Functional expression of carnitine/organic cation transporter OCTN1 in mouse brain neurons: Possible involvement in neuronal differentiation

The aim of the present study is to clarify the functional expression and physiological role in brain neurons of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring antioxidant ergothioneine (ERGO) as a substrate in vivo. After intracerebroventricular administration, the distribution of [ 3H]ERGO in several brain regions of octn1 -/- mice was much lower than that in wild-type mice, whereas extracellular marker [ 14C]mannitol exhibited similar distribution in the two strains. The [ 3H]ERGO distribution in wild-type mice was well correlated with the amount of ERGO derived from food intake and the OCTN1 mRNA level in each brain region. Immunohistochemical analysis revealed colocalization of OCTN1 with neuronal cell markers microtubule-associated protein 2 (MAP2) and βIII-tubulin in mouse brain and primary cultured cortical neurons, respectively. Moreover, cultured cortical neurons exhibited time-dependent and saturable uptake of [ 3H]ERGO. These results demonstrate that OCTN1 is functionally expressed in brain neurons. The addition of ERGO simultaneously with serum to culture medium of cortical neurons attenuated mRNA and protein expressions of MAP2, βIII-tubulin and synapse formation marker synapsin I, and induced those of sex determining region Y-box 2 (Sox2), which is required to maintain the properties of undifferentiated neural stem cells. In neuronal model Neuro2a cells, knockdown of OCTN1 by siRNA reduced the uptake of [ 3H]ERGO with concomitant up-regulation of oxidative stress marker HO-1 and Sox2, and down-regulation of neurite outgrowth marker GAP43. Interestingly, the siRNA knockdown decreased the number of differentiated Neuro2a cells showing long neurites, but increased the total number of cells. Thus, OCTN1 is involved in cellular differentiation, but inhibits their proliferation, possibly via the regulation of cellular oxidative stress. This is the first evidence that OCTN1 plays a role in neuronal differentiation and proliferation, which are required for brain development. Crown Copyright © 2012

Pharmacokinetic modeling of hepatocyte growth factor in experimental animals and humans

Hepatocyte growth factor (HGF) is under development for treatment of renal failure. This study was designed to clarify changes in HGF pharmacokinetics in renal failure and to establish a pharmacokinetic model applicable to single and repeated doses. The plasma concentration profile in mice with glycerol-induced acute renal failure was similar to that in normal mice, indicating a minimal contribution of kidney to systemic clearance of HGF. Nevertheless, accumulation of fluorescein-4-isocyanate-labeled HGF in renal tubules in both cases suggests the occurrence of efficient endocytosis of HGF in kidney. A pharmacokinetic model including plasma and liver compartments was constructed, incorporating both high- and low-affinity receptors for association and subsequent endocytosis of HGF because HGF is eliminated via specific receptor c-Met and heparin-like substance. The model well explained the plasma concentration profiles at all doses examined after bolus injection in animals and humans, and those during infusion in rodents. It includes externalization of receptors, which is negatively regulated by HGF, and can explain the gradual increase in trough concentration during repeated dosing in monkeys. Overall pharmacokinetic profiles of HGF are governed by at least two receptors and are well described by this pharmacokinetic model, which should assist in safe management of clinical trials. © 2012 Wiley Periodicals, Inc

Update on the Keio collection of Escherichia coli single-gene deletion mutants

The Keio collection (Baba et al, 2006) has been established as a set of single‐gene deletion mutants of Escherichia coli K‐12. These mutants have a precisely designed deletion from the second codon from the seventh to the last codon of each predicted ORF. Further information is available at http://sal.cs.purdue.edu:8097/GB7/index.jsp or http://ecoli.naist.jp/. The distribution is now being handled by the National Institute of Genetics of Japan (http://www.shigen.nig.ac.jp/ecoli/pec/index.jsp). To date more than 4 million samples have been distributed worldwide. As we described earlier (Baba et al, 2006), gene amplification during construction is likely to have led to a small number of mutants with genetic duplications

The Escherichia coli K-12 ORFeome: a resource for comparative molecular microbiology

<p>Abstract</p> <p>Background</p> <p>Systems biology and functional genomics require genome-wide datasets and resources. Complete sets of cloned open reading frames (ORFs) have been made for about a dozen bacterial species and allow researchers to express and study complete proteomes in a high-throughput fashion.</p> <p>Results</p> <p>We have constructed an open reading frame (ORFeome) collection of 3974 or 94% of the known <it>Escherichia coli </it>K-12 ORFs in Gateway^®entry vector pENTR/Zeo. The collection has been used for protein expression and protein interaction studies. For example, we have compared interactions among YgjD, YjeE and YeaZ proteins in <it>E. coli</it>, <it>Streptococcus pneumoniae</it>, and <it>Staphylococcus aureus</it>. We also compare this ORFeome with other Gateway-compatible bacterial ORFeomes and show its utility for comparative functional genomics.</p> <p>Conclusions</p> <p>The <it>E. coli </it>ORFeome provides a useful resource for functional genomics and other areas of protein research in a highly flexible format. Our comparison with other ORFeomes makes comparative analyses straighforward and facilitates direct comparisons of many proteins across many genomes.</p

Direct inhibition and down-regulation by uremic plasma components of hepatic uptake transporter for sn-38, an active metabolite of irinotecan, in humans

Purpose: Clinical study has previously revealed that plasma concentration of 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan, was higher in patients with end-stage renal failure than those with normal kidney function although SN-38 is mainly eliminated in the liver. Here, we focused on inhibition by uremic toxins of hepatic SN-38 uptake and down-regulation of uptake transporter(s) by uremic plasma in humans. Methods: We evaluated SN-38 uptake and its inhibition by uremic toxins, 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), indoxyl sulfate (Indox), hippuric acid (HA) and indole acetate (IA), with cryopreserved human hepatocytes and HEK293 cells stably expressing hepatic uptake transporters, organic anion transporting polypeptides (OATPs). We also collected plasma samples from patients with severe renal failure to examine their effects on mRNA level of OATPs in primary cultured human hepatocytes. Results: SN-38 was taken up by hepatocytes, which showed biphasic saturation patterns. The SN-38 uptake by hepatocytes was significantly inhibited by a uremic toxin mixture including clinically relevant concentrations of CMPF, Indox, HA and IA. Kinetic analyses for OATP-mediated transport revealed that the uptake of SN-38 by OATP1B1 was the highest, followed by OATP1B3. Among the uremic toxins, CMPF exhibited most potent inhibition of OATP1B1-mediated SN-38 uptake and directly inhibited the uptake of SN-38 also in hepatocytes. In addition, gene expression of OATP1B1 and OATP1B3 in hepatocytes was significantly down-regulated by the treatment with the uremic plasma. Conclusions: OATP1B1-mediated hepatic uptake of SN-38 was inhibited by uremic toxins, and gene expression of OATP1B1 was down-regulated by uremic plasma. © 2013 Springer Science+Business Media New York

Localization of Xenobiotic Transporter OCTN1/SLC22A4 in Hepatic Stellate Cells and Its Protective Role in Liver Fibrosis

Xenobiotic transporters play key roles in disposition of certain therapeutic agents, although limited information is available on their roles other than pharmacokinetic issues. Here, suppressive effect of multispecific organic cation transporter OCTN1/SLC22A4 on liver fibrosis was proposed in liver injury models. After injection of hepatotoxins such as dimethylnitrosamine (DMN) or concanavalin A, hepatic fibrosis, and oxidative stress, evaluated in terms of Sirius red and 4-hydroxy-2-nonenal staining, respectively, were more severe in liver of octn1/slc22a4 gene knockout (octn1-/-) mice than that in wild-type mice. DMN treatment markedly increased α-smooth muscle actin and F4/80, markers of activated stellate and Kupffer cells, respectively, in liver of octn1-/-, but had less effect in wild-type mice. Thus, octn1/slc22a4 gene deletion results in more severe hepatic fibrosis, oxidative stress, and inflammation. DMN-treated wild-type mice showed increased Octn1 staining and hepatic concentration of its food-derived antioxidant ergothioneine (ERGO). The upregulated Octn1 was co-localized with α-smooth muscle actin. Functional expression of Octn1 was demonstrated in activated human hepatic stellate cell lines, LI90 and LX-2. Provision of ERGO-rich feed ameliorated DMN-induced liver fibrosis and oxidative stress. Overall, Octn1 is upregulated in activated stellate cells, resulting in increased delivery of its substrate antioxidant ERGO and a protective effect against liver fibrosis. © 2016 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.Embargo Period 12 month

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