Podocytes in the urine may be a useful marker of disease activity in diabetic nephropathy. Trandolapril may be effective for podocyte injury.

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A protein containing a heme-binding PAS (PAS is from the protein names in which imperfect repeat sequences were first recognized: PER, ARNT, and SIM) domain from Escherichia coli has been implied a direct oxygen sensor (Ec DOS) enzyme. In the present study, we isolated cDNA for the Ec DOS full-length protein, expressed it in E. coli, and examined its structure-function relationships for the first time. Ec DOS was found to be tetrameric and was obtained as a 6-coordinate low spin ferric heme complex. Its ␣-helix content was calculated as 53% by CD spectroscopy. The redox potential of the heme was found to be ؉67 mV versus SHE. Mutation of His-77 of the isolated PAS domain abolished heme binding, whereas mutation of His-83 did not, suggesting that His-77 is one of the heme axial ligands. Ferrous, but not ferric, Ec DOS had phosphodiesterase (PDE) activity of nearly 0.15 min ؊1 with cAMP, which was optimal at pH 8.5 in the presence of Mg 2؉ and was strongly inhibited by CO, NO, and etazolate, a selective cAMP PDE inhibitor. Absorption spectral changes indicated tight CO and NO bindings to the ferrous heme. Therefore, the present study unequivocally indicates for the first time that Ec DOS exhibits PDE activity with cAMP and that this is regulated by the heme redox state.Heme proteins and enzymes perform a broad range of functions. Well known examples include O 2 storage with myoglobin, O 2 carriage with hemoglobin, mediators of electron transfer with cytochromes, and catalytic activation of heme ligands with P450s and peroxidases (1-3). Recently, a new class of heme enzymes involved in intramolecular signal transduction is emerging, known as heme-based sensors (4 -6). Almost of all the heme-based sensors contain two different functional domains as follows: one is an N-terminal heme domain, which acts a sensor, and the other is a catalytic domain such as a histidine kinase or a soluble guanylate cyclase. These heme sensor enzymes use the heme for mediating transcriptional and regulatory events associated with the presence of gaseous molecules such as CO, NO, and O 2 (4 -6). In these enzymes, the ligand association or dissociation from the heme iron leads to protein conformational changes, which transmit signals to the other domain where they initiate catalytic function or DNA binding. For example, the CooA 1 protein from Rhodospirillum rubrum is a CO sensor heme protein that regulates the expression of the coo genes associated with CO-dependent growth (Refs. 7 and 8 and references therein). Soluble guanylate cyclase is an NO sensor heme protein that regulates conversion of 5Ј-GTP to the intracellular second messenger, cGMP (Refs. 9 and 10 and references therein). Hem-AT-Bs and Hem-AT-Hs are oxygen sensors in which the hemes are thought to mediate signal transduction for methylation of the chemotaxis proteins (11, 12).The Fix proteins, FixL and FixJ, of Rhizobium meliloti are well characterized as biological oxygen sensors and regulate the expression of the nitrogen fixation genes of a plant symbiotic bacterium, Sino...

No abstract

SummaryThe derivation of self-renewing tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would shorten the time needed to engineer mature cell types in vitro and would have broad reaching implications for the field of regenerative medicine. Here we report the directed differentiation of human iPSCs into putative airway basal cells (“iBCs”), a population resembling the epithelial stem cell of lung airways. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato) we track and purify these cells over time, as they first emerge from iPSC-derived foregut endoderm as developmentally immature NKX2-1GFP+ lung progenitors which then augment a TP63 program during subsequent proximal airway epithelial patterning. These cells clonally proliferate, initially as NKX2-1GFP+/TP63tdTomato+ immature airway progenitors that lack expression of the adult basal cell surface marker NGFR. However, in response to primary basal cell medium, NKX2-1GFP+/ TP63tdTomato+ cells upregulate NGFR and display the molecular and functional phenotype of airway basal stem cells, including the capacity to clonally self-renew or undergo multilineage ciliated and secretory epithelial differentiation in air-liquid interface cultures. iBCs and their differentiated progeny recapitulate several fundamental physiologic features of normal primary airway epithelial cells and model perturbations that characterize acquired and genetic airway diseases. In an asthma model of mucus metaplasia, the inflammatory cytokine IL-13 induced an increase in MUC5AC+ cells similar to primary cells. CFTR-dependent chloride flux in airway epithelium generated from cystic fibrosis iBCs or their syngeneic CFTR-corrected controls exhibited a pattern consistent with the flux measured in primary diseased and normal human airway epithelium, respectively. Finally, multiciliated cells generated from an individual with primary ciliary dyskinesia recapitulated the ciliary beat and ultrastructural defects observed in the donor. Thus, we demonstrate the successful de novo generation of a tissue-resident stem cell-like population in vitro from iPSCs, an approach which should facilitate disease modeling and future regenerative therapies for a variety of diseases affecting the lung airways.

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Behavioral neuroendocrinology has benefited tremendously from the use of a wide range of model organisms that are ideally suited for particular questions. However, in recent years the ability to manipulate the genomes of laboratory strains of mice has led to rapid advances in our understanding of the role of specific genes, circuits and neural populations in regulating behavior. While genome manipulation in mice has been a boon for behavioral neuroscience, the intensive focus on the mouse restricts the diversity in behavioral questions that can be investigated using state-of-the-art techniques. The CRISPR/Cas9 system has great potential for efficiently generating mutants in non-traditional animal models and consequently to reinvigorate comparative behavioral neuroendocrinology. Here we describe the efficient generation of oxytocin receptor (Oxtr) mutant prairie voles (Microtus ochrogaster) using the CRISPR/Cas9 system, and describe initial behavioral phenotyping focusing on behaviors relevant to autism. Oxtr mutant male voles show no disruption in pup ultrasonic vocalization, anxiety as measured by the open field test, alloparental behavior, or sociability in the three chamber test. Mutants did however show a modest elevation in repetitive behavior in the marble burying test, and an impairment in preference for social novelty.

Anabaenopeptins I (1) and J (2), two new ureido bond-containing cyclic peptides, were isolated from the cultured cyanobacterium Aphanizomenon flos-aquae (NIES-81) as potent carboxypeptidase-A (CPA) inhibitors. The gross structures of 1 and 2 were established by spectroscopic analysis, including the 2D NMR techniques. The absolute configurations of 1 and 2 were determined by spectral and chemical methods. Anabaenopeptins I and J inhibited CPA with IC(50) values of 5.2 and 7.6 ng/mL, respectively.