In Vitro Reconstruction of Bacterial ß-Barrel Membrane Protein Assembly Using E. coli Microsomal (Mid-Density) Membrane.

The in vitro reconstruction assay enables us to evaluate in detail the insertion and proper protein folding (together termed assembly) of ß-barrel membrane proteins. Here, we introduce an in vitro reconstitution experiments using isolated membrane fractions from Escherichia coli (E. coli). Membrane fractions isolated from E. coli cells and disrupted by sonication, which we have termed E. coli microsomal (mid-density) membrane (EMM), are ideal for biochemical experiments, as they can be harvested by high-speed centrifugation and do not require ultra-centrifugation. EMM pretreated with detergent can assemble externally supplemented ß-barrel membrane proteins via intact ß-barrel assembly machinery (BAM) complex retained in EMM. This method not only allows assembly analysis with inexpensive equipment but it also can be applied to drug screening using assembly as an indicator with high reproducibility. In this chapter, we introduce our method of evaluating assembled ß-barrel membrane proteins by demonstrating four representative ß-barrel membrane proteins: E. coli major porins OmpA and OmpF; enterohemorrhagic E. coli (EHEC) autotransporter EspP, and Haemophilus influenzae (H. influenzae) adhesin Hia.

Predicting the pathogenicity of missense variants based on protein instability to support diagnosis of patients with novel variants of ARSL.

Rare diseases are estimated to affect 3.5%-5.9% of the population worldwide and are difficult to diagnose. Genome analysis is useful for diagnosis. However, since some variants, especially missense variants, are also difficult to interpret, tools to accurately predict the effect of missense variants are very important and needed. Here we developed a method, "VarMeter", to predict whether a missense variant is damaging based on Gibbs free energy and solvent-accessible surface area calculated from the AlphaFold 3D protein model. We applied this method to the whole-exome sequencing data of 900 individuals with rare or undiagnosed disease in our in-house database, and identified four who were hemizygous for missense variants of arylsulfatase L (ARSL; known as the genetic cause of chondrodysplasia punctata 1, CPDX1). Two individuals had a novel Ser89 to Asn (Ser89Asn) or Arg469 to Trp (Arg469Trp) substitution, respectively predicted as "damaging" or "benign"; the other two had an Arg111 to His (Arg111His) or Gly117 to Arg (Gly117Arg) substitution, respectively predicted as "damaging" or "possibly damaging" and previously reported in patients showing clinical manifestations of CDPX1. Expression and analysis of the missense variant proteins showed that the predicted pathogenic variants (Ser89Asn, Arg111His, and Gly117Arg) had complete loss of sulfatase activity and reduced protease resistance due to destabilization of protein structure, while the predicted benign variant (Arg469Trp) had activity and protease resistance comparable to those of wild-type ARSL. The individual with the novel pathogenic Ser89Asn variant exhibited characteristics of CDPX1, while the individual with the benign Arg469Trp variant exhibited no such characteristics. These findings demonstrate that VarMeter may be used to predict the deleteriousness of variants found in genome sequencing data and thereby support disease diagnosis.

Terminal bridging of siRNA duplex at the ribose 2' position controls strand bias and target sequence preference.

Small interfering RNA (siRNA) and short hairpin RNA (shRNA) are widely used as RNA interference (RNAi) reagents. Recently, truncated shRNAs that trigger RNAi in a Dicer-independent manner have been developed. We generated a novel class of RNAi reagent, designated enforced strand bias (ESB) RNA, in which an siRNA duplex was chemically bridged between the 3' terminal overhang region of the guide strand and the 5' terminal nucleotide of the passenger strand. ESB RNA, which is chemically bridged at the 2' positions of ribose (2'-2' ESB RNA), functions in a Dicer-independent manner and was highly effective at triggering RNAi without the passenger strand-derived off-target effect. In addition, the 2'-2' ESB RNA exhibited a unique target sequence preference that differs from siRNA and silenced target sequences that could not be effectively suppressed by siRNA. Our results indicate that ESB RNA has the potential to be an effective RNAi reagent even when the target sequence is not suitable for siRNA.

Morphological difference of Escherichia coli non-heme ferritin iron cores reconstituted in the presence and absence of inorganic phosphate.

The iron core of Escherichia coli ferritin was reconstituted in the presence and absence of phosphate. The core formed in the presence of phosphate contained phosphate in amounts comparable to the iron content. The size distribution of the core was analyzed by analytical ultracentrifugation. A continuous size distribution was observed in the presence of phosphate, whereas a multimodal distribution was found in the absence of phosphate. In the presence of phosphate, the core size observed by electron microscopy was consistent with the inner diameter of ferritin. In contrast to this, clusters of several smaller particles were observed in the absence of phosphate. The small-angle X-ray scattering was measured under contrast matching conditions to obtain information on the iron core shape. A fringe was observed in the scattering profile in the presence of phosphate, but it was not observed in the absence of phosphate. Combining all results, we conclude that a hollow spherical core was formed in the presence of phosphate, while several small particles were formed within the inner cavity in the absence of phosphate.

In vitro assembly of Haemophilus influenzae adhesin transmembrane domain and studies on the electrostatic repulsion at the interface.

Haemophilus influenzae adhesin (Hia) belongs to the trimeric autotransporter family, and it mediates the adherence of these bacteria to the epithelial cells of host organisms. Hia is composed of the passenger domain, which is a virulence factor, and the translocator domain, which anchors the passenger domain into the outer membrane. The Hia transmembrane domain forms a transmembrane ß-barrel of 12 ß-strands, four of which are provided from each subunit. The ß-barrel has a pore that is traversed by three α-helices, one of which is provided from each subunit. This domain has a unique arginine arrangement inside the ß-barrel. The side chains of the arginine residues protrude from the ß-strands of three subunits toward the center of the barrel and are close to each other. Mutation of this arginine residue revealed the importance of the electrostatic repulsion between the three arginines. Electrostatic repulsion is considered to prevent misfolding and/or misassembly. The arginine clusters at the interface were found in several proteins and might generally play an important role in the assembly of the oligomer.

Optimization of Haemophilus influenzae adhesin transmembrane domain expression in Escherichia coli.

To obtain a high yield of the transmembrane domain of Haemophilus influenzae adhesin (HiaTD) in Escherichia coli, we attempted to express the HiaTD with and without a signal sequence using a T7 expression system. The expression level of HiaTD after induction was followed by quantification of the purified HiaTD, flow cytometric analysis of the outer membrane integrated HiaTD, and immunoblotting assay of fractionated cell lysate. In the expression system with a signal sequence, although the amount of cell-surface-expressed HiaTD increased over time, the number of HiaTD-expressing cells decreased, probably because of plasmid instability. As a result, the amount of purified HiaTD reached a plateau at 2 h postinduction. Although expression without the signal sequence provides a large amount of proteins as inclusion bodies in some membrane proteins, HiaTD expressed without a signal sequence was not observed as inclusion bodies and seemed to be assembled into the outer membrane during or after cell lysis.

Electrostatic Repulsion between Unique Arginine Residues Is Essential for the Efficient in Vitro Assembly of the Transmembrane Domain of a Trimeric Autotransporter.

Haemophilus influenzae adhesin (Hia) belongs to the trimeric autotransporter family and mediates the adherence of these bacteria to the epithelial cells of host organisms. Hia contains a passenger and a transmembrane domain. The transmembrane domain forms a 12-stranded ß-barrel in which four strands are provided by each subunit. The ß-barrel has a pore that is traversed by three α-helices. This domain has a unique arginine cluster, in which the side chains of the three arginine residues located at position 1077 (Arg1077) protrude into the pore of the ß-barrel. This arrangement seems to be unfavorable for assembly, because of repulsion between the positive charges. In this study, we investigated the in vitro assembly of the Hia transmembrane minimum domain (mHiaTD) and found that the dissociated mHiaTD reassembled in detergent solution. To investigate the role of Arg1077 in trimer assembly, we generated mutant proteins in which Arg1077 was replaced with methionine or lysine. The reassembly kinetics of the mutants was compared with that of the wild-type protein. The methionine mutant showed misassembly, whereas the lysine mutant showed reversible assembly, similar to that observed for the wild-type protein. These results show that electrostatic repulsion between the positive charges of Arg1077 is important for preventing the formation of misassembled oligomers by the mHiaTD in vitro.

Contemporary sex differences among patients with acute coronary syndrome treated by emergency percutaneous coronary intervention.

Acute coronary syndrome (ACS) is an important cause of mortality and morbidity in the general population. Recent advances in percutaneous coronary intervention (PCI) and optimal medical treatment have helped to improve the prognosis of patients with ACS. The previous reports indicated that women with ACS have a higher risk of adverse outcomes. However, sex differences in clinical outcomes with contemporary coronary revascularization and medical therapy for ACS have not been elucidated. We analyzed data from 676 consecutive patients with ACS (female, n = 166; male, n = 510) who were treated by emergency PCI between 2011 and 2014 at Juntendo Shizuoka Hospital. The patients were grouped according to sex. We defined major adverse cardiovascular events as a composite of all-cause death and ACS recurrence at 1 year and compared rates of major adverse cardiac events (MACE) between the groups. Women were older (75.4 ± 11.0 vs. 66.2 ± 12.2 years) and had a higher rate of multi-vessel disease, chronic kidney disease, and Killip IV at presentation. The cumulative rate of MACE at 1 year was significantly higher among women than men (17.5 vs. 10.2 %, p = 0.02, log-rank test). However, the association between women and a higher risk of MACE was attenuated after adjusting for age (HR 1.25, 95 % CI 0.77-2.00, p = 0.36) and other variables (HR 0.93, 95 % CI 0.36-2.44, p = 0.88). Adjustment for age and other risk factors attenuated sex differences in mid-term clinical outcomes among patients with ACS after emergency PCI.

Effects of extremely low-frequency electromagnetic fields (ELF-EMF) exposure on B6C3F1 mice.

OBJECTIVE: Long-term exposure study was conducted to investigate the effects of extremely low-frequency electromagnetic field on the tumor promotion process and fertility. METHODS: Ten pregnant C57BL/6NCrj mice were exposed to 50 Hz field 500 mG for 1 week (12 h per day), and 24 male and 42 female B6C3F1mice born from them were further exposed up to 15.5 months. As a control group, 10 pregnant mice were bred without exposure, and 30 produced male and 32 female mice were observed without exposure for the same period. RESULTS: Mean body weights of exposed groups of male and female mice were decreased significantly than those of the control groups. In exposed mice, there was no increased incidence of liver and lung tumor. In female mice, the incidence of chronic myeloid leukemia [3/42 (7%)] in the exposed group was significantly greater than in the control group. The size of seminiferous tubules in the EMF exposed groups were significantly less than the control group. CONCLUSIONS: These data support the hypothesis that long-term exposure of 50 Hz magnetic fields is a significant risk factor for neoplastic development and fertility in mice.

Control of HCV Replication With iMIRs, a Novel Anti-RNAi Agent.

MicroRNAs (miRNAs) serve important roles in regulating various physiological activities through RNA interference (RNAi). miR-122 is an important mediator of RNAi that is known to control hepatitis C virus (HCV) replication and is being investigated in clinical trials as a target for anti-HCV therapy. In this study, we developed novel oligonucleotides containing non-nucleotide residues, termed iMIRs, and tested their abilities to inhibit miR-122 function. We compared the inhibitory effects of iMIRs and locked nucleic acids (LNAs) on HCV replication in OR6 cells, which contained full-length HCV (genotype 1b) and a luciferase reporter gene. We found that RNA-type iMIRs with bulge-type, imperfect complementarity with respect to miR-122 were 10-fold more effective than LNAs in inhibiting HCV replication and functioned in a dose-dependent manner. Moreover, iMIR treatment of OR6 cells reduced HCV replication without inducing interferon responses or cellular toxicity. Based on these results, we suggest that iMIRs can inhibit HCV replication more effectively than LNAs and are therefore promising as novel antiviral agents.

Telomeric g-tail length and hospitalization for cardiovascular events in hemodialysis patients.

BACKGROUND AND OBJECTIVES: Telomeric G-tails play a pivotal role in maintaining the intramolecular loop structure of telomeres. Previous in vitro studies have suggested that the erosion of telomeric G-tails triggers cellular senescence, leading to organ dysfunction and atherosclerosis. The authors recently established a method to measure telomeric G-tail length using a hybridization protection assay. Using this method, this study investigated whether telomeric G-tail length could be used as a novel predictor for future cardiovascular events in hemodialysis patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A prospective observational study was performed involving a cohort of 203 Japanese hemodialysis patients to examine the lengths of telomeric G-tails and total telomeres and subsequent cardiovascular events during a median follow-up period of 48 months. The lengths of telomeric G-tails and total telomeres were also measured in 203 participants who did not have CKD and who were age- and sex-matched to hemodialysis patients. RESULTS: The lengths of telomeric G-tails and total telomeres were significantly shorter in hemodialysis patients than in control subjects. Telomeric G-tails, but not total telomeres, were independently and negatively associated with clinical history of cardiovascular disease. During follow-up, 80 cardiovascular events occurred. Total telomere length did not predict cardiovascular events. However, the length of telomeric G-tails was associated with new-onset cardiovascular events (hazard ratio per log luminescence signals, 0.12; 95% confidence interval, 0.12 to 0.50) that persisted after adjustment for age, sex, diabetes mellitus, clinical history of cardiovascular disease, inflammation, use of vitamin D, and serum levels of phosphate and intact parathyroid hormone. CONCLUSIONS: Longer telomeric G-tail length is associated with a lower risk of future cardiovascular events in hemodialysis patients.

Nuclear Aurora B and cytoplasmic Survivin expression is involved in lymph node metastasis of colorectal cancer.

The chromosomal passenger complex (CPC) is a key regulator of chromosome segregation and cytokinesis, and consists of Aurora B kinase, INCENP, Survivin and Borealin. Aurora B is a member of a family of serine/threonine protein kinases, and Survivin belongs to the inhibitors of apoptosis (IAP) gene family, and is also a member of the CPC family. Aurora B and Survivin have also been reported to be overexpressed in various human cancers; however, as yet no studies have investigated the co-expression of Survivin and Aurora B in colorectal carcinoma. Therefore, in the present study, the correlation between Aurora B and Survivin expression was investigated using immunohistochemistry and the associated pathological features in colorectal carcinoma were analyzed. Our present findings showed that nuclear Aurora B and cytoplasmic Survivin expression are strongly associated with and involved in lymph node metastasis in colorectal cancer. Therefore, we suggest that nuclear Aurora B and cytoplasmic Survivin are useful diagnostic markers and therapeutic targets in colorectal carcinoma.

Efficient DNA strand displacement by a W-shaped nucleoside analogue (WNA-ßT) containing an ortho-methyl-substituted phenyl ring.

Molecules that can target duplex DNA with sequence selectivity have the potential to be useful tools in genomic research and also as therapeutic agents. Homopurine-homopyrimidine stretches in duplex DNA can be recognized by homopurine or homopyrimidine TFOs (triplex-forming oligonucleotides) through the formation of triplex DNA. We have previously developed bicyclic nucleoside analogues (WNAs) for the formation of stable triplexes in the formation of stable antiparallel triplexes containing a TA or a CG interrupting site. In this study, we investigated the effects on triplex DNA formation of ortho-, meta-, and para-methyl substituent groups on the aromatic ring of the WNA analogue. It was found that the homopurine TFO containing meta- and para-methyl-substituted WNA-ßT (mMe-WNA-ßT, pMe-WNA-ßT) stabilized triplexes containing a TA interrupting site or a GC site, respectively. Interestingly, the ortho-methyl-substituted WNA-ßT (oMe-WNA-ßT) efficiently promoted DNA strand displacement to form the TFO/pyrimidine duplex. A detailed investigation showed that the duplex was in the antiparallel orientation and that its formation took place prior to triplex formation with the need for a magnesium cation. NOESY measurements indicated a significant difference in the rotation flexibilities of the phenyl rings of WNA-ßTs: that is, the conformation of the ortho-methylated phenyl ring was stable in a temperature-independent manner. It was speculated that the initial formation of a ternary complex was followed by strand displacement and then the formation of the TFO/pyrimidine duplex together with the TFO(2)/pyrimidine triplex formation during the early stage, and that the equilibrium shifted to the triplex during the later stage. Although the detailed role is still uncertain, the fixed phenyl ring of oMe-WNA-ßT might play a role in the displacement reaction.

Chronic administration with rotenone does not enhance MPTP neurotoxicity in C57BL/6 mice.

Systematic administration of rotenone as one of pesticides is known to produce degeneration of nigral dopaminergic neurons and motor deficits in experimental animals. Here, we investigated to determine whether systematic administration of rotenone causes the increased susceptibility in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Rotenone was injected into MPTP-treated mice over a period of 4 weeks. Thereafter, we evaluated the effect of rotenone 1, 3, and 6 weeks after the cessation of treatment with rotenone. In the present study with HPLC analysis, rotenone did not enhance MPTP-induced dopaminergic neurotoxicity in mice. Furthermore, MPTP + rotenone (9 mg/kg)-treated mice exhibit a significant loss of motor activity 1 day after the cessation of treatment with rotenone, However, no significant change of motor activity was found in MPTP-treated and MPTP + rotenone (9 mg/kg)-treated animals 6 weeks after the cessation of treatment with 0.5% carboxymethyl cellulose or rotenone. Our Western blot analysis study demonstrated that the change of tyrosine hydroxylase and glial fibrillary acidic protein protein levels in MPTP-treated mice was similar than that in MPTP + rotenone-treated animals. These results suggest that rotenone did not enhance MPTP neurotoxicity in mice. Our findings suggest that rotenone is not a reliable model for PD. Thus, our findings provide further valuable information for the pathogenesis of PD for exposure to agricultural pesticides.

Recognition of CG interrupting site by W-shaped nucleoside analogs (WNA) having the pyrazole ring in an anti-parallel triplex DNA.

We have previously developed W-shaped nucleoside analogs (WNA) for recognition of TA and CG interrupting sites, which are the intrinsic limitation for the formation of a stable triplex DNA by the natural triplex-forming oligonucleotide (TFO). However, the stabilization effect of WNA is dependent on the neighboring nucleobases at both sides of the WNA analogs within the TFO. Considering that the base is located at the hindered site constructed of three bases of the target duplex and the TFO, it was expected that replacement of the pyrimidine base of the WNA analog with a smaller pyrazole ring might avoid steric repulsion to produce a greater stability for the triplex. In this study, the new WNA analogs bearing the pyrazole ring, 3-aminopyrazole (AP), and 4-methyl-3-pyrazole-5-on (MP) were synthesized, incorporated into the TFOs, then their stabilizing effects on the triplexes were evaluated. A remarkable success was illustrated by the fact that the TFO containing WNA-betaAP in the 3'G-WNA-G-5' sequence formed a stable triplex with selectivity to the CG interrupting site where the previous WNA-betaC did not induce the triplex formation.

Intracellular localization of survivin determines biological behavior in colorectal cancer.

Survivin is a bifunctional protein that suppresses apoptosis and regulates cell division and is highly expressed in various human cancers. Recently, the intracellular localization of survivin in tumors has been suggested as a prognostic marker, but the molecular mechanisms are not understood. The aims of the present study were to investigate the different localization of survivin expression in colorectal carcinoma and expression of survivin relationships with clinicopathological factors and patient survival. Immunohistochemical analyses of 142 cases of advanced colorectal cancer showed that 109 (76.8%) cases expressed survivin in the nucleus and 29 cases (20.4%) in the cytoplasm. Cytoplasmic survivin overexpression was associated with a poor prognosis, but nuclear survivin overexpression was associated with a better prognosis. Subcellular distribution of survivin in five cases of cancerous or surrounding normal tissues derived from fresh biopsy of non-fixed samples of colorectal cancer patients was further demonstrated by Western blotting. Survivin was primarily found in the insoluble fraction. Interestingly, regardless of survivin protein levels in the insoluble fraction, patients who had cancerous tissue expressing cytoplasmic and nuclear soluble survivin suffered from lymph nodes metastases. These data suggest that the function of cytoplasmic survivin might be important for malignant progress and the levels of cytoplasmic and nuclear soluble survivin might be more relevant for prognostic factors for colorectal cancer than the total amount of survivin.

Role of nuclear transcription factor kappa B (NF-kappaB) for MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine)-induced apoptosis in nigral neurons of mice.

The biochemical and cellular changes that occur following treatment with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine) are remarkably similar to that seen in idiopathic Parkinson's disease. In this study, we investigated the time course changes of NF-kappaB (Nuclear factor kappa B) p65 protein and apoptosis in the substantia nigra after MPTP treatment in mice. Four administrations of MPTP at 2 h intervals showed a significant and severe decrease of the number of TH (tyrosine hydroxylase) immunopositive neurons in the substantia nigra of mice from 5 h up to 21 days posttreatment. Densities of DAT (dopamine transporter) immunoreactivity were also significantly decreased in nigral neurons of mice from 1 up to 21 days after MPTP treatment. GFAP (glial fibrillary acidic protein) immunopositive cells were increased significantly in the substantia nigra from 5 h up to 21 days after MPTP treatment. In contrast, isolectin B(4) positive microglia were increased markedly in the substantia nigra only 3 and 7 days after MPTP treatment. On the other hand, a significant increase of NF-kappaB p65 immunoreactivity was observed mainly in glial cells of the substantia nigra from 5 h to 3 days after MPTP treatment. A significant increase of ssDNA (single stranded DNA) immunopositive apoptotic neurons was also observed in the substantia nigra from 5 h to 3 days after MPTP treatment. These results demonstrate that dopaminergic neuronal loss may be caused by apoptosis due to increased cytokines and apoptosis-related proteins via the activation of NF-kappaB in reactive astrocytes of the substantia nigra after MPTP treatment in mice. Thus our findings suggest that the inhibition of NF-kappaB activation in astrocytes may be useful intervention in Parkinson's disease and other neurogenerative disorders where apoptosis or inflammation plays a key role in disease pathogenesis.

Formation of a stable triplex incorporating a CG interrupting site by a new WNA derivative containing 3-aminopyrazole as a nucleobase.

Triplex-forming oligonucleotides (TFOs) bind within the major groove of duplex DNA in a sequence-specific manner, and have attracted much interest as genomic tools. However, as the triplex DNA is formed by the interaction between the TFOs and homopurine/homopyrimidine sequences of the target duplex DNA, the stable triplex formation is prevented by one pyrimidine base in the homopurine strand. Previously, we developed the nucleoside analogues (WNA: W-shaped nucleoside analogues) that furnish an aromatic ring as a stacking part and a nucleobase as a recognition part onto the bicyclic skeleton. Selective recognition of a TA and a CG interrupting site has been achieved by WNA-beta T and WNA-beta C, respectively. In the subsequent study, it was found that the triplex formation by the WNA analogues depend on its neighbouring bases within the TFO. In this paper, we describe the synthesis and the evaluation of the triplex forming ability of WNA-beta 3AP, having 3-aminopyrazole (3AP) as a nucleobase. It is remarkable that the TFO containing the WNA-beta 3AP recognizes the CG interrupting site with high selectivity in the TFO sequence of 3'-GZG-5', in which the previous WNA-beta C did not show the stabilizing effect.

Comparative pharmacological study of free radical scavenger, nitric oxide synthase inhibitor, nitric oxide synthase activator and cyclooxygenase inhibitor against MPTP neurotoxicity in mice.

The biochemical and cellular changes that occur following the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are remarkably similar to that seen in idiopathic Parkinson's disease(PD). There is growing evidence indicating that reactive oxygen species (ROS), reactive nitrogen species (RNS) and inflammation are a major contributor to the pathogenesis and progression of PD. Hence, we investigated whether 7-nitroindazole [neuronal nitric oxide synthase (nNOS) inhibitor], edaravone (free radical scavenger), minocycline [inducible NOS (iNOS) inhibitor], fluvastatin [endothelial NOS (eNOS) activator], pitavastatin (eNOS activator), etodolac [cyclooxygenase-2 (COX-2) inhibitor] and indomethacin (COX inhibitor) can protect against MPTP neurotoxicity in mice under the same conditions. For the evaluation of each drug, the levels of dopamine, DOPAC and HVA were quantified using HPLC with an electrochemical detector. Four administrations of MPTP at 1-h intervals to mice produced marked depletion of dopamine, DOPAC (3,4-dihydroxyphenylacetic acid) and HVA (homovanilic acid) in the striatum after 5 days. 7-Nitroindazole prevented dose-dependently a significant reduction in dopamine contents of the striatum 5 days after MPTP treatment. In contrast, edaravone, minocycline, fluvastatin, pitavastatin, etodolac and indomethacin did not show the neuroprotective effect on MPTP-induced striatal dopamine, DOPAC and HVA depletions after 5 days. The present study demonstrates that the overexpression of nNOS may play a major role in the neurotoxic processes of MPTP, as compared with the production of ROS, the overexpression of iNOS, the modulation of eNOS and the involvement of inflammatory response. Thus our pharmacological findings provide further information for progressive neurodegeneration of the nigrostriatal dopaminergic neuronal pathway.

Effective strand invasion ODN incorporating a new bicyclic nucleoside analogue (WNA).

Efficient and specific targeting of DNA sequences by synthetic ligands is a major goal in chemical biology. Triplex-forming oligonucleotides (TFOs) are sequence-specific DNA-binding agents, but are limited to homopurine/homopyrimidine target sequences. We have previously reported two useful analogues (WNA: W-shaped nucleoside analogues), WNA-bT and WNAbC, which recognize a TA and a CG interrupting site forming triplexes with high stability and selectivity, respectively. However, their ability to form triplexes depended on their neighbouring bases in the TFO. Subsequent studies have shown that the sequence-dependency of the WNA analogues, for the formation of triplexes, has been partially solved by use of a WNA analogue bearing a substituted aromatic ring. Investigations into the effects of the substituted aromatic ring of WNA derivatives on the stability of triplexes led to the discovery of strand invasion by the TFO incorporating the new WNA analogue to form a highly stable duplex.