Ashitaba (Angelica Keiskei) Exudate Prevents Increases in Plasminogen Activator Inhibitor-1 Induced by Obesity in Tsumura Suzuki Obese Diabetic Mice.

Angelica keiskei koidzumi (ashitaba) is consumed as a traditional folk medicine and health food in Japan. Ashitaba extract contains abundant flavonoids containing chalcones. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of tissue plasminogen activator. Excessive amounts of PAI-1 in plasma disrupt the fibrinolytic balance and promote a prothrombotic state with which thrombosis and cardiovascular diseases are associated. In the present study, we investigated the effects of ashitaba yellow exudate (AE) on enhanced PAI-1 levels in Tsumura Suzuki obese diabetic (TSOD) mice. AE significantly decreased food efficiency and plasma PAI-1 in TSOD mice but did not affect lean control Tsumura Suzuki nonobese (TSNO) mice. AE also decreased some parameters in the plasma, such as glucose, insulin, tumor necrosis factor alpha (TNF-α) and gains in body weight, subcutaneous, mesenteric fat weight in TSOD mice but had little effect on these parameters in TSNO mice. Levels of adipose PAI-1 were significantly higher in TSOD than in TSNO mice. Major sources of plasma PAI-1 are thought to be adipose tissue and liver. AE significantly suppressed PAI-1 protein levels in the livers of both TSOD and TSNO mice. These results suggest that AE decreased plasma PAI-1 levels by suppressing both the adipose tissue retention of PAI-1 protein and liver PAI-1 production in TSOD mice. Supplementing the diet with AE might help to prevent thrombotic diseases or alleviate the risk of thrombotic diseases as well as to suppress metabolic state in obese individuals.

Two chalcones, 4-hydroxyderricin and xanthoangelol, stimulate GLUT4-dependent glucose uptake through the LKB1/AMP-activated protein kinase signaling pathway in 3T3-L1 adipocytes.

4-Hydroxyderricin (4HD) and xanthoangelol (XAG) are major components of n-hexane/ethyl acetate (5:1) extract of the yellow-colored stem juice of Angelica keiskei. 4-Hydroxyderricin and XAG have been reported to increase glucose transporter 4 (GLUT4)-dependent glucose uptake in 3T3-L1 adipocytes, but the detailed mechanism of this phenomenon remains unknown. This present study was aimed at clarifying the detailed mechanism by which 4HD and XAG increase GLUT4-dependent glucose uptake in 3T3-L1 adipocytes. Both 4HD and XAG increased glucose uptake and GLUT4 translocation to the plasma membrane. 4-Hydroxyderricin and XAG also stimulated the phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK) and its downstream target acetyl-CoA carboxylase. In addition, phosphorylation of liver kinase B1 (LKB1), which acts upstream of AMPK, was also increased by 4HD and XAG treatment. Small interfering RNA knockdown of LKB1 attenuated 4HD- and XAG-stimulated AMPK phosphorylation and suppressed glucose uptake. These findings demonstrate that 4HD and XAG can increase GLUT4-dependent glucose uptake through the LKB1/AMPK signaling pathway in 3T3-L1 adipocytes.

Bone marrow-derived mononuclear cell transplantation in spinal cord injury patients by lumbar puncture.

PURPOSE: This study was conducted to assess the safety and feasibility of intrathecal transplantation of autologous bone marrow-derived mononuclear cells for the treatment of patients with spinal cord injury. METHODS: Ten patients were included in the study. Approximately 120 ml of bone marrow aspirate was obtained from bilateral iliac bone of patients with spinal cord injury. Isolation of mononuclear cells was performed using Ficoll density-gradient centrifugation. Bone marrow mononuclear cells were transplanted into cerebrospinal fluid by lumbar puncture. Functional tests were performed prior to the cell transplantation and six months after cell transplantation. The patients were carefully observed for up to six months. RESULTS: In 5 patients with AIS A prior to cell transplantation, 1 patient converted to AIS B six months after cell transplantation. In 5 patients with AIS B, 1 patient converted to AIS D and 2 patients to AIS C. MRI did not show any complication. Two patients showed slight anemia after aspiration of bone-marrow cells, which returned to normal level within a several weeks. CONCLUSION: The results of this study suggest that this method may be safe and feasible.

Inhibition of angiotensin I converting enzyme by subtilisin NAT (nattokinase) in natto, a Japanese traditional fermented food.

Angiotensin I converting enzyme (ACE) was inhibited by the culture medium of Bacillus subtilis subsp. natto, which ferments boiled soy beans to natto, a Japanese traditional food. Subtilisin NAT (nattokinase) produced by B. subtilis also inhibited ACE, and the inhibition was markedly stimulated by heat treatment of subtilisin at 120 °C for 15 min. Inhibition of ACE by subtilisin was of a mixed type: the decrease in V(max) and the increase in K(m) value. SDS-polyacrylamide gel electrophoresis showed that heat treatment of subtilisin caused inactivation with fragmentation of the enzyme protein into small peptides. The inhibitory action of subtilisin was not due to an enzymatic action of protease, but may be ascribed to the potent ACE-inhibitory peptides such as LY and FY, amino acid sequences in subtilisin. HPLC-MS analysis of heat-inactivated subtilisin confirmed that LY and FY were liberated by fragmentation of the enzyme. Inhibition of ACE by subtilisin and its degradation peptides such as LY and FY may participate in the suppression of blood pressure by ingestion of natto.

Antihypertensive effects of continuous oral administration of nattokinase and its fragments in spontaneously hypertensive rats.

To determine whether the antihypertensive effect of nattokinase is associated with the protease activity of this enzyme, we compared nattokinase with the fragments derived from nattokinase, which possessed no protease activity, in terms of the effect on hypertension in spontaneously hypertensive rats (SHR). In the continuous oral administration test, the groups were given a basic diet alone (control), the basic diet containing nattokinase (0.2, 2.6 mg/g diet) or the basic diet containing the fragments derived from nattokinase (0.2, 0.6 mg/g diet). The group fed the basic diet containing high-dosage nattokinase (2.6 mg/g diet) showed significant reductions in systolic blood pressure (SBP), diastolic blood pressure (DBP) and plasma fibrinogen level, compared with control group and no influence on activities of renin and angiotensin-converting enzyme (ACE, EC 3.4.15.1), and plasma angiotensin II level in the renin-angiotensin system. The treatment of the basic diet containing high-dosage fragments (0.6 mg/g diet) significantly decreased SBP, DBP and plasma angiotensin II level in plasma but the treatment did not influence on plasma fibrinogen level. These results suggest that nattokinase and its fragments are different from each other in the mechanism to reduce hypertension. Nattokinase, retained its protease activity after absorbance across the intestines, may decrease blood pressure through cleavage of fibrinogen in plasma. The fragments, which absorbed as nattokinase-degradation products, prevents the elevation of plasma angiotensin II level to suppress hypertension.

Xanthoangelols isolated from Angelica keiskei inhibit inflammatory-induced plasminogen activator inhibitor 1 (PAI-1) production.

The folk medicine Angelica keiskei (Ashitaba) exhibits antitumor, antioxidant and antidiabetic activities and it has recently attracted attention as a health food. Ashitaba is thought to have antithrombotic properties, but this has not yet been scientifically proven. The elevation of plasma plasminogen activator inhibitor 1 (PAI-1), an inhibitor of fibrinolysis results in a predisposition to the risk of thrombosis. The present study showed that Ashitaba exudates injected intraperitoneally and orally administered over long-term suppressed the lipopolysaccharide (LPS) induced PAI-1 increase in mouse plasma. We also found that xanthoangelol, xanthoangelols B and D, the components of Ashitaba exudates, significantly inhibited TNFα-induced PAI-1 production from human umbilical vein endothelial cells (HUVECs). These findings suggest that Ashitaba can decrease elevated PAI-1 production, and that daily consumption of Ashitaba product might maintain anticoagulant status by inhibiting elevations in PAI-1 under inflammatory conditions.

A multicentre trial of bucillamine in the treatment of early rheumatoid arthritis (SNOW study).

In this study, we enrolled early rheumatoid arthritis (RA) patients at multiple institutes who fulfilled the American Rheumatism Association 1987 revised criteria for the classification of RA, and followed the clinical results of disease-modifying anti-rheumatic drug (DMARD) treatment prospectively. With the aim of developing therapeutic guidelines using the disease activity score 28 (DAS28) as disease indices, we investigated the usefulness of bucillamine (BUC), one of the most widely used DMARDs in Japan. Eighty-one patients with early RA who had not previously been treated with DMARDs were suitable for BUC therapy as first-choice treatment. After 24 months of treatment, at least moderate improvement was seen in 87.5% of patients using the DAS28 erythrocyte sedimentation rate (ESR). After 24 months of BUC therapy, 7 patients (43.8%) met the remission criterion of DAS28 (ESR) <2.6. The 24-month BUC continuation rate was 60.5% (49/81, monotherapy + combination therapy), of which 59.2% (29/49) were on BUC monotherapy. From the efficacy and safety viewpoints alike, BUC was useful as first-choice treatment for early RA.

Inverted repeat of a large segment unveiled on the right arm of Saccharomyces cerevisiae chromosome II.

By means of gene disruption analyses, Saccharomyces cerevisiae strain YPH499 was shown to have two, and only two, copies of ATP3 that encodes the gamma-subunit of H+-dependent ATP synthase and locates on the right arm of chromosome II. Linkage analyses of the two distinguishably marked copies of ATP3 indicated that the distance between them was about 43 cM. Since YBR030W, an ORF proximal to ATP3 by a distance of 17 kbp, was also found to be duplicated, we marked them with two distinguishable nutritional markers, which were also distinguishable from those used for marking the two copies of ATP3, and achieved four-point linkage analyses; CEN2 marked with an appropriate nutritional marker gene was included as a reference point. And, the following linkage map was deduced: CEN2- [11 cM]-YBR030Wa- [8 cM]-ATP3a-[47 cM]-ATP3b- [55 cM]-YBR030Wb. From this map, we suspected that a segment spanning at least YBR030W-ATP3 would be inversely duplicated on the right arm of chromosome II. We then carried out chromosome fragmentation analyses, using several laboratory strains including YPH499, and obtained data in accord with our speculation for all strains, although the distance between the two copies of ATP3 varied from 48 kbp to 192 kbp among the strains examined.

Topographical and cytopathological lesion analysis of the white matter in Binswanger's disease brains.

The purpose of the present study was to document the topographical and cytopathological lesions in the white matter (WM) of Binswanger's disease (BD) brains. Subcortical WM lesions in each lobe and fiber bundle lesions related to the medial thalamic and hippocampal structures in clinicopathologically proven BD brains were evaluated by Klüver-Barrera staining using a grading score. Lesions in the frontal subcortical WM of BD brains, brains from non-neurological patients, and brains with cerebral hemorrhage or large cortical infarcts were also examined immunohistochemically using molecular markers for axonal flow damage: amyloid precursor protein (APP); and for demyelinating axonopathy: encephalitogenic peptide (EP). Our results indicated that the WM lesions in BD were significantly more prominent in the frontal periventricular and subcortical regions as compared with other subcortical WM lesions, in the order of the parietal, occipital and temporal lobes. Fiber bundle lesions in the capsular genu, including the anterior thalamic peduncle, were also significantly more prominent in BD brains as compared with the other bundle lesions. Furthermore, the frequency of damaged nerve fibers labeled by the EP antiserum and APP immunoreactive fibers was significantly higher in BD brains as compared with the control brains. The grading scores for the WM damage correlated significantly with those for the APP and EP immunoreactive fibers in all brains, including the control brains. The axonal damage in the frontal WM lesions of the BD brains was clearly revealed in our study using immunohistochemistry for APP and EP.

Studies on the ATP3 gene of Saccharomyces cerevisiae: presence of two closely linked copies, ATP3a and ATP3b, on the right arm of chromosome II.

In this paper, we present evidence that there are two closely linked copies of the ATP3 gene coding for the gamma subunit of the F(1)F(0)-ATPase complex (EC3.6.1.34) in four laboratory strains of Saccharomyces cerevisiae, even though the yeast genome project has reported that ATP3 is a single-copy gene on chromosome II. We previously reported that the gene dosage (three copies) of ATP1 and ATP2 is coincident with the subunit number of F(1)-alpha and F(1)-beta, but that the gene dosage of ATP3 was not consistent with the subunit stoichiometry of F(1)F(0)-ATPase. By applying long PCR and gene walking analyses, we estimated that the two copies of ATP3 were approximately 20 kb apart, and we designated that which is proximal to the centromere ATP3a, while we named that which is distal ATP3b. The nucleotide sequences of the two copies of ATP3 were identical to the reported sequence in the W303-1A, W303-1B and LL20 strains, while only the DC5 strain had a single base substitution in its ATP3a. With the exception of this substitution, the other nucleotide sequences were identical to the upstream 860 bp and the downstream 150 bp. The differences between ATP3 with the single base substitution (Ser(308) to Phe) and ATP3 without the substitution on the complementation of the ATP3 disruptant and on the maintenance of the mitochondrial DNA were observed, suggesting that Atp3ap and Atp3bp in the DC5 strain might have different functions. However, it should not always be necessary for yeast cells to carry different types of ATP3 because the other three strains carry the same type of ATP3. It was also demonstrated that the disruption of the ATP3 genes basically leads to a loss of wild-type mtDNA, but the stability of the mtDNA is not dependent on the ATP3 alone.

Spontaneous muscle action potentials fail to develop without fetal-type acetylcholine receptors.

In mammals, two combinations of muscle nicotinic acetylcholine receptors (AChRs) are used: alpha2betagammadelta (gamma-AChR) or alpha2betaepsilondelta (epsilon-AChR). After birth, gamma-AChRs are replaced by epsilon-AChRs (gamma/epsilon-switch). The two receptors have different conductances and open times. During perinatal period, the long open time gamma-AChRs generate random myofiber action potentials from uniquantal miniature end-plate potentials (mEPPs). epsilon-AChRs are suitable for strong adult muscle activities. Since the effect of the gamma/epsilon-switch on neuromuscular development was unclear, despite the many differences in channel characteristics, we carried out this study to generate gamma-subunit-deficient mice. Homozygotes born alive survived for 2 days in a stable condition, and were able to move their forelimbs. Endplate AChRs included epsilon-subunits, and muscle fibers had multiple neuromuscular junctions. Both pre- and postsynapses were abnormal and spontaneous action potentials generated from mEPPs were totally absent. Results suggest a requirement for gamma-AChRs in mediating synaptically-induced action potential activity critical for neuromuscular development.

Repair of facial nerve with alginate sponge without suturing: an experimental study in cats.

A bioabsorbable material, alginate, was used to repair a defect in the facial nerve. In five cats a 5-mm gap was created in the dorsal ramus of the facial nerve on one side selected at random, which was repaired by implantation of alginate sponge without suturing. In the control group, two cats had a similar nerve injury but without implantation of alginate. Behavioural, electrophysiological, and histological examinations were made over a period of 16 weeks postoperatively. Movement of the upper eyelids and electrophysiological function were restored 12 weeks postoperatively, and many myelinated axons were observed both in the gap of facial nerve and its branches 16 weeks after operation, whereas no alginate residue was detected remaining within gap. In the control group, no movement or electrophysiological restoration was recorded, and there were few regenerated axons accompanied by a large amount of scar tissue. The nerve repaired with alginate showed remarkable regeneration. These results suggest that alginate is a promising material for facial nerve repair, and sutureless repair with alginate is a possible option for treating defects in the facial nerve.

Deterioration in learning and memory of inferential tasks for evaluation of transitivity and symmetry in aged SAMP8 mice.

This study examined age-dependent deficits in the learning and memory of inferential tasks, using an established senescence-accelerated mouse model in age-related brain dysfunction (SAMP8) and its genetically related inbred strain (SAMR1). The mice learned two sets of nonspatial odor-odor pairs by association learning successively (i.e., A-->B, X-->Y, then B-->C, Y-->Z). They were tested in transitive inference (i.e., A-->C, X-->Z) and symmetrical inference (i.e., C-->B, Z-->Y). In the probe test of A-->C, X-->Z transitive inference, 1-month-old SAMP8 and control SAMR1 at the same age significantly chose the alternative based on transitive inference, but 4- and 7-month-old SAMP8 performed at a random chance level, in comparison with unambiguous inference by control SAMR1 at the same ages. During the test of C-->B, Z-->Y symmetrical inference, SAMP8 at 1 month of age made errors as frequently as control SAMR1 at the same age, but SAMP8 at 4 and 7 months of age made more errors than SAMR1 at the same ages. At 4 and 7 months of age, SAMP8 made more errors than 1-month-old SAMP8. Control SAMR1 did not show such an age-related deficient. These results indicate that SAMP8 mice have age-related learning and memory deficits in the ability to perform inferential tasks. Age-related hippocampal dysfunction is suggested to be the cause of these age-related deficits in old SAMP8 mice during the performance of inferential tasks mediated by declarative memory.