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The role of hair cells, cilia and ciliary motility in otolith formation in the zebrafish otic vesicle

Stooke-Vaughan, Georgina A; Huang, Peng; Hammond, Katherine L; Schier, Alexander F; Whitfield, Tanya T
Otoliths are biomineralised structures required for the sensation of gravity, linear acceleration and sound in the zebrafish ear. Otolith precursor particles, initially distributed throughout the otic vesicle lumen, become tethered to the tips of hair cell kinocilia (tether cilia) at the otic vesicle poles, forming two otoliths. We have used high-speed video microscopy to investigate the role of cilia and ciliary motility in otolith formation. In wild-type ears, groups of motile cilia are present at the otic vesicle poles, surrounding the immotile tether cilia. A few motile cilia are also found on the medial wall, but most cilia (92-98%) in the otic vesicle are immotile. In mutants with defective cilia (iguana) or ciliary motility (lrrc50), otoliths are frequently ectopic, untethered or fused. Nevertheless, neither cilia nor ciliary motility are absolutely required for otolith tethering: a mutant that lacks cilia completely (MZovl) is still capable of tethering otoliths at the otic vesicle poles. In embryos with attenuated Notch signalling [mindbomb mutant or Su(H) morphant], supernumerary hair cells develop and otolith precursor particles bind to the tips of all kinocilia, or bind directly to the hair cells' apical surface if cilia are absent [MZovl injected with a Su(H)1+2 morpholino]. However, if the first hair cells are missing (atoh1b morphant), otolith formation is severely disrupted and delayed. Our data support a model in which hair cells produce an otolith precursor-binding factor, normally localised to tether cell kinocilia. We also show that embryonic movement plays a minor role in the formation of normal otoliths.
PMCID:3328178
PMID: 22461562
ISSN: 0950-1991
CID: 876822

Connexin43, and the regulation of intercalated disc function

Delmar M; Liang FX
PMCID:3289726
PMID: 22056332
ISSN: 1556-3871
CID: 150000

Cardiac connexins, mutations and arrhythmias

Delmar, Mario; Makita, Naomasa
PURPOSE OF REVIEW: Connexins are the pore forming subunits of gap junction channels. They are essential for cardiac action potential propagation. Connexins are modified at the transcriptional or posttranslational levels under pathological states such as cardiac hypertrophy or ischemia, thus contributing to the arrhythmogenic substrate. However, the relation between nucleotide substitutions in the connexin gene and the occurrence of cardiac arrhythmias remains largely unexplored. RECENT FINDINGS: Recent studies have reported an association between nucleotide substitutions in the connexin40 (Cx40) and connexin43 (Cx43) genes (GJA5 and GJA1, respectively) and cardiac arrhythmias. Of note, however, germline mutations in Cx43 are considered causative of oculodentodigital dysplasia, a pleiotropic syndrome wherein cardiac manifestations are notoriously absent. SUMMARY: Here, we review some of the current knowledge on the association between cardiac connexins and inherited arrhythmias.
PMID: 22382502
ISSN: 0268-4705
CID: 164342

Clinical features and genetic basis of 63 patients with progressive cardiac conduction defect [Meeting Abstract]

Makita, N; Makiyama, T; Seki, A; Nogami, A; Ohkubo, K; Watanabe, I; Shimizu, W; Watanabe, H; Sumitomo, N; Horie, M; Delmar, M
Introduction: Progressive cardiac conduction disturbance (PCCD) is a hereditary disorder of the His-Purkinje system that often leads to complete heart block, pacemaker implantation, or sudden death. Although mutations in genes SCN5A, SCN1B and TRPM4 have been identified in some PCCD pedigrees, large scale studies of its clinical features, prognosis, and genetic basis are not available. We studied a population of 63 Japanese PCCD patients (47 probands and 16 family members; male 37, female 26) without underlying structural heart diseases. Despite the assumption that PCCD predominates in the elderly, the age of onset of the proband showed a wide-range distribution (59.6+/-23.7 years) with two peaks in the 2nd and 6th decade of life. Patients were followed for a variable period of time (0.3 to 33 years; 11.6+/-10.0 years). Progressive aggravation of the conduction disturbance and cardiac dysfunction with LVEF<35% was evident in 46% and 33% of patients, respectively. Pacemaker or ICD was implanted in 44 patients (70%). Six patients, three with device therapy and three without, died suddenly. Genetic screening revealed mutations in SCN5A (coding for Nav1.5; n=9) and in GJA5 (coding for connexin40; n=1). Heterologous expression of the GJA5 mutant in N2A cells resulted in marked reduction of junctional conductance and diffuse localization of Cx40-immunoreactive proteins in the vicinity of the plasma membrane without formation of gap junctions. The proband of the GJA5 mutation and his mother died at the age of 10 and 30, respectively, indicating an early-onset and malignant variant of PCCD. Moreover, mutations were identified in LMNA(n=9), the gene encoding lamin A/C, an inner nuclear membrane protein involved in a number of reported cases of dilated cardiomyopathy. The electrophysiological defects preceded the cardiac dysfunction in all 9 LMNA carriers and in 2 family members, presumably due to an age-dependent enhancement of apoptosis in cells of the conduction system. These data show that the PCCD is a heterogeneous disease that can be associated with defects in the amino acid sequence of integral membrane proteins involved in cell excitability and/or action potential propagation. A separate group may represent a prodromal stage of lamin A/C-related dilated cardiomyopathy. Methods: N/A Results:N/A Conclusions: N/A
EMBASE:70738731
ISSN: 1547-5271
CID: 166948

Picking ChIP-seq peak detectors for analyzing chromatin modification experiments

Micsinai, M; Parisi, F; Strino, F; Asp, P; Dynlacht, BD; Kluger, Y
Numerous algorithms have been developed to analyze ChIP-Seq data. However, the complexity of analyzing diverse patterns of ChIP-Seq signals, especially for epigenetic marks, still calls for the development of new algorithms and objective comparisons of existing methods. We developed Qeseq, an algorithm to detect regions of increased ChIP read density relative to background. Qeseq employs critical novel elements, such as iterative recalibration and neighbor joining of reads to identify enriched regions of any length. To objectively assess its performance relative to other 14 ChIP-Seq peak finders, we designed a novel protocol based on Validation Discriminant Analysis (VDA) to optimally select validation sites and generated two validation datasets, which are the most comprehensive to date for algorithmic benchmarking of key epigenetic marks. In addition, we systematically explored a total of 315 diverse parameter configurations from these algorithms and found that typically optimal parameters in one dataset do not generalize to other datasets. Nevertheless, default parameters show the most stable performance, suggesting that they should be used. This study also provides a reproducible and generalizable methodology for unbiased comparative analysis of high-throughput sequencing tools that can facilitate future algorithmic development.
PMCID:3351193
PMID: 22307239
ISSN: 0305-1048
CID: 162561

A comparative analysis of the osteogenic effects of BMP-2, FGF-2, and VEGFA in a calvarial defect model

Behr, Bjorn; Sorkin, Michael; Lehnhardt, Marcus; Renda, Andrea; Longaker, Michael T; Quarto, Natalina
The utilization of growth factors for bone regeneration is a widely studied field. Since the approval of bone morphogenetic protein-2 (BMP-2) for therapeutic use in humans, the concept of utilizing growth factors for bone regeneration in translational medicine has become even more attractive. Despite many studies published on individual growth factors in various bone models, comparative analysis is largely lacking. The aim of our study was to compare three different proosteogenic factors under identical in vivo conditions. Thus, we tested the bone regeneration capacity of the three different growth factors BMP-2, fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor A (VEGFA) in a calvarial defect model. We demonstrated that BMP-2 and VEGFA had similar bone healing capacities, resulting in complete calvarial healing as early as week 3. FGF-2 also showed a significantly higher bone regeneration capacity; however, the healing rate was lower than with BMP-2 and VEGFA. Interestingly, these findings were paralleled by an increased angiogenic response upon healing in BMP-2- and VEGFA-treated calvarial defects as compared with FGF-2. Immunohistochemistry for proliferating and osteoprogenitor cells revealed activity at different points after surgery among the groups. In conclusion, we demonstrated an efficient bone regeneration capacity of both BMP-2 and VEGFA, which was superior to FGF-2. Moreover, this study highlights the efficient bone regeneration of VEGFA, which was comparable with BMP-2. These data provide a valuable comparative analysis, which can be used to further optimize growth factor-based strategies in skeletal tissue engineering.
PMCID:3338108
PMID: 22195699
ISSN: 1937-3341
CID: 1216952

Attractive guidance: How the chemokine SDF1/CXCL12 guides different cells to different locations

Lewellis, Stephen W; Knaut, Holger
During the development and adult life of multicellular organisms cells move from one location to another as they assemble into organs, seal a wound or fight pathogens. For navigation, migrating cells follow cues that guide them to their final position. Frequently, a single cue simultaneously guides different cells to different positions. Recent studies of one such cue-the chemokine SDF1-suggest strategies for how the animal achieves this task without causing erroneous migration.
PMCID:3345092
PMID: 22414535
ISSN: 1084-9521
CID: 166787

The degradation of apolipoprotein B100: Multiple opportunities to regulate VLDL triglyceride production by different proteolytic pathways

Fisher, Edward A
Very low density lipoproteins (VLDL) are a major secretory product of the liver. They serve to transport endogenously synthesized lipids, mainly triglycerides (but also some cholesterol and cholesteryl esters) to peripheral tissues. VLDL is also the precursor of LDL. ApoB100 is absolutely required for VLDL assembly and secretion. The amount of VLDL triglycerides secreted by the liver depends on the amount loaded onto each lipoprotein particle, as well as the number of particles. Each VLDL has one apoB100 molecule, making apoB100 availability a key determinant of the number of VLDL particles, and hence, triglycerides, that can be secreted by hepatic cells. Surprisingly, the pool of apoB100 in the liver is typically regulated not by its level of synthesis, which is relatively constant, but by its level of degradation. It is now recognized that there are multiple opportunities for the hepatic cell to intercept apoB100 molecules and to direct them to distinct degradative processes. This mini-review will summarize progress in understanding these processes, with an emphasis on autophagy, the most recently described pathway of apoB100 degradation, and the one with possibly the most physiologic relevance to common metabolic perturbations affecting VLDL production. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.
PMCID:3593638
PMID: 22342675
ISSN: 0006-3002
CID: 166878

Development stage-specific proteomic profiling uncovers small, lineage specific proteins most abundant in the Aspergillus Fumigatus conidial proteome

Suh, Moo-Jin; Fedorova, Natalie D; Cagas, Steven E; Hastings, Susan; Fleischmann, Robert D; Peterson, Scott N; Perlin, David S; Nierman, William C; Pieper, Rembert; Momany, Michelle
BACKGROUND: The pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals such as leukemia and bone marrow transplant patients. Germination of inhaled conidia (asexual spores) in the host is critical for the initiation of infection, but little is known about the underlying mechanisms of this process. RESULTS: To gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development. Four different stages were examined: dormant conidia, isotropically expanding conidia, hyphae in which germ tube emergence has just begun, and pre-septation hyphae. To enrich for glycan-linked cell wall proteins we used an alkaline cell extraction method. Shotgun proteomic resulted in the identification of 375 unique gene products with high confidence, with no evidence for enrichment of cell wall-immobilized and secreted proteins. The most interesting discovery was the identification of 52 proteins enriched in dormant conidia including 28 proteins that have never been detected in the A. fumigatus conidial proteome such as signaling protein Pil1, chaperones BipA and calnexin, and transcription factor HapB. Additionally we found many small, Aspergillus specific proteins of unknown function including 17 hypothetical proteins. Thus, the most abundant protein, Grg1 (AFUA_5G14210), was also one of the smallest proteins detected in this study (M.W. 7,367). Among previously characterized proteins were melanin pigment and pseurotin A biosynthesis enzymes, histones H3 and H4.1, and other proteins involved in conidiation and response to oxidative or hypoxic stress. In contrast, expanding conidia, hyphae with early germ tubes, and pre-septation hyphae samples were enriched for proteins responsible for housekeeping functions, particularly translation, respiratory metabolism, amino acid and carbohydrate biosynthesis, and the tricarboxylic acid cycle. CONCLUSIONS: The observed temporal expression patterns suggest that the A. fumigatus conidia are dominated by small, lineage-specific proteins. Some of them may play key roles in host-pathogen interactions, signal transduction during conidial germination, or survival in hostile environments.
PMCID:3424117
PMID: 22545825
ISSN: 1477-5956
CID: 309662

Intracellular modulation of signaling pathways by annexin a6 regulates terminal differentiation of chondrocytes

Minashima, Takeshi; Small, William; Moss, Stephen E; Kirsch, Thorsten
Annexin A6 (AnxA6) is highly expressed in hypertrophic and terminally differentiated growth plate chondrocytes. Rib chondrocytes isolated from newborn AnxA6-/- mice showed delayed terminal differentiation as indicated by reduced terminal differentiation markers, including alkaline phosphatase, matrix metalloproteases-13, osteocalcin, and runx2, and reduced mineralization. Lack of AnxA6 in chondrocytes led to a decreased intracellular Ca(2+) concentration and protein kinase C alpha (PKCalpha) activity, ultimately resulting in reduced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activities. The 45 C-terminal amino acids of AnxA6 (AnxA6(1-627)) were responsible for the direct binding of AnxA6 to PKCalpha. Consequently, transfection of AnxA6-/- chondrocytes with full-length AnxA6 rescued the reduced expression of terminal differentiation markers, whereas transfection of AnxA6-/- chondrocytes with AnxA6(1-627) did not or only partially rescued the decreased mRNA levels of terminal differentiation markers. In addition, lack of AnxA6 in matrix vesicles, which initiate the mineralization process in growth plate cartilage, resulted in reduced alkaline phosphatase activity and Ca(2+) and inorganic phosphate (P(i)) content and the inability to form hydroxyapatite-like crystals in vitro. Histological analysis of femoral, tibial, and rib growth plates from newborn mice revealed that the hypertrophic zone of growth plates from newborn AnxA6-/- mice was reduced in size. In addition, reduced mineralization was evident in the hypertrophic zone of AnxA6-/- growth plate cartilage, although apoptosis was not altered compared with wild type growth plates. In conclusion, AnxA6 via its stimulatory actions on PKCalpha and its role in mediating Ca(2+) flux across membranes regulates terminal differentiation and mineralization events of chondrocytes.
PMCID:3340232
PMID: 22399299
ISSN: 0021-9258
CID: 166502