Wafer-scale nanowell array patterning based electrochemical impedimetric immunosensor

We have reported that nanowell array (NWA) can enhance electrochemical detection of molecular binding events by controlling the binding sites of the captured molecules. Using NWA biosensor based amperometric analysis, we have detected biological macromolecules such as DNA, protein or aptamers at low concentrations. In this research, we developed an impedimetric immunosensor based on wafer-scale NWA for electrochemical detection of stress-induced-phosphoprotein-1 (STIP-1). In order to develop NWA sensor through the cost-effective combination of high-throughput nanopattern, the NWA electrode was fabricated on Si wafer by krypton-fluoride (KrF) stepper semiconductor process. Finally, 12,500,000 ea nanowell with a 500 nm diameter was fabricated on 4 mm × 2 mm substrate. Next, by using these electrodes, we measured impedance to quantify antigen binding to the immunoaffinity layer. The limit of detection (LOD) of the NWA was improved about 100-fold compared to milli-sized electrodes (4 mm × 2 mm) without an NWA. These results suggest that wafer-scale NWA immunosensor will be useful for biosensing applications because their interface response is appropriate for detecting molecular binding events.     

Endothelial Transient Receptor Potential Conical Channel (TRPC)-3 Activation Induces Vasogenic Edema Formation in the Rat Piriform Cortex Following Status Epilepticus

Transient receptor potential canonical channel (TRPC) is a nonselective cation channel permeable to Ca²⁺, which express in many cell types, including neurons. However the alterations in TRPC receptor expressions in response to status epilepticus (SE) have not been explored. Therefore, the present study was designated to elucidate the roles of TRPC3 in neuronal death and vasogenic edema within the rat piriform cortex (PC) following SE. In non-SE animals, TRPC3 immunoreactivity was abundantly detected in the PC. Following SE, TRPC3 immunoreactivity was increased in neurons. Furthermore, TRPC3 expression was detected in endothelial cells that did not contain it in non-SE animals. Loss of SMI-71 (a blood–brain barrier antigen) immunoreactivity was also observed in TRPC3 positive endothelial cells. In addition, FJB positive neurons and vasogenic edema were noticeably detected in the PC. To directly determine whether TRPC3 activation is correlated to SE-induced vasogenic edema formation and neuronal damages in the PC, the effect of Pyr-3 (a TRPC3 antagonist) on SE-induced insults were investigated. Pyr-3 infusion effectively attenuated vasogenic edema in the PC as compared to the vehicle. Therefore, our findings indicate that TRPC3 activation/overexpression induced by SE may involve BBB disruption and neuronal damages in the rat PC following SE. Therefore, the present study was TRPC3 may play an important role in SE-induced vasogenic edema formation through BBB disruptions in the rat PC.     

The Reverse Roles of Transient Receptor Potential Canonical Channel-3 and -6 in Neuronal Death Following Pilocarpine-Induced Status Epilepticus

Transient receptor potential canonical channel (TRPC) is a nonselective cation channel permeable to Ca²⁺, which is expressed in many cell types, including neurons. However, the alterations in TRPC receptor expressions in response to status epilepticus (SE) have not been explored. Therefore, the present study was designated to elucidate the roles of TRPC3 and TRPC6 in neuronal death following SE. In non-SE animals, TRPC3 and TRPC6 immunoreactivity was abundantly detected in the dendrites of pyramidal cells and the cell bodies of dentate granule cells. Following SE, TRPC3 expression was significantly elevated in CA1-, CA3 pyramidal cells, and dentate granule cells, while TRPC6 expression was reduced in these regions. Pyrazole-3 (a TRPC3 inhibitor) effectively prevented up-regulation of neuronal TRPC3 expression induced by SE. Hyperforin (a TRPC6 activator) effectively prevented down-regulation of neuronal TRPC6 expression induced by SE. In addition, both Pyr3 and hyperforin effectively protected neuronal damages from SE. Therefore, the present study yields novel information regarding the role of TRPC3 and 6 in epileptogenic insults and suggests that TRPC 3 and 6 may be involved in neurodegeneration following SE.     

Detection of Spiroplasma from the mite Macrocheles sp. (Acari; Macrochelidae) ectoparasitic to the fly Drosophila hydei (Diptera; Drosophilidae): a possible route of horizontal transmission?

Some members of the genus Spiroplasma are vertically transmitted endosymbionts of insects. Among them, Spiroplasma sp. Dhd, a member of the Spiroplasma poulsonii clade, is highly prevalent among worldwide populations of Drosophila hydei. Here we found that 53 out of 3,763 wild-caught D. hydei (1.4 %) were ectoparasitized by the mite that belong to the genus Macrocheles. Many of the ectoparasitized flies (79 %) had a single mite, but some flies had up to five mites. Among 59 mites subjected to Spiroplasma-specific PCR, 15 individuals were found to be positive. Infection status of Spiroplasma in flies and the associated mites were incongruent. Partial nucleotide sequences of the Spiroplasma P58 gene suggest that some of the mites are infected with a Spiroplasma, which is identical or closely related to Spiroplasma sp. Dhd. This finding provides a potential route of horizontal Spiroplasma transmission between D. hydei individuals in natural populations. In addition, a Spiroplasma strain that does not form a monophyletic group with S. poulsonii was also found from a mite individual.     

Effects of Ethanol Exposure During Early Pregnancy in Hyperactive, Inattentive and Impulsive Behaviors and MeCP2 Expression in Rodent Offspring

Prenatal exposure to alcohol has consistently been associated with adverse effects on neurodevelopment, which is collectively called fetal alcohol spectrum disorder (FASD). Increasing evidence suggest that prenatal exposure to alcohol increases the risk of developing attention deficit/hyperactivity disorder-like behavior in human. In this study, we investigated the behavioral effects of prenatal exposure to EtOH in offspring mice and rats focusing on hyperactivity and impulsivity. We also examined changes in dopamine transporter and MeCP2 expression, which may underlie as a key neurobiological and epigenetic determinant in FASD and hyperactive, inattentive and impulsive behaviors. Mouse or rat offspring born from dam exposed to alcohol during pregnancy (EtOH group) showed hyper locomotive activity, attention deficit and impulsivity. EtOH group also showed increased dopamine transporter and norepinephrine transporter level compared to control group in the prefrontal cortex and striatum. Prenatal exposure to EtOH also significantly decreased the expression of MeCP2 in both prefrontal cortex and striatum. These results suggest that prenatal exposure to EtOH induces hyperactive, inattentive and impulsive behaviors in rodent offspring that might be related to global epigenetic changes as well as aberration in catecholamine neurotransmitter transporter system.     

Effect of GABA Receptor Agonists or Antagonists Injected Spinally on the Blood Glucose Level in Mice

The possible roles of gamma-amino butyric acid (GABA) receptors located in the spinal cord for the regulation of the blood glucose level were studied in ICR mice. We found in the present study that intrathecal (i.t.) injection with baclofen (a GABA_B receptor agonist; 1–10 μg/5 μl) or bicuculline (a GABA_A receptor antagonist; 1–10 μg/5 μl) caused an elevation of the blood glucose level in a dose-dependent manner. The hyperglycemic effect induced by baclofen was more pronounced than that induced by bicuculline. However, muscimol (a GABA_A receptor agonist; 1–5 μg/5 μl) or phaclofen (a GABA_B receptor antagonist; 5–10 μg/5 μl) administered i.t. did not affect the blood glucose level. Baclofen–induced elevation of the blood glucose was dose-dependently attenuated by phaclofen. Furthermore, i.t. pretreatment with pertussis toxin (PTX; 0.05 or 0.1 μg/5 μl) for 6 days dose-dependently reduced the hyperglycemic effect induced by baclofen. Our results suggest that GABA_B receptors located in the spinal cord play important roles for the elevation of the blood glucose level. Spinally located PTX-sensitive G-proteins appear to be involved in hyperglycemic effect induced by baclofen. Furthermore, inactivation of GABA_A receptors located in the spinal cord appears to be responsible for tonic up-regulation of the blood glucose level.     

Identification of Aniline-assimilating Bacteria

Twenty bacterial strains, grown on aniline as a sole source of carbon and nitrogen, and isolated from soil, were identified by morphological and biochemical tests, and cell wall analyses. Eight isolates were Rhodococcus erythropolis (Gray and Thornton) Goodfellow and Alderson, five were Pseudomonas maltophilia Hugh and Ryschenkow and seven were DAB (diaminobutyric acid)-type coryneform bacteria. The Rhodococcus spp. maintained the ability to assimilate aniline, but the pseudomonads and coryneform bacteria readily lost the ability, when grown in the absence of aniline.     

Targeted delivery of a phosphopeptide prodrug inhibits the proliferation of a human glioma cell line

Peptides are ideal candidates for developing therapeutics. Polo-like kinase 1 is an important regulatory protein in the cell cycle and contains a C-terminal polo-box domain, which is the hallmark of this protein family. We developed a peptide inhibitor of polo-like kinase 1 that targets its polo-box domain. This new phosphopeptide, cRGDyK-S-S-CPLHSpT, preferentially penetrates the cancer cell membrane mediated by the integrin receptor, which is expressed at high levels by cancer cells. In the present study, using high performance liquid chromatography and mass spectroscopy, we determined the stability of cRGDyK-S-S-CPLHSpT and its cleavage by glutathione under typical conditions for cell culture. We further assessed the ability of the peptide to inhibit the proliferation of the U87MG glioma cell line. The phosphorylated peptide was stable, and the disulfide bond of cRGDyK-S-S-CPLHSpT was cleaved in 50 mM glutathione. This peptide inhibited the growth of cancer cells and changed their morphology. Therefore, we conclude that the phosphopeptide shows promise as a prodrug and has a high potential to act as an anticancer agent by inhibiting polo-like kinase 1 by binding its polo-box domain. These findings indicate the therapeutic potential of PLHSpT and peptides similarly targeted to surface receptors of cancer cells and to the functional domains of regulatory proteins.