Optimization of adeno-associated virus vector-mediated gene transfer to the respiratory tract.

An efficient adeno-associated virus (AAV) vector was constructed for the treatment of respiratory diseases. AAV serotypes, promoters and routes of administration potentially influencing the efficiency of gene transfer to airway cells were examined in the present study. Among the nine AAV serotypes (AAV1-9) screened in vitro and four serotypes (AAV1, 2, 6, 9) evaluated in vivo, AAV6 showed the strongest transgene expression. As for promoters, the cytomegalovirus (CMV) early enhancer/chicken ß-actin (CAG) promoter resulted in more robust transduction than the CMV promoter. Regarding delivery routes, intratracheal administration resulted in strong transgene expression in the lung, whereas the intravenous and intranasal administration routes yielded negligible expression. The combination of the AAV6 capsid and CAG promoter resulted in sustained expression, and the intratracheally administered AAV6-CAG vector transduced bronchial cells and pericytes in the lung. These results suggest that AAV6-CAG vectors are more promising than the previously preferred AAV2 vectors for airway transduction, particularly when administered into the trachea. The present study offers an optimized strategy for AAV-mediated gene therapy for lung diseases, such as cystic fibrosis and pulmonary fibrosis.

Intrinsic and extrinsic regulatory mechanisms are required to form and maintain a lens of the correct size and shape.

Understanding how tissues and organs acquire and maintain an appropriate size and shape remains one of the most challenging areas in developmental biology. The eye lens represents an excellent system to provide insights into regulatory mechanisms because in addition to its relative simplicity in cellular composition (being made up of only two forms of cells, epithelial and fiber cells), these cells must become organized to generate the precise spheroidal arrangement that delivers normal lens function. Epithelial and fiber cells also represent spatially distinct proliferation and differentiation compartments, respectively, and an ongoing balance between these domains must be tightly regulated so that the lens achieves and maintains appropriate dimensions during growth and ageing. Recent research indicates that reciprocal inductive interactions mediated by Wnt-Frizzled and Notch-Jagged signaling pathways are important for maintaining and organizing these compartments. The Hippo-Yap pathway has also been implicated in maintaining the epithelial progenitor compartment and regulating growth processes. Thus, whilst some molecules and mechanisms have been identified, further work in this important area is needed to provide a clearer understanding of how lens size and shape is regulated.

Relationships between transepidermal water loss, cutaneous microcirculatory function and autonomic nervous activity.

OBJECTIVE: Several studies have shown that a deterioration of skin properties, an impaired cutaneous microcirculatory function and an imbalance of autonomic nervous activity are observed in smokers and in patients with diabetes mellitus or Raynaud's phenomenon. These observations suggest that skin properties are associated with cutaneous microcirculatory function and autonomic nervous activity in pathological conditions. However, there is no published evidence to support the concept that these two functions have any relationship with skin properties even in healthy subjects. To investigate the hypothesis that these properties are related, we conducted a survey of healthy adult subjects to investigate the relationships between cutaneous microcirculatory function and autonomic nervous activity and skin properties. METHODS: The hydration of the stratum corneum and transepidermal water loss (TEWL) were investigated as skin properties, and the responsiveness of skin blood flow (SkBF) to local warming was examined as an index of cutaneous microcirculatory function in 19 healthy adult male subjects. Electrocardiograms were monitored for 24 h and heart rate variability was analysed considering low-frequency power (LF: 0.04-0.15 Hz), high-frequency power (HF: 0.15-0.40 Hz) and a ratio of low- to high-frequency power (LF/HF) as indices of autonomic nervous activity; HF is an index of parasympathetic activity, whereas LF/HF is an index of sympathovagal balance. The relationships between those indices were then analysed. RESULTS: A moderate negative correlation was found between TEWL and the relative maximum rate of increases in the responsiveness of SkBF on local warming. A moderate positive and a moderate negative correlation were observed between TEWL and LF/HF or HF, respectively. Moreover, a moderate negative and a moderate positive correlation were shown between the responsiveness of SkBF and LF/HF or HF, respectively. The hydration of the stratum corneum showed no correlations with any indices of microcirculation or autonomic nervous activity. CONCLUSION: These results indicate that skin barrier function, cutaneous microcirculatory function and autonomic nervous activity are mutually associated in healthy adults.

Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

Interactions between lens epithelial and fiber cells reveal an intrinsic self-assembly mechanism.

How tissues and organs develop and maintain their characteristic three-dimensional cellular architecture is often a poorly understood part of their developmental program; yet, as is clearly the case for the eye lens, precise regulation of these features can be critical for function. During lens morphogenesis cells become organized into a polarized, spheroidal structure with a monolayer of epithelial cells overlying the apical tips of elongated fiber cells. Epithelial cells proliferate and progeny that shift below the lens equator differentiate into new fibers that are progressively added to the fiber mass. It is now known that FGF induces epithelial to fiber differentiation; however, it is not fully understood how these two forms of cells assemble into their characteristic polarized arrangement. Here we show that in FGF-treated epithelial explants, elongating fibers become polarized/oriented towards islands of epithelial cells and mimic their polarized arrangement in vivo. Epithelial explants secrete Wnt5 into the culture medium and we show that Wnt5 can promote directed behavior of lens cells. We also show that these explants replicate aspects of the Notch/Jagged signaling activity that has been shown to regulate proliferation of epithelial cells in vivo. Thus, our in vitro study identifies a novel mechanism, intrinsic to the two forms of lens cells, that facilitates self-assembly into the polarized arrangement characteristic of the lens in vivo. In this way the lens, with its relatively simple cellular composition, serves as a useful model to highlight the importance of such intrinsic self-assembly mechanisms in tissue developmental and regenerative processes.

A new objective histological scale for studying human photoaged skin.

BACKGROUND: A quantitative understanding of the histological alteration of the skin is important for assessing the severity of photoaging. METHODS: We performed Elastica-van Gieson staining and immunohistochemistry for decorin on 34 facial skin sections. We evaluated the alteration of collagen fibers and decorin (a modulator for collagen fibrillogenesis), according to the 5 grades of morphological change in elastic fibers that was established by Kligman (1969). The objectivity of a stage (Stages I-VI), which was established in this study, was evaluated using weighted kappa statistical analysis based on the degree of agreement in stage determination by 11 observers using a blind procedure. Correlation between the crow's-feet-area wrinkles grades of another 26 women and stages was also analyzed. RESULTS: The initial alteration of elastic fibers was observed in the deep dermis. Decorin was not detected in very severely altered skin. Based on the combination of changes in the elastic fibers, collagenic fibers, and decorin, skin tissues were categorized into 6 stages according to severity. The statistical analysis showed almost perfect agreement between observers. Significant positive correlation between stages and wrinkle scores was found. CONCLUSIONS: We propose a new objective histological scale that is useful for assessing the severity of photoaging.

Activation of TRPV4 strengthens the tight-junction barrier in human epidermal keratinocytes.

The transient receptor potential cation channel, subfamily V (TRPV), is expressed in the epidermis and considered to be a sensor of extrinsic stimuli such as temperature and other physical or chemical factors. In this study, we examined whether or not the activation of TRPVs by their agonists alters the epidermal tight junction (TJ) function in cultured human epidermal keratinocytes. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that mRNA for TRPV1, 3 and 4 were expressed in differentiated keratinocytes in which TJs had formed. Stimulation of the keratinocytes with a TRPV4 agonist (4α-phorbol 12, 13-didecanoate, 4α-PDD) strengthened the TJ-associated barrier, analyzed by means of transepithelial electric resistance measurements and flux measurements of the paracellular tracer. Stimulation with TRPV1 and TRPV3 agonists did not have the same result. Simultaneously, the 4α-PDD-stimulated keratinocytes showed an upregulation of TJ structural proteins, occludin and claudin-4, and TJ regulatory factors, phospho-atypical PKCζ/ι. It was also observed that the amounts of occludin and phospho-atypical PKCζ/ι complex were higher in 4α-PDD stimulated keratinocytes. In conclusion, we demonstrated that the activation of TRPV4 strengthened the TJ-associated barrier of epidermal cells. It was also suggested that the upregulation of TJ structural proteins and/or the posttranslational modification of TJ structural proteins by phospho-atypical PKCζ/ι are responsible for the enhancement of TJ function. Our study supports the hypothesis that TJs change their function in response to a change in the external environment sensed through TRPVs.

Potential responders to FOLFOX therapy for colorectal cancer by Random Forests analysis.

BACKGROUND: Molecular characterisation using gene-expression profiling will undoubtedly improve the prediction of treatment responses, and ultimately, the clinical outcome of cancer patients. METHODS: To establish the procedures to identify responders to FOLFOX therapy, 83 colorectal cancer (CRC) patients including 42 responders and 41 non-responders were divided into training (54 patients) and test (29 patients) sets. Using Random Forests (RF) algorithm in the training set, predictor genes for FOLFOX therapy were identified, which were applied to test samples and sensitivity, specificity, and out-of-bag classification accuracy were calculated. RESULTS: In the training set, 22 of 27 responders (81.4% sensitivity) and 23 of 27 non-responders (85.1% specificity) were correctly classified. To improve the prediction model, we removed the outliers determined by RF, and the model could correctly classify 21 of 23 responders (91.3%) and 22 of 23 non-responders (95.6%) in the training set, and 80.0% sensitivity and 92.8% specificity, with an accuracy of 69.2% in 29 independent test samples. CONCLUSION: Random Forests on gene-expression data for CRC patients was effectively able to stratify responders to FOLFOX therapy with high accuracy, and use of pharmacogenomics in anticancer therapy is the first step in planning personalised therapy.

Analysis of regulatory review times of new drugs in Japan: association with characteristics of new drug applications, regulatory agency, and pharmaceutical companies.

WHAT IS KNOWN AND OBJECTIVE: Various factors have been reported to be associated with the duration of regulatory review of new drug applications (NDAs). We investigated potential links between the review times in Japan and the attributes of NDAs, the regulatory agency and pharmaceutical companies. METHODS: We analysed new drugs approved in 2000-2009 in Japan using a proprietary database collected through annual surveys to pharmaceutical companies. Regression models in which individual firms were treated as either a fixed effect or a random effect were applied to examine factors associated with the overall review time and the duration of each step of the review. RESULTS AND DISCUSSION: The fixed effect model analysis using variations within each firm indicated that new molecular entities that were submitted to the Pharmaceuticals and Medical Devices Agency (PMDA), priority reviews and pre-NDA consultations were associated with a shorter overall review time, whereas additional studies during the review resulted in a longer review. In the random effect model analysis using both within- and between-firm variations, use of end-of-phase 2 consultations and foreign clinical data also had negative coefficients, suggesting the effect of these two vary among firms. Analysis of each step of the review process revealed NDAs reviewed by the Committee on Drugs under the Ministry of Health, Labour and Welfare, and the number of NDAs assigned to a review team were significantly linked with their duration, whereas consultation services and the number of reviewers had no relation. WHAT IS NEW AND CONCLUSION: Factors associated with each step of the review process as well as the differences in attributes and strategies among pharmaceutical companies should be considered to further improve the speed, quality and efficiency of the regulatory review.

Effects of intermittent Pringle's manoeuvre on cirrhotic compared with normal liver.

BACKGROUND: Although patients with liver cirrhosis are supposed to tolerate ischaemia-reperfusion poorly, the exact impact of intermittent inflow clamping during hepatic resection of cirrhotic compared with normal liver remains unclear. METHODS: Intermittent Pringle's manoeuvre was applied during minor hepatectomy in 172 patients with a normal liver, 59 with chronic hepatitis and 97 with liver cirrhosis. To assess hepatic injury, delta (D)-aspartate aminotransferase (AST) and D-alanine aminotransferase (ALT) (maximum level minus preoperative level) were calculated. To evaluate postoperative liver function, postoperative levels of total bilirubin, albumin and cholinesterase (ChE), and prothrombin time were measured. RESULTS: Significant correlations between D-AST or D-ALT and clamping time were found in each group. The regression coefficients of the regression lines for D-AST and D-ALT in patients with normal liver were significantly higher than those in patients with cirrhotic liver. Irrespective of whether clamping time was 45 min or less, or at least 60 min, D-AST and D-ALT were significantly lower in patients with cirrhosis than in those with a normal liver. Parameters of hepatic functional reserve, such as total bilirubin, prothrombin time, albumin and ChE, were impaired significantly after surgery in patients with a cirrhotic liver. CONCLUSION: Patients with liver cirrhosis had a smaller increase in aminotransferase levels following portal triad clamping than those with a normal liver. However, hepatic functional reserve in those with a cirrhotic liver seemed to be affected more after intermittent inflow occlusion.

Microdose study of a P-glycoprotein substrate, fexofenadine, using a non-radioisotope-labelled drug and LC/MS/MS.

OBJECTIVE: Fexofenadine is a P-glycoprotein substrate of low bioavailability. It is primarily excreted into faeces as a parent drug via biliary excretion. The predictability from microdose data for the drug absorbed via transporters such as P-glycoprotein is not known. Therefore, this study assessed the predictability of therapeutic-dose pharmacokinetics of fexofenadine from microdosing data using non-radioisotope-labelled drug and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). METHOD: In a single dose, randomized, two-way crossover study, eight subjects received a microdose (100 microg) or a therapeutic dose (60 mg) of fexofenadine. Blood samples were collected until 12 h after dosing, and assayed using LC/MS/MS. RESULTS: Plasma concentration-time curves of fexofenadine between microdose and therapeutic dose were similar. The mean +/- SD of C(max) normalized to 60 mg dose after microdose and therapeutic dose were 379 +/- 147 and 275 +/- 145 ng/mL respectively. The mean AUC(last) normalized to 60 mg dose after microdose and therapeutic dose were 1914 +/- 738 and 1431 +/- 432 ng/h/mL respectively. The mean dose-adjusted C(max) and AUC(last) after microdose were higher compared with those after therapeutic dose. Individual plots of C(max) and AUC(last) normalized to 60 mg dose, were similar for microdose and therapeutic dose. None of the pharmacokinetic parameters were statistically different using anova. Overall, the microdose pharmacokinetics profile was similar to, and hence predictive of, that of the therapeutic dose. CONCLUSION: For the P-glycoprotein substrate fexofenadine, the predictability of therapeutic-dose pharmacokinetics from microdose data was good. A microdose study using a non-radioisotope-labelled drug and LC/MS/MS is convenient, and has the potential to aid the early selection of drug candidates.

Interleukin-1 receptor-related protein ST2 suppresses the initial stage of bleomycin-induced lung injury.

Acute lung injury has a range of causes, and occasionally leads to lethal respiratory failure. Despite advances in treatment, acute lung injury continues to have a high mortality rate, and thus a new therapeutic approach is needed. ST2 is an interleukin (IL)-1 receptor-related protein, and its expression is induced by various inflammatory responses. Recently, ST2 has been speculated to exert anti-inflammatory effects; therefore, we investigated the role of the ST2 in the murine model of acute lung injury. To elucidate the function of ST2 in vivo, mice that transiently overexpressed ST2 protein were prepared using the hydrodynamic gene transfer method, and lung injury was induced by intratracheal administration of bleomycin. In bleomycin-treated ST2-overexpressing mice, the increase of neutrophils in the bronchoalveolar lavage fluid (BALF) was markedly suppressed. Additionally, the levels of tumour necrosis factor-alpha and IL-6, as well as the concentration of albumin, in BALF were reduced compared with those of controls. Furthermore, the pulmonary architecture in ST2-overexpressing mice remained almost normal, and the survival rate was significantly improved. From these results, we concluded that ST2 has the potential to suppress the initial stage of acute lung injury, and therefore it may be a useful reagent for the treatment of acute lung injury.

A novel integrin-linked kinase-binding protein, affixin, is involved in the early stage of cell-substrate interaction.

Focal adhesions (FAs) are essential structures for cell adhesion, migration, and morphogenesis. Integrin-linked kinase (ILK), which is capable of interacting with the cytoplasmic domain of beta1 integrin, seems to be a key component of FAs, but its exact role in cell-substrate interaction remains to be clarified. Here, we identified a novel ILK-binding protein, affixin, that consists of two tandem calponin homology domains. In CHOcells, affixin and ILK colocalize at FAs and at the tip of the leading edge, whereas in skeletal muscle cells they colocalize at the sarcolemma where cells attach to the basal lamina, showing a striped pattern corresponding to cytoplasmic Z-band striation. When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia. Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers. The coexpression of ILK enhances this effect. These results provide evidence suggesting that affixin is involved in integrin-ILK signaling required for the establishment of cell-substrate adhesion.

MKBP, a novel member of the small heat shock protein family, binds and activates the myotonic dystrophy protein kinase.

Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as alphaB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by alphaB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM.

Method for predicting the risk of drug-drug interactions involving inhibition of intestinal CYP3A4 and P-glycoprotein.

To develop a method to predict the risk of drug-drug interactions involving the inhibition of intestinal CYP3A4 or P-glycoprotein, data from clinical drug-drug interaction studies of CYP3A4 and/or P-glycoprotein substrates were analysed. The ratio of inhibitor dose (Dose(i)) to inhibition constant (K(i)), termed the drug-interaction number, was used to index intestinal drug-drug interaction. From the analysis, it was found that (1) CYP3A4 inhibitors with a drug-interaction number below 2.8 L have a low risk of interacting with substrates which exhibit intestinal first-pass metabolism and those with a drug-interaction number above 9.4 L have a high risk; (2) P-glycoprotein inhibitors with a drug-interaction number below 10.8 L have a low risk of interacting with P-glycoprotein substrates and those with a drug-interaction number above 27.9 L have a high risk; and (3) the drug-interaction number indexes, 2.8 L and 9.4 L for CYP3A4 and 10.8 L and 27.9 L for P-glycoprotein were validated by data from dual CYP3A4/P-glycoprotein substrates. In conclusion, the drug-interaction number is useful for classifying the risk of drug-drug interactions involving the inhibition of intestinal CYP3A4 and P-glycoprotein. This drug-interaction number-based approach is similar to the method that the US Food and Drug Administration (USFDA) recently proposed in the draft guidance for predicting P-glycoprotein-mediated drug-drug interaction.

Canine oocyte maturation in culture: significance of estrogen and EGF receptor gene expression in cumulus cells.

We examined the role of cumulus cells regarding in vitro maturation of canine oocytes, and investigated estrogen and epidermal growth factor (EGF) receptor gene expression and action on nuclear maturation. Canine cumulus-oocyte complexes (COC) were collected from anestrous and diestrous bitches; only COC with vitelline diameter >100 microm were used. In Experiment 1, expression of estrogen receptor (ER) alpha, ERbeta and EGF-receptor (EGF-R) were determined by reverse transcription-polymerase chain reaction (RT-PCR), using mRNA from the oocyte or cumulus cell. Transcripts for the ERbeta and EGF-R were detected in oocytes and cumulus cells, but no message was detected for ERalpha. In Experiment 2, intact COC and the denuded oocytes were cultured in TCM199 medium supplemented with various concentrations of estradiol-17beta (E(2); 0-10 microg/mL) or EGF (0-100 ng/mL) for 72 h; nuclear maturation was then evaluated. In oocytes cultured within intact COC, the rate of germinal vesicle breakdown (GVBD) was higher in the 1 microg/mL E(2) supplemented group (P<0.05), and the rate of metaphase I (MI) was higher in the 10 ng/mL EGF supplemented group, than in the non-supplemented group (P<0.05). However, supplementation of E(2) or EGF to denuded oocytes failed to promote nuclear maturation. In Experiment 3, intact COC were cultured in TCM199 supplemented with 1 microg/mL E(2), 10 ng/mL EGF, and 10% fetal bovine serum (FBS) for 72 h, and nuclear maturation was evaluated. There was no significant difference in the rate of metaphase II (MII) between the medium only, E(2)+EGF, and FBS supplement groups. When E(2) and EGF in combination with FBS were supplemented, the rate of MII was higher than in other groups (P<0.05). We inferred that cumulus cells were involved in mediating the stimulatory effects of E(2) and EGF on nuclear maturation of canine oocytes, and that E(2) and EGF in combination with FBS promoted the completion of oocyte meiotic maturation.

Effects of pharmacological lesion of the nucleus retroambiguus region on the pharyngeal phase of swallowing.

Pharyngeal swallowing is controlled by synaptic interactions within a swallowing central pattern generator (sw-CPG) that is composed of a dorsal and a ventral swallowing group (VSG). Here, we used electrical stimulation (10 s) of the superior laryngeal nerve (SLN; 20 Hz; pulse width: 100 µs) to explore the role of the VSG in an arterially-perfused brainstem preparation of rats. To investigate the effects of pharmacological lesion (local microinjection of an GABA(A)-R agonist) of the nucleus retroambiguus (NRA), a designated component of the VSG, we recorded phrenic (PNA) and vagal nerve (VNA) activities. Control SLN stimulation with stepwise increasing stimulus intensities (from 20 µA to 160 µA) elicited robust suppression of PNA and evoked sequential swallowing activity in the VNA. Lesioning of the NRA had no effect on the pattern of pharyngeal swallowing, but significantly increased the sensory gating of SLN inputs. We conclude that the NRA is not part of the VSG, but appears to have important roles for the central gating of swallowing.

Effects of edaravone, a free-radical scavenger, on bleomycin-induced lung injury in mice.

Reactive oxygen species play an important role in the pathogenesis of acute lung injury and pulmonary fibrosis. The present authors hypothesise that edaravone, a free-radical scavenger, is able to attenuate bleomycin (BLM)-induced lung injury in mice by decreasing oxidative stress. Lung injury was induced in female ICR mice by intratracheal instillation of 5 mg x kg(-1) of BLM. Edaravone (300 mg x kg(-1)) was administered by intraperitoneal administration 1 h before BLM challenge. Edaravone significantly improved the survival rate of mice treated with BLM from 25 to 90%, reduced the number of total cells and neutrophils in bronchoalveolar lavage fluid (BALF) on day 7, and attenuated the concentrations of lipid hydroperoxide in BALF and serum on day 2. The fibrotic change in the lung on day 28 was ameliorated by edaravone, as evaluated by histological examination and measurement of hydroxyproline contents. In addition, edaravone significantly increased the prostaglandin E(2) concentration in BALF on day 2. In summary, edaravone was shown to inhibit lung injury and fibrosis via the repression of lipid hydroperoxide production and the elevation of prostaglandin E(2) production in the present experimental murine system.

DOPA cyclohexyl ester, a DOPA antagonist, blocks the depressor responses elicited by microinjections of nicotine into the nucleus tractus solitarii of rats.

Nicotinic cholinergic receptors play a role in cardiovascular regulation in the lower brain stem. Herein, we present evidence that l-3,4-dihydroxyphenylalanine (DOPA), a putative neurotransmitter in the central nervous system, is involved in the depressor response to microinjection of nicotine into the nucleus tractus solitarii (NTS). Microinjection of nicotine into the medial area of the NTS led to decreases in arterial blood pressure and heart rate in anesthetized rats. Mecamylamine, a nicotinic receptor antagonist, microinjected into NTS, blocked the depressor and bradycardic responses to nicotine. Nicotine-induced depressor and bradycardic responses were blocked by DOPA cyclohexyl ester (DOPA CHE), an antagonist for DOPA. DOPA CHE did not modify the action of carbachol on excitatory postsynaptic potential in rat cortical slices. These results suggest that endogenous DOPA is involved in nicotine-induced depressor responses in the NTS of anesthetized rats.

Misreporting of dietary energy, protein, potassium and sodium in relation to body mass index in young Japanese women.

OBJECTIVE: Although under-reporting of dietary intake is more common in persons with a high body mass index (BMI), it is not well known whether or not misreporting is selective for different foods (and hence energy and nutrients), particularly in non-Western populations. We examined misreporting of dietary intake against biomarkers and its relation with BMI in young Japanese women. DESIGN: Cross-sectional study. SUBJECTS: A total of 353 female Japanese dietetic students aged 18-22 years (mean BMI: 21.4 kg/m(2), mean fat intake: 29.8% of energy). METHODS: Misreporting of dietary energy, protein, potassium and sodium (assessed by a self-administered diet history questionnaire) was examined against respective biomarkers (estimated energy expenditure and 24-h urinary excretion). Reporting accuracy was calculated as the ratio of reported intake to that estimated from corresponding biomarkers (complete accuracy: 1.00). RESULTS: Mean reporting accuracy of absolute intake (amount per day) varied considerably (0.86-1.14). Reporting accuracy of absolute intake decreased with increasing BMI (P for trend <0.001). However, no association was observed between reporting accuracy of energy-adjusted values and BMI (P for trend >0.15), indicating that BMI-dependent misreporting was canceled by energy adjustment. This was owing to positive correlation between the reporting accuracy of energy intake and that of absolute intake of the three nutrients (Pearson correlation coefficient: 0.49-0.67, P<0.0001). CONCLUSIONS: Although differential misreporting of absolute intake was associated with BMI, differential misreporting of energy-adjusted value was not. These findings support the use of energy-adjusted values in the investigation of diet-disease relationships among lean populations with a low-fat intake.