Cloning and functional characterization of human sodium-dependent organic anion transporter (SLC10A6)

We have cloned human sodium-dependent organic anion transporter (SOAT) cDNA, which consists of 1502 bp and encodes a 377-amino acid protein. SOAT shows 42% sequence identity to the ileal apical sodium-dependent bile acid transporter ASBT and 33% sequence identity to the hepatic Na(+)/taurocholate-cotransporting polypeptide NTCP. Immunoprecipitation of a SOAT-FLAG-tagged protein revealed a glycosylated form at 46 kDa that decreased to 42 kDa after PNGase F treatment. SOAT exhibits a seven-transmembrane domain topology with an outside-to-inside orientation of the N-terminal and C-terminal ends. SOAT mRNA is most highly expressed in testis. Relatively high SOAT expression was also detected in placenta and pancreas. We established a stable SOAT-HEK293 cell line that showed sodium-dependent transport of dehydroepiandrosterone sulfate, estrone-3-sulfate, and pregnenolone sulfate with apparent K(m) values of 28.7, 12.0, and 11.3 microm, respectively. Although bile acids, such as taurocholic acid, cholic acid, and chenodeoxycholic acid, were not substrates of SOAT, the sulfoconjugated bile acid taurolithocholic acid-3-sulfate was transported by SOAT-HEK293 cells in a sodium-dependent manner and showed competitive inhibition of SOAT transport with an apparent K(i) value of 0.24 mum. Several nonsteroidal organosulfates also strongly inhibited SOAT, including 1-(omega-sulfooxyethyl)pyrene, bromosulfophthalein, 2- and 4-sulfooxymethylpyrene, and alpha-naphthylsulfate. Among these inhibitors, 2- and 4-sulfooxymethylpyrene were competitive inhibitors of SOAT, with apparent K(i) values of 4.3 and 5.5 microm, respectively, and they were also transported by SOAT-HEK293 cells.     

Establishment of the Amplified Fragment Length Polymorphism (AFLP) technique for genotyping of pollen beetle (Meligethes aeneus) - a noxious insect pest on oilseed rape (Brassica napus)

In order to conduct studies concerning genetic variability of pollen beetles (Meligethes aeneus), a genotyping protocol was established. No genome information is available for pollen beetles so the amplified fragment length polymorphism (AFLP) technique was chosen since it does not depend on any prior sequence information of the samples and also is a sensitive and robust technique. However, several modifications were needed in order to adapt the method for analysis of pollen beetles. Basic modifications included (i) alterations of DNA purification, (ii) use of two six-cutter restriction enzymes, (iii) and modified PCR conditions. This protocol resulted in a favourable number of fragments of an appropriate size range for standard gel analysis by a DNA sequencer applicable to a single insect and even body parts enabling different assays to be conducted on a single specimen. Pollen beetles from different areas of Sweden were analysed to verify the reproducibility and efficacy of the protocol as well as for phenetic analysis. The high reproducibility of the modified AFLP protocol allows it to be used as a reliable tool for genotype analysis of pollen beetles.     

The heparan sulfate proteoglycan (HSPG) glypican‐3 mediates commitment of MC3T3‐E1 cells toward osteogenesis

Heparan sulfate (HS) sugar chains attached to core proteoglycans (PGs) termed HSPGs mediate an extensive range of cell–extracellular matrix (ECM) and growth factor interactions based upon their sulfation patterns. When compared with non‐osteogenic (maintenance media) culture conditions, under established osteogenic culture conditions, MC3T3‐E1 cells characteristically increase their osteogenic gene expression profile and switch their dominant fibroblast growth factor receptor (FGFR) from FGFR1 (0.5‐fold decrease) to FGFR3 (1.5‐fold increase). The change in FGFR expression profile of the osteogenic‐committed cultures was reflected by their inability to sustain an FGF‐2 stimulus, but respond to BMP‐2 at day 14 of culture. The osteogenic cultures decreased their chondroitin and dermatan sulfate PGs (biglycan, decorin, and versican), but increased levels of the HS core protein gene expression, in particular glypican‐3. Commitment and progress through osteogenesis is accompanied by changes in FGFR expression, decreased GAG initiation but increased N‐ and O‐sulfation and reduced remodeling of the ECM (decreased heparanase expression) resulting in the production of homogenous (21 kDa) HS chain. With the HSPG glypican‐3 expression strongly upregulated in these processes, siRNA was used to knockdown this gene to examine the effect on osteogenic commitment. Reduced glypican‐3 abrogated the expression of Runx2, and thus differentiation. The reintroduction of this HSPG into Runx2‐null cells allowed osteogenesis to proceed. These results demonstrate the dependence of osteogenesis on specific HS chains, in particular those associated with glypican‐3. J. Cell. Physiol. 220: 780–791, 2009. © 2009 Wiley‐Liss, Inc.     

Pheno-and genotyping of a placental detoxication system in ecologically unfavorable regions of Ukraine

During 1991-1993 period a study of detoxifying activity of the fetoplacental barrier and genotyping of the major detoxifying enzymes in it (CYP1A1 Ile462Val, GSTP1 Ile104Val, GSTM1 present/absent) was undertaken in different regions of Ukraine that were radioactively contaminated with summary effective equivalent annual expositional doses (SEEAED approximately 1.7 mSv (Group I) and 0.1-0.4 mSv (Group III), chemically polluted Zaporizzhia, monitored for ambient levels of benzo(a)pyrene (BP) (Group II) and Poltava that was judged as "clean" one (Group IV). Glutathione-S-transferase (GSTase) and glutathionereductase (GSSG-Rase) activities of cytosol and concentration of thiobarbituric acid reacting compounds (TBA-reactants) and reduced low-molecular weight thiols (rLMW thiols) were used as phenotype parameters. Cytosolic GSTase activities were nearly two times less in the samples from radioactively contaminated area (Group I, SEEAED approximately 1.7 mSv) and in chemically polluted area (Group II, mean BP level 12.3 ng/m3), compared with the groups III and IV. The highest level of TBA-reactants indicative of lipid peroxidation in response to radiation was observed in Group I, while the lowest level in Group IV. The level of rLMW thiols was 2.5-4 times more in Group II comparative with Groups I, III and IV. The frequency of the genotypes in all the investigated samples corresponds to that reported for Caucasians. For the combined exposure groups, individuals with the CYP1A1 (Ile462Val) genotype (n = 5) had significantly higher levels of GST, GSSG-R and TBA reacting compounds compared to individuals with the Ile462Ile genotype (n = 14 for TBA-reactants and n = 24--for GST and GSSG-R). Despite the challenge of small numbers of individuals, stratification by exposure group for Groups I, II and III indicated significantly higher GST levels in CYP1A1 (Ile462Val) variants from Groups II and III (n = 3) compared to the Ile462Ile variants (n = 17). The data demonstrate contributions by both exposure and genotype on the detoxification of radiation and chemical damage in the human placenta.     

Different pathways of homologous recombination are used for the repair of double-strand breaks within tandemly arranged sequences in the plant genome

Different DNA repair pathways that use homologous sequences in close proximity to genomic double-strand breaks (DSBs) result in either an internal deletion or a gene conversion. We determined the efficiency of these pathways in somatic plant cells of transgenic Arabidopsis lines by monitoring the restoration of the beta-glucuronidase (GUS) marker gene. The transgenes contain a recognition site for the restriction endonuclease I-SceI either between direct GUS repeats to detect deletion formation (DGU.US), or within the GUS gene to detect gene conversion using a nearby donor sequence in direct or inverted orientation (DU.GUS and IU.GUS). Without expression of I-SceI, the frequency of homologous recombination (HR) was low and similar for all three constructs. By crossing the different lines with an I-SceI expressing line, DSB repair was induced, and resulted in one to two orders of magnitude higher recombination frequency. The frequencies obtained with the DGU.US construct were about five times higher than those obtained with DU.GUS and IU.GUS, irrespective of the orientation of the donor sequence. Our results indicate that recombination associated with deletions is the most efficient pathway of homologous DSB repair in plants. However, DSB-induced gene conversion seems to be frequent enough to play a significant role in the evolution of tandemly arranged gene families like resistance genes.