Miracle-dz/UniFAR-FTUs huggingface dataset:
5‐Aminolevulinic Acid–based Photochemical Internalization of the Immunotoxin MOC31‐gelonin Generates Synergistic Cytotoxic Effects In Vitro ¶

Photochemical internalization (PCI) is a novel method for the endosomal or lysosomal release of membrane‐impermeable molecules into the cytosol of target cells. This novel technology is based on the photodynamically induced rupture of endocytic vesicles preloaded with molecules of therapeutic interest. PCI of the ribosome‐inactivating plant toxin gelonin and the immunotoxin monoclonal antibody 31 (MOC31)‐gelonin has been performed previously by the use of the endocytic vesicle–localizing photosensitizers TPPS2a and AlPcS2a and light, demonstrating synergistic toxicity against the more than 20 different cell lines tested, most of them of neoplastic origin. In this study we demonstrate that 5‐aminolevulinic acid (5‐ALA)–induced protoporphyrin IX (PpIX) is also capable of inducing PCI of MOC31‐gelonin in the human colon adenocarcinoma cell line WiDr. The cells were incubated with 1 mM 5‐ALA for up to 8 h in serum‐free medium and from 24 to 96 h in serum‐containing medium. Fluorescence microscopical studies indicate a partial plasma membrane localization of PpIX when 5‐ALA was applied under serum‐free conditions. This plasma membrane localization was not seen when 5‐ALA was given in the presence of serum. There was a granular component of the PpIX localization in addition to a diffuse cytoplasmic localization. The granular component resembled the localization of the fluorescent dye conjugate Alexa‐gelonin and the lysosomal localizing dye acridine orange. Our present results provide evidence for an endocytic vesicle–associated fraction of PpIX after 5‐ALA incubation of the WiDr cells. We demonstrate that PCI, by combining 5‐ALA, MOC31‐gelonin and light, induces a synergistic cytotoxic effect against the WiDr cells.
Heterospecific transcription as a criterion for the investigation of the molecular evolution in bacteria

In this thesis I present studies on the synthesis and function of E.coli RNA polymerase subunits in different gram-negative bacteria. The primary purpose of this study was to explore the extent to which RNA polymerase function has been conserved in the course of evolution. The plasmid pZDlOQ, constructed in vivo, provides a vehicle for rpoBC transfer from E.coli to many bacteria. This plasmid is the product of the integration of XdrpoBC18, which carries rpoBC genes, into the plasmid RP4 Xatt. The plasmids pZD44 and pZD23 are derived from pZDlOO and have deletion removing some of XdrpoBClB DNA. In addition, pZD44 retains and pZD23 lacks the determintcat ampicillin resistance. The plasmid pZD23 is a better tool for my purpose as it is not subjected to zygotic induction during transfer. In vivo as well as in vitro studies provided structural information about these plasmids and Rn. First, the orientation of transfer of RP4 is in an anticlockwise direction; in otherwords, rpoBC plasmids transfer at O_rpoBC_Apr_Tcr_Kmt. Second, the repressor gene(s) for the transfer of RP4 is located between the single EcoRI site and Ap gene. Third, the incompatibility, mc, gene(s) of RP4 is located between AR r and Tcr gene. The Enterobacteriaceae bacteria chosen for this study are: C.freundii, E.aerogenes, E.coli, H.alvei mØller, K.pneumoniae, P.morganii, P.vulgaris, S.marcescens and S.typhimurium. The non-Enterobacteriaceae bacteria are: A.vinelandii, P.aeruginosa and R.leguminosarulfl. Heterospecific gene expression can differentiate between the genetically related sies to E.coli and one could conclude that it is possible to use this criterion for the investigation of the genetic relatedness between bacterial species. In addition, promoters are highly conserved parts of the DNA and thus the C + C content of the DNA does not affect heterospecific gene expression.
Bleeding disorders in Noonan Syndrome

To the Editor: We appreciate the opportunity to respond to the issues raised by Dr. Sharathkumar. In regard to patients with Noonan Syndrome (NS), we recommended initial screening to include PTT/aPTT to accommodate facility preference, as either is adequate. The decision not to include PT in initial testing was made because NS patients have not been found to have disorders of the extrinsic pathway [1]. We agree that many institutions have replaced the bleeding time with the platelet function analyzer (PFA)-100 test, but felt the discussion to choose one over the other was beyond the scope of our paper. Additionally, controversy remains regarding the clinical usefulness of the PFA-100 [2,3]. Although our paper does not focus on the treatment of von Willibrand disease (VWD), we agree that new advances in the treatment of this disorder are being made and that, at present, intermediate purity viral inactivated FVIII concentrate with a high von Willebrand factor (VWF) ratio would be the most appropriately targeted treatment for VWD [4]. However, since 93.3% of patients with NS and a bleeding complication of an unknown etiology will be secondary to a factor deficiency, thrombocytopenia, or a platelet dysfunction [1], it seems reasonable and cost-effective first to treat with FFP and platelets reserving the use of cryoprecipitate if that treatment fails. It is not true as Dr. Sharathkumar states in his letter that cryoprecipitate does not contain VWF [5]. Cryoprecipitate has been recommended as a treatment option for patients with VWD when VWF-containing factor concentrates are not available [6]. Only after a failure to correct with the above agents would the use of the expensive and not as easily obtainable FVIII concentrate as described above be used. Finally, it was our recommendation, as stated under Screening Recommendations, that the investigation of bleeding disorders in NS patients be undertaken in consultation with a hematologist if any of the initial screening tests are abnormal or if clinical concern for a bleeding diathesis persists [1].
RESEARCH NOTE

Abstract— Fluorescence microscopy has been utilized to investigate the effects of UV irradiation on the organization of keratin intermediate filaments in normal human epidermal keratinocytes. Sun lamp irradiation induced the condensation of keratin intermediate filaments into the perinuclear region and inhibited the reorganization of keratin filaments normally induced by Ca2+. Exposure to UVC appeared to disrupt keratin filaments similarly, whereas UVA had no discernible effect.
Quantitative Model Calculation of the Time‐dependent Protoporphyrin IX Concentration in Normal Human Epidermis After Delivery of ALA by Passive Topical Application or Iontophoresis ¶

We present a mathematical layer model to quantitatively calculate the diffusion of 5‐aminolevulinic acid (ALA) in the skin in vivo, its uptake into the cells and its conversion to protoporphyrin IX (PpIX) and subsequently to heme. The model is a modification and extension of a recently presented three‐compartment model. The diffusion of ALA in the skin (epidermis, dermis) is described by the time‐dependent diffusion equation, and the sink in this equation accounts for ALA uptake in the cells. As boundary conditions, we use the ALA flux across the human stratum corneum (SC) in vitro during passive or iontophoretic ALA delivery as measured in vitro. Besides the diffusion equation, the model includes three additional equations, similar in form to those of the three‐compartment model but with a different interpretation. Our additional equations are supposed to describe, respectively, the conversion of ALA in the cytoplasm to some intermediate compound in the mitochondria and the conversion of the latter to PpIX and of PpIX to heme. The first conversion is a process of the Michaelis–Menten type, the other two are first‐order rate processes. When fitted to the published data of PpIX fluorescence from normal human skin following iontophoresis of ALA, the model yields the tissue concentration of PpIX as a function of time after ALA application. The computed concentrations are in good agreement with the published phototoxic concentrations of PpIX in the tissues obtained from extraction. The model parameters obtained from the fit are subsequently used to compute the PpIX concentration in normal human skin after 4 h topical application of 10, 20 and 40% ALA. This again yields the PpIX concentrations in tissue, in good agreement with the published values. The saturation of the PpIX concentration as a function of applied ALA concentration is calculated and agrees with clinical observations on the effectiveness of photodynamic therapy. Photobleaching is simulated, with subsequent resynthesis of PpIX in qualitative agreement with experiment. Finally, the model predicts that only 2.5–3.5% of the ALA entering the skin after passing the SC is converted to PpIX. The layered model is a considerable simplification of real skin, but its successful qualitative and quantitative reproduction of experimental data may encourage further studies to test and refine the model to improve our understanding of the kinetics of ALA and the synthesis of PpIX in the skin.
Effects of Fluence Rate on Cell Survival and Photobleaching in Meta‐Tetra‐(hydroxyphenyl)chlorin–photosensitized Colo 26 Multicell Tumor Spheroids ¶

We report the influence of fluence rate on the photobleaching and cell survival in Colo 26 multicell spheroids photosensitized by meta‐tetra‐(hydroxyphenyl)chlorin (mTHPC). Photosensitizer degradation and therapeutic efficacy increased dramatically and progressively when the fluence rate was reduced over the range from 90 to 5 mW cm−2. These experimental results were compared to a mathematical model of photobleaching based on self‐sensitized singlet oxygen reactions with the photosensitizer ground state. This model incorporates photophysical parameters obtained from microelectrode measurements of oxygen depletion at the surface of mTHPC‐sensitized spheroids and was refined by including the inhomogeneous distribution of mTHPC in spheroids and oxygen depletion in the bulk medium. Since the model is consistent with the experimental data we conclude that the fluence rate dependence of the cell survival and of mTHPC photobleaching is due to photochemical oxygen consumption and a predominantly singlet oxygen‐mediated mechanism of mTHPC photobleaching. The threshold dose of reacting singlet oxygen was calculated to be 7.9 ± 2.2 mM in this system.
FEDERAL UNIVERSITY OF JUIZ DE FORA GRADUATE PROGRAM OF CHEMISTRY MASTER IN CHEMISTRY

The Nuclear Magnetic Resonance is a widely used technique and plays a key role in many different areas, especially in elucidation of reaction mechanisms and designing of new drugs. In this context, the use of Quantum Chemistry methods is an important tool in NMR study. However, theoretical calculations of NMR parameters are, most of the times, expensive and imprecise. Therefore, one of the greatest challenges in Computational Chemistry is developing protocols with low computational cost that yield satisfactory results. In this work, the 1J(195Pt-15N) coupling constant was studied in Pt(II) complexes in order to develop a computational protocol for the calculation of the constant with affordable cost and satisfactory results using the all-electron relativistic basis set NMR-DKH, previously proposed for calculation of 195Pt chemical shift. Several topics were tested and analyzed in order to develop the protocol, such as DFT functionals and modifications to the basis set. The final computational protocol for predicting the 1J(195Pt-15N) coupling constant in Pt(II) complexes included a working set of 82 coupling constants for 57 Pt(II) complexes. Furthermore, it was applied for a testing set of 16 coupling constants in 14 Pt(II) complexes. The protocol was based on nonrelativistic calculations at PBEPBE/NMR-DKH level. The MAD was 36 Hz corresponding to the MRD of 10.4%, considering all 98 coupling constants for 71 Pt(II) complexes.
cis‐Urocanic Acid does not Induce the Expression of Immunosuppressive Cytokines in Murine Keratinocytes ¶ †

trans‐Urocanic acid (UCA) acts as a chromophore for UV radiation in the epidermis and isomerizes to cis‐UCA which then initiates some of the changes leading to UV‐induced immunosuppression. The mechanism of the immunomodulation by cis‐UCA is unknown at present, but one possibility is that the interaction between cis‐UCA and keratinocytes causes the release of immunosuppressive cytokines locally. To test this hypothesis, PAM‐212 cells, a murine keratinocyte cell line, were incubated with 0.10–100 μg/mL trans‐ and cis‐UCA for 6 or 24 h, respectively. The expression of interleukin (IL)‐10, transforming growth factor (TGF)‐β and tumor necrosis factor (TNF)‐α messenger RNA (mRNA) was then measured by reverse transcription‐polymerase chain reaction in comparison with the mRNA for the house‐keeping gene, β‐actin. No change or significant induction of any of the cytokine messages occurred. However, the expression of IL‐10 messenger RNA (mRNA) was induced 24 h after UVB irradiation (300 J/m2) and that of TNF‐α mRNA occurred 6 h after treatment with phorbol myristate acetate. The expression of IL‐10 protein was also examined by immunostaining in both PAM‐212 cells and B16‐F10 murine melanoma cells between 3 and 48 h after incubation with 10 and 100 μg/mL cis‐ and trans‐UCA. No alteration was seen with either isomer at either concentration. In contrast, UVB irradiation of both cell lines resulted in a marked increase in intracellular IL‐10 protein at 24 and 48 h. Therefore the upregulation of the immunosuppressive cytokines, IL‐10, TNF‐α and TGF‐β, in keratinocytes is unlikely to be the mechanism by which cis‐UCA induces immunosuppression in mice.
Protein Changes in HL60 Leukemia Cells Associated with 5‐Aminolevulinic Acid–based Photodynamic Therapy. Early Effects on Endoplasmic Reticulum Chaperones ¶

Using two‐dimensional electrophoresis we investigated the effect of 5‐aminolevulinic acid (ALA)–based photodynamic therapy (PDT; induction with 1 mM ALA for 4 h followed by blue light dose of 18 J/cm2) on the protein expression in HL60 leukemia cells. ALA‐PDT resulted in extensive qualitative and quantitative changes in the protein pattern of HL60 cell lysates. Of more than 1350 protein spots recognized on the protein maps of ALA‐induced cells, seven proteins were enhanced and 17 suppressed following irradiation. Three of these, calreticulin presursor, p58 microsomal protein (ERp57) and protein disulfide isomerase (p55) have been identified by matrix‐assisted laser desorption and ionization‐mass spectrometry and the pI/molecular weight parameters of the affected proteins were estimated by computer analysis. The findings suggest participation of endoplasmic reticulum Ca2+‐binding chaperones and/or Ca2+ signaling in ALA‐PDT mediated cytotoxicity.
Spectroscopic Properties of Fluoroquinolone Antibiotics and Nanosecond Solvation Dynamics in Aerosol‐OT Reverse Micelles

Among fluoroquinolone antibiotics, ofloxacin (OFL) and norfloxacin (NOR) have piperazinyl groups but flumequine (FLU) does not have this substitutent. The emission spectra of OFL and NOR are strong, broad structureless bands with large Stokes' shifts in water but the emission intensities are very weak in organic solvents. Thus we find that these compounds exist as different chemical species in various solvents. A continuous red shift in the emission bands for OFL and NOR is observed as the water concentration within the aerosol‐OT (AOT; sodium 1,4‐bis[2‐ethylhexyl]sulfosuccinate) micelle increases or temperature of this solution rises. From the fluorescence anisotropy measurements of OFL and NOR, we assume the intramolecular charge transfer after excitation from the nitrogen of the piperazinyl group to the keto oxygen. Theoretical calculations further support this observation. Multifrequency phase and modulation experiments and time‐resolved emission spectra clearly show the occurrence of intramolecular charge transfer and the subsequent nanosecond water reorganization around OFL or NOR in the AOT micelle. Upon increasing the water concentration within the AOT micelle, the relaxation rate increases because of the large amount of free water. The emission spectra of FLU do not exhibit any significant response to the physical properties of their environment.
Optical Properties and Nondestructive Estimation of Anthocyanin Content in Plant Leaves ¶

Absorption and reflectance spectra of maple (Acer platanoides), cotoneaster (Cotoneaster alaunica), dogwood (Cornus alba) and pelargonium (Pelargonium zonale) leaves with a wide range of pigment content and composition were studied in visible and near‐infrared spectra in order to reveal specific anthocyanin (Anth) spectral features in leaves. Comparing absorption spectra of Anth‐containing and Anth‐free leaves with the same chlorophyll (Chl) content, absorption spectra of Anth in leaves were derived. The main spectral feature of Anth absorption in vivo was a peak around 550 nm; the peak magnitude was closely related to Anth content. A quantitative nondestructive technique was developed to subtract Chl contribution to reflectance in this spectral region and retrieve Anth content from reflectance over a wide range of pigment content and composition. Anth reflectance index in the form ARI = (R550)−1− (R700)−1, where (R550)−1 and (R700)−1 are inverse reflectances at 550 and 700 nm, respectively, allowed an accurate estimation of Anth accumulation, even in minute amounts, in intact senescing and stressed leaves.
Assignment of the Lowest QY‐state and Spectral Dynamics of the CP29 Chlorophyll a/b Antenna Complex of Green Plants: A Hole‐burning Study ‡

Low‐temperature absorption, fluorescence and persistent nonphotochemical hole‐burned spectra are reported for the CP29 chlorophyll (Chl) a/b antenna complex of photosystem II of green plants. The absorption‐origin band of the lowest Qy‐state lies at 678.2 nm and carries a width of ∼130 cm−1 that is dominated by inhomogeneous broadening at low temperatures. Its absorption intensity is equivalent to that of one of the six Chl a molecules of CP29. The absence of a significant satellite hole structure produced by hole burning, within the absorption band of the lowest state, indicates that the associated Chl a molecule is weakly coupled to the other Chl and, therefore, that the lowest‐energy state is highly localized on a single Chl a molecule. The electron–phonon coupling of the 678.2 nm state is weak with a Huang–Rhys factor S of 0.5 and a peak phonon frequency (ωm) of ∼20 cm−1. These values give a Stokes shift (2Sωm) in good agreement with the measured positions of the absorption band at 678.2 nm and a fluorescence‐origin band at 679.1 nm. Zero‐phonon holes associated with the lowest state have a width of ∼0.05 cm−1 at 4.2 K, corresponding to a total effective dephasing time of ∼400 ps. The temperature dependence of the zero‐phonon holewidth indicates that this time constant is dominated at temperatures below 8 K by pure dephasing/spectral diffusion due to coupling of the optical transition to the glass‐like two‐level systems of the protein. Zero‐phonon holewidths obtained for the Chl b bands at 638.5 and 650.0 nm, at 4.2 K, lead to lower limits of 900 ± 150 fs and 4.2 ± 0.3 ps, respectively, for the Chl b→ Chl a energy‐transfer times. Downward energy transfer from the Chl a state(s) at 665.0 nm occurs in 5.3 ± 0.6 ps at 4.2 K.
Differential Activation of ERK 1/2 and JNK in Normal Human Fibroblast‐like Cells in Response to UVC Radiation Under Different Oxygen Tensions ¶

The mechanisms by which mitogen‐activated protein kinases (MAPK) respond to the input of UV‐induced signal transduction pathways and the resulting biological functions are not well understood. We investigated whether the level of oxygen tension of culture was responsible for the differential activation of MAPK and different cellular outcomes in UVC‐irradiated cells. The intracellular oxidative level of normal human fibroblast‐like cells in a normal atmosphere (normoxic, 20% O2) was increased within 30 min after UVC irradiation. When cells were cultured at lower oxygen tension in the presence of an antioxidant N‐acetyl‐l‐cysteine (NAC) or under physiologically hypoxic (5% O2) conditions, the elevation of the oxidative level by UV‐irradiation was significantly reduced. Among MAPK, extracellular‐signal related kinase (ERK) 1/2 was activated by UV regardless of the oxidative level, while c‐Jun N‐terminal kinase (JNK) activation was inhibited in NAC‐treated and in hypoxic cultures. In addition, in cultures at lower oxygen tension, there was less apoptosis and cell survival was enhanced. These results suggest that UV‐induced oxidative stress was responsible for intracellular signaling through the JNK pathway. Furthermore, the balance between ERK1/2 and JNK activities after UV irradiation under different oxygen tensions possibly modified cellular outcome in response to UV.
Monitoring cell cycle modulated gene expression in plants by means of a whole-genome-based approach

A cDNA-AFLP-based transcript profiling method has been optimized and applied in a genome-wide screen of cell cycle modulated genes in synchronized tobacco BY2 cells. In subsequent global and in-depth analyses, abundant as well as scarce messengers could be visualized. In addition to known genes, several transcript tags corresponding to putative novel sequences have been identified, demonstrating the sensitivity and the power of the technology for discovering new genes in organisms for which no sequence information is available. Furthermore, the cDNA-AFLP technique has been demonstrated to be highly specific and competent to discriminate between very homologous gene copies, what is an important prerequisite for detailed gene expression studies. Apart from being an efficient method for gene discovery, we have also verified that cDNA-AFLP is an appropriate tool for determining quantitative gene expression profiles. Based on the profiles, several discrete transcript patterns were recognized and subtle temporal differences in transcript accumulation during cell cycle progression could reliably be distinguished. Based on the obtained results, it can be concluded that cDNA-AFLP is a valid tool for genome-wide expression analysis at both the qualitative and quantitative level. The contents of this chapter are submitted to Nature Biotechnology by Breyne, Dreesen, Cannoot, Rombaut, Vandepoele, Rombauts, and Zabeau under the title “Cell cycle modulated gene expression in plants monitored by cDNA-AFLP-based transcript profiling” The data were also presented as a poster at the 6th meeting of the ISPMB in Quebec (Canada), June 2000