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.     

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.     

Implication of mouse Vps26b-Vps29-Vps35 retromer complex in sortilin trafficking

The retromer complex, which mediates retrograde transport from endosomes to the trans-Golgi network, is a heteropentameric complex that contains a multifunctional cargo recognition heterotrimer consisted of the vacuolar protein sorting (Vps) subunits Vps26, Vps29, and Vps35. In mammals, there are two different isoforms of Vps26, Vps26a and Vps26b, that localize to the endosome, and to the plasma membrane, respectively. To elucidate the biological significance of the Vps26b isoform, we generated Vps26b knockout mice and studied their molecular, histological, and behavioral phenotypes. We found that the loss of Vps26b results in no significant defects in the behavior, body size, and health of the mice. Vps26b-deficient mice showed a severe reduction of Vps35 protein at cellular level and lacked the Vps26b-Vps29-Vps35 retromer complex, despite the normal presence of the Vps26a-Vps29-Vps35 retromer complex. Relatively, the amount of sortilin was increased approximately 20% in the Vps26b-deficient mice, whereas the sorLA was normal. These results suggest that mouse Vps26b-Vps29-Vps35 retromer complex is implicated in the transport of sortilin from endosomes to the trans-Golgi network (TGN).     

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.     

Extramedullary hematopoiesis in a case of benign mixed mammary tumor in a female dog: cytological and histopathological assessment

Backgroud  

Extramedullary hematopoiesis (EMH) is defined as the presence of hematopoietic stem cells such as erythroid and myeloid lineage plus megakaryocytes in extramedullary sites like liver, spleen and lymph nodes and is usually associated with either bone marrow or hematological disorders. Mammary EMH is a rare condition either in human and veterinary medicine and can be associated with benign mixed mammary tumors, similarly to that described in this case.     

The use of sparse CT datasets for auto-generating accurate FE models of the femur and pelvis

The finite element (FE) method when coupled with computed tomography (CT) is a powerful tool in orthopaedic biomechanics. However, substantial data is required for patient-specific modelling. Here we present a new method for generating a FE model with a minimum amount of patient data. Our method uses high order cubic Hermite basis functions for mesh generation and least-square fits the mesh to the dataset. We have tested our method on seven patient data sets obtained from CT assisted osteodensitometry of the proximal femur. Using only 12 CT slices we generated smooth and accurate meshes of the proximal femur with a geometric root mean square (RMS) error of less than 1 mm and peak errors less than 8 mm. To model the complex geometry of the pelvis we developed a hybrid method which supplements sparse patient data with data from the visible human data set. We tested this method on three patient data sets, generating FE meshes of the pelvis using only 10 CT slices with an overall RMS error less than 3 mm. Although we have peak errors about 12 mm in these meshes, they occur relatively far from the region of interest (the acetabulum) and will have minimal effects on the performance of the model. Considering that linear meshes usually require about 70-100 pelvic CT slices (in axial mode) to generate FE models, our method has brought a significant data reduction to the automatic mesh generation step. The method, that is fully automated except for a semi-automatic bone/tissue boundary extraction part, will bring the benefits of FE methods to the clinical environment with much reduced radiation risks and data requirement.     

Insulin-mimetic and insulin-sensitizing activities of a pentacyclic triterpenoid insulin receptor activator

Five pentacyclic triterpenoids isolated from Campsis grandiflora were tested for insulin-mimetic and insulin-sensitizing activity. The compounds enhanced the activity of insulin on tyrosine phosphorylation of the IR (insulin receptor) beta-subunit in CHO/IR (Chinese-hamster ovary cells expressing human IR). Among the compounds tested, CG7 (ursolic acid) showed the greatest enhancement and CG11 (myrianthic acid) the least. We characterized the effect of CG7 further, and showed that it acted as an effective insulin-mimetic agent at doses above 50 mug/ml and as an insulin-sensitizer at doses as low as 1 mug/ml. Additional experiments showed that CG7 increased the number of IRs that were activated by insulin. This indicates that a major mechanism by which CG7 enhances total IR auto-phosphorylation is by promoting the tyrosine phosphorylation of additional IRs. CG7 not only potentiated insulin-mediated signalling (tyrosine phosphorylation of the IR beta-subunit, phosphorylation of Akt and glycogen synthase kinase-3beta), but also enhanced the effect of insulin on translocation of glucose transporter 4 in a classical insulin-sensitive cell line, 3T3-L1 adipocytes. The results of the present study demonstrate that a specific pentacyclic triterpenoid, CG7, exerts an insulin-sensitizing effect as an IR activator in CHO/IR cells and adipocytes. The enhancement of insulin activity by CG7 may be useful for developing a new class of specific IR activators for treatment of Type 1 and Type 2 diabetes.     

RSC mobilizes nucleosomes to improve accessibility of repair machinery to the damaged chromatin

Repair of DNA double-strand breaks (DSBs) protects cells and organisms, as well as their genome integrity. Since DSB repair occurs in the context of chromatin, chromatin must be modified to prevent it from inhibiting DSB repair. Evidence supports the role of histone modifications and ATP-dependent chromatin remodeling in repair and signaling of chromosome DSBs. The key questions are, then, what the nature of chromatin altered by DSBs is and how remodeling of chromatin facilitates DSB repair. Here we report a chromatin alteration caused by a single HO endonuclease-generated DSB at the Saccharomyces cerevisiae MAT locus. The break induces rapid nucleosome migration to form histone-free DNA of a few hundred base pairs immediately adjacent to the break. The DSB-induced nucleosome repositioning appears independent of end processing, since it still occurs when the 5'-to-3' degradation of the DNA end is markedly reduced. The tetracycline-controlled depletion of Sth1, the ATPase of RSC, or deletion of RSC2 severely reduces chromatin remodeling and loading of Mre11 and Yku proteins at the DSB. Depletion of Sth1 also reduces phosphorylation of H2A, processing, and joining of DSBs. We propose that RSC-mediated chromatin remodeling at the DSB prepares chromatin to allow repair machinery to access the break and is vital for efficient DSB repair.