Controlled conductivity at low pH in Protein L chromatography enables separation of bispecific and other antibody formats by their binding valency.

The complex molecular formats of recent therapeutic antibodies, including bispecific antibodies, antibody fragments, and other fusion proteins, makes the task of purifying the desired molecules in a limited number of purification steps more and more challenging. Manufacturing these complicated biologics can be substantially improved in the affinity capture stage if the simple bind-and-elute mode is accompanied by targeted removal of the impurities, such as mis-paired antibodies and oligomers or aggregates. Here, we report a method, based on the binding valency to Protein L resin, of separating proteins during the elution step by simply controlling the conductivity at low pH.

Transport of carnitine and acetylcarnitine by carnitine/organic cation transporter (OCTN) 2 and OCTN3 into epididymal spermatozoa.

Carnitine and acetylcarnitine are important for the acquisition of motility and maturation of spermatozoa in the epididymis. In this study, we examined the involvement of carnitine/organic cation transporter (OCTN) in carnitine and acetylcarnitine transport in epididymal spermatozoa of mice. Uptake of both compounds by epididymal spermatozoa was time-dependent and partially Na(+)-dependent.

Viral envelope protein gp64 transgenic mouse facilitates the generation of monoclonal antibodies against exogenous membrane proteins displayed on baculovirus.

We have been investigating the functional display of multipass membrane protein such as transporter or G-protein coupled receptor on the budded baculovirus (BV). We tested the use of a viral envelope protein gp64 transgenic mouse for the direct immunization of these membrane proteins displayed on BVs. The gp64 transgenic mice showed only a weak response to virus compared to wild type BALB/c mice.

Organic anion transporting polypeptide-C mediates arsenic uptake in HEK-293 cells.

Arsenic is an established human carcinogen. The role of aquaglyroporins (AQPs) in arsenic disposition was recently identified. In order to examine whether organic anion transporting polypeptide-C (OATP-C) also plays a role in arsenic transport, OATP-C cDNA was transfected into cells of a human embryonic kidney cell line (HEK-293).

Recovery of functional peptide transporter PepT1 in budded baculovirus fraction.

Transporters play a critical role in many physiological and pathological states and expression of the functional transporter protein is essential in exploring its kinetics and developing effective drugs. We describe here the recovery of functional transporter protein in the baculovirus fraction. We introduced a gene encoding human peptide transporter PepT1, important for the absorption of protein hydrolytic products or peptide-mimetic drugs, into a baculovirus vector.

Carnitine/xenobiotics transporters in the human mammary gland epithelia, MCF12A.

The barrier function of the human mammary gland collapses if challenged with cationic drugs, causing their accumulation in milk. However, underlying molecular mechanisms are not well understood. To gain insight into the mechanism, we characterized transport of organic cations in the MCF12A human mammary gland epithelial cells, using carnitine and tetraethylammonium (TEA) as representative nutrient and xenobiotics probes, respectively.

OCTN2-mediated transport of carnitine in isolated Sertoli cells.

Carnitine is extensively accumulated in epididymis. Carnitine is also accumulated in testis at higher concentration than in the plasma and is used in spite of the presence of the blood-testis barrier. In this study, we examined the characteristics of carnitine transport in primary-cultured rat Sertoli cells, which constitute a part of the blood-testis barrier.

Involvement of OCTN1 (SLC22A4) in pH-dependent transport of organic cations.

OCTN1 (SLC22A4) transports cationic compounds such as tetraethylammonium in a pH-sensitive and sodium-independent manner in cultured cells, and is expressed in wide variety of tissues, including kidney, muscle, placenta, heart, and others. This study focused on the clarification of its subcellular distribution in kidney and on its driving force to throw light on the pharmacological and physiological roles of OCTN1. Uptake of [14C]tetraethylammonium by membrane vesicles prepared from HEK293 cells stably transfected with human OCTN1 cDNA was osmolarity-sensitive, and the Km of tetraethylammonium was 1.

Expression of organic cation transporter OCTN1 in hematopoietic cells during erythroid differentiation.

Objective: Organic cation/carnitine transporter, OCTN1 (SLC22A4) shows a relatively broad tissue distribution and transports organic cations in a pH-dependent manner. However, its physiological role remains to be clarified. To understand the physiological role of OCTN1, tissue expression of OCTN1 in human and mice was characterized.

A novel therapeutic approach for thrombocytopenia by minibody agonist of the thrombopoietin receptor.

Antibodies have brought valuable therapeutics in the clinical treatment of various diseases without serious adverse effects through their intrinsic features such as specific binding to the target antigen with high affinity, clinical safety as serum proteins, and long half-life. Agonist antibodies, furthermore, could be expected to maximize the value of therapeutic antibodies. Indeed, several IgG/IgM antibodies have been reported to induce cellular growth/differentiation and apoptosis.

Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma.

For detection of hepatocellular carcinoma (HCC) in patients with liver cirrhosis, serum alpha-fetoprotein has been widely used, but its sensitivity has not been satisfactory, especially in small, well-differentiated HCC, and complementary serum marker has been clinically required. Glypican-3 (GPC3), a heparan sulfate proteoglycan anchored to the plasma membrane, is a good candidate marker of HCC because it is an oncofetal protein overexpressed in HCC at both the mRNA and protein levels. In this study, we demonstrated that its NH(2)-terminal portion [soluble GPC3 (sGPC3)] is cleaved between Arg(358) and Ser(359) of GPC3 and that sGPC3 can be specifically detected in the sera of patients with HCC.

Involvement of organic anion transporting polypeptides in the transport of troglitazone sulfate: implications for understanding troglitazone hepatotoxicity.

Troglitazone is a thiazolidinedione insulin sensitizer drug that is metabolized mainly to a sulfate conjugate (M-1) in humans. It was reported to cause hepatotoxicity, although the cause has not been fully clarified. The objective of this study was to identify whether organic anion transporting polypeptide (OATP) transporters expressed at the basolateral membrane of human hepatocytes participate in troglitazone-associated hepatotoxicity.

Functional characterization of pH-sensitive organic anion transporting polypeptide OATP-B in human.

The pH-sensitive activity of human organic anion transporting polypeptide OATP-B, which is expressed at the apical membrane of human small intestinal epithelial cells, was functionally characterized. When initial uptake of estrone-3-sulfate, a typical substrate of OATP, was studied kinetically, we observed an increase in V(max) with decrease of pH from 7.4 to 5.

Contribution of organic anion transporting polypeptide OATP-C to hepatic elimination of the opioid pentapeptide analogue [D-Ala2, D-Leu5]-enkephalin.

The objective of this study was to examine the transport activity of the human organic anion transporter OATP-C (SLC21A6) for oligopeptides that are eliminated rapidly from the systemic circulation. We focused on an opioid peptide analogue, [D-Ala(2), D-Leu(5)]-enkephalin (DADLE), a linear pentapeptide modified to be stable. [(3)H]DADLE was taken up by rat isolated hepatocytes in a saturable manner and highly accumulated in the liver after intravenous administration to rats.

Involvement of human organic anion transporting polypeptide OATP-B (SLC21A9) in pH-dependent transport across intestinal apical membrane.

Some organic anions are absorbed from the gastrointestinal tract through carrier-mediated transport mechanism(s), which may include proton-coupled transport, anion exchange transport, and others. However, the molecular identity of the organic anion transporters localized at the apical membrane of human intestinal epithelial cells has not been clearly demonstrated. In the present study, we focused on human organic anion transporting polypeptide OATP-B and examined its subcellular localization and functionality in the small intestine.

Interaction of activator of G-protein signaling 3 (AGS3) with LKB1, a serine/threonine kinase involved in cell polarity and cell cycle progression: phosphorylation of the G-protein regulatory (GPR) motif as a regulatory mechanism for the interaction of GPR motifs with Gi alpha.

Activator of G-protein signaling 3 (AGS3) has a modular domain structure consisting of seven tetratricopeptide repeats (TPRs) and four G-protein regulatory (GPR) motifs. Each GPR motif binds to the alpha subunit of Gi/Go (Gialpha > Goalpha) stabilizing the GDP-bound conformation of Galpha and apparently competing with Gbetagamma for GalphaGDP binding. As an initial approach to identify regulatory mechanisms for AGS3-G-protein interactions, a yeast two-hybrid screen was initiated using the TPR and linker region of AGS3 as bait.

Studies on functional sites of organic cation/carnitine transporter OCTN2 (SLC22A5) using a Ser467Cys mutant protein.

The organic cation/carnitine transporter OCTN2 mediates transport of carnitine and organic cations in Na(+)-dependent and Na(+)-independent manners, respectively. However, the mechanism of molecular recognition of different substrates has not been clarified yet. We previously found a single amino acid change in OCTN2, Ser467Cys (S467C), in the Japanese population and observed a decreased carnitine transport but unchanged organic cation transport compared with wild type.

Genetic polymorphisms of human organic anion transporters OATP-C (SLC21A6) and OATP-B (SLC21A9): allele frequencies in the Japanese population and functional analysis.

Genetic polymorphisms of human organic anion transporting polypeptides OATP-C (SLC21A6) and OATP-B (SLC21A9) in the Japanese population were analyzed. The allele frequencies of OATP-C*1a, OATP-C*1b (N130D), OATP-C*1c (R152K and D241N), and OATP-C*5 (V174A) were 35.2, 53.

Role of Lkb1, the causative gene of Peutz-Jegher's syndrome, in embryogenesis and polyposis.

Peutz-Jeghers syndrome (PJS) is a dominantly inherited human disorder characterized by gastrointestinal hamartomatous polyposis and mucocutaneous melanin pigmentation. LKB1 (STK11) serine/threonine kinase is the product of the causative gene of PJS, which has been mapped to chromosome 19p13.3.