Expression patterns of members of the isocitrate dehydrogenase gene family in murine inner ear.

Age-related hearing loss (ARHL) is characterized by an age-dependent decline of auditory function characterized by with loss of sensory hair cells, spiral ganglion neurons, and stria vascularis (SV) cells in the cochlea of the inner ear. Aging and age-related diseases result from accumulated oxidative damage caused by reactive oxygen species (ROS) generated by mitochondria. The isocitrate dehydrogenase (IDH) family includes three enzymes in human cells: IDH1, IDH2, and IDH3. Although all three enzymes catalyze the same enzymatic reaction, that is, oxidative decarboxylation of isocitrate to produce α-ketoglutarate, each IDH enzyme has unique features. We identified and characterized IDH expression in the cochlea and vestibule of the murine inner ear. We examined the mRNA expression levels of Idh family members in the cochlea and vestibule using reverse transcription-PCR (RT-PCR) and detected expression of IDH family members in both tissues. We also used immunohistochemistry to localize IDH family members within the cochlea and vestibule of the adult mouse inner ear. IDH1 was detected throughout the cochlea. IDH2 was expressed specifically in the hair cells, spiral ganglion, and stria vascularis. IDH3α was found in the cell bodies of neurons of the spiral ganglion, the stria vascularis, and in types II, IV, and V cells of the spiral ligament in a pattern that resembled the location of the Na+, K+-ATPase ion channel. We postulate that the IDH family participates in transporting K+ ions in the cochlea. In the vestibule, all IDH family members were detected in both hair cells and the vestibular ganglion. We hypothesize that IDH1, IDH2, and IDH3 function to protect proteins in the inner ear from oxidative stress during K+ recycling.

Safety and efficacy of tuberculin skin testing with microneedle MicronJet600™ in healthy adults.

SETTING: Intradermal injection using a syringe and needle is generally accepted as the most accurate method for the tuberculin skin test (TST). However, the Mantoux technique using a conventional needle is often difficult to perform reliably, affecting testing results and safety. OBJECTIVE: We evaluated the efficacy and safety of a novel intradermal injection device, the MicronJet600(TM) microneedle, compared with conventional injection in terms of skin reactivity to the TST. DESIGN: A prospective, open-label clinical study was conducted. The TST was administered by both methods in the same subject. For pain assessment, participants filled in a visual analogue scale (VAS) after each TST. Any side effects due to TST or injections were observed. RESULTS: TST reaction rates (cut-off ⩾5 mm) from microneedles and needles were respectively 44.0% and 47.2%, with no significant difference between the two. Furthermore, agreement of positivity between the two methods was excellent with both 5 mm and 10 mm cut-off values. However, the level of pain experienced when microneedles were used for TST was significantly lower than with conventional needles. No adverse effects were attributed to the MicronJet device. CONCLUSION: The novel microneedle device used for TST in this study was effective, safe and less painful in healthy adult volunteers.

HOXB13 downregulates intracellular zinc and increases NF-κB signaling to promote prostate cancer metastasis.

Characteristically, prostate cancer (PCa) cells exhibit marked decrease in intracellular zinc; however, the mechanism responsible is not clearly understood. HOXB13 is involved in PCa progression and is overexpressed in castration-resistant PCa. DNA microarray analysis of LNCaP Pca cells showed that ZnT zinc output transporters were strikingly upregulated among androgen-independent HOXB13 target genes. Furthermore, exogenous HOXB13 caused intracellular zinc concentrations to fall in PCa cells, stimulated NF-κB-mediated signaling by reducing inhibitor of NF-κB alpha (IκBα) and enhanced the nuclear translocation of RelA/p65. Human prostate tumors also exhibited strong inverse correlation between the protein expressions of HOXB13 and IκBα. Consequently, HOXB13 stimulated PCa cell invasion, and this was inhibited by the suppression of ZnT4. In addition, studies in a PC3 orthotopic mouse model of PCa metastasis showed that HOXB13 is a strong metastatic stimulator. Taken together, these results show that HOXB13 promotes PCa invasion and metastasis by decreasing intracellular zinc levels, thus stimulating NF-κB signals, and suggest that HOXB13 acts as a modulator of intracellular zinc levels that promotes the malignant characteristics of PCa.

Development of a groEL gene-based species-specific multiplex polymerase chain reaction assay for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus.

AIMS: To develop an effective multiplex polymerase chain reaction (PCR) for the simultaneous detection of three important Vibrio species, Vibrio cholerae (Vc), V. parahaemolyticus (Vp) and V. vulnificus (Vv) using the groEL gene, a potential phylogenetic marker. METHODS AND RESULTS: Three species-specific primer sets were designed to target Vc, Vp and Vv. A total of 131 Vibrio and non-Vibrio strains were used to determine the specificity and sensitivity of primers. The primers produced specific PCR fragments from all target species strains and did not cross-react with other Vibrio and non-Vibrio species. This PCR method showed good efficiency in detecting coexisting target species in the same sample with a detection limit of 100 pg of Vc, Vp and Vv from mixed purified DNA. Detection of three target species was also possible from artificially inoculated shellfish, flounder and sea water. CONCLUSIONS: The groEL gene is a potential marker for accurate simultaneous detection of Vc, Vp and Vv and could be used to detect these species in environmental and clinical samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This newly developed multiplex PCR is a useful and cost-effective method that is applicable in a disease-outbreak prediction system and may provide an effective tool for both the epidemiologist and ecologist.

Application of groEL gene for the species-specific detection of Vibrio parahaemolyticus by PCR.

AIMS: Vibrio parahaemolyticus is a significant cause of human gastrointestinal disorders and is transmitted through ingestion of raw or undercooked contaminated seafood. We used the groEL gene for the species-specific detection of V. parahaemolyticus from artificially inoculated shellfish, fish and seawater. METHODS AND RESULTS: The nucleotide sequences of 24 Vibrio and seven non-Vibrio spp. were compared, and less conserved regions were selected for the designing of primer sets. To detect V. parahaemolyticus specifically, PCR conditions were standardized and tested to evaluate the specificity of primers. A 510-bp band was appeared only from V. parahaemolyticus by PCR. Notably, the detection was shown to be functional at high annealing temperature above 68°C. The groEL primers detected 100 pg and 1 ng of DNA purified from V. parahaemolyticus culture and artificially infected oyster tissue, respectively. CONCLUSIONS: The groEL gene is a potential marker for the species-specific detection of V. parahaemolyticus and could be used to detect this bacterium in contaminated food by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR using primers designed from groEL gene provide an efficient method for the accurate identification of V. parahaemolyticus from contaminated samples.

Novel anti-apoptotic mechanism of A20 through targeting ASK1 to suppress TNF-induced JNK activation.

The zinc-finger protein A20 has crucial physiological functions as a dual inhibitor of nuclear factor-κB (NF-κB) activation and apoptosis in tumor necrosis factor (TNF) receptor 1 signaling pathway. Although the molecular basis for the anti-NF-κB function of A20 has been well elucidated, the anti-apoptotic function of A20 is largely unknown. Here, we report a novel mechanism underlying the anti-apoptotic function of A20: A20 blocks TNF-induced apoptosis through suppression of c-jun N-terminal kinase (JNK) by targeting apoptosis signal-regulating kinase1 (ASK1). First, the ectopic expression of A20 drastically inhibits TNF-induced JNK activation and apoptosis in multiple cell types including those deficient of NF-κB activation. Unexpectedly, the blunting effect of A20 on TNF-induced JNK activation is not mediated by affecting the TNFR1 signaling complex formation. Instead, A20 interacts with ASK1, an important MAPKK kinase in the JNK signaling cascade. More importantly, overexpression of wild-type A20, but not of mutant A20 (ZnF4; C624A, C627A), promotes degradation of the ASK1 through the ubiquitin-proteasome system. Taken together, the results from this study reveal a novel anti-apoptotic mechanism of A20 in TNF signaling pathway: A20 binds to ASK1 and mediates ASK1 degradation, leading to suppression of JNK activation and eventually blockage of apoptosis.

Caspase-8 has an essential role in resveratrol-induced apoptosis of rheumatoid fibroblast-like synoviocytes.

OBJECTIVE: Resveratrol is a naturally occurring polyphenol, which possesses chemotherapeutic potential through its ability to trigger apoptosis. The objective of this study was to investigate the major determinant for the apoptotic cell death induction by resveratrol in fibroblast-like synoviocytes (FLS) derived from patients with RA. METHODS: The effect of resveratrol on apoptotic cell death was quantified in a population of subG1 in RA FLS by flow cytometry. The underlying signalling mechanism for apoptotic death was examined by analysing mitochondrial membrane potential, activation of the caspase cascade and translocation of Bid. RESULTS: We show that activation of caspase-8 is essential for triggering resveratrol-induced apoptotic signalling via the involvement of the mitochondrial pathway in RA FLS. Our findings also suggest that this enhanced apoptosis caused by resveratrol occurred in RA FLS irrespective of p53 status. Exposure to resveratrol caused extensive apoptotic cell death, along with a caspase-dependent (activation of caspase-9 and -3, poly ADPribose polymerase (PARP) cleavage and mitochondrial cytochrome c release) or caspase-independent [translocation of apoptosis-inducing factor (AIF) to the nucleus] signalling pathway. Analysis of upstream signalling events affected by resveratrol revealed that the activated caspase-8 triggered mitochondrial apoptotic events by inducing Bid cleavage without any alteration in the levels of Bax, Bcl-xL or Bcl2. The caspase-8 inhibitor or over-expression of crmA abrogated cell death induced by resveratrol and prevented processing of the downstream cascade. CONCLUSION: The results suggest that resveratrol causes activation of caspase-8, which in turn results in modulation of mitochondrial apoptotic machinery to promote apoptosis of RA FLS.

Small and large deformation rheology for hard wheat flour dough as influenced by mixing and resting.

The effects of mixing and resting on the physicochemical properties of doughs prepared with strong and weak hard wheat flours were investigated, specifically concerning aspects related to their rheological behavior and molecular mobility. Small deformation dynamic tests showed that, during the initial resting period, the complex modulus G* decreased and phase angle decreased for undermixed dough, whereas overmixed dough showed opposite trends. G* values for optimally mixed dough did not vary during the resting period investigated. This was more obvious for the strong dough. Large deformation tests more clearly showed differences among optimal, under-, and overmixed dough, and also between doughs prepared with strong and weak flour. Optimally mixed dough exhibited the highest peak stress and strain for both samples. In addition, the peak stress of dough prepared with the strong flour was higher than that of dough prepared with weak flour. Inconsistent results between small and large deformation tests implied that small and large deformation tests reflected different structural aspects of dough. NMR measurements were performed to estimate the relaxation properties of the sample upon resting. Decreased water mobility during resting, indicated by decreasing T(1) relaxation time, was possibly attributed to increasing molecular interactions caused by continued hydration. Evidence of additional molecular interactions created by mixing was also observed.

Structural characterization of rice starch in rice cake modified by Thermus scotoductus 4-alpha-glucanotransferase (TS alpha GTase).

Rice cake was produced with a thermostable 4-alpha-glucanotransferase from Thermus scotoductus (TS alpha GTase). Starch molecular fine structure, texture, and retrogradation for the enzymatically prepared rice cake were investigated and compared to those for control rice cake. The amylose content in TS alpha GTase-treated rice cakes decreased, whereas branched and linear malto-oligosaccharides ranging from maltose to maltoheptaose increased slightly. The average molecular weight of the enzyme-treated rice starch in rice cake decreased as amylopectin macromolecules were cleaved and reorganized into small amylopectin clusters. The number of shorter side chains (degree of polymerization [DP] < 9) increased, whereas the number of longer side chains (DP > 10) decreased through the disproportionation reaction of TS alpha GTase. After 24 h of storage at 4 degrees C, the enzyme-treated samples demonstrated significantly lower melting enthalpy of retrograded starch (0.4 mJ/mg) compared to that of the control (1.4 mJ/mg). The results indicated that TS alpha GTase treatment effectively inhibited starch retrogradation in rice cakes. It is suggested that the reduction of amylose content, the rearrangement of amylopectin, and the production of malto-oligosaccharides caused by TS alpha GTase treatment are responsible for the ineffective molecular reassociation of rice starch in rice cake.

Focused ion beam induced deflections of freestanding thin films.

Prominent deflections are shown to occur in freestanding silicon nitride thin membranes when exposed to a 50 keV gallium focused ion beam for ion doses between 10(14) and 10(17) ions/cm(2). Atomic force microscope topographs were used to quantify elevations on the irradiated side and corresponding depressions of comparable magnitude on the back side, thus indicating that what at first appeared to be protrusions are actually the result of membrane deflections. The shape in high-stress silicon nitride is remarkably flattopped and differs from that in low-stress silicon nitride. Ion beam induced biaxial compressive stress generation, which is a known deformation mechanism for other amorphous materials at higher ion energies, is hypothesized to be the origin of the deflection. A continuum mechanical model based on this assumption convincingly reproduces the profiles for both low-stress and high-stress membranes and provides a family of unusual shapes that can be created by deflection of freestanding thin films under beam irradiation.

Bright spatial solitons on a partially incoherent background

We present the first observation of incoherent antidark spatial solitons in noninstantaneous nonlinear media. This new class of soliton states involves bright solitons on a partially incoherent background of infinite extent. In the case where the nonlinearity is of the Kerr type, their existence is demonstrated analytically by means of an exact solution. Computer simulations and experiments indicate that these incoherent antidark solitons can propagate in a stable fashion provided that the spatial coherence of their background is reduced below the incoherent modulation instability threshold.