PAI-1 deficiency attenuates the fibrogenic response to ureteral obstruction.

BACKGROUND: Progressive renal disease is characterized by the induction of plasminogen activator inhibitor-1 (PAI-1), suggesting that impaired activity of the renal plasmin cascade may play a role in renal fibrosis. METHODS: To test this hypothesis, the severity of renal fibrosis caused by unilateral ureteral obstruction (UUO) was compared in PAI-1 wild-type (+/+) and PAI-1 deficient (-/-) mice. The extent of interstitial inflammation and fibrosis, renal plasminogen activator and plasmin activity, and renal expression of profibrotic genes was evaluated after 3, 7, and 14 days of UUO. RESULTS: Renal PAI-1 mRNA levels increased 8- to 16-fold in the +/+ mice after UUO surgery, and PAI-1 protein was detected in kidney homogenates. Interstitial fibrosis was significantly attenuated in -/- mice compared with +/+ mice at day 7 and day 14, based on the interstitial area stained with picrosirius red and total kidney collagen content. However, neither the mean renal plasminogen activator nor plasmin activities were increased in -/- mice compared with +/+ mice. The number of interstitial macrophages were significantly lower in the -/- mice three and seven days after UUO; interstitial myofibroblasts were significantly fewer at three days. At the same time points, this altered interstitial cellularity was associated with a significant reduction in renal mRNA levels for transforming growth factor-beta and procollagens alpha 1(I) and alpha 1(III). CONCLUSIONS: These studies establish an important fibrogenic role for PAI-1 in the renal fibrogenic response. The results demonstrate that one important fibrosis-promoting function of PAI-1 is its role in the recruitment of fibrosis-inducing cells, including myofibroblasts and macrophages.

Structure-function studies of the BTB/POZ transcriptional repression domain from the promyelocytic leukemia zinc finger oncoprotein.

The evolutionarily conserved BTB/POZ domain from the promyelocytic leukemia zinc finger (PLZF) oncoprotein mediates transcriptional repression through the recruitment of corepressor proteins containing histone deacetylases in acute promyelocytic leukemia. We have determined the 2.0 A crystal structure of the BTB/POZ domain from PLZF (PLZF-BTB/POZ), and have carried out biochemical analysis of PLZF-BTB/POZ harboring site-directed mutations to probe structure-function relationships. The structure reveals a novel alpha/beta homodimeric fold in which dimer interactions occur along two surfaces of the protein subunits. The conservation of BTB/POZ domain residues at the core of the protomers and at the dimer interface implies an analogous fold and dimerization mode for BTB/POZ domains from otherwise functionally unrelated proteins. Unexpectedly, the BTB/POZ domain forms dimer-dimer interactions in the crystals, suggesting a mode for higher-order protein oligomerization for BTB/POZ-mediated transcriptional repression. Biochemical characterization of PLZF-BTB/POZ harboring mutations in conserved residues involved in protein dimerization reveals that the integrity of the dimer interface is exquisitely sensitive to mutation and that dimer formation is required for wild-type levels of transcriptional repression. Interestingly, similar mutational analysis of residues within a pronounced protein cleft along the dimer interface, which had been implicated previously for interaction with corepressors, has negligible effects on dimerization or transcriptional repression. Together, these studies form a structure-function framework for understanding BTB/POZ-mediated oligomerization and transcriptional repression properties.

Identification of a novel cis-acting element for fibroblast-specific transcription of the FSP1 gene.

The FSP1 gene encodes a filament-binding S100 protein with paired EF hands that is specifically expressed in fibroblasts. This led us to look for cis-acting elements in the FSP1 promoter that might engage nuclear transcription factors unique to fibroblasts. The first exon of FSP1 is noncoding, therefore, a series of luciferase reporter minigenes were created containing varying lengths of 5'-flanking sequence, the first intron, and the noncoding region of the second exon. A position and promoter-dependent proximal element between -187 and -88 bp was shown to be active in fibroblasts but not in epithelium. Sequence in the first intron from +777 to +964 had an enhancing effect that was not cell type specific. Hsv TK reporter constructs driven by this promoter/intron cassette in transgenic mice were coexpressed appropriately with FSP1 in tissue fibroblasts. Gel mobility shift competitor assays identified a novel domain, FTS-1 (fibroblast transcription site-1; TTGAT from -177 to -173 bp), that specifically interacts with nuclear extracts from fibroblasts. The necessity of this binding site was confirmed by site-specific mutagenesis. Database searches also turned up putative FTS-1 sites in the early promoter regions of other fibroblast expressed proteins, including the alpha1 and alpha2(I), and alpha1(III) collagens and the alphaSM-actin gene. We hypothesize that the selective engagement of FTS-1 elements may contribute to the mesenchymal phenotype of fibroblasts and perhaps other dedifferentiated cells.

Cloning and expression of primate Daxx cDNAs and mapping of the human gene to chromosome 6p21.3 in the MHC region.

Murine Daxx, a protein that binds to Fas and enhances Fas-mediated apoptosis through a signal transduction pathway involving the Jun amino-terminal kinase, was recently described. Here we report the cloning of human and monkey Daxx cDNAs, the widespread expression of Daxx mRNA in human tissues, and the mapping of the human Daxx gene to 6p21.3 in the major histocompatibility complex (MHC) region. The location of the Daxx gene, which is implicated in the pathway for deletion of autoreactive lymphocytes, in the MHC region may shed light on the genetic basis of autoimmune diseases.

Overexpression, purification, characterization, and crystallization of the BTB/POZ domain from the PLZF oncoprotein.

The BTB/POZ domain defines a conserved region of about 120 residues and has been found in over 40 proteins to date. It is located predominantly at the N terminus of Zn-finger DNA-binding proteins, where it may function as a repression domain, and less frequently in actin-binding and poxvirus-encoded proteins, where it may function as a protein-protein interaction interface. A prototypic human BTB/POZ protein, PLZF (promyelocytic leukemia zinc finger) is fused to RARalpha (retinoic acid receptor alpha) in a subset of acute promyelocytic leukemias (APLs), where it acts as a potent oncogene. The exact role of the BTB/POZ domain in protein-protein interactions and/or transcriptional regulation is unknown. We have overexpressed, purified, characterized, and crystallized the BTB/POZ domain from PLZF (PLZF-BTB/POZ). Gel filtration, dynamic light scattering, and equilibrium sedimentation experiments show that PLZF-BTB/POZ forms a homodimer with a Kd below 200 nM. Differential scanning calorimetry and equilibrium denaturation experiments are consistent with the PLZF-BTB/POZ dimer undergoing a two-state unfolding transition with a Tm of 70.4 degrees C, and a DeltaG of 12.8 +/- 0.4 kcal/mol. Circular dichroism shows that the PLZF-BTB/POZ dimer has significant secondary structure including about 45% helix and 20% beta-sheet. We have prepared crystals of the PLZF-BTB/POZ that are suitable for a high resolution structure determination using x-ray crystallography. The crystals form in the space group I222 or I212121 with a = 38.8, b = 77.7, and c = 85.3 A and contain 1 protein subunit per asymmetric unit with approximately 40% solvent. Our data support the hypothesis that the BTB/POZ domain mediates a functionally relevant dimerization function in vivo. The crystal structure of the PLZF-BTB/POZ domain will provide a paradigm for understanding the structural basis underlying BTB/POZ domain function.

DNA recognition by splicing variants of the Wilms' tumor suppressor, WT1.

The Wilms' tumor suppressor, WT1, is a zinc finger transcriptional regulator which exists as multiple forms owing to alternative mRNA splicing. The most abundant splicing variants contain a nine-nucleotide insertion encoding lysine, threonine, and serine (KTS) in the H-C link region between the third and fourth WT1 zinc fingers which disrupts binding to a previously defined WT1-EGR1 binding site. We have identified WT1[+KTS] binding sites in the insulin-like growth factor II gene and show that WT1[+KTS] represses transcription from the insulin-like growth factor II P3 promoter. The highest affinity WT1[+KTS] DNA binding sites included nucleotide contacts involving all four WT1 zinc fingers. We also found that different subsets of three WT1 zinc fingers could bind to distinct DNA recognition elements. A tumor-associated, WT1 finger 3 deletion mutant was shown to bind to juxtaposed nucleotide triplets for the remaining zinc fingers 1, 2, and 4. The characterization of novel WT1 DNA recognition elements adds a new level of complexity to the potential gene regulatory activity of WT1. The results also present the possibility that altered DNA recognition by the dominant WT1 zinc finger 3 deletion mutant may contribute to tumorigenesis.

Mucin secretion in germfree rats fed fiber-free and psyllium diets and bacterial mass and carbohydrate fermentation after colonization.

The effect of psyllium on mucin secretion was determined by comparing water-soluble and -insoluble fractions of excreta from germfree rats fed a fiber-free (FF) diet or a diet containing psyllium seed husk (PS). Excreta from the same rats after colonization with a rat mixed cecal culture were separated into water-soluble, plant, and bacterial fractions to compare the remaining carbohydrate and the mass of bacteria. The sugar composition and water solubility of carbohydrate in excreta from germfree rats fed FF diets indicated that a primary fermentable substrate was mucin. PS increased fecal excretion of mucin-derived sugars almost threefold in germfree rats. Fecal carbohydrate was reduced from 619 to 237 mumol/g of dry feces and mostly in the bacterial fraction when rats fed an FF diet were colonized. The total sugar content and the amount of muramic acid, but not bacterial counts and mass, indicated that PS increased fecal bacteria. Fractionation of excreta from PS-fed rats was complicated by a gel which, based on sugar composition, was PS. Sugar composition of the water-soluble fraction from excreta from PS-fed rats suggested that it contained some bacterial component, possibly exopolysaccharides and some of the PS, but not mucin. PS digestibility ranged from 60 to 80%, depending on what fecal fraction was used for output. Because of the presence of PS-derived sugars in the gel and soluble fraction, it was not possible to determine which, if any, of the PS digestibilities was correct.

Effect of copper and cobalt addition on digestion and growth in heifers fed diets containing alfalfa silage or corn crop residues.

Copper and cobalt were added to the diet of heifers in excess of NRC recommendations for these minerals. In Experiment 1, the rate of DM disappearance from dacron bags placed in the rumen was increased by the additional dietary Cu and Co for alfalfa hay (14.6 vs. 8.4%/h) and corn cobs (5.8 vs. 3.1%/h) but did not affect the DM disappearance rate for corn crop residue silage (3.6 vs. 3.4%/h). In Experiment 2, additional Cu and Co increased DM disappearance rate of corn crop residue silage (6.2 vs. 3.4%/h) but did not influence the rate of alfalfa hay (8.6 vs. 7.6%/h). In Experiment 3, a random design with a 2 x 2 factorial arrangement was used. The diet consisted of 45% corn crop residue silage, 35% alfalfa silage containing 60 or 40% DM, 18% high moisture corn, and 2% vitamin and mineral mix with or without added Cu and Co, respectively. Each of the four diets was group-fed to 9 light and 11 heavy Holstein heifers. Growth rate was lower with Cu and Co supplementation, but DM digestibility was improved by the additional Cu and Co. These results indicate that addition of Cu and Co above the NRC requirements may aid in digestion of low quality forages.

Effect of forage source on retention of digesta markers applied to corn gluten meal and brewers grains for heifers.

Effect of forage source on retention time of dietary protein supplements in the rumen was measured, and solid and liquid markers of digesta passage were compared. Two markers for liquid, two for each of the forages, and two for each of the supplemental proteins were compared for estimating retention time. Eight Holstein heifers were allocated to a single-reversal design with two periods of 29 d each. Heifers were fed either alfalfa hay or corn husks and leaves combined with one of two resistant protein sources: corn gluten meal or wet brewers grains. Average rumen liquid retention times, measured with Co-EDTA or Cr-EDTA, were similar (16.2 to 16.9 h). Liquid retention times differed when alfalfa or corn residue was fed (14.1 and 18.9 h, respectively). Rumen retention times were similar for Sm and Ce (24.9 to 26.3 h) and across markers for forage sources (25.1 to 26.1 h). Total tract mean retention time was higher for corn residue than for hay (57.8 vs. 48.8 h). Rumen retention times for La and Yb (27.1 and 26.0 h) applied to protein sources were similar and were not different when applied to corn gluten meal or brewers grains (26.3 and 26.8 h). Forage source did not have an effect on the retention time of protein supplements. Low daily DMI of 2.22 and 1.91% of BW for hay and corn residue diets explain the rather long retention times of the protein supplements. Both Co-EDTA and Cr-EDTA were effective as liquid phase markers.

Utilization of ensiled corn crop residues by Holstein heifers.

Corn crop residues were harvested 1 to 2 d after harvest of high moisture corn. Half the harvested residues were treated with an aqueous solution of ammonia before ensiling to give 34 g of ammonia/kg of residue DM. The untreated half was the control silage. Both silages were ensiled in bunker silos. More residues were harvested 2.5 wk later from the same field and combined with Brassica napus L. before ensiling in a bag, and this constituted the third forage treatment. Three diets containing 48.8, 46.5, and 63.8% residue silage (DM basis), with the latter containing corn crop residue and brassica in a 3:1 ratio, were designated as control, ammoniated, and brassica, respectively. The other ingredients of the diet were alfalfa silage, cracked corn, and urea. The diets were fed to Holstein heifers averaging 186 kg (light BW) and 372 kg (heavy BW). Weight gains were similar at 84 d for heifers fed the control and the ammoniated residue diets. Light and heavy heifers gained 619 and 631 g/d with the control diet and 678 and 631 g/d with the ammoniated residue diets. At 70 d, the heavy heifers fed control, ammoniated, and brassica diets had weight gains of 629, 671, and 786 g/d, respectively. Digestion coefficients were similar between the control and ammoniated residue diets: DM (62.6 and 58.7%), NDF (56.7 and 56.2%), and ADF (56.3 and 55.8%). Ammonia treatment of corn crop residue silage had no effect on animal performance or digestibility. Brassica appeared to improve animal performance when it was mixed with corn crop residue silage.

Effect of cellulose fine structure on kinetics of its digestion by mixed ruminal microorganisms in vitro.

The digestion kinetics of a variety of pure celluloses were examined by using an in vitro assay employing mixed ruminal microflora and a modified detergent extraction procedure to recover residual cellulose. Digestion of all of the celluloses was described by a discontinuous first-order rate equation to yield digestion rate constants and discrete lag times. These kinetic parameters were compared with the relative crystallinity indices and estimated accessible surface areas of the celluloses. For type I celluloses having similar crystallinities and simple nonaggregating particle morphologies, the fermentation rate constants displayed a strong positive correlation (r2 = 0.978) with gross specific surface area; lag time exhibited a weaker, negative correlation (r2 = 0.930) with gross specific surface area. Crystallinity was shown to have a relatively minor effect on the digestion rate and lag time. Swelling of microcrystalline cellulose with 72 to 77% phosphoric acid yielded substrates which were fermented slightly more rapidly than the original material. However, treatment with higher concentrations of phosphoric acid resulted in a more slowly fermented substrate, despite a decrease in crystallinity and an increase in pore volume. This reduced fermentation rate was apparently due to the partial conversion of the cellulose from the type I to the type II allomorph, since mercerized (type II) cellulose was also fermented more slowly, and only after a much longer lag period. The results are consistent with earlier evidence for the cell-associated nature of cellulolytic enzymes of ruminal bacteria and suggest that ruminal microflora do not rapidly adapt to utilization of celluloses with altered unit cell structures.

Neutral sugar composition and gravimetric yield of plant and bacterial fractions of feces.

Separating dietary fiber from other polysaccharides in digesta and feces is necessary to understand its mechanisms of action. A gravimetric method that separates fecal plant and bacterial matter based on size and density was evaluated and modified to determine the plant and bacterial mass of lyophilized whole and blended rat and human feces. Three screen mech combinations (150 and 75 microns, 150 and 35 microns, 35 microns) were used with rat feces. Filtration of a homogenized rat fecal slurry sequentially through 150- and 35-microns-mesh screens versus 150- and 75-microns-mesh screens decreased the gravimetric recovery of bacteria from congruent to 35 to congruent to 25% of fecal dry weight and increased the plant fraction weight. Neutral sugar composition, determined by gas chromatography of alditol acetates, and bacterial counts of the fractions suggested that the decreased yield of bacterial fraction represented removal of plant material and not a loss of bacteria. Rat excreta contained 29.5% (dry weight) total neutral sugar, 88% of which was recovered in the plant material. Human feces containing wheat bran, fractionated with the 150- and 35-microns-mesh screens, was 21% neutral sugar, congruent to 65% of which was in the plant fraction. The plant fractions had more xylose and arabinose and less glucose than the bacterial fractions. Processing samples in a Waring blender had no adverse effect on the rat or human fecal bacterial counts. The use of this gravimetric method in combination with the sugar analysis of the fractions provided a better measure of plant and bacteria than only gravimetric yield.

Processed oat hulls as potential dietary fiber sources in rats.

Processed oat hull products were evaluated as potential dietary fiber sources. Three levels, 5, 10 and 15%, of processed oat hulls, bleached oat hulls or oat hulls coated with starch, were added to purified diets and fed to groups of rats for 6 wk. Control diets consisted of 5, 10 or 15% alpha-cellulose or commercial nonpurified diet. None of the oat hull products at the three levels tested had any negative effect on rat growth. Fresh and dry fecal weights increased linearly as the concentration of dietary fiber increased and were highly correlated with fiber intake (r = 0.95). Apparent digestibility of neutral detergent fiber in all diets was low and apparent calcium absorption was not consistently affected by any diet. None of the oat hull test diets lowered plasma or hepatic cholesterol levels, a finding consistent with the failure to detect mixed-linkage beta-glucans in any of the processed oat hull products. Detailed analysis of the processed oat hull fibers also indicated that they were greater than 95% insoluble fiber and high in cellulose and xylans. Light-microscopy histology of kidney, spleen, pancreas, stomach, duodenum, ileum and colon was normal. The extent of hepatocellular destruction produced by the cholesterol (1%) and cholic acid (0.2%) added to the diet to induce hypocholesterolemia was independent of the kind and amount of dietary fiber.