OCT (22-oxa-calcitriol), a vitamin D analog, has been reported to show strong inhibitory effects on mesangial cell proliferation in vitro.In the present study, we report a study of the effect of OCT on anti-thy-1 glomerulonephritis. Both OCT and 1,25(OH) 2 D 3 significantly inhibited mesangial cell proliferation, the degree of glomerulosclerosis, and albuminuria at day 8 compared to the disease control group. The OCTtreated group showed normal calcium levels but the 1,25(OH) 2 D 3 -treated group showed higher levels. The disease control group showed a marked increase of type I and type IV collagens, and ␣-smooth muscle actin (␣-SMA) compared to the normal group. The treatment of OCT or 1,25(OH) 2 D 3 significantly reduced the expression of these proteins. The mRNA of the glomeruli of anti-thy-1 model expressed significantly higher levels of type I and type IV collagens, and ␣-SMA at day 8 compared to normal rats. Treatment with OCT or 1,25(OH) 2 D 3 inhibited the mRNA expressions of type I and type IV collagens, as well as that of ␣-SMA. These data demonstrate that OCT inhibits mesangial cell proliferation and extracellular matrix expansion with a low calcemic activity. Disease control rats showed significantly increased levels of transforming growth factor-␤1 protein in the glomeruli, but treatment with OCT or 1,25(OH) 2 D 3 markedly reduced this expression. The levels of mRNA in glomeruli were also consistent with these protein levels. Therefore, the suppressive effect of OCT may be mediated by inhibition of transforming growth factor-␤1. The present results suggest that OCT has potential for use in therapeutic strategy for the treatment of glomerulonephritis without inducing hypercalcemia. (Am J Pathol 2001, 158:1733-1741) A number of progressive glomerular diseases are characterized by initial mesangial cell proliferation, which is followed by glomerulosclerosis, and finally develops to end-stage kidney disease. Examples of this process are membranoproliferative glomerulonephritis, diabetic nephropathy, and IgA nephropathy.1,2 Nonanticoagulant heparin, a potent in vitro inhibitor of mesangial cell proliferation, inhibits mesangial cell proliferation and matrix expansion in mesangioproliferative glomerulonephritis. 3Treatment with anti-platelet-derived growth factor also blocks mesangial cell proliferation in vivo, preventing the development of glomerulosclerosis. 4 This suggests that mesangial cell proliferation may play an important role in the development of glomerular lesions. Therefore, the search for agents capable of inhibiting mesangial cell proliferation is clinically important for progressive glomerular change.Recently, 1,25(OH) 2 D 3 was shown to have a preventive effect in progressive glomerular damage in a renal ablation model. 5 However, 1,25(OH) 2 D 3 has an adverse effect in that its use leads to hypercalcemia and hyperphosphatemia, which eventually represent risk factors for the progression of renal injury. It is, therefore, difficult to use 1,25(OH) 2 D 3 for the practical treatment of patie...

Site-specific recombination requires conserved DNA sequences specific to each system, and system-specific proteins that recognize specific DNA sequences. The site-specific recombinases seem to fall into at least two families, based on their protein structure and chemistry of strand breakage. One of these is the resolvase-invertase family, members of which seem to form a serine-phosphate linkage with DNA. Members of the other family, called the integrase family, contain a conserved tyrosine residue that forms a covalent linkage with the 3'-phosphate of DNA at the site of recombination. Structural comparison of integrases shows that these proteins share a highly conserved 40-residue motif. V-(D)-J recombination of the immunoglobulin gene requires conserved recombination signal sequences (RS) of a heptamer CACTGTG and a T-rich nonamer GGTTTTTGT, which are separated by a spacer sequence of either 12 or 23 bases We have recently purified, almost to homogeneity, a protein that specifically binds to the immunoglobulin J kappa RS containing the 23-base-pair spacer sequence. By synthesizing probes on the basis of partial amino-acid sequences of the purified protein, we have now isolated and characterized the complementary DNA of this protein. The amino-acid sequence deduced from the cDNA sequence reveals that the J kappa RS-binding protein has a sequence similar to the 40-residue motif of integrases of phages, bacteria and yeast, indicating that this protein could be involved in V-(D)-J recombination as a recombinase.

Organic anion transporters in the kidney proximal tubule play an essential role in eliminating a wide range of organic anions including endogenous compounds, xenobiotics, and their metabolites, thereby preventing their potentially toxic effects within the body. We have previously cloned a cDNA encoding an organic anion transporter from mouse kidney (mOAT) (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J. G., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478; Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519 -1524). In the present study, we assessed the potential for regulation of this transporter by heterologous expression of mOAT in the pig proximal tubule-like cell line, LLC-PK 1 . We report here that both protein phosphatase (PP1/PP2A) inhibitor, okadaic acid, and protein kinase C (PKC) activators down-regulate mOAT-mediated transport of para-aminohippuric acid (PAH), a prototypic organic anion, in a time-and concentrationdependent manner. However their mechanisms of action for this down-regulation are distinct. Okadaic acid modulated PAH transport, at least in part, through phosphorylation/dephosphorylation of mOAT; phosphoamino acid analysis indicated this phosphorylation occurs on serine. In contrast, PKC activation induced a decrease in the maximum transport velocity (V max ) of PAH transport without direct phosphorylation of the transporter protein. Together these results provide the first demonstration that regulation of organic anion transport by mOAT is likely to be tightly controlled directly and indirectly by phosphatase PP1/PP2A and PKC. Our results also suggest that kinases other than PKC are involved in this process.Renal organic anion transport plays a vital role in the elimination of a wide variety of potentially toxic and negatively charged waste products of metabolism, drugs, environmental pollutants, and their metabolites from the body. The transport mechanisms responsible for this elimination have been extensively studied (3-5). Based on these studies, it has been suggested that the transport of organic anions is a complex process involving distinctly different proteins at the apical and basolateral membranes of the proximal tubule cells. Organic anions are transported across the basolateral membrane into the cell in exchange for intracellular dicarboxylates, which are subsequently returned into the cell via a sodium-dependent dicarboxylate transporter. Once inside the cell, organic anions are subject to intracellular binding and sequestration within vesicular structures. Finally, luminal exit is thought to occur by anion exchange and/or facilitated diffusion (3-5).We (1, 2) and others (6 -11) have recently cloned the organic anion transporter cDNA from kidneys of multiple species. Using computer modeling based on hydropathy analysis, the predicted proteins share several common features, including 12 putative membrane-spanning segments, a cluster of potential glycosylation sites located in the first extracellular...

Organic anion transporters play an essential role in eliminating a wide range of organic anions including endogenous compounds, xenobiotics, and their metabolites from kidney, thereby preventing their potentially toxic effects within the body. The goal of this study was to extend our previous study on the functional characterization and posttranslational modification of a mouse kidney organic anion transporter (mOAT), in a mammalian cell system, COS-7 cells. The transporter-mediated p-aminohippurate (PAH) uptake was saturable, probenecid-sensitive, and inhibited by a wide range of organic anions including vitamins, antihypertensive drugs, anti-tumor drugs, and anti-inflammatory drugs. Tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited the transport activity. Immunofluorescence provided evidence that most of the protein remained in the intracellular compartment in tunicamycin-treated cells. Diethyl pyrocarbonate (DEPC), a histidine residue-specific reagent, completely blocked PAH transport. The inhibitory effect by DEPC was significantly protected (90%) by pretreating the cells with excess unlabeled PAH, suggesting that the histidine residues may be close to the PAH binding sites. Finally, in situ mRNA localization was studied in postnatal mouse kidney. The expression was observed in proximal tubules throughout development. We conclude that COS-7 cells may be useful in pharmacological and molecular biological studies of this carrier. The carbohydrate moieties are necessary for the proper trafficking of mOAT to the plasma membrane, and histidine residues appear to be important for the transport function.

A human neoplastic B cell line SSK41 that expresses IgM on its surface switches spontaneously to IgG-producing cells. The SSK41 line contains a single immunoglobulin heavy-chain locus, the constant region (C) genes of which retain the germline configuration. The IgG-producing SSK41 line was purified by sorting, and shown to have undergone S-S recombination with deletion of the C mu gene. This line produced secretory and membrane-bound forms of gamma-chain mRNA. From cDNA libraries of a mixed population of IgM+/IgG+ SSK41 cells, we have isolated cDNA clones encoding the mature membrane-bound and secretory forms of the mu and gamma 1 heavy chains, all of which share the same variable region sequence. cDNA clones containing the mature gamma 3 chain were identified as well. We also isolated cDNA clones containing C gamma 1 and C gamma 3 sterile transcripts from the SSK41 line. These sterile transcripts contained additional exon sequences designated 'I' which were localized upstream of the C gamma 1 and C gamma 3 switch regions and homologous to murine counterparts. The I sequences were precisely spliced to the 5' ends of the corresponding C gamma exon sequences. These features of germline CH transcripts, i.e. the isotype specificity to class switching, location of exons, and sequences per se, are highly conserved between man and mouse.