Characterization of SPATA5-related encephalopathy in early childhood.

Mutations in SPATA5 have recently been shown to result in a phenotype of microcephaly, intellectual disability, seizures, and hearing loss in childhood. Our aim in this report is to delineate the SPATA5 syndrome as a clinical entity, including the facial appearance, neurophysiological, and neuroimaging findings. Using whole-exome sequencing and Sanger sequencing, we identified three children with SPATA5 mutations from two families. Two siblings carried compound heterozygous mutations, c.989_991del (p.Thr330del) and c.2130_2133del (p.Glu711Profs*21), and the third child had c.967T>A (p.Phe323Ile) and c.2146G>C (p.Ala716Pro) mutations. The three patients manifested microcephaly, psychomotor retardation, hypotonus or hypertonus, and bilateral hearing loss from early infancy. Common facies were a depressed nasal bridge/ridge, broad eyebrows, and retrognathia. Epileptic spasms or tonic seizures emerged at 6-12 months of age. Interictal electroencephalography showed multifocal spikes and bursts of asynchronous diffuse spike-wave complexes. Augmented amplitudes of visually evoked potentials were detected in two patients. Magnetic resonance imaging revealed hypomyelination, thin corpus callosum, and progressive cerebral atrophy. Blood copper levels were also elevated or close to the upper normal levels in these children. Clinical delineation of the SPATA5-related encephalopathy should improve diagnosis, facilitating further clinical and molecular investigation.

Efficacy and immunomodulatory actions of ONO-4641, a novel selective agonist for sphingosine 1-phosphate receptors 1 and 5, in preclinical models of multiple sclerosis.

ONO-4641 is a next-generation sphingosine 1-phosphate (S1P) receptor agonist selective for S1P receptors 1 and 5. The objective of the study was to characterize the immunomodulatory effects of ONO-4641 using preclinical data. ONO-4641 was tested in both in-vitro pharmacological studies as well as in-vivo models of transient or relapsing-remitting experimental autoimmune encephalomyelitis (EAE). In vitro, ONO-4641 showed highly potent agonistic activities versus S1P receptors 1 and 5 [half maximal effective concentration (EC(50) ) values of 0·0273 and 0·334 nM, respectively], and had profound S1P receptor 1 down-regulating effects on the cell membrane. ONO-4641 decreased peripheral blood lymphocyte counts in rats by inhibiting lymphocyte egress from secondary lymphoid tissues. In a rat experimental autoimmune encephalomyelitis (EAE) model, ONO-4641 suppressed the onset of disease and inhibited lymphocyte infiltration into the spinal cord in a dose-dependent manner at doses of 0·03 and 0·1 mg/kg. Furthermore, ONO-4641 prevented relapse of disease in a non-obese diabetic mouse model of relapsing-remitting EAE. These observations suggest that ONO-4641 may provide therapeutic benefits in the treatment of multiple sclerosis.

GATA-3 induces T helper cell type 2 (Th2) cytokine expression and chromatin remodeling in committed Th1 cells.

Committed T helper type 1 (Th1) and Th2 effector cells, resulting from chronic antigenic stimulation in interleukin (IL)-12 and IL-4, are implicated in the pathology of autoimmune and allergic diseases. Committed Th1 cells cannot be induced to change their cytokine profiles in response to antigenic stimulation and Th2 cytokine-inducing conditions. Here, we report that ectopic expression of GATA-3 induced Th2-specific cytokine expression not only in developing Th1 cells but also in otherwise irreversibly committed Th1 cells and a Th1 clone, HDK1. Moreover, cAMP, an inhibitor of cytokine production by Th1 cells, markedly augmented Th2 cytokine production in GATA-3-expressing Th1 cells. Ectopic expression of GATA-3 in developing Th1 cells, but not in Th1 clone HDK1, induced endogenous GATA-3, suggesting an autoregulatory mechanism for maintenance of GATA-3 expression in Th2 cells. Structure-function analyses of GATA-3 revealed that the NH(2)-terminal transactivation domain and the COOH-terminal zinc finger domain of GATA-3 were critical, whereas the NH(2)-terminal zinc finger domain was dispensable for the induction of IL-4. Both zinc fingers, however, were required for IL-5 induction. A Th2-specific DNaseI-hypersensitive site of the IL-4 locus was detected in GATA-3-expressing Th1 cells. Thus, GATA-3 can change the phenotype of committed Th1 cells, previously considered to be irreversible.

HAADF-STEM analysis of layered double perovskite La(2)CuSnO(6) grown epitaxially.

Structural observation of layered double perovskite oxide La(2)CuSnO(6) thin films grown epitaxially on SrTiO(3) is reported by high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). Particularly the transition layer at the interface was observed, and the first B site layer at the interface was found to be almost formed by the Cu atomic layer as the random structure, followed by formation of the layered structure. In addition, HAADF-STEM images indicate that the thin film is not single crystalline, but some irregular structures were observed to grow around the interface near atomic steps of the substrate of SrTiO(3). Therefore, the steps largely affect the growth process of the thin film.

Local analysis of the edge dislocation core in BaTiO(3) thin film by STEM-EELS.

The a <100> edge dislocation core formed in an epitaxial BaTiO(3) (BTO) thin film grown on a substrate was investigated by scanning transmission electron microscopy combined with electron energy-loss spectroscopy. Elemental analysis using core-loss spectrum indicates that the atomic ratios of O/Ti and Ba/Ti are decreased at the dislocation core. The near-edge fine structure of the oxygen K-edge recorded from the dislocation core differs slightly from that of relaxed BTO region, which suggests that Ba-O bonding is decreased at the dislocation core. The structure of the dislocation core is discussed using a high-angle annular dark-field image and the electron energy-loss spectroscopy results.

Effects of electron channeling in HAADF-STEM intensity in La2CuSnO6.

Atomic resolution imaging using the high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) can be applied to analyze the atomic structures of materials directly. This technique provides incoherent Z-contrast with the atomic number of the constituent elements. In the present work, unique contrasts that make intuitively interpreting the HAADF-STEM image in double perovskite oxide La(2)CuSnO(6) difficult were observed. Multislice simulation confirmed that this occurred as an effect of the channeling process of electrons in combination with the effect of Debye-Waller factors. This was confirmed because in the La(2)CuSnO(6) crystal, two independent Sn atoms and four independent La atoms in the unit cell had different Debye-Waller factors, and the La columns consisted of pairs of columns with a small separation, whereas the Sn atoms were arranged straight. Furthermore, the image contrast was examined by mutislice simulation, and two atomic La columns were separated in a projected plane and appeared as one column contrast using multislice simulation. As a result, the HAADF intensity did not decrease constantly with the increase in column separation, with the exception of a very thin sample, which could be interpreted by the specific change in the electron-channeling process.

A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) mainly stimulates proliferation and maturation of myeloid progenitor cells. Although the signal transduction pathways triggered by GM-CSF receptor (GMR) have been extensively characterized, the roles of GMR signals in differentiation have remained to be elucidated. To examine the relationship between receptor expression and differentiation of hemopoietic cells, we used transgenic mice (Tg-mice) that constitutively express human (h) GMR at almost all stages of hemopoietic cell development. Proliferation and differentiation of hemopoietic progenitors in bone marrow cells from these Tg-mice were analyzed by methylcellulose colony formation assay. High affinity GMR interacts with GM-CSF in a species-specific manner, therefore one can analyze the effects of hGMR signals on differentiation of mouse hemopoietic progenitors using hGM-CSF. Although mouse (m) GM-CSF yielded only GM colonies, hGM-CSF supported various types of colonies including GM, eosinophil, mast cell, erythrocyte, megakaryocyte, blast cell, and mixed hemopoietic colonies. Thus, the effects of hGM-CSF on colony formation more closely resembled mIL-3 than those of mGM-CSF. In addition, hGM-CSF generated a much larger number of blast cell colonies and mixed cell colonies than did mIL-3. hGM-CSF also generated erythrocyte colonies in the absence of erythropoietin. Therefore, GM-CSF apparently has the capacity to promote growth of cells of almost all hemopoietic cell lineages, if functional hGMR is present.

Hyaluronan activates cell motility of v-Src-transformed cells via Ras-mitogen-activated protein kinase and phosphoinositide 3-kinase-Akt in a tumor-specific manner.

We investigated the production of hyaluronan (HA) and its effect on cell motility in cells expressing the v-src mutants. Transformation of 3Y1 by v-src virtually activated HA secretion, whereas G2A v-src, a nonmyristoylated form of v-src defective in cell transformation, had no effect. In cells expressing the temperature-sensitive mutant of v-Src, HA secretion was temperature dependent. In addition, HA as small as 1 nM, on the other side, activated cell motility in a tumor-specific manner. HA treatment strongly activated the motility of v-Src-transformed 3Y1, whereas it showed no effect on 3Y1- and 3Y1-expressing G2A v-src. HA-dependent cell locomotion was strongly blocked by either expression of dominant-negative Ras or treatment with a Ras farnesyltransferase inhibitor. Similarly, both the MEK1 inhibitor and the kinase inhibitor clearly inhibited HA-dependent cell locomotion. In contrast, cells transformed with an active MEK1 did not respond to the HA. Finally, an anti-CD44-neutralizing antibody could block the activation of cell motility by HA as well as the HA-dependent phosphorylation of mitogen-activated protein kinase and Akt. Taken together, these results suggest that simultaneous activation of the Ras-mitogen-activated protein kinase pathway and the phosphoinositide 3-kinase pathway by the HA-CD44 interaction is required for the activation of HA-dependent cell locomotion in v-Src-transformed cells.

Tumor induction by a single subcutaneous injection of sterigmatocystin in newborn mice.

Sterigmatocystin, a mycotoxin produced by Aspergillus versicolor, Aspergillus sydowi, Aspergillus nidulans, and a species of Bipolaris, was given to newborn BALB/c X DBA/2F1 (hereafter referred to as CD2F1) mice by a single s.c. administration in 1% gelatin suspension. In an acute toxicity study, the maximum tolerated dose of sterigmatocystin was 5 mug/g body weight. In a chronic study, a single s.c. injection of 5, 1, or 0.5 mug/g body weight gave rise to high incidences of lung and liver adenomas when the animals were killed at the end of 1 year. The incidence of both tumors in mice at the dose of 5 mug/g body weight was statistically significant, and the incidences of lung tumor in female mice and of liver tumor in male mice at the dose of 1 mug/g body weight were also statistically significant, compared with tumors in control mice. Other tumors also were induced in treated mice (two malignant lymphomas and one adenoma of the submaxillary gland), in contrast to a zero incidence in vehicle control mice. These results confirm that a small quantity of sterigmatocystin induces tumors of lung and liver and that the dose of sterigmatocystin is related to the incidence of tumors in mice.

The Ras-mitogen-activated protein kinase pathway is critical for the activation of matrix metalloproteinase secretion and the invasiveness in v-crk-transformed 3Y1.

To search for the intracellular signaling pathway critical for the secretion of matrix metalloproteinases (MMP), we studied the effects of dominant negative Ras (S17N Ras) and dominant negative MEK1 (MEK1AA) expression in v-crk-transformed 3Y1. Expression of either S17N Ras or MEK1AA dramatically suppressed the augmented secretion of MMP-2 and MMP-9 in v-crk-transfected 3Y1. Similarly, a Ras farnesyltransferase inhibitor, manumycin A, and a MEK1 inhibitor, U0126, suppressed MMP secretion in a dose-dependent manner, whereas a PI3 kinase inhibitor, wortmannin, could not. In addition, the suppression of MMP secretion by S17N Ras showed good correlation with the inhibition of in vitro invasiveness of the cells. In contrast, expression of dominant negative C3G did not suppress MMP secretion, although it substantially blocked the c-Jun N-terminal kinase activation. Taken together, the Ras-MEK1 pathway, but not the C3G-JNK pathway, seems to play a key role in the activation of MMP secretion and, hence, the invasiveness of v-crk-transformed cells.

Suppression of cell spreading by v-Crk requires Ras-MEK-MAP kinase signaling.

We investigated the attachment and spreading of v-Crk-transformed cells, v-Crk3Y1, on fibronectin. Transformation by v-Crk virtually suppressed the spreading, but not the attachment, of cells on fibronectin. This suppression of cell spreading was not correlated with the suppression of integrin alpha5 and beta1 expression. However, the spreading of v-Crk3Y1 on fibronectin was dramatically restored by either expression of dominant-negative Ras or treatment with manumycin A, a Ras farnesyltransferase inhibitor. Moreover, both expression of dominant-negative MEK1 and treatment of cells with U0126, a MEK1 inhibitor, restored the cell spreading of v-Crk3Y1. In contrast, neither treatment with LY294002, a PI3K inhibitor, nor expression of dominant-negative C3G showed no effect on cell spreading on fibronectin. Taken together, our results suggest that, among multiple signaling pathways activated by v-Crk, the Ras-MEK1-MAP kinase cascade plays a pivotal role in the suppression of cell spreading on fibronectin, but C3G and the PI3 kinase do not.

v-Src suppresses SHPS-1 expression via the Ras-MAP kinase pathway to promote the oncogenic growth of cells.

We investigated the effect of cell transformation by v-src on the expression and tyrosine phosphorylation of SHPS-1, a putative docking protein for SHP-1 and SHP-2. We found that transformation by v-src virtually inhibited the SHPS-1 expression at mRNA level. While nontransforming Src kinases including c-Src, nonmyristoylated forms of v-Src had no inhibitory effect on SHPS-1 expression, transforming Src kinases including wild-type v-Src and chimeric mutant of c-Src bearing v-Src SH3 substantially suppressed the SHPS-1 expression. In cells expressing temperature sensitive mutant of v-Src, suppression of the SHPS-1 expression was temperature-dependent. In contrast, tyrosine phosphorylation of SHPS-1 was rather activated in cells expressing c-Src or nonmyristoylated forms of v-Src. SHPS-1 expression in SR3Y1 was restored by treatment with herbimycin A, a potent inhibitor of tyrosine kinase, or by the expression of dominant negative form of Ras. Contrary, active form of Mekl markedly suppressed SHPS-1 expression. Finally, overexpression of SHPS-1 in SR3Y1 led to the drastic reduction of anchorage independent growth of the cells. Taken together, our results suggest that the suppression of SHPS-1 expression is a pivotal event for cell transformation by v-src, and the Ras-MAP kinase cascade plays a critical role in the suppression.

The investigation of the structure of collagen in growth process by X-ray analysis and electron microscopy.

The structure of collagen in growth process has been investigated by X-ray analysis and electron microscopy using laying hen leg tendon collagen. On unstretching, the orientation of the band structure becomes better with age in birds younger than 4.5 months and is complete at ages above 4.5 months, whereas on stretching, the orientation is complete independent of age. This result suggests that the band structure may be complete by hatching. At approx. 4.5 months of age, the laying hen leg tendon collagen begins to harden from the lower end portion of the leg. On a macroscopic level, this hardening means that the collagen changes from the soft and flexible state to the rigid state. The higher-order structure of the hardened collagen consists of two domains: one is the domain in which the band structure remains, though it is not as clear as that of the collagen in the soft tendon, and another is the domain in which the band structure disappears and slender streaks having widths of 5-10 nm run along the fibril axis. It can be considered that this hardening might be caused by the increase of cross-links accompanied by calcification.

Molecular cloning and characterization of a novel human gene (NESCA) which encodes a putative adapter protein containing SH3.

A full-length cDNA encoding a novel protein was isolated and sequenced from a human placental cDNA library. This cDNA consists of 1990 bp and has a predicted open reading frame encoding 433 amino acids. It possesses an Src homology 3 (SH3) motif, a leucine zipper motif and no catalytic domain, suggesting that it seems to be an adapter protein. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 1q21-22.

Altered state dependence of c-type inactivation in the long and short forms of human Kv1.5.

Evidence from both human and murine cardiomyocytes suggests that truncated isoforms of Kv1.5 can be expressed in vivo. Using whole-cell patch-clamp recordings, we have characterized the activation and inactivation properties of Kv1.5DeltaN209, a naturally occurring short form of human Kv1.5 that lacks roughly 75% of the T1 domain. When expressed in HEK 293 cells, this truncated channel exhibited a V(1/2) of -19.5 +/- 0.9 mV for activation and -35.7 +/- 0.7 mV for inactivation, compared with a V(1/2) of -11.2 +/- 0.3 mV for activation and -0.9 +/- 1.6 mV for inactivation in full-length Kv.15. Kv1.5DeltaN209 channels exhibited several features rarely observed in voltage-gated K(+) channels and absent in full-length Kv1.5, including a U-shaped voltage dependence of inactivation and "excessive cumulative inactivation," in which a train of repetitive depolarizations resulted in greater inactivation than a continuous pulse. Kv1.5DeltaN209 also exhibited a stronger voltage dependence to recovery from inactivation, with the time to half-recovery changing e-fold over 30 mV compared with 66 mV in full-length Kv1.5. During trains of human action potential voltage clamps, Kv1.5DeltaN209 showed 30-35% greater accumulated inactivation than full-length Kv1.5. These results can be explained with a model based on an allosteric model of inactivation in Kv2.1 (Klemic, K.G., C.-C. Shieh, G.E. Kirsch, and S.W. Jones. 1998. Biophys. J. 74:1779-1789) in which an absence of the NH(2) terminus results in accelerated inactivation from closed states relative to full-length Kv1.5. We suggest that differential expression of isoforms of Kv1.5 may contribute to K(+) current diversity in human heart and many other tissues.

Study of mammalian selenocysteyl-tRNA synthesis with [75Se]HSe.

The mechanisms of the synthesis of mammalian selenocysteyl-(Scy)-tRNA were studied using [75SE]H2Se. H2Se was prepared from [75Se]selenite, glutathione, NADPH and glutathione reductase, and was purified by chromatography. It was confirmed that this H2Se was a Se donor in the reaction of the synthesis of Scy-tRNA. [75Se]Scy, liberated from aminoacyl-tRNA, was analyzed by TLC on silica gel an subsequent autoradiography. The activity of Scy-tRNA synthesis was found in the supernatant at 105,000 x g of the murine liver extract, but not in the precipitate. The supernatant was chromatographed on DEAE-cellulose, and the activity was eluted at a concentration of 0.17 M KCl. This position is at the front shoulder of the peak of seryl-tRNA synthetase which was eluted at 0.20 M KCl. Major serine tRNA(IGA) is not a substrate on which to synthesize Scy-tRNA, but natural opal suppressor serine tRNA is. On a chromatographic pattern of a Scy-tRNA preparation on Sephacryl S-200, the radioactivity of 75Se was eluted at the tRNA peak. This showed that Scy bound to tRNA. The active protein fraction from DEAE-cellulose did not contain tRNA kinase, therefore Scy-tRNA must be directly synthesized from seryl-tRNA, not through phosphoseryl-tRNA. This mechanism is similar to that seen in Escherichia coli [1991, J. Biol. Chem. 266, 6324].

The 5' untranslated region of the human cellular glutathione peroxidase gene is indispensable for its expression in COS-7 cells.

We studied the expression of the human cellular glutathione peroxidase (GPx) gene, from which a key enzyme containing selenocysteine (Scy) at the active site is produced. Expression of some human GPx gene mutants in COS-7 cells revealed that the 5' untranslated region (utr) was necessary for expression of the GPx gene, since mutant genes having 10 base pairs (bps) at the 5'utr (the complete had 311 bps) expressed GPx at very low levels. The genes with 311 or 408 bps at the 5'utr were better expressed than those having 257 bps. The GPx gene having 133 bps at the 3'utr (80 bps shorter than the entire length) was highly expressed. This deletion did not influence expression. We constructed some mutants in which 3 bases were altered at the upstream region of the Scy UGA codon in the frame of the GPx gene, by site-directed mutagenesis. GPx expression decreased but the expression was restored. Therefore, the upstream region of the in-frame Scy codon was not essential in the Scy decoding mechanisms. Finally, the 5'utr was essential for the expression of GPx gene. However, the deletion of a part of the 3'utr and the site-directed mutation upstream of the Scy codon did not show drastic effects on the expression.

Effects of exercise training and etidronate treatment on bone mineral density and trabecular bone in ovariectomized rats.

This study was designed to assess the effects of exercise training (Tr) following an etidronate treatment (E) on bone mineral density (BMD) of the femur and trabecular bone of the tibia in ovariectomized (ovx) rats. Female Wistar rats were ovariectomized (ovx) or sham-operated (sham) at 15 weeks of age and divided into five experimental groups: sham; ovx; ovx + E; ovx + Tr; ovx + E + Tr. Etidronate treatment of 5 mg/kg, 5 days/week was administered for 2 weeks and exercised on a treadmill for 30 m/min, 60 min/day, 5 days/week for 10 weeks. BMD of the femur and the trabecular bone area of the proximal tibia were significantly (p < 0.05) higher in E and/or Tr compared to ovx groups. However, the cortical region was not affected significantly by ovariectomy. The area partially filled with the trabecular bone at the constant width was observed only in the E rats. The number of osteoclasts in E group was significantly lower (p < 0.05) than in the ovx and ovx + Tr groups. The ovx + Tr rats had a higher number of osteoblasts (p < 0.05) than the ovx and ovx + E groups. There was a significant interaction between ovx + Tr and ovx + E on BMD in the proximal region of the femur (p < 0.05) and trabecular bone area of the tibia (p < 0.001). These results suggest that the etidronate treatment for 2 weeks beforehand influenced the effects of subsequent exercise training on maintaining the BMD in the proximal femur and the trabecular bone area of the tibia.

Smoking cessation increases the resistance of low-density lipoprotein to oxidation.

The effects of smoking cessation on the susceptibility to oxidation of low-density lipoprotein (LDL) was investigated in 14 men who quit smoking for 3 months. LDL was isolated and susceptibility of LDL to V-70 (4-methoxy-2,4-dimethylvalerinitrile)-mediated oxidation was assessed by measuring conjugated diene production at 234 nm, the lag phase being a measure of the resistance of LDL to oxidation. The mean duration of the lag phase became 1.9-fold longer after 3 months (P < 0.001). The result suggests that the increase in resistance of LDL to oxidation contributes to the reduction of the risk of coronary heart disease by smoking cessation.

Vitrification of hematopoietic progenitor cells obtained from human cord blood.

Human cord blood mononuclear cells (MNCs) purified by Ficoll-Hypaque gradient centrifugation were cryopreserved in a vitreous state. The vitrification solution was MEM-alpha medium containing 4-8 mol/l (M) ethylene glycol (EG). A pellet of 1.5-2.0 x 10(6) MNCs was added with 10 microliters of 1 M EG and then 50 microliters of high concentration EG at room temperature (the final concentration was 4-8 M EG). The cell suspension was directly plunged into liquid nitrogen, stored for various periods and rapidly warmed in a water bath at 37 degrees C. The vitrification solution was removed by addition of MEM-alpha medium containing 1 M sucrose. The present experiments demonstrated that a vitrification solution consisting of 8 M EG produced the highest recovery rate for MNCs (89.5 +/- 8.5%), CFU-GM (66.6 +/- 20.8%) and BFU-E (66.5 +/- 22.8%) and the highest Trypan blue viability (98.7 +/- 0.4%). This ultrarapid cryopreservation method may be useful for the preservation of hematopoietic progenitor cells.