Biomedical subjects

Hongtao Yu

Publications and source records attributed to Hongtao Yu.

At least 55 records · Page 3Linked to original sources

PubMed2004

Photomutagenicity of 16 polycyclic aromatic hydrocarbons from the US EPA priority pollutant list.

The photomutagenicity of 16 polycyclic aromatic hydrocarbons (PAHs), all on the United States Environmental Protection Agency (US EPA) priority pollutant list, was studied. Concomitant exposing the Salmonella typhimurium bacteria strain TA102 to one of the PAHs and light (1.1 J/cm2 UVA+2.1 J/cm2 visible) without the activation enzyme S9, strong photomutagenic response is observed for anthracene, benz[a]anthracene, benzo[ghi]perylene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, and pyrene. Under the same conditions, acenaphthene, acenaphthylene, benzo[k]fluoranthene, chrysene, and fluorene are weakly photomutagenic. Benzo[b]fluoranthene, fluoranthene, naphthalene, phenanthrene, and dibenz[a,h]anthracene are not photomutagenic. These results indicate that PAHs can be activated by light and become mutagenic in Salmonella TA102 bacteria. At the same time, the mutagenicity for all the 16 PAHs was examined with the standard mutagenicity test with 10% S9 as the activation system. Benzo[b]fluoranthene, benzo[k]fluoranthene, chrysene, acenaphthylene, and fluorene are weakly mutagenic, while the rest of the PAHs are not. In general, the photomutagenicity of PAHs in TA102 does not correlate with their S9-activated mutagenicity in either TA102 or TA98/TA100 since they involve different activation mechanisms.

Animals↗

PubMed2004

DNA damage produced in HaCaT cells by combined fluoranthene exposure and ultraviolet A irradiation.

Fluoranthene is a polycyclic aromatic hydrocarbon (PAH) and a principal constituent of PAH-contaminated aquatic systems. In the present study, fluorescein diacetate uptake and the Comet assay were used to assess the cytotoxicity and genotoxicity of fluoranthene in HaCaT (human adult low calcium high temperature) cells in the presence or absence of ultraviolet A (UVA) irradiation. Exposure of cells to 0.1, 0.25, 0.75, 2, and 5 microM fluoranthene alone for 30 min or to 6.1 +/- 0.07 J/cm2 UVA alone did not cause cytotoxicity or cellular DNA damage. However, concomitant exposure to both caused a nonlinear dose-response in cytotoxicity to HaCat cells. The same exposure conditions also resulted in a dose-responsive DNA damage in HaCaT cells. Because DNA damage mainly was detected at relatively high levels of cytotoxicity, we cannot rule out the possibility that it occurred as a consequence of cellular toxicity mechanisms.

Cell Line↗

PubMed2004

Functional analysis of the spindle-checkpoint proteins using an in vitro ubiquitination assay.

The spindle checkpoint helps to ensure the fidelity of chromosome segregation during mitosis and meiosis. In response to sister chromatids not properly attached to the mitotic spindle, this checkpoint blocks the activity of a large ubiquitin protein ligase complex, called the anaphase-promoting complex (APC) or cyclosome. This chapter describes the detailed protocols of an in vitro ubiquitination assay reconstituted with purified APC, cofactors, other ubiquitination enzymes, and the spindle-checkpoint proteins. This assay is extremely useful in dissecting the biochemical functions of various spindle-checkpoint proteins.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2004

Photochemical transformation and phototoxicity of 1-aminopyrene.

1-Aminopyrene (1-AP) is an environmental mutagen and a metabolite of 1-nitropyrene (1-NO2P). On light irradiation, 1-AP transforms into oxidation products with a half-life of 7.1 min in 10% methanolic buffer. The presence of DNA or free-radical/ singlet oxygen scavengers 1,4-dithiothreitol, histidine, or NaN3 slows down 1-AP photochemical reaction. The photoproducts identified include 1-hydroxyaminopyrene, 1-nitrosopyrene, 1-NO2P, 1-amino-x-hydroxypyrene, and three covalent dimers. Since it is known that 1-NO2P and 1-nitrosopyrene are genotoxic and 1-hydroxyaminopyrnene can react with DNA to form covalent adducts, we used the Mutatox test to assess the toxicity of 1-AP and its photoproducts. It was found that the lowest-observed-effect concentrations for 1-AP, 1-AP photoproducts, and 1-NO2P are 1.25 microM, 10 microM, and NA (no mutagenic response was seen at this concentration range) in direct medium (no S-9) and NA, 5 microM, and 0.625 microM in S-9 medium, respectively. Therefore, 1-AP photoproducts are more genotoxic than 1-AP itself in the S-9 medium and more mutagenic than 1-NO2P in the direct medium. Thus, 1-NO2P alone cannot account for all the mutagenicity of the photoproducts. Irradiation of 1-AP together with DNA leads to covalent DNA adduct formation possibly via the 1-hydroxyaminopyrene intermediate. In this study, ultraviolet-A (UVA) was used at approximately the same magnitude as the outdoor UVA irradiance. Considering the half-life of 1-AP in the test solutions in this study, the aquatic biota (including humans) near the surface layer of a static water body are most likely subjected to the photoinduced toxicity of the study compound. The biota at the lower depths will also be affected if turbulence becomes a significant factor in enhancing the exposure risk for aquatic organisms.

DNA Damage↗

PubMed2004

Arsenic-induced genotoxic and cytotoxic effects in human keratinocytes, melanocytes and dendritic cells.

Arsenical keratosis and skin cancer are among the most common health effects associated with acute and chronic exposures to arsenic. This study examines the acute and chronic dose-responses of arsenic in established human cell lines using keratinocytes (HaCaT), melanocytes (CRL1675) and dendritic cells (THP-1 + A23187). Chronic conditions were established by treating the three cell lines with at least 8 passages in 0.2 microg/mL arsenic trioxide. Cytotoxicity was assessed using the fluorescein diacetate assay after 72 hrs of exposure. Single cell gel electrophoresis (Comet assay) was used to measure DNA damage. Acute exposure to arsenic had LD10 and LD25 values of 0.38 microg/mL and 3.0 microg/mL for keratinocytes; 0.19 microg/mL and 0.38 microg/mL for melanocytes; and 0.38 microg/mL and 0.75 microg/mL for dendritic cells. Cytotoxicity assays for chronically exposed cells resulted in LD10, and LD25 values of 0.4 microg/mL and 0.8 microg/mL for keratinocytes; 0.10 microg/mL and 0.20 microg/mL for melanocytes; and 0.10 microg/mL and 1.0 microg/mL for dendritic cells. The Comet assay showed that arsenic was highly genotoxic to the three cell lines. No significant differences (p > 0.05) in DNA cleavage were observed between acute and chronic exposures. In acute exposure arsenic genotoxicity was more severe with dendritic cells while melanocytes were more sensitive to arsenic cytotoxicity. Similarly, chronically exposed dendritic cells showed the maximum genotoxic damage while melanocytes were more sensitive to arsenic cytotoxicity. In conclusion, this research shows that arsenic is dermatotoxic, showing a high degree of genotoxicity and cytotoxicity to skin cells.

Animals↗

PubMed2003

Identification of two novel components of the human NDC80 kinetochore complex.

Proper kinetochore function is essential for the accurate segregation of chromosomes during mitosis. Kinetochores provide the attachment sites for spindle microtubules and are required for the alignment of chromosomes at the metaphase plate (chromosome congression). Components of the conserved NDC80 complex are required for chromosome congression, and their disruption results in mitotic arrest accompanied by multiple spindle aberrations. To better understand the function of the NDC80 complex, we have identified two novel subunits of the human NDC80 complex, termed human SPC25 (hSPC25) and human SPC24 (hSPC24), using an immunoaffinity approach. hSPC25 interacted with HEC1 (human homolog of yeast Ndc80) throughout the cell cycle and localized to kinetochores during mitosis. RNA interference-mediated depletion of hSPC25 in HeLa cells caused aberrant mitosis, followed by cell death, a phenotype similar to that of cells depleted of HEC1. Loss of hSPC25 also caused multiple spindle aberrations, including elongated, multipolar, and fractured spindles. In the absence of hSPC25, MAD1 and HEC1 failed to localize to kinetochores during mitosis, whereas the kinetochore localization of BUB1 and BUBR1 was largely unaffected. Interestingly, the kinetochore localization of MAD1 in cells with a compromised NDC80 function was restored upon microtubule depolymerization. Thus, hSPC25 is an essential kinetochore component that plays a significant role in proper execution of mitotic events.

Amino Acid Sequence↗

PubMed2003

Identification of 6-aminochrysene photoproducts and study of the effect of a humic acid and riboflavin on its photolysis.

The effect of the photosensitizer riboflavin (0, 10, 25, 50, 100 microM) and a river humic acid (0, 20, 40, 80 ppm) on the photolysis of 6-aminochrysene (6AC) in 10 mM PBS solution (pH 7.0) was studied. The presence of riboflavin significantly enhanced photolysis rate of 6AC. The photo-transformation half-life of 6AC was 1 or 36 min, respectively, in the presence or absence of riboflavin. The humic acid inhibited the photo-transformation rate of 6AC. The photo-transformation half-lives of 6AC were 37, 56, 92 min at 20, 40, 80 ppm humic acid, respectively. By using LC-MS, the main 6AC photoproduct identified was 5,6-chrysene-quinone along with some minor products. Both 6AC and 5,6-chrysene-quinone exhibited photoinduced cytotoxicity. A photochemical transformation pathway for 6AC was derived.

Chrysenes↗

PubMed2003

Degradation of origin recognition complex large subunit by the anaphase-promoting complex in Drosophila.

The initiation of DNA synthesis is thought to occur at sites bound by a heteromeric origin recognition complex (ORC). Previously, we have shown that in Drosophila, the level of the large subunit, ORC1, is modulated during cell cycle progression and that changes in ORC1 concentration alter origin utilization during development. Here, we investigate the mechanisms underlying cell cycle-dependent degradation of ORC1. We show that signals in the non-conserved N-terminal domain of ORC1 mediate its degradation upon exit from mitosis and in G1 phase by the anaphase-promoting complex (APC) in vivo. Degradation appears to be the result of direct action of the APC, as the N-terminal domain is ubiquitylated by purified APC in vitro. This regulated proteolysis is potent, sufficient to generate a normal temporal distribution of protein even when transcription of ORC1 is driven by strong constitutive promoters. These observations suggest that in Drosophila, ORC1 regulates origin utilization much as does Cdc6 in budding yeast.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2003

Photomutagenicity of cosmetic ingredient chemicals azulene and guaiazulene.

The photomutagenicity of the popular skin conditioning agents azulene and guaiazulene were tested in Salmonella typhimurium TA98, TA100 and TA102. Following irradiation with UVA and/or visible light, both azulene and guaiazulene exhibited mutagenicity 4-5-fold higher than the spontaneous background mutation. In contrary, naphthalene, a structural isomer of azulene, was not photomutagenic under the same conditions. Azulene was photomutagenic when irradiated with UVA light alone, visible light alone, or a combination of UVA and visible light. Azulene and guaiazulene are not mutagenic when the experiment is conducted with the exclusion of light. Therefore, extreme care must be taken when using cosmetic products with azulene/guaiazulene as ingredients since after applying these products on the skin, exposure to sunlight is inevitable.

Azulenes↗

PubMed2003

FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation.

Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ) regulates its degradation and the proper function of the clock. The mechanism by which FRQ undergoes degradation has not been established. Here we show that FRQ is likely ubiquitylated in vivo, and its proper degradation requires FWD1, an F-box/WD-40 repeat-containing protein. In the fwd1 disruption strains, FRQ degradation is severely impaired, resulting in the accumulation of hyperphosphorylated FRQ. Furthermore, the circadian rhythms of gene expression and the circadian conidiation rhythms are abolished in these fwd1 mutants. Finally, FRQ and FWD1 interact physically in vivo, suggesting that FWD1 is the substrate-recruiting subunit of an SCF-type ubiquitin ligase responsible for FRQ ubiquitylation and degradation. Together with the recent finding that Slimb (the Drosophila homolog of FWD1) is involved in the degradation of the Period protein in flies, our results indicate that FWD1 regulates the degradation of FRQ in Neurospora and is an evolutionarily conserved component of the eukaryotic circadian clock.

Amino Acid Sequence↗

PubMed2003

Regulation of CDC14: pathways and checkpoints of mitotic exit.

Progression of the mitotic cell cycle is driven by fluctuations of the cyclin-dependent kinase (Cdk) activities. Entry into mitosis is promoted by the elevated activity of Cdk1 associated with B-type cyclins. Conversely, exit from mitosis requires the inactivation of Cdk1 and the dephosphorylation of at least a subset of Cdk1 substrates. The Cdc14 family of phosphatases antagonizes the action of Cdk1, and is thus a major player in controlling the mitotic exit. We review recent discoveries in several model systems that have shed light on the function of Cdc14 and propose a general framework within which Cdc14 plays conserved roles in regulating the exit from mitosis and cytokinesis.

Cell Cycle↗

PubMed2003

Two distinct pathways for inhibiting pds1 ubiquitination in response to DNA damage.

The presence of DNA damage activates a conserved cellular response known as the DNA damage checkpoint pathway. This pathway induces a cell cycle arrest that persists until the damage is repaired. Consequently, the failure to arrest in response to DNA damage is associated with genomic instability. In budding yeast, activation of the DNA damage checkpoint pathway leads to a mitotic cell cycle arrest. Following the detection of DNA damage, the checkpoint signal is transduced via the Mec1 kinase, which in turn activates two kinases, Rad53 and Chk1 that act in parallel pathways to bring about the cell cycle arrest. The downstream target of Rad53 is unknown. The target of Chk1 is Pds1, an inhibitor of anaphase initiation whose degradation is a prerequisite for mitotic progression. Pds1 degradation is dependent on its ubiquitination by the anaphase-promoting complex/cyclosome ubiquitin ligase, acting in conjunction with the Cdc20 protein (APC/CCdc20). Previous studies showed that the Rad53 and Chk1 pathways independently lead to Pds1 stabilization but the mechanism for this was unknown. In the present study we show that both the Chk1 and the Rad53 pathways inhibit the APC/CCdc20-dependent ubiquitination of Pds1 but they affect different steps of the process: the Rad53 pathway inhibits the Pds1-Cdc20 interaction whereas Chk1-dependent phosphorylation of Pds1 inhibits the ubiquitination reaction itself. Finally, we show that once the DNA damage is repaired, Pds1 dephosphorylation is involved in the recovery from the checkpoint induced cell cycle arrest.

Cdc20 Proteins↗

PubMed2002

Riboflavin-photosensitized degradation of atrazine in a freshwater environment.

The effect of the photosensitizer riboflavin (0, 10, 50, 100 microM) on the fate of atrazine (10 mg/l) in a freshwater environment was studied. It was found that at 100 microM riboflavin significantly enhanced the degradation of atrazine and more than 80% of atrazine in a natural water environment was depleted in 72 h. The relative contribution of microbial assemblages and the freshwater matrix to the degradation of atrazine and the degradation kinetics of atrazine were compared under different experimental conditions. The products and pathways of atrazine transformation were studied with GC-MS and HPLC with a photodiode array detector. The results show that dealkylation and alkyl chain oxidation are involved in the degradation of atrazine.

Atrazine↗

PubMed2002

Regulation of APC-Cdc20 by the spindle checkpoint.

The spindle checkpoint ensures the fidelity of chromosome segregation in mitosis and meiosis. In response to defects in the mitotic apparatus, it blocks the activity of the anaphase-promoting complex, a large ubiquitin ligase required for chromosome segregation. Recent studies indicate that the spindle checkpoint monitors both the attachment of chromosomes to the mitotic spindle and the tension across the sister chromatid generated by microtubules. Upon checkpoint activation, checkpoint protein complexes containing BubR1(Mad3), Bub3, Mad2 and Cdc20 directly bind to the anaphase-promoting complex and inhibit its ligase activity. Therefore, the checkpoint proteins form a complex intracellular signalling network to inhibit the anaphase-promoting complex.

Anaphase↗

PubMed2002

The Mad2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or Cdc20.

Mad2 participates in spindle checkpoint inhibition of APC(Cdc20). We show that RNAi-mediated suppression of Mad1 function in mammalian cells causes loss of Mad2 kinetochore localization and impairment of the spindle checkpoint. Mad1 and Cdc20 contain Mad2 binding motifs that share a common consensus. We have identified a class of Mad2 binding peptides with a similar consensus. Binding of one of these ligands, MBP1, triggers an extensive rearrangement of the tertiary structure of Mad2. Mad2 also undergoes a similar striking structural change upon binding to a Mad1 or Cdc20 binding motif peptide. Our data suggest that, upon checkpoint activation, Mad1 recruits Mad2 to unattached kinetochores and may promote binding of Mad2 to Cdc20.

Calcium-Binding Proteins↗

PubMed2002

Synthesis and fluorescence study of 7-azaindole in DNA oligonucleotides replacing a purine base.

The fluorescence spectroscopy of 7-azaindole (7aIn) incorporated in DNA oligonucleotides is investigated. Incorporation of 7aIn into DNA oligonucleotides is accomplished through standard solid-phase phosphoramidite chemistry. Fluorescence emission of the 7aIn chromophore shifts slightly to the red (from 386 nm to 388 nm) upon glycosylation at the N-1 position, but its relative fluorescence quantum yield increases 23 times, from 0.023 to 0.53. Upon incorporation into DNA, the fluorescence emission of 7aIn is greatly quenched with fluorescence quantum yields of 0.020 and 0.016 in single and double strand DNA, respectively. The fluorescence emission for 7aIn in DNA oligonucleotides shifts to the blue with an emission maximum at 379 nm. Both the strong fluorescence quenching and the blue shift of the emission spectrum signify that 7aIn is stacked with neighboring DNA bases in both single and double strand DNA. As the duplex DNA melts due to temperature increase, the fluorescence of the 7aIn chromophore increases, indicating the transition from the less fluorescent duplex DNA to the more fluorescent single strand DNA. Since this fluorescent 7aIn is a structural analog of purine, its fluorescence property may be utilized as a probe for studying nucleic acid structure and dynamics.

Amides↗

PubMed2002

UVA light-induced DNA cleavage by isomeric methylbenz[a]anthracenes.

UVA light-induced DNA single strand cleavage by a set of 12 monomethyl substituted benz[a]anthracenes (MBAs) along with their parent compound, benz[a]anthracene (BA), and the potent carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA), was studied. On the basis of the relative DNA single strand photocleavage efficiency of the fourteen compounds, they are divided into three groups: (1) strong DNA cleavers, 4-MBA, 5-MBA, 6-MBA, 8-MBA, 9-MBA, 10-MBA, and BA; (2) medium DNA cleavers, 1-MBA, 2-MBA, 3-MBA, and 11-MBA; and (3) weak DNA cleavers, 7-MBA, 12-MBA, and DMBA. The relative DNA photocleavage efficiency parallels very well with the energy gap between the highest-occupied-molecular-orbital (HOMO) and the lowest-unoccupied-molecular-orbital (LUMO) of each MBA, indicating that the DNA cleavage is related to their excited-state properties. The 7 and 12 positions of BA are two unique sites. Methyl substitution at either 7 or 12 (or both) positions lowers the HOMO-LUMO gap and greatly diminishes the DNA photocleavage efficiency. UVA light-induced photodegradation of selected MBAs reveals that methyl substitution at either 7 or 12 (or both) positions greatly enhances the degradation rate. Photodegradation of 7-MBA, 12-MBA, and DMBA yields products that are much less effective in mediating DNA cleavage. Photodegradation of other MBAs, exemplified by 5-MBA, yields a photooxidation product 5-MBA-7,12-quinone which is relatively stable under light and is a stronger DNA photocleaver than 5-MBA itself. The higher efficiency of DNA photocleavage for MBAs with methyl substitution at positions other than 7 or 12 is due, at least in part, to the formation of 7,12-quinone. Light-induced DNA single strand cleavage efficiency for several MBAs parallels the light-induced toxicity observed by other research groups, suggesting that light-induced DNA cleavage of MBAs are the source for phototoxicity. Since some PAHs such as coal tar are used commercially as creams, therapeutic agents, or ointments, or those roofers and asphalt workers that are subject to contamination with PAHs, the combination of PAHs and light (in the skin) may present a greater health risk to humans.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2002

Distinguishing "looped-out" and "stacked-in" DNA bulge conformation using fluorescent 2-aminopurine replacing a purine base.

The conformation of a bulged DNA base, whether looped-out of the DNA helix or stacked-in between the flanking bases, can be distinguished using fluorescence spectroscopy of an inserted fluorescent base. If 2-aminopurine, a structural analog of adenine and guanine, is placed in duplex DNA as the bulged base replacing an adenine or guanine, it loops out of the DNA helix into solution. This is determined by the decrease or increase of 2-aminopurine fluorescence during DNA thermomelting: if the 2-aminopurine base stacks into the helix, its fluorescence increases or remains about the same during DNA duplex melting, but if the 2-aminopurine base loops out of the helix, its fluorescence decreases upon melting of the DNA duplex.

2-Aminopurine↗