Photoreaction Dynamics of a Full-Length Protein YtvA and Intermolecular Interaction with RsbRA.

YtvA from Bacillus subtilis is a sensor protein that responds to blue light stress and regulates the activity of transcription factor σB. It is composed of the N-terminal LOV (light-oxygen-voltage) domain, the C-terminal STAS (sulfate transporter and anti-sigma factor antagonist) domain, and a linker region connecting them. In this study, the photoreaction and kinetics of full-length YtvA and the intermolecular interaction with a downstream protein, RsbRA, were revealed by the transient grating method. Although N-YLOV-linker, which is composed of the LOV domain of YtvA with helices A'α and Jα, exhibits a diffusion change due to the rotational motion of the helices, the YtvA dimer does not show the diffusion change. This result suggests that the STAS domain inhibits the rotational movement of helices A'α and Jα. We found that the YtvA dimer formed a heterotetramer with the RsbRA dimer probably via the interaction between the STAS domains, and we showed the diffusion change upon blue light illumination with a time constant faster than 70 µs. This result suggests a conformational change of the STAS domains; i.e., the interface between the STAS domains of the proteins changes to enhance the friction with water by the rotation structural change of helices A'α and Jα of YtvA.

Comparison of signaling profiles in the low dose range following low and high LET radiation.

During space travel astronauts will be exposed to a very low, mixed field of radiation containing different high LET particles of varying energies, over an extended period. Thus, defining how human cells respond to these complex low dose exposures is important in ascertaining risk. In the current study, we have chosen to investigate how low doses of three different ion's at various energies uniquely change the kinetics of three different phospho-proteins. A normal hTERT immortalized fibroblast cell line, 82-6, was exposed to a range of lower doses (0.05-0.5 Gy) of radiation of different qualities and energies (Si 1000 MeV/u, Si 300 MeV/u, Si 173 MeV/u, Si 93 MeV/u, Fe 1000 MeV/u, Fe 600 MeV/u, Fe 300 MeV/u, Ti 300 MeV/u, Ti 326 MeV/u, Ti 386 MeV/u), covering a wide span of LET's. Exposed samples were analyzed for the average intensity of signal as a fold over the geometric mean level of the sham controls. Three phospho-proteins known to localize to DNA DSBs following radiation (γH2AX, pATF2, pSMC1) were studied. The kinetics of their response was quantified by flow cytometery at 2 and 24 h post exposure. These studies reveal unique kinetic patterns based on the ion, energy, fluence and time following exposure. In addition, γH2AX phosphorylation patterns are uniquely different from phospho-proteins known to be primarily phosphorylated by ATM. This latter finding suggests that the activating kinase(s), or the phosphatases deactivating these proteins, exhibit differences in their response to various radiation qualities and/ or doses of exposure. Further studies will be needed to better define what the differing kinetics for the kinases activated by the unique radiation qualities plays in the biological effectiveness of the particle.

Phosphoproteomics Reveals the Biosynthesis of Secondary Metabolites in Catharanthus roseus under Ultraviolet-B Radiation.

Ultraviolet (UV)-B radiation acts as an elicitor to enhance the production of secondary metabolites in medicinal plants. To investigate the mechanisms, which lead to secondary metabolites in Catharanthus roseus under UVB radiation, a phosphoproteomic technique was used. ATP content increased in the leaves of C. roseus under UVB radiation. Phosphoproteins related to calcium such as calmodulin, calcium-dependent kinase, and heat shock proteins increased. Phosphoproteins related to protein synthesis/modification/degradation and signaling intensively changed. Metabolomic analysis indicated that the metabolites classified with pentoses, aromatic amino acids, and phenylpropanoids accumulated under UVB radiation. Phosphoproteomic and immunoblot analyses indicated that proteins related to glycolysis and the reactive-oxygen species scavenging system were changed under UVB radiation. These results suggest that UVB radiation activates the calcium-related pathway and reactive-oxygen species scavenging system in C. roseus. These changes lead to the upregulation of proteins, which are responsible for the redox reactions in secondary metabolism and are important for the accumulation of secondary metabolites in C. roseus under UVB radiation.

Thymoquinone Enhances the Effect of Gamma Knife in B16-F10 Melanoma Through Inhibition of Phosphorylated STAT3.

BACKGROUND: Patients with brain metastasis from melanoma have a dismal prognosis with poor survival time. Gamma Knife (GK) is an effective treatment to control brain metastasis from melanoma. Thymoquinone (TQ) has emerged as a potential therapeutic option due to its antiproliferative effects on various cancers. The purpose of the study was to assess the effect of GK on B16-F10 melanoma cells in vitro and intracerebral melanoma in vivo, and its synergistic effect in combination with TQ. METHODS: The effects of GK and combination treatment of GK and TQ were studied on B16-F10 melanoma cells by evaluating cytotoxicity with an adenosine triphosphate assay, apoptosis by acridine orange staining, and genotoxicity by comet assay. Western blot analysis was performed to investigate the expression of STAT3, p-STAT3 (Tyr705), JAK2, p-JAK2, caspase-3, Bax, Bcl-2, survivin, and ß-actin. Expression of inflammatory cytokines was assessed by enzyme-linked immunosorbent assay. GK alone and in combination with TQ was assessed in an established intracerebral melanoma tumor in mice. RESULTS: The effects of GK on cytotoxicity, genotoxicity, and apoptosis were enhanced by TQ in B16-F10 melanoma cells. GK induced apoptosis through inhibition of p-STAT3 expression, which in turn regulated pro- and antiapoptotic proteins such as caspase-3, Bax, Bcl-2, and survivin. Adding TQ to GK irradiation further enhanced this apoptotic effect of GK irradiation. GK was shown to reduce the levels of tumor-related inflammatory cytokines in B16-F10 melanoma cells. This effect was more pronounced when TQ was added to GK irradiation. GK with 15 Gy increased the survival of mice with intracerebral melanoma compared with untreated mice. However, despite the additive effect of TQ in addition to GK irradiation on B16-F10 melanoma cells in vitro, TQ did not add any significant survival benefit to GK treatment in mice with intracerebral melanoma. CONCLUSIONS: Our findings suggest that TQ would be a potential therapeutic agent in addition to GK to enhance the antitumor effect of irradiation. Further studies are required to support our findings.

DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity.

Radiotherapy is under investigation for its ability to enhance responses to immunotherapy. However, the mechanisms by which radiation induces anti-tumour T cells remain unclear. We show that the DNA exonuclease Trex1 is induced by radiation doses above 12-18 Gy in different cancer cells, and attenuates their immunogenicity by degrading DNA that accumulates in the cytosol upon radiation. Cytosolic DNA stimulates secretion of interferon-ß by cancer cells following activation of the DNA sensor cGAS and its downstream effector STING. Repeated irradiation at doses that do not induce Trex1 amplifies interferon-ß production, resulting in recruitment and activation of Batf3-dependent dendritic cells. This effect is essential for priming of CD8+ T cells that mediate systemic tumour rejection (abscopal effect) in the context of immune checkpoint blockade. Thus, Trex1 is an upstream regulator of radiation-driven anti-tumour immunity. Trex1 induction may guide the selection of radiation dose and fractionation in patients treated with immunotherapy.

Expressão de DMP-1, DSPP, VEGF e FGF2 em células derivadas da polpa de dentes decíduos após o uso do laser de baixa intensidade / FGF2, VEGF, DSPP, and DMP-1 expression in cells of the pulp deciduous teeth with a low intensity laser

O objetivo do presente trabalho foi avaliar o efeito de diferentes densidades de energia do Laser de Baixa Intensidade na viabilidade e proliferação celular de fibroblastos derivados da polpa de dentes decíduos humanos e na expressão de RNAm para DMP- 1, DSPP, VEGF e FGF-2. Amostras de fibroblastos pulpares da polpa de dentes decíduos humanos foram obtidas de um Biorrepositório. Foram utilizadas células entre a 4ª e a 7ª passagem, irradiadas com Laser de Baixa Intensidade (InGaAlP) de acordo com os seguintes grupos experimentais: Grupo 1: 1,2 J/cm2 - 05 mW - 10s; Grupo 2: 2,5 J/cm2 - 05 mW - 20s; Grupo 3: 3,7 J/cm2 - 05 mW - 30s; Grupo 4: 5,0 J/cm2 - 05 mW - 40s; Grupo 5: 6,2 J/cm2 - 05 mW - 50s; Grupo 6: 2,5 J/cm2 - 10 mW - 10s; Grupo 7: 3,7 J/cm2 - 15 mW - 10s; Grupo 8: 5,0 J/cm2 - 20 mW - 10s; Grupo 9: 6,2 J/cm2 - 25 mW - 10s; Controle Negativo: DMEM 1% SFB ­ não irradiado; Controle Positivo: DMEM 10% SFB ­ não irradiado. As técnicas utilizadas para as análises de viabilidade e proliferação celular foram MTT e CV. A técnica utilizada para avaliação da expressão de RNAm para os alvos DMP-1, DSPP, VEGF e FGF-2 foi RT-PCR. Os resultados foram analisados pelo método ANOVA a dois critérios, seguido pelo teste de Tukey (p<0,05). Para o teste MTT, na comparação intragrupos observou-se que houve diferença estatisticamente significativa entre os períodos 6h, 12h e 24h, diminuindo a viabilidade com o passar do tempo, exceto para o Grupo 1. Na comparação intergrupos, o MTT mostrou menor viabilidade para o controle negativo em comparação com os outros grupos (p<0,05), exceto com grupo 5 (5mW/50 seg). Observou-se que os grupos com maiores potências (10mW, 15mW, 20mW e 25mW), menores tempos de aplicação (10 segundos) e densidades de energia entre 2,5 J/cm2 e 6,2 J/cm2, apresentaram estatisticamente maior viabilidade que o grupo com menor potência (5mW), maior tempo de aplicação (50 segundos) e densidade de energia de 6,2 J/cm2. Para o teste CV não houve diferença intragrupos, mas houve diferença intergrupos entre os controles positivo e negativo. Para a expressão de RNAm por RTPCR, os fatores de crescimento VEGF e FGF-2 foram expressos em grande quantidade no primeiro período experimental, enquanto que DMP-1 e DSPP não foram expressos de maneira significativa. De acordo com os resultados obtidos, frente as diferentes densidades de energia, sugere-se que a terapia a laser de baixa intensidade manteve os fibroblastos viáveis e aumentou a expressão de RNAm para VEGF e FGF-2.(AU)

This study aimed to evaluate the effect of different energy densities of Low Level Laser (LLL) on cell viability and proliferation of fibroblasts from the pulp of human primary teeth (DHPF) and on the RNAm expression of DMP-1, DSPP, VEGF and FGF-2. DHPF were obtained from a biorepository and used at passages 4th to 7th. The cells were irradiated with LLL (InGaAlP) according to the following experimental groups: Group 1: 1.2 J/cm2 - 05 mW - 10s; Group 2: 2.5 J/cm2 - 05 mW - 20s; Group 3: 3.7 J/cm2 - 05 mW - 30s; Group 4: 5.0 J/cm2 - 05 mW - 40s; Group 5: 6.2 J/cm2 - 05 mW - 50s; Group 6: 2.5 J/cm2 - 10 mW - 10s; Group 7: 3,7 J/cm2 - 15 mW - 10s; Group 8: 5.0 J/cm2 - 20 mW - 10s; Group 9: 6.2 J/cm2 - 25 mW - 10s; Negative Control: DMEM 1% SFB ­ not irradiated; Positive Control: DMEM 10% SFB ­ not irradiated. The techniques used to evaluate the cell viability/proliferation were MTT and Crystal Violet (CV) assays. RT-PCR was used to verify the RNAm expression of DMP-1, DSPP, VEGF, and FGF-2. Two-way ANOVA, followed by Tukey test (p<0.05) was used to analyze the results. In the intragroup comparison, MTT assay revealed statistically significant differences among the periods of 6h, 12h, and 24h, with viability reduction as time went by, except for Group 1. In the intergroup comparison, the MTT assay showed that the negative control had statistically lower viability than that of the other groups (p<0.05), except for Group 5 (5mW/50 s). The groups with higher powers (10mW, 15mW, 20mW, and 25mW), shortest application periods (10 s), and energy densities between 2.5 J/cm2 and 6.2 J/cm2 exhibited statistically higher viability than that of the group with small power (5mW), longer application period (50 s), and energy density of 6.2 J/cm2 . CV assay did not show intergroup statistically differences. In the intragroup comparison, CV assay revealed statistically significant differences between positive and negative controls (p<0.05). RT-PCR revealed increased RNAm expression of the growth factors VEGF and FGF-2 at the first experimental period, while DMP-1 and DSPP was not significant. Based on the results and different energy densities used, LLL maintained DHPF viability and increased the RNAm expression of VEGF and FGF-2.(AU)

GaAlAs laser-induced pulp mineralization involves dentin matrix protein 1 and osteopontin expression.

OBJECTIVES: GaAlAs lasers induce pulp mineralization by promoting reparative dentinogenesis. This study analyzed the expression of dentin matrix protein 1 (DMP1) and osteopontin in GaAlAs laser-irradiated rat molars, to examine the hypothesis that these proteins play a role in the laser-induced reparative dentinogenic process. MATERIALS AND METHODS: The mesial surfaces of the upper first molars of 8-week-old Wistar rats were irradiated with a pulsed GaAlAs laser. After 1-14 days, mRNA expression of DMP1 and osteopontin in the coronal pulp was analyzed using real-time PCR. DMP1, osteopontin, and heat shock protein 25 (HSP25) were immunolocalized at 1-21 days. RESULTS: The pulp exhibited a degenerative zone in its mesial portion on days 1-3, and progressive formation of reparative dentin lined with HSP25-immunoreactive odontoblast-like cells, from day 7 onwards. DMP1 and osteopontin mRNA expression were significantly upregulated on days 1-7 and 3-7, respectively. From day 7 onwards, DMP1 and osteopontin immunoreactivity colocalized along the boundary between the primary and reparative dentin. CONCLUSION: GaAlAs laser irradiation of rat molars induced upregulated DMP1 and osteopontin mRNA expression in the coronal pulp, followed by the formation of reparative dentin and the colocalization of DMP1 and osteopontin immunoreactivity at the site at which this tissue first appeared.

Implementation of Ultraviolet Photodissociation on a Benchtop Q Exactive Mass Spectrometer and Its Application to Phosphoproteomics.

Proteomics applications performed on the popular benchtop Q Exactive Orbitrap mass spectrometer have so far relied exclusively on higher collision-energy dissociation (HCD) fragmentation for peptide sequencing. While this fragmentation technique is applicable to a wide range of biological questions, it also has limitations, and all questions cannot be addressed equally well. Here, we demonstrate that the fragmentation capabilities of the Q Exactive mass spectrometer can be extended with ultraviolet photodissociation (UVPD) fragmentation, complete with synchronization triggering to make it compatible with liquid chromatography (LC)/tandem mass spectrometry (MS/MS) workflows. We show that UVPD not only is directly compatible with LC/MS workflows but also, when combined with these workflows, can result in higher database scores and increased identification rates for complex samples as compared to HCD methods. UVPD as a fragmentation technique offers prompt, high-energy fragmentation, which can potentially lead to improved analyses of labile post-translational modifications. Techniques like HCD result in substantial amounts of modification losses, competing with fragmentation pathways that provide information-rich ion fragments. We investigate here the utility of UVPD for identification of phosphorylated peptides and find that UVPD fragmentation reduces the extent of labile modification loss by up to ∼60%. Collectively, when integrated into a complete workflow on the Q Exactive Orbitrap, UVPD provides distinct advantages to the analysis of post-translational modifications and is a powerful and complementary addition to the proteomic toolbox.

Essential role of the A'α/Aß gap in the N-terminal upstream of LOV2 for the blue light signaling from LOV2 to kinase in Arabidopsis photototropin1, a plant blue light receptor.

Phototropin (phot) is a blue light (BL) receptor in plants and is involved in phototropism, chloroplast movement, stomata opening, etc. A phot molecule has two photo-receptive domains named LOV (Light-Oxygen-Voltage) 1 and 2 in its N-terminal region and a serine/threonine kinase (STK) in its C-terminal region. STK activity is regulated mainly by LOV2, which has a cyclic photoreaction, including the transient formation of a flavin mononucleotide (FMN)-cysteinyl adduct (S390). One of the key events for the propagation of the BL signal from LOV2 to STK is conformational changes in a Jα-helix residing downstream of the LOV2 C-terminus. In contrast, we focused on the role of the A'α-helix, which is located upstream of the LOV2 N-terminus and interacts with the Jα-helix. Using LOV2-STK polypeptides from Arabidopsis thaliana phot1, we found that truncation of the A'α-helix and amino acid substitutions at Glu474 and Lys475 in the gap between the A'α and the Aß strand of LOV2 (A'α/Aß gap) to Ala impaired the BL-induced activation of the STK, although they did not affect S390 formation. Trypsin digested the LOV2-STK at Lys603 and Lys475 in a light-dependent manner indicating BL-induced structural changes in both the Jα-helix and the gap. The digestion at Lys603 is faster than at Lys475. These BL-induced structural changes were observed with the Glu474Ala and the Lys475Ala substitutes, indicating that the BL signal reached the Jα-helix as well as the A'α/Aß gap but could not activate STK. The amino acid residues, Glu474 and Lys475, in the gap are conserved among the phots of higher plants and may act as a joint to connect the structural changes in the Jα-helix with the activation of STK.

Phototherapy up-regulates dentin matrix proteins expression and synthesis by stem cells from human-exfoliated deciduous teeth.

OBJECTIVES: The aim of this study was to evaluate the effects of infrared LED (850nm) irradiation on dentin matrix proteins expression and synthesis by cultured stem cells from human exfoliated deciduous teeth (SHED). METHODS: Near-exfoliation primary teeth were extracted (n=3), and SHED cultures were characterized by immunofluorescence using STRO-1, CD44, CD146, Nanog and OCT3/4 antibodies, before experimental protocol. The SHEDs were seeded (3×10(4) cells/cm(2)) with DMEM containing 10% FBS. After 24-h incubation, the culture medium was replaced by osteogenic differentiation medium, and the cells were irradiated with LED light at energy densities (EDs) of 0 (control), 2, or 4J/cm(2) (n=8). The irradiated SHEDs were then evaluated for alkaline phosphatase (ALP) activity, total protein (TP) production, and collagen synthesis (SIRCOL™ Assay), as well as ALP, collagen type I (Col I), dentin sialophosphoprotein (DSPP), and dentin matrix acidic phosphoprotein (DMP-1) gene expression (qPCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (α=0.05). RESULTS: Increased ALP activity and collagen synthesis, as well as gene expression of DSPP and ALP, were observed for both EDs compared with non-irradiated cells. The ED of 4J/cm(2) also increased gene expression of COL I and DMP-1. CONCLUSIONS: In conclusion, infrared LED irradiation was capable of biostimulating SHEDs by increasing the expression and synthesis of proteins related with mineralized tissue formation, with overall better results for the energy dose of 4J/cm(2). CLINICAL SIGNIFICANCE: Phototherapy is an additional approach for the clinical application of LED in Restorative Dentistry. Infrared LED irradiation of the cavity's floor could biostimulate subjacent pulp cells, improving local tissue healing.

Signaling kinetics of cyanobacterial phytochrome Cph1, a light regulated histidine kinase.

Cyanobacterial phytochrome 1 (Cph1) is a red/far-red light regulated histidine kinase, which together with its response regulator (Rcp1) forms a two-component light signaling system in Synechocystis 6803. In the present study we followed the in vitro autophosphorylation of Cph1 and the subsequent phosphotransfer to Rcp1 in different ionic milieus and following different light treatments. Both processes were red/far-red reversible with activity manifested in the Pr ground state (in darkness or after far-red irradiation) and with strongest activities being exhibited in the presence of Mn(2+). In vivo and in vitro assembled holoproteins in the Pr state displayed at least 4-fold higher efficiencies (k(cat)/K(m)) for autophosphorylation and phosphotransfer than the apoprotein or the holoprotein at photoequilibrium in red light. The reduced activities observed following red light treatments were consistent with the Pfr state being enzymatically inactive. Thus, both the rate of kinase autophosphorylation and the rate of phosphotransfer regulate the phosphorylation state of the response regulator, consistent with the rotary switch model regulating accessibility of the histidine target.

In vivo phosphorylation site mapping and functional characterization of Arabidopsis phototropin 1.

Phototropins (phot1 and phot2) are blue-light receptor kinases controlling a range of responses that optimize the photosynthetic efficiency of plants. Light sensing is mediated by two flavin-binding motifs, known as LOV1 and LOV2, located within the N-terminal region of the protein. Photoexcitation via LOV2 leads to activation of the C-terminal kinase domain and consequently receptor autophosphorylation. However, knowledge of the in-vivo phosphorylation sites for Arabidopsis phototropins is lacking and has impeded progress in elucidating the functional significance of receptor phosphorylation. We have purified phot1 from Arabidopsis and identified the in-vivo sites of receptor phosphorylation by liquid chromatography tandem mass spectrometry. Arabidopsis-derived phot1 binds flavin mononucleotide as chromophore and is phosphorylated at four major sites located upstream of LOV2 (Ser(58), Ser(85), Ser(350), and Ser(410)), three of which are induced by blue light. Nevertheless, structure-function analysis indicates that the biological activity of phot1 can be attributed to a modular unit comprising the LOV2-kinase region of the protein. Thus, peptide regions upstream of LOV2, including the sites of receptor phosphorylation identified here, do not appear to be important for receptor signaling. By contrast, these regions may be necessary for maximizing stomatal performance and possibly light-induced relocalization of phot1.

Pulsed electromagnetic fields rapidly modulate intracellular signaling events in osteoblastic cells: comparison to parathyroid hormone and insulin.

Pulsed electromagnetic field (PEMF) devices are approved for the healing of bone nonunions, but there is a lack of understanding as to their mechanism of action at the cell and molecular level. Intermittent parathyroid hormone (PTH) therapy is currently utilized for treatment of osteoporosis, and is also being investigated for the purpose of augmenting fracture healing. Insulin and IGF-1 are also thought to play important anabolic roles in osteogenesis. In this report, signaling pathways activated by acute PTH or insulin treatments were compared to those activated by PEMF treatment in osteoblast-like cells. Some signaling molecules like the extracellular response kinases 1/2 (Erk1/2) and the cAMP response element binding protein (CREB) were activated by insulin and PTH, respectively, but not by PEMF treatment. Other signaling molecules like the insulin receptor substrate-1 (IRS-1), the S6 ribosomal subunit kinase, and the endothelial nitric oxide synthase (eNOS) were phosphorylated by PTH, insulin, and PEMF to the same relative extent and within the same time frame. IRS-1, eNOS, and S6 have been implicated in bone anabolism, and our results suggest that the anabolic effects of PEMF may be mediated, in part, through the activation of these proteins.

Phosphoproteome profiling of human skin fibroblast cells in response to low- and high-dose irradiation.

A hallmark of the response to high-dose radiation is the up-regulation and phosphorylation of proteins involved in cell cycle checkpoint control, DNA damage signaling, DNA repair, and apoptosis. Exposure of cells to low doses of radiation has well documented biological effects, but the underlying regulatory mechanisms are still poorly understood. The objective of this study is to provide an initial profile of the normal human skin fibroblast (HSF) phosphoproteome and explore potential differences between low- and high-dose irradiation responses at the protein phosphorylation level. Several techniques including Trizol extraction of proteins, methylation of tryptic peptides, enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC), nanoflow reversed-phase HPLC (nano-LC)/electrospray ionization, and tandem mass spectrometry were combined for analysis of the HSF cell phosphoproteome. Among 494 unique phosphopeptides, 232 were singly phosphorylated, while 262 peptides had multiple phosphorylation sites indicating the overall effectiveness of the IMAC technique to enrich both singly and multiply phosphorylated peptides. We observed approximately 1.9-fold and approximately 3.6-fold increases in the number of identified phosphopeptides in low-dose and high-dose samples respectively, suggesting both radiation levels stimulate cell signaling pathways. A 6-fold increase in the phosphorylation of cyclin dependent kinase (cdk) motifs was observed after low- dose irradiation, while high-dose irradiation stimulated phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT/RSK motifs 8.5- and 5.5-fold, respectively. High- dose radiation resulted in the increased phosphorylation of proteins involved in cell signaling pathways as well as apoptosis while low-dose and control phosphoproteins were broadly distributed among biological processes.

Targeting of C-terminal binding protein (CtBP) by ARF results in p53-independent apoptosis.

ARF encodes a potent tumor suppressor that antagonizes MDM2, a negative regulator of p53. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF or p53 singly null mice, results in a broadened tumor spectrum and decreased tumor latency. To investigate the mechanism of p53-independent tumor suppression by ARF, potential interacting proteins were identified by yeast two-hybrid screen. The antiapoptotic transcriptional corepressor C-terminal binding protein 2 (CtBP2) was identified, and ARF interactions with both CtBP1 and CtBP2 were confirmed in vitro and in vivo. Interaction with ARF resulted in proteasome-dependent CtBP degradation. Both ARF-induced CtBP degradation and CtBP small interfering RNA led to p53-independent apoptosis in colon cancer cells. ARF induction of apoptosis was dependent on its ability to interact with CtBP, and reversal of ARF-induced CtBP depletion by CtBP overexpression abrogated ARF-induced apoptosis. CtBP proteins represent putative targets for p53-independent tumor suppression by ARF.

Does irradiation affect the protein composition of saliva?

The purpose of this study was to compare the relative amount of low molecular weight salivary proteins in patients with head and neck tumours treated with radiotherapy and healthy subjects. Reverse-phase high-pressure liquid chromatography was used for protein separation. Nine protein fractions (including acidic and basic proline-rich proteins (PRPs), cystatins, histatins and statherin) were identified in saliva from irradiated patients as well as healthy subjects. However, compared with non-irradiated healthy subjects, the fraction of acidic PRPs was significantly reduced in irradiated patients. These data indicate an alteration of the relative amount of low molecular weight salivary proteins in irradiated patients besides the reduction of salivary flow.

High LET-induced H2AX phosphorylation around the Bragg curve.

We investigated the spatial distribution of the induction of the phosphorylated form of the histone protein H2AX (gamma-H2AX), known to be activated by DSBs. Following irradiation of human fibroblast cells with 600 MeV/nucleon silicon and 600 MeV/nucleon iron ions we observed the formation of gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in an x/y plane. Polyethylene shielding was used to achieve a Bragg curve distribution with beam geometry parallel to the monolayer of cells. We present data that highlights the formation of immunofluorescent gamma-H2AX tracks showing the ion trajectories across the Bragg peak of irradiated human fibroblast cells. Qualitative analyses of these distributions indicated potentially increased clustering of DNA damage before the Bragg peak, enhanced gamma-H2AX distribution at the peak, and provided visual evidence of high-linear energy transfer particle traversal of cells beyond the Bragg peak in agreement with one-dimensional transport approximations. Spatial assessment of gamma-H2AX fluorescence may provide direct insights into DNA damage across the Bragg curve for high charge and energy ions including the biological consequences of shielding and possible contributors to bystander effects.

Ultraviolet radiation induces phosphorylation and ubiquitin-mediated degradation of DeltaNp63alpha.

DeltaNp63alpha, a homologue of the tumor suppressor p53, acts as a transcriptional repressor with dominant negative effects towards p53. Additionally, DeltaNp63alpha is overexpressed in a number of squamous cell carcinomas, suggesting a potential role in oncogenesis. However, the mechanisms regulating p63 have yet to be elucidated. The goal of the current study was to determine the effect of various genotoxic stresses on DeltaNp63alpha posttranslational modification and stability in normal and transformed squamous epithelial cells. We found that DeltaNp63alpha protein levels decreased after ultraviolet radiation and paclitaxel treatment of both normal and transformed cells. After UV and paclitaxel treatment, DeltaNp63alpha phosphorylation was significantly modulated. Additionally, DeltaNp63alpha protein levels were regulated in a proteasome-dependent manner in control and UV treated cells with increased DeltaNp63alpha ubiquitination after UV treatment or proteasome inhibition. Our studies provide insight to a mechanism for DeltaNp63alpha regulation during normal cell proliferation and, in particular, after stress. Further, the inverse regulation of p53 and DeltaNp63alpha protein levels after cell stress through opposing regulation of proteasome-mediated degradation may allow for rapid transcriptional changes of specific target genes that are consistent with the roles of these family members in tumor suppression and cell growth.

Focused microwave irradiation of the brain preserves in vivo protein phosphorylation: comparison with other methods of sacrifice and analysis of multiple phosphoproteins.

At any point in time, net protein phosphorylation represents the contribution of protein kinase and protein phosphatase activities affecting a specific site on a given substrate. Preservation of phosphorylated proteins in neural tissues has traditionally included flash-freezing or fresh tissue processing following tissue isolation. Rapid heat inactivation of protein kinases and phosphatases by focused microwave irradiation sacrifice represents another method to preserve, in vivo, brain protein phosphorylation state. In this study, we compared preservation of the phosphorylation state of a variety of phosphoproteins in the brain following sacrifice of mice by decapitation, decapitation into liquid nitrogen and focused microwave irradiation. We found that microwave irradiation generally provided the highest and most consistent levels of protein phosphorylation, regardless of the substrates examined in striatum and hippocampus. In general, flash-freezing resulted in the least preservation of phospho-state with ERK1/2 and CREB showing almost complete dephosphorylation. When regions of freshly decapitated brains were homogenized and incubated on ice for 30 min, ERK1/2 phosphorylation was completely lost, whereas it was well preserved in microwaved samples left at room temperature for 2 h. Loss of ERK1/2 phosphorylation in the fresh samples could not be attributed to substrate proteolysis. Our results indicate that focused microwave irradiation sacrifice may be required to achieve biologically relevant data for the in vivo protein phosphorylation state of many phosphoproteins.

Response regulator homologues have complementary, light-dependent functions in the Arabidopsis circadian clock.

TIMING OF CAB EXPRESSION 1 ( TOC1) functions with CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) in a transcriptional feedback loop that is important for the circadian clock in Arabidopsis thaliana (L.) Heynh. TOC1 and its four paralogues, the Arabidopsis PSEUDO-RESPONSE REGULATOR (PRR) genes, are expressed in an intriguing daily sequence. This was proposed to form a second feedback loop, similar to the interlocking clock gene circuits in other taxa. We show that prr9 and prr5 null mutants have reciprocal period defects for multiple circadian rhythms, consistent with subtly altered expression patterns of CCA1 and TOC1. The period defects are conditional on light quality and combine additively in double-mutant plants. Thus PRR9 and PRR5 modulate light input to the circadian clock but are neither uniquely required for rhythm generation nor form a linear series of mutual PRR gene regulation.