Superoxide-responsive fluorogenic molecular probes for optical bioimaging of neurodegenerative events in Alzheimer's disease.

Since oxidative stress has been recognized as a major factor contributing to the progression of several neurodegenerative disorders, reactive oxygen species (ROS) including superoxide have received great attention as a representative molecular marker for the diagnosis of Alzheimer's disease (AD). Here, superoxide-sensitive fluorogenic molecular probes, benzenesulfonylated resorufin derivatives (BSRs), were newly devised for optical bioimaging of oxidative events in neurodegenerative processes. BSRs, fluorescence-quenched benzenesulfonylated derivatives of resorufin, were designed to recover their fluorescence upon exposure to superoxide through a selective nucleophilic uncaging reaction of the benzenesulfonyl cage. Among BSRs, BSR6 presented the best sensitivity and selectivity to superoxide likely due to the optimal reactivity matching between the nucleophilicity of superoxide and its electrophilicity ascribed to the highly electron-withdrawing pentafluoro-substitution on the benzenesulfonyl cage. Fluorescence imaging of inflammatory cells and animal models presented the potential of BSR6 for optical sensing of superoxide in vitro and in vivo. Furthermore, microglial cell (Bv2) imaging with BSR6 enabled the optical monitoring of intracellular oxidative events upon treatment with an oxidative stimulus (amyloid beta, Aß) or the byproduct of oxidative stress (4-hydroxynonenal, HNE).

MutY DNA Glycosylase Protects Cells From Tumor Necrosis Factor Alpha-Induced Necroptosis.

Numerous studies have implied that mutY DNA glycosylase (MYH) is involved in the repair of post-replicative mispairs and plays a critical role in the base excision repair pathway. Recent in vitro studies have shown that MYH interacts with tumor necrosis factor receptor type 1-associated death domain (TRADD), a key effector protein of tumor necrosis factor receptor-1 (TNFR1) signaling. The association between MYH and TRADD is reversed during tumor necrosis factor alpha (TNF-α)- and camptothecin (CPT)-induced apoptosis, and enhanced during TNF-α-induced survival. After investigating the role of MYH interacts with various proteins following TNF-α stimulation, here, we focus on MYH and TRADD interaction functions in necroptosis and its effects to related proteins. We report that the level of the MYH and TRADD complex was also reduced during necroptosis induced by TNF-α and zVAD-fmk. In particular, we also found that MYH is a biologically important necrosis suppressor. Under combined TNF-α and zVAD-fmk treatment, MYH-deficient cells were induced to enter the necroptosis pathway but primary mouse embryonic fibroblasts (MEFs) were not. Necroptosis in the absence of MYH proceeds via the inactivation of caspase-8, followed by an increase in the formation of the kinase receptor- interacting protein 1 (RIP1)-RIP3 complex. Our results suggested that MYH, which interacts with TRADD, inhibits TNF-α necroptotic signaling. Therefore, MYH inactivation is essential for necroptosis via the downregulation of caspase-8. J. Cell. Biochem. 118: 1827-1838, 2017. © 2017 Wiley Periodicals, Inc.

Visualization of the physical and functional interaction between hMYH and hRad9 by Dronpa bimolecular fluorescence complementation.

BACKGROUND: Human MutY glycosylase homolog (hMYH), a component of the base excision repair pathway, is responsible for the generation of apurinic/apyrimidinic sites. Rad9-Rad1-Hus1 (9-1-1) is a heterotrimeric protein complex that plays a role in cell cycle checkpoint control and DNA repair. In humans, hMYH and 9-1-1 interact through Hus1 and to a lesser degree with Rad1 in the presence of DNA damage. In Saccharomyces pombe, each component of the 9-1-1 complex interacts directly with SpMYH. The glycosylase activity of hMYH is stimulated by Hus1 and the 9-1-1 complex and enhanced by DNA damage treatment. Cells respond to different stress conditions in different manners. Therefore, we investigated whether Rad9 interacted with hMYH under different stresses. Here, we identified and visualized the interaction between hRad9 and hMYH and investigated the functional consequences of this interaction. RESULTS: Co-IP and BiFC indicates that hMYH interacts with hRad9. As shown by GST-pull down assay, this interaction is direct. Furthermore, BiFC with deletion mutants of hMYH showed that hRad9 interacts with N-terminal region of hMYH. The interaction was enhanced by hydroxyurea (HU) treatment. mRNA and protein levels of hMYH and hRad9 were increased following HU treatment. A marked increase in p-Chk1 (S345) and p-Cdk2 (T14, Y15) was observed. But this phosphorylation decreased in siMYH- or siRad9-transfected cells, and more pronounced decrease observed in co-transfected cells. CONCLUSIONS: Our data reveal that hRad9 interacts directly with N-terminal region of hMYH. This interaction is enhanced by HU treatment. Knockdown of one or both protein result in decreasing Chk1 and Cdk2 phosphorylation. Since both protein functions in the early detection of DNA damage, we suggest that this interaction occurs early in DNA damage pathway.

Ubiquitin ligase CHIP induces TRAF2 proteasomal degradation and NF-κB inactivation to regulate breast cancer cell invasion.

Transcriptional factor nuclear factor-kappaB (NF-κB) plays a crucial role in human breast cancer cell invasion and metastasis. The carboxyl terminus of Hsc70-interacting protein (CHIP) is a U-box-type ubiquitin ligase that induces ubiquitination and proteasomal degradation of its substrate proteins. In this study, we investigated the role of CHIP in the NF-κB pathway in the invasion of MDA-MB-231 cells, a highly aggressive breast cancer cell line. We showed that overexpression of CHIP significantly inhibits the invasion of the MDA-MB-231 cells. The overexpression of CHIP suppressed expression of urokinase plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) in MDA-MB-231 cells. Moreover, CHIP strongly inhibited the nuclear localization and the transcriptional activity of NF-κB. The activation of the IkappaB kinase complex (IKK) was also blocked by CHIP overexpression. Importantly, CHIP overexpression resulted in a significant decrease in the level of TNF receptor-associated factor 2 (TRAF2), an upstream key player in the NF-κB pathway. However, the level of TRAF2 was restored after treatment with a proteasome inhibitor, MG-132. Moreover, CHIP overexpression promoted the ubiquitination of TRAF2. We also found cell invasion significantly decreased in cells transfected with TRAF2 small interfering RNA (siRNA). In contrast, when CHIP expression was suppressed by siRNA in poorly invasive MCF-7 cells, cell invasion significantly increased in conjunction with enhanced NF-κB activation and TRAF2 levels. Taken together, these results suggest that CHIP regulates NF-κB-mediated cell invasion via the down-regulation of TRAF2.

Matrix metalloproteinase-1 induces cleavage of exogenous alphaB-crystallin transduced by a cell-penetrating peptide.

Cell-penetrating peptides (CPPs), including TAT-CPP, have been used to deliver exogenous proteins into living cells. Although a number of proteins fused to TAT-CPP can be delivered into various cells, little is known about the proteolytic cleavage of TAT-fusion proteins in cells. In this study, we demonstrate that a small heat shock protein (sHSP), alphaB-crystallin (αB-crystallin), delivered by TAT-CPP is susceptible to proteolytic cleavage by matrix metalloproteinase-1 (MMP-1) in cardiac myoblast H9c2 cells. Recombinant TAT-αB-crystallin was efficiently transduced into H9c2 cells. For a few hours following protein transduction, generation of a 14-kDa fragment, a cleavage band of TAT-αB-crystallin, increased in a time-dependent manner. This fragment was observed only in detergent-insoluble fractions. Interestingly, treatment with MMP inhibitors blocked the cleavage of TAT-αB-crystallin. In test tubes, recombinant MMP-1 processed TAT-αB-crystallin to generate the major cleavage fragment 14-kDa, as observed in the cells treated with TAT-αB-crystallin. The N-terminal sequences of the 14-kDa fragment were identified as Leu-Arg-Ala-Pro-Ser-Trp-Phe, indicating that this fragment is generated by cleavage at Phe54-Leu55 of αB-crystallin. The MMP-1-selective inhibitor abolished the production of 14-kDa fragments in cells. In addition, the cleaved fragment of TAT-αB-crystallin was significantly reduced in cells transfected with MMP-1 siRNA. Moreover, the enzymatic activity of MMP-1 was markedly increased in TAT-αB-crystallin-treated cells. TAT-αB-crystallin has a cytoprotective effect on H9c2 cells under hypoxic insult, moreover, MMP-1-selective inhibitor treatment led to even increased cell viability. These results suggest that MMP-1 is responsible for proteolytic cleavage of TAT-αB-crystallin during its intracellular transduction in H9c2 cells.

Effects of quercetin on human α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated ion currents.

Quercetin is a low molecular weight flavonoid found in dietary fruits and vegetables. Quercetin, like other flavonoids, has demonstrated neuroprotective effects in vitro and in vivo. However, relatively little is known about how quercetin achieves its neuroprotective abilities. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is one of several excitatory receptors, which play an important role in postsynaptic neurotransmission. Over-stimulation of ionotropic glutamate receptor including AMPA receptors is closely associated with excitatory neurotoxicities. In the present study, we investigated the effects of quercetin on the glutamate-induced inward current (IGlu) in Xenopus oocytes that heterologously express human AMPA receptor and stargazin, an auxiliary subunit of AMPA receptor. IGlu was measured using the two-electrode voltage clamp technique. In oocytes injected with cRNAs coding AMPA receptor (GluR1) and stargazin, quercetin inhibited IGlu in a reversible and concentration-dependent manner. The IC50 was 84.9+/-15.0 microM. Quercetin action on IGlu was attenuated by increasing glutamate concentration, and was membrane holding potential-dependent. These results show a possibility that quercetin interacts with AMPA receptor, which was heterologously expressed in Xenopus oocytes and that quercetin action on IGlu of AMPA receptor could be one of contributions of quercetin-mediated neuroprotections.

Recognition memory impairments and amyloid-beta deposition of the retrosplenial cortex at the early stage of 5XFAD mice.

Early diagnosis and treatment of AD are critical for delaying its progression. The present study, therefore, examined the cognitive status and neuropathological characteristics of 4-month-old 5X familial AD (5XFAD) transgenic (Tg) mice, as an early stage of AD animal model. The novel object recognition task was performed with retention tests at varying intervals (i.e., 10 min, 1 h, 4 h, and 24 h) to measure the retention capacity of recognition memory of 5XFAD mice. At the 4h retention interval, 5XFAD mice exhibited worse performances than non-Tg control mice. Therefore, using amyloid-beta (Aß) 42- and 4G8-immunoreactive plaques, the accumulation of Aß was examined in the gray and white matter of the system that was necessary for the retention of recognition memory, with a focus on the hippocampus and retrosplenial cortex. The expression of ionized calcium-binding adapter molecule-1 (Iba-1) was also examined to measure microglial activation. The immunohistological analysis of Aß and Iba-1 revealed that the retrosplenial cortex was the most affected region in the brains of 4-month-old 5XFAD mice. These findings indicate that the cognitive and neuropathological characteristics of 4-month-old 5XFAD mice would provide a research platform for studying early diagnosis and treatment of AD.

Modulation of Ca(v)3.1 T-type Ca2+ channels by the ran binding protein RanBPM.

In order to study the currently unknown cellular signaling pathways of Ca(v)3.1 T-type Ca(2+) channels (Ca(v)3.1 channels), we performed a yeast two-hybrid screening using intracellular domains of Ca(v)3.1 alpha1 subunit as bait. After screening the human brain cDNA library, several proteins, including RanBPM, were identified as interacting with Ca(v)3.1 channels. RanBPM was found to bind to the cytoplasmic intracellular loop between transmembrane domains I and II of Ca(v)3.1 channels. Using whole-cell patch-clamp techniques, we found that Ca(v)3.1 currents were increased by the expression of RanBPM in HEK293/Ca(v)3.1 cells. We next examined whether RanBPM affected the biophysical properties and plasma membrane expression of Ca(v)3.1 channels. Furthermore, we showed that the PKC activator inhibited Ca(v)3.1 currents, an effect that was abolished by the expression of RanBPM. These results suggest that RanBPM could be a key regulator of Ca(v)3.1 channel-mediated signaling pathways.

Detection of 8-oxoguanine and apurinic/apyrimidinic sites using a fluorophore-labeled probe with cell-penetrating ability.

BACKGROUND: Reactive oxygen species (ROS) produce different lesions in DNA by ROS-induced DNA damage. Detection and quantification of 8-oxo-7,8-dihydroguanine (8-oxoG) within cells are important for study. Human ribosomal protein S3 (hRpS3) has a high binding affinity to 8-oxoG. In this study, we developed an imaging probe to detect 8-oxoG using a specific peptide from hRpS3. Transactivator (TAT) proteins are known to have cell-penetrating properties. Therefore, we developed a TAT-S3 probe by attaching a TAT peptide to our imaging probe. RESULTS: A DNA binding assay was conducted to confirm that our probe bound to 8-oxoG and apurinic/apyrimidinic (AP) sites. We confirmed that the TAT-S3 probe localized in the mitochondria, without permeabilization, and fluoresced in H2O2-treated HeLa cells and zebrafish embryos. Treatment with Mitoquinone (MitoQ), a mitochondria-targeted antioxidant, reduced TAT-S3 probe fluorescence. Additionally, treatment with O8, an inhibitor of OGG1, increased probe fluorescence. A competition assay was conducted with an aldehyde reaction probe (ARP) and methoxyamine (MX) to confirm binding of TAT-S3 to the AP sites. The TAT-S3 probe showed competitive binding to AP sites with ARP and MX. CONCLUSIONS: These results revealed that the TAT-S3 probe successfully detected the presence of 8-oxoG and AP sites in damaged cells. The TAT-S3 probe may have applications for the detection of diseases caused by reactive oxygen species.

Human ribosomal protein S3 (hRpS3) interacts with uracil-DNA glycosylase (hUNG) and stimulates its glycosylase activity.

Human ribosomal protein S3 (hRpS3) is a small ribosomal subunit showing apurinic/apyrimidinic (AP) lyase activity and has been suggested to play a role in the cellular DNA-damage response pathway. However, the functional interactions between hRpS3 and other base excision repair (BER) DNA glycosylases have not been reported. We identified, for the first time, the interaction between hRpS3 and human uracil-DNA glycosylase (hUNG) and investigated the functional consequences of this interaction. hRpS3 was shown to interact with hUNG in co-immunoprecipitation assay using transiently transfected HEK293 cells and GST pull-down assay using microbial expression systems. In an assay using a 5'-end-radiolabeled 39-mer oligonucleotide duplex containing a U/G mismatch, hRpS3 dramatically stimulated the uracil-excision activity of hUNG, whereas hRpS3 alone had no cleavage activity. Pre-incubation of hRpS3 with the U/G mismatch containing DNA duplex also increased the hUNG uracil-excision activity; however, hRpS3 did not increase the DNA binding activity of hUNG in a trapping assay of hUNG and the U/G mismatch containing DNA duplex using UV cross-linking. hRpS3 has been suggested to stimulate the uracil-excision activity of hUNG by enhancing its dissociation from AP sites and increasing its turn-over rate. The disruption of hRpS3 by small-interfering RNA (siRNA-hRpS3) transfection reduced the uracil-excision activity preserved in cell extracts, whereas the supplement of purified hRpS3 retained uracil-excision activity. These results strongly suggest that hRpS3 may be involved in the uracil-excision pathway, probably by participating in the DNA repair mechanism to remove uracil generated by the deamination of cytosine in DNA, and by preventing C/G-->T/A transition mutations.

Chronic Cerebral Hypoperfusion Induces Alterations of Matrix Metalloproteinase-9 and Angiopoietin-2 Levels in the Rat Hippocampus.

Angiogenic factors contribute to cerebral angiogenesis following cerebral hypoperfusion, and understanding these temporal changes is essential to developing effective treatments. The present study examined temporal alterations in angiogenesis-related matrix metalloproteinase-9 (MMP-9) and angiopoietin-2 (ANG-2) expression in the hippocampus following bilateral common carotid artery occlusion (BCCAo). Male Wistar rats (12 weeks of age) were randomly assigned to sham-operated control or experimental groups, and expression levels of MMP-9 and ANG-2 were assessed after BCCAo (1 week, 4 weeks, and 8 weeks), using western blotting. Protein expression increased 1 week after BCCAo and returned to control levels at 4 and 8 weeks. In addition, immunofluorescence staining demonstrated that the MMP-9- and ANG-2-positive signals were primarily observed in the NeuN-positive neurons with very little labeling in non-neuronal cells and no labeling in endothelial cells. In addition, these cellular locations of MMP-9- and ANG-2-positive signals were not altered over time following BCCAo. Other angiogenic factors such as vascular endothelial growth factor and hypoxia-inducible factor did not differ from controls at 1 week; however, expression of both factors increased at 4 and 8 weeks in the BCCAo group compared to the control group. Our findings increase understanding of alterations in angiogenic factors during the progression of cerebral angiogenesis and are relevant to developing effective temporally based therapeutic strategies for chronic cerebral hypoperfusion-associated neurological disorders such as vascular dementia.

Apoptotic and Anti-Inflammatory Effects of Eupatorium japonicum Thunb. in Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by synovitis, hyperplasia, and the destruction of bone and cartilage. A variety of immunosuppressive biological agents have been developed because the pathogenesis of RA is related predominantly to the inflammatory response. However, rheumatoid arthritis fibroblast-like synovial cells (RAFLS), which are known to play an important role in RA progression, exhibit resistance to immunosuppressants through cancer-like properties. In this study, we identified a novel therapeutic compound for RA, which reduced inflammation and the abnormal proliferation of RAFLS in natural product library made from Korean native plants. Eupatorium japonicum Thunb. (EJT) extract, a component of the natural product library, most effectively reduced viability through the induction of ROS-mediated apoptosis in a dose-dependent manner. In addition, the increased ROS induced the expression of ATF4 and CHOP, key players in ER stress-mediated apoptosis. Interestingly, EJT extract treatment dose-dependently reduced the expression of IL-1ß and the transcription of MMP-9, which were induced by TNF-α treatment, through the inhibition of NF-κB and p38 activation. Collectively, we found that EJT extract exerted apoptotic effects through increases in ROS production and CHOP expression and exerted anti-inflammatory effects through the suppression of NF-κB activation, IL-1ß expression, and MMP-9 transcription.

Angelica gigas Nakai Has Synergetic Effects on Doxorubicin-Induced Apoptosis.

Natural products are valuable sources for drug discovery because they have a wide variety of useful chemical components and biological properties. A quick reevaluation of the potential therapeutic properties of established natural products was made possible by the recent development of the methodology and improvement in the accuracy of an automated high-throughput screening system. In this study, we screened natural product libraries to detect compounds with anticancer effects using HeLa cells. Of the 420 plant extracts screened, the extract of Angelica gigas Nakai (AGN) was the most effective in reducing cell viability of HeLa cells. Markers of apoptosis, such as exposure of phosphatidylserine and cleavage of caspase-7 and PARP, were increased by treatment with the AGN extract. Treatment of the AGN extract increased expression of PKR as well as ATF4 and CHOP, the unfolded protein response genes. In addition, cotreatment of doxorubicin and the AGN extract significantly increased doxorubicin-induced apoptosis in HeLa cells. Decursin and decursinol angelate, which were known to have anticancer effects, were the main components of the AGN extract. These results suggest that the extract of AGN containing, decursin and decursinol angelate, increases doxorubicin susceptibility.

A distinct TthMutY bifunctional glycosylase that hydrolyzes not only adenine but also thymine opposite 8-oxoguanine in the hyperthermophilic bacterium, Thermus thermophilus.

Oxidative damage represents a major threat to genomic stability because the major product of DNA oxidation, 8-oxoguanine (GO), frequently mispairs with adenine during replication. We were interested in finding out how hyperthermophilic bacteria under goes the process of excising mispaired adenine from A/GO to deal with genomic oxidative damage. Herein we report the properties of an Escherichia coli MutY (EcMutY) homolog, TthMutY, derived from a hyperthermophile Thermus thermophilus. TthMutY preferentially excises on A/GO and G/GO mispairs and has additional activities on T/GO and A/G mismatches. TthMutY has significant sequence homology to the A/G and T/G mismatch recognition motifs, respectively, of MutY and Mig.MthI. A substitution from Tyr112 to Ser or Ala (Y112S and Y112A) in the putative thymine-binding site of TthMutY showed significant decrease in DNA glycosylase activity. A mutant form of TthMutY, R134K, could form a Schiff base with DNA and fully retained its DNA glycosylase activity against A/GO and A/G mispair. Interestingly, although TthMutY cannot form a trapped complex with substrate in the presence of NaBH(4), it expressed AP lyase activity, suggesting Tyr112 in TthMutY may be the key residue for AP lyase activity. These results suggest that TthMutY may be an example of a novel class of bifunctional A/GO mismatch DNA glycosylase that can also remove thymine from T/GO mispair.

Acacetin-induced apoptosis of human breast cancer MCF-7 cells involves caspase cascade, mitochondria-mediated death signaling and SAPK/JNK1/2-c-Jun activation.

The mechanism of acacetin-induced apoptosis of human breast cancer MCF-7 cells was investigated. Acacetin caused 50% growth inhibition (IC50) of MCF-7 cells at 26.4% 0.7% M over 24 h in the MTT assay. Apoptosis was characterized by DNA fragmentation and an increase of sub-G1 cells and involved activation of caspase-7 and PARP (poly-ADP-ribose polymerase). Maximum caspase 7 activity was observed with 100 microM acacetin for 24 h. Caspase 8 and 9 activation cascades mediated the activation of caspase 7. Acacetin caused a reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c and apoptosis inducing factor (AIF) into the cytoplasm, enhancing ROS generation and subsequently resulting in apoptosis. Pretreatment of cells with N-acetylcysteine (NAC) reduced ROS generation and cell growth inhibition, and pretreatment with NAC or a caspase 8 inhibitor (Z-IETD-FMK) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c and AIF. Stress-activated protein kinase/c-Jun NH4-terminal kinase 1/2 (SAPK/ JNK1/2) and c-Jun were activated by acacetin but extracellular-regulated kinase 1/2 (Erk1/2) nor p38 mitogen-activated protein kinase (MAPK) were not. Our results show that acacetin-induced apoptosis of MCF-7 cells is mediated by caspase activation cascades, ROS generation, mitochondria-mediated cell death signaling and the SAPK/JNK1/2-c-Jun signaling pathway, activated by acacetin-induced ROS generation.

Human glycine alpha1 receptor inhibition by quercetin is abolished or inversed by alpha267 mutations in transmembrane domain 2.

Quercetin, one of the flavonoids, is a compound of low molecular weight found in fruits and vegetables. Besides its antioxidative effect, quercetin also shows a wide range of diverse neuropharmacological actions. However, the cellular mechanisms of quercetin's actions, especially on ligand-gated ion channels and synaptic transmissions, are not well studied. We investigated the effect of quercetin on the human glycine alpha1 receptor channel expressed in Xenopus oocytes using a two-electrode voltage clamp technique. Application of quercetin reversibly inhibited glycine-induced current (I(Gly)). Quercetin's inhibition depends on its dose, with an IC(50) of 21.5+/-.2 microM. The inhibition was sensitive to membrane voltages. Site-directed mutations of S267 to S267Y but not S267A, S267F, S267G, S267K, S267L and S267T at transmembrane domain 2 (TM2) nearly abolished quercetin-induced inhibition of I(Gly). In contrast, in site-directed mutant receptors such as S267 to S267I, S267R and S267V, quercetin enhanced I(Gly) compared to the wild-type receptor. The EC(50) was 22.6+/-1.4, 25.5+/-4.2, and 14.5+/-3.1 microM for S267I, S267R and S267V, respectively. These results indicate that quercetin might regulate the human glycine alpha(1) receptor via interaction with amino acid residue alpha267 and that alpha267 plays a key role in determining the regulatory consequences of the human glycine alpha1 receptor by quercetin.

Alzheimer's disease drug discovery from herbs: neuroprotectivity from beta-amyloid (1-42) insult.

OBJECTIVE: To comparatively evaluate selected herbs for their ability to protect neuronal cells from direct betaA(1-42) insult. DESIGN: Twenty-seven (27) herbs were selected, extracted with aqueous methanol (90%) and chloroform, and the extracts were evaluated for their ability to protect PC12 rat pheochromocytoma and primary neuronal cells from betaA(1-42) insult using both 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction assay and lactate dehydrogenase efflux assay. RESULTS: Curcuma aromatia (ul-keum) and Zingiber officinale (ginger) extracts effectively protected cells from betaA(1-42) insult, followed by Ginkgo biloba (ginkgo), Polygonatum sp. (King Solomon's seal), Cinnamum cassia (Chinese cinnamon), Rheum coreanum (Korean rhubarb), Gastrodia elata (gastrodia), and Scutellaria baicalensis (skullcap). Several extracts showed cytotoxicity at high concentration (approximately 150 microg/mL), whereas other extracts did not at all protect cells from betaA(1-42) insult. CONCLUSION: Selective herbs may be potentially important resources to discover drug candidates against the onset of Alzheimer's disease.

Bacterial beta-lactamase fragmentation complementation strategy can be used as a method for identifying interacting protein pairs.

We investigated the applicability of the TEM-1 beta- lactamase fragment complementation (BFC) system to develop a strategy for the screening of protein-protein interactions in bacteria. A BFC system containing a human Fas-associated death domain (hFADD) and human Fas death domain (hFasDD) was generated. The hFADD-hFasDD interaction was verified by cell survivability in ampicillin-containing medium and the colorimetric change of nitrocefin. It was also confirmed by His pull-down assay using cell lysates obtained in selection steps. A coiled-coil helix coiled-coil domain-containing protein 5 (CHCH5) was identified as an interacting protein of human uracil DNA glycosylase (hUNG) from the bacterial BFC cDNA library strategy. The interaction between hUNG and CHCH5 was further confirmed with immunoprecipitation using a mammalian expression system. CHCH5 enhanced the DNA glycosylase activity of hUNG to remove uracil from DNA duplexes containing a U/G mismatch pair. These results suggest that the bacterial BFC cDNA library strategy can be effectively used to identify interacting protein pairs.

New role of human ribosomal protein S3: Regulation of cell cycle via phosphorylation by cyclin-dependent kinase 2.

Human ribosomal protein S3 (hRpS3) is a component of the 40S ribosomal subunit that associated in protein synthesis. hRpS3 has additional ribosomal functions such as DNA repair, transcription, metastasis, and apoptosis via interaction with numerous signaling molecules and has different modifications. Cyclin-dependent kinases (CDKs) are heterodimeric serine/threonine protein kinases that regulate cell cycle progression. Among its members, the Cdk1-cyclin B complex is known to control cell progression in the G2/M phase, while Cdk2-cyclin E/A complexes function in G1/S and S/G2 transition. In our previous study, we observed interaction between hRpS3 and Cdk1. The present study investigated the interaction between hRpS3 and Cdk2. Cdk2 phosphorylated hRps3 at amino acid residues S6 and T221 during the S-phase. Furthermore, hRpS3 knockdown delayed cell cycle progression by modulating the expression of cell cycle-related proteins, including cyclin B1 and cyclin E1. These findings suggest that hRpS3 is involved in Cdk2-mediated cell cycle regulation.

Endonuclease IV enhances base excision repair of endonuclease III from Methanobacterium thermoautotrophicum.

Damaged DNA strands are repaired by base excision (BER) in organisms, a process initiated by repair enzymes, which include DNA glycosylases and endonucleases. We expressed and characterized two putative endonuclease genes from Methanobacterium thermoautotrophicum, Mt0764 and Mt1010, encoding homologues of endonuclease III (endo III) and endonuclease IV (endo IV) of Escherichia coli. The Mt0764 and Mt1010 proteins showed endo III activity by removing thymine glycol from DNA strand and AP endonuclease activity, respectively. The Mt0764 protein not only cleaved the oligonucleotide duplex, containing a thymine glycol/adenine pair efficiently, but also showed activity on the 8-oxoguanine-containing oligonucleotide duplex. In this study, we report upon the stimulation of endo III activity by endo IV using two recombinant proteins (Mt1010 and Mt0764) from M. thermoautotrophicum. Mt1010 stimulated the DNA glycosylase activity of Mt0764 for DNA substrates containing 8-oxoguanine residues and increasing the formation of the Mt0764 protein-DNA complex. The interaction between Mt1010 and Mt0764 was observed by using an in vitro binding assay. These results suggest that association between endo III and endo IV may occur in vivo, and this contributes to efficient base excision repair for the oxidative damage of DNA.