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Spectroscopic evaluation of the effect of a red microalgal polysaccharide on herpes-infected Vero cells.

The sulfated polysaccharide obtained from a species of red microalga has proved to be a potent antiviral agent against various members of the herpes family. In the present study, we used microscopic Fourier transform infrared spectroscopy (FT-IR) to investigate differences between normal cells, those infected with herpes viruses, and infected cells treated with red microalgal polysaccharide. FT-IR enables the characterization of cell or tissue pathology based on characteristic molecular vibrational spectra of the cells. The advantage of microscopic FT-IR spectroscopy over conventional FT-IR spectroscopy is that it facilitates inspection of restricted regions of cell cultures or tissue. Our results showed significant spectral differences at early stages of infection between infected and noninfected cells, and between infected cells treated with the polysaccharide and those not treated. In infected cells, there was an impressive decrease in sugar content and a considerable increase in phosphate levels in conjunction with the infection progress. Our results also proved that sugars penetrated and accumulated inside cells treated with the red microalgal polysaccharide. These could have been sugar fragments of low molecular weight present in the polysaccharide solution, despite purification by dialysis. Such sugar accumulation might be responsible for a breakdown in the internal steps of the viral replication cycle.

Animals↗

Effect of fatty acids on phase behavior of hydrated dipalmitoylphosphatidylcholine bilayer: saturated versus unsaturated fatty acids.

The effect of some fatty acids on the phase behavior of hydrated dipalmitoylphosphatidylcholine (DPPC) bilayer was investigated with special interest in possible difference between saturated and unsaturated fatty acids. The phase behavior of hydrated DPPC bilayer was followed by a differential scanning calorimetry and a Fourier transform infrared spectroscopy. The addition of palmitic acid (PA) increased the bilayer phase transition temperature with the increase of the PA content in the mixture. In addition, DPPC molecules in gel phase bilayer became more rigid in the presence of PA compared with those in the absence of PA. This effect of PA on the phase behavior of hydrated DPPC bilayer is common to other saturated fatty acids, stearic acid, myristic acid, and also to unsaturated fatty acid with trans double bond, elaidic acid. Contrary to these fatty acids, oleic acid (OA), the unsaturated fatty acid with cis double bond in the acyl chain, exhibited quite different behavior. The effect of OA on the bilayer phase transition temperature was rather small, although a slight decrease in the temperature was appreciable. Furthermore, the IR spectral results demonstrated that the perturbing effect of OA on the gel phase bilayer of DPPC was quite small. These results mean that OA does not disturb the hydrated DPPC bilayer significantly.

1,2-Dipalmitoylphosphatidylcholine↗

The identification of polar organic compounds found in consumer products and their toxicological properties.

Exposure to volatile organic compounds (VOCs) in the indoor environment has received substantial research attention in the past several years, with the goal of better understanding the impact of such exposures on human health and well-being. Many VOCs can arise from consumer products used within the indoor environment. The VOCs emitted from five representative consumer products were collected onto Tenax-GC and subjected to thermal desorption and analysis by gas chromatography, in combination with low-resolution mass spectrometry (MS), high-resolution MS, and matrix-isolation Fourier transform infrared spectroscopy for structural characterization. An emphasis was placed on the polar organic compounds often used to provide fragrance in these products. The structures of a number of these compounds were confirmed, and an electronic literature search was carried out on them to determine any known toxic properties. The search revealed that many of the VOCs possess toxic properties when studied at acute, relatively high-level exposures. In addition, toxic effects were reported for a few of the chemicals, such as benzaldehyde, alpha-terpineol, benzyl acetate, and ethanol, at relatively low dose levels of 9-14 mg/kg. In general, the data were unclear as to the effect of chronic, low-level exposures. The widespread use of such chemicals suggests that the health effects of chronic exposures need to be determined. Validated analytical methods for the quantitative characterization of polar organic compounds at low concentrations will be required to make such work possible.

Air Pollution, Indoor↗

Properties of amorphous calcium carbonate and the template action of vaterite spheres.

The fast mixing of aqueous solutions of calcium chloride and sodium carbonate could immediately result in amorphous calcium carbonate (ACC). Under vigorous stirring, the formed ACC in the precipitation system will dissolve first and, then, transform within minutes to produce crystalline forms of vaterite and calcite. After that, the solution-mediated mechanism dominates the transformation of the thermodynamically unstable vaterite into the thermodynamically stable calcite. Although ACC is the least stable form of the six anhydrous phases of calcium carbonate (CaCO(3)), it could be, however, produced and stabilized by a variety of organisms. To better understand the formation-transformation mechanism of ACC and vaterite into calcite, ex-situ methods (i.e., scanning electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction spectroscopy) were used to characterize the formation-transformation process of ACC and vaterite in aqueous systems without organic additives, showing that ACC sampled at different conditions has different properties (i.e., lifetime, morphology, and spectrum characterization). It is also very interesting to capture the obviously polycrystalline particles of CaCO(3) during the transformation process from vaterite to calcite, which suggests the formation mechanism for the calcite superstructure with multidimensional morphology.

Calcium Carbonate↗

Mechanism(s) of in vitro percutaneous absorption enhancement of tamoxifen by enhancers.

The effects of enhancers (5% terpenes; i.e., eugenol, limonene, and menthone) in combination with 50% propylene glycol in water (50% PG) on the in vitro percutaneous absorption of tamoxifen through the porcine epidermis, on biophysical changes in the stratum corneum (SC) lipids, on macroscopic barrier properties, and on binding of the drug to the SC were investigated. These enhancers in combination with 50% PG significantly increased (p<0.05) the permeability coefficient of tamoxifen in comparison with that of the control (50% PG in water). Fourier transform infrared spectroscopy (FT-IR) was employed to investigate the biophysical changes in the SC lipids. The FT-IR results showed that treatment of the SC with 5% terpenes/50% PG did not shift the asymmetric and symmetric C-H stretching absorbances peak positions to higher wavenumbers but resulted in a decrease in the peak heights and areas in comparison with the untreated SC. Treatment with menthone and limonene in combination with 50% PG significantly increased (p<0.05) the partition coefficient of tamoxifen in comparison with treatment with 50% PG alone. Also, exposure of the SC to 5% terpenes in combination with 50% PG significantly increased (p < 0.05) the in vitro transepidermal water loss (TEWL) in comparison with 50% PG alone. Thus, an enhancement by menthone, eugenol, and limonene in the permeability of the SC to tamoxifen is due to lipid extraction and macroscopic barrier perturbation. Moreover, the effective diffusion coefficient of tamoxifen through the epidermis was enhanced following the treatment with either 5% eugenol/50%PG or 5% limonene/50%PG compared with 50%PG alone.

Animals↗

Solution effects on the surface reactions of three bioactive glass compositions.

The in vitro five-stage surface reactions of two bioactive glass compositions, 45S5 and 52S4.6, and one bioinert glass, 60S3.8, exposed to three simulated body fluids (SBF) were analyzed using Fourier Transform infrared Spectroscopy (FTIR). There was little effect of SBF composition on ion exchange, silica hydrolysis, and silica polymerization (stages 1-3) of glass with silica content up to 52 wt%. However, calcium and phosphate ions in SBF accelerated the formation of an amorphous calcium-phosphate (a-CP) layer (stage 4), and crystallization (stage 5) of the hydroxycarbonate apatite (HCAp) layer. The magnesium ions had a retardation effect on the kinetics of stages 4 and 5, but little effect on stages 1-3. In SBF solutions which contained calcium and phosphate ions an amorphous calcium-phosphate (a-CaP) layer formed on even a 60S3.8 glass which was not bioactive in vivo. However, the a-CaP layer did not crystallize to form HCAp. Thus, there is a significant contribution from the ions present in the SBF solutions to the HCAP formation and crystallization of HCAp on bioactive glasses. Also, silanol repolymerization is necessary for rapid crystallization of HCAp.

Apatites↗

Comparative study of the retrogradation of intermediate water content waxy maize, wheat, and potato starches.

The retrogradation of extruded starches from three different botanical sources was studied in concentrated conditions (34 +/- 1% water) at 25 degrees C using differential scanning calorimetry (DSC) and isothermal calorimetry, Fourier transform infrared spectroscopy (FTIR), and wide-angle X-ray scattering. Potato starch showed the highest rate of retrogradation (approximately 0.17 h(-1)) followed by waxy maize (approximately 0.12 h(-1)), while the retrogradation of wheat starch was the slowest (approximately 0.05 h(-1)). In addition to the kinetics, the extent of molecular order in the retrograded samples was studied in detail in terms of "short-range" (helical) and "long-range" (crystalline) distance scales. The amylopectin crystallinity indices were essentially the same (approximately 47-51% amylopectin basis) for the three starches. However, significant differences were found in the enthalpy of melting measured by DSC after "full" retrogradation (potato, 11.6 +/- 0.7; waxy maize, 9.0 +/- 0.5; and wheat, 6.1 +/- 0.3 J/g of amylopectin). The degree of short-range molecular order in the retrograded state determined by FTIR was waxy maize > potato > wheat. The effect of amylopectin average chain length and the polymorphism of the crystalline phase were taken into account to explain the differences in the retrogradation enthalpies.

Amylopectin↗

The stability of insulin in crystalline and amorphous solids: observation of greater stability for the amorphous form.

PURPOSE: Generalizations based upon behavior of small molecules have established that a crystalline solid is generally much more stable toward chemical degradation than is the amorphous solid. This study examines the validity of this generalization for proteins using biosynthetic human insulin as the model protein. METHODS: Amorphous insulin was prepared by freeze drying the supernate from a suspension of zinc insulin crystals adjusted to pH 7.1. Storage stability at 25 degrees C and 40 degrees C were compared for the freeze dried material, the dried suspended crystals, and the starting batch of crystals. Samples were equilibrated at selected relative humidities between zero and 75% to obtain samples at various water contents. Assays for dimer formation were performed by size exclusion HPLC and assays for deamidated product were carried out by reverse phase HPLC. Degradation was found to be linear in square root of time, and the slopes from % degradation vs. square root of time were used to define the rate constants for degradation. Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) were used to characterize the state of the protein in the solids. RESULTS: As expected based upon previous results, the primary degradation pathways involve deamidation at the AsnA21 site and co-valent dimer formation, presumably involving the A-21 site. Contrary to expectations, amorphous insulin is far more stable than crystalline insulin under all conditions investigated. While increasing water content increases the rate of degradation of crystalline insulin, rate constants for degradation in the amorphous solid are essentially independent of water content up to the maximum water content studied (approximately 15%). CONCLUSIONS: Based upon the FTIR and DSC data, both crystalline and amorphous insulin retain some higher order structure when dried, but the secondary structure is significantly perturbed from that characteristic of the native solution state. However, neither DSC nor FTIR data provide a clear interpretation of the difference in stability between the amorphous and crystalline solids. The mechanism responsible for the superior stability of amorphous insulin remains obscure.

Calorimetry, Differential Scanning↗

FT-IR spectroscopic analysis of normal and cancerous tissues of esophagus.

AIM: To investigate the special Fourier transform infrared spectroscopy (FT-IR) spectra in normal and cancerous tissues of esophagus. METHODS: Twenty-seven pairs of normal and cancerous tissues of esophagus were studied by using FT-IR and the special spectra characteristics were analyzed in different tissues. RESULTS: Different spectra were found in normal and cancerous tissues. The peak at 1 550/cm was weak and wide in cancerous tissues but strong and high in normal tissues. The ratio of I 1 647/I 1 550 was 2.0 in normal tissues and 2.36 in cancerous tissues (P<0.05). The ratio of I 1 550/I 1 080 was 4.5 in normal tissues and 3.4 in cancerous tissues (P<0.01). The peak at 1453 /cm was higher than at 1 402/cm in normal tissue and lower than at 1 402/cm in cancerous tissues. CONCLUSION: The results indicate that FTIR may be used in clinical diagnosis.

Case-Control Studies↗

Temporary ex vivo inhibition of the expression of the human oncogene HER2 (NEU) by a triple helix-forming oligonucleotide.

A 28-base phosphodiester triple helix-forming oligonucleotide, mostly G and A containing, targeted to a polypurine tract interrupted by a purine-pyrimidine inversion, situated upstream from the TATA box of the promoter of the human HER2 gene, was conceived by computer modeling. The "energetically best choice" was oligo 28(C), which formed the triple helix in vitro, as proved by gel retardation and Fourier transform infrared spectroscopy. When administered as a complex with lipofectin, fluorescence confocal microscopy and electrophoresis confirmed the delivery and persistence of this unprotected oligonucleotide inside MCF7 (breast cancer) cells. At a concentration of 2 microM, the oligonucleotide reduced within 6 h the HER2 mRNA level to 42% (Northern blot) but did not interfere with the transcription of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. During the first day of administration at 0.22 microM, it lowered to 59% the HER2 protein in treated, as compared to nontreated, cells (ELISA). The effect was sequence specific when compared to that of five different negative controls, and it was target selective when compared to the expression of a related, nontargeted protein, the epidermal growth factor receptor. By day 2, the inhibitory effect was overcome by replenishment reactions.

Base Sequence↗

Nanoscale biomimetics: fabrication and optimization of stability of peptide-based thin films.

The method of thin film preparation known as layer-by-layer assembly is of growing interest for current and envisioned developments in bionanotechnology. Here, cysteine-containing 32mer peptides have been designed, synthesized, purified, and used to prepare polypeptide films. A range of methods-quartz crystal microbalance, Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and high-performance liquid chromatography-have been used to probe the effect of ionic strength and polymer secondary structure in solution on peptide self-assembly, and on secondary structure formation and disulfide bond cross-linking in the multilayer film. The amount of designed peptide deposited per adsorption step of film fabrication increased with increasing ionic strength, as with conventional polyelectrolytes. Secondary structure content changed from random coil to beta sheet on incorporation of peptides into a film. "Peptide-inherent" cross-linking by disulfide bond formation increased film stability at acidic pH. Conditions for disulfide stabilization have been optimized. The results contribute to exploration of the physical basis of peptide self-assembly broaden the scope of applications of layer-by-layer assembly, particularly where biocompatibility and stability are key design concerns, and provide a basis for mass production of custom polypeptide thin films of high stability, even in harsh environments.

Amino Acid Sequence↗

Solid state interactions between the proton pump inhibitor omeprazole and various enteric coating polymers.

The influence of the acidic film formers Eudragit L 100, HPMCAS-HF, HP-55, and shellac on the stability of the acid-labile proton pump inhibitor omeprazole in solid drug-polymer blends at accelerated storage conditions (40 degrees C/75% RH) was determined by fourier transform infrared spectroscopy (FTIR), modulated temperature differential scanning calorimetry (MTDSC), and high performance liquid chromatography (HPLC). As expected, acidic polymers caused a degradation of omeprazole which was manifested by discolorations and increasing amounts of degradation products. However, MTDSC curves and FTIR spectra did not show additional peaks resulting from the omeprazole degradation products. These methods appeared to be not sensitive enough to separate analytically the drug and polymer signals from those of the decomposition products. With HPLC a sufficient quantification of the degradation products was possible. HP-55 caused the highest degree of omeprazole degradation, followed by shellac, HPMCAS-HF, and Eudragit L 100. No correlation with the microenvironmental pH values generated by the acidic polymers at the applied storage conditions was found. The melting process and the dissolution of acidic impurities were figured out as possible reasons for the more pronounced decomposition of the drug in presence of HP-55 and shellac.

Anti-Ulcer Agents↗

Surface properties of superparamagnetic iron oxide MR contrast agents: ferumoxides, ferumoxtran, ferumoxsil.

The surface properties of the active ingredients in AMI colloidal, superparamagnetic iron oxide magnetic resonance (MR) contrast agents are described. Scanning electron microscopy/energy dispersive X-ray elemental analyses and diffuse reflectance Fourier transform infrared spectroscopy (FTIR) spectra of ferumoxsil (AMI-121 drug substance) were consistent with the presence of a monolayer of H2NCH2CH2NHCH2CH2CH2Si(O-)3 siloxane monomer or dimer. The X-ray photoelectron spectra (XPS) of ferumoxsil are also consistent with complete coverage of the iron oxide surface with a monolayer of siloxane. The static secondary ion mass spectra (SSIMS) of ferumoxsil showed that the siloxane film is covalently bonded (i.e., Si-O-Fe bonds) to the iron oxide surface. The FTIR of ferumoxides (AMI-25) and Ferumoxtran (AMI-227) showed only adsorbed dextran. The XPS spectra of the dextran-coated colloids showed that Ferumoxtran has a thicker layer of dextran than ferumoxides iron oxide particles (approximately 5 and approximately 3 nm, respectively). The SSIMS spectra of these dextran-coated colloids showed only low mass fragments due to the adsorbed dextran. The nature of the interactions of the dextran coating with the iron oxide surfaces of ferumoxides and Ferumoxtran is discussed.

Contrast Media↗

Preparation of budesonide- and indomethacin-hydroxypropyl-beta-cyclodextrin (HPBCD) complexes using a single-step, organic-solvent-free supercritical fluid process.

The purpose of this study was to determine whether budesonide- and indomethacin-hydroxypropyl-beta-cyclodextrin (HPBCD) complexes could be formed using a supercritical fluid (SCF) process. The process involved the exposure of drug-HPBCD mixtures to supercritical carbon dioxide (SC CO2). The ability of the SCF process to form complexes was assessed by determining drug dissolution, drug crystallinity, and drug-excipient interactions. Drug dissolution was assessed using a HPLC assay. Crystallinity was assessed using powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC). Drug-excipient interactions were characterized using Fourier transform infrared spectroscopy (FTIR). Scanning electron microscopy (SEM) was used to determine any morphological changes. SC CO2 process did not alter the dissolution rate of pure drugs but resulted in two- and three-fold higher dissolution rates for budesonide- and indomethacin-HPBCD mixtures, respectively. SCF-processed mixtures exhibited a disappearance of the crystalline peaks of the drugs (PXRD), a partial or complete absence of the melting endotherm of the drugs (DSC), and a shift in the C=O stretching of the carboxyl groups of the drugs (FTIR), consistent with the loss of drug crystallinity and formation of intermolecular bonds with HPBCD. SEM indicated no discernible drug crystals upon physical mixing with or without SCF processing. Thus, budesonide- and indomethacin-HPBCD complexes with enhanced dissolution rate can be formed using a single-step, organic solvent-free SC CO2 process.

2-Hydroxypropyl-beta-cyclodextrin↗

Lipid and protein changes due to freezing in Dunning AT-1 cells.

Defining the process of cellular injury during freezing, at the molecular level, is important for cryosurgical applications. This work shows changes to both membrane lipids and protein structures within AT-1 Dunning prostate tumor cells after a freezing stress which induced extreme injury and cell death. Cells were frozen in an uncontrolled fashion to -20 or -80 degrees C. Freezing resulted in an increase in the gel to liquid crystalline phase transition temperature (T(m)) of the cellular membranes and an increase in the temperature range over which the transition occurred, as determined by Fourier transform infrared spectroscopy (FTIR). Thin layer chromatography (TLC) analysis of total lipid extracts showed free fatty acids (FFA) in the frozen samples, indicating a change in the lipid composition. The final freezing temperature had no effect on the thermotropic response of the membranes or on the FFA content of the lipid fraction. The overall protein secondary structure as determined by FTIR showed only slight changes after freezing to -20 degrees C, in contrast to a strong and apparently irreversible denaturation after freezing to -80 degrees C. Taken together, these results suggest that the decrease in viability between control and frozen cells can be correlated with small changes in the membrane lipid composition and membrane fluidity. In addition, loss of cell viability is associated with massive protein denaturation as observed in cells frozen to -80 degrees C, which was not observed in samples frozen to -20 degrees C.

Adenocarcinoma↗

In vitro percutaneous absorption enhancement of propranolol hydrochloride through porcine epidermis by terpenes/ethanol.

The purpose of this study was to investigate the mechanism(s) of percutaneous absorption enhancement of propranolol hydrochloride (PHCL) across porcine epidermis by terpenes (e.g. menthone and limonene) in combination with ethanol. The in vitro percutaneous absorption experiments were performed using Franz diffusion cells. The solubility of PHCL in control and enhancer solutions was determined through high-performance liquid chromatography. Partitioning of PHCL to powdered SC from control or enhancer solutions was also determined in order to investigate the binding of PHCL to the SC from the SC/enhancer system. Fourier transform infrared spectroscopy (FT-IR) was employed to study the biophysical changes in stratum corneum (SC) lipids. The in vitro macroscopic barrier properties were investigated by measuring transepidermal water loss (TEWL) using Tewameter. Five percent menthone or limonene in combination with ethanol (EtOH) (menthone/EtOH or limonene/EtOH) significantly increased (P<0.05) the flux of PHCL through porcine epidermis in comparison to the control (EtOH). The partitioning of PHCL to the SC from the SC/enhancer system was also significantly greater than the SC/control system. The above enhancers showed a decrease in the peak heights and areas for both asymmetric and symmetric C-H stretching absorbances in comparison with the untreated SC, indicating the SC lipids extraction. Menthone/EtOH and limonene/EtOH enhanced (P<0.05) the in vitro TEWL through the epidermis in comparison to the control. Thus, an enhancement in the flux of PHCL by menthone/EtOH and limonene/EtOH is due to SC lipid extraction, macroscopic barrier perturbation, and improvement in the partitioning of the drug to the SC.

Animals↗

Improvement of stability and cell adhesion properties of polyelectrolyte multilayer films by chemical cross-linking.

Poly(L-lysine)/hyaluronan (PLL/HA) films were chemically cross-linked with a water soluble carbodiimide (EDC) in combination with a N-hydroxysulfo-succinimide (NHS) to induce amide formation. Fourier transform infrared spectroscopy confirms the conversion of carboxylate and ammonium groups into amide bonds. Quartz crystal microbalance-dissipation reveals that the cross linking reaction is accompanied by a change in the viscoelastic properties of the films leading to more rigid films. After the cross-linking reaction, both positively and negatively ending films exhibit a negative zeta potential. It is shown by fluorescence recovery after photobleaching measured by confocal laser scanning microscopy that cross-linking dramatically reduces the diffusion of the PLL chains in the network. Cross linking also renders the films highly resistant to hyaluronidase, an enzyme that naturally degrades hyaluronan. Finally, the adhesion of chondrosarcoma cells on the films terminating either with PLL or HA is also investigated. Whereas the non cross-linked films are highly resistant to cell adhesion, the cells adhere and spread well on the cross-linked films.

Cell Adhesion↗

Interaction of metal ions with caffeine and theophylline: stability and structural features.

The interactions of caffeine and theophylline with divalent cadmium, mercury, strontium and barium ions were studied in aqueous solution and physiological pH. Fourier transform infrared spectroscopy (FTIR) and absorption spectra were used to determine the cation binding mode and association constants. Spectroscopic results showed that Cd(2+), Hg(2+), Sr(2+) and Ba(2+) bind strongly to caffeine and theophylline. Direct and indirect (through metal hydration shell) interactions were observed for caffeine and theophylline with Cd(2+), Hg(2+), Sr(2+) and Ba(2+) through O6 and N9 (caffeine) and O6, N9 and N7 atoms (theophylline). The overall binding constants are:k(Cd-caffeine) = 1.24 x 10(5) M(-1), k(Hg-caffeine) = 1.74 x 10(5) M(-1), k(Sr- caffeine) = 3.3 x 10(4) M(-1), k(Ba-caffeine) = 1.8 x 10(4) M(-1), k(Cd-theophylline) = 5.75 x 10(5) M(-1), k(Hg-theophylline) = 2.14 x 10(5) M(-1), k(Sr-theophylline) = 4.6 x 10(4) M(-1), k(Ba-theophylline) = 3 x 10(4) M(-1). These k values are evidence for weak and strong cation interactions in these metal complexes.

Caffeine↗