Bioavailability of Glucosyl Hesperidin in Rats

Glucosyl hesperidin (G-hesperidin) is a water-soluble derivative of hesperidin. We compared the absorption and metabolism of G-hesperidin with those of hesperidin in rats. After oral administration of G-hesperidin or hesperidin to rats, hesperetin was detected in sera hydrolyzed with β-glucuronidase, but it was not detectable in unhydrolyzed sera. Serum hesperetin was found more rapidly in rats administered G-hesperidin than in those administered hesperidin. The area under the concentration-time curve for hesperetin in the sera of rats administered G-hesperidin was approximately 3.7-fold greater than that of rats administered hesperidin. In the urine of both administration groups, hesperetin and its glucuronide were found. Urinary excretion of metabolites was higher in rats administered G-hesperidin than in those administered hesperidin. These results indicate that G-hesperidin presents the same metabolic profile as hesperidin. Moreover, it was concluded that G-hesperidin is absorbed more rapidly and efficiently than hesperidin, because of its high water solubility.     

Ligand‐induced formation of transient dimers of mammalian 12/15‐lipoxygenase: A key to allosteric behavior of this class of enzymes?

Mammalian lipoxygenases (LOXs) have been implicated in cellular defense response and are important for physiological homeostasis. Since their discovery, LOXs have been believed to function as monomeric enzymes that exhibit allosteric properties. In aqueous solutions, the rabbit 12/15-LOX is mainly present as hydrated monomer but changes in the local physiochemical environment suggested a monomer-dimer equilibrium. Because the allosteric character of the enzyme can hardly be explained using a single ligand binding-site model, we proposed that the binding of allosteric effectors may shift the monomer-dimer equilibrium toward dimer formation. To test this hypothesis, we explored the impact of an allosteric effector [13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid] on the structural properties of rabbit 12/15-LOX by small-angle X-ray scattering. Our data indicate that the enzyme undergoes ligand-induced dimerization in aqueous solution, and molecular dynamics simulations suggested that LOX dimers may be stable in the presence of substrate fatty acids. These data provide direct structural evidence for the existence of LOX dimers, where two noncovalently linked enzyme molecules might work in unison and, therefore, such mode of association might be related to the allosteric character of 12/15-LOX. Introduction of negatively charged residues (W181E + H585E and L183E + L192E) at the intermonomer interface disturbs the hydrophobic dimer interaction of the wild-type LOX, and this structural alteration may lead to functional distortion of mutant enzymes.     

Intestinal Transit Time During Infection with Eimeria nieschulzi in Rats*

SYNOPSIS. Experiments were designed to test whether or not intestinal transit time increases significantly during severe coccidiosis in the rat. Intraduodenal catheters were surgically implanted into 25 rats. Six to 12 days after surgery 11 rats were inoculated orally with 10⁴ sporulated oocysts of Eimeria nieschulzi Dieben, and 11 were inoculated with 10⁶ oocysts; 3 rats were retained as uninfected controls. At 2, 4, 8, 9, and 16 days postinoculation (PI) Na₂⁵¹CrO₄ was injected through the catheter into the duodenum of fasted rats and allowed to progress through the small bowel for 15 min, at which time the rats were killed. The distribution of ⁵¹Cr in the gut was plotted as a function of gut length. The leading edge of radioactivity traversed 70% of the gut length in controls, and ～ 50–60% in parasitized rats on days 2, 4, 8, and 9 PI. Also, a reflux of gut contents, as evidenced by radioactivity in the stomach, occurred early (PI days 2 & 4) in rats infected with 10⁴ oocysts and throughout patency in rats infected with 10⁶ oocysts. A 2nd study was undertaken to determine if chemically induced suppression of gut transit time during early infection would enhance infectivity as measured by increased parasite fecundity. Nine rats were injected subcutaneously with an antidiarrheal agent, Loperamide®, known to slow small bowel motility significantly. Another group of 9 control rats was injected with the ethanol-propylene glycol solvent. Ten min after injection, all rats were inoculated per os with 10⁴E. nieschulzi oocysts. The daily number of oocysts discharged/rat was followed from PI days 5–11. Patency began for all rats on PI day 7. The total number of oocysts discharged by the drugged rats as compared with controls was not significantly different.     

Localization of a base-paired interaction between small nuclear RNAs U4 and U6 in intact U4/U6 ribonucleoprotein particles by psoralen cross-linking

The small nuclear RNAs U4 and U6 display extensive sequence complementarity and co-exist in a single ribonucleoprotein particle. We have investigated intermolecular base-pairing between both RNAs by psoralen cross-linking, with emphasis on the native U4/U6 ribonucleoprotein complex. A mixture of small nuclear ribonucleoproteins U1 to U6 from HeLa cells, purified under non-denaturing conditions by immune affinity chromatography with antibodies specific for the trimethylguanosine cap of the small nuclear RNAs was treated with aminomethyltrioxsalen. A psoralen cross-linked U4/U6 RNA complex could be detected in denaturing polyacrylamide gels. Following digestion of the cross-linked U4/U6 RNA complex with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate cross-linked fragments. These fragments were analysed by chemical sequencing methods and their positions identified within RNAs U4 and U6. Two overlapping fragments of U4 RNA, spanning positions 52 to 65, were cross-linked to one fragment of U6 RNA (positions 51 to 59). These fragments show complementarity over a contiguous stretch of eight nucleotides. From these results, we conclude that in the native U4/U6 ribonucleoprotein particle, both RNAs are base-paired via these complementary regions. The small nuclear RNAs U4 and U6 became cross-linked in the deproteinized U4/U6 RNA complex also, provided that small nuclear ribonucleoproteins were phenolized at 0 degree C. When the phenolization was performed at 65 degrees C, no cross-linking could be detected upon reincubation of the dissociated RNAs at lower temperature. These results indicate that proteins are not required to stabilize the mutual interactions between both RNAs, once they exist. They further suggest, however, that proteins may well be needed for exposing the complementary RNA regions for proper intermolecular base-pairing in the course of the assembly of the U4/U6 RNP complex from isolated RNAs. Our results are discussed also in terms of the different secondary structures that the small nuclear RNAs U4 and U6 may adopt in the U4/U6 ribonucleoprotein particle as opposed to the isolated RNAs.     

The complete mitochondrial genome of the wild silkworm moth, Actias selene

The complete mitochondrial genome (mitogenome) of Actias selene (Lepidoptera: Saturniidae) was determined to be 15,236 bp, including 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a control region. The arrangement of 13 PCGs was similar to that of other sequenced lepidopterans. The AT skew of the mitogenome of A. selene was slightly negative, indicating a higher number of T compared to A nucleotides. The nucleotide composition of the mitogenome of A. selene was also biased toward A+T nucleotides (78.91%). All PCGs were initiated by ATN codons, except for the gene encoding cytochrome c oxidase subunit 1 (cox1), which may be initiated by the TTAG, as observed in other lepidopterans. Three genes, including cox1, cox2, and nad5, had incomplete stop codons consisting of just a T. With an exception for trnS1(AGN), all the other tRNA genes displayed a typical clover-leaf structure of mitochondrial tRNA. The A+T-rich region of the mitogenome of A. selene was 339 bp in length, and contains several features common to the Lepidopteras, including non-repetitive sequences, a conserved structure combining the motif ATAGA and an 18-bp poly-T stretch and a poly-A element upstream of trnM gene. Phylogenetic analysis showed that A. selene was close to Saturniidae.     

Protective effects of activated vitamin D receptor on radiation‐induced intestinal injury

Radiation‐induced intestinal injury (RIII) is a common complication after radiation therapy in patients with pelvic, abdominal, or retroperitoneal tumours. Recently, in the model of DSS (Dextran Sulfate Sodium Salt) ‐induced intestinal inflammatory injury, it has been found in the study that transgenic mice expressing hVDR in IEC (Intestinal Epithelial Cell) manifest highly anti‐injury properties in colitis, suggesting that activated VDR in the epithelial cells of intestine may inhibit colitis by protecting the mucosal epithelial barrier. In this study, we investigated the effect of the expression and regulation of VDR on the protection of RIII, and the radiosensitivity in vitro experiments, and explored the initial mechanism of VDR in regulating radiosensitivity of IEC. As a result, we found that the expression of VDR in intestinal tissues and cells in mice can be induced by ionizing radiation. VDR agonists are able to prolong the average survival time of mice after radiation and reduce the radiation‐induced intestinal injury. For lack of vitamin D, the radiosensitivity of intestinal epithelial cells in mice increased, which can be reduced by VDR activation. Ensuing VDR activation, the radiation‐induced intestinal stem cells damage is decreased, and the regeneration and differentiation of intestinal stem cells is promoted as well. Finally, on the basis of sequencing analysis, we validated and found that VDR may target the HIF/PDK1 pathway to mitigate RIII. We concluded that agonism or upregulation of VDR expression attenuates radiation‐induced intestinal damage in mice and promotes the repair of epithelial damage in intestinal stem cells.     

Interactions of a Bacterial RND Transporter with a Transmembrane Small Protein in a Lipid Environment

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Effect of serum,fibronectin, and laminin on adhesion of rabbit intestinal epithelial cells in culture

Rabbit intestinal epithelial cells, obtained after a limited hyaluronidase digestion, were incubated in medium with or without calf serum, on bacteriological plastic dishes. The dishes, either plain or coated with an air-dried type I collagen film, were pretreated with medium alone or with medium containing purified laminin or purified fibronectin. Cells did not attach in significant numbers to untreated bacteriological plastic, even in the presence of serum. Cells did attach to collagen-coated dishes, and were judged viable on the basis of their incorporation of radiolabeled leucine into cell protein. Cell adhesion to the collagen substrate increased in proportion to the concentration of serum in the medium, with maximal attachment at 5% serum or greater. Pretreatment of plain or collagen-coated dishes with increasing amounts of fibronectin enhanced cell adhesion in a concentration-dependent manner. Either serum, or fibronectin-free serum in the medium enhanced cell attachment to substrates pretreated with cither fibronectin or laminin. Thus, intestinal epithelial cells appear to possess surface receptors for both laminin and fibronectin. The evidence further suggests that calf serum may contain factors, other than fibronectin, capable of enhancing intestinal epithelial cell attachment to collagen substrates.     

RNA binding is more critical to the suppression of silencing function of Cucumber mosaic virus 2b protein than nuclear localization

Previously, we found that silencing suppression by the 2b protein and six mutants correlated both with their ability to bind to double-stranded (ds) small RNAs (sRNAs) in vitro and with their nuclear/nucleolar localization. To further discern the contribution to suppression activity of sRNA binding and of nuclear localization, we have characterized the kinetics of in vitro binding to a ds sRNA, a single-stranded (ss) sRNA, and a micro RNA (miRNA) of the native 2b protein and eight mutant variants. We have also added a nuclear export signal (NES) to the 2b protein and assessed how it affected subcellular distribution and suppressor activity. We found that in solution native protein bound ds siRNA, miRNA, and ss sRNA with high affinity, at protein:RNA molar ratios ~2:1. Of the four mutants that retained suppressor activity, three showed sRNA binding profiles similar to those of the native protein, whereas the remaining one bound ss sRNA at a 2:1 molar ratio, but both ds sRNAs with 1.5-2 times slightly lower affinity. Three of the four mutants lacking suppressor activity failed to bind to any sRNA, whereas the remaining one bound them at far higher ratios. NES-tagged 2b protein became cytoplasmic, but suppression activity in patch assays remained unaffected. These results support binding to sRNAs at molar ratios at or near 2:1 as critical to the suppressor activity of the 2b protein. They also show that cytoplasmically localized 2b protein retained suppressor activity, and that a sustained nuclear localization was not required for this function.