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Many pharmaceuticals and dietary foods have been reported to inhibit cholesterol biosynthesis, mainly by inhibiting the presqualene enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase rather than a postsqualene enzyme. In this study, we examined the inhibitory effects of Latilactobacillus sakei UONUMA on cholesterol biosynthesis, especially postsqualene, in human HepG2 hepatoma cells. We quantified cholesterol and its precursors, and the mRNA and protein levels of enzymes involved in cholesterol biosynthesis. Three L. sakei UONUMA strains exhibited new inhibitory effects on cholesterol biosynthesis and inhibited the mRNA level of sterol-delta24-reductase (DHCR24), which is involved in the postsqualene cholesterol biosynthesis pathway. These strains will be useful for the prevention and treatment of hyperlipidemia.

Current treatment approaches for hyperlipidemia rely mainly on reducing the cholesterol level by inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), which is involved in the presqualene pathway of cholesterol biosynthesis. Finding a compound that instead targets the postsqualene pathway could aid in the treatment of hyperlipidemia and synergistically reduce the cholesterol level when used in conjunction with HMGCR inhibitors. Ergosterol is a fungal sterol that is converted to brassicasterol by 7-dehydrocholesterol reductase (DHCR7). DHCR7 is also a cholesterol biosynthesis enzyme, and thus ergosterol may cause the accumulation of 7-dehydrocholesterol, a precursor of cholesterol and vitamin D

Many pharmaceuticals and dietary foods have been reported to inhibit cholesterol biosynthesis, mainly by inhibiting the presqualene enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase rather than a postsqualene enzyme. In this study, we examined the inhibitory effects of Latilactobacillus sakei UONUMA on cholesterol biosynthesis, especially postsqualene, in human HepG2 hepatoma cells. We quantified cholesterol and its precursors, and the mRNA and protein levels of enzymes involved in cholesterol biosynthesis. Three L. sakei UONUMA strains exhibited new inhibitory effects on cholesterol biosynthesis and inhibited the mRNA level of sterol-delta24-reductase (DHCR24), which is involved in the postsqualene cholesterol biosynthesis pathway. These strains will be useful for the prevention and treatment of hyperlipidemia.

Proportional navigation (PN) is a widely-used guidance law for missiletarget engagement. The goal of the missile intercept problem is to reduce the closest distance between the missile and target by diminishing the line-of-sight rate (LOS rate). In general, PN guidance law necessitates information of the LOS rate and missile velocity. The closing velocity (relative approaching speed to the target) instead of the missile velocity is an additional option for effective guidance. However, there are cases where a sensing device for measuring target motions that can be mounted on a missile is limited. In this paper, we propose a novel guidance law on the basis of proportional navigation (PN) using only line-of-sight (LOS) rate information. In this paper, an uncertainty and disturbance estimator (UDE) is applied to estimate such target motions including velocity change or unpredictable movement etc. The UDE works also for compensating uncertain modeled dynamics such as a missile’s bearing uncertainty and velocity changes. The proposed guidance law is referred to as uncertainty and disturbance-compensated intercept guidance. Numerical simulations with some engagement scenarios are presented taking account of the velocity changes of the missile to demonstrate the potential of the proposed guidance law.

Growth hormone (GH) replacement therapy improves hypercholesterolemia in patients with GH deficiency, suggesting that GH modulates cholesterol metabolism.We examined GH effects on lipid profiles and cholesterol-related markers reflecting hepatic and cerebral cholesterol metabolism in small-for-gestational age (SGA) children without catch-up growth.This study examined SGA children without catch-up growth (n = 22) and healthy children (controls, n = 11). Based on parents' choice, 11 SGA children received GH at 0.23 to 0.25 mg/kg/d for 6 months, and at 0.34 to 0.36 mg/kg/d for the subsequent 6 months (GH (+) group). The other SGA children received no GH (GH (-) group, n = 11). We ascertained baseline and posttreatment lipid profiles and cholesterol-related markers reflecting hepatic and cerebral cholesterol metabolism.Baseline lipid profiles of SGA children and controls were similar. Serum 24S-hydroxycholesterol (marker for cerebral cholesterol metabolism) concentration was 19% lower in SGA children than in controls (P .05). Compared with baseline, the GH (+) group low-density lipoprotein-cholesterol concentration had decreased by 6.6% during 6 months and 8.8% during 12 months (P .01), whereas the high-density lipoprotein-cholesterol concentration had increased by 1.7% (P = .07) and 3.3% (P .01). Serum 7α-hydroxycholesterol (marker for hepatic cholesterol elimination) concentration had increased by 34% at 6 months and 35% at 12 months (P .01). In addition, 24S-hydroxycholesterol increased by 25% and 26% (P .001). No marker for cholesterol synthesis or absorption changed. The GH (-) group lipid profiles and oxysterols remained unchanged during the observation period.GH activates hepatic and cerebral cholesterol metabolism in SGA children without catch-up growth.

γδT cell receptor (TCR)-positive T cells, which control the innate immune system, display anti-tumor immunity as well as other non-immune-mediated anti-cancer effects. γδT cells expanded ex vivo by nitrogen-containing bisphosphonate (N-BP) treatment can kill tumor cells. N-BP inhibits farnesyl pyrophosphate synthase in the mevalonate pathway, resulting in the accumulation of isopentenyl pyrophosphate (IPP), which is a stimulatory antigen for γδT cells. We have previously observed that as they get closer, migrating γδT cells increase in speed toward target multiple myeloma (MM) cells. In the present study, we investigated the γδT cell chemotactic factors involving using a micro total analysis system-based microfluidic cellular analysis device. The addition of supernatant from RPMI8226 MM cells treated with the N-BP zoledronic acid (ZOL) or the addition of IPP to the device induced chemotaxis of γδT cells and increased the speed of migration compared to controls. Analysis of the ZOL-treated RPMI8226 cell supernatant revealed that it contained IPP secreted in a ZOL-dose-dependent manner. These observations indicate that IPP activates the chemotaxis of γδT cells toward target MM cells treated with ZOL.

Significance Bietti’s crystalline dystrophy (BCD) is an autosomal recessive, progressive chorioretinal degenerative disease. Retinal pigment epithelium (RPE) cells are impaired in patients with BCD, but the underlying mechanisms of RPE cell damage have not yet been determined because cells from lesions cannot be readily acquired from patients with BCD. In the present study, we successfully generated a human in vitro model of BCD, BCD patient-specific iPSC-RPE cells, and demonstrated that the accumulation of free cholesterol caused RPE cell damage and subsequent cell death via the induction of lysosomal dysfunction and impairment of autophagy flux in BCD-affected cells. We believe these findings provide evidence of the possible therapeutic efficacy of reducing intracellular free cholesterol in BCD., Bietti’s crystalline dystrophy (BCD) is an intractable and progressive chorioretinal degenerative disease caused by mutations in the CYP4V2 gene, resulting in blindness in most patients. Although we and others have shown that retinal pigment epithelium (RPE) cells are primarily impaired in patients with BCD, the underlying mechanisms of RPE cell damage are still unclear because we lack access to appropriate disease models and to lesion-affected cells from patients with BCD. Here, we generated human RPE cells from induced pluripotent stem cells (iPSCs) derived from patients with BCD carrying a CYP4V2 mutation and successfully established an in vitro model of BCD, i.e., BCD patient-specific iPSC-RPE cells. In this model, RPE cells showed degenerative changes of vacuolated cytoplasm similar to those in postmortem specimens from patients with BCD. BCD iPSC-RPE cells exhibited lysosomal dysfunction and impairment of autophagy flux, followed by cell death. Lipidomic analyses revealed the accumulation of glucosylceramide and free cholesterol in BCD-affected cells. Notably, we found that reducing free cholesterol by cyclodextrins or δ-tocopherol in RPE cells rescued BCD phenotypes, whereas glucosylceramide reduction did not affect the BCD phenotype. Our data provide evidence that reducing intracellular free cholesterol may have therapeutic efficacy in patients with BCD.

Objective Cholesterol and diet-derived oxidized cholesterol are absorbed in the small intestine and eliminated by bile acids. We determined whether ezetimibe, a selective cholesterol absorption inhibitor, changes serum oxidized cholesterol levels. Methods We measured levels of plant sterols, cholesterol precursors, and oxysterols by gas chromatography-mass spectrometry in 47 hypercholesterolemics and 32 controls. Twenty-four hypercholesterolemics received 10 mg ezetimibe/day for 4 weeks. Results Plant sterols were 30–42% higher in hypercholesterolemics than in controls and positively correlated with low-density lipoprotein-cholesterol (LDL-C). Ezetimibe decreased plant sterols by 21–53%, but did not change bile acid synthesis markers. 7β-hydroxycholesterol, a marker for non-enzymatic oxidation of cholesterol, was 66% higher in hypercholesterolemics than controls. Ezetimibe decreased 7β-hydroxycholesterol levels by 15% regardless of LDL-C reduction. Conclusions Ezetimibe decreases serum oxidized cholesterol generated by non-enzymatic reactions without impairing bile acid synthesis. Ezetimibe may maintain cholesterol excretion into bile and alleviate the diet-derived oxidative burden.

Aims We investigated the pharmacological effects of saponins isolated from ginseng root and their metabolites, which occur by hydrolysis of the sugar moieties connecting the aglycone of saponins in the digestive tract, on the production of corticosteroids in bovine adrenal fasciculata cells in vitro. Main methods The levels of corticosteroids produced from adrenal corticotropic hormone (ACTH)-stimulated bovine adrenal fasciculata cells were determined under the presence or absence of ginseng saponins (ginsenosides) and their metabolites using fluorometry, gas-chromatography–mass spectrometry, and sweeping-micellar electrokinetic capillary chromatography. Key findings An end metabolite of the protopanaxatriol saponins in ginseng, 20(s)-protopanaxatriol (M4), strongly reduced ACTH-stimulated cortisol production. M4 significantly inhibited the production of cortisol induced by different stimuli, alamethicin, dibutyryl cyclic AMP, forskolin, and 22(R)-hydroxycholesterol, a membrane-permeable cholesterol. However, it did not affect the production of cortisol by either pregnenolone, a precursor of cortisol synthesis, or cyclic AMP. Furthermore, M4 significantly inhibited the production of pregnenolone, progesterone, deoxycorticosterone, cortisol, and corticosterone in a dose-dependent manner. Significance Results strongly suggest that protopanaxatriol saponins in ginseng are prodrugs metabolized in the digestive tract so that the end metabolite, M4, produces inhibitory activity of corticosteroid production in the adrenal fasciculata cells in vivo. The results also suggest that M4 inhibits the conversion from cholesterol to pregnenolone because the production of pregnenolone was reduced.

When carbohydrate metabolism is impaired, fatty acid metabolism is activated. Excess acetyl-coenzyme A (CoA) is generated from fatty acids by β-oxidation and is used for the formation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) and subsequently for acetoacetate. High levels of secreted ketone bodies (acetoacetate and 3β-hydroxybutyrate) lower the pH of blood and urine, resulting in ketoacidosis. HMG-CoA lyase in hepatic cells is a rate-limiting enzyme catalyzing the cleavage of HMG-CoA to acetoacetate, and thus inhibition of this enzyme results in reduced acetoacetate production, in other words, impaired ketoacidosis. Inhibition of HMG-CoA lyase activity possibly prevents ketoacidosis and should be the therapeutic target. Polyphenols are common and abundant dietary constituents with beneficial effects on human health. We examined the inhibitory effects of dietary polyphenols on HMG-CoA lyase activity in cellular extracts of human hepatoma HepG2 cells. Of the nine representative dietary polyphenols tested, (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), and gallic acid (GA) effectively inhibited HMG-CoA lyase activity. Lineweaver-Burk analysis revealed that EGC and EGCG are likely to be mixed-type noncompetitive inhibitors. Pyrogallol with the gallyl structure also inhibited HMG-CoA lyase activity, suggesting that the gallyl moiety of polyphenols is important for the inhibition of HMG-CoA lyase activity.

The mechanisms for the cytotoxicity of staphylococcal Panton-Valentine leukocidin (PVL), a pore-forming toxin consisting of LukS-PV and LukF-PV, in human immune cells are still unclear. Because LukS-PV binds to ganglioside GM1, a constituent of detergent-resistant membrane microdomains (DRMs) of the plasma membrane, the role of DRMs in PVL cytotoxicity was examined in human polymorphonuclear neutrophils (PMNs), monocytes, HL-60 cells, and THP-1 cells. PVL binding capacities in HL-60 and THP-1 cells were higher than those in PMNs and monocytes; however, the PVL concentration to obtain more than 80% cell lysis in HL-60 cells was 10 times higher than that in PMNs and PVL even at such concentration induced

Micafungin is an echinocandin antifungal agent that is important for treating candidiasis in emergency and intensive care medicine; however, current methods for measuring micafungin plasma levels are lengthy and complicated. We report a simple quantitative method using column-switching high-performance liquid chromatography (HPLC) for determining micafungin in human plasma samples. Human plasma was directly injected into a column-switching HPLC system with a MAYI-ODS pre-column to remove the plasma matrix. The calibration curve for micafungin showed good linearity in the range 0.1-10 µg/mL in human plasma. The mean relative standard deviation value of the intra-day and inter-day precision was less than 7.3%. More than 450 successive, accurate measurements were made before the system had to be washed with ammonium acetate solution. The therapeutic micafungin level in patients' plasma was successfully measured using this method. Because the pretreatment is simple and reproducible, our method can be used for routine therapeutic monitoring.

24S-ヒドロキシコレステロールは脳細胞に対して毒性を有するため，毒性の軽減はアルツハイマー病治療の補助療法につながると考えられる．本論文では，ポリフェノールがこの毒性に対して軽減作用を示し，抗酸化能の強さの順に軽減作用を認め，ポリフェノールの摂取はアルツハイマー病治療の補助療法として有効と示唆された.

We developed and validated a sensitive and convenient high-performance liquid chromatography (HPLC) method for the specific determination of ketone bodies (acetoacetate and D-3-hydroxybutyrate) in human plasma. p-Nitrobenzene diazonium fluoroborate (diazo reagent) was used as a precolumn derivatization agent, and 3-(2-hydroxyphenyl) propionic acid was used as an internal standard. After the reaction, excess diazo reagent and plasma proteins were removed by passing through a solid-phase cartridge (C(18)). The derivatives retained on the cartridge were eluted with methanol, introduced into the HPLC system, and then detected with UV at 380 nm. A calibration curve for acetoacetate standard solution with a 20-microl injection volume showed good linearity in the range of 1 to 400 microM with a 0.9997 correlation coefficient. For the determination of D-3-hydroxybutyrate, it was converted to acetoacetate before reaction with the diazo reagent by an enzymatic coupling method using D-3-hydroxybutyrate dehydrogenase and lactate dehydrogenase. A calibration curve for D-3-hydroxybutyrate standard solution also showed good linearity in the range of 1.5 to 2000 microM with a 0.9988 correlation coefficient. Analytical recoveries of acetoacetate and D-3-hydroxybutyrate in human plasma were satisfactory. The method was successfully applied to samples from diabetic patients, and results were consistent with those obtained using the thio-NAD enzymatic cycling method used in clinical laboratories.

To promote the proper use of voriconazole (VRCZ),we monitored VRCZ blood concentrations,and investigated the clinical significance of doing this.On comparing C/D (blood VRCZ concentration/VRCZ dose) values for different VRCZ administration methods we found that there was no significant difference between the intravenous drip and oral administration methods (t-test : P=0.607)in this respect.There was also no correlation between the dose of VRCZ per unit body weight and its blood concentration (variance analysis : R2=0.012,P=0.449).Though total bilirubin (T-Bil) levels were correlated with C/D values (variance analysis : R2=0.104,P=0.020),serum creatinine clearance (CCr) and C/D values (variance analysis : R2=0.062,P=0.078)were not correlated.Furthermore,C/D values in males were significantly higher than those in females (t-test : P=0.001).Approximately 25% of the patients had a blood concentration trough value exceeding 4.5μg/mL,which indicates an enhanced risk of liver dysfunction,and this suggests that the measurement of blood VRCZ concentrations is clinically important for preventing side effects.

A 53-year-old woman ingested about 300 mL of 95% methanol. After immediate ethanol antagonist therapy and hemodialysis, she recovered completely. Few days later, the plasma concentration of methanol and formate was measured. A gas chromatography was used for the plasma methanol concentration measurement, and a colorimetric method was used for plasma formate concentration measurement (Formate Colorimetric Assay Kit; BioVision, California, USA). Patient's plasma methanol concentration before hemodialysis was 676.9 mg/dL and plasma formate concentration was 16.9 mg/dL. By removing blood methanol and formate using hemodialysis before formate accumulations in the body, the patient was discharged without any sequelae. We were able to obtain correlation between a gas chromatography and colorimetric method without gas chromatography-mass spectrometry, with good correlation coefficients. The sensitivity was sufficient for analyzing blood sample. Monitoring formate concentration is useful in determining the treatment and evaluating the prognosis of methanol poisoning. We suggest that this colorimetric method is useful in a facility with no access to a gas chromatography in order to measure a plasma formate concentration.

Toxic shock syndrome toxin 1 (TSST-1), produced by Staphylococcus aureus (including methicillin-resistant S. aureus ), is a superantigenic toxin responsible for toxic shock syndrome as well as neonatal TSS-like exanthematous disease. TSST-1 exhibits its deleterious effects by leading to the abnormal proliferation of, e.g., Vβ2 + T cells and overproduction of proinflammatory cytokines. In the present study we examined the inhibitory effect of a Chinese herbal extract, anisodamine, on TSST-1 using human peripheral blood mononuclear cells (PBMCs). Anisodamine inhibited the production of proinflammatory cytokines better than interleukin-10 (an anti-inflammatory cytokine). The inhibitory effect of anisodamine was greater than that of any tropane alkaloid examined. Anisodamine acted directly on both monocytes and T cells in human PBMCs, and the effect was confirmed at the transcriptional level. Inhibition of NF-κB activation was also demonstrated. In contrast, no significant inhibition of Vβ2 + T-cell proliferation was observed. In mice injected with TSST-1, anisodamine treatment significantly decreased serum proinflammatory cytokine levels and prevented TSST-1-induced death. These results suggest that anisodamine specifically acts against the production of cytokines (inflammatory cytokines in particular) and not against Vβ2 + T-cell proliferation and that anisodamine may have a beneficial effect on TSST-1-associated disease.

Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), produces superantigenictoxins, such as toxic shock syndrome toxin-1 (TSST-1). TSST-1 abnormally activates T cells to overproduce inflammatory cytokines (such as tumor necrosis factor-alpha, interleukin-2, and interferon-gamma) leading to shock. In this study, we investigated the inhibitory effect of antimicrobial agents and anisodamine (a Chinese herbal extract) on TSST-1-induced cytokine production. Among the macrolides and related agents examined, azithromycin and rokitamycin showed the greatest inhibitory activity against the TSST-1-induced cytokine production. This inhibitory effect was similar to that of anisodamine, which, however, had no inhibitory activity against bacterial growth. Vancomycin, teicoplanin, arbekacin, and linezolid (anti-MRSA and related agents) had no significant inhibitory effect on cytokine production. The inhibitory effect of the drugs on cell proliferation was not significant. These data indicate that some antimicrobial agents, e.g., azithromycin and rokitamycin, manifest anti-superantigenic toxin activity through the inhibition of cytokine production, just like anisodamine.

Background The proportion of poor metabolizers (PMs) of cytochrome P450 (CYP) 2C19 is much higher in the Japanese population than in European populations. Cycling probe technology (CPT) is a simple signal amplification technique for targeting specific DNA sequences. CPT utilizes a chimeric DNA-RNA-DNA probe that is cleaved by the enzyme ribonuclease (RNase H). In this study, using CPT, we aimed to detect the CYP2C19 gene polymorphism from noninvasive samples to determine extensive metabolizers (EMs) and PMs of CYP2C19. Methods DNA samples were extracted from hair, buccal mucosa, and blood cells. Primers and cycling probes were designed specifically for region G636A for exon 4 and G681A for exon 5, reported to be gene polymorphisms of CYP2C19. Results DNA extracted from hair follicle cells and buccal epithelial cells was the same as that collected from invasive blood sampling. The genotype of CYP2C19 was successfully identified as either EM or PM in 71 samples, producing identical results to those for the TaqMan method, except in three samples. Conclusions We successfully detected the two gene polymorphisms of CYP2C19 from noninvasive samples using a simple DNA extraction method and CPT.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is associated with hemolytic uremic syndrome (HUS). High inflammatory cytokine [interleukin (IL)-6 and IL-8] levels and low anti-inflammatory cytokine (IL-10) levels are indicators of a high risk for developing HUS in STEC-infected children. In this study, we investigated inhibitory action of telithromycin, a ketolide, against STEC and against Stx and lipopolysaccharide (LPS). Telithromycin inhibited in vitro STEC growth without inducing Stx phage, in marked contrast to norfloxacin. Stx markedly induced inflammatory (but not anti-inflammatory) cytokine production in human peripheral blood monocytes, while LPS induced both inflammatory and anti-inflammatory cytokine production. Telithromycin selectively inhibited the IL-6 and IL-8 production from Stx-stimulated (but not LPS-stimulated) monocytes. The drug did not significantly inhibit IL-10 production. Our data suggest that Stx plays a crucial role in the stimulation of inflammatory cytokines and such inflammatory response is inhibited by telithromycin, an anti-bacterial agent.

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the human intestinal mucosa, produces Stx from phage, and causes the development of hemolytic-uremic syndrome via Stx-induced inflammatory cytokine production. Azithromycin exhibited strong in vitro activity against STEC without inducing Stx-converting phage, in marked contrast to norfloxacin. Azithromycin decreased the tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 production from Stx-treated human peripheral mononuclear cells or monocytes to a greater extent than did clarithromycin. In Stx-injected mice, azithromycin significantly suppressed Stx-induced TNF-α, IL-1β, and IL-6 levels in serum and improved the outcome as assessed by survival rate. In the STEC oral infection experiment using immature mice immediately after weaning (weaned immature-mouse model), all mice died within 7 days postinfection. Azithromycin administration gave the mice 100% protection from killing, while ciprofloxacin administration gave them 67% protection. The data suggest that azithromycin (at least at higher concentrations) has a strong effect on Stx production by STEC and on the Stx-induced inflammatory host response and prevents death in mice. Azithromycin may have a beneficial effect on STEC-associated disease.

Ceftazidime-resistant Enterobacter aerogenes was isolated from blood cultures of three patients with fever. DNA analysis using pulsed-field gel electrophoresis and ribosomal RNA gene restriction digest pattern analysis revealed that the strains were clonally similar to each other with a 79.3-96.0% homology. The same strain of E. aerogenes was isolated from a three-way stopcock connected to the indwelling catheter in one of the patients at a concentration of 45 cfu/mL. A similar strain was also isolated from the urine of one other patient on the same floor. The data suggest that E. aerogenes caused septicaemia via low bacterial contamination of a three-way stopcock in a peripheral drip intravenous infusion system in at least one patient, and that the outbreak of E. aerogenes infections was due to clonally-related strains.

Modulating steroid hormone levels is a curative and preventive measure for Cushing's syndrome, aldosteronism, and various stress-triggered symptoms. Polyphenols have been reported to inhibit steroidogenic enzymes such as 3β-hydroxysteroid dehydrogenase (3β-HSD) and aromatase. However, evidence for their inhibitory effects is fragmentary because it has been determined in studies with small groups of steroid hormones. To investigate the effects of steroids on complete steroidogenic pathways, comprehensive analysis of steroid hormones is necessary. Here we cultured forskolin-stimulated NCI-H295R, a human adrenocortical carcinoma cell line, in the presence of a polyphenol and employed GC-MS to simultaneously determine the levels of nine steroid hormones (pregnenolone, progesterone, deoxycorticosterone, aldosterone, 17α-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, and estradiol) in cell culture supernatant. We found that daidzein, genistein, apigenin, hesperetin, naringenin, and eriodictyol significantly reduced deoxycorticosterone and androstenedione levels (p

Phenylketonuria (PKU) possibly leads to hypocholesterolemia and lowered vitamin D (VD) status. Metabolism of oxysterols linking with those of cholesterol and VD has never been examined in PKU.Blood oxysterols along with blood phenylalanine, lipids and VD were examined for 33 PKU adults aged 21-38 years and 20 age-matched healthy controls.Total- and low-density cholesterols, and 25-hydroxy VD(3) were decreased significantly in the PKU group (cholesterols, 10% decrease; 25-hydroxy VD(3) 35% decrease vs. the control group). 24S-hydroxycholesterol (24S-OHC) eliminating brain cholesterol, and 27-OHC and 7α-hydroxycholesterol (7α-OHC) representing peripheral and hepatic cholesterol elimination, respectively, were significantly decreased in PKU group: 24S-OHC, 25% decrease, p.01; 27-OHC and 7α-OHC, 35-40% decrease, p.001. 7β-Hydroxycholesterol (7β-OHC) reflecting oxidative stress was increased significantly in PKU group (p.05). 7α-OHC and 27-OHC levels in PKU group always showed similar values, regardless of other parameters while the 24S-OHC and 7β-OHC levels decreased and increased, respectively, showing significant correlations with phenylalanine level (p.005). 27-OHC level showed a significant positive correlation with the 25-hydroxy VD(3) level in this group (p.001).Blood oxysterol changes predominate over blood cholesterol changes and influence on VD status in adult PKU patients.

Micafungin is an echinocandin antifungal agent that is important for treating candidiasis in emergency and intensive care medicine; however, current methods for measuring micafungin plasma levels are lengthy and complicated. We report a simple quantitative method using column-switching high-performance liquid chromatography (HPLC) for determining micafungin in human plasma samples. Human plasma was directly injected into a column-switching HPLC system with a MAYI-ODS pre-column to remove the plasma matrix. The calibration curve for micafungin showed good linearity in the range 0.1-10 µg/mL in human plasma. The mean relative standard deviation value of the intra-day and inter-day precision was less than 7.3%. More than 450 successive, accurate measurements were made before the system had to be washed with ammonium acetate solution. The therapeutic micafungin level in patients' plasma was successfully measured using this method. Because the pretreatment is simple and reproducible, our method can be used for routine therapeutic monitoring.

The effects of Lactobacillus johnsonii La1 (LC1) on Helicobacter pylori colonization in the stomach were investigated. H. pylori colonization and gastritis in LC1-inoculated Mongolian gerbils were significantly less intense than those in the control animals. LC1 culture supernatant (>10-kDa fraction) inhibited H. pylori motility and induced bacterial aggregation in human gastric epithelial cells, suggesting the potential of clinical use of LC1 product.

Helicobacter pylori in Vladivostok, Far Eastern Russia, was investigated during 2004 to 2009. The genotype cagA(+) vacA(+) (s1/m1 or m2) accounted for 74.7%, with cagA(-) vacA(+) (s2/m2) at 11.2%. The CagA EPIYA type was mainly Western ABC, with minor types (ABCCC and novel AAABC) or non-Western/non-East Asia type (AB). Regarding drug resistance, metronidazole resistance was the highest, with a marked decrease in 6 years (from 71.4% to 30.8%); in contrast, levofloxacin and clarithromycin resistance increased. The data indicate that in Vladivostok, H. pylori was mainly the Western (not East Asian) type and dynamic changes in drug resistance occurred during 6 years.

A method for the simultaneous determination of pregnenolone and 17α-hydroxypregnenolone by high-performance liquid chromatography with an immobilized cholesterol oxidation enzyme reactor was developed. Pregnenolone and 17α-hydroxypregnenolone were converted to progesterone and 17α-hydroxyprogesterone, respectively, by the immobilized enzyme packed into the reactor column, and could thus be monitored by UV absorption at 240 nm. The calibration curves for pregnenolone and 17α-hydroxypregnenolone were linear in the range of 0.4-10 and 0.3-10 μg/ml with a correlation coefficient of 0.9993 and 0.9998, respectively. The detection limit at a signal-to-noise ratio of 3 was 0.12 and 0.08 μg/ml for pregnenolone and 17α-hydroxypregnenolone, respectively. The conversion rate of pregnenolone to progesterone and 17α-hydroxypregnenolone to 17α-hydroxyprogesterone was 90.6% and 99.3%, respectively. Intra-day and inter-day precision (in terms of percentage coefficient of variation) were less than 9.3%, with accuracy greater than 94.8%. This method was successfully applied to the simultaneous determination of pregnenolone and 17α-hydroxypregnenolone secreted into the culture medium of bovine adrenal fasciculata cells and of both analytes produced within the cells.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was collected from children with bullous impetigo in 2003 and 2004. One strain collected in 2003 was Panton-Valentine leucocidin (PVL) positive. In 2004, a multiple-drug-resistant PVL(+) CA-MRSA strain was isolated from an athlete with a cutaneous abscess. These strains were analyzed by multilocus sequence typing, spa typing, agr typing, coagulase typing, staphylococcal cassette chromosome mec (SCCmec) typing, PCR assay for 30 virulence genes, drug susceptibility testing, pulsed-field gel electrophoresis, and for plasmids. The two Japanese PVL(+) CA-MRSA strains belonged to the globally extant ("pandemic") sequence type 30 (ST30) with SCCmec IV. A transmissible, multiple-drug resistance plasmid emerged in such ST30 strains. The PVL(-) CA-MRSA strains ("domestic" CA-MRSA) accumulated for bullous impetigo, exhibiting new genotypes. Hospital-acquired MRSA of ST91 (but not pandemic ST5) shared common features with the PVL(-) CA-MRSA strain.

A new category of methicillin-resistant Staphylococcus aureus (MRSA), called community-acquired MRSA (CA-MRSA), has emerged worldwide. In contrast to previous MRSA, most CA-MRSA carries the Panton-Valentine leukocidin (PVL) genes (lukPVSF) as a virulence genetic trait. Sequence analysis of the lukPVSF gene of a Japanese isolate demonstrated that the gene has more similarity to methicillin-susceptible S. aureus from France than MRSA from the United States. Based on the sequences, we developed a real-time PCR assay for the three key genes of CA-MRSA; that is, lukPVSF, mecA (for methicillin resistance), and spa (for S. aureus). Dual or triple assay for lukPVSF, mecA, and spa in one test tube became possible. The detection limit of the assay with probe and SYBR Green methods was between 2.7 and 2.7 × 101 CFU/ml. The assay detected PVL-positive MRSA in clinical (blood) isolates.

Large-scale nosocomial outbreaks of Serratia marcescens septicaemia in Japan have had a fatality rate of 20-60% within 48 h. As a countermeasure, a real-time PCR assay was constructed for the rapid diagnosis of S. marcescens septicaemia. This assay indeed detected S. marcescens in clinical blood specimens (at ca. 10(2)CFU ml(-1)), at a frequency of 0.5% in suspected cases of septicaemia. In mice, the assay provided estimates of blood S. marcescens levels at various infectious stages: namely, 10(7) to 10(8)CFU ml(-1) at a fatal stage (resulting in 100% death), 10(4)-10(5)CFU ml(-1) at a moderately fatal stage (resulting in 50% or more death), and10(3)CFU ml(-1) at a mild stage (resulting in 100% survival), consistent with actual CFU measurements. Blood bacterial levels could be an important clinical marker that reflects the severity of septicaemia. The simultaneous detection of S. marcescens and the carbapenem resistance gene was also demonstrated.

Clarithromycin-resistant Helicobacter pylori (CRHP) is increasing worldwide, especially in children. We report a family case in which both the mother and child were infected with CRHP. DNA analysis revealed that all of the mother's and daughter's isolates were indistinguishable, suggesting that the same CRHP strain spread between the family members. The spread of CRHP within families may be increasing.

Shiga toxin (Stx)-producing Escherichia coli (STEC), an important cause of hemolytic uremic syndrome, was completely killed by (60)Co irradiation at 1 x l0(3) gray (1 kGy) or higher. However, a low dose of irradiation (0.1-0.3 kGy) markedly induced Stx phage from STEC. Stx production was observed in parallel to the phage induction. Inactivation of Stx phage required a higher irradiation dose than that for bacterial killing. Regarding Stx, cytotoxicity was susceptible to irradiation, but cytokine induction activity was more resistant than Stx phage. The findings suggest that (1). although (60)Co irradiation is an effective means to kill the bacteria, it does induce Stx phage at a lower irradiation dose, with a risk of Stx phage transfer and emergence of new Stx-producing strains, and (2). irradiation differentially inactivates some activities of Stx.