Nonspecific interaction of Escherichia coli pyrenyl RNA polymerase holoenzyme with synthetic polynucleotides as monitored by fluorescence spectroscopy

A derivative of RNA polymerase containing approximately 2 pyrene equiv per enzyme molecule has been used to study the interaction of RNA polymerase with poly[d(A-T)].poly[d(A-T)] and poly[d-(G-C)].poly[d(G-C)]. As monitored by fluorescence spectroscopy, pyrenyl RNA polymerase displays a unique set of conformational changes with each synthetic polynucleotide as a function of temperature. An increase in the fluorescence intensity was observed for both polynucleotides at 5 degrees C. A decrease was observed in the case of poly[d(A-T)].poly[d(A-T)] at 25 and 37 degrees C, whereas no discernible perturbation was observed in the case of poly[d(G-C)].poly[d(G-C)]. Different salt dependencies were observed for the interaction of pyrenyl RNA polymerase with these polynucleotides at 5 and 25 degrees C. Further characterization of these interactions as well as correlation of the observed fluorescence changes to the corresponding open and closed complexes was carried out with heparin. The interaction between pyrenyl RNA polymerase and poly[d-(A-T)].poly[d(A-T)] at 25 degrees C was quantified by using two different methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of ribosomal RNA cistron number in a strain of Neurospora crassa with a duplication of the nucleolus organizer region

Some progeny from a cross of the translocation mutant T(VL→IVL)AR33 with wild-type Neurospora crassa are double nucleolus organizer (DNO) strains, usually displaying two distinct nucleolus organizer regions. The DNO strain is sterile but displays the same growth response as normal laboratory strains of Neurospora. We used DNA-DNA hybridization techniques to quantify the number of rRNA cistrons in the DNO mutant and its vegetative progeny. Comparisons of the rate of hybridization of genomic DNA from the parental AR33 strain and from the DNO strain showed that hybridization was more rapid for the DNO strain than for the parental strain. Successive vegetative progeny of the DNO strain displayed hybridization rates intermediate to those of the original DNO strain and the parental single nucleolus strain, indicating that the number of rRNA cistrons had decreased during vegetative propagation. Estimates of rRNA cistron number obtained from comparisons of the amount of single copy DNA and rDNA hybridized to genomic DNO and AR33 DNA at saturation indicate that the parental AR33 strain contains 225 copies of the rRNA repeat unit, while the DNO strain has approx. 440 copies. The number of rRNA cistrons decreases gradually in the successive vegetative progeny, approximating the parental haploid value by the eleventh vegetative transfer.     

Denaturation and renaturation studies of benzo[a]pyrene metabolite modified DNAs

Evidence from absorbance, fluorescence, and circular dichroism (CD) measurements strongly suggests that adduct conformations at the binding sites are grossly different before and after thermal denaturation of (+)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]py ren e [(+)-anti-BPDE] modified DNAs. This conclusion is reached through the following observations: (1) upon melting and cooling, the (+)-anti-BPDE-modified DNA exhibits pronounced hypochromic effects with concomitant spectral red shifts for the pyrenyl absorbance; (2) the pyrenyl CD spectrum reverses sign upon thermal denaturation-renaturation; (3) the fluorescence emission spectra resulting from excitations at 353 nm (10 nm to the red of hydrolyzed and unbound anti-BPDE) exhibit enhanced intensities and spectral red shifts for the thermally denatured and cooled adducts; and (4) in contrast to the absence of a shoulder prior to melting, the postmelt adducts exhibit a prominent 355-nm maximum (evidence of stacking interactions) in the excitation spectrum when 384-387-nm emission is monitored. Studies with synthetic polynucleotides further reveal that (+)-anti-BPDE-modified poly(dG).poly(dC) exhibits the greatest nonreversible renaturation at the binding sites, possibly as a consequence of pyrenyl self-stacking. This, coupled with the previous findings that this polymer suffers the most extensive (+)-anti-BPDE modification, appears to suggest that (dG)n . (dC)n regions may be responsible for such observed effects in native DNA.     

Side reaction of pyrenylalanine-peptides containing NG-tosylarginine during detosylation with hydrogen fluoride

Formation of by-products of pyrenylalanine-peptides was observed during the cleavage of a tosyl group in pyrenylalanine-peptides containing an Arg(Tos) residue with HF. NMR and fluorescence experiments showed that by-products were compounds in which a pyrenyl group was modified with a tosyl group(s). The side reaction was little suppressed by the addition of usual scavengers such as anisole, 1,2-ethanedithiol, thioanisole and p-cresol. Under the conditions used for the cleavage of N alpha-Boc group, i.e., trifluoroacetic acid or 4 M HCl in dioxane, the pyrenylalanine residue was stable. No side reaction was, furthermore, observed in the reductive cleavage with sodium in liquid ammonia. These results suggest that the side reaction may be due to an electrophilic attack of tosyl cations to the electron-rich pyrenyl group in the pyrenylalanine residue under the HF-mediated acidic conditions.     

Conformational behavior of polyethylenimine derivatives revealed by fluorescence spectroscopy

Conformation behavior of polyethylenimine has been examined by studies of the fluorescence characteristics of derivatives of the polymer containing pyrenyl ligands. Excimer formation within the macromolecular matrix serves as a sensitive probe of group proximities. The experimental observations combined with nearest neighbor analyses lead to quantitative assessments of the extent of interaction between polymer side chains.     

Exchange dynamics of nitric oxide in the human nose.

Nasal nitric oxide (NO) exchange dynamics are poorly understood but potentially are of importance, inasmuch as they may provide insight into the NO-related physiology of the bronchial tree. In healthy human volunteers, NO output was assessed by isolating the nasal cavity through elevation of the soft palate and application of tight-fitting nasal olives. Mean NO output was 334 nl/min and was a positive function of gas flow. With the use of a mathematical model and the introduction of nonzero concentrations of NO, the diffusing capacity for NO in the nose (DNO) and the mucosal NO concentration (Cw) were determined. DNO ranged from 0.52 to 2.98 x 10(-3) nl x s(-1) x ppb(-1) and Cw from 1,236 to 8,947 ppb. Cw declined with increasing gas flow, while DNO was constant. NO output declined with luminal hypoxia, particularly at oxygen tensions <10%. Measurement of nasal DNO and Cw is easy using this method, and the range of intersubject values of Cw raises the possibility of interindividual differences in NO-dependent nasal physiology.     

Parinaroyl and pyrenyl phospholipids as probes for the lipid surface layer of human low density lipoproteins

A simple protocol employing lipid transfer proteins was developed to label human low density lipoprotein (LDL) in a controlled manner with parinaroyl and pyrenyl phosphatidylcholines. In order to study the lipid fluidity in the surface lipid layer of LDL, the temperature-dependence of both polarization (parinaroyl probes) and excimer to monomer (E/M) intensity ratio (pyrenyl probes) were analyzed. A series of pyrenyl phosphatidylcholines containing a pyrenyl fatty acid varying from 6 to 14 carbons in length at the sn-2 position were inserted into LDL to investigate the lateral distribution of different phosphatidylcholines in the lipoprotein surface at 37 degrees C. Both polarization and E/M vs. temperature plots displayed discontinuities in the region of 22-32 degrees C, which coincides with the melting of the neutral lipid core, indicating that the latter induces an ordered to more disordered phase transition in the surface lipid layer. Determination of the E/M intensity ratio as a function of pyrene lipid concentration in LDL showed a linear relationship for the pyrenyl hexanoate and octanoate species, whereas a slope discontinuity was observed for the lipids containing a longer pyrenyl chain. These data suggest that two lipid domains with distinct properties exist in the surface layer and secondly, pyrenyl lipids partition between these domains in a chainlength-dependent manner. This is consistent with measurement of the tryptophan to pyrene energy transfer efficiency vs. pyrenyl lipid concentration, which showed a biphasic relationship for the long-chain pyrenyl lipids. These measurements further indicate that two surface lipid domains correspond to the protein-lipid boundary and the bulk lipid phase, respectively. The fact that relatively small changes in chainlength have a marked influence on the partitioning of pyrenyl lipids between the boundary and the bulk phase suggests also that native phospholipid species may not be randomly distributed in the surface lipid layer of LDL.     

A spectrofluorimetric investigation of calf thymus DNA modified by BP diolepoxide and 1-pyrenyloxirane

7β,8α-Dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP diolepoxide, $\text{1}$) and 1-pyrenyloxirane ($\text{2}$) bind chemically to calf thymus DNA. The fluorescence efficiency of pyrenyl groups in mutagen modified DNA varies appreciably with its conformation and decreases in the order: pyrenees, modified denatured DNA and modified native DNA. A particularly interesting observation is that the fluorescence efficiency of mutagen modified DNA intensifies substantially upon denaturation. Our results suggest that the pyrenyl groups in mutagen modified DNA are intercalated between the base pairs of DNA. Since both $\text{1}$ and $\text{2}$ are powerful frame-shifting mutagens for S. typhimurium TA-98, the intercalative covalent binding of these compounds to DNA may provide a molecular basis for their mutagenic activity.     

The photophysics of green fluorescent protein: influence of the key amino acids at positions 65, 203, and 222

The three amino acids S65, T203, and E222 crucially determine the photophysical behavior of wild-type green fluorescent protein. We investigate the impact of four point mutations at these positions and their respective combinations on green fluorescent protein's photophysics using absorption spectroscopy, as well as steady-state and time-resolved fluorescence spectroscopy. Our results highlight the influence of the protein's hydrogen-bonding network on the equilibrium between the different chromophore states and on the efficiency of the excited-state proton transfer. The mutagenic approach allows us to separate different mechanisms responsible for fluorescence quenching, some of which were previously discussed theoretically. Our results will be useful for the development of new strategies for the generation of autofluorescent proteins with specific photophysical properties. One example presented here is a variant exhibiting uncommon blue fluorescence.     

pH-dependent fluorescence of uncharged benzothiazole-based dyes binding to DNA.

Uncharged benzothiazole-based dyes were synthesized, and their fluorescence properties were examined. These fluorescent dyes showed an intense fluorescence in aqueous solution in the presence of DNA. In addition, the fluorescence intensities were pH dependent, while those of the positively charged dyes were nearly independent of pH. The pH-dependent photophysical behavior suggests that interaction of protonated dyes with DNA results in high intensities of fluorescent emission.     

Fluorescence characteristics and photophysical parameters of aggregates of light-harvesting chlorophyll a/b complexes

Changes in the fluorescence characteristics and molecular photophysical parameters induced by disaggregation of light-harvesting chlorophyll a/b complexes isolated from pea were studied. Disaggregation was achieved by increasing the concentration of the detergent triton X-100 (concentration range 0-230 microM, chlorophyll concentration 2.45 microg/ml). Laser fluorimetry methods were used to measure the molecular photophysical parameters. It was shown that the decrease in aggregate size is accompanied by a decrease in fluorescence at 700 nm at 77 K, a fall of the singlet-singlet annihilation rate (by more than two orders), and the growth of fluorescence life time (from 160 ps to 3.2 ns).     

Fluorescence characteristics and photophysical parameters of light-harvesting chlorophyll <Emphasis Type="Italic">a/b</Emphasis> complex aggregates

Fluorescence characteristics and molecular photophysical parameters of light-harvesting chlorophyll a/b complexes isolated from pea were studied in relation to their aggregation state. The aggregate size was varied by changing the Triton X-100 concentration from 0 to 0.23 mM at a chlorophyll concentration of 2.45 μg/ml. Molecular photophysical parameters were determined with laser fluorimetry. Dispersion of large aggregates into smaller ones drastically decreased the intensity of low-temperature (77 K) fluorescence at 700 nm, reduced the singlet-singlet annihilation rate by more than two orders of magnitude, and prolonged the fluorescence lifetime from 0.16 to 3.2 ns.     

Fluorescence assay of the specificity of human plasma and bovine liver phospholipid transfer proteins

The specificities of a human plasma and bovine liver phospholipid transfer protein were studied using a fluorescence assay based on the transfer of pyrenyl phospholipids. This method was used previously to determine the mechanism of spontaneous transfer of phospholipids between model lipoproteins (Massey, J.B., Gotto, A.M., Jr. and Pownall, H.J. (1982) Biochemistry 21, 3630-3636). The pyrenyl phospholipids varied in the headgroup moiety; pyrenyl phosphatidylcholines contained different fatty acyl chains in the sn-1 position. Model high-density lipoproteins (R-HDL) consisting of apolipoprotein A-I and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were used as donor and acceptor particles. As previously shown, the bovine liver protein mediated the transfer of only phosphatidylcholine. In contrast, the human plasma protein transferred all species studied which included a phosphatidylserine, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, sphingomyelin, galactosylcerebroside, and a diacylglycerol. The activity of these transfer proteins was only slightly affected by changes in the acyl chain composition of the transferring lipid. Pyrenyl and radioactive ([3H]POPC) phospholipids were transferred with equal rates by the human transfer protein, suggesting that this protein has similar binding characteristics for pyrenyl and natural phospholipids. Spontaneous phospholipid transfer occurs by the aqueous diffusion of monomeric lipid where the rate is highly dependent on fatty acyl chain composition. In this study, no correlation between the rate of spontaneous transfer and protein-mediated transfer was found. The apparent Km values for R-HDL and low-density lipoprotein (LDL), when used as acceptors, were similar when based on the number of acceptor particles. The apparent Vmax for the bovine liver protein was identical for R-HDL and LDL but for the plasma protein Vmax was slightly higher for R-HDL. These results suggest that, like the bovine liver protein, the plasma protein functions as a phospholipid-binding carrier that exchanges phospholipids between membrane surfaces. The assay of lipid transfer proteins by pyrenyl-labeled lipids is faster and easier to perform than other current methods, which require separation of donor and acceptor particles, and is suitable for studies on the function and mechanism of action of lipid transfer proteins.     

A spectroscopic and photophysical study on molecular recognition via hydrogen-bonding and pi-pi stacking interactions.

Spectroscopic and photophysical properties of a Kemp's tricarboxylic acid derivative having an anthracene chromophore (I) upon recognition of 9-butyladenine (BA) in chloroform were studied in detail. Molecular recognition of BA by I via hydrogen-bonding and pi-pi stacking interactions were sensed successfully on the basis of absorption and fluorescence spectroscopies, by which the binding constant of the I:BA complex was determined to be 240 M(-1). The fluorescence quantum yield and lifetime of I in the absence of BA were 0.24 and 5.6 ns, respectively, while those in the presence of an enough amount of BA increased to 0.35 and 13 ns, respectively. These values demonstrated that the nonradiative decay rate constant of I decreased from 13.6 x 10(7) to 5.0 x 10(7) s(-1) upon binding with BA. Such changes in the photophysical properties of I before and after complexation with BA were discussed in terms of hydrogen-bonding and pi-pi stacking interactions between I and BA. In particular, intramolecular hydrogen-bonding between the amide and imide groups in was shown to play important roles in determining the photophysical characteristics of I before complexation, while intermolecular hydrogen-bonding between I and BA governed the excited-state properties of the I:BA complex. The change in the hydrodynamic diameter of I before and after complexation with BA was also discussed on the basis of the results by fluorescence dynamic anisotropy measurements.     

A glutathione-based hydrogel and its site-selective interactions with water

Glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) is ubiquitous biological tripeptide with multiple functions and possible therapeutic uses. The oxidized disulfide form (GSSG) self-assembles into fibrillar aggregates and gels in organic solvents, but not in solvent mixtures with high water content. Here, the disulfide bond has been replaced with a pyrenyl moiety in order to test the ability of GSH to direct noncovalent self-assembly in H2O, when combined with a hydrophobic driving force for aggregation. The resulting GSH-pyrene forms gels in 95% H2O:5% DMSO. The gamma-glutamyl group is critical for gelation, as it is with GSSG organo-gels, inasmuch as neither S-(pyrenyl)-cysteinyl-glycine nor the iodo-acetamido-pyrene precursor gels under any conditions studied. Circular dichroism and fluorescence spectroscopy indicate that the pyrene moieties cluster within the gels. Scanning and transmission electron microscopy reveal fibrous networks with individual strands of approximately 50-100 nm diameter. Saturation transfer difference (STD) NMR studies demonstrate that water interacts strongly with GSH-borne protons in both solution and gel states, but only the gels include water-pyrenyl interactions with significant residence times.     

Synthesis, photophysical effects, and DNA targeting properties of oxazole yellow-peptide bioconjugates

The design of a dsDNA-sensitive fluorescent bioconjugate capable of targeting a specific DNA sequence with high efficiency is described. The bioconjugate has the molecular recognition features of the polypeptide from a DNA-binding protein and the dsDNA-dependent fluorescence of an intercalating dye. The DNA sequence selectivity of the probe was characterized, as were the changes in photophysical properties of the dye upon covalent linkage to the peptide to assess whether such bioconjugates could function as molecular probes of gene sequences. The oxazole yellow-peptide bioconjugate exhibits DNA recognition and binding affinity comparable to the native Hin recombinase protein. Examination of photophysical effects to dye conjugation indicates a negligible affect on the fluorescence quantum yield. Fluorescence studies indicate this molecular probe is useful to determine the presence of a given DNA target sequence and gives negligible fluorescence in the absence of a given target site. Using the synthetic route described here, bioconjugates could be designed using different combinations of DNA recognition polypeptides and cyanine dyes to generate an array of sequence specific and wavelength specific probes.     

Evolution of influence: signaling in a lycaenid-ant interaction

Some phytophagous insects gain defense from natural enemies by associating with otherwise potentially harmful top predators. Many lycaenid butterfly caterpillars are involved in such interactions with ants: larvae provide carbohydrate rewards from the dorsal nectary organ (DNO) to associated ants in return for protection from natural enemies. The stability of these interactions involves signals that identify the lycaenid caterpillar as a mutualist. However, larvae of some lycaenid species, such as Lycaena xanthoides, are found in close association with ants but do not possess the reward producing DNO. Evaluating the relationship in a phylogenetic framework, we show that the association between L. xanthoides and ants likely evolved from a non-ant-associated ancestor. Behavioral trials also show that L. xanthoides larvae are capable of influencing ant behavior to increase ant tending when faced with a simulated predator attack, without providing DNO-derived rewards to ant associates. These results demonstrate that the DNO is not necessary to maintain associations between lycaenid larvae and ants. Third-party interactions may affect the evolution of mutualisms and consideration of underlying evolutionary history is necessary to understand contemporary species associations.     

Study of interaction of long-chain n-alcohols with fluid DOPC bilayers by a lateral pressure sensitive fluorescence probe

The excimer 1,2-dipyrenedecanoyl-sn-glycero-3-phosphatidylcholine (dipy10PC) fluorescence probe was used to determine effects of aliphatic alcohols (CnH2n+1OH, n = 12-18 is the even number of carbons in alkyl chain) on fluid dioleoylphosphatidylcholine (DOPC) +dioleoylphosphatidylserine (DOPS) bilayers in multilamellar vesicles at molar ratio DOPC/DOPS = 24.7. The excimer to monomer fluorescence intensity ratio increases with the increase of CnH2n+1OH/DOPC molar ratio and decreases with the CnH2n+1OH alkyl chain length n at a constant CnH2n+1OH/DOPC = 0.4 molar ratio. These effects indicate changes in the bilayer lateral pressure on the level of pyrenyl moieties location.     

Lateral diffusivity of lipid analogue excimeric probes in dimyristoylphosphatidylcholine bilayers.

The lateral mobility of pyrenyl phospholipid probes in dimyristoylphosphatidylcholine (DMPC) vesicles was determined from the dependence of the pyrene monomeric and excimeric fluorescence yields on the molar probe ratio. The analysis of the experimental data makes use of the milling crowd model for two-dimensional diffusivity and the computer simulated random walks of probes in an array of lipids. The fluorescence yields for 1-palmitoyl-2-(1'-pyrenedecanoyl)phosphatidylcholine (py10PC) in DMPC bilayers are well fitted by the model both below and above the fluid-gel phase transition temperature (Tc) and permit the evaluation of the probe diffusion rate (f), which is the frequency with which probes take random steps of length L, the host membrane lipid-lipid spacing. The lateral diffusion coefficient is then obtained from the relationship D = fL2/4. In passing through the fluid-gel phase transition of DMPC (Tc = 24 degrees C), the lateral mobility of py10PC determined in this way decrease only moderately, while D measured by fluorescence photobleaching recovery (FPR) experiments is lowered by two or more orders of magnitude in gel phase. This difference in gel phase diffusivities is discussed and considered to be related either to (a) the diffusion length in FPR experiments being about a micrometer or over 100 times greater than that of excimeric probes (approximately 1 nm), or (b) to nonrandomicity in the distribution of the pyrenyl probes in gel phase DMPC. At 35 degrees C, in fluid DMPC vesicles, the diffusion rate is f = 1.8 x 10(8) s-1, corresponding to D = 29 microns2 s-1, which is about three times larger than the value obtained in FPR experiments. The activation energy for lateral diffusion in fluid DMPC was determined to be 8.0 kcal/mol.     

Folic acid conjugates with photosensitizers for cancer targeting in photodynamic therapy: Synthesis and photophysical properties

Recent researches in photodynamic therapy have focused on novel techniques to enhance tumour targeting of anticancer drugs and photosensitizers. Coupling a photosensitizer with folic acid could allow more effective targeting of folate receptors which are over-expressed on the surface of many tumour cells. In this study, different folic acid–OEG-conjugated photosensitizers were synthesized, characterized and their photophysical properties were evaluated. The introduction of an OEG does not significantly improve the hydrophilicity of the FA–porphyrin. All the FA-targeted photosensitizers present good to very good photophysical properties. The best one appears to be Ce6. Molar extinction coefficient, fluorescence and singlet oxygen quantum yields were determined and were compared to the corresponding photosensitizer alone.