Study on the Effect of Residual Polymer Superplasticizer on the Properties of Graphene-Cement Composites.

Graphene, renowned for its exceptional mechanical, thermal, and electrical properties, is being explored as a cement nanofiller in the construction field. However, the limited water dispersibility of graphene requires the use of polymer superplasticizers, such as polycarboxylate ether (PCE). Previous studies have investigated the mechanisms by which PCE facilitates the dispersion of graphene within cement nanocomposites. However, such studies have made minimal progress, indicating a lack of understanding of the effect of residual PCE (rPCE) remaining in aqueous solution without binding to graphene. In this study, the effects of rPCE on the dispersion of graphene and the mechanical properties of graphene-cement composites (GCCs) were systematically analyzed. For this purpose, the content of rPCE was accurately measured through the centrifugation process and thermal analysis of graphene dispersion with PCE, and the result was 78.0 wt.% compared to graphene. The optical microscopy, particle size analysis, and contact angle measurement of the graphene dispersions with and without rPCE confirmed that rPCE is crucial for the dispersion of graphene and the enhancement of the interfacial affinity between graphene and cement. Additionally, the compressive strength of GCC with rPCE exhibited a substantial enhancement of approximately 10% (68.36 MPa) compared to plain cement (62.33 MPa). The effectiveness of rPCE in enhancing compressive strength correlated with the uniform dispersion of graphene within GCC and the promotion of cement hydration, as evidenced by field emission scanning electron microscopy and X-ray diffraction, respectively.

Reinforcement of Cement Nanocomposites through Optimization of Mixing Ratio between Carbon Nanotube and Polymer Dispersing Agent.

Carbon nanotubes (CNTs), known for their exceptional mechanical, thermal, and electrical properties, are being explored as cement nanofillers in the construction field. However, due to the limited water dispersion of CNTs, polymer dispersing agents like polycarboxylate ether (PCE) and sulfonated naphthalene formaldehyde (SNF) are essential for uniform dispersion. In a previous study, PCE and SNF, common cement superplasticizers, effectively dispersed CNTs in cement nanocomposites. However, uncertainties remained regarding the extent to which all dispersing agents interacted efficiently with CNTs. Therefore, this research quantitatively assessed CNT interaction with dispersing agents through dispersion and centrifugation. Approximately 37% of PCE and 50% of SNF persisted compared to CNT after centrifugation. The resulting cement nanocomposites, with optimized mixing ratios, exhibited enhanced compressive strength of about 14% for CNT/PCE (78.13 MPa) and 12.3% for CNT/SNF (76.97 MPa) compared to plain cement (68.52 MPa). XRD results linked strength reinforcement to increased cement hydrate from optimized CNT dispersion. FE-SEM analysis revealed that CNTs were positioned within the pores of the cement. These optimized cement nanocomposites hold promise for improved safety in the construction industry.

Enzymatic characterization of a novel recombinant 1,3-α-3,6-anhydro-L-galactosidase specific for neoagarobiose hydrolysis into monosaccharides.

Two GH117 family α-neoagarobiose hydrolases (GH117A α-NABH and GH117B α-NABH) from the freshwater agar-degrading Cellvibrio sp. KY-GH-1 were expressed and purified as recombinant His-tagged proteins using an Escherichia coli expression system to compare activities. The amino acid sequence of GH117A α-NABH (364 amino acids, 40.9 kDa) showed 35% identity with that of GH117B α-NABH (392 amino acids, 44.2 kDa). GH117A α-NABH, but not GH117B α-NABH, could hydrolyze neoagarobiose (NA2) into monosaccharides 3,6-anhydro-L-galactose (L-AHG) and D-galactose. The presence of GH117A α-NABH homologues in all of the agar-degrading bacteria aligned suggests that GH117A α-NABH hydrolyzing NA2 into L-AHG and D-galactose is an essential component of the agar-degrading enzyme machinery. For GH117A α-NABH-catalyzed hydrolysis, NA2 was the sole substrate among various neoagaro-oligosaccharides (NA2~NA18). GH117A α-NABH appeared to exist as a dimer, and optimal enzymatic temperature and pH were 35 °C and 7.5, respectively. GH117A α-NABH was stable up to 35 °C and at pH 7.5 and unstable beyond 35 °C and outside pH 7.0~7.5. The kinetic parameters Km, Vmax, kcat, and kcat/Km for NA2 were 16.0 mM, 20.8 U/mg, 14.2 s-1, and 8.9 × 102 s-1 M-1, respectively. Combined addition of 5 mM MnSO4 and 10 mM tris(2-carboxyethyl)phosphine enhanced the enzyme activity by 2.4-fold. The enzyme-mediated hydrolysis of 5.0% NA2 into monosaccharide and purification of L-AHG from hydrolysis products by Sephadex G-10 column chromatography recovered ~ 192 mg L-AHG from 400 mg NA2 (~ 92% of the theoretical maximum yield). These results indicate that the recombinant GH117A α-NABH is NA2-specific and useful to produce L-AHG from NA2. KEY POINTS: • Recombinant GH117A α-NABH (364 aa, 40.9 kDa) purified from E. coli forms a dimer. • The enzyme hydrolyzes only NA2 among various neoagaro-oligosaccharides (NA2~NA18). • The enzyme completely hydrolyzes up to 5% NA2 into monomers under optimal conditions.

G1 Cell Cycle Arrest and Extrinsic Apoptotic Mechanisms Underlying the Anti-Leukemic Activity of CDK7 Inhibitor BS-181.

In vitro antitumor activity of the CDK7 inhibitor BS-181 against human T-ALL Jurkat cells was determined. Treatment of Jurkat clones (JT/Neo) with BS-181 caused cytotoxicity and several apoptotic events, including TRAIL/DR4/DR5 upregulation, c-FLIP down-regulation, BID cleavage, BAK activation, ΔΨm loss, caspase-8/9/3 activation, and PARP cleavage. However, the BCL-2-overexpressing Jurkat clone (JT/BCL-2) abrogated these apoptotic responses. CDK7 catalyzed the activating phosphorylation of CDK1 (Thr161) and CDK2 (Thr160), and CDK-directed retinoblastoma phosphorylation was attenuated in both BS-181-treated Jurkat clones, whereas only JT/BCL-2 cells exhibited G1 cell cycle arrest. The G1-blocker hydroxyurea augmented BS-181-induced apoptosis by enhancing TRAIL/DR4/DR5 upregulation and c-FLIP down-regulation. BS-181-induced FITC-annexin V-positive apoptotic cells were mostly in the sub-G1 and G1 phases. BS-181-induced cytotoxicity and mitochondrial apoptotic events (BAK activation/ΔΨm loss/caspase-9 activation) in Jurkat clones I2.1 (FADD-deficient) and I9.2 (caspase-8-deficient) were significantly lower than in A3 (wild-type). Exogenously added recombinant TRAIL (rTRAIL) markedly synergized BS-181-induced apoptosis in A3 cells but not in normal peripheral T cells. The cotreatment cytotoxicity was significantly reduced by the DR5-blocking antibody but not by the DR4-blocking antibody. These results demonstrated that the BS-181 anti-leukemic activity is attributed to extrinsic TRAIL/DR5-dependent apoptosis preferentially induced in G1-arrested cells, and that BS-181 and rTRAIL in combination may hold promise for T-ALL treatment.

Enhancement of Quercetin-Induced Apoptosis by Cotreatment with Autophagy Inhibitor Is Associated with Augmentation of BAK-Dependent Mitochondrial Pathway in Jurkat T Cells.

A flavonoid antioxidant quercetin promotes dose-dependent activation of the ATM-CHK-p53 pathway, downregulation of antiapoptotic survivin, and upregulation of proapoptotic NOXA in human T cell acute lymphoblastic leukemia Jurkat clones (J/Neo and J/BCL-XL). However, the downregulation of antiapoptotic BAG3 and MCL-1 occurred in J/Neo cells but not in J/BCL-XL cells overexpressing BCL-XL. Additionally, several BCL-XL-sensitive intrinsic mitochondrial apoptotic events including apoptotic sub-G1 cell accumulation, TUNEL-positive DNA fragmentation, BAK activation, mitochondrial membrane potential (Δψm) loss, caspase-9/caspase-8/caspase-3 activation, and PARP cleavage were induced only in J/Neo cells. Both cytosolic and mitochondrial ROS levels were elevated in quercetin-treated J/Neo cells; however, the ROS elevations were almost completely abrogated in J/BCL-XL cells, suggesting the ROS elevations were downstream of BCL-XL-sensitive mitochondrial damage and dysfunction. Wild-type A3, FADD-deficient I2.1, and caspase-8-deficient I9.2 Jurkat clones exhibited similar susceptibilities to the cytotoxicity of quercetin, excluding an involvement of extrinsic pathway in triggering the apoptosis. The autophagic events such as attenuation of AKT-mTOR pathway, formation of acridine orange-stainable acidic vesicular organelles, conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to LC3-II, and downregulation of p62/SQSTM1 level were detected in quercetin-treated J/Neo and J/BCL-XL cells, regardless of BCL-XL overexpression. Cotreatment with the autophagy inhibitor (3-methyladenine, LY294002, or chloroquine) resulted in a significant enhancement of quercetin-induced BAK activation and subsequently the mitochondrial damage-mediated apoptosis pathway by augmenting the downregulation of BAG3 and MCL-1 levels in J/Neo cells. These results demonstrated that quercetin induces intrinsic apoptosis and cytoprotective autophagy, and autophagy inhibition can potentiate BAK-dependent apoptotic activity of quercetin in Jurkat T cells.

l-Serine protects mouse hippocampal neuronal HT22 cells against oxidative stress-mediated mitochondrial damage and apoptotic cell death.

The cytoprotective mechanism of l-serine against oxidative stress-mediated neuronal apoptosis was investigated in mouse hippocampal neuronal HT22 cells. Treatment with the reactive oxygen species (ROS) inducer 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) increased cytosolic and mitochondrial ROS and apoptosis, without necrosis, in HT22 cells. ROS-mediated apoptosis was accompanied by the induction of the endoplasmic reticulum (ER) stress-mediated apoptotic pathway, involving CHOP/GADD153 upregulation, JNK and p38 MAPK activation, and caspase-12 and caspase-8 activation, and subsequent induction of the mitochondrial apoptotic pathway through BAK and BAX activation, mitochondrial membrane potential (Δψm) loss, caspase-9 and caspase-3 activation, PARP cleavage, and nucleosomal DNA fragmentation. However, the DMNQ-caused ROS elevation and ER stress- and mitochondrial damage-induced apoptotic events were dose-dependently suppressed by co-treatment with l-serine (7.5-20 mM). Although DMNQ reduced both the intracellular glutathione (GSH) level and the ratios of reduced GSH to oxidized GSH (GSSG), the reduction was restored by co-treatment with l-serine. Co-treatment with GSH or N-acetylcysteine also blocked DMNQ-caused ROS elevation and apoptosis; however, co-treatment with the GSH synthesis inhibitor buthionine sulfoximine significantly promoted ROS-mediated apoptosis and counteracted the protection by l-serine. In HT22 cells, DMNQ treatment appeared to tilt the mitochondrial fusion-fission balance toward fission by down-regulating the levels of profusion proteins (MFN1/2 and OPA1) and inhibitory phosphorylation of profission protein DRP1 at Ser-637, resulting in mitochondrial fragmentation. These DMNQ-caused alterations were prevented by l-serine. A comparison of mitochondrial energetic function between DMNQ- and DMNQ/l-serine-treated HT22 cells showed that the DMNQ-caused impairment of the mitochondrial energy generation capacity was restored by l-serine. These results demonstrate that l-serine can protect neuronal cells against oxidative stress-mediated apoptotic cell death by contributing to intracellular antioxidant GSH synthesis and maintaining the mitochondrial fusion-fission balance.

Cytoprotective effect of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (CMEP-NQ) is mediated by the inhibition of BAK-dependent mitochondrial apoptosis pathway.

The inhibitory mechanism of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (CMEP-NQ) against apoptosis induced by the microtubule-damaging agents (MDAs), nocodazole (NOC) and 2-methoxyestradiol (2-MeO-E2), or a DNA-damaging agent (DDA), camptothecin (CPT) were investigated in human Jurkat T cell clones (J/Neo and J/BCL-XL cells). Treatment of J/Neo cells with NOC, 2-MeO-E2, or CPT caused cytotoxicity and apoptotic DNA fragmentation but these events were significantly attenuated in the presence of CMEP-NQ. Although not only MDA (NOC or 2-MeO-E2)-induced mitotic arrest, CDK1 activation, and BCL-2, BCL-XL and BIM phosphorylation, but also DDA (CPT)-induced S-phase arrest and ATM-CHK1/CHK2-p53 pathway activation were not or were barely affected in the presence of CMEP-NQ, the levels of anti-apoptotic BAG3 and MCL-1, which were markedly downregulated after MDA- or DDA-treatment, were rather elevated by CMEP-NQ. Under the same conditions, MDA- or DDA-induced mitochondrial apoptotic events including BAK activation, mitochondrial membrane potential (Δψm) loss, caspase-9 activation, and PARP cleavage were significantly inhibited by CMEP-NQ. While MDA- or DDA-induced sub-G1 peak and Δψm loss were abrogated in J/BCL-XL cells, MDA-induced mitotic arrest and DDA-induced S-arrest were more apparent in J/BCL-XL cells than in J/Neo cells. Simultaneously, the induced cell cycle arrest in J/BCL-XL cells was not significantly disturbed by CMEP-NQ. MDA- or DDA-treatment caused intracellular reactive oxygen species (ROS) production; however, MDA- or DDA-induced ROS production was almost completely abrogated in J/BCL-XL cells. MDA- or DDA-induced ROS production in J/Neo cells was significantly suppressed by CMEP-NQ, but the suppressive effect was hardly observed in J/BCL-XL cells. Together, these results show that CMEP-NQ efficiently protects Jurkat T cells from apoptotic cell death via the elevation of BAG3 and MCL-1 levels, which results in the inhibition of intrinsic BAK-dependent mitochondrial apoptosis pathway, as does the overexpression of BCL-XL.

Pharmacologic Inhibition of Autophagy Sensitizes Human Acute Leukemia Jurkat T Cells to Acacetin-Induced Apoptosis.

Exposure of Jurkat T cell clone (J/Neo cells) to acacetin (5,7-dihydroxy-4'-methoxyflavone), which is present in barnyard millet (Echinochloa esculenta (A. Braun)) grains, caused cytotoxicity, enhancement of apoptotic sub-G1 rate, Bak activation, loss of mitochondrial membrane potential (Δψ), activation of caspase-9 and caspase-3, degradation of poly(ADP-ribose) polymerase, and FITC-Annexin V-stainable phosphatidylserine exposure on the external surface of the cytoplasmic membrane without accompanying necrosis. These apoptotic responses were abrogated in Jurkat T cell clone (J/Bcl-xL) overexpressing Bcl-xL. Under the same conditions, cellular autophagic responses, including suppression of the Akt-mTOR pathway and p62/SQSTM1 down-regulation, were commonly detected in J/Neo and J/Bcl-xL cells; however, formation of acridine orange-stainable acidic vascular organelles, LC3-I/II conversion, and Beclin-1 phosphorylation (Ser-15) were detected only in J/Neo cells. Correspondingly, concomitant treatment with the autophagy inhibitor (3-methyladenine or LY294002) appeared to enhance acacetin-induced apoptotic responses, such as Bak activation, Δψ loss, activation of caspase-9 and caspase-3, and apoptotic sub-G1 accumulation. This indicated that acacetin could induce apoptosis and cytoprotective autophagy in Jurkat T cells simultaneously. Together, these results demonstrate that acacetin induces not only apoptotic cell death via activation of Bak, loss of Δψ, and activation of the mitochondrial caspase cascade, but also cytoprotective autophagy resulting from suppression of the Akt-mTOR pathway. Furthermore, pharmacologic inhibition of the autophagy pathway augments the activation of Bak and resultant mitochondrial damage-mediated apoptosis in Jurkat T cells.

Inhibition of autophagy enhances dynamin inhibitor-induced apoptosis via promoting Bak activation and mitochondrial damage in human Jurkat T cells.

Treatment of Jurkat T cells with the dynamin inhibitor, myristyl trimethyl ammonium bromides (MiTMAB) caused cytokinesis impairment and apoptotic DNA fragmentation along with down-regulation of anti-apoptotic BAG3 and Mcl-1 levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9 and -3, and PARP cleavage, without accompanying necrosis. Bcl-xL overexpression completely abrogated these MiTMAB-induced mitochondrial damage and resultant caspase cascade activation, except for impaired cytokinesis and down-regulated BAG3 and Mcl-1 levels. Additionally, autophagic responses including Akt-mTOR pathway inhibition, formation of acridine orange-stainable acidic vesicular organelles, LC3-I/II conversion, and p62/SQSTM1 down-regulation were detected regardless of Bcl-xL overexpression. The autophagy inhibitors 3-methyladenine and LY294002 enhanced MiTMAB-induced apoptotic sub-G1 peak, BAG3 and Mcl-1 down-regulation, Bak activation, Δψm loss, and caspase activation. These results indicate that MiTMAB-caused cytokinesis failure leads to concomitant induction of apoptosis and cytoprotective autophagy, and suggest that inhibition of autophagy is a promising strategy to augment antitumor activity of MiTMAB.

Kaempferol Activates G2-Checkpoint of the Cell Cycle Resulting in G2-Arrest and Mitochondria-Dependent Apoptosis in Human Acute Leukemia Jurkat T Cells.

The effect of kaempferol (3,5,7,4-tetrahydroxyflavone), a flavonoid compound that was identified in barnyard millet (Echinochloa crus-galli var. frumentacea) grains, on G2-checkpoint and apoptotic pathways was investigated in human acute leukemia Jurkat T cell clones stably transfected with an empty vector (J/Neo) or a Bcl-xL expression vector (J/Bcl-xL). Exposure of J/Neo cells to kaempeferol caused cytotoxicity and activation of the ATM/ATR-Chk1/Chk2 pathway, activating the phosphorylation of p53 (Ser-15), inhibitory phosphorylation of Cdc25C (Ser-216), and inactivation of cyclin-dependent kinase 1 (Cdk1), with resultant G2- arrest of the cell cycle. Under these conditions, apoptotic events, including upregulation of Bak and PUMA levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9, -8, and -3, anti-poly (ADP-ribose) polymerase (PARP) cleavage, and accumulation of apoptotic sub-G1 cells, were induced without accompanying necrosis. However, these apoptotic events, except for upregulation of Bak and PUMA levels, were completely abrogated in J/Bcl-xL cells overexpressing Bcl-xL, suggesting that the G2-arrest and the Bcl-xL-sensitive mitochondrial apoptotic events were induced, in parallel, as downstream events of the DNA-damage-mediated G2-checkpoint activation. Together these results demonstrate that kaempferol-mediated antitumor activity toward Jurkat T cells was attributable to G2-checkpoint activation, which caused not only G2-arrest of the cell cycle but also activating phosphorylation of p53 (Ser-15) and subsequent induction of mitochondriadependent apoptotic events, including Bak and PUMA upregulation, Bak activation, Δpsim loss, and caspase cascade activation.