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.

Amino acid sequence of rabbit muscle aldolase and the structure of the active center.

Elucidation of the amino acid sequence of fructose-1,6-bis-phosphate aldolase from rabbit muscle has made it possible to assign the positions of the functional groups known to play specific roles in the catalytic activity, and also to locate the buried, exposed, and active site cysteine residues. The results indicate that the middle portion of the polypeptide chain, including Cys-134, Cys-149, Cys-177, and Cys-l99, is buried in the native structure, with regions containing Cys-72, Lys-107, Lys-227, Cys-336, His-359, and the COOH-terminal residue (Tyr-361) folded into the active center of the enzyme, at or near the surface of the enzyme molecule.

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.

Regulation by physical training of enzyme activity and gene expression of branched-chain 2-oxo acid dehydrogenase complex in rat skeletal muscle.

We examined the effects of short-term (5 weeks) and long-term (12 weeks) physical training on actual and total activities, protein content and mRNA abundance of branched-chain 2-oxo acid dehydrogenase complex in rat skeletal muscle. The actual and total activities were significantly increased approximately 60% and approximately 40%, respectively, by long-term training. No effects of short-term training on activities were observed. The increase in the total activity corresponded to increased protein content of the E1 alpha and E2 components of the complex. On the other hand, mRNA abundance for E1 alpha and E2 were not affected by the training, but that for E1 beta was slightly, but significantly increased by both short-term and long-term trainings. These divergent alterations of the message levels for the subunits of the complex suggest that posttranslational regulatory mechanisms determine the amount of the complex in skeletal muscle. Since the complex is located in the mitochondrial matrix space, mitochondrial biogenesis in response to the training was examined by determining the content of mitochondrial DNA in the muscle. The mitochondrial DNA was proportionally increased with the total activity as well as the protein content of the complex, suggesting that expression of branched-chain 2-oxo acid dehydrogenase complex in skeletal muscle in response to physical training is associated with mitochondrial biogenesis.

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.

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.

Photo-oxidation of histidine residues in rat M1- and L-type pyruvate kinases.

Rat M1- and L-type pyruvate kinases were inactivated by photo-oxidation mediated by methylene blue at pH 8.0 and O degrees C according to first-order kinetics. The pH profiles of the inactivation rates of these isozymes showed that amino acid residues having a pK value of 7.0-7.5 were involved in the inactivation. Three histidine residues per subunit of M1- or L-type enzyme were destroyed by the photo-oxidation with complete loss of the enzyme activities. However, two of the three photo-oxidized histidine residues in the L-type enzyme were more important in the inactivation reaction. The kinetics of the partially inactivated L-type enzyme suggests that complete inactivation is achieved via an intermediate form having low affinity for phosphoenolpyruvate (PEP). These observations revealed the involvement of essential histidine residues of two different kinds in the catalytic mechanism of the L-type enzyme. In the photo-oxidation of M1-type enzyme, no intermediate form was observed. Addition of PEP or pyruvate to the reaction mixture markedly prevented the photo-oxidative inactivation of only the M1-type enzyme in the presence of K+ and Mg2+; the addition of ADP or ATP was ineffective even in the presence of both metal ions. This protective effect of PEP was counteracted by further addition of ATP but not by ADP. However, photo-oxidative inactivation of the L-type enzyme was not prevented even by the addition of PEP in the presence of both metal ions, owing to the low affinity for PEP at 0 degrees C, in spite of the presence of fructose 1,6-bisphosphate (Fru-1,6-P2).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequence around a cysteine residue in the active center of jack bean urease.

Cysteine residues in the active center of jack bean urease [EC 3.5.1.5] were modified with 14C-labeled diazonium-1H-tetrazole (DHT). The labeled enzyme was carboxymethylated with iodoacetic acid, and then hydrolyzed with trypsin. The tryptic digest was subjected to gel filtration on Sephadex G-50, yielding two radioactive fractions. The [14C]DHT-labeled peptide having a lower molecular weight, which was determined to be approximately 1,000 by the method of gel filtration, was further purified to homogeneity by ion-exchange chromatography on DEAE-Sephadex A-25. [14C]DHT-labeled cysteine was identified as cysteic acid after performic acid oxidation, and the amino acid sequence of the low-molecular-weight [14C]DHT-labeled peptide was determined to be Phe-Glu-Pro-Gly-Asp-Cys-Asn-Ser-Thr-Phe-Lys.

Binding of retinol-binding protein obtained from human urine with vitamin A derivatives and terpenoids.

Retinol-binding protein(RBP) was purified from fresh urine of patients suffering from Itai-Itai disease. The purified preparation contained two types of apo-RBP(Apo I and II) in equal amounts as major components (about 85% of the total RBP). The corresponding two retinol-binding forms (Holo I and II) were present as minor components (about 15% of the total)...

Insulin activation of pyruvate dehydrogenase complex is enhanced by exercise training.

We studied the effects of exercise training on the activity of the pyruvate dehydrogenase (PDH) complex in rat gastrocnemius muscle (experiment 1) and the response of the complex to glucose and insulin infusion (euglycemic clamp) in trained and sedentary rats (experiment 2). In experiment 1, half of the rats were randomly allocated as sedentary animals and the other half were trained by voluntary running exercise for 8 weeks. The total activity of the PDH complex was not affected by exercise training, and the activity state (proportion of the active form) of the PDH complex was decreased from 15.0%+/-2.4% to 7.5%+/-1.1% by exercise training. The activity of 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase ([3-HADH] an enzyme in beta-oxidation) was significantly higher in trained versus sedentary rats. In experiment 2, sedentary and trained rats were starved for 24 hours before performing the euglycemic clamp. Glucose and insulin infusion was performed by a euglycemic clamp (insulin infusion rate, 6 mU/kg/min) for 90 minutes. The PDH complex was inactivated to less than 1% in both sedentary and trained rats after 24 hours of starvation. The glucose infusion rate (GIR) during the euglycemic clamp was higher in trained versus sedentary rats. The euglycemic clamp resulted in activation of the PDH complex in both sedentary and trained rats, but the response of the PDH complex to the euglycemic clamp was significantly higher in trained rats (5.8%+/-0.5%) than in sedentary rats (2.9%+/-0.5%). These results suggest that exercise training promotes fatty acid oxidation in association with suppression of glucose oxidation in skeletal muscle under resting conditions, but increases the rate of carbohydrate oxidation when glucose flux into muscle cells is stimulated by insulin.

Exercise training prevents maturation-induced decreases in insulin receptor substrate-1 and phosphatidylinositol 3-kinase in rat skeletal muscle.

We have previously reported that exercise training prevents a maturation-induced decrease in insulin sensitivity and suggested that an improvement of insulin sensitivity by exercise training was attributable, in part, to an increase in insulin-sensitive GLUT-4 on the skeletal muscle plasma membrane. In this study, we examined the effects of maturation and exercise training on the gene expression and protein content of the components of post-insulin receptor signal transduction in rat skeletal muscle. Rats aged 3 weeks were sedentary or trained by voluntary running through 4 or 27 weeks of age, and then the rats in both the sedentary and trained groups were killed and the gastrocnemius muscle was immediately removed for analysis of mRNA and protein content. The concentration of mRNA and protein for insulin receptor substrate-1 (IRS-1) in sedentary rats significantly decreased with maturation (49% and 63%, respectively, at age 27 weeks v age 4 weeks), but in trained rats they did not decrease with maturation. Although the level of phosphatidylinositol 3-kinase (PI 3-kinase) mRNA in sedentary rats was not altered with maturation, PI 3-kinase protein in sedentary rats significantly decreased with maturation (73% at 27 weeks v 4 weeks). However, PI 3-kinase protein in trained rats did not decrease with maturation. These results suggest that the prevention of maturation-induced decreases in the protein content of IRS-1 and PI 3-kinase is involved in the mechanisms responsible for the improvement of insulin sensitivity by exercise training, and exercise training may affect transcriptional regulation of the IRS-1 gene and posttranscriptional regulation of PI 3-kinase expression.

Exercise training prevents maturation-induced decrease in insulin sensitivity.

We examined the effects of exercise training initiated before maturation or after maturation on insulin sensitivity and glucose transporter GLUT-4 content in membrane fractions of skeletal muscle. Female Wistar rats (4 wk of age) were divided into sedentary and exercise-trained groups. At 12 wk of age, a subset of the trained animals (Tr) was killed along with a subset of sedentary controls (Sed). One-half of the remaining sedentary animals remained sedentary (Sed-Sed) while the other half began exercise training (Sed-Tr). The remaining rats in the original trained group continued to train (Tr-Tr). Euglycemic clamp (insulin infusion rate at 6 mU.kg body wt-1. min-1) was performed at 4, 12, and 27 wk. After euglycemic clamp in all animals except the 4-wk-old, hindlimb (gastrocnemius and part of quadriceps) muscles were removed for preparation of membrane fractions. In sedentary rats, glucose infusion rate (GIR) during euglycemic clamp was decreased from 15.9 mg.kg-1.min-1 at 4 wk of age to 9.8 mg.kg-1.min-1 at 12 wk of age and 9.1 mg.kg-1.min-1 at 27 wk of age. In exercise-trained rats, the GIR was not significantly decreased by maturation (at 12 wk) and further aging (at 27 wk). Initiation of exercise after maturation restored the GIR at 27 wk of age to the same levels as these for the corresponding exercise-trained rats. GLUT-4 content in plasma and intracellular membrane fractions of hindlimb muscle obtained just after euglycemic clamp showed the same trend as the results of GIR. These results suggest that exercise training prevented the maturation-induced decrease in insulin sensitivity. Improvement of insulin sensitivity caused by exercise training was attributed, at least in part, to the increase in insulin-sensitive GLUT-4 on the plasma membrane in skeletal muscle.

Additive effects of estrogen deficiency and diabetes on bone mineral density in rats.

We investigated the combined effects of estrogen deficiency and diabetes on bone mineral density (BMD) and bone metabolism in rats. Ten-week-old, female rats were randomly divided into four groups: controls (C), an ovariectomized group (O), a streptozotocin-induced diabetic group (S), and a combined ovariectomy and streptozotocin-induced diabetic group (OS). The BMD of the lumbar spine and the femur were measured before grouping and at 23 weeks old. At the end of the experiment, blood samples were obtained via cardiac puncture, and bone gla protein (BGP), tartrate-resistant acid phosphatase (TRAP) and 1,25-dihydroxyvitamin D levels were measured. The rats in the C, O, S, and OS groups, in that order, had higher levels of BMD of the lumbar spine and femur at 23 weeks of age. The BGP levels in the S and OS groups were significantly lower than in C and O groups. Significantly higher 1,25-dihydroxyvitamin D was observed in the O group compared with the C, S and OS groups. No differences were obtained in TRAP among four groups. Our data suggest that the combined effects of estrogen deficiency and diabetes on BMD are not synergistic or counteractive but additive.

Selective potentiation of IGE-dependent histamine release from rat peritoneal mast cells by stem cell factor.

Effect of stem cell factor on histamine release from rat peritoneal mast cells was studied. Although stem cell factor did not evoke histamine release by itself, it clearly potentiated histamine release from sensitized mast cells caused by antigen, anti-IgE and concanavalin A. However, stem cell factor did not affect histamine release caused by compound 48/80, calcium ionophore A23187 and substance P. Although maximum potentiation of antigen-induced histamine release by stem cell factor was accomplished after 1-10 minute-preincubation, potentiation was decline after a longer incubation period. Potentiation of histamine release by phosphatidylserine and non-mast cells in the rat peritoneal cavity was incubation time-dependent. Potentiation by stem cell factor was additive to that by phosphatidylserine or non-mast cells. These results indicate that stem cell factor selectively potentiates IgE-dependent histamine release from rat peritoneal mast cells, and suggest that the mechanism involved is distinct from that of phosphatidylserine or non-mast cells in the rat peritoneal cavity.

Effects of aging on the activities of pyruvate dehydrogenase complex and its kinase in rat heart.

Effects of aging on the activities of heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase were examined using 7, 35 and 60 wk old rats. Aging did not affect the total activity of pyruvate dehydrogenase complex but decreased the activity state (percentage of active form) of the complex in rats under the fed condition (52%, 36% and 26% for 7, 35 and 60 wk old rats, respectively). This decrease in the complex activity with aging was suggested to be associated with an age-related decrease in the blood glucose disposal. Starvation for 24 h decreased the activity state to less than 3% in all of the age groups. The activity of pyruvate dehydrogenase kinase associated with the complex was not related to the alteration in the activity state of the complex; the kinase activity was slightly lower in 60 wk old rats than in the younger rats under the fed condition and activation of the kinase by starvation was greater in the younger rats. The mechanism for the decrease in activity of pyruvate dehydrogenase complex was discussed on the basis of glucose and fatty acid utilization of heart muscle cells.

Activities of liver pyruvate dehydrogenase complex and 3-hydroxyacyl-CoA dehydrogenase in sand rat (Psammomys obesus).

The sand rat (Psammomys obesus) is an animal model for non-insulin dependent diabetes mellitus, which is induced by a regular chow diet. The total activity of liver pyruvate dehydrogenase complex in the sand rats under normoglycemic and normoinsulinemic conditions was one half as high as that in the albino rats, but the activity of liver 3-hydroxyacyl-CoA dehydrogenase was more than 4 times greater in the former than in the latter, suggesting a low capacity for glucose oxidation and a high capacity for fatty acid oxidation in the sand rats. These metabolic conditions may be related to the predisposition of the animals towards diabetes. Diet-induced diabetes in the sand rats resulted in decreasing the active form of liver pyruvate dehydrogenase complex and in increasing the activity of liver 3-hydroxyacyl-CoA dehydrogenase, suggesting that the diabetic conditions further suppress glucose oxidation and promote fatty acid oxidation.

The abundance of mRNAs for pyruvate dehydrogenase kinase isoenzymes in brain regions of young and aged rats.

The abundance of mRNAs for pyruvate dehydrogenase kinase (PDK) isoenzymes in four brain regions of young (10 wk) and aged (50 wk) rats was investigated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNAs for PDK1, 2, and 4 were detected in all the regions examined. The level of PDK2 mRNA was the most abundant among the isoenzymes in all the brain regions when judged from the PCR cycles. The level of PDK1 mRNA was relatively high in cerebellum and cerebral cortex compared to medulla oblongata and hippocampus. Aging decreased the levels of mRNAs for PDK1 and 2 in cerebellum and increased the PDK2 mRNA in hippocampus and cerebral cortex. The level of PDK4 mRNA was not affected by aging. These results provide the first evidence suggesting that there is the regional difference in the abundance of mRNAs for PDK isoenzymes in rat brain and that the levels of mRNAs for the isoenzymes were affected by aging.

Case report of lymphoepithelioma-like carcinoma of the lung--lymphoid population consisting of cytotoxic T cells in resting state.

The present report describes a case of lymphoepithelioma-like carcinoma (LELC) of the lung and presents immunohistochemical and in situ hybridization (ISH) studies of the tumor. A 39-year-old Chinese woman, who was born in China and emigrated to Japan at the age of 29, suffered from a cough for 2 years and received a middle and lower lobectomy with mediastinal lymph node dissection after induction chemotherapy. The tumor consisted of undifferentiated carcinoma and areas of more differentiated squamous cell carcinoma with an intense lymphoid infiltrate. Serological studies and ISH studies showed EBV infection of the tumor. The immunophenotype of tumor-infiltrating T-lymphocytes (TITL) of the present case was examined immunohistochemically and was compared with that of an LELC case reported previously. Most CD3-positive T cells of TITL in both cases were labeled with both CD8 and TIA-1 but not with granzyme-B, indicating the TITL to be cytotoxic T lymphocytes (CTL) in the resting state. The lack of CTL activation at the tumor site might have been due to local inhibition of EBV-specific CTL responses such as T-cell anergy. Because the EBV-specific CTL derived from peripheral blood lymphocytes, in contrast to the TITL, may not be influenced by either tumor-produced suppressor factors or negative regulatory T cells, they may inhibit the hematogenous metastasis of EBV-positive LELC, possibly resulting in a better prognosis. Because LELC of the lung responded to preoperative chemotherapy in the present study, it may be useful for reducing the local tumor burden and facilitate subsequent local therapy, although the mechanism of chemosensitivity of LELC remains unknown.