STUDIES OF THE ROLE OF THE COAT PROTEIN IN THE U.V. PHOTOINACTIVATION OF U(1) AND U(2) STRAINS OF TMV*

Abstract— Two properties of the u.v. inactivation process in the u.v. sensitive U(2) strain have been investigated: (1) The increased binding of protein to RNA induced by irradiation of the virus at 254 nm; (2) The action spectrum for u.v. inactivation of U(2) between 250 nm and 285 nm. The extent of the u.v. induced binding of protein to RNA is similar to that previously found in the resistant U(1) strain, thereby eliminating the possibility that the capacity for this binding phenomenon bears any correlation to the difference in u.v. sensitivities of these two viruses at 254 nm. The results indicate that the radiation induced interaction of protein and RNA in U(1) and U(2) are probably similar. The action spectrum for U(2) resembles the absorption spectrum of the RNA between 250 nm and 285 nm implicating the RNA as the primary absorber leading to inactivation of the virus in this region of the spectrum. Quantum yields calculated for U(2) virus and free TMV-RNA irradiated at 254 nm reveal that the irradiated free RNA may be as much as 1–4 times more sensitive to inactivation at this wavelength than RNA in the intact virus. It is concluded that the coat protein of U(2) probably offers some protection to the enclosed RNA against u.v. damage at 254 nm, therefore, the difference in u.v. sensitivity between U(1) and U(2) TMV at this wavelength is a consequence of a difference in the degree of protection offered by the respective coat proteins to the enclosed RNA.     

Modeling and conformation analysis of β-cyclodextrin complexes

Summary A series of -cyclodextrin complexes containing various guest molecules was studied using computer-aided molecular modeling and conformation analysis techniques. The geometry of each complex was studied using crystallographic data. The positions of the glycosidic O4 atoms indicate that the -cyclodextrin molecules are elliptically distorted. This distortion can be related to the van der Waals volume of the guest molecules. This correlation is different for aromatic and non-aromatic guest compounds. Rigid body docking experiments demonstrated that in crystal structures the guest molecule occupies a position in the cavity of nearly minimum interaction energy when there are no other molecules having interactions with the guest molecule. From the crystallographic data several rules could be deduced which seem to determine the conformation of -cyclodextrin molecules in complexes. A procedure was developed to construct -cyclodextrin molecules that are able to encompass guest molecules having a given van der Waals volume.     

PHOTOBIOLOGY OF RNA BACTERIOPHAGES—I. ULTRAVIOLET INACTIVATION AND PHOTOREACTIVATION STUDIES*

Abstract— Biologically active f2-RNA, Obtained from bacteriophage f2, was inactivated by ultraviolet (u.v) light (2537 Å) with a quantum yield of 3.3 ± 0.3 times 10^-3 when assayed in the dark with protoplasts of an F- strain of E. coli k12. Assay under “black light” gave a quantum yield of 2.7 ± 0.5 times 10^-3 which was just enough lower to suggest that 17 per cent photorecovery of the u.v. lesions has taken place. Intact phage f2 was inactivated by u.v. radiation with a quantum yield of 0.7 ± 0.12 times 10^-3, Thus the whole phage is much less sensitive than the free RNA. No evidence of photorecovery was found in u.v.-irradiated RNA phage 7S assayed in its host Pseudomonas aeruginosa.     

The effect of ultraviolet light on RNA metabolism in bromouracil deoxyriboside-grown HeLa cells

Abstract— The presence of 5-bromouracil deoxyriboside (BrUdR) in the DNA of HeLa cells has profound effects on RNA metabolism after u.v. irradiation. In normally grown cells 200 ergs/mm² depresses RNA synthesis by about 30 per cent while in BrUdR-grown cells the same exposure to u.v. depresses RNA synthesis by 95 per cent. When BrUdR-grown cells are u.v. -irradiated after being labeled with ³H-uridine, the normal autoradiographic pattern, where label shifts from nucleus to cytoplasm, fails to occur. Also, in lieu of the increase in RNA specific activity that is observed in unirradiated cells for a few hours after 20-min pulse-labeling, there occurs a constant decrease in specific activity after the irradiation.     

THE U.V. SENSITIVITY OF BACTERIA: ITS RELATION TO THE DNA REPLICATION CYCLE

Abstract— A striking increase in the shoulder of the u.v. survival curve but no change in the limiting slope is obtained when cultures of Escherichia coli strain TAU complete the DNA replication cycle in the absence of concommitant protein synthesis prior to irradiation. The u.v. sensitivity of protein synthesis or RNA synthesis is not altered significantly by this treatment.
In contrast to the result for strain TAU, there is no significant change in the u.v. survival curve for the u.v. sensitive E. coli B_s-1 when its DNA replication cycle is completed under similar conditions.
Following a period of inhibited protein synthesis there is a delay in the reinitiation of the normal DNA replication cycle when protein synthesis resumes. This delay would allow time for an intracellular repair system to operate before the attempted resumption of normal replication. Strain B_s-1, which is deficient in this repair system, would not be expected to benefit from such a delay, as consistent with the observed results. A model is presented to account for lethality due to attempted DNA replication during a period of repair synthesis. The maximum survival for a given u.v. dose would be predicted for a culture which has completed the normal DNA replication cycle prior to irradiation and which is not permitted to reinitiate the cycle until all possible repair synthesis is completed.     

SUPPRESSION OF PHAGE T4 BY CO-INFECTION WITH U.V.-INACTIVATED HOMOLOGOUS PHAGE

Abstract— Unirradiated phage T4v₁ may fail to produce viable progeny in cells which are co-infected with u.v.-irradiated homologous particles. The extent of this effect, called suppression , is positively correlated with the multiplicity of infection of the irradiated phage and with the U.V. dose. The suppression reaches a maximum level at about 30–600 lethal hits. Quantitative evaluation of the results shows that in some complexes one irradiated phage particle is sufficient to suppress an unirradiated phage. Two hypotheses are discussed to explain the results. (a) Lethal u.v.-damages are incorporated into the unirradiated phage genome by genetic recombination; ( b ) Genetic subunits (e.g. cistrons or operons) of the u.v.-irradiated phages produce informationally incorrect messenger RNA molecules, which compete with the correct ones from the unirradiated phage in the protein-synthetizing system. Hypothesis (6) appears to be more adequate to explain the experimental results.     

INACTIVATION BY ULTRAVIOLET RADIATION AT DIFFERENT WAVELENGTHS OF THE RNA ISOLATED FROM POTATO VIRUS X

Abstract— –The action spectra and quantum yields for photoreactivable, non-photoreactivable and total damage caused by u.v. in the RNA isolated from potato virus X differ from those for similar types of damage in the whole virus. The differences result from the virus protein partly protecting the RNA from damage, and the degree of protection depends on the wavelength of u.v. and on the salt concentration of the irradiated solution. The action spectra for the different types of damage in the RNA all resemble the absorption spectrum of the RNA, but do not exactly parallel it. The photoreactivable sectors of the RNA and of the whole virus are greater at 290 nm than at 230 nm but, whereas that of the virus increases rectilinearly, that of the RNA has a pronounced minimum at about 250 nm. At wavelengths longer than 240 nm, the photoreactivable sector of the virus exceeds that of the RNA, because, at these wavelengths, the virus protein protects the RNA more against non-photoreactivable damage than against photoreactivable damage.     

Mechanisms of antioxidant action: Metal complexes as u.v. stabilisers in polyethylene

In order to evaluate the importance of u.v. screening, peroxide decomposition and excited state quenching in the mechanism of action of a variety of metal complex stabilisers, screening and additive studies were carried out in high density polyethylene and in model hydrocarbons. All the metal complexes studied were found to be u.v. screening agents when contained in a separate phase between the u.v. source and the polymer and all were rather more effective than a typical commercial u.v. absorber (2-hydroxy-4-octyl-oxybenzophenone, HOBP). However, when used as additives, all the stabilisers had an additional function. In the case of the transition metal dibutyl dithiocarbamates (NiDBC and FeDBC), the additional stabilisation mechanism is consistent with their known peroxide decomposing behaviour. In the case of the 2-hydroxy-acetophenone oxime complexes (NiOx and CuOx), the initial function is a stoichiometric reaction with hydroperoxides to give non-radical products. An amine complex of nickel bis-octylphenol sulphide (NiBOPS) appeared to behave primarily as a screen. Only one of the metal complexes (FeOx) appeared to be capable of functioning as an excited state quencher and this complex is effectively sensitised by triplet carbonyl to give a photo-pro-oxidant effect in cumene. It is concluded that the concept of excited state quenching, although it may occur with some metal complexes, is not a sufficient criterion of u.v. stabilising activity.     

Mechanisms of antioxidant action: Synergism between tin stabilizers and polymer-bound antioxidants in the u.v. stabilization of PVC

It is shown that a conventional tin stabilizer (dibutyltin maleate) (DBTM) forms an effective synergistic u.v. stabilizing combination when used in conjunction with a bound phenolic antioxidant (BHBM) and a bound u.v. stabilizer (EBHPT). These additives become bound to the polymer through sulphur during processing in the presence of DBTM and it is suggested that the sulphide of the adduct makes its own contribution to u.v. stabilization by acting as a peroxide decomposing (PD-C) antioxidant during photo-oxidation. However, the 2-hydroxybenzophenone function appears to be an essential part of the system and is probably involved in quenching of excited states of the intermediate sulphoxides which precede the formation of the PD-C antioxidant.     

Alignment effect of N2(A3Σu+) in the energy transfer reaction of aligned N2(A3Σu+) + NO(X2Π) → NO(A2Σ+) + N2(X1Σg+)

Steric effect in the energy transfer reaction of N(2)(A(3)Σ(u)(+)) + NO(X(2)Π) → NO(A(2)Σ(+)) + N(2)(X(1)Σ(g)(+)) has been studied under crossed beam conditions at a collision energy of ~0.07 eV by using an aligned N(2)(A(3)Σ(u)(+)) beam prepared by a magnetic hexapole. The emission intensity of NO(A(2)Σ(+)) has been measured as a function of the magnetic orientation field direction (i.e., alignment of N(2)(A(3)Σ(u)(+))) in the collision frame. A significant alignment effect on the energy transfer probability is observed. The shape of the steric opacity function turns out to be most reactive at the oblique configuration of N(2)(A(3)Σ(u)(+)) with an orientation angle of γ(v(R)) ~ 45° with respect to the relative velocity vector (v(R)), which has a good correlation with the spatial distribution of the 2pπ(g)* molecular orbital of N(2)(A(3)Σ(u)(+)). We propose the electron exchange mechanism in which the energy transfer probability is dominantly controlled by the orbital overlap between N(2)(2pπ(g)*) and NO(6σ).     

Photobiology of RNA bacteriophages. II. U.V.-irradiation of f2: effects on extracellular stages of infection and on early replication

Abstract— The effect of u.v. irradiation (2537 Å) on the RNA bacteriophage f2 has been studied with respect to the adsorption of f2 to E. coli K12 (male strain), the penetration of f2-RN A into the host cell and the conversion of the phage nucleic acid to the double-stranded replicative intermediate. The biological parameter most sensitive to u.v. was the plaque-forming ability of the phage. Its loss could be attributed to several factors. (1). A binding of capsid protein to phage nucleic acid interfering with host penetration by the f2-RNA. (2). Desorption of some irradiated phage at 37° from their attachment sites on the host. (3). Molecular alterations in the RNA preventing formation of the replicative intermediate within the host. The relationship of these factors to the lack of photoreactivability of irradiated f2 is discussed.     

The effects of thermal processing on PVC—IX. A polymer reactive u.v. stabilizer as a thermal stabilizer for PVC

It is shown that a thiol u.v. stabilizer (4-ethoxymercaptoaceto-2-hydroxybenzophenone, EBHPT) becomes efficiently bound to PVC during normal processing at 170° in the presence of dibutyltin maleate (DBTM). This combination is an effective melt stabilizer and shows synergism as a heat stabilizer at 140°. This is shown to be due to the protection of DBTM by the PD antioxidant function in EBHPT and a synergistic optimum is observed. In the absence of DBTM or in the presence of a thiotin stabilizer binding of the u.v. stabilizer is incomplete.     

Photochemistry of tobacco mosaic virus RNA. Effect of HCN

Abstract— In attempting to sort out possible mechanisms of photoreactivation of tobacco mosaic virus RNA (TMV-RNA) inactivated by ultraviolet radiation (u.v.) in buffer of ionic strength 0.25, we have investigated the effect of HCN on the quantum yield for u.v. inactivation of TMV-RNA and on the percent photoreactivation of inactivated TMV-RNA. Some photo-products produced by irradiation of model substances, polyuridylic acid (poly U) and polycytidylic acid (poly C), in the presence of HCN have also been studied. The ratio of the quantum yield for inactivation of TMV-RNA in the presence of HCN to that in the absence of HCN is 1.5, under non-photoreactivating conditions. By comparison, the ratio of the initial rates of loss of uracil residues in poly U under comparable conditions is 1.6; by contrast, the rate of loss of cytosine residues in poly C is unaffected by HCN. This similarity of ratios between poly U and TMV-RNA suggests that two of the mechanisms of u.v. inactivation of TMV-RNA at high ionic strength are akin to known reactions of uracil residues in poly U, i.e. hydrate and dimer formation. The photohydration reaction in poly U, as measured by the heat reversal of hydrated residues to uracil residues, is almost abolished by HCN, and the rate of dimerization, as measured by the appearance of dimer containing oligonucleotides following enzymatic hydrolysis of irradiated poly U, is reduced to half by HCN. HCN does not affect the rate of hydration of cytosine residues in poly C. Since photoreactivation of RNA inactivated in presence of HCN is only 60 per cent of that in absence of HCN it is suggested that uracil dimers are somehow involved in photoreactivation of TMV-RNA inactivated at high ionic strength.     

THEORETICAL ASPECTS OF THE U.V. INACTIVATION OF PROTEINS CONTAINING DISULFIDE BONDS

Equations are proposed for the estimation of quantum yields for cystine destruction and disulfide protein inactivation during u.v.-irradiation in acidic and neutral solutions. The formulas permit a discussion of energy transfer from excited aromatic amino acids to cystines and/or of chemical reactions between excited tryptophans or tyrosines and cystines. The results are discussed with regard to general aspects of the photo-biochemistry of enzymes.     

THEORETICAL ASPECTS OF THE U.V. INACTIVATION OF PROTEINS CONTAINING DISULFIDE BONDS

Abstract Equations are proposed for the estimation of quantum yields for cystine destruction and disulfide protein inactivation during u.v.-irradiation in acidic and neutral solutions. The formulas permit a discussion of energy transfer from excited aromatic amino acids to cystines and/or of chemical reactions between excited tryptophans or tyrosines and cystines. The results are discussed with regard to general aspects of the photo-biochemistry of enzymes.     

Poly-α-methyleneglutaronitrile. Characteristic features of some anionic polymers

For (initiator)/(monomer) ratios exceeding 10^-3, the anionic polymerization of α-methyleneglutaronitrile in homogeneous solution in DMF (initiated by lithium-naphthalene at -35° or by diphenylmethyl-lithium at -70°) leads to fairly white polymers with yields higher than 75 per cent. Infra-red and u.v. spectrometric studies and flash pyrolysis experiments show that the nitrile functions are involved in the polymerization only to a very small extent. On the other hand, molecular weight and sedimentation measurements and hydrodynamic behaviour of the polymer in DMF solution suggest a broad polydispersity for the samples, and a very high branching density for their higher molecular weight fractions. These characteristic features are to be correlated with those of anionic polyacrylonitrile; they may be tentatively attributed to transfer to polymer at the acidic methylene group a to the nitrile function.     

THE EFFECTS OF HOST-CELL REACTIVATION ON ASSAY OF U.V.-IRRADIATED HAEMOPHILUS INFLUENZAE TRANSFORMING DNA

Abstract— The host cell reactivation (HCR) mechanism in Haemophilus influenzae cells is inhibited by sub-microgram concentrations of acriflavine (as is already known to be true for Escherichia coli ). Exposure of these cells to similar concentrations of the drug during bacterial transformation increases the apparent ultraviolet light (u.v.) sensitivity of previously irradiated transforming DNA, indicating a repair of this DNA after uptake by the cells under normal conditions. Repair is inhibited by applying acriflavine at any time up to one hour after competent cells contact the irradiated transforming DNA. The fraction of the u.v. damage repaired by HCR is very different for different genetic markers. Those markers which are most u.v. sensitive when assayed in the absence of acriflavine are most poorly repaired, suggesting that this is the reason for their higher sensitivity. For all markers the fraction of the damage repairable by in vitro photoreactivation is approximately constant, and strongly overlaps the damage repairable by HCR. The degree of HCR achieved can be altered by experimental treatment of the H. influenzae DNA prior to transformation. Thus treatment of irradiated DNA with an enzyme from Micrococcus lysodeikticus –known to attack u.v. damaged, but not undamaged DNA–prevents subsequent intracellular repair of the same u.v. lesions whose repair is inhibited by acriflavine. Similarly, partial replacement of the thymine in transforming DNA by 5-bromouracil (BU) strongly inhibits repair of subsequent u.v. damage. As in bacteriophage, the BU effect is relieved if the u.v. exposure occurs in the presence of cysteamine. It is clear that intracellular repair must be considered in interpreting experiments with u.v.-irradiated transforming DNA.     

Investigation of electronic structure of allenyl ethers by means of photoelectron and UV spectroscopy

A study of the photoelectron spectra of alkyl allenyl ethers has demonstrated their similarity to spectra of alkyl vinyl ethers, with the exception of a band in the 10.0–10.3 eV interval due to ionization of the -orbital of the allenyl fragment C=C. According to data obtained by photoelectron spectroscopy and MNDO quantum-chemical calculations, alkyl allenyl ethers with straightchain substituents exist primarily in the s-cis conformation. The stable conformation of allenyl tert-butyl ether is the s-trans form. The long-wave absorption bands in the UV spectra of alkyl allenyl ethers at 255–275 and 208–213 nm are assigned to electronic transitions of the ^* and ^{* *} types; these are quite insensitive to s-cis/s-trans isomerism.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 1, pp. 81–88, January, 1990.The authors are extremely grateful to V. K. Turchaninov for assistance in interpreting the spectra.     

ESR study of some methyl methoxy benzoates. Additivity model of substituent effects

The radical anions of several methyl methoxy benzoates are obtained by an in situ electrochemical method. ESR spectra of these radicals and u.v. spectra of their parent compounds are analyzed in order to study the change of conformation in the ortho substituted esters, when these are reduced. The effects of the substituents on the hyperfine coupling constants (HCCs) are analyzed by means of an additivity model.     

DISTRIBUTIONS OF FREE RADICALS AMONG AMINO ACIDS FROM LYOPHILIZED ULTRAVIOLET-IRRADIATED PROTEINS

Abstract— The effects of u.v.-irradiation at 254 nm upon lyophilized ribonuclease, lysozyme, insulin, and chymotrypsinogen have been investigated by electron spin resonance (ESR). enzymatic assay, and labeling of free radical sites with tritiated hydrogen sulfide (HST). The ESR signal of the irradiated protein diminishes on exposure to HST, and tritium becomes covalently bound to carbon. The distribution of tritium among the amino acids of each protein. studied as an indicator of the carbon free radical distribution, differs markedly from those observed previously to result from exposure to gamma radiation, electrical discharge. or hydrogen atoms. However, the earlier observation that the tritium distribution is influenced by protein conformation holds true as well for u.v.-irradiation. Moreover, the distributions of tritium among the amino acids of u.v.-irradiated proteins indicate a broad scattering of free radicals. Tyrosine and phenylalanine, residues that absorb light energy in the region of the wavelength employed, are not particularly important as radical carriers. Thus, for ribonuclease, these residues incorporated 3.8 and 1.5 per cent of the total tritium, but they absorb 51 and 12 per cent of the light, respectively. These results, together with the observed low recoveries of methionine, an amino acid that does not absorb at 254 nm, add weight to the concept that a migration of energy ensues after the initial absorption of light energy and that photolytic damage may thus be due to destruction of amino acids other than those initially absorbing the u.v.-radiation.