Anti-Angiogenic Activity of Selected Receptor Tyrosine Kinase Inhibitors, PD166285 and PD173074: Implications for Combination Treatment with Photodynamic Therapy

Angiogenesis, the formation of new blood vessels from an existing vasculature, is requisite for tumor growth. It entails intercellular coordination of endothelial and tumor cells through angiogenic growth factor signaling. Interruption of these events has implications in the suppression of tumor growth. PD166285, a broad-spectrum receptor tyrosine kinase (RTK) inhibitor, and PD173074, a selective FGFR₁TK inhibitor, were evaluated for their anti-angiogenic activity and anti-tumor efficacy in combination with photodynamic therapy (PDT). To evaluate the anti-angiogenic and anti-tumor activities of these compounds, RTK assays, in vitro tumor cell growth and microcapillary formation assays, in vivo murine angiogenesis and anti-tumor efficacy studies utilizing RTK inhibitors in combination with photodynamic therapy were performed. PD166285 inhibited PDGFR--, EGFR-, and FGFR₁TKs and c-src TK by 50% (IC₅₀) at concentrations between 7–85nM. PD173074 displayed selective inhibitory activity towards FGFR₁TK at 26nM. PD173074 demonstrated (>100 fold) selective growth inhibitory action towards human umbilical vein endothelial cells compared with a panel of tumor cell lines. Both PD166285 and PD173074 (at 10nM) inhibited the formation of microcapillaries on Matrigel-coated plastic. In vivo anti-angiogenesis studies in mice revealed that oral administration (p.o.) of either PD166285 (1–25 mg/kg) or PD173074 (25–100 mg/kg) generated dose dependent inhibition of angiogenesis. Against a murine mammary 16c tumor, significantly prolonged tumor regressions were achieved with daily p.o. doses of PD166285 (5–10 mg/kg) or PD173074 (30–60 mg/kg) following PDT compared with PDT alone (p<0.001). Many long-term survivors were also noted in combination treatment groups. PD166285 and PD173074 displayed potent anti-angiogenic and anti-tumor activity and prolonged the duration of anti-tumor response to PDT. Interference in membrane signal transduction by inhibitors of specific RTKs (e.g. FGFR₁TK) should result in new chemotherapeutic agents having the ability to limit tumor angiogenesis and regrowth following cytoreductive treatments such as PDT.     

Characterizing the influence of structure and route of exposure on the disposition of dithiocarbamates using toluene-3,4-dithiol analysis of blood and urinary carbon disulfide metabolites.

Differences in the toxicities observed for dithiocarbamates have been proposed to result from the influence of nitrogen substitution, oxidation state, and route of exposure. To better characterize the fate of dithiocarbmates in vivoas a function of structure and route of exposure, rats were administered equimolar doses of carbon disulfide (CS2), N-methyldithiocarbamate, pyrrolidine dithiocarbamate, N,N-diethyldithiocarbamate, or disulfiram daily for five days, either po or ip, and sequential blood samples obtained. Protein dithiocarbamates formed by the in vivo release of CS2, parent dithiocarbamate, and protein-bound mixed disulfides were assessed in plasma and hemolysate by measuring toluene trithiocarbonate generated upon treatment with toluene-3, 4-dithiol (TdT). To aid in determining the bioavailability of CS2 from the administered dithiocarbamates, the urinary CS2 metabolites, 2-thiothiazolidine-4-carboxylic acid (TTCA) and 2-thiothiazolidin-4-ylcarbonylglycine (TTCG), were also determined. The levels of TdT-reactive moieties detected depended upon both the compound administered and the route of exposure. Parent dithiocarbamates, with the exception of disulfiram, were eliminated from blood within 24 h; but protein associated TdT-reactive moieties persisted and accumulated with repeated exposure, regardless of the route of exposure. N-Methyldithiocarbamate demonstrated the greatest potential to produce intracellular globin modifications, presumably through its unique ability to generate a methylisothiocyanate metabolite. Urinary excretion of TTCA and TTCG was more sensitive than TdT analysis for detecting dithiocarbamate exposure, but TdT analysis appeared to be a better indicator of in vivo release of CS2 by dithiocarbamates than were urinary CS2 metabolites. These data suggest that CS2 is a more important metabolite, following oral exposure, than are other routes of exposure, e.g., inhalation or dermal. In addition, data also suggest that acid stability, nitrogen substitution, and route of exposure are important factors governing the toxicity observed for a particular dithiocarbamate.     

Effect of Preparation Taper,Height and Marginal Design Under Varying Occlusal Loading Conditions on Cement Lute Stress: A Three Dimensional Finite Element Analysis

The functional surfaces of the porcelain fused to metal fixed partial dentures are often abraded to adjust occlusion, such restorations are often found to fail in service. This study was therefore conducted to study the effect of surface abrasion on flexural strength of glazed porcelain fused to metal samples. It was also the aim of this study to find the effect of re-glazing on flexural strength of abraded samples. A total of ninety glazed porcelain fused to metal bar samples of the dimension 15 mm × 2 mm × 1.5 mm were fabricated. These samples were then divided into three groups (30 samples each) according to the surface treatments: group A-glazed (control); group B-abraded and group C-abraded and then re-glazed (self-glazed). Flexural strength was measured by using three point bend test on universal testing machine (texture analyser) with a cross-head speed of 0.6 mm/min. Peak force at the time of failure for all the samples was recorded. Statistical analysis found that mean flexural strength was highest for group A-80.65 ± 12.81 MPa; as compared to group B-74.18 ± 10.74 MPa and group C-77.85 ± 9.39 MPa. Student’s t test indicated that the difference in the flexural strength between groups A and B was significant while it was non-significant between groups B and C and also between groups A and C. The ‘f’ test indicated that the difference between the groups was non-significant. This study therefore showed that there is a marked decrease in the flexural strength of the porcelain fused to metal restorations after occlusal abrasion. The study also found that reglazing of these restorations may not restore their flexural strength significantly.     

Structural elucidation of NASICON (Na3Al2P3O12) based glass electrolyte materials: effective influence of boron and gallium

Understanding the conductivity variations induced by compositional changes in sodium super ionic conducting (NASICON) glass materials is highly relevant for applications such as solid electrolytes for sodium (Na) ion batteries. In the research reported in this paper, NASICON-based NCAP glass (Na_2.8Ca_0.1Al₂P₃O₁₂) was selected as the parent glass. The present study demonstrates the changes in the Na⁺ ion conductivity of NCAP bulk glass with the substitution of boron (NCABP: Na_2.8Ca_0.1Al₂B_0.5P_2.7O₁₂) and gallium (NCAGP: Na_2.8Ca_0.1Al₂Ga_0.5P_2.7O₁₂) for phosphorus and the resulting structural variations found in the glass network. For a detailed structural analysis of NCAP, NCABP and NCAGP glasses, micro-Raman and magic angle spinning-nuclear magnetic resonance (MAS-NMR) spectroscopic techniques (for ³¹P, ²⁷Al, ²³Na, ¹¹B and ⁷¹Ga nuclei) were used. The Raman spectrum revealed that the NCAP glass structure is more analogous to the AlPO₄ mesoporous glass structure. The ³¹P MAS-NMR spectrum illustrated that the NCAP glass structure consists of a high concentration of Q⁰ (3Al) units, followed by Q⁰ (2Al) units. The ²⁷Al MAS-NMR spectrum indicates that alumina exists at five different sites, which include AlO₄ units surrounded by AlO₆ units, Al(OP)₄, Al(OP)₅, Al(OAl)₆ and Al(OP)₆, in the NCAP glass structure. The ³¹P, ²⁷Al and ¹¹B MAS-NMR spectra of the NCABP glass revealed the absence of B–O–Al linkages and the presence of B₃–O–B₄–O–P₄ linkages which further leads to the formation of borate and borophosphate domains. The ⁷¹Ga MAS-NMR spectrum suggests that gallium cations in the NCAGP glass compete with the alumina cations and occupy four (GaO₄), five (GaO₅) and six (GaO₆) coordinated sites. The Raman spectrum of NCAGP glass indicates that sodium cations have also been substituted by gallium cations in the NCAP glass structure. From impedance analysis, the dc conductivity of the NCAP glass (∼3.13 × 10⁻⁸ S cm⁻¹) is slightly decreased with the substitution of gallium (∼2.27 × 10⁻⁸ S cm⁻¹) but considerably decreased with the substitution of boron (∼1.46 × 10⁻⁸ S cm⁻¹). The variation in the conductivity values are described based on the structural changes of NCAP glass with the substitution of gallium and boron.

Understanding the conductivity variations induced by compositional changes in sodium super ionic conducting (NASICON) glass materials is highly relevant for applications such as solid electrolytes for sodium (Na) ion batteries.     

Dicarbonyl Electrophiles Mediate Inflammation-Induced Gastrointestinal Carcinogenesis

1. Download : Download high-res image (348KB)
2. Download : Download full-size image

     

Multiscale model of light harvesting by photosystem II in plants

The first step of photosynthesis in plants is the absorption of sunlight by pigments in the antenna complexes of photosystem II (PSII), followed by transfer of the nascent excitation energy to the reaction centers, where long-term storage as chemical energy is initiated. Quantum mechanical mechanisms must be invoked to explain the transport of excitation within individual antenna. However, it is unclear how these mechanisms influence transfer across assemblies of antenna and thus the photochemical yield at reaction centers in the functional thylakoid membrane. Here, we model light harvesting at the several-hundred-nanometer scale of the PSII membrane, while preserving the dominant quantum effects previously observed in individual complexes. We show that excitation moves diffusively through the antenna with a diffusion length of 50 nm until it reaches a reaction center, where charge separation serves as an energetic trap. The diffusion length is a single parameter that incorporates the enhancing effect of excited state delocalization on individual rates of energy transfer as well as the complex kinetics that arise due to energy transfer and loss by decay to the ground state. The diffusion length determines PSII’s high quantum efficiency in ideal conditions, as well as how it is altered by the membrane morphology and the closure of reaction centers. We anticipate that the model will be useful in resolving the nonphotochemical quenching mechanisms that PSII employs in conditions of high light stress.The first step of photosynthesis is light harvesting, the absorption and conversion of sunlight into chemical energy. In photosynthetic organisms, the functional units of light harvesting are self-assembled arrays of pigment–protein complexes called photosystems. Antenna complexes absorb and transfer the nascent excitation energy to reaction centers, where long-term storage as chemical energy is initiated (1). In plants, photosystem II (PSII) flexibly responds to changes in sunlight intensity on a seconds to minutes time scale. In dim light, under ideal conditions, PSII harvests light with a >80% quantum efficiency (2), whereas, in intense sunlight PSII dissipates excess absorbed light safely as heat via nonphotochemical quenching pathways (3). The ability of PSII to switch between efficient and dissipative states is important for optimal plant fitness in natural sunlight conditions (4). Understanding how PSII’s function arises from the structure of its constituent pigment−protein complexes is a prerequisite for systematically engineering the light-harvesting apparatus in crops (5–7) and could be useful for designing artificial materials with the same flexible properties (8, 9).Recent advances have established structure−function relationships within individual pigment−protein complexes, but not how these relationships affect the functioning of the dynamic PSII (grana) membrane (10). Electron microscopy and fitting of atomic resolution structures (11) place the pigment−protein complexes in the grana membrane in close proximity, enabling long-range transport. Indeed, connectivity of excitation between different PSII reaction centers has been discussed since 1964 (12), suggesting that the functional unit for PSII must involve a large area of the membrane. Two limiting cases have been used to model PSII light harvesting: The lake model assumes perfect connectivity between reaction centers across the membrane; alternatively, the membrane can be described as a collection of disconnected “puddles” of pigments that each contain one reaction center (1, 13). At present, however, resolving the spatiotemporal dynamics within the grana membrane on the relevant length (tens to hundreds of nanometers) and time (1 ps to 1 ns) scales experimentally is not possible. Structure-based modeling of the grana membrane, however, can access this wide range of length and time scales.The dense packing of the major light-harvesting antenna (LHCII, discs), which is a trimeric complex, and PSII supercomplexes (PSII-S, pills) in the grana membrane is shown in Fig. 1 A and B. PSII-S is a multiprotein complex (14) that contains the PSII core reaction center dimer, along with several minor light-harvesting complexes and LHCII (Fig. 1A, Inset). Electronic excited states in LHCII and PSII-S are delocalized over several pigments (15–17), making conventional Förster theory inadequate to describe the excitation dynamics. On the protein length scale, generalized Förster (18, 19) calculations between domains of tightly coupled chlorophylls agree very well with more exact methods [e.g., the zeroth-order functional expansion of the quantum-state diffusion model (ZOFE) approximation to non-Markovian quantum state diffusion (20)] for simulating the excitation population dynamics (21, 22). This agreement suggests that the primary quantum phenomenon involved in PSII energy transfer is the site basis coherence that arises from excited states delocalized across a few (approximately three to four) pigments.Open in a separate windowFig. 1.Accurate simulation of chlorophyll fluorescence dynamics from thylakoid membranes using structure-based modeling of energy transfer in PSII. (A and B) The representative mixed (A) and segregated (B) membrane morphologies generated using Monte Carlo simulations and used throughout this work. PSII-S are indicated by the light teal pills, and LHCII, which are trimeric complexes, are indicated by the light grey-green circles. The segregated membrane forms PSII-S arrays and LHCII pools. As shown schematically in A (Bottom) existing crystal structures of PSII-S (14) and LHCII (24) were overlaid on these membrane patches to establish the locations of all chlorophyll pigments. The light teal and light grey-green dashed lines outline the excluded area associated with PSII-S and LHCII trimers respectively, in the Monte Carlo simulations. The chlorophyll pigments are indicated in green, and the protein is depicted by the grey cartoon ribbon. PSII-S is a twofold symmetric dimer of pigment−protein complexes that are outlined by black lines. LHCII-S (strongly bound LHCII), CP26, CP29, CP43, and CP47 are antenna proteins, and RC indicates the reaction center. The inhomogeneously averaged rates of energy transfer between strongly coupled clusters of pigments were calculated using generalized Förster theory. (C) Simulated fluorescence decay of the mixed membrane (solid black line) and the PSII component of experimental fluorescence decay data from thylakoid membranes from ref. 26 (red, dotted line). Inset shows the lifetime components and amplitudes of the simulated decay as calculated using our model with a Gaussian convolution (σ = 20 ps) (black line) or by fitting to three exponential decays (green bars).Here, we construct a generalized Förster model for the ∼10⁴ pigments covering the few-hundred-nanometer length scale of the grana membrane that correctly incorporates the dynamics occurring within and between complexes on the picosecond time scale. We show how delocalized excited states, or excitons, in individual complexes affect light harvesting on the membrane length scale. The formation of excitons is sufficient to explain the high quantum efficiency of PSII in dim light. The model, by being an accurate representation of the complex kinetic network that underlies PSII light harvesting, provides mechanistic explanations for long-observed biological phenomena and sets the stage for developing a better understanding of PSII light harvesting in high light conditions.