Novel (p)ppGpp0 suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp⁰ strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp⁰ strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp⁰ suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp⁰ backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.     

Irisin/FNDC5: A participant in camel metabolism

The quantification, localization, production, function, and regulation of irisin/FNDC5 in camel species have not been previously studied. The objective of this study was to detect the irisin content in Arabian camel blood and tissues and study the gene expression of FNDC5 and PGC-1α in camel skeletal muscles and white adipose tissue depots under basal conditions. To monitor if exercise influences blood and tissue irisin protein levels as well as FNDC5 and PGC-1α gene expression levels, we analyzed irisin concentrations in the serum, skeletal muscles (soleus and gastrocnemius), and white adipose tissues (hump, subcutaneous, visceral, epididymal, and perirenal) in both control (n = 6) and exercised group (n = 6) using ELISA and determined the cellular localization of irisin/FNDC5 and the mRNA levels of FNDC5 and PGC-1α in skeletal muscles and adipose tissues via immunohistochemistry and real-time PCR, respectively. The possible regulatory roles of exercise on some hormones and metabolites as well as the detection of links between serum irisin and other circulating hormones (insulin, leptin, and cortisol) and metabolites (glucose, free fatty acids, triglycerides, and ATP) were explored for the first time in camels. Our results indicated that exercise induces tissue-specific regulation of the camel irisin, FNDC5, and PGC-1α levels, which subsequently regulates the circulating irisin level. Significant associations were detected between the levels of irisin/FNDC5/PGC-1α in camels and the metabolic and hormonal responses to exercise. Our study suggested that irisin regulates, or is regulated by, glucose, FFA, insulin, leptin, and cortisol in camels. The novel results of the present study will serve as baseline data for camels.     

SLC26A4 Targeted to the Endolymphatic Sac Rescues Hearing and Balance in Slc26a4 Mutant Mice

Mutations of SLC26A4 are a common cause of human hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in a variety of epithelial cells in the cochlea, the vestibular labyrinth and the endolymphatic sac. Slc26a4 ^Δ/Δ mice are devoid of pendrin and develop a severe enlargement of the membranous labyrinth, fail to acquire hearing and balance, and thereby provide a model for the human phenotype. Here, we generated a transgenic mouse line that expresses human SLC26A4 controlled by the promoter of ATP6V1B1. Crossing this transgene into the Slc26a4 ^Δ/Δ line restored protein expression of pendrin in the endolymphatic sac without inducing detectable expression in the cochlea or the vestibular sensory organs. The transgene prevented abnormal enlargement of the membranous labyrinth, restored a normal endocochlear potential, normal pH gradients between endolymph and perilymph in the cochlea, normal otoconia formation in the vestibular labyrinth and normal sensory functions of hearing and balance. Our study demonstrates that restoration of pendrin to the endolymphatic sac is sufficient to restore normal inner ear function. This finding in conjunction with our previous report that pendrin expression is required for embryonic development but not for the maintenance of hearing opens the prospect that a spatially and temporally limited therapy will restore normal hearing in human patients carrying a variety of mutations of SLC26A4.     

Mutation of GDP-Mannose-4,6-Dehydratase in Colorectal Cancer Metastasis

Fucosylation is a crucial oligosaccharide modification in cancer. The known function of fucosylation in cancer is to mediate metastasis through selectin ligand-dependent processes. Previously, we found complete loss of fucosylation in the colon cancer cell line HCT116 due to a mutation in the GDP-fucose synthetic enzyme, GDP-mannose-4,6-dehydratase (GMDS). Loss of fucosylation led to escape of cancer cells from tumor immune surveillance followed by tumor progression and metastasis, suggesting a novel function of fucosylation in tumor progression pathway. In the present study, we investigated the frequency of GMDS mutation in a number of clinical colorectal cancer tissue samples: 81 samples of primary colorectal cancer tissue and 39 samples of metastatic lesion including liver and lymph node. Four types of deletion mutation in GMDS were identified in original cancer tissues as well as metastatic lesions. The frequency of GMDS mutation was slightly higher in metastatic lesions (12.8%, 5/39 samples) than in original cancer tissues (8.6%, 7/81 samples). No mutation of the GMDS gene was observed in normal colon tissues surrounding cancer tissues, suggesting that the mutation is somatic rather than in the germline. Immunohistochemical analysis revealed complete loss of fucosylation in three cases of cancer tissue. All three cases had GMDS mutation. In one of three cases, loss of fucosylation was observed in only metastatic lesion, but not its original colon cancer tissue. These data demonstrate involvement of GMDS mutation in the progression of colorectal cancer.     

Yamogenin in fenugreek inhibits lipid accumulation through the suppression of gene expression in fatty acid synthesis in hepatocytes

Yamogenin is a diastereomer of diosgenin, which we have identified as the compound responsible for the anti-hyperlipidemic effect of fenugreek. Here, we examined the effects of yamogenin on the accumulation of triacylglyceride (TG) in hepatocytes, because yamogenin is also contained in fenugreek. It was demonstrated that yamogenin also inhibited TG accumulation in HepG2 hepatocytes and suppressed the mRNA expression of fatty acid synthesis-related genes such as fatty acid synthase and sterol response element-binding protein-1c. Indeed, yamogenin also antagonized the activation of the liver X receptor (LXR) in luciferase ligand assay similar to diosgenin. However, yamogenin could not exert such effects in the presence of T0901713, a potent agonist of LXR. These findings indicate that the effects of yamogenin on TG accumulation would be weaker than those of diosgenin, suggesting that the structural difference between yamogenin and diosgenin would be important for the inhibition of LXR activation.     

Validity of ultrasound muscle thickness measurements for predicting leg skeletal muscle mass in healthy Japanese middle-aged and older individuals

Background

The skeletal muscle mass of the lower limb plays a role in its mobility during daily life. From the perspective of physical resources, leg muscle mass dominantly decreases after the end of the fifth decade. Therefore, an accurate estimate of the muscle mass is important for the middle-aged and older population. The present study aimed to clarify the validity of ultrasound muscle thickness (MT) measurements for predicting leg skeletal muscle mass (SM) in the healthy Japanese middle-aged and older population.

Findings

MTs at four sites of the lower limb and the bone-free lean tissue mass (LTM) of the right leg were determined using brightness-mode ultrasonography and dual-energy X-ray absorptiometry (DXA), respectively, in 44 women and 33 men, 52- to 78-years old. LTM was used as a representative variable of leg skeletal muscle mass. In the model-development group (30 women and 22 men), regression analysis produced an equation with R² and standard error of the estimate (SEE) of 0.958 and 0.3 kg, respectively: LTM (kg) = 0.01464 × (MT_SUM×L) (cm²) - 2.767, where MT_SUM is the sum of the product of MTs at four sites, and L is length of segment where MT is determined. The estimated LTM (7.0 ± 1.7 kg) did not significantly differ from the measured LTM (7.0 ± 1.7 kg), without a significant systematic error on a Bland-Altman plot. The application of this equation for the cross-validation group (14 women and 11 men) did not yield a significant difference between the measured (7.2 ± 1.6 kg) or estimated (7.2 ± 1.6 kg) LTM and systematic error.

Conclusion

The developed prediction equation may be useful for estimating the lean tissue mass of the lower extremity for the healthy Japanese middle-aged and older population.     

Impact of Robotic Assistance on Precision of Vitreoretinal Surgical Procedures

Purpose

To elucidate the merits of robotic application for vitreoretinal maneuver in comparison to conventional manual performance using an in-vitro eye model constructed for the present study.

Methods

Capability to accurately approach the target on the fundus, to stabilize the manipulator tip just above the fundus, and to perceive the contact of the manipulator tip with the fundus were tested. The accuracies were compared between the robotic and manual control, as well as between ophthalmologists and engineering students.

Results

In case of manual control, ophthalmologists were superior to engineering students in all the 3 test procedures. Robotic assistance significantly improved accuracy of all the test procedures performed by engineering students. For the ophthalmologists including a specialist of vitreoretinal surgery, robotic assistance enhanced the accuracy in the stabilization of manipulator tip (from 90.9 µm to 14.9 µm, P = 0.0006) and the perception of contact with the fundus (from 20.0 mN to 7.84 mN, P = 0.046), while robotic assistance did not improve pointing accuracy.

Conclusions

It was confirmed that telerobotic assistance has a potential to significantly improve precision in vitreoretinal procedures in both experienced and inexperienced hands.     

Lung Surfactant Levels are Regulated by Ig-Hepta/GPR116 by Monitoring Surfactant Protein D

Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta^+/+ and Ig-Hepta^−/− mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i) balanced synthesis of surfactant lipids and proteins and (ii) surfactant secretion, and (iii) a stimulating effect on recycling (uptake) in response to elevated levels of Sp-D in alveolar space.     

Association between Smoking Status and Obesity in a Nationwide Survey of Japanese Adults

Objective

A positive association between the number of cigarettes smoked per day and obesity has been reported, whereas how other smoking-related indices, such as pack-years and duration of smoking, are related with obesity has been less investigated. We analyzed the age-adjusted cross-sectional association between smoking and obesity in a general Japanese population.

Methods

We used data from a nationwide epidemiological study of Japanese adults (N = 23,106). We compared the prevalence of obesity (defined as body mass index ≥ 25kg/m²) among groups classified by smoking behavior, pack-years, number of cigarettes per day, duration of smoking, and duration and time of smoking cessation.

Results

In men, current smokers had a lower odds ratio (OR) for obesity of 0.80 (95% confidence interval (CI): 0.72–0.88) compared to non-smokers, whereas past smokers had a higher OR of 1.23 (95% CI: 1.09–1.37) compared to current smokers. In women, there were no differences in obesity between the three groups classified by smoking behavior. However, in both sexes, the prevalence of obesity tended to increase with pack-years and the number of cigarettes per day, but not with duration of smoking in current and past smokers. Further, in male smokers, the risks for obesity were markedly higher in short-term heavy smokers compared with long-term light smokers, even with the same number of pack-years. Regarding the impact of smoking cessation, female past smokers who quit smoking at an age > 55-years had an elevated OR of 1.60 (95% CI:1.05–2.38) for obesity.

Conclusions

In a general Japanese population, obesity is progressively associated with pack-years and number of cigarettes per day, but not with the duration of smoking. When investigating the association between obesity and cigarette smoking, the daily smoking burden and the duration of smoking require to be independently considered.