Comprehensive validation of early diagnostic algorithms for myocardial infarction in the emergency department.

OBJECTIVE: To comprehensively evaluate diagnostic algorithms for myocardial infarction using a high-sensitivity cardiac troponin I (hs-cTnI) assay. PATIENTS AND METHODS: We prospectively enrolled patients with suspected myocardial infarction without ST-segment elevation from nine emergency departments in Japan. The diagnostic algorithms evaluated: (i) based on hs-cTnI alone, such as the European Society of Cardiology (ESC) 0/1-h or 0/2-h and High-STEACS pathways; or (ii) used medical history and physical findings, such as the ADAPT, EDACS, HEART, and GRACE pathways. We evaluated the negative predictive value (NPV), sensitivity as safety measures, and proportion of patients classified as low or high-risk as an efficiency measure for a primary outcome of type 1 myocardial infarction or cardiac death within 30 days. RESULTS: We included 437 patients, and the hs-cTnI was collected at 0 and 1 hours in 407 patients and at 0 and 2 hours in 394. The primary outcome occurred in 8.1% (33/407) and 6.9% (27/394) of patients, respectively. All the algorithms classified low-risk patients without missing those with the primary outcome, except for the GRACE pathway. The hs-cTnI-based algorithms classified more patients as low-risk: the ESC 0/1-h 45.7%; the ESC 0/2-h 50.5%; the High-STEACS pathway 68.5%, than those using history and physical findings (15-30%). The High-STEACS pathway ruled out more patients (20.5%) by hs-cTnI measurement at 0 hours than the ESC 0/1-h and 0/2-h algorithms (7.4%). CONCLUSIONS: The hs-cTnI algorithms, especially the High-STEACS pathway, had excellent safety performance for the early diagnosis of myocardial infarction and offered the greatest improvement in efficiency.

Wireless Multichannel Neural Recording With a 128-Mbps UWB Transmitter for an Implantable Brain-Machine Interfaces.

Simultaneous recordings of neural activity at large scale, in the long term and under bio-safety conditions, can provide essential data. These data can be used to advance the technology for brain-machine interfaces in clinical applications, and to understand brain function. For this purpose, we present a new multichannel neural recording system that can record up to 4096-channel (ch) electrocorticogram data by multiple connections of customized application-specific integrated circuits (ASICs). The ASIC includes 64-ch low-noise amplifiers, analog time-division multiplexers, and 12-bit successive approximation register ADCs. Recorded data sampled at a rate of 1 kS/s are multiplexed with time division via an integrated multiplex board, and in total 51.2 Mbps of raw data for 4096 ch are generated. This system has an ultra-wideband (UWB) wireless unit for transmitting the recorded neural signals. The ASICs, multiplex boards, and UWB transmitter unit are designed with the aim of implanting them. From preliminary experiments with a human body-equivalent liquid phantom, we confirmed 4096-ch UWB wireless data transmission at 128 Mbps for distances below 20 mm .

Synthesis of DNA fragments containing 2'-deoxy-4'-selenonucleoside units using DNA polymerases: comparison of dNTPs with O, S and Se at the 4'-position in replication.

4'-SelenoDNA fragments were synthesized for the first time using 4'-selenothymidine triphosphate (SeTTP) by taking advantage of its bioequivalence against DNA polymerases. DNA fragments each with a homologous element (O, S or Se) at the 4'-position of the thymidine units were effectively amplified using KOD Dash DNA polymerase.

Permanent acceptance of mouse cardiac allografts with CD40 siRNA to induce regulatory myeloid cells by use of a novel polysaccharide siRNA delivery system.

The CD40/CD154 co-stimulatory pathway is crucial in alloimmune response. We developed a novel small interfering RNA (siRNA) delivery system with a poly-dA extension at the 5'-end of the siRNA sense strand that was stably incorporated into 1,3-ß-glucan (schizophyllan, SPG). This was captured and incorporated into dendritic cells (DCs) through its receptor, Dectin-1, specifically silencing CD40 genes (siCD40) to exert immunoregulatory activity. siCD40/SPG-treated CBA mice permanently accepted B10 fully mismatched cardiac allografts. Consistent with graft survival, the infiltration of CD4(+), CD8(+) T cells into the graft was lower, and that the numbers of CD40(low)CD11c(+) DCs cells and CD4(+)Foxp3(+)cells were increased in both the graft and in the recipient spleen. In addition, naive CBA recipients given an adoptive transfer of splenocytes from the primary recipients with siCD40/SPG accepted a heart graft from donor-type B10, but not third-party Balb/c mice. In conclusion, the treatment with siCD40/SPG targeting DCs could generate antigen-specific Tregs, resulting in the permanent acceptance of mouse cardiac allografts. These findings have important implications for clarifying the mechanism underlying the induction of tolerance in DCs, and also highlight the potential of immunomodulation and the feasibility of siRNA-based clinical therapy in the transplantation field.

Antimicrobial properties and mechanism of volatile isoamyl acetate, a main flavour component of Japanese sake (Ginjo-shu).

AIMS: To evaluate the antimicrobial properties of the main Ginjo-flavour components of sake, volatile isoamyl acetate and isoamyl alcohol. METHODS AND RESULTS: Volatile isoamyl acetate and isoamyl alcohol both inhibited growth of the five yeast and 10 bacterial test strains. The minimum inhibitory dose and minimum bactericidal (fungicidal) dose of isoamyl acetate were higher than those of isoamyl alcohol. Escherichia coli and Acetobacter aceti were markedly sensitive to isoamyl acetate and isoamyl alcohol. In E. coli exposed to isoamyl acetate for 5 h, changes in expression were noted in proteins involved in sugar metabolism (MalE, MglB, TalB and PtsI), tricarboxylic acid cycle (AceA, Pfl and AcnB) and protein synthesis (EF-Tu, EF-G, and GlyS). Expression of acid and alcohol stress-response proteins was altered in E. coli exposed to isoamyl acetate. Esterase activity was detected in E. coli, suggesting that isoamyl acetate was hydrolyzed to acetic acid and isoamyl alcohol. Acetic acid and isoamyl alcohol damaged E. coli cell membranes and inactivated membrane proteins, impairing respiration. CONCLUSIONS: Volatile isoamyl acetate and isoamyl alcohol were effective in inactivating various micro-organisms, and antimicrobial mechanism of volatile isoamyl acetate against E. coli was clarified based on proteome analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report to examine the antimicrobial mechanism of volatile organic compound using proteome analysis combining two-dimensional difference gel electrophoresis with peptide mass fingerprinting.

Multichannel neural recording with a 128 Mbps UWB wireless transmitter for implantable brain-machine interfaces.

To realize a low-invasive and high accuracy BMI (Brain-machine interface) system, we have already developed a fully-implantable wireless BMI system which consists of ECoG neural electrode arrays, neural recording ASICs, a Wi-Fi based wireless data transmitter and a wireless power receiver with a rechargeable battery. For accurate estimation of movement intentions, it is important for a BMI system to have a large number of recording channels. In this paper, we report a new multi-channel BMI system which is able to record up to 4096-ch ECoG data by multiple connections of 64-ch ASICs and time division multiplexing of recorded data. This system has an ultra-wide-band (UWB) wireless unit for transmitting the recorded neural signals to outside the body. By preliminary experiments with a human body equivalent liquid phantom, we confirmed 4096-ch UWB wireless data transmission at 128 Mbps mode below 20 mm distance.

Diurnal and circadian oscillations in expression of kisspeptin, kisspeptin receptor and gonadotrophin-releasing hormone 2 genes in the grass puffer, a semilunar-synchronised spawner.

In seasonally breeding animals, the circadian and photoperiodic regulation of neuroendocrine system is important for precisely-timed reproduction. Kisspeptin, encoded by the Kiss1 gene, acts as a principal positive regulator of the reproductive axis by stimulating gonadotrophin-releasing hormone (GnRH) neurone activity in vertebrates. However, the precise mechanisms underlying the cyclic regulation of the kisspeptin neuroendocrine system remain largely unknown. The grass puffer, Takifugu niphobles, exhibits a unique spawning rhythm: spawning occurs 1.5-2 h before high tide on the day of spring tide every 2 weeks, and the spawning rhythm is connected to circadian and lunar-/tide-related clock mechanisms. The grass puffer has only one kisspeptin gene (kiss2), which is expressed in a single neural population in the preoptic area (POA), and has one kisspeptin receptor gene (kiss2r), which is expressed in the POA and the nucleus dorsomedialis thalami. Both kiss2 and kiss2r show diurnal variations in expression levels, with a peak at Zeitgeber time (ZT) 6 (middle of day time) under the light/dark conditions. They also show circadian expression with a peak at circadian time 15 (beginning of subjective night-time) under constant darkness. The synchronous and diurnal oscillations of kiss2 and kiss2r expression suggest that the action of Kiss2 in the diencephalon is highly dependent on time. Moreover, midbrain GnRH2 gene (gnrh2) but not GnRH1 or GnRH3 genes show a unique semidiurnal oscillation with two peaks at ZT6 and ZT18 within a day. The cyclic expression of kiss2, kiss2r and gnrh2 may be important in the control of the precisely-timed diurnal and semilunar spawning rhythm of the grass puffer, possibly through the circadian clock and melatonin, which may transmit the photoperiodic information of daylight and moonlight to the reproductive neuroendocrine centre in the hypothalamus.

Hypothalamic brain-derived neurotrophic factor regulates glucagon secretion mediated by pancreatic efferent nerves.

Understanding the molecular mechanism of the regulation of glucagon secretion is critical for treating the dysfunction of α cells observed in diabetes. Glucagon-like peptide (GLP)-1 analogues reduce plasma glucagon and are assumed to contribute to their action to lower blood glucose. It has previously been demonstrated that the central administration of brain-derived neurotrophic factor (BDNF) improves glucose metabolism by a mechanism independent of feeding behaviour in obese subjects. Using male rats, we examined whether BDNF influences glucagon secretion from α cells via the the central nervous system. We investigate whether: (i) the central infusion of BDNF stimulates glucagon and/or insulin secretion via the pancreatic efferent nerve from the hypothalamus; (ii) the intraportal infusion of GLP-1 regulates glucose metabolism via the central and peripheral nervous system; and (iii) BDNF receptor and/or BDNF-positive fibres are localised near α cells of islets. The portal glucagon level decreased with the central administration of BDNF (n = 6, in each; P < 0.05); in contrast, there was no significant change in portal insulin, peripheral glucagon and insulin levels with the same treatment. This reduction of glucagon secretion was abolished by pancreatic efferent denervation (n = 6, in each; P < 0.05). In an immunohistochemical study, pancreatic α cells were stained specifically with BDNF and tyrosine-related kinase B, a specific receptor for BDNF, and α cells were also co-localised with BDNF. Moreover, intraportal administration of GLP-1 decreased glucagon secretion, as well as blood glucose, whereas it increased the BDNF content in the pancreas; these effects were inhibited with the central infusion of BDNF antibody (n = 6, in each; P < 0.05). BDNF and GLP-1 affect glucose metabolism and modulate glucagon secretion from pancreatic α cells via the central and peripheral nervous systems.

Phase II multi-institutional prospective randomised trial comparing S-1+paclitaxel with S-1+cisplatin in patients with unresectable and/or recurrent advanced gastric cancer.

BACKGROUND: A combination of S-1 and cisplatin has been shown to be effective with acceptable safety for the first-line treatment of far-advanced gastric cancer in Japan. This is the first randomised phase II trial to compare S-1+paclitaxel with S-1+cisplatin in this setting. METHODS: Patients with unresectable and/or recurrent advanced gastric cancer were randomly assigned to receive one of the two regimens: S-1 (40 mg m(-2) twice daily) on days 1-14 plus paclitaxel (60 mg m(-2)) on days 1, 8, and 15 of a 4-week cycle (S-1+paclitaxel) or S-1 (40 mg m(-2) twice daily) on days 1-21 plus cisplatin (60 mg m(-2)) on day 8 of a 5-week cycle (S-1+cisplatin). The primary end point was the response rate (RR). Secondary end points included progression-free survival (PFS), overall survival (OS), and safety. RESULTS: A total of 83 patients were eligible for safety and efficacy analyses. In the S-1+paclitaxel and S-1+cisplatin groups, RRs (52.3% vs 48.7%; P=0.74) and median PFS (9 vs 6 months; P=0.50) were similar. The median OS was similar in the S-1+paclitaxel and S-1+cisplatin groups (16 vs 17 months; P=0.84). The incidence of grade 3 or higher haematological toxicity was 19.0% with S-1+paclitaxel and 19.5% with S-1+cisplatin. The incidence of grade 3 or higher non-haematological toxicity was 14.2% with S-1+paclitaxel and 17.1% with S-1+cisplatin. CONCLUSION: S-1+paclitaxel was suggested to be a feasible and effective non-platinum-based regimen for chemotherapy in patients with advanced gastric cancer. Our results should be confirmed in multicenter, phase III-controlled clinical trials.

Psychological stress during in vitro fertilization and embryo transfer is influenced by the patients' background and gender.

Purpose: This study evaluated the changes in psychological stress during in vitro fertilization and embryo transfer (IVF-ET) and the relationship of such stress to the patients' background and gender. Methods: Sixty couples undergoing IVF-ET were administered the State-Trait Anxiety Inventory-JYZ (STAI) test at six different points during IVF-ET procedures. Anxiety scores at each time point were recorded and analyzed according to gender, fertility status, and duration of treatment. Results: The median state anxiety score for women increased following induction until oocyte collection, after which it temporarily declined and then increased again until the pregnancy test. No such changes were noted in men. Scores for women who had undergone a shorter period of IVF treatments were higher while state and trait anxiety in men increased with a prolonged treatment period. Unsuccessful treatment increased the state and trait anxiety of women. Conclusions: Psychological stress changed periodically depending on the duration of the patients' treatment and fertility status also influenced anxiety levels. These findings will prove helpful in guiding psychological therapy and counseling for couples attempting to conceive by in vitro fertilization.

High-affinity ligands of Siglec receptors and their therapeutic potentials.

Sialic acids are one of the important constituents of glycoconjugates in the deuterostome lineage of animals and microorganisms. Siglecs (Sialic acid-binding immunoglobulin like lectins) are a family of cell-surface receptor proteins that recognize sialylated glycoconjugates as ligands. To date, 15 Siglecs have been described in humans and are mainly known as regulators of the immune system. Several of the Siglecs are emerging as potential targets for the treatment of some inflammatory, autoimmune, allergic, neurodegenerative and infectious diseases. In addition to antibody mediated therapy, high-affinity ligand-based probes of Siglec receptors would represent invaluable tools to effectively address therapeutic opportunities of Sialic acid-mediated Siglec recognition. This review discusses some aspects of structure and function of Siglec receptors, and concisely summarizes up-to-date progress on the identification of sialic acid based high-affinity ligands of certain well explored Siglec receptors.

Synchronised expressions of LPXRFamide peptide and its receptor genes: seasonal, diurnal and circadian changes during spawning period in grass puffer.

Among the RFamide peptide family, the LPXRFamide peptide (LPXRFa) group regulates the release of various pituitary hormones and, recently, LPXRFa genes were found to be regulated by photoperiod via melatonin. As a first step towards investigating the role of LPXRFa on reproductive function in grass puffer (Takifugu niphobles), which spawns in semilunar cycles, genes encoding LPXRFa and its receptor (LPXRFa-R) were cloned, and seasonal, diurnal and circadian changes in their absolute amounts of mRNAs in the brain and pituitary were examined by quantitative real-time polymerase chain reaction. The grass puffer LPXRFa precursor contains two putative RFamide peptides and one possible RYamide peptide. LPXRFa and LPXRFa-R genes were extensively expressed in the diencephalon and pituitary. The expression levels of both genes were significantly elevated during the spawning periods in both sexes in the brain and pituitary, although they were low in the spawning fish just after releasing eggs and sperm. The treatment of primary pituitary cultures with goldfish LPXRFa increased the amounts of follicle-stimulating hormone ß- and luteinising hormone ß-subunit mRNAs. In the diencephalon, LPXRFa and LPXRFa-R genes showed synchronised diurnal and circadian variations with one peak at zeitgeber time 3 and circadian time 15, respectively. The correlated expression patterns of LPXRFa and LPXRFa-R genes in the diencephalon and pituitary and the possible stimulatory effects of LPXRFa on gonadotrophin subunit gene expression suggest the functional significance of the LPXRFa and LPXRFa-R system in the regulation of lunar-synchronised spawning of grass puffer.

Differential impacts of CYP2C19 gene polymorphisms on the antiplatelet effects of clopidogrel and ticlopidine.

We examined the influence of CYP2C19 polymorphisms on the antiplatelet effects of clopidogrel and ticlopidine. The platelet aggregation induced by 20 µmol/l adenosine diphosphate (ADP) and CYP2C19 single-nucleotide polymorphisms (*2 and *3) was determined in patients with coronary artery disease (CAD) who were taking aspirin alone (n = 21), aspirin plus clopidogrel (n = 97), or aspirin plus ticlopidine (n = 47). The degree of platelet aggregation in the clopidogrel group, although not in the ticlopidine group, depended on the CYP2C19 polymorphism, and the maximal platelet aggregation in poor metabolizers (PMs) taking clopidogrel was equivalent to that in the group taking aspirin alone. After being switched from clopidogrel to ticlopidine, all seven of the PMs showed markedly lower platelet aggregation. These results suggest that CYP2C19 polymorphisms have a profound impact on the antiplatelet effect of clopidogrel but not on that of ticlopidine. Ticlopidine may be an effective therapeutic option for CYP2C19 PMs.

Morphological changes and viability of Cryptosporidium parvum sporozoites after excystation in cell-free culture media.

Cryptosporidium parvum, belonging to the phylum Apicomplexa, is a major cause of waterborne gastroenteritis throughout the world. The sporozoites are thought to invade host enterocytes using an active process termed gliding motility. However, the biological and morphological changes within the sporozoites during this process are not fully understood. In the present study, excysted sporozoites of C. parvum were analysed ultrastructurally in vitro and their viability was evaluated using fluorescent dyes. The sporozoites excysted from oocysts changed morphologically from banana-shaped to rod-shaped and finally to a rounded shape, in culture media in 3 h. Transmission microscopy revealed that the distance between the apical end and the nucleus was markedly reduced, dense granules were present close to the rhoptry in the apical region, amylopectin granules were absent, and membranes of round sporozoites were less clear. A fluorescent assay showed that the rate of survival decreased from 89% to 56% at 0-3 h (84.3% for banana-shaped and 49.2% for rod-shaped sporozoites). Therefore, post-excysted sporozoites in vitro underwent morphological changes and a rapid loss of viability. This staining method is useful, inexpensive and provides an alternative to more costly and intensive flow cytometric assays or infectivity assays with host cells in vitro.

Toxicological evaluation of L-proline in a 90-day feeding study with Fischer 344 rats.

L-proline (L-Pro) is a non-essential amino acid, and has become widely used as supplements and health foods, recently. A subchronic oral toxicity study of L-Pro was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.625%, 1.25%, 2.5% and 5.0% of L-Pro for 90 days. No treatment-related clinical signs and mortality were noted. We observed no clear treatment-related effects with regard to body weight, food intake or urinalysis data. The average daily water intakes of the treated female groups were significantly increased compared to the controls. The hematology (red blood cell parameter) and serum biochemistry (glucose, blood urea nitrogen, creatinine or uric acid) of the treated male and/or female groups were lower than those of the control groups. However, these changes were lacked dose-dependence, and no abnormalities were found in corresponding pathological findings. In conclusion, the no-observed-adverse-effect-level (NOAEL) for L-Pro was determined to be a dietary dose of 5.0% (2772.9 mg/kg body weight/day for males and 3009.3mg/kg body weight/day for females) under the present experimental conditions.

Pyrazinamide resistance in multidrug-resistant Mycobacterium tuberculosis isolates in Japan.

Thirty-six multidrug-resistant (MDR) Mycobacterium tuberculosis isolates collected in Japan were examined for pyrazinamide susceptibility and pyrazinamidase activity, and analysed by pncA sequencing and a hybridization-based line probe assay (LiPA), which was used to detect pncA mutations for the rapid identification of pyrazinamide-resistant isolates. Pyrazinamide resistance was found in 19 (53%) of them. All pyrazinamide-resistant isolates had no pyrazinamidase activity and at least one mutation in pncA. Among the pncA mutations, 11 had not been previously reported. The results of the LiPA were fully consistent with the DNA sequencing results. A majority of MDR M. tuberculosis isolates in Japan were resistant to pyrazinamide.

Prognostic significance of the expression of MUC1 and collagen type IV in advanced gastric carcinoma.

BACKGROUND: Scirrhous gastric carcinoma is characterized by excessive deposition of collagen in the stroma. However, the clinical significance of this fibrosis of the stomach has not been clarified. The aim of this study was to examine the fibrotic mechanism in several histological types of gastric carcinoma, and the combination of MUC1 and collagen type IV as a possible predictor of patient survival. METHODS: One hundred and two paraffin-embedded specimens of gastric carcinoma were examined by immunohistochemical staining using monoclonal antibodies against collagen type IV and MUC1. RESULTS: Collagen type IV-positive expression was significantly associated with depth of wall penetration (P = 0.025) and stage (P = 0.023). There was a significant relationship between MUC1-positive expression and interstitial collagen type IV-positive expression (P = 0.035). Survival was shorter for patients with the combination of MUC1-positive expression and interstitial collagen type IV-negative expression than for those with other expression patterns. CONCLUSION: In patients with differentiated-type advanced gastric carcinoma, the combination of MUC1-positive and interstitial collagen type IV-negative expression may be a marker of unfavourable prognosis.

Clock gene expression in peripheral leucocytes of patients with type 2 diabetes.

AIM/HYPOTHESIS: Recent studies have demonstrated relationships between circadian clock function and the development of metabolic diseases such as type 2 diabetes. We investigated whether the peripheral circadian clock is impaired in patients with type 2 diabetes. METHODS: Peripheral leucocytes were obtained from eight patients with diabetes and six comparatively young non-diabetic volunteers at 09:00, 15:00, 21:00 and 03:00 hours (study 1) and from 12 male patients with diabetes and 14 age-matched men at 09:00 hours (study 2). Transcript levels of clock genes (CLOCK, BMAL1 [also known as ARNTL], PER1, PER2, PER3 and CRY1) were determined by real-time quantitative PCR. RESULTS: In study 1, mRNA expression patterns of BMAL1, PER1, PER2 and PER3 exhibited 24 h rhythmicity in the leucocytes of all 14 individuals. The expression levels of these mRNAs were significantly (p < 0.05) lower in patients with diabetes than in non-diabetic individuals at one or more time points. Moreover, the amplitudes of mRNA expression rhythms of PER1 and PER3 genes tended to diminish in patients with diabetes. In study 2, leucocytes obtained from patients with diabetes expressed significantly (p < 0.05) lower transcript levels of BMAL1, PER1 and PER3 compared with leucocytes from control individuals, and transcript expression was inversely correlated with HbA(1c) levels (rho = -0.47 to -0.55, p < 0.05). CONCLUSIONS/INTERPRETATION: These results suggest that rhythmic mRNA expression of clock genes is dampened in peripheral leucocytes of patients with type 2 diabetes. The impairment of the circadian clock appears to be closely associated with the pathophysiology of type 2 diabetes in humans.

Simultaneous monitoring by real-time polymerase chain reaction of epstein-barr virus, human cytomegalovirus, and human herpesvirus-6 in juvenile and adult liver transplant recipients.

Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus-6 (HHV-6) cause symptomatic diseases in liver transplant recipients. The loads of these viruses, the associations between viral DNAemia, serologic status, and acute rejection reactions were investigated in a group of 17 juvenile and 17 adult recipients of living donor liver transplantation (LDLT) for a median of 8 weeks posttransplantation. At least 1 plasma sample from 15/34 (44.1%) patients was positive for CMV DNA. For most of the CMV-positive patients, the CMV DNA appeared in the second week of LDLT, and disappeared by the eighth week. A minimum of 200 EBV DNA copies/mug peripheral blood mononuclear cell DNA (defined as positive for EBV) was detected in 5/34 (14.7%) patients, and the number of EBV-positive children was significantly greater than the number of EBV-positive adults. In most of the EBV-positive patients, the EBV loads increased after 4 weeks posttransplantation. Plasma HHV-6 was detected in 7/34 (20.6%) patients. HHV-6 DNA appeared for a short period from the second week of LDLT. In addition, 8 of the 19 virus-positive recipients carried 2 viruses, with the combination of CMV and HHV-6 being the most frequent. Serologic status seemed to be an important factor for all 3 viral infections. The rate of acute cellular rejection was not significantly higher in the CMV-, EBV-, or HHV-6-positive groups. Simultaneous monitoring for 3 herpesviruses revealed the impact of these viruses on LDLT recipients.

Human CYP3A4-introduced HepG2 cells: in vitro screening system of new chemicals for the evaluation of CYP3A4-inhibiting activity.

1. The aims were to attest whether HepG2-GS-3A4, a cell line into which the human CYP3A4 gene was introduced, can be used for a screening of chemicals that will inhibit CYP3A4 activity. 2. The capacity of the cells for metabolizing CYP3A4 substrates in vitro was evaluated. Also determined was the effect of CYP3A4 inhibitors and non-inhibitors on nifedipine hydroxylation. Western blot, immunohistochemostry and determination of beta-nicotinamide adenine dinucleotide phosphate (NADPH)-reductase activity were performed. 3. HepG2-GS-3A4 selectively metabolized substrates of CYP3A4 (diazepam, nordiazepam, lidocaine, atorvastatin, and nifedipine) to a greater degree than control. The metabolites were easily detected in the culture medium. Values of V(max) of HepG2-GS-3A4 were about 30- to 100-fold higher than those of the control, while values of K(m) were comparable. Pre-incubation of cimetidine and ketoconazole significantly inhibited nifedipine hydroxylation, while addition of inhibitors specific to other isoforms of CYPs had no substantial effect. The HepG2-GS-3A4 expressed a higher amount of CYP3A4 protein and mRNA than control. Most NADPH reductase activity was detected in microsomal fractions. 4 In conclusion, HepG2-GS-3A4 sufficiently and selectively metabolize substrates of CYP3A4, and inhibitors of CYP3A4 reduced the metabolism. Because the metabolites were easily detected in the culture medium, this cell might be useful for the new and easy screening of new drugs for the evaluation of CYP3A4-inhibiting activity in vitro.