Identification of a novel Cochlin isoform in the perilymph: insights to Cochlin function and the pathogenesis of DFNA9

The COCH gene mutated in DFNA9, an autosomal dominant hereditary sensorineural hearing loss and vestibular disorder, encodes Cochlin. Previously, we reported three bovine Cochlin isoforms, p63s, p44s, and p40s, which exhibit significant molecular heterogeneity in vivo. Here we have characterized Cochlin isoforms by generating four isoform-specific anti-Cochlin antibodies. The same three Cochlin isoforms, p63s, p44s, and p40s, were detected in human and cow inner ear tissue; however, p44s and p40s were not detected in perilymph. We identified a novel short 16kDa isoform in human perilymph and a 18-23kDa isoform in cow perilymph, named Cochlin-tomoprotein (CTP), corresponding to the N-terminus of full-length Cochlin (p63s) and the LCCL domain. Notably, CTP contains all of the known mutation sites associated with DFNA9. The pathogenesis of DFNA9 is not fully clarified as yet, and this novel perilymph-associated CTP isoform might provide mechanistic clues to how mutations in the COCH gene damage the inner ear function.     

Diversity in the oligomeric channel structure of the multidrug efflux pumps in Pseudomonas aeruginosa

MexAB-OprM, the multidrug efflux pump of Pseudomonas aeruginosa, contributes to the high resistance of this organism to a wide variety of antibiotics. To investigate the structure and function of OprM, the outer membrane channel of MexAB-OprM, we examined the oligomeric states of OprM and its homologues OprJ and OprN. These proteins were treated with crosslinking reagent after their reconstitution into liposome membranes. The crosslinked products indicated that OprM and OprN formed trimers, while OprJ unexpectedly appeared to form a tetramer. In order to test whether differences in oligomeric structure might be intimately related to channel function, we examined the channel-forming activity of these proteins by liposome swelling assay. However, no significant differences in channel characteristics were detected among OprM, OprJ, and OprN. We proposed the probable explanation for the diversity in the oligomeric structure of the channel proteins.     

雌激素及其受体在内耳发育和功能上的作用

雌激素在生殖系统、认知记忆系统、骨骼和神经的发育及其功能维持等多种生理功能中扮演了重要的作用。近年来,在内耳发育及其功能研究过程中,许多学者发现在听力和平衡系统功能上的性别差异可能归根因于不同性别的雌激素水平差异。这些研究表明,雌激素及其受体在内耳发育、听力和平衡系统功能维持上也具有重要作用。该文用一个新的视角聚焦于雌激素及其受体在内耳发育和功能上的研究进展。该综述能为进一步研究雌激素在听力和平衡系统中的作用机制及相关疾病的临床治疗提供参考。     

Functional analysis of spoT, relA and dksA genes on quinolone tolerance in Pseudomonas aeruginosa under nongrowing condition

To assess the contribution of ppGpp in antibiotic tolerance to quinolone in Pseudomonas aeruginosa, knockout mutants of the genes involved or linked with the stringent response, such as relA, spoT and dksA, were constructed and investigated for their antibiotic susceptibility to quinolones. The survival of the dksA and spoT mutants in the presence of 8 microg/ml of ofloxacin and 1 microg/ml of ciprofloxacin were shown to be approximately 20-180 and 10-40 times respectively, higher than the same for the wild type strain. The intracellular levels of ppGpp determined with high performance liquid chromatography (HPLC) demonstrated that spoT and dksA mutants possess higher basal levels of ppGpp. The data suggest that elevated basal levels of ppGpp may be responsible for rendering these mutants tolerant to quinolones and expand the importance of ppGpp as an antimicrobial target in P. aeruginosa.     

Evaluation of the biofilm-forming ability and genetic typing for clinical isolates of Pseudomonas aeruginosa by enterobacterial repetitive intergenic consensus-based PCR

Biofilm formation is an important phenotype associated with chronic Pseudomonas aeruginosa infections. In the present study, a total of 48 P. aeruginosa strains isolated from clinical specimens were examined for their biofilm-forming ability using a microtiter plate method. The different biofilm-forming abilities were demonstrated among the strains; however, most strains formed a larger biofilm than strain PAO1, a reference strain. The genetic typing was also carried out by enterobacterial repetitive intergenic consensus-based polymerase chain reaction. Although they were divided into five groups (A to E), most of the strains showing the higher biofilm-forming ability were found to be in groups D and E, suggesting a significant relationship between the biofilm-forming ability and the genetic group.     

Selection of phages and conditions for the safe phage therapy against Pseudomonas aeruginosa infections

Virologica Sinica - The emergence of multidrug-resistant bacterial pathogens forced us to consider the phage therapy as one of the possible alternative approaches to treatment. The purpose of this...     

Induction of apoptosis in macrophage cell line,J774, by the cell-free supernatant from Pseudomonas aeruginosa

Pseudomonas aeruginosa is able to secrete many virulence factors that are cytotoxic towards eukaryotic cells. To investigate the effect of the bacterium on macrophages, we obtained cell-free supernatants from P. aeruginosa (Pa) IID1117 (elastase-positive and protease-positive) and Pa IID1130 (elastase-positive and protease-negative). After 6 hr of incubation with the cell-free supernatant from the Pa IID1117 strain, the viability of J774 macrophages was shown to be significantly reduced (47.5+/-11%), but not Pa IID1130 (96.4+/-1.6%) at a concentration of 10% (v/v) compared to control J774 macrophages without any supernatant (97.2+/-1.7%) by the detection of trypan blue dye exclusion. The death of cells was further demonstrated to be due to apoptosis characterized by chromatin condensation and apoptotic bodies by Hoechst 33258 staining, DNA fragmentation by agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL). An activated subunit was found to be released from procaspase-3 in cell lysate. But in the presence of protease inhibitor, the apoptosis was completely blocked. The findings indicate that the Pa IID1117 strain is capable of inducing apoptosis in J774 macrophages. The apoptosis induced by the cell-free supernatant from Pa IID1117 strain is suggested to be dependent on protease, but not elastase.     

Caspase-1-Like Regulation of the proPO-System and Role of ppA and Caspase-1-Like Cleaved Peptides from proPO in Innate Immunity

Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.     

High-level fluoroquinolone resistance in Pseudomonas aeruginosa due to interplay of the MexAB-OprM efflux pump and the DNA gyrase mutation

Fluoroquinolone resistance in Pseudomonas aeruginosa is mainly attributable to the constitutive expression of the xenobiotic efflux pump and mutation in DNA gyrase or topoisomerase IV. We constructed cells with a double-mutation in gyrA and mexR encoding DNA gyrase and repressor for the mexAB-oprM operon, respectively. The mutant showed 1,024 times higher fluoroquinolone resistance than cells lacking the MexAB-OprM. Cells with a single mutation in gyrA and producing a wild-type level of the MexAB-OprM efflux pump showed 128 times higher fluoroquinolone resistance than cells lacking the MexAB-OprM. In contrast, a single mutation in gyrA or mexR caused only 4 and 64 times higher resistance, respectively. These findings manifested the interplay between the MexAB-OprM efflux pump and the target mutation in fluoroquinolone resistance.     

The Measurement of Swimming Velocity of Vibrio cholerae and Pseudomonas aeruginosa Using the Video Tracking Method

The swimming velocities of two monotrichous flagellated bacteria were measured by a computer-assisted video tracking method. Tracing the moving path of the individual bacterium revealed that the bacterial cell did not swim continuously in a straight direction, but frequently changed swimming direction and velocity. The average swimming velocities calculated from the 3-sec path were 75.4 ±9.4 μm/sec in four strains of Vibrio cholerae and 513 ±8.4 μm/sec in five strains of Pseudomonas aeruginosa. These results suggest that V. cholerae swim faster than P. aeruginosa at 30 C in nutrient broth. This method is useful for a detailed analysis of bacterial movement and moving patterns in different environmental conditions.     

Identification of two novel missense WFS1 mutations,H696Y and R703H,in patients with non-syndromic low-frequency sensorineural hearing loss

Non-syndromic low-frequency sensorineural hearing loss(LFSNHL) is an unusual type of hearing loss in which frequencies≤2000 Hz predominantly are affected.To date,different mutations in two genes,DIAPH1 and WFS1,have been found to be associated with LFSNHL. Here,we report a five-generation Chinese family with postlingual and progressive LFSNHL.We mapped the disease locus to a 2.5 Mb region on chromosome 4p16 between markers SNP_A-2167174 and D4S431,overlapping with the DFNA6/14/38 locus.Sequencing of cand...     

Molecular cloning and characterization of the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa.

We cloned and characterized the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa PAO1. The oprQ gene was composed of 1,275 base pairs including a sequence encoding for the signal sequence and a mature protein with a Mr of 44,602. Computer-aided alignment and hydropathy analyses of the predicted amino acid sequences suggested that OprE3 is a transmembrane protein homologous to outer membrane proteins of P. aeruginosa such as OprD2 (OprD) porin and OprE1 (OprE) porin. Susceptibility to several antibiotics of the strains lacking or overproducing OprE3 was indistinguishable from that of the wild-type strain, suggesting that OprE3 is unlikely involved in the diffusion of carbapenems and other beta-lactam antibiotics.     

Study of the inhibitory effect of Quercus Coccifera's aqueous extract on Staphylococcus aureus and Pseudomonas aeruginosa In vitro

The use of therapeutic herbs has become of great importance these days due to the increase in drug resistance. From a long time ago the Venus'' navel plant has been used to treat infections. In this study the antibacterial effect of the aqueous extract from the Quercus coccifera (jaft) herb, under laboratory conditions. This study was carried out experimentally. After collecting the Venus navel herb, it was dried in a warm dry environment away from direct sunlight in the shade. The alcoholic extract was prepared using a standard method. Clinical samples of staphylococcus aureus and pseudomonas aeruginosa were acquired from Ilam''s health care institutes. The inhibitory effect of the extracts was analysed in the Mueller Hinton using the disk diffusion method for both bacteria. Then MIC and MBC of the extracts was determined using the Macro broth dilution method. At its highest concentration the aqueous extract had an inhibition zone of 27.2 and 23.7 mm on staphylococcus aureus and pseudomonas aeruginosa consecutively. The MIC and MBC for staphylococcus aureus were 10 and 12.5 µg/ml and for pseudomonas aeruginosa they were 10 and 17.5 µg/m consecutively. The results of this study show the strong antimicrobial effect of jaft''s aqueous extract on staphylococcus aureus and pseudomonas aeruginosa and if more studies are based on this topic it could be a substitute for common antibiotics.     

How Transmembrane Inner Ear (TMIE) plays role in the auditory system: A mystery to us

Different cellular mechanisms contribute to the hearing sense, so it is obvious that any disruption in such processes leads to hearing impairment that greatly influences the global economy and quality of life of the patients and their relatives. In the past two decades, transmembrane inner ear (TMIE) protein has received a great deal of research interest because its impairments cause hereditary deafness in humans. This evolutionarily conserved membrane protein contributes to a fundamental complex that plays role in the maintenance and function of the sensory hair cells. Although the critical roles of the TMIE in mechanoelectrical transduction or hearing procedures have been discussed, there are little to no review papers summarizing the roles of the TMIE in the auditory system. In order to fill this gap, herein, we discuss the important roles of this protein in the auditory system including its role in mechanotransduction, olivocochlear synapse, morphology and different signalling pathways; we also review the genotype-phenotype correlation that can per se show the possible roles of this protein in the auditory system.     

Distinct function of Pseudomonas aeruginosa type IV pili disclosed in the bacterial pass-through of membrane filter with smaller pore sizes

Membrane filter pass-through ability of Pseudomonas aeruginosa was analyzed with isogenic mutants. A flagellum-deficient fliC mutant required two-times longer time (12 hr) to pass through a 0.45-microm pore size filter. With 0.3- and 0.22-microm filters, however, the fliC mutant showed no remarkable disability. Meanwhile a pilA mutant defective in twitching motility failed to pass through the 0.22-microm filter. Complementation of the mutant with pilA gene on a plasmid restored the twitching motility and the 0.22-microm filter pass-through activity. Thus, the distinctive role of P. aeruginosa type IV pili in infiltration into finer reticulate structures was indicated.     

Antibacterial properties of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide-specific monoclonal antibody (MAb) in a murine thigh infection model: combined effects of MAb and ceftazidime

A murine monoclonal antibody (MAb) specific for the Pseudomonas aeruginosa immunotype 1 (It-1) lipopolysaccharide (LPS) O-side chain was evaluated in terms of its in vitro bactericidal opsonophagocytic activity and in vivo bacterial killing in a mouse thigh infection model. An immunoglobulin (Ig) G2a MAb Ld3-2F2, specific for It-1 LPS, mediated in vitro complement-dependent opsonophagocytic killing at a concentration of 10 microg/ml. MAb-mediated, complement-dependent killing also occurred in the absence of neutrophils at serum concentrations in excess of 20%. A remarkable synergy was observed in opsonophagocytic assays between MAb Ld3-2F2 (0.5 microg/ml) and ceftazidime (1/4 MIC). The administration of MAb Ld3-2F2 at a level of 1 microg resulted in a significant decrease in the number of bacteria in the thigh muscles of normal mice, while 100 microg of the same MAb was required for one log of reduction in the number of bacteria at the same site in neutropenic mice. The combined therapy with MAb Ld3-2F2 and ceftazidime provided a significant reduction in the density of bacteria in the thigh muscle at 9 hr post-infection in normal and neutropenic mice as compared with those after treatment alone or with no treatment (P< 0.01). These favorable in vitro and in vivo interactions of an LPS-specific IgG MAb and ceftazidime strongly support their potential for use in therapy, combined with an LPS-reactive MAb and parenteral antipseudomonas beta-lactam antibiotics in the therapy of systemic Pseudomonas infections in normal and neutropenic hosts.     

Unique presence of 2-methylthio-ribosylzeatin in the transfer ribonucleic acid of the bacterium Pseudomonas aeruginosa

Analysis of ³⁵S labled nucleosides prepared from tRNA of Pseudomonas aeruginosa by phosphocellulose column chromatography, thin layer chromatography and Sephadex LH-20 column chromatography revealed the presence of 2-methylthioribosylzeatin in it.     

GPR107, a G-protein-coupled Receptor Essential for Intoxication by Pseudomonas aeruginosa Exotoxin A,Localizes to the Golgi and Is Cleaved by Furin

A number of toxins, including exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation, and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan G-protein-coupled receptor. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin, and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport.