A probable relationship between characteristic accumulation of doxorubicin and P-glycoprotein transporter in rat liver

Correlations between the distribution of anthracycline antibiotics doxorubicin (DX) and daunorubicin (DR) in the liver of rats injected with a single i.v. injection of each drug and the reported distribution of P-glycoprotein transporter for the drug was histochemically examined. Immunocytochemical studies for DX or DR using monoclonal antibody that equally detects both drugs, as well as conventional electron microscopy were employed. DX persisted for more than 5 days in the cytoplasm and nuclei of the hepatocytes in a characteristic granular morphology on the bile capillaries. Meanwhile, DR distributed in almost the same pattern, but more rapidly decreased to a level that almost no granular morphology in the hepatocytes was visible 24 h after the injection. Also, unknown large cells that strongly reacted with the antibody appeared in the hepatic sinusoids near the interlobular triads rather than the central vein. The accumulation sites of DX or DR on the bile capillaries seems to correspond to specific sites where the ATP-binding cassette transport protein P-glycoprotein for the anthracycline occurs, suggesting a possibility that DX or DR is actually and actively excreted at these sites, possibly through P-glycoprotein. This is supported by conventional electron microscopic studies, since no specific subcellular organelles such as lysosomes assemble in the vicinity of the bile capillaries of the hepatocytes.     

Simultaneous bioanalysis of L-arginine, L-citrulline, and dimethylarginines by LC-MS/MS

Purposel-Arginine (ARG) is converted to nitric oxide (NO) and l-citrulline (CIT) by endothelial nitric oxide synthase which is competitively inhibited by asymmetric dimethylarginine (ADMA). We have developed a liquid chromatography–mass spectrometric method for the simultaneous determination of endogenous ARG, labeled ARG (¹⁵N₄-ARG), CIT, ADMA, and its inactive isomer, symmetric dimethylarginine (SDMA) in biological samples.MethodsConcentrations of unlabeled ARG, ¹⁵N₄-ARG, CIT, ADMA, and SDMA in EA.hy926 human endothelial cell lysate, cell incubation media, rat plasma or rat urine were measured by hydrophilic-interaction liquid chromatography electrospray tandem mass spectrometry. ¹³C₆-ARG, D₄-CIT and D₇-ADMA were used as internal standards for ARG and ¹⁵N₄-ARG, CIT, and dimethylarginines, respectively.ResultsThe calibration curves of ARG, ¹⁵N₄-ARG, CIT, ADMA, and SDMA were linear and independent of several sample matrices. Intra- and inter-day variabilities for the quantification of all the compounds were below 15% in quality control samples. Application of this method to determine the uptake as well as efflux of these compounds was illustrated through in vitro cell study by exposing human endothelial cells to ¹⁵N₄-ARG, which allowed the observation of generation of ¹⁵N₃-CIT and ¹⁵N₃-ARG in the cell lyate. Use of these isotopes adds insights into the cellular handling of endogenous vs. exogenous ARG. Application of this method for rat plasma and rat urine assays was demonstrated after ARG oral supplementation in rats.ConclusionAn LC–MS/MS method was developed to quantify 6 ARG-related compounds simultaneously, utilizing 3 separate internal standards. This assay allows concurrent monitoring of uptake, efflux and metabolic processes when isotope-labeled ARG and CIT are measured, and can be applied for determination of these compounds in rat plasma and rat urine.     

Planar cell polarity in the mammalian eye lens

The major role of the eye lens is to transmit and focus images onto the retina. For this function, the lens needs to develop and maintain the correct shape, notably, the precise curvature and high-level order and organization of its elements. The lens is mainly comprised of highly elongated fiber cells with hexagonal cross-sectional profiles that facilitate regular packing. Collectively, they form concentrically arranged layers around the anterior-posterior polar axis, and their convex curvature contributes to the spheroidal shape of the lens. Although the lens has been a popular system for developmental studies, little is known about the mechanism(s) that underlies the development of its exquisite three-dimensional cellular architecture. In this review, we will describe our recent work, which shows how planar cell polarity (PCP) operates in lens and contributes to its morphogenesis. We believe that the lens will be a useful model system to study PCP in general and gain insights into mechanisms that generate high-level cellular order during development.     

Systemic transplantation of human adipose-derived stem cells stimulates bone repair by promoting osteoblast and osteoclast function

Systemic transplantation of adipose-derived stem cells (ASCs) is emerging as a novel therapeutic option for functional recovery of diverse damaged tissues. This study investigated the effects of systemic transplantation of human ASCs (hASCs) on bone repair. We found that hASCs secrete various bone cell-activating factors, including hepatocyte growth factor and extracellular matrix proteins. Systemic transplantation of hASCs into ovariectomized mice induced an increased number of both osteoblasts and osteoclasts in bone tissue and thereby prevented bone loss. We also observed that conditioned medium from hASCs is capable of stimulating proliferation and differentiation of osteoblasts via Smad/extracellular signal-regulated kinase (ERK)/JNK (c-jun NH(2) -terminal kinase) activation as well as survival and differentiation of osteoclasts via ERK/JNK/p38 activation in vitro. Overall, our findings suggest that paracrine factors secreted from hASCs improve bone repair and that hASCs can be a valuable tool for use in osteoporosis therapy.     

Genome-wide DNA methylation profiles in noncancerous gastric mucosae with regard to Helicobacter pylori infection and the presence of gastric cancer

Background and Aims: To determine genome‐wide DNA methylation profiles induced by Helicobacter pylori (H. pylori) infection and to identify methylation markers in H. pylori‐induced gastric carcinogenesis. Methods: Gastric mucosae obtained from controls (n = 20) and patients with gastric cancer (n = 28) were included. A wide panel of CpG sites in cancer‐related genes (1505 CpG sites in 807 genes) was analyzed using Illumina bead array technology. Validation of the results of Illumina bead array technique was performed using methylation‐specific PCR method for four genes (MOS, DCC, CRK, and PTPN6). Results: The Illumina bead array showed that a total of 359 CpG sites (269 genes) were identified as differentially methylated by H. pylori infection (p < .0001). The correlation between methylation‐specific PCR and bead array analysis was significant (p < .0001, Spearman coefficient = 0.5054). Methylation profiles in noncancerous gastric mucosae of the patients with gastric cancer showed quite distinct patterns according to the presence or absence of the current H. pylori infection; however, 10 CpG sites were identified to be hypermethylated and three hypomethylated in association with the presence of gastric cancer regardless of H. pylori infection (p < .01). Conclusions: Genome‐wide methylation profiles showed a number of genes differentially methylated by H. pylori infection. Methylation profiles in noncancerous gastric mucosae from the patients with gastric cancer can be affected by H. pylori‐induced gastritis. Differentially methylated CpG sites in this study needs to be validated in a larger population using quantitative methylation‐specific PCR method.     

Screening and characterization of a cellulase gene from the gut microflora of abalone using metagenomic library

A metagenomic fosmid library was constructed using genomic DNA isolated from abalone intestine. Screening of a library of 3,840 clones revealed a 36 kb insert of a cellulase positive clone (pAMHElO). A shotgun clone library was constructed using the positive clone (pAMHElO) and further screening of 3,840 shotgun clones with an approximately 5 kb insert size using a Congo red overlay revealed only one cellulase positive clone (pAMHL9). The pAMHL9 consisted of a 5,293-bp DNA sequence and three open reading frames (ORFs). Among the three ORFs, cellulase activity was only shown in the recombinant protein (CelAMll) coded by ORF3, which showed 100% identity with outer membrane protein A from Vibrio alginolyticus 12G01, but no significant sequence homology to known cellulases. The expressed protein (CelAMll) has a molecular weight of approximately 37 kDa and the highest CMC hydrolysis activity was observed at pH 7.0 and 37°C. The carboxymethyl cellulase activity was determined by zymogram active staining and different degraded product profiles for CelAMll were obtained when cellotetraose and cellopentaose were used as the substrates, while no substrate hydrolysis was observed on oligosaccharides such as cellobiose and cellotriose.     

Production of cephalosporin C using crude glycerol in fed-batch culture of <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis> M35

In this study, cephalosporin C production by Acremonium chrysogenum M35 cultured with crude glycerol instead of rice oil and methionine was investigated. The addition of crude glycerol increased cephalosporin C production by 6-fold in shake-flask culture, and also the amount of cysteine. In fed-batch culture without methionine, crude glycerol resulted only in overall improvement in cephalosporin C production (about 700%). In addition, A. chrysogenum M35 became highly differentiated in fed-batch culture with crude glycerol, compared with the differentiation in batch culture. The results presented here suggest that crude glycerol can replace methionine and plant oil as cysteine and carbon sources during cephalosporin C production by A. chrysogenum M35.     

Biocontrol of moth pests in apple orchards: Preliminary field study of application potential for mass trapping

Many moth pheromones and synthetic attractants have been used to monitor or decrease pest populations. However, due to their low economic efficiencies and narrow target specificities these methods are of limited use for trapping pests in large agricultural fields. In an effort to address this problem, we selected oak (Quercus serrata) sawdust media fermented by the yeast strain Pichia anomala, and examined its ability to attract lepidopteran moth pests for mass trapping in an apple (Malus domestica) orchard. A total of 57 taxa were trapped, including 42 species of lepidopteran pests. The most frequently caught lepidopteran pests included Adoxophyes orana (40.0 ± 3.5 individuals/trap/week), Oraesia excavata (35.5 ± 2.5), and Adris tyrannus amurensis (35.5 ± 2.5). Notably, less fruit damage was recorded in the experimental orchard versus a trap-free neighboring control orchard. These results suggest that adult moths might be effectively attracted and mass captured using P. anomala-fermented oak sawdust media as bait.