Expression levels of drug-metabolizing enzyme, transporter, and nuclear receptor mRNAs in a novel three-dimensional culture system for human hepatocytes using micro-space plates

We evaluated a novel three-dimensional primary culture system using micro-space plates to determine the expression levels of 61 target (drug-metabolizing enzymes, transporters, and nuclear receptors) mRNAs in human hepatocytes. We measured mRNA expression levels of many target genes in four lots of cryopreserved human hepatocyte primary cells after 120 h of culture and compared differences in mRNA expression levels between cultures using traditional plates and those using micro-space plates. In this study, we show that the mRNA levels of many experimental targets in human hepatocytes before inoculation resemble the levels inside the human liver. Furthermore, we show that the rate of change of expression levels of many target mRNAs relative to the value before inoculation of the hepatocytes into micro-space plates was relatively smaller than the rate of change in hepatocytes inoculated into traditional plates. Pharmacokinetics-related examinations using this system are possible within a time frame of 120 h. We report that this novel three-dimensional culture system reproduces mRNA expression levels that are nearer to those in the liver in vivo and is an excellent platform for maintaining mRNA expression levels of drug-metabolizing enzymes and transporters when compared to common monolayer cultures.     

High-level expression of podoplanin in benign and malignant soft tissue tumors: immunohistochemical and quantitative real-time RT-PCR analysis

Podoplanin is a 38 kDa mucin-type transmembrane glycoprotein that was first identified in rat glomerular epithelial cells (podocytes). It is expressed in normal lymphatic endothelium, but is absent from vascular endothelial cells. D2-40 is a commercially available mouse monoclonal antibody which binds to an epitope on human podoplanin. D2-40 immunoreactivity is therefore highly sensitive and specific for lymphatic endothelium. Recent investigations have shown widespread applications of immunohistochemical staining with D2-40 in evaluating podoplanin expression as an immunohistochemical marker for diagnosis and prognosis in various tumors. To determine whether the podoplanin (D2-40) antibody may be useful for the diagnosis of soft tissue tumors, 125 cases, including 4 kinds of benign tumors, 15 kinds of malignant tumors and 3 kinds of tumor-like lesions were immunostained using the D2-40 antibody. Total RNA was extracted from frozen tumor tissue obtained from 41 corresponding soft tissue tumor patients and 12 kinds of soft tissue tumor cell lines. Quantitative real-time PCR reactions were performed. Immunohistochemical and quantitative real-time RT-PCR analyses demonstrated the expression of the podoplanin protein and mRNA in the majority of benign and malignant soft tissue tumors and tumor-like lesions examined, with the exception of alveolar soft part sarcoma, embryonal and alveolar rhabdomyosarcoma, extraskeletal Ewing's sarcoma/peripheral primitive neuro-ectodermal tumor and lipoma, which were completely negative for podoplanin. Since it is widely and highly expressed in nearly all kinds of soft tissue tumors, especially in spindle cell sarcoma, myxoid type soft tissue tumors and soft tissue tumors of the nervous system, podoplanin is considered to have little value in the differential diagnosis of soft tissue tumors.     

Bactericidal effects of 2.94 µm and 1.67 µm laser

The bactericidal effects of lasers with wavelengths of 1.67 and 2.94 μm on cariogenic Streptococcus mutans were investigated. Temperature during irradiation was also measured to determine the mechanism underlying the bactericidal effects of the lasers. An aliquot of 2 μl cell suspension of S. mutans JC-2 strain was placed on anhydrous quartz or dentin plate, covering an area of approximately 5.0 mm in diameter to a depth of approximately 0.1 mm. Cell suspension was then irradiated at a power of 0.8 W (3.1 J/cm2) at a rate of 40 pps for 30 sec. After irradiation, the plate was put into a bottle containing PBS and vigorously voltated. Solution was serially diluted and inoculated on MS agar. After incubation anaerobically for 72 hr, colony forming units on the agar were counted. The experimental group, the number of bacteria decreased significantly compared to the control group under all conditions. No significant differences were observed in effect of wavelength or plate on bactericidal activity. In conclusion, laser irradiation at a wavelength of 1.67 μm for 30 sec showed a bactericidal effect on S. mutans, suggesting that this wavelength is more useful than 2.94 μm due to greater tissue penetration.     

Remnant lipoprotein metabolism is improved more when body weight is reduced by exercise than by dietary restriction

BACKGROUND: Remnant lipoprotein levels during weight-loss programs have not been examined in detail. We investigated the effects of reducing body weight by exercise and by dietary restriction on lipid metabolism including remnant-like particle-cholesterol (RLP-C) and apolipoprotein B48 (apo B48). METHODS: Six adult male volunteers participated in 2 experiments over 10 days. The first 5 days were an adjustment period (energy intake, 2656+/-150 kcal/day; mean+/-SEM). During the second 5 days, the participants either exercised on a bicycle ergometer to consume 886+/-50 kcal/day or reduced their energy intake to 1770+/-100 kcal/day. RESULTS: Serum triglyceride (TG) and RLP-C levels were significantly lower, whereas the increase in high density lipoprotein-cholesterol (HDL-C) level was significantly larger after exercise than after dietary restriction. We measured changes (Delta) in serum levels and identified positive correlations between Delta insulin and Delta TG, Delta apo B48 (p<0.05) and Delta RLP-C (p=0.050) only during exercise. CONCLUSIONS: Reducing body weight by exercise has more beneficial effects on lipid metabolism than a dietary approach, and improved insulin resistance accompanies the decrease of serum remnant lipoprotein levels.     

A novel beta-myosin heavy chain gene mutation, p.Met531Arg, identified in isolated left ventricular non-compaction in humans, results in left ventricular hypertrophy that progresses to dilation in a mouse model

Mutations in the betaMHC (beta-myosin heavy chain), a sarcomeric protein are responsible for hypertrophic and dilated cardiomyopathy. However, the mechanisms whereby distinct mutations in the betaMHC gene cause two kinds of cardiomyopathy are still unclear. In the present study we report a novel betaMHC mutation found in a patient with isolated LVNC [LV (left ventricular) non-compaction] and the phenotype of a mouse mutant model carrying the same mutation. To find the mutation responsible, we searched for genomic mutations in 99 unrelated probands with dilated cardiomyopathy and five probands with isolated LVNC, and identified a p.Met531Arg mutation in betaMHC in a 13-year-old girl with isolated LVNC. Next, we generated six lines of transgenic mice carrying a p.Met532Arg mutant alphaMHC gene, which was identical with the p.Met531Arg mutation in the human betaMHC. Among these, two lines with strong expression of the mutant alphaMHC gene were chosen for further studies. Although they did not exhibit the features characteristic of LVNC, approx. 50% and 70% of transgenic mice in each line displayed LVH (LV hypertrophy) by 2-3 months of age. Furthermore, LVD (LV dilation) developed in approx. 25% of transgenic mice by 18 months of age, demonstrating biphasic changes in LV wall thickness. The present study supports the idea that common mechanisms may be involved in LVH and LVD. The novel mouse model generated can provide important information for the understanding of the pathological processes and aetiology of cardiac dilation in humans.     

Association between sleep duration and hemoglobin A1c level

ObjectiveThe association between sleep habits and hemoglobin A_1c (HbA_1c) level has not been sufficiently examined. In the present study of residents in a local community, the associations between sleep duration and HbA_1c level were examined.MethodsSelf-administered questionnaires were mailed to 1062 residents in a rural community in Japan, and completed questionnaires were collected. At the time of collection, the fasting plasma glucose and HbA_1c levels were measured using peripheral blood samples. For the analyses, values that were considered to represent high levels were a fasting plasma glucose level of ⩾126 mg/dl and a HbA_1c level of ⩾6.5%. Logistic regression analyses were performed to examine the associations between sleep duration and high fasting plasma glucose or high HbA_1c levels.ResultsThe prevalence of high fasting plasma glucose and high HbA_1c levels was significantly high (p < 0.01) in subjects with a short or a long sleep duration. Logistic regression analyses demonstrated a significant association between high HbA_1c level and sleep duration. The adjusted odds ratios for a high HbA_1c level showed high values with regard to both short and long sleep durations.ConclusionsHbA_1c level showed a U-shaped association with sleep duration. These results suggest that there may be an appropriate range of sleep duration in individuals with glucose tolerance disorders. It is expected that the present findings will contribute to the treatment and prevention of diabetes mellitus.     

Frequency features of mechanomyographic signals of human soleus muscle during quiet standing

The purpose of the present study was to determine whether the frequency features of the MMG signals during quiet standing reflect body sway as well as recurring muscle activity. Twenty healthy men maintained quiet standing in a barefoot position with their eyes open or closed. During quiet standing, MMG detected using uniaxial piezoresistive accelerometer and surface electromyogram (EMG) signals were recorded from the soleus (SOL) muscle, and the center of mass (CoM) displacement (CoMdis) in the anteroposterior direction was measured by a high-resolution laser displacement sensor. In addition, CoMdis was time-differentiated to yield CoM velocity (CoMvel). Cross-spectral analysis revealed that significant coherency spectra from MMG to CoMdis and from MMG to rectified EMG of SOL were observed below 2Hz and 8-12Hz frequency band, respectively. Furthermore, we revealed that the trajectories of MMG and the calculated dMMG/dt were significantly correlated to CoMdis and CoMvel, respectively. These results suggest that kinematic and physiological parameters of postural control during quiet standing can be quantified by frequency features of the MMG.     

The relationship between passive ankle plantar flexion joint torque and gastrocnemius muscle and achilles tendon stiffness: implications for flexibility

STUDY DESIGN: Experimental laboratory study. OBJECTIVES: We tested the hypothesis that the muscle fibers and the connective tendinous structures, combined in series, provide the resistance to passive joint movement at the ankle. We also determined the relative association between passive joint torque and each of these 2 elements. BACKGROUND: The reason for individual variation in joint flexibility or tightness is not clearly understood, but the influence of musculotendinous stiffness has been inferred. METHODS AND MEASURES: Each of the subjects (6 women and 6 men) was seated with the right knee extended and right ankle positioned at a 30 degrees , 20 degrees , 10 degrees , 0 degrees , -10 degrees , -20 degrees , and -30 degrees (0, neutral position, positive values reflecting plantar flexion) angle while passive plantar flexion torque was measured. The distal muscle-tendon junction of the medial gastrocnemius was visualized by ultrasonography, and its positional change was defined as muscle belly length change. The whole muscle-tendon unit length change was estimated from joint angle changes, from which Achilles tendon length change was estimated. RESULTS: Both the muscle belly and tendon were significantly elongated as the ankle was dorsiflexed (at 0 degrees the mean +/- SD muscle belly elongation was 10.3% +/- 1.8 %, and the tendon elongation was 2.8% +/- 1.2 %, of the initial length at 30 degrees of ankle plantar flexion), from which stiffness indices were determined both for muscle belly and tendon. The passive torque at 0 degrees , -10 degrees , -20 degrees , and -30 degrees was significantly correlated with the stiffness indices of the Achilles tendon (at 0 degrees , r2 = 0.70 and 0.62 for overall and specific stiffness, respectively; P<.05). A tendon stiffness index, separately obtained from tendon lengthening during maximal isometric contraction, was also correlated with passive ankle plantar flexion torque at 0 degrees , -10 degrees , -20 degrees , and -30 degrees (at 0 degrees , r2 = 0.76; P<.05). The specific stiffness index of the muscle belly was correlated (r2 = 0.47, P<.05) with the passive ankle plantar flexion torque at 0 degrees , but its overall stiffness index was not (r2 = 0.32, P>.05). CONCLUSION: Results suggest that extensibility of the muscle-tendon unit of the Achilles tendon for the most part is related to passive ankle plantar flexion joint torque.