The wavelength dependence of 8-methoxypsoralen photosensitization of radiation-enchanced reactivation in a mammalian cell-virus system.

The combined effect of 8-methoxypsoralen (8-MOP) and ultraviolet (UV) radiation on the ability of an irradiated mammalian cell (CV-1) to reactivate UV-irradiated mammalian virus (Herpes simplex) was tested. Prior treatment of cells with 8-MOP was found to increase Radiation-Enhanced Reactivation (RER) at one wavelength (297 nm) in the far ultraviolet but not at others (240-289 nm). This same treatment induced RER in the near UV (302-370 nm) and the visible region (380-400 nm). An action spectrum for the photo-sensitized induction of this cellular parameter was obtained. This action spectrum is consistent with the absorption spectrum for 8-MOP and the theory that damage to DNA is, at least in part, responsible for Radiation-Enhanced Reactivation.     

Interpretation of the absorption and circular dichroic spectra of oriented purple membrane films.

The absorption and circular dichroic (CD) spectra of purple membrane films in which the plane of the membranes is oriented perpendicular to the incident beam are compared with the solution spectra. This enables one to relate structural features of the purple membrane to a coordinate system as defined by a normal to the membrane plane and two mutually perpendicular in-plane axes. The film and solution absorption spectra were similar except for a relative depression in the 200 - 225-nm region of the film spectrum. However, the CD spectra showed significant differences in the visible region, where the biphasic band in the solution spectrum was replaced by a single positive band at 555 nm in the film spectrum and in the far ultraviolet region, where the 208-nm band was deleted from the film spectra of the native and regenerated membranes. Moreover, a small shoulder occurred at 208 nm in the film spectrum of the bleached membrane. The near ultraviolet spectra also showed differences, whereas the 317-nm band remained essentially the same for both spectra. Based on excitonic interpretations of the visible and far ultraviolet spectra the following conclusions were reached: (a) a relatively strong in-plane monomeric interaction occurs between te retinyl chromophore and apoprotein; (b) the helical axes of the native and regenerated membrane proteins are oriented primarily normal to the membrane plane; and (c) the helical axes of the bleached membrane proteins are tilted more in-plane than the axes of the native or regenerated membrane. Additional conclusions were that an interaction occurs between an in-plane magnetic dipole moment of the retinyl chromophore and probably an in-plane electric dipole moment of a nearby aromatic amino acid(s), and that although the membrane is anisotropic with respect to coupling between electric and magnetic moments of the aromatic amino acids, the transition dipole moments of the aromatic amino acids are not preferentially oriented in either direction.     

Circular dichroism and the conformational properties of soybean beta-amylase

The conformational properties of soybean β-amylase were investigated by the circular dichroism probe and measurement of enzyme activity. The enzyme exhibited a positive circular dichroism band at 192 nm, a negative band at 222 nm, and a shoulder near 210 nm. Analysis of the spectrum in the far ultraviolet zone indicated the presence of approximately 30% of α helix and 5–10% of β-pleated sheet, the rest of the polypeptide main chain possessing aperiodic structure. In the near ultraviolet reagion, the enzyme protein showed at least six positive peaks at 259, 265, 273, 281, 292, and 297 nm. The positive bands at 292 and 297 nm remained unaltered on acetylation of the enzyme by N-acetylimidazole and were assigned to tryptophanyl chromophores. These bands were affected in intensity in the presence of maltose or cycloheptaamylose, which indicates that some tryptophan residues are situated at the binding sites. The native conformation of soybean β-amylase was found to be sensitive to pH variation (below pH 5 and above pH 10), sodium dodecyl sulfate, guanidine hydrochloride, and heating to 50–55 °C. Complete disorganization of the secondary structure was attained by 6 m guanidine hydrochloride. Sodium dodecyl sulfate was effective in disturbing the tertiary structure of the enzyme but did not affect significantly the secondary structure. Enzymatic inactivation was paralleled by the decrease of circular dichroism bands in the near ultraviolet region as produced by the denaturants. It is concluded that the uniquely folded structure of the enzyme contains some less rigid domains and a rigid core stabilized by hydrophobic interactions, electrostatic interactions, and hydrogen bonds.     

Cholera toxin does not impair hormonal inhibition of adenylate cyclase and concomitant stimulation of a GTPase in adipocyte membranes

Factor H of the human complement system exhibits an unusual circular dichroism spectrum. The CD spectrum of Factor H exhibits a positive extreme at 230 nm and a negative extreme at 190 nm. No apparent alpha-helical or beta-sheet conformations were present in the native protein structure. However, when the disulfide bridges are reduced, followed either by reoxidation or alkylation, the structure of Factor H is modified so that it now exhibits conventional protein secondary structure as determined from its CD spectra in the far ultraviolet region. Factor H also fails to mediate its regulatory function of inhibiting the alternative pathway convertase once the disulfides have been ruptured and conformational rearrangement has occurred. CD studies indicate that minor conformational changes take place when Factor H and C3b associate in free solution.     

Heme-linked spectral changes of the protein moiety of hemoproteins in the near ultraviolet region

Spectral changes of hemoproteins in the near ultraviolet region on binding to a ligand and on oxidation-reduction of the heme-iron were studied by computer-controlled spectrophotometry. Near ultraviolet difference spectra between the low spin and high spin forms of ferric hemoproteins were classified into three groups: Those showing two absorption peaks having maxima at around 285 and 295 nm, those showing a peak at around 275 nm, and those showing a peak at around 300 nm. No corresponding absorption peak was observed with model heme complexes of low molecular weight. The intensity of the peak in cyanide difference spectra of catalase and horseradish peroxidase in the near ultraviolet region was dependent on the concentration of added cyanide and paralleled the intensity of the spectral changes in the Soret region. The spectral changes in both the near ultraviolet and Soret regions developed within 6 ms after the addition of cyanide. Difference spectra between the reduced and oxidized forms of cytochrome c, cytochrome oxidase-cyanide complex, hemoglobin, and lactoperoxidase-cyanide complex showed a characteristic peak at around 285-290 nm. Various difference spectra of hemoglobin in the near ultraviolet region were also measured. The observed positions, shapes, combinations, and relative intensities of the peaks were compared with those of solvent perturbation difference spectra and pH difference spectra of proteins and aromatic amino acids and also with the diacetylchitobiose-induced difference spectrum of lysozyme. The kinds of aromatic amino acid residues possibly responsible for the observed difference peaks were discussed on the basis of the results of the comparison. Based on the results obtained, the common occurrence of a heme-linked functional response of the hemoprotein conformation was suggested.     

Effect of far and near ultraviolet radiation on the DNA content in the unicellular flagellate alga Dunaliella salina]

Effects of far (254 nm) and near (above 300 nm) ultraviolet radiation on DNA in Dunaliella salina were compared. DNA synthesis was delayed by both the kinds of UV radiation applied in isoeffective doses (ld0 and LD90), but the extent of inhibition of DNA synthesis was much stronger after action of far UV rays.     

Discovery of signaling effect of UV-B/C light in the extended UV-A/blue-type action spectra for step-down and step-up photophobic responses in the unicellular flagellate algaEuglena gracilis

Summary Cultures of unicellular algal flagellateEuglena gracilis grown in different conditions were subjected to action spectroscopy for step-down and step-up photophobic responses, respectively. The spectral region was extended into the UV-B/C as well as in the UV-A and visible regions with the Okazaki Large Spectrograph as the monochromatic light source. The photophobic responses of the cells were measured with an individual-cell assay method with the aid of a computerized video motion analyzer. In the UV-A and visible regions, the shapes of the action spectra were the so-called UV-A/blue type. In the newly studied UV-B/C region, new action peaks were found at 270 nm for the step-down response and at 280 nm for the step-up one. The absorption spectrum of flavin adenine dinucleotide (FAD) appeared to fit the action spectrum for the step-up response, whereas the shape of the step-down action spectrum, which has a UV-A peak (at 370 nm) higher than the blue peak (at 450 nm), appeared to be mimicked by the absorption spectrum of a mixed solution of 6-biopterin and FAD. These observations might also account for the fact that the UV-B/C peak wavelength at 270 nm of the action spectrum for the step-down response is shorter by 10 nm than the action spectrum for the step-up response at 280 nm.Abbreviations FAD flavin adenine dinucleotide - FWHM spectral full width at half maximum - NIBB National Institute for Basic Biology - OLS Okazaki Large Spectrograph - PFB paraflagellar body - UV-A ultraviolet light of spectral region between 320 and 400 nm - UV-B/C ultraviolet light of spectral region between 190 and 320 nm     

Natural DNA sequences can form left-handed helices in low salt solution under conditions of topological constraint

The conformation of DNA that originates from association of complementary single-stranded circles (form V DNA) is investigated in solution at low salt concentration. It is shown that circular dichroism extended to the far ultraviolet region (down to 165 nm) represents a powerful tool for determination of the handedness of double helical DNAs in solution. The positive intense band at 186 nm followed by a strong negative band around 170 nm is characteristic of all right-handed helical forms (B,A) of DNA, whereas the circular dichroism spectrum of the Z form of poly[d(G-C)] of opposite helical sense represents a quasi inversion of these far ultraviolet bands. Thus, form V DNA is found to represent a co-existence of left-handed Z-type and right-handed B double helical stretches in addition to negative superturns. The Raman spectrum of form V DNA provides further support for the contribution of a left-handed double helical conformation, as shown by comparison to the high resolution Raman spectra of poly[d(G-C)] in the Z and B forms.The analysis of present spectroscopic data and the analysis of occurrence of alternating [d(G-C)] purine-pyrimidine sequences in the form V DNA used strongly suggest that in DNA of natural sequence, topological constraint may generate left-handed double helices, a conformation thought so far to be limited to the alternating [d(G-C)] sequences. Such structure could play a role in recognition and regulation of gene expression.     

Corynebacterium sarcosine oxidase: a unique enzyme having covalently-bound and noncovalently-bound flavins

A sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating), EC, 1.5.3.1) was purified to homogeneity from Corynebacterium sp. U-96 by the use of ionic exchange chromatographies and gel filtrations. The enzyme contained two mol FAD per mol enzyme (one covalently-bound and one noncovalently-bound; mol. wt., 174,000). The “semiapoenzyme”, which contains the covalently-bound FAD alone, was prepared by the acid-ammonium sulfate treatment. The semiapoenzyme had practically no activity for sarcosine oxidation, but retained intact back-bone structure judging from the circular dichroic spectrum in the far ultraviolet region. On the contrary, the circular dichroic spectrum of the semiapoenzyme in the visible region (a large negative band around 443 nm) was quite distinct from that of the holoenzyme (positive bands at 387, 456 and 489 nm).     

Circular dichroism studies on coat proteins of some strains and mutants of tobacco mosaic virus

1. Tobacco mosaic virus (TMV) protein has in near ultraviolet a complex but well resolved circular dichroism (CD) spectrum at room temperature. There are seven positive bands at 248, 252, 257, 265, 274, 281 and 291 nm, and a negative one at 296 nm. The CD spectrum is pH-dependent. The shape of the pH-dependence curves and the comparison with CD spectra of model compounds suggest that the bands at 248, 252 and 257 nm are mainly caused by phenylalanyl, those at 265, 274 and 281 nm by tyrosyl, and those at 291 and 296 nm by tryptophanyl side chains. 2. Only insignificant changes of the tertiary structure seem to occur between pH 6.5 and 8.5. Changes in ellipticity of TMV protein during the pH-induced polymerization reaction suggest that: (1) tyrosyl residues are involved in the binding of subunits, (2) phenylalanyl residues seem to be transferred to a less rigid environment, and (3) tryptophanyl residues are not essential for the reaction. 3. The proteins of several TMV strains and mutants studied have similar far ultraviolet CD spectra and apparently do not differ significantly in their structure. Their near ultraviolet CD spectra are, however, different. Replacements involving aliphatic amino acids do not change considerably the near ultraviolet CD spectra. On the other hand, replacements involving aromatic amino acids have a great effect on the spectra rendering possible identification of CD bands and recognition of the aromatic amino acid residues responsible for optical activity.     

The relative importance of ultraviolet and longer wavelengths in the response properties of fly visual systems

A generalized analysis of the generator potential responses of R1-6 cells of Calliphora provides remarkable information on the visual properties for the Diptera. This shows that, although these cells have two peak response sensivities for monochromatic stimuli at 350 and 480 nm under single color stimulus conditions, and when the background illumination is either zero or in the region of 450–560 nm, the sensitivity to ultraviolet light is practically eliminated for background illumination in either the ultraviolet or the region around 600 nm or when any simultaneous dynamic stimulus in the region of 480–550 nm is also applied. These results seem somewhat perplexing to an understanding of the behavioral vision properties. It also is not consistant with the concept that the ultraviolet response is initiated by a sensitizing pigment within these cells that transfers energy to the rhodopsin-metarhodopsin process. However, it strengthens other evidence that the limited condition of ultraviolet responses comes from interaction from R7,8 cells but does not play an important behavioral role in the visual system fed from cells R1-6. As discussed in this paper, any high level pattern recognition controlling behavioral response to ultraviolet stimuli comes from the R7,8 cell system.     

Peculiar ultraviolet absorbing moieties of human osteoarthritic cartilage

The ultraviolet absorbing components of human cartilage have been measured by microspectrophotometry. The characteristics of the chondrocytes appeared to be identical, irrespective of the pathology. However the matrix of osteoarthritis cartilage contained components that absorbed maximally in the region of 270 to 250 nm; such components were not found in the matrix of cartilage of non-arthritic joints. Substances that absorb maximally in this region of the ultraviolet could generate free radicals.     

Circular dichroism of cephalopod rhodopsin and its intermediates in the bleaching and photoregeneration process.

In the bleaching process of cephalopod rhodopsin, a new intermediate was found in the conversion process from lumirhodopsin to metarhodopsin. This intermediate of octopus has an absorption peak at about 475 nm and has been named as M475. The circular dichroism value of M475 is too small to be evaluated. On the other hand, lumirhodopsin shows a negative CD at 470 nm, a positive CD at 350 nm and a large positive CD band with three peaks at 280, 287 and 295 nm. Such a large CD band in the ultraviolet region is not observed in rhodopsin, M475 and metarhodopsin. This CD seems to be mainly due to tryptophan and tyrosine residues restricted in free rotation in the protein moiety of lumirhodopsin. The intermediate in the photoregeneration process of cephalopod rhodopsin, P380, has a positive CD band at the main peak, 380 nm, and also a large positive CD band in the ultraviolet region like lumirhodopsin.     

The effects of light intensity and spectral quality on growth and shoot initiation in tobacco callus

The effects of eight different narrow band-emitting fluorescent lamps (371-750 nm) and four commercial broad band-emitting fluorescent sources upon growth and shoot initiation in tobacco callus (Nicotiana tabacum var. Wisconsin 38) have been characterized. Wavelength and intensity are equally important parameters in determining morphogenic changes. Near ultraviolet light (371 nm) was found to stimulate (0.024 mw/cm²) or inhibit (above 0.15 mw/cm²) callus growth and shoot initiation, depending on the light intensity. Stimulation of growth and shoot production occurs also in blue light region, but at higher intensity than in the near ultraviolet. Red and far red light (up to 1.7 mw/cm²) do not appear to affect callus growth or stimulate shoot initiation. The enhancement of callus growth and the stimulation of shoot initiation are controlled by the same near ultraviolet-absorbing photoreceptor system present in a small enough concentration that it cannot be recognized in the absorption spectrum of the intact tissue. Carotenoids, porphyrins, and phytochrome associated with the high irradiance response do not appear to qualify as the photoreceptor. Flavonoids are possible candidates. Radiation emitted by fluorescent lamps outside the near visible region was determined, and we concluded that energy levels were not sufficient to affect the reported results. The spectral output of several commercial lamps in the visible and near visible regions is such that there could be different effects on growth and development of tissue cultures.     

Circular dichroism of mammalian follitropins and the effects of treatment with N-bromosuccinimide

The circular dichroism of the tryptophan containing glycoprotein hormone, follitropin, displays bands in the near ultraviolet which are absent in homologous, tryptophan-free hormones. In the far ultraviolet, the dichroism is very similar to the other glycoprotein hormones with little or no indication of α-helix. The single tryptophan of follitropin is in a domain of the β subunit sequences of these hormones which is highly conserved from hormone to hormone. Without prior dissociation of the follitropin into subunits, no change is seen in circular dichroism, absorption at 280 nm, fluorescence emission or hormonal activity after treatment with N-bromosuccinimide. In contrast, these properties change when intact human lutropin is studied; its tryptophan residue is a position different than in follitropin. These results support the proposal that the domain containing the tryptophan in follitropin is in or near a region of subunit-subunit contact in the glycoprotein hormones.     

Spectral sensitivity in the eyes of male and female Lutzomyia longipalpissandflies

Abstract. Using electroretinogram recordings, the response of Lutzomyia longipalpis sandfly eyes to a range of wavelengths of light was measured, and spectral sensitivity determined. The eyes of both male and female adult sandflies were found to respond maximally to light in the ultraviolet region (at 340 nm) with a secondary peak in the blue-green-yellow region at 520 nm for females and 546 nm for males. The Mann-Whitney U test showed no significant differences between males and females at corresponding wavelengths.     

Interaction of proteins with detergents: Binding of cationic detergents with lysozyme

Binding studies of cationic detergents such as cetyl trimethylammonium bromide, Cetylpyridinium bromide and dodecyl trimethylammonium bromide with lysozyme were carried out by equilibrium dialysis, ultraviolet difference and circular dichroism techniques at 25 C. Binding isotherms at pH 5·0, 7·0 and 9·0 show cooperative binding at all concentrations of detergents and the number of available binding sites in lysozyme increases with pH. Gibbs free energy of binding calculated on the basis of Wymans’ binding potential concept increases with pH indicating increased binding strength at higher pH. The ultraviolet difference spectra of the detergent complexes with lysozyme at pH 7·0 and 9·0 in the region of 250–300 nm indicate the involvement of aromatic amino acid residues as probable binding sites and also the carboxylate groups since the binding is cooperative. The circular dichroism spectra also indicate the involvement of aromatic amino acid residues in the binding of these detergents. This is substantiated by the decrease in the intensity of the aromatic positive bands in the near ultraviolet region. The increase in the magnitude of [θ]_{222 nm} values in the far ultraviolet region with the increase in the concentration of the detergent in the complex indicates conformational changes resulting in an increase of α-helical content producing a more ordered structure of lysozyme.These binding studies show that at pH 7·0 and 9·0, hydrophobic interactions play a major role, while at pH 5·0 only electrostatic interactions play prominent role in the binding of these detergents. Paper presented at the International Symposium on Biomolecular Structure and Interactions held at the Molecular Biophysics Unit, Indian Institute of Science, Bangalore, during 17–22, December 1984.     

Perithecial Formation in Gelasinospora reticulispora: IV. Action Spectra for the Photoinduction

Action spectra for photoinduction of perithecia after different lengths of dark period were determined with apically growing mycelium of a sordariaceous fungus Gelasinospora reticulispora. When hyphae were exposed to monochromatic light in near ultraviolet and visible regions, reciprocity between intensity and exposure time was observed within the range of incident energy used. The resulting action spectrum determined after a dark period of 48 hours showed a peak at 460 nm with shoulders at 420 and 480 nm and another peak at 370 nm, indicating minima at 410, 430, and 470 nm. After 72 hours darkness the spectrum was very similar to the above, except that the major peak shifted to 450 nm and the near ultraviolet region was somewhat less effective. In both cases, wavelengths longer than 520 nm showed no effect.     

Intensity and spectral emission as factors affecting the efficacy of an insect electrocutor trap towards the house-fly

Competitive tests were used to determine how the quantitative and spectral characteristics of an electrocutor trap light source affected the attraction of the house-fly, Musca domestica L. It was found that an increase in the radiant flux (F_e) of the trap lamps due to an increase in radiant area (A), caused a much larger increase in catch than if radiant flux was increased through higher radiant emittance (M_c). The results from electroretinograms recorded in response to different levels of M_e were consistent with the idea that at a given wavelength the attractiveness of a lamp is attributable to the quantitative output perceived by the fly. Of nine fluorescent lamps, the most attractive had peak emission at 340 nm. A blue lamp (peak emission 419 nm) attracted less than a third as many flies as the UV emitting lamps, and a white lamp (peak emission 585 nm) attracted fewer than a quarter as many. The corresponding photoreceptor responses were measured using the electroretinogram. At wavelengths above 400 nm the attractiveness of a lamp to a fly appears to be lower relatively than the photoreceptor response. Within the ultraviolet region (300 nm–400 nm) attractiveness is again attributable to the quantitative output perceived by the fly. It is concluded that there is a genuine behavioural preference for lamp emissions in the ultraviolet region.     

Effect of counterions on the conformation of hyaluronic acid.

To study the effect of metal ions on the conformation of hyaluronic acid, circular dichroism (CD) and optical rotatory dispersion (ORD), along with viscosity measurements of the Na, Li, Ca, and Mg salts of the polymer, were carried out. With divalent cations, the results show a decrease in CD minima at 210 nm and an increase in ORD troughs at 220 nm, as compared to monovalent ions. To account for this behavior, the ORD in the visible range corresponding to the observed CD bands was directly calculated from the Moscowitz equation using Kronig-Kramer's transform. The background rotation was found to be more levorotatory in bivalent than in monovalent cations. The ORD spectra of various metal hyaluronates differ significantly from each other in the far ultraviolet region, especially at lower pH values. The values of intrinsic viscosities of these hyaluronates, on the other hand, are almost the same in the pH range of 1–3. These results indicate a local conformation variation rather than any appreciable change in the chain conformation of the molecule in the presence of different counterions.