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We used one of the porphyrin compounds to investigate the selective sensitivity of the different neurones in network to free radical (FR) damage. In general, interneurones are much more resistant to FR damage as compared with other components of the network.
Some porphyrin compounds: P-NO2 and CVRIV were screened for cytotoxic activity against HT-29, LOVO, human tumor cell lines. The new radiometric assay was used for all cell lines. The soft agar cloning system was also utilized. The tested compounds decrease the growth index, measured in the radiometric assay, as 14CO2 production, and similarly depress the growth of tumor colonies on soft agar in the clonogenic assay. The cytotoxic effects of the compounds tested by these different methods were analysed statistically and resulted quantitatively similar. Based on these findings the radiometric assay represents a method, simple and rapid, which can be used as the clonogenic assay to screen new anticancer drugs.
Present-day developments in oncological applications of laser therapy are adaptive laser immunotherapy (ALIT) and photodynamic therapy (PDT). ALIT (helium-neon laser) was used in 35 ENT-cancer patients to irradiate immunocompetent leukocytes isolated from blood in an Amino cell separator. The use of ALIT in the combined treatment of our patients improved their health condition. Cytological, immunochemical and immunological examinations of blood revealed an increased count of activated lymphocytes, normalization of acute-phase proteins, stimulation of cell-mediated immunity and nonspecific resistance. The pharmacokinetics and photodynamic activity of porphyrin compounds in mice with inoculated tumors were investigated. Experimental observations demonstrated that the application of photodynamic therapy of hematoporphyrins in ENT-oncology seems very promising.
In this study, the light-emission properties of carious and sound root surfaces were investigated under a wide range of excitation wavelengths. Human molar teeth with exposed root surfaces containing light- and dark-discolored root caries (n = 3 of each) were selected. Emission spectra were recorded from carious and corresponding sound root surface areas from each tooth by using a fluorescence spectrophotometer at excitation wavelengths from 360 nm up to 580 nm, in steps of 20 nm. The spectra were corrected for fluctuations in detector sensitivity and excitation light intensity, and normalized to peak intensity. Excitation spectra were recorded for selected emission wavelengths that showed maximum intensity. Light- and dark-discolored root surface caries showed distinct fluorescence emission bands between 600 and 700 nm that were not present in sound root surface areas. These bands were strongest for excitation wavelengths between 390 and 420 nm. The excitation spectra of root caries revealed maximum excitation at around 405 nm, which is equivalent to the Soret band of porphyrin compounds. The emission spectra of both types of root caries lesions were shifted towards longer wavelengths (red shift at half maximum) when compared to the spectra of corresponding sound root surfaces. The red shift for dark-discolored root caries was higher than for light-discolored lesions at all excitation wavelengths.
This study was carried out to evaluate the clinical characteristics of autofluorescence in oral squamous cell carcinoma (SCC) and analyze the fluorescent substances using high-performance liquid chromatography (HPLC). Fifty of 55 oral SCCs (91%) emitted orange or red fluorescence, which was recorded by fluorescence photography. The intensity of the fluorescence significantly correlated with the T and N categories of the cancers, but did not show statistical difference for the types of clinical appearance and primary sites. Protoporphyrin and coproporphyrin were identified as the fluorescent substance in the SCC samples, and the elution patterns on HPLC revealed some porphyrin compounds as specific to oral cancer. These results suggest that the autofluorescence in oral SCC correlates with the progression of lesions, and that fluorescent substances such as protoporphyrin are produced in association with the cancerous tissue.
Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrP(CWD)) was used as an indicator of CWD infection. Although no PrP(CWD) was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrP(CWD)-positive clone out of 51. This clone, designated MDB(CWD), has maintained stable PrP(CWD) production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrP(CWD)-positive subclones out of 30, one of which was designated MDB(CWD2). The MDB(CWD2) cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrP(CWD) accumulation in MDB(CWD) cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrP(CWD) inhibitors and suggests that these compounds have potential to be active against CWD in vivo.
1-Acyldipyrromethanes are important precursors in rational syntheses of diverse porphyrinic compounds. 1-Acyldipyrromethanes are difficult to purify, typically streaking upon chromatography and giving amorphous powders upon attempted crystallization. A solution to this problem has been achieved by reacting the 1-acyldipyrromethane with a dialkylboron triflate (e.g., Bu2B-OTf or 9-BBN-OTf) to give the corresponding B,B-dialkyl-B-(1-acyldipyrromethane)boron(III) complex. The reaction is selective for a 1-acyldipyrromethane in the presence of a dipyrromethane. The 1-acyldipyrromethane-boron complexes are stable to routine handling, are soluble in common organic solvents, are hydrophobic, crystallize readily, and chromatograph without streaking. The 1-acyldipyrromethane can be liberated in high yield from the boron complex upon treatment with 1-pentanol. Alternatively, the 1-acyldipyrromethane-boron complex can be used in the formation of a trans-A2B2-porphyrin. In summary, the boron-complexation strategy has broad scope and greatly facilitates the isolation of 1-acyldipyrromethanes.
The range of compounds, both paramagnetic and ferromagnetic, with potential utility as tracers for enhancing contrast in magnetic resonance imaging is very large. Fortunately, the changes in 1/T1 and 1/T2 of water protons in solution--and ultimately in tissue--induced by these agents arise from variations of only a few physical parameters. In the present work, we first consider the properties of Gd-DTPA2- in solution in some detail, including its effects on both inner and outer sphere relaxation of solvent protons. Then, by varying one or another physical parameter, we derive behavior that characterizes several classes of contrast agents with markedly different properties. These include small macrocycles, such as Gd-DOTA-; larger complexes, including oligomers and conjugates of small paramagnetic complexes with protein; magnetized particles, such as magnetite, as a function of their size; and highly anisotropic porphyrin compounds. Predictions are compared with data from the literature.
A newly developed microscope using acousto-optic tunable filters (AOTFs) was used to generate in vivo hemoglobin saturation (SO2) and oxygen tension (PO2) maps in the cerebral cortex of mice. SO2 maps were generated from the spectral analysis of reflected absorbance images collected at different wavelengths, and PO2 maps were generated from the phosphorescence lifetimes of an injected palladium-porphyrin compound using a frequency-domain measurement. As the inspiratory O2 was stepped from hypoxia (10% O2), through normoxia (21% O2), to hyperoxia (60% O2), measured SO2 and PO2 levels rose accordingly and predictably throughout. A plot of SO2 versus PO2 in different arterial and venous regions of the pial vessels conformed to the sigmoidal shape of the oxygen-hemoglobin dissociation curve, providing further validation of the two mapping procedures. The study demonstrates the versatility of the AOTF microscope for in vivo physiologic investigation, allowing for the generation of nearly simultaneous SO2 and PO2 maps in the cerebral cortex, and the frequency-domain detection of phosphorescence lifetimes. This class of study opens up exciting new possibilities for investigating the dynamics of hemoglobin and O2 binding during functional activation of neuronal tissues.
A comparison has been made of the in vitro light-activated drug cytotoxicities of two different porphyrin compounds, Photofrin II and TPPS4. An early passage human colon adenocarcinoma cell line, WiDr, has been exposed to either drug for 24 h, the excess drug washed from the cells and the cells irradiated with light using quartz-tungsten-halogen lamps. Neither light nor drug alone under the experimental conditions employed was toxic to WiDr cells. Together, considerable cytotoxicity could be seen and the shapes of the cell survival curves following exposure to either drug then irradiation with light, were similar. For equal amounts of drug in the medium, Photofrin II was a more efficient photosensitizer of WiDr cells than TPPS4, and differences in cellular uptake could only partly explain this. When the experimental procedure was changed by reducing the temperature of irradiation, a reduction in photosensitizing efficiency could be demonstrated. This was more pronounced for Photofrin II, and was seen as a change in the slope of the final portion of the survival curve; and as a change in the shoulder for TPPS4. Two different batches of the two drugs were compared and shown to give slightly different results for Photofrin II (change in shoulder) but not to differ for TPPS4.
To determine whether the prosthetic group of cytochrome c is synthesized and linked to the apoprotein in the cytosol or in connexion with the endoplasmic reticulum, we have studied the incorporation in vitro of delta-amino[(14)C]laevulinate into porphyrin compounds and cytochrome c by the cell sap from rat liver. The radioactive precursor was incorporated into a trichloroacetic acid-precipitable form partially resistant to extractions by acid solvents, suggesting the existence of a fraction covalently linked to protein. The activity was proportional to the amount of protein incubated, did not increase substantially by supplementation with the microsomal fraction and an energy source, and was very low in the pH5 fraction. Addition of increasing amounts of haemin inhibited the incorporation, as with purified delta-aminolaevulinate dehydratase. [(14)C]Protoporphyrin IX was identified by paper chromatography, together with a shoulder running as protohaem IX. The cell sap in the absence of ribosomes was also able to incorporate radioactivity into purified cytochrome c, and the addition of ribosomes significantly enhanced the activity. The precursors of haem c were synthesized in the soluble system by the known haem-synthetic pathway, as shown by the kinetics of labelling of the coproporphyrin, protoporphyrin and haem fractions, and the activities were concentrated in the precipitate obtained between 40 and 60% saturation with (NH(4))(2)SO(4). The presence of ferrochelatase was indicated by the incorporation of (55)Fe into proto- and haemato-haem identified by paper chromatography. It is concluded that the cell sap from rat liver contains the complete set of enzymes for the synthesis from delta-aminolaevulinate of haem c and its linkage to a small pool of free apoprotein c present in soluble form. This suggests that an ancillary pathway of haem synthesis occurs in the cytosol for at least the formation of the prosthetic group, which is linked post-translationally to that pool of apoprotein c synthesized by free polyribosomes.
The response of the human gynecological carcinoma cell-lines HEC-1-A (endometrial carcinoma) and OvCar-3 (ovarian carcinoma) to photodynamic therapy in vitro was examined. The porphyrin compound Photosan III (Ph III) was used for photosensitization of the cells after incubation times of 24 h (HEC-1-A) and 48 h (HEC-1-A and OvCar-3). The Ph III doses varied from 0-10 micrograms/ml medium. Irradiation was performed with laser light at 630 nm. Irradiation doses up to 20 J/cm2 were applied at an irradiance of 40-100 mW/cm2. Cell vitality of the untreated control groups and of the therapy group was determined 48 h after irradiation, using the trypan blue exclusion test. The experimental results show that treatment of OvCar-3 cells with 10 J/cm2 resulted in a decrease in vitality dependent on photosensitizer dose (0-5 micrograms/ml, 48 h incubation time) but independent of the irradiance (40-100 mW/cm2). Complete cell death was observed after application of irradiation doses in the range of 5-20 J/cm2 combined with drug concentrations of 10-2.5 micrograms/ml, at a fixed incubation time of 48 h. HEC-1-A cells did not survive photodynamic therapy with 10 J/cm2 after incubation with 5 micrograms/ml for 48 h. After a shorter incubation time of 24 h, 10 micrograms/ml Ph III was necessary for the same effect. There was a maximum decrease in cell vitality when measured 48 h after irradiation. This was not improved at 72 h.
In this work, an electron paramagnetic resonance (EPR) strategy to study the heme-pocket structure of low-spin ferric heme proteins is optimized. Frozen solutions of ferric mouse neuroglobin (mNgb) are analyzed by means of electron spin echo envelope modulation and pulsed electron-nuclear double resonance techniques. The hyperfine and nuclear quadrupole couplings of the directly coordinating heme and histidine nitrogens are derived and are discussed in comparison with known data of other ferric porphyrin compounds. In combination with the hyperfine matrices of the imidazole protons, the 14N EPR parameters reveal structural information on the heme pocket of mNgb that is in agreement with previous X-ray diffraction data on neuroglobins.
The complete iron atom vibrational spectrum has been obtained by refinement of normal mode calculations to nuclear inelastic x-ray absorption data from (nitrosyl)iron(II)tetraphenylporphyrin, FeTPP(NO), a useful model for heme dynamics in myoglobin and other heme proteins. Nuclear resonance vibrational spectroscopy (NRVS) provides a direct measurement of the frequency and iron amplitude for all normal modes involving significant displacement of (57)Fe. The NRVS measurements on isotopically enriched single crystals permit determination of heme in-plane and out-of-plane modes. Excellent agreement between the calculated and experimental values of frequency and iron amplitude for each mode is achieved by a force-field refinement. Significantly, we find that the presence of the phenyl groups and the NO ligand leads to substantial mixing of the porphyrin core modes. This first picture of the entire iron vibrational density of states for a porphyrin compound provides an improved model for the role of iron atom dynamics in the biological functioning of heme proteins.
OBJECTIVES: Tiny papillary tumors and flat urothelial lesions such as dysplasia or carcinoma in situ can easily be missed during routine cystoscopy. Various methods for in vivo detection of fluorescing agents (preferentially localized in malignant tissue) have been developed. Most of them are based on systemically administered synthetic porphyrin compounds and require sensitive detection devices and image processing units for fluorescence visualization. The usefulness of intracellularly accumulated endogenous protoporphyrin IX (PPIX), induced by 5-aminolevulinic acid (ALA), for diagnosis of early bladder cancer and the correlation with cystoscopic, microscopic, and fluorescence findings was investigated. METHODS: ALA was instilled intravesically in 68 patients, followed by fluorescence cystoscopy with violet light from a krypton ion laser that produced fluorescence excitation. There were 299 biopsies obtained from fluorescing and nonfluorescing areas of the bladder. RESULTS: ALA-induced fluorescence could be easily observed with the naked eye during cystoscopy under violet light illumination. All tumor lesions were sharply marked with brightly shining red fluorescence. Correlation of fluorescence and microscopic findings gave a sensitivity of 100% and a specificity of 68.5%. There were 26 malignant or precancerous lesions that were missed during routine cystoscopy but were detected only by ALA-induced fluorescence. CONCLUSIONS: Labeling of urothelial lesions by PPIX fluorescence induced by intravesically instilled ALA seems to be a promising diagnostic procedure for malignant lesions that are difficult to visualize with standard cystoscopy.
G-quadruplexes are a family of secondary DNA structures formed in the presence of monovalent cations that consist of four-stranded structures in which Hoogsteen base-pairing stabilizes G-tetrad structures. These structures are proposed to exist in vivo, although direct confirmatory evidence is lacking. Guanine-rich regions of DNA capable of forming G-quadruplex structures are found in a variety of chromosomal regions, including telomeres and promoter regions of DNA. In this review, we describe the design of three separate groups of G-quadruplex-interactive compounds and their interaction with G-quadruplex DNA. Using the first group of compounds (anthraquinones), we describe experiments that provide the proof of concept that a G-quadruplex is required for inhibition of telomerase. Using the second group of compounds (perylenes), we describe the structure of a G-quadruplex-ligand complex and its effect on the dynamics of formation and enzymatic unwinding of the quadruplex. For the third group of compounds (porphyrins), we describe the experiments that relate the biological effects to their interactions with G-quadruplexes.
A new boronated porphyrin compound (STA-BX909) was developed as a possible agent for boron neutron capture therapy. The boron concentration was measured by an in vivo rat experimental brain tumor model and an in vitro cell culture study. This agent was compared to sodium borocaptate (BSH) which has been used in clinical trials of boron neutron capture therapy. In the 9L rat brain tumor model, STA-BX909 achieved a higher boron tumor/blood ratio 24 h after injection in comparison to BSH. A boron concentration study in cultured glioma cell lines (U-251, U-87, 9L) demonstrated an increased boron concentration as a function of exposure time to STA-BX909, while the boron concentration remained stable with increasing exposure time to BSH. Use of a colony forming assay with thermal neutron irradiation revealed more cytotoxicity with STA-BX909 than BSH when the same concentration of 10B was administered. We concluded that STA-BX909 may be an effective drug for use in boron neutron capture therapy and that it merits further investigation.
The substrate-bound form of the enzyme heme oxygenase (HO), which catalyzed the stereospecific alpha-meso bridge cleavage of hemin to yield biliverdin IX alpha, has been investigated by 1H NMR in both its primarily high-spin and its cyanide-inhibited low-spin forms. Both derivatives yield 1H NMR spectra indicative of extensive heterogeneity that is largely resolved when a 2-fold-symmetric hemin substrate is bound. The structural origin of the heterogeneity is shown to result from approximately 1:1 isomeric binding of the native hemin substrate in the binding pocket. The substrate orientational disorder is about the alpha,gamma-meso axis, as established on the basis of 2D NMR experiments that identify characteristic aromatic van der Waals contact in the substrate binding pocket. The isomeric substrate-HO complexes exhibit differential cyanide affinity, and the ratio of isomers is sensitive to the hemin 2,4-substituents. The assignment of hemin signals by isotopic labeling and 2D NMR methods reveals a contact shift pattern that reflects an unusual hemin electronic structure that is characterized by large differences in delocalized spin density for the two positions within a given pyrrole, rather than the more conventional large differences between adjacent pyrroles. This pattern of spin density delocalized primarily to the pyrrole positions adjacent to the alpha,gamma-meso axis can be rationalized by postulating a direct electronic perturbation of the hemin by the protein matrix in the form of an anionic side chain close to the alpha-meso carbon. Similar influences on hemin electronic structure, in the form of chemical substitution of the meso positions, have been observed in iron porphyrin compounds and successfully modeled by simple molecular orbital theory (Tan et al., 1994). This is interpreted as evidence for a direct electronic effect by HO to activate the alpha-meso position for electrophilic rather than nucleophilic attack. The unique contact shift pattern is present to different degrees for the two hemin orientations, is strongly pH dependent, and is largely abolished at acidic pH. Portions of several heme pocket residues are located and it is shown that the pattern of the dipolar shifts for these residues, which likely reflects the distal steric influence on the tilt of the coordinated cyanide, differs significantly for the two substrate orientations.(ABSTRACT TRUNCATED AT 400 WORDS)