Clinical and histological characterization of transient dermal pain triggered by sweating stimuli.

BACKGROUND: Tingling dermal pain triggered by sweating impairs the lives of patients with cholinergic urticaria and generalized anhidrosis. However, dermal pain evoked by sweating stimuli has been under investigated. METHODS: To clarify characteristics of tingling dermal pain on sweating, we retrospectively evaluated clinical and histopathological manifestations in 30 patients having the main problem of dermal pain on sweating, and the efficacy of treatments. RESULTS: Dermal pain upon sweating affected mostly young males. It accompanied eruptions upon sweating and/or hypohidrosis in 24 patients, while 6 patients had dermal pain independently of hypohidrosis or eruptions. Dermal pain appeared immediately upon exposure to sweating stimuli, and disappeared within mostly 30 or 10 min. Hypohidrosis was not necessarily generalized but localized or absent. Histological analysis revealed that dermal pain could occur even without morphological changes and inflammation of sweat glands. Hypersensitivity to sweat contents was found only in 26% of patients. Sweat histamine and increase of plasma histamine after thermal induction in patients were significantly higher than those in healthy subjects. Effectiveness of steroid pulse therapy was demonstrated for dermal pain with hypohidrosis. Medications acting on nervous systems and regular sweat-inducing activities for promoting perspiration were also effective. CONCLUSIONS: Short-lasting tingling dermal pain appears immediately upon exposure to sweating stimuli, regardless of developing eruptions and/or presence of hypohidrosis, but possibly in association with sweat and plasma histamine.

Transcriptional priming as a conserved mechanism of lineage diversification in the developing mouse and human neocortex.

How the rich variety of neurons in the nervous system arises from neural stem cells is not well understood. Using single-cell RNA-sequencing and in vivo confirmation, we uncover previously unrecognized neural stem and progenitor cell diversity within the fetal mouse and human neocortex, including multiple types of radial glia and intermediate progenitors. We also observed that transcriptional priming underlies the diversification of a subset of ventricular radial glial cells in both species; genetic fate mapping confirms that the primed radial glial cells generate specific types of basal progenitors and neurons. The different precursor lineages therefore diversify streams of cell production in the developing murine and human neocortex. These data show that transcriptional priming is likely a conserved mechanism of mammalian neural precursor lineage specialization.

Dorsal-to-Ventral Cortical Expansion Is Physically Primed by Ventral Streaming of Early Embryonic Preplate Neurons.

Despite recent studies elucidating the molecular mechanisms underlying cortical patterning and map formation, very little is known about how the embryonic pallium expands ventrally to form the future cortex and the nature of the underlying force-generating events. We find that neurons born at embryonic day 10 (E10) in the mouse dorsal pallium ventrally stream until E13, thereby superficially spreading the preplate, and then constitute the subplate from E14. From E11 to E12, the preplate neurons migrate, exerting pulling and pushing forces at the process and the soma, respectively. At E13, they are morphologically heterogeneous, with ∼40% possessing corticofugal axons, which are found to be in tension. Ablation of these E10-born neurons attenuates both deflection of radial glial fibers (by E13) and extension of the cortical plate (by E14), which should occur ventrally, and subsequently shrinks the postnatal neocortical map dorsally. Thus, the preplate stream physically primes neocortical expansion and arealization.

Distinct Neocortical Progenitor Lineages Fine-tune Neuronal Diversity in a Layer-specific Manner.

How the variety of neurons that organize into neocortical layers and functional areas arises is a central question in the study of cortical development. While both intrinsic and extrinsic cues are known to influence this process, whether distinct neuronal progenitor groups contribute to neuron diversity and allocation is poorly understood. Using in vivo genetic fate-mapping combined with whole-cell patch clamp recording, we show that the firing pattern and apical dendritic morphology of excitatory neurons in layer 4 of the barrel cortex are specified in part by their neural precursor lineage. Further, we show that separate precursors contribute to unique features of barrel cortex topography including the intralaminar position and thalamic innervation of the neurons they generate. Importantly, many of these lineage-specified characteristics are different from those previously measured for pyramidal neurons in layers 2-3 of the frontal cortex. Collectively, our data elucidate a dynamic temporal program in neuronal precursors that fine-tunes the properties of their progeny according to the lamina of destination.

Synaptic transmission from subplate neurons controls radial migration of neocortical neurons.

The neocortex exhibits a six-layered structure that is formed by radial migration of excitatory neurons, for which the multipolar-to-bipolar transition of immature migrating multipolar neurons is required. Here, we report that subplate neurons, one of the first neuron types born in the neocortex, manage the multipolar-to-bipolar transition of migrating neurons. By histochemical, imaging, and microarray analyses on the mouse embryonic cortex, we found that subplate neurons extend neurites toward the ventricular side of the subplate and form transient glutamatergic synapses on the multipolar neurons just below the subplate. NMDAR (N-methyl-d-aspartate receptor)-mediated synaptic transmission from subplate neurons to multipolar neurons induces the multipolar-to-bipolar transition, leading to a change in migration mode from slow multipolar migration to faster radial glial-guided locomotion. Our data suggested that transient synapses formed on early immature neurons regulate radial migration.

Synthesis and basic evaluation of 7α-(3-[18F]fluoropropyl)-testosterone and 7α-(3-[18F]fluoropropyl)-dihydrotestosterone.

OBJECTIVE: 7α-Substituted androgen derivatives may have the potential to visualize androgen receptors with positron emission tomography. In the present study, we synthesized fluoropropyl derivatives of 7α-(3-[18F]fluoropropyl)-testosterone ([18F]7) and 7α-(3-[18F]fluoropropyl)-dihydrotestosterone ([18F]15), and characterized their in vitro binding, in vivo biodistribution, and performed blocking studies in mature androgen deprived male rats. METHODS: We synthesized [18F]7 and [18F]15. In vitro binding to recombinant rat AR ligand binding domain protein was determined using a competitive radiometric ligand-binding assay with the high-affinity synthetic androgen [17α-methyl-3H]-methyltrienolone ([3H]R1881). In vivo biodistribution was performed in mature male rats treated with diethylstilbestrol (chemical castration). A blocking study was performed by co-administration of dihydrotestosterone (36 µg/animal). RESULTS: 7α-(3-Fluoropropyl)-testosterone (7) and 7α-(3-fluoropropyl)-dihydrotestosterone (15) showed competitive binding to recombinant rat AR ligand binding domain protein. The IC50 value of 15 (13.0 ± 3.3 nM) was higher than 7 (47.8 ± 10.0 nM). In contrast to the AR binding affinity, the ventral prostate uptake of [18F]7 and [18F]15 at 2 h post-injection was similar (0.07 % injected dose/g of tissue). A blocking study indicated that specific binding of [18F]15 is observed in the ventral prostate. [18F]7 and [18F]15 showed moderate levels of bone uptake, which indicates moderate metabolic de-fluorination in rodents. CONCLUSION: [18F]15 is better than [18F]7 in terms of radiochemical yield, in vitro binding affinity, prostate specific binding and stability against in vivo metabolic de-fluorination. However, the net uptake level of [18F]15 in prostate might be insufficient for in vivo visualization. Although [18F]7 and [18F]15 improved in vivo stability against de-fluorination, other basic characterization data in rodents were not superior to the current standard tracer 16ß-[18F]fluoro-5α-dihydrotestosterone. It is also revealed that the shorter side chain length of 7α-[18F]fluoromethyl-dihydrotestosterone is superior to the longer three carbon chain in [18F]15, in terms of net prostate uptake and in vivo metabolic stability.

Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy.

Fatty acids are taken up by cells and incorporated into complex lipids such as neutral lipids and glycerophospholipids. Glycerophospholipids are major constituents of cellular membranes. More than 1000 molecular species of glycerophospholipids differ in their polar head groups and fatty acid compositions. They are related to cellular functions and diseases and have been well analyzed by mass spectrometry. However, intracellular imaging of fatty acids and glycerophospholipids has not been successful due to insufficient resolution using conventional methods. Here, we developed a method for labeling fatty acids with bromine (Br) and applied scanning X-ray fluorescence microscopy (SXFM) to obtain intracellular Br mapping data with submicrometer resolution. Mass spectrometry showed that cells took up Br-labeled fatty acids and metabolized them mainly into glycerophospholipids in CHO cells. Most Br signals observed by SXFM were in the perinuclear region. Higher resolution revealed a spot-like distribution of Br in the cytoplasm. The current method enabled successful visualization of intracellular Br-labeled fatty acids. Single-element labeling combined with SXFM technology facilitates the intracellular imaging of fatty acids, which provides a new tool to determine dynamic changes in fatty acids and their derivatives at the single-cell level.-Shimura, M., Shindou, H., Szyrwiel, L., Tokuoka, S. M., Hamano, F., Matsuyama, S., Okamoto, M., Matsunaga, A., Kita, Y., Ishizaka, Y., Yamauchi, K., Kohmura, Y., Lobinski, R., Shimizu, I., Shimizu, T. Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy.

Synthesis of 24,24-Difluoro-1,3-cis-25-dihydroxy-19-norvitamin D3 Derivatives and Evaluation of Their Vitamin D Receptor-Binding Affinity.

Two vitamin D3 derivatives, namely 24,24-difluoro-1ß,3ß,25-dihydroxy-19-norvitamin D3 (6a) and 24,24-difluoro-1α,3α,25-dihydroxy-19-norvitamin D3 (6b), were synthesized via a convergent route employing Julia-Kocienski olefination as a key step. Compounds 6a and b bound to vitamin D receptor (VDR) with IC50 values of 64.8 and 57.6 nM, respectively, exhibiting about 400- and 30-fold greater binding affinity than the corresponding non-fluorinated derivatives 5a and b.

Optimization of the alkyl side chain length of fluorine-18-labeled 7α-alkyl-fluoroestradiol.

INTRODUCTION: Several lines of evidence suggest that 7α-substituted estradiol derivatives bind to the estrogen receptor (ER). In line with this hypothesis, we designed and synthesized (18)F-labeled 7α-fluoroalkylestradiol (Cn-7α-[(18)F]FES) derivatives as molecular probes for visualizing ERs. Previously, we successfully synthesized 7α-(3-[(18)F]fluoropropyl)estradiol (C3-7α-[(18)F]FES) and showed promising results for quantification of ER density in vivo, although extensive metabolism was observed in rodents. Therefore, optimization of the alkyl side chain length is needed to obtain suitable radioligands based on Cn-7α-substituted estradiol pharmacophores. METHODS: We synthesized fluoromethyl (23; C1-7α-[(18)F]FES) to fluorohexyl (26; C6-7α-[(18)F]FES) derivatives, except fluoropropyl (C3-7α-[(18)F]FES) and fluoropentyl derivatives (C5-7α-[(18)F]FES), which have been previously synthesized. In vitro binding to the α-subtype (ERα) isoform of ERs and in vivo biodistribution studies in mature female mice were carried out. RESULTS: The in vitro IC50 value of Cn-7α-FES tended to gradually decrease depending on the alkyl side chain length. C1-7α-[(18)F]FES (23) showed the highest uptake in ER-rich tissues such as the uterus. Uterus uptake also gradually decreased depending on the alkyl side chain length. As a result, in vivo uterus uptake reflected the in vitro ERα affinity of each compound. Bone uptake, which indicates de-fluorination, was marked in 7α-(2-[(18)F]fluoroethyl)estradiol (C2-7α-[(18)F]FES) (24) and 7α-(4-[(18)F]fluorobutyl)estradiol (C4-7α-[(18)F]FES) (25) derivatives. However, C1-7α-[(18)F]FES (23) and C6-7α-[(18)F]FES (26) showed limited uptake in bone. As a result, in vivo bone uptake (de-fluorination) showed a bell-shaped pattern, depending on the alkyl side chain length. C1-7α-[(18)F]FES (23) showed the same levels of uptake in uterus and bone compared with those of 16α-[(18)F]fluoro-17ß-estradiol. CONCLUSIONS: The optimal alkyl side chain length of (18)F-labeled 7α-fluoroalkylestradiol was the shortest: C1-7α-[(18)F]FES. Our results indicate that shorter chain lengths within the 4-Å ligand binding cavities of ERα are suitable for 7α-fluoroalkylestradiol derivatives.

Cell-cycle-independent transitions in temporal identity of mammalian neural progenitor cells.

During cerebral development, many types of neurons are sequentially generated by self-renewing progenitor cells called apical progenitors (APs). Temporal changes in AP identity are thought to be responsible for neuronal diversity; however, the mechanisms underlying such changes remain largely unknown. Here we perform single-cell transcriptome analysis of individual progenitors at different developmental stages, and identify a subset of genes whose expression changes over time but is independent of differentiation status. Surprisingly, the pattern of changes in the expression of such temporal-axis genes in APs is unaffected by cell-cycle arrest. Consistent with this, transient cell-cycle arrest of APs in vivo does not prevent descendant neurons from acquiring their correct laminar fates. Analysis of cultured APs reveals that transitions in AP gene expression are driven by both cell-intrinsic and -extrinsic mechanisms. These results suggest that the timing mechanisms controlling AP temporal identity function independently of cell-cycle progression and Notch activation mode.

Radiation-Induced RhoGDIß Cleavage Leads to Perturbation of Cell Polarity: A Possible Link to Cancer Spreading.

The equilibrium between proliferation and apoptosis is tightly balanced to maintain tissue homeostasis in normal tissues and even in tumors. Achieving and maintaining such a balance is important for cancer regrowth and spreading after cytotoxic treatments. Caspase-3 activation and tumor cell death following anticancer therapy as well as accompanying cell death pathways are well characterized, but their association to homeostasis of cancerous tissue and tumor progression remains poorly understood. Here we proposed a novel mechanism of cancer spreading induced by caspase-3. RhoGDIß, known as a direct cleavage substrate of caspase-3, is overexpressed in many epithelial cancers. The N-terminal-truncated RhoGDIß (ΔN-RhoGDIß) is accumulated in caspase-3-activated cells. Stable expression of ΔN-RhoGDIß in HeLa cells did not induce apoptosis, but impaired directional cell migration in a wound-healing assay accompanied by a perturbed direction of cell division at the wound edge. Subcellular protein fractionation experiments revealed that ΔN-RhoGDIß but not wild-type RhoGDIß was present in the detergent-soluble cytoplasmic and nuclear fractions and preferentially associated with Cdc42. Furthermore, Cdc42 activity was constitutively inhibited by stable expression of ΔN-RhoGDIß, resulting in increased radiation-induced compensatory proliferation linking to RhoA activation. Thus, ΔN-RhoGDIß dominant-negatively regulates Cdc42 activity and contributes to loss of polarity-related functions. The caspase-3-cleaved RhoGDIß is a possible determinant to promote cancer spreading due to deregulation of directional organization of tumor cell population and inhibition of default equilibrium between proliferation and apoptosis after cytotoxic damage. J. Cell. Physiol. 231: 2493-2505, 2016. © 2016 Wiley Periodicals, Inc.

(R)-[¹¹C]Emopamil as a novel tracer for imaging enhanced P-glycoprotein function.

INTRODUCTION: 2-Isopropyl-5-[methyl-(2-phenylethyl)amino]-2-phenylpentanenitrile (emopamil; EMP) is a calcium channel blocker of the phenylalkylamine class, with weak substrate properties for P-glycoprotein (P-gp). A weak substrate for P-gp would be suitable for measuring enhanced P-gp function. This study was performed to synthesise (R)- and (S)-[(11)C]EMP and characterise their properties as P-gp tracers. METHODS: We synthesised (R)- and (S)-[(11)C]EMP and compared their biodistribution, peripheral metabolism, and effects of the P-gp inhibitor cyclosporine A (CsA, 50 mg/kg). We compared the brain pharmacokinetics of (R)-[(11)C]EMP and (R)-[(11)C]verapamil [(R)-[(11)C]VER] at baseline and CsA pretreatment with small animal positron emission tomography (PET). RESULTS: (R)- and (S)-[(11)C]EMP were synthesised from (R)- and (S)-noremopamil, respectively, by methylation with [(11)C]methyl triflate in the presence of NaOH at room temperature. (R)- and (S)-[(11)C]EMP yields were ~30%, with specific activity>74 GBq/µmol and radiochemical purity>99%. (R)-[(11)C]EMP showed significantly greater uptake in the mouse brain than (S)-[(11)C]EMP. Both showed homogeneous non-stereoselective regional brain distributions. (R)- and (S)-[(11)C]EMP were rapidly metabolised to hydrophilic metabolites. Unchanged plasma (S)-[(11)C]EMP level was significantly lower than that of (R)-[(11)C]EMP 15 minutes post-injection, whilst>88% of radioactivity in the brain was intact at 15 minutes post-injection and was non-stereoselective. CsA pretreatment increased brain activity ~3-fold in mice, but was non-stereoselective. The baseline area-under-the-curve (AUC) of brain radioactivity (0-60 minutes) of (R)-[(11)C]EMP was 2-fold higher than that of (R)-[(11)C]VER, but their AUCs after CsA pretreatment were comparable. CONCLUSIONS: (R)-[(11)C]EMP is a novel tracer for imaging P-gp function with higher baseline uptake than (R)-[(11)C]VER.

Synthesis and evaluation of 7α-(3-[(18)F]fluoropropyl) estradiol.

INTRODUCTION: Several lines of evidence suggest that C-7α-substituted estradiol derivatives are well tolerated by estrogen receptor (ER). In line with this hypothesis, we are interested in the design and synthesis of C-7α-substituted estrogens as molecular probes to visualize ER function. METHODS: We have synthesized 7α-(3-[(18)F]fluoropropyl) estradiol (C3-7α-[(18)F]FES) as a potential radiopharmaceutical for ER imaging by positron emission tomography (PET). In vitro receptor binding and in vivo biodistribution and blocking studies in mature female mice, and in vivo metabolite analysis were carried out. Furthermore, in vivo ER-selective uptake was confirmed using ER-positive T-47D and ER-negative MDA-MB-231 tumor-bearing mice. We also compared the in vivo biodistribution of C3-7α-[(18)F]FES with 16α-[(18)F]FES. RESULTS: C3-7α-[(18)F]FES was produced in moderate yields (30.7%±15.1%, decay corrected) with specific activity of 32.0±18.1GBq/µmol (EOS). The in vitro binding affinity of C3-7α-FES to the ERα isoform was sufficient and equivalent to that of estradiol. C3-7α-[(18)F]FES showed selective uptake in ER-rich tissues, such as the uterus (4.7%ID/g±1.2%ID/g at 15minutes) and ovary (4.0%ID/g±1.0%ID/g at 5minutes). The tissue time activity curves of these organs showed reversible kinetics, indicating suitability for quantitative analysis. The highest contrast was obtained at 120minutes after injection of C3-7α-[(18)F]FES in the uterus (uterus/blood=18, uterus/muscle=17.3) and ovary (ovary/blood=6.3, ovary/muscle=6.0). However, the level of selective uptake of C3-7α-[(18)F]FES was significantly lower than that of 16α-[(18)F]FES. Most radioactivity in the uterus was detected in unchanged form, although peripherally C3-7α-[(18)F]FES was rapidly degraded to hydrophilic metabolites. In accordance with this peripheral metabolism, gradual increases in bone radioactivity were observed, indicating defluorination. Coinjection with estradiol dose-dependently inhibited C3-7α-[(18)F]FES uptake in the uterus and ovary. The in vivo IC50 values of estradiol in the uterus and ovary were 34.4 and 38.5nmol/kg, respectively. Furthermore, in vivo tumor uptake of C3-7α-[(18)F]FES was significantly higher (unpaired t test with Welch's correction; p=0.015) in ER-positive T-47D tumors (2.3%ID/g±0.4%ID/g) than ER-negative MDA-MB-231 tumors (0.9%ID/g±0.1%ID/g). CONCLUSIONS: Although extensive metabolism was observed in rodents, C3-7α-[(18)F]FES showed promising results for quantitative analysis of ER density in vivo. However, the selective uptake of C3-7α-[(18)F]FES was lower than that of 16α-[(18)F]FES. Further optimizations and structure-activity relationship studies of the C-7α-substituted estradiol are needed.

Positive regulation of Rho GTPase activity by RhoGDIs as a result of their direct interaction with GAPs.

BACKGROUND: Rho GTPases function as molecular switches in many different signaling pathways and control a wide range of cellular processes. Rho GDP-dissociation inhibitors (RhoGDIs) regulate Rho GTPase signaling and can function as both negative and positive regulators. The role of RhoGDIs as negative regulators of Rho GTPase signaling has been extensively investigated; however, little is known about how RhoGDIs act as positive regulators. Furthermore, it is unclear how this opposing role of GDIs influences the Rho GTPase cycle. We constructed ordinary differential equation models of the Rho GTPase cycle in which RhoGDIs inhibit the regulatory activities of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) by interacting with them directly as well as by sequestering the Rho GTPases. Using this model, we analyzed the role of RhoGDIs in Rho GTPase signaling. RESULTS: The model constructed in this study showed that the functions of GEFs and GAPs are integrated into Rho GTPase signaling through the interactions of these regulators with GDIs, and that the negative role of GDIs is to suppress the overall Rho activity by inhibiting GEFs. Furthermore, the positive role of GDIs is to sustain Rho activation by inhibiting GAPs under certain conditions. The interconversion between transient and sustained Rho activation occurs mainly through changes in the affinities of GDIs to GAPs and the concentrations of GAPs. CONCLUSIONS: RhoGDIs positively regulate Rho GTPase signaling primarily by interacting with GAPs and may participate in the switching between transient and sustained signals of the Rho GTPases. These findings enhance our understanding of the physiological roles of RhoGDIs and Rho GTPase signaling.

tRNA modifying enzymes, NSUN2 and METTL1, determine sensitivity to 5-fluorouracil in HeLa cells.

Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD). The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU) whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer.

Ferret-mouse differences in interkinetic nuclear migration and cellular densification in the neocortical ventricular zone.

The thick outer subventricular zone (OSVZ) is characteristic of the development of human neocortex. How this region originates from the ventricular zone (VZ) is largely unknown. Recently, we showed that over-proliferation-induced acute nuclear densification and thickening of the VZ in neocortical walls of mice, which lack an OSVZ, causes reactive delamination of undifferentiated progenitors and invasion by these cells of basal areas outside the VZ. In this study, we sought to determine how VZ cells behave in non-rodent animals that have an OSVZ. A comparison of mid-embryonic mice and ferrets revealed: (1) the VZ is thicker and more pseudostratified in ferrets. (2) The soma and nuclei of VZ cells were horizontally and apicobasally denser in ferrets. (3) Individual endfeet were also denser on the apical (ventricular) surface in ferrets. (4) In ferrets, apicalward nucleokinesis was less directional, whereas basalward nucleokinesis was more directional; consequently, the nuclear density in the periventricular space (within 16 µm of the apical surface) was smaller in ferrets than in mice, despite the nuclear densification seen basally in ferrets. These results suggest that species-specific differences in nucleokinesis strategies may have evolved in close association with the magnitudes and patterns of nuclear stratification in the VZ.