Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.     

Reprofiling of pyrimidine-based DAPK1/CSF1R dual inhibitors: identification of 2,5-diamino-4-pyrimidinol derivatives as novel potential anticancer lead compounds

Abstract

Hybridization of reported weakly active antiproliferative hit 5-amino-4-pyrimidinol derivative with 2-anilino-4-phenoxypyrimidines suggests a series of 2,5-diamino-4-pyrimidinol derivatives as potential antiproliferative agents. Few compounds belonging to the proposed series were reported as CSF1R/DAPK1 inhibitors as anti-tauopathies. However, the correlation between CSF1R/DAPK1 signalling pathways and cancer progression provides motives to reprofile them against cancer therapy. The compounds were synthesised, characterized, and evaluated against M-NFS-60 cells and a kinase panel which bolstered predictions of their antiproliferative activity and suggested the involvement of diverse molecular targets. Compound 6e, the most potent in the series, showed prominent broad-spectrum antiproliferative activity inhibiting the growth of hematological, NSCLC, colon, CNS, melanoma, ovarian, renal, prostate and breast cancers by 84.1, 52.79, 72.15, 66.34, 66.48, 51.55, 55.95, 61.85, and 60.87%, respectively. Additionally, it elicited an IC₅₀ value of 1.97?µM against M-NFS-60 cells and good GIT absorption with P_e value of 19.0?±?1.1?×?10^?6?cm/s (PAMPA-GIT). Molecular docking study for 6e with CSF1R and DAPK1 was done to help to understand the binding mode with both kinases. Collectively, compound 6e could be a potential lead compound for further development of anticancer therapies.     

Insecticidal activity of recombinant baculovirus co-expressing Bacillus thuringiensis crystal protein and Kunitz-type toxin isolated from the venom of bumblebee Bombus ignitus

To improve the insecticidal activity of Autographa californica nucleopolyhedrovirus (AcMNPV), using co-expression of Bacillus thuringiensis crystal protein and a Kunitz-type toxin isolated from bumblebee Bombus ignitus venom, a recombinant AcMNPV, ApPolh5-3006BiKTI, expressing Bi-KTI under the control of early promoter from Cotesia plutellae bracovirus (CpBV) was constructed. In this recombinant virus, B. thuringiensis cry1-5 crystal protein gene was introduced into the genome by the fusion of polyhedrin-cry1-5 under the control of polyhedrin gene promoter. RT-PCR analysis indicated that both Bi-KTI and polyhedrin-cry1-5 fusion protein were successfully expressed from the infected cells. In addition, SDS-PAGE revealed that polyhedrin-cry1-5 fusion protein expressed by recombinant viruses was occluded into the polyhedra. ApPolh5-3006BiKTI showed an improved insecticidal activity against larvae of Plutella xylostella and Spodoptera exigua. At low dosage rates, it was more effective against S. exigua than on P. xylostella, but more rapid insecticidal activity was shown in P. xylostella. These results strongly suggest that co-expression of Bt toxin and Kunitz-type toxins could be successfully applied to improve the insecticidal activity of baculoviruses.     

Biosynthesis of capsinoid is controlled by the Pun1 locus in pepper

Pungency in pepper (Capsicum annuum L.) has unique characteristics due to the alkaloid compound group, capsaicinoids, which includes capsaicin. Although capsaicinoids have been proved to have pharmacological and physiological effects on human health, the application of capsaicinoids has been limited because of their pungency. Capsinoids found in non-pungent peppers share closely related structures with capsaicinoids and show similar biological effects. Previous studies demonstrated that mutations in the p-AMT gene were related to the production of capsinoids; however, the pathway of capsinoid synthesis has not yet been fully elucidated. In this study, we performed genetic analysis to determine the mechanism of capsinoid synthesis using a F6 recombinant inbred line population. In this population, the presence/absence of capsinoids co-segregated with the genotype of the Pun1 locus, without exception. In addition, we screened the patterns of capsinoid synthesis and the correlation between the Pun1 locus and capsinoid synthesis in p-AMT mutant accessions. In Capsicum germplasms, we selected amino-acid-substituted mutants in the PLP binding domain of the p-AMT gene. Capsinoids were not synthesized with the recessive pun1 gene, regardless of the p-AMT genotype, and no relationship was found between p-AMT mutant type and capsinoid content. We concluded that the Pun1 gene, which is responsible for capsaicinoid synthesis, also controls capsinoid synthesis.     

Therapeutic angiogenesis of three-dimensionally cultured adipose-derived stem cells in rat infarcted hearts

Background aimsTo successfully treat myocardial infarction (MI), blood must be resupplied to the ischemic myocardium by inducing angiogenesis. Many studies report enhanced angiogenesis using stem cells; however, the therapeutic efficacy of cell transplant remains low because transplanted cells may not survive, be retained at the site of transplant, or develop into vascular tissue. In this study, we assessed the therapeutic potential of three-dimensional cell masses (3DCM) composed of human adipose-derived stem cells (hASC) in a rat MI model.MethodsFor formation of 3DCM, hASC were cultured on a substrate with immobilized fibroblast growth factor 2. The morphology and phenotypes of 3DCM were analyzed 1 day after culture. The cells (hASC and 3DCM, 5 × 10⁵ cells) were injected into ischemic regions after ligation of the left coronary artery (n = 6 in each group). Cell retention ratio, therapeutic efficacy and vascularization were evaluated 4 weeks after transplant.ResultsA spheroid-type 3DCM, which included vascular cells (CD34⁺/CD31⁺/KDR⁺/α-SMA⁺) with high production of human vascular endothelial growth factor, was obtained. Infarct size and cardiomyocyte apoptosis were reduced in the 3DCM-injected group compared with the hASC-injected group. The retention ratio of hASC was 14-fold higher in the 3DCM-injected group. Many transplanted cells differentiated into endothelial and smooth muscle cells and formed vascular networks incorporated into host vessels.ConclusionsTransplant of 3DCM may be useful for angiogenic cell therapy to treat MI.     

Prevalence of and Factors Associated with Albuminuria in the Korean Adult Population: The 2011 Korea National Health and Nutrition Examination Survey

Background

Microalbuminuria is associated with increased risk of renal disease and cardiovascular diseases even in non-diabetic subjects. High incidence rates of microalbuminuria have been found in a number of population-based studies. However, the prevalence and risk factors associated with microalbuminuria in the general population in Korea are unclear.

Objectives

The present study was performed to estimate the prevalence of microalbuminuria and investigate the associated risk factors in the general adult population using the Fifth Korea National Health and Nutrition Examination Survey (KNHANES V-2) data from 2011.

Methods

A total of 5,202 participants (mean age, 45.6 years; men, 2,337; women, 2,865) were included in the analysis. Microalbuminuria was evaluated in participants of KNHANES V-2 based on the urine albumin–creatinine ratio. Estimated glomerular filtration rate was calculated using the Modification of Diet in Renal Disease study equation.

Results

The weighted prevalence of microalbuminuria was 5.2% (95% CI, 4.4–6.1) in the general population. The prevalence of albuminuria is increased with age. After adjustment for age and sex, the presence of albuminuria was associated with increased waist circumference, systolic and diastolic blood pressure, aspartate aminotransferase, triglyceride, fasting plasma glucose, and the presence of hypertension and diabetes. In logistic regression analyses, older age, female sex, diabetes, hypertension, and serum aspartate aminotransferase were independently associated with the presence of albuminuria.

Conclusion

The prevalence of microalbuminuria was found to be 5.2%, and conventional risk factors for cardiovascular diseases are closely related to the presence of microalbuminuria in Korea. Microalbuminuria may be a useful marker to identify individuals with increased risk of cardiovascular disease.     

Magnetic Resonance Angiography Signal Intensity as a Marker of Hemodynamic Impairment in Intracranial Arterial Stenosis

Background

Intracranial arterial stenosis (ICAS) is the predominant cause of ischemic stroke and transient ischemic attack in Asia. Change of signal intensities (SI) across an ICAS on magnetic resonance angiography (MRA) may reflect its hemodynamic severity.

Methods

In-patients with a symptomatic single ICAS detected on 3D time-of-flight MRA were recruited from 2 hospitals. Baseline and 1-year follow-up data were collected. Signal intensity ratio (SIR) [ =  (mean post-stenotic SI -mean background SI)/(mean pre-stenotic SI - mean background SI)] was evaluated on baseline MRA to represent change of SIs across an ICAS. Acute infarct volume was measured on baseline diffusion-weighted images (DWI). Relationships between SIR and baseline characteristics as well as 1y outcomes were evaluated.

Results

Thirty-six subjects (86.1% males, mean age 55.0) were recruited. Overall, mean SIR was 0.84±0.23. Mean SIRs were not significantly different between the 23 (63.9%) anatomically severe stenoses and the 13 (36.1%) anatomically moderate stenoses (0.80±0.23 versus 0.92±0.21, p = 0.126). SIR was significantly, linearly and negatively correlated to acute infarct volume on DWI (Spearman correlation coefficient −0.471, p = 0.011). Two patients (5.6%) had recurrent ischemic strokes at 1y, not related to SIR values.

Conclusions

Change of signal intensities across an ICAS on MRA may reflect its hemodynamic and functional severity. Future studies are warranted to further verify the relationships between this index and prognosis of patients with symptomatic ICAS.     

Direct Detection of Disease-Associated Prions in Brain and Lymphoid Tissue Using Antibodies Recognizing the Extreme N-terminus of PrPC

A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrP^C). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrP^Sc followed by detection with a europium-labelled anti-PrP^C antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrP^Sc in cases of vCJD.