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Cephalonoplos segetum has been used as an herbal remedy, and is considered to have anti-inflammatory potential. However, its biological mechanism in this treatment process remains unknown. Therefore, the anti-inflammatory activity of the ethyl acetate fraction of C. segetum extracts (CSE-EA), more active than C. segetum extracts (CSE) in murine macrophages, was investigated. Production levels of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) by lipopolysaccharide (LPS)-induced RAW 264.7 macrophages were measured by ELISA. In addition, protein expression levels of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2, and the phosphorylation of mitogenactivated protein kinases (MAPKs) in the LPS-induced macrophages were investigated by Western blotting. The CSE-EA (50, 100 or 200 μg/mL) significantly inhibited NO, PGE2, TNF-α, and IL-1β production in LPS-induced macrophages in a dose-dependent manner with 50% inhibitory concentration values of 80.4, 104.7, 91.3, and 46.7 μg/mL, respectively. Similarly, CSE-EA reduced protein expression of iNOS and COX-2 and led to the attenuated activation of kinases ERK1/2 and JNK in the macrophages. Results of the present study suggest that the anti-inflammatory effects of CSE-EA are likely due to the down-regulation of NO, PGE2 TNF-α, and IL-1β and the reduced expression of iNOS and COX-2 via suppression of MAPK signaling pathways in LPS-induced murine macrophages.

Definitive identification of original plant species is important for standardizing herbal medicine. The herbal medicines Cynanchi Wilfordii Radix (Baekshuoh in Korean and Beishuwu in Chinese) and Polygoni Multiflori Radix (Hashuoh in Korean and Heshuwu in Chinese) are often misidentified in the Korean herbal market due to morphological similarities and similar names. Therefore, we developed a reliable molecular marker for the identification of Cynanchi Wilfordii Radix and Polygoni Multiflori Radix. We used random amplified polymorphic DNA (RAPD) analysis of three plant species, Polygoni multiflorum, Cynanchum wilfordii, and Cynanchum auriculatum, to obtain several species-specific RAPD amplicons. From nucleotide sequences of these RAPD amplicons, we developed six sequence characterized amplification region (SCAR) markers for distinguishing Polygoni Multiflori Radix and Cynanchi Wilfordii Radix. Furthermore, we established SCAR markers for the simultaneous discrimination of the three species within a single reaction by using multiplex-PCR. These SCAR markers can be used for efficient and rapid authentication of these closely related species, and will be useful for preventing the distribution of adulterants.

Aims of study Although the flowers of Chrysanthemum indicum Linne (Asteraceae) have long been used in traditional Korean and Chinese medicine to treat inflammatory diseases, the underlying mechanism(s) by which these effects are induced remains to be defined. We investigated the effects of a 70% ethanolic extract of C. indicum (CIE) on the activities of cellular signaling molecules that mediate inflammatory responses. Materials and methods Production of NO, PGE2, TNF-α, and IL-1β by ELISA, mRNA and protein expression of iNOS and COX-2, phosphorylation of MAPKs, and activation of NF-κB by RT-PCR and Western blotting were examined in LPS-induced RAW 264.7 macrophages. Results The CIE strongly inhibited NO, PGE2, TNF-α, and IL-1β production, and also significantly inhibited mRNA and protein expression of iNOS and COX-2 in LPS-induced RAW 264.7 macrophages, in a dose-dependent manner. Furthermore, the CIE clearly suppressed nuclear translocation of NF-κB p65 subunits, which correlated with an inhibitory effect on IκBα phosphorylation. The CIE also attenuated the activation of ERK1/2 and JNK in a dose-dependent manner. Conclusion Our results suggest that the anti-inflammatory properties of CIE might result from the inhibition of inflammatory mediators, such as NO, PGE2, TNF-α, and IL-1β, via suppression of MAPKs and NF-κB-dependent pathways.

The roots of both Aralia continentalis and Angelica biserrata, known as /lsDokwhal/rs in Korea, have been used widely as a traditional oriental medicine to treat inflammation and thrombosis. However, the pharmacological differences between A. continentalis and A. biserrata have not been fully established. In the present study, we investigated and compared the inhibitory effects of 70% ethanolic extracts of A. continentalis (ACE) and A. biserrata (ABE) on the production of inflammatory mediators and secondary swelling from chemically induced ear edema. In RAW264.7 macrophages, both ACE and ABE significantly inhibited the release of nitric oxide, prostaglandin E2, interlukin-1beta, and tumor necrosis factor-alpha in a dose dependent manner. In addition, the swelling from TPA-induced edema in mouse ears was reduced by ACE and ABE. Overall, ACE showed stronger activities than ABE in vitro and in vivo. Our results indicate that A. continentalis roots possess stronger anti-inflammatory activity than A. Biserrata roots.

Aims of study Although Asparagus cochinchinensis Merrill (Liliaceae) has long been used in traditional Korean and Chinese medicine to treat inflammatory diseases, the underlying mechanism(s) by which these effects are induced remains to be defined. We investigated the effects of 70% ethanolic extract from Asparagus cochinchinensis Merrill (ACE) on skin inflammation in mice. Materials and methods Production of pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β), activation of myeloperoxidase, and histological assessment were examined in acute and chronic skin inflammation using 12- O -tetradecanoyl-phorbol-13-acetate (TPA)-induced mouse ear edema. We also performed acetic acid-induced vascular permeability test. Results ACE inhibited topical edema in the mouse ear, following administration at 200 mg/kg (i.p.), leading to substantial reductions in skin thickness and tissue weight, inflammatory cytokine production, neutrophil-mediated myeloperoxidase (MPO) activity, and various histopathological indicators. Furthermore, ACE was effective at reducing inflammatory damage induced by chronic TPA exposure and evoked a significant inhibition of vascular permeability induced by acetic acid in mice. Conclusion These results demonstrate that ACE is an effective anti-inflammatory agent in murine phorbol ester-induced dermatitis, and suggest that the compound may have therapeutic potential in a variety of immune-related cutaneous diseases.

We previously reported that transgenic (TG) mice over-expressing translationally controlled tumor protein (TCTP) developed systemic arterial hypertension at about 6 weeks after birth. In the present study, we identified, using proteomics technologies, 24 other proteins that were differentially expressed in the heart of TCTP over-expressing TG mice. These 24 proteins are involved in a variety of biological processes such as reactive oxygen species metabolism, cytoskeleton organization, fatty acid metabolism, amino acid metabolism and energy metabolism. We determined protein expression levels of the peroxiredoxin (Prx)2, Prx3, myosin light chain 1, stress protein (heat shock protein) 25K, and T-complex protein 1 alpha subunit by western blot analysis. Over-expression of TCTP probably regulates the expression of other proteins which play a pivotal role in a variety of cellular functions in TCTP over-expressing TG mice.

Estrogens, a group of steroid compounds functioning as the primary female sex hormone, play an important role in the development and progression of breast cancer. In this study, using a novel annealing control primer-based GeneFishing PCR technology, five differentially expressed genes (DEGs), expressed using 10nM mitogenic estrogens, -estradiol (E2) and -hydroxyestrone (-OHE1), were selected from the estrogen receptor (ER)-positive MCF-7 human breast cancer cells. The DEGs, MRPL42, TUBA1B, SSBP1, KNCT2, and RUVBL1, were identified by comparison with the known genes via direct sequencing and sequence homology search in BLAST. Quantitative real-time PCR data showed that two DEGs, tubulin and kinetochore associated 2, were greater than 2-fold upregulated by E2 or -OHE1. Both genes could be new biomarkers for the treatment and prognosis of cancers, and further study may provide insights into the molecular mechanisms underlying development and progression of breast cancer.

Background: Down syndrome (DS; trisomy 21) is the most common chromosomal aberration, with an incidence of 1/700 live births, and presents with mental retardation, typical phenotype and a series of associated pathological conditions. Although overexpression of genes encoded on chromosome 21 was proposed to lead to the phenotype, this has not been fully addressed at the protein level. Aim: It was therefore the aim of our study to determine protein levels of several chromosome 21 gene products in fetal DS brain. We selected interferon (IFN) α/β receptor β chain (IFNAR2), glutamate receptor 5 (ionotropic kainate 1), h2 calponin, claudin-8 and oligodendrocyte transcription factor 1 for quantification in fetal brain of the early second trimester using immunoblotting. Results: We observed significant and about twofold overexpression of the 20-kD immunoreactive IFN receptor α/β band in the fetal cortex of DS brain (p = 0.0293), while the 70-kD immunoreactive band was overexpressed but did not reach statistical significance (p = 0.065). When data were normalized to the housekeeping protein β-actin, a significant increase in IFN receptor levels was observed. Levels of the aforementioned proteins were comparable between DS and controls. Conclusion: Overexpression of IFNAR2 in fetal DS brain may increase the responsiveness of DS cells, leading to altered neuronal excitability, neuronal growth and survival as well as cytoskeleton derangement.

Histamine-N-methyltransferase (HMT) inactivates the neurotransmitter histamine. Central histaminergic deficits may contribute to the cognitive impairment of neurodegenerative disorders including Alzheimer's disease (AD) and Down syndrome (DS). However, there is no evidence for histaminergic deficits in Pick's disease (PiD). HMT levels were measured in the frontal cortex and cerebellum of brains of patients with AD, DS, and PiD, and normal aged subjects using proteomics techniques. In frontal cortex, HMT was significantly decreased in DS, but significantly increased in PiD compared with controls. HMT levels were comparable in cerebellum of all groups. Elevated HMT in PiD could lead to increased histamine degradation that in turn would be in agreement with impaired cognitive functions of PiD. Decreased HMT in DS would be compatible with findings of decreased histamine synthesis, thus reflecting a compensation mechanism to antagonize reduced synthesis by decreased degradation.

Asparagus cochinchinensis Merrill (Liliaceae) has been traditionally used for the treatment of cancer in Korea and China, but its anticancer activity and underlying mechanism remain to be defined. Anticancer activities were investigated on fractions obtained from A. cochinchinensis 70% ethanol extract (ACE-EtOH) in human hepatocellular carcinoma HepG2 cells. Ethylacetate fraction from A. cochinchinensis extract (ACE-EA), more effective than other fractions, induced apoptosis of HepG2 (IC50=72.33±0.34 μg/mL), as revealed by apoptotic feature observation, increased capase-3 activity and Poly ADP ribose polymerase cleavage, and decreased expression of X-linked inhibitor of apoptosis protein in a dose-dependent manner. Protein levels of autophagyrelated molecules, microtubule-associated protein 1 light chain 3 α and beclin 1, appeared to be induced by ACE-EA, suggesting ACE-EA exhibits anti-cancer activity with induction via both apoptosis and autophagy signaling pathways in HepG2 cells.

Glehnia littoralis (Umbelliferae) has been used traditionally in Korean, Japanese, and Chinese medicine for the treatment of immune-related diseases; however, its anti-inflammatory activity and underlying mechanism remain to be defined. We investigated the anti-inflammatory effect and inhibitory mechanism on inflammation by the methylene chloride fraction from Glehnia littoralis extract (MCF-GLE), which was more effective than Glehnia littoralis extract (GLE). MCF-GLE inhibited 12-O-Tetradecanoyl-phorbol-13-acetate (TPA)–induced inflammation in an inflammatory edema mouse model. Also, MCF-GLE strongly inhibited the releases of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) and significantly suppressed the mRNA and protein expression of inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-stimulated RAW 264.7 macrophage cells in a dose-dependent manner. Furthermore, MCF-GLE suppressed NF-κB activation and IκB-α degradation. MCF-GLE also attenuated the activation of ERK and JNK in a dose-dependent manner. These results indicate that MCF-GLE has an inhibitory effect on the in vivo and in vitro inflammatory reaction and is a possible therapeutic agent. Our results suggest that the anti-inflammatory properties of MCF-GLE may result from the inhibition of pro-inflammatory mediators, such as NO, PGE2, TNF-α, and IL-1β via suppression of NF-κB– and mitogen-activated protein kinases-dependent pathways. Keywords:: inflammation, Glehnia littoralis, NF-κB, mitogen-activated protein kinase (MAPK)

Aim of the study Although Chrysanthemum indicum Linne (Compositae) has long been used in traditional Korean, Chinese, Japanese medicine to treat various immune-related diseases the underlying mechanism(s) by which these effects are induced remains to be defined in vivo model system. We investigated the effects of 70% ethanolic extract from Chrysanthemum indicum Linne (CIE) on skin inflammation in mice. Materials and methods Production of pro-inflammatory cytokines (TNF-α and IL-1β), activation of myeloperoxidase, and histological assessment were examined in acute and chronic skin inflammation using 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced mouse ear edema. Results CIE inhibited topical edema in the mouse ear, following administration at 200 mg/kg (i.p.), leading to substantial reductions in skin thickness and tissue weight, inflammatory cytokine production, neutrophil-mediated myeloperoxidase activity, and various histopathological indicators. Furthermore, CIE was effective at reducing inflammatory damage induced by chronic TPA exposure. Conclusions These results demonstrate that CIE is an effective anti-inflammatory agent in murine phorbol ester-induced dermatitis, and suggest that the extract may have therapeutic potential in a variety of immune-related cutaneous diseases.

Aspirin is a popular nonsteroidal anti-inflammatory drug, but some patients suffer from hypersensitivity to it. This prompted us to identify the factors or molecules related to these responses.A commercially available DNA microarray was used to study changes in gene expression in human peripheral blood mononuclear cells (PBMCs) after aspirin treatment. The PBMCs were collected from a patient with aspirin-intolerant asthma and one normal healthy control.We identified 61 and 107 genes respectively induced and repressed by aspirin treatment in the PBMCs derived from the normal control. In the patient showing aspirin-induced asthma responses, 31 genes were up-regulated and 6 were down-regulated after aspirin treatment. Among these,1 gene was expressed with the same pattern in the control and the patient. In contrast, 19 genes showed different expression patterns, and it turned out that most of them were involved in immune responses, cell growth/proliferation, transcription/translation, and signaling pathways.These results show the molecules involved in hypersensitivity to aspirin and may lead to a better understanding of adverse responses to aspirin. Furthermore, they can provide clues for identifying novel therapeutic and/or preventive molecular targets of the adverse effects of aspirin.

Down Syndrome (DS, trisomy 21) is the most common genetic cause of mental retardation. The completed sequencing of genes encoded on chromosome 21 provides excellent basic information, however the molecular mechanisms leading to the phenotype of DS remain to be elucidated. Although overexpression of chromosome 21 encoded genes has been documented information at the protein expression level is mandatory as it is the proteins that carry out function. We therefore decided to evaluated expression level of seven proteins whose genes are encoded on chromosome 21: DSCR4, DSCR5, DSCR6; KIR4.2, GIRK2, KCNE1 and KCNE2 in fetal cortex brain of DS and controls at the early second trimester of pregnancy by Western blotting. beta-actin and neuron specific enolase (NSE) were used to normalise cell loss and neuronal loss. DSCR5 (PIG-P), a component of glycosylphosphatidylinositol- N-acetylglucosaminyltransferase (GPI-GnT), was overexpressed about twofold, even when levels were normalised with NSE. DSCR6 was overexpressed in addition but when normalised versus NSE, levels were comparable to controls. DSCR4 was not detectable in fetal brain. Potassium channels KIR4.2 and GIRK2 were comparable between DS and controls, whereas KCNE1 and KCNE2 were not detectable. Quantification of these proteins encoded on chromosome 21 revealed that not all gene products of the DS critical region are overexpressed in DS brain early in life, indicating that the DS phenotype cannot be simply explained by the gene dosage effect hypothesis. Overexpression of PIG-P (DSCR5) may lead to or represent impaired glycosylphosphatidylinositol- N-acetylglucosaminyltransferase mediated posttranslational modifications and subsequent anchoring of proteins to the plasma membrane.

Down syndrome (DS) is the most significant genetic disorder with mental retardation and is caused by trisomy 21. The phenotype of DS is thought to result from overexpression of a gene(s) located on the triplicated chromosome (region). An increasing body of evidence that challenge this "gene dosage effect" hypothesis, however, has been reported indicating that this hypothesis still remains to be elucidated. The availability of the complete sequence of genes on chromosome 21 could have an immediate impact on DS research, but no conclusions can be drawn from nucleic acid levels. This made us evaluate protein levels of six proteins, gene products, encoded on chromosome 21 (T-cell lymphoma invasion and metastasis inducing Tiam1 protein, holocarboxylase synthetase, human interferon-regulated resistance GTP-binding protein MxA, Pbx regulating protein 1, autoimmune regulator, and pericentrin) in fetal cortex from DS and controls at 18-19 weeks of gestational age using Western blot technique. None of the investigated proteins showed overexpression in DS compared to controls. Our present data showing unaltered expression of six proteins on chromosome 21 in fetal DS brain suggest that the existence of the trisomic state is not involved in abnormal development of fetal DS brain and that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are in the process of quantifying all gene products of chromosome 21 and our first results do not support the gene dosage hypothesis.

There is a series of about 12 transcription factors expressed on chromosome 21. These transcription factors (TFs) are major candidates for playing a pathogenetic role for the abnormal wiring of the brain in fetal Down Syndrome (DS) as approximately 5,000 TFs are developmentally involved in the complex architecture of the human brain. TF derangement in DS has been already reported and we decided to contribute to the problem by studying four TFs encoded on chromosome 21 in fetal DS brain.

Down syndrome (trisomy of chromosome 21) (DS) is the most common genetic cause of mental retardation. In our study we employed immunoblotting to evaluate protein expression of reduced folate carrier (hRFC), encoded by a gene localised on chromosome 21, in fetal DS brain. We observed increased expression of hRFC-immunoreactive band with an apparent MW of approximately 150kDa, whereas the other bands (MWs ∼60 and 50kDa), were comparable to control. In conclusion, we suggest that aberrant hRFC expression may well have a role in the already observed deterioration of folate metabolism in DS. Moreover, no alterations of expression level of p53 and Sp1, supposed to play a role in the regulation of hRFC, suggest that regulation of hRFC expression in fetal life by these proteins is highly unlikely, at least by changes in their protein level.

Fatty acid binding proteins (FABPs) are thought to play a role in the binding, targeting and transport of long-chain fatty acids, and at least three types of FABPs are found in human brain; heart type (H)-FABP, brain type (B)-FABP and epidermal type (E)-FABP. Although all three FABPs could be involved in normal brain function in prenatal and postnatal life, a neurobiological role of FABPs in neurodegenerative diseases has not been reported yet. These made us evaluate the protein levels of FABPs in brains from patients with Down syndrome (DS) and Alzheimer’s disease (AD) and fetal cerebral cortex with DS using two-dimensional (2-D) gel electrophoresis with subsequent matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS) identification and specific software for quantification of proteins. In adult brain, B-FABP was significantly increased in occipital cortex of DS, and H-FABP was significantly decreased in DS (frontal, occipital and parietal cortices) and AD (frontal, temporal, occipital and parietal cortices). In fetal brain, B-FABP and epidermal E-FABP levels were comparable in controls and DS. We conclude that aberrant expression of FABPs, especially H-FABP may alter membrane fluidity and signal transduction, and consequently could be involved in cellular dysfunction in neurodegenerative disorders.

The peripheral and central nervous system are harbouring herpes simplex virus type 1 (HSV-1) and this virus has been proposed to be implicated in the aetiology of Alzheimer's disease (AD). We tested whether the HSV-1 genome is found indeed in the brain of controls, patients with AD and Down syndrome (DS) and whether HSV-1 infectious proteins in brain were induced. Moreover, we tested whether interleukin (IL)-6, a marker for neuroinflammation, is found in brains of AD and DS. HSV-1 glycoprotein D gene, as well as viral phosphoprotein and glycoprotein were detected in all brain samples. IL-6 was detectable in seven out of the eight AD and all of the eight DS patients, but only three out of ten controls in the frontal cortex. IL-6 in cerebellum was detectable in all AD and DS patients, but only three out of nine controls. In conclusion, we propose that the detection of HSV-1 genome and HSV-1 inducible protein IL-6 not only shows the presence in human brain, but may indicate a role for HSV-1 in the process of neuroinflammation and apoptosis, known to occur in both neurodegenerative disorders, AD and DS.

Stathmin, distributed in neurons with high abundance, acts as an intracellular relay, integrating various transduction pathways triggered by extracellular signals and it is involved in physiological regulation of microtubule destabilization. Stathmin has been also shown to be a critical molecule in pathology of neurodegeneration such as Alzheimer’s disease (AD), particularly, in neurofibrillary tangle (NFT) formation. Here we evaluated protein levels of stathmin in adult brain from patients with AD and Down syndrome (DS) showing AD-like pathology by applying proteomic technologies with two-dimensional (2-D) gel electrophoresis, matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS) identification and specific software for quantification of proteins. Significantly decreased protein levels of stathmin were observed in frontal (2.12 ± 1.17, n = 6) and temporal (3.05 ± 2.81, n = 10) cortices of AD compared to controls (frontal cortex: 4.41 ± 1.70, n = 8; temporal cortex: 5.26 ± 2.26, n = 13). Stathmin was also significantly decreased in frontal (2.47 ± 1.11, n = 7) and temporal (2.02 ± 1.18, n = 9) cortices of DS. We also investigated stathmin levels in fetal brain. Stathmin was not significantly changed between fetal DS brain and controls. We suggest that the decreased protein level of stathmin in brains is associated with tangle formation and microtubule instability in DS as well as AD, but stathmin is not involved in the abnormal development of fetal DS brain.