DIRECT PHOTO-AFFINITY LABELING WITH CYCLIC NUCLEOTIDES: ACTION SPECTRA

Abstract— When [³H]-cyclic adenosine 3',5'-monophosphate and [³H]-cyclic guanosine 3',5'-monophos-phate are irradiated at varying UV wavelengths in the presence of cyclic nucleotide receptor proteins, photo-incorporation is maximal in the region of 280 nm. This suggests that covalent coupling occurs by means of photo-activation of an entity other than the ligand cyclic nucleotide. The action spectra more closely resemble the absorbtion spectrum of the reacting protein, making it likely that absorhtion of radiant energy by components of the protein receptor leads to photo-affinity labeling.     

DIRECT PHOTO-AFFINITY LABELING OF CYCLIC NUCLEOTIDE BINDING PROTEINS WITH GUANOSINE-3'',5''-MONOPHOSPHATE

Abstract— Radioactivity from [³H]-guanosine-3',5'-monophosphate was shown to become stably linked to proteins in testis extracts during ultraviolet irradiation. Adenosine-3',5'-monophosphate competitively inhibited incorporation into these proteins. Guanosine-3',5'-monophosphate had a lower affinity for binding sites than did adenosine-3',5'-monophosphate. Similar peptides were photo-labeled with [³H]-guanosine-3',5'-monophosphate and [3H]-adenosine-3',5'-monophosphate. Guanosine-3',5'-monophosphate was more efficiently incorporated than was adenosine-3',5'-monophosphate. Extracts from a number of tissues incorporated both cyclic nucleotides photochemically.     

PHOTODYNAMIC THERAPY-INDUCED HYPOXIA IN RAT TUMORS and NORMAL TISSUES

Abstract The administration of misonidazole (MISO) to Fischer x Copenhagen rats whose R3327-H prostate tumors were treated with photodynamic therapy (PDT) produced enhanced tumor growth delays and cures. This potentiation of PDT by MISO was previously observed with R3327-AT tumors and was postulated to result from drug cytotoxicity of naturally-occurring and PDT-induced hypoxic cells. Radioactively-labelled MISO has been developed as a marker for tissue p0₂ at the cellular level and [³H]MISO was administered to R3327-AT and R3327-H tumor-bearing rats before and after standard PDT treatments. The amount of ³H in tissues 24 h after drug administration was a measure of'bound MISO'which reflects average tissue oxygenation. [³H]MISO retained in R3327-AT tumors was ˜4x and in liver tissue ˜2x that retained in muscle, heart, brain and R3327-H tumors (1x). Tumors treated with Photofrin II and lased with 1000 J showed a 6-fold increase in retained [³H]MISO in R3327-H tumors and a 2-fold increase in retained [³H]MISO in R3327-AT tumors. The absolute levels of retained ³H in both tumors after PDT were similar. These data provide direct evidence that PDT induces rapid hypoxia in both tumors. When the gastrocnemius muscle of the rat leg was similarly treated, the amount of [³H]MISO retained was ˜4x greater than that in untreated muscle. This result suggests that PDT-induced hypoxia is not selective to just tumor tissue. These data suggest that the hypoxia-inducing property of PDT might be exploited in combination with hypoxic cell cytotoxins to produce improved tumor responses and cures.     

Novel 3',5'-cyclic nucleotide analogue: adenosine 3',5'-cyclic boranomonophosphate

A general procedure for the first synthesis of a 3',5'-cyclic boranomonophosphate was established. Specifically, adenosine 3',5'-cyclic boranomonophosphosphate (cyclic AMPB, 4c), a P-borane (BH(3)) analogue of adenosine 3',5'-cyclic monophosphate (cAMP), was synthesized via a phosphite approach in good yield. The method is also applicable for syntheses of natural cAMP and its phosphorothioate analogue. The two diasteromers of cyclic AMPB 4c were separated, and their chemical structures were established via spectroscopic methods.     

ATP-GTP-BINDING PROTEINS AND ENDOGENOUS ADP-RIBOSYL TRANSFERASE IN Lemna paucicostata 441

Abstract— A crude extract containing membrane components of Lemna paucicostata was treated with 1% Lubrol PX and fractionated by gel nitration. Binding activities to non-hydrolyzable analogues of ATP, [³⁵S]ATPγS (adenosine 5'[;γ-thio]triphosphate) and that of GTP, [³⁵S]GTPγS (guanosine 5'[γ-thiojtriphosphate) were detected in some fractions, and these activities were prevented in the presence of 0.1 mM ATP or GTP. ATP and GTP were 2 to 3 orders of magnitude more effective than CTP or UTP in preventing this binding activity. These fractions showed ATPase and GTPase activities with 1 nM [γ-³²P]ATP or [γ–³²P]GTP substrate. Analyses by sodium dodecyl sulfate polyacrylamide gel electrophoresis of these fractions after binding with [³⁵S]ATPγS or [³⁵S]GTP-γ S revealed that these fractions contained [³⁵S]ATPγS and [³⁵S]GTPγS binding proteins with molecular weights of 53 000 and 60 000, respectively. Both of these proteins were [³²P]ADP-ribosylated by endogenous ADP-ribosyl transferase. Three proteins with molecular weights of 11 000, 12 000 and 13 000 which could bind [³⁵S]ATP7S or [-³⁵S]GTP-γ S were ADP-ribosylated by endogenous ADP-ribosyl transferase. Pertussis toxin stimulated ADP-ribosylation of these proteins. Four proteins with molecular weight of 37 000, 50 000, 80 000 and 115 000 with P^SS]ATP7S and [^,3S]GTP7S binding activities were also detected. The signal transduction of light to underlying clock mechanism in Lemna may be controlled by ATP-GTP-binding proteins and by the ADP-ribosylation of these proteins.     

Mass spectrometric identification of Rab23 phosphorylation as a response to challenge by cytidine 3',5'-cyclic monophosphate in mouse brain

While the functions and mechanisms of action of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) are well established and are the basis of the action of a large number of successful pharmaceuticals, the role of a third naturally occurring cyclic nucleotide, cytidine 3',5'-cyclic monophosphate (cCMP), remains to be elucidated. Immobilized metal affinity chromatography (IMAC) was used to selectively extract proteins phosphorylated in mouse brain in response to challenge by cAMP, cGMP and cCMP, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToFMS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) of tryptic digests to identify Rab23 as the first protein reported to be phosphorylated only in response to cCMP.     

Regulation of cAMP-mediated photosignaling by a phytochrome in the cyanobacterium Anabaena cylindrica

Changes in cellular adenosine 3',5'-cyclic monophosphate (cAMP) content induced by monochromatic light of various wavelengths were determined in the cyanobacterium Anabaena cylindrica. Irradiation with monochromatic red light caused a rapid decrease in cAMP content. In contrast, far-red light caused a rapid increase in its content. The effects of red and far-red light were reversible, suggesting the involvement of a prototype phytochrome as the photoreceptor for cAMP-mediated light-responsive signal transduction.     

FUNCTIONAL ROLE OF CALCIUM IN PHOTORECEPTOR CELLS

Abstract— Ca-uptake by disc membranes prepared from frog rod photoreceptor outer segments was examined. Ca-uptake study revealed two affinity sites which were saturated with 10–⁵ M and 10–³ M of ATP. When disc membranes in 20 m M Tris-HCl buffer (pH 7.5) were stored at -20°C for 6 h, more than 95% of Ca-uptake activity was lost. Ca-uptake activity was, however, preserved if the disc membrane suspension was mixed with 1–10m M ATP and stored at -20°C. Furthermore the reactivation of Ca-uptake was observed if disc membranes, which had lost Ca-uptake ability by storing at 4°C for 3 h, were mixed with 10 m M ATP and then frozen at -20°C for 5 h or 28 h (ATP-induced ATP-dependent Ca-uptake). When the contents of ATP bound to disc membranes were measured during a brief aging at 37°C, the decrement of bound ATP content was correlated well with the decreasing of Ca-uptake activity. Carbonylcyanide m -chlorophenylhydrazone (CCCP), a conductor of protons, inhibited Ca-uptake activity and half maximal inhibition was achieved at 2 × 10–⁸ M. When 10–⁶ M of CCCP was added to the ⁴⁵Ca-accumulated disc membranes, rapid release of ⁴⁵Ca from the disc membranes was observed. These results suggest that ATP may play a role in the Ca-pump regulation in disc membranes and a [H⁺] gradient across disc membrane may be linked to Ca-uptake mechanism.     

FORMATION OF THYMINE DIMERS IN MAMMALIAN SKIN BY ULTRAVIOLET RADIATION IN VIVO

Abstract— Ultraviolet radiation of 220–300 nm is known to produce cyclobutyl pyrimidine dimers in extracellular DNA, in bacteria, and in mammalian cells in culture. The formation in vivo of such dimers in mammalian skin has remained inferential. We report that one of the important and recognizable biologic events that occurs in mammalian skin during irradiation is the formation of thymine dimers. [³H]-labelled thymidine was applied to the epilated skin of guinea pigs to label their DNA. Animals were irradiated individually, using wavelengths of either 254, 285–350, or 320–400 nm. Immediately after irradiation, epidermis was separated from the rest of the skin and homogenized; DNA and RNA were isolated. Irradiation with wavelengths of 285–350 nm, which included the sunburn-producing spectrum (i.e., 290–320 nm), produced thymine dimers (1·7–2·6 per cent of the total [³H]-thymine incorporated into DNA). Irradiation with 254nm also produced fewer dimers (0·46–1·2 percent); and 320–400 nm produced none. The dimer could be cleaved by 250 nm radiation to form thymine. The epidermal cell damage by ultraviolet radiation, particularly by the sunburn-producing spectrum (290–320 nm), may be related to the formation of such dimers.     

Photosensitive signal transduction induces membrane hyperpolarization in Paramecium bursaria

The protozoan ciliate Paramecium bursaria exhibits membrane hyperpolarization in response to photostimulation, accompanied with an increased swimming speed. The external addition of cyclic nucleotide phosphodiesterase (PDE) inhibitors, either theophylline (1,3-dimethylxanthine) or 3-isobutyl-1-methylxanthin (IBMX), increased in both amplitudes of the membrane hyperpolarization and the increase in swimming speed. Moreover, the addition of membrane permeable cyclic nucleotide analogs, either 8-bromo-adenosine 3',5'-cyclic monophosphate (Br-cAMP) or 8-Br-guanosine 3',5'-cyclic monophosphate (Br-cGMP), increased these amplitudes. On the other hand, the addition of l-cis-diltiazem, known to block the conductance of cyclic nucleotide-gated channels, partially decreased both amplitudes of the membrane hyperpolarization and the increase in swimming speed. An enzyme immunoassay of cellular cyclic nucleotide contents showed that photostimulation induced a rapid increase in adenosine 3',5'-cyclic monophosphate (cAMP), but little increase in guanosine 3',5'-cyclic monophosphate (cGMP), raising the possibility that a rapid increase in cAMP mediates the light-induced hyperpolarization in P. bursaria.     

PHOTOADDITION OF CHLORPROMAZINE TO GUANOSINE-5'-MONOPHOSPHATE

Abstrart—The photochemistry of chlorpromazine (CPZ) with guanosine-5'-monophosphate (GMP) was studied as a model for the photoaddition of CPZ to DNA. Irradiation of CPZ with calf thymus DNA produced a product emitting at 520 nm, whereas with GMP emission was at 495 nm. HPLC separation of photolysis mixtures of [³H]CPZ with GMP and [¹⁴C]GMP with CPZ indicated that three photoadducts were formed. One of the adducts fluoresced at 500 nm and appeared to be the product detected but not separated by Fujita et al. (Photochem. Photobiol . 1981, 34 , 101–105). A second adduct emitted at 460 nm, and the third was nonfluorescent. The photoadduct emitting at 500 nm was characterized by UV, fluorescence, and NMR to be an adduct from coupling of the C-8 position of guanine to the C-2 position of the phenothiazine ring of CPZ. The cation radical of CPZ (CPZ ⁺) does not appear to be an intermediate since enzymatically generated CPZ ⁺ formed a product that eluted with a retention time close to that of the photoadducts, but did not emit at 520 nm.     

The Structure of the Aggregate Form of Bacteriochlorophyll c Showing the Qy Absorption above 740 nm as Determined by the Ring-current Effects on 1H and 13C Nuclei and by 1H–1H Intermolecular NOE Correlations

¹³C-enriched bacteriochlorophyll c (S[I, E] BChl c _F) was suspended in a 1:3 mixture of methylene chloride and carbon tetrachloride to form an aggregate showing the Q_y absorption above 740 nm; changes in the ¹³C chemical shifts were traced when methanol was titrated to dissolve the aggregate, and then, the changes were correlated to the ring-current effects due to the neighboring macrocycles in the aggregate. A pair of aggregate structures has been proposed based on the ring-current effects on both ¹H and ¹³C nuclei; the monomeric units are stacked together to form an inclined column with different sliding directions, in which the y-axis of the molecule is parallel to the long axis of the column. In order to confirm this pair of models, the ring-current effects on the ¹H and ¹³C nuclei were calculated based on both the magnetic-dipole and the loop-current approximations. Further, an application of three-dimensional F1 ¹³C-edited F3 ¹³C-filtered heteronuclear single-quantum correlation-nuclear Overhauser effect spectroscopy to the above aggregate consisting of a 1:1 mixture of ¹³C-labeled and unlabeled BChl c succeeded in detecting selectively the ¹H–¹H intermolecular nuclear Overhauser effect correlations, which established the coexistence of the above pair of stacked structures in the aggregate.     

PHOTOCHEMISTRY OF trans-[Rh(BIS(DIPHENYLPHOSPHINO)ETHANE)2X2][PF6]: TRANSIENT ABSORBANCE KINETIC STUDIES OF METAL-HALOGEN BOND HOMOLYSIS

Abstract— Laser flash photolysis of trans -[Rh(dppe)₂X₂][PF₆] (X=Br and I; dppe=bis(diphenylphosphino)ethane) in CH₂Cl₂ or CH₃CN produces the d⁷ Rh(II) radicals, [Rh(dppe)₂X]⁺, and halogen atoms. The kinetics of the disappearance of [Rh(dppe)₂X]⁺ radicals in CH₂Cl₂ or CH₃CN were mixed order: H-atom abstraction from solvent to produce the rhodium hydrides, [RhH(dppe)₂X][PF₆], and Rh/X recombination. In the poor H-atom donor solvent, benzonitrile, Rh/Br recombination was observed to be uncomplicated by competing H-atom abstraction. The hydride complexes [RhH(dppe)²X][PF₆], formed by H-atom abstraction were completely characterized by ³¹P{¹H}-NMR, ¹H-NMR, and mass specrometry. Cyclohexene was used as an effective trap for photogenerated Br atoms and yielded bromocyclohexane and 3-bromocyclohexene in a relative yield, 1:9. The photochemical mechanism is discussed in light of the transient absorbance and trapping studies.     

Contribution of the cAMP-Dependent Signal Pathway to Circadian Synchrony of Motility and Resting Membrane Potential in Paramecium

It is known that the ciliated protozoan Paramecium multimicronucleatum has synchronized circadian rhythms of motility, resting membrane potential and cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) concentrations. The present study shows that (1) extracellularly added 4 m M tetraethylammonium (TEA)⁺ (a K⁺ channel blocker) almost completely abolishes the diurnal oscillation of intracellular cAMP concentrations; (2) even 32 m^M TEA⁺ fails to abolish the circadian motility rhythm; but (3) the motility rhythm is highly damped when 4 m^M TEA⁺ and 100 μ^M CdCl₂ (a Ca²⁺ channel blocker) are added simultaneously. A cAMP analogue ( N ⁶-monobutyryl-cAMP) added extracellularly accelerates swimming velocity. Both a K⁺ channel blocker ( e.g . TEA⁺) and an inhibitor (trifluoperazine) of adenylate cyclase (AC) suppress cAMP formation, supporting the hypothesis that AC in Paramecium has dual functions, as a K⁺ channel and as an enzyme for cAMP formation. It is hypothesized that the circadian synchrony is due to circadian fluctuations of AC causing separate circadian changes both in ciliary motion and membrane potential through a cAMP-dependent signal pathway that forms a sophisticated network of second messengers to govern the synchrony together with Ca²⁺- and cGMP-dependent pathways in a manner antiphasic and/or complementary to one another.     

PRIOR EXPOSURE OF HUMAN CELLS TO NEAR UV-RADIATION GIVES A DECREASE IN THE AMOUNT OF THE UNSCHEDULED DNA-SYNTHESIS INDUCED BY FAR UV-RADIATION

Pretreatment of human cells with near UV radiation (UVA) in fluences exceeding 5 × 10⁴ Jm⁻² caused a decrease in the amount of the unscheduled DNA synthesis induced by far UV radiation (UVC). The DNA repair synthesis, as measured by the incorporation of [³H] -thymidine, is reduced by nearly a factor of 2 for a UVA radiation exposure of 1.5 × 10⁵ Jm⁻². Since solar UVA fluence rate is rather independent of latitude, this figure corresponds to a UVA exposure time of 50-60 min from noon sunlight in the summer time.     

UVA self-photosensitized oxygenation of beta-ionone

The steady-state UVA (350 nm) photolysis of ( E )-β-ionone ( 1 ) in aerated toluene solutions was studied by ¹H NMR spectroscopy. The formation of the 1,2,4-trioxane ( 2 ) and 5,8-endoperoxide ( 5 ) derivatives in the ratio of 4:1 was observed. Time-resolved laser induced experiments at 355 nm, such as laser-flash photolysis, photoacoustic and singlet oxygen ¹O₂ phosphorescence detection, confirmed the formation of the excited triplet state of 1 with a quantum yield Φ _T = 0.50 as the precursor for the generation of singlet oxygen ¹O₂ ( Φ _Δ = 0.16) and the isomeric α-pyran derivative ( 3 ), which was a reaction intermediate detected by NMR. In turn, the reaction of ¹O₂ with 1 and 3 occurred with rate constants of 1.0 × 10⁶ and 2.5 × 10⁸  m ⁻¹s⁻¹ to yield the oxygenated products 5 and 2 , respectively, indicating the relevance of the fixed s-cis configuration in the α-pyran ring in the concerted [2+4] cycloaddition of ¹O₂.     

Synthesis and Photoreaction of 5-Aikenyloxypsoralen Derivatives

Abstract— In order to investigate the intramolecular "quenching" of the photoexcitation of some 5-alkenyloxypsoralen derivatives, we have prepared model compounds in which a psoralen moiety was linked at position 5 to a terminal double bond via a polymethylenic chain of various length (n = 2-9). The isolation and characterization of photocycloadducts obtained for each compound after irradiation at 365 nm in a polar solvent was performed. The results on the photoreactivity of this series of compounds show that the 3,4-pyrone double bond of 5-alkoxypsoralens is the most reactive. Four kinds of intramolecular photocycloadducts between the 3,4-pyrone double bond and the chain unsaturation were obtained according to the length of the linking chain: cis-syn, trans-syn, cis-anti and trans-anti. Their structures were established by a combination of ¹H and ¹³C NMR and fully assigned by ^lH NOE (nuclear Overhauser effect) and ¹H-¹³C HMQC (heteronuclear multiquantum correlation) spectroscopies. No traces of 4',5' adducts were detected.     

ALTERATIONS IN DNA IRRADIATED WITH ULTRAVIOLET RADIATION—I. THE FORMATION PROCESS OF CYCLOBUTYLPYRIMIDINE DIMERS: CROSS SECTIONS, ACTION SPECTRA and QUANTUM YIELDS

Abstract— We have determined the dimerization and monomerization cross sections of the Thy < > Thy (cyclobutyl dimer of thymine and thymine) and the Cyt < > Thy (cyclobutyl dimer of cytosine and thymine) dimers in Escherichia coti [³H]-DNA ([³H]-thymine labeled DNA) at five wavelengths in the range 240–300 nm. It may be concluded from the dimerization action spectra for the two dimers that the excitation of Thy (thymine) is mainly responsible for the photochemical dimerization reaction in both cases. The calculated quantum yields of dimerization and monomerization are also presented in this paper and several questions, raised by the results obtained at 300 nm, are discussed.     

LEAKAGE OF 86Rb+ AFTER ULTRAVIOLET IRRADIATION OF Escherichia coli K-12

Abstract— Stationary phase cultures of a DNA repair proficient Escherichia coli K-12 strain showed a release of intracellular material as assessed by three different methods (260 nm absorption; [methyl-³H]thymidine leakage and ⁸⁶Rb⁺ leakage) after broad-band (Black-Light Blue) near-UV radiation but not after far-UV (254 nm) radiation. As a control response for membrane damage to cells, this leakage of intracellular material was also determined by each method after mild-heat (52°C) treatment of E. coli K-12. An action spectrum for the release of ⁸⁶Rb⁺ from E. coli K-12 after irradiation with monochromatic wavelengths, from 254 to 405 nm, is also presented. The action spectrum for lethality (F₃₇ values) obtained for this strain, shows that leakage of ⁸⁶Rb⁺ occurs at fluences equivalent to or slightly less than fluences causing inactivation at wavelengths above 305 nm. In contrast, at wavelengths below 305 nm, leakage of ⁸⁶Rb⁺ from irradiated cells can be induced but only at fluences significantly greater than was required to cause cell inactivation. These results indicate, therefore, that near-UV radiation can induce a damaging effect on the cell's permeability barrier which may be significant in causing the death of the cell, whereas the effect is not significant in causing the death of cells by far-UV radiation where DNA damage is known to be the main cause of lethality.     

4',5'-SUBSTITUTED METHYLANGELICINS: PHOTOCYCLOADDUCTS WITH PYRIMIDINE BASES OF DNA

The isolation and characterization of photocycloadducts with pyrimidine bases from DNA samples irradiated (365 nm) in the presence of four 4',5'-substituted methylangelicins was performed. All these furocoumarins yielded mainly the cis-syn furan-side cycloadduct with thymine. For 4',5'-dimethyl-, 5,4',5'-trimethyl- and 6,4',5'-trimethylangelicin this adduct was accompanied by two pyrone-side adducts ( cis-syn and cis-anti ), whereas the 4,4',5'-trimethyl derivative gave the furan-side adduct with cytosine.
The characterization of the regio- and stereochemistry of the adducts was accomplished by ¹H NOE (nuclear Overhauser effect) and ¹H-¹³C HMBC (heteronuclear multiple-bond connectivity) spectroscopies.
The formation of different cycloadducts in DNA by the various derivatives highlights the role of the methyl groups in determining the regio- and stereochemistry of the cycloaddition.