DHCR7 links cholesterol synthesis with neuronal development and axonal integrity.

The DHCR7 enzyme converts 7-DHC into cholesterol. Mutations in DHCR7 can block cholesterol production, leading to abnormal accumulation of 7-DHC and causing Smith-Lemli-Opitz syndrome (SLOS). SLOS is an autosomal recessive disorder characterized by multiple malformations, including microcephaly, intellectual disability, behavior reminiscent of autism, sleep disturbances, and attention-deficit/hyperactivity disorder (ADHD)-like hyperactivity. Although 7-DHC affects neuronal differentiation in ex vivo experiments, the precise mechanism of SLOS remains unclear. We generated Dhcr7 deficient (dhcr7-/-) zebrafish that exhibited key features of SLOS, including microcephaly, decreased neural stem cell pools, and behavioral phenotypes similar to those of ADHD-like hyperactivity. These zebrafish demonstrated compromised myelination, synaptic anomalies, and neurotransmitter imbalances. The axons of the dhcr7-/- zebrafish showed increased lysosomes and attenuated autophagy, suggesting that autophagy-related neuronal homeostasis is disrupted.

Differentiation Capacity of Porcine Skeletal Muscle-Derived Stem Cells as Intermediate Species between Mice and Humans.

Large animal experiments are important for preclinical studies of regenerative stem cell transplantation therapy. Therefore, we investigated the differentiation capacity of pig skeletal muscle-derived stem cells (Sk-MSCs) as an intermediate model between mice and humans for nerve muscle regenerative therapy. Enzymatically extracted cells were obtained from green-fluorescence transgenic micro-mini pigs (GFP-Tg MMP) and sorted as CD34+/45- (Sk-34) and CD34-/45-/29+ (Sk-DN) fractions. The ability to differentiate into skeletal muscle, peripheral nerve, and vascular cell lineages was examined via in vitro cell culture and in vivo cell transplantation into the damaged tibialis anterior muscle and sciatic nerves of nude mice and rats. Protein and mRNA levels were analyzed using RT-PCR, immunohistochemistry, and immunoelectron microscopy. The myogenic potential, which was tested by Pax7 and MyoD expression and the formation of muscle fibers, was higher in Sk-DN cells than in Sk-34 cells but remained weak in the latter. In contrast, the capacity to differentiate into peripheral nerve and vascular cell lineages was significantly stronger in Sk-34 cells. In particular, Sk-DN cells did not engraft to the damaged nerve, whereas Sk-34 cells showed active engraftment and differentiation into perineurial/endoneurial cells, endothelial cells, and vascular smooth muscle cells, similar to the human case, as previously reported. Therefore, we concluded that Sk-34 and Sk-DN cells in pigs are closer to those in humans than to those in mice.

Synthesis of Three-Dimensional (Di)Azatricyclododecene Scaffold and Its Application to Peptidomimetics.

A novel sp3 carbon-rich tricyclic 3D scaffold-based peptide mimetic compound library was constructed to target protein-protein interactions. Tricyclic framework 7 was synthesized from 9-azabicyclo[3,3,1]nonan-3-one (11) via a gold(I)-catalyzed Conia-ene reaction. The electron-donating group on the pendant alkyne of cyclization precursor 12 b-e was the key to forming 6-endo-dig cyclized product 7 with complete regioselectivity. Using the synthetic strategy for regioselective construction of bridged tricyclic framework 7, a diazatricyclododecene 3D-scaffold 8 a, which enables the introduction of substituents into the scaffold to mimic amino acid side chains, was designed and synthesized. The peptide mimetics 21 a-u were synthesized via step-by-step installation of three substituents on diazatricyclododecene scaffold 8 a. Compounds 21 a-h were synthesized as α-helix peptide mimics of hydrophobic ZZxxZ and ZxxZZ sequences (Z=Leu or Phe) and subjected to cell-based assays: antiproliferative activity, HIF-1 transcriptional activity which is considered to affect cancer malignancy, and antiviral activity against rabies virus. Compound 21 a showed the strongest inhibitory activity of HIF-1 transcriptional activity (IC50 =4.1±0.8 µM), whereas compounds 21 a-g showed antiviral activity with IC50 values of 4.2-12.4 µM, suggesting that the 3D-scaffold 8 a has potential as a versatile peptide mimic skeleton.

Potential involvement of drought-induced Ran GTPase CLRan1 in root growth enhancement in a xerophyte wild watermelon.

Enhanced root growth is known as the survival strategy of plants under drought. Previous proteome analysis in drought-resistant wild watermelon has shown that Ran GTPase, an essential regulator of cell division and proliferation, was induced in the roots under drought. In this study, two cDNAs were isolated from wild watermelon, CLRan1 and CLRan2, which showed a high degree of structural similarity with those of other plant Ran GTPases. Quantitative RT-PCR and promoter-GUS assays suggested that CLRan1 was expressed mainly in the root apex and lateral root primordia, whereas CLRan2 was more broadly expressed in other part of the roots. Immunoblotting analysis confirmed that the abundance of CLRan proteins was elevated in the root apex region under drought stress. Transgenic Arabidopsis overexpressing CLRan1 showed enhanced primary root growth, and the growth was maintained under osmotic stress, indicating that CLRan1 functions as a positive factor for maintaining root growth under stress conditions.

Potato yield enhancement through intensification of sink and source performances.

The combined total annual yield of six major crops (maize, rice, wheat, cassava, soybean, and potato; Solanum tuberosum L.) amounts to 3.1 billion tons. In recent years, staple crops have begun to be used as substitutes for fossil fuel and feedstocks. The diversion of crop products to fuels and industrial feedstocks has become a concern in many countries because of competition for arable lands and increased food prices. These concerns are definitely justified; however, if plant biotechnology succeeds in increasing crop yields to double the current yields, it will be possible to divert the surplus to purposes other than food without detrimental effects. Maize, rice, wheat, and soybean bear their sink organs in the aerial parts of the plant, and potato in the underground parts. Plants with aerial storage organs cannot accumulate products beyond their capacity to support the weight of these organs. In contrast, potato has heavy storage organs that are supported by the soil. In this mini-review, we introduce strategies of intensifying potato productivity and discuss recent advances in this research area.

The effects of corticotropin-releasing factor on motor learning.

Corticotropin-releasing factor (CRF) is mainly secreted from the hypothalamic paraventricular nuclei and plays a crucial role in stress-related responses. Recent studies have reported that CRF is a neuromodulator in the central nervous system. In the cerebellum, CRF is essential for the induction of long-term depression (LTD) at the parallel fiber-Purkinje cell synapses. Given that LTD is thought to be one of the fundamental mechanisms of motor learning, CRF may affect motor learning. However, the role of CRF in motor learning in vivo remains unclear. In this study, we aimed to examine the role of CRF in motor learning. This was achieved through a series of behavioral experiments involving the in vivo administration of CRF and its antagonists. Rats injected with CRF directly into the cerebellum exhibited superior performance on the rotarod test, especially during initial training phases, compared to control subjects. Conversely, rats receiving a CRF receptor antagonist demonstrated reduced endurance on the rotating rod compared to controls. Notably, CRF mRNA expression levels in the cerebellum did not show significant variance between the CRF-injected and control groups. These findings imply a critical role of endogenous CRF in cerebellar motor learning and suggest that exogenous CRF can augment this process. (199 words).

A novel mRNA decay inhibitor abolishes pathophysiological cellular transition.

In cells, mRNA synthesis and decay are influenced by each other, and their balance is altered by either external or internal cues, resulting in changes in cell dynamics. We previously reported that it is important that an array of mRNAs that shape a phenotype are degraded before cellular transitions, such as cellular reprogramming and differentiation. In adipogenesis, the interaction between DDX6 and 4E-T had a definitive impact on the pathway in the processing body (PB). We screened a library of α-helix analogs with an alkaloid-like backbone to identify compounds that inhibit the binding between DDX6 and 4E-T proteins, which occurs between the α-helix of structured and internally disordered proteins. IAMC-00192 was identified as a lead compound. This compound directly inhibited the interaction between DDX6 and 4E-T. IAMC-00192 inhibited the temporal increase in PB formation that occurs during adipogenesis and epithelial-mesenchymal transition (EMT) and significantly suppressed these cellular transitions. In the EMT model, the half-life of preexisting mRNAs in PBs was extended twofold by the compound. The novel inhibitor of RNA decay not only represents a potentially useful tool to analyze in detail the pathological conditions affected by RNA decay and how it regulates the pathological state. The identification of this inhibitor may lead to the discovery of a first-in-class RNA decay inhibitor drug.

Involvement of activator protein-1 family members in ß-catenin and p300 association on the genome of PANC-1 cells.

Wnt/ß-catenin is believed to regulate different sets of genes with different coactivators, cAMP response element-binding protein (CREB)-binding protein (CBP) or p300. However, the factors that determine which coactivators act on a particular promoter remain elusive. ICG-001 is a specific inhibitor for ß-catenin/CBP but not for ß-catenin/p300. By taking advantage of the action of ICG-001, we sought to investigate regulatory mechanisms underlying ß-catenin coactivator usage in human pancreatic carcinoma PANC-1 cells through combinatorial analysis of chromatin immunoprecipitation-sequencing and RNA-sequencing. CBP and p300 preferentially bound to regions with the TCF motif alone and with both the TCF and AP-1 motifs, respectively. ICG-001 increased ß-catenin binding to regions with both the TCF and AP-1 motifs, flanking the genes induced by ICG-001, concomitant with the increments of the p300 and AP-1 component c-JUN binding. Taken together, AP-1 possibly coordinates ß-catenin coactivator usage in PANC-1 cells. These results would further our understanding of the canonical Wnt/ß-catenin signaling divergence.

The influence of stimulus and behavioral histories on predictive control of smooth pursuit eye movements.

The smooth pursuit system has the ability to perform predictive feedforward control of eye movements. This study attempted to examine how stimulus and behavioral histories of past trials affect the control of predictive pursuit of target motion with randomized velocities. We used sequential ramp stimuli where the rightward velocity was fixed at 16 deg/s while the leftward velocity was either fixed (predictable) at one of seven velocities (4, 8, 12, 16, 20, 24, or 28 deg/s) or randomized (unpredictable). As a result, predictive pursuit responses were observed not only in the predictable condition but also in the unpredictable condition. Linear mixed-effects (LME) models showed that both stimulus and behavioral histories of the previous two or three trials influenced the predictive pursuit responses in the unpredictable condition. Intriguingly, the goodness of fit of the LME model was improved when both historical effects were fitted simultaneously rather than when each type of historical data was fitted alone. Our results suggest that predictive pursuit systems allow us to track randomized target motion using weighted averaging of the information of target velocity (stimulus) and motor output (behavior) in past time sequences.

Comprehensive exploration of chemical space using trisubstituted carboranes.

A total of 42 trisubstituted carboranes categorised into five scaffolds were systematically designed and synthesized by exploiting the different reactivities of the twelve vertices of o-, m-, and p-carboranes to cover all directions in chemical space. Significant inhibitors of hypoxia inducible factor transcriptional activitay were mainly observed among scaffold V compounds (e.g., Vi-m, and Vo), whereas anti-rabies virus activity was observed among scaffold V (Va-h), scaffold II (IIb-g), and scaffold IV (IVb) compounds. The pharmacophore model predicted from compounds with scaffold V, which exhibited significant anti-rabies virus activity, agreed well with compounds IIb-g with scaffold II and compound IVb with scaffold IV. Normalized principal moment of inertia analysis indicated that carboranes with scaffolds I-V cover all regions in the chemical space. Furthermore, the first compounds shown to stimulate the proliferation of the rabies virus were found among scaffold V carboranes.

Effects of smooth pursuit and second-order stimuli on visual motion prediction.

The purpose of this study was to determine whether smooth pursuit eye movements affect visual motion prediction using a time-to-contact task where observers anticipate the exact instant that a partially occluded target would coincide with a stationary object. Moreover, we attempted to clarify the influence of second-order motion on visual motion prediction during smooth pursuit. One target object moved to another stationary object (6 deg apart) at constant velocity of 3, 4, and 5 deg/s, and then the two objects disappeared 500 ms after the onset of target motion. The observers estimated the moment the moving object would overlap the stationary object and pressed a button. For the pursuit condition, both a Gaussian window and a random dots texture moved in the same direction at the same speed for the first-order motion, whereas a Gaussian window moved over a static background composed of random dots texture for the second-order motion. The results showed that the constant error of the time-to-contact shifted to a later response for the pursuit condition compared to the fixation condition, regardless of the object velocity. In addition, during smooth pursuit, the constant error for the second-order motion shifted to an earlier response compared to the first-order motion when the object velocity was 3 deg/s, whereas no significant difference was found at 4 and 5 deg/s. Therefore, our results suggest that visual motion prediction using a time-to-contact task is affected by both eye movements and motion configuration such as second-order motion.

Selective engagement of plasticity mechanisms for motor memory storage.

The number and diversity of plasticity mechanisms in the brain raises a central question: does a neural circuit store all memories by stereotyped application of the available plasticity mechanisms, or can subsets of these mechanisms be selectively engaged for specific memories? The uniform architecture of the cerebellum has inspired the idea that plasticity mechanisms like cerebellar long-term depression (LTD) contribute universally to memory storage. To test this idea, we investigated a set of closely related, cerebellum-dependent motor memories. In mutant mice lacking Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV), the maintenance of cerebellar LTD is abolished. Although memory for an increase in the gain of the vestibulo-ocular reflex (VOR) induced with high-frequency stimuli was impaired in these mice, memories for decreases in VOR gain and increases in gain induced with low-frequency stimuli were intact. Thus, a particular plasticity mechanism need not support all cerebellum-dependent memories, but can be engaged selectively according to the parameters of training.

Branched chitins as nonnatural immunomodulatory biopolymers: secretions of TNF-alpha and NO by direct stimulation of macrophages.

In view of the interesting properties of branched polysaccharides occurring in nature, biological activities of nonnatural branched chitins having beta-1,6-N-acetyl-D-glucosamine branches on the poly(beta-1,4-N-acetyl-D-glucosamine) backbone have been studied. The immunostimulatory activities of the branched chitins were determined and compared with those of lentinan, a beta-1,3-D-glucan having beta-1,6-D-glucose branches, using the mouse macrophagelike cell line RAW264.7 in vitro. The secretions of the tumor necrosis factor and nitric oxide proved to be significantly higher with the branched chitins than with lentinan. Moreover, when interferon-gamma was used in conjunction with the branched chitins on macrophage treatment, a marked augmentation of nitric oxide production was observed. These results are interpreted as the direct stimulation of macrophages by the branched chitins, and the distinctive activities suggest the possibility of developing new types of polysaccharide antitumor agents.

ß-catenin signaling inhibitors ICG-001 and C-82 improve fibrosis in preclinical models of endometriosis.

Endometriosis exhibits unique characteristics, such as fibrosis, resistance to apoptosis, and promotion of cell proliferation; however, its pathophysiology is not fully understood. Recurrence rates after treatment are high, and the progression risk continues until menopause; hence, more effective therapy for endometriosis is needed. CREB-binding protein (CBP)/ß-catenin signaling inhibitors have demonstrated antifibrogenetic effects in liver, lung, and skin diseases. The present study evaluated the effects of two CBP/ß-catenin signaling inhibitors, ICG-001 and C-82, on the progression of endometriosis using endometriotic cyst stromal cells from the ovary and normal endometrial stromal cells from the uterus. ICG-001 was also evaluated in a mouse model. ICG-001 and C-82 inhibited cell proliferation, fibrogenesis, and cell migration, and promoted apoptosis in vitro. ICG-001 inhibited the growth of endometriotic lesions in the mouse model. CBP/ß-catenin signaling plays an important role in the pathophysiology of endometriosis. Inhibiting the CBP/ß-catenin signal can be a therapeutic target for endometriosis.

Selective Inhibition of ß-Catenin/Co-Activator Cyclic AMP Response Element-Binding Protein-Dependent Signaling Prevents the Emergence of Hapten-Induced Atopic Dermatitis-Like Dermatitis.

BACKGROUND: The canonical Wnt/ß-catenin signaling pathway is a fundamental regulatory system involved in various biological events. ICG-001 selectively blocks the interaction of ß-catenin with its transcriptional co-activator cyclic AMP response element-binding protein (CBP). Recent studies have provided convincing evidence of the inhibitory effects of ICG-001 on Wnt-driven disease models, such as organ fibrosis, cancer, acute lymphoblastic leukemia, and asthma. However, the effects of ICG-001 in atopic dermatitis (AD) have not been investigated. OBJECTIVE: To investigate whether ß-catenin/CBP-dependent signaling was contributed in the pathogenesis of AD and ICG-001 could be a therapeutic agent for AD. METHODS: We examined the effects of ICG-001 in an AD-like murine model generated by repeated topical application of the hapten, oxazolone (Ox). ICG-001 or vehicle alone was injected intraperitoneally every day during the development of AD-like dermatitis arising from once-daily Ox treatment. RESULTS: Ox-induced AD-like dermatitis characterized by increases in transepidermal water loss, epidermal thickness, dermal thickness accompanied by increased myofibroblast and mast cell counts, and serum levels of thymic stromal lymphopoietin and thymus and activation-regulated chemokine, and decreases in stratum corneum hydration, were virtually normalized by the treatment with ICG-001. Elevated serum levels of periostin tended to be downregulated, without statistical significance. CONCLUSION: These results suggest that ß-catenin/CBP-dependent signaling might be involved in the pathogenesis of AD and could be a therapeutic target.

Quality evaluation of Angelica acutiloba Kitagawa roots by 1H NMR-based metabolic fingerprinting.

The prices of Angelica acutiloba Kitagawa (yamato-toki) and A. acutiloba Kitagawa var. sukiyamae Hikino (hokkai-toki) are now mainly determined according to the sensory quality determined by experts in addition to the physical properties. This method provides a low reliability result for differentiating and qualifying their qualities. In addition, the quality in terms of pharmacological efficiency is not taken into account for consideration in the ordinary sensory method. A combination of a 1H NMR technique and a multivariate analysis was preliminarily applied for the quality evaluation of both toki roots with regard to their geographical and variety differences. A broad range of metabolites was detected by a single-run 1H NMR spectrometry. Partial least-squares discrimination analysis (PLS-DA), a pattern recognition method, was applied to the 1H NMR spectra of aqueous extracts of toki samples having different sensory qualities. The PLS-DA result showed a clear clustering corresponding to the cultivation area between toki samples cultivated in Hokkaido (Japan) and those cultivated in the southern part of China and the Nara prefecture (Japan), while there was no separation corresponding to the toki's variety and sensory qualities, indicating the inconsistency of the sensory evaluation result. The chemical metabolites contributing to the discrimination of toki samples in relation to pharmacological and sensory properties were reported for the first time. A reliable multivariate calibration model used to predict the sensory quality was successfully carried out by PLS regression.

Tailoring the peptide-binding specificity of an RNA by combinations of specificity-altering mutations.

In this study, the ability to tailor the peptide-binding specificity of an RNA was investigated. First, variants of the Rev-response element (RRE) RNA with different specificities toward the natural binding partner, Rev, and two RRE-binding aptamers, the RSG-1.2 and the Kl peptides, were identified. Next, hybrid RRE mutants with combinations of two sets of specificity-altering substitutions were tested for peptide-binding specificity. It was shown that in most cases the results of the combination of individual mutations were of an additive nature, therefore providing a way to manipulate the peptide-binding specificity of an RNA in a predictable manner.

Optogenetic Control of Synaptic AMPA Receptor Endocytosis Reveals Roles of LTD in Motor Learning.

Long-term depression (LTD) of AMPA-type glutamate receptor (AMPA receptor)-mediated synaptic transmission has been proposed as a cellular substrate for learning and memory. Although activity-induced AMPA receptor endocytosis is believed to underlie LTD, it remains largely unclear whether LTD and AMPA receptor endocytosis at specific synapses are causally linked to learning and memory in vivo. Here we developed a new optogenetic tool, termed PhotonSABER, which enabled the temporal, spatial, and cell-type-specific control of AMPA receptor endocytosis at active synapses, while the basal synaptic properties and other forms of synaptic plasticity were unaffected. We found that fiberoptic illumination to Purkinje cells expressing PhotonSABER in vivo inhibited cerebellar motor learning during adaptation of the horizontal optokinetic response and vestibulo-ocular reflex, as well as synaptic AMPA receptor decrease in the flocculus. Our results demonstrate that LTD and AMPA receptor endocytosis at specific neuronal circuits were directly responsible for motor learning in vivo. VIDEO ABSTRACT.

Bis(allixinato)oxovanadium(IV) complex is a potent antidiabetic agent: studies on structure-activity relationship for a series of hydroxypyrone-vanadium complexes.

There is an urgent medical need for orally effective drugs to replace insulin injections for the treatment of diabetes mellitus. Vanadium complexes with insulin-mimetic activities have recently been proposed as candidates as new antidiabetic drugs. Following in vitro and in vivo studies on a group of bis(3-hydroxy-4-pyronato)oxovanadium(IV) (1) complexes with VO(O4) coordination mode, bis(allixinato)oxovanadium(IV) (3) which contains allixin, a garlic component, was found to be the most potent antidiabetic agent among them. Complex 3 with a high in vitro insulin-mimetic activity in terms of both free fatty acid (FFA)-release inhibitory and glucose-uptake enhancing activities in isolated rat adipocytes exhibited a high hypoglycemic effect in type 1 diabetic model mice by both intraperitoneal injections and oral administrations. Complex 3 is thus proposed to be one of the most effective candidates for antidiabetic therapy.

Unusual temperature dependence of enantioselectivity in asymmetric reductions by chiral NADH models.

[reaction: see text] Unusual stereoselectivity changes, i.e., enhancement and inversion of enantioselectivity with increasing temperature, were observed in the asymmetric reduction of methyl benzoylformate with chiral 1,4-dihydropyridines possessing amino acid residues as ligating chiral auxiliaries. The differential activation parameters, DeltaDeltaH(S-R) and DeltaDeltaS(S-R), obtained from the Eyring plots demonstrate that the entropy term controls the enantiodifferentiating step, accounting for the observed unique temperature dependencies.