Genomic analysis of the function of the transcription factor gata3 during development of the Mammalian inner ear

We have studied the function of the zinc finger transcription factor gata3 in auditory system development by analysing temporal profiles of gene expression during differentiation of conditionally immortal cell lines derived to model specific auditory cell types and developmental stages. We tested and applied a novel probabilistic method called the gamma Model for Oligonucleotide Signals to analyse hybridization signals from Affymetrix oligonucleotide arrays. Expression levels estimated by this method correlated closely (p<0.0001) across a 10-fold range with those measured by quantitative RT-PCR for a sample of 61 different genes. In an unbiased list of 26 genes whose temporal profiles clustered most closely with that of gata3 in all cell lines, 10 were linked to Insulin-like Growth Factor signalling, including the serine/threonine kinase Akt/PKB. Knock-down of gata3 in vitro was associated with a decrease in expression of genes linked to IGF-signalling, including IGF1, IGF2 and several IGF-binding proteins. It also led to a small decrease in protein levels of the serine-threonine kinase Akt2/PKB beta, a dramatic increase in Akt1/PKB alpha protein and relocation of Akt1/PKB alpha from the nucleus to the cytoplasm. The cyclin-dependent kinase inhibitor p27(kip1), a known target of PKB/Akt, simultaneously decreased. In heterozygous gata3 null mice the expression of gata3 correlated with high levels of activated Akt/PKB. This functional relationship could explain the diverse function of gata3 during development, the hearing loss associated with gata3 heterozygous null mice and the broader symptoms of human patients with Hearing-Deafness-Renal anomaly syndrome

Biocompatible Polyhydroxyethylaspartamide-based Micelles with Gadolinium for MRI Contrast Agents

Biocompatible poly-[N-(2-hydroxyethyl)-d,l-aspartamide]-methoxypoly(ethyleneglycol)-hexadecylamine (PHEA-mPEG-C16) conjugated with 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid-gadolinium (DOTA-Gd) via ethylenediamine (ED) was synthesized as a magnetic resonance imaging (MRI) contrast agent. Amphiphilic PHEA-mPEG-C16-ED-DOTA-Gd forms micelle in aqueous solution. All the synthesized materials were characterized by proton nuclear magnetic resonance (1H NMR). Micelle size and shape were examined by dynamic light scattering (DLS) and atomic force microscopy (AFM). Micelles with PHEA-mPEG-C16-ED-DOTA-Gd showed higher relaxivities than the commercially available gadolinium contrast agent. Moreover, the signal intensity of a rabbit liver was effectively increased after intravenous injection of PHEA-mPEG-C16-ED-DOTA-Gd

Serum amyloid A inhibits RANKL-induced osteoclast formation

When mouse bone marrow-derived macrophages were stimulated with serum amyloid A (SAA), which is a major acute-phase protein, there was strong inhibition of osteoclast formation induced by the receptor activator of nuclear factor kappaB ligand. SAA not only markedly blocked the expression of several osteoclast-associated genes (TNF receptor-associated factor 6 and osteoclast-associated receptor) but also strongly induced the expression of negative regulators (MafB and interferon regulatory factor 8). Moreover, SAA decreased c-fms expression on the cell surface via shedding of the c-fms extracellular domain. SAA also restrained the fusion of osteoclast precursors by blocking intracellular ATP release. This inhibitory response of SAA is not mediated by the well-known SAA receptors (formyl peptide receptor 2, Toll-like receptor 2 (TLR2) or TLR4). These findings provide insight into a novel inhibitory role of SAA in osteoclastogenesis and suggest that SAA is an important endogenous modulator that regulates bone homeostasis.open

BRCA1 Interacts with Smad3 and Regulates Smad3-Mediated TGF-β Signaling during Oxidative Stress Responses

BRCA1 is a key regulatory protein participating in cell cycle checkpoint and DNA damage repair networks. BRCA1 plays important roles in protecting numerous cellular processes in response to cell damaging signals. Transforming growth factor-beta (TGF-beta) is a potent regulator of growth, apoptosis and invasiveness of tumor cells. TFG-beta activates Smad signaling via its two cell surface receptors, the TbetaRII and ALK5/TbetaRI, leading to Smad-mediated transcriptional regulation.Here, we report an important role of BRCA1 in modulating TGF-beta signaling during oxidative stress responses. Wild-type (WT) BRCA1, but not mutated BRCA1 failed to activate TGF-beta mediated transactivation of the TGF-beta responsive reporter, p3TP-Lux. Further, WT-BRCA1, but not mutated BRCA1 increased the expression of Smad3 protein in a dose-dependent manner, while silencing of WT-BRCA1 by siRNA decreased Smad3 and Smad4 interaction induced by TGF-beta in MCF-7 breast cancer cells. BRCA1 interacted with Smad3 upon TGF-beta1 stimulation in MCF-7 cells and this interaction was mediated via the domain of 298-436aa of BRCA1 and Smad3 domain of 207-426aa. In addition, H(2)O(2) increased the colocalization and the interaction of Smad3 with WT-BRCA1. Interestingly, TGF-beta1 induced Smad3 and Smad4 interaction was increased in the presence of H(2)O(2) in cells expressing WT-BRCA1, while the TGF-beta1 induced interaction between Smad3 and Smad4 was decreased upon H(2)O(2) treatment in a dose-dependent manner in HCC1937 breast cancer cells, deficient for endogenous BRCA1. This interaction between Smad3 and Smad4 was increased in reconstituted HCC1937 cells expressing WT-BRCA1 (HCC1937/BRCA1). Further, loss of BRCA1 resulted in H(2)O(2) induced nuclear export of phosphor-Smad3 protein to the cytoplasm, resulting decreased of Smad3 and Smad4 interaction induced by TGF-beta and in significant decrease in Smad3 and Smad4 transcriptional activities.These results strongly suggest that loss or reduction of BRCA1 alters TGF-beta growth inhibiting activity via Smad3 during oxidative stress responses

Epigenetic understanding of gene-environment interactions in psychiatric disorders: a new concept of clinical genetics

Epigenetics is a mechanism that regulates gene expression independently of the underlying DNA sequence, relying instead on the chemical modification of DNA and histone proteins. Although environmental and genetic factors were thought to be independently associated with disorders, several recent lines of evidence suggest that epigenetics bridges these two factors. Epigenetic gene regulation is essential for normal development, thus defects in epigenetics cause various rare congenital diseases. Because epigenetics is a reversible system that can be affected by various environmental factors, such as drugs, nutrition, and mental stress, the epigenetic disorders also include common diseases induced by environmental factors. In this review, we discuss the nature of epigenetic disorders, particularly psychiatric disorders, on the basis of recent findings: 1) susceptibility of the conditions to environmental factors, 2) treatment by taking advantage of their reversible nature, and 3) transgenerational inheritance of epigenetic changes, that is, acquired adaptive epigenetic changes that are passed on to offspring. These recently discovered aspects of epigenetics provide a new concept of clinical genetics

13[C]-Urea Breath Test as a Novel Point-of-Care Biomarker for Tuberculosis Treatment and Diagnosis

BACKGROUND: Pathogen-specific metabolic pathways may be detected by breath tests based on introduction of stable isotopically-labeled substrates and detection of labeled products in exhaled breath using portable infrared spectrometers. METHODOLOGY/PRINCIPAL FINDINGS: We tested whether mycobacterial urease activity could be utilized in such a breath test format as the basis of a novel biomarker and diagnostic for pulmonary TB. Sensitized New-Zealand White Rabbits underwent bronchoscopic infection with either Mycobacterium bovis or Mycobacterium tuberculosis. Rabbits were treated with 25 mg/kg of isoniazid (INH) approximately 2 months after infection when significant cavitary lung pathology was present. [(13)C] urea was instilled directly into the lungs of intubated rabbits at selected time points, exhaled air samples analyzed, and the kinetics of delta(13)CO(2) formation were determined. Samples obtained prior to inoculation served as control samples for background (13)CO(2) conversion in the rabbit model. (13)CO(2), from metabolic conversion of [(13)C]-urea by mycobacterial urease activity, was readily detectable in the exhaled breath of infected rabbits within 15 minutes of administration. Analyses showed a rapid increase in the rate of (13)CO(2) formation both early in disease and prior to treatment with INH. Following INH treatment, all evaluable rabbits showed a decrease in the rate of (13)CO(2) formation. CONCLUSIONS/SIGNIFICANCE: Urea breath testing may provide a useful diagnostic and biomarker assay for tuberculosis and for treatment response. Future work will test specificity for M. tuberculosis using lung-targeted dry powder inhalation formulations, combined with co-administering oral urease inhibitors together with a saturating oral dose of unlabeled urea, which would prevent the delta(13)CO(2) signal from urease-positive gastrointestinal organisms

Trauma Hemorrhagic Shock-Induced Lung Injury Involves a Gut-Lymph-Induced TLR4 Pathway in Mice

Injurious non-microbial factors released from the stressed gut during shocked states contribute to the development of acute lung injury (ALI) and multiple organ dysfunction syndrome (MODS). Since Toll-like receptors (TLR) act as sensors of tissue injury as well as microbial invasion and TLR4 signaling occurs in both sepsis and noninfectious models of ischemia/reperfusion (I/R) injury, we hypothesized that factors in the intestinal mesenteric lymph after trauma hemorrhagic shock (T/HS) mediate gut-induced lung injury via TLR4 activation.The concept that factors in T/HS lymph exiting the gut recreates ALI is evidenced by our findings that the infusion of porcine lymph, collected from animals subjected to global T/HS injury, into naïve wildtype (WT) mice induced lung injury. Using C3H/HeJ mice that harbor a TLR4 mutation, we found that TLR4 activation was necessary for the development of T/HS porcine lymph-induced lung injury as determined by Evan's blue dye (EBD) lung permeability and myeloperoxidase (MPO) levels as well as the induction of the injurious pulmonary iNOS response. TRIF and Myd88 deficiency fully and partially attenuated T/HS lymph-induced increases in lung permeability respectively. Additional studies in TLR2 deficient mice showed that TLR2 activation was not involved in the pathology of T/HS lymph-induced lung injury. Lastly, the lymph samples were devoid of bacteria, endotoxin and bacterial DNA and passage of lymph through an endotoxin removal column did not abrogate the ability of T/HS lymph to cause lung injury in naïve mice.Our findings suggest that non-microbial factors in the intestinal mesenteric lymph after T/HS are capable of recreating T/HS-induced lung injury via TLR4 activation

The Wor1-like Protein Fgp1 Regulates Pathogenicity, Toxin Synthesis and Reproduction in the Phytopathogenic Fungus Fusarium graminearum

WOR1 is a gene for a conserved fungal regulatory protein controlling the dimorphic switch and pathogenicity determents in Candida albicans and its ortholog in the plant pathogen Fusarium oxysporum, called SGE1, is required for pathogenicity and expression of key plant effector proteins. F. graminearum, an important pathogen of cereals, is not known to employ switching and no effector proteins from F. graminearum have been found to date that are required for infection. In this study, the potential role of the WOR1-like gene in pathogenesis was tested in this toxigenic fungus. Deletion of the WOR1 ortholog (called FGP1) in F. graminearum results in greatly reduced pathogenicity and loss of trichothecene toxin accumulation in infected wheat plants and in vitro. The loss of toxin accumulation alone may be sufficient to explain the loss of pathogenicity to wheat. Under toxin-inducing conditions, expression of genes for trichothecene biosynthesis and many other genes are not detected or detected at lower levels in Δfgp1 strains. FGP1 is also involved in the developmental processes of conidium formation and sexual reproduction and modulates a morphological change that accompanies mycotoxin production in vitro. The Wor1-like proteins in Fusarium species have highly conserved N-terminal regions and remarkably divergent C-termini. Interchanging the N- and C- terminal portions of proteins from F. oxysporum and F. graminearum resulted in partial to complete loss of function. Wor1-like proteins are conserved but have evolved to regulate pathogenicity in a range of fungi, likely by adaptations to the C-terminal portion of the protein