Infrared and ultraviolet spectral signatures and conformational preferences of jet-cooled serotonin

The ultraviolet and infrared spectroscopy of single conformations of neutral serotonin (5-hydroxytryptamine) have been studied in the gas phase using a combination of methods including laser-induced fluorescence, resonance-enhanced two-photon ionization, UV-UV hole-burning spectroscopy, and resonant ion-dip infrared spectroscopy. By comparison to its close analogue tryptamine, for which firm assignments to seven low-energy conformations have been made, UV and IR transitions due to eight conformations of serotonin are observed and assigned. The ultraviolet spectrum divides into two subsets of transitions separated from one another by approximately 230 cm-1 ascribable to syn and anti conformations of the 5-OH group. These two subsets are also distinguishable via their 5-OH stretch fundamentals, with the anti-OH subset shifted by approximately 4-5 cm-1 to lower frequency than those due to syn-OH conformers. The existing firm assignments for tryptamine play a decisive role in assignments in serotonin, where the alkyl CH stretch infrared spectrum is diagnostic of the conformation of the ethylamine side chain. Conformer A of serotonin (SERO(A)), with S1 <-- S0 origin transition at 32584 cm-1, is assigned to Gpy(out)/anti-OH, SERO(B) at 32548 cm-1 to Gpy(up)/anti, SERO(C) at 32545 cm-1 to Gph(out)/anti, SERO(D) at 32560 cm-1 to Anti(py)/anti, SERO(E) at 32537 cm-1 to Anti(up)/anti, SERO(F) at 32353 cm-1 to Gpy(out)/syn, SERO(G) at 32313 cm-1 to Gpy(up)/syn, and SERO(H) at 32282 cm-1 to Gph(out)/syn. The conformational preferences of serotonin differ from those of tryptamine most notably in the selective stabilization observed for the Gph(out)/anti-OH conformer SERO(C), which makes it the second-most intense transition in the ultraviolet spectrum, surpassed only by the Gpy(out)/anti-OH conformer SERO(A).     

Infrared spectral characteristics of noncyclic carbonylureas

Noncyclic diacylimides (noncyclic carbonylureas) have NH frequencies higher than cyclic diacylimides. In solid state spectra, two bands at 3240-3220 cm⁻¹ and 3140-3120 cm⁻¹, and in chloroform spectra, one band at 3480-3380 cm⁻¹ appear consistently in all compounds under discussion. Consequently, these bands could be considered as characteristic of noncyclic diacylimides, and furthermore, the occurrence of several weak bands in the region 3300–3100 cm⁻¹ along with regular NH bands may be considered as characteristic of noncyclic diacylimides. By contrast, cyclic diacylimides in the solid state show two widely separated carbonyl bands in the 1790-1720 cm⁻¹ and 1710-1670 cm⁻¹ regions, whereas in noncyclic diacylimides these bands appear as one strong band at 1670-1660 cm⁻¹ with a weak shoulder at 1690-1680 cm⁻¹. Furthermore, the compounds behave like secondary amides, as there appear amide I, II and III bands regularly. The amide II band seems to be characteristic of noncyclic diacylimides, as the band is missing in cyclic diacylimides.     

Sphingosine mediates FTY720-induced apoptosis in LLC-PK1 cells

FTY720, a synthetic sphingoid base analog, was examined as a new sphingosine kinase inhibitor, which converts endogenous sphingosine into its phosphate form. With 20 microM of FTY720, sphingosine accumulated in the LLC-PK(1) cells in a time- and dose-dependent manner. The FTY720 treated cells showed a high concentration of fragmented DNA, a high caspase-3 like activity and TUNEL staining cells. It was also found that the sphingosine and sphinganine level increased in a time- and dose-dependent manner within 12 h after the FTY720 treatment. The sphingosine kinase activity was reduced by FTY720 as much as other sphingosine kinase inhibitors, N, N-dimethylsphingosine (DMS), dl-threo-dihydrosphingosine (DHS). The fragmented DNA content as a result of the 20 microM of FTY720 treatment and by 5 microM of the exogenously added BSA-sphingosine complex indicated typical apoptosis. Under similar conditions, the accumulated sphingosine concentration in all the cells was almost identical even though the sphingosine distribution inside the cells was somewhat different. These results indicate that the FTY720 induced apoptosis is associated with the inhibition of the sphingosine kinase activity and is strongly associated with the successive accumulation of sphingosine.     

Infrared spectral study of noncrystalline rodlike polymers

Oriented and unoriented films of noncrystalline, wholly-aromatic, rodlike polyamides, and polyesteramides were examined by infrared spectroscopy. The results indicate that orientation, accomplished by means of mechanical stretching, approaches 80% in both polymer classes. Examination of the NH and the CO stretching region revealed an increase in the population of nonhydrogen bonded species (plateauing at ～ 85%) as orientation increases. This unusual result may be caused by interchain steric interactions which may also be responsible for both the noncrystalline morphology and the non-lyotropic behavior exhibited by these polymers.     

Infrared spectral profiles in liquids and atom-diatom interactions

Molecular dynamics simulations of the infrared spectrum of a generic simple polar diatomic in a liquid nonpolar solvent allow to reproduce the different prototypical experimental line shapes of this kind of systems. This is feasible by using different solute-solvent anisotropic potentials at fixed thermodynamic conditions. In the limit cases, the rotation of the diatomic is explained in terms of a quasifree motion or a rotational diffusion evolution and the spectra show a doublet structure formed by P and R branches or a unique collapsed branch, respectively. When the profile contains three branches, including an intense Q branch in the vicinity of the center of the band, rotational evolution presents a particular hindering that can be understood by studying the influence on rotational spectral densities of the different time scales involved in rotational relaxation. Cancellation/enhancement effects among spectral density terms arising from intermediate and long times (0.4-1 ps) are essential to understand rotational hindering.     

Discrimination of zone-specific spectral signatures in normal human prostate using Raman spectroscopy

The prostate gland is the most common site of pathology in human males. Using the urethra as an anatomical reference point, it can be divided into three distinct zones known as the transition zone (TZ), peripheral zone (PZ) and central zone (CZ). The pathological conditions of benign prostatic hypertrophy and/or prostate adenocarcinoma are highly prevalent in this gland. This preliminary study set out to determine whether biochemical intra-individual differences between normal prostate zones could be identified using Raman spectroscopy with subsequent exploratory analyses. A normal (benign) prostate transverse tissue section perpendicular to the rectal surface and above the verumontanum was obtained in a paraffin-embedded block. A 10-μm-thick slice was floated onto a gold substrate, de-waxed and analysed using Raman spectroscopy (200 epithelial-cell and 140 stromal spectra/zone). Raman spectra were subsequently processed in the 1800-367 cm(-1) spectral region employing principal component analysis (PCA) to determine whether wavenumber-intensity relationships expressed as single points in hyperspace might reveal biochemical differences associated with inter-zone pathological susceptibility. Visualisation of PCA scores plots and their corresponding loadings plots highlighted 781 cm(-1) (cytosine/uracil) and 787 cm(-1) (DNA) as the key discriminating factors segregating PZ from less susceptible TZ and CZ epithelia (P < 0.001). Conversely, 1459 cm(-1) (lipids and proteins) and 1003 cm(-1) (phenylalanine) were identified as the key biochemical factor distinguishing TZ from CZ epithelia (P < 0.05). All stromal zones were discriminated by the protein/lipid region (1459 cm(-1) and 1100 cm(-1)) with DNA/RNA region (781 cm(-1) and 787 cm(-1)) only highlighted between PZ and CZ (P < 0.05). This novel approach identifies biochemical markers that may have aetiological functional roles towards susceptibility of human prostate zones to specific pathological conditions.     

Role of p21CIP1 as a determinant of SC-560 response in human HCT116 colon carcinoma cells

SC-560, a structural analogue of celecoxib, induces growth inhibition in a wide range of human cancer cells in a cyclooxygenase (COX)-independent manner. Since SC-560 suppresses the growth of cancer cells mainly by inducing cell cycle arrest, we sought to examine the role of p21CIP1, a cell cycle regulator protein, in the cellular response against SC-560 by using p21(+/+) and p21(-/-) isogenic HCT116 colon carcinoma cells. In HCT116 (p21(+/+)) cells, SC-560 dose-dependently induced growth inhibition and cell cycle arrest at the G1 phase without significant apoptosis induction. SC-560-induced cell cycle arrest was accompanied by upregulation of p21CIP1. However, the extent of SC-560-induced accumulation at the G1 phase was approximately equal in the p21(+/+) and the p21(-/-) cells. Nonetheless, the growth inhibition by SC-560 was increased in p21(-/-) cells than p21(+/+)cells. SC-560-induced reactive oxygen species (ROS) generation did not differ between p21(+/+) and p21(-/-) cells but the subsequent activation of apoptotic caspase cascade was more pronounced in p21(-/-) cells compared with p21(+/+) cells. These results suggest that p21CIP1 blocks the SC-560-induced apoptotic response of HCT116 cells. SC-560 combined with other therapy that can block p21 CIP1 expression or function may contribute to the effective treatment of colon cancer.     

Photodynamically induced effects in colon carcinoma cells (WiDr) by endogenous photosensitizers generated by incubation with 5-aminolaevulinic acid.

Human adenocarcinoma cells of the line WiDr have been treated with 2 mM 5-aminolaevulinic acid (5-ALA) in the presence of 10% foetal calf serum. The treatment induces a linear accumulation of protoporphyrin IX (PpIX) for at least 7.5 h. After 7.5 h of incubation about 45% of the PpIX accumulated is cell-bound, while the rest is found in the medium (25%) or lost from the cells during washing with phosphate-buffered saline (30%). Exposure to white light at an intensity of 30 W/m2 for 18 min results in 95% reduction of clonogenicity in cells treated with 2 mM 5-ALA for 3.5 h. The enzymatic activities of enzymes located in cytosol (glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase) and lysosomes (acid phosphatase and beta-glucuronidase) are not influenced by a 5-ALA and light treatment inactivating about 35% of the cells. The MTT assay, which reflects mitochondrial dehydrogenase activity, but not succinate dehydrogenase, is partly inhibited by the same treatment. Treatment with 5-ALA in the absence of light increases O2 consumption by a factor of two, while the O2 consumption is inhibited when 5-ALA treatment is combined with exposure to light. In addition, 5-ALA and light exposure enhance accumulation of rhodamine 123 by 40% and reduce the intracellular ATP level by 25%. Confocal laser scanning microscopical analysis indicates granular perinuclear localization of the PpIX formed by 5-ALA treatment. In conclusion, photodynamic treatment using 5-ALA as a prodrug induces damage to mitochondrial function without inhibiting lysosomal and cytosolic marker enzymes.     

Infrared action spectra of Ca2+(H2O)(11-69) exhibit spectral signatures for condensed-phase structures with increasing cluster size

Infrared laser action spectroscopy is used to characterize divalent calcium ions solvated by up to 69 water molecules. The spectrum for Ca(2+)(H2O)12 indicates that in the predominant structure, eight inner-shell water molecules solvate the metal ion and donate one hydrogen bond to one of four second-shell, double-acceptor water molecules. Eight-coordinate solvation is consistent with results from many condensed-phase studies, and contrasts with results for smaller gas-phase clusters that are most consistent with six-coordinate solvation. Each water molecule in this structure of Ca(2+)(H2O)12 coordinates with two other members of the cluster. With increasing cluster size, the number of two-coordinate water molecules decreases, whereas that of three-coordinate water molecules increases. The number of one-coordinate water molecules increases until n approximately 18, but they are essentially depleted by n approximately = 30. Spectra of the largest clusters, which have effective concentrations of divalent calcium that are less than 1 M, exhibit only subtle changes with increasing cluster size. The bonded-OH regions of these spectra are similar to, but blue-shifted from that of bulk water, whereas the free-OH regions are well-resolved and indicate that the surfaces of these clusters are well-structured. These results comprise the most extensive vibrational spectroscopic study yet performed on metal ion hydration in the gas phase and provide insights into metal ion solvation in bulk and interfacial environments.     

Infrared photoelectric effects in polyethylene

We have observed a flow of charge on illumination of low-density polyethylene with infrared light in the wavelength range 1.6–0.7 μ by using silver electrodes. This flow is reversed on removal of the light, is proportional to the light intensity, and varies little with wavelength in this spectral region. There is no significant response to light of visible wavelengths. The effect decreases as the temperature is increased up to 60°C, though the time constant remains fixed. There are qualitative differences between the responses obtained with gold and with silver electrodes, and quantitative changes when the ambient air pressure is altered. The behavior suggests that during illumination, the dynamic equilibrium between carriers in the electrodes and those in the material close to the electrodes undergoes a reversible adjustment.     

Comparative study using Raman microspectroscopy reveals spectral signatures of human induced pluripotent cells more closely resemble those from human embryonic stem cells than those from differentiated cells

Raman microspectroscopy is a non-destructive, label-free optical technique that offers information-rich molecular analysis of living cells. We report here the first Raman spectral study of human induced pluripotent stem cells (hiPSCs), and compare their Raman features to those of human embryonic stem cells (hESCs) and differentiated progeny of hESCs. Raman spectra from 687 cm(-1) to 1073 cm(-1) were collected from living hiPSCs, hESCs and hESCs non-specifically differentiated for 20 days. Spectra of hiPSCs and hESCs were found to be highly similar, and both were distinguishable from differentiated hESCs in terms of relative Raman peak intensities and variances. Principal component analysis (PCA) of the spectra demonstrated a clear discrimination between hiPSCs and differentiated hESCs. These results suggested that reprogramming returned human somatic cells to a state where the overall cellular composition was similar to that of human embryonic stem cells. Some metabolic differences between the two groups of pluripotent cells could be inferred, however it was unclear whether or not these differences were related to reprogramming.     

Two-dimensional infrared spectral signatures of 3(10)- and alpha-helical peptides

Two-dimensional infrared (2D IR) spectra of Calpha-alkylated model octapeptides Z-(Aib)8-OtBu, Z-(Aib)5-L-Leu-(Aib)2-OMe, and Z-[L-(alphaMeVal)]8-OtBu have been measured in the amide I region to acquire 2D spectral signatures characteristic of 3(10)- and alpha-helical conformations. Phase-adjusted 2D absorptive spectra recorded with parallel polarizations are dominated by intense diagonal peaks, whereas 2D rephasing spectra obtained at the double-crossed polarization configuration reveal cross-peak patterns that are essential for structure determination. In CDCl3, all three peptides are of the 3(10)-helix conformation and exhibit a doublet cross-peak pattern. In 1,1,1,3,3,3-hexafluoroisopropanol, Z-[L-(alphaMeVal)]8-OtBu undergoes slow acidolysis and 3(10)-to-alpha-helix transition. In the course of this conformational change, its 2D rephasing spectrum evolves from an elongated doublet, characteristic of a distorted 3(10)-helix, to a multiple-peak pattern, after becoming an alpha-helix. The linear IR and 2D absorptive spectra are much less informative in discerning the structural changes. The experimental spectra are compared to simulations based on a vibrational exciton Hamiltonian model. The through-bond and through-space vibrational couplings are modeled by ab initio coupling maps and transition dipole interactions. The local amide I frequency is evaluated by a new approach that takes into account the effects of hydrogen-bond geometry and sites. The static diagonal and off-diagonal disorders are introduced into the Hamiltonian through statistical models to account for conformational fluctuations and inhomogeneous broadening. The sensitivity of cross-peak patterns to different helical conformations and the chain length dependence of the spectral features for short 3(10)- and alpha-helices are discussed.     

Infrared microspectroscopy of Oral Squamous Cell Carcinoma: Spectral signatures of cancer grading

In the present paper, we compared the histopathological and vibrational analyses of different tissue sections of Oral Squamous Cell Carcinoma (OSCC) at various malignancy grades, in order to unambiguously identify them. To achieve reliable results, healthy and dysplastic samples were also taken into account. FT-IR microspectroscopy is considered an effective tool for studying different molecular structures occurring in tumoral tissues and offers an interesting alternative to detect biochemical changes in a non-subjective way. In particular, on an adequate number of tissue sections affected by three different grades of OSSC (well G1, moderately G2, and poorly G3 differentiated), as well as on dysplastic and healthy tissues (all obtained from surgical resection), the chemical maps were acquired on meaningful areas containing both epithelial and connective structures. The multivariate analysis (Hierarchical Cluster Analysis, HCA, and Principal Component Analysis, PCA), performed separately on epithelial and connective spectral data, afforded to a good segregation for the different morphological structures. By analysing the representative spectra of healthy, dysplastic and tumoral epithelia and connectives, modifications were pin-pointed in the position of bands and absorbance band ratios usually associated with carcinogenesis. Above all, the changes in the protein pattern (with modifications in the length of side chains and in secondary structures), and in carbohydrates and nucleic acids moieties were associated with specific spectral markers of this pathology. The vibrational investigation led to a satisfactory understanding of these lesions so contributing to an early diagnosis, when the sole morphological inspection may result troublesome.     

Infrared spectral observations of chlorophyll-a and ethyl chlorophyllide-a in the solid state

Abstract— Infrared spectra of ethyl chlorophyllide-a and chlorophlyll-a in Nujol mulls show a shift of the C₉ ketone carbonyl absorption band to higher frequencies when the pigments are subjected to drying processes. The ketone band can then be reversed back to lower frequencies by exposing the material to moisture. These observations indicate that the nature of chlorophyll aggregation in the solid state varies with the water content of the pigment.     

Alterations in mitochondrial and apoptosis-regulating gene expression in photodynamic therapy-resistant variants of HT29 colon carcinoma cells

Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragment-associated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3' region Mbol complementary DNA, glutamate dehydrogenase, hepatoma-derived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondrial and apoptosis-regulating genes contributes to PDT resistance.     

Studies on the structure, stability, and spectral signatures of hydride ion-water clusters

The gas-phase structure, stability, spectra, and electron density topography of H(-)W(n) clusters (where n = 1-8) have been calculated using coupled-cluster CCSD(T) and M?ller-Plesset second-order perturbation (MP2) theory combined with complete basis set (CBS) approaches. The performance of various density functional theory (DFT) based methods such as B3LYP, M05-2X, M06, M06-L, and M06-2X using 6-311++G(d,p), and aug-cc-pVXZ (aVXZ, where X = D, T, and Q) basis sets has also been assessed by considering values calculated using CCSD(T)/CBS limit as reference. The performance of the functionals has been ranked based on the mean signed/unsigned error. The comparison of geometrical parameters elicits that the geometrical parameters predicted by B3LYP/aVTZ method are in good agreement with those values obtained at MP2/aVTZ level of theory. Results show that M05-2X functional outperform other functionals in predicting the energetics when compared to CCSD(T)/CBS value. On the other hand, values predicted by M06-2X, and M06 methods, are closer to those values obtained from MP2/CBS approach. It is evident from the calculations that H(-)W(n) (where n = 5-8) clusters adopt several interesting structural motifs such as pyramidal, prism, book, Clessidra, cubic, cage, and bag. The important role played by ion-water (O-H···H(-)) and water-water (O-H···O) interactions in determining the stability of the clusters has also been observed. Analysis of the results indicates that the most stable cluster is made up of minimum number of O-H···H(-) interaction in conjugation with the maximum number of O-H···O interactions. The Bader theory of atoms in molecules (AIM) and natural bond orbital (NBO) analyses has also been carried out to characterize the nature of interactions between hydride ion and water molecules. It can be observed from the vibrational spectra of H(-)W(n) clusters, the stretching frequencies involving ion-water interaction always exhibit larger redshift and intensities than that of water-water (inter solvent) interactions.     

Infrared spectral comparison of 5-aminolevulinic acid and its hexyl ester

5-aminolevulinic acid (ALA) and its hexyl ester (ALA-H) are bioorganic molecules, now used as drugs in the study and clinical application of photodynamic therapy (PDT). Their infrared spectra were reported in first time here. The spectral characteristic was found well correlated to their structure feature. The strong peaks of C=O, C-H(2) and O-H band were shown in ALA spectrum. While in case of ALA-H, besides the vibration modes of C=O and CH(2) the additional CH(3) infrared peaks appeared, which correspond with their structural difference. Thus the infrared spectrum could be used to detect and distinguish ALA and ALA-H, which have potential for the mechanism study of ALA and ALA-H based PDT in biological system. Using the infrared spectrum as the probe, the thermal effect on structure stability was detected. Below the temperature of 80 degrees C, the ALA and ALA-H are thermally stable in structure. When temperature reached 120 degrees C, the serious structure breaking (thermal decomposition) happened for both ALA and ALA-H.     

Infrared spectral changes identified during different stages of herpes viruses infection in vitro

Microscopic Fourier transform infrared spectroscopy (FTIR) which is based on the characteristic molecular vibrational spectra of cells was previously applied for the identification of various biological samples. In the present study, FTIR spectroscopy was used for the characterization of different stages during the development of herpes viruses infection. Vero cells in culture were infected with high and low doses of different herpes viruses [herpes simplex virus types 1 and 2 (HSV-1, -2) or varicella-zoster virus (VZV)], and cellular changes were observed by optical and electron microscopy and analyzed by FTIR microscopy at different periods of time post-infection. Specific different spectral changes were observed at various stages of the viral infection development. The spectral intensity in the 1220-1260 cm(-1) region (mainly attributed to phosphate levels) was considerably increased in all infected cells compared to normal uninfected cells during the early stages of the viral infection development. However, at the late stages of the viral infection development (when all the cells in the infected culture lost their spindle shape and became circular) the spectral intensities in this region significantly decreased in the infected compared to the control cells. In addition, the peak at 1023 cm(-1), attributed to carbohydrates, almost fully disappeared at early stages of the viral infection development, whereas at late stages of the infection it raised to an equivalent or higher level than that of the uninfected control cells. These results support the potential of developing FTIR microspectroscopy as a simple, reagent free method for the early detection and accurate differentiation of different stages during the development of herpes virus infection.