The longitudinal effect of the aldehyde dehydrogenase 2*2 allele on the risk for nonalcoholic fatty liver disease.

Aldehyde dehydrogenase 2 (ALDH2) detoxifies toxic aldehydes and has a key role in protecting the liver. An elevated gamma-glutamyl transferase (GGT) level is related to oxidative stress and nonalcoholic fatty liver disease (NAFLD). We herein investigated the association between inactive ALDH2*2 allele (rs671) and the risk of NAFLD, including the relationship to the GGT level. A retrospective follow-up study (mean 5.4±1.1 years) was conducted among 341 Japanese health screening program participants. The receiver operating characteristic curve indicated that the GGT level predicted the development of NAFLD (area under the curve: 0.65, P<0.05) with a cutoff value of 25.5 IUl(-1). The longitudinal risk of NAFLD was higher in the ALDH2*2 allele carriers than in the noncarriers (odds ratio (OR): 2.30, 95% confidence interval (CI): 1.21-4.40), and the risk was further increased among the *2 allele carriers with GGT values ⩾25.5 IUl(-1) (OR: 4.28, 95% CI: 1.80-10.19). On the other hand, there were no significant changes in the subjects' body weight and body mass index during observation period. The ALDH2*2 allele, in relation to the GGT level, may potentially be a novel risk factor for NAFLD.

Cytochrome P450 2C19 polymorphisms and valproic acid-induced weight gain.

OBJECTIVES: Cytochrome P450 (CYP) 2C19 plays a role in the biotransformation of clinically relevant drugs as well as endogenous compounds, including sex hormones, which are known to be modulators of food intake and energy balance in humans. We attempted to investigate the influence of CYP2C19 polymorphisms on valproic acid (VPA)-induced weight gain. MATERIALS AND METHODS: This retrospective longitudinal study included 85 VPA-treated and 93 carbamazepine (CBZ)-treated (as a reference) young patients with epilepsy. The body mass index (BMI) gap between the patient's BMI and the cutoff value for being overweight was calculated in each patient during the follow-up period. The longitudinal associations of the CYP2C19 genotype with the BMI gap and risk for becoming overweight during VPA or CBZ therapy were examined retrospectively using the generalized estimating equations approach and the Kaplan-Meier method. RESULTS: During the follow-up period, the values of the BMI gap were significantly greater (P = 0.002 or P = 0.005) and the cumulative incidence of becoming overweight tended to be higher (P = 0.032) in the VPA-treated female patients with one or two loss-of-function CYP2C19 alleles than in the females without the loss-of-function CYP2C19 alleles. No associations were observed among the VPA-treated male patients and CBZ-treated male and female patients (P > 0.05). CONCLUSIONS: This is the first report to show a relationship between the CYP2C19 polymorphism and VPA-induced weight gain in female patients with epilepsy. Further investigations are needed to verify these findings.

Associations between the common HNF1A gene variant p.I27L (rs1169288) and risk of type 2 diabetes mellitus are influenced by weight.

AIM: The common variants p.I27L (rs1169288), p.A98V (rs1800574) and p.S487N (rs2464196) of the hepatocyte nuclear factor 1-α (HNF1A) gene have been inconsistently associated with impaired glucose tolerance and/or an increased risk of type 2 diabetes mellitus (T2DM). The present study aimed to investigate whether these associations are affected by weight. METHODS: A cross-sectional analysis was conducted among 861 Japanese subjects (males: 65.5%; 61.8±12.3years) attending a health-screening programme. Interactive effects between HNF1A variants and weight status on risk of T2DM or dysglycaemic status were determined. RESULTS: The 27L variant carriers were at higher risk of T2DM and dysglycaemic status than non-carriers, but only in normal-weight subjects [odds ratio (OR): 2.04, P=0.03 and OR: 2.56, P=0.01, respectively]. An interactive effect of the p.I27L (rs1169288) variant and weight status on the risk of dysglycaemic status was found (P=0.04). Age, but not body mass index (BMI), was a risk factor for dysglycaemic status in the 27L carriers (OR: 1.05, P=0.0003), whereas BMI was a risk factor in non-carriers (OR: 1.23, P=0.008). No carriers of 98V were identified, and 487N was not associated with either T2DM or dysglycaemic status in our study population. CONCLUSION: These findings suggest that the HNF1A p.I27L (rs1169288) variant may be a significant risk factor of T2DM in normal-weight subjects and that earlier inconsistent results may have been due, in part, to subjects' weight status. Further investigations in larger cohorts are needed to verify these findings.

Gender differences in the incidence and progression of diabetic retinopathy among Japanese patients with type 2 diabetes mellitus: a clinic-based retrospective longitudinal study.

A clinic-based retrospective longitudinal study conducted for 5.8 ± 2.5 years, including 383 (M/F 245/138) Japanese patients with type 2 diabetes mellitus showed that females exhibit a significantly higher prevalence of proliferative diabetic retinopathy (DR) at baseline and that female gender is an independent risk factor for the development of DR.

Effects of interferon-α-transduced tumor cell vaccines and blockade of programmed cell death-1 on the growth of established tumors.

Interferon-alpha (IFN-α) has strong antitumor effects, and IFN-α gene therapy has been used clinically against some cancers. In this study, we evaluated the efficacy of the combination of IFN-α-transduced tumor cell vaccines and programmed cell death 1 (PD-1) blockade, and investigated the mechanisms of the antitumor effects of the combined therapy. A poorly immunogenic murine colorectal cancer cell line, MC38, was transduced to overexpress IFN-α. In a therapeutic model, parental tumor-bearing mice were inoculated with MC38-IFNα cells and an anti-PD-1 antagonistic antibody. Analyses of immunohistochemistry and tumor-specific lysis were performed. The outgrowth of the established tumors was significantly reduced in mice treated with the combination of IFN-α and anti-PD-1. Immunohistochemical analyses of the therapeutic model showed marked infiltration of CD4(+) cells and CD8(+) cells in the established MC38 tumors of mice treated with both IFN-α and anti-PD-1. Significant tumor-specific cytolysis was detected when splenocytes of mice that were treated with both IFN-α and anti-PD-1 were used as effector cells. These results suggest that blockade of the PD-1 PD-ligand enhanced the Th1-type antitumor immune responses induced by IFN-α. The combination of IFN-α gene-transduced tumor cell vaccines and PD-1 blockade may be a possible candidate for a cancer vaccine for clinical trials.

Treatment with BBB022A or rolipram stabilizes the blood-brain barrier in experimental autoimmune encephalomyelitis: an additional mechanism for the therapeutic effect of type IV phosphodiesterase inhibitors.

We examined the treatment effects of two structurally distinct phosphodiesterase type IV (PDE IV) inhibitors, BBB022 and rolipram, in murine and rat models of experimental autoimmune encephalomyelitis (EAE). Based on our data, we propose a mechanism of action which may supplement immunomodulatory effects of PDE IV inhibitors. In particular, PDE inhibitors promote elevation of intracellular cAMP levels, increasing the electrical resistance of endothelial monolayers by stabilizing intercellular junctional complexes. Such an effect on central nervous system (CNS) vascular endothelium has the potential to reduce disease severity in EAE, because both inflammatory cells and humoral factors readily cross a disrupted blood-brain barrier (BBB). In this report, we demonstrate the capacity of BBB022 and rolipram to decrease clinical severity of EAE. further, PDE IV inhibitors significantly reduced BBB permeability in the spinal cords of mice with EAE. These results provide evidence that PDE IV-inhibitors may exert therapeutic effects in EAE by modifying cerebrovascular endothelial permeability, reducing tissue edema as well as entry of inflammatory cells and factors.

Species differences and mechanism of the epimerization of a new MAO-A inhibitor.

1. (5R)-3-[2-((1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl]-5-metho xymethyl-2-oxazolidinone (E2011) has two chiral centers in its structure. In vivo optical inversion of the hydroxy group at one of the chiral centers converts E2011 to a diastereoisomer (ER-20593). Pharmacokinetic parameters of E2011 and ER-20593 were determined after administration of E2011 to rat at 10 mg/kg, and the plasma concentration ratios of E2011 to ER-20593 were almost constant after Tmax of the plasma concentrations. 2. E2011 and ER-20593 were separately administered orally to six species in addition to rat, and the species differences in both directions of epimerization (i.e. from E2011 to ER-20593 and from ER-20593 to E2011) were studied by measuring the plasma concentrations of both compounds. In mouse, guinea pig, dog, and squirrel monkey, the epimerization of E2011 to ER-20593 did not occur, but the epimerization of ER-20593 to E2011 did. In rat, pig and rhesus monkey, the inversion of E2011 to ER-20593 occurred, but the ratios of this inversion were smaller than those for the inversion in the opposite direction. E2011 underwent about 15% inversion to ER-20593 in rat, which was the largest inversion in the seven species examined. 3. To study the mechanism of the epimerization, deuterium-labelled E2011 and ER-20593 (created by substituting the proton at the chiral center of the parent compounds for deuterium) were orally administered (separately) to rat and dog, and the concentration ratios and molecular weights of E2011 and ER-20593 in the plasma were determined by hplc and FAB(+)-mass spectrometry respectively. The results indicated that the major mechanism of the epimerization was oxidation to the carbonyl form followed by reduction to the original epimer and/or the other epimer. 4. The carbonyl form of E2011 (CO-E2011) was reduced to E2011 and ER-20593 (alcohol forms) by liver cytosol and microsomes from rat and dog in vitro with NADH or NADPH. The resultant epimeric ratios (E2011:ER-20593) were consistent with the in vivo results in rat and dog. 5. In conclusion, species differences in the epimerization of E2011 would result from product stereoselectivity of the reductase activity with the carbonyl intermediate.

Protection against blood-brain barrier disruption in focal cerebral ischemia by the type IV phosphodiesterase inhibitor BBB022: a quantitative study.

We examined the effect of BBB022, a type IV phosphodiesterase inhibitor, on blood-brain barrier (BBB) integrity after transient middle cerebral artery occlusion (MCAo) in rats. Male Sprague-Dawley rats were anesthetized with halothane and subjected to 120 min of temporary MCAo by retrograde intraluminal insertion of a nylon suture coated with poly-L-lysine. The drug (BBB022 in saline, 1 mg kg-1 h-1, i.v.) or vehicle (0.9% saline, 1-2 ml kg-1 h-1) was administered by infusion after the onset of MCAo. Four animal groups were studied: Groups A and B were treated by infusion of vehicle or drug over 5 h, and groups C and D over 48 h. Damage to the BBB was judged by extravasation of Evans blue (EB) dye, which was administered i.v. at 3 h after the onset of MCAo in groups A and B; and at 46 h in groups C and D. Fluorometric quantitation of EB was performed 1 or 2 h later in six brain regions. In the 5-h infusion series (group B), BBB022 decreased dye extravasation in the ipsilateral cortex, striatum and hemisphere (hemisphere mean+/-S.E.M. : 41.2+/-5.4 vs. 82.4+/-9.2 microg/g, p=0.005) compared to the vehicle-treated group (A). The 48-h infusion of BBB022 (group D) also decreased dye extravasation in the ipsilateral cortex (7.4+/-2. 5 vs. 29.0+/-8.3 microg/g, p=0.05), striatum (17.2+/-2.2 vs. 50. 8+/-12.1 microg/g, p=0.03) and hemisphere (30.7+/-4.0 vs. 93.2+/-18 microg/g, p=0.01) compared to the vehicle-treated group (C). BBB022 also significantly improved the neurological score at 3 and 5 h after MCAo (in the 5-h infusion group) and at 60 min, 24 h and 48 h (in the 48-h infusion group) compared to the vehicle groups. These data indicate that BBB022 prevents ischemic damage to the BBB after focal cerebral ischemia in rats.

E2011 a novel, selective and reversible inhibitor of monoamine oxidase type A.

E2011, (5R)-3-[2-((1S)-3-cyano-1hydroxypropyl)benzothiazol- 6-yl]-5-methoxymethyl-2-oxazolidine, is a novel inhibitor of monoamine oxidase type A (MAO-A). We have characterized the neurochemical and pharmacological profiles of E2011 and compared them with those of known inhibitors of MAO-A. E2011 potently inhibited MAO-A with more than 30,000 times higher selectivity for MAO-A relative to MAO-B in rat brain homogenate. E2011 did not affect putative neural receptors or reuptake of biogenic amines into synaptosomes of rat brain, which suggests that it is specific to monoaminergic systems. In vivo, E2011 at a dose of 0.3 mg/kg p.o. exhibited potent MAO-A inhibitory activity, whereas MAO-B inhibition was not observed even at 100 mg/kg p.o. E2011 inhibited monoamine metabolism in the rat brain, but the effect disappeared 24 h after administration. Like other reversible MAO-A inhibitors, E2011 did not show a cumulative inhibitory effect during repeated administration for 7 days. However, inhibition of MAO-A by E2011 in ex vivo experiments appeared to be less potent than that by moclobemide. The MAO-A inhibition by E2011 was partially but significantly reversed by dialysis at 4 degrees C for 24 h, which indicates that E2011 could be dissociated from the enzyme. These findings suggest that E2011 is a reversible and highly selective inhibitor of MAO-A. The potency of inhibition by highly reversible MAO-A inhibitors such as E2011 is likely to be underestimated in ex vivo studies because of dilution of the homogenate in the assay system.

[Three patients from two families with familial Creutzfeldt-Jakob disease having a point mutation in the prion protein gene at codon 200 (Glu-->Lys)].

We present three patients with Creutzfeldt-Jakob disease (CJD). They lived in Fuji city and its neighboring towns in the eastern part of Shizuoka prefecture. Patient 1 and patient 2 were cousins. Patient 1 developed the illness at the age of 50 in 1987 and died 13 months later. Patient 2 became ill at the age of 73 in 1989 and died seven months later. Patient 3 was related to a familial CJD cases in Yamanashi prefecture, known as Akai's "H" family (Akai et al in 1979, Yamamoto et al in 1986). She became ill at the age of 78 in 1990 and died four months later. Their clinical features were common; rapidly progressive dementia, generalized myoclonus, and periodic synchronous discharges on electroencephalographies. They were autopsied and neuropathologically diagnosed as typical CJD. Molecular genetic analysis of the prion protein (PrP) gene was performed on patient 2 and patient 3 using their frozen brain sections. The results showed a point mutation in the PrP gene at codon 200; GAG to AAG (Glu-->Lys). The eastern part of Shizuoka prefecture is adjacent to Yamanashi prefecture where a large number of patients with CJD including familial cases has been found during the recent 15 years. This study suggests that the patients with CJD in both Yamanashi and Shizuoka prefecture should be re-evaluated by analysis of the PrP gene.

Quantitative EEG in patients with presenile and senile dementia of the Alzheimer type.

EEG data obtained from 27 patients with presenile Alzheimer's disease (AD) and 28 patients with senile dementia of the Alzheimer type (SDAT) were compared with data from 30 age- and sex-matched controls. Both patient groups exhibited more pronounced delta and theta activity and less prominent alpha and beta activity than the controls. AD, however, was accompanied by more severe slowing than SDAT. The slowing was distributed in the left temporal and frontal regions in AD, and bilaterally in the frontal regions in SDAT. As the severity of the dementia increased, delta activity alone increased in AD, whereas, there were significantly greater increases in both delta and theta activity and decreases in alpha and beta activity in SDAT. These EEG differences appear to be related to the degree of brain damage and the speed of progression of the disease process.

[Experience of air-CT cisternography].

Ten patients were examined with air-CT cisternography. Two acoustic neurinomas protruding into the cistern were visualized as filling defects in the air. Air-CT cisternography identified the 2 tumors with a sensitivity rate of 100%. This retrospective study is aimed to assess the diagnostic efficacy of Air-CT in relation to MRI, enhanced CT and other radiographic studies.

[EEG background activity in patients with dementia of the Alzheimer type--with special reference to analysis by t-statistic significance probability mapping (SPM) in Alzheimer's disease and senile dementia].

EEG power amplitude and power ratio data obtained from 15 (3 men and 12 women) patients with Alzheimer's disease (AD) and 8 (2 men and 6 women) with senile dementia of Alzheimer type (SDAT) were compared with similar data from 40 age- and sex-matched normal controls. Compared with the healthy controls, both patient groups demonstrated increased EEG background slowing, and it indicated more slower in AD than in SDAT. Moreover, both groups showed characteristic findings respectively on EEG topography and t-statistic significance probability mapping (SPM). The differences between AD and their controls indicated high slowing with reductions in alpha 2, beta 1 and beta 2 activity. The SPMs of power ratio in theta and alpha 2 bands showed most prominent significance in the right posterior-temporal region and delta and beta bands did in the frontal region. Severe AD indicated only frontal delta slowing compared to mild AD. The differences between SDAT and their controls indicated only mild slowing in delta and theta bands. The SPM of power amplitude showed occipital slowing, whereas the SPM of power ratio showed the slowing in the frontal region. Judging from both topographic findings, these were considered to denote diffuse slow tendency. In summary, these results presumed that in AD, cortical damages followed by EEG slowing with reductions of alpha 2 and beta bands originated rapidly and thereafter developed subcortical (non-specific area in thalamus) changes with frontal delta activity on SPM. On the other hand, in SDAT, diffuse cortico-subcortical damages with diffuse slowing on EEG topography were caused gradually.

Potentiation of contractile response and increase in tissue sodium content induced by aconitine in the guinea-pig vas deferens.

Aconitine potentiated the contractile response of the guinea-pig vas deferens and increased the tissue Na and Ca content. These effects were abolished in the presence of tetrodotoxin. These results suggest that aconitine causes an increasing Na+ permeability of the smooth muscle membrane to increase Ca2+ availability and thus induces potentiation.

Dehydropipernonaline, an amide possessing coronary vasodilating activity, isolated from Piper longum L.

An amide (dehydropipernonaline) that has coronary vasorelaxant activity was isolated from the fruit of Piper longum L. This substance was characterized on the basis of spectroscopic data.

Geographutoxin II, a novel peptide inhibitor of Na channels of skeletal muscles and autonomic nerves.

Geographutoxin II (GTX II, 3 X 10(-9) to 10(-7) M) from a cone shell Conus geographus inhibited twitch responses of the isolated mouse diaphragm to direct stimulation in a dose-dependent manner. The contraction of the diaphragm induced by grayanotoxin I or veratridine was abolished by GTX II (3 X 10(-7) M), whereas the contractile response to KCI or caffeine was not affected. GTX II induced similar effects on isolated bullfrog sartorius muscles, but required higher concentrations (6 X 10(-7) to 3 X 10(-6) M). GTX II (greater than 10(-6) M) inhibited or abolished the action potential evoked in sartorius muscles markedly. In the isolated guinea pig vas deferens and ileum, GTX II caused a dose-dependent inhibition of the twitch responses to indirect nerve stimulation at concentrations of 3 X 10(-8) to 10(-6) M and 10(-7) to 10(-6) M, respectively. But the toxin had no effect on the dose contractile-response curves for norepinephrine, carbamylcholine or KCI in the vas deferens and for carbamylcholine or histamine in the ileum. GTX II (5 X 10(-8) to 10(-6) M) decreased norepinephrine release induced by veratridine from the vas deferens in a dose-dependent manner. These results suggest that GTX II blocks the voltage-sensitive Na channels in the cell membrane of skeletal muscles and autonomic nerves and these may play an important role in the mechanism of inhibitory effects of GTX II on contractile responses of these tissues to electrical stimulation.

Isolation of evodiamine, a powerful cardiotonic principle, from Evodia rutaecarpa Bentham (Rutaceae).

The crude acetone extract of the fruits of Evodia rutaecarpa Bentham (Rutaceae) exhibited a positive inotropic effect on the guinea pig isolated left atria. The extract was subject to bioassay-directed fractionation to yield the powerful cardiotonic agent evodiamine.