Supplementation with Korean Red Ginseng Improves Current Perception Threshold in Korean Type 2 Diabetes Patients: A Randomized, Double-Blind, Placebo-Controlled Trial.

BACKGROUND: Many Type 2 diabetes (T2DM) patients in Korea take Korean Red Ginseng (KRG) for various reasons. In this study, we investigated the effects of KRG administration on diabetic peripheral neuropathy in T2DM patients. METHODS: This study was a randomized, double-blind, placebo-controlled trial. Participants were randomly allocated to either the placebo or KRG group and took corresponding tablets for 24 weeks. The primary outcomes were changes in current perception threshold (CPT) at week 24. Secondary outcomes were altered fasting plasma glucose, HbA1c, and various metabolic and inflammatory markers at week 24. RESULTS: Sixty-one patients completed the study. The CPT of the lower extremities at various frequencies exhibited significant improvements at week 24 in the KRG group. Other metabolic parameters were not altered after 24 weeks in both groups. In the subgroup analysis, CPT levels were improved in those with a longer diabetes duration or who already had neuropathy at the beginning of the study, and insulin resistance was improved in patients with a shorter diabetes duration. CONCLUSION: Twenty-four week administration of KRG in T2DM patients resulted in a significant improvement in neuropathy, especially in those with a longer diabetes duration. A further, larger population study with a longer follow-up period is warranted to verify the effects of KRG on diabetic neuropathy.

Coherent Raman Imaging of Live Muscle Sarcomeres Assisted by SFG Microscopy.

In this study, we used spectrally focused coherent anti-Stokes Raman scattering (spCARS) microscopy assisted by sum-frequency generation (SFG) to monitor the variations in the structural morphology and molecular vibrations of a live muscle of Caenorhabditis elegans. The subunits of the muscle sarcomeres, such as the M-line, myosin, dense body, and α-actinin, were alternatively observed using spCARS microscopy for different sample orientations, with the guidance of a myosin positional marker captured by SFG microscopy. Interestingly enough, the beam polarization dependence of the spCARS contrasts for two parallel subunits (dense body and myosin) showed a ~90° phase difference. The chemically sensitive spCARS spectra induced by the time-varying overlap of two pulses allowed (after a robust subtraction of the non-resonant background using a modified Kramers-Krönig transformation method) high-fidelity detection of various genetically modified muscle sarcomeres tuned to the C-H vibration (2800-3100 cm-1). Conversely, SFG image mapping assisted by phase-retrieved spCARS spectra also facilitated label-free monitoring of the changes in the muscle content of C. elegans that are associated with aging, based on the hypothesis that the C-H vibrational modes could serve as qualitative chemical markers sensitive to the amount and/or structural modulation of the muscle.

Effects of addition of Ta and Y ions to InZnO thin film transistors by sol-gel process.

We have investigated the effects of the addition of tantalum (Ta) and yttrium (Y) ions to InZnO thin film transistors (TFTs) using the sol-gel process. TaInZnO and YInZnO TFTs had significantly lower off current and higher on-to-off current ratio than InZnO TFTs. Ta and Y ions have strong affinity to oxygen and so suppress the formation of free electron carriers in thin films; they play an important role in enhancing the electrical characteristic due to their high oxygen bonding ability. The optimized TaInZnO and YInZnO TFTs showed high on/off ratio and low subthreshold swing.

Inhibition of hepatitis C virus (HCV) replication by specific RNA aptamers against HCV NS5B RNA replicase.

This study identified specific and avid RNA aptamers consisting of 2'-hydroxyl- or 2'-fluoropyrimidines against hepatitis C virus (HCV) NS5B replicase, an enzyme that is essential for HCV replication. These aptamers acted as potent decoys to competitively impede replicase-catalyzed RNA synthesis activity. Cytoplasmic expression of the 2'-hydroxyl aptamer efficiently inhibited HCV replicon replication in human liver cells through specific interaction with, and sequestration of, the target protein without either off-target effects or escape mutant generation. A selected 2'-fluoro aptamer could be truncated to a chemically manufacturable length of 29 nucleotides (nt), with increase in the affinity to HCV NS5B. Noticeably, transfection of the truncated aptamer efficiently suppressed HCV replication in cells without escape mutant appearance. The aptamer was further modified through conjugation of a cholesterol or galactose-polyethylene glycol ligand for in vivo availability and liver-specific delivery. The conjugated aptamer efficiently entered cells and inhibited genotype 1b subgenomic and genotype 2a full-length HCV JFH-1 RNA replication without toxicity and innate immunity induction. Importantly, a therapeutically feasible amount of the conjugated aptamer was delivered in vivo to liver tissue in mice. Therefore, cytoplasmic expression of 2'-hydroxyl aptamer or direct administration of chemically synthesized and ligand-conjugated 2'-fluoro aptamer against HCV NS5B could be a potent anti-HCV approach.

Prevalence of human papillomavirus and Chlamydia trachomatis infection among women attending cervical cancer screening in the Republic of Korea.

Cervical cancer screening with the conventional Papanicolaou test is recommended for the women aged 30 years and more in Korea. Cervical infection with human papillomavirus (HPV) is the most important cause of cervical cancer and Chlamydia trachomatis (CT) is the most common bacterial sexually transmitted infection and may also be associated with risk of cervical cancer. A cross-sectional study of women attending the National Cervical Cancer Screening Program in Busan and Suwon was carried out. Exfoliated cervical cells were collected, and questionnaires were administered to 4595 women. High-risk HPV types and CT were tested by Hybrid Capture 2 (HC2). HPV genotyping of 355 high-risk HPV-positive women at HC2 was performed using linear array. Age-standardized prevalence of high-risk HPV types and CT was 10.4% (95% confidence interval: 9.5-11.3) and 4.3% (95% confidence interval: 3.7-4.8). That 68.5% of women were high-risk HPV-positive at HC2 was confirmed by Linear Array whereas 17.5% seemed to be infected with only low-risk HPV types, not normally detected by HC2. Korean women showed a relatively high prevalence of high-risk HPV and a rather low prevalence of CT. As in cancer-free women in other Asian populations, HPV 52, 58, and 39 were detected more frequently than HPV 16. Cross-reaction of HC2 with low-risk HPV types is of some concern, were the test to be used in primary screening.

Toll-like receptor 9-stimulated monocyte chemoattractant protein-1 is mediated via JNK-cytosolic phospholipase A2-ROS signaling.

Monocyte chemoattractant protein-1 (MCP-1) influences monocyte migration into sites of inflammation. This study highlights the importance of cytosolic phospholipase A2 (cPLA2)-mediated reactive oxygen species (ROS) signaling processes in the regulation of MCP-1 release as a result of toll-like receptor (TLR) activation. In macrophages, activation of TLR9 induced MCP-1 and cPLA2-phosphorylated arachidonic acid (AA) release. Inhibition of cPLA2 blocked CpG-induced MCP-1 and AA release. Although CpG stimulates phosphorylation of ERK, p38 and JNK, only inhibition of the JNK signaling pathways attenuated MCP-1 release, suggesting that the TLR9-mediated MCP-1 release was dependent upon the JNK pathway. TLR9 activation also stimulated ROS generation, while inhibition of NADPH oxidases (Noxs) blocked CpG-induced MCP-1 release. The CpG treatment increased macrophage Nox1 mRNA level, however it had no effect on macrophage Nox2 mRNA level. Overall, these results suggest that CpG enhances ROS generation through cPLA2-dependent pathways, which results in MCP-1 release.

Activation of toll-like receptor 4 modulates vascular endothelial growth factor synthesis through prostacyclin-IP signaling.

In this study, we show that activation of toll-like receptor (TLR)4 by lipopolysaccharide (LPS) induces cyclooxygenase-2 (COX-2) expression, which results in prostaglandin (PG)I2 formation in macrophages. The LPS-stimulated COX-2 expression and PGI2 release were accompanied by production of the potent angiogenic cytokine, vascular endothelial growth factor (VEGF), and these effects were suppressed by NS-398, which is a COX-2 inhibitor. Direct addition of iloprost (an analogue of PGI2) for IP receptor also induced the production of VEGF, whereas DP, FP, and TP receptor agonists did not. Inhibition of IP protein expression by micro interfering RNA blocked LPS-induced VEGF production. Additionally, macrophages transiently caused Akt phosphorylation after stimulation with LPS, and inhibition of Akt phosphorylation blocked the production of VEGF and COX-2 expression in response to LPS. Overall, this study demonstrated that engagement of TLR4 with LPS induces production of PGI2 via Akt and generates VEGF through IP receptor.

Suppression of P-glycoprotein expression by antipsychotics trifluoperazine in adriamycin-resistant L1210 mouse leukemia cells.

Multidrug resistance (MDR) to unrelated chemotherapeutic drugs can be mediated by overexpression of P-glycoprotein (P-gp), the mdr gene product. Trifluoperazine (TFP), a phenothiazine derivative antipsychotics, is known to reverse MDR of tumor cell lines by blocking P-gp efflux function. In the present study, we evaluated the effect of TFP on the expression of P-gp in multidrug-resistant L1210/Adr mouse leukemic cell lines, which are characterized by overexpession of P-gp. We found that TFP induced the downregulation of P-gp protein and mdr1b mRNA in a dose- and time-dependent manner in L1210/Adr cells. TFP reduction of mdr1b mRNA was paralleled by transcriptional suppression of the mdr1b promoter. Moreover, TFP restored the adriamycin-induced apoptosis in L1210/Adr cells. These results suggest that TFP may have utility as an adjuvant in the therapy of leukemia for the reversal of P-gp-dependent MDR as well as for the management of psychological symptoms in the cancer patients.

Involvement of glycogen synthase kinase-3beta in hydrogen peroxide-induced suppression of Tcf/Lef-dependent transcriptional activity.

Hydrogen peroxide (H(2)O(2)) mediates induction of cytotoxicity in various cell types. GSK-3beta has been found to participate in a number of signaling pathways, including cell proliferation and cell death. In the present study, we show that GSK-3beta is rapidly dephosphorylated and activated in response to H(2)O(2) treatment. H(2)O(2) also dephosphorylates Akt/PKB in a dose- and time-dependent manner. Overexpression of Akt/PKB attenuates H(2)O(2)-induced dephosphorylation of GSK-3beta. Ectopic expression of Dvl-1, a component of Wnt signaling, stimulates Akt/PKB and inhibits dephosphorylation of GSK-3beta by H(2)O(2). Furthermore, H(2)O(2) causes the reduction of beta-catenin level and LiCl-mediated activation of Tcf/Lef-dependent transcription activity. These findings suggest that GSK-3beta is involved in H(2)O(2)-mediated inhibition of Tcf/Lef-dependent transcriptional activity.

Implication of Egr-1 in trifluoperazine-induced growth inhibition in human U87MG glioma cells.

The early growth response gene-1 (Egr-1) is a tumor suppressor which plays an important role in cell growth, differentiation and apoptosis. Egr-1 has been shown to be down-regulated in many types of tumor tissues. Trifluoperazine (TFP), a phenothiazine class of antipsychotics, restored serum-induced Egr-1 expression in several cancer cell lines. We investigated the effect of Egr-1 expression on the TFP-induced inhibition of cell growth. Ectopic expression of Egr-1 enhanced the TFP-induced antiproliferative activity and downregulated cyclin D1 level in U87MG glioma cells. Our results suggest that antipsychotics TFP exhibits antiproliferative activity through up-regulation of Egr-1.

Isolation of specific and high-affinity RNA aptamers against NS3 helicase domain of hepatitis C virus.

Hepatitis C virus (HCV)-encoded nonstructural protein 3 (NS3) possesses protease, NTPase, and helicase activities, which are considered essential for viral proliferation. Thus, HCV NS3 is a good putative therapeutic target protein for the development of anti-HCV agents. In this study, we isolated specific RNA aptamers to the helicase domain of HCV NS3 from a combinatorial RNA library with 40-nucleotide random sequences using in vitro selection techniques. The isolated RNAs were observed to very avidly bind the HCV helicase with an apparent Kd of 990 pM in contrast to original pool RNAs with a Kd of >1 microM. These RNA ligands appear to impede binding of substrate RNA to the HCV helicase and can act as potent decoys to competitively inhibit helicase activity with high efficiency compared with poly(U) or tRNA. The minimal binding domain of the ligands was determined to evaluate the structural features of the isolated RNA molecules. Interestingly, part of binding motif of the RNA aptamers consists of similar secondary structure to the 3'-end of HCV negative-strand RNA. Moreover, intracellular NS3 protein can be specifically detected in situ with the RNA aptamers, indicating that the selected RNAs are very specific to the HCV NS3 helicase. Furthermore, the RNA aptamers partially inhibited RNA synthesis of HCV subgenomic replicon in Huh-7 hepatoma cell lines. These results suggest that the RNA aptamers selected in vitro could be useful not only as therapeutic and diagnostic agents of HCV infection but also as a powerful tool for the study of HCV helicase mechanism.

The inhibition of nitric oxide synthase enhances PSA-NCAM expression and CREB phosphorylation in the rat hippocampus.

It is well known that nitric oxide (NO) acts downstream of NMDA receptor activation, which regulates the neural plasticity in the brain. In the present study, the effect of L-NAME, a non-selective nitric oxide synthase (NOS) inhibitor, on neural plasticity in the hippocampus was investigated. L-NAME increased the expression of PSA-NCAM and pCREB in the adult rat hippocampus. The co-localization of PSA-NCAM and pCREB indicates a possible relationship between the two in the granule cell layer in the dentate gyrus. Our results demonstrate that NO, as a subsignal of NMDA receptors, could be involved in the structural plasticity of the granule cell layer in the dentate gyrus by regulating the expression of PSA-NCAM and pCREB in the hippocampus.

7-Nitroindazole upregulates phosphorylated cAMP response element binding protein, polysialylated-neural cell adhesion molecule and tryptophan hydroxylase expression in the adult rat hippocampus.

We examined the effect of nitric oxide (NO) on the expression of phosphorylated cAMP response element binding (pCREB) protein and on the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), in the hippocampus of the adult rat, by administering 7-nitroindazole (7-NI) for 5 days, to block neuronal nitric oxide synthase (nNOS) activity. It was found that PSA-NCAM and pCREB immunoreactive cells were increased two-fold in the dentate gyrus by this treatment. In addition, the immunoreactivities of serotonin (5-HT, 5-hydroxytryptamine), known to be involved in neural plasticity, and of tryptophan hydroxylase immunoreactive-fibers in the dentate gyrus were both upregulated by 7-NI. Our results indicate that nNOS inhibition upregulates neural plasticity and increases 5-HT in the rat hippocampus, and suggest the existence of regulatory relationships between NO, neural plasticity and 5-HT.

Methyl-beta-cyclodextrin inhibits cell growth and cell cycle arrest via a prostaglandin E(2) independent pathway.

Methyl-beta-cyclodextrin, a cyclic oligosaccharide known for its interaction with the plasma membrane induces several events in cells including cell growth and anti-tumor activity. In this study, we have investigated the possible role of cyclooxygenase 2 (COX-2) in cell growth arrest induced by methyl-beta-cyclodextrin in Raw264.7 macrophage cells. Methyl-beta-cyclodextrin inhibited cell growth and arrested the cell cycle, and this cell cycle arrest reduced the population of cells in the S phase, and concomitantly reduced cyclin A and D expressions. Methyl-beta-cyclodextrin in a dose- and time-dependent manner, also induced COX-2 expression, prostaglandin E(2) (PGE(2)) synthesis, and COX-2 promoter activity. Pretreatment of cells with NS398, a COX-2 specific inhibitor completely blocked PGE(2) synthesis induced by methyl-beta-cyclodextrin, however inhibition on cell proliferation and cell cycle arrest was not effected, suggesting non-association of COX-2 in the cell cycle arrest. These results suggest that methyl-beta-cyclodextrin induced cell growth inhibition and cell cycle arrest in Raw264.7 cells may be mediated by cyclin A and D1 expression.

Phosphatidic acid regulates systemic inflammatory responses by modulating the Akt-mammalian target of rapamycin-p70 S6 kinase 1 pathway.

Macrophages are pivotal effector cells in the innate immune system. When microbial products bind to pathogen recognition receptors, macrophages are activated and release a broad array of mediators, such as cytokines, that orchestrate the inflammatory responses of the host. Phosphatidic acid (PA) has been implicated as an important metabolite of phospholipid biosynthesis and in membrane remodeling and has been further suggested to be a crucial second messenger in various cellular signaling events. Here we show that PA is an essential regulator of inflammatory response. Deleterious effects of PA are associated with the secretion of proinflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and the production of nitric oxide, prostaglandin E2, which are predominantly released by macrophage Raw264.7 cells. Furthermore, the administration of PA to mice increased the serum cytokine level. Moreover, direct or lipopolysaccharide-induced PA accumulation by macrophages led to the Akt-dependent activation of the mammalian target of rapamycin-p70 S6 kinase 1, a process required for the induction of inflammatory mediators. These findings demonstrate the importance of the role of PA in systemic inflammatory responses, and provide a potential usefulness as specific targets for the development of therapies.

Ribozyme-mediated selective induction of new gene activity in hepatitis C virus internal ribosome entry site-expressing cells by targeted trans-splicing.

Although hepatitis C virus (HCV) causes worldwide health problems, efficient and specific therapy is not available so far. In this study, we describe a new genetic approach to the specific HCV therapy that is based upon trans-splicing ribozymes that can selectively replace HCV transcripts with a new RNA that exerts anti-HCV activity. We have developed a group I intron-based ribozyme targeting the internal ribosome entry site (IRES) of HCV with high fidelity and specificity. The ribozyme was designed to trans-splice its 3' tagging sequence comprising a new coding RNA, such as firefly luciferase transcript, that is linked to the 3' part of the HCV 5' UTR encompassing the downstream sequence of the targeted residue in the IRES. This ribozyme was then demonstrated to induce HCV IRES-dependent translation of the firefly luciferase gene selectively in HCV IRES-expressing cells with trans-splicing reaction. Moreover, a specific ribozyme with the coding sequence of the diphtheria toxin A chain in place of the firefly luciferase selectively triggered expression of the cytotoxin in cells expressing HCV IRES and specifically activated apoptosis of the cells. These results suggest that the trans-splicing ribozyme could be a potent anti-HCV agent to deliver therapeutic new gene activities specifically and selectively in HCV-infected cells.

The chronic inhibition of nitric oxide synthase enhances cell proliferation in the adult rat hippocampus.

We investigated the effect of chronic blocking nitric oxide synthase (NOS), an enzyme producing NO from L-arginine, on granule cell proliferation in the dentate gyrus of adult rats under normal conditions. We treated 7-nitroindazole (7-NI) for 5, 15, and 25 days or N-nitro-L-arginine-methyl ester (L-NAME) for 25 days to block NOS activity and subsequently injected 5-bromo-2-deoxyuridine (BrdU) to detect proliferating cells. The BrdU-immunoreactive (IR) cell number was significantly increased in the 7-NI 15 and 25 day treated group, but not in the control or in the 7-NI 5 day treated group. L-NAME treatment for 25 days significantly increased BrdU-IR cells versus the control and 7-NI 25 day treated group. In addition, nissl staining showed no cell death occurred in the dentate gyrus after 7-NI or L-NAME 25 day treatments. Our results demonstrate that chronic inhibition of NOS increases cell proliferation and has no effect on cell death in the dentate gyrus of the rat hippocampus, which suggests that NO may regulate cell proliferation in the dentate gyrus.

Akt as a mediator of secretory phospholipase A2 receptor-involved inducible nitric oxide synthase expression.

The induction of inducible NO synthase (iNOS) by group IIA phospholipase A(2) (PLA(2)) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA(2) up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA(2) receptor (sPLA(2)R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA(2) stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA(2), Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA(2)-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA(2), and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA(2)-induced reporter activity of the iNOS promoter. M-type sPLA(2)R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA(2)R enhanced group IIA PLA(2)-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA(2) catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA(2) induces nitrite production by involving of M-type sPLA(2)R, which then mediates signal transduction events that lead to PI3K/Akt activation.

Negative regulatory role of overexpression of PLC gamma 1 in the expression of early growth response 1 gene in rat 3Y1 fibroblasts.

The early growth response 1 (Egr-1) gene product is a transcription factor that functions as an oikis factor. Loss of Egr-1 expression is closely associated with tumor formation. Phospholipase Cgamma1 (PLCgamma1) is overexpressed in some tumors, and its overexpression causes anchorage-independent growth. Here we report that overexpression of PLCgamma1 and SH2-SH3 domain of PLCgamma1 decreased induction of Egr-1 and the Egr-1-regulated genes TSP-1 and PAI-1. Results from the nuclear run-on assay and transfection experiment with the proximal 455 base pair region of the Egr-1 promoter (-454 to +1) showed that Egr-1 transcriptional activity was suppressed in PLCgamma1-3Y1 cells whereas decay of Egr-1 mRNA was similar in both cell lines. Serum response element- and ternary complex factor Elk-1-mediated transcriptional activation of the reporter gene in response to EGF were also inhibited in PLCgamma1-3Y1 cells. Pretreatment with the protein synthesis inhibitor cycloheximide (CHX) partially abrogated the serum-induced suppression of Egr-1 transcription in PLCgamma1-3Y1 cells, suggesting that a CHX-sensitive factor(s) is involved in the suppression of Egr-1 transcription in PLCgamma1-3Y1 cells. Our results demonstrated that overexpression of PLCgamma1 functions as a negative modulator of the tumor suppressor Egr-1 gene expression, possibly through inhibition of Elk-1-dependent transcriptional activity.

Inhibition of neuronal nitric oxide synthase increases adrenalectomy-induced granule cell death in the rat dentate gyrus.

Recent studies have shown that the expression of neuronal nitric oxide synthase (NOS) mRNA is increased after adrenalectomy (ADX). However, the role of increased NO production after ADX in the dentate gyrus is unknown. In this study, the relationship between NO inhibition and apoptosis in the dentate gyrus after ADX was examined. 7-Nitroindazole (7-NI; 30 mg/kg, i.p.), a selective inhibitor of neuronal NOS, was injected 1 day before ADX and subsequently once every 24 h. Then 4 days after ADX, dentate granule cell death was evaluated using silver impregnation and Nissl staining methods. Inhibition of neuronal NOS by 7-NI increased the number of dying granule cells approximately 4-fold in the dentate gyrus of the ADX rats, compared to vehicle-injected ADX controls. These results suggest that increased NO production after ADX may play an endogenous neuroprotective role in the dentate gyrus.