Clustering autism: using neuroanatomical differences in 26 mouse models to gain insight into the heterogeneity.

Autism is a heritable disorder, with over 250 associated genes identified to date, yet no single gene accounts for >1-2% of cases. The clinical presentation, behavioural symptoms, imaging and histopathology findings are strikingly heterogeneous. A more complete understanding of autism can be obtained by examining multiple genetic or behavioural mouse models of autism using magnetic resonance imaging (MRI)-based neuroanatomical phenotyping. Twenty-six different mouse models were examined and the consistently found abnormal brain regions across models were parieto-temporal lobe, cerebellar cortex, frontal lobe, hypothalamus and striatum. These models separated into three distinct clusters, two of which can be linked to the under and over-connectivity found in autism. These clusters also identified previously unknown connections between Nrxn1α, En2 and Fmr1; Nlgn3, BTBR and Slc6A4; and also between X monosomy and Mecp2. With no single treatment for autism found, clustering autism using neuroanatomy and identifying these strong connections may prove to be a crucial step in predicting treatment response.

Pharmacokinetics and disposition of CS-8958, a long-acting prodrug of the novel neuraminidase inhibitor laninamivir in rats.

CS-8958, a prodrug of laninamivir (R-125489), is currently under development as an inhaled anti-influenza drug. In this study, the pharmacokinetics and disposition of CS-8958 were characterized in rats. After intratracheal administration of 14C-CS-8958, radioactivity was retained over long periods in the target tissues (trachea and lung) as its active metabolite R-125489 - 19.12% of the dose was retained in the lung at 24 h. After intratracheal administration of CS-8958, plasma R-125489 concentration was slowly eliminated, and its half-life (14.1 h) was considerably longer than that after intravenous administration of R-125489. The radioactivity of intratracheally administered 14C-CS-8958 was mainly excreted into the urine (67.5% of dose), and this excretion lasted over long periods. R-125489 accounted for most of the urinary radioactivity recovered after 24 h. These results demonstrated that CS-8958 administered intratracheally to rats was converted/hydrolysed to R-125489 in the target tissues, and that the R-125489 was slowly excreted into the urine via an absorption rate-limiting process. Such distinctive pharmacokinetics attributed to the slow release of R-125489 suggests the potential for a long-acting anti-influenza drug.

Bulk electronic structure of the antiferromagnetic superconducting phase in ErNi2B2C.

We have performed temperature- (T-)dependent laser-photoemission spectroscopy of the antiferromagnetic (AF) superconductor ErNi2B2C to study the electronic-structure evolution reflecting the interplay between antiferromagnetism and superconductivity. The spectra at the superconducting (SC) phase show a very broad spectral shape. A T-dependent SC gap shows a sudden deviation from the BCS prediction just below TN. This observation can be explained well by the theoretical model and thus represents the characteristic bulk electronic structure of the AF SC phase for the first time.

A mouse model of a human multiple GIST family with KIT-Asp820Tyr mutation generated by a knock-in strategy.

Several families exhibiting multiple gastrointestinal stromal tumours (GISTs) and germline c-kit gene mutations at exons 8, 11, 13, or 17 have been reported. These patients also exhibit diffuse hyperplasia of the interstitial cells of Cajal (ICCs) as a pre-existing lesion of multiple GISTs. We generated a mouse model of a family with germline c-kit gene mutation at exon 17, and compared the phenotypes between the mice and humans. The mouse counterpart (KIT-Asp818Tyr) of the human KIT-Asp820Tyr mutation was transmitted into germline by a knock-in strategy. Mating of male and female heterozygotes (KIT-Asp818Tyr/+) resulted in the generation of homozygotes (KIT-Asp818Tyr/KIT-Asp818Tyr). Histological examination revealed that all heterozygotes had both a small KIT-positive mesenchymal tumour at the caecum, consistent with GIST, and KIT-positive diffuse spindle-shaped cell proliferation in the distal oesophagus, stomach, proximal duodenum, and colon consistent with ICC hyperplasia. All homozygotes exhibited a larger caecal tumour and more prominent spindle-shaped cell proliferation compared with the heterozygous mice, and they usually died within 10 weeks after birth, likely due to ileus. The small intestine of both genotypes showed no apparent morphological abnormality, and autonomous contraction of the ileal segments appeared normal. Western blotting demonstrated that the caecal tumours expressed phosphorylated KIT, MAPK, Stat1, and Stat5. These mutant mice are considered to be useful for further investigation of the mechanism of GIST development as a result of ICC hyperplasia and for assessment of the in vivo effects of drugs against molecular targets.

Therapeutic RNA interference of malignant melanoma by electrotransfer of small interfering RNA targeting Mitf.

Microphthalmia-associated transcription factor (Mitf) is critically involved in melanin synthesis as well as differentiation of cells of the melanocytic lineage. Some earlier studies suggested that Mitf is also essential in the survival of melanoma cells, but this notion remains controversial. We synthesized short interfering RNA (siRNA) duplexes corresponding to the mitf sequence and transfected them into B16 melanoma. Lipid-mediated transfection in vitro of Mitf-specific siRNA resulted in specific downregulation of Mitf and of the tyrosinase that is a transcriptional target of Mitf. This treatment also remarkably reduced the viability of melanoma cells by inducing apoptosis. To examine the potential feasibility of RNAi therapy against melanoma, B16 cells were subcutaneously injected into syngenic mice and siRNA was transfected into the pre-established tumor by means of electroporation. The Mitf-specific siRNA drastically reduced outgrowth of subcutaneous melanoma, while nonspecific siRNA failed to affect tumor progression. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-based analysis of tumor specimens demonstrated that the tumor cells transfected with Mitf-siRNA effectively underwent apoptosis in vivo. The present results indicate that Mitf plays important roles in melanoma survival. Intratumor electrotransfer of Mitf-specific siRNA may provide a powerful strategy for therapeutic intervention of malignant melanoma.

Role(s) of nucleoli and phosphorylation of ribosomal protein S6 and/or HSP27 in the regulation of muscle mass.

Effects of 14 days of hindlimb unloading or synergist ablation-related overloading with or without deafferentation on the fiber cross-sectional area, myonuclear number, size, and domain, the number of nucleoli in a single myonucleus, and the levels in the phosphorylation of the ribosomal protein S6 (S6) and 27-kDa heat shock protein (HSP27) were studied in rat soleus. Hypertrophy of fibers (+24%), associated with increased nucleolar number (from 1-2 to 3-5) within a myonucleus and myonuclear domain (+27%) compared with the preexperimental level, was induced by synergist ablation. Such phenomena were associated with increased levels of phosphorylated S6 (+84%) and HSP27 (+28%). Fiber atrophy (-52%), associated with decreased number (-31%) and domain size (-28%) of myonuclei and phosphorylation of S6 (-98%) and HSP27 (-63%), and with increased myonuclear size (+19%) and ubiquitination of myosin heavy chain (+33%, P > 0.05), was observed after unloading, which inhibited the mechanical load. Responses to deafferentation, which inhibited electromyogram level (-47%), were basically similar to those caused by hindlimb unloading, although the magnitudes were minor. The deafferentation-related responses were prevented and nucleolar number was even increased (+18%) by addition of synergist ablation, even though the integrated electromyogram level was still 30% less than controls. It is suggested that the load-dependent maintenance or upregulation of the nucleolar number and/or phosphorylation of S6 and HSP27 plays the important role(s) in the regulation of muscle mass. It was also indicated that such regulation was not necessarily associated with the neural activity.

Ubiquitous V-shape density of states in a mixed state of clean limit type II superconductors.

It is demonstrated theoretically and experimentally that the low energy density of states N(E) is described by a singular V-shape form N(E)=N(0)(H)+alpha|E|+O(E2) for all clean superconductors in a vortex state, irrespective of the underlying gap structure. The linear term alpha|E| which has not been recognized so far is obtained by exactly evaluating the vortex contribution. Based on microscopic Eilenberger theory N(E) is evaluated for the isotropic gap, line, and point-node gaps to yield a V-shape N(E). Scanning tunneling spectroscopy-STM experiments on NbSe2 and YNi2B2C give direct evidence for this. We provide arguments on the significance of this finding and on the relevance to other experiments.

Differential effects of sex steroid hormones on the expression of multiple first exons including a novel first exon of prolactin receptor gene in the rat liver.

In addition to the known four alternative first exons E1(1), E1(2), E1(3) and E1(4) of the rat prolactin receptor (PRL-R) gene, a novel first exon, E1(5), was identified by cDNA cloning of the 5'-end region of PRL-R mRNA in the rat liver. Genomic fragments containing E1(5) and its 5'- or 3'-flanking regions were also cloned from rat kidney genomic DNA. A sequence search for E1(5) revealed that E1(5) is located 49 kb upstream of exon 2 of the PRL-R gene in rat chromosome 2q16. RT-PCR analysis revealed that E1(5) was preferentially expressed in the liver, brain and kidney. Expression profiles of E1(2)-, E1(3)- and E1(5)-PRL-R mRNAs in the liver of male and female rats at 5 days of age and those at 8 weeks of age were examined by RT-PCR. The levels of E1(2)-PRL-R mRNA in the female rat increased remarkably in rats at 8 weeks of age compared with those at 5 days of age, and the levels of E1(5)-PRL-R mRNA in the male rat decreased markedly at 8 weeks of age compared with those at 5 days of age. In the female rat, the levels of E1(2)-PRL-R mRNA at 8 weeks of age decreased with ovariectomy performed at 4 weeks of age and recovered with the administration of beta-oestradiol. On the contrary, the levels of E1(5)-PRL-R mRNA increased with ovariectomy and decreased with the oestrogen treatment. In the male rat liver, the levels of E1(2)-PRL-R mRNA at 8 weeks of age increased strikingly with castration performed at 4 weeks of age and became undetectable with the administration of testosterone. The levels of E1(5)-PRL-R mRNA increased slightly with castration and were restored by testosterone treatment. Removal of gonadal tissues and sex steroid hormone treatment had no effect on the expression levels of E1(3)-PRL-R mRNA in both female and male rat livers. These results indicated that the expression of the PRL-R gene in the liver is regulated by the differential effects of sex steroid hormones on the transcription of the multiple first exons including the novel one.

Retreatment of cerebral aneurysms after guglielmi detachable coil embolization.

SUMMARY: Of 175 patients with 181 aneurysms initially treated with Guglielmi Detachable Coils (GDC), 25 were retreated. All retreatments except one were performed on previously ruptured aneurysms. Thirteen aneurysms were retreated because of recurrence, and 12 aneurysms were retreated to complete initial insufficient embolization. Sixteen patients underwent re-embolization and 9 patients were operated upon surgically. No complications related to the retreatment were experienced. We consider that repeat embolization should be attempted before considering surgical treatment in case that additional therapy is required. However, it is difficult to retreat aneurysms having wide necks. In regard to surgical clipping, aneurysms without a coil in the neck are easier to treat with primary clipping, whereas aneurysms with a coil mass in the neck are difficult to surgical clip. We have never used temporary clipping and coil extraction if the distance between the coil and the parent artery was wider than 2 mm. Emerging new embolic agents or devices and technical improvement might decrease the need for retreatment and increase long-term efficacy after endovascular treatment.

Reentrant vortex lattice transformation in fourfold symmetric superconductors.

The physics behind the rhombic-->square-->rhombic flux line lattice transformation in increasing fields is clarified on the basis of Eilenberger theory. We demonstrate that this reentrance observed in LuNi2B2C is due to intrinsic competition between the superconducting gap and Fermi surface anisotropies. The calculations not only reproduce it but also predict the not yet found lock-in transition to a square lattice with different orientation in a higher field. In view of the physical origin given, this sequence of transitions is rather generic to occur in fourfold symmetric superconductors.

The efficacy of acetic acid for glycogen repletion in rat skeletal muscle after exercise.

We examined the effect of acetic acid, the main component of vinegar, on glycogen repletion by using swimming-exercised rats. Rats were trained for 7 days by swimming. After an overnight fast, they were subjected to a 2-hr swimming exercise. Immediately afterward, they were given by gavage 2 ml of one of the following solutions: 30 % glucose only or 30 % glucose with 0.4 % acetic acid. Rats were sacrificed by decapitation before, immediately after exercise and 2 hours after the feeding. Exercise significantly decreased soleus and gastrocnemius glycogen content, and feeding significantly increased liver, soleus and gastrocnemius glycogen content. In soleus muscle, acetate feeding significantly increased glycogen content and the ratio of glycogen synthase in the I form (means +/- SEM: 4.04 +/- 0.41 mg/g-tissue and 47.0 +/- 0.7 %, respectively) in contrast to no acetate feeding (3.04 +/- 0.29 mg/g-tissue and 38.1 +/- 3.4 %, respectively). Thus, these findings suggest that the feeding of glucose with acetic acid can more speedily accelerate glycogen repletion in skeletal muscle than can glucose only.

Testis-specific and developmentally induced expression of a ghrelin gene-derived transcript that encodes a novel polypeptide in the mouse.

Ghrelin is a novel growth hormone-releasing peptide isolated from rat stomach. In the present study, we report expression of a ghrelin gene-derived transcript (GGDT) in the mouse testis. Analysis of GGDT cDNA revealed that the 68 bp sequence at the 5'-end was unique and the remaining 252 bp sequence was identical with the sequence encoded by exons 4 and 5 of mouse ghrelin gene. The 5'-unique sequence encoded 12 amino acid residues being in-frame with the C-terminal 42 amino acid sequence of mouse ghrelin. The 54-amino-acid polypeptide encoded by GGDT contained no apparent signal peptide sequence but possessed a nuclear localization signal-like sequence. Ghrelin mRNA was extensively expressed in the stomach, while GGDT was expressed only in the testis. The 5'-unique sequence of GGDT was identified between exons 3 and 4 of the ghrelin gene, indicating that GGDT was generated by alternative usage of the 68 bp exon as the testis-specific first exon. The GGDT expression in the testis was initiated and increased after 2 weeks of postnatal period. These results indicate that the expression of GGDT is regulated in testis-specific and developmental stage-specific manners.

Acetic acid feeding enhances glycogen repletion in liver and skeletal muscle of rats.

To investigate the efficacy of the ingestion of vinegar in aiding recovery from fatigue, we examined the effect of dietary acetic acid, the main component of vinegar, on glycogen repletion in rats. Rats were allowed access to a commercial diet twice daily for 6 d. After 15 h of food deprivation, they were either killed immediately or given 2 g of a diet containing 0 (control), 0.1, 0.2 or 0.4 g acetic acid/100 g diet for 2 h. The 0.2 g acetic acid group had significantly greater liver and gastrocnemius muscle glycogen concentration than the control group (P < 0.05). The concentrations of citrate in this group in both the liver and skeletal muscles were >1.3-fold greater than in the control group (P > 0.1). In liver, the concentration of xylulose-5-phosphate in the control group was significantly higher than in the 0.2 and 0.4 g acetic acid groups (P < 0.01). In gastrocnemius muscle, the concentration of glucose-6-phosphate in the control group was significantly lower and the ratio of fructose-1,6-bisphosphate/fructose-6-phosphate was significantly higher than in the 0.2 g acetic acid group (P < 0.05). This ratio in the soleus muscle of the acetic acid fed groups was <0.8-fold that of the control group (P > 0.1). In liver, acetic acid may activate gluconeogenesis and inactivate glycolysis through inactivation of fructose-2,6-bisphosphate synthesis due to suppression of xylulose-5-phosphate accumulation. In skeletal muscle, acetic acid may inhibit glycolysis by suppression of phosphofructokinase-1 activity. We conclude that a diet containing acetic acid may enhance glycogen repletion in liver and skeletal muscle.

Organization of the mouse ghrelin gene and promoter: occurrence of a short noncoding first exon.

Ghrelin is a growth hormone-releasing peptide recently discovered in the stomach of rat and human as an endogenous ligand for growth hormone-secretagogue receptor. In the present study, a full-length cDNA for mouse ghrelin has been cloned from the stomach using the oligo-capping and rapid amplification methods, and the organization of its gene and promoter has been analyzed. The mouse ghrelin cDNA was 521 bp long, consisting of 44 bp 5'-noncoding region, 354 bp coding region encoding a pre-proghrelin composed of 117 amino acid residues and 123 bp 3'-noncoding region. The genomic sequence analysis has revealed that the mouse ghrelin gene consists of 5 exons and 4 introns. The first exon was revealed to be only 19 bp long presented at the noncoding region of cDNA. The identical 19 bp sequence was also found as the first exon at the 5'-end of full-length rat ghrelin cDNA obtained from the stomach. A TATA box-like sequence, TATATAA was localized 24 bp upstream of the transcription start site of the mouse ghrelin gene. The sequence of the 5'-promoter region of mouse ghrelin gene including the TATA-like sequence and short exon 1 was highly homologous to that of reported human ghrelin gene. These findings suggest that the structure of the promoter region including the short noncoding first exon and its transcriptional regulation are conserved among the mammalian ghrelin genes.

Recovery of purification-associated reduction in antigen-induced histamine release from rat peritoneal mast cells.

Immunoglobulin E (IgE)-dependent histamine release from purified rat peritoneal mast cells (PMC) is very low in comparison to that from a non-purified preparation (PEC). The reduced histamine release from PMC is recovered or potentiated by reconstitution with separated non-mast cells (NMC). In the present study, further characterization was undertaken to elucidate the mechanisms involved. Sensitized mast cells were recovered from peritoneal cavities of rats, and purified by density gradient centrifugation with Percoll. Effects of NMC reconstitution, membrane fraction of NMC, NMC incubation supernatant, adhesion molecules and extracellular matrix proteins on IgE-dependent histamine release from PMC were examined. IgE-dependent histamine release was significantly potentiated by NMC reconstitution to PMC. The potentiation was dependent on the concentration of NMC reconstituted and reached a plateau after 30 min incubation. Increasing concentration of PMC did not affect the histamine release. Membrane fraction prepared from NMC also potentiated PMC histamine release in a dose-dependent manner. The potentiation reached a plateau in 5 min. Furthermore, incubation supernatant of NMC potentiated PMC histamine release. Antibodies against intercellular adhesion molecule (ICAM)-1, lymphocyte function-associated antigen (LFA)-1, very late activation antigen (VLA)-1, VLA-4 and VLA-6, and fibronectin did not affect the potentiation of PMC histamine release by NMC reconstitution. Fibronectin, laminin and collagen failed to potentiate PMC histamine release. These results indicate that the membrane component(s) of NMC in the rat peritoneal cavity seems to modulate IgE-dependent histamine release from peritoneal mast cells of rats, and that the active molecule(s) may be released from NMC. Adhesion molecules such as ICAM-1, LFA-1 and VLA are not involved.

Effects of troglitazone and voluntary running on insulin resistance induced high fat diet in the rat.

It is well known that troglitazone and voluntary running have the capacity to improve insulin resistance. The purpose of this study was to evaluate the combination effect of troglitazone and voluntary running on insulin action. Female rats aged 7 weeks were divided into high-fat diet (HF), high-fat diet + troglitazone (0.3% in diet; Tg), high-fat diet + voluntary running (for 3 wks; Tr), high-fat diet + troglitazone + voluntary running (Tg-Tr), and control (C) groups. A sequential euglycemic clamp experiment with two different insulin infusion rates of 3.0 (L-clamp) and 30.0 mU/kg BW/min (H-clamp) was performed on these rats after an overnight fast. Blood glucose concentrations were kept at fasting levels by periodic adjustment of the intravenous glucose infusion rate during the clamp experiment. Glucose infusion rates (GIRs) calculated from 60 to 90, 150 to 180 min were regarded as an index of whole body insulin action. After the clamp experiment, we determined the amount of glycogen content in the gastrocnemius muscle. Fat feeding markedly reduced GIRs in both L- and H- clamp experiments compared with C. Troglitazone treatment did not improve high-fat induced insulin resistance. In both L- and H-clamp experiments, GIRs were increased by voluntary running compared with HF, and reached the same levels as in C. GIRs of Tg-Tr were not greater than those of Tr. Glycogen content in gastrocnemius muscle showed the same trend as the results for GIRs. Therefore, the combination effect of troglitazone and voluntary running on insulin action was not found, but the effect of voluntary running was shown in fat-induced insulin resistance.

Taxon-specific zeta -crystallin in Japanese tree frog (Hyla japonica) lens.

The present study demonstrated that the 38-kDa protein, instead of rho-crystallin (36 kDa), is expressed taxon specifically in the lens of Japanese tree frog (Hyla japonica). The 38-kDa protein was distinguished from rho-crystallin expressed in the lenses of bullfrog (Rana catesbeiana) and European common frog (Rana temporaria) immunochemically. Although the N terminus of the 38-kDa protein was blocked, the analyses of partial amino acid sequences showed that the protein was zeta-crystallin. Analysis of cDNA sequence encoding zeta-crystallin of the tree frog lens demonstrated that the deduced protein consisted of 329 amino acids including initial methionine and having 62.2 and 62.9% identity with zeta-crystallin of camel and guinea pig lenses, respectively. The molecular mass of the deduced structure was calculated to be 35,564 Da. zeta-Crystallin of the tree frog lens exhibited the intrinsic enzymatic activity of quinone reductase (EC, NADPH:quinone oxidoreductase). The crystallin specifically catalyzed the reduction of 9,10-phenanthrenequinone (Km, 42 microm) using NADPH (Km, 60 microm) as a cofactor. The enzymatic activity was inhibited by dicumarol, anti-coagulant drug, with IC50 of 4 microm. On gel filtration chromatography, the crystallin was recovered as 150-kDa molecular mass complex, indicating that the crystallin was homotetramer consisting of 38-kDa subunits. The crystallin gene was expressed specifically in the lens. These results show that taxon-specific crystallins such as zeta- and rho-crystallins may be available for the biochemical discrimination of Hyla- and Rana groups among frogs.