Genetic identification of two major modifier loci of polycystic kidney disease progression in pcy mice.

Unlike the uniform disease progression in inbred animals, polycystic kidney disease (PKD) progression within human families can be highly variable. This may be due to environmental or genetic factors or both. To determine if PKD severity can be influenced by modifier genes, we carried out an intercross between DBA/2-pcy/pcy and Mus m. castaneous involving 3,105 6-wk-old F2 mice. Large differences in PKD severity were observed in this cross. In addition, 23/ 800 phenotypically normal mice were pcy/pcy genotypically. These results demonstrated that PKD progression in pcy/ pcy mice is a quantitative trait that is strongly modulated by modifier genes. Whole genome quantitative trait loci mapping of 114 selected pcy/pcy mice (68 with the mild PKD and 46 with severe PKD) identified two loci, MOP1 and MOP2 that strongly modulate PKD progression. MOP1 (max LOD score = 10.3 at D4Mit111) and MOP2 (max LOD score = 13.8 at D16Mit1) accounted for 36.7 and 46.8% of the phenotypic variance, respectively. Two-factor ANOVA of the phenotypes and genotypes of all 673 pcy/pcy mice from our cross indicated that MOP1 and MOP2 alleles regulate PKD progression in a complex additive manner. Characterization of these novel modifying loci may provide additional insights into the pathogenesis of polycystic kidney diseases.

Arrested testis development in the cpk mouse may be the result of abnormal steroid metabolism.

Ke 6 is a 17beta-hydroxysteroid dehydrogenase that is expressed in several somatic tissues as well as the female reproductive tissues. We previously correlated a dramatic reduction in the expression of the Ke 6 gene with the development of recessive polycystic kidney disease, in three murine models, the cpk, jck and pcy mice. We also determined that in one of the murine models, the cpk mouse, the female reproductive organs fail to mature properly and remain arrested at an early stage of development. In this study, we report the expression of the Ke 6 protein in normal male reproductive tissues by immunofluorescent staining. We determined in the cpk mouse that the testes similar to the immature ovaries, is also under-developed and arrested at an early developmental stage. Direct measurement of 17betaHSD activity showed a conspicuous reduction in sex steroid metabolism in the cpk/cpk testes. Our findings suggest that estrogen/androgen metabolism play an important role in the development of the urogenital system.

Microtubule active taxanes inhibit polycystic kidney disease progression in cpk mice.

Homozygous cpk/cpk mice develop polycystic kidney disease and die of uremia between the fourth and fifth weeks of age. Cpk/cpk mice treated weekly with paclitaxel (Taxol) can live to over six months of age. This dramatic moderation of polycystic kidney disease progression has been postulated to be a result of paclitaxel's ability to stabilize microtubules. In this study, the ability of taxanes with differing abilities to promote spontaneous in vitro assembly of tubulin dimers into microtubules were tested for their ability to inhibit the progression of polycystic kidney disease in polycystic cpk/cpk mice. We found that taxanes that are active in promoting microtubule assembly, including paclitaxel, 10-deactyl-taxol and cephalomannine increased the survival of polycystic cpk/cpk mice significantly longer than control animals. In contrast, the microtubule inactive taxane baccatin-III has no effect on the progression of renal failure in cpk/cpk mice. We conclude that the ability to promote microtubule assembly may be necessary for paclitaxel and related taxanes to modulate the progression of polycystic kidney progression in cpk/cpk mice.

Ultrastructure of Bacteroides species: Bacteroides asaccharolyticus, Bacteroides fragilis, Bacteroides melaninogenicus subspecies melaninogenicus, and B. melaninogenicus subspecies intermedius.

Representative strains of two subspecies of Bacteroides melaninogenicus (subspecies melaninogenicus and subspecies intermedius) and Bacteroides asaccharolyticus as well as B. asaccharolyticus strain 536B isolated from a human perirectal abscess and Bacteroides fragilis ATCC 25285 were examined by glutaraldehyde-osmium fixation, ruthenium red fixation and staining, and thorium hydroxyde staining as well as by the physical preparative techniques of critical point drying--transmission electron microscopy (CPD--TEM) and scanning electron microscopy (SEM). All strains, with the exception of B. fragilis 25285, possessed an electron-dense material external to their outer membranes. Ruthenium red staining further revealed a layer, external to the surface of the outer membrane, that was distinct for each species examined. Thorium hydroxide, as well as CPD--TEM and SEM, showed the cells to be interconnected by thin fibers that not only connected adjacent cells but also traversed several microns to connect cell aggregates.

Transforming growth factor alpha and a PC12-derived growth factor induce neurites in PC12 cells and enhance the survival of embryonic brain neurons.

We have identified and characterized a 5000-Da protein that induces neurite outgrowth from PC12 pheochromocytoma cells, enhances the survival of embryonic rat brain neurons in primary culture, and induces the multiplication of embryonic rat brain astrocytes in primary culture. The factor is produced by a flat cell PC12 variant that expresses the activated ras oncogene after transfection of the gene. The factor resembles transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) in that it induces anchorage-independent colony formation of normal rat kidney cells in soft agar and competes with EGF for binding to the EGF receptor. Rat TGF alpha and human TGF alpha also induce neurite outgrowth from PC12 and enhance the survival of embryonic brain neurons. The PC12 variant-derived factor can be distinguished from TGF alpha and EGF immunologically and by migration rates on reversed-phase high-performance liquid chromatography.

Taxol inhibits progression of congenital polycystic kidney disease.

Polycystic kidney diseases (PKD) are the most common hereditary diseases of the human kidney and account for ten per cent of patients requiring renal transplantation or dialysis. Renal cyst formation has been attributed to enhanced cell proliferation, unbalanced cell death, abnormal targeting of membrane proteins, aberrant kidney development and tubular obstruction, but there is no treatment that blocks the formation and enlargement of renal cysts. We have now developed an in vitro model of spontaneous cyst formation that distinguishes polycystic kidney epithelium from its normal counterpart. Inhibitors of DNA, RNA and protein synthesis did not prevent in vitro cyst formation, but this was reversibly inhibited by ouabain, amiloride and the microtubule-specific agents colchicine, vinblastine and taxol. The cpk mouse is a well-characterized recessive PKD model and we find that cpk/cpk mice develop PKD and die from uraemia by 4-5 weeks of age, but when treated weekly with taxol they survive for more than 200 days with minimal loss of renal function, show limited collecting-dust cyst enlargement, and attain adult size. Our results indicate that the microtubule cytoskeleton has a central role in the pathogenesis of PKD in cpk mice and that taxol may also be useful in treating human PKD.

Chemical synthesis in protein engineering: total synthesis, purification and covalent structural characterization of a mitogenic protein, human transforming growth factor-alpha.

Successful approaches to protein engineering required that the desired analogs be easily and rapidly obtained in sufficient quantities and purities for unambiguous structural and functional characterizations. Chemical synthesis is the method of choice for engineering small peptides. We now demonstrate that with improved methodologies and instrumentation, total chemical synthesis can be used to produce a small protein in a form suitable for engineering studies. Active human transforming growth factor-alpha (TGF-alpha), a 50 amino acid long protein with three disulfide bonds, has been synthesized and purified in multiple tens of mg amounts in less than 7 days. The purified human TGF-alpha migrated as a single band on SDS-polyacrylamide gels, ran as a single sharp major band at pI = 6.2 on isoelectric focusing gels, displayed an MW = 5546.2 (Th.5546.3) by mass spectrometry, contained three disulfide bonds and had EGF receptor binding, mitogenic and soft agar colony formation activities. The locations of disulfide bonds were found to be analogous to those found in epidermal growth factor (EGF) and in human TGF-alpha expressed in bacteria.

Differential phosphorylation of the progesterone receptor by insulin, epidermal growth factor, and platelet-derived growth factor receptor tyrosine protein kinases.

Purified preparations of insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF-activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites.

Microscale structure analysis of a high-molecular-weight, hydrophobic membrane glycoprotein fraction with platelet-derived growth factor-dependent kinase activity.

General methods for the study of the primary structure of picomole quantities of large, hydrophobic membrane glycoproteins with blocked amino-termini have been developed. Three techniques designed to be used in concert with each other are described: first, modified protein preparation and fragmentation techniques; secondly, a simple but very selective two-dimensional reversed-phase high-performance liquid chromatography system for the resolution of complex mixtures of small to medium-sized tryptic peptides on Vydac C4, C18 and diphenyl columns and thirdly, a two-dimensional separation method for large, denaturated (CNBr) polypeptide fragments by size-exclusion high-performance liquid chromatography, combined with either reversed-phase high-performance liquid chromatography (C4) or sodium dodecyl sulphate polyacrylamide gel electrophoresis in conjunction with electroblotting and autoradiography. These methods were applied to studies of the platelet-derived growth factor receptor. Starting with 500 pmoles of purified protein, a total of 232 amino acids were sequenced.

Progesterone receptor subunits are high-affinity substrates for phosphorylation by epidermal growth factor receptor.

Purified preparations of epidermal growth factor (EGF) receptor were used to test hen oviduct progesterone receptor subunits as substrates for phosphorylation catalyzed by EGF receptor. Both the 80-kilodalton (kDa) (A) and the 105-kDa (B) progesterone receptor subunits were phosphorylated in a reaction that required EGF and EGF receptor. No phosphorylation of progesterone receptor subunits was observed in the absence of EGF receptor, even when Ca2+ was substituted for Mg2+ and Mn2+. Phospho amino acid analysis revealed phosphorylation at tyrosine residues, with no phosphorylation detectable at serine or threonine residues. Two-dimensional maps of phosphopeptides generated from phosphorylated 80- or 105-kDa subunits by tryptic digestion revealed similar patterns, with resolution of two major, several minor, and a number of very minor phosphopeptides. The Km of progesterone receptor for phosphorylation by EGF-activated EGF receptor was 100 nM and the Vmax was 2.5 nmol/min per mg of EGF receptor protein at 0 degrees C. The stoichiometry of phosphorylation/hormone binding for progesterone receptor subunits was 0.31 at ice-bath temperature and approximately 1.0 at 22 degrees C.

Fibroblasts of rabbit kidney in culture. II. Paracrine stimulation of papillary fibroblasts by PDGF.

To examine the role of tubulointerstitial cell interaction in the regulation of fibroblast growth, fibroblasts from the rabbit renal cortex (CF) and papilla (PF) were cocultured with epithelial cells from the same tissue location. Inner medullary collecting duct epithelial cells (IMCDE) or IMCDE-conditioned medium stimulated DNA synthesis in PF, whereas proximal tubule epithelium (PTE) had no effect on the proliferation of CF. PF and CF showed a similar mitogenic response to exogenous epidermal growth factor and insulin-like growth factor 1 (IGF-I). Transforming growth factor-beta 1 inhibited growth of both cell types, and basic fibroblast growth factor (bFGF) had no effect on proliferation of either cell type. In contrast, platelet-derived growth factor (PDGF) was a potent mitogen for PF but was only weakly mitogenic for CF. Both CF and PF expressed a similar number of a single-affinity class of PDGF receptors (Kd, 2-4 x 10(-10) M). Assay for growth factor activity in conditioned medium from IMCDE and PTE showed that only IMCDE produced detectable PDGF. IMCDE-stimulated proliferation of PF was partially blocked by an antibody to PDGF, whereas antibodies to IGF-I had no neutralizing effect. The data suggest a role for PDGF in the regulation of interstitial fibroblast proliferation by IMCDE in the renal papilla. This paracrine system may be important in the pathogenesis of some forms of interstitial fibrosis of the kidney.