Dissecting the pretransitional conformational changes in aminoacylase I thermal denaturation

Aminoacylase I (ACYI) catalyzes the stereospecific hydrolysis of L-acylamino acids and is generally assumed to be involved in the final step of the degradation of intracellular N-acetylated proteins. Apart from its crucial functions in intracellular amino acid metabolism, ACYI also has substantial commercial importance for the optical resolution of N-acylated DL-amino acids. As a zinc-dependent enzyme, ACYI is quite stable against heat-induced denaturation and can be regarded as a thermostable enzyme with an optimal temperature for activity of approximately 65 degrees C. In this research, the sequential events in ACYI thermal denaturation were investigated by a combination of spectroscopic methods and related resolution-enhancing techniques. Interestingly, the results from fluorescence and infrared (IR) spectroscopy clearly indicated that a pretransitional stage existed at temperatures from 50 degrees C to 66 degrees C. The thermal unfolding of ACYI might be a three-state process involving an aggregation-prone intermediate appearing at approximately 68 degrees C. The pretransitional structural changes involved the partial unfolding of the solvent-exposed beta-sheet structures and the transformation of about half of the Class I Trp fluorophores to Class II. Our results also suggested that the usage of resolution-enhancing techniques could provide valuable information of the step-wise unfolding of proteins.     

Paraquat resistance of transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene

Transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene, which encodes a membrane transporter mediating resistance to paraquat, were generated. Transgenic plants displayed higher resistance against paraquat than wild-type plants, as estimated by plant viability, ion leakage and chlorophyll loss, but no resistance against other active oxygen generators, such as H2O2 and menadione. Moreover, lower levels of paraquat accumulated in transgenic plants, compared to wild-type plants, indicating that the PqrA protein detoxifies paraquat either via increased efflux or decreased uptake of the herbicide, but not by removing active oxygen species. The results collectively demonstrate that the bacterial paraquat resistance gene, pqrA, can be functionally expressed in plant cells, and utilized for the development of paraquat-resistant crop plants.     

Enrichment of electrochemically active bacteria using a three-electrode electrochemical cell

Electrochemically active bacteria were successfully enriched in an electrochemical cell using a positively poised working electrode. The positively poised working electrode (+0.7 V vs. Ag/AgCl) was used as an electron acceptor for enrichment and growth of electrochemically active bacteria. When activated sludge and synthetic wastewater were fed to the electrochemical cell, a gradual increase in amperometric current was observed. After a period of time in which the amperometric current was stabilized (generally 8 days), linear correlations between the amperometric signals from the electrochemical cell and added BOD (biochemical oxygen demand) concentrations were established. Cyclic voltammetry of the enriched electrode also showed prominent electrochemical activity. When the enriched electrodes were examined with electron microscopy and confocal scanning laser microscopy, a biofilm on the enriched electrode surface and bacterium-like particles were observed. These experimental results indicate that the electrochemical system in this study is a useful tool for the enrichment of an electrochemically active bacterial consortium and could be used as a novel microbial biosensor.     

Role of osmolytes as chemical chaperones during the refolding of aminoacylase.

The refolding and reactivation of aminoacylase is particularly difficult because of serious off-pathway aggregation. The effects of 4 osmolytes--dimethylsulphoxide, glycerol, proline, and sucrose--on the refolding and reactivation of guanidine-denatured aminoacylase were studied by measuring aggregation, enzyme activity, intrinsic fluorescence spectra, 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra, and circular dichroism (CD) spectra. The results show that all the osmolytes not only inhibit aggregation but also recover the activity of aminoacylase during refolding in a concentration-dependent manner. In particularly, a 40% glycerol concentration and a 1.5 mol/L sucrose concentration almost completely suppressed the aminoacylase aggregation. The enzyme activity measurements revealed that the influence of glycerol is more significant than that of any other osmolyte. The intrinsic fluorescence results showed that glycerol, proline, and sucrose stabilized the aminoacylase conformation effectively, with glycerol being the most effective. All 4 kinds of osmolytes reduced the exposure of the hydrophobic surface, indicating that osmolytes facilitate the formation of protein hydrophobic collapse. The CD results indicate that glycerol and sucrose facilitate the return of aminoacylase to its native secondary structure. The results of this study suggest that the ability of the various osmolytes to facilitate the refolding and renaturation of aminoacylase is not the same. A survey of the results in the literature, as well as those presented here, suggests that although the protective effect of osmolytes on protein activity and structure is equal for different osmolytes, the ability of osmolytes to facilitate the refolding of various proteins differs from case to case. In all cases, glycerol was found to be the best stabilizer and a folding aid.     

Analysis of the BRAFV600E Mutation in Central Nervous System Tumors

BRAF^V600E mutations are involved in the development of melanoma, colon cancer, and papillary thyroid carcinoma. These mutations are also found in primary brain tumors at low to moderate frequencies. In this study, we investigated a series of brain tumors to determine the prevalence and associated clinicopathologic features of BRAF^V600E mutations. By direct sequencing, we analyzed 223 brain tumors, including 51 gangliogliomas (GGs), 45 pilocytic astrocytomas (PAs), 12 pleomorphic xanthoastrocytomas (PXAs), 35 glioblastomas (GBs), 28 anaplastic astrocytomas (AAs), 44 oligodendroglial tumors (ODGs), 3 anaplastic oligoastrocytomas, and 5 diffuse astrocytomas. Thirty-six cases (16.1%) exhibited the BRAF^V600E mutation, including 66.7% of PXAs, 23.5% of GGs, 15.6% of PAs, and 9.7% of the malignant gliomas; the latter included 14.3% of AAs, 8.6% of GBs, and 4.5% of ODGs. Copy number aberration at the 7q34 (BRAF) locus was found in 73.1% of PAs and 50% of PXAs. 9p Homozygous deletion was found in 66.7% of PXAs, but it was not correlated with the BRAF^V600E mutation. Patients' age, sex, histologic grade, and progression-free survival were also not correlated with the BRAF^V600E mutation. The BRAF^V600E mutation in brain tumors did not have prognostic value but is certainly a diagnostic marker and therapeutic target, not only for pediatric low-grade gliomas but also for malignant gliomas, even though the rate of mutation was not high. These results should be verified in a larger study with more cases and a longer follow-up period to overcome the limitation of small sample size.     

Cerebral Blood Volume Calculated by Dynamic Susceptibility Contrast-Enhanced Perfusion MR Imaging: Preliminary Correlation Study with Glioblastoma Genetic Profiles

Purpose

To evaluate the usefulness of dynamic susceptibility contrast (DSC) enhanced perfusion MR imaging in predicting major genetic alterations in glioblastomas.

Materials and Methods

Twenty-five patients (M:F = 13∶12, mean age: 52.1±15.2 years) with pathologically proven glioblastoma who underwent DSC MR imaging before surgery were included. On DSC MR imaging, the normalized relative tumor blood volume (nTBV) of the enhancing solid portion of each tumor was calculated by using dedicated software (Nordic TumorEX, NordicNeuroLab, Bergen, Norway) that enabled semi-automatic segmentation for each tumor. Five major glioblastoma genetic alterations (epidermal growth factor receptor (EGFR), phosphatase and tensin homologue (PTEN), Ki-67, O6-methylguanine-DNA methyltransferase (MGMT) and p53) were confirmed by immunohistochemistry and analyzed for correlation with the nTBV of each tumor. Statistical analysis was performed using the unpaired Student t test, ROC (receiver operating characteristic) curve analysis and Pearson correlation analysis.

Results

The nTBVs of the MGMT methylation-negative group (mean 9.5±7.5) were significantly higher than those of the MGMT methylation-positive group (mean 5.4±1.8) (p = .046). In the analysis of EGFR expression-positive group, the nTBVs of the subgroup with loss of PTEN gene expression (mean: 10.3±8.1) were also significantly higher than those of the subgroup without loss of PTEN gene expression (mean: 5.6±2.3) (p = .046). Ki-67 labeling index indicated significant positive correlation with the nTBV of the tumor (p = .01).

Conclusion

We found that glioblastomas with aggressive genetic alterations tended to have a high nTBV in the present study. Thus, we believe that DSC-enhanced perfusion MR imaging could be helpful in predicting genetic alterations that are crucial in predicting the prognosis of and selecting tailored treatment for glioblastoma patients.     

Therapeutic effect of anti-C-X-C motif chemokine 10 (CXCL10) antibody on C protein-induced myositis mouse

Introduction

C-X-C motif chemokine 10 (CXCL10) is a chemokine that plays a critical role in the infiltration of T cells in autoimmune diseases and is reported to be expressed in muscle tissue of polymyositis. To determine the therapeutic efficacy of CXCL10 blockade, we investigated the role of CXCL10 and the effect of anti-CXCL10 antibody treatment in C protein-induced myositis (CIM), an animal model of polymyositis.

Methods

CIM was induced with human skeletal muscle C protein fragment in female C57BL/6 mice. Immunohistochemistry of CXCL10 and C-X-C motif chemokine receptor 3 (CXCR3) and measurement of serum CXCL10 were performed. Cell surface markers and interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in CIM lymph node cells was investigated by flow cytometry. Mice with CIM were treated with anti-CXCL10 antibody or control antibody (anti-RVG1) and the inflammation in muscle tissue was assessed.

Results

Immunohistochemistry showed increased expression of CXCL10 and CXCR3 in the inflammatory lesions of muscle in CIM. Especially, CD8+ T cells invading myofiber expressed CXCR3. Serum level of CXCL10 was increased in CIM compared to the level in normal mice (normal mouse, 14.3 ± 5.3 pg/ml vs. CIM, 368.5 ± 135.6 pg/ml, P < 0.001). CXCR3 positivity in CD8+ T cells was increased compared to that of CD4+ T cells in the lymph node cells of CIM (CXCR3+ among CD8+ T cell, 65.9 ± 2.1% vs. CXCR3+ among CD4+ T cell, 23.5 ± 4.7%, P <0.001). Moreover, IFN-γ+ cells were increased among CXCR3+CD8+ T cells compared to CXCR3–CD8+ T cells (CXCR3+CD8+ T cell, 28.0 ± 4.2% vs. CXCR3-CD8+ T cell, 9.5 ± 1.5%, P = 0.016). Migration of lymph node cells was increased in response to CXCL10 (chemotactic index was 1.91 ± 0.45). CIM mice treated with anti-CXCL10 antibody showed a lower inflammation score in muscles than those with anti-RVG1 (median, anti-CXCL10 treatment group, 0.625 vs. anti-RVG1 treatment group, 1.25, P = 0.007).

Conclusions

CXCL10/CXCR3 expression was increased in the inflammation of CIM model and its blockade suppressed inflammation in muscle.     

Prognosis Prediction of Measurable Enhancing Lesion after Completion of Standard Concomitant Chemoradiotherapy and Adjuvant Temozolomide in Glioblastoma Patients: Application of Dynamic Susceptibility Contrast Perfusion and Diffusion-Weighted Imaging

Purpose

To assess the prognosis predictability of a measurable enhancing lesion using histogram parameters produced by the normalized cerebral blood volume (nCBV) and normalized apparent diffusion coefficient (nADC) after completion of standard concomitant chemoradiotherapy (CCRT) and adjuvant temozolomide (TMZ) medication in glioblastoma multiforme (GBM) patients.

Materials and Methods

This study was approved by the institutional review board (IRB), and the requirement for informed consent was waived. A total of 59 patients with newly diagnosed GBM who received standard CCRT with TMZ and adjuvant TMZ for six cycles underwent perfusion-weighted and diffusion-weighted imaging. Twenty-seven patients had a measurable enhancing lesion and 32 patients lacked a measurable enhancing lesion based on the Response Assessment in Neuro-Oncology (RANO) criteria in the follow-up MRI, which was performed within 3 months after adjuvant TMZ therapy was completed. We measured the nCBV and nADC histogram parameters based on the measurable enhancing lesion. The progression free survival (PFS) was analyzed by the Kaplan-Meier method with the use of the log-rank test.

Results

The median PFS of patients lacking measurable enhancing lesion was longer than for those with measurable enhancing lesions (17.6 vs 3.3 months, P<.0001). There was a significant, positive correlation between the 99^th percentile nCBV value of a measurable enhancing lesion and the PFS (P = .044, R² = .152). In addition, the median PFS was longer in patients with a 99^th percentile nCBV value ≧4.5 than it was in those with a value <4.5 (4.4 vs 3.1 months, P = .036).

Conclusion

We found that the nCBV value can be used for the prognosis prediction of a measurable enhancing lesion after the completion of standard treatment for GBM, wherein a high 99^th percentile nCBV value (≧4.5) suggests a better PFS for GBM patients.     

Prognosis of Glioblastoma With Oligodendroglioma Component is Associated With the IDH1 Mutation and MGMT Methylation Status

Glioblastoma (GBM) with oligodendroglioma component (GBMO) is a newly described GBM subtype in the 2007 World Health Organization classification. However, its biological and genetic characteristics are largely unknown. We investigated the clinicopathological and molecular features of 34 GBMOs and compared the survival rate of these patients with those of patients with astrocytoma, oligodendroglioma, anaplastic oligoastrocytoma (AOA), and conventional GBMs in our hospital. GBMO could be divided into two groups based on the presence of an IDH1 mutation. The IDH1 mutation was more frequently found in secondary GBMO, which had lower frequencies of EGFR amplification but higher MGMT methylation than the wild type IDH1 group, and patients with mutant IDH1 GBMO were on average younger than those with wild-type IDH1. Therefore, GBMO is a clinically and molecularly heterogeneous subtype, largely belonging to a proneural and classical subtype of GBM. The survival rate of GBMO patients itself was worse than that of AOA patients but not significantly better than that of conventional GBM patients. GBMO survival was independent of the dominant histopathological subtype i.e., astrocyte-dominant or oligodendroglioma -dominant, but it was significantly associated with the IDH1 mutation and MGMT methylation status. Therefore, GBMO should be regarded as a separate entity from AOA and must be classified as a subtype of GBM. However, further study is needed to determine whether it is a pathologic variant or a pattern of GBM because GBMO has a similar prognosis to conventional GBMs.