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Aim: The extensive development of nanoparticles (NPs) and their widespread employment in daily life have led to an increase in environmental concentrations of substances that may pose a biohazard to humans. The aim of this work was to examine the effects of zinc oxide nanoparticles (ZnO-NPs) on the host’s pulmonary immune system response to nontypeable Haemophilus influenzae (NTHi) infection. Materials & Methods: A murine infection model was employed to assess pulmonary inflammation and bacterial clearance in response to exposure to ZnO-NPs. The molecular mechanisms underlying ZnO-NP-impaired macrophage activation were investigated. Results: Treatment with ZnO-NPs impaired macrophage activation, leading to a delay in NTHi clearance in the bronchial alveolar lavage fluids and lungs. Exposure to ZnO-NPs followed by NTHi challenge decreased levels of nitric oxide compared with NTHi infection alone. The effects of ZnO-NPs involved downregulation of NTHi-activated expression of inducible nitric oxide synthase and the translocation of active NF-kB into the nucleus. Conclusion: These results demonstrate that exposure to ZnO-NPs can impair innate immune responses and attenuate macrophage responses to bacterial infection. Original submitted 17 December 2013; Revised submitted 3 March 2014

Methadone (MTD) is widely used for detoxification of heroin addicts and also in pain management programs. Information about the distribution of methadone between blood, plasma, and alternative specimens, such as oral fluid (OF), is needed in clinical, forensic, and traffic medicine when analytical results are interpreted. We determined MTD and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in blood, plasma, blood cells, and OF by gas chromatography–mass spectrometry (GC-MS) after adding deuterium-labeled internal standards. The analytical limits of quantitation for MTD and EDDP by this method were 20 and 3 ng/mL, respectively. The amounts of MTD and EDDP were higher in plasma (80.4 % and 76.5 %) compared with blood cells (19.6 % and 23.5 %) and we found that repeated washing of blood cells with phosphate–buffered saline increased the amounts in plasma (93.6 % and 88.6 %). Mean plasma/blood concentration ratios of MTD and EDDP in spiked samples (N = 5) were 1.27 and 1.21, respectively. In clinical samples from patients (N = 46), the concentrations of MTD in plasma and whole blood were highly correlated (r = 0.92, p

Idiopathic nephrotic syndrome (INS) is the most frequent type of nephrotic syndrome that occurs in children. Its response to treatment with steroids varies. The aim of this study was to analyze the correlation between steroid metabolism-related genes and the response to steroid treatment. The patient cohort comprised 74 children with INS, of whom were 58 steroid-sensitive (SS) cases and 16 steroid-resistant (SR) cases. The genetic polymorphisms analyzed were those of the CYP3A5 gene (A6986G) and ABCB1 gene (C1236T, G2677T/A, and C3435T), and the polymorphisms between SS and SR children were compared. C1236T in ABCB1 was associated with steroid resistance in INS children [odds ratio (OR) 2.65, 95 % confidence interval (CI) 1.01–6.94; p = 0.042] The frequency of the T allele was significantly higher in SR subjects than in SS subjects (0.81 vs. 0.62, respectively). A6986G in CYP3A5 showed a trend of association, but this association did not reach statistical significance (OR 2.63, 95 % CI 0.94–7.37; p = 0.059). No significant correlation was found between treatment response and G2677T/A or C3435T in ABCB1. Our results indicate that among our pediatric patients with INS the C1236T polymorphism in the ABCB1 gene was associated with steroid resistance, while the A6986G polymorphism in the CYP3A5 gene showed a trend of association, but did not reach statistical significance, requiring further analysis.

Limulus amebocyte lysate (LAL) assay and gas chromatography-mass spectrum (GC-MS) analysis are usually used for the quantification of lipopolysaccharides (LPS) from the environment. The LAL assay measures endotoxin units to represent the LPS biological function but GC-MS analysis measures decanoic (C(10:0)) and dodecanoic (C(12:0)) 3-hydorxy fatty acids (3-OH FA) concentration to represent the LPS chemical composition. A study was carried out using these methods to evaluate their degree of correlation. Using the culture supernatants of 30 independent strains of Pseudomonas aeruginosa, the bacterial supernatants gave of 0.53+/-0.45 and 2.49+/-1.75mg/l of C(10:0) and C(12:0) 3-OH FA, respectively, compared to 17.96+/-13.28mg/l of LPS with the LAL assay (1ng/ml of LPS congruent with0.78EU/ml). The 3-OH FA concentration relative to the endotoxin unit dose in the supernatants exhibited a positive correlation (r(2)=0.5182, C(10:0); r(2)=0.4359, C(12:0)). When supernatants having a high level of endotoxin were used to treat peritoneal exudates cells, nitric oxide (NO) was generated in a dose-dependent manner (r(2)=0.6174). To determine if either C(10:0) or C(12:0) 3-OH FA can act as an indictor of LPS quantity was correlated with this immunostimulatory effect, the correlation of these 3-OH FA concentrations against the produced NO levels was evaluated. This also exhibited a positive correlation; however, the two indicators of 3-OH FA gave different dose-responsible performances (r(2)=0.3211, C(10:0); r(2)=0.4527, C(12:0)).

Elevated temperatures induce coral bleaching. To investigate subcellular changes in heat-stressed corals, we examined heat-shock proteins (hsps) and the concentration of calcium in coral cells at elevated temperatures. After heat treatment, three hsps with approximate molecular weights of 70, 60 and 35 kDa were prominent. The 35 kDa heat-shock protein was further demonstrated to be heme oxygenase by immunoblotting, suggesting the involvement of oxidative stress in coral cells during heat treatment. Since the expression of hsps needs the activation of a calcium signal, the change of intracellular calcium concentration in coral cells was examined by a FURA 2 fluorescence method under heat treatment. Intracellular calcium concentration increased at high temperatures with or without the presence of ambient calcium. The extent of the calcium concentration increase was constant for at least 6 hours. This result indicates the existence of an active calcium signal in coral cells at elevated temperatures.

Background/Purpose Active efflux is known to play a major role in the resistance of many bacteria to antibiotics. To evaluate the possibility of overcoming resistance by suppressing the efflux, we determined the effect of reserpine, an efflux pump inhibitor. Methods Intracellular accumulations and the minimal inhibitory concentrations (MICs) of ciprofloxacin in M. tuberculosis H37Rv and 16 clinical isolates were determined, compared, and analyzed. Nine of the clinical isolates were resistant to isoniazid and rifampin (multiple-drug resistant MDR). Five of these were resistant to ciprofloxacin. Results A reserpine-inhibited efflux system was identified in the H37Rv control and 10:1 (90.9%) of ciprofloxacin-susceptible and 4:1 (80%) of ciprofloxacin-resistant clinical isolates. The MIC of ciprofloxacin decreased in the presence of reserpine in 3/10 (30%) of the ciprofloxacin-susceptible and 2/4 (50%) of the MDR ciprofloxacin-resistant strains that expressed efflux pumps. Two of the efflux-positive, ciprofloxacin-resistant strains in which the MIC of ciprofloxacin was not decreased by reserpine were found to carry a D94A gyrA mutation. In contrast, two strains with the D94G gyrA mutation were susceptible to ciprofloxacin in the presence of reserpine. An efflux-negative strain, highly resistant to multiple antibiotics, was found to have a novel G247S mutation that differs from known mutations in the QRDR region of the gyrA gene. Conclusion These findings indicate t hat reserpine can increase intracellular concentrations of ciprofloxacin, but is unable to overcome other mechanisms of resistance in clinical isolates.

Three mutants of rat hsc70 were constructed, overexpressed in Escherichia coli, purified, and characterized. First, site-directed mutation was utilized to substitute Asn for Asp-10. The recombinant protein, hsc70(D10N), loses not only its peptide-stimulated ATPase activity but also its basal ATPase activity. The measured dissociation constants of ATP (0.3 microM) and S-peptide (5 microM) for hsc70(D10N), however, are virtually identical to those of hsc70. The intrinsic fluorescence spectra of hsc70(D10N) also remain largely unchanged. Therefore, the overall structure of the hsc70 protein is most likely intact after mutation. Second, the entire C-terminal peptide-binding domain was deleted and the resultant mutant contains only the N-terminal ATPase domain of hsc70. This recombinant protein, Nt-hsc70, is a peptide-independent ATPase. The ATPase activity at 37 degrees C of the Nt-hsc70, 270 pmol/h/micrograms of protein, is comparable to that of maximally peptide-activated hsc70. Third, the Asp-10 of Nt-hsc70 was replaced by Asn. Despite that this mutant, Nt-hsc70(D10N), is capable of binding ATP and it loses the capability to hydrolyze ATP. Taken together, these results indicate that aspartyl residue 10 of hsc70 is essential for ATP hydrolysis. Purified hsc70 and its mutants autophosphorylate in vitro at a substoichiometric level. On average, less than 1% of the hsc70 and Nt-hsc70 proteins are phosphorylated. Although the amount of phosphate incorporated into hsc70(D10N) and Nt-hsc70(D10) is reduced, a significant level of phosphorylation can still be achieved in these two site-directed mutants. Hence, autophosphorylation of hsc70 and its mutants is not correlated with their ability to hydrolyze ATP.

A total of 141 independent strains of Pseudomonas aeruginosa with different heterogeneities in the exo gene ( exoS , exoT , exoU , and exoY ) background were examined for their pathogenic roles. Results indicated that the exoU gene is the major contributor to cytotoxicity in Madin-Darby canine kidney cells but is not related to bacterial colonization in mice.

Insulin-like growth factor-binding protein-1 (IGFBP-1) is one of six soluble binding proteins that regulate the actions of the insulin-like growth factors (IGFs). Liver is the major source of IGFBP-1 in non-pregnant humans. In normal physiology, IGFBP-1 transcription is potently inhibited by insulin and serum levels are limited by a rapid clearance rate. Elevated levels of IGFBP-1 in liver disease have been attributed to insulin resistance; however, the relationships between these analytes have not been defined. We studied insulin, proinsulin and IGFBP-1 in normal subjects (NL, N=47, 43+/-12 yr), cirrhosis (CIR, N=29, 54+/-14 yr), hepatocellular carcinoma (HCC, N=42, 61+/-11 yr), and other liver tumors (TUM, N=8, 60+/-17 yr). All three analytes were significantly increased in liver disease (mean+/-SEM; p-values relative to normals): IGFBP-1 (NL 24+/-4 ng/ml; CIR 235+/-53, p

Apoptin, a chicken anemia virus-encoded protein, induces apoptosis in human tumor cells but not in normal cells. The tumor-specific activity of Apoptin is correlated with its nuclear localization in tumor cells. In an attempt to elucidate the molecular mechanism of Apoptin-induced apoptosis, we identified human Hippi, the protein interactor and apoptosis co-mediator of Huntingtin interacting protein 1, as one of the Apoptin-associated proteins by yeast two-hybrid screen. We also demonstrated that Hippi could interact with Apoptin both in vitro and in human cells. Furthermore, subcellular localization studies showed that Hippi and Apoptin perfectly colocalized in the cytoplasm of normal human HEL cells, whereas in cancerous HeLa cells most Apoptin and Hippi were located separately in the nucleus and cytoplasm and, thus, showed only a modest colocalization. Mapping studies indicate that Hippi binds within the self-multimerization domain of Apoptin, and Apoptin binds to the C-terminal half of Hippi, including its death effector domain-like motif. Our results suggest that the Apoptin-Hippi interaction may play a role in the suppression of apoptosis in normal cells.

Benzidine (BZ) and its six structural analogues (2-aminobiphenyl [2-ABP], 4-aminobiphenyl [4-ABP], 3,3'-diaminobenzidine [DABZ], 3,3'-dichlorobenzidine [DCBZ] 3,3'-dimethoxybenzidine [DEBZ], and 3,3'-dimthylbenzidine [DMBZ]) were examined for DNA damage in human lymphocytes using the alkaline comet assay. All the tested compounds showed a distinct disparity in their respective DNA-damaging capacities with an order of DABZ > BZ > DCBZ > 2-ABP > DEBZ > 4-ABP > DMBZ when lymphocytes were exposed to these chemicals for 2 h. Results show that the DNA-damaging effects of these compounds had no bearing on some physicochemical parameters including oxidation potentials, the energy differences between the lowest unoccupied molecular orbital and the highest occupied molecular orbital, ionization potentials, dipole moment, and relative partition coefficient. On the other hand, the free radical scavengers, including catalase, SOD, BHT, EDTA, and histidine exerted varying degrees of inhibitory effects on the DNA damage caused by benzidine. This suggests that genotoxicity in lymphocytes caused by benzidine proceeded via a reactive oxygen species (ROS)-mediated mechanism.

The existence of specific immunoglobulin E (IgE) allows us to determine the allergens that cause the allergic disease. For the purposes of allergen avoidance and immunotherapy, the measurement of specific IgE is widely applied in clinical laboratories. However, if IgE from the serum of an allergic patient exhibits reactivity to multiple allergens, it would cause a problem. The present study analyzes whether the serum IgE with multiple reactivity is due to the presence of unique IgE against the common epitope shared by different allergens or the presence of multiple IgEs against different epitopes on different allergens. The quantitative-competitive inhibition tests and the immunoblotting were applied to analyze the immunosimilarity among examined allergens. The result shows that the competitive inhibition of IgE binding between shrimp and crab allergens is higher than those between either shrimp and cockroach or between crab and cockroach. Furthermore, the results of immunoblotting are consistent with those of quantitative-competitive inhibition tests. These results allow us to detect the cross-reactivity for atopic IgE against multiple allergens.

This paper presents the finding of the possible cause of the high false-positive rate in acid-fast staining in histological examinations. Using acid-fast staining, culture, and PCR, acid-fast bacilli were detected in 83.7% of 49 hospital tap water samples and nontuberculous mycobacteria (NTM) were detected in 20.4% of the same 49 samples. The 10 NTM isolates were also identified to the species level using PCR-restriction fragment length polymorphism. Our findings indicate that NTM in hospital tap water are the possible cause of false positives in acid-fast staining and of nosocomial infection in immunocompromised patients.

The efficacy and sensitivity of selenite brilliant green sulfa enrichment (SBG) broth for the isolation of Salmonella from fecal specimens were evaluated by using both clinical and artificially infected (artificial) fecal specimens. An examination of 1,588 clinical fecal specimens found Salmonella in 296 specimens, including 89 cases detected by the direct-plating xylose-lysine-desoxycholate method and an additional 207 cases detected after enrichment with SBG broth. Therefore, the recovery of Salmonella with SBG broth is increased 3.3-fold over that by the direct-plating method alone. Furthermore, the isolation rate of Salmonella is higher when using SBG broth than when using gram-negative (GN) broth or GN broth supplemented with sodium selenite. To determine the sensitivity for the recovery of Salmonella , artificial specimens containing various amounts of Salmonella were prepared and analyzed. The results indicated that the sensitivity is also higher with SBG broth than with GN broth. Moreover, the optimal incubation period for SBG broth can be extended to 24 h. In conclusion, the SBG enrichment method provides a higher recovery rate of Salmonella from fecal specimens.