Intracellular expression of the Salmonella plasmid virulence protein,SpvB, causes apoptotic cell death in eukaryotic cells

The spv genes carried on the Salmonella virulence plasmid are commonly associated with severe systemic infection in experimental animals. The SpvB virulence-associated protein has been shown to ADP-ribosylate actin, and this enzymatic activity is essential for virulence in mice. Here, we present evidence that intracellular expression of SpvB protein induces not only disruption of actin filaments but also apoptotic cell death in eukaryotic cells.     

Involvement of the essential metal transporter Zip14 in hepatic Cd accumulation during inflammation

Upregulation of Zip14 contributes to hepatic zinc (Zn) and non-transferrin-bound iron (Fe) uptake during infection and inflammation. We investigated whether this essential metal transporter is also involved in hepatic cadmium (Cd) uptake under these conditions. An injection of lipopolysaccharide (LPS), turpentine oil (Tur) and n-hexane (Hex) resulted in an decrease in plasma Zn and Fe concentrations to 25–50% and an increase in hepatic concentrations of both metals to 150–200% of control mice. LPS significantly increased plasma interleukin (IL)-6 levels more rapidly than Tur or Hex. Tur or Hex significantly increased hepatic Zip14 mRNA expression and decreased ferroportin 1 mRNA expression following continuous increase of IL-6 level. Hepatic Cd and Zn concentrations increased significantly after repeated injections of Cd in Tur- or Hex-treated mice fed a control diet. Treatment with Tur or Hex additionally increased hepatic Cd accumulation in Zn-deficient mice, unlike in Fe-deficient mice. These results suggest that Zn transporters, such as Zip14, may be involved in hepatic Cd uptake during inflammation.     

Survival of Vibrio anguillarum in freshwater environments: adaptation or debilitation?

Vibriosis caused by Vibrio anguillarum seriously damages freshwater fish (Salmoniforms) almost every year in Lake Biwa, Japan. This pathogen requires NaCl for its growth. When the pathogen was exposed to sterilized aged lake water, it lost its culturability without loosing respiratory activity. Although this physiological state appears to be "viable but nonculturable" (VBNC), the cells could not be resuscitated even in host fish bodies. As these cells totally lost their pathogenicity, the VBNC-like state is probably on the pathway to cell death. Environmental factors such as darkness, coldness, and anaerobiosis helped the pathogen to survive in freshwater without reaching a nonculturable state. Received: October 4, 1999 / Accepted: January 19, 2000     

Subchronic exposure to ethyl tertiary butyl ether resulting in genetic damage in Aldh2 knockout mice

Ethyl tertiary butyl ether (ETBE) is biofuel additive recently used in Japan and some other countries. Limited evidence shows that ETBE has low toxicity. Acetaldehyde (AA), however, as one primary metabolite of ETBE, is clearly genotoxic and has been considered to be a potential carcinogen. The aim of this study was to evaluate the effects of ALDH2 gene on ETBE-induced genotoxicity and metabolism of its metabolites after inhalation exposure to ETBE. A group of wild-type (WT) and Aldh2 knockout (KO) C57BL/6 mice were exposed to 500 ppm ETBE for 1–6 h, and the blood concentrations of ETBE metabolites, including AA, tert-butyl alcohol and 2-methyl-1,2-propanediol, were measured. Another group of mice of WT and KO were exposed to 0, 500, 1750, or 5000 ppm ETBE for 6 h/day with 5 days per weeks for 13 weeks. Genotoxic effects of ETBE in these mice were measured by the alkaline comet assay, 8-hydroxyguanine DNA-glycosylase modified comet assay and micronucleus test. With short-term exposure to ETBE, the blood concentrations of all the three metabolites in KO mice were significantly higher than the corresponding concentrations of those in WT mice of both sexes. After subchronic exposure to ETBE, there was significant increase in DNA damage in a dose-dependent manner in KO male mice, while only 5000 ppm exposure significantly increased DNA damage in male WT mice. Overall, there was a significant sex difference in genetic damage in both genetic types of mice. These results showed that ALDH2 is involved in the detoxification of ETBE and lack of enzyme activity may greatly increase the sensitivity to the genotoxic effects of ETBE, and male mice were more sensitive than females.     

ROC Analysis of Detection of Metastatic Pulmonary Nodules on Digital Chest Radiographs with Temporal Subtraction

RATIONALE AND OBJECTIVES: The authors' purpose was to evaluate the effect of temporal subtraction on digital chest radiographs in the detection of metastatic pulmonary nodules. MATERIALS AND METHODS: The study included 21 cases with metastatic pulmonary nodule and 21 cases without metastatic nodule. Eleven radiologists, including eight residents and three certified radiologists, provided their confidence levels for the presence or absence of pulmonary nodules without and with temporal subtraction. Their performances without and with temporal subtraction were evaluated by means of receiver operating characteristic analysis with both independent and sequential tests. RESULTS: For the independent test, the radiologists' Az (area under the receiver operating characteristic curve) values were 0.871 without and 0.954 with temporal subtraction, compared with 0.882 and 0.955, respectively, for the sequential test. Diagnosis accuracy was significantly improved with the use of temporal subtraction. There was no significant difference in Az values between the independent and sequential tests. CONCLUSION: Temporal subtraction is useful in the detection of metastatic pulmonary nodules, and this technique augments the value of digital chest radiography.     

The radiographic study in the relationship of the glenohumeral joint.

Accurate reproduction of anatomic relationship is important in non-constrained prosthetic arthroplasty. The accurate lateral glenohumeral offset, which indicates a parameter of the lever arm of the deltoid and supraspinatus muscles, is one of the most important elements in achieving the efficient shoulder functions after prosthetic reconstruction. However, to our knowledge, there has been no detailed study on the influence of minute changes in the neck shaft angle, within the normal range, on lateral glenohumeral offset. In this study, we evaluated the relationship between the neck shaft angle and various geometric measurement values in the glenohumeral joint. Radiographs of 471 shoulders without osseous lesions in the glenohumeral joint and greater tuberosity were reviewed. There were 265 males and 206 females, and a mean age was 53.5 years old. The final diagnosis was impingement syndrome in 269 patients, rotator cuff tear in 147, and control group in 55 patients. Eight parameters were measured on true anteroposterior radiographs. The lateral glenohumeral offset correlated with the radius of the curvature of the humeral head and the humeral head diameter. The neck shaft angle was not correlated with the offset value. However, when the offset value was divided by the humeral head diameter, to eliminate the affection of the size of the humeral head toward the offset, a correlation was observed with the neck shaft angle. Based on these correlations, we could obtain the equation of the lateral glenohumeral offset according to the neck shaft angle. Using the equation, when the neck shaft angle is decided, the lateral glenohumeral offset can be estimated based on the correlation between the parameters, which achieve the efficient shoulder functions after prosthetic components. Therefore, inaccurate determination of the neck shaft angle in the humeral head component design and selection may induce dysfunction of the abductor muscles.     

Magnetic resonance lymphography of sentinel lymph nodes in patients with breast cancer using superparamagnetic iron oxide: a feasibility study

Background

The sentinel lymph node (SLN) biopsy technique using superparamagnetic iron oxide (SPIO) as a tracer instead of radioisotopes has been described. To further advance this technique, we evaluated preoperative SPIO-MR sentinel lymphography to facilitate the accurate identification of the lymphatic pathways and primary SLN.

Methods

A prospective study was performed in ten patients with breast cancer and clinically negative axillary lymph nodes. None of the patients received preoperative chemotherapy. After 1.6 ml of SPIO (ferucarbotran) was injected in the subareolar breast tissue, sentinel axillary lymph nodes were detected by MRI in T2*-weighted gradient echo images and resected using the serial SPIO-SLN biopsy procedure with a handheld magnetometer.

Results

In one patient, gadolinium-enhanced MR imaging was performed at the same time as SPIO-MR lymphography, and this patient was excluded from further analysis. In all patients (9/9) SLNs were detected by SPIO-MR sentinel lymphography and successfully identified at surgery. The number of SLNs detected by lymphography (mean 2.7) significantly correlated with SLNs identified at surgery (mean 2.2). One patient had nodal metastases. In one patient, skin color changed to brown at the injection site and resolved spontaneously. There were no severe reactions to the procedure or complications in any patient.

Conclusions

This is the first study to evaluate SPIO both as a contrast material in MR sentinel lymphography and as a tracer in SLN biopsy using an integrated method. The acquired three-dimensional imaging demonstrated excellent image quality and usefulness to identify SLN in conjunction with SLN biopsy.     

Period is involved in the proliferation of human pancreatic MIA-PaCa2 cancer cells by TNF-alpha

Recent studies have found alterations to clock genes in several forms of cancer. Period (Per) gene plays an important role in the circadian system, the cell cycle, the induction of apoptosis, and DNA damage. However, the functions of Per in pancreatic cancer have not yet been elucidated. Here, we show that tumor necrosis factor-alpha (TNF-alpha) suppressed the expression of both Per1 and Per3 in MIA-PaCa2 cells, a human pancreatic cancer cell line. The levels of these proteins were 10% lower in the cells treated with 10 ng/mL TNF-alpha. Cell proliferation showed a 15%, 14%, and 16% decrease at 0.4, 2.0, and 10 ng/mL of TNF-alpha, respectively. In MTS-assays, MIA-PaCa2 cells transfected with siRNA against Per1 showed a 19% reduction in proliferation. However, the knockdown of Per3 did not significantly inhibit cell proliferation. The results suggest Per1 to be involved in the inhibition of the proliferation of MIA-PaCa2 cells by TNF-alpha.     

Lung toxicity of 16 fine particles on intratracheal instillation in a bioassay model using f344 male rats

We have developed a bioassay model to estimate toxicity of fine particles in the lungs at an early stage after intratracheal instillation (Yokohira et al. 2005; Yokohira et al. 2007). The present experiment was conducted to improve the model by estimating appropriate doses based on dose-dependent toxicity of instilled quartz (4 mg to 0 mg) as a positive control and assessing the impact of powdered particles without suspension (Experiment 1). In addition, examination of the toxicity of a series of particles was performed with the developed bioassay (Experiments 2A, 2B, and 2C). The materials chosen were sixteen particles, including nanoparticles and diesel powder. Histopathological and immunohistochemical analysis of bromodeoxyuridine (BrdU) incorporation and inducible nitric oxide synthase (iNOS) were performed after exposure of the lungs. A dose of 2 mg quartz suspended in 0.2 mL saline was suggested to be most appropriate for sensitive detection of acute and subchronic inflammatory changes. Although some materials, including nanoparticles, demonstrated toxicity that was too strong for sensitive assessment, the ranking order could be given as follows: CuO > quartz > neutralized Na2PdCl4 > NiO > hydrotalcite > MnO2 > diesel > titanium dioxide (in Experiment 2B) > beta-cyclodextrin > diesel standard > titanium dioxide (in Experiment 2A) > CaCO3.     

Immunochemical detection of Nepsilon-(carboxyethyl)lysine using a specific antibody

Methylglyoxal (MG) is generated through the Embden-Meyerhof and polyol pathways, and it rapidly reacts with proteins to form advanced glycation end products (AGE) such as N(epsilon)-(carboxyethyl)lysine (CEL). In the present study, polyclonal and monoclonal antibodies specific for CEL were prepared to estimate CEL content in aldehydes-modified proteins and the pathological localization in human kidneys. Polyclonal CEL-specific antibody was prepared by removing cross-reactive antibodies against N(epsilon)-(carboxymethyl) lysine (CML), one of the major AGE structures, using CML-conjugated affinity chromatography. Monoclonal CEL-specific antibody (CEL-SP) was obtained by immunization with CEL-bovine serum albumin, followed by successive screening according to CEL-RNase-positive but CML-RNase-negative criteria. A non-competitive ELISA showed that both the polyclonal and monoclonal CEL-specific antibodies significantly reacted with CEL-proteins but not with CML-proteins. A competitive ELISA also demonstrated that CEL-SP does not show cross-reactivity against CEL analogues such as CML, carboxymethylarginine (CMA) and S-carboxymethylcysteine (CMC), thus indicating that antibody is able to recognize the difference of one methyl group between carboxymethyl group and carboxyethyl group. Furthermore, CEL-SP significantly reacted with human serum albumin modified with MG but not with glyoxal or 3-deoxyglucosone, and its reactivity was highly correlated with the CEL content, which was determined by high performance liquid chromatography. Immunohistochemical studies using CEL-SP provided evidence that CEL-modified proteins accumulate in distal tubular epithelial cells of the diabetic rat. These results demonstrate that a specific antibody against CEL can be a powerful tool for detecting CEL both in vitro and in vivo.