An ab initio and DFT study of trifluoromethylation using Umemoto's reagent.

Trifluoromethylation using Umemoto's reagent is an important transformation that allows the preparation of compounds bearing trifluoromethyl groups. To investigate the mechanism of this reaction, ab initio and density functional theory (DFT) calculations were carried out using pyrrole, aniline, sodium acetylacetonate, and sodium methyl acetoacetate as nucleophiles. At the highest level of theory examined (i.e., CCSD(T)/6-311+G(d,p)//M06-2X/6-311+G(d,p)), the energy barriers for the forward process (ΔE‡1) of both the backside and frontside attack of pyrrole on a model Umemoto reagent (i.e., S-(trifluoromethyl)dimethylsulfonium, CF3DMS) were predicted to be 135.9 and 192.3 kJ mol-1, respectively, while values of 131.9 and 188.2 kJ mol-1 were obtained at the MP2/6-311+G(d,p)//M06-2X/6-31+G(d,p) level. These outcomes suggest that the reaction proceeds via the backside mechanism. Using the MP2 method, the investigation of the trifluoromethylation of pyrrole and sodium acetoacetate with the sulfonium moiety of Umemoto's reagent, S-(trifluoromethyl)dibenzothionium, revealed that this reaction would also occur through the backside mechanism, thereby indicating that this pathway remains feasible despite solvent effects. Finally, computational investigations revealed that the simple single-electron transfer mechanism, which should occur between Umemoto's reagent and nucleophiles, did not take place during this reaction.

An ab initio and DFT study of the autoxidation of THF and THP.

Tetrahydropyran (THP) is known to undergo autoxidation much more slowly than tetrahydrofuran (THF). To investigate the difference in reactivity in the autoxidation of these two ethers, ab initio and DFT calculations were carried out. At the BHandHLYP/aug-cc-pVDZ//BHandHLYP/cc-pVDZ level of theory, the energy barrier for hydrogen abstraction from THP is predicted to be 104.1 kJ mol(-1), whereas that for THF is calculated as 94.1 kJ mol(-1). Including solvation effects in the calculations lowers these barriers to 98.0 (THP) and 84.4 kJ mol(-1) (THF); the energy barrier for the process involving THP is smaller by 14 kJ mol(-1) than that for THF. While scanning the potential energy surface for the radical coupling process between the THP (or THF) radical with molecular oxygen, an energy barrier of 11.2 kJ mol(-1) (BHandHLYP/6-311G**) was found for the process involving the THP radical, although no barrier was found for the reaction involving THF. Analysis of the Kohn-Sham singly occupied molecular orbitals (SOMOs) in the hydroperoxy complexes reveals that the SOMO of the THP complex would be blocked by the neighbouring hydrogen atoms in the THP ring. These factors would work together to delay the autoxidation of THP.

Effects of C-reactive protein on CC chemokine receptor 2-mediated chemotaxis of monocytes.

Periodontal infections can increase patients' serum C-reactive protein (CRP) level, which is a predictive marker of future cardiovascular events. Serum CRP may be a key mediator associating periodontitis with cardiovascular disease. It is not yet clarified whether the chemotactic activity of monocytes changes with increased serum CRP. This study investigated the influence of CRP on monocyte chemotaxis and the effects of CRP on CC chemokine receptor 2 (CCR2) expression by monocytes in vitro. Monocyte cell line THP-1 was cultured with human recombinant CRP of different final concentrations, which were 2, 4, 6, 8, and 10 mg/L, respectively. After 24 h incubation, Transwell chambers were applied to analyze the chemotactic activity of pretreated monocytes. Flow cytometry analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR) were applied to detect the CCR2 protein and gene expression levels. In Transwell chambers, more cells were attracted in CRP-pretreated groups than that of blank control with no CRP (p<0.05). The chemotaxis activity was stronger in higher CRP concentration groups than lower ones (p<0.05). The CCR2 protein and mRNA expression was increased in a CRP concentration-dependent manner (p<0.05). CRP stimulation may induce CCR2 overexpression on monocytes and then promote the chemotaxis ability of monocytes. This result suggests that increased serum CRP concentration of periodontitis patients may be associated with high risk of cardiovascular disease.

Caspase inhibitors increase the rate of recovery of neural stem/progenitor cells from post-mortem rat brains stored at room temperature.

To match the demand of regenerative medicine for nerve system, collection of stem cells from the post-mortem body is one of the most practical ways. In this study, the storage condition of the post-mortem body was examined. We prepared neural stem/progenitor cells (NSPCs) from post-mortem rat brains stored at different temperatures. When brains were stored at 4 degrees C, for one week, we were able to obtain neurospheres (a spheroid body containing NSPCs) by stimulation of cells with epidermal growth factor (EGF). Incremental increases in storage temperature decreased the rate of appearance of neurospheres. Within 48 h at 15 degrees C, 24 h at 25 degrees C, in both condition, we were able to recover NSPCs from post-mortem rat brains. At 15 degrees C, 90% of neurosphere-forming activity was lost within 24 h. However, even after 24 h at 25 degrees C, 2% neurosphere-forming activity remained. After 6 h of death, there was very little difference between the rates of NSPC recovery at 4 degrees C and 25 degrees C. Addition of caspase inhibitors to both the rat brain storage solution and the NSPC culture medium increased the rate of neurosphere-forming activity. In particular, an inhibitor of caspase-8 activity increased the NSPC recovery rate approximately three-fold, with no accompanying detrimental effects on neural differentiation in vitro.

In Vitro Screening of Exogenous Factors for Human Neural Stem/Progenitor Cell Proliferation Using Measurement of Total ATP Content in Viable Cells.

One of the newest and most promising methods for treating intractable neuronal diseases and injures is the transplantation of ex vivo-expanded human neural stem/progenitor cells (NSPCs). Human NSPCs are selectively expanded as free-floating neurospheres in serum-free culture medium containing fibroblast growth factor 2 (FGF2) and/or epidermal growth factor (EGF); however, the culture conditions still need to be optimized for performance and cost before the method is used clinically. Here, to improve the NSPC culture method for clinical use, we used an ATP assay to screen the effects of various reagents on human NSPC proliferation. Human NSPCs responded to EGF, FGF2, and leukemia inhibitory factor (LIF) in a dose-dependent manner, and the minimum concentrations eliciting maximum effects were 10 ng/ml EGF, 10 ng/ml FGF2, and 5 ng/ml LIF. EGF and LIF were stable in culture medium without NSPCs, although FGF2 was degraded. In the presence of human NSPCs, however, FGF2 and LIF were both degraded very rapidly, to below the estimated minimum concentration on day 3, but EGF remained above the minimum concentration for 5 days. Adding supplemental doses of each growth factor during the incubation promoted human NSPC proliferation. Among other supplements, insulin and transferrin promoted human NSPC growth, but progesterone, putrescine, selenite, D-glucose, and lactate were not effective and were cytotoxic at higher concentrations. Supplementing with conditioned medium from human NSPCs significantly increased human NSPC proliferation, but using a high percentage of the medium had a negative effect. These findings suggest that human NSPC culture is regulated by a balance in the culture medium between decreasing growth factor levels and increasing positive or negative factors derived from the NSPCs. Thus, in designing culture conditions for human NSPCs, it is useful to take the individual properties of each factor into consideration.

In vitro screening of exogenous factors for human neural stem/progenitor cell proliferation using measurement of total ATP content in viable cells.

One of the newest and most promising methods for treating intractable neuronal diseases and injures is the transplantation of ex vivo-expanded human neural stem/progenitor cells (NSPCs). Human NSPCs are selectively expanded as free-floating neurospheres in serum-free culture medium containing fibroblast growth factor 2 (FGF2) and/or epidermal growth factor (EGF); however, the culture conditions still need to be optimized for performance and cost before the method is used clinically. Here, to improve the NSPC culture method for clinical use, we used an ATP assay to screen the effects of various reagents on human NSPC proliferation. Human NSPCs responded to EGF, FGF2, and leukemia inhibitory factor (LIF) in a dose-dependent manner, and the minimum concentrations eliciting maximum effects were 10 ng/ml EGF, 10 ng/ ml FGF2, and 5 ng/ml LIF. EGF and LIF were stable in culture medium without NSPCs, although FGF2 was degraded. In the presence of human NSPCs, however, FGF2 and LIF were both degraded very rapidly, to below the estimated minimum concentration on day 3, but EGF remained above the minimum concentration for 5 days. Adding supplemental doses of each growth factor during the incubation promoted human NSPC proliferation. Among other supplements, insulin and transferrin promoted human NSPC growth, but progesterone, putrescine, selenite, D-glucose, and lactate were not effective and were cytotoxic at higher concentrations. Supplementing with conditioned medium from human NSPCs significantly increased human NSPC proliferation, but using a high percentage of the medium had a negative effect. These findings suggest that human NSPC culture is regulated by a balance in the culture medium between decreasing growth factor levels and increasing positive or negative factors derived from the NSPCs. Thus, in designing culture conditions for human NSPCs, it is useful to take the individual properties of each factor into consideration.

Increased regional cerebral blood flow in the contralateral thalamus after successful motor cortex stimulation in a patient with poststroke pain.

The mechanisms underlying poststroke pain have not been clearly identified. Although motor cortex stimulation (MCS) sometimes reduces poststroke pain successfully, the exact mechanism is not yet known. For further investigation of the neural pathways involved in the processing of poststroke pain and in pain reduction by MCS, the authors used positron emission tomography (PET) scanning to determine significant changes in regional cerebral blood flow (rCBF). This 58-year-old right-handed man suffered from right-sided poststroke pain for which he underwent implantation of a stimulation electrode in the right motor cortex. After 30 minutes of stimulation, his pain was remarkably reduced (Visual Analog Scale scores decreased 8 to 1) and he felt warmth in his left arm. The rCBF was studied using PET scanning with 15O-labeled water when the patient was in the following states: before MCS (painful condition, no stimulation) and after successful MCS (painless condition, no stimulation). The images were analyzed using statistical parametric mapping software. State-dependent differences in global blood flow were covaried using analysis of covariance. Comparisons of the patient's rCBF in the painful condition with that in the painless condition revealed significant rCBF increases in the left rectus gyrus (BA11), left superior frontal lobe (BA9), left anterior cingulate gyms (BA32), and the left thalamus (p < 0.05, corrected). On the other hand, there were significant decreases in rCBF in the right superior temporal gyrus (BA22, p < 0.01, corrected) and the left middle occipital gyrus (BA19, p < 0.05, corrected). The efficacy of MCS was mainly related to increased synaptic activity in the thalamus, whereas the activations in the rectus gyrus, anterior cingulate gyrus, and superior frontal cortex as well as the inactivation of the superior temporal lobe may be related to emotional processes. This is the first report in which the contralateral thalamus was significantly activated and pain relief was achieved using MCS.

Quantitative follow-up analysis by computed tomographic imaging in neonatal hydrocephalus.

We sought a simple and accurate method to monitor neonatal hydrocephalic infants using standard computed tomographic scans. Volume measurements were made by means of pixel counting using a personal computer and a drawing device, as a graphic tablet system, over computed tomographic scans of six infants with neonatal hydrocephalus and four age-matched control infants. The mean value (763.9 +/- 83.3 cm(3)) of the volume of the cranium in the hydrocephalic group was two times higher than that in the age-matched control infants (360.4 +/- 41.4 cm(3)), P < 0.00001. Sequential changes of the ventricular/intracranial volume ratio steadily decreased after cerebrospinal fluid diversion by means of a "two-step procedure" as early in postnatal life as feasible. The mean value (0.67 +/- 0.12) of the lateral ventricle/intracranial volume ratio at birth improved to 12 months of age (0.26 +/- 0.14), P < 0.05. This study has documented, by means of quantitative analysis of serial scans, a statistically significant increase in the neonatal hydrocephalic brain volume after cerebrospinal fluid shunting.

Neural Cell Adhesion Molecule L1 Promotes Regeneration of Injured Optic Nerve of Rats.

Neural cell adhesion molecule L1 is a member of the immunoglobulin superfamily; it plays an important role in neurite outgrowth in vitro. We present evidence that the transected optic nerve in adult rats was promoted to regenerate by transplanted L1-expressing cells that were embedded in the Matrigel matrix. To obtain the maximum effects, a very careful operative procedure by which both stumps remained attached and the blood supply was preserved was essential. Visual evoked potential was partially recovered and traced fibers were seen to pass through the lesion site 7 weeks after optic nerve transection. Immunohistochemical analysis demonstrated that neurofilament-positive fibers were present around the site of experimental lesion. Our in vivo study demonstrated that L1 promoted the regeneration of the lesioned optic nerve in rats under strictly controlled environmental conditions.