Unmethylated and methylated CpG dinucleotides distinctively regulate the physical properties of DNA

In eukaryotic cells, DNA has to bend significantly to pack inside the nucleus. Physical properties of DNA such as bending flexibility and curvature are expected to affect DNA packaging and partially determine the nucleosome positioning patterns inside a cell. DNA CpG methylation, the most common epigenetic modification found in DNA, is known to affect the physical properties of DNA. However, its detailed role in nucleosome formation is less well‐established. In this study, we evaluated the effect of defined CpG patterns (unmethylated and methylated) on DNA structure and their respective nucleosome‐forming ability. Our results suggest that the addition of CpG dinucleotides, either as a (CG)_n stretch or (CGX₈)_n repeats at 10 bp intervals, lead to reduced hydrodynamic radius and decreased nucleosome‐forming ability of DNA. This effect is more predominant for a DNA stretch ((CG)₅) located in the middle of a DNA fragment. Methylation of CpG sites, surprisingly, seems to reduce the difference in DNA structure and nucleosome‐forming ability among DNA constructs with different CpG patterns. Our results suggest that unmethylated and methylated CpG patterns can play very different roles in regulating the physical properties of DNA. CpG methylation seems to reduce the DNA conformational variations affiliated with defined CpG patterns. Our results can have significant bearings in understanding the nucleosome positioning pattern in living organisms modulated by DNA sequences and epigenetic features. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 517–524, 2014.     

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.     

Expression and identification of a minor extracellular fibrinolytic enzyme (Vpr) from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> KCTC 3014

Previously, three extracellular proteases, Vpr, PepT, and subtilisin were identified from Bacillus subtilis KCTC 3014. To confirm the activity of Vpr, two recombinant Vpr proteins, full Vpr with TTG (pGST-fTTG-Vpr) and full Vpr with ATG (pGST-fATG-Vpr) as an initiation codon were expressed using a pGEX-2T vector encoding glutathione S-transferase (GST) in Escherichia coli. Vpr was produced in two forms, occurring as four spots on a 2-DE gel, 68 and 75 kDa proteins with similar pI values (4.0 ∼ 4.5). Activity was detected in a fibrin zymography at the expected molecular size of 68 kDa (mature form) processed from full Vpr. However, the recombinant 75 kDa of GST-fVpr did not exhibit activity. Replacement of the TTG codon with ATG led to 1.9-fold increased enzyme activity in 68 kDa. Interestingly, the expression of GSTVpr resulted in the proteolytic degradation of the protein and no GST fusion Vpr protein was detected.     

Effect of reactor surface on production of bacterial cellulose and water soluble oligosaccharides by <Emphasis Type="Italic">Gluconacetobacter hansenii</Emphasis> PJK

The effect of agar plates on the bacterial cellulose (BC) production in a static culture was investigated in order to find the role of agar component as a surface modifying agent. Two types of surface modified reactors (SMRs: SMRD and SMRB) were prepared by coating the bottom of the reactors with agar dissolved in distilled water and basal medium, respectively. The SMRs were used for BC and water soluble oligosaccharides (WSOS) production. Control was done by the same procedure using reactors without agar plate. In both types of SMRs, the maximum production rate was observed after the second day of cultivation compared to third day of cultivation in the case of the control. The maximum productions of BC 5.308 and 5.472 g/L were observed at the first batch using SMRs prepared with agar dissolved in distilled water (SMRDs) and SMRs prepared with agar dissolved in a basal medium (SMRBs), respectively. Similarly, in the daily-culture and successive batch strategy experiments the maximum amount of WSOS produced in the SMRs was almost double that of the control. The highest water holding capacity value 92.21 g/g was observed for BC formed in the SMRs prepared with 3.0% of agar. FTIR and XRD analyses were carried out to study the structural features of the prepared BC.     

New route for synthesizing poly(ethylene glycol)-acrylic acid hydrogels using γ-irradiation for drug delivery carriers

Biocompatible and pH-responsive poly(ethylene glycol) (PEG)-acrylic acid (AAc) hydrogels were prepared by new technique using γ-irradiation for controlled oral drug delivery. The gel fraction was over 80% and the equal amounts of PEG and AAc blended hydrogel had efficient insulin loading using equilibrium swelling. These hydrogels exhibited unique pH-responsive characteristics in which interpolymer complexes were formed in acidic media and dissociated in neutral or basic environments. The insulin release from the gel was significantly retarded in acidic media while rapid release occurred under neutral/basic conditions. At the high pH solution, the gels swelled rapidly and over 70% of the insulin loaded was released over a period of 10 h. Within 2 h of administration of the insulin-containing gels, significant blood glucose reduction effects were observed in diabetic rats. The blood glucose reduction lasted for up to 10 h following administration.     

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.     

2′, 3′-<Emphasis Type="Italic">O</Emphasis>-(4-benzoylbenzoyl)–ATP-mediated calcium signaling in rat glioma C6 cells: role of the P2Y<Subscript>2</Subscript> nucleotide receptor

In this study, we examined the response of glioma C6 cells to 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) and showed that the BzATP-induced calcium signaling does not involve the P2X₇ receptor activity. We show here that in the absence of extracellular Ca²⁺, BzATP-generated increase in [Ca²⁺]_i via Ca²⁺ release from intracellular stores. In the presence of calcium ions, BzATP established a biphasic Ca²⁺ response, in a manner typical for P2Y receptors. Brilliant Blue G, a selective antagonist of the rat P2X₇ receptor, did not reduce any of the two components of the Ca²⁺ response elicited by BzATP. Periodate-oxidized ATP blocked not only BzATP- but also UTP-induced Ca²⁺ elevation. Moreover, BzATP did not open large transmembrane pores. What is more, a cross-desensitization between UTP and BzATP occurred, which clearly shows that in glioma C6 cells BzATP activates most likely the P2Y₂ but not the P2X₇ receptors.     

Comparative analysis of the secretory proteome of human adipose stromal vascular fraction cells during adipogenesis

Adipogenesis is a complex process that is accompanied by a number of molecular events. In this study, a proteomic approach was adopted to identify secretory factors associated with adipogenesis. A label‐free shotgun proteomic strategy was implemented to analyze proteins secreted by human adipose stromal vascular fraction cells and differentiated adipocytes. A total of 474 proteins were finally identified and classified according to quantitative changes and statistical significances. Briefly, 177 proteins were significantly upregulated during adipogenesis (Class I), whereas 60 proteins were significantly downregulated (Class II). Changes in the expressions of several proteins were confirmed by quantitative RT‐PCR and immunoblotting. One obvious finding based on proteomic data was that the amounts of several extracellular modulators of Wnt and transforming growth factor‐β (TGF‐β) signaling changed during adipogenesis. The expressions of secreted frizzled‐related proteins, dickkopf‐related proteins, and latent TGF‐β‐binding proteins were found to be altered during adipogenesis, which suggests that they participate in the fine regulation of Wnt and TGF‐β signaling. This study provides useful tools and important clues regarding the roles of secretory factors during adipogenic differentiation, and provides information related to obesity and obesity‐related metabolic diseases.     

OsZIP5 is a plasma membrane zinc transporter in rice

Zinc is essential for normal plant growth and development. To understand its transport in rice, we characterized OsZIP5, which is inducible under Zn deficiency. OsZIP5 complemented the growth defect of a yeast Zn-uptake mutant, indicating that OsZIP5 is a Zn transporter. The OsZIP5-GFP fusion protein was localized to the plasma membrane. Transgenic plants overexpressing the gene grew less well. Overexpression of the gene decreased the Zn concentration in shoots, but caused it to rise in the roots. Knockout plants showed no visible phenotypic changes under either normal or deficient conditions. However, they were tolerant to excess Zn and contained less Zn. In contrast, overexpressing transgenics were sensitive to excess Zn. These results indicate that OsZIP5 plays a role in Zn distribution within rice.     

Defensins play a crucial role in protecting mice against oral Shigella flexneri infection

An earlier study revealed that 4-day-old mice, but not older mice, were infected with invasive Shigella strains. Here we attempted to determine the underlying mechanism that induces inflammation in the intestines of neonate mice after oral Shigella infection. Wild-type BALB/c mice of different ages (7, 14, and 35 days old) were orally administered GFP-expressing Shigella flexneri 5a M90T strain (5 × 10⁹ CFU) and analyzed for colonization 6 h following infection. We found that Shigella localized in the epithelium, lamina propria, and crypt regions of the small intestines of 7-day-old BALB/c mice. Microarray analysis revealed that expression levels of cryptdin and various types of cryptdin-related mRNA (e.g., cryptrs-2, -5, -7, -12 and lysozyme) in the small intestines were significantly lower in 7-day-old than in older mice regardless of Shigella infection status. Interestingly, matrix metalloprotease-7 (matrilysin)-deficient (MAT^−/−) mice of B6 background had more colonies and more severe symptoms of inflammation in the intestines than did wild-type B6 mice after oral Shigella challenge. This suggests that cryptdin-related antimicrobial molecules are indispensable for efficient protection against oral Shigella infection.