Olanzapine increases hepatic glucose production through the activation of hypothalamic adenosine 5'-monophosphate-activated protein kinase.

AIMS: To investigate the mechanism of the metabolic disturbance induced by the atypical antipsychotic olanzapine, we examined whether adenosine 5'-monophosphate-activated protein kinase (AMPK) in the hypothalamus and hepatic glucose production are involved in the effect of olanzapine. METHODS: Male 6-week-old ICR mice were used. Blood glucose levels were determined by the glucose oxidase method. The mRNA levels of gluconeogenic or glycolytic enzymes were measured by reverse transcription polymerase chain reaction (RT-PCR). AMPK expression was measured by Western blotting. RESULTS: Systemic injection of olanzapine increased blood glucose levels in both unfasted and fasted mice. However, the increase in fasted mice was less than that in unfasted mice. Central administration of olanzapine also increased the blood glucose levels in unfasted mice, but not in fasted mice. In a pyruvate tolerance test, olanzapine significantly increased blood glucose levels. In addition, olanzapine increased the mRNA levels of glucose-6-phosphatase (G6Pase), a gluconeogenic enzyme, in the liver. Furthermore, olanzapine increased phosphorylated AMPK in the hypothalamus of unfasted mice, and olanzapine-induced hyperglycaemia was inhibited by the AMPK inhibitor compound C. Central administration of the AMPK activator AICAR significantly increased G6Pase mRNA levels in the liver and blood glucose levels. Moreover, both olanzapine- and AICAR-induced hyperglycaemia were attenuated by the ß-adrenergic receptor antagonist propranolol, suggesting that olanzapine and AICAR induce hepatic glucose production through the sympathetic nervous system. CONCLUSIONS: Our results indicate that olanzapine activates AMPK in the hypothalamus, which increases hepatic glucose production via the sympathetic nervous system.

Bifidobacterium breve B-3 exerts metabolic syndrome-suppressing effects in the liver of diet-induced obese mice: a DNA microarray analysis.

We previously reported that supplementation with Bifidobacterium breve B-3 reduced body weight gain and accumulation of visceral fat in a dose-dependent manner, and improved serum levels of total cholesterol, glucose and insulin in a mouse model of diet-induced obesity. In this study, we investigated the expression of genes in the liver using DNA microarray analysis and q-PCR to reveal the mechanism of these anti-obesity effects in this mouse model. Administration of B. breve B-3 led to regulated gene expression of pathways involved in lipid metabolism and response to stress. The results indicate that these regulations in the liver are related to the anti-metabolic syndrome effects of B. breve B-3.

Pyridoxal-aminoguanidine adduct is more effective than aminoguanidine in preventing neuropathy and cataract in diabetic rats.

We examined the ability of a pyridoxal-aminoguanidine adduct with both antiglycation and antioxidant activities in vitro to protect against neuropathy and cataract in streptozotocin-diabetic rats and compared the result with that of aminoguanidine. In vivo antiglycation and antioxidant activities were also compared between the adduct and aminoguanidine. Diabetic rats were given either of the compounds in their drinking water (9 mM) for 7 weeks. Neither compound affected body weight, blood glucose level or urine volume. The adduct, but not aminoguanidine, significantly improved motor nerve conduction velocity. The time to develop cataract was longer in adduct-treated rats than in untreated and aminoguanidine-treated rats. The increase in opacification of lenses in culture medium containing high glucose levels (55.5 mM) was more efficiently attenuated by the adduct than by aminoguanidine. Adduct and aminoguanidine similarly lowered glycated hemoglobin levels. The level of urinary 8-hydroxy-2'-deoxyguanosine, a marker of oxidative DNA damage, and the level of liver malondialdehyde plus 4-hydroxy-2-alkenals, a marker of tissue lipid peroxidation, both of which were elevated by diabetes, were significantly reduced by the adduct but not by aminoguanidine. These findings indicate that the pyridoxal-aminoguanidine adduct is superior to aminoguanidine in preventing diabetic neuropathy and cataracts, and we suggest that this may be at least partly due to the higher antioxidant activity of the former.

Comparison of post-translational modifications of alpha A-crystallin from normal and hereditary cataract rats.

In order to investigate the relationship between lens opacities and the various modifications of lens proteins, we analyzed and compared the properties of lens proteins of 85-day old normal Wistar rats and the hereditary cataract model, ICR/f rats. The present study identified many differences between normal and mutant lens proteins. In the ICR/f mutant rats, the relative amounts of gamma-crystallin decreased and high molecular weight (HMW) protein increased. Racemization and isomerization of Asp-151 of alpha A-crystallin was observed in the mutant ICR/f rats, and Met-1 of alpha A-crystallin was oxidized to methionine sulfoxide. These modifications were not found in the age-matched normal rats. These tendencies are consistent with aged and cataractous human lenses.

Effect of calpain on hereditary cataractous rat, ICR/f.

The crystallins in the lenses of ICR/f mutation rat, a known hereditary cataract model, were analyzed during cataractogenesis. Opacification of the mutant lenses was found to be accompanied by changes in crystallin structure and composition, including several deletions of the N-terminals of beta-crystallins and low molecular weight alpha- crystallins. Because similar deletions were observed when the soluble fraction of normal lens protein was incubated with calpain, we considered that calpain could be related to the deletions in mutant lenses. Although measurement of the content of calpain protein by the ELISA method revealed no significant difference between mutant and normal lenses, it was found that the concentrations of Ca2+ and K+ were different between the two lenses and that calpain activity was dependent on both ion concentrations. Endogenous m-calpain in the soluble fraction from normal lenses was activated by addition of 1 mm calcium chloride in the presence of 50 mm KCl (the same concentration as in mutant lenses), and insoluble protein was found in the fraction 1 d after calpain activation. On the other hand, the presence of 120 mm KCl (the concentration in normal lenses) inhibited calpain activity and prevented this insolubilization. These results suggest that calpain in mutant lenses is involved in the proteolysis of crystallins and the progression of cataract formation.

Calcium requirement estimated by balance study in elderly Japanese people.

The recommended dietary allowance (RDA) for calcium (Ca) of Japanese adults is proposed to be 600 mg/day, which is lower than those of other countries. In this report we estimated the Ca requirement and the RDA for Ca in elderly Japanese utilizing a Ca balance method. Subjects were 10 men aged 65-72 years and 10 women aged 62-77 years. Following a 14 day adaptation period, each participant was subjected to a low Ca diet (Ca 250 mg as a meal) for 6 days. After an interval of 2 weeks or more, another 14 day adaptation period was set and then a high Ca diet (Ca 250 mg as a meal and 600 mg as CaCO3) was served to the subjects for 6 days. Ca balance was calculated at each dose of Ca intake. Ca requirement was estimated by the intersection of the average Ca intake-retention diagram. Daily Ca requirement was 702 mg in the men and 788 mg in the women. The Ca requirement values were multiplied by 1.2 to obtain the RDA for Ca. As a result, RDA for Ca was 842 mg/day for men and 946 mg/day for women. When these values were normalized with the body weight, the RDA for Ca of Japanese and Caucasian women was similar (18.1 and 18.5 mg/kg body weight per day, respectively). Our results suggest the difference in Ca balance between the genders and among populations may be ascribed at least partly to differences in body size. In addition, body weight should be considered when comparing the RDAs among different populations.

DNA microarray analysis of cyanobacterial gene expression during acclimation to high light.

DNA microarrays bearing nearly all of the genes of the unicellular cyanobacterium Synechocystis sp PCC 6803 were used to examine the temporal program of gene expression during acclimation from low to high light intensity. A complete pattern is provided of gene expression during acclimation of a photosynthetic organism to changing light intensity. More than 160 responsive genes were identified and classified into distinct sets. Genes involved in light absorption and photochemical reactions were downregulated within 15 min of exposure to high light intensity, whereas those associated with CO(2) fixation and protection from photoinhibition were upregulated. Changes in the expression of genes involved in replication, transcription, and translation, which were induced to support cellular proliferation, occurred later. Several unidentified open reading frames were induced or repressed. The possible involvement of these genes in the acclimation to high light conditions is discussed.

The circuit of Papez in mesial temporal sclerosis: MRI.

We looked at abnormalities in the circuit of Papez in patients with the mesial temporal sclerosis (MTS). We reviewed the MRI studies of 15 patients with probable MTS, seeking changes in the fornix, mamillary body, mamillothalamic tract, thalamus and cingulate and parahippocampal gyri. We correlated any abnormalities with each other and with clinical severity. Atrophy and/or signal change in one or more structures in the circuit of Papez were found in five patients. They involved the parahippocampal gyri in all five, the fornices in four, mamillary bodies in three, the thalamus in two and the cingulate gyrus in one. Changes in the fornix, mamillary body, thalamus or cingulate gyrus were always accompanied by hippocampal and parahippocampal atrophy. The patients with abnormalities of the circuit of Papez did not have more severe epilepsy than those without. Changes in the parahippocampal gyrus, including the entorhinal cortex and subiculum, in which forniceal fibres originate, may be crucial in causing abnormalities more distally in the circuit.

Induction of virus-specific cytotoxic T lymphocytes by in vivo electric administration of peptides.

Generally, major histocompatibility complex (MHC) class I presentation of peptide antigens only occur for proteins' which are actively synthesized and processed intracellularly, so that immunization with a cytotoxic T lymphocyte (CTL) target peptide does not usually elicit effective CTL responses. In the present study, we explored the use of epitope peptides by in vivo electroporation to introduce directly into the cytoplasm for the vaccine elicitation of virus-specific CTLs in a mouse system. BALB/c mice were immunized with human immunodeficiency virus (HIV) env (P18, residues 311-320) or hepatitis C virus (HCV) NS5 (P17, residues 2423-2434) with or without electric pulses. Effector cells against peptide-labeled target cells were elicited in mice immunized with peptides with electric administration but not without electric administration. Moreover, cytolytic activities of CTL against peptide-labeled target cells were enhanced by the addition of plasmid having the immunostimulatory sequence (ISS) or cDNA of the B7-1 molecule in electric administration of peptides. The results of the present study suggest that a peptide vaccine against a virus using electric administration is effective in eliciting virus specific CTLs.

Does post-translational modification influence chaperone-like activity of alpha-crystallin? I. Study on phosphorylation.

It is difficult to isolate derivatives of alpha-crystallin with only one type of post-translational modification, because this protein is subjected to several different types of modification. In the present study using bovine lens proteins, we isolated mono-phosphorylated alphaB-crystallin with no other post-translational modifications. Using this material, we demonstrated that mono-phosphorylation reduced the activity of alphaB-crystallin by approximately 30%. Our results confirmed that investigation of the correlation between chaperone-like activities of alpha-crystallin and post-translational modification is important to understand the mechanism of cataract formation.

A eukaryotic-type protein kinase, SpkA, is required for normal motility of the unicellular Cyanobacterium synechocystis sp. strain PCC 6803.

The genome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 comprises many open reading frames (ORFs) which putatively encode eukaryotic-type protein kinase and protein phosphatase. Based on gene disruption analysis, a region of the hypothetical ORF sll1575, which retained a part of the protein kinase motif, was found to be required for normal motility in the original isolate of strain PCC 6803. Sequence determination revealed that in this strain sll1575 was part of a gene (designated spkA) which harbored an entire eukaryotic-type Ser/Thr protein kinase motif. Strain ATCC 27184 and a glucose-tolerant strain derived from the same isolate as the PCC strain had a frameshift mutation dividing spkA into ORFs sll1574 and sll1575. The structural integrity of spkA agreed well with the motility phenotype, determined by colony morphology on agar plates. The spkA gene was expressed in Escherichia coli as a His-tagged protein, which was purified by Ni2+ affinity chromatography. With [gamma-32P]ATP, SpkA was autophosphorylated and transferred the phosphate group to casein, myelin basic protein, and histone. SpkA also phosphorylated several proteins in the membrane fraction of Synechocystis cells. These results suggest that SpkA is a eukaryotic-type Ser/Thr protein kinase and regulates cellular motility via phosphorylation of the membrane proteins in Synechocystis.

Characterization of genes encoding multi-domain proteins in the genome of the filamentous nitrogen-fixing Cyanobacterium anabaena sp. strain PCC 7120.

Computational analysis of gene structures in the genome of Anabaena sp. PCC 7120 revealed the presence of a large number of genes encoding proteins with multiple functional domains. This was most evident in the genes for signal transduction pathway and the related systems. Comparison of the putative amino acid sequences of the gene products with those in the Pfam database indicated that and PAS domains which may be involved in signal recognition were extremely abundant in Anabaena: 87 GAF domains in 62 ORFs and 140 PAS domains in 59 ORFs. As for the two-component signal transduction system, 73, 53, and 77 genes for simple sensory His kinases, hybrid His kinases and simple response regulators, respectively, many of which contained additional domains of diverse functions, were presumptively assigned. A total of 52 ORFs encoding putative Hanks-type Ser/Thr protein kinases with various domains such as WD-repeat, GAF and His kinase domains, as well as genes for presumptive protein phosphatases, were also identified. In addition, genes for putative transcription factors and for proteins in the cAMP signal transduction system harbored complex gene structures with multiple domains.

Siblings of Schwartz-Jampel syndrome with abnormal muscle computed tomographic findings.

Schwartz-Jampel syndrome (SJS) is a disorder characterized by myotonia, joint contractures, skeletal abnormalities, facial dysmorphism and growth retardation. We present two boys of ages 4 and 8 years with SJS. Their clinical, electromyographic and histopathological findings were similar to those described, except for computed tomography (CT) images that revealed diffuse high attenuation in sternocleidomastoid muscles and low attenuation in the paraspinal, quadriceps, sartorius, soleus and gastrocnemius muscles. This is the first report describing abnormal muscle CT findings associated with SJS. Additional studies of muscle CT might help to improve understanding of the pathogenesis of SJS.

Bone marrow transplantation for the treatment of X-linked adrenoleukodystrophy.

The effects of bone marrow transplantation (BMT) in X-linked childhood adrenoleukodystrophy (ALD) are described in four Japanese patients. Two older patients, 10-year-old boys with IQ 60 and difficulty in school, had favourable results. IQ levels and MRI findings were generally maintained after BMT. One patient showed improvement of gait disturbance. They have both attended ordinary schools after BMT, although a learning disorder persists. On the other hand, two other younger patients with a rapid course and indeterminate IQ at BMT showed deterioration of neurological functions. Indication for BMT seems to be a maintained IQ level, preferably higher than 80, since it seems to be difficult to normalize IQ level after BMT. Younger patients have higher risk of developing a rapidly progressive form of the disease. Identification of presymptomatic boys, and serial and careful follow-up by neuropsychological and neuroradiological studies, are essential prerequisites to successful BMT in X-ALD.

Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by an intrahepatic inoculation with an expression plasmid.

We assessed the possibility of intrahepatic inoculation with a plasmid encoding hepatitis C virus (HCV) proteins to elicit HCV-specific cytotoxic T lymphocytes (CTL) in mice as a conventional animal model of HCV infection. BALB/c mice were intrahepatically or intramuscularly inoculated with an expression plasmid DNA encoding HCV structural proteins under the control of the elongation factor 1-alpha promoter. Expressions of HCV-core protein and envelope proteins (E1 and E2) in hepatocytes were detected immunohistochemically 6 days after inoculation. CTL responses were examined using target cells either pulsed with a specific peptide or infected with a recombinant vaccinia virus expressing HCV structural protein. Both intrahepatically and intramuscularly DNA-inoculated mice developed CD8(+), MHC class I-restricted CTL responses that recognized the peptide pulsed as well as HCV proteins expressing target cells. These studies demonstrated the usefulness of a murine model of HCV infection induced by direct intrahepatic DNA inoculation for understanding the immunopathogenic mechanisms in HCV infection.

Successful control with bromide of two patients with malignant migrating partial seizures in infancy.

A 3-month-old male and a 4-month-old female infant with intractable seizures were diagnosed as having malignant migrating partial seizures in infancy (MMPSI) with developmental arrest on the basis of characteristics of symptoms, clinical courses and EEGs. We treated these two patients with potassium bromide (80 mg/kg) after conventional antiepileptic drugs failed to adequately control the seizures. The potassium bromide therapy resulted in complete control of seizures in one patient, and more than 95% reduction in seizure frequency in the other.

Post-translational modification of alphaB-crystallin of normal human lens.

The current study reports several post-translational modifications of alphaB-crystallin in normal human lenses. The isoforms of post-translational modified alphaB-crystallin were isolated from the normal human lenses of >70-age group by ion exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoform modifications were determined in detail by fast atom bombardment mass spectroscopy and amino acid sequence analysis. As the criterion of non-modified alphaB-crystallin, alphaB-crystallin from 1-d-old infant lenses were used. The modifications found in this study involve oxidation of the N-terminal methionine-1 residue, phosphorylation of the serine-59 residue, and truncation of four amino acids from the C-terminal position of the crystallin. The oxidation of methionine-1 was found in the early stage of human life in 1-d-old lens, although other modification of alphaB-crystallin were usually only found in old lenses (>70-age group).

Crystallin proteins in lenses of hereditary cataractous rat, ICR/f.

ICR/f mutation in rat, an inherited disorder, is characterized by the development of cataracts. In this study, we analyzed and compared the crystallins in normal and cataractous rat lenses using gel filtration and two-dimensional gel electrophoresis, and determined the transglutaminase activities and Ca2+ content in the mutant and normal lenses. The Ca2+ content about 10-fold and the activity of transglutaminase was about 1.8-fold higher in the cataractous lenses than in the normal lenses. Analysis of the cataractous lens proteins showed a remarkable decrease in gamma-, betaB1-, betaA3-, and betaA4-crystallin content, accompanied with some increase in alpha-crystallin (or its aggregate). Higher molecular weight proteins were also observed in the cataractous lenses, with molecular masses which correspond to those of cross-linked dimers (43 to 55 kDa) of beta-crystallins. We consider that the mutation accelerates the aggregation of the crystallins, which is associated with their cross-linking by transglutaminase.

A single immunization with a plasmid encoding hepatitis C virus (HCV) structural proteins under the elongation factor 1-alpha promoter elicits HCV-specific cytotoxic T-lymphocytes (CTL).

Recent studies have raised the possibility that DNA-based vaccination may prove useful for generating virus-specific cytotoxic T-lymphocytes (CTL) responses. Recently, a plasmid containing the human elongation factor 1alpha(EF1-alpha) promoter, pEF321, was reported to be a versatile expression vector for gene expression in mammalian cells in vitro. In the present study, we assessed the capability of a novel plasmid, pEFCE1E2, encoding hepatitis C virus (HCV) structural proteins (core, E1 and E2) under the EF1-alpha promoter to generate CTL against HCV in vivo. BALB/c mice were immunized with the pEFCE1E2 but not with a plasmid possessing the same cDNA under the cytomegalovirus developed HCV-specific effector cells by a single immunization. These effector cells elicited by pEFCE1E2 immunization were CD8(+) and major histocompatibility complex class I restricted. These studies provided evidence for the potential utility of the EF1-alpha promoter for development of DNA vaccines against HCV infections.