A novel pathway of chronic allograft rejection mediated by NK cells and alloantibody.

Chronic allograft vasculopathy (CAV) in murine heart allografts can be elicited by adoptive transfer of donor specific antibody (DSA) to class I MHC antigens and is independent of complement. Here we address the mechanism by which DSA causes CAV. B6.RAG1(-/-) or B6.RAG1(-/-)C3(-/-) (H-2(b)) mice received B10.BR (H-2(k)) heart allografts and repeated doses of IgG2a, IgG1 or F(ab')(2) fragments of IgG2a DSA (anti-H-2(k)). Intact DSA regularly elicited markedly stenotic CAV in recipients over 28 days. In contrast, depletion of NK cells with anti-NK1.1 reduced significantly DSA-induced CAV, as judged morphometrically. Recipients genetically deficient in mature NK cells (γ-chain knock out) also showed decreased severity of DSA-induced CAV. Direct NK reactivity to the graft was not necessary. F(ab')(2) DSA fragments, even at doses twofold higher than intact DSA, were inactive. Graft microvascular endothelial cells responded to DSA in vivo by increased expression of phospho-extracellular signal-regulated kinase (pERK), a response not elicited by F(ab')(2) DSA. We conclude that antibody mediates CAV through NK cells, by an Fc dependent manner. This new pathway adds to the possible mechanisms of chronic rejection and may relate to the recently described C4d-negative chronic antibody-mediated rejection in humans.

An unexpected counter-regulatory role of IL-10 in B-lymphocyte-mediated transplantation tolerance.

Monoclonal antibody against the CD45RB protein induces stable transplantation tolerance to multiple types of allograft. We have previously established that this tolerance protocol relies on the regulatory function of B lymphocytes for its effect. B lymphocytes have also been reported to participate in immune regulation in several other settings. In most of these systems, the regulatory function of B lymphocytes depends on the production of IL-10. Therefore, we investigated the role of IL-10 in the anti-CD45RB model of B-cell-mediated transplantation tolerance. Surprisingly, using antibody-mediated neutralization of IL-10, IL-10-deficient recipients and adoptive transfer of IL-10-deficient B lymphocytes, we found that IL-10 actually counter-regulates tolerance induced by anti-CD45RB. Furthermore, neutralization of IL-10 reduced the development of chronic allograft vasculopathy compared to anti-CD45RB alone and reduced the production of graft reactive alloantibodies. These data suggest that the participation of regulatory B lymphocytes in transplantation tolerance may be distinct from how they operate in other systems. Identifying the specific B lymphocytes that mediate transplantation tolerance and defining their mechanism of action may yield new insights into the complex cellular network through which antigen-specific tolerance is established and maintained.

Complement independent antibody-mediated endarteritis and transplant arteriopathy in mice.

Complement fixation, as evidenced by C4d in the microvasculature, is a widely accepted criterion of antibody-mediated rejection. Complement fixation has been shown to be essential in acute antibody-mediated rejection, but its role in chronic rejection has not been addressed. Previous studies showed that passive transfer of complement fixing monoclonal IgG2a anti-H-2Kk into B6.RAG1-/- KO recipients of B10.BR hearts led to progressive chronic transplant arteriopathy (CTA) over 14-28 days, accompanied by C4d deposition. The present studies were designed to test whether complement was required for these lesions. We report that a noncomplement fixing donor-specific alloantibody (DSA, monoclonal IgG1 anti-H-2Kk) injected into B6.RAG1-/- KO recipients of B10.BR hearts also promotes CTA, without C4d deposition. Furthermore, a passive transfer of DSA (monoclonal IgG2a anti-H-2Kk) initiated endarteritis followed by CTA in B6.RAG1-/- mice genetically deficient in the third component of complement (RAG1-/-C3-/-). These studies indicate that antibody to class I MHC antigens can trigger chronic arterial lesions in vivo without complement participation, in contrast to acute antibody-mediated rejection. This pathway may be relevant to C4d-negative chronic rejection sometimes observed in patients with DSA, and argues that lack of C4d deposition does not exclude antibody-mediated chronic rejection.

Viral infection induces de novo lesions of coronary allograft vasculopathy through a natural killer cell-dependent pathway.

Viral infections including those due to cytomegalovirus have been associated with accelerated cardiac allograft vasculopathy (CAV) in clinical trials and some animal models. Evidence demonstrating a direct causal relationship between such infections and de novo formation of coronary vascular lesions is lacking. Heterotopic murine cardiac transplants were performed in a parental to F1 combination in animals lacking both T- and B-lymphocytes (RAG(-/-)). Coronary vasculopathy developed almost exclusively in the presence of recipient infection with lymphocytic choriomeningitis virus but not in uninfected controls. This process was also dependent upon the presence of natural killer (NK) cells as depletion of NK cells abrogated the process. These data show that a viral infection in its native host, and not previously implicated in the production of CAV, can contribute to the development of advanced coronary vascular lesions in cardiac allotransplants in mice. These data also suggest that virus-induced CAV can develop via an NK-cell-dependent pathway in the absence of T- and B-lymphocytes.

Generation of Ag-specific cytotoxic T lymphocytes by DC transfected with in vitro transcribed influenza virus matrix protein (M1) mRNA.

BACKGROUND: Application of DC transfected with tumor Ag RNA is promising for DC-based tumor immunotherapy. In this study, Ag-specific cytotoxic T lymphocytes (CTL) were generated by priming lymphocytes with DC transfected with in vitro transcribed (IVT) influenza virus matrix protein M1 (M1) mRNA. METHODS: Human UC blood-CD34+ cell-derived DC were transfected with IVT mRNA encoding either the enhanced green fluorescence protein (EGFP), or M1 by square-wave electroporation. DC were confirmed to have typical morphology and phenotype. DC transfected with IVT EGFP mRNA were analyzed with the FACScan flow cytometer, to confirm the efficiency of this transfection method. On Days 7, 14, 21 and 28 after the start of DC culture, DC were harvested and electroporated with M1 mRNA. The transfected DC were co-cultured with autologous UC blood CD34- cells. One week after the fourth priming of autologous CD34 negative cells with M1 mRNA electroporated DC, Ag-specific CTL activity was evaluated. To prepare target cells, M1 mRNA was added to autologous DC 48 h prior to CTL assays. RESULTS: Our CTL assays results indicate that UC blood CD34+ cell-derived DC transfected with M1 mRNA by electroporation stimulated Ag-specific CTL responses that are capable of recognizing and lysing autologous DC loaded with M1 mRNA. M1 mRNA transfected DC-primed CTL showed a significant cytotoxic activity against M1 mRNA loaded autologous DC, while nearly baseline cytotoxic activity was recorded for the M1 mRNA unloaded DC. DISCUSSION: Our results showed that mRNA-transfected DC are potent stimulators of T-cell immunity in vitro. In addition, mRNA-loaded DC can function as targets in CTL cytotoxicity assays, which offer a practical substitute for tumor cells in assays to test the immunological effects of specific Ags.

Maize non-photosynthetic ferredoxin precursor is mis-sorted to the intermembrane space of chloroplasts in the presence of light.

Preprotein translocation across the outer and inner envelope membranes of chloroplasts is an energy-dependent process requiring ATP hydrolysis. Several precursor proteins analyzed so far have been found to be imported into isolated chloroplasts equally well in the dark in the presence of ATP as in the light where ATP is supplied by photophosphorylation in the chloroplasts themselves. We demonstrate here that precursors of two maize (Zea mays L. cv Golden Cross Bantam) ferredoxin isoproteins, pFdI and pFdIII, show distinct characteristics of import into maize chloroplasts. pFdI, a photosynthetic ferredoxin precursor, was efficiently imported into the stroma of isolated maize chloroplasts both in the light and in the dark. In contrast pFdIII, a non-photosynthetic ferredoxin precursor, was mostly mis-sorted to the intermembrane space of chloroplastic envelopes as an unprocessed precursor form in the light but was efficiently imported into the stroma and processed to its mature form in the dark. The mis-sorted pFdIII, which accumulated in the intermembrane space in the light, could not undergo subsequent import into the stroma in the dark, even in the presence of ATP. However, when the mis-sorted pFdIII was recovered and used for a separate import reaction, pFdIII was capable of import into the chloroplasts in the dark. pFNRII, a ferredoxin-NADP+ reductase isoprotein precursor, showed import characteristics similar to those of pFdIII. Moreover, pFdIII exhibited similar import characteristics with chloroplasts isolated from wheat (Pennisetum americanum) and pea (Pisum sativum cv Alaska). These findings suggest that the translocation of precursor proteins across the envelope membranes of chloroplasts may involve substrate-dependent light-regulated mechanisms.

Intradiscal pressure after intradiscal injection of hypertonic saline: an experimental study.

Although chemonucleolysis with chymopapain is a long-established treatment for lumbar intervertebral disc herniation, serious complications have been reported. Accordingly, alternative substances for chemonucleolysis have been sought. The main beneficial effect of chemonucleolysis derives from the decrease in intradiscal pressure. Several previous studies have investigated the relationship between physiological saline injection and disc mechanics in cadaveric specimens [2, 5, 16]. However, no previous study has assessed the intradiscal pressure after intradiscal injection of "hypertonic saline" in living animals. The present study compared the changes in intradiscal pressure after intradiscal injection of hypertonic saline with those after chymopapain injection. The lumbar intervertebral discs of 26 living rabbits were examined: 10% hypertonic saline was injected in ten rabbits, and chymopapain (10 pikokatal units) was injected intradiscally in another ten, with the remaining six being used as controls. The intradiscal pressure was measured at 1, 4, and 12 weeks after injection. The intradiscal pressure of the hypertonic saline-injected group at 4 weeks was significantly lower than that of the control group, but by 12 weeks it had recovered. On the other hand, that of the chymopapain-injected group remained significantly lower than that of the control group at 12 weeks. The results of this study found that hypertonic saline injected into the intervertebral discs temporarily decreased the intradiscal pressure.

Biliary excretion of 17beta-estradiol 17beta-D-glucuronide is predominantly mediated by cMOAT/MRP2.

PURPOSE: The mechanism for the biliary excretion of 17beta-estradiol 17beta-D-glucuronide (E(2)17betaG), a cholestatic metabolite of estradiol, is still controversial. The purpose of the present study is to examine the transport of E(2)17betaG across the bile canalicular membrane. METHODS: We examined the uptake of [3H]E(2)17betaG by isolated canalicular membrane vesicles (CMVs) prepared from Sprague-Dawley (SD) rats and Eisai Hyperbilirubinemic rats (EHBR) whose canalicular multispecific organic anion transporter/multidrug resistance associated protein 2 (cMOAT/MRP2) function is hereditarily defective. Also, in vivo biliary excretion of intravenously administered [3H]E(2)17betaG was examined. RESULTS: In CMVs prepared from SD rats, but not from EHBR, a marked ATP-dependent uptake of [3H]E(2)17betaG was observed. Moreover, E(2)17betaG competitively inhibited the ATP-dependent uptake of [3H]2,4-dinitrophenyl-S-glutathione (DNP-SG). In addition, no significant inhibitory effect of verapamil (100 microM) and PSC-833 (5 microM) on the uptake of [3H]E(2)17betaG was observed. In vivo, the biliary excretion of intravenously administered [3H]E(2)17betaG was severely impaired in EHBR while the biliary excretion of [3H]E(2)17betaG in SD rats was reduced by administering a cholestatic dose (10 micromol/kg) unlabeled E(2)17betaG, but not by PSC-833 (3 mg/kg). CONCLUSIONS: The transport of E(2)17betaG across the bile canalicular membrane is predominantly mediated by cMOAT/MRP2.

Effects of holmium(HO)-YAG laser irradiation on rabbit lumbar discs.

To study the effect of laser irradiation on normal lumbar discs, a 2100 nm Holmium (HO)-YAG laser irradiation was applied to the 83 lumbar discs of 23 adult rabbits. The extent of disc vaporization, the temperature changes in the surrounding tissues, and changes in the radiograph and MRI findings were assessed after laser irradiation. When laser irradiation was delivered to the discs, the disc weight decreased linearly with the increase in total laser energy, indicating steady vaporization of disc material. The temperature was highest at the site of the guide needle. Laser irradiation was delivered at 0.5 J/pulse or 1.4 J/pulse x 5 pulses/sec to the intervertebral discs, and radiographs and T2-weighted MRI of the irradiated discs were investigated at 1, 4, and at 12 weeks after irradiation. At 1 week after irradiation at 0.5 and 1.4 J/pulse, the radiographs showed a decrease in the disc height. At 12 weeks after irradiation at 0.5 J/pulse, the disc height had restored to normal, while the decrease was persistent after irradiation at 1.4 J/pulse. At 1 week after irradiation, MRI showed a decrease in the signal intensity of discs treated at 0.5 J/pulse, but the decrease was recovered at 12 weeks. After irradiation at 1.4 J/pulse, the decrease in signal intensity was also recovered by 12 weeks, but the recovery was less than the recovery after treatment at 0.5 J/pulse. Laser irradiation is applicable for the treatment of intervertebral discs, but it is necessary to select the optimal operating conditions. It may also be necessary to change the power of irradiation according to the pathological condition of the disc being treated.

Molecular cloning and characterization of maize Toc34, a regulatory component of the protein import machinery of chloroplast.

Several components of the chloroplast protein import machinery have been identified mostly in the C3 dicot plant pea. We describe here two homologous cDNA sequences of Toc34, a regulatory component of the import machinery, from maize, a C4 monocot plant. The deduced amino acid sequences of two maize Toc34 proteins, named ZmToc34-1 and ZmToc34-2, show 95% identity to each other and about 60% to those of previously identified Toc34 proteins. The GTP-binding motif and the presumed carboxyl terminal membrane anchor are also conserved in the ZmToc34 proteins. The maize Toc34, like that of pea, is tightly associated with the outer envelope membrane of chloroplast and exposed to the cytosol.

Characterization of inducible nature of MRP3 in rat liver.

We found previously that expression of multidrug resistance-associated protein (MRP) 3 is induced in a mutant rat strain (Eisai hyperbilirubinemic rats) whose canalicular multispecific organic anion transporter (cMOAT/MRP2) function is hereditarily defective and in normal Sprague-Dawley (SD) rats after ligation of the common bile duct. In the present study, the inducible nature of MRP3 was examined, using Northern and Western blot analyses, in comparison with that of other secondary active [Na(+)-taurocholic acid cotransporting polypeptide (Ntcp), organic anion transporting polypeptide 1 (oatp1), and organic cation transporter (OCT1)] and primary active [P-glycoprotein (P-gp), cMOAT/MRP2, and MRP6] transporters. alpha-Naphthylisothiocyanate treatment and common bile duct ligation induced expression of P-gp and MRP3, whereas expression of Ntcp, oatp1, and OCT1 was reduced by the same treatment. Although expression of MRP3 was also induced by administration of phenobarbital, that of cMOAT/MRP2, MRP1, and MRP6 was not affected by any of these treatments. Moreover, the mRNA level of MRP3, but not that of P-gp, was increased in SD rats after administration of bilirubin and in Gunn rats whose hepatic bilirubin concentration is elevated because of a defect in the expression of UDP-glucuronosyl transferase. However, the MRP3 protein level was not affected by bilirubin administration. Although the increased MRP3 mRNA level was associated with the increased concentration of bilirubin and/or its glucuronides in mutant rats and in SD rats that had undergone common bile duct ligation or alpha-naphthylisothiocyanate treatment, we must assume that factor(s) other than these physiological substances are also involved in the increased protein level of MRP3.

ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (Mrp3).

We have previously shown that cloned rat multidrug resistance-associated protein 3 (Mrp3) has the ability to transport organic anions such as 17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG) and has a different substrate specificity from MRP1 and MRP2 in that glutathione conjugates are poor substrates for Mrp3 (Hirohashi, T., Suzuki, H., and Sugiyama, Y. (1999) J. Biol. Chem. 274, 15181-15185). In the present study, the involvement of Mrp3 in the transport of endogenous bile salts was investigated using membrane vesicles from LLC-PK1 cells transfected with rat Mrp3 cDNA. The ATP-dependent uptake of [(3)H]taurocholate (TC), [(14)C]glycocholate (GC), [(3)H]taurochenodeoxycholate-3-sulfate (TCDC-S), and [(3)H]taurolithocholate-3-sulfate (TLC-S) was markedly stimulated by Mrp3 transfection in LLC-PK1 cells. The extent of Mrp3-mediated transport of bile salts was in the order, TLC-S > TCDC-S > TC > GC. The K(m) and V(max) values for the uptake of TC and TLC-S were K(m) = 15.9 +/- 4.9 microM and V(max) = 50.1 +/- 9.3 pmol/min/mg of protein and K(m) = 3.06 +/- 0.57 microM and V(max) = 161.9 +/- 21.7 pmol/min/mg of protein, respectively. At 55 nM [(3)H]E(2)17betaG and 1.2 microM [(3)H]TC, the apparent K(m) values for ATP were 1.36 and 0.66 mM, respectively. TC, GC, and TCDC-S inhibited the transport of [(3)H]E(2)17betaG and [(3)H]TC to the same extent with an apparent IC(50) of approximately 10 microM. TLC-S inhibited the uptake of [(3)H]E(2)17betaG and [(3)H]TC most potently (IC(50) of approximately 1 microM) among the bile salts examined, whereas cholate weakly inhibited the uptake (IC(50) approximately 75 microM). Although TC and GC are transported by bile salt export pump/sister of P-glycoprotein, but not by MRP2, and TCDC-S and TLC-S are transported by MRP2, but not by bile salt export pump/sister of P-glycoprotein, it was found that Mrp3 accepts all these bile salts as substrates. This information, together with the finding that MRP3 is extensively expressed on the basolateral membrane of human cholangiocytes, suggests that MRP3/Mrp3 plays a significant role in the cholehepatic circulation of bile salts.

Function and expression of multidrug resistance-associated protein family in human colon adenocarcinoma cells (Caco-2).

Several organic anions are actively extruded from intestinal epithelial cells into the lumen and vascular sides. To examine the role of the multidrug resistance-associated protein (MRP) family in the intestinal efflux of organic anions, the function and expression of these proteins were investigated with Caco-2, a human adenocarcinoma cell line that retains many of the characteristics of normal enterocytes. [(3)H]2,4-Dinitrophenyl-S-glutathione (DNP-SG) and [(3)H]17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG), typical substrates for MRP1 and cMOAT (canalicular multispecific organic anion transporter)/MRP2, were taken up into brush-border membrane vesicles (BBMVs) from Caco-2 in an ATP-dependent manner, with K(m) values of 16.9 +/- 7.2 and 9.4 +/- 1.2 microM, respectively. The uptake of [(3)H]DNP-SG into BBMVs was osmotically sensitive and stimulated to some extent by other nucleotide triphosphates (GTP, CTP, and UTP) but not by ADP or AMP. An ATPase inhibitor, vanadate, inhibited the ATP-dependent uptake of [(3)H]DNP-SG to some extent. Reverse-transcriptase polymerase chain reaction resulted in the amplification of MRP1, MRP3, and MRP5. Northern blot analysis indicated extensive expression of cMOAT/MRP2 and MRP3 and only minimal expression of MRP1 and MRP5. Although cMOAT/MRP2 was continuously expressed throughout the culture period, MRP3 was not expressed immediately after the confluent state was reached. Collectively, the presence of ATP-dependent transport systems for DNP-SG and E(2)17betaG was demonstrated in Caco-2 cells. Because cMOAT/MRP2 and MRP3 may be expressed on brush-border and basolateral membranes in epithelial cells, respectively, the transport activity associated with BBMVs may result from the function of cMOAT/MRP2.

Involvement of an organic anion transporter (canalicular multispecific organic anion transporter/multidrug resistance-associated protein 2) in gastrointestinal secretion of glutathione conjugates in rats.

We investigated the role of cMOAT/MRP2 (canalicular multispecific organic anion transporter/multidrug resistance-associated protein 2) in the intestinal secretion of organic anions by comparing the behavior in Sprague-Dawley (SD) rats and Eisai hyperbilirubinemic rat (EHBR) whose cMOAT/MRP2 is hereditarily defective. After i.v. administration of 1-chloro-2,4-dinitrobenzene (30 micromol/kg), the biliary and intestinal excretion of its glutathione conjugate 2, 4-dinitrophenyl-S-glutathione (DNP-SG), a substrate for cMOAT/MRP2, was significantly reduced in EHBR compared with SD rats. This result also was confirmed by Ussing chamber studies; DNP-SG showed 1.5-fold greater serosal-to-mucosal flux compared with the mucosal-to-serosal flux in SD rats, whereas a similar flux was observed in both directions in EHBR. In addition, metabolic inhibitors reduced the preferential serosal-to-mucosal flux of DNP-SG in SD rats. In everted sac studies, intestinal secretion clearance, defined as the efflux rate of DNP-SG into the mucosal side divided by the area under the curve on the serosal side, was significantly lower in the jejunum of EHBR than that in SD rats. Northern blot analyses demonstrated the highest mRNA level of cMOAT/MRP2 in the jejunum, which is in good agreement with the results of the everted sac studies. These results suggest that cMOAT/MRP2 is involved in the secretion of organic anions in the small intestine.

Chloroplast chaperonins: evidence for heterogeneous assembly of alpha and beta Cpn60 polypeptides into a chaperonin oligomer.

Higher plant chloroplasts contain two chaperonin 60 family proteins, Cpn60alpha and Cpn60beta, which are known to have divergent amino acid sequences. Although Cpn60alpha and Cpn60beta are present in roughly equal amounts and copurify in their native states, a heterogeneous ensemble of the chaperonin oligomer has not yet been demonstrated. We separately purified Cpn60alpha and Cpn60beta proteins from spinach leaves as the monomeric form. Either antibody raised against one chaperonin 60 protein could coimmunoprecipitate the other chaperonin 60 protein in their oligomeric state but not in its monomeric state, suggesting that the chloroplast Cpn60alpha and Cpn60beta polypeptides actually reside in the same chaperonin oligomer in the stroma. Moreover, the chaperonin oligomers migrated as at least two distinct bands on the native gel electrophoresis, each of which contained both chaperonin 60 proteins. These results suggest that chloroplast chaperonin oligomers might be composed of at least two distinct sets of two chaperonin proteins.

Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3).

We have previously cloned rat MRP3 as an inducible transporter in the liver (Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068-1075). In the present study, the function of rat MRP3 was investigated using membrane vesicles isolated from LLC-PK1 and HeLa cell population transfected with corresponding cDNA. The ATP-dependent uptake of both 17beta estradiol 17-beta-D-glucuronide ([3H]E217betaG) and glucuronide of [14C] 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), but not that of [3H]leukotriene C4 and [3H]2, 4-dinitrophenyl-S-glutathione, was markedly stimulated by MRP3 transfection in both cell lines. The Km and Vmax values for the uptake of [3H]E217betaG were 67 +/- 14 microM and 415 +/- 73 pmol/min/mg of protein, respectively, for MRP3-expressing membrane vesicles and 3.0 +/- 0.7 microM and 3.4 +/- 0.4 pmol/min/mg of protein, respectively, for the endogenous transporter expressed on HeLa cells. [3H]E217betaG had also a similar Km value for MRP3 when LLC-PK1 cells were used as the host. All glucuronide conjugates examined (E3040 glucuronide, 4-methylumbelliferone glucuronide, and naphthyl glucuronide) and methotrexate inhibited MRP3-mediated [3H]E217betaG transport in LLC-PK1 cells. Moreover, [3H]methotrexate was transported via MRP3. The inhibitory effect of estrone sulfate, [3H]2,4-dinitrophenyl-S-glutathione, and [3H]leukotriene C4 was moderate or minimal, whereas N-acetyl-2,4-dinitrophenylcysteine had no effect on the uptake of [3H]E217betaG. The uptake of [3H]E217betaG was enhanced by E3040 sulfate and 4-methylumbelliferone sulfate. Thus we were able to demonstrate that several kinds of organic anions are transported via MRP3, although the substrate specificity of MRP3 differs from that of MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for MRP3.

Involvement of a chloroplast homologue of the signal recognition particle receptor protein, FtsY, in protein targeting to thylakoids.

We isolated an Arabidopsis thaliana cDNA whose translated product shows sequence similarity to the FtsY, a bacterial homologue of SRP receptor protein. The Arabidopsis FtsY homologue contains a typical chloroplast transit peptide. The in vitro-synthesized 37 kDa FtsY homologue was imported into chloroplasts, and the processed 32 kDa polypeptide bound peripherally on the outer surface of thylakoids. Antibodies raised against the FtsY homologue also reacted with a thylakoid-bound 32 kDa protein. The antibodies inhibited the cpSRP-dependent insertion of the light-harvesting chlorophyll alb-binding protein into thylakoid membranes suggesting that the chloroplast FtsY homologue is involved in the cpSRP-dependent protein targeting to the thylakoid membranes.

cDNA sequence and overexpression of chloroplast chaperonin 21 from Arabidopsis thaliana.

Higher plant chloroplasts contain a 21-kDa protein, chaperonin 21 (Cpn21), that is a functional homolog of the chaperonin 10 (Cpn10). The chloroplast Cpn21 polypeptide consists of two Cpn10-like domains fused together in tandem. We describe here the cDNA sequence of the Cpn21 (AtCpn21) precursor protein from Arabidopsis thaliana. The deduced amino acid sequence of the AtCpn21 precursor protein, 253 amino acids long, shows 61% identity with the spinach Cpn21 protein. The AtCpn21 precursor protein contains the typical chloroplast transit peptide of 51 amino acids at its aminoterminus and the two Cpn10-like domains which exhibits 46% sequence identity to each other. The predicted mature-sized polypeptide of AtCpn21 was expressed in Escherichia coli as a soluble 21-kDa protein. Gel-filtration and chemical cross-linking analyses showed that the recombinant mature AtCpn21 protein forms a stable homo-oligomer composed of three or four polypeptides.

cDNA cloning and inducible expression of human multidrug resistance associated protein 3 (MRP3).

Previously, we cloned rat MRP3 as a candidate for an inducible transporter for the biliary excretion of organic anions [Hirohashi et al. (1998) Mol. Pharmacol. 53, 1068-10751. In the present study, we cloned human MRP3 (1527 amino acids) from Caco-2 cells. Human MRP3 is predominantly expressed in liver, small intestine and colon; hepatic expression of MRP3 was observed in humans but not in normal rats. In HepG2 cells, the expression of MRP3 was induced by phenobarbital. These results suggest that MRP3 may act as an inducible transporter in the biliary and intestinal excretion of organic anions.

Hepatic expression of multidrug resistance-associated protein-like proteins maintained in eisai hyperbilirubinemic rats.

The biliary excretion of several organic anions is mediated by the canalicular multispecific organic anion transporter (cMOAT), which is hereditarily defective in mutant rats such as Eisai hyperbilirubinemic rats (EHBR). In addition, using a kinetic study with isolated canalicular membrane vesicles, we recently suggested the presence of ATP-dependent organic anion transporter(s) other than cMOAT in EHBR [Pharm Res (NY) 12:1746-1755 (1995); J Pharmacol Exp Ther 282:866-872 (1997)]. The aim of this study is to provide a molecular basis for the presence of multiplicity in the biliary excretion of organic anions in rats. Based on the homology with human multidrug resistance-associated protein (hMRP), two cDNA fragments encoding the carboxyl-terminal ATP-binding cassette region were amplified by reverse transcription-polymerase chain reaction from EHBR liver. These fragments exhibited approximately 70% amino acid identity with hMRP and rat cMOAT;, therefore, they were designated MRP-like proteins (MLP-1 and MLP-2). The cloned full length cDNA of MLP-1 and -2 from the Sprague-Dawley (SD) rat liver and colon cDNA library was composed of 1502 and 1523 amino acids, respectively, had the characteristics of ATP-binding cassette transporters, and exhibited homology with hMRP and rat cMOAT. Northern blot analysis indicated that MLP-1 is expressed predominantly in the liver in both SD rats and EHBR, whereas hepatic expression of MLP-2 was observed only in EHBR. In addition, MLP-2 was markedly induced by ligation of the bile duct in SD rat liver. In both SD rats and EHBR, MLP-2 was expressed predominantly in the duodenum, jejunum, and colon. These findings suggest that MLP-1 and MLP-2 might be novel members of the MRP family responsible for the excretion of organic anions from these epithelial cells, and that MLP-2 is an inducible one.