Volatile organic compounds emission in the rubber products manufacturing processes.

Volatile organic compounds (VOCs) emission from rubber products manufacture processes, mixing, shaping and vulcanization were investigated in four rubber products factories in China. The source emission air was passively sampled by pre-vacuumized stainless steel canister and analyzed by gas chromatography-mass spectrometry-flame ionization detection (GC/MS-FID). The species profile of 107 VOCs in the emission processes were obtained. We calculated the photochemical ozone formation potential (OFP) and carcinogenic risk (CR) of the VOCs for each manufacture process. The results showed that mixing process mainly released dichloromethane (14.53%), carbon disulfide (CS2) (6.88%), styrene (5.72%), 4-methyl-2-pentanone (5.22%) and naphthalene (3.69%) for solvents used and raw rubber degradation in the process. The C6-C8 alkanes, especially heptane and isomers of heptane (44.71%), were dominated in shaping process. The major species released from vulcanization process were carbon disulfide (29.72%), naphthalene (8.17%), acetone (7.73%) and dichloromethane (4.26%). VOCs emitted from vulcanization process had the highest OFP, which contributed by naphthalene, m/p-xylene, o-xylene and carbon disulfide. VOCs emission from mixing process had the highest CR, and 1,2-dibromoethane, 1,2-dichlorethane and 1,3-butadiene were the main contributors to CR. We also estimated the total VOCs emissions into the atmosphere from tires manufacturing in China, which were 7.58 × 105 t in 2018 and contributed about 9% of total industry processes VOCs emissions.

Chained multimediator model of sexual orientation disclosure, sexual minority stigma, sexual minority identity, social support, and resilience among ymsms.

INTRODUCTION: This study aimed to investigate sexual orientation disclosure and mental health among young men who have sex with men (YMSMs). To this end, we constructed a chained multimediator model of sexual minority stigma, sexual minority identity, social support, and resilience, with the moderator of sexual orientation disclosure. METHODS: We conducted a cross-sectional survey of 345 YMSMs in Nanning, China. Bivariate analysis was used to evaluate factors associated with sexual orientation disclosure. Sexual minority stigma was used to predict identity, with social support as the step 1 mediator and resilience as the step 2 mediator. Sexual minority identity was analyzed using a chained moderated mediation model; sexual orientation disclosure was included as a moderator in all models to control its confounding effect. RESULTS: The average age of YMSMs was 20.0 ± 1.3 years. Bivariate analysis indicated that YMSMs who disclosed sexual orientation may have experienced less stigma (15.49 ± 3.02 vs 16.21 ± 2.74), obtained more social support (65.98 ± 11.18 vs 63.19 ± 11.13), had strong psychological resilience (37.40 ± 8.57 vs 35.39 ± 7.73), and had a more positive self-identity (104.12 ± 21.10 vs 95.35 ± 16.67); differences between subgroups were statistically significant (p < 0.05). Sexual minority stigma, perceived stigma, and enacted stigma were significantly associated with social support and resilience. The association between sexual minority stigma and sexual minority identity was significantly mediated by social support (indirect effect [95% CI] = - 3.307 [- 4.782, - 1.907]). Resilience significantly mediated the same association for identity (- 2.544 [- 4.052, - 1.114]). The chained relationship from sexual minority stigma to social support, resilience, and identity was also significant, with an indirect effect of - 0.404 [- 0.621, - 0.249]. CONCLUSION: Among YMSMs in China, sexual minority stigma affects sexual minority identity through social support and resilience. Given the psychological effects of stigma, social support and resilience must be considered to better promote positive self-identity and mental health among YMSMs.

Individual and network factors associated with HIV self-testing among men who have sex with men in resource-limited settings in China.

BACKGROUND: HIV self-testing (HIVST) is a potential strategy to overcome challenges of HIV testing among men who have sex with men (MSM). However, for resource-limited settings, technology and diagnostic devices are lagging. Hence, we estimated the status and correlates of HIVST among MSM in resource-limited settings in China to inform the development of HIVST to reach United Nations Programme on HIV and AIDS (UNAIDS) targets to end HIV by 2030. METHODS: A cross-sectional study was conducted among MSM in Nanning, Guangxi, China, between August 2019 and January 2020. The HIVST status was collected and data on social network features, sociodemographic information, risk behaviours, etc. were compared between prior- and non-HIVST MSM. Logistic regression analyses were conducted to examine the correlates of HIVST. RESULTS: The prevalence of HIVST among 446 MSM was 40.4% (95% confidence interval [CI] 35.8-44.9%). The main component of sociocentric network contains more prior-HIVST MSM (38.3%) than non-HIVST MSM (28.6%, P =0.031). More MSM with individual features such as substance use during anal sex (22.8% vs 15.4%, P =0.049) and multiple sexual partners (76.1% vs 59.4%, P <0.001) were detected among prior-HIVST MSM. In multivariable analysis, prior HIVST was associated with the strong strength of ego-alter ties in the egocentric network (adjusted odds ratio [aOR] 1.72; 95% CI 1.09-2.71), HIV-infected partners (aOR, 7.17; 95% CI, 1.40-36.60), and vaginal intercourse (aOR, 0.38; 95% CI, 0.17-0.85). CONCLUSIONS: HIVST coverage among MSM in resource-limited settings is suboptimal. Integrating social networks into testing services may be viable to promote HIVST in MSM within resource-limited settings.

The effect of catalase on migration and invasion of lung cancer cells by regulating the activities of cathepsin S, L, and K.

Abundant clinical evidences indicate that up-regulation of several cathepsins in many human cancers is correlated with malignant progression and poor patient prognosis. In addition, a decrease in catalase activity or accumulation of hydrogen peroxide correlates with cancer metastasis. Recent studies indicate that cathepsin activation and expression can be modulated via H2O2 treatment. However, the actual relationship between catalase and cathepsins is not yet fully understood. In the present study, we found that catalase expression (or activity) was higher, while intracellular and extracellular Cat S, Cat L, and Cat K activities were lower in the non-invasive CL1-0 cells compared to the highly invasive CL1-5 cells. After CL1-0 cells were transfected with catalase-shRNA, the corresponding ROS (H2O2) level and Cat S, Cat L, or Cat K expression (or activity) was up-regulated, accompanied by an increase in cell migration and invasion. On the other hand, ROS (H2O2) level, cathepsin S, L, and K activities, cell migration and invasion were decreased in catalase-overexpressed CL1-5 cells. It is suggested that catalase may regulate cathepsin activity by controlling the production of ROS (H2O2), leading to variation in migration and invasion ability of lung cancer cells.

Toll-like receptor 9 agonist enhances anti-tumor immunity and inhibits tumor-associated immunosuppressive cells numbers in a mouse cervical cancer model following recombinant lipoprotein therapy.

BACKGROUND: Although cytotoxic T lymphocytes (CTLs) play a major role in eradicating cancer cells during immunotherapy, the cancer-associated immunosuppressive microenvironment often limits the success of such therapies. Therefore, the simultaneous induction of cancer-specific CTLs and reversal of the immunosuppressive tumor microenvironment may be more effectively achieved through a single therapeutic vaccine. A recombinant lipoprotein with intrinsic Toll-like receptor 2 (TLR2) agonist activity containing a mutant form of E7 (E7m) and a bacterial lipid moiety (rlipo-E7m) has been demonstrated to induce robust CTL responses against small tumors. This treatment in combination with other TLR agonists is able to eliminate large tumors. METHODS: Mouse bone marrow-derived dendritic cells (DCs) were employed to determine the synergistic production of pro-inflammatory cytokines upon combination of rlipo-E7m and other TLR agonists. Antigen-specific CTL responses were investigated using immunospots or in vivo cytolytic assays after immunization in mice. Mice bearing various tumor sizes were used to evaluate the anti-tumor effects of the formulation. Specific subpopulations of immunosuppressive cells in the tumor infiltrate were quantitatively determined by flow cytometry. RESULTS: We demonstrate that a TLR9 agonist (unmethylated CpG oligodeoxynucleotide, CpG ODN) enhances CTL responses and eradicates large tumors when combined with rlipo-E7m. Moreover, combined treatment with rlipo-E7m and CpG ODN effectively increases tumor infiltration by CTLs and reduces the numbers of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) in the tumor microenvironment. CONCLUSION: These findings suggest that the dramatic anti-tumor effects of the recombinant lipoprotein together with CpG ODN may reflect the amplification of CTL responses and the repression of the immunosuppressive environment. This promising approach could be applied for the development of additional therapeutic cancer vaccines.

Effects of novel human cathepsin S inhibitors on cell migration in human cancer cells.

Elevated cathepsin S (Cat S) level is correlated with higher migration ability in tumor cells. This study investigates the inhibitory effect of novel synthetic α-ketoamide compounds on cathepsin activity and cancer cell migration. The effect of several α-ketoamide compounds on the activity of recombinant cathepsins (Cat S, Cat L and Cat K) was examined. Two highly metastatic cancer cell lines were incubated with three Cat S-specific compounds (6n, 6 w and 6r) to analyze their effect on cellular Cat S activity and cell migration. At a 100 nM concentration, compounds 6n, 6r and 6 w effectively inhibited Cat S activity. Cat S activity and cell migration were significantly reduced in CL1-3 cells after treatment with either 6n or 6 w at 5 µM. Similar results were also obtained when A2058 cells were treated with 6n. These results highlight the therapeutic potential of α-ketoamide compounds, especially 6n and 6 w, to prevent or delay cancer metastasis.

Proteomic and redox-proteomic study on the role of glutathione reductase in human lung cancer cells.

Glutathione reductase (GR), a cytosolic protein, plays a vital role in maintaining a correct redox status in cells. However, comprehensive investigations of GR-modulated cellular responses, including protein level alteration and redox regulation, have yet to be performed. In this study, we cultured a human lung adenocarcinoma line transfected with empty pLKO.1 vector as a control, CL1-0shControl, and its GR-knockdown derivative, CL1-0shΔGR, to evaluate differential protein level alteration and redox regulation of these two cell lines. We identified 34 spots that exhibited marked changes in intensities, and 13 proteins showing significant changes in thiol reactivity, in response to GR depletion. Several proteins involved in redox regulation, calcium signaling, cytoskeleton regulation, and protein folding showed significant changes in expression, whereas proteins involved in redox regulation, protein folding, and glycolysis displayed changes in thiol reactivity. Interestingly, GR knockdown induces peroxiredoxin-1 overexpression in the air-exposed tissue and high oxygen consuming tissue such as cornea and liver, but not in the low oxygen consuming tissues such as breast and uterine. In summary, we used a comprehensive lung adenocarcinoma based proteomic approach for identifying GR-modulated protein expression alteration and redox modification. Based on our research, this is the first comprehensive proteomic and redox-proteomic analysis used to investigate the role of GR in a mammalian cell model.

Family outcome disparities between sexual minority and heterosexual families: a systematic review and meta-analysis.

BACKGROUND: The number of children in sexual minority parent families has increased. This systematic review aims to synthesise the evidence of disparities in family outcomes between sexual minority and heterosexual families and to identify specific social risk factors of poor family outcomes. METHODS: We systematically searched PubMed, the Web of Science, Embase, the Cochrane Library and APA PsycNet for original studies that compared family outcomes between sexual minority and heterosexual families. Two reviewers independently selected studies and assessed the risk of bias of included studies. Narrative synthesis and meta-analysis were conducted to synthesise evidence. RESULTS: Thirty-four articles were included. The narrative synthesis results revealed several significant findings for children's gender role behaviour and gender identity/sexual orientation outcomes. Overall, 16 of 34 studies were included in the meta-analyses. The quantitative synthesis results suggested that sexual minority families may perform better in children's psychological adjustment and parent-child relationship than heterosexual families (standardised mean difference (SMD) -0.13, 95% CI -0.20 to -0.05; SMD 0.13, 95% CI 0.06 to 0.20), but not couple relationship satisfaction (SMD 0.26, 95% CI -0.13 to 0.64), parental mental health (SMD 0.00, 95% CI -0.16 to 0.16), parenting stress (SMD 0.01, 95% CI -0.20 to 0.22) or family functioning (SMD 0.18, 95% CI -0.11 to 0.46). CONCLUSION: Most of the family outcomes are similar between sexual minority and heterosexual families, and sexual minority families have even better outcomes in some domains. Relevant social risk factors of poor family outcomes included stigma and discrimination, poor social support and marital status, etc. The next step is to integrate multiple aspects of support and multilevel interventions to reduce the adverse effects on family outcomes with a long-term goal of influencing policy and law making for better services to individuals, families, communities and schools.

Induction of oxidative DNA damage by flavonoids of propolis: its mechanism and implication about antioxidant capacity.

Propolis from beehives is commonly used as a home remedy for various purposes including as a topical antiseptic. Despite its antioxidant capacity, propolis induces oxidative DNA damage. In exploring the underlying mechanism, we found that the induction of oxidative DNA damage is attributed to the hydrogen peroxide (H(2)O(2)) produced by propolis. The formation of H(2)O(2) can take place without the participation of cells but requires the presence of transition metal ions such as iron. Flavonoids such as galangin, chrysin, and pinocembrin that are commonly detected in propolis have the capacity to induce oxidative DNA damage, and that capacity correlates with the production of H(2)O(2), suggesting the involvement of flavonoids in propolis in this process. On the basis of these results, we propose that the flavonoids of propolis serve as temporary carriers of electrons received from transition metal ions that are relayed to oxygen molecules to subsequently generate superoxide and H(2)O(2). In addition, propolis induces oxidative DNA damage that is subject to repair, and propolis-treated cells show a lower level of DNA damage level when challenged with another oxidative agent such as amoxicillin. This is reminiscent of an adaptive response that might contribute to the beneficial effects of propolis.

Usability of dual HIV/syphilis self-testing among men who have sex with men in China: study protocol for a three-arm randomised controlled trial.

INTRODUCTION: HIV self-testing (HIVST) provides a key measure for the early detection of HIV infection in men who have sex with men (MSM). However, dual HIV/syphilis self-testing in the MSM population has not been studied. We describe a randomised controlled trial to evaluate the effect of dual HIV/syphilis self-testing on the testing frequency among MSM in China. METHODS AND ANALYSIS: This randomised controlled trial will be implemented in Guangxi, China. 330 MSM, including 255 frequent testers and 75 less frequent testers, will be recruited and randomly assigned in a 1:1:1 ratio into one of three arms: a site-based testing arm, a single HIVST arm and a dual HIV/syphilis self-testing arm. Participants in the single HIVST arm and dual HIV/syphilis self-testing arm will receive two free finger-prick-based HIVST or HIV/syphilis self-testing kits at enrolment. The data will be collected at five separate times: baseline, 3 months, 6 months, 9 months and 12 months. The primary outcome is the mean frequency of HIV testing used by MSM after intervention comparing each group during the study period. The secondary outcome includes changes in sex behaviours (eg, number of male sex partners and the proportion of consistent condom use) and the mean number of HIV tests used by the social network members over the study period. ETHICS AND DISSEMINATION: The study protocol was reviewed and approved by the Medical Ethics Committee of Guangxi Medical University, China (20210173). The study results will be disseminated through conferences and academic journals. TRIAL REGISTRATION NUMBER: ChiCTR2100050898.

High baseline body mass index predicts recovery of CD4+ T lymphocytes for HIV/AIDS patients receiving long-term antiviral therapy.

The relationship between baseline BMI and CD4+ T cells during follow-up in HIV patients in China requires further evaluation. We conducted a retrospective cohort study based on adult AIDS patients who underwent or received antiretroviral therapy from 2003 to 2019 in Guangxi, China. BMI was divided into categories and compared, and after adjusting for BMI being related to the change in CD4 lymphocyte count, with normal weight as the reference group, the BMI before treatment was positively correlated with the changes in CD4+ T cells at different time periods. Among them, obese patients had significant CD4+ cell gain. In patients with pretreatment CD4+ T lymphocyte counts <200 cells/µL, a higher BMI was associated with an increased likelihood of achieving immunologic reconstitution [≥350 cells/µL: AHR: 1.02(1.01, 1.04), P = 0.004; ≥500 cells/µL: AHR: 1.03 (1.01, 1.05), P = 0.004]. Underweight in HIV patients was a risk factor for poor viral suppression [AHR: 1.24 (1.04, 1.48), P = 0.016]. Our study demonstrated that HIV/AIDS patients receiving ART with higher baseline BMI had better immune reconstitution and that baseline BMI could be an important predictor of immune reconstitution in patients receiving ART. Baseline BMI was not associated with virological failure, but a lower baseline BMI indicated poor viral suppression during follow-up.

(-)-Epigallocatechin-3-gallate decreases thrombin/paclitaxel-induced endothelial tissue factor expression via the inhibition of c-Jun terminal NH2 kinase phosphorylation.

Patients with paclitaxel-eluting stents are concerned with stent thrombosis caused by premature discontinuation of dual antiplatelet therapy or clopidogrel resistance. This study investigates the effect of (-)-epigallocatechin-3-gallate (EGCG) on the expression of thrombin/paclitaxel-induced endothelial tissue factor (TF) expressions in human aortic endothelial cells (HAECs). EGCG was nontoxic to HAECs at 6h up to a concentration of 25 micromol/L. At a concentration of 25 micromol/L, EGCG pretreatment potently inhibited both thrombin-stimulated and thrombin/paclitaxel-stimulated endothelial TF protein expression. Thrombin and thrombin/paclitaxel-induced 2.6-fold and 2.9-fold increases in TF activity compared with the control. EGCG pretreatment caused a 29% and 38% decrease in TF activity on thrombin and thrombin/paclitaxel treatment, respectively. Real-time polymerase chain reaction (PCR) showed that thrombin and thrombin/paclitaxel-induced 3.0-fold and 4.6-fold TF mRNA expressions compared with the control. EGCG pretreatment caused an 82% and 72% decrease in TF mRNA expression on thrombin and thrombin/paclitaxel treatment, respectively. The c-Jun terminal NH2 kinase (JNK) inhibitor SP600125 reduced thrombin/paclitaxel-induced TF expression. Furthermore, EGCG significantly inhibited the phosphorylation of JNK to 49% of thrombin/paclitaxel-stimulated HAECs at 60min. Immunofluorescence assay did not show an inhibitory effect of EGCG on P65 NF-kappaB nuclear translocation in the thrombin/paclitaxel-stimulated endothelial cells. In conclusion, EGCG can inhibit TF expression in thrombin/paclitaxel-stimulated endothelial cells via the inhibition of JNK phosphorylation. The unique property of EGCG may be used to develop a new drug-eluting stent by co-coating EGCG and paclitaxel.

Identification and biochemical characterization of a unique Mn2+-dependent UMP kinase from Helicobacter pylori.

Uridine monophosphate (UMP) kinase converts UMP to the corresponding UDP in the presence of metal ions and ATP and is allosterically regulated by nucleotides such as UTP and GTP. Although the UMP kinase reported to date is Mg(2+)-dependent, we found in this study that the UMP kinase of Helicobacter pylori had a preference for Mn(2+) over Mg(2+), which may be related to a conformational difference between the Mn(2+)-bound and Mg(2+)-bound UMP kinase. Similar to previous findings, the UMP kinase activity of H. pylori UMP kinase was inhibited by UTP and activated by GTP. However, a relatively low GTP concentration (0.125 mM) was required to activate H. pylori UMP kinase to a level similar to other bacterial UMP kinases using a higher GTP concentration (0.5 mM). In addition, depending on the presence of either Mg(2+) or Mn(2+), a significant difference in the level of GTP activation was observed. It is therefore hypothesized that the Mg(2+)-bound and Mn(2+)-bound H. pylori UMP kinase may be activated by GTP through different mechanisms.

Transformational Leadership and Employees' Thriving at Work: The Mediating Roles of Challenge-Hindrance Stressors.

Based on previous research, the conceptual model presenting the interaction between transformational leadership, challenge-hindrance stressors and thriving at work was constructed and used to generate the hypotheses for the study. Data were obtained from 542 questionnaires distributed across different organizations. The participants included ordinary employees, grassroots middle and senior managers from China. The major findings are as follows. First, transformational leadership directly is positively related to challenge stressors and thriving at work. Second, challenge stressors are positively relate to thriving at work, while hindrance stressors are negatively relate to thriving at work. Furthermore, challenge stressors mediate the relationship between transformational leadership and thriving at work. Given these findings, the study examined the moderating effect of supervisor developmental feedback on the relationship between transformational leadership and thriving at work. Results reveal that supervisor developmental feedback plays a positive regulatory role between challenge stressors and thriving at work. Additionally, it is shown that the mediating effect of challenge stressors on the relationship between transformational leadership and thriving at work is moderated by supervisor developmental feedback.

Paclitaxel induces up-regulation of tissue factor in human aortic endothelial cells.

Patients who underwent paclitaxel-eluting stent implantation are at a risk of developing late stent thrombosis. However, it is unclear whether paclitaxel alone can modulate tissue factor (TF) expression in human aortic endothelial cells (HAEC). HAEC were stimulated with paclitaxel. Western blotting, real-time PCR, and a chromogenic TF activity assay were done. In HAEC, while paclitaxel (10 (-5) mol/L to 10 (-9) mol/L) treatment for 5 h up-regulated the expression of TF in a dose-dependent manner, paclitaxel cotreatment with thrombin further enhanced it. While paclitaxel (10 (-5) mol/L) itself induced a 3.7-fold enhancement in TF activity, its cotreatment along with thrombin elicited a 7.6-fold increase in TF activity. Paclitaxel also caused an 8.1-fold increase in TF mRNA expression, and paclitaxel cotreatment with thrombin caused a 13.6-fold enhancement in TF mRNA expression. In summary, paclitaxel alone can up-regulate endothelial TF expression. These findings are significant for the patients receiving paclitaxel-eluting stents, and they may provide opportunities to develop novel therapeutic strategies for DES thrombosis.

Kinetic and structural properties of triosephosphate isomerase from Helicobacter pylori.

Triosephosphate isomerase (TIM) catalyzes the interconversion between dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate in the glycolysis-gluconeogenesis metabolism pathway. The Helicobacter pylori TIM gene (HpTIM) was cloned, and HpTIM was expressed and purified. The enzymatic activity of HpTIM for the substrate GAP was determined (K(m) = 3.46 +/- 0.23 mM and k(cat) = 8.8 x 10(4) min(-1)). The crystal structure of HpTIM was determined by molecular replacement at 2.3 A resolution. The overall structure of HpTIM was (beta/alpha)beta(beta/alpha)(6), which resembles the common TIM barrel fold, (beta/alpha)(8); however, a helix is missing after the second beta-strand. The conformation of loop 6 and binding of phosphate ion suggest that the determined structure of HpTIM was in the "closed" state. A highly conserved Arg-Asp salt bridge in the "DX(D/N)G" motif of most TIMs is absent in HpTIM because the sequence of this motif is "(211)SVDG(214)." To determine the significance of this salt bridge to HpTIM, four mutants, including K183S, K183A, D213Q, and D213A, were constructed and characterized. The results suggest that this conserved salt bridge is not essential for the enzymatic activity of HpTIM; however, it might contribute to the conformational stability of HpTIM.

Crystal structure of Helicobacter pylori spermidine synthase: a Rossmann-like fold with a distinct active site.

Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the transfer of the aminopropyl group from decarboxylated S-adenosylmethionine to putrescine during spermidine biosynthesis. Helicobacter pylori PAPT (HpPAPT) has a low sequence identity with other PAPTs and lacks the signature sequence found in other PAPTs. The crystal structure of HpPAPT, determined by multiwavelength anomalous dispersion, revealed an N-terminal beta-stranded domain and a C-terminal Rossmann-like domain. Structural comparison with other PAPTs showed that HpPAPT has a unique binding pocket between two domains, numerous non-conserved residues, a less acidic electrostatic surface potential, and a large buried space within the structure. HpPAPT lacks the gatekeeping loop that facilitates substrate binding in other PAPTs. PAPTs are essential for bacterial cell viability; thus, HpPAPT may be a potential antimicrobial drug target for H. pylori owing to its characteristic PAPT sequence and distinct conformation.

Kinetic and structural properties of inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori.

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of pyrophosphate (PPi) to orthophosphate (Pi) and controls the level of PPi in cells. PPase plays an essential role in energy conservation and provides the energy for many biosynthetic pathways. The Helicobacter pylori pyrophosphatase (HpPPase) gene was cloned, expressed, purified, and found to have a molecular weight of 20 kDa. The K(m) and V (max) of HpPPase were determined as 214.4 microM and 594 micromol Pi min(-1) mg(-1), respectively. PPi binds Mg(2+) to form a true substrate that activates the enzyme. However, free PPi could be a potent inhibitor for HpPPase. The effects of the inhibitors NaF, ATP, iminodiphosphate, and N-ethylmaleimide on HpPPase activity were evaluated. NaF showed the highest inhibition of the enzyme. Crystal structures of HpPPase and the PPi-HpPPase complex were determined. HpPPase comprises three alpha-helices and nine beta-strands and folds as a barrel structure. HpPPase forms a hexamer in both the solution and crystal states, and each monomer has its own PPi-binding site. The PPi binding does not cause a significant conformational change in the PPi-HpPPase complex, which might represent an inhibition state for HpPPase in the absence of a divalent metal ion.

Restoration of UV sensitivity in UV-resistant HeLa cells by antisense-mediated depletion of damaged DNA-binding protein 2 (DDB2).

Damaged DNA-binding activity comprises two major protein components, DDB1 and DDB2, which are implicated in the repair of ultraviolet (UV) radiation-induced DNA damage. The possible role of DDB2 as a determinant of cellular sensitivity to UV was investigated. The abundance of DDB2 in UV-resistant HeLa cell lines was increased compared with that in the parental UV-sensitive cells. Stable transfection of the resistant cells with DDB2 antisense cDNA resulted in marked depletion of DDB2 protein and restored cellular sensitivity to UV-induced apoptosis. Whereas the extent of UV-induced activation of apoptosis executioners, including DNA fragmentation factor, and caspase-3 were reduced in the UV-resistant cells compared with those apparent in the sensitive cells, depletion of DDB2 from the resistant cells restored the normal activation patterns for these proteins. In contrast, overexpressing DDB2 in DDB2-depleted cells with recombinant adenovirus, which carries ddb2 cDNA, markedly inhibited the extent of UV-induced activation of DNA fragmentation factor, and caspase-3. Interestingly, a mutated form of DDB2, which is defective in interacting with DDB1 and binding to UV-damaged DNA, also markedly inhibited the activation of apoptosis executioners. These results indicate that DDB2 is a modulator of UV-induced apoptosis, and that UV resistance can be overcome by inhibition of DDB2. The findings also suggest that modulation of UV-induced apoptosis by DDB2 may be independent of DNA repair.

Site-directed mutations of the gatekeeping loop region affect the activity of Escherichia coli spermidine synthase.

Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), and plays a crucial role in cell proliferation and differentiation. The gatekeeping loop identified in the structure of spermidine synthase was predicted to contain residues important for substrate binding, but its correlation with enzyme catalysis has not been fully understood. In this study, recombinant Escherichia coli spermidine synthase (EcSPDS) was produced and its enzyme kinetics was characterized. Site-directed mutants of EcSPDS were obtained to demonstrate the importance of the amino acid residues in the gatekeeping loop. Substitution of Asp158 and Asp161 with alanine completely abolished EcSPDS activity, suggesting that these residues are absolutely required for substrate interaction. Reduction in enzyme activity was observed in the C159A, T160A, and P165Q variants, indicating that hydrophobic interactions contributed by Cys159, Thr160, and Pro165 are important for enzyme catalysis as well. On the other hand, replacement of Pro162 and Ile163 had no influence on EcSDPS activity. These results indicate that residues in the gatekeeping loop of spermidine synthase are indispensable for the catalytic reaction of EcSPDS. To the best of our knowledge, this is the first functional study on the gatekeeping loop of EcSPDS by site-directed mutagenesis.