Yasuhide Hibino

Chronic Treatment with a Water-Soluble Extract from the Culture Medium of Ganoderma lucidum Mycelia Prevents Apoptosis and Necroptosis in Hypoxia/Ischemia-Induced Injury of Type 2 Diabetic Mouse Brain

Type 2 diabetes mellitus has been known to increase systemic oxidative stress by chronic hyperglycemia and visceral obesity and aggravate cerebral ischemic injury. On the basis of our previous study regarding a water-soluble extract from the culture medium of Ganoderma lucidum mycelia (designed as MAK), which exerts antioxidative and neuroprotective effects, the present study was conducted to evaluate the preventive effects of MAK on apoptosis and necroptosis (a programmed necrosis) induced by hypoxia/ischemia (H/I) in type 2 diabetic KKAy mice. H/I was induced by a combination of unilateral common carotid artery ligation with hypoxia (8% O2 for 20 min) and subsequent reoxygenation. Pretreatment with MAK (1 g/kg, p.o.) for a week significantly reduced H/I-induced neurological deficits and brain infarction volume assessed at 24 h of reoxygenation. Histochemical analysis showed that MAK significantly suppressed superoxide production, neuronal cell death, and vacuolation in the ischemic penumbra, which was accompanied by a decrease in the numbers of TUNEL- or cleaved caspase-3-positive cells. Furthermore, MAK decreased the expression of receptor-interacting protein kinase 3 mRNA and protein, a key molecule for necroptosis. These results suggest that MAK confers resistance to apoptotic and necroptotic cell death and relieves H/I-induced cerebral ischemic injury in type 2 diabetic mice.

Anticarcinogenic actions of water-soluble and alcohol-insoluble fractions from culture medium of Lentinus edodes mycelia

The water-soluble (LEM) and alcohol-insoluble (LAP and LAP1) fractions were prepared from the culture medium of Lentinus edodes mycelia which was composed of bagasse and rice bran. LEM suppressed rat hepatocarcinogenesis and its cell proliferation of rat-ascites hepatoma to about 50% or less of each control group. LAP also suppressed cell proliferation at almost the same rate. LAP1 induced many small cells in the ascites and significantly raised the survival rate of hepatoma-bearing rats. Thus, anticarcinogenic action was revealed in LAP or LAP1 fractions which were mainly composed of xylose-containing polysaccharide and protein.

Anticarcinogenic action of an alcohol-insoluble fraction (LAP1) from culture medium of Lentinus edodes mycelia

From the culture medium of Lentinus edodes mycelia, water-soluble material (LEM) was prepared and further fractionated by alcohol precipitation and gel filtration on Sepharose 6B. The resulting fraction of xylose-rich proteoglycan at the void volume was designated as LAP1. The 25% and 50% survival rates of hepatoma-bearing rats were raised by intraperitoneal (i.p.) administration of LAP1 at doses of 3-10 mg/kg (an optimum dose, 3 mg/kg). This fraction did not suppress in vitro cell proliferation of the hepatoma. Moreover, the i.p. administration of LAP1 significantly augmented the activity of macrophage-migration inhibition of the splenic cells from hepatoma-bearing rats in the early stage after transplantation. Thus, the anticarcinogenic action of LAP1 would partly be interpreted by host-dependent immunomodulation.

Purification and characterization of nuclear scaffold proteins which bind to a highly repetitive bent DNA from rat liver

Our previous work (Hibino et al. (1992) Biochem. Biophys. Res. Commun. 184, 853-858) has shown that the binding affinities of a highly repetitive DNA component for rat nuclear scaffold proteins, P123 and P130, depend on the degree of sequence-directed bending of the helix axis. In the present experiment, these proteins have been purified and finally isolated by DNA-Sepharose column chromatography. The pI values of P123 and P130 were 7.2 and 8.1, respectively. The southwestern blotting revealed that a highly repetitive bent DNA (370-bp XmmI fragment) from rat liver binds readily to the isolated proteins under a hypotonic condition (50 mM NaCl) and that the level of the binding affinity for each protein was lowered with increasing NaCl concentration. The sedimentation analysis predicted that direct interaction between the XmnI fragment and P123 or P130 results in the formation of a complex which consists of two of the fragments and one molecule of the protein, alternatively, one of the fragment and three molecules of the proteins. Distamycin A, an antibiotic which binds specifically to AT-rich DNA, removed the bend in the XmnI fragment and inhibited binding of the fragment to P123 or P130, whereas neither removal of the bend nor binding inhibition was observed with chromomycin A3, an antibiotic specific for GC-rich sites in DNA. These results imply that AT-rich regions in a highly repetitive DNA component cause bending of the helix axis to be recognized by some of nuclear scaffold proteins.

Orally Administrated Ascorbic Acid Suppresses Neuronal Damage and Modifies Expression of SVCT2 and GLUT1 in the Brain of Diabetic Rats with Cerebral Ischemia-Reperfusion

Diabetes mellitus is known to exacerbate cerebral ischemic injury. In the present study, we investigated antiapoptotic and anti-inflammatory effects of oral supplementation of ascorbic acid (AA) on cerebral injury caused by middle cerebral artery occlusion and reperfusion (MCAO/Re) in rats with streptozotocin-induced diabetes. We also evaluated the effects of AA on expression of sodium-dependent vitamin C transporter 2 (SVCT2) and glucose transporter 1 (GLUT1) after MCAO/Re in the brain. The diabetic state markedly aggravated MCAO/Re-induced cerebral damage, as assessed by infarct volume and edema. Pretreatment with AA (100 mg/kg, p.o.) for two weeks significantly suppressed the exacerbation of damage in the brain of diabetic rats. AA also suppressed the production of superoxide radical, activation of caspase-3, and expression of proinflammatory cytokines (tumor necrosis factor-α and interleukin-1β) in the ischemic penumbra. Immunohistochemical staining revealed that expression of SVCT2 was upregulated primarily in neurons and capillary endothelial cells after MCAO/Re in the nondiabetic cortex, accompanied by an increase in total AA (AA + dehydroascorbic acid) in the tissue, and that these responses were suppressed in the diabetic rats. AA supplementation to the diabetic rats restored these responses to the levels of the nondiabetic rats. Furthermore, AA markedly upregulated the basal expression of GLUT1 in endothelial cells of nondiabetic and diabetic cortex, which did not affect total AA levels in the cortex. These results suggest that daily intake of AA attenuates the exacerbation of cerebral ischemic injury in a diabetic state, which may be attributed to anti-apoptotic and anti-inflammatory effects via the improvement of augmented oxidative stress in the brain. AA supplementation may protect endothelial function against the exacerbated ischemic oxidative injury in the diabetic state and improve AA transport through SVCT2 in the cortex.

Antidepressant-like effects of a water-soluble extract from the culture medium of Ganoderma lucidum mycelia in rats

BackgroundGanoderma lucidum is a popular medicinal mushroom used for promoting health and longevity in Asian countries. Previously, we reported that a water-soluble extract from a culture medium of Ganoderma lucidum mycelia (MAK) exerts antioxidative and cerebroprotective effects against ischemia–reperfusion injury in vivo. Here, we evaluated the antidepressant and anxiolytic activities of MAK in rats.MethodsMAK (0.3 or 1 g/kg, p.o.) was administered in the experimental animals 60 min before the forced swimming, open-field, elevated plus-maze, contextual fear-conditioning, and head twitch tests. Additionally, the mechanisms involved in the antidepressant-like action of MAK were investigated by the serotonin precursor 5-hydroxy-L-tryptophan (5-HTP)- or 5-HT2A agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)-induced head twitch responses.ResultsTreatment with MAK (1 g/kg) exhibited antidepressant-like effects in the forced swimming test, attenuated freezing behavior in the contextual fear-conditioning test, and decreased the number of head twitches induced by DOI, but not with 5-HTP. No significant response was observed in locomotion or anxiety-like behavior, when the animals were evaluated in the open-field or elevated plus-maze test, respectively.ConclusionsThese data suggest that MAK has antidepressant-like potential, which is most likely due to the antagonism of 5-HT2A receptors, and possesses anxiolytic-like effects toward memory-dependent and/or stress-induced anxiety in rats.

Productions of interferon-γ and nitrite are induced in mouse splenic cells by a heteroglycan-protein fraction from culture medium of Lentinus edodes mycelia

A xylose-rich heteroglycan-protein fraction (LAP1) was prepared from a solid culture medium in which Lentinus edodes mycelia were growing actively. Mouse splenic cells (SPs) were incubated with [3H]TdR in the presence of LAP1. The incubated SPs were fractionated into plastic adherent splenic cells (ADs), nylon-column effluent splenic cells (NEs) and sIg-expressed splenic cells (SIs), which are rich in Mac-1+, Thy-1.2+ and Ly-5+ cells, respectively. The incorporation of [3H]TdR in response to LAP1 was enhanced in each of the fractionated cell populations. Northern or dot blot hybridization showed that productions of IFN-gamma and its receptor mRNAs are induced predominantly in NEs. In another experiment, SPs were fractionated into ADs, NEs and SIs. Then, NE-AD, SI-AD and NE-SI mixtures were prepared and incubated in the same manner. A significant incorporation of [3H]TdR was shown only in the NE-AD mixture. The enzyme-linked immunosorbent assay showed that IFN-gamma production in response to LAP1 is induced in SPs or in the NE-AD mixture, but not in NEs alone. The level of the production was about 5 times higher in the mixture than in SPs after a 72 h incubation. Moreover, LAP1 was capable of inducing NO2- production in SPs. Thus, the present studies imply that this heteroglycan-protein fraction stimulates productions of IFN-gamma and nitrite in mouse splenic cells, augmenting antitumor immune response(s).

Affinity of a highly repetitive bent DNA for nuclear scaffold proteins from rat liver

A highly repetitive component in rat nuclear DNA was isolated by HindIII digestion and cloned. A self-ligated tandem dimer of the 370-bp cloned component was digested with each of DraI, HindIII and XmnI. The resulting 370-bp restriction fragments exhibited anomalously slow gel electrophoretic mobilities. Of them, the XmnI fragment had the slowest mobility. This suggested that sequence-directed bending of the helix axis is the strongest in this fragment. Moreover, the 370-bp restriction fragments had selective affinities for the nuclear scaffold proteins, P123 and P130. The affinity levels of XmnI fragment were higher than those of DraI or HindIII fragment. These results implied that the affinity of a highly repetitive component for the nuclear scaffold proteins depends on the degree of sequence-directed bending of the helix axis.

Effects of Etanercept against Transient Cerebral Ischemia in Diabetic Rats

Diabetes mellitus is known to exacerbate acute cerebral ischemic injury. Previous studies have demonstrated that infarction volumes caused by transient cerebral ischemia were greater in diabetic rats than in nondiabetic rats. Tumor necrosis factor-α (TNF-α) is a proinflammatory protein produced in the brain in response to cerebral ischemia that promotes apoptosis. Etanercept (ETN), a recombinant TNF receptor (p75)-Fc fusion protein, competitively inhibits TNF-α. Therefore, we evaluated the neuroprotective effects of chronic or acute treatment with ETN on cerebral injury caused by middle cerebral artery occlusion/reperfusion (MCAO/Re) in rats with streptozotocin-induced diabetes. Furthermore, we evaluated the effects of ETN against the apoptosis and myeloperoxidase activity. Single administration of ETN before MCAO significantly suppressed exacerbation of cerebral damage in nondiabetic rats, as assessed by infarct volume. In contrast, the diabetic state markedly aggravated MCAO/Re-induced cerebral damage despite ETN treatment within 24 h before MCAO. However, the damage was improved by repeated administration of ETN at 900 μg/kg/daily in rats in an induced diabetic state. These results suggested that repeated administration of ETN can prevent exacerbation of cerebral ischemic injury in the diabetic state and is mainly attributed to anti-inflammatory effects.

Base sequences of highly repetitive components in nuclear DNAs from rat liver and rat-ascites hepatoma.

A 370-bp highly repetitive component in each of the nuclear DNAs from rat liver (RL) and rat-ascites hepatoma (AH) was isolated by HindIII digestion and cloned in pUC9. Ten of the resulting clones were arbitrarily selected and sequenced. Heterogeneity of size was found in 7 of the RL clones (366-369 bp), but in only 2 of the AH clones (369 bp). The sequence homology was 64.6% among the RL clones; 80.3% among the AH clones. The base compositions were AT-rich, ranging from 61.1% to 64.7%. Many A and/or T runs consisting of 2-5 bases were interspersed throughout each sequence. The restriction sites reported previously, EcoRI, HaeIII, HindIII, HinfI and HphI sites, were confirmed in almost all of the clones. In the present experiment, 12 kinds of the sites were further found in both RL and AH clones.