Neuroprotection from Excitotoxic Injury by Local Administration of Lipid Emulsion into the Brain of Rats.

Lipid emulsion was recently shown to attenuate cell death caused by excitotoxic conditions in the heart. There are key similarities between neurons and cardiomyocytes, such as excitability and conductibility, which yield vulnerability to excitotoxic conditions. However, systematic investigations on the protective effects of lipid emulsion in the central nervous system are still lacking. This study aimed to determine the neuroprotective effects of lipid emulsion in an in vivo rat model of kainic acid-induced excitotoxicity through intrahippocampal microinjections. Kainic acid and/or lipid emulsion-injected rats were subjected to the passive avoidance test and elevated plus maze for behavioral assessment. Rats were sacrificed at 24 h and 72 h after kainic acid injections for molecular study, including immunoblotting and qPCR. Brains were also cryosectioned for morphological analysis through cresyl violet staining and Fluorojade-C staining. Anxiety and memory functions were significantly preserved in 1% lipid emulsion-treated rats. Lipid emulsion was dose-dependent on the protein expression of ß-catenin and the phosphorylation of GSK3-ß and Akt. Wnt1 mRNA expression was elevated in lipid emulsion-treated rats compared to the vehicle. Neurodegeneration was significantly reduced mainly in the CA1 region with increased cell survival. Our results suggest that lipid emulsion has neuroprotective effects against excitotoxic conditions in the brain and may provide new insight into its potential therapeutic utility.

Analgesic effects of FAAH inhibitor in the insular cortex of nerve-injured rats.

The insular cortex is an important region of brain involved in the processing of pain and emotion. Recent studies indicate that lesions in the insular cortex induce pain asymbolia and reverse neuropathic pain. Endogenous cannabinoids (endocannabinoids), which have been shown to attenuate pain, are simultaneously degraded by fatty acid amide hydrolase (FAAH) that halts the mechanisms of action. Selective inhibitor URB597 suppresses FAAH activity by conserving endocannabinoids, which reduces pain. The present study examined the analgesic effects of URB597 treatment in the insular cortex of an animal model of neuropathic pain. Under pentobarbital anesthesia, male Sprague-Dawley rats were subjected to nerve injury and cannula implantation. On postoperative day 14, rodents received microinjection of URB597 into the insular cortex. In order to verify the analgesic mechanisms of URB597, cannabinoid 1 receptor (CB1R) antagonist AM251, peroxisome proliferator-activated receptor alpha (PPAR alpha) antagonist GW6471, and transient receptor potential vanilloid 1 (TRPV1) antagonist Iodoresiniferatoxin (I-RTX) were microinjected 15 min prior to URB597 injection. Changes in mechanical allodynia were measured using the von-Frey test. Expressions of CB1R, N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), and TRPV1 significantly increased in the neuropathic pain group compared to the sham-operated control group. Mechanical threshold and expression of NAPE-PLD significantly increased in groups treated with 2 nM and 4 nM URB597 compared with the vehicle-injected group. Blockages of CB1R and PPAR alpha diminished the analgesic effects of URB597. Inhibition of TRPV1 did not effectively reduce the effects of URB597 but attenuated expression of NAPE-PLD compared with the URB597-injected group. In addition, optical imaging demonstrated that neuronal activity of the insular cortex was reduced following URB597 treatment. Our results suggest that microinjection of FAAH inhibitor into the insular cortex causes analgesic effects by decreasing neural excitability and increasing signals related to the endogenous cannabinoid pathway in the insular cortex.

Design, Synthesis, Photophysical Properties, Biological Estimation and Molecular Docking Studies of Novel Schiff Base Derivatives as Potential Urease Inhibitors.

A quinoline functionalized two novel fluorescent Schiff bases, N-(quinolin-2-ylmethylene) anthracen-1-amine (SB1) and 2-(quinolin-2-ylmethyleneamino) benzene thiol (SB2) were synthesized and confirmed by using 1H NMR, IR and GC-MS techniques. The spectroscopic properties were examined by absorption spectroscopy and fluorescence spectroscopy. The absorption and fluorescence spectra of the probes (SB1 and SB2) were measured in a variety of solvents. Both the compounds were tested for urease inhibitory activity. The synthesized compound SB2 proved to be the most effective screening for enzyme inhibitory activity with IC50 = 0.111 µM than SB1 (IC50 = 0.287 µM). Molecular docking studies were performed to delineate the binding affinity and conformational positions of chemical compounds within the active region of the target protein. In-vitro analysis depicts the potency of SB1 in free radical scavenging as compared to the reference drug vitamin C.

Synthesis and Studies of Fluorescein Based Derivatives for their Optical Properties, Urease Inhibition and Molecular Docking.

Herein, we design and synthesized new fluorescein based derivatives by insitu formation of fluorescein ester and further treated with corresponding hydrazide and amine to yield respective compounds i.e. FB1, FB2, FB3 and FB4. The spectral purity and characterization was done by using IR, NMR and Mass spectroscopies. The synthesized derivatives were examined for their photophysical properties by using variety of organic solvents and results were discussed in details. The structural diversity of synthesized compounds motivate us to evaluate these compounds for urease inhibition. The compound FB3 (IC50 = 0.0456 µM) shows 100 fold more active against Jack bean urease than standard drug thiourea (IC50 = 4.7455 µM). Other synthesized compounds showed potent activity. Free radical percentage scavenging assay further supported the capacity of compounds to urease inhibition. While, molecular docking simulations helps to examine the molecular interactions of active compounds FB1, FB2, FB3 and FB4 within the binding site of urease enzyme.

Synthesis, Photophysical Properties and Application of New Porphyrin Derivatives for Use in Photodynamic Therapy and Cell Imaging.

New derivatives of tetrakis(4-carboxyphenyl) porphyrin were designed, synthesized and characterized by IR, proton NMR and mass spectroscopy. The ground and excited state nature of new derivatives were examined using UV-Vis. absorption and fluorescence spectroscopy, fluorescence quantum yield and fluorescence lifetime studies. The singlet oxygen quantum yield of each synthesized derivative of porphyrin was estimated for their further efficacy as potential photosensitizer in biological studies. The significant photophysical data of all synthesized derivatives was supplementary accessed to examine the cell imaging and cytotoxicity against two cancer cell lines viz. MBA-MD-231 and A375. The fluorescence lifetime, fluorescence quantum yield and efficiency of singlet oxygen generation suggests alkyl amine and alkyl hydrazide linked new porphyrin photosensitizers can be useful for PDT agent in cancer treatment.

Plasticity-Related PKMζ Signaling in the Insular Cortex Is Involved in the Modulation of Neuropathic Pain after Nerve Injury.

The insular cortex (IC) is associated with important functions linked with pain and emotions. According to recent reports, neural plasticity in the brain including the IC can be induced by nerve injury and may contribute to chronic pain. Continuous active kinase, protein kinase Mζ (PKMζ), has been known to maintain the long-term potentiation. This study was conducted to determine the role of PKMζ in the IC, which may be involved in the modulation of neuropathic pain. Mechanical allodynia test and immunohistochemistry (IHC) of zif268, an activity-dependent transcription factor required for neuronal plasticity, were performed after nerve injury. After ζ-pseudosubstrate inhibitory peptide (ZIP, a selective inhibitor of PKMζ) injection, mechanical allodynia test and immunoblotting of PKMζ, phospho-PKMζ (p-PKMζ), and GluR1 and GluR2 were observed. IHC demonstrated that zif268 expression significantly increased in the IC after nerve injury. Mechanical allodynia was significantly decreased by ZIP microinjection into the IC. The analgesic effect lasted for 12 hours. Moreover, the levels of GluR1, GluR2, and p-PKMζ were decreased after ZIP microinjection. These results suggest that peripheral nerve injury induces neural plasticity related to PKMζ and that ZIP has potential applications for relieving chronic pain.

Molecular epidemiology of norovirus infections in children with acute gastroenteritis in South Korea in November 2005 through November 2006.

Norovirus infections were detected in 114 of 762 children with acute gastroenteritis in South Korea from November 2005 to November 2006. Seasonality peaks in December, March, and October were also assessed in this study. We identified seven noroviral genotypes (GI-6, GII-2, GII-3, GII-4, GII-5, GII-6, and GII-8) and a C1-120 strain showing low identity (79.3%) with GII-13 and GII-17.

Selective detection of Co2+ by fluorescent nano probe: Diagnostic approach for analysis of environmental samples and biological activities.

Nowadays scientist over the world are engaging to put forth improved methods to detect metal ion in an aqueous medium based on fluorescence studies. A simple, selective and sensitive method was proposed for detection of Co2+ ion using fluorescent organic nanoparticles. We synthesized a fluorescent small molecule viz. 4,4'-{benzene-1,4-diylbis-[(Z)methylylidenenitrilo]}dibenzoic acid (BMBA) to explore its suitability as sensor for Co2+ ion and biocompatibility in form of nanoparticles. Fluorescence nanoparticles (BMBANPs) prepared by simple reprecipitation method. Aggregation induced enhanced emission of BMBANPs exhibits the narrower particle size of 68nm and sphere shape morphology. The selective fluorescence quenching was observed by addition of Co2+ and does not affected by presence of other coexisting ion solutions. The photo-physical properties, viz. UV-absorption, fluorescence emission, and lifetime measurements are in support of ligand-metal interaction followed by static fluorescence quenching phenomenon in emission of BMBANPs. Finally, we develop a simple analytical method for selective and sensitive determination of Co2+ ion in environmental samples. The cell culture E. coli, Bacillus sps., and M. tuberculosis H37RV strain in the vicinity of BMBANPs indicates virtuous anti-bacterial and anti-tuberculosis activity which is of additional novel application shown by prepared nanoparticles.

Facile Synthesis, Crystal Structure, DFT Calculation and Biological Activities of 4-(2-fluorophenyl)-3-(3-methoxybenzyl)-1H-1,2,4-triazol-5 (4H)-one (5).

BACKGROUND: In the past few decades, design, synthesis, and characterization of novel heterocyclic compounds with auspicious biological profile received the considerable attention of the scientific community. Among them, the small and simple organic molecular backbone like triazole moiety have a broad spectrum of applications in the medicinal as well as diagnostic areas. OBJECTIVE: The objective of present study was synthesis, characterization, and exploration of biological profile of 4-(2-fluorophenyl)-3-(3-methoxybenzyl)-1H-1,2,4-triazole-5(4H)-one (5). The tautomeric interconversion of the molecule was observed by the single crystal XRD and DFT analysis. METHODS: N-(2-fluorophenyl)-2-[2-(3-methoxyphenyl)acetyl]hydrazine carboxamide (4) was synthesized by the condensation of 2-(3-methoxyphenyl)acetohydrazide (3) with 1-fluoro-2- isocyanatobenzene. The dehydrocyclization of compound (4) yielded target compound (5) by refluxing in 2 N aqueous sodium hydroxide solutions. The target molecule was characterized by FTIR, 1H NMR, 13C NMR, single crystal X-ray diffraction analysis and DFT calculation. The enzymatic assay measurements were carried out by using a microplate reader (OPTI Max, Tunable Microplate Reader; Wavelength range: 340-850 nm; for 96-well plates) while DFT calculation was performed by Gaussian 09 package. RESULTS: The XRD result and DFT calculations showed that molecule 5 predominantly exists in thione conformation and crystallized in the triclinic system of P-1 space group. Furthermore, for the practical applicability of synthesized compound 5, the in vitro acetylcholinesterase as well as α-glucosidase inhibition activities were performed and found moderate enzyme inhibition potential comparable with that of reference inhibitors. CONCLUSION: This study might be helpful for future design and development of potent enzyme inhibitor to control Alzheimer's as well as diabetic disease. The DFT and single crystal XRD analysis data might be helpful for understanding the mechanism of drug binding and its mode of action.

Inhibition of Mammalian Target of Rapamycin (mTOR) Signaling in the Insular Cortex Alleviates Neuropathic Pain after Peripheral Nerve Injury.

Injury of peripheral nerves can trigger neuropathic pain, producing allodynia and hyperalgesia via peripheral and central sensitization. Recent studies have focused on the role of the insular cortex (IC) in neuropathic pain. Because the IC is thought to store pain-related memories, translational regulation in this structure may reveal novel targets for controlling chronic pain. Signaling via mammalian target of rapamycin (mTOR), which is known to control mRNA translation and influence synaptic plasticity, has been studied at the spinal level in neuropathic pain, but its role in the IC under these conditions remains elusive. Therefore, this study was conducted to determine the role of mTOR signaling in neuropathic pain and to assess the potential therapeutic effects of rapamycin, an inhibitor of mTORC1, in the IC of rats with neuropathic pain. Mechanical allodynia was assessed in adult male Sprague-Dawley rats after neuropathic surgery and following microinjections of rapamycin into the IC on postoperative days (PODs) 3 and 7. Optical recording was conducted to observe the neural responses of the IC to peripheral stimulation. Rapamycin reduced mechanical allodynia and downregulated the expression of postsynaptic density protein 95 (PSD95), decreased neural excitability in the IC, thereby inhibiting neuropathic pain-induced synaptic plasticity. These findings suggest that mTOR signaling in the IC may be a critical molecular mechanism modulating neuropathic pain.

In vivo voltage-sensitive dye imaging of the insular cortex in nerve-injured rats.

The insular cortex (IC) is a pain-related brain region that receives various types of sensory input and processes the emotional aspects of pain. The present study was conducted to investigate spatiotemporal patterns related to neuroplastic changes in the IC after nerve injury using voltage-sensitive dye imaging. The tibial and sural nerves of rats were injured under pentobarbital anesthesia. To observe optical signals in the IC, rats were re-anesthetized with urethane 7days after injury, and a craniectomy was performed to allow for optical imaging. Optical signals of the IC were elicited by peripheral electrical stimulation. Neuropathic rats showed a significantly higher optical intensity following 5.0mA electrical stimulation compared to sham-injured rats. A larger area of activation was observed by 1.25 and 2.5mA electrical stimulation compared to sham-injured rats. The activated areas tended to be larger, and the peak amplitudes of optical signals increased with increasing stimulation intensity in both groups. These results suggest that the elevated responsiveness of the IC to peripheral stimulation is related to neuropathic pain, and that neuroplastic changes are likely to be involved in the IC after nerve injury.

Use of rpoB sequences for phylogenetic study of Mycoplasma species.

rpoB sequences encoding the beta-subunit of RNA polymerase were determined in 26 Mycoplasma species for phylogenetic study. The portion of rpoB DNA used in this study showed a high degree of variation in terms of size and sequence among species. The rpoB phylogenies inferred from amino acid and nucleotide sequences were used to divide the mycoplasmas into two groups, a 'pneumoniae group' and a 'hominis group', which was consistent with the result from 16S rDNA sequence analysis. However, phylogenetic relationships within these groups differed in the two gene trees, which were supported by the incongruence length difference (ILD) test. This indicates that multiple gene sequences must be applied to infer accurate phylogenetic relationships among the mycoplasmas. The rpoB sequence, and especially the deduced amino acid sequence, offers a good alternative marker.

Detection and identification of Legionella pneumophila by PCR-restriction fragment length polymorphism analysis of the RNA polymerase gene (rpoB).

The partial RNA polymerase beta-subunit coding gene (rpoB) sequences of 38 Legionella species (59 reference strains) were used to select both Legionella genus-specific and Legionella pneumophila species-specific primers to amplify the 347-bp and 217-bp DNAs, respectively. Enzyme restriction sites for PCR-restriction fragment length polymorphism (PCR-RFLP) analysis were also generated by a computer program. Thirty-eight Legionella species were well differentiated by the identification scheme for Legionella genus-specific PCR-RFLP using HaeIII, AluI, CfoI, PstI, and MaeII. The most common and important pathogenic species, L. pneumophila, was differentiated into two subspecies (L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri) by both Legionella genus-specific PCR-RFLP and L. pneumophila species-specific PCR-RFLP using BamHI. Eighty-two Korean culture isolates could also be easily identified by both PCR-RFLP methods as 68 strains of L. pneumophila subsp. pneumophila, 11 strains of L. pneumophila subsp. fraseri, and three novel strains that were separately confirmed by 16S rDNA and rpoB sequence analysis. These results suggest that the rpoB PCR-RFLP for Legionella is a simple and convenient method, not only for specific detection, but also for the rapid identification of Legionella species.

Identification of Mycobacterium tuberculosis by PCR-linked reverse hybridization using specific rpoB oligonucleotide probes.

A reverse probe hybridization method using two different Mycobacterium tuberculosis-specific rpoB DNA probes in combination was evaluated for the identification of M. tuberculosis culture isolates. Among the 384 isolates tested, 354 strains were identified as M. tuberculosis, which included 37 rifampin-resistant strains, and 30 were nontuberculous mycobacteria (NTM). This result was in accord with partial rpoB sequence analysis and IS6110 polymerase chain reaction (PCR) results, but not with the results of biochemical testing, which produced two false negative results. Because of its high level of sensitivity and specificity, we suggest that M. tuberculosis-specific rpoB probes immobilized on micro-titer well plates or on other solid matrixes can be used efficiently for the rapid and convenient identification of M. tuberculosis.

Inhibition of herpesvirus-6B RNA replication by short interference RNAs.

RNA interference (RNAi) is a process of sequence-specific gene silencing, which is initiated by double-stranded RNA (dsRNA). RNAi may also serve as an antiviral system in vertebrates. This study describes the inhibition of herpesvirus-6B (HHV-6B) replication by short interference RNAs (siRNAs) that are targeted to the U38 sequence that encodes DNA polymerase. When virus-infected SupT1 cells were treated by siRNA, these cells blocked the cytopathic effect (CPE) and detected the HHV-6B antibody-negative in indirect immunofluorescence assays (IFA). Our result suggests that RNAi can efficiently block Herpesvirus-6B replication.

Characteristics of HIV-Tat protein transduction domain.

The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD), which contains rich arginine and lysine residues, is responsible for the highly efficient transduction of protein through the plasma membrane. In addition, it can be secreted from infected cells and has the ability to enter neighboring cells. When the PTD of Tat is fused to proteins and exogenously added to cells, the fusion protein can cross plasma membranes. Recent reports indicate that the endogenously expressed Tat fusion protein can demonstrate biodistribution of several proteins. However, intercellular transport and protein transduction have not been observed in some studies. Therefore, this study examined the intercellular transport and protein transduction of the Tat protein. The results showed no evidence of intercellular transport (biodistribution) in a cell culture. Instead, the Tat fusion peptides were found to have a significant effect on the transduction and intercellular localization properties. This suggests that the HIV-1 PTD passes through the plasma membrane in one direction.

PCR-linked reverse DNA hybridization using oligonucleotide-specific probes of rpoB for identification of Mycobacterium avium and Mycobacterium intracellulare.

A PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes (Avp and Intp) of Mycobacterium avium and Mycobacterium intracellulare, respectively, were evaluated for the differentiation and identification of M. avium and M. intracellulare culture isolates. Among the 504 culture isolates tested by this method, 48 strains showed positive results for M. avium and 60 strains showed positive results for M. intracellulare. The other 396 culture isolates showed negative results for both M. avium and M. intracellulare. These results were consistent with those obtained from partial rpoB (306 bp) sequence analysis and biochemical tests. The negative strains obtained by this DNA hybridization method were identified as M. tuberculosis (366 strains), M. peregrinum (11 strains), M. abscessus (9 strains), M. fortuitum (8 strains), and M. flavescens (2 strains) by rpoB DNA sequence analysis. Due to the high sensitive and specific result obtained by this assay, we suggest that this PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes of M. avium and M. intracellulare would be used for the rapid and precise method for differentiation and identification of M. avium and M. intracellulare.

Anti-HIV-1 efficacy of extracts from medicinal plants.

The anti-HIV-1 activities of butanol, hexane, chloroform and water extracts from four widely used folk medicinal plants (Sophora flavescens, Tulipa edulis, Herba ephedra, and Pachyma hoelen Rumph) were evaluated in this study. The hexane extract of Pachyma hoelen Rumph, PH-4, showed effective inhibition against HIV-1. The 50% effective concentration (EC(50)) of PH-4 was 37.3 microg/ml in the p24 antigen assay and 36.8% in the HIV-1 recombinant RT activity test (at 200 microg/ml). In addition, the PH-4 showed the protective effect on the infected MT-4 cells, with a 58.2% rate of protection. The 50% cytotoxic concentration (CC(50)) of PH-4 was 100.6 microg/ml. These results suggest that PH-4 from Pachyma hoelen Rumph might be the candidate for the chemotherapy agent against HIV-1 infection with further study.

Characterization of a G11,P[4] strain of human rotavirus isolated in South Korea.

A novel human rotavirus strain, CUK-1, containing a G11 type combined with a P[4] type was isolated from a 1-year-old female patient with fever and severe diarrhea at Our Lady of Mercy Hospital in Incheon, South Korea. This CUK-1 strain showed the highest degree of nucleic acid similarity (98.7% and 93%) to G11 Dhaka6 and P[4] RV 5, respectively. This novel combined type of CUK-1 rotavirus strain (G11,P[4]) was uncovered from humans and is reported on here for the first time.

Inducible vesicular stomatitis virus (VSV) L cell line for packaging of recombinant VSV.

Recently, recombinant vesicular stomatitis viruses (VSV) have been developed as high-level expression vectors which serve as effective vaccine vectors in animals. An ideal approach for VSV vector production would be the development of stable packaging cell lines that can produce vector particles without transfection step. In this report, we describe generation of an inducible cell line that expresses the VSV polymerase gene (L) under the control of the reverse tetracycline-controlled transactivator (rtTA) system as a first step to make VSV-based packaging cell lines. Integrated polymerase (L) gene was controlled by an rtetR-dependent promoter in the rtTA-producing BHK cell line. When the cell lines were cultured in the presence of tet (tetracycline) or tetracycline derivative doxycycline, the recombinant VSV and wild type VSV were replicated, whereas in the absence of tet or tetracycline derivative doxycycline, the recombinant VSV was not replicated. Viral supernatants were harvested, diluted, and monitored by plaque assay for the presence of infectious VSV. Plaques of VSV containing an additional sequence encoding the EGFP protein allowed rapid detection of infection. Our results suggest wide applications of other surrogate viruses based on VSV. The availability of stable packaging cell lines represents a step toward the use of a VSV vector delivery system that can allow scale-up production of vector-stocks for gene therapy.