Heightened epithelial Na+ channel-mediated Na+ absorption in a murine polycystic kidney disease model epithelium lacking apical monocilia

The Tg737°^rpk autosomal recessive polycystic kidney disease (ARPKD) mouse carries a hypomorphic mutation in the Tg737 gene. Because of the absence of its protein product Polaris, the nonmotile primary monocilium central to the luminal membrane of ductal epithelia, such as the cortical collecting duct (CCD) principal cell (PC), is malformed. Although the functions of the renal monocilium remain elusive, primary monocilia or flagella on neurons act as sensory organelles. Thus we hypothesized that the PC monocilium functions as a cellular sensor. To test this hypothesis, we assessed the contribution of Polaris and cilium structure and function to renal epithelial ion transport electrophysiology. Properties of Tg737°^rpk mutant CCD PC clones were compared with clones genetically rescued with wild-type Tg737 cDNA. All cells were grown as polarized cell monolayers with similarly high transepithelial resistance on permeable filter supports. Three- to fourfold elevated transepithelial voltage (V_te) and short-circuit current (I_sc) were measured in mutant orpk monolayers vs. rescued controls. Pharmacological and cell biological examination of this enhanced electrical end point in mutant monolayers revealed that epithelial Na⁺ channels (ENaCs) were upregulated. Amiloride, ENaC-selective amiloride analogs (benzamil and phenamil), and protease inhibitors (aprotinin and leupeptin) attenuated heightened V_te and I_sc. Higher concentrations of additional amiloride analogs (ethylisopropylamiloride and dimethylamiloride) also revealed inhibition of V_te. Cell culture requirements and manipulations were also consistent with heightened ENaC expression and function. Together, these data suggest that ENaC expression and/or function are upregulated in the luminal membrane of mutant, cilium-deficient orpk CCD PC monolayers vs. cilium-competent controls. When the genetic lesion causes loss or malformation of the monocilium, ENaC-driven Na⁺ hyperabsorption may explain the rapid emergence of severe hypertension in a majority of patients with ARPKD. cilia; hypertension; ion transport; epithelial cells     

Disease-Causing 7.4 kb Cis-Regulatory Deletion Disrupting Conserved Non-Coding Sequences and Their Interaction with the FOXL2 Promotor: Implications for Mutation Screening

To date, the contribution of disrupted potentially cis-regulatory conserved non-coding sequences (CNCs) to human disease is most likely underestimated, as no systematic screens for putative deleterious variations in CNCs have been conducted. As a model for monogenic disease we studied the involvement of genetic changes of CNCs in the cis-regulatory domain of FOXL2 in blepharophimosis syndrome (BPES). Fifty-seven molecularly unsolved BPES patients underwent high-resolution copy number screening and targeted sequencing of CNCs. Apart from three larger distant deletions, a de novo deletion as small as 7.4 kb was found at 283 kb 5′ to FOXL2. The deletion appeared to be triggered by an H-DNA-induced double-stranded break (DSB). In addition, it disrupts a novel long non-coding RNA (ncRNA) PISRT1 and 8 CNCs. The regulatory potential of the deleted CNCs was substantiated by in vitro luciferase assays. Interestingly, Chromosome Conformation Capture (3C) of a 625 kb region surrounding FOXL2 in expressing cellular systems revealed physical interactions of three upstream fragments and the FOXL2 core promoter. Importantly, one of these contains the 7.4 kb deleted fragment. Overall, this study revealed the smallest distant deletion causing monogenic disease and impacts upon the concept of mutation screening in human disease and developmental disorders in particular.     

The apolipoprotein A-I mimetic peptide 4F prevents defects in vascular function in endotoxemic rats

High density lipoprotein (HDL) and apolipoprotein A-I (apoA-I) reduce inflammatory responses to lipopolysaccharide (LPS). We tested the hypothesis that the apoA-I mimetic peptide 4F prevents LPS-induced defects in blood pressure and vascular reactivity. Systolic blood pressure (SBP) was measured in rats at baseline and 6 h after injection of LPS (10 mg/kg) or saline vehicle. Subgroups of LPS-treated rats also received 4F (10 mg/kg) or scrambled 4F (Sc-4F). LPS administration reduced SBP by 35% compared with baseline. 4F attenuated the reduction in SBP in LPS-treated rats (17% reduction), while Sc-4F was without effect. Ex vivo studies showed a reduced contractile response to phenylephrine (PE) in aortae of LPS-treated rats (ED₅₀ = 459 ± 83 nM) compared with controls (ED₅₀ = 57 ± 6 nM). This was associated with nitric oxide synthase 2 (NOS2) upregulation. 4F administration improved vascular contractility (ED₅₀ = 60 ± 9 nM), reduced aortic NOS2 protein, normalized plasma levels of NO metabolites, and reduced mortality in LPS-treated rats. These changes were associated with a reduction in plasma endotoxin activity. In vivo administration of ¹⁴C-4F and Bodipy-LPS resulted in their colocalization and retention in the HDL fraction. It is proposed that 4F promotes the localization of LPS to the HDL fraction, resulting in endotoxin neutralization. 4F may thus prevent LPS-induced hemodynamic changes associated with NOS2 induction.     

Production and characterization of liquid-core capsules made from cross-linked acrylamide copolymers for biotechnological applications

A novel chemistry has been developed for the production of capsules composed of a hydrophobic liquid core surrounded by a cross-linked polyacrylamide/alginate membrane. These liquid-core capsules may be used in capsular perstraction for the removal of inhibitory products from bioprocesses and bioconversions. They have the advantage of having a high surface area to promote rapid mass transfer, while separation of the organic core phase from the aqueous environment by the capsule membrane prevents the formation of stable emulsions and potential problems associated with toxicity of the organic phase for microbial cells or enzymes. Monodisperse spherical liquid-core capsules of between 800 microm and 1.6 mm diameter, with high mechanical resistance, have been prepared by co-extrusion, using the jet break-up technique. Capsules produced from a solution of MBA/total monomer (5%) were found to be more elastic and have a higher burst force when exposed to chelating agents such as phosphate or citrate. The mechanical resistance was unaffected by buffer solutions in the pH range 4-9 and after sterilization at 121 degrees C for 20 min. Capsules having membranes composed of a copolymer of acrylamide and N-hydroxymethylacrylamide exhibited even higher mechanical stability toward chelating agents.     

Two paralogous families of a two-gene subtilisin operon are widely distributed in oral treponemes

Certain oral treponemes express a highly proteolytic phenotype and have been associated with periodontal diseases. The periodontal pathogen Treponema denticola produces dentilisin, a serine protease of the subtilisin family. The two-gene operon prcA-prtP is required for expression of active dentilisin (PrtP), a putative lipoprotein attached to the treponeme's outer membrane or sheath. The purpose of this study was to examine the diversity and structure of treponemal subtilisin-like proteases in order to better understand their distribution and function. The complete sequences of five prcA-prtP operons were determined for Treponema lecithinolyticum, "Treponema vincentii," and two canine species. Partial operon sequences were obtained for T. socranskii subsp. 04 as well as 450- to 1,000-base fragments of prtP genes from four additional treponeme strains. Phylogenetic analysis demonstrated that the sequences fall into two paralogous families. The first family includes the sequence from T. denticola. Treponemes possessing this operon family express chymotrypsin-like protease activity and can cleave the substrate N-succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide (SAAPFNA). Treponemes possessing the second paralog family do not possess chymotrypsin-like activity or cleave SAAPFNA. Despite examination of a range of protein and peptide substrates, the specificity of the second protease family remains unknown. Each of the fully sequenced prcA and prtP genes contains a 5' hydrophobic leader sequence with a treponeme lipobox. The two paralogous families of treponeme subtilisins represent a new subgroup within the subtilisin family of proteases and are the only subtilisin lipoprotein family. The present study demonstrated that the subtilisin paralogs comprising a two-gene operon are widely distributed among treponemes.     

Enzymatic oxidation of p-nitrophenol.

A Moraxella sp. capable of growth with p-nitrophenol was isolated from activated sludge. Differential centrifugation of crude cell extracts gave a membrane preparation that oxidized p-nitrophenol to hydroquinone and nitrite. Enzymatic activity was dependent on the presence of oxygen and reduced pyridine nucleotides and was stimulated by the addition of flavin adenine dinucleotide. Experiments with ¹⁸O₂ showed that the incoming hydroxyl group was derived from molecular oxygen. The soluble fraction prepared from crude cell extracts oxidized hydroquinone to a product whose spectral properties were identical to those reported for γ-hydroxymuconic semialdehyde.     

The charge heterogeneity of soluble human galactosyltransferases isolated from milk, amniotic fluid and malignant ascites.

UDP-galactose: N-acetylglucosamine galactosyltransferase was isolated from pooled human milk, pooled amniotic fluid and from two different individual samples of malignant ascites. The purification procedure involving two successive affinity chromatography steps on N-acetylglucosamine--agarose and alpha-lactalbumin--agarose yielded an enzyme preparation homogeneous by size. Under non-denaturing conditions the ascites and amniotic fluid enzymes had identical electrophoretic mobility, but they moved faster than the milk enzyme. Isoelectric analysis in the presence and absence of urea resolved the milk enzyme into at least 13 different forms, nine of which had the same isoelectric points after refocusing. All enzyme forms showed similar activity when free N-acetylglucosamine, ovalbumin, sialic-acid-free ovine submaxillary mucin and glucose, in the presence of alpha-lactalbumin, were used as acceptor substrates. Comparative isoelectric focusing of the three galactosyltransferases revealed identical patterns of the amniotic and ascites enzymes, but only partial overlap with the milk enzyme, which was less negatively charged. Neuraminidase treatment of ascites and milk galactosyltransferases produced very similar focusing patterns. The possible structural basis for this charge heterogeneity is briefly discussed.