Laser desorption 7.87 eV postionization mass spectrometry of antibiotics in Staphylococcus epidermidis bacterial biofilms

This paper describes the development of laser desorption 7.87 eV vacuum UV (VUV) postionization MS to detect antibiotics within intact bacterial colony biofilms. As >99% of the molecules ejected by laser desorption are neutrals, VUV photoionization of these neutrals can provide significantly increased signal as compared to the detection of directly emitted ions. Postionization with VUV radiation from the molecular fluorine laser single photon ionizes laser desorbed neutrals with ionization potentials below the 7.87 eV photon energy. Antibiotics with structures indicative of sub-7.87 eV ionization potentials were examined for their ability to be detected by 7.87 eV laser desorption postionization MS. Tetracycline, sulfadiazine, and novobiocin were successfully detected neat as dried films physisorbed on porous silicon oxide substrates. Tetracycline and sulfadiazine were then detected within intact Staphylococcus epidermidis colony biofilms, the former with LOD in the micromolar concentration range.     

Laser desorption mass spectrometry for microbial DNA analysis.

Recently, we demonstrated that a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) can be used to determine the molecular weight of polymerase chain reaction (PCR) products of intact 16S rRNA regions and to profile their restriction digests. This is the first time that MALDI-TOF MS with ultraviolet (UV) photoionization has been used to analyze a PCR product of approximately 1600 nucleotides in length.     

Aspects of secondary and quaternary structure of surfactant protein A from canine lung

The results of a large number of studies indicate that pulmonary surfactant contains a unique protein whose principal isoform has a molecular weight of about 30,000, and whose presence in surfactant is associated with important metabolic and physicochemical properties. This protein, SP-A, as isolated from canine surfactant, contains a domain of 24 repeating triplets of Gly-X-Y, similar to that found in collagens. These studies were undertaken to determine whether SP-A forms a collagen-like triple helix when in solution, and to describe certain aspects of its size and shape. Our experiments were done on SP-A extracted by two different methods from canine surfactant, and on SP-A produced by molecular cloning. The results from all three preparations were similar. The circular dichroism of the complete protein was characterized by a relatively large negative ellipticity at 205 nm, with a negative shoulder ranging from 215 to 230 nm. There was no positive ellipticity, and the spectrum was not characteristic of collagen. Trypsin hydrolysis resulted in a fragment with peak negative ellipticity at about 200 nm, without the negative shoulder. Further hydrolysis of this fragment with pepsin resulted in a CD spectrum similar to that of collagen. The spectrum of the collagen-like fragment was reversibly sensitive to heating to 50 degrees C, and was irreversibly lost after treatment with bacterial collagenase. SP-A migrated on molecular sieving gels with an equivalent Stokes radius of 110 to 120 A, and had a sedimentation coefficient of 14 S. Using these data we calculate a molecular weight of about 700,000. The hydrodynamic characteristics can be approximated as a prolate ellipsoid of revolution having an axial ratio of about 20. We conclude that SP-A aggregates into a complex of 18 monomers, which may form six triple-helices. The shape of the complex is considerably more globular than collagen and is not consistent with end-to-end binding of the helices to form fibrous structures.     

Scanning Microphotolysis: Three-Dimensional Diffusion Measurement and Optical Single-Transporter Recording

Scanning microphotolysis (SCAMP) is a combination of fluorescence microphotolysis and confocal laser scanning microscopy. A laser scanning microscope is equipped with an optical switch able to modulate the power or/and wavelength of the laser beam in less than a microsecond while a dedicated computer program is employed to precisely coordinate scanning process and laser beam modulation. By these means it becomes possible to vary the power or/and wavelength of the laser beam during scanning at a precision of one resolution element. Patterns of almost arbitrary design can be written into the object by photolysis, e.g., photobleaching or photoactivation. The dissipation of the photolysis pattern by diffusion or other types of molecular transport can be followed at confocal resolution and used to characterize the transport process. SCAMP can be employed in conjunction with single-photon or multiphoton excitation. Furthermore, it can be easily installed on virtually any confocal laser scanning microscope. We summarize at first the conceptual and practical basis of SCAMP. Then, two novel applications are discussed: (i) measurements of translational diffusion coefficients in truly three-dimensional systems at diffraction-limited resolution, and (ii) optical recording of single transporters in membrane patches.     

Circular dichroism studies on the binding of type I substrates and reverse type I compounds to rabbit liver microsomal cytochrome P-450.

Liver microsomes from control, 3-methylcholanthrene-treated, and phenobarbital-treated New Zealand White rabbits were examined for differences detectable by circular dichroism (CD) spectroscopy. Addition of the Type I substrate cyclohexane to phenobarbital microsomes decreases the negative ellipticity at about 418 nm and concomitantly increases the negative ellipticity at about 395 nm. Cyclohexane added to microsomes from control or 3-methylcholanthrene-treated animals shows little or no CD changes in these wavelength regions. The effect by cyclohexane is completely reversed by the subsequent addition of butanol-1. Addition of benzo[a]pyrene to phenobarbital microsomes also decreases the negative ellipticity at about 418 nm, and this effect can be completely reversed with the subsequent addition of butanol-1. The ellipticity at about 395 nm is reversed in sign and is markedly increased by benzo[a]pyrene, however, and this effect is not changed with the subsequent addition of butanol-1. Restoring the cyclohexane- or benzo[a]pyrene-induced changes by the subsequent addition of alcohol is proportional to the aliphatic chain length, with 4 or more carbon atoms being maximally effective. Primary alcohols inhibit aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2) activity of phenobarbital microsomes, and the inhibitory effect is enhanced with increasing chain length of the alcohols; 4 or more carbon atoms being maximally effective. Stimulation of monooxygenase metabolism of cyclohexane or benzo[a]pyrene by NADPH results in restoration of the negative ellipticity band at about 418 nm, whereas the ellipticity peak at about 395 nm remains unchanged. More negative ellipticity at about 210 and 222 nm is found in phenobarbital microsomes than in control or 3-methylcholanthrene microsomes and cyclohexane addition in vitro increases these negative ellipticity peaks in phenobarbital microsomes but not in control or 3-methylcholanthrene microsomes.These results show that with CD studies one can detect directly both high spin (negative ellipticity peak at 385–395 nm) and low spin (negative ellipticity peak at about 418 nm) P-450 iron in liver microsomes from control, 3-methylcholanthrene-treated, or phenobarbital-treated rabbits. These data are consistent with a weak ligand such as oxygen, rather than a stronger ligand such as nitrogen, in the sixth position of 6-coordinated (low spin) ferric iron in P-450 in vivo. Type I substrates such as cyclohexane or benzo[a]pyrene, when bound to P-450, change low spin P-450 iron to the high spin state. Cyclohexane-bound high spin P-450 iron in vitro is more easily converted to low spin iron by butanol-1 than is benzo[a]pyrene-bound high spin P-450 iron. Liver microsomal proteins from phenobarbital-treated rabbits have a higher helical content than those from either control or 3-methylcholanthrene-treated rabbits. Cyclohexane addition in vitro increases this helical character only in phenobarbital microsomes, indicating that one or more forms of phenobarbital-induced P-450 apoproteins is (are) more specific for cyclohexane binding and metabolism than control or 3-methylcholanthrene-induced forms of P-450.     

Molecular Beam Mass Spectrometry With Tunable Vacuum Ultraviolet (VUV) Synchrotron Radiation

Tunable soft ionization coupled to mass spectroscopy is a powerful method to investigate isolated molecules, complexes and clusters and their spectroscopy and dynamics^1-4. Fundamental studies of photoionization processes of biomolecules provide information about the electronic structure of these systems. Furthermore determinations of ionization energies and other properties of biomolecules in the gas phase are not trivial, and these experiments provide a platform to generate these data. We have developed a thermal vaporization technique coupled with supersonic molecular beams that provides a gentle way to transport these species into the gas phase. Judicious combination of source gas and temperature allows for formation of dimers and higher clusters of the DNA bases. The focus of this particular work is on the effects of non-covalent interactions, i.e., hydrogen bonding, stacking, and electrostatic interactions, on the ionization energies and proton transfer of individual biomolecules, their complexes and upon micro-hydration by water^{1, 5-9}.We have performed experimental and theoretical characterization of the photoionization dynamics of gas-phase uracil and 1,3-dimethyluracil dimers using molecular beams coupled with synchrotron radiation at the Chemical Dynamics Beamline¹⁰ located at the Advanced Light Source and the experimental details are visualized here. This allowed us to observe the proton transfer in 1,3-dimethyluracil dimers, a system with pi stacking geometry and with no hydrogen bonds¹. Molecular beams provide a very convenient and efficient way to isolate the sample of interest from environmental perturbations which in return allows accurate comparison with electronic structure calculations^{11, 12}. By tuning the photon energy from the synchrotron, a photoionization efficiency (PIE) curve can be plotted which informs us about the cationic electronic states. These values can then be compared to theoretical models and calculations and in turn, explain in detail the electronic structure and dynamics of the investigated species ^{1, 3}.     

Two ways to inactivate the Ki‐67 protein—Fragmentation by nanoparticles,crosslinking with fluorescent dyes

Light can manipulate molecular biological processes with high spatial and temporal precision and optical manipulation has become increasingly popular during the last years. In combination with absorbing dyes or gold nanoparticles light is a valuable tool for cell and protein inactivation with high precision. Here we show distinct differences in the underlying mechanisms whether gold nanoparticles or fluorescent dyes are used for the inactivation of the Ki‐67 protein. The proliferation‐associated protein Ki‐67 was addressed by the antibody MIB‐1. In vitro studies showed a fragmentation of the Ki‐67 protein after laser irradiation of 15 nm gold nanoparticle antibody conjugates with nanosecond pulsed laser, while continuous wave (cw) irradiation of fluorescein isothiocyanate (FITC)‐ and Alexa 488‐labeled antibodies led to specific crosslinking of Ki‐67. The irradiation energy for the gold nanoparticles was above cavitation bubble formation threshold. We observed a fragmentation of the target protein and also of the gold particles. The understanding of the underlying inactivation mechanisms is important for the application and further development of these two techniques, which can harness nanotechnology to introduce molecular selectivity to biological systems.     

Circular dichroism and the pH-induced β-coil transition of poly(S-carboxymethyl-L-cysteine) and its side-chain homolog

The CD of aqueous solutions of poly(S-carboxymethyl-L -cysteine) and poly(S-carboxyethyl-L -cysteine) has been measured at different pH, and the pH-induced β-coil transition is observed by changes in residue ellipticity of dichroic bands around 200 and 225 nm. The residue ellipticity at 200 nm of the former polypeptide is twice as large as that of the latter, when the β-conformation is formed in solution. However, the β-conformation of the latter polypeptide is more stable against electrostatic repulsion than that of the former. The transition curve of poly(S-carboxymethyl-L -cysteine) has also been determined for different molecular weights. The curves were found to be completely coincident with one another if the degree of polymerization were higher than about 100. Such a transition curve is generally divided into three steps: initiation, cooperative formation, and rearrangement of hydrogen bonds. The cooperative step is very sharp, occurring at a constant pH. These steps become agglomerated into two or one when the polypeptide concentration or added salt concentration is increased.     

Molecular weight and related properties of lily amylose determined by monitoring of elution from TSK-GEL PW high performance gel chromatography columns by the low-angle laser light scattering technique and precision differential refractometry

Amylose was fractionated according to its molecular weight by high performance gel chromatography using columns of a TSK-GEL PW series. Elution from the columns was monitored with a low-angle laser light scattering photometer and a precision differential refractometer. The following results were obtained indicating that the procedure is highly efficient for characterizing an amylose preparation with respect to its molecular weight: 1) the weight-average molecular weight of lily amylose used as a test material was determined to be 786,000 +/- 26,000 (n = 7); 2) the molecular weight distribution curve of the amylose was worked out from the chromatographic data; and 3) based on the concept of the universal calibration curve, the Mark-Houwink-Sakurada equation of the amylose was presumed to be [eta] = 2.27 X 10(-4)M0.62 (dl/g). The technique saves time and sample significantly compared with the conventional ones, and consequently enables the characterization of amylose in aqueous solvents without either the degradation or association peculiar to the amylose molecule.     

What does inductance plethysmography really measure?

Inasmuch as it has been claimed that inductance plethysmography can measure cross-sectional area changes, we tested this assumption. We present experimental and computed relationships between self-inductance (L) of coils and areas (A) included inside for a coil with a well-defined side wavy pattern (triangular or sinusoidal) and for a real belt (Respitrace) placed on elliptical or rectangular configurations. The results are applied to the physiological field using measurements obtained from a computed tomography experiment. We demonstrate that the L-A relationships vary not only with shape or ellipticity of the cross section but also with the wavy pattern shape. This last parameter is critical because it is difficult to actually control. When the coil wavy pattern remains steady, there are some physiological situations where inductance plethysmography can more accurately estimate area changes: when the configuration shape is constant, the correspondence between delta L and delta A is almost linear with a shape-dependent sensitivity; when the configuration is nearly circular (ellipticity in the range 0.8-1), the relative error in delta A estimation is less than 5%.     

Photoionization two-stream instability in a collisional plasma

An analysis is made of how electron collisions influence the development of photoionization two-stream instability of a plasma produced in the irradiation of matter by a short X-ray pulse from a free-electron laser. The cases of a weakly ionized and a completely ionized plasma are discussed.     

Kinetic resolution of peptide bond and side chain far-UV circular dichroism during the folding of hen egg white lysozyme.

The kinetics of regain of the native ellipticity in the far- and near-UV spectra have been investigated during the refolding at pH 7.8 and 20 degrees C of guanidine-unfolded, nonreduced hen egg white lysozyme. Stopped-flow studies showed that the ellipticities at 260 and 289.5 nm exhibit biphasic kinetics with rate constants of about 50 s-1 and 2.5 s-1 for the rapid and slow phase, respectively. The ellipticity in the far-UV obeyed triphasic kinetics. In addition to a rapid and a slow phase with rate constants similar to those observed in the near-UV, a "burst" of ellipticity was shown to occur in the dead time of the experiments. The effects of low pH and of concentrations of guanidine ranging from 0.075 to 1.5 M on the rapid and slow rate constants were studied. Under all conditions investigated, the rate constants observed in the far- and near-UV for a given phase were the same, thus suggesting that the molecular events observed in the two regions of the UV spectrum are either identical or strongly coupled. Continuous-flow experiments at different wavelengths between 214 and 240 nm under conditions where the dead time for the observation was only 4 ms, followed by a detailed analysis of the kinetics of ellipticity change at each wavelength, provided the spectrum of the molecular species formed at the end of the burst phase. This spectrum was found to closely fit that predicted from the secondary structure of native lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression microdissection adapted to commercial laser dissection instruments

Laser-based microdissection facilitates the isolation of specific cell populations from clinical or animal model tissue specimens for molecular analysis. Expression microdissection (xMD) is a second-generation technology that offers considerable advantages in dissection capabilities; however, until recently the method has not been accessible to investigators. This protocol describes the adaptation of xMD to commonly used laser microdissection instruments and to a commercially available handheld laser device in order to make the technique widely available to the biomedical research community. The method improves dissection speed for many applications by using a targeting probe for cell procurement in place of an operator-based, cell-by-cell selection process. Moreover, xMD can provide improved dissection precision because of the unique characteristics of film activation. The time to complete the protocol is highly dependent on the target cell population and the number of cells needed for subsequent molecular analysis.     

Effect of 5-iodo-2′-deoxyuridine on physical properties and nonhistone chromosomal proteins of chromatin from Burkitt somatic cell hybrids

A Burkitt somatic cell hybrid line, D98/HR-1, contains latent Epstein-Barr virus genome which can be induced to express by 5-iodo-2′-deoxyuridine (IdUrd). Chromatins were isolated from confluent monolayers of D98/HR-1 grown in the presence and absence of IdUrd and its effect on the chromatin was studied by circular dichroism, ethidium bromide binding capacity, and template activity. IdUrd-Treated chromatin consistently exhibits a 20 to 25% decrease in positive ellipticity of circular dichroism at 275 nm. Scatchard plots of spectrophotometric titration of chromatin with ethidium bromide indicate a 25% reduction of the number of primary binding sites in IdUrd-treated chromatin which corresponds with a 25% reduction in template activity of the same chromatin. These effects are reversible when IdUrd-treated cells were released into IdUrd-free medium for 3 h. However, the differences in circular dichroism spectra and dye binding capacity are not observed between DNA isolated from IdUrd-treated and nontreated cells. These results suggest that chromosomal proteins or modification of protein-DNA interactions are responsible for the alterations. Polyacrylamide gel electrophoretic analysis of the chromosomal proteins reveals that a class of nonhistone chromosomal protein in the high molecular weight region (above 130K) is lacking in IdUrd-treated cells.     

Dihydrofolate reductase from soybean seedlings: a fluorescence and circular dichroism study.

The fluorescence emission spectrum of soybean dihydrofolate reductase suggests that the emitting tryptophan residues are situated in a hydrophobic microenvironment. The dissociation constants determined from fluorescence and circular dichroism data reveal that the soybean enzyme has a lower affinity for substrates and substrate analogs than that determined for dihydrofolate reductases isolated from other sources. The binding of methotrexate to the soybean enzyme does not affect the binding of NADPH. Similarly, the binding of NADPH has no effect on subsequent methotrexate binding. Polarimetric study indicates that the enzyme has a low (ca. 5%) α-helical content. Addition of dihydrofolate to the soybean enzyme results in the generation of a positive ellipticity band at 298 nm with a molar ellipticity, [θ], of 186,000, whereas the binding of folate induces a negative ellipticity band at 280 nm with [θ] of ?181,000. The qualitative and quantitative differences in the circular dichroism of the enzyme-dihydrofolate and enzyme-folate complexes indicate that the mode of binding of these ligands may be different. The formation of an enzyme-NADPH complex is accompanied by a negative Cotton effect at 270 nm. These studies indicate that the binding of substrates or inhibitors causes significant conformational changes in the enzyme and also leads to the formation of a number of spectroscopically identifiable complexes.     

Binding of progesterone by rat uterus in vitro

Circular dichroism (CD) spectra have been determined for chromatin fractions obtained by ECTHAM-cellulose chromatography. The molecular ellipticity at the positive long wavelength maximum is about 3000 deg cm²/dmol for early-eluted chromatin fractions, thought to be relatively repressed $\text{in vivo}$, and 5000–6000 deg cm²/dmol for late-eluted chromatin fractions, those thought to be preferentially transcribable $\text{in vivo}$. CD bands in the peptide bond spectral region also differ for the two chromatin fractions, early-eluted chromatin having a more helical conformation for proteins. In addition to previously known differences in protein content, the biological activity of a native chromatin fraction can now be correlated with the conformation of its DNA.     

Femtosecond charge separation in organized assemblies: free-radical reactions with pyridine nucleotides in micelles

Femtosecond laser UV pulse-induced charge separation and electron transfer across a polar interface have been investigated in anionic aqueous micells (sodium lauryl sulfate) containing an aromatic hydrocarbon (phenothiazine). The early events of the photoejection of the electron from the micellized chromophore and subsequent reaction of electron with the aqueous perimicellar phase have been studied by ultrafast infrared and visible absorption spectroscopy. The charge separation (chromophore +...e-) inside the micelle occurs in less than 10(-13) s (100 fs). The subsequent thermalization and localization of the photoelectron in the aqueous phase are reached in 250 fs. This results in the appearance of an infrared band assigned to a nonrelaxed solvated electron (presolvated state). This transient species relaxes toward the fully solvated state of the electron in 270 fs. In anionic aqueous micelles containing pyridine dinucleotides at high concentration (0.025-0.103 M), a single electron transfer can be initiated by femtosecond photoionization of phenothiazine. The one-electron reduction of the oxidized pyridine dinucleotide leads to the formation of a free pyridinyl radical. The bimolecular rate constant of this electron transfer depends on both the pH of the micellar system and the concentration of oxidized acceptor. The free-radical reaction is analyzed in terms of the time dependence of a diffusion-controlled process. In the first 2 ps following the femtosecond photoionization of PTH inside the micelle, an early formation of a free pyridinyl radical is observed. This suggests that an ultrafast free-radical reaction with an oxidized form of pyridine nucleotide can be triggered by a single electron transfer in less than 5 X 10(11) s-1.     

Chemical and physical properties of the disulfides of bovine neurophysin-II.

Bovine neurophysin-II is shown to be very susceptible to partial reduction in the absence of urea. Reduction of an average of one disulfide leads to major changes in conformation and disulfide optical activity, manifest in part by pronounced far-uv ellipticity changes, complete loss of the 248-nm ellipticity band, and a shift of the 278-nm ellipticity band to shorter wavelengths with loss of half its intensity; the reduction process generates a mixture of products and appears to be accompanied by disulfide interchange. The circular dichroism data indicate that the disulfide(s) most susceptible to reduction or interchange are either the principal contributors to the 248- and 278-nm ellipticity bands or that the optical activity of other disulfides is dependent on their integrity. Peptides that bind to the hormone-binding site of neurophysin-II protect against reduction. On reoxidation of partially reduced neurophysin-II there is only a partial return of the native circular dichroism spectrum and electrophoretic behavior. The percentage of native protein in samples reoxidized following different degrees of reduction was estimated by comparison of the circular dichroism spectra of these samples with those of the fractionated native and denatured components of monoreduced-reoxidized neurophysin. Under our reoxidation conditions, less than 50% native protein was found in monoreduced-reoxidized neurophysin and less than 10% native protein was found in completely reduced-reoxidized neurophysin. The results are interpreted with qualified reference to a model in which one or more disulfides are "strained" in the native state and in which the native protein is unstable relative to species in which the disulfides are differently paired.     

The radical ions and photoionization of bile pigments

The semireduced, semioxidized, and OH(.)-adduct radicals of bilirubin (BR) and biliverdin (BV) have been characterized using pulse radiolysis techniques. Laser flash photolysis (265-nm) of these pigments led to monophotonic photoionization with quantum yields of 0.08 for BR and 0.03 for BV. No evidence for triplet formation or for photoisomerization was found after 265-nm laser excitation. However, 347-nm excitation of BR in chloroform led to simultaneous photoisomerization and radical formation, but the radicals are thought to have originated from a pathway other than photoionization. The relevance of these observations to BR photoreactivity is discussed. BR radical ions in alkaline solution did not react with tryptophan (TrpH), but the semioxidized TrpH radical oxidized BR with k = 4.3 X 10(8) dm3 mole-1 sec-1. When human serum albumin (HSA) was oxidized using radiolytically generated azide radicals, a radical transformation involving TrpH and TyrOH residues occurred with k = 3.8 X 10(3) sec-1. When BR was complexed with the protein the transformation rate was reduced to 1.6 X 10(3) sec-1. This was interpreted in terms of a conformational change in the protein. Identification of the probable residues involved provided information about the primary BR binding site which was consistent with an earlier report.     

On the Onset of Cracks in Arteries¹

We present a theoretical approach to study the onset of failure localization into cracks in arterial wall. The arterial wall is a soft composite comprising hydrated ground matrix of proteoglycans reinforced by spatially dispersed elastin and collagen fibers. As any material, the arterial tissue cannot accumulate and dissipate strain energy beyond a critical value. This critical value is enforced in the constitutive theory via energy limiters. The limiters automatically bound reachable stresses and allow examining the mathematical condition of strong ellipticity. Loss of the strong ellipticity physically means inability of material to propagate superimposed waves. The waves cannot propagate because material failure localizes into cracks perpendicular to a possible wave direction. Thus, not only the onset of a crack can be analyzed but also its direction. We use the recently developed constitutive theories of the arterial wall including 8 and 16 structure tensors to account for the fiber dispersion. We enhance these theories with energy limiters. We examine the loss of strong ellipticity in uniaxial tension and pure shear in circumferential and axial directions of the arterial wall. We find that the vanishing longitudinal wave speed predicts the appearance of cracks in the direction perpendicular to tension. We also find that the vanishing transverse wave speed predicts the appearance of cracks in the the direction inclined (non-perpendicular) to tension. The latter result is counter-intuitive yet it is supported by recent experimental observations.