Genetic Analysis of Ligation-Induced Neointima Formation in an F2 Intercross of C57BL/6 and FVB/N Inbred Mouse Strains

Objective

Proliferation and migration of vascular smooth muscle cells (SMCs) are central for arterial diseases including atherosclerosis and restenosis. We hypothesized that the underlying mechanisms may be modeled by carotid ligation in mice. In FVB/N inbred mice, ligation leads to abundant neointima formation with proliferating media-derived SMCs, whereas in C57BL/6 mice hardly any neointima is formed. In the present study, we aimed to identify the chromosomal location of the causative gene variants in an F2 intercross between these two mouse strains.

Methods and Results

The neointimal cross-sectional area was significantly different between FVB/N, C57BL/6 and F1 female mice 4 weeks after ligation. Carotid artery ligation and a genome scan using 800 informative SNP markers were then performed in 157 female F2 mice. Using quantitative trait loci (QTL) analysis, we identified suggestive, but no genome-wide significant, QTLs on chromosomes 7 and 12 for neointimal cross-sectional area and on chromosome 14 for media area. Further analysis of the cross revealed 4 QTLs for plasma cholesterol, which combined explained 69% of the variation among F2 mice.

Conclusions

We identified suggestive QTLs for neointima and media area after carotid ligation in an intercross of FVB/N and C57BL/6 mice, but none that reached genome-wide significance indicating a complex genetic architecture of the traits. Genome-wide significant QTLs for total cholesterol levels were identified on chromosomes 1, 3, 9, and 12.     

Modulation of Sweet Taste by Umami Compounds via Sweet Taste Receptor Subunit hT1R2

Although the five basic taste qualities—sweet, sour, bitter, salty and umami—can be recognized by the respective gustatory system, interactions between these taste qualities are often experienced when food is consumed. Specifically, the umami taste has been investigated in terms of whether it enhances or reduces the other taste modalities. These studies, however, are based on individual perception and not on a molecular level. In this study we investigated umami-sweet taste interactions using umami compounds including monosodium glutamate (MSG), 5’-mononucleotides and glutamyl-dipeptides, glutamate-glutamate (Glu-Glu) and glutamate-aspartic acid (Glu-Asp), in human sweet taste receptor hT1R2/hT1R3-expressing cells. The sensitivity of sucrose to hT1R2/hT1R3 was significantly attenuated by MSG and umami active peptides but not by umami active nucleotides. Inhibition of sweet receptor activation by MSG and glutamyl peptides is obvious when sweet receptors are activated by sweeteners that target the extracellular domain (ECD) of T1R2, such as sucrose and acesulfame K, but not by cyclamate, which interact with the T1R3 transmembrane domain (TMD). Application of umami compounds with lactisole, inhibitory drugs that target T1R3, exerted a more severe inhibitory effect. The inhibition was also observed with F778A sweet receptor mutant, which have the defect in function of T1R3 TMD. These results suggest that umami peptides affect sweet taste receptors and this interaction prevents sweet receptor agonists from binding to the T1R2 ECD in an allosteric manner, not to the T1R3. This is the first report to define the interaction between umami and sweet taste receptors.     

Differential Impact of Constrictive Physiology after Pericardiocentesis in Malignancy Patients with Pericardial Effusion

Background

Echocardiographic signs of constrictive physiology (CP) after pericardiocentesis are frequently observed in malignancy patients. The purpose of the current study was to explore whether features of CP after pericardiocentesis have prognostic impact in malignancy patients with pericardial effusion (PE).

Methods

We retrospectively reviewed 467 consecutive patients who underwent pericardiocentesis at our institution from January 2006 to May 2014. Among them, 205 patients with advanced malignancy who underwent comprehensive echocardiography after the procedure comprised the study population. Co-primary end points were all-cause mortality (ACM) and repeated drainage (RD) for PE. Patients were divided into four subgroups according to cytologic result for malignant cells and CP (positive cytology with negative CP, both positive, both negative, and negative cytology with positive CP).

Results

CP after pericardiocentesis was present in 106 patients (50%) at median 4 days after the procedure. During median follow-up of 208 days, ACM and RD occurred in 162 patients (79%) and 29 patients (14%), respectively. Cox regression analysis revealed that independent predictors for ACM were male gender and positive cytology (all, p < 0.05). For RD, predictors were positive cytology, the absence of cardiac tamponade, and negative CP after pericardiocentesis (all, p < 0.05). When the patients were divided into four subgroups, patients with negative cytology and positive CP demonstrated the most favorable survival (hazard ratio [HR]: 0.39, p = 0.005) and the lowest RD rates (HR: 0.07, p = 0.012).

Conclusion

CP after pericardiocentesis is common, but does not always imply poor survival or the need for RD in patients with advanced malignancies. On the contrary, the presence of CP in patients with negative cytology conferred the most favorable survival and the lowest rate of RD. Comprehensive echocardiographic evaluation for CP after pericardiocentesis would be helpful for predicting prognosis in patients with advanced malignancies.     

Incidence of Alternaria Species Associated with Watermelon Leaf Blight in Korea

Alternaria leaf blight is one of the most common diseases in watermelon worldwide. In Korea, however, the Alternaria species causing the watermelon leaf blight have not been investigated thoroughly. A total of 16 Alternaria isolates was recovered from diseased watermelon leaves with leaf blight symptoms, which were collected from 14 fields in Korea. Analysis of internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and RNA polymerase II second largest subunit (RPB2) were not competent to differentiate the Alternaria isolates. On the contrary, analysis of amplicon size of the histone H3 (HIS3) gene successfully differentiated the isolates into three Alternaria subgroups, and further sequence analysis of them identified three Alternaria spp. Alternaria tenuissima, A. gaisen, and A. alternata. Representative Alternaria isolates from three species induced dark brown leaf spot lesions on detached watermelon leaves, indicating that A. tenuissima, A. gaisen, and A. alternata are all causal agents of Alternaria leaf blight. Our results indicate that the Alternaria species associated watermelon leaf blight in Korea is more complex than reported previously. This is the first report regarding the population structure of Alternaria species causing watermelon leaf blight in Korea.     

DNA Methylation Effects on Tetra-Nucleosome Compaction and Aggregation

DNA CpG methylation has been associated with chromatin compaction and gene silencing. Whether DNA methylation directly contributes to chromatin compaction remains an open question. In this study, we used fluorescence fluctuation spectroscopy (FFS) to evaluate the compaction and aggregation of tetra-nucleosomes containing specific CpG patterns and methylation levels. The compactness of both unmethylated and methylated tetra-nucleosomes is dependent on DNA sequences. Specifically, methylation of the CpG sites located in the central dyad and the major grooves of DNA seem to have opposite effects on modulating the compactness of tetra-nucleosomes. The interactions among tetra-nucleosomes, however, seem to be enhanced because of DNA methylation independent of sequence contexts. Our finding can shed light on understanding the role of DNA methylation in determining nucleosome positioning pattern and chromatin compactness.     

Physiological determinants of the candidate physical ability test in firefighters

The purpose of this study was to examine the relative importance of physiological characteristics during firefighting performance, as assessed by the Candidate Physical Ability Test (CPAT). Subjects included career and volunteer firefighters aged 18-39 (N = 33). Upper- and lower-body strength, muscle endurance, lower body muscle power, body composition analysis, aerobic capacity, anaerobic fitness, and the heart rate (HR) and blood pressure response to stair climbing were assessed to determine the physiological characteristics of the subjects. To quantify firefighting performance, the CPAT was administered by members of the fire service. Absolute and relative mean power during the Wingate anaerobic cycling test (WAnT), relative peak power during the WAnT, and absolute maximal oxygen uptake (VO2max) were significantly higher in those who passed the CPAT (N = 18), compared to those who failed (N = 15; p < 0.01). Mean power during the WAnT, fatigue index during WAnT, absolute VO2max, upper body strength, grip strength, and the HR response to stair climbing were significantly related to CPAT performance time (p < 0.01). Absolute VO2max and anaerobic fatigue resistance during WAnT best predicted CPAT performance (Adj. R2 = 0.817; p < 0.001). Performance on the ceiling breach and pull was the only CPAT task that was not significantly related to the physiological characteristics assessed. Measures of anaerobic and cardiovascular fitness best predict overall CPAT performance, and individual task performance. Remedial programs aimed at improving firefighting performance should target anaerobic and aerobic fitness qualities.     

Efficient constitutive expression of thermostable 4-α-glucanotransferase in Bacillus subtilis using dual promoters

4-α-Glucanotransferases possess strong transglycosylation activity which has been used in various carbohydrate chemistry fields. Due to safety issues of the recombinant enzymes we chose Bacillus subtilis as an expression host to produce a thermostable 4-α-glucanotransferase from Thermus scotoductus (TSαGT). The HpaII promoter in the Gram-positive bacterial vector pUB110 was used first to express TSαGT gene in B. subtilis. However, the activity of TSαGT in B. subtilis was only 4% of that in our previous Escherichia coli system. Two expression systems constructed by sequential alignment of another constitutive promoter for either α-amylase from B. subtilis NA64 or maltogenic amylase from Bacillus licheniformis downstream of the HpaII promoter elevated the TSαGT productivity by 11- and 12-fold, respectively, compared to the single HpaII promoter system. In conclusion, the dual promoter systems in this study were much better than the single promoter system to express the TSαGT gene in B. subtilis.     

β-propeller phytase hydrolyzes insoluble Ca(2+)-phytate salts and completely abrogates the ability of phytate to chelate metal ions

Phytate is an antinutritional factor that influences the bioavailability of essential minerals by forming complexes with them and converting them into insoluble salts. To further our understanding of the chemistry of phytate's binding interactions with biologically important metal cations, we determined the stoichiometry, affinity, and thermodynamics of these interactions by isothermal titration calorimetry. The results suggest that phytate has multiple Ca(2+)-binding sites and forms insoluble tricalcium- or tetracalcium-phytate salts over a wide pH range (pH 3.0-9.0). We overexpressed the β-propeller phytase from Hahella chejuensis (HcBPP) that hydrolyzes insoluble Ca(2+)-phytate salts. Structure-based sequence alignments indicated that the active site of HcBPP may contain multiple calcium-binding sites that provide a favorable electrostatic environment for the binding of Ca(2+)-phytate salts. Biochemical and kinetic studies further confirmed that HcBPP preferentially recognizes its substrate and selectively hydrolyzes insoluble Ca(2+)-phytate salts at three phosphate group sites, yielding the final product, myo-inositol 2,4,6-trisphosphate. More importantly, ITC analysis of this final product with several cations revealed that HcBPP efficiently eliminates the ability of phytate to chelate several divalent cations strongly and thereby provides free minerals and phosphate ions as nutrients for the growth of bacteria. Collectively, our results provide significant new insights into the potential application of HcBPP in enhancing the bioavailability and absorption of divalent cations.