HPLC ANALYSIS OF 4'',5''-MONOADDUCT FORMATION IN CALF THYMUS DNA and SYNTHETIC POLYNUCLEOTIDES TREATED WITH UVA and 8-METHOXYPSORALEN

Abstract— 8-methoxypsoralen monoadduct formation in calf thymus DNA irradiated with subbands of ultraviolet A light has been quantitated by HPLC analysis of the enzymatic hydrolysates of the DNA. Normalization of the yield of monoadducts for the variation in source output and the absorptivity of 8-MOP at each of the irradiating wavelengths showed that the 4',5'-furan monoadduct was the principal photoproduct and the efficiency of its formation was independent of irradiating wavelength. Synthetic polynucleotides irradiated with ultraviolet A light demonstrated a base composition and sequence dependence for 8-MOP photoreactivity: (poly(dAdT.dAdT) > poly(dA.dT) > poly(dGdC.dGdC) in both the B and Z forms > pofy(dT).     

THE PHOTOREACTIVITY OF PYRIMIDINE-PURINE SEQUENCES IN SOME DEOXYDINUCLEOSIDE MONOPHOSPHATES AND ALTERNATING DNA COPOLYMERS

Abstract— A reversed-phase HPLC system has been developed which separates the common nucleo-bases from the 6-methylimidazo[4,5- b ]pyridin-5-one (6-MIP) produced on acid hydrolysis of a thymine-adenine photoadduct (TA*) that is formed between adjacent thymine and adenine bases in UV-irradiated polydeoxyribonucleotides. By measuring the relative amounts of adenine and 6-MIP in acid hydrolysates, this system has been used to investigate how polynucleotide conformation affects the yield of TA* in poly(dA-dT) irradiated at 254 nm. The photoreactivity of other pyrimidine-purine sequences has been examined with the deoxydinucleoside monophosphates d(TpI) and d(m⁵CpA) and with the alternating DNA copolymers poly(dA-dU), poly(dI-dC), poly(dG-dC) and poly(dA-dC).poly(dG-dT). Samples were irradiated at 254 nm in aqueous solution and in ice, and at wavelengths >290 nm with acetone as photosensitizer. A photoproduct resembling TA*, and giving 6-MIP on acid hydrolysis, was isolated from d(TpI) irradiated at 254 nm in solution or in ice; d(m⁵CpA) was comparatively unreactive. Acid hydrolysates of the irradiated DNA copolymers were screened by HPLC and by TLC and paper electrophoresis, for the presence of imidazo[4,5- b ]pyridin-5-one, 6-MIP, or other species attributable to specific photoproduct formation. By this criterion, however, none of the copolymers showed evidence of significant photoreactivity in either their single- or double-stranded conformational states. The formation of mixed pyrimidine-purine photoadducts in DNA is therefore probably restricted to T-A doublets.     

ULTRAVIOLET LIGHT IRRADIATION OF DEFINED-SEQUENCE DNA UNDER CONDITIONS OF CHEMICAL PHOTOSENSITIZATION

Abstract— The formation of cyclobutane pyrimidine dimers and UV light-induced (6-4) products was examined under conditions of triplet state photosensitization. DNA fragments of defined sequence were irradiated with 313 nm light in the presence of either acetone qr silver ion. UV irradiation in the presence of both silver ion and acetone enhanced the formation of TT cyclobutane dimers, yet no (6-4) photoproducts were formed at appreciable levels. When photoproduct formation was also measured in pyrimidine dinucleotides, only cyclobutane dimers were formed when the dinucleotides were exposed to 313 nm light in the presence of photosensitizer. The relative distribution of each type of cyclobutane dimer formed was compared for DNA fragments that were irradiated with 254, 313, or 313 nm UV light in the presence of acetone. The dimer distribution for DNA irradiated with 254 and 313 nm UV light were very similar, whereas the distribution for DNA irradiated with 313 nm light in the presence of acetone favored TT dimers. Alkaline labile lesions at guanine sites were also seen when DNA was irradiated with 313 nm light in the presence of acetone.     

Phototoxicity testing by online irradiation and HPLC

A high-performance liquid chromatography (HPLC) system was developed for the determination of drug photostability and phototoxicity based on an automated column-switching system with aqueous online UV-A irradiation and hyphenated organic separation of the drug and its photoproducts. The photoreactor is built with an poly(ethylene-co-tetrafluoroethylene) (ETFE) reaction coil knitted around a UV-A light source. The chromatographic separation was performed with two special C₁₈ columns, which are also suitable for using with pure water as eluent. Degradation of chlorpromazine (CPZ) by ultraviolet light was investigated at pH 7 and pH 3. Furthermore chlorpromazine was irradiated in the presence of guanosine-5-monophosphate (GMP) in pH 7 buffered solution, leading to a new photoproduct. In the pH 3 irradiation studies of CPZ and GMP, no reaction was detected between the molecules.     

AN EXPERIMENTAL STUDY OF THE CHANGES IN PIGMENTATION IN HUMAN SKIN IN VIVO WITH VISIBLE AND NEAR INFRARED LIGHT

Abstract— The skin of the lower inner arm of volunteers was irradiated, with a 390–1700 nm light source, through a fiber optic bundle for times of up to 1.2 × 10⁴ s and with powers of up to 0.35 W/cm². Simultaneously with the irradiation, spectra (390–720 nm) of the remitted intensity were measured, while a 5.0 cm in diameter area of the skin around the fiber bundle was maintained at constant temperature, within 0.2°C. The generation of a photoproduct was observed and measured as changes in the remitted intensity within 600 s (10 min) of the start of irradiation.
The photoproduct formed was characterized by a weak absorption in the blue part of the spectrum (400–450 nm), leading to a bluish appearance in the irradiated area only. The color change appears as a two step process. It starts with a "soluble" photoproduct, which disappears, within 24 h after irradiation, and an "insoluble" photoproduct which appears with irradiation greater than 3 ×10³ s (50 min). No spectral differences were detected between the two photoproducts. The "insoluble" photoproduct persists for periods of up to 8 weeks. The color change in the skin is immediate and there is no erythema associated with this color change.     

OLIGONUCLEOTIDE PHOTOPRODUCTS FORMED BY PHOTOLYSIS OF POLYRIBOBROMOURIDYLIC ACID

Abstract— Polyribobromouridylic acid was irradiated with 313 nm light at an exposure of ˜ 190 pE/cm*. Oligonucleotides found after RNase hydrolysis of the photolysed poly-rBrU were isolated by DEAE-cellulose chromatography and partially characterized. The dmucleo-tide fraction, found in highest amounts, was not susceptible to hydrolysis by KOH or snake venom phosphodiesterase and may contain a coupled photoproduct. The properties of the dinucleotide were not those of a molecule containing a cyclobutane-type dimer, but were compatible with the properties of a coupled product similar to 5–5'-diuracil. The trinucleotide fraction probably consisted of more than one component. One component may contain a dimeric photoproduct. The tetranucleotide material was sensitive to cleavage into fragments by KOH, and could consist of adjacent photoproducts of the types found in the di- and trinucleotide fractions. The photoproducts formed over a range of lower doses of light were found to have properties similar to those found at high doses.     

PHOTOPRODUCTS OF BROMOURACIL-LABELED DNA AND THE STRUCTURE OF 5-BROMODEOXYURIDYLYL-(3' → 5')-THYMIDINE PHOTOPRODUCT

Abstract— –An attempt was made to identify some of the ultraviolet (u.v.) photoproducts of 5-bromouracil-labeled DNA (BrU-DNA). Two synthetic dinucleotides, 5-bromodeoxyuridylyl-(3' →5 ')-thymidine (BrdUpT) and 5-bromodeoxyuridylyl-(3' → 5')-deoxycytidine (BrdUpdC), were prepared. Each gave a single u.v. photoproduct which in turn gave a single acid hydrolysis product. 2-¹⁴C-BrU-DNA. prepared from E. coli B3, was irradiated (275–280 nm), hydrolyzed, and paper chromatographed in four systems. Comparison with the two synthetic photoproducts showed that if present at all, BrdUpT and BrdUpdC photoproducts could account for no more than 10 and 3.5 per cent respectively of the total photoproducts. At 55 per cent conversion of BrU into photoproducts, the major ¹⁴C-photoproduct was uracil (78 per cent); the remaining 22 per cent was made up of at least six products, three of which were reversed by 232 nm irradiation.
The debrominated cyclobutane structure proposed by Haug for BrdUpT photoproduct has been shown to be incorrect. It was found to contain one atom of bromine per molecule. On the basis of nuclear magnetic resonance and u.v. spectra, two possible structures are proposed for the photoproduct, each containing an eight-membered ring.     

The Effect of Chain Architecture on the Dynamics of Copolymers in a Homopolymer Matrix: Lattice Monte Carlo Simulations using the Bond‐Fluctuation Model

Summary: The effects of copolymer sequence distribution on the dynamics of a copolymer in a homopolymer matrix are studied using computer simulations within the framework of the bond‐fluctuation model on blends containing low concentrations (10%) of copolymers dispersed in a homopolymer matrix. The sequence distribution of the two copolymer components was changed while maintaining the overall copolymer composition at 50/50. Our results indicate that copolymers with disordered sequence distributions exhibit dynamics that are faster than that of a homopolymer melt, while those with ordered sequence distributions exhibit a tendency to form aggregates that lead to slower dynamics as well as phase separation. Analysis of the structure suggests that copolymers with an alternating sequence distribution form large aggregates that are short‐lived, while diblocks form permanent micelle‐like structures. Analysis of the local composition around a copolymer molecule indicates that aggregation between copolymer chains has a direct impact on the local composition. This in turn has a significant impact on system dynamics. Our results indicate that the dynamics of random, random‐blocky, and alternating copolymers are nearly identical and are faster than that of a homopolymer melt. However, alternating copolymers form aggregates and hence are not uniformly distributed throughout the matrix phase. Thus, alternating copolymers are at a disadvantage in their ability to be effective compatibilizers. From a dynamic perspective, copolymers with random and random‐blocky copolymers seem to be ideal compatibilizers since they are distributed uniformly throughout the system and move rapidly through the matrix phase.

Snapshots of aggregates of alternating copolymer chains. Dark and bright spheres represent A and B monomers, respectively.     

THE FATE OF PYRIMIDINE DIMERS IN THE DNA OF ULTRAVIOLET-IRRADIATED CHINESE HAMSTER CELLS

Abstract —As an aid to understanding the relationship between dimer repair and cellular recovery, we have studied dimer removal and replication of dimer-containing DNA in Chinese hamster ovary (CHO) cells irradiated with ultraviolet light (254 nm). These investigations demonstrated that (1) dimers are not excised as polynucleotides of less than 500,000 mol. wt, (2) fractionation of the ultraviolet dose does not enhance dimer excision, (3) dimer-containing DNA is replicated in ultraviolet-irradiated CHO cells, and (4) the dimers are conserved in the replicated DNA. These findings support the proposed mechanism of bypass of photoproducts during DNA replication in mammalian cells.     

PHOTOPRODUCT FORMATION IN DNA AT LOW TEMPERATURes.

Abstract— The temperature dependence of thy mine photoproduct formation in Escherichia culi DNA dissolved either in water or in a 50 per cent ethylene glycol solution was studied at temperatures between + 25 and — 196°C. At low temperatures, the formation of thymine dimer was strongly inhibited. A dose of 1 × 10⁴ ergs/mm² at 280 nm converted 2 per cent of the thymine to dimer at 25°C as compared with 0.2 per cent at — 196°C. In addition, a new thymine photo-product which was both nonphotoreversible and nonphotoreactivable was found at low temperatures. On the basis of its chromatographic mobility, this new photoproduct was assumed to be the same as that isolated from irradiated spores of Bacillus megaterium . Extensive irradiation at 254 nrn of DNA at — 120°C resulted in a yield of > 23 per cent for the 'spore-type' photoproduct as compared with 6 per cent for the thymine dimer. In poly d(AT), irradiated at low temperature, no spore-type photoproduct was found; this suggests that adjacent thymine residues are necessary for the formation of the spore-type photoproduct.     

Photo-thermal properties of poly(4-tert-butoxycarbonyloxystyrene) bearing imino sulfonate units

Copolymers of 4-tert-butoxycarbonyloxystyrene (BOS) and 9-fluorenilideneimino p-styrenesulfonate (FISS) were synthesized. FISS units in copolymers became p-styrenesulfonic acid units upon ultraviolet irradiation. The irradiated copolymers thermally decomposed to poly(hydroxystyrene) by liberating tert-butoxycarbonyl (BOC) units at temperatures where the unirradiated copolymers were stable. The thermal decomposition of the copolymers catalyzed by sulfonic acid formed photochemically was studied by thermogravimetry. The pseudo first-order rate constant (k) and the activation energy for the acid catalyzed thermal decomposition of BOC units in copolymers were evaluated. The thermolysis of the irradiated copolymer system was compared with that of the irradiated blended system of poly(4-tert-butoxycarbonyloxystyrene) (PBOCST) and 9-fluorenilideneimino p-toluene-sulfonate (FITS). © 1993 John Wiley & Sons, Inc.     

SINGLE-STRAND BREAKS IN THE DNA OF THE uvrA AND uvrB STRAINS OF ESCHERICHIA COLI K-12 AFTER ULTRAVIOLET IRRADIATION

Abstract— DNA single-strand breaks were produced in uvrA and uvrB strains of E. coli K-12 after UV (254 nm) irradiation. These breaks appear to be produced both directly by photochemical events, and by a temperature-dependent process. Cyclobutane-type pyrimidine dimers are probably not the photoproducts that lead to the temperature-dependent breaks, since photoreactivation had no detectable effect on the final yield of breaks. The DNA strand breaks appear to be repairable by a process that requires DNA polymerase I and polynucleotide ligase, but not the recA, recB, recF, lexA 101 or uvrD gene products. We hypothesize that these temperature-dependent breaks occur either as a result of breakdown of a thermolabile photoproduct, or as the initial endonucleolytic event of a uvrA , uvrB -independent excision repair process that acts on a UV photoproduct other than the cyclobutane-type pyrimidine dimer.     

SOME ASPECTS OF MORPHOLOGIES AND INTERFACES IN COPOLYMER/HOMOPOLYMER BLENDS

Based on a series of morphological studies of blends of homopolymer (Homo) and a variety of block and graft copolymers (Cop), the nature of phase separation, interface, emulsification and inner morphology of copolymer-dispersed phase etc. in the blends are discussed. In the cases of Cop AB/Homo A/Homo B systems, in which one homopolymer forms matrix, it is observed that the dispersed homopolymer phase is exclusively associated with Cop AB, i.e. no Homo A-Homo B interface exists. This phenomenon is believed to be caused by minimizing the interfacial energy of the systems. Meanwhile, preferential solubilization or anchoring of the like chains of copolymer into homopolymer matrix leads to stabilization of the dispersed phase in the matrix. In addition, regular variation of the inner morphology of the dispersed copolymer phase with the composition and molecular parameters of the component polymers is observed. When the two components have comparable proportions, alternating concentric shells are the most common feature which is associated with minimizing the interfacial energy in the Cop/Homo systems.     

Formation and flocculation of graft copolymer micelles

Criteria for formation and flocculation of micelles from pure graft copolymers were investigated in single selective solvents by turbidimetry with the use of two series of graft copolymers from poly(vinyl acetate) (PVAC), i.e., PVAC–styrene graft copolymers with one branch and PVAC–methyl methacrylate graft copolymers with one and several branches. These graft copolymers could be completely coagulated through two processes in the selective solvents which had widely different ? temperatures. The first process is the formation of micelles. One sequence, i.e., either backbone or branch of the graft copolymers, becomes desolvated under conditions similar to those for the corresponding homopolymer. This results in formation of the core of the micelle, the other soluble sequence extending from the surface of the core into the solvent phase. As the soluble chains cover the micelle core, no macroscopic phase separation occurs, but a stable dispersion is formed. The second process is that the micelle becomes too unstable to exist as dispersed when the solvency of the medium for the soluble sequence decreases to a certain degree. As a result, flocculation of the micelle finally takes place.     

Using neutron reflectivity to determine the dynamic properties of a copolymer in a homopolymer matrix

A protocol for using neutron reflectivity to monitor the dynamic properties of a copolymer in a homopolymer matrix is described. This technique may be used to monitor a broad range of systems, as long as the copolymer and homopolymer form a miscible blend at low copolymer concentrations. Moreover, with knowledge of the Flory–Huggins interaction parameter between the copolymer and homopolymer, the molecular dynamic parameters of the copolymer, such as the tracer diffusion coefficient, segmental friction factor, and longest relaxation time, can be quantitatively determined. This technique is demonstrated by the determination of these parameters for a series of styrene/methyl methacrylate alternating copolymers dispersed in a matrix of deuterated poly(methyl methacrylate). Interestingly, the segmental friction factor of these alternating copolymers is significantly different from that of similar diblock copolymers. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 3235–3247, 2004     

CHANGES IN cAMP AND cGMP CONCENTRATION, BIREFRINGENT FIBRILS AND CONTRACTILE ACTIVITY ACCOMPANYING UV AND BLUE LIGHT PHOTOAVOIDANCE IN PLASMODIA OF AN ALBINO STRAIN OF Physarum polycephalum

Abstract— Photoavoidance by plasmodia of an albino strain of Physarum polycephalum was studied. When the organism was irradiated locally, the protoplasm moved away from the irradiated region. The action spectrum for this avoidance showed three peaks at about 260, 370 and 460 nm. The organism was about one hundred times as sensitive to far UV as to near UV and blue light, and high intensity far-UV caused the gelation of the protoplasm. Irradiation with UV or blue light increased the mean level or the amplitude of oscillation in intracellular cAMP and cGMP concentrations. Upon UV irradiation, birefringent fibrils, presumably microfilaments of F-actin, became thick and numerous, and the plasmodial strand generated a strong tensile force. It is postulated that UV or blue light brings about an increased concentration of cyclic nucleotides which leads to an enhanced local development of contractile fibrils which squeeze protoplasmic sol from the area, resulting in photoavoidance.     

TRANSFER OF PYRIMIDINE DIMERS DURING BACTERIAL CONJUGATION

When male strains of Escherichia coli are irradiated with 254 nm ultraviolet (UV) light and mated with suitable females, DNA is transferred at almost the normal frequency (Howard-Flanders et al. , 1968). Cole (1971) observed that an episome damaged by UV irradiation of the male parent could be transferred to a recipient and restored to activity by administering photoreactivating light. He therefore deduced that UV lesions are transferred to the recipients during bacterial conjugation. The object of this Research Note is to report experiments providing direct evidence of the transfer of pyrimidine dimers -the main lesions of UV irradiation.     

Formation of singlet oxygen by urocanic acid by UVA irradiation and some consequences thereof

Singlet oxygen-initiated decomposition of urocanic acid (UCA) (3-(1H-imidazol-4(5)-yl)-2-propenoic acid) was used to successfully confirm the report that UCA generates singlet oxygen when irradiated with ultraviolet A light (UVA). The UCA-generated singlet oxygen converts UCA to one or more products that then catalyze the further destruction of the UCA with UVA light by singlet oxygen formation. Some nicking of the phiX-174 supercoiled plasmid DNA was observed when UCA was irradiated with UVA to complete destruction of the starting material, and the product mixture was then mixed with the plasmid in the dark. More extensive nicking was seen when the photoproduct mixture and the plasmid were irradiated with UVA light. An "aged" (4 days) solution of UCA photoproduct no longer caused nicking in the dark but retained the capability to nick the plasmid when irradiated. There is evidence for the presence of hydroperoxides in the UCA photolysis product mixture, and the quenching studies with 2-propanol indicate that free radicals are involved in the plasmid-nicking photochemistry. Singlet oxygen does not appear to play a role in the nicking of the plasmid.     

Synthesis and Self-Association of Stimuli-Responsive Diblock Copolymers by Living Cationic Polymerization

The living cationic polymerization of several functional monomers in the presence of an added base is investigated as a possible preparation of a new series of water-soluble or stimuli-responsive copolymers. Under appropriate conditions, the polymerization allows the selective preparation of polymers with various shapes and different sequence distributions of monomer units, including stimuli-responsive block copolymers, gradient copolymers, poly(vinyl alcohol) graft copolymers, and star-shaped polymers. The stimuli-induced self-association of the diblock copolymers is also examined. An aqueous solution of the diblock copolymer with a thermo-sensitive segment undergoes rapid physical gelation upon warming to the critical temperature to give a transparent gel, and returns sensitively to the solution state upon cooling. The sharp transition of stimuli-responsive segments with highly controlled primary structure turns out to play an important role in the self-association. Small-angle neutron scattering, dynamic light scattering, and electron microscopy studies reveal that the physical gelation involves a thermosensitive micellization of diblock copolymers (core size: 18-20 nm) and subsequent micelle macrolattice formation (bcc symmetry). Based on the gelation mechanism, several stimuli-responsive gelation systems are achieved using other stimuli such as the addition of a selective solvent or compound, cooling, pH change, and irradiation with ultraviolet light.     

CHANGE IN THE BASE COMPOSITION OF MESSENGER RNA OF ESCHERICHIA COLI BY ULTRAVIOLET IRRADIATION AND ITS RECOVERY

Abstract— The base composition of messenger RNA in Escherichia coli B/r and B _8–1 irradiated with ultraviolet (u.v.) light has been examined. The experimental results are as follows: (1) the synthesis of rapidly labeled RNA does not stop in ultraviolet irradiated bacteria. (2) The rapidly labeled RNA in irradiated cells shows a change in base composition corresponding to the formation of pyrimidine dimers in DNA molecules. The mole per cent of adenine component is increased with ultraviolet dose. The ratio of purine/pyrimidine becomes larger and the GC content smaller. (3) The base composition of the rapidly labeled RNA in irradiated bacteria reversed to that in unirradiated cells, when the irradiated cells were reactivated by experimental procedures for photoreactivation or dark reactivation. The reversion in the base composition corresponds well to the decrease in the amount of thymine dimers in DNA molecules. (4) The mechanism of the change in the base composition of rapidly labeled RNA caused by ultraviolet irradiation is discussed.