Tricellulin is a tight-junction protein necessary for hearing

The inner ear has fluid-filled compartments of different ionic compositions, including the endolymphatic and perilymphatic spaces of the organ of Corti; the separation from one another by epithelial barriers is required for normal hearing. TRIC encodes tricellulin, a recently discovered tight-junction (TJ) protein that contributes to the structure and function of tricellular contacts of neighboring cells in many epithelial tissues. We show that, in humans, four different recessive mutations of TRIC cause nonsyndromic deafness (DFNB49), a surprisingly limited phenotype, given the widespread tissue distribution of tricellulin in epithelial cells. In the inner ear, tricellulin is concentrated at the tricellular TJs in cochlear and vestibular epithelia, including the structurally complex and extensive junctions between supporting and hair cells. We also demonstrate that there are multiple alternatively spliced isoforms of TRIC in various tissues and that mutations of TRIC associated with hearing loss remove all or most of a conserved region in the cytosolic domain that binds to the cytosolic scaffolding protein ZO-1. A wild-type isoform of tricellulin, which lacks this conserved region, is unaffected by the mutant alleles and is hypothesized to be sufficient for structural and functional integrity of epithelial barriers outside the inner ear.     

Improved mass spectrometric proteomic profiling of the secretome of rat vascular endothelial cells

Serum albumin contamination of cells cultured in vitro significantly impedes the mass spectrometric analysis of proteins secreted by the cells. Here we report a novel washing and culturing technique for rat vascular endothelial cells that considerably reduces the concentration of the commonly used additive for cell culture, bovine serum albumin (BSA), in the secretome of these cells. Cells are rinsed stringently and cultured for 24 h in serum-free media without appreciably impeding cell growth or viability. The percentage of BSA scans identified by tandem mass spectrometry (LC-MS/MS) in stringently rinsed cells (average 13.2%) was significantly lower than either the moderately rinsed or no rinse cell treatments (average 35.2% and 45.2% respectively). Furthermore, the stringent wash treatment allowed the confident identification of a larger portion of the secretome of rat endothelial cells by LC-MS/MS.     

Imatinib mesylate inhibits platelet derived growth factor stimulated proliferation of rheumatoid synovial fibroblasts

Synovial fibroblast is the key cell type in the growth of the pathological synovial tissue in arthritis. Here, we show that platelet-derived growth factor (PDGF) is a potent mitogen for synovial fibroblasts isolated from patients with rheumatoid arthritis. Inhibition of PDGF-receptor signalling by imatinib mesylate (1muM) completely abrogated the PDGF-stimulated proliferation and inhibited approximately 70% of serum-stimulated proliferation of synovial fibroblasts. Similar extent of inhibition was observed when PDGF was neutralized with anti-PDGF antibodies, suggesting that imatinib mesylate does not inhibit pathways other than those mediated by PDGF-receptors. No signs of apoptosis were detected in synovial fibroblasts cultured in the presence of imatinib. These results suggest that imatinib mesylate specifically inhibits PDGF-stimulated proliferation of synovial fibroblasts, and that inhibition of PDGF-receptors could represent a feasible target for novel antirheumatic therapies.     

Antibacterial Co(II), Ni(II), Cu(II) and Zn(II) complexes of Schiff bases derived from fluorobenzaldehyde and triazoles

Antibacterial Schiff bases derived from 1,2,4-triazoles as well as their metal complexes incorporating cobalt(II), nickel(II), copper(II) and zinc(II) have been synthesized and characterized. Physico-chemical studies suggest that an octahedral geometry for the cobalt(II), nickel(II) and zinc(II)and square-planer geometry for the copper(II) complexes. These complexes have been screened for antibacterial activity against three Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis) and two Gram-negative (Salmonella typhi and Pseudomonas aeruginosa) bacterial strains, and results compared with the activity of the free ligands. The metal complexes were found to be more potent against one or more bacterial strains than the free ligands.     

Zygomatic implant-retained fixed complete denture for an elderly patient

Dental rehabilitation of a completely edentulous geriatric patient has always been a challenge to the clinician, especially in treating those with higher expectations and demands. Treatment duration and the amount of residual alveolar bone available are often important considerations when planning for dental implant-based fixed treatment for these patients. With the introduction of zygomatic implants, a graftless alternative solution has emerged for deficient maxillary bone with provision for immediate loading. This article describes the treatment of a completely edentulous elderly patient using zygomatic implants in conjunction with conventional implants. The implants were immediately loaded using a definitive acrylic resin fixed denture reinforced with a cast metal framework, to provide function and aesthetics.     

Role of Somatodendritic and Postsynaptic 5-HT1A Receptors on Learning and Memory Functions in Rats

Memory impairment is a major problem afflicting mankind. The association between memory functions and neurotransmitter functions is of great interest for understanding brain function. Serotonergic pathways play an important role in the modulation of memory functions but the importance of its receptor types and subtypes on memory functions is still unclear. Activation and blockade of various serotonin (5-HT) receptors has been reported to alter cognitive processes and 5-HT receptor antagonism could be beneficial in the treatment of cognitive diseases. The role of 5-HT on memory functions is complicated. Among the 5-HT receptors subtypes, 5-HT(1A) receptors are of special interest because these receptors are present in the brain areas involved in learning and memory functions such as hippocampus and cortex. The present study was therefore designed to investigate the effect of activation and blockade of somatodendritic and/or postsynaptic 5-HT(1A) receptor on learning and memory functions in rats using modified version of water maze. In this study, 8-OH-DPAT (8-hydroxy-2-(di-N-propylamino) tetralin) at 0.3?mg/kg significantly enhanced learning acquisition (LA), short-term memory (STM) and long term memory (LTM) of rats pre-injected with saline suggesting that the activation of pre-synaptic 5-HT(1A) receptors by its agonist enhanced the memory functions of rats. Conversely, rats injected with 8-OH-DPAT at 1.0?mg/kg exhibited impaired LA and STM and had no effect on LTM. It was also shown in this study that blockade of 5-HT(1A) receptors by spiperone enhanced LA, had no effect on STM but impaired the LTM, which showed that the blockade of 5-HT(1A) receptors by its antagonist exerts different effect on different types of memory. This study suggests that 5-HT(1A) receptor could be used as a significant pharmacological target for the treatment of CNS diseases. Unraveling the role of serotonin in cognition and memory disorders could provide better therapy and it may lead to new insights in our understandings of learning and memory.     

Role of Ca(2+) in proteolysis-inducing factor (PIF)-induced atrophy of skeletal muscle

Proteolysis-inducing factor (PIF) induces muscle loss in cancer cachexia through a high affinity membrane bound receptor. This study investigates the mechanism by which the PIF receptor communicates to intracellular signalling pathways. C(2)C(12) murine myoblasts were used as a model using PIF purified from MAC16 tumours. Calcium imaging was determined using fura-4-acetoxymethyl ester (Fura-4-AM). PIF induced a rapid rise in Ca(2+)(i), which was completely attenuated by a anti-receptor antibody, or peptides representing 20 mers of the N-terminus of the PIF receptor. Other agents catabolic for skeletal muscle including angiotensin II (AngII) tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) also induced a rise in Ca(2+)(i), but this was not attenuated by anti-PIF-receptor antibody. The rise in Ca(2+)(i) induced by PIF and AngII was completely attenuated by the Zn(2+) chelator D-myo-inositol-1,2,6-triphosphate, and this was reversed by administration of exogenous Zn(2+). The Ca(2+)(i) rise induced by PIF was independent of the presence of extracellular Ca(2+), and attenuated by the Ca(2+) pump inhibitor thapsigargin, suggesting that the Ca(2+)(i) rise was due to release from intracellular stores. This rise in Ca(2+)(i) induced by PIF was attenuated by both the phospholipase C inhibitor U73122 and 2-APB, an inhibitor of the inositol 1,4,5-triphosphate receptor, suggesting the involvement of a G-protein. Binding of the PIF to its receptor in skeletal muscle triggers a rise in Ca(2+)(i), which initiates a signalling cascade leading to a depression in protein synthesis, and an increase in protein degradation.     

NADPH oxidase 4 mediates upregulation of type 4 phosphodiesterases in human endothelial cells

The protective actions of prostacyclin (PGI(2) ) are mediated by cyclic AMP (cAMP) which is reduced by type 4 phosphodiesterases (PDE4) which hydrolyze cAMP. Superoxide (O2(-)) from NADPH oxidase (Nox) is associated with impaired PGI(2) bioactivity. The objective of this study, therefore, was to study the relationship between Nox and PDE4 expression in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with the thromboxane A(2) analog, U46619, 8-isoprostane F(2α) (8IP), or tumor necrosing factor alpha (TNFα) [±iloprost (a PGI(2) analog)] and the expression of PDE4A, B, C, and D and splice variants thereof assessed using Western blotting and qPCR and mRNA silencing of Nox4 and Nox5. Effects on cell replication and angiogenesis were also studied. U46619, 8IP, and TNFα increased the expression of Nox 4 and Nox 5 and all PDE4 isoforms as well as cell replication and tubule formation (index of angiogenesis), effects inhibited by mRNA silencing of Nox4 (but not Nox5) and iloprost and rolipram. These data demonstrate that upregulation of Nox4 leads to an upregulation of PDE4A, B, and D and increased hydrolysis of cAMP which in turn augments cell replication and angiogenesis. This mechanism may be central to vasculopathies associated with endothelial dysfunction since the PGI(2)-cAMP signaling axis plays a key role in mediating functions that include hemostasis and angiogenesis.     

Biophysical characterization of the proton-coupled oligopeptide transporter YjdL

Proton-coupled oligopeptide transporters (POTs) utilize the electrochemical proton gradient to facilitate uptake of di- or tripeptide molecules. YjdL is one of four POTs found in Escherichia coli. It has shown an extraordinary preference for di- rather than tripeptides, and is therefore significantly different from prototypical POTs such as the human hPepT1. Nonetheless YjdL contains several highly conserved POT residues, which include Glu388 that is located in the putative substrate binding cavity. Here we present biophysical characterization of WT-YjdL and Glu388Gln. Isothermal titration calorimetrical studies exhibit a K_d of 14 μM for binding of Ala-Lys to WT-YjdL. Expectedly, no binding could be detected for the tripeptide Ala-Ala-Lys. Surprisingly however, binding could not be detected for Ala-Gln, although earlier studies indicated inhibitory potencies of Ala-Gln to be comparable to Ala-Lys (IC₅₀ values of 0.6 compared to 0.3 mM). Finally, Ala-Lys binding to Glu388Gln was also undetectable which may support a previously suggested role in interaction with the ligand peptide N-terminus.     

Oligonucleotide-stabilized fluorescent silver nanoclusters for turn-on detection of melamine

Fluorescence nanoclusters have been used for the determination of melamine for the first time. The method is based on the fluorescence turn-on of oligonucleotide-stabilized silver nanoclusters (DNA-Ag NCs) by melamine. The enhancement factors (I-I(0))/I(0) increase linearly with melamine concentrations over the range 5.0×10(-8)-7.0×10(-6) M (R(2)=0.998). The detection limit is 1.0×10(-8) M, which is approximately 2000 times lower than the US Food and Drug Administration estimated melamine safety limit of 20.0 μM. Furthermore, the milk samples spiked with melamine are analyzed with excellent recoveries.