SPECTROSCOPIC ANALYSIS OF BACTERIOCHLOROPHYLLS IN VITRO AND IN VIVO*

Abstract— Chromatophores from Rhodopseudonionas spheroides were treated with potassium iridic chloride so as to destroy the major complement of bacteriochlorophyll (BChl) without harming the photochemically active P870. A band at 802 mμ, attributed to a pigment P800, survived this treatment along with P870. Extraction of such chromatophores with a mixture of acetone and methanol removed the absorption bands of P800 and P870; a corresponding amount of BChl was found in the extract. The yield of BChl was too great to have been derived from either P800 or P870 alone; analysis of extinction cofficients and band areas of these pigments indicates that they are both specialized fornis of BChl, in a molecular ratio of 2P800:1P870. Bleaching of P870, without attenuation of the absorption band of P800, could be effected by adding potassium ferricyanide to the iridic chloride-treated chromatophores. Extraction of chromatophores in this condition gave a reduced yield of BChl, consistent with a 2:1 ratio of P800 to P870 under the assumption that the BChl in the extract was derived in this case from P800 alone. An absorption band at 600 mμ in iridic chloride-treated chromatophores, characteristic of BChl and ascribed to P800 and P870, is partly bleached and shifted to shoiter wavelengths upon illumination. This reversible effect, and a similar one near 375 mμ (corresponding to the Soret band maximum of BChl), has the combined attributes of the blue-shift of P800 and the bleaching of P870 seen in a spectrally resolved form near 800 and 865 mμ respectively. The 600 mμ band is bleached by about 30 per cent, again consistent with a ratio of 2P800:1P870. These data, in conjunction with information published elsewhere, support the view that two molecules of P800 and one of P870 are associated jointly with a photosynthetic reaction center. It was observed that the long wave absorption bands of BChl in vivo are sometimes narrower than the narrowest bands that have been observed for BChl in dilute organic solutions. Sharpness of these bands is most conspicuous in some forms absorbing near 800 mμ.     

A COMPARISON OF IN VIVO AND IN VITRO TESTING OF SUNSCREENING FORMULAS

Abstract— Seven commercially available sunscreens were compared by three different methods. Absorbance spectra were measured for each product in isopropanol solution and also on hairless mouse epidermis. In vivo tests were performed on human volunteers using a Xe arc solar simulator. Sun Protection Factors (SPF) were calculated by each method for each product tested and the results compared. By all methods used, the combination of 7% octyl dimethyl para-aminobenzoic acid and 3% oxybenzone provided the most protection from U.V. light. While estimates of the effectiveness of all products were much too high when calculated by the isopropanol solution method, the hairless mouse epidermis technique seems to be an accurate tool for predicting product efficacy in vivo .     

FLUENCE-RATE DEPENDENCE OF MONOPHOTONIC REACTIONS OF NUCLEIC ACIDS IN VITRO AND IN VIVO

The Bunsen-Roscoe law, also known as the reciprocity law ( E = f(F) with F = I t ) has only limited validity for monophotonic reactions of nucleic acids. Especially at low fluence rates, the extent of in vitro and in vivo photoreactions of nucleic acids in the far-UV and near-UV range is a function of the fluence and of the fluence rate ( E = f (F;I)). In vitro experiments with poly(dA)poly(dT) clearly show that the far-UV (254 nm) response, indicated by the changes of the ellipticity at 315 nm, does not obey the Bunsen-Roscoe law at low fluence rates in the range between 1 W m^-2 and 20 W m^-2. In vivo experiments with Escherichia coli revealed very similar anomalies. Studying the growth delay after irradiation with far-UV light at 280 nm or near-UV light at 334 nm, we have confirmed the lack of reciprocity in both spectral ranges. The failure of the Bunsen-Roscoe law for the 280 nm and 334 nm UV irradiation effect at low fluence rates was in the range O < I < 40 W m^-2. In both cases reciprocity occurred at higher fluence rates (40 < I < 100 W m^-2).     

PHOTOTOXICITY FROM BENOXAPROFEN: IN VIVO AND IN VITRO STUDIES

Abstract Benoxaprofen (2-[4-chlorophenyl]-α-methyl-5-benzoxazole acetic acid), a non-steroidal antiinflammatory drug, was found to provoke phototoxicity reactions in humans. Exposure of the skin of benoxaprofen-treated subjects to either solar simulating radiation or a broad UV-A wavelength band produced intense itching and burning sensations, followed by the development of a classical wheal and flare response within 2-4 min. Phototoxicity was related to both the UV radiation fluence and the dose of orally administered benoxaprofen. Wavelengths between 320 and 340nm were active in provoking urticaria. In vitro studies demonstrated that benoxaprofen and UV irradiation produced a dose-dependent lysis of sheep erythrocytes which did not appear to be dependent on the presence of oxygen. Red cells were not lysed by pre-irradiated benoxaprofen arguing against the production of stable lytic photoproducts. Human neutrophils were lysed by benoxaprofen and UV light which resulted in the liberation of both cytoplasmic and lysosomal enzymes. Benoxaprofen failed to induce photolysis of human platelets and photoactivation of complement in human serum. It is suggested that phototoxic urticaria provoked by benoxaprofen may be due to a direct photolytic effect on human dermal mast cells.     

IRREVERSIBLE PHOTOBINDING TO SKIN CONSTITUENTS AFTER SYSTEMIC ADMINISTRATION OF CHLORPROMAZINE TO WISTAR RATS

From in vitro experiments it is known that chlorpromazine binds to protein and DNA/ RNA upon UV-irradiation. In the present study the possible photobinding of chlorpromazine (or its metabolites) in vivo was examined. Tritium labeled drug was administered intraperitoneally to female albino Wistar rats after which they were irradiated with light with maximum intensity at 310, 370 or 420 nm. After homogenization, unbound radioactivity in tissue of several organs was removed by dialysis. In the ears, eyes and skin of the back irreversibly bound radioactivity could be detected after irradiation with 310- and 370- but not with 420 nm light. Binding in the skin of the back after UVA irradiation was examined in more detail by separating epidermal lipids, DNA/RNA and proteins by a selective extraction/precipitation method. Radioactivity appeared to be bound to lipids and proteins but not to DNA/RNA.     

KINETICS OF DNA-INDUCED BREAKS BY REDUCTONE TREATMENT: IN VITRO AND IN VIVO STUDIES

Abstract— Reductone, a keto-aldehyde that blocks repair of UV-induced DNA damage also produces DNA breaks. These breaks are observed either in irradiated or unirradiated wild type and uvrA strains of Escherichia coli. DNA breakage has also been observed after in vitro treatment of T4 phage DNA. The results suggest that the breaking ability of reductone is a result of a direct attack on DNA.     

THE EFFECT OF APPLIED THICKNESS ON SUNSCREEN PROTECTION: IN VIVO AND IN VITRO STUDIES

The effect of applied thickness of sunscreen on the degree of protection against ultraviolet exposure has been investigated using an in vitro model (excised human epidermis) and phototesting in human subjects. The agreement between the protection factors obtained by each technique was excellent. It is shown that the influence of applied thickness on sunscreen protection factors is much less than would be predicted by Beer's law.     

OPTICAL PROPERTIES OF PHYTOCHROME IN THE BLUE- SPECTRAL REGION AS MEASURED IN VIVO AND IN VITRO

In the blue spectral region, the phototransformation difference spectrum of oat phytochrome extracted as P_fr differs from that of phytochrome extracted as P_r. The difference absorbance maximum for phytochrome extracted as P_fr is at 420 nm, while that extracted as P_r is at 412 nm. The phototransformation difference spectrum measured in the blue in oat coleoptile tips without inner leaves, corresponds very well with that of phytochrome as extracted in its P_fr form. There is, however, a slight apparent attenuation of the blue difference band relative to those in the red-far-red. In coleoptile tissue containing inner leaves, the blue difference band is relatively even more highly attenuated. A similar attenuation is observed in the blue, in the protochlorophyllide to chlorophyllide phototransformation difference spectrum. In the spectrum measured with excised coleoptile without inner leaves, there is a small attenuation, while in coleptile tissue with inner leaves the attentuation is nearly 9-fold. These data suggest that the observed attenuation is probably artifactual. Neither instrumental non-linearity nor fluorescence induced by the measuring beam could explain the observed attenuation. It is suggested that the observed attenuation is probably mainly the result of wavelength dependent scatter amplification, the amplification in the blue being attenuated by the high background absorption of other pigments in this region.     

ANALYTICAL MODELING FOR THE OPTICAL PROPERTIES OF THE SKIN WITH IN VITRO AND IN VIVO APPLICATIONS

Abstract— Analytical modeling that interrelates the optical properties of multilayered structures is applied to the skin. The mathematical approach is based on relations of diffuse reflectance and transmittance of a multilayered system and the diffuse reflectance and transmittance of each component layer. The formula can also be derived from the Kubelka–Munk theory of radiation transfer. Using both collimated and diffuse incident irradiance, the applicability of the model to human epidermis over the UV and visible region has been verified. The model has been applied to calculate the absorption and scattering coefficients of human epidermis in vitro , and to estimate the epidermal transmittance under simulated in vivo condition.     

MONOFUNCTIONAL COVALENT PHOTOBINDING OF DICTAMNINE, A FUROQUINOLINE ALKALOID, TO DNA AS TARGET IN VITRO

Abstract— The naturally occurring furoquinoline alkaloid, dictamnine, is phototoxic to Gram-positive bacteria, yeasts and filamentous fungi in the presence of long-wave UV light. Prior to photobinding to DNA dictamnine forms a complex with the macromolecule in the dark. Photobinding to DNA is shown with [³H]-dictamnine. In contradiction to an earlier finding, dictamnine proved to be incapable of forming interstrand crossiinkages with native DNA. This fact was ascertained by the use of methods additional to hydroxyapatite column chromatography, e.g. alkaline sucrose gradient centrifugation and hyperchromicity tests. The alkaloid therefore represents a new monofunctional photoreagent towards DNA in vitro. It is assumed that it binds through its furyl-C=C to DNA. The inability to form crosslinks may be attributed to the stability of the lateral aromatic nucleus. As no photoreactivity against human erythrocytes could be observed, it is possible that nucleic acids represent the major target of damage in vivo.     

FREE RADICAL PRODUCTION BY CHLORPROMAZINE SULFOXIDE, AN ESR SPIN-TRAPPING AND FLASH PHOTOLYSIS STUDY

Abstract— Using the spin-trapping technique we have investigated the photolysis of chlorpromazine sulfoxide and promazine sulfoxide. Photolysis of these sulfoxides in aqueous solution resulted in a species which is capable of oxidizing ascorbate, cysteine, glutathione, NADH, and azide by one electron, in addition to extracting hydrogen atoms from ethyl alcohol and dimethyl sulfoxide. These oxidations were not dependent on the presence of dissolved oxygen. The oxidizing species is proposed to be the hydroxyl free radical arising from the homolytic cleavage of the S-O bond of the sulfoxide. Flash photolysis of the chlorpromazine and promazine sulfoxides demonstrated the formation of cation radicals consistent with the loss of the hydroxyl radical from the sulfoxides. In addition we present a simple direct method for the quantitative synthesis of promazine and chlorpromazine sulfoxides from the parent promazine derivatives.     

IRRADIATION-ENHANCED PHYTOCHROME PELLETABILITY IN AVENA: IN VIVO DEVELOPMENT OF A POTENTIAL TO PELLET AND THE ROLE OF Mg2+ IN ITS EXPRESSION IN VITRO

The enhanced phytochrome pelletability that results from in vivo irradiation of Avena shoots may be divided into two operationally defined sequential stages: the in vivo development of a “potential to pellet” and the “expression” of this potential in vitro. Kinetic studies confirm previous findings that the generation of this “potential to pellet” is a very rapid (complete in < 10 s, 25°C), genuinely intracellular process, itself photoreversibly induced by P_fr. In addition, it is shown that the sustained development of the “potential to pellet”, that proceeds in the dark at 0°C following a red pulse, requires P_fr continually in the cell over the entire development period. Far red light immediately terminates further development of the red-induced “potential” at any point during the development phase. No immediate reduction is observed, however, in that level of “potential pelletability” already attained at the time of the far red pulse. This indicates that the level of “potential pelletability” established in vivo is insensitive to the form of the pigment at extraction regardless of the level reached. “Expression” of the “potential to pellet” refers to the actual detection in homogenates of an enhanced physical association of phytochrome with pelletable material. Maximum “expression” requires the presence of a divalent cation in the medium during homogenization. Rapid posthomogenization addition of Mg²⁺ to Mg²⁺-free extracts sustains enhanced pelletability but with rapidly declining effectiveness over the fmt 1–2 min after extraction. The rate of decline is faster if the phytochrome is present as P_fr than as P_r in the homogenate. Neither these nor previous data permit a distinction to be made between (a) preservation by the cation of a pre-existing intracellular interaction, and (b) a Mg²⁺-mediated induction of an artifactual, in vitro association predetermined in the cell by a genuine phyto-chrome-controlled process. Various formalistic models are discussed in the context of these and other data.     

THE PHOTOCHEMICAL INTERACTION OF DEOXYRIBONUCLEIC ACID AND PROTEIN IN VIVO AND ITS BIOLOGICAL IMPORTANCE*,†

Summary When bacteria are irradiated with u.v. light there is a dose dependent decrease in the amount of DNA that can subsequently be extracted free of protein with detergent. This appears to be due to the crosslinking of the DNA with protein and the precipitation of the linked DNA when the denatured proteins are precipitated in the procedure used for the isolation of the DNA. The type of linkage between the DNA and the protein is unknown except that it resists the sequential attack of 2% sodium lauryl sulfate and 0.5 M KCI or 55% CsCI. The main evidence that the loss of DNA in vivo is due to the crosslinking of DNA and protein is that the crosslinking of DNA and protein can be demonstrated in vitro . X-rays do not crosslink DNA and protein in vivo , but acridine orange and visible light cause the crosslinking of DNA and protein both in vivo and in vitro .
By pulse labeling the DNA of bacteria with tritated thymine it can be shown that newly synthesized DNA is most sensitive to crosslinking and that this sensitivity shows a cyclic response keyed to the generation time of the bacteria. Under conditions of thymine starvation where the intrinsic sensitivity of the cells to killing by u.v. is markedly increased, there is a parallel increase in the sensitivity of the DNA of these cells to be crosslinked to protein. The similarity in the time sequence of these two events strongly suggests that the crosslinking may play an important role in the loss of viability following u.v. irradiation.     

THE CONTRIBUTION OF RHODOSPZRZLLUM RUBRUM FERREDOXIN II TO IN VIVO EPR SIGNALS AND ITS ROLE IN ELECTRON TRANSPORT

Evidence is presented that Fd II, an iron-sulfur protein containing 4 irons and 4 acid-labile sulfurs, is responsible for a number of signals previously reported detectable in cells of R. rubrum. When oxidized, Fd II exhibits a g = 2.012 EPR signal which is readily detected in R. rubrum cells. In our hands, Fd II is photochemically reduced to an EPR-silent product contrary to the results of other investigators. However. in the presence of reducing agents. the reduced form is apparently denatured upon freezing. The denatured form exhibits EPR signals similar to some also previously observed in whole cells. Fd II catalyzes the ascorbate-DCPIP linked photoreduction of NAD with R. rubrum chromatophores even in the presence of an inhibitor which suppresses the formation of pH and emf gradients. This result raises the possibility of a role for Fd II in non-cyclic electron transport in R. rubrum.     

IN VITRO AND IN VIVO PROTECTION AGAINST PHOTOTOXIC SIDE EFFECTS OF PHOTODYNAMIC THERAPY BY RADIOPROTECTIVE AGENTSWR–2721 ANDWR–77913

Abstract— The experimental radioprotective agentsS–2-(3-aminopropylamino) ethyl phosphorothioate(WR–2721) and 3-amino-2-hydroxypropyl phosphorothioate(WR–77913) are also protective against photosensitized oxidation. They reduce porphyrin-induced photopolymerization of lens cytosol proteins in vitro, and phototoxic damage to mouse skin in vivo. The phototoxic dose modification factor (DMF) was 1.5 which is similar to that found in ionizing radiation. Part of the mechanism by which sulfhydryls afford this protection is by accelerating photobleaching of the porphyrins.     

PHOTOCHEMICAL INTERACTIONS OF CHLORPROMAZINE WITH PHOSPHOLIPIDS AND CHOLESTEROL IN ARTIFICIAL AND NATURAL MEMBRANES

Abstract— Dipalmitoylphosphatidyl-choline (DPPC) liposomes do not modify the nature of the photoproducts formed by UV irradiation of chlorpromazine. However, when cholesterol is added to DPPC liposomes, new photoproducts are formed which demonstrate the photoaddition of chlorpromazine to cholesterol. Similar results are obtained with phosphatidyl-serine and phosphatidyl-inositol. The reactivity of irradiated chlorpromazine towards lipidic components of myelin, taken as an example of biological membrane, is also demonstrated.     

CAROTENOID PIGMENTS AND THE IN VIVO SPECTRUM OF BACTERIOCHLOROPHYLL

Abstract— The isolation of a mutant, strain PM-9, of Rhodopseudonionus spheroides with an abnormal complement of carotenoid pigments is described.
PM-9 accumulates phytoene, phytofluene, ζ-carotene and neurosporene. Semi-aerobic cultures form more ζ-carotene and neurosporene relative to total carotenoids than do photo-synthetic cultures.
PM-9 is killed on exposure to light and oxygen.
By making use of the effect of oxygen on the nature of the carotenoids in PM-9, we have shown that these pigments do not directly influence the in vivo spectrum of bacteriochlorophyll.
Diphenylamine inhibits the synthesis of coloured carotenoids in Rhodopseudonionos gelatinosa but does not change the bacteriochlorophyll spectrum.     

聚芳醚亚砜的合成与表征

以二苯醚和二氯亚砜为主要单体 ,在无水AlCl3/CH2 ClCH2 Cl/NMP催化剂溶剂体系中进行低温溶液共聚 ,合成聚芳醚亚砜 (PPOS) ,研究了AlCl3/NMP用量及单体摩尔比对聚合反应的影响 .用IR、DSC、TG和X ray等对PPOS进行结构和性能的分析与表征 ,研究结果表明 :PPOS属无定形聚合物 ,具有较高的玻璃化转变温度 (Tg) ,软化温度在 2 6 0℃左右 ,在加热过程中出现二个热分解温度