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Protein structures respond to changes in their chemical and physical environment. However, studying such conformational changes is notoriously difficult, as many structural biology techniques are also affected by these parameters. Here, the use of photo-crosslinking, coupled with quantitative crosslinking mass spectrometry (QCLMS), offers an opportunity, since the reactivity of photo-crosslinkers is unaffected by changes in environmental parameters. In this study, we introduce a workflow combining photo-crosslinking using sulfosuccinimidyl 4,4'-azipentanoate (sulfo-SDA) with our recently developed data-independent acquisition (DIA)-QCLMS. This novel photo-DIA-QCLMS approach is then used to quantify pH-dependent conformational changes in human serum albumin (HSA) and cytochrome C by monitoring crosslink abundances as a function of pH. Both proteins show pH-dependent conformational changes resulting in acidic and alkaline transitions. 93% and 95% of unique residue pairs (URP) were quantifiable across triplicates for HSA and cytochrome C, respectively. Abundance changes of URPs and hence conformational changes of both proteins were visualized using hierarchical clustering. For HSA we distinguished the N-F and the N-B form from the native conformation. In addition, we observed for cytochrome C acidic and basic conformations. In conclusion, our photo-DIA-QCLMS approach distinguished pH-dependent conformers of both proteins.

Embryos cryopreservation is a widely used technology for genetic resources storage. Cryopreservation suppresses cell respiration, but very little is known about the changes that occur with mitochondria at low temperatures. We used Raman spectroscopy to investigate photoresponse and redox state of cytochromes in the respiratory electron transport chain (ETC) in early mouse embryos during cooling. Redox state of cytochromes was probed by the intensity of cytochrome resonance Raman lines. Photoinduced reactions of cytochromes were used to study the changes in the rates of redox reactions. It is found that the rate of cytochrome photoresponse detected by Raman spectra abruptly changes when embryos are cooled below -50 °C. Raman mapping revealed that the average intensity of cytochrome Raman peaks at -65 °C is higher than at -40 °C. Cytochrome b reduction was found in embryos frozen below -50 °C when irradiated with 532 nm laser radiation. These effects were observed for cells frozen in aqueous solutions of two different cryoprotectants: glycerol and propylene glycol. Raman spectroscopy of cytochromes reveals the abrupt changes in the ETC work of frozen mouse embryos at temperatures below -50 °C. We suggest that similar phenomena can be found in various cell types., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: The impairment of the salivary glands is a permanent side effect of 131 I ablation therapy for patients with differentiated thyroid cancer. Effective and safe treatments for protecting the salivary glands against 131 I are currently not available. Mitochondria are susceptible to ionizing radiation, but alterations after 131 I exposure are unknown. Here, we investigated the mechanisms of 131 I damage in submandibular glands (SMGs) and evaluated the cytoprotective effect of phenylephrine (PE) against mitochondrial radiation damage., Methods and Materials: Rats were randomly divided into 4 groups: control, PE alone, 131 I alone, and 131 I with PE pretreatment. The mitochondrial structure of SMGs was observed under transmission electron microscopy. Apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Cytochrome c, cleaved-caspase 3, SIRT1, NAMPT, and PGC-1α protein levels were determined with Western blot and immunohistochemistry. Levels of mitochondrial membrane potential, nicotinamide adenine dinucleotide (NAD), and adenosine triphosphate (ATP) were measured with relevant kits., Results: After exposing rat SMGs to 131 I, the mitochondrial membrane structures were destroyed, the mitochondrial membrane potential decreased, the release of cytochrome c increased, and cleaved-caspase 3 and cell apoptosis were activated. Moreover, the expression of SIRT1, NAMPT, and PGC-1α was downregulated, and the levels of NAD and ATP decreased. In contrast, PE alleviated the 131 I-induced mitochondrial damages and upregulated the expression of SIRT1/NAMPT/PGC-1α and the levels of NAD and ATP., Conclusions: These findings demonstrate that 131 I impairs the salivary glands via the downregulation of SIRT1/NAMPT/PGC-1α signal pathways, which disturbs mitochondrial homeostasis. PE alleviated the 131 I damage in SMGs at the mitochondrial level, suggesting that PE could be used as a potential radioprotector for patients with differentiated thyroid cancer with radiation sialadenitis., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: Our primary hypothesis was that red-to-near infrared (R-NIR) irradiation would have an effect on the kinetics parameters of the reaction of cytochrome c with isolated cytochrome c oxidase (CCO), and that the magnitude and direction of these changes could be interpreted in the context of the reaction schemes proposed by other authors. New values for the milimolar extinction coefficients of cytochrome c were also determined., Background Data: Definitive answers to the fundamental processes involved in red-to-near infrared photobiomodulation (R-NIR-PBM) have not been obtained. The consensus is that the electron transport chain enzyme CCO is the target for R-NIR-PBM. This work was undertaken to explore the effect of R-NIR on the activity of isolated CCO., Methods: Scans for cytochrome c were obtained in both reduced and oxidized states, and values for the extinction coefficients were calculated. Activity assays were performed by following the oxidation state of cytochrome c at 550 or 415 nm. R-NIR effects on CCO activity were evaluated by pre-irradiating the enzyme at 670 or 830 nm, or by irradiating the reaction mixture with 660 nm light., Results: Milimolar extinction coefficients (L -1 cm -1 ) were: ɛ 550red  = 29.1 ± 0.4, ɛ 550ox  = 8.60 ± 0.15, ɛ 415red  = 140 ± 2, and ɛ 415ox  = 89.0 ± 1.1. Reduced-oxidized extinction coefficients were: δɛ 550red-ox  = 20.5 ± 0.2, and δɛ 415red-ox  = 51.0 ± 2.0. The second order rate constants k' for irradiated CCO did not show a statistically significant difference from controls., Conclusions: The oxidation of cytochrome c by isolated CCO has not been shown to be affected by R-NIR irradiation, whether applied prior to or concurrently with the enzymatic assays. This lack of effect by R-NIR calls into question the CCO activity model of R-NIR photobiomodulation.

Background: There have been several Radio Frequency (RF) field researches on various populations and groups of different ages in recent years. However, the most important group for research has been declared as the pregnant women and their babies., Objective: The aim of the study was to analyse the effect on apoptotic factors of RF fields on newborn rabbit liver tissues., Materials and Methods: Cytochrome c and AIF (Apoptosis Inducing Factor) levels were measured by western blot and caspase 1, 3 and 9 activities were measured by colorimetric method., Results: Cytochrome c and AIF levels were not altered, but all caspase activities were increased in female infant rabbits that exposed to 1800 MHz GSM-like RF signals when they reached 1 month of age and caspase 1 and caspase 3 levels were decreased in male infant rabbits that exposed to 1800 MHz GSM-like RF signals between 15th and 22nd days of the gestational period. Results showed that 1800 MHz GSM-like RF exposure might lead to apoptosis in infant rabbit's liver tissues., Conclusion: According to the results, we suggest that postnatal RF exposure causes caspase dependent apoptosis in female infant rabbits liver tissues (Tab. 1, Fig. 2, Ref. 27).

With the particular conjugation structure in the heme prosthetic group, Cyt c shows unusual functions similar to chlorophyll while irradiated by specific wavelength of UV-Vis lights. To further reveal mechanism of the photo-irradiation of Cyt c, we then studied various external factors that may influence the photo induced process. The absorbance intensity increase of band (317 nm) and Q band (520 nm and 549 nm)indicated Cyt c in phosphate-buffered saline within N2 atmosphere was photoreduced to Fe(II) Cyt c. Irradiated by 410 nm, the photoreduction process was facilitated by Met. But Trp, Tyr and Phe impeded the process due to their light absorbance abilities. In addition, the results of fluorescence and CD spectra indicated that the microenvironment polarity of Trp residue varied during the photoreduction process. And the secondary structure of Cyt c changed with lower α-helix/βsheet ratio. The photoreduction mechanism of Cyt c was intramolecular electron transfer and porphyrin cation radicals were generated. The protein structure of Cyt c changed as well as part of the photoreduction.

Objective: The objective of this study was to investigate the correlation between in vivo δ-tocotrienol (DT3) pharmacokinetics, pharmacodynamics and radiation protection, and to evaluate the effect of DT3 pre-treatment on radiation-induced alterations in apoptotic and autophagic pathways., Methods: We evaluated pharmacokinetics (plasma, 0.5 to 12 h) and pharmacodynamics (peripheral blood indices; day 3, 7, 10 and 14) after a single subcutaneous injection of 300 mg kg(-1) DT3 in unirradiated CD2F1 mice. Next, we monitored 30-day post-irradiation survival (9.25 Gy) and haematopoietic recovery of DT3-treated mice (7 Gy) exposed to cobalt-60 γ-irradiation. The effects of DT3 on irradiated bone marrow apoptosis and autophagy were determined by analyses of key caspases (3, 7, 9 and 8), beclin-1 and light chain 3 conversion., Results: Plasma concentration of DT3 reached ∼195 µM (Cmax) 1 h after injection (Tmax), and DT3 was eliminated from plasma 12 h later. In unirradiated mice, DT3 significantly increased white blood cells (WBCs), neutrophils, lymphocytes (day 3 post DT3 injection) and platelets (day 7) by 1.5- to 2-fold, over vehicle-treated control. DT3 pre-treatment improved 30-day survival to 100% (∼15% in control) and accelerated recovery of reticulocytes, platelets, WBCs, neutrophils, lymphocytes and monocytes in peripheral blood. DT3 reduced activation of caspase-8, caspase-3 and caspase-7, inherent to apoptosis, while increasing autophagy-related beclin-1 expression in irradiated bone marrow., Conclusion: These data indicate that DT3 stimulates multilineage haematopoiesis, protects against radiation-induced apoptosis downstream of the mitochondria and stimulates cytoprotective autophagy. Apart from a potent antioxidant activity, DT3 may elicit survival advantage following irradiation by enhancing haematopoiesis and modulating signalling pathways.

Singlet oxygen, (1)O(2), is produced by absorption of red light by the phthalocyanine dye Pc 4, followed by energy transfer to dissolved triplet molecular oxygen, (3)O(2). In tissues, Pc 4 concentrates in lipid bilayers, and particularly in mitochondrial membranes, because of its positive charge. Illumination of cells and tissues with red light after uptake of Pc 4 results in cell death. The potential initial chemical steps that result in cellular dysfunction have been characterized in this study. Both unsaturated acyl chains of phospholipids and proteins are identified as targets of oxidation. Tetra-linoleoyl cardiolipin was oxidized in both liposomes and mitochondria after Pc 4-mediated (1)O(2) generation. Evidence for the formation of both mono- and bis-hydroperoxide adducts of single linoleoyl side chains is provided by ESI-MS and ESI-MS/MS. Similarly, illumination of Pc 4 in liposomes and mitochondria resulted in cytochrome c oxidation as detected by oxidation of His 26 in the peptide H(26)*KTGPNLHGLFGK, further supporting the potential use of this peptide as a biomarker for the presence of mitochondrial oxidative stress characteristic of (1)O(2) in vivo (J. Kim et al., Free Radic. Biol. Med. 44:1700-1711; 2008). These observations provide evidence that formation of lipid hydroperoxides and/or protein oxidation can be the initial chemical steps in Pc 4-mediated induction of apoptosis in photodynamic therapy., (Copyright 2010 Elsevier Inc. All rights reserved.)

Aims: Irradiation-induced damage to pulmonary endothelial cells is thought to be an important mediator of the pathogenesis of radiation pneumonopathy. Tetrahydropalmatine (THP) has been shown to have a protective effect against oxidative stress. This study was designed to investigate the potential radioprotective effect of THP against irradiation-induced endothelial cellular damage and to elucidate the underlying mechanisms., Main Methods: Human EA.hy926 cells were treated with THP and irradiation. Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For the detection of apoptosis, morphological observation, flow cytometry and a caspase-3 activity assay were employed. The expression of cytochrome-c and Bax/Bcl-2 protein were detected by western blot analysis. Generation of reactive oxygen species (ROS) was measured by flow cytometry. Malondialdehyde (MDA), lactate dehydrogenase (LDH), glutathione (GSH) and superoxide dismutase (SOD) were measured to assess cellular oxidative stress induced injury., Key Findings: Preincubation of EA.hy926 cells with THP before gamma-radiation resulted in significant inhibition of apoptosis and enhancement of cell viability, as revealed by morphological observation, flow cytometry and MTT assay. THP significantly reduced intracellular ROS formation, levels of intracellular MDA and LDH, and enhanced the production of intracellular antioxidants (GSH and SOD) in EA.hy926 cells. Meanwhile, THP also inhibited the decrease of intracellular mitochondrial membrane potential (psim), caspase-3 activation, cytochrome-c release and reduced Bax/Bcl-2 ratio in THP pretreated, irradiated cells., Significance: Our findings demonstrated THP could effectively protect endothelial cells against gamma-irradiation injury, which could potentially be applied to the prevention of endothelial cell dysfunctions associated with ionizing irradiation-induced lung injury., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Treatment of head and neck cancers is still rather poor and worldwide new treatment options are sought. Sensitizing radioresistant tumours by combining irradiation with other therapeutics to induce apoptosis are widely investigated. We examined whether chicken anaemia virus-derived apoptin protein would have a beneficial effect on irradiation of radiosensitive SCC61 and radioresistant SQD9 human head and neck squamous carcinoma cell lines. In both cell lines, concurrent exposure to irradiation and apoptin resulted in analysed mitochondrial cytochrome c release and in cleavage of caspase-3, whereas irradiation alone of SQD9 cells under identical conditions did not. Moreover, in comparison with the irradiation, only the synchronized treatment of apoptin and irradiation resulted in increased cell death in especially the radioresistant SQD9 cells, as measured by means of a colony survival assay. Our data reveal that apoptin treatment represents an effective way for enhancing radiotherapy of tumours responding poorly to radiotherapy.

Agents that inhibit histone deacetylases (HDAC inhibitors) have been shown to enhance radiation response. The aim of this study was to evaluate the effects of low, minimally cytotoxic concentrations of the HDAC inhibitor, valproic acid (VPA), on radiation response of colorectal cancer cells. Cell lines LS174T and an isogenic pair of HCT116, which differed only for the presence of wild-type p53, were exposed to ionizing radiation (IR) alone, VPA alone, or the combination. Clonogenic survival, gamma-H2AX induction, apoptosis, changes in mitochondrial membrane potential, and mitochondrial levels of p53 and Bcl-2 family proteins were assessed. In vivo studies monitored tumor growth suppression after therapy in mice bearing HCT116/p53(+/+) and HCT116/p53(-/-) tumor xenografts. VPA led to radiosensitization, which was dependent on p53 status. A decrease in clonogenic survival, an increase in apoptosis, and an increase in levels of gamma-H2AX were observed after VPA+IR, compared to IR alone, in wild-type p53 cells (LS174T and HCT116/p53(+/+)), as opposed to p53 null cells (HCT116/p53(-/-)). Exposure to VPA resulted in enhancement of IR-induced mitochondrial localizations of Bax and Bcl-xL, mitochondrial membrane potential, and cytochrome c release only in wild-type p53 cell lines. VPA also enhanced tumor growth suppression after IR only in wild-type p53 xenografts. These data suggest that VPA may have an important role in enhancing radiotherapy response in colorectal cancer, particularly in tumors with the wild-type p53 genotype.

Arsenic compounds have been used as anti-cancer agents in traditional Chinese medicine. Ionizing radiation (IR) is one of the most effective tools in the clinical treatment of cancer. The induction of apoptotic cell death is a significant mechanism of tumor cells under the influence of radio-/chemotherapy, and resistance to these treatments has been linked to some cancer cell lines with a low propensity for apoptosis. A combination of different anti-tumoral treatment modalities is advantageous in limiting non-specific toxicity often observed by an exceedingly high dose of single regimen. The present study aimed at investigating the enhanced effects and mechanisms in cell cycle distribution and apoptosis of U937 cells, a human pre-monocytic leukemia cell line lacking functional p53 protein, after combination treatment with irradiation and As(2)O(3). Our results indicated that combined treatment led to activation of cdc-2, which is related to the expression of cyclin B. In addition, combined treatment increased apoptotic cell death in U937 cells, which is correlated with the induction of mitotic arrest, the increase in intracellular reactive oxygen species (ROS) generation, the decrease in B-cell leukemia/lymphoma 2 (Bcl-2) and B-cell leukemia/lymphoma XL (Bcl-XL) levels, the loss of mitochondria membrane potential, and the activation of caspase-3. We found that combining radiation and As(2)O(3) may be an effective strategy against p53-deficient leukemia cells.

PDT has been used in the treatment of malignant brain tumors for the last 2 decades. It is based on the interaction of a photosensitizer (PS) and light of an appropriate wavelength, with generation of oxygen species, mainly singlet oxygen. Brain is particularly susceptible to oxidative stress; therefore the study of PDT effects on cerebral mitochondria might provide mechanistic insights into the action of the therapy, contributing to its optimization. In the present study, we addressed the mitochondrial toxicity of the second generation PS, zinc phthalocyanine tetrasulfonate (ZnPcS4), on rat brain isolated mitochondria, by investigating both intrinsic toxicity and photodynamic action. At higher concentrations (15 and 25 microM/mg protein) ZnPcS4 caused (a) inhibition of state-3 respiration and (b) decrease of RCR and ADP/O. Electrochemical potential, state-4 respiration and Ca2+ retention capacity were not affected. Cytochrome c release was not observed. Coupled with 600 or 1800 mJ/cm2 laser irradiation, ZnPcS4 (5 microM/mg protein) caused more intense effects on state 3, RCR and ADP/O; moreover state-4 respiration and membrane potential were affected. Besides that, Ca2+ and cytochrome c release were induced. Cyclosporine A (CsA) decreased Ca2+ release and ameliorated the electrochemical potential, suggesting that membrane permeability transition (MPT) might be involved in the photodynamic effect. The low intrinsic toxicity and the high photodynamic effect on rat brain mitochondria induced by ZnPcS4, allied to its improved photophysical properties, might indicate its potential for the treatment of malignant brain tumors.

We have measured the electronic circular dichroism (ECD) of the ferri- and ferro-states of several natural cytochrome c derivatives (horse heart, chicken, bovine, and yeast) and the Y67F mutant of yeast in the region between 300 and 750 nm. Thus, we recorded the ECD of the B- and Q-band region as well as the charge-transfer band at approximately 695 nm. The B-band region of the ferri-state displays a nearly symmetric couplet at the B0-position that overlaps with a couplet 790 cm-1 higher in energy, which we assigned to a vibronic side-band transition. For the ferro-state, the couplet is greatly reduced, but still detectable. The B-band region is dominated by a positive Cotton effect at energies lower than B0 that is attributed to a magnetically allowed iron-->heme charge-transfer transition as earlier observed for nitrosyl myoglobin and hemoglobin. The Q-band region of the ferri-state is poorly resolved, but displays a pronounced positive signal at higher wavenumbers. This must result from a magnetically allowed transition, possibly from the methionine ligand to the dxy-hole of Fe3+. For the ferro-state, the spectra resolve the vibronic structure of the Qv-band. A more detailed spectral analysis reveals that the positively biased spectrum can be understood as a superposition of asymmetric couplets of split Q0 and Qv-states. Substantial qualitative and quantitative differences between the respective B-state and Q-state ECD spectra of yeast and horse heart cytochrome c can clearly be attributed to the reduced band splitting in the former, which results from a less heterogeneous internal electric field. Finally, we investigated the charge-transfer band at 695 nm in the ferri-state spectrum and found that it is composed of at least three bands, which are assignable to different taxonomic substates. The respective subbands differ somewhat with respect to their Kuhn anisotropy ratio and their intensity ratios are different for horse and yeast cytochrome c. Our data therefore suggests different substate populations for these proteins, which is most likely assignable to a structural heterogeneity of the distal Fe-M80 coordination of the heme chromophore.

Far infrared dielectric response is used to characterize the collective mode density of states for cytochrome c as a function of oxidation state and hydration using terahertz time domain spectroscopy. A strong absorbance and refractive index increase was observed with the oxidation. A simple phenomenological fitting using a continuous distribution of oscillators reproduces the frequency dependence of the complex dielectric response as well as demonstrates quantitative agreement with a uniform increase in either mode density or polarizability with oxidation in the 5-80 cm(-1) frequency range. Hydration dependence measurements find that a difference in the equilibrium water content for ferri and ferro cytochrome c is not sufficient to account for the large change in terahertz response. The large dielectric increase at terahertz frequencies with oxidation suggests either a significant global softening of the potential and/or a significant increase in polarizability with oxidation.

N-dodecyl-N'-(2-phosphonoethyl)-1,4,5,8-naphthalenetetracarboxylic diimide (DNDI) is a novel naphthalenic diimide with amphiphilic character. DNDI was synthesized through the sequential reaction of 1,4,5,8-naphthalenetetracarboxylic dianhydride, first with dodecylamine and then with 2-aminoethylphosphonic acid. Fluorescence measurements showed that DNDI forms excimers in water at sufficiently high concentrations. The fluorescence quantum yield of DNDI in diluted solutions is sensitive to the polarity of the microenvironment, decreasing as going from water to less polar solvents. This property allowed to monitor the incorporation of DNDI into cetyl trimethyl ammonium bromide (CTAB) micelles, with a binding constant of 1.2x10(4) M-1. UV irradiation (365 nm) of solutions containing DNDI and the redox protein cytochrome c (cyt c) resulted in the reduction of the heme iron from the Fe(III) to the Fe(II) state, a reaction that was inhibited by the incorporation of DNDI into CTAB micelles. DNDI formed host-guest complexes with alpha-cyclodextrin (alpha-CD) through the inclusion of the dodecyl group, resulting in an increased aqueous solubility of the compound.

Objective: To answer the question whether mitochondrial permeability transition (MPT) participates in blue light-induced damage to human retinal pigment epithelium (RPE) cells, this study was directed at assessing the effect of blue light on mitochondrial membrane potential (delta psi(m)) and cytochrome C (Cyt C) of cultured human RPE cells., Methods: Human RPE cells were exposed to blue light (wave length 470-490 nm); delta psi(m) was measured by rhodamine 123 staining and subsequent flow cytometry. Three groups were investigated: Group A (exposure to different intensity of blue light); group B (exposure to identical intensity for different duration); group C (exposure to identical intensity and duration, different prolongation of post-exposure culture). Cyt C activity was assayed by ELISA. Caspase-3 was detected by colorimetric assay. In these aspects, two groups were investigated: Group I [(2000+/-500) 1x for 6 h]; Group II [(2000+/-500) 1x for 12 h]., Results: When human RPE cells were exposed to blue light, the more pronounced decrease of delta psi(m) was consistent with the increase of light intensity in group A. Pronounced decrease of delta psi(m) was seen at 6 h and 12 h of exposure duration in group B. At 6 h prolongation of post-exposure culture in group C, the decrease of delta psi(m) was observed, lasting 48 h. The concentration of Cyt C was detected; no significant changes were found at 6 h and 12 h prolongation of post-exposure culture, but a significant increase was found at 24 h and 36 h post-exposure in the two groups. The increase was more significant in Group II than in Group I at 24 h post-exposure. The activity change of caspase-3 was not found in the two groups., Conclusion: Blue light exposure over threshold can induce damage to human RPE cells, probably by triggering the mitochondrial permeability transition, which results in the decrease of delta psi(m) and the release of cytochrome C.

The crystal structures of high-molecular-weight cytochrome c (HMC) from Desulfovibrio vulgaris Hildenborough in the transient and reduced states have been determined at 2.8 A resolution. An absorption spectrum measured with microspectrophotometer indicated that about 86% of the hemes were reduced after 45 min irradiation of the X-ray beam. Further exposure for 90 min did not significantly change the spectrum. These results suggest that HMC in the crystalline state is easily reduced by illumination of the X-ray beam from synchrotron radiation.

We performed pressure-tuning hole-burning experiments on a modified cytochrome c protein in a glycerol/buffer glass. The shift and the broadening of the holes were investigated for various frequencies within the inhomogeneous band. On the basis of a simple model, we were able to estimate the interaction range between chromophore and protein. It is approximately 4.5 A. The parameters that enter the model are the compressibility, the static mean-square displacement, the inhomogeneous width, and the average spectral shift per pressure. From this result and from our experiments on pressure-induced denaturing, we conclude that water molecules have to be brought very close to the chromophore during the denaturation process.

Ultra thin film of photosensitive polyimide having benzene and sulfonyloxyimide moieties in the main chain was prepared using a Langmuir-Blodgett (LB) technique, and then micro array pattern of the polyimide LB film on a gold substrate was obtained by deep UV lithographic technique. In order to array cytochrome c molecules along the micro-patterned gold substrate, the well-characterized monolayer of cytochrome c was immobilized with a mixed monolayer of 11-mercaptoundecanoic acid (11-MUDA) and decanethiol. The redox activity and electron transfer between cytochrome c molecular center and gold electrode interface for the self-assembled cytochrome c monolayer were investigated by cyclic voltammetry measurement. Biomolecular photodiode consisting of cytochrome c and green fluorescent protein (GFP) onto the patterned gold substrate was fabricated by self-assembly process. The integration and morphology of cytochrome c and GFP were studied from the measurements of atomic force microscopy (AFM) and fluorescence emission. Especially, current-voltage characteristics of the protein multilayers were investigated by scanning tunneling microscopy (STM) and its application in biomolecular photodiode was also examined.

Commercial cytochrome c (Cyt c) was irradiated with Co-60 gamma-rays in the dose range of up to 3.0 kGy to investigate the enhancement of the nitrite reducing activity of Cyt c. The optimum irradiation dose to induce nitrite reducing activity for 30 muM Cyt c solution was 1.0 kGy under an O(2) atmosphere. The nitrite reducing activity of Cyt c irradiated at this dose was approximately 45-fold that of unirradiated Cyt c and ca. 1.2-fold that of nitrite reductase. The irradiation treatment resulted in unfolding of the peptide chain, exposure of the heme group, oxidation of methionine to methionine sulfoxide, dissociation of the sixth ligand (Met), and occurrence of autoxidation in Cyt c. Sepharose-immobilized irradiated Cyt c had a similar activity to that in solution. The resin retained the activity after five uses even after 1 year of storage. The irradiated Cyt c will be able to be used as a substitute for nitrite reductase.

We investigated the capacity of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] to protect human keratinocytes against the hazardous effects of ultraviolet B (UVB)-irradiation, recognized as the most important etiological factor in the development of skin cancer. Cytoprotective effects of 1,25(OH)(2)D(3) on UVB-irradiated keratinocytes were seen morphologically and quantified using a colorimetric survival assay. Moreover, 1,25(OH)(2)D(3) suppressed UVB-induced apoptotic cell death. An ELISA, detecting DNA-fragmentation, demonstrated that pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM for 24 h reduced UVB-stimulated apoptosis by 55-70%. This suppression required pharmacological concentrations 1,25(OH)(2)D(3) and a preincubation period of several hours. In addition, 1,25(OH)(2)D(3) also inhibited mitochondrial cytochrome c release (90%), a hallmark event of UVB-induced apoptosis. Furthermore, we demonstrated that 1,25(OH)(2)D(3) reduced two important mediators of the UV-response, namely, c-Jun-NH(2)-terminal kinase (JNK) activation and interleukin-6 (IL-6) production. As shown by Western blotting, pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM diminished UVB-stimulated JNK activation with more than 30%. 1,25(OH)(2)D(3) treatment (1 microM) reduced UVB-induced IL-6 mRNA expression and secretion with 75-90%. Taken together, these findings suggest the existence of a photoprotective effect of active vitamin D(3) and create new perspectives for the pharmacological use of active vitamin D compounds in the prevention of UVB-induced skin damage and carcinogenesis., (Copyright 2003 Wiley-Liss, Inc.)

Apoptotic response of keratinocytes to UVB irradiation has physiological significance on photocarcinogenesis. Here, we show that the sustained release of Smac/DIABLO from mitochondria is an important event for the onset of apoptosis in keratinocytes exposed to UVB irradiation. In human keratinocyte HaCaT cells, UVB irradiation at 500 J/m(2), but not at 150 J/m(2), induces apoptosis. Significant activations of caspases-9 and -3, and slight activation of caspase-7 were observed only in 500 J/m(2) UVB irradiated HaCaT cells. Correspondingly, the cleavage of PARP, a substrate of caspases-3 and -7, was detected in cells irradiated at 500 J/m(2) UVB, but not at 150 J/m(2). However, with both 150 and 500 J/m(2) UVB irradiation, cytochrome c, an activator of caspase-9 via the formation of apoptosome, was released from mitochondria to the cytosol at the same extent. In contrast, significant amounts of Smac/DIABLO are released from mitochondria to the cytosol only with 500 J/m(2) UVB irradiation, and that the level of XIAP is decreased. These results suggest that the extent of Smac/DIABLO efflux from mitochondria is a determinant whether a cell will undergo apoptosis or survival.

QLT0074 is a newly introduced, porphyrin-derivative for use in photodynamic therapy (PDT). In the current study, the intracellular distribution of QLT0074 and the mode of cell death induced by photosensitization with this compound in vitro were assessed for transformed human HaCaT keratinocytes. Fluorescence microscopy studies indicated a distribution of the drug to the cytoplasm, nuclear membrane and mitochondria of these cells. In the absence of light, QLT0074 produced no evidence of apoptosis-related biochemical changes or affected cell viability. When combined with blue light exposure, cytotoxicity was exerted in a QLT0074- and light-dose-related manner. Appearance of the mitochondrial protein cytochrome c in the cytosolic fraction and expression of the apoptosis-associated mitochondrial 7A6 antigen were demonstrable following photosensitization at nano-molar levels of QLT0074. Evidence of processing of the apoptosis-effector molecules caspase-3, -6, -7, -8 and -9 as well as cleavage of the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) were demonstrable subsequent to cytochrome c release after PDT. Treatment with the anti-oxidant pyrrolidine dithiocarbamate (PDTC) inhibited cytochrome c release, caspase-3 activation and PARP cleavage associated with PDT thereby supporting the contention that QLT0074 induces apoptosis through the generation of reactive oxygen species upon light activation. QLT0074 is a potent photosensitizer with the capacity to directly initiate apoptosis by acting upon mitochondria.

The focus of the present study is to better understand the complex factors influencing intermolecular electron transfer (ET) in biological molecules using a model system involving free-base coproporphyrin (COP) complexed with horse heart cytochrome c (Cc). Coproporphyrin exhibits bathochromic shifts in both the Soret and visible absorption bands in the presence of Cc and an absorption difference titration reveals a 1:1 complex with an association constant of 2.63 +/- 0.05 x 10(5) M(-1). At 20 degrees C, analysis of time-resolved fluorescence data reveals two lifetime components consisting of a discrete lifetime at 15.0 ns (free COP) and a Gaussian distribution of lifetimes centered at 2.8 ns (representing (1)COP --> Cc ET). Temperature-dependent, time-resolved fluorescence data demonstrate a shift in singlet lifetime as well as changes in the distribution width (associated with the complex). By fitting these data to semiclassical Marcus theory, the reorganizational energy (lambda) of the singlet state electron transfer was calculated to be 0.89 eV, consistent with values for other porphyrin/Cc intermolecular ET reactions. Using nanosecond transient absorption spectroscopy the temperature dependences of the forward and thermal back ET originating from triplet state were examined ((3)COP --> Cc ET). Fits of the temperature dependence of the rate constants to semiclassical Marcus theory gave lambda of 0.39 eV and 0.11 eV for the forward and back triplet ET, respectively (k(f) = (7.6 +/- 0.3) x 10(6) s(-1), k(b) = (2.4 +/- 0.3) x 10(5) s(-1)). The differing values of lambda for the forward and back triplet ET demonstrate that these ET reactions do not occur within a static complex. Comparing these results with previous studies of the uroporphyrin:Cc and tetrakis (4-carboxyphenyl)porphyrin:Cc complexes suggests that side-chain flexibility gives rise to the conformational distributions in the (1)COP --> Cc ET whereas differences in overall porphyrin charge regulates gating of the back ET reaction (reduced Cc --> COP(+)).

Oxidative stress has been shown to be associated with apoptosis (programmed cell death) in a number of cell systems. We earlier reported in vitro cultured Spodoptera frugiperda (Sf9) cells as a model system to study oxidative stress induced apoptosis (J Biosciences 24 (1999) 13) and the inhibition of UV-induced apoptosis by the baculovirus antiapoptotic p35 protein that acts as a sink to sequester reactive oxygen species (Proc Natl Acad Sci USA 96 (1999) 4838). We now show that UV-induced apoptosis in Sf9 cells, is preceded by the release of mitochondrial cytochrome c into the cytosol and consequent activation of Sf-caspase-1. The inhibitory effect of different antioxidants including scavengers of oxygen radicals such as butylated hydroxyanisole (BHA), alpha tocopherol acetate, benzoate and reduced-glutathione (GSH) on ultra violet B (UVB)-induced apoptosis in cultured Sf9 cells was assessed. Both, cytochrome c release as well as Sf-caspase-1 activation was inhibited by pre-treatment with antioxidants such as BHA and alpha tocopherol acetate, suggesting that these antioxidants inhibit apoptosis by acting quite upstream in the apoptosis cascade at the mitochondrial level, as well as downstream at the caspase level.