Curcumin Oligosaccharides (Gluco-oligosaccharides) Penetrate the Blood-Brain Barrier in Mouse Brain: Glycoside (Polysaccharide) Modification Approach for Brain Drug Delivery Across the Blood-Brain Barrier and Tumor Drug Delivery

In order to expand our drug delivery technique by glycosylation of chemical into brain delivery, it was demonstrated that oligosaccharide and monosaccharide modifications of curcumin enhanced its crossing ability of the blood-brain barrier (BBB) in mice. The brain sample prepared by glycosidase-catalyzed hydrolysis of brain tissue homogenates of mice, to which curcumin gluco-oligosaccharides were intraperitoneally injected, contained curcumin at 116 ng/1 g of tissue of brain, indicating that curcumin modified with gluco-oligosaccharides residues can smoothly cross the BBB in mouse brain. The brain samples of mice, which were treated with curcumin monosaccharide or curcumin itself, contained curcumin at 18 ng and 0 ng per 1 g of tissue of brain, respectively. On the other hand, after the administration of curcumin gluco-oligosaccharides to C57BL mouse with a large tumor for 5 days, the tumor disappeared.

Analysis of experimental mouse PRNP genetic polymorphisms and their susceptibility to prion diseases

Research studies showed that the polymorphisms in prion protein gene (PRNP) were associated with susceptibility to prion diseases in several animals, including humans and mouse. Several mouse strains carried natural PRNP mutations which had been identified and these could provide as animal models for human prion diseases. In this study, the genetic polymorphisms of PRNP in six common mouse strains were investigated. The experimental mice included KM mouse, ICR mouse, DBA mouse, C3H/He mouse, C57BL mouse and BALB mouse. The results showed only one new polymorphism was identified compared with the reference sequence. The identified new mutation site was C564T and it was homozygous, but this locus did not result in amino acid change. Sequence analyses suggested that these six mouse strains were susceptible to prion diseases and are suitable as susceptibility models of prion diseases.

Endothelial Activation by Sickle Mouse Red Cells and Their Endothelial Adhesion In Vivo is Abolished by Catalase Treatment of Sickle Cells, but Not Quiescent Endothelium, Implying a Role of Heme-Mediated Peroxide Generation in Sickle Cell Adhesion.

Abstract 902 Multiple adhesion molecules, expressed on sickle red blood cells (SS RBCs) and activated endothelium, have been implicated in SS RBC adhesion to vascular endothelium. Moreover, intrinsic differences among heterogeneous SS RBC subpopulations, involving differences in red cell adhesion molecules and cell deformability, may contribute to their adhesive and obstructive properties and lead to postcapillary obstruction. However, the role of SS RBCs in endothelium activation and adhesion has not been evaluated despite the insightful studies of Hebbel and coworkers (JCI, 1982) demonstrating that SS RBCs generate excessive amounts of reactive oxygen species due to the presence of unstable hemoglobin S (HbS) and autoxidation of iron in heme. RBCs from transgenic-knockout sickle (BERK) mice similarly show a pronounced increase in heme degradation (Nagababu et. al. Blood Cells Mol Dis, 2008). We hypothesize that hypoxic conditions in venules (oxygen tension,∼30 mm Hg) will accelerate autoxidation of RBC membrane-bound HbS and release H2O2 that will be transferred to adjoining endothelium resulting in its activation (i.e., up-regulation of endothelial adhesion molecules) and SS RBC adhesion. To test the hypothesis that HbS-containing red cells from BERK mice will result in activation of quiescent endothelium in normal mice, we infused FITC (fluorescein isothiocynate)-labeled BERK red cells into congenic C57BL mice. BERK mice, expressing exclusively human βS- and α-globins, have been extensively backcrossed onto C57BL background. Intravital observations were made in the cremaster muscle microcirculatory bed. A single bolus of 150 μl of FITC-labeled BERK RBCs (Hct 30%) was infused into the recipient C57BL mouse via the jugular vein over a period of 5 min to avoid any shear related platelet aggregation. Infusion of FITC-labeled control (C57BL) mouse RBCs into C57BL recipient mice resulted in rare or no RBC adhesion, suggesting that there was no activating effect on endothelium. In contrast, infusion of BERK mouse RBCs into C57BL mice resulted in time-dependent increase in adhesion to venular endothelium. Adhesion became discernable after 3 minutes and showed a 3-5 fold increase after 5-min compared with the number of adherent RBCs at 3 min (P<0.01). Next, we investigated if the infusion of BERK mouse RBCs would induce increased endothelial oxidants. To this end, the cremaster preparation was suffused for 15 min with 123 dihydrorhodamine (DHR), a H2O2-sensitive probe (10 μl/L), followed by a bolus infusion of BERK mouse RBCs, and time-dependent changes in DHR fluorescence intensity were monitored in venules, the sites of adhesion. Infusion of BERK mouse RBCs, but not C57BL RBCs, resulted in time-dependent increase in the fluorescence intensity (ΔI) in venular endothelium, with almost 5-fold increase in DHR intensity after 5 min of BERK RBC infusion (P<0.001) compared with ΔI at 1 min. When infusion of catalase (900 U/mouse) into recipient C57BL mice was followed 30 min later by a bolus of FITC-labeled BERK mouse RBCs, BERK RBC adhesion and pronounced DHR fluorescence in endothelium were observed, demonstrating that intravascular infusion of catalase had little effect on oxidant generation by BERK mouse RBCs. In contrast, infusion of BERK RBCs pre-treated with catalase (100 U in 0.2 ml RBC suspension, 9-fold less catalase per mouse) to quench RBC generated H2O2 inhibited endothelial DHR fluorescence and BERK RBC adhesion. These results strongly suggest an obligatory role of heme-mediated peroxide generation by SS RBC in endothelial activation and SS RBC adhesion, and support the notion that heme-mediated oxidant generation may play a vital role in endothelial dysfunction in sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

Analysis of specific factors generating 2-cell block in AKR mouse embryos

SummaryThe phenomenon of the developmental arrest at the 2-cell stage of 1-cell embryos from some mouse strains during in vitro culture is known as the 2-cell block. We investigated the specific factors involved in the 2-cell block of AKR embryos by means of a modified culture system, the production of reconstructed embryos by pronuclear exchange and a cross experiment. In a culture medium with phosphate, 94.6% of 1-cell embryos from the C57BL mouse strain developed to the blastocyst stage, but 95.7% of embryos from the AKR mouse strain showed 2-cell block. Phosphate-free culture medium rescued the 2-cell block of AKR embryos and accelerated the first cell cycle of the embryos. Co-culture with BRL cells and a BRL-conditioned medium fractionated below 30 kDa also rescued the 2-cell block of AKR embryos. Examinations of in vitro development of reconstructed embryos and of embryos from F1 females between AKR and C57BL strains clearly demonstrated that the AKR cytoplast caused the 2-cell block. In the backcrossed female progeny between (AKR × C57BL) F1 males and AKR females, about three-quarters of the embryos were of the 2-cell blocking phenotype and about one-quarter were of the non-blocking phenotype. These results suggest that two genes are responsible for the 2-cell block of AKR embryos.

Positively Charged Alpha-Chains Can Stimulate K-Cl Cotransport in Transgenic Mouse Red Cells.

Transgenic mice were generated with human α-chain anti-sickling mutations at contact sites for the HbS polymer. Some of these mice were found to have elevated K-Cl cotransport. Elevation of K-Cl cotransport in patients with homozygous HbS or SC disease increases red cell MCHC and contributes to pathology. In contrast to C57Bl mouse red cells (mRBC) and mRBC expressing only HbA that have little volume-stimulated K-Cl cotransport, we previously reported that HbC under all conditions, and HbS + γ, in the absence of mouse globins, are able to stimulate the activity of K-Cl cotransport in mRBC. These observations support the contention that HbS and HbC stimulate K-Cl cotransport activity in both mouse and human red cells and may do so via the positive charge on the human β-chain. We report here that positively charged α-chains also stimulate K-Cl cotransport in mRBC. Mice expressing α-chain mutants were generated: α49 (HbSavaria, α49S→R, +1 positive charge vs human α) with no human β-globin; α49 and βS; α49 and NY1 (that expresses human α and βS); α49–114, that expresses both α49 and α114 (HbChiapas α114P→R, +2 positive charge vs human α) with no human β-globin; α49–114 and βS; α20 (HbLeLamentin, α20 H→Q, -1 negative charge vs human α) and βS; and α20–114 (that has no average charge difference from human α) with no human β-globin. Mice were bred with α- and β-KO mice and mice expressing the NY1 transgene to produce mice expressing various levels of mutant α, human α, βS, and mouse globins. Density gradients detected increased red cell density relative to wild type mice (C57Bl) in mice expressing α-globins with a positive charge relative to human α. To isolate the effect of charged α-globin, we first examined mice with only mutated α-globins and no βS. We previously measured volume-stimulated K-Cl cotransport in C57Bl and HbAKO mice (that only express HbA) as 2.0±0.9 and 2.4±1.7 mmol/L cells x hr (FU) respectively. We found a similar value (2.4±1.1 FU) in mRBC expressing either 32% or 100% α20–114 with no βS (no charge difference from human α). mRBC expressing α49 at 44 or 100% with no βS had an average value of 12.9±3.3 FU; similarly, mRBC expressing 48% α114-49 with no βS averaged 9.4±1.2 FU. The volume-stimulated K-Cl cotransport was both chloride and okadaic acid sensitive. These results demonstrate that positively charged α-chains stimulate K-Cl cotransport while mutant α-chains without a charge difference do not. We have also examined mRBC expressing α49 or α49–114 with βS or NY1 and found that all mice exhibited increased K-Cl cotransport with the exception of mRBC from founder α49–114βS mice that express 31% mutant α and 9% βS. We conclude that a positive charge in excess of that found on human α can stimulate K-Cl cotransport and result in increased MCHC. In the presence of βS, this effect results in mouse red cells with properties similar to human SC disease and prevents the birth of mice that are fully knocked out, due to polymer formation, despite the presence of anti-sickling mutations. In constrast, mice with α20βS (−1 vs human α) were viable with exclusively human globins. These observations could only have been made by creating mouse models and imply that charge must also be considered when anti-sickling globins are proposed.