Association of Left Vertebral Artery Hypoplasia with Posterior Circulation Stroke and the Functional Outcome of Patients with Atrial Fibrillation-Related Cardioembolic Stroke.

BACKGROUND AND PURPOSE: A cardiogenic embolus could reach the posterior circulation through the right vertebral artery because of a relatively larger diameter in cases of left vertebral artery hypoplasia. Hence, we investigated whether left vertebral artery hypoplasia is associated with cardiac embolisms with atrial fibrillation in the posterior circulation and its functional outcomes. MATERIALS AND METHODS: In this monocentric retrospective study, patients with acute cardioembolic stroke with atrial fibrillation were enrolled and underwent CT or neck MRA, which visualized the aortic arch and subclavian arteries. The laterality and size of vertebral artery hypoplasia were recorded. Posterior circulation stroke, basilar artery occlusion, and the functional outcomes after 3 months were investigated. RESULTS: This study included 407 patients; the patients with left vertebral artery hypoplasia experienced a higher rate of posterior circulation stroke (19 versus 73; 42.2% versus 20.2%; P = .001) and basilar artery occlusion (5 versus 10; 11.1% versus 2.8%; P = .005) than the patients without left vertebral artery hypoplasia. Multivariate analysis revealed that left vertebral artery hypoplasia showed an association with lower odds of achieving a good functional outcome 3 months after the stroke (OR = 0.4; 95% CI, 0.2-0.9; P = .027). CONCLUSIONS: Patients with cardioembolic stroke and left vertebral artery hypoplasia had posterior circulation stroke, basilar artery occlusion, and poor functional outcomes after 3 months.

Consequences of Metabolic Disruption in Alzheimer's Disease Pathology.

Alzheimer's disease (AD) is an irreversible, progressive disease that slowly destroys cognitive function, such as thinking, remembering, and reasoning, to a level that one cannot carry out a daily living. As people live longer, the risk of developing AD has increased to 1 in 10 among people who are older than 65 and to almost 1 in 2 among those who are older than 85 according to a 2019 Alzheimer's Association report. As a most common cause of dementia, AD accounts for 60-80% of all dementia cases. AD is characterized by amyloid plaques and neurofibrillary tangles, composed of extracellular aggregates of amyloid-ß peptides and intracellular aggregates of hyperphosphorylated tau, respectively. Besides plaques and tangles, AD pathology includes synaptic dysfunction including loss of synapses, inflammation, brain atrophy, and brain hypometabolism, all of which contribute to progressive cognitive decline. Recent genetic studies of sporadic cases of AD have identified a score of risk factors, as reported by Hollingworth et al. (Nat Genet 43:429-435, 2001) and Lambert et al. (Nat Genet 45:1452-1458, 2013). Of all these genes, apolipoprotein E4 (APOE4) still presents the biggest risk factor for sporadic cases of AD, as stated in Saunders et al. (Neurology 43:1467-1472, 1993): depending on whether you have 1 or 2 copies of APOE4 allele, the risk increases from 3- to 12-fold, respectively, in line with Genin et al. (Mol Psychiatry 16:903-907, 2011). Besides these genetic risk factors, having type 2 diabetes (T2D), a chronic metabolic disease, is known to increase the AD risk by at least 2-fold when these individuals age, conforming to Sims-Robinson et al. (Nat Rev Neurol 6:551-559, 2010). Diabetes is reaching a pandemic scale with over 422 million people diagnosed worldwide in 2014 according to World Health Organization. Although what proportion of these diabetic patients develop AD is not known, even if 10% of diabetic patients develop AD later in their life, it would double the number of AD patients in the world. Better understanding between T2D and AD is of paramount of importance for the future. The goal of this review is to examine our current understanding on metabolic dysfunction in AD, so that a potential target can be identified in the near future.

Neutrophil pyroptosis mediates pathology of P. aeruginosa lung infection in the absence of the NADPH oxidase NOX2.

Nod-like receptor family, CARD domain-containing 4 (NLRC4) inflammasome activation is required for efficient clearance of intracellular pathogens through caspsase-1-dependent pyroptosis in macrophages. Although neutrophils have a critical role in protection from Pseudomonas aeruginosa infection, the mechanisms regulating inflammasome-mediated pyroptosis in neutrophils and its physiological role are largely unknown. We sought to determine the specific mechanisms regulating neutrophil pyroptosis in P. aeruginosa strain PAO1 (PAO1) lung infection and to identify the pathological role of this process. Nox2-/- models with reduced neutrophil antibacterial activity exhibited increased neutrophil pyroptosis, which was mediated by flagellin, a pathogenic PAO1 component. We also demonstrate that PAO1-induced pyroptosis depended on NLRC4 and Toll-like receptor 5 (TLR5) in neutrophils generated from Nlrc4-/- or Tlr5-/- mice. Our study reveals previously unknown mechanisms and physiological role of neutrophil pyroptosis during P. aeruginosa lung infection. Furthermore, our findings regarding neutrophil pyroptosis in the context of neutrophil dysfunction may explain the causes of acute and/or chronic infectious diseases discovered in immune-compromised patients.

Octanal-induced inflammatory responses in cells relevant for lung toxicity: expression and release of cytokines in A549 human alveolar cells.

Inhalation is an important route of aldehyde exposure, and lung is one of the main targets of aldehyde toxicity. Octanal is distributed ubiquitously in the environment and is a component of indoor air pollutants. We investigated whether octanal exposure enhances the inflammatory response in the human respiratory system by increasing the expression and release of cytokines and chemokines. The effect of octanal in transcriptomic modulation was assessed in the human alveolar epithelial cell line A549 using oligonucleotide arrays. We identified a set of genes differentially expressed upon octanal exposure that may be useful for monitoring octanal pulmonary toxicity. These genes were classified according to the Gene Ontology functional category and Kyoto Encyclopedia of Genes and Genomes analysis to explore the biological processes related to octanal-induced pulmonary toxicity. The results show that octanal affects the expression of several chemokines and inflammatory cytokines and increases the levels of interleukin 6 (IL-6) and IL-8 released. In conclusion, octanal exposure modulates the expression of cytokines and chemokines important in the development of lung injury and disease. This suggests that inflammation contributes to octanal-induced lung damage and that the inflammatory genes expressed should be studied in detail, thereby laying the groundwork for future biomonitoring studies.

New electrohydrodynamic flow caused by the Onsager effect.

We report a new type of electrohydrodynamic (EHD) flow generated around a circular cylinder and a spherical particle in a dielectric liquid under dc and ac electric fields. The EHD flow is observed for various combinations of dielectric liquids and polar additives. We suggest that the EHD flow is caused by a gradient of electrical conductivity produced by a nonuniform electric field and subsequent generation of free charge in the bulk liquid. Analytical and numerical analyses which are based on the leaky-dielectric model show good agreement with experimental results.

Low frequency mutation of the Ephrin receptor A3 gene in hepatocellular carcinoma.

EphA3 is a component of the Eph/ephrin tyrosine kinase system, which participates in vasculature development. This receptor/ligand system is associated with various signaling pathways related to cell growth and viability, cytoskeletal organization, cell migration, and anti-apoptosis. Accumulated evidence suggests that aberrant regulation of EphA3 and its genetic alterations are implicated in the development and progression of various cancers. However, despite a high incidence of EphA3 over-expression, no such investigation has been performed in hepatocellular carcinoma. Thus, we investigated genetic alterations of the EphA3 gene in 73 cases of hepatocellular carcinoma by single-strand conformational polymorphism and sequencing. One novel D219V missense mutation was found in the extracellular domain of EphA3, and two genetic alterations in the intracellular sterile-alpha-motif (SAM) domain of EphA3 appeared to be polymorphisms. Although the functional assessments of this mutant are incomplete, it is believed that this novel EphA3 mutation may contribute to the development of hepatocellular carcinoma.

Mutational analysis of JAK1 gene in human hepatocellular carcinoma.

UNLABELLED: The Janus kinase 1 (JAK1) gene encodes a cytoplasmic tyrosine kinase that is noncovalently associated with a variety of cytokine receptors and plays a nonredundant role in cell proliferation, survival, and differentiation. The mutated forms of JAK1 often altered the activation of JAK1 and then changed the activation of JAK1/STAT pathways, and this may contribute to cancer development and progression. Thus, to investigate whether genetic mutations of JAK1 gene are associated in hepatocellular carcinoma (HCC) progression, we analyzed genetic alterations of JAK1 gene in 84 human HCCs by single-strand conformational polymorphism (SSCP) and direct sequencing. Of 24 exons of JAK1 gene, 12 exons were previously reported to have mutations, we searched genetic alteration of JAK1 in these exons. Overall, one missense mutation (1.2%) was found. In addition, 12 cases (14%) were found to have single nucleotide polymorphism (14%) in exon 14. Taken together, we found one novel missense mutation of JAK1 gene in hepatocellular carcinomas with some polymorphisms. Although the functional assessment of this novel mutant remains to be completed, JAK1 mutation may contribute to the tumor development in liver cancer. KEYWORDS: JAK1 gene, hepatocellular carcinoma, mutation.

Capillary electrophoresis of high-molecular chitosan: the natural carbohydrate biopolymer.

Due to its high resolving power and diverse application range, capillary electrophoresis (CE) has been successfully applied to the analysis of carbohydrates. In this paper, a method for the determination of high-molecular chitosan (Mr 200,000) using CE is presented. We studied the optimal condition of buffer pH and type, and column type for determination of chitosan. Optimal CE performance was found when employing 100 mM triethylamine (TEA)-phosphate buffer, pH 2.0 and untreated fused-silica capillary (50 microm x 27 cm) for the chitosan analysis. Under optimum conditions, excellent linear responses were obtained in the concentration range of 1.25-20 microM, with a linear correlation coefficient of 0.9983. The standard deviations of the migration time and peak area were found to be 2.5 and 6.4%, respectively. This method could be readily applied to chitosan determination in real biological samples and commercial products.

The effect of methyl methanesulfonate (MMS)-induced excision repair on p53-dependent apoptosis in human lymphoid cells.

The tumor suppressor p53 product has been shown to play an important role in preventing carcinogenesis by at least two different mechanisms, by evoking cell cycle arrest and eliciting DNA repair on one hand, or by eliminating damaged cells by induction of apoptosis on the other hand. As a first step toward understanding the relationship between protective responses and apoptosis after genotoxic stress, we examined the effect of DNA strand breaks generated from repair processes in respect to acute cellular responses against DNA damage, and on p53-dependent apoptosis in human lymphoid cells. We used two isogenic cell lines, TK6 harboring wild-type p53, and WI-L2-NS, which carries a mutant p53. A significant difference in sensitivity was observed at 50 microg/ml methyl methane-sulfonate (MMS) between the two cell lines used. In addition, a clear p53-mediated contribution to apoptosis in MMS-induced cell death was observed. However, we did not observe any differences in repair of MMS-lesions, as determined by comet assay, between the two cell lines. These data suggest that the differences in apoptosis induction in the two lines are not a reflection of differences in strand-break frequency or repair capacity.

Salsolinol, a naturally occurring tetrahydroisoquinoline alkaloid, induces DNA damage and chromosomal aberrations in cultured Chinese hamster lung fibroblast cells.

Salsolinol (SAL) is a tetrahydroisoquinoline neurotoxin that has been speculated to contribute to pathophysiology of Parkinson's disease and chronic alcoholism. The compound is also found in certain beverages and food stuffs, including soy sauce, beer and bananas. Despite potential human exposure to SAL and its endogenous formation, little is known about the genotoxic or carcinogenic potential of this substance. In the present investigation, SAL induced DNA damage in cultured Chinese hamster lung (CHL) fibroblasts as assessed by single cell gel electrophoresis (Comet). CHL cells treated with SAL also exhibited higher frequencies of chromosomal aberrations than did vehicle-treated controls. Our recent study has revealed that SAL in combination with Cu(II) causes the strand scission in phiX174 supercoiled DNA [Neurosci. Lett. 238 (1997) 95]. In line with this notion, addition of cupric ion potentiated the DNA damaging and clastogenic activity of SAL. Antioxidant vitamins, such as Vitamin C and Vitamin E, and reduced glutathione inhibited clastogenicity of SAL, suggesting the involvement of reactive oxygen species (ROS) in SAL-induced DNA damage and genotoxicity in CHL cells.

Identification of propentofylline metabolites in rats by gas chromatography/mass spectrometry.

Propentofylline (PPF, 3-methyl-1-(5-oxohexyl)-7-propylxanthine) has been reported to be a compound for treatment of both vascular dementia and dementia of the Alzheimer type. The short half-life (about 15 min) of PPF at the terminal elimination phase and poor bioavailability after oral administration of PPF to rabbits (Kim et al., 1992) suggest in part that this drug takes the extensive first-pass metabolism in the liver. In addition, the metabolic pathway for PPF remains unclear. The objective of this experiment is to identify urinary metabolites of PPF in rats. For the identification of the metabolites, rat urine was collected after oral administration of 100 mg/kg PPF. PPF metabolite, 3-methyl-1-(5-hydroxyhexyl)-7-propylxanthine, was synthesized and confirmed by gas chromatography/mass spectroscopy (GC/MS) and 1H nuclear magnetic resonance spectroscopy. The urinary metabolites of PPF were extracted with diethyl ether and identified by electron impact and chemical ionization GC/MS. One urinary metabolite was confirmed to be 3-methyl-1-(5-hydroxyhexyl)-7-propylxanthine by synthesized authentic compound. Several metabolites of monohydroxy- and dihydroxy-PPF were identified based on mass fragmentation of both intact and trimethylsilylated derivatives of PPF metabolites and the novel structure of these metabolites is suggested based on mass spectra.

Genetic engineering of drought resistant potato plants by introduction of the trehalose-6-phosphate synthase (TPS1) gene from Saccharomyces cerevisiae.

In yeast, trehalose-6-phosphate synthase is a key enzyme for trehalose biosynthesis, encoded by the structural gene TPS1. Trehalose affects sugar metabolism as well as osmoprotection against several environmental stresses, such as heat and desiccation. The TPS1 gene of Saccharomyces cerevisiae was engineered under the control of the CaMV 35S promoter for constitutive expression in transgenic potato plants by Ti-plasmid of Agrobacterium-mediated transformation. The resulting TPS1 transgenic potato plants exhibited various morphological phenotypes in culture tubes, ranging from normal to severely retarded growth, including dwarfish growth, yellowish lancet-shaped leaves, and aberrant root development. However, the plants recovered from these negative growth effects when grown in a soil mixture. The TPS1 transgenic potato plants showed significantly increased drought resistance. These results suggest that the production of trehalose not only affects plant development but also improves drought tolerance.

Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.

Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.

Inhibition of activation of nuclear factor kappaB is responsible for inhibition of inducible nitric oxide synthase expression by higenamine, an active component of aconite root.

Effects of higenamine on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression (RAW 264.7 cells), on vascular reactivity in vitro and in vivo (rats), and on survival rates (mice) and serum nitrite/nitrate levels (rats) were investigated by using last lipopolysaccharide (LPS) plus interferon (IFN)-gamma. Higenamine concentration-dependently inhibited NO production and inducible NO synthase mRNA in RAW 264.7 cells, in which the IC(50) was 53 microM. Higenamine (10 mg/kg i.p.) administered 90 min before LPS (5 mg/kg i.v.) prevented not only LPS-induced hypotension but also pressor response to norepinephrine (1 microgram/kg) in rats. Incubation of thoracic aorta with LPS (300 ng/ml) for 8 h in vitro resulted in suppression of the vasoconstrictor effects to phenylephrine, which was prevented by coincubation with higenamine. The survival rate to endotoxin in mice was significantly (P <.01) increased by the presence of higenamine in the LPS-treated group up to 48 h. Serum nitrite/nitrate levels were significantly (P <.05) reduced by higenamine in LPS-treated rats. Finally, higenamine inhibited the activation of nuclear factor kappaB in RAW 264.7 cells due to LPS + IFN-gamma by mobility shift assays. Taken together, these data strongly suggest that higenamine inhibits iNOS expression by inhibiting nuclear factor kappaB activation by LPS + IFN-gamma, which may be beneficial in inflammatory diseases in which enhanced formation of NO is the main causative factor. Furthermore, due to positive inotropic action, higenamine may be more effective in a condition where myocardial contractility is likely to depress, such as in septic shock and/or endotoxin-induced inflammatory disorders.

Isolation of csm1 encoding a class V chitin synthase with a myosin motor-like domain from the rice blast fungus, Pyricularia oryzae.

A gene encoding a chitin synthase with a myosin motor-like domain (csm1) was isolated from Pyricularia oryzae using a PCR fragment amplified from a fungal chitin synthase conserved region. The deduced amino acid sequence of csm1 is homologous to that of CsmA of Aspergillus nidulans (65% identity). The putative gene product of csm1 is consisted of the myosin motor-like domain and a chitin synthase domain as in A. nidulans csmA. The chitin synthase domain of its C-terminus was also homologous to Aspergillus fumigatus ChsE (61.4% identity) and Ustilago maydis Chs6 (48.6% identity) that encode class V chitin synthases. Northern analysis demonstrated that the csm1 was expressed throughout the mycelial growth of P. oryzae. This is the first report on the isolation of the gene encoding a class V chitin synthase with the myosin motor-like domain from P. oryzae.

Effect of p53 tumor suppressor on nucleotide excision repair in human colon carcinoma cells treated with 4-nitroquinoline 1-oxide.

In probing the mechanism of nucleotide excision repair (NER) in response to 4-nitroquinoline 1-oxide (4NQO)-induced DNA damage, the effect of p53 tumor suppressor was investigated. The effect of p53 protein on the repair of damaged DNA was examined by comet assay. Expression of p53 and p21(Waf1/Cip1) proteins was measured by the Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry, respectively. Compared to RKO cells having the wild-type p53 gene, increased cytotoxicity by 4NQO was observed in RKOmp53 cells with a mutation in p53 protein. DNA single strand breaks (SSB), indicative of the DNA repair, were considerably increased in 4NQO-treated RKO cells. Also, the expression of p53 and p21 proteins was significantly increased in 4NQO-treated RKO cells. In RKOmp53 cells, no effect of 4NQO on p21 expression was observed. Our findings suggest that 4NQO-induced NER is p53-dependent and involves up-regulation of its downstream regulator, p21(Waf1/Cip1) proteins.

Mild hyperthermia-induced apoptosis is dependent on p53 in human lymphoid cells.

Mild hyperthermia is known to enhance apoptosis. The p53 tumor-suppressor gene product has been shown to function in apoptosis in response to genotoxic stress. However, there is little information regarding the mechanism of p53-dependent apoptosis induced by heat stress. In present study, a p53 contribution in mild hyperthermia-induced apoptosis was investigated in human lymphoid system. After 30-minute exposure at 44 degrees C, the accumulation of p53 protein was clearly observed in TK6 and ML-1 cells. Using comet assay, the more significant sensitivity to hyperthermic apoptosis was found in TK6 (wild-type p53) than in WI-L2-NS (mutated in p53). Furthermore, the significantly rapid shifting from early apoptotic phase to late apoptotic was observed in heat-induced p53 TK6 cells. These findings suggest that p53-dependent apoptosis is efficaciously induced by mild hyperthermia as non-genotoxic stress in human lymphoid system.

Pharmacokinetics of propentofylline and the quantitation of its metabolite hydroxypropentofylline in human volunteers.

Propentofylline (PPF, 3-methyl-1-(5-oxohexyl)-7-propylxanthine) has been reported to be effective for the treatment of both vascular dementia and dementia of the Alzheimer type. The pharmacological effects of PPF may be exerted via the stimulation of nerve growth factor, increased cerebral blood flow, and inhibition of adenosine uptake. The objectives of this experiment are to determine the kinetic behavior of PPF, to identify, and to quantify its metabolite in human. Blood samples were obtained from human volunteers following oral administration of 200 mg of PPF tablets. For the identification and quantification of the metabolite, 3-methyl-1-(5-hydroxyhexyl)-7-propylxanthine (PPFOH), PPFOH was synthesized and identified by gas chromatography/mass spectroscopy (GC/MS) and 1H-nuclear magnetic resonance spectroscopy. The molecular weight of synthesized metabolite is 308 dalton. The PPF and PPFOH in plasma were extracted with diethyl ether and identified by electron impact GC/MS. The plasma concentrations of PPF and PPFOH were determined by gas chromatography/nitrogen phosphorus detector in plasma and their pharmacokinetic parameters were determined. The mean half-life of PPF was 0.74 hr. The areas under the curve (AUCs) of PPF and PPFOH were 508 and 460 ng.hr/ml, respectively. Cmax of PPF was about 828.4 ng/ml and the peak concentration was achieved at about 2.2 hr (Tmax). These results indicate that PPF is rapidly disappeared from blood due to extensive metabolism into PPFOH.

Screening of new bioactive materials from microbial extracts of soil microorganism (I). Antimicrobial activity from 200 samples using microdilution assay.

The microdilution assay recommended by NCCLS (National Committee for Clinical Laboratory Standards) is one of the standardized methods of antibiotic susceptibility test. This method has been widely used clinically to obtain MIC values of antibiotics on pathogenic microorganisms. It is more convenient, rapid and simple to test many samples than other test methods such as agar diffusion assay and broth macrodilution assay. The screening of antimicrobial agents from microbial extracts is too laborious in its process. Therefore, a number of screening methods having more simple procedure have been developed. In our laboratory, we applied microdilution assay for screening the antimicrobial agents. This assay showed dose-response results and was more sensitive than disc diffusion assay in our system. We tested 200 samples of microbial extracts originated from 100 microbial strains and selected several samples as potential candidates. In this report, we show that the microdilution assay is more convenient method in screening of antibiotic susceptibility than those previously reported.