Picosecond transient absorption study of photodissociated carboxy hemoglobin and myoglobin.

The optical transient absorption spectra at 30 ps and 6.5 ns after photolysis are compared for both carboxy hemoglobin (HbCO) and carboxy myoglobin (MbCO). Both 355- and 532-nm excitation pulses were used. In all cases the shapes of the optical difference spectra thus generated are stationary over the complete time-scale studied. The photolysis spectra for MbCO are not significantly different from the equilibrium difference spectra generated on the same picosecond spectrometer when measured to an accuracy of +/- 0.5 nm. In addition, spectral parameters for delegated HbCO generated on the same spectrometer but detected by two different techniques, either by a Vidicon detector or point by point with photomultiplier tubes, are reported; the results are different from some of the previously reported picosecond experiments.     

Extraterrestrial Handedness: A Reply

Recent investigations of stable isotope ratios of amino acids from the Murchison meteorite have shown them to be of unambiguous extraterrestrial origin, and examinations of their enantiomeric compositions, where terrestrial contamination can be excluded, have found a consistent excess of L-enantiomers. One explanation for this observation has been the asymmetric photolysis of racemic extraterrestrial amino acids by circularly polarized light (CPL) in the synchrotron radiation from orbiting electrons around the pulsar remnants of supernovae. Mason (1997) has attempted to discredit this mechanism on the grounds that circular dichroism (CD) bands for optically active molecules alternate in sign and sum to zero over the entire spectrum, and hence enantioselective photochemical reactions cannot be induced by broad band CPL. We submit arguments disputing this conclusion and present reasons for expecting that broad band CPL synchrotron radiation would be quite capable of inducing asymmetric photolysis, particularly in aliphatic amino acids.     

Circular dichroism and circular polarization of luminescence of reduced nicotinamide adenine dinucleotide in solution and bound to dehydrogenases.

The CD (circular dichroism) and CPL (circular polarization of luminescence) spectra of NADPH in aqueous solution were studied and found to be markedly different. The spectra were not affected by cleavage of the coenzyme molecule with phosphodiesterase. The differences are thus not due to the existence of extended and folded conformations of NADPH and it is concluded that they originate in excited state conformational changes of the nicotinamide--ribose fragment. Opposite signs of both the CD and CPL spectra were observed for NADH bound to horse liver alcohol dehydrogenase and to beef heart lactate dehydrogenase indicating structural differences between the nicotinamide binding sites. The binding of substrate analogues to enzyme--coenzyme complexes did not affect the CD spectra and hence no significant conformational changes are induced upon formation of the ternary complexes. No changes in the CPL spectrum of NADH bound to lactate dehydrogenase were observed upon adding oxalate to form the ternary complex. Marked differences were found between the CPL spectra of binary and ternary complexes with liver alcohol dehydrogenase, while the CD spectra of these complexes were identical. It is concluded that a conformational change of the excited NADH molecule occurs in the binary but not in the ternary complex involving LADH, thus indicating an increased rigidity of the latter complex.     

Ultraviolet, circular dichroism, fluorescence, and circularly polarized luminescence spectra of regioregular poly-[3-((s)-2-methylbutyl)-thiophene] in solution

Ultraviolet (UV), Circular Dichroism (CD), Fluorescence, and Circularly Polarized Luminescence (CPL) spectra were recorded for mixed chloroform/methanol and chloroform/butanol solutions of regioregular poly[3-((S)-2-methylbutyl)-thiophene]. An evaluation of the chiral and nonchiral components in solution is proposed on the basis of the UV and CD spectra. The fluorescence and CPL spectra are observed in a wavelength range where some UV and CD activity still appears. For this reason, the observed CPL features are strongly influenced by CD. A general procedure for evaluating true CPL spectra is proposed, when absorption and emission coexist. Chirality 24:725-730, 2012. ? 2012 Wiley Periodicals, Inc.     

On the determination of empirical absolute chiral structure: chiroptical spectrum correlations for D3 lanthanide (III) complexes

Circularly polarized luminescence (CPL) from selected transitions of Eu(III) in resolved single crystals of Na3[Eu(ODA)3].2NaClO4.6H2O are compared to CPL results obtained from solutions containing perturbed racemic mixtures of Eu(2,6-pyridine-dicarboxylate)3 (3-) and enantiomerically pure d-f helicate LambdaLambda-(-)EuCr(L8)3] in order to determine an empirical relationship between helicity and CPL spectra. Comparison of the CPL results, even for the magnetic dipole allowed transitions of Eu(III) where one measures large chiral discrimination, shows that the signs and magnitudes do not correlate with the overall helicity of the Eu(III) site. It is concluded that the symmetry of the Eu(III) site in LambdaLambda-(-)EuCr(L8)3 is not close enough to D3 to allow for the confirmation of the presumed spectra:structure correlation.     

Conformational changes induced in a homogeneous anti-type III pneumococcal antibody by oligosaccharides of increasing size.

The circular polarization of luminescence (CPL) emitted by tryptophan residues was used as a sensitive probe for measuring ligand-induced structural changes in a homogeneous type III pneumococcal antibody. A series of oligosaccharide ligands of increasing size derived from type III polysaccharide by partial acid hydrolysis was assayed. Ligand-induced changes in the circular polarization of fluorescence of the antibody were observed for all antigens tested, including tetra-, hexa-, and octasaccharides and a 16-residue oligomer, the largest changes being recorded upon interaction with the intact soluble type III pneumococcal (SIII) polysaccharide. When Fab' or F(ab')2 fragments were used instead of the antibody IgG for binding of SIII polysaccharide, the extent of conformational changes was decreased. This suggests interactions between Fab and Fc portions in the IgG molecule and subsequent conformational changes in Fc part upon antigen binding. Reduction of interchain disulfide bonds abolished the additional spectral changes attributed to the Fc part but not the changes observed in the Fab part, thus suggesting that the presence of the interchain disulfide bond in the hinge region is required for maximal CPL changes to occur. Small monovalent ligands, i.e., the tetra-, hexa-, and octasaccharides, were capable of inducing CPL changes in the Fab part of the antibody molecule as well as CPL changes attributed to the Fc portion. A multivalent ligand containing about 16 sugar residues appears to be the minimal antigenic size required for triggering conformational changes attributed to the Fc part, similar to those seen in the interaction with the whole polysaccharide antigen.     

Cationic poly(ethyleneglycol) lipids incorporated into pre-formed vesicles enhance binding and uptake to BHK cells

This paper describes a new method for enhancing the interaction of liposomes with cells. A novel class of cationic poly(ethyleneglycol) (PEG)-lipid (CPL) conjugates have been characterized for their ability to insert into pre-formed vesicles and enhance in vitro cellular binding and uptake of neutral and sterically-stabilized liposomes. The CPLs, which consist of a distearoylphosphatidylethanolamine (DSPE) anchor, a fluorescent dansyl moiety, a heterobifunctional PEG polymer (M(r) 3400), and a cationic headgroup composed of lysine derivatives, have been described previously [Bioconjug. Chem. 11 (2000) 433]. Five separate CPL, possessing 1-4 positive charges in the headgroup (referred to as CPL(1)-CPL(4), respectively), were incubated (as micellar solutions) in the presence of neutral or sterically-stabilized cationic large unilamellar vesicles (LUVs), and were found to insert into the external leaflet of the LUVs in a manner dependent on temperature, time, CPL/lipid ratio, and LUV composition. For CPL/lipid molar ratios < or =0.1, optimal insertion levels of approximately 70% of initial CPL were obtained following 3 h at 60 degrees C. The insertion of CPL resulted in aggregation of the LUVs, as assessed by fluorescence microscopy, which could be prevented by the presence of 40 mM Ca(2+). The effect of CPL-insertion on the binding of LUVs to cells was examined by fluorescence microscopy and quantified by measuring the ratio of rhodamine fluorescence to protein concentration. Neither control LUVs or LUVs containing CPL(2) displayed significant uptake by BHK cells. However, a 3-fold increase in binding was observed for LUVs possessing CPL(3), while for CPL(4)-LUVs values as high as 10-fold were achieved. Interestingly, the increase in lipid uptake did not correlate with total surface charge, but rather with increased positive charge density localized at the CPL distal headgroups. These results suggest that incorporation of CPLs into existing liposomal drug delivery systems may lead to significant improvements in intracellular delivery of therapeutic agents.     

Photoreactive derivatives of corticotropin. 2. Preparation and characterization of 2-nitro-4(5)-azidophenylsulfenyl derivatives of corticotropin

Two new photoreactive arylsulfenyl chlorides, 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) and 2-nitro-5-azidophenylsulfenyl chloride (2,5-NAPS-Cl), have been synthesized and used for the selective modification of corticotropin (ACTH). Both reagents reacted rapidly with N-acetyltryptophanamide and ACTH under acidic conditions. The NAPS derivatives of ACTH were purified by partition chromatography and characterized by absorption spectra, amino acid analysis, and peptide mapping. The spectral changes caused by photolysis as well as the kinetics of photolysis are described. Tritiated 2,5-NAPS-ACTH was attached covalently to a pituitary protein fraction FI by photolysis. The photolabeling of FI was blocked in the presence of excess ACTH.     

A survey of homopteran species (Auchenorrhyncha) from coffee shrubs and poró and laurel trees in shaded coffee plantations,in Turrialba,Costa Rica

A survey of homopteran species (Auchenorryncha) was conducted in coffee plantations with no shade (C), and in those with shade of either poró (Erythrina poeppigiana) (CP) or poró plus laurel (Cordia alliodora) (CPL), in Turrialba, Costa Rica. A total of 130 species in ten families were collected, dominated by Cicadellidae (82 species). Species richness was highest in the CP system (88), followed by CPL (74) and C systems (60). Five most common species for all systems were Fusigonalia lativittata, Hebralebra nicaraguensis, Neocoelidia sp., Oliarus sp. and Clastoptera sp. Diversification of the coffee agroecosystem favors some species while limiting others, and have no effect on the majority of species. Thus, only F. lativittata, Neocoelidia sp. and Scaphytopius ca. latidens were well represented in all systems, but were more abundant in coffee shrubs. Additionally, the following were the dominant species in each system: Graphocephala sp. 1 (C), F. lativittata (CP) and H. nicaraguensis (CPL). Four species abundant on laurel trees, including H. nicaraguensis, appeared almost exclusively on these tree species. Species similarity was highest on the CP and CPL systems (51% of the species in common), followed by the C and CP (39%) and the C and CPL systems (38%). These findings show that even disturbed systems can harbor many insect species, so that they deserve attention from conservation advocates and biologists.     

Distal cationic poly(ethylene glycol) lipid conjugates in large unilamellar vesicles prepared by extrusion enhance liposomal cellular uptake

Cationic poly(ethylene glycol)-lipid conjugates (CPLs), a class of lipid designed to enhance the interaction of liposomes with cells, possess the following architectural features: 1) a hydrophobic lipid anchor of distearoylphosphatidylethanolamine (DSPE); 2) a hydrophilic spacer of poly(ethylene glycol); and 3) a cationic head group prepared with 0, 1, 3, or 7 lysine residues located at the distal end of the PEG chain, giving rise to CPL possessing 1, 2, 4, or 8 positive charges, respectively (CPL1 to CPL8). Previously we have described the synthesis of CPL, have characterized the postinsertion of CPL into PEG-containing LUVs and SPLP (stabilized plasmid-lipid particles), have shown significant increases in the binding of CPL-LUV to cells, and have observed dramatically enhanced transfection (up to a million-fold) of cells with CPL-SPLP in the presence of calcium [Chen et al. (2000) Bioconjugate Chem. 11, 433-437; Fenske et al. (2001) Biochim. Biophys. Acta 1512, 259-272; Palmer et al. (2003) Biochim. Biophys. Acta 1611, 204-216]. In the present study, we examine a variety of CPL properties (such as polarity and CMC) and characterize CPL-vesicular systems formed by extrusion and examine their interaction with cells. While CPL polarity was observed to increase dramatically with increasing charge number, CMC values were all found to be low, in the range of other PEGylated lipids, and exhibited only a small increase, going from CPL1 (1.3 microM) to CPL8 (2 microM). The CPLs were almost quantitatively incorporated into large unilamellar vesicles (LUVs) prepared by the extrusion method and were evenly distributed across the lipid bilayer. Lower levels of incorporation were obtained when CPLs were incubated with preformed liposomes (DSPC/Chol, 55:45) at 60 degrees C. The binding of CPL-LUVs to BHK cells in vitro was found to be dependent on the distal charge density of the CPL rather than total surface charge. Liposomes possessing CPL4 or CPL8 were observed to bind efficiently to cell surfaces and enhance cellular uptake in BHK cells (as observed with both lipid and aqueous content markers), whereas those possessing CPL1 or CPL2 exhibited little or no binding. These results suggest new directions for the design of liposomal systems capable of in vivo delivery of both conventional and genetic (plasmid and antisense) drugs.     

Does the use of DM-nitrophen,nitr-5, or diazo-2 interfere with the measurement of indo-1 fluorescence?

Emission spectra of the photolabile Ca2+ chelators DM-nitrophen, nitr-5, and diazo-2 were studied alone, and in the presence of indo-1, to investigate potential interactions that would make the simultaneous manipulation and ratiometric measurement of the intracellular Ca2+ concentration difficult. Neither diazo-2 nor its photoproduct were found to be significantly fluorescent, and consequently concentrations of diazo-2 up to 20 times that of indo-1 did not distort the emission spectra of indo-1. DM-nitrophen was scarcely fluorescent, but its fluorescence did increase upon photolysis. In contrast to diazo-2 and DM-nitrophen, nitr-5 itself was found to be quite fluorescent, and this fluorescence was significantly increased upon photolysis. Thus, combined use of nitr-5 and indo-1 poses the most difficulty. The emission spectra of all the investigated compounds were used to define experimental conditions and calibration procedures that make possible simultaneous measurement and manipulation of the intracellular Ca2+ concentration.     

Factors of humoral resistance in workers exposed to vinyl chloride with a view to smoking habits

Serum samples were assayed in 110 workers (59 smokers and 51 non-smokers) at PVC manufacturing factory, the results being compared with those obtained in a group of age-matched healthy controls. Non-smokers had significantly higher levels of immunoglobulins (IgG, IgA, IgM), while in smokers there was an increase in IgM only. Lysozyme levels (LYS) were elevated in all exposed subjects, but there was a highly significant decrease in the total protein (TP) content. Alfa-2-macroglobulin (A2M) and orosomucoid (ORO) were also elevated in exposed workers. A significant increase was found in ceruloplasmin (CPL), with smokers having higher levels than non-smokers. No difference was detected between the examined groups in transferrin (TRF) and alfa-1-antitrypsin (A1AT). Exposure duration did not correlate with any of the assayed parameters. The positive correlation of IgA levels and inverse correlation of CPL with age in the control group could not be confirmed in the exposed subjects where a significant inverse correlation between age and AlAT levels, which was found to be positive in exposed non-smokers, proved to be inverse in the subgroup of smokers. The authors discuss possible explanations of the reported findings.     

The circular polarization of fluorescence of the ionophore lasalocid A (X-537A) and some of its metal complexes

The conformation of the ionophore lasalocid A (X-537A) and its complexes with metal ions was probed by the circular polarization of their luminescence (CPL). The CPL of each complex in methanol was found to be different than when in n-hexane. Furthermore, the different metal ion complexes investigated had a different CPL spectrum in each solvent. These findings indicate wide variability in the conformation of the complexes depending on the metal ion and the solvent. From the spectral behaviour of the CPL it was concluded that at least some of the complexes exist in more than one form in solution. A comparison between the CPL and CD spectra indicates a change in the conformation of the ionophore in the vicinity of the salicylate chromophore upon electronic excitation.     

Conformational role of His-12 in C-peptide of ribonuclease A

Possible interactions of the His-12 ring with other side chain and backbone groups of C-peptide lactone (CPL) are discussed. The works published so far are critically reviewed and compared with the latest results obtained by the authors. The main new conclusion is that in the helical conformation of CPL, the Phe-8 and His-12 rings are clustered together. Studies of Phe-8----Ala analogs of CPL and calculations of ring current effects satisfactorily explain the observed environmental shifts of Phe-8 and His-12 protons in NMR spectra of CPL. Interaction between both rings is favorable for alpha-helix formation, but cannot explain an increase in helix stability related with protonation of His-12. This effect arises from favorable interactions of the charged His+-12 ring with the helix backbone.     

Bivalency of plasminogen monoclonal antibodies is required for plasminogen bridging to fibrin and enhanced plasmin formation

Binding of plasminogen to fibrin and cell surfaces is essential for fibrinolysis and pericellular proteolysis. We used surface plasmon resonance and enzyme kinetic analyses to study the effect of two mAbs (A10.2, CPL15) on plasminogen binding and activation at fibrin surfaces. A10.2 is directed against the lysine-binding site (LBS) of kringle 4, whereas CPL15 recognises a region in kringle 1 outside the LBS. In the presence of CPL15 and A10.2 mAbs, binding of plasminogen (K(d)=1.16+/-0.22 micromol/l) to fibrin was characterised by a mAb concentration-dependent bell-shaped isotherm. A progressive increase in the concentration of mAbs at the surface was also detected, and reached a plateau corresponding to the maximum of plasminogen bound. These data indicated that at low mAb concentration, bivalent plasminogen-mAb-plasminogen ternary complexes are formed, whereas at high mAb concentration, a progressive shift to monovalent plasminogen-mAb binary complexes is observed. Plasmin formation in the presence of mAbs followed a similar bell-shaped profile. Monovalent Fab fragments of mAb A10.2 showed no effect on the binding of plasminogen, confirming the notion that a bivalent mAb interaction is essential to increase plasminogen binding and activation at the surface of fibrin.     

Seeking the mechanism responsible for fluoroquinolone photomutagenicity: a pulse radiolysis,steady-state,and laser flash photolysis study

The mechanism responsible for the remarkable photomutagenicity of fluoroquinolone (FQ) antibiotics remains unknown. For this reason, it was considered worthwhile to study in detail the interactions between DNA and a dihalogenated FQ such as lomefloxacin (LFX; one of the most photomutagenic FQs) and its N-acetyl derivative ALFX. Studies of photosensitized DNA damage by (A)LFX, such as formation of DNA single-strand breaks (SSBs), together with pulse radiolysis, laser flash photolysis, and absorption and fluorescence measurements, have shown the important effects of the cationic character of the piperazinyl ring on the affinity of this type of drug for DNA. Hence, the formation of SSBs was detected for LFX, whereas ALFX and ciprofloxacin (a monofluorated FQ) needed a considerably larger dose of light to produce some damage. In this context, it was determined that the association constant (K_a) for the binding of LFX to DNA is ca. 2×10³ M⁻¹, whereas in the case of ALFX it is only ca. 0.5×10³ M⁻¹. This important difference is attributed to an association between the cationic peripheral ring of LFX and the phosphate moieties of DNA and justifies the DNA SSB results. The analysis of the transient species detected and the photomixtures has allowed us to establish the intermolecular processes involved in the photolysis of FQ in the presence of DNA and 2'-deoxyguanosine (dGuo). Interestingly, although a covalent binding of the dihalogenated FQ to dGuo occurs, the photodegradation of FQ…DNA complexes did not reveal any significant covalent attachment. Another remarkable outcome of this study was that (A)LFX radical anions, intermediates required for the onset of DNA damage, were detected by pulse radiolysis but not by laser flash photolysis.