(1/82) Intercalation of proflavine and a platinum derivative of proflavine into double-helical Poly(A).
The equilibria and kinetics of the interactions of proflavine (PR) and its platinum-containing derivative [PtCl(tmen)(2)HNC(13)H(7)(NHCH(2)CH(2))(2)](+) (PRPt) with double-stranded poly(A) have been investigated by spectrophotometry and Joule temperature-jump relaxation at ionic strength 0.1 M, 25 degrees C, and pH 5.2. Spectrophotometric measurements indicate that base-dye interactions are prevailing. T-jump experiments with polarized light showed that effects due to field-induced alignment could be neglected. Both of the investigated systems display two relaxation effects. The kinetic features of the reaction are discussed in terms of a two-step series mechanism in which a precursor complex DS(I) is formed in the fast step, which is then converted to a final complex in the slow step. The rate constants of the fast step are k(1) = (2.5 +/- 0.4) x 10(6) M(-1) s(-1), k(-1) = (2.4 +/- 0.1) x 10(3) s(-1) for poly(A)-PR and k(1) = (2.3 +/- 0.1) x 10(6) M(-1) s(-1), k(-1) = (1.6 +/- 0.2) x 10(3) s(-1) for poly(A)-PRPt. The rate constants for the slow step are k(2) = (4.5 +/- 0.5) x 10(2) s(-1), k(-2) = (1.7 +/- 0.1) x 10(2) s(-1) for poly(A)-PR and k(2) = 9.7 +/- 1.2 s(-1), k(-2) = 10.6 +/- 0.2 s(-1) for poly(A)-PRPt. Spectrophotometric measurements yield for the equilibrium constants and site size the values K = (4.5 +/- 0.1) x 10(3) M(-1), n = 1.3 +/- 0.5 for poly(A)-PR and K = (2.9 +/- 0.1) x 10(3) M(-1), n = 2.3 +/- 0.6 for poly(A)-PRPt. The values of k(1) are similar and lower than expected for diffusion-limited reactions. The values of k(-1) are similar as well. It is suggested that the formation of DS(I) involves only the proflavine residues in both systems. In contrast, the values of k(2) and k(-2) in poly(A)-PRPt are much lower than in poly(A)-PR. The results suggest that in the complex DS(II) of poly(A)-PRPt both proflavine and platinum residues are intercalated. In addition, a very slow process was detected and ascribed to the covalent binding of Pt(II) to the adenine. (+info)
(2/82) Low dielectric response in enzyme active site.
The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of alpha-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. (+info)
(3/82) Specific binding of Hoechst 33258 to site 1 thymidylate synthase mRNA.
The translational initiator codon in thymidylate synthetase (TS) mRNA is located in a stem-loop structure with a CC bubble. TS is an important target for anticancer drugs. Aminoglycoside antibiotics have been shown to specifically bind to TS mRNA site 1 constructs and, furthermore, specific binding requires the non-duplex CC bubble region. It is shown here that DNA intercalating agents and DNA minor groove-binding drugs also bind to a TS mRNA site 1 construct. This binding is competitive with aminoglycosides, suggesting that the binding sites overlap. Hoechst 33258 binds with a dissociation constant of 60 nM, a value significantly lower than the approximately 1 microM values found for aminoglycosides. Footprinting and direct binding studies show that the CC bubble is important for binding of the Hoechst compound. However, the exact structure of the bubble is unimportant. Interestingly, mutations in regions adjacent to the bulge also affect binding. These studies point to the important role of non-duplex RNA structures in binding of the DNA minor groove binder Hoechst 33258. (+info)
(4/82) Ozonation of mutagenic and carcinogenic polyaromatic amines and polyaromatic hydrocarbons in water.
The Salmonella-microsome assay for mutagenesis was used to determine the effect of ozone on the mutagenesis of selected carcinogens and mutagens in water. Short periods of ozonation were shown to completely inactivate the mutagenicity of several polyaromatic amine mutagens including acriflavine, proflavine, and beta-naphthylamine. Selected polyaromatic hydrocarbons were also sensitive to ozonation. Kinetic studies revealed that the mutagenicity of benzo(a)pyrene, 3-methylcholanthrene, and 7,12-dimethylbenz(a)anthracene was destroyed after short periods of ozonation. To correlate loss of mutagenicity with loss of carcinogenicity, two polyaromatic hydrocarbons were treated with ozone, extracted from water with hexane, and tested for carcinogenicity in mice. When 7,12-dimethyl-benz(a)anthracene and 3-methyl-cholanthrene were treated with ozone, there was a substantial reduction in carcinogenicity compared to control groups treated with oxygen alone. However, a small number of tumors developed in the group of animals receiving a hexane extract of ozonated 7,12-dimethylbenz(a)anthracene. This activity may be due to breakdown products of 7,12-dimethylbenz(a)anthracene that are not mutagenic. (+info)
(5/82) Comparison of the treatment of herpes genitalis in men with proflavine photoinactivation, idoxuridine ointment, and normal saline.
36 male patients with genital infection by HSV confirmed by culture were each allocated to one of three treatment groups: (1) Proflavine photoinactivation, (2) 0.5 per cent. idoxuridine ointment (IDU), (3) Normal saline. They were assessed objectively at each attendance by measurement of the lesions with an operating microscope fitted with a measuring grid in one eyepiece. Material for culture for HSV was taken at each visit; the presence of symptoms (pain, discomfort, and irritation) was noted. The areas of lesions in the proflavine photoinactivation group remained larger significantly longer than in the other groups, the healing time was slower, and HSV could be isolated for longer. It is concluded that proflavine photoinactivation is of no greater value than 0.5 per cent. IDU or normal saline in the treatment of genital infection by HSV in the male. (+info)
(6/82) Effect of DNA delay mutations of bacteriophage T4 on genetic recombination.
Studies have been made of the effect of the DNA delay mutations of bacteriophage T4 on growth and genetic recombination in a number of Escherichia coli hosts. DNA delay mutations in genes 39, 52, 58 (61), and 60 result in abnormally high recombination frequencies. These high recombination frequencies are discussed in the context of other observations. (+info)
(7/82) Intrinsic and extrinsic light responses of Salmonella typhimurium and Escherichia coli.
Exposure to intense light in the region between 390 and 530 nm has been shown to have three effects on the motility of Salmonella typhimurium and Escherichia coli. Short pulses of light initiate continuous tumbling. Longer exposures to light induce smooth swimming, and prolonged exposures induced paralysis. The tumbling response is intimately connected with the chemical gradient-sensing apparatus of the bacterium and can be overcome by strong temporal gradients of attractant. Some mutants of S. typhimurium which are defective in the tumble-generating mechanism for chemotaxis are also unable to tumble in intense light. This intrinsic light effect can be mimicked by the addition of external dyes (the classical photodynamic effect), but it can be shown that the two phenomena are distinct. The extrinsic (photodynamic) effect can be inhibited by histidine or by anaerobic conditions, whereas the intrinsic effect is not. The observation that the extrinsic effect can also produce the three types of light responses listed above suggests a common pathway after an intial event on either an endogenous or an externally added photoreceptor. (+info)
(8/82) Effect of acriflavin on the kinetoplast of Leishmania tarentolae. Mode of action and physiological correlates of the loss of kinetoplast DNA.
The loss of kinetoplast DNA in Leishmania tarentolae, which occurs in the presence of low concentrations of acriflavin, was found to be a result of selective inhibition of replication of this DNA. Nuclear DNA synthesis was relatively unaffected and cell and kinetoplast division proceeded normally for several generations. An approximately equal distribution of parental kinetoplast DNA between daughter kinetoplasts resulted in a decrease in the average amount of DNA per kinetoplast. The final disappearance of the stainable kinetoplast DNA occurred at a cell division in which all the remaining visible kinetoplast DNA was retained by one of the daughter cells. The selective inhibition of kinetoplast DNA synthesis was caused by a selective localization of acriflavin in the kinetoplast. The apparent intracellular localization of dye and the extent of uptake at a low dye concentration could be manipulated, respectively, by varying the hemin (or protoporphyrin IX) concentration in the medium and by adding red blood cell extract (or hemoglobin). Hemin and protoporphyrin IX were found to form a complex with acriflavin. During growth in acriflavin, cells exhibited an increasing impairment of colony-forming ability and rate of respiration. No change in the electrophoretic pattern of total cell soluble proteins was apparent. The data fit the working hypothesis that the loss of kinetoplast DNA leads to a respiratory defect which then leads to a decrease in biosynthetic reactions and eventual cell death. A possible use of the selective localization of acriflavin in the kinetoplast to photooxidize selectively the kinetoplast DNA is suggested. (+info)