Abstract Mechanosensory hair cells are essential for audition in vertebrates, and many species, have the capacity regeneration when damaged. Regeneration is robust fish lateral line system as new can reappear after damage induced by waterborne aminoglycoside antibiotics, platinum‐based drugs, heavy metals. Here, we characterize loss reappearance of zebrafish larvae treated with copper sulfate using diverse molecular markers. Transgenic that express green fluorescent protein different cell...

Significance Maintenance of homeostasis is dependent on intercellular communication via secreted hormones that bind G protein-coupled receptors. Many these receptors activate an enzyme called protein kinase A (PKA) modifies cell function by covalently attaching phosphate groups to proteins. To comprehensively identify PKA substrates, we used genome editing (CRISPR-Cas9) delete from kidney epithelial cells followed large-scale mass spectrometry measure phosphorylation changes throughout the...

Vasopressin regulates water excretion, in part, by controlling the abundances of channel aquaporin-2 (AQP2) protein and regulatory proteins renal collecting duct. To determine whether vasopressin-induced alterations abundance result from modulation production, degradation, or both, we used mass spectrometry with dynamic stable isotope labeling cell culture to achieve a proteome-wide determination half-lives relative translation rates mpkCCD cells. Measurements were made at steady state...

Abstract Vasopressin-mediated regulation of renal water excretion is defective in a variety balance disorders humans. It occurs part through long-term mechanisms that regulate the abundance aquaporin-2 channel collecting duct cells. Here, we use deep DNA sequencing mouse cells to ask whether vasopressin signaling selectively increases Aqp2 gene transcription or it triggers broadly targeted transcriptional network. ChIP-Seq quantification binding sites for RNA polymerase II was combined with...

Vasopressin controls osmotic water transport in the renal collecting duct through regulation of aquaporin-2 (AQP2). We carried out bioinformatic analysis quantitative proteomic data from accompanying article to investigate mechanisms involved. The experiments used stable isotope labeling by amino acids cell culture cultured mpkCCD cells quantify each protein species five differential-centrifugation (DC) fractions with or without vasopressin analog 1-desamino-8-d-arginine-vasopressin (dDAVP)....

The sensory organs of the zebrafish lateral-line system (neuromasts) originate from migrating primordia that move along precise pathways. posterior primordium, which deposits neuromasts on body and tail embryo, migrates horizontal myoseptum otic region to tip tail. This migration is controlled by chemokine SDF1, expressed prospective pathway, its receptor CXCR4, cells. In this report, we describe another gene heterogeneously in cells, tacstd. codes for a membrane protein homologous TACSTD1/2...

The peptide hormone arginine vasopressin (AVP) plays a critical role in regulating salt and water transport the mammalian kidney. Recent studies have also demonstrated that AVP can promote cell survival neuronal cells through V1 receptors. current study addresses whether inhibit apoptosis kidney collecting duct via V2 receptors explores downstream signaling pathways this phenomenon. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling analysis caspase cleavage assays...

The mouse mpkCCD cell line is a continuous cultured epithelial with characteristics of renal collecting duct principal cells. This widely used to study transport and its regulation. To provide data resource useful for experimental design interpretation in studies using cells, we have carried out "deep" proteomic profiling these cells three levels fractionation (differential centrifugation, SDS-PAGE, HPLC) followed by tandem mass spectrometry identify quantify proteins. analysis all resulting...

Protein carbamylation is a post-translational modification that can occur in the presence of urea. In solution, urea equilibrium with ammonium cyanate, and occurs when cyanate ions react amino groups lysines, arginines, protein N-termini, as well sulfhydryl cysteines. The concentration elevated renal inner medulla compared other tissues. Due to high concentration, we hypothesized endogenously within rat medulla. Using immunoblotting kidney cortical medullary homogenates carbamyl-lysine...

The control of renal water excretion occurs in part by regulation transcription response to vasopressin cells the collecting duct. A systems biology-based approach understanding transcriptional duct depends on knowledge what factors and other regulatory proteins are present cells' nuclei. goal this article is report comprehensive proteomic profiling cellular fractions enriched nuclear from native inner medullary (IMCD) rat. Multidimensional separation procedures state-of-the art protein mass...

Biological information is growing at a rapid pace, making it difficult for individual investigators to be familiar with all that relevant their own research. Computers are beginning used extract and curate biological information; however, the complexity of human language in research papers continues critical barrier full automation knowledge extraction. Here, we report manually curated base vasopressin actions renal epithelial cells designed readable either by humans or computer programs...

ABSTRACT The recovery of injured or lost sensory neurons after trauma, disease aging is a major scientific challenge. Upon hearing loss balance disorder, regeneration mechanosensory hair cells has been observed in fish, some amphibians and under special circumstances birds, but absent adult mammals. In aquatic vertebrates, are not only present the inner ear also neuromasts lateral line system. zebrafish neuromast an almost unlimited capacity to regenerate cells. This remarkable ability...

Long-term exposure of collecting duct cells to vasopressin increases the abundance aquaporin-2 (AQP2) as well that other proteins. To discover mechanisms involved, we carried out large-scale measurements protein half-lives (t1/2) in cultured renal mpkCCD(clone 11) at steady state using metabolic labeling with stable isotopes (SILAC) coupled mass spectrometry (LC-MS/MS). A mass-balance model assuming first-order degradation kinetics, allowed calculation t1/2 for over 3000 The shortest half...

We hypothesize that the high urea concentration in renal inner medulla (IM) is associated with physiological carbamylation of proteins. Using a carbamylation‐specific antibody, immunoblotting revealed multiple bands kidney antidiuretic rats gradient from cortex to IM, suggestive large‐scale protein carbamylation. To test specificity we incubated BSA 1 M PBS or alone for 4 days. The antibody recognized band consistent carbamylated presence but not absence urea. confirm widespread rat carried...

Vasopressin regulates the water channel aquaporin‐2 in collecting duct cells. Identification of mechanisms involved depends on knowledge genes expressed. To obtain a comprehensive proteome list for vasopressin‐sensitive mpkCCD cells, we used protein mass spectrometry to identify proteins after two levels fractionation: differential centrifugation followed by SDS‐PAGE. After slicing resulting gels into 43 consecutive blocks, gel blocks were subjected in‐gel trypsinization LC‐MS/MS. filtering...

Sickle cell anemia was a rare disease in Chile, especially adults, however the recent immigration wave from Haiti is changing this scenario. We report 29 year old black female with non-disclosed history of sickle anemia. She transfused two units red blood cells, found unconscious and jaundice five days later admitted to hospital. On admission she had hemoglobin 3.3 g/dL, total bilirubin 5.08 mg/dL, LDH 1,306 Ui/L. again, worsening her condition. An alloimmunization delayed hemolytic reaction...

The peptide hormone vasopressin regulates transport in the renal collecting duct part through regulation of gene expression. To obtain a global profile genes regulated by vasopressin, we performed ChIP‐seq analysis RNA polymerase II and acetylated histone H3K27 binding sites associated with transcriptionally active genes. Cultured mpkCCD cells were treated either analog dDAVP (0.1 nM) or vehicle for 24 hours, then processed chromatin IP. DNA‐protein complexes immunoprecipitated using pol...

Blood pressure regulation depends in part on control of ion and water transport the renal collecting duct by vasopressin other hormones. To identify genes that are transcriptionally regulated cultured mouse cells (mpkCCD), we carried out both RNA-seq (n=9) ChIP-seq for RNA polymerase II (Pol-II, n=3) after 24-hr treatment with analog dDAVP or vehicle (Illumina HiSeq2000 sequencer). analysis showed significant changes 33 transcripts 8393 quantified (Volcano plot thresholds: p<0.05...

Vasopressin regulates the expression of many genes in renal collecting duct. To identify mediators, we have carried out LC‐MS/MS profiling cytoplasm, soluble nuclear compartment, and pellet duct mpkCCD cells. Stable isotope labeling (SILAC) was used for relative quantification tryptic peptides vasopressin‐treated (1h dDAVP, 0.1nM) compared to control There were no measurable differences total protein content. We identified 5776 corresponding 1074 proteins compartment. 49 either decreased or...

Modern systems biology utilizes protein mass spectrometry (MS) to quantify thousands of proteins in a given tissue. The alternative MS, western blotting, is low‐throughput, but gives quantitative information as function molecular weight, providing critical about modifications. Here, we describe an approach that combines the advantages both: “pseudo‐western blots” (pWBs). Protein extracts are separated by standard SDS‐PAGE, and each lane sliced into 40 successive blocks followed in‐gel...