repeats have been reported in a subset of gastric adenomas, however the inactivation profiles of DNA mismatch repair genes in MSI-positive gastric adenomas have not been characterized. To address the origin of MSI in gastric adenomas, expressions of hMLH1 and hMSH2 were explored in 86 gastric adenomas. Gastric carcinomas, of which 16 were MSI-positive and 22 MSI-negative, were used as controls. MSI was found in 15 (17%) of gastric adenomas. Absent or decreased hMLH1 expression by immunohistochemistry was noted in most of the MSI-positive adenomas (13/15, 87%) and carcinomas (14/16, 88%), and all of these tumours showed methylation of the hMLH1 gene promoter. In contrast, rare inactivation of hMLH1 expression was found in MSI-negative adenomas (3/71, 4%) and carcinomas (2/22, 9%). Intense expression of hMSH2 gene product was observed in most of the gastric adenomas and carcinomas regardless of MSI status. These findings indicate that the inactivation of hMLH1 gene expression by promoter methylation is an early event and might be the origin of MSI-positive gastric adenomas.

(2)H MAS NMR studies of the manganese dioxide tunnel structures and hydroxides used as cathode materials in primary batteries.

Variable-temperature (2)H MAS NMR spectroscopy was used to investigate the local environments and mobility of deuterons in the manganese dioxide tunnel structures. Five systems were investigated: electrolytic manganese dioxide (EMD), the model compounds groutite and manganite, and deuterium intercalated ramsdellite and pyrolusite. Ruetschi deuterons, located in the cation vacancy sites in EMD, were detected by NMR and give rise to a resonance at 150 ppm at room temperature. These deuterons are rigid on the (2)H MAS NMR time scale (i.e., the correlation time for motion, tau(c), is >10(-3) s) at room temperature, but start to become mobile above 150 degrees C. No Coleman protons (in the so-called 1 x 1 and 1 x 2 tunnels in EMD) were observed. Much larger (2)H NMR hyperfine shifts of approximately 300 and approximately 415 ppm were observed for the deuterons in the tunnel structures of manganite and groutite, which could be explained by considering the different bonding arrangements for deuterons in the 1 x 1 and 1 x 2 tunnels. The smaller shift of the EMD deuterons was primarily ascribed to the smaller number of manganese ions in the deuterium local coordination sphere. Experiments performed as a function of intercalation level for ramsdellite suggest that the 1 x 1 tunnels are more readily intercalated in highly defective structures. The almost identical shifts seen as a function of intercalation level for deuterons in both 1 x 1 and 1 x 2 tunnels are consistent with the localization of the e(g) electrons near the intercalated deuterium atoms. A Curie-Weiss-like temperature dependence for the hyperfine shifts of EMD and groutite was observed with temperature, but very little change in the shift of the manganite deuterons was observed, consistent with the strong antiferromagnetic correlations that exist above the Néel temperature for this compound. These different temperature dependences could be used to identify manganite-like domains within the sample of groutite, which could not be detected by X-ray diffraction.

Emergence of YMDD motif mutant of hepatitis B virus during short-term lamivudine therapy in South Korea.

BACKGROUND/AIMS: The emergence of a YMDD mutant resistant to lamivudine therapy has been reported in patients with hepatitis B treated with long-term lamivudine therapy. However, it is not well known whether the YMDD mutant could be detected early in lamivudine therapy in hepatitis B virus (HBV) endemic areas. The aim of this study was to investigate the emergence of the YMDD mutant during short-term lamivudine therapy in South Korea. METHODS: We prospectively investigated the emergence of the YMDD mutant by the nested PCR assay using restriction fragment length polymorphism in 28 patients with chronic hepatitis B who were treated with 100 mg of lamivudine daily for 12 weeks. RESULTS: The YMDD mutant was detected in 17 (60.7%) out of 28 patients at week 12, and the only type of mutation found was the YIDD mutation. When we carried out the nested PCR serially in five patients, YIDD mutants were detected as early as 2 weeks by the nested PCR assay. The nested PCR results were in concordance with DNA sequencing in one patient's serial samples. CONCLUSIONS: YMDD mutants in HBV were detected within a few weeks during lamivudine therapy in South Korea, which suggests that the YMDD mutant may exist even before lamivudine therapy in HBV endemic areas.

HWY-289, a novel semi-synthetic protoberberine derivative with multiple target sites in Candida albicans.

The antifungal properties of 515 synthetic and semi-synthetic protoberberines were investigated. HWY-289 was chosen for further study because it exhibited the most significant anti-Candida activity (MICs were 1.56 mg/L for Candida albicans and Candida krusei; 6.25 mg/L for Candida guilliermondii) but did not demonstrate toxicity in rats. HWY-289 inhibited the incorporation of L-[methyl-(14)C]methionine into the C-24 of ergosterol in whole cells of C. albicans (IC(50) 20 microM). However, HWY-289 (100 microM) had no effect on mammalian cholesterol biosynthesis in rat microsomes while miconazole (100 microM) was a potent inhibitor of cholesterol biosynthesis under identical assay conditions. A second major target site for HWY-289 was identified that involves cell wall biosynthesis in C. albicans. HWY-289 was a potent inhibitor of the chitin synthase isozymes CaCHS1 and CaCHS2, with IC(50) values of 22 microM for each enzyme. The effect was highly specific in that HWY-289 had no significant effect on C. albicans CaCHS3 (IC(50) > 200 microM). Thus, HWY-289 compared favourably with well-established antifungal agents as an inhibitor of the growth of Candida species in vitro, and may have considerable potential as a new class of antifungal agent that lacks toxic side effects in the human host.

An ischemic skin lesion after chemoembolization of the right internal mammary artery in a patient with hepatocellular carcinoma.

A huge nodular hepatocellular carcinoma located at the anterior superior portion of the left lobe in a patient with hepatocellular carcinoma was treated with transcatheter arterial chemoembolization through the left hepatic artery. Three months later, however, there was a re-elevation of the serum alpha-fetoprotein level and evidence of a marginal recurrence at the left side of the previously embolized tumor was noted on the postembolization computed tomographic scan. Although the hepatic artery was intact in the second hepatic arteriography, we found that the right internal mammary artery was feeding the recurred hepatocellular carcinoma. This internal mammary artery was successfully treated with Lipiodol-transcatheter arterial chemoembolization. However, an ischemic lesion occurred in the skin of the anterior chest and abdominal wall several days after internal mammary artery embolization. We report here a very rare case of ischemic skin lesion on the anterior chest and abdominal wall following transcatheter arterial chemoembolization of the right internal mammary artery. This internal mammary artery was embolized because it had developed a collateral tumor feeding vessel following the initial chemoembolization of a hepatocellular carcinoma.

Cholesterol biosynthesis from lanosterol. A concerted role for Sp1 and NF-Y-binding sites for sterol-mediated regulation of rat 7-dehydrocholesterol reductase gene expression.

The 7-dehydrocholesterol reductase (Dhcr7) is the terminal enzyme in the pathway of cholesterol biosynthesis. We have previously reported that sterol depletion in vivo caused a significant induction of both liver mRNA and enzyme activity of Dhcr7 (Bae, S.-H., Lee, J. N., Fitzky, B. U., Seong, J., and Paik, Y.-K. (1999) J. Biol. Chem. 274, 14624-14631). In this paper, we also observed liver cell-specific sterol-mediated Dhcr7 gene induction in vitro by sterol depletion in rat hepatoma cells, suggesting the presence of sterol-mediated regulatory elements in the Dhcr7 gene. To understand the mechanisms responsible for regulating Dhcr7 expression, we have isolated the 5'-flanking region of the gene encoding rat Dhcr7 and have characterized the potential regulatory elements of the gene that are responsible for sterol-mediated regulation. The Dhcr7 promoter contains binding sites for Sp1 (at -177, -172, -125, and -20), NF-Y (at -88 and -51), and SREBP-1 or ADD1 (at -33). Deletion analysis of the Dhcr7 gene promoter (-1053/+31), employing a nested series of Dhcr7-luciferase constructs, demonstrated that the -179 upstream region of the gene is necessary and sufficient for optimal efficient sterol-regulated transcription. DNase I footprinting and electrophoretic mobility shift assay showed that the SRE1/E box (-33/-22) involved in sterol response of many sterol-related enzyme genes was protected specifically by the overexpressed recombinant ADD1. Mutational analysis for the functional relationship between the identified cis-elements in this region indicate that one of the binding sites for Sp1 (GC box at -125) and NF-Y (CCAAT box at -88) plays a cooperative role in the sterol-mediated activation, in which the latter site also acts as a co-regulator for SREBP-activated Dhcr7 promoter activity. We believe that Dhcr7 is the first enzyme characterized with a sterol-regulatory function in the post-lanosterol pathway. This may be important for understanding the coordinated control of cholesterol biosynthesis as well as the molecular mechanism of Smith-Lemli-Opitz syndrome-related protein in mammals.

Cholesterol biosynthesis from lanosterol: molecular cloning, chromosomal localization, functional expression and liver-specific gene regulation of rat sterol delta8-isomerase, a cholesterogenic enzyme with multiple functions.

Sterol Delta(8)-isomerase (SI) (EC 5.3.3.5), also known as emopamil binding protein or sigma receptor, catalyses the conversion of the 8-ene isomer into the 7-ene isomer in the cholesterol biosynthetic pathway in mammals. Recently, mutations of SI have been found to be associated with Conradi-Hünermann syndrome in humans. To investigate the in vitro and in vivo modes of molecular regulation of SI and its role in cholesterol biosynthesis in mammals, we isolated a full-length cDNA encoding rat SI. The deduced amino-acid sequence of rat SI predicts a 230-residue protein (26737 Da) with 87% and 80% amino-acid identity to mouse and human counterparts. The rat SI gene was mapped to chromosome 12q1.2 using fluorescence in situ hybridization (FISH). The biological function of the cloned rat SI cDNA was verified by overexpressing recombinant Myc-SI in Saccharomyces cerevisiae. It showed a characteristic pattern of inhibition on exposure to trans-2-[4-(1,2-diphenylbuten-1-yl)phenoxy]-N,N-dimethylethylamine (tamoxifen; IC(50)=11.2 microM) and 3beta-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A; IC(50)=4.2 microM), two well known potent inhibitors of SI. Northern-blot analysis of 3-week-old rats compared with 2-year-old rats showed that SI mRNA expression in both age groups was restricted to liver, where a 70% reduction in mRNA levels was observed in 2-year-old rats. The FISH studies revealed ubiquitous expression of SI mRNA in rat hepatocytes. The in vitro studies showed that the SI mRNA was highly suppressed by 25-hydroxycholesterol in H4IIE cells. Treatment of H4IIE cells grown in medium supplemented with fetal bovine serum with tamoxifen for 24 h resulted in a dose-dependent induction of SI mRNA, with a concomitant suppression of sterol regulatory element binding protein-1 mRNA. Interestingly, this effect was not seen in emopamil-treated cells. The in vivo experiments also indicate that both mRNA expression and enzymic activity of SI in liver were induced approx. 3-fold in rats fed 5% (w/w) cholestyramine plus 0.1% (w/w) lovastatin in normal chow for 2 weeks. With this newly cloned rat SI cDNA, it becomes possible to gain molecular understanding of previously unknown and tamoxifen-mediated gene regulation of SI that is involved in cholesterol metabolism, ischaemia and genetic diseases.

Physical stability of the blue pigments formed from geniposide of gardenia fruits: effects of pH, temperature, and light.

Fruits of Gardenia jasminoides contain geniposide which can be transformed to blue pigments by a simple modification. Colorless geniposide obtained from gardenia fruits by charcoal and silica gel column chromatographies was hydrolyzed with beta-glucosidase to yield genipin. The resulting genipin was transformed to blue pigments by reaction with amino acids (glycine, lysine, or phenylalanine). The stability of the blue pigments against heat, light, and pH was studied to examine the blue dye for possible use as a value-added food colorant. Thermal degradation reactions at temperatures of 60-90 degrees C were carried out at different pH levels within the range 5.0-9.0 (pH 5.0, acetate buffer; pH 7.0, phosphate buffer; and pH 9.0, CHES buffer). The blue pigments remained stable after 10 h at temperatures of 60-90 degrees C, and in some cases, more new pigments formed. The pigments were more stable at alkaline pH than neutral and acidic pH. Similarly, the pigments were stable under light irradiance of 5000-20 000 lux. In this case, pH effect was not significant.

Solution structure of a designed amphipathic antimicrobial synthetic peptide, PGAa.

A designed peptide, PGAa showed an excellent antifungal activity as well as an efficient bactericidal activity toward gram-positive, especially in the pathogenic yeast Candida albicans 28838. The solution structures of PGAa have been determined both in 40% TFE/water solution and DPC micelle by CD and NMR spectroscopy. Based on NOEs, vicinal coupling constants, backbone amide exchange rates, and chemical shift indices, PGAa formed a long amphipathic alpha-helical conformation in both TFE and DPC micelle environments, spanning the residues Ile(2)-Ala(19) in TFE and Lys(5)-Ala(19) in DPC micelle, respectively. Solution structures suggested that the hydrophobic residues would interact with the fatty acyl chains of the lipid bilayer, while the positively charged side-chains exposed to aqueous environments. Therefore, we conclude that the alpha-helical structure as well as the highly amphiphatic nature of PGAa peptide may play a critical role in its antimicrobial activity as well as selectivities in different species.

Enzymatic conversion of precarthamin to carthamin by a purified enzyme from the yellow petals of safflower.

Precarthamin, a yellow precursor of carthamin, was efficiently isolated from the yellow petals of safflower (Carthamus tinctorius L. ) with Sephadex LH-20 column chromatography and preparative HPLC, and identified with UV-vis and NMR spectrometry. The UV-vis spectrum of precarthamin showed lambda(max) of 238 and 406 nm in MeOH. The molar extinction coefficients of precarthamin at 406 nm in MeOH and 50 mM citrate buffer (pH 5.0) were 59 000 M(-)(1) cm(-)(1) and 45 400 M(-)(1) cm(-)(1), respectively. The isolated and structurally identified precarthamin was converted to a red pigment by a homogeneously purified enzyme from the immature petals of safflower in 50 mM citrate buffer (pH 5.0). The enzymatically converted red pigment was identified as carthamin, a red pigment of safflower by TLC, HPLC, and UV-vis spectral analysis.