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The atom-centered density matrix propagation (ADMP) method is an extended Lagrangian approach to ab initio molecular dynamics, which includes the density matrix in an orthonormalized atom-centered Gaussian basis as additional, fictitious, electronic degrees of freedom, classically propagated along with the nuclear ones. A high adiabaticity between the nuclear and electronic subsystems is mandatory in order to keep the trajectory close to the Born-Oppenheimer (BO) surface. In this regard, the fictitious electronic mass μ , being a symmetric, nondiagonal matrix in its most general form, represents a free parameter, exploitable to optimize the propagation of the electronic density. Although mass-weighting schemes in ADMP exist, a systematic procedure to define an optimal value of the fictitious masses is not available yet. In this work, in order to rationally evaluate the electronic mass, fictitious electronic normal modes are defined through the diagonalization of the Hessian of the electronic density matrix. If the same frequency is imposed on all such modes (compatible with the chosen integration time step), then the corresponding μ matrix can be calculated and then employed for the following propagation. Analysis of several ADMP test simulations reveals that such Hessian-based mass-weighting approach is able to ensure, together with a 0.1/0.2 fs time steps, a high separation between the (real) nuclear and the (fictitious) electronic frequencies, which determines a high adiabaticity. This high, unprecedented, accuracy in the propagation leads, in turn, to low errors in the estimated nuclear vibrational frequencies, making the ADMP method totally comparable to a fully converged BO molecular dynamics simulation but more computationally efficient. This work, therefore, contributes to a further development of the ADMP ab initio molecular dynamics method, aimed at improving its accuracy through a more rational evaluation of the fictitious electronic mass parameter.

In this work, we computed and analyzed, by means of density-based descriptors, the real-time evolution of both the locally excited (LE) and charge-transfer (CT) excited states for the planar and twisted conformations of the DMABN (4-( N , N -dimethylamino)benzonitrile) molecule using real-time time-dependent density functional theory (DFT) and three different exchange-correlation energy functionals (EXC) belonging to the same family (the PBE one). Our results based on the analysis of density-based descriptors show that the underlying EXC modifies the evolution in time of the density. In particular, comparing the frequency of density reorganization computed with the three functionals (PBE, PBE0, and LC-PBE), we found that the frequency of electronic interconversion of the individual determinants involved during the dynamics increases from PBE to PBE0 and to LC-PBE. This allows us to show that there is a correlation between the delocalization of the electronic density and the frequency of reorganization. In particular, the greater the mean hole-electron distance during the dynamics, the lower is the frequency of density reorganization.

We present a combined experimental and theoretical study of the photophysics of 5-benzyluracil (5BU) in methanol, which is a model system for interactions between nucleic acids and proteins. A molecular dynamics study of 5BU in solution through efficient DFT-based hybrid ab initio potentials revealed a remarkable conformational flexibility - allowing the population of two main conformers - as well as specific solute-solvent interactions, which both appear as relevant factors for the observed 5BU optical absorption properties. The simulated absorption spectrum, calculated on such an ensemble, enabled a molecular interpretation of the experimental UV-Vis lowest energy band, which is also involved in the induced photo-reactivity upon irradiation. In particular, the first two excited states (mainly involving the uracil moiety) both contribute to the 5BU lowest energy absorption. Moreover, as a key finding, the nature and brightness of such electronic transitions are strongly influenced by 5BU conformation and the microsolvation of its heteroatoms.

Here is presented the ultrafast hole-electron dynamics of photoinduced metal to ligand charge-transfer (MLCT) states in a Ru(II) complex, [Ru(dcbpy) 2 (NCS) 2 ] 4- (dcbpy = 4,4'-dicarboxy-2,2'-bipyridine), a photoactive molecule employed in dye sensitized solar cells. Via cutting-edge computational techniques, a tailored computational protocol is here presented and developed to provide a detailed analysis of the electronic manifold coupled with nuclear vibrations to better understand the nonradiative pathways and the resulting overall dye performances in light-harvesting processes (electron injection). Thus, the effects of different vibrational modes were investigated on both the electronic levels and charge transfer dynamics through a theoretical-computational approach. First, the linear response time-dependent density functional (LR-TDDFT) formalism was employed to characterize excitation energies and spacing among electronic levels (the electronic layouts). Then, to understand the ultrafast (femtosecond) charge dynamics on the molecular scale, we relied on the nonperturbative mean-field quantum electronic dynamics via real-time (RT-) TDDFT. Three vibrational modes were selected, representative for collective nuclear movements that can have a significant influence on the electronic structure: two involving NCS - ligands and one involving dcbpy ligands. As main results, we observed that such MLCT states, under vibrational distortions, are strongly affected and a faster interligand electron transfer mechanism is observed along with an increasing MLCT character of the adiabatic electronic states approaching closer in energy due to the vibrations. Such findings can help both in providing a molecular picture of multidimensional vibro-electronic spectroscopic techniques, used to characterize ultrafast coherent and noncoherent dynamics of complex systems, and to improve dye performances with particular attention to the study of energy or charge transport processes and vibronic couplings.

Electronic properties and absorption spectra are the grounds to investigate molecular electronic states and their interactions with the environment. Modeling and computations are required for the molecular understanding and design strategies of photo-active materials and sensors. However, the interpretation of such properties demands expensive computations and dealing with the interplay of electronic excited states with the conformational freedom of the chromophores in complex matrices (i.e., solvents, biomolecules, crystals) at finite temperature. Computational protocols combining time dependent density functional theory and ab initio molecular dynamics (MD) have become very powerful in this field, although they require still a large number of computations for a detailed reproduction of electronic properties, such as band shapes. Besides the ongoing research in more traditional computational chemistry fields, data analysis and machine learning methods have been increasingly employed as complementary approaches for efficient data exploration, prediction and model development, starting from the data resulting from MD simulations and electronic structure calculations. In this work, dataset reduction capabilities by unsupervised clustering techniques applied to MD trajectories are proposed and tested for the ab initio modeling of electronic absorption spectra of two challenging case studies: a non-covalent charge-transfer dimer and a ruthenium complex in solution at room temperature. The K-medoids clustering technique is applied and is proven to be able to reduce by ∼100 times the total cost of excited state calculations on an MD sampling with no loss in the accuracy and it also provides an easier understanding of the representative structures (medoids) to be analyzed on the molecular scale.

Photoinduced charge transfer (CT) excited states and their relaxation mechanisms can be highly interdependent on the environment effects and the consequent changes in the electronic density. Providing a molecular interpretation of the ultrafast (subpicosecond) interplay between initial photoexcited states in such dense electronic manifolds in condensed phase is crucial for improving and understanding such phenomena. Real-time time-dependent density functional theory is here the method of choice to observe the charge density, explicitly propagated in an ultrafast time domain, along with all time-dependent properties that can be easily extracted from it. A designed protocol of analysis for real-time electronic dynamics to be applied to time evolving electronic density related properties to characterize both in time and in space CT dynamics in complex systems is here introduced and validated, proposing easy to be read cross-correlation maps. As case studies to test such tools, we present the photoinduced charge-transfer electronic dynamics of 5-benzyluracil, a mimic of nucleic acid/protein interactions, and the metal-to-ligand charge-transfer electronic dynamics in water solution of [Ru(dcbpy) 2 (NCS) 2 ] 4- , dcbpy = (4,4'-dicarboxy-2,2'-bipyridine), or "N3 4- ", a dye sensitizer for solar cells. Electrostatic and explicit ab initio treatment of solvent molecules have been compared in the latter case, revealing the importance of the accurate modeling of mutual solute-solvent polarization on CT kinetics. We observed that explicit quantum mechanical treatment of solvent slowed down the charge carriers mobilities with respect to the gas-phase. When all water molecules were modeled instead as simpler embedded point charges, the electronic dynamics appeared enhanced, with a reduced hole-electron distance and higher mean velocities due to the close fixed charges and an artificially increased polarization effect. Such analysis tools and the presented case studies can help to unveil the influence of the electronic manifold, as well as of the finite temperature-induced structural distortions and the environment on the ultrafast charge motions.

Charge-transfer dynamics and interligand electron transfer (ILET) phenomena play a pivotal role in dye-sensitizers, mostly represented by the Ru-based polypyridyl complexes, for TiO 2 and ZnO-based solar cells. Starting from metal-to-ligand charge-transfer (MLCT) excited states, charge dynamics and ILET can influence the overall device efficiency. In this letter, we focus on N3 4- dye ( [Ru(dcbpy) 2 (NCS) 2 ] 4- , dcbpy = 4,4'-dicarboxy-2,2'-bipyridine) to provide a first direct observation with high time resolution (<20 fs) of the ultrafast electron exchange between bpy-like ligands. ILET is observed in water solution after photoexcitation in the ∼400 nm MLCT band, and assessment of its ultrafast time-scale is here given through a real-time electronic dynamics simulation on the basis of state-of-the-art electronic structure methods. Indirect effects of water at finite temperature are also disentangled by investigating the system in a symmetric gas-phase structure. As main result, remarkably, the ILET mechanism appears to be based upon a purely electronic evolution among the dense, experimentally accessible, MLCT excited states manifold at ∼400 nm, which rules out nuclear-electronic couplings and proves further the importance of the dense electronic manifold in improving the efficiency of dye sensitizers in solar cell devices., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

The interplay between light absorption and the molecular environment has a central role in the observed photophysics of a wide range of photoinduced chemical and biological phenomena. The understanding of the interplay between vibrational and electronic transitions is the focus of this work, since it can provide a rationale to tune the optical properties of charge transfer (CT) materials used for technological applications. A clear description of these processes poses a nontrivial challenge from both the theoretical and experimental points of view, where the main issue is how to accurately describe and probe drastic changes in the electronic structure and the ultrafast molecular relaxation and dynamics. In this work we focused on the intermolecular CT reaction that occurs upon photon absorption in a π-stacked model system in dichloromethane solution, in which the 1-chloronaphthalene (1ClN) acts as the electron donor and tetracyanoethylene (TCNE) is the electron acceptor. Density functional theory calculations have been carried out to characterize both the ground-state properties and more importantly the low-lying CT electronic transition, and excellent agreement with recently available experimental results [Mathies, R. A.; et al. J. Phys. Chem. A 2018, 122 (14), 3594] was obtained. The minima of the ground state and first singlet excited state have been accurately characterized in terms of spatial arrangements and vibrational Raman frequencies, and the CT natures of the first two low-lying electronic transitions in the absorption spectra have been addressed and clarified too. Finally, by modeling the possible coordination sites of the TCNE electron acceptor with respect to monovalent ions (Na + , K + ) in an implicit solution of acetonitrile, we find that TCNE can accommodate a counterion in two different arrangements, parallel and orthogonal to the C═C axis, leading to the formation of a contact ion pair. The nature of the counterion and its relative position entail structural modifications of the TCNE radical anion, mainly the central C═C and C≡N bonds, compared to the isolated case. An important red shift of the C═C stretching frequency was observed when the counterion is orthogonal to the double bond, to a greater extent for Na + . On the contrary, in the second case, where the counterion ion lies along the internuclear C═C axis, we find that K + polarizes the electron density of the double bond more, resulting in a greater red shift than with Na + .

Environmental effects can drastically influence the optical properties and photoreactivity of molecules, particularly in the presence of polar and/or protic solvents. In this work we investigate a negatively charged Ru(II) complex, [Ru(dcbpy) 2 (NCS) 2 ] 4- [dcbpy = (4,4'-dicarboxy-2,2'-bipyridine)], in water solution, since this system belongs to a broader class of transition-metal compounds undergoing upon photo-excitation rapid and complex charge transfer (CT) dynamics, which can be dictated by structural rearrangement and solvent environment. Ab initio molecular dynamics (AIMD) relying on a hybrid quantum/molecular mechanics scheme is used to probe the equilibrium microsolvation around the metal complex in terms of radial distribution functions of the main solvation sites and solvent effects on the overall equilibrium structure. Then, using our AIMD-based generalized normal mode approach, we investigate how the ligand vibrational spectroscopic features are affected by water solvation, also contributing to the interpretation of experimental Infra-Red spectra. Two solvation sites are found for the ligands: the sulfur and the oxygen sites can interact on average with ∼4 and ∼3 water molecules, respectively, where a stronger interaction of the oxygen sites is highlighted. On average an overall dynamic distortion of the C 2 symmetric gas-phase structure was found to be induced by water solvation. Vibrational analysis reproduced experimental values for ligand symmetric and asymmetric stretchings, linking the observed shifts with respect to the gas-phase to a complex solvent distribution around the system. This is the groundwork for future excited-state nuclear and electronic dynamics to monitor non-equilibrium processes of CT excitation in complex environments, such as exciton migration in photovoltaic technologies.

The EU and its Member States have articulated a sustainability vision 'to live well within the limits of our planet' by 2050. In this context, consumers play a key role, being able to drive seafood production sustainability and responsibility according to their behaviour, also in relation to their attitudes towards health, nutrition and well-being. On the basis of these premises, this research explores Italian consumers' attitudes towards health and sustainability in relation to seafood, in order to segment different target of consumers. The framework used in this study is mainly focused on a quantitative exploratory data collection based on an online survey. Three groups of consumers were identified based on general health interest, perceived benefits of eating seafoods and attitude towards seafoods: Health seekers who eat seafood for duty; Health seekers and seafood lovers; Low commitment to health and indifferent to seafood. Differences among groups related to socio-demographic characteristics, sustainability attitudes, intentions and interest in information about seafood products were also investigated. In particular, the first two groups are more familiar with sustainable seafood products and more interested in information on these products than the third, both in terms of product origin and seasonality. Consumers belonging to second group show a higher probability to buy seafood products considering this characteristic than the other two groups. Based on the results obtained, a strategic plan could be developed to achieve relevant goals in education, communication and sustainability labelling related with seafood products. Following a preliminary scouting carried out with all the seafood stakeholders, a specific territory to test this approach has been already identified in Torre del Cerrano (Italy). These results have a strong implication for policy makers and educational institutions as they identify differences in attitudes and perceptions among consumers that are crucial in order to design the right communication strategy strategies as well as messages content., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The excited state proton transfer (ESPT) reaction from the photoacid 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS or pyranine) to an acetate molecule has been investigated in explicit aqueous solution via excited state ab initio molecular dynamics simulations based on hybrid quantum/molecular mechanics (QM/MM) potentials. In all the trajectories, the direct proton transfer has been observed in the excited state within 1 ps. We find that the initial structural configuration extracted from the ground state distribution strongly affects the ESPT kinetics. Indeed, the relative orientation of the proton donor-acceptor pair and the presence of a water molecule hydrogen bonded to the phenolic acid group of the pyranine are the key factors to facilitate the ESPT. Furthermore, we analyze the vibrational fingerprints of the ESPT reaction, reproducing the blue shift of the acetate CO stretching ( CO ac ), from 1666 to 1763 cm -1 testifying the transformation of acetate to acetic acid. Finally, our findings suggest that the acetate CC stretching ( CC ac ) is also sensitive to the progress of the ESPT reaction. The CC ac stretching is indeed ruled by the two vibrational modes (928 and 1426 cm -1 ), that in the excited state are alternately activated when the proton is shared or bound to the donor/acceptor, respectively.

In the search for new therapeutic strategies to contrast SARS-CoV-2, we here studied the interaction of polydatin (PD) and resveratrol (RESV)-two natural stilbene polyphenols with manifold, well known biological activities-with Spike, the viral protein essential for virus entry into host cells, and ACE2, the angiotensin-converting enzyme present on the surface of multiple cell types (including respiratory epithelial cells) which is the main host receptor for Spike binding. Molecular Docking simulations evidenced that both compounds can bind Spike, ACE2 and the ACE2:Spike complex with good affinity, although the interaction of PD appears stronger than that of RESV on all the investigated targets. Preliminary biochemical assays revealed a significant inhibitory activity of the ACE2:Spike recognition with a dose-response effect only in the case of PD.

The Green Fluorescent Protein (GFP) is a widely studied chemical system both for its large amount of applications and the complexity of the excited state proton transfer responsible of the change in the protonation state of the chromophore. A detailed investigation on the structure of the chromophore environment and the influence of chromophore form (either neutral or anionic) on it is of crucial importance to understand how these factors could potentially influence the protein function. In this study, we perform a detailed computational investigation based on the analysis of ab-initio molecular dynamics simulations, to disentangle the main structural quantities determining the fine balance in the chromophore environment. We found that specific hydrogen bonds interactions directly involving the chromophore (or not), are correlated to quantities, such as the volume of the cavity in which the chromophore is embedded and that it is importantly affected by the chromophore protonation state. The cross-correlation analysis performed on some of these hydrogen bonds and the cavity volume, demonstrates a direct correlation among them and we also identified the ones specifically involved in this correlation. We also found that specific interactions among residues far in the space are correlated, demonstrating the complexity of the chromophore environment and that many structural quantities have to be taken into account to properly describe and understand the main factors tuning the active site of the protein. From an overall evaluation of the results obtained in this work, it is shown that the residues which a priori are perceived to be spectators play instead an important role in both influencing the chromophore environment (cavity volume) and its dynamics (cross-correlations among spatially distant residues)., (Copyright © 2020 Coppola, Perrella, Petrone, Donati and Rega.)

Solute-solvent interactions are proxies for understanding how the electronic density of a chromophore interacts with the environment in a more exhaustive way. The subtle balance between polarization, electrostatic, and non-bonded interactions need to be accurately described to obtain good agreement between simulations and experiments. First principles approaches providing accurate configurational sampling through molecular dynamics may be a suitable choice to describe solvent effects on solute chemical-physical properties and spectroscopic features, such as optical absorption of dyes. In this context, accurate energy potentials, obtained by hybrid implicit/explicit solvation methods along with employing nonperiodic boundary conditions, are required to represent bulk solvent around a large solute-solvent cluster. In this work, a novel strategy to simulate methanol solutions is proposed combining ab initio molecular dynamics, a hybrid implicit/explicit flexible solvent model, nonperiodic boundary conditions, and time dependent density functional theory. As case study, the robustness of the proposed protocol has been gauged by investigating the microsolvation and electronic absorption of the anionic green fluorescent protein chromophore in methanol and aqueous solution. Satisfactory results are obtained, reproducing the microsolvation layout of the chromophore and, as a consequence, the experimental trends shown by the optical absorption in different solvents., (© 2020 Wiley Periodicals LLC.)

Electrons and protons are the main actors in play in proton coupled electron transfer (PCET) reactions, which are fundamental in many biological (i.e., photosynthesis and enzymatic reactions) and electrochemical processes. The mechanism, energetics and kinetics of PCET reactions are strongly controlled by the coupling between the transferred electrons and protons. Concerted PCET reactions are classified according to the electronical adiabaticity degree of the process. To discriminate among different mechanisms, we propose a new analysis based on the use of electron density based indexes. We choose, as test case, the 3-Methylphenoxyl/phenol system in two different conformations to show how the proposed analysis is a suitable tool to discriminate between the different degree of adiabaticity of PCET processes. The very low computational cost of this procedure is extremely promising to analyze and provide evidences of PCET mechanisms ruling the reactivity of many biological and catalytic systems., (© 2020 Wiley Periodicals, Inc.)

Fe(III)-Mimochrome VI (MC6) is a recently reported artificial heme-peptide conjugate system with a high peroxidase-like activity. By design, its structure features a five-coordinated Fe(III)-deuteroporphyrin active site, embedded in a compact α-helix-heme-α-helix "sandwich" motif. Up to now, no detailed MC6 structural characterization is available. In this work we propose a theoretical investigation based on molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) optimizations, aimed to shed light on several Fe(III)-MC6 structural features and to validate the de novo designed fold. Key structural elements were analyzed to achieve indirect insight relevant to understand Fe(III)-MC6 catalytic performances in solution. Extensive MD simulations showed a partial stability of the "sandwich" fold in water solution. The smaller peptide chain bonded to the heme revealed a high conformational freedom, which promoted the exposition of the heme distal side to the solvent. Regarding the accessibility of water molecules, even in Fe(III)-MC6 "closed" structure the heme cavity appeared hydrated, suggesting an easy accessibility by exogenous ligands. Fe(III)-MC6 structure in both high and low spin states was then further characterized through hybrid QM/MM optimizations. In particular, an accurate description of the active site structure was obtained, allowing a direct comparison of Fe(III)-MC6 coordination environment with that observed in the Horseradish Peroxidase crystal structures. Our results suggest a structural similarity between Fe(III)-MC6 and the natural enzyme. This study supports the interpretation of data from experimental Fe(III)-MC6 structural and functional characterization and the rational design of new artificial mimics with improved catalytic performances., (© 2018 Wiley Periodicals, Inc.)

Background: Latex allergy is mediated by residual IgE-binding proteins found on latex products. While most reported reactions involve latex gloves, the allergenic potential of other latex containing devices is unknown., Objective: To assess the allergenic potential of latex urologic catheters., Methods: Two identical lots of urologic catheters were extracted in sterile saline and analyzed for latex protein content using sensitive ELISA and Western blot assays. The extracts were tested by skin prick testing in a population of 47 latex-allergic patients., Results: Latex proteins were detected by ELISA assay, however, the Western Blot method was not sensitive enough to measure latex proteins in these catheter extracts. Skin prick testing using a standard latex reagent (Bencard) demonstrated the test population to be extremely sensitive to latex as 68% of the population reacted to a 1/100,000 dilution. Using undiluted catheter extracts, only 11% of these latex-allergic patients reacted to catheter lot A and 2% to catheter lot C, by prick skin test. Eight percent of the control patients had a positive latex prick skin test and none reacted to the catheter extract., Conclusions: The data suggest that processing and leaching of these specific latex urologic catheters resulted in a very low latex allergen content. The low prevalence of skin test reactivity in a population of latex-allergic patients suggests that these catheters would be an unusual cause of allergic reactions. It is not possible, however, to determine whether latex-allergic patients could safely use latex urologic catheters.

A series of nitrocoumarin and nitrochromene derivatives have been prepared and shown to inhibit the phosphatidylinositol-specific phospholipase C(PLC)(IC50 < 10 micrograms/mL) isolated from human melanoma. The inhibition of PLC by nitrocoumarin 4a was time-dependent and irreversible. The inhibition of PLC was shown to interfere with inositide metabolism in whole cells (IC50 = 4 micrograms/mL) in a manner consistent with their proposed mode of activity. Finally, the compounds were shown to be growth inhibitory to cultured melanoma cells (ID50 = 2 micrograms/mL), suggesting that PLC may be an attractive new target for chemotherapeutic intervention.

The novel anticancer drug candidate brequinar sodium (DuP 785, NSC 368390, 6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinoline- carboxylic acid sodium salt) was shown previously to be an inhibitor of dihydroorotate dehydrogenase, the fourth enzyme of the de novo pyrimidine biosynthetic pathway. Brequinar sodium inhibits the activity of this enzyme isolated from mammalian sources only but not those forms isolated from yeast or bacteria, which also use ubiquinone as the cofactor. Brequinar sodium also does not inhibit the activity of a soluble Zymobacterium oroticum dihydroorotate dehydrogenase which uses NAD+ as a cofactor. Brequinar sodium inhibits L1210 dihydroorotate dehydrogenase with mixed inhibition kinetics with respect to either the substrate (dihydroorotate) or the cofactor (ubiquinone Q6) with Ki' values in the 5-8 nM range. Our results suggest that brequinar sodium inhibits dihydroorotate dehydrogenase by binding to the enzyme at a unique site that is distinct from the dihydroorotate or the ubiquinone-binding site. This binding site appears to be unique to the mammalian enzyme, because brequinar sodium does not inhibit the yeast, Escherichia coli, or Z. oroticum forms of the enzyme.

Phospholipase C was purified from human melanoma grown as solid tumors in nude mice. The specific activity of the pure enzyme was approx. 100 mumol/min per mg; its apparent molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 150 kDa. The enzyme required calcium for activity and was activated by deoxycholate in the presence of the substrate phosphatidylinositol. The melanoma phospholipase C has a distinctly different substrate preference than those identified from normal tissues; it prefers phosphatidylinositol to phosphatidylinositol bisphosphate. The tumor enzyme was approx. 4-5-fold more active using phosphatidylinositol than phosphatidylinositol bisphosphate as the substrate.

Phosphoinositide phospholipase C activity was investigated in human melanoma grown as solid tumor xenografts in nude mice. The enzyme was dependent on calcium for activity and was stimulated by the detergent deoxycholate. The pH optimum was 5.5 in the absence of detergent, and in the presence of deoxycholate two pH maxima were present, 5.5 and 7.2. Phospholipase C activity was inhibited by the sulfhydryl reagent dithionitrobenzoate with an IC50 in the micromolar range. Phospholipase C activity was distributed widely in mouse tissues. The enzyme showed a progressive increase in activity from heart, liver, lung, colon, spleen, to brain tissue. Mouse and human melanomas grown as solid tumors had higher phospholipase C activity than mouse brain. The relatively high activity of this enzyme in melanoma may suggest a biological role for phospholipase C in solid tumor growth.

Ornithine decarboxylase was purified at least 1500-fold from mouse epidermis pretreated with five consecutive doses of 12-O-tetradecanoylphorbol-13-acetate and 3-isobutyl-1-methylxanthine at 3- to 4-day intervals. Following DEAE-cellulose chromatography and ammonium sulfate precipitation, ornithine decarboxylase was purified further by affinity chromatography. Ornithine decarboxylase was then radioactively labeled by covalently binding [3H]-alpha-difluromethylornithine to the enzyme following polyacrylamide gel electrophoresis under non-denaturing conditions. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver staining of protein, a band was identified that corresponded to a molecular weight of approx. 56,000, coincident with a peak of radioactivity. This is the first study to purify ornithine decarboxylase from mouse epidermis.

The present study was undertaken to determine the effect of the potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on gamma radiation-induced DNA repair in resting (Go) lymphocytes. Mezerein, a non-promoter but a co-mitogen in lymphocytes, was used as a control agent. It was previously proposed that a possible mechanism for the action of tumor promoters was through the inhibition of DNA repair processes. Our results indicate that TPA does not inhibit DNA repair following gamma irradiation of Go lymphocytes. These data support the hypothesis that the tumor promoting ability of TPA is not a result of impaired repair of potentially mutagenic lesions in DNA.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) binds reversibly and with high affinity and specificity to nuclear macromolecules in mouse epidermis. The dissociation constants determined from Scatchard analysis of epidermal nuclei and nuclear macromolecules are 3.58 +/- 0.66 (S.E.) and 2.18 +/- 0.54 nM, respectively. The solubilization of TPA receptors from epidermal nuclei by DNase I was examined. Following a 20-min digestion at 22 degrees, more than a 2-fold increase in specific TPA binding was observed in the supernatant relative to non-nuclease-treated nuclei (0.71 versus 0.32 pmol/mg protein, respectively). Our data indicate that epidermal nuclei contain saturable and specific TPA-binding macromolecules and that these binding components may be associated with regions of chromatin that are preferentially susceptible to nucleolytic cleavage. These data suggest the existence of nuclear receptors for the phorbol ester tumor promoters. These observations may necessitate a more critical assessment of plasma membrane binding as the sole binding site responsible for triggering the multistep process of tumor promotion in mouse epidermis.

Rat liver and hepatoma nuclei were incubated in vitro with [3H]nicotinamide adenine dinucleotide to allow synthesis of a polymer of adenosine diphosphoribose subunits joined in an 1',2' ribose-ribose linkage. The addition of 1 mM spermine altered the adenosine 5'-diphosphate (ADP) ribosylation patterns of nuclear proteins in hepatoma, host liver, and regenerating liver. Spermine-treated nuclei showed a greater incorporation of ADP-ribose into H1 histones and nonhistone nuclear proteins with isoelectric points between pH 3.0 and 6.0 when separated on polyacrylamide gels. Conversely, a large reduction in ADP ribosylation was seen in core histones (H2A, H2B, and H3) from the same nuclei. The proportion of ADP-ribose incorporated into histones was reduced in the nuclei from proliferating cells relative to their respective control livers. These results imply that polyamines, which are higher in concentration in rapidly dividing cells, may elicit a regulatory function by causing the preferential ADP ribosylation of H1 histones, as well as the more acidic of the nuclear proteins.

The dorsal skin of female CD-1 mice was treated with triethylenemelamine (TEM) to determine whether this agent acted either as a complete carcinogen or as an initiator of carcinogenesis. A dose of 0.01-1.0 mumol of TEM applied once a week for 32 weeks to the skin of the backs of mice did not produce any detectable tumors. A dose of 2.5 mumol applied once a week over the same period produced only a single papilloma in a group of 20 mice. However, when mice were treated with a single dose of 1 mumol of TEM followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) twice a week, 88% of the mice produced papillomas after 28 weeks. Using the same protocol, a single application of hexamethylmelamine (HMM), pentamethylmelamine (PMM), or melamine followed by promotion with TPA had no significant tumor initiating activity. These data suggest that TEM acts primarily as an initiator of two-stage carcinogenesis.

EZ-FIT, an interactive microcomputer software package, has been developed for the analysis of enzyme kinetic and equilibrium binding data. EZ-FIT was designed as a user-friendly menu-driven package that has the facility for data entry, editing, and filing. Data input permits the conversion of cpm, dpm, or optical density to molar per minute per milligram protein. Data can be fit to any of 14 model equations including Michaelis-Menten, Hill, isoenzyme, inhibition, dual substrate, agonist, antagonist, and modified integrated Michaelis-Menten. The program uses the Nelder-Mead simplex and Marquardt nonlinear regression algorithms sequentially. A report of the results includes the parameter estimates with standard errors, a Student t test to determine the accuracy of the parameter values, a Runs statistic test of the residuals, identification of outlying data, an Akaike information criterion test for goodness-of-fit, and, when the experimental variance is included, a chi 2 statistic test for goodness-of-fit. Several different graphs can be displayed: an X-Y, a Scatchard, an Eadie-Hofstee, a Lineweaver-Burk, a semilogarithmic, and a residual plot. A data analysis report and graphs are designed to evaluate the goodness-of-fit of the data to a particular model.

The human promyelocytic leukemia cell line HL-60 can be induced to differentiate into macrophage-like cells by nanomolar concentrations of phorbol esters. A phorbol ester-resistant variant R1B6 obtained by culturing HL-60 cells with increasing concentrations of 12-O-tetradecanoylphorbol-13-acetate, is reversibly resistant. These cells have been growing continuously in the presence of phorbol esters for more than 1 yr, but when the phorbol ester is removed, the cells gradually regain their sensitivity and express characteristics of macrophage-like cells upon readdition of phorbol ester. The concentration of phorbol ester receptors in R1B6 is about one-third that in the parental HL-60 cells. The reversion of the variants to sensitivity to phorbol esters is associated with the up regulation of the cytosol and membrane phorbol ester receptors. When partially purified, these receptor populations contain protein kinase C activity, in support of the identity of protein kinase C and the receptor. This study demonstrates that a phenotypic change in a clonal cell population correlates with the up regulation of the phorbol ester receptor-calcium-activated phospholipid-dependent protein kinase. This variant cell line is a useful model for analyzing the relationship between phorbol ester binding and protein kinase C during differentiation of HL-60 cells.