GM1 Induced the inflammatory response related to the Raf-1/MEK1/2/ERK1/2 pathway in co-culture of pig mesenchymal stem cells with RAW264.7

Pig-human xenotransplantation can trigger cell-mediated immune responses. We explored the role of gangliosides in inflammation related to immune rejection in xenotransplantation. Co-culture of xenogeneic cells (pig-MSCs and RAW264.7) was used to emulate xenotransplantation conditions. MTT assay results indicated that cell viability was significantly decreased in pADMSCs co-cultured with RAW264.7 cells. GM1 and GM3 were highly expressed in pADMSCs co-cultured with RAW264.7 cells. pADMSCs co-cultured with RAW264.7 cells strongly expressed pro-inflammatory proteins such as COX-2, iNOS, p50, p65, pIκBα, and TNF-α. GM1-knockdown pADMSCs co-cultured with RAW 264.7 cells did not show significantly altered cell viability, but pro-inflammatory proteins were markedly inhibited. Co-culture of pADMSCs with RAW264.7 cells induced significant phosphorylation (p) of JNK1/2 and pERK1/2. However, pERK1/2 and pJNK1/2 were decreased and MEK1/2 and Raf1 were suppressed in GM1-knockdown pADMSCs co-cultured with RAW264.7 cells. Thus, the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways were significantly upregulated in response to increases of GM1 in co-cultured xenogeneic cells. However, the inflammatory response was suppressed in co-culture of GM1-knockdown pADMSCs with RAW264.7 cells via down-regulation of the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways. Therefore, the ganglioside GM1 appears to play a major role in the inflammatory response in xenotransplantation via the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways.     

Effects of medium components on L-ornithine production byBrevibacterium ketoglutamicum

Effects of yeast extract, and ammonium sulfate were investigated on the production of L-ornithine by an arginine auxotroph.Brevibacterium ketoglutamicum in flask and batch cultures. Yeast extract as an arginine source and ammonium sulfate as an inorganic nitrogen source had significant effects on L-ornithine, production and cell growth. L-ornithine production was repressed by the excessive addition of arginine. Reversion of auxotrophic cells to the wild type was observed when the initial yeast extract concentration was too low. There existed optimum concentrations of yeast extract and ammonium sulfate for L-ornithine production. The effects of yeast extract and ammonium sulfate concentrations on the Leudeking-Piret model parameters were examined to analyze, the relationship between cell growth and L-ornithine production.     

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.     

Role of Glu445 in the substrate binding of β-glucosidase

A previously uncharacterized gene in Neosartorya fischeri was cloned and expressed in Escherichia coli. It was found to encode a β-glucosidase (NfBGL1) distinguishable from other BGLs by its high turnover of p-nitrophenyl β-d-glucopyranoside (pNPG). Molecular determinants for the substrate recognition of NfBGL1 were studied through an initial screening of residues by sequence alignment, a second screening by homology modeling and subsequent site-directed mutagenesis to alter individual screened residues. A conserved amino acid, E445, in the substrate binding pocket of wild-type NfBGL1 was identified as an important residue affecting substrate affinity. Replacement of E445 with amino acids other than aspartate significantly decreased the catalytic efficiency (k_cat/K_m) of NfBGL1 towards pNPG, mainly through decreased binding affinity. This was likely due to the disruption of hydrogen bonding between the substrate and the carboxylate oxygen of the residue at position 445. Density functional theory (DFT) based studies suggested that an acidic amino acid at position 445 raises the substrate affinity of NfBGL1 through hydrogen bonding. The residue E445 is completely conserved indicating that this position can be considered as a crucial determinant for the substrate binding among GHs tested.     

Fenobam promoted the neuroprotective effect of PEP-1-FK506BP following oxidative stress by increasing its transduction efficiency

We examined the ways in which fenobam could promote not only the transduction of PEP-1-FK506BP into cells and tissues but also the neuroprotective effect of PEP-1-FK506BP against ischemic damage. Fenobam strongly enhanced the protective effect of PEP-1-FK506BP against H₂O₂-induced toxicity and DNA fragmentation in C6 cells. In addition, combinational treatment of fenobam with PEP-1-FK506BP significantly inhibited the activation of Akt and MAPK induced by H₂O₂, compared to treatment with PEP-1-FK506BP alone. Interestingly, our results showed that fenobam significantly increased the transduction of PEP-1-FK506BP into both C6 cells and the hippocampus of gerbil brains. Subsequently, a transient ischemic gerbil model study demonstrated that fenobam pretreatment led to the increased neuroprotection of PEP-1-FK506BP in the CA1 region of the hippocampus. Therefore, these results suggest that fenobam can be a useful agent to enhance the transduction of therapeutic PEP-1-fusion proteins into cells and tissues, thereby promoting their neuroprotective effects. [BMB Reports 2013; 46(11): 561-566]     

Characterization of a bifunctional HPr kinase/phosphorylase from Leuconostoc mesenteroides SY1

The hprK gene encoding bifunctional HPrK/P (kinase/ phosphorylase) was cloned from L. mesenteroides SY1, a strain isolated from kimchi. hprK was transcribed as a monocistronic gene. His-tagged HPrH16A and HPrK/P were produced in E. coli BL21(DE3) using pET26b(+) and purified. HPrK/P phosphorylation assay with purified proteins showed that the kinase activity of HPrK/P increased at slightly acidic pHs. Divalent cations such as Mg2+ and Mn2+ and glycolytic intermediates such as fructose-1, 6-bisphosphate (FBP) and phosphoenolpyruvate (PEP) increased the kinase activity of HPrK/P, but inorganic phosphate strongly inhibited it. Kinetic studies for the kinase activity of HPrK/P showed that the apparent Km values were 0.18 and 14.57 microM for ATP and HPr, respectively. The Km value for the phosphorylase activity of HPrK/P was 14.16 microM for P-Ser-HPr (HPr phosphorylated at the serine residue).     

Characterization, metabolites and gas formation of fumarate reducing bacteria isolated from Korean native goat (Capra hircus coreanae)

Fumarate reducing bacteria, able to convert fumarate to succinate, are possible to use for the methane reduction in rumen because they can compete for H₂ with methanogens. In this, we isolated fumarate reducing bacteria from a rumen of Korean native goat and characterized their molecular properties [fumarate reductase A gene (frdA)], fumarate reductase activities, and productions of volatile fatty acids and gas. Eight fumarate reducing bacteria belonging to Firmicutes were isolated from rumen fluid samples of slaughtered Korean black goats and characterized their phylogenetic positions based on 16S rRNA gene sequences. PCR based analyses showed that only one strain, closely related to Mitsuokella jalaludinii, harbored frdA. The growths of M. jalaludinii and Veillonella parvula strains were tested for different media. Interestingly, M. jalaludinii grew very well in the presence of hydrogen alone, while V. parvula grew well in response of fumarate and fumarate plus hydrogen. M. jalaludinii produced higher levels of lactate (P≤0.05) than did V. parvula. Additionally, M. jalaludinii produced acetate, but not butyrate, whereas V. parvula produced butyrate, not acetate. The fumarate reductase activities of M. jalaludinii and V. parvula were 16.8 ± 0.34 and 16.9 ± 1.21 mmol NADH oxidized/min/mg of cellular N, respectively. In conclusion, this showed that M. jalaludinii can be used as an efficient methane reducing agent in rumen.     

Absolute Lymphocyte Count as a Predictor of Mortality in Emergency Department Patients with Paraquat Poisoning

Background

Paraquat (PQ) is a potent, highly toxic and widely used herbicide. The major medical problems associated with PQ are accidental or suicidal ingestion. There are several prognostic markers of PQ poisoning, with the serum PQ concentration considered to be the best indicator of outcome. However, the measurement of such markers is limited in many hospitals.

Objective

The present study was conducted to investigate the association of absolute lymphocyte count (ALC) and the 30-day mortality rate in patients with PQ poisoning.

Methods

We performed a retrospective analysis of patients admitted to the emergency department after paraquat poisoning between January 2010 and April 2013. Independent risk factors including ALC for 30-day mortality were determined. The ALC was categorized in quartiles as ≤1700, 1700 to 3200, 3200 to 5000, and >5000. Univariate and multivariate Cox proportional hazard analysis were performed to determine the independent risk factors for mortality.

Results

A total of 136 patients were included in the study, and the 30-day mortality was 73.5%. ALC was significantly higher in nonsurvivors than in survivors. The highest ALC quartile (ALC>5000; hazard ratio, 2.58; 95% CI, 1.08–6.21) was associated with increased mortality in multivariate analysis. In addition, old age, lower arterial PaCO₂, increased peripheral neutrophil count, and high serum levels of creatinine were associated with mortality.

Conclusion

The absolute lymphocyte count is associated with the 30-day mortality rate in patients with paraquat poisoning.     

Grazing impact of heterotrophic dinoflagellates and ciliates on common red-tide euglenophyte Eutreptiella gymnastica in Masan Bay, Korea

The euglenophyte Eutreptiella gymnastica is a common red tide causative species. However, there have been no studies on the grazing impact of heterotrophic protists on this species. To investigate the grazing impact of heterotrophic protists on E. gymnastica, we measured daily the abundances of E. gymnastica and co-occurring potential heterotrophic protistan grazers in Masan Bay, Korea, in August 2004 when an E. gymnastica red tide occurred. In addition, we tested whether the common heterotrophic dinoflagellates Gyrodinium dominans, Oxyrrhis marina, Pfiesteria piscicida, Polykrikos kofoidii, Protoperidinium bipes, and Stoeckeria algicida and the naked ciliates Strobilidium sp. (30–40 μm in cell length) and Strombidinopsis sp. (70–100 μm in cell length) were able to feed on E. gymnastica. We also measured their growth and ingestion rates on E. gymnastica as a function of prey concentration. Finally, we calculated the grazing coefficients by combining field data on the abundance of the heterotrophic dinoflagellate and ciliate grazers and co-occurring E. gymnastica with laboratory data on ingestion rates obtained in this study. The maximum abundance of E. gymnastica in Masan Bay in August, 2004 was 7575 cells ml⁻¹, while those of Gyrodinium spp., P. kofoidii, P. bipes, the naked ciliates (≤50 μm in cell length), and naked ciliates (>50 μm in cell length) were 50, 9, 58, 32, and 3 cells ml⁻¹, respectively. The maximum growth rate of G. dominans on E. gymnastica (1.13 d⁻¹) was higher than that of O. marina (0.81 d⁻¹) or P. bipes (0.77 d⁻¹). However, E. gymnastica did not support positive growth of P. kofoidii, Strobilidium sp., and Strombidinopsis sp. (−0.04 ∼ −2.8 d⁻¹). The maximum ingestion rates of G. dominans, P. kofoidii, P. bipes, O. marina, and Strobilidium sp. on E. gymnastica (2.1–2.7 ng C predator⁻¹ d⁻¹) were similar, but they were much lower than that of Strombidinopsis sp. (156 ng C predator⁻¹ d⁻¹). The calculated grazing coefficients for P. bipes, small heterotrophic Gyrodinium spp. (25–35 μm in cell length), naked ciliates (≤50 μm in cell length), P. kofoidii, and naked ciliates (>50 μm in cell length) on E. gymnastica were up to 0.77, 0.61, 0.22, 0.07 and 0.03 d⁻¹, respectively (i.e., up to 54%, 46%, 20%, 7%, and 3% of E. gymnastica populations were removed by the population of each of these heterotrophic protistan grazers in 1 d, respectively). The results of the present study suggest that P. bipes, small heterotrophic Gyrodinium spp., and naked ciliates (≤50 μm in cell length) sometimes have considerable potential grazing impact on the populations of E. gymnastica.     

A resource for discovering specific and universal biomarkers for distributed stem cells

Specific and universal biomarkers for distributed stem cells (DSCs) have been elusive. A major barrier to discovery of such ideal DSC biomarkers is difficulty in obtaining DSCs in sufficient quantity and purity. To solve this problem, we used cell lines genetically engineered for conditional asymmetric self-renewal, the defining DSC property. In gene microarray analyses, we identified 85 genes whose expression is tightly asymmetric self-renewal associated (ASRA). The ASRA gene signature prescribed DSCs to undergo asymmetric self-renewal to a greater extent than committed progenitor cells, embryonic stem cells, or induced pluripotent stem cells. This delineation has several significant implications. These include: 1) providing experimental evidence that DSCs in vivo undergo asymmetric self-renewal as individual cells; 2) providing an explanation why earlier attempts to define a common gene expression signature for DSCs were unsuccessful; and 3) predicting that some ASRA proteins may be ideal biomarkers for DSCs. Indeed, two ASRA proteins, CXCR6 and BTG2, and two other related self-renewal pattern associated (SRPA) proteins identified in this gene resource, LGR5 and H2A.Z, display unique asymmetric patterns of expression that have a high potential for universal and specific DSC identification.