Hydroxyzine, promethazine and thioridazine interaction with phospholipid monomolecular layers at the air-water interface

In this work the interaction of Hydroxyzine, Promethazine and Thioridazine with Langmuir films of dipalmitoylphosphatidylcholine (dpPC) and dipalmitoylphosphatidic acid (dpPA), is studied. Temporal variations in lateral surface pressure (pi) were measured at different initial pi (pi(i)), subphase pH and drug-concentration. Drugs with the smallest (PRO) and largest (HYD) molecular size exhibited the lowest adsorption (k(a)) and the highest desorption (k(d)) rate constant values, respectively. The affinity binding constants (K(b)) obtained in monolayers followed the same profile (K(b,PRO) < K(b,HYD) < K(b,THI)) of the egg-PC/water partition coefficients (P) determined in bilayers. The drug concentration required to reach the half-maximal Deltapi at pi(i) = 14 mN/m (K(0.5)), was very sensitive to pH. The maximal increment in pi upon drug incorporation into the monolayer (deltapi(max)) will depend on the phospholipid collapse pressure (pi(c)), the monolayers's compressibility and drug's size, shape and charge. The higher pi(c) of dpPC lead to higher pi(cut-off) values (maximal pi allowing drug penetration), if compared with dpPA. In dpPC and dpPA pi(cut-off) decreased as a function of the molecular size of the uncharged drugs. In dpPA, protonated drugs became electrostatically trapped at the monolayer surface hence drug penetration, monolayer deformation and pi increase were impaired and the correlation between pi(cut-off) and drug molecular size was lost.     

Incorporation of the chlorophyll d-binding light-harvesting protein from Acaryochloris marina and its localization within the photosynthetic apparatus of Synechocystis sp. PCC6803

The gene encoding a chlorophyll d-binding light-harvesting protein, pcbA from Acaryochloris marina (now called as accessory Chlorophyll Binding Protein CBPII) marked with a His-tag was transformed into the genome of Synechocystis PCC6803. Protein gel electrophoresis and western blotting confirmed that this foreign chlorophyll d-binding protein CBPII was expressed and integrated into the thylakoid membrane and bound with chlorophyll a, the only type of chlorophyll present in Synechocystis PCC 6803. Native electrophoresis suggested that CBPII interacts with photosystem II of Synechocystis PCC 6803. Surprisingly, spectral analyses showed that the phycobiliproteins were suppressed in the transformed Synechocystis pcbA⁺, with a lower ratio of phycobilins to chlorophyll a. These results suggest that there are competitive interactions between the external antenna system of phycobiliproteins and the integral antenna system of chlorophyll-bound protein complexes.     

On the species-specific biotransformation of dibenzo[a,l]pyrene

We were aimed at investigating the activation of the carcinogenic polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P) in Chinese hamster V79 cells that express single human, rat or fish cytochrome P450 (CYP) enzymes. DB[a,l]P is detectable in environmental samples and has been characterized as the most potent carcinogenic species among all PAHs as yet tested in rodent bioassays. Metabolite profiles and metabolite-dependent cytotoxic and clastogenic activities were monitored. The total turnover of CYP-mediated transformation of DB[a,l]P was as follows: human CYP1B1>fish CYP1A1 approximately human CYP1A1>rat CYP1A2>rat CYP1A1. By contrast, enzyme forms that are not classified as being members of family CYP1, such as CYP2A6, 2E1, 2B1, and 3A4, failed to catalyze any detectable conversion of this substrate. All CYP1A1 enzymes tested formed both the K-region trans-8,9- and the trans-11,12-dihydrodiol, whereas human CYP1B1 failed to catalyze K-region activation. In cells expressing human or fish CYP1A1, human CYP1B1, and rat CYP1A2, the (-)-trans-11,12-dihydrodiol was formed enantiospecifically. DB[a,l]P-dependent cytotoxicities (EC(50)) were found in the following order: human CYP1A1 (12 nM)>fish CYP1A1 (30 nM)>human CYP1B1 (45 nM)>other forms. In addition, an appreciable micronuclei formation was detected in human CYP1A1- and 1B1-expressing cells during exposure to DB[a,l]P. Our study demonstrates that human CYP1A1, 1B1 and fish CYP1A1 are able to transform DB[a,l]P into genotoxic derivatives in appreciable amounts. In contrast, CYP enzymes from rat predominantly target the K-region of DB[a,l]P and thus are serving more a rather protective route of biotransformation. Together our data suggest that humans might be more susceptible to DB[a,l]P-induced carcinogenicity than rats.     

Crystal structures of SULT1A2 and SULT1A1∗3: Insights into the substrate inhibition and the role of Tyr149 in SULT1A2

The cytosolic sulfotransferases (SULTs) in vertebrates catalyze the sulfonation of endogenous thyroid/steroid hormones and catecholamine neurotransmitters, as well as a variety of xenobiotics, using 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as the sulfonate donor. In this study, we determined the structures of SULT1A2 and an allozyme of SULT1A1, SULT1A1∗3, bound with 3′-phosphoadenosine 5′-phosphate (PAP), at 2.4 and 2.3 Å resolution, respectively. The conformational differences between the two structures revealed a plastic substrate-binding pocket with two channels and a switch-like substrate selectivity residue Phe247, providing clearly a structural basis for the substrate inhibition. In SULT1A2, Tyr149 extends approximately 2.1 Å further to the inside of the substrate-binding pocket, compared with the corresponding His149 residue in SULT1A1∗3. Site-directed mutagenesis study showed that, compared with the wild-type SULT1A2, mutant Tyr149Phe SULT1A2 exhibited a 40 times higher K_m and two times lower V_max with p-nitrophenol as substrate. These latter data imply a significant role of Tyr149 in the catalytic mechanism of SULT1A2.     

Oligomerization of the SPP1 scaffolding protein

Viral scaffolding proteins direct polymerization of major capsid protein subunits into icosahedral procapsid structures. The scaffolding protein of bacteriophage SPP1 was engineered with a C-terminal hexahistidine tag (gp11-His₆) and purified. The protein is an α-helical-rich molecule with a very elongated shape as found for internal scaffolding proteins from other phages. It is a 3.3 S tetramer of 93.6 kDa at micromolar concentrations. Intersubunit cross-linking of these tetramers generated preferentially covalently bound dimers, revealing that gp11-His₆ is structurally a dimer of dimers. Incubation at temperatures above 37 °C correlated with a reduction of its α-helical content and a less effective intersubunit cross-linking. Complete loss of secondary structure was observed at temperatures above 60 °C. Refolding of gp11-His₆ thermally denatured at 65 °C led to reacquisition of the protein native ellipticity spectrum but the resulting population of molecules was heterogeneous. Its hydrodynamic behavior was compatible with a mix of 3.3 S elongated tetramers (∼ 90%) and a smaller fraction of 2.4 S dimers (∼ 10%). This population of gp11-His₆ was competent to direct polymerization of the SPP1 major capsid protein gp13 into procapsid-like structures in a newly developed assembly assay in vitro. Although native tetramers were active in assembly, refolded gp11-His₆ showed enhanced binding to gp13 revealing a more active species for interaction with the major capsid protein than native gp11-His₆.     

Effect of different salt ions on the propensity of aggregation and on the structure of Alzheimer's abeta(1-40) amyloid fibrils

The formation of amyloid fibrils and other polypeptide aggregates depends strongly on the physico-chemical environment. One such factor affecting aggregation is the presence and concentration of salt ions. We have examined the effects of salt ions on the aggregation propensity of Alzheimer's Abeta(1-40) peptide and on the structure of the dissolved and of the fibrillar peptide. All salts examined promote aggregation strongly. The most pronounced effect is seen within the cationic series, i.e. for MgCl2. Evaluation of different possible explanations suggests that Abeta(1-40) aggregation depends on direct interaction between ions and Abeta(1-40) peptide, and correlates with ion-induced changes of the surface tension. Salts have profound effects on the fibril structure. In the presence of salts, fibrils are associated with smaller diameters, narrower crossover distances and lower amide I maxima. Since Abeta(1-40) aggregation responds to salts in a manner unlike that for other polypeptides, such as glucagon, beta2-microglobulin or alpha-synuclein; these data argue that there is no fully uniform way in which salts affect aggregation of different polypeptide chains. These observations are important for understanding and predicting aggregation on the basis of simple physico-chemical properties.     

S100 proteins interact with the N-terminal domain of MDM2

S100 proteins interact with the transactivation domain and the C-terminus of p53. Further, S100B has been shown to interact with MDM2, a central negative regulator of p53. Here, we show that S100B bound directly to the folded N-terminal domain of MDM2 (residues 2-125) by size exclusion chromatography and surface plasmon resonance experiments. This interaction with MDM2 (2-125) is a general feature of S100 proteins; S100A1, S100A2, S100A4 and S100A6 also interact with MDM2 (2-125). These interactions with S100 proteins do not result in a ternary complex with MDM2 (2-125) and p53. Instead, we observe the ability of a subset of S100 proteins to disrupt the extent of MDM2-mediated p53 ubiquitylation in vitro.

Structured summary

MINT-7905256: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A6 (uniprotkb:P06703) by surface plasmon resonance (MI:0107)MINT-7905063: MDM2 (uniprotkb:Q00987) and s100A1 (uniprotkb:P23297) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905376: s100A4 (uniprotkb:P26447) and MDM2 (uniprotkb:Q00987) physically interact (MI:0915) by competition binding (MI:0405)MINT-7905130: s100A6 (uniprotkb:P06703) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905207: s100A6 (uniprotkb:P06703) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905043: s100B (uniprotkb:P04271) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905196: p53 (uniprotkb:P04637) and s100A4 (uniprotkb:P26447) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905358: p53 (uniprotkb:P04637) and s100A4 (uniprotkb:P26447) physically interact (MI:0915) by fluorescence polarization spectroscopy (MI:0053)MINT-7905220: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100B (uniprotkb:P04271) by surface plasmon resonance (MI:0107)MINT-7905104: s100A4 (uniprotkb:P26447) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905229: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A1 (uniprotkb:P23297) by surface plasmon resonance (MI:0107)MINT-7905317, MINT-7905162: s100B (uniprotkb:P04271) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905238: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A2 (uniprotkb:P29034) by surface plasmon resonance (MI:0107)MINT-7905174, MINT-7905308: s100A1 (uniprotkb:P23297) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905247: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A4 (uniprotkb:P26447) by surface plasmon resonance (MI:0107)MINT-7905090: s100A2 (uniprotkb:P29034) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905142, MINT-7905326: MDM2 (uniprotkb:Q00987) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905185, MINT-7905347: s100A2 (uniprotkb:P29034) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)     

Application of drug metabolising mutants of cytochrome P450 BM3 (CYP102A1) as biocatalysts for the generation of reactive metabolites

Recently, several mutants of cytochrome P450 BM3 (CYP102A1) with high activity toward drugs have been obtained by a combination of site-directed and random mutagenesis. In the present study, the applicability of these mutants as biocatalysts in the production of reactive metabolites from the drugs clozapine, diclofenac and acetaminophen was investigated. We showed that the four CYP102A1 mutants used in this study formed the same metabolites as human and rat liver microsomes, with an activity up to 70-fold higher compared to human enzymes. Using these CYP102A1 mutants, three novels GSH adducts of diclofenac were discovered which were also formed in incubations with human liver microsomes. This work shows that CYP102A1 mutants are very useful tools for the generation of high levels of reference metabolites and reactive intermediates of drugs. Producing high levels of those reactive metabolites, that might play a role in adverse drug reactions (ADRs) in humans, will facilitate their isolation, structural elucidation, and could be very useful for the toxicological characterization of novel drugs and/or drug candidates.     

The solution structure of an N-terminally truncated version of the yeast CDC24p PB1 domain shows a different beta-sheet topology

Phox and Bem1 (PB1) domains mediate protein-protein interactions via the formation of homo- or hetero-dimers. The C-terminal PB1 domain of yeast cell division cycle 24 (CDC24p), a guanine-nucleotide exchange factor involved in cell polarity establishment, is known to interact with the PB1 domain occurring in bud emergence MSB1 interacting 1 (BEM1p) during the regulation of the yeast budding process via its OPR/PC/AID (OPCA) motif. Here, we present the structure of an N-terminally truncated version of the Sc CDC24p PB1 domain. It shows a different topology of the beta-sheet than the long form. However, the C-terminal part of the structure shows the conserved PB1 domain features including the OPCA motif with a slight rearrangement of helix alpha1. Residues which are important for the heterodimerization with BEM1p are structurally preserved.     

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b₆f with the electron transfer proteins plastocyanin and cytochrome c₆. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c₆ from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c₆ were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge–charge interactions pre-orient cytochrome c₆ with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.     

The MUC1-C oncoprotein binds to the BH3 domain of the pro-apoptotic BAX protein and blocks BAX function

The pro-apoptotic BAX protein contains a BH3 domain that is necessary for its dimerization and for activation of the intrinsic apoptotic pathway. The MUC1 (mucin 1) heterodimeric protein is overexpressed in diverse human carcinomas and blocks apoptosis in the response to stress. In this study, we demonstrate that the oncogenic MUC1-C subunit associates with BAX in human cancer cells. MUC1-C·BAX complexes are detectable in the cytoplasm and mitochondria and are induced by genotoxic and oxidative stress. The association between MUC1-C and BAX is supported by the demonstration that the MUC1-C cytoplasmic domain is sufficient for the interaction with BAX. The results further show that the MUC1-C cytoplasmic domain CQC motif binds directly to the BAX BH3 domain at Cys-62. Consistent with binding to the BAX BH3 domain, MUC1-C blocked BAX dimerization in response to (i) truncated BID in vitro and (ii) treatment of cancer cells with DNA-damaging agents. In concert with these results, MUC1-C attenuated localization of BAX to mitochondria and the release of cytochrome c. These findings indicate that the MUC1-C oncoprotein binds directly to the BAX BH3 domain and thereby blocks BAX function in activating the mitochondrial death pathway.     

Cooperation of binding sites at the hydrophilic domain of cell-surface sulfatase Sulf1 allows for dynamic interaction of the enzyme with its substrate heparan sulfate

Background

Sulf1 is a cell-surface sulfatase removing internal 6-O-sulfate groups from heparan sulfate (HS) chains. Thereby it modulates the activity of HS-dependent growth factors. For HS interaction Sulf1 employs a unique hydrophilic domain (HD).

Methods

Affinity-chromatography, AFM-single-molecule force spectroscopy (SMFS) and immunofluorescence on living cells were used to analyze specificity, kinetics and structural basis of this interaction.

Results

Full-length Sulf1 interacts broadly with sulfated glycosaminoglycans (GAGs) showing, however, higher affinity toward HS and heparin than toward chondroitin sulfate or dermatan sulfate. Strong interaction depends on the presence of Sulf1-substrate groups, as Sulf1 bound significantly weaker to HS after enzymatic 6-O-desulfation by Sulf1 pretreatment, hence suggesting autoregulation of Sulf1/substrate association. In contrast, HD alone exhibited outstanding specificity toward HS and did not interact with chondroitin sulfate, dermatan sulfate or 6-O-desulfated HS. Dynamic SMFS revealed an off-rate of 0.04/s, i.e., ~ 500-fold higher than determined by surface plasmon resonance. SMFS allowed resolving the dynamics of single dissociation events in each force–distance curve. HD subdomain constructs revealed heparin interaction sites in the inner and C-terminal regions of HD.

Conclusions

Specific substrate binding of Sulf1 is mediated by HD and involves at least two separate HS-binding sites. Surface plasmon resonance K_D-values reflect a high avidity resulting from multivalent HD/heparin interaction. While this ensures stable cell–surface HS association, the dynamic cooperation of binding sites at HD and also the catalytic domain enables processive action of Sulf1 along or across HS chains.

General significance

HD confers a novel and highly dynamic mode of protein interaction with HS.     

A new temperature-insensitive allele of the Arabidopsis AXR6/CUL1 locus derived from a missense mutation in the C-terminal RBX1 binding region

We isolated a new recessive allele at the AUXIN RESISTANT6/CULLIN1 (AXR6/CUL1) locus, axr6–101, from an EMS-mutagenized population of Arabidopsis thaliana, the Landsberg erecta ecotype. axr6–101 is auxin resistant and semi-dwarf similar to the other recessive axr6 mutants. The axr6–101 phenotype is caused by the E716K substitution of the CUL1 protein, which is likely to affect its ability to bind to the C-terminal RING domain of RING-box 1 (RBX1). The previously reported allele of AXR6, cul1–7, is caused by a substitution at T510 that binds to the N-terminal β-strand of RBX1. Although cul1–7 shows temperature-sensitive phenotype, the axr6–101 phenotype is largely unaffected by temperature. axr6–101 may provide an important genetic resource for study of the structure−function relationship of the CUL1 protein.     

Structure of the C-terminal domain of the pro-apoptotic protein Hrk and its interaction with model membranes

The protein harakiri (Hrk) is a pro-apoptotic BH3-only protein which belongs to the Bcl-2 family. Hrk appears associated to the mitochondrial outer membrane, apparently by a putative transmembrane domain, where it exerts its function. In this work we have identified a 27mer peptide supposed to be the putative membrane domain of the protein at the C-terminal region, and used infrared and fluorescence spectroscopies to study its secondary structure as well as to characterize its effect on the physical properties of phospholipid model membranes. The results presented here showed that the C-terminal region of Hrk adopts a predominantly α-helical structure whose proportion and destabilization capability varied depending on phospholipid composition. Moreover it was found that the orientation of the α-helical component of this C-terminal Hrk peptide was nearly perpendicular to the plane of the membrane. These results indicate that this domain is able of inserting into membranes, where it adopts a transmembrane α-helical structure as well as it considerably perturbs the physical properties of the membrane.     

Bcs1p can rescue a large and productive cytochrome bc1 complex assembly intermediate in the inner membrane of yeast mitochondria

The yeast cytochrome bc₁ complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc₁ complex, some ancillary proteins, such as the chaperone Bcs1p, are actively involved. The deletion of the nuclear gene encoding this chaperone caused the arrest of the bc₁ assembly and the formation of a functionally inactive bc₁ core structure of about 500-kDa. This immature bc₁ core structure could represent, on the one hand, a true assembly intermediate or, on the other hand, a degradation product and/or an incorrect product of assembly. The experiments here reported show that the gradual expression of Bcs1p in the yeast strain lacking this protein was progressively able to rescue the bc₁ core structure leading to the formation of the functional homodimeric bc₁ complex. Following Bcs1p expression, the mature bc₁ complex was also progressively converted into two supercomplexes with the cytochrome c oxidase complex. The capability of restoring the bc₁ complex and the supercomplexes was also possessed by the mutated yeast R81C Bcsp1. Notably, in the human ortholog BCS1L, the corresponding point mutation (R45C) was instead the cause of a severe bc₁ complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc₁ core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc₁ complex and gives new insights into the molecular mechanisms involved in the last steps of bc₁ assembly.     

Spectral and kinetic resolution of the bc1 complex components in situ: A simple and robust alternative to the traditional difference wavelength approach

The kinetics of the cytochrome (cyt) components of the bc₁ complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The “traditional” set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c_tot (cyt c₁ + cyt c₂), cyt b_L, cyt b_H, and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c₁ and c₂ is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c₁, c₂, b_L, and b_H) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc₁ complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc₁ complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.     

A novel photoactive GAF domain of cyanobacteriochrome AnPixJ that shows reversible green/red photoconversion

We report the discovery of a novel cyanobacteriochrome, the green/red photoreceptor AnPixJ (All1069), isolated from the heterocyst-forming cyanobacterium Anabaena (Nostoc) sp. PCC 7120. Cyanobacteriochromes are a recently emerging tetrapyrrole-based photoreceptor superfamily that are distantly related to the conventional red/far-red photoreceptor phytochromes (Phys). The chromophore-binding domains of AnPixJ produced in cyanobacterial and Escherichia coli cells both showed a reversible and full photoconversion between a green-absorbing form (λ_max = 543 nm) and a red-absorbing form (λ_max = 648 nm). Denaturation analysis revealed that the green-absorbing form and the red-absorbing form covalently ligated phycocyanobilin with E-configuration and Z-configuration at the C15C16 double bond, respectively. Time-resolved spectral analysis showed the formation of the first intermediate state peaking at 680 nm from the dark-stable red-absorbing form. This step resembles the first photoconversion step from the red-absorbing form to the red-shifted lumi-R intermediate state of the Phys. These results suggest that the Pr of AnPixJ is almost equivalent to that of the Phys and starts a primary photoreaction with Z-to-E isomerization in a mechanism similar to that in the Phys, but is finally photoconverted to the unique green-absorbing form.     

Investigation of the genotoxicity of dibenzo[c,p]chrysene in human carcinoma MCF-7 cells in culture

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that have been linked to certain human cancers. The fjord region PAH dibenzo[a,l]pyrene exhibits the highest levels of carcinogenic activity of all PAH as yet tested in rodent tumor models. Another hexacyclic aromatic hydrocarbon, dibenzo[c,p]chrysene (DBC), is a unique PAH that possesses one bay region and two fjord regions within the same molecule. Due to its structure, which is a merger of the fjord region PAHs benzo[c]phenanthrene, benzo[c]chrysene, and benzo[g]chrysene, DBC is of considerable research interest. In order to investigate the pathway of regioselective metabolism we have studied the cytotoxicity, metabolic activation and DNA adduct formation of DBC in human mammary carcinoma MCF-7 cells in culture. The cytotoxicity assay indicated undisturbed cell proliferation even at concentrations as high as 4.5 microM (1.5 micro g/ml) DBC. Concurrently, DNA adducts were detected in MCF-7 cells treated with DBC only in low amounts (0.6 pmol adducts/mg DNA). On the contrary, exposure to anti-DBC-1,2-diol-3,4-epoxide and anti-DBC-11,12-diol-13,14-epoxide, two putatively genotoxic metabolites of DBC, resulted in high levels of DNA adducts (33 and 51 pmol adducts/mg DNA, respectively). Although DBC was not efficiently transformed into DNA-reactive metabolites in MCF-7 cells in culture, the results from our study indicate that the two fjord region diol-epoxide derivatives of DBC may serve as ultimate genotoxic metabolites once they are enzymatically generated under certain circumstances in vitro or in vivo.     

New features of Asian Crassostrea oyster mitochondrial genomes: A novel alloacceptor tRNA gene recruitment and two novel ORFs

A feasible way to perform evolutionary analyses is to compare characters divergent enough to observe significant differences, but sufficiently similar to exclude saturation of the differences that occurred. Thus, comparisons of invertebrate mitochondrial (mt) genomes at low taxonomic levels can be extremely helpful in investigating patterns of variation and evolutionary dynamics of genomes, as intermediate stages of the process may be identified. Fortunately, in this study, we newly sequenced the mt genome of the eighth member of Asian Crassostrea oysters which can provide necessary intermediate characters for us to believe that the variation of Crassostrea mt genomes is considerably greater than previously acknowledged. Several new features of Asian Crassostrea oyster mitochondrial genomes were revealed, and our results are particularly significant as they 1) suggest a novel model of alloacceptor tRNA gene recruitment, namely "vertical" tRNA gene recruitment, which can be successfully used to explain the origination of the unusually additional trnK and trnQ genes (annotated as trnK(2) and trnQ(2) respectively) in the mt genomes of the five Asian oysters, and we speculate that this recruitment progress may be a common phenomenon in the evolution of the tRNA multigene family; 2) reveal the existence of two additional, lineage-specific, mtDNA-encoded genes that may originate from duplication of nad2 followed by rapid evolutionary change. Each of these two genes encodes a unique amino terminal signal peptide, thus each might possess an unknown function; and 3) identify for the first time the atp8 gene in oysters. The present study thus gives further credence to the comparison of congeneric bivalves as a meaningful strategy to investigate mt genomic evolutionary trends in genome organization, tRNA multigene family, and gene loss and/or duplication that are difficult to undertake at higher taxonomic levels. In particular, our study provides new evidence for the identification and characterization of ORFs in the "non-coding region" of animal mt genomes.