Anti-oxidant and anti-inflammatory activities of macelignan in murine hippocampal cell line and primary culture of rat microglial cells

Epidemiological studies suggest that the treatments of anti-inflammatory agents and anti-oxidants slow the progress of neurological diseases. Lignans are anti-oxidants and phytoestrogens found in a variety of plants. In this study, we investigated the neuroprotective effect of macelignan on glutamate-induced neurotoxicity and reactive oxygen species (ROS) in murine hippocampal HT22 cell line. Macelignan significantly attenuated the ROS production and neurotoxicity induced by glutamate in HT22 cell. Also, the properties of macelignan as an anti-inflammatory agent were investigated in microglials activation by lipopolysaccharide (LPS). It potently suppressed the expression of cyclooxygenase-2 and inducible nitric oxide synthase, that consequently resulted in the reduction of nitric oxide in LPS-treated microglial cells. It also significantly suppressed the production of pro-inflammatory cytokine tumor necrosis factor-alpha and interleukin-6. These results suggest that macelignan possesses therapeutic potentials against neurodegenerative diseases with oxidative stress and neuroinflammation.     

Receptor-type protein tyrosine phosphatase beta (RPTP-beta) directly dephosphorylates and regulates hepatocyte growth factor receptor (HGFR/Met) function

Protein tyrosine phosphorylation is a ubiquitous, fundamental biochemical mechanism that regulates essential eukaryotic cellular functions. The level of tyrosine phosphorylation of specific proteins is finely tuned by the dynamic balance between protein tyrosine kinase and protein tyrosine phosphatase activities. Hepatocyte growth factor receptor (also known as Met), a receptor protein tyrosine kinase, is a major regulator of proliferation, migration, and survival for many epithelial cell types. We report here that receptor-type protein tyrosine phosphatase β (RPTP-β) specifically dephosphorylates Met and thereby regulates its function. Expression of RPTP-β, but not other RPTP family members or catalytically inactive forms of RPTP-β, reduces hepatocyte growth factor (HGF)-stimulated Met tyrosine phosphorylation in HEK293 cells. Expression of RPTP-β in primary human keratinocytes reduces both basal and HGF-induced Met phosphorylation at tyrosine 1356 and inhibits downstream MEK1/2 and Erk activation. Furthermore, shRNA-mediated knockdown of endogenous RPTP-β increases basal and HGF-stimulated Met phosphorylation at tyrosine 1356 in primary human keratinocytes. Purified RPTP-β intracellular domain preferentially dephosphorylates purified Met at tyrosine 1356 in vitro. In addition, the substrate-trapping mutant of RPTP-β specifically interacts with Met in intact cells. Expression of RPTP-β in human primary keratinocytes reduces HGF induction of VEGF expression, proliferation, and motility. Taken together, the above data indicate that RPTP-β is a key regulator of Met function.     

Inhibition of matrix metalloproteinase-9 prevents neutrophilic inflammation in ventilator-induced lung injury

Neutrophils are considered to play a central role in ventilator-induced lung injury (VILI). However, the pulmonary consequences of neutrophil accumulation have not been fully elucidated. Matrix metalloproteinase-9 (MMP-9) had been postulated to participate in neutrophil transmigration. The purpose of this study was to investigate the role of MMP-9 in the neutrophilic inflammation of VILI. Male Sprague-Dawley rats were divided into three groups: 1) low tidal volume (LVT), 7 ml/kg of tidal volume (VT); 2) high tidal volume (HVT), 30 ml/kg of VT; and 3) HVT with MMP inhibitor (HVT+MMPI). As a MMPI, CMT-3 was administered daily from 3 days before mechanical ventilation. Degree of VILI was assessed by wet-to-dry weight ratio and acute lung injury (ALI) scores. Neutrophilic inflammation was determined from the neutrophil count in the lung tissue and myeloperoxidase (MPO) activity in the bronchoalveolar lavage fluid (BALF). MMP-9 expression and activity were examined by immunohistochemical staining and gelatinase zymography, respectively. The wet-to-dry weight ratio, ALI score, neutrophil infiltration, and MPO activity were increased significantly in the HVT group. However, in the HVT+MMPI group, pretreatment with MMPI decreased significantly the degree of VILI, as well as neutrophil infiltration and MPO activity. These changes correlated significantly with MMP-9 immunoreactivity and MMP-9 activity. Most outcomes were significantly worse in the HVT+MMPI group compared with the LVT group. In conclusion, VILI mediated by neutrophilic inflammation is closely related to MMP-9 expression and activity. The inhibition of MMP-9 protects against the development of VILI through the downregulation of neutrophil-mediated inflammation.     

Plant regeneration from protoplasts of Japanese lawngrass

Embryogenic callus of Japanese lawngrass (Zoysia japonica Steud.) was induced from sterile mature seeds on LS medium with 5 mg / l of 2,4-D. Embryogenic callus selected visually under microscope was proliferated in liquid N6 medium with amino acids (N6-AA medium). Protoplasts were isolated from suspension cells by the treatment of enzyme mixture containing pectolyase Y-23 and cultured in K8p medium with 2 mg / l of 2,4-D at the density of 10⁶ / ml. Plants were regenerated by transferring the protoplasts derived callus to MS medium and incubating at 28 °C under light for two months. Plantlets were successfully transplanted in the soil.Abbreviations 2,4-D 2,4-dichrolophenoxyacetic acid - MES 2-(N-Morpholino) ethanesulfonic acid     

Incorporation of plant residue-derived carbon into the microeukaryotic community in a rice field soil revealed by DNA stable-isotope probing

The microbial decomposition of plant residue is a central part of the carbon cycle in soil ecosystems. Here, we explored the microeukaryotic community responsible for the uptake of plant residue carbon in a rice field soil through DNA-based stable-isotope probing (SIP) using dried rice callus labelled with (13) C as a model substrate. Molecular fingerprinting with PCR-DGGE showed that the total eukaryotic community in soil under drained (upland) conditions distinctly changed within 3 days after the callus was applied and stable thereafter. The predominant group of eukaryotes that incorporated callus carbon were fungi affiliated with the Mucoromycotina (Mortierella), Ascomycota (Galactomyces, Eleutherascus, Gibberella and Fusarium) and Zoopagomycotina (Syncephalis). 'Fungus-like' protists such as Pythium (stramenopiles) and Polymyxa (Cercozoa) were also involved in carbon flow from the callus. Some of these fungi and 'fungus-like' protists took up soil organic matter with time, which suggested a priming effect of the callus on the eukaryotic community. Our results demonstrated the usefulness of SIP not only to trace the carbon flow from fresh organic matter but also to study the effect of fresh organic matter on the utilization of soil organic matter by the microbial community.     

Phylogenetic and taxonomic incongruence involving nuclear and mitochondrial markers in Korean populations of the freshwater snail genus Semisulcospira (Cerithioidea: Pleuroceridae)

We investigated the population genetic structure and phylogenetic relationships of four morphospecies of Semisulcospira sampled from multiple South Korean drainages. One, S. extensa, displayed modest levels of genetic diversity and formed a species-specific clade for both mitochondrial (mt) and nuclear markers. In contrast, the other three, S. coreana, S. gottschei, and S. libertina, were polyphyletic for both mt and nuclear markers. They formed, together with other nominal congeners (S. forticosta, S. multicincta, S. nodiperda, and S. tegulata), a taxonomically heterogeneous species complex containing population-level admixtures of genotypes from numerically predominant modal clades in addition to rare, phylogenetically divergent, mt and nuclear genotypes. The modal mt haplotypes exhibited far more geographic than taxonomic congruence and typically co-clustered into drainage-specific tip clades, irrespective of nominal taxonomic status. The evolutionary origins of the observed non-extensa phylogenetic heterogeneity are unclear at present although the available data do not support paralogous mt marker phenomena or the presence of cryptic species. We cannot distinguish among retention of ancestral polymorphisms or recticulate evolutionary origins as explanatory mechanisms and it may require the genetic characterization of Semisulcospira lineages throughout much of their collective east Asian range to address this issue. Based on the available data, we consider it best to view all of our non-extensa Korean study taxa as a single polymorphic species complex of the type species S. libertina. There is considerable evidence that similarly complex genetic structuring, at least for mt, may be typical of many other freshwater cerithioidean snail taxa. In light of our Semisulcospira results, we suggest that meaningful molecular phylogenetic characterization of freshwater cerithioidean lineages may require the use of both mt and nuclear markers together with population level sampling of all nominal taxa within regional drainages.     

Cellular localization of integrin isoforms in phenylephrine-induced hypertrophic cardiac myocytes

Cardiac hypertrophy is characterized by remodeling of the extracellular matrix (ECM). Integrins are cell-surface molecules that link the ECM to the cellular cytoskeleton where they play roles as signaling molecules and transducers of mechanical force. To clarify the possible roles of integrins in cardiac myocyte hypertrophy, we investigated the cellular localization and expression of ECM proteins and integrins in both normal cardiac myocytes and phenylephrine-induced hypertrophic myocytes. Addition of phenylephrine (PE) to cultured neonatal cardiac myocytes induced sarcomeric organization, increase in cell size, and synthesis of the hypertrophic marker, atrial natriuretic factor (ANF). In particular, fibronectin and collagen underwent dramatic localization changes during PE-induced cardiac hypertrophy. Significant changes were noted in the cellular localization of the respective collagen and fibronectin receptors, integrin alpha1 and alpha5, from diffuse to a sarcomeric banding pattern. Expression levels of integrins were also increased during hypertrophy. Treatment with okadaic acid (OA), an inhibitor of protein phosphatase 2A (PP2A), resulted in inhibition of hypertrophic response. These results suggest that dephosphorylation of integrin beta1 may be important in the induction of cardiac hypertrophy.     

Effects of matrix metalloproteinase inhibitor on LPS-induced goblet cell metaplasia

Bacterial infections of the lung are known to induce inflammatory responses, which lead to mucus hypersecretion. Moreover, mucin synthesis in the airways has been reported to be regulated by neutrophilic inflammation-induced epidermal growth factor receptor (EGFR) expression and its activation. Furthermore, matrix metalloproteinases (MMPs), especially MMP-9, have been reported to promote the transmigration of activated neutrophils. In this study, we investigated the associations between lipopolysaccharide (LPS)-induced goblet cell (GC) metaplasia and EGFR expression and the effects of MMP inhibitor (MMPI). Various concentrations of LPS were instilled into the tracheas of pathogen-free Sprague-Dawley rats, and airways were examined at different times after LPS instillation. To examine the role of MMP-9, we treated rats 3 days before LPS instillation and daily thereafter with MMPI. Neutrophilic infiltration, Alcian blue/periodic acid-Schiff (AB/PAS) staining, and immunohistochemical staining for MUC5AC, EGFR, and MMP-9 were performed. The instillation of LPS increased AB/PAS and MUC5AC staining in time- and dose-dependent manners, and treatment with MMPI significantly prevented GC metaplasia. The instillation of LPS into the trachea also induced neutrophilic infiltration and EGFR and MMP-9 expression in the airway epithelium, and MMPI was found to significantly prevent neutrophil recruitment, GC metaplasia, and EGFR and MMP-9 expression. This study demonstrates that the MMP-9 and EGFR cascades are associated with LPS-induced mucus hypersecretion.     

Habitual sleep initiation time and metabolic syndrome in middle-aged and elderly adults

This study aimed to examine the association between habitual types of sleep initiation time and metabolic syndrome. A total of 2674 participants aged 40 to 69 years (48.73% men, mean age 48.33 ± 7.18 years), who were free of cardiovascular disease or cancer and were not shift workers, participated in this population-based cross-sectional study embedded within the Korean Genome Epidemiology Study (KoGES). Based on at baseline and the last visit, the study participants were classified into four types of sleep initiation time: persistent late sleep (PLS), persistent usual sleep (PUS), persistent early sleep (PES), and non-persistent sleep (NPS) types. Metabolic syndrome was defined as having three or more of the following five criteria: abdominal obesity, impaired glucose intolerance, high blood pressure, high triglyceride, and low high-density lipoprotein-cholesterol. Among the 2674 study participants, the prevalence of metabolic syndrome was 865 (32.35%). To estimate the association between sleep initiation time and the risk of having metabolic syndrome, we constructed multivariable logistic regression models. After adjusting for covariates including sleep duration, the participants of the PLS type were 1.87 times more likely to have metabolic syndrome (odds ratio = 1.87, 95% confidence interval 1.07–3.27) than those of the PES type. In conclusion, in this population-based cross-sectional study, we observed that the PLS type of sleep initiation time had a significantly increased risk of metabolic syndrome as compared to the PES type, even after adjusting for covariates.

     

PKCε Promotes HuD-Mediated Neprilysin mRNA Stability and Enhances Neprilysin-Induced Aβ Degradation in Brain Neurons

Amyloid-beta (Aβ) peptide accumulation in the brain is a pathological hallmark of all forms of Alzheimer’s disease. An imbalance between Aβ production and clearance from the brain may contribute to accumulation of neurotoxic Aβ and subsequent synaptic loss, which is the strongest correlate of the extent of memory loss in AD. The activity of neprilysin (NEP), a potent Aβ-degrading enzyme, is decreased in the AD brain. Expression of HuD, an mRNA-binding protein important for synaptogenesis and neuronal plasticity, is also decreased in the AD brain. HuD is regulated by protein kinase Cε (PKCε), and we previously demonstrated that PKCε activation decreases Aβ levels. We hypothesized that PKCε acts through HuD to stabilize NEP mRNA, modulate its localization, and support NEP activity. Conversely, loss of PKCε-activated HuD in AD leads to decreased NEP activity and accumulation of Aβ. Here we show that HuD is associated with NEP mRNA in cultures of human SK-N-SH cells. Treatment with bryostatin, a PKCε-selective activator, enhanced NEP association with HuD and increased NEP mRNA stability. Activation of PKCε also increased NEP protein levels, increased NEP phosphorylation, and induced cell surface expression. In addition, specific PKCε activation directly stimulated NEP activity, leading to degradation of a monomeric form of Aβ peptide and decreased Aβ neuronal toxicity, as measured by cell viability. Bryostatin treatment also rescued Aβ-mediated inhibition of HuD-NEP mRNA binding, NEP protein expression, and NEP cell membrane translocation. These results suggest that PKCε activation reduces Aβ by up-regulating, via the mRNA-binding protein HuD, Aβ-degrading enzymes such as NEP. Thus, PKCε activation may have therapeutic efficacy for AD by reducing neurotoxic Aβ accumulation as well as having direct anti-apoptotic and synaptogenic effects.