Changes occurring in nutritional components (phytochemicals and free amino acid) of raw and sprouted seeds of white and black sesame (Sesamum indicum L.) and screening of their antioxidant activities.

The present study is the first to investigate the germination properties regarding phytochemicals, amino acids, total phenolics, and antioxidant capacities of white and black sesame seeds. Nutritional components and antioxidant effects showed considerable differences. Sesamine and sesamolin composition decreased (white: 4.21→1.72, 3.57→1.57 mg/g; black: 2.43→0.58, 1.36→0.45 mg/g) during germination. Moreover, catechin displayed the predominant composition in sprouted seeds with values of 13.50 mg/g (white) and 19.09 (black) mg/g followed by (-)-epicatechin and sinapic acid. Total phenolics increased by approximately 4 times upon germination, i.e., 503.1±27.1→ 2085.0±56.7 (white) and 645.8±31.5→2480.1±49.5 (black), mg GAE/g. Amino acids also remarkably increased in sprouted white (7.04→31.69mg/g) and black (6.55→26.97mg/g) seeds, with individual composition occurring in the following order: asparagine>arginine>tryptophan>leucine>alanine. In particular, arginine and tryptophan exhibited the greatest variations. The antioxidant effects against DPPH radical were stronger in sprouted seeds depending on the phytochemicals. Therefore, sprouted sesame can be utilized as an excellent source for functional foods.

Comparison of nutritional components (isoflavone, protein, oil, and fatty acid) and antioxidant properties at the growth stage of different parts of soybean [Glycine max (L.) Merrill].

This is the first study to investigate antioxidant capacities of isoflavones prepared using microwave-assisted hydrolysis method from different parts (seeds, leaves, leafstalks, pods, stems and roots) of soybean at growth stages. In addition, the fluctuations in the isoflavone, protein, fatty acid, and oil contents in R6-R8 (R6: beginning; R7: beginning maturity; R8: full maturity) seeds were confirmed. The R7 seeds exhibited the most predominant contents of isoflavones (1218.1±7.3 µg/g) in the following order: daidzein (48%)>genistein (35%)>glycitein (17%). The second highest isoflavone content was found in the leaves (1052.1±10.4 µg/g), followed by R8 seeds>roots>R6 seeds>leafstalks> pods; the stems exhibited the lowest isoflavone content (57.2±1.7 µg/g). Interestingly, daidzein showed the highest individual isoflavone content with remarkable variations (57.2-766.8 µg/g), representing 46-100% of the total isoflavone content. R8 exhibited higher protein, fatty acid, and oil contents than R6 or R7. Moreover, the antioxidant capacities against two radicals in different parts of soybean plant showed considerable differences depending upon the isoflavone content. Our results suggested that soybean leaves and seeds might be useful materials for functional foods.

Inhibitory evaluation of sulfonamide chalcones on β-secretase and acylcholinesterase.

The action of ß-secretase (BACE1) is strongly correlated with the onset of Alzheimer's disease (AD). Aminochalcone derivatives were examined for their ability to inhibit BACE1. Parent aminochalcones showed two digit micromolar IC(50)s against BACE1. Potency was enhanced 10-fold or more by introducing benzenesulfonyl derivatives to the amino group: 1 (IC(50) = 48.2 µM) versus 4a (IC(50) = 1.44 µM) and 2 (IC(50) = 17.7 µM) versus 5a (IC(50) = 0.21 µM). The activity was significantly influenced by position and number of hydroxyl groups on the chalcone B-ring: 3,4-dihydroxy 5a (IC(50) = 0.21 µM) > 4-hydroxy 4a (IC(50) = 1.44 µM) > 2,4-dihydroxy 6 (IC(50) = 3.60 µM) > 2,5-dihydroxy 7 (IC(50) = 16.87 µM) > des hydroxy 4b (IC(50) = 168.7 µM). Lineweaver-Burk and Dixon plots and their secondary replots indicate that compound 5a was a mixed inhibitor with reversible and time-dependent behavior. Potent BACE1 inhibitors 4a,c,f, 5a-c showed moderate inhibition against two other enzymes implicated in AD pathogenesis, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), with IC(50)s ranging between 56.1 ~ 95.8 µM and 19.5 ~ 79.0 µM, respectively.

Potent inhibition of bacterial neuraminidase activity by pterocarpans isolated from the roots of Lespedeza bicolor.

Bacterial neuraminidase has been highlighted as a key enzyme for pathogenic infection and sepsis. Six pterocarpans displaying significant levels of neuraminidase inhibitory activity were isolated from the root bark of Lespedeza bicolor. The isolated compounds were identified as three new pterocarpans (1-3) together with known compounds erythrabyssin II (4), lespebuergine G4 (5), and 1-methoxyerythrabyssin II (6). The new compounds were characterized as bicolosin A (1), bicolosin B (2), and bicolosin C (3). All compounds inhibited bacterial neuraminidase in a dose-dependent manner with significant inhibition (IC(50)=0.09-3.25 µM). All neuraminidase inhibitors screened were found to exhibit noncompetitive kinetics. The three most potent neuraminidase inhibitors (1, 3 and 6) feature a methoxy substitution on C-1.

Amyloid-beta dynamics are regulated by orexin and the sleep-wake cycle.

Amyloid-beta (Abeta) accumulation in the brain extracellular space is a hallmark of Alzheimer's disease. The factors regulating this process are only partly understood. Abeta aggregation is a concentration-dependent process that is likely responsive to changes in brain interstitial fluid (ISF) levels of Abeta. Using in vivo microdialysis in mice, we found that the amount of ISF Abeta correlated with wakefulness. The amount of ISF Abeta also significantly increased during acute sleep deprivation and during orexin infusion, but decreased with infusion of a dual orexin receptor antagonist. Chronic sleep restriction significantly increased, and a dual orexin receptor antagonist decreased, Abeta plaque formation in amyloid precursor protein transgenic mice. Thus, the sleep-wake cycle and orexin may play a role in the pathogenesis of Alzheimer's disease.

Tyrosinase inhibitory polyphenols from roots of Morus lhou.

Twelve polyphenols (1-12) possessing tyrosinase inhibitory properties were isolated from the methanol (95%) extract of Morus lhou. The isolated compounds consisted of four flavanones (1-4), four flavones (5-8), and four phenylbenzofuranes (9-12). Moracin derivative 12 proved to be new a compound which was fully characterized. Compounds 1-12 were evaluated for both monophenolase and diphenolase (the two steps catalyzed by tyrosinase) inhibition to identify the structural characteristics required for mushroom tyrosinase inhibition. We observed that all parent compounds (1, 5, and 9) possessing an unsubstituted resorcinol group were highly effective inhibitors of monophenolase activity (IC(50) values of 1.3, 1.2, and 7.4 microM). The potency of the inhibitors diminished with alkyl substitution on either the aromatic ring or the hydroxyl functions. Interestingly, flavone 5 was shown to possess only monophenolase inhibitory activity, but flavanone 1 and phenylbenzofuran 9 inhibited diphenolase as well as monophenolase significantly. The inhibitory mode of these species was also dependent upon the skeleton: phenylbenzofuran 9 manifested a simple competitive inhibition mode for monophenolase and diphenolase; on the other hand flavanone 1 (monophenolase, k(3) = 0.1966 min(-1) microM(-1), k(4) = 0.0082 min(-1), and K(i)(app) = 0.0468 microM; diphenolase, k(3) = 0.0014 min(-1) microM(-1), k(4) = 0.0013 min(-1), and K(i)(app) = 0.8996 microM) and flavone 5 both showed time-dependent inhibition against monophenolase. Compound 1 operated according to the simple reversible slow binding model whereas compound 5 operated under the enzyme isomerization model.

Endocytosis is required for synaptic activity-dependent release of amyloid-beta in vivo.

Aggregation of amyloid-beta (Abeta) peptide into soluble and insoluble forms within the brain extracellular space is central to the pathogenesis of Alzheimer's disease. Full-length amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce Abeta. Abeta is subsequently released into the brain interstitial fluid (ISF). We hypothesized that synaptic transmission results in more APP endocytosis, thereby increasing Abeta generation and release into the ISF. We found that inhibition of clathrin-mediated endocytosis immediately lowers ISF Abeta levels in vivo. Two distinct methods that increased synaptic transmission resulted in an elevation of ISF Abeta levels. Inhibition of endocytosis, however, prevented the activity-dependent increase in Abeta. We estimate that approximately 70% of ISF Abeta arises from endocytosis-associated mechanisms, with the vast majority of this pool also dependent on synaptic activity. These findings have implications for AD pathogenesis and may provide insights into therapeutic intervention.

Acute stress increases interstitial fluid amyloid-beta via corticotropin-releasing factor and neuronal activity.

Aggregation of the amyloid-beta (Abeta) peptide in the extracellular space of the brain is critical in the pathogenesis of Alzheimer's disease. Abeta is produced by neurons and released into the brain interstitial fluid (ISF), a process regulated by synaptic activity. To determine whether behavioral stressors can regulate ISF Abeta levels, we assessed the effects of chronic and acute stress paradigms in amyloid precursor protein transgenic mice. Isolation stress over 3 months increased Abeta levels by 84%. Similarly, acute restraint stress increased Abeta levels over hours. Exogenous corticotropin-releasing factor (CRF) but not corticosterone mimicked the effects of acute restraint stress. Inhibition of endogenous CRF receptors or neuronal activity blocked the effects of acute stress on Abeta. Thus, behavioral stressors can rapidly increase ISF Abeta through neuronal activity in a CRF-dependent manner, and the results suggest a mechanism by which behavioral stress may affect Alzheimer's disease pathogenesis.

Sulfonamide chalcone as a new class of alpha-glucosidase inhibitors.

Chalcones 1-20, a new class of glycosidase inhibitors, were synthesized, and their glycosidase inhibitory activities were investigated. Non-aminochalcones 1-12 had no inhibitory activity, however, aminochalcones 13-20 had strong glycosidase (alpha-glucosidase, alpha-amylase, and beta-amylase) inhibitory activities. In particular, sulfonamide chalcones 17-20 had more potent alpha-glucosidase inhibitory activity than aminated chalcone 13-16. 4'-(p-Toluenesulfonamide)-3,4-dihydroxy chalcone 20 (IC(50)=0.4microM) was the best inhibitor against alpha-glucosidase, and these sulfonamide chalcones showed non-competitive inhibition.

Histone deacetylase-1 represses transcription by interacting with zinc-fingers and interfering with the DNA binding activity of Sp1.

Sp1 activates the transcription of many cellular and viral genes, and histone deacetylase 1 (HDAC1) removes the acetyl group of nucleosomal core histones. Treatment of cells with the histone deacetylase 1 inhibitor, TSA, robustly activates the transcription of the Sp1-dependent promoters, suggesting the inhibition of Sp1 activity which is critical in the activation of transcription, by HDAC1. We assessed the protein-protein interactions occurring between Sp1 and HDAC1, and the transcriptional regulatory mechanism controlled by this interaction. In vitro GST fusion pull down assays, co-immunoprecipitation, and mammalian two-hybrid assays revealed that the HDAC1 noncatalytic domain (a.a. 237-482) interacts directly with the zinc-finger DNA binding domain of Sp1. DNase I footprinting revealed that this interaction prevents the binding of Sp1 zinc-fingers to the target GC-box. Gal4-HDAC1 fusion, targeted proximally to the GC-boxes, potently repressed the transcription of pG5-5x(GC)-Luc, in which Sp1 potently activates transcription. This repression of transcription does not involve the deacetylase activity of HDAC1, and is accomplished by the direct protein-protein interactions which occur between the Sp1 zinc-finger DNA binding domain and HDAC1, which interferes with the promoter GC-box binding of Sp1.

Transcriptional activity of Sp1 is regulated by molecular interactions between the zinc finger DNA binding domain and the inhibitory domain with corepressors, and this interaction is modulated by MEK.

Sp1 activates the transcription of many cellular and viral genes with the GC-box in either the proximal promoter or the enhancer. Sp1 is composed of several functional domains, such as the inhibitory domain (ID), two serine/threonine-rich domains, two glutamine-rich domains, three C2H2-type zinc finger DNA binding domains (ZFDBD), and a C-terminal D domain. The ZDDBD is the most highly conserved domain among the Sp-family transcription factors and plays a critical role in GC-box recognition. In this study, we investigated the protein-protein interactions occurring at the Sp1ZFDBD and the Sp1ID, and the molecular mechanisms controlling the interaction. Our results found that Sp1ZFDBD and Sp1ID repressed transcription once they were targeted to the proximal promoter of the pGal4 UAS reporter fusion gene system, suggesting molecular interaction with the repressor molecules. Indeed, mammalian two-hybrid assays, GST fusion protein pull-down assays, and co-immunoprecipitation assays showed that Sp1ZFDBD and Sp1ID are able to interact with corepressor proteins such as SMRT, NcoR, and BCoR. The molecular interactions appear to be regulated by MAP kinase/Erk kinase kinase (MEK). The molecular interactions between Sp1ID and the corepressor might explain the role of Sp1 as a repressor under certain circumstances. The siRNA-induced degradation of the corepressors resulted in an up-regulation of Sp1-dependent transcription. The cellular context of the corepressors and the regulation of molecular interaction between corepressors and Sp1ZFDBD or Sp1ID might be important in controlling Sp1 activity.

FBI-1 enhances transcription of the nuclear factor-kappaB (NF-kappaB)-responsive E-selectin gene by nuclear localization of the p65 subunit of NF-kappaB.

The POZ domain is a highly conserved protein-protein interaction motif found in many regulatory proteins. Nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of a variety of genes in response to infection, inflammation, and stressful conditions. We found that the POZ domain of FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) interacted with the Rel homology domain of the p65 subunit of NF-kappaB in both in vivo and in vitro protein-protein interaction assays. FBI-1 enhanced NF-kappaB-mediated transcription of E-selectin genes in HeLa cells upon phorbol 12-myristate 13-acetate stimulation and overcame gene repression by IkappaB alpha or IkappaB beta. In contrast, the POZ domain of FBI-1, which is a dominant-negative form of FBI-1, repressed NF-kappaB-mediated transcription, and the repression was cooperative with IkappaB alpha or IkappaB beta. In contrast, the POZ domain tagged with a nuclear localization sequence polypeptide of FBI-1 enhanced NF-kappaB-responsive gene transcription, suggesting that the molecular interaction between the POZ domain and the Rel homology domain of p65 and the nuclear localization by the nuclear localization sequence are important in the transcription enhancement mediated by FBI-1. Confocal microscopy showed that FBI-1 increased NF-kappaB movement into the nucleus and increased the stability of NF-kappaB in the nucleus, which enhanced NF-kappaB-mediated transcription of the E-selectin gene. FBI-1 also interacted with IkappaB alpha and IkappaB beta.

Regulation of the proapoptotic activity of huntingtin interacting protein 1 by Dyrk1 and caspase-3 in hippocampal neuroprogenitor cells.

Dual specific protein kinase Dyrks are thought to play a key role in the regulation of cell growth in a variety of cellular systems. Interestingly, human Dyrk1 is mapped to the Down's syndrome (DS) critical region on chromosome 21, and thought to be a candidate gene responsible for the mental retardation of DS patients. Huntingtin-interacting protein 1 (Hip-1), a proapoptotic mediator, is implicated as a molecular accomplice in the pathogenesis of Huntington's disease. In the present study we found that Dyrk1 selectively binds to and phosphorylates Hip-1 during the neuronal differentiation of embryonic hippocampal neuroprogenitor (H19-7) cells. The Dyrk1-mediated phosphorylation of Hip-1, in response to bFGF, resulted in the blockade of Hip-1-mediated neuronal cell death as well as the enhancement of neurite outgrowth. Furthermore, the addition of etoposide to proliferating H19-7 cells caused the diminished binding of Hip-1 to Dyrk1 and the levels of phosphorylated Hip-1 remarkably decreased. Simultaneously, the dissociated Hip-1 from Dyrk1 bound to caspase-3 in response to etoposide, which led to its activation and consequently cell death in H19-7 cells. These data suggest that the phosphorylation of Hip-1 by Dyrk1 has a dual role in regulating neuronal differentiation and cell death. The interaction between Dyrk1 and Hip-1 appeared to be differentially modulated by different kinds of stimuli, such as bFGF and etoposide in H19-7 cells.

Artificial zinc finger fusions targeting Sp1-binding sites and the trans-activator-responsive element potently repress transcription and replication of HIV-1.

Tat activates transcription by interacting with Sp1, NF-kappaB, positive transcription elongation factor b, and trans-activator-responsive element (TAR). Tat and Sp1 play major roles in transcription by protein-protein interactions at human immunodeficiency virus, type 1 (HIV-1) long terminal repeat. Sp1 activates transcription by interacting with cyclin T1 in the absence of Tat. To disrupt the transcription activation by Tat and Sp1, we fused Sp1-inhibiting polypeptides, zinc finger polypeptide, and the TAR-binding mutant Tat (TatdMt) together. A designed or natural zinc finger and Tat mutant fusion was used to target the fusion to the key regulatory sites (GC box and TAR) on the long terminal repeat and nascent short transcripts to disrupt the molecular interaction that normally result in robust transcription. The designed zinc finger and TatdMt fusions were targeted to the TAR, and they potently repressed both transcription and replication of HIV-1. The Sp1-inhibiting POZ domain, TatdMt, and zinc fingers are key functional domains important in repression of transcription and replication. The designed artificial zinc fingers were targeted to the high affinity Sp1-binding site, and by being fused with TatdMt and POZ domain, they strongly block both Sp1-cyclin T1-dependent transcription and Tat-dependent transcription, even in the presence of excess expressed Tat.