Separation of enantiomers of chiral fullerene derivatives through enantioselective encapsulation within an adaptable helical cavity of syndiotactic poly(methyl methacrylate) with helicity memory.

Optically active left (M)- and right (P)-handed helical syndiotactic poly(methyl methacrylate)s (M- and P-st-PMMAs) with a helicity memory enantioselectively encapsulated the racemic C60 derivatives, such as 3,4-fulleroproline tert-butyl ester (rac-1) and tetraallylated C60 (rac-2), as well as the C60-bound racemic 310-helical peptides (rac-3) within their helical cavities to form peapod-like inclusion complexes and a unique "helix-in-helix" superstructure, respectively. The enantiomeric excess (ee) and separation factor (enantioselectivity) (α) of the analyte 1 (ee = 23%-25% and α = 2.35-2.50) encapsulated within the helical cavities of the M- and P-st-PMMAs were higher than those of the analytes 2 and 3 (ee = 4.3%-6.0% and α = 1.28-1.50). The optically pure (S)- and (R)-1 were found to more efficiently induce an excess one-handed helical conformation in the st-PMMA backbone than the optically pure (S)- and (R)-1-phenylethylamine, resulting in intense mirror-image vibrational circular dichroism (VCD) spectra in the PMMA IR regions. The excess one-handed helices induced in the st-PMMAs complexed with (S)- and (R)-1 were memorized after replacement with the achiral C60, and the complexes exhibited induced electric CDs in the achiral C60 chromophore regions.

Precise size separation of water-soluble red-to-near-infrared-luminescent silicon quantum dots by gel electrophoresis.

Gel electrophoresis, which is a standard method for separation and analysis of macromolecules such as DNA, RNA and proteins, is applied for the first time to silicon (Si) quantum dots (QDs) for size separation. In the Si QDs studied, boron (B) and phosphorus (P) are simultaneously doped. Codoping induces a negative potential on the surface of a Si QD and makes it dispersible in water. Si QDs with different B and P concentrations and grown at different temperatures (950 °C-1200 °C) are studied. It is shown that native polyacrylamide gel electrophoresis can separate codoped Si QDs by size. The capability of gel electrophoresis to immobilize size-separated QDs in a solid matrix makes detailed analyses of size-purified Si QDs possible. For example, the photoluminescence (PL) studies of the dried gel of Si QDs grown at 1100 °C demonstrate that a PL spectrum of a Si QD solution with the PL maximum around 1.4 eV can be separated into more than 15 spectra with the PL maximum changing from 1.2 to 1.8 eV depending on the migration distance. It is found that the relationship between the PL peak energy and the migration distance depends on the growth temperature of Si QDs as well as the B and P concentration. For all the samples with different impurity concentrations and grown at different temperatures, a clear trend is observed in the relationship between the full width at half maximum (FWHM) and the peak energy of the PL spectra in a wide energy range. The FWHM increases with the increasing peak energy and it is nearly twice larger than those observed for undoped Si QDs. The large PL FWHM of codoped Si QDs suggests that excitons are further localized in codoped Si QDs due to the existence of charged impurities.

Clinical significance of anti-DNA/N-methyl-D-aspartate receptor 2 antibodies in de novo and post-steroid cases with neuropsychiatric systemic lupus erythematosus.

BACKGROUND: Anti-DNA/N-methyl-D-aspartate receptor 2 (NR2) antibodies (anti-DNA/NR2 antibodies) are a subset of anti-DNA autoantibodies that cross-react with the extracellular domain of the GluN2A/GluN2B subunits of NR2. These antibodies induce apoptosis of hippocampus neurons and psychiatric disorder in mice and humans. Neuropsychiatric system lupus erythematosus (NPSLE) can develop after initiation of corticosteroids (post-steroid neuropsychiatric manifestation: PSNP) or before treatment (de novo NPSLE); however, pathophysiological differences between these subtypes remain unclear. The objective of this study was to clarify the prevalence of anti-DNA/NR2 antibodies in patients with NPSLE. METHODS: This study involved a cohort of patients with NPSLE admitted to our hospital. NPSLE patients were classified into two groups, de novo NPSLE and PSNP-SLE. Serum anti-DNA antibodies and anti-DNA/NR2 antibodies were measured by enzyme-linked immunosorbent assays. RESULTS: Serum samples were obtained from 24 patients with de novo NPSLE, 25 with PSNP-SLE and 76 healthy controls (HC). The level of anti-DNA/NR2 antibodies in patients with de novo NPSLE and PSNP-SLE were also higher than those in HC. Positive correlation between anti-DNA antibodies and anti-DNA/NR2 antibodies were found in PSNP-SLE, but was not significant in de novo NPSLE. CONCLUSION: The levels of anti-DNA/NR2 antibodies in PSNP-SLE were similar to those in de novo NPSLE. Anti-DNA/NR2 antibodies in PSNP-SLE were suggested as a dominant subset of anti-DNA antibodies, indicating that anti-DNA/NR2 antibodies may be a predictive factor in PSNP-SLE.

Selegiline ameliorates depression-like behaviors in rodents and modulates hippocampal dopaminergic transmission and synaptic plasticity.

Selegiline, an irreversible inhibitor of monoamine oxidase (MAO)-type B, is widely prescribed for Parkinson's disease and, at higher doses, for major and atypical depression, whereby it is non-selectively inhibitory to both MAO-A and MAO-B activities. MAO inhibitors have been considered to function as antidepressants through MAO-A inhibition. We have previously reported that selegiline exerts antidepressant-like effects in the mouse forced swim test (FST) via dopamine D1 receptor activation. Our objective was to elucidate the mechanisms underlying the antidepressant-like effects of selegiline. We also tested another propargylamine MAO-B inhibitor, rasagiline. Triple subcutaneous injection (at 24, 5, and 1 h prior to behavioral testing) with selegiline (10 mg/kg/injection), but not rasagiline (1, 3, or 10 mg/kg/injection), reduced the immobility time in the mouse FST and rat tail suspension test. In the hippocampus and prefrontal cortex of mice subjected to the FST, selegiline and rasagiline completely inhibited MAO-B activities. However, selegiline suppressed MAO-A activities and monoamine turnover rates at a lesser degree than rasagiline at the same doses, indicating that the antidepressant-like effects of selegiline are independent of MAO-A inhibition. Moreover, selegiline, but not rasagiline, increased the hippocampal dopamine content. A single subcutaneous administration of 10 mg/kg selegiline, but not of rasagiline, significantly prevented hippocampal CA1 long-term potentiation impairment, induced by low-frequency stimulation prior to high-frequency stimulation in rats. These results suggest that the antidepressant-like effects of selegiline are attributable to enhancement of dopaminergic transmission and prevention of the impairment of synaptic plasticity in the hippocampus.

Determination of Serum 25-Hydroxyvitamin D3 by LC/MS/MS and Its Monthly Variation in Sapporo Indoor Workers.

25-Hydroxyvitamin D3 (25(OH)D3) as the metabolite of vitamin D, is connected with various of diseases, and important to people with limited sunshine. Thus, the investigation of serum 25-hydroxyvitamin D and its variation in these people is necessary. In this study, a simple, precise, and accurate method for serum 25(OH)D3 determination by LC/MS/MS was developed. Serum samples were obtained monthly for one year from 11 male and 11 female indoor workers in Sapporo, Japan, and the overall 25(OH)D3 concentration was 12.9 ± 4.7 ng/mL. The 25(OH)D3 in females was significantly lower than that in males (14.0 ± 5.0 vs. 11.9 ± 4.3 ng/mL). The serum 25(OH)D3 concentration in males and females were both strongly correlated to UV-B radiation (r2 = 0.8477 and 0.7384, respectively), with a two-month's lag. Also the monthly change in 25(OH)D3 in males was more significant than that in females.

The effect of DN (dominant-negative) Ku70 and reoxygenation on hypoxia cell-kill: evidence of hypoxia-induced potentially lethal damage.

PURPOSE: To study the effect of DN (dominant-negative) Ku70 and reoxygenation on the hypoxia-induced cell-kill. MATERIALS AND METHODS: Cell lines were human colorectal carcinoma HCT8 and HT29 cells and their respective derivatives, v-HCT8 and v-HT29 infected with DNKu70-containing adenovirus. Cells were plated in glass tubes and made hypoxic by flushing N(2) gas containing 0, 0.1 or 0.5% O(2). Cell survival was determined by colony formation assay immediately after 0-96 h hypoxia. To reoxygenate medium were replaced fresh following 48 or 72 h in hypoxia and cells were incubated in aerobic environment for 2-24 h before survival assay. RESULTS: When incubated in hypoxia, cells lost reproductive capability ∼ exponentially as a function of time in hypoxia, and depending on the O(2) concentration. DNKu70 rendered cells more prone to hypoxia-induced cell-kill. Following reoxygenation cell survival increased rapidly but without detectable cell proliferation during first 24 hours. This evinced hypoxia-induced potentially lethal damage (PLD) that was repairable upon reoxygenation. DNKu70 did not significantly inhibit this repair. CONCLUSION: Hypoxia-induced cell lethality was facilitated by DNKu70, but substantially repaired upon reoxygenation. This may have negative impact on the effect of reoxygenation in cancer therapy.

Response to multiple radiation doses of human colorectal carcinoma cells infected with recombinant adenovirus containing dominant-negative Ku70 fragment.

PURPOSE: To investigate the effect of recombinant replication-defective adenovirus containing dominant-negative Ku70 fragment on the response of tumor cells to multiple small radiation doses. Our ultimate goal is to demonstrate the feasibility of using this virus in gene-radiotherapy to enhance the radiation response of tumor cells. METHODS AND MATERIALS: Human colorectal HCT8 and HT29 carcinoma cells were plated in glass tubes, infected with virus (25 multiplicity of infection), and irradiated with a single dose or zero to five doses of 3 Gy each at 6-h intervals. Hypoxia was induced by flushing with 100% nitrogen gas. The cells were trypsinized 0 or 6 h after the final irradiation, and cell survival was determined by colony formation. The survival data were fitted to linear-quadratic model or exponential line. RESULTS: Virus infection enhanced the radiation response of the HCT8 and HT29 cells. The virus enhancement ratio for single-dose irradiation at a surviving fraction of 0.1 was approximately 1.3 for oxic and hypoxic HCT8 and 1.4 and 1.1 for oxic and hypoxic HT29, respectively. A similar virus enhancement ratio of 1.2-1.3 was observed for both oxic and hypoxic cells irradiated with multiple doses; however, these values were smaller than the values found for dominant-negative Ku70-transfected Rat-1 cells. This difference has been discussed. The oxygen enhancement ratio for HCT8 and HT29 receiving fractionated doses was 1.2 and 2.0, respectively, and virus infection altered them slightly. CONCLUSION: Infection of recombinant replication-defective adenovirus containing dominant-negative Ku70 fragment enhanced the response of human colorectal cancer cells to single and multiple radiation doses.

Noninvasive molecular imaging of hypoxia in human xenografts: comparing hypoxia-induced gene expression with endogenous and exogenous hypoxia markers.

Tumor hypoxia is important in the development and treatment of human cancers. We have developed a novel xenograft model for studying and imaging of hypoxia-induced gene expression. A hypoxia-inducible dual reporter herpes simplex virus type 1 thymidine kinase and enhanced green fluorescence protein (HSV1-TKeGFP), under the control of hypoxia response element (9HRE), was stably transfected into human colorectal HT29 cancer cells. Selected clones were further enriched by repeated live cell sorting gated for hypoxia-induced eGFP expression. Fluorescent microscopy, fluorescence-activated cell sorting, and radioactive substrate trapping assays showed strong hypoxia-induced expression of eGFP and HSV1-tk enzyme in the HT29-9HRE cells in vitro. Sequential micropositron emission tomography (PET) imaging of tumor-bearing animals, using the hypoxic cell tracer (18)F-FMISO and the reporter substrate (124)I-FIAU, yielded similar tumor hypoxia images for the HT29-9HRE xenograft but not in the parental HT29 tumor. Using autoradiography and IHC, detailed spatial distributions in tumor sections were obtained and compared for the following hypoxia-associated biomarkers in the HT29-9HRE xenograft: (124)I-FIAU, (18)F-FMISO, Hoechst (perfusion), lectin-TRITC (functional blood vessels), eGFP, pimonidazole, EF5, and CA9. Intratumoral distributions of (124)I-FIAU and (18)F-FMISO were similar, and eGFP, pimonidazole, EF5, and CA9 colocalized in the same areas but not in well-perfused regions that were positive for Hoechst and lectin-TRITC. In enabling the detection of hypoxia-induced molecular events and mapping their distribution in vivo with serial noninvasive positron emission tomography imaging, and multiple variable analysis with immunohistochemistry and fluorescence microscopy, this human xenograft model provides a valuable tool for studying tumor hypoxia and in validating existing and future exogenous markers for tumor hypoxia.

Response to multiple radiation doses of fibroblasts over-expressing dominant negative Ku70.

PURPOSE: To evaluate the response of cells over-expressing dominant negative (DN) Ku70 to single and multiple small radiation doses. METHODS AND MATERIALS: Clones of fibroblasts over-expressing DNKu70, DNKu70-7, DNKu70-11, and parental Rat-1 cells were irradiated under oxic or hypoxic conditions with single or multiple doses. Cells were trypsinized 0 or 6 h after irradiation to determine surviving fraction (SF). RESULTS: Oxic DNKu70-7 or -11 cells trypsinized 6 h after irradiation were 1.52 or 1.25 and 1.28 or 1.15 times more sensitive than oxic Rat-1 at SF of 0.5 and 0.1, respectively. Hypoxic DNKu70-7 or -11 cells trypsinized 6 h after irradiation were 1.44 or 1.70 and 1.33 or 1.51 times more sensitive than hypoxic Rat-1 at SF of 0.5 and 0.1, respectively. To the multiple doses, oxic and hypoxic DNKu70-7 or -11 cells were 1.35 or 1.37 and 2.23 or 4.61 times more sensitive than oxic and hypoxic Rat-1, respectively, resulting in very small oxygen enhancement ratios. Namely, enhancement caused by DNKu70 under hypoxia after multiple doses was greater than that under oxic conditions and greater than that after single dose. CONCLUSIONS: Over-expression of DNKu70 enhances cells' response to radiation given as a single dose and as multiple small doses. The enhancement after multiple doses was stronger under hypoxic than under oxic conditions. These results encourage the use of DNKu70 fragment in a gene-radiotherapy.

Antidepressant-like effects of selegiline in the forced swim test.

Although selegiline, a monoamine oxidase (MAO)-B inhibitor, is reported to exert antidepressant effects in depressant patients, evidence in rodents for effects of selegiline is quite limited. The purpose of the present study was to assess effects of selegiline in the forced swim test (FST) and on locomotor activity, and to investigate whether MAO inhibition or stimulation of receptors contributes to antidepressant-like effects of selegiline. Drugs were subcutaneously administrated. The single administration of reference drug nortriptyline at 5 mg/kg reduced locomotor activity without effects in FST and brain MAO activities. But nortriptyline repeatedly given 24, 5 and 1 h before behavioral tests significantly decreased an immobility time in FST without effects in motor activities, and showed weak brain MAO-B inhibition. Single and following repeated (24, 5 and 1 h before behavioral tests) administrations of selegiline at 10 mg/kg significantly decreased the immobility time in FST, with little motor stimulant effect. In contrast, (+)-methamphetamine caused a marked decrease in immobility time and an increase in locomotor activity. Selegiline at 1 and 3 mg/kg, which failed to decrease immobility time, markedly inhibited brain total-MAO and MAO-B activities. A dopamine D1 receptor antagonist SCH 23390 completely blocked antidepressant-like effects of selegiline, but not dopamine D2, serotonergic or noradrenergic receptor antagonists. These results suggest that selegiline exerts the antidepressant-like effects by prolonging escape-directed behavior rather than by a motor stimulant effect and D1 receptor activation contributes to its effect.

Effects of selegiline alone or with donepezil on memory impairment in rats.

Selegiline, a monoamine oxidase-B inhibitor, is reported to improve memory and learning in dementia of Alzheimer's type. However, only a few studies have reported its use in animal models. Here, we evaluated the effects of selegiline only or its combined use with donepezil, a selective acetylcholinesterase inhibitor on memory impairment, using a Morris water maze. Selegiline dose-dependently attenuated ethylcholine aziridinium ion-induced memory impairment. Co-administration of selegiline and donepezil, at doses that do not exert efficacy individually, significantly ameliorated scopolamine+p-chlorophenylalanine-induced memory deficits. These results suggest that selegiline improves memory impairment mediated by the cholinergic system, and provide evidence of the usefulness of co-treatment with selegiline and donepezil for treating spatial deficits in dementia.