Marin Kosović

FT-IR spectroscopy of lipoproteins—A comparative study

FT-IR spectra, in the frequency region 4000-600 cm(-1), of four major lipoprotein classes: very low density lipoprotein (VLDL), low density lipoprotein (LDL) and two subclasses of high density lipoproteins (HDL(2) and HDL(3)) were analyzed to obtain their detailed spectral characterization. Information about the protein domain of particle was obtained from the analysis of amide I band. The procedure of decomposition and curve fitting of this band confirms the data already known about the secondary structure of two different apolipoproteins: apo A-I in HDL(2) and HDL(3) and apo B-100 in LDL and VLDL. For information about the lipid composition and packing of the particular lipoprotein the well expressed lipid bands in the spectra were analyzed. Characterization of spectral details in the FT-IR spectrum of natural lipoprotein is necessary to study the influence of external compounds on its structure.

Expression of Secreted Frizzled-Related Protein 1 and 3, T-cell Factor 1 and Lymphoid Enhancer Factor 1 in Clear Cell Renal Cell Carcinoma

Frequency and mortality of renal cell carcinoma (RCC) are increasing for decades. However, the molecular background of RCC tumorigenesis is still poorly understood. In current study we investigated the expression of TCF/LEF and SFRP family members (SFRP1 and SFRP3) to gain a better understanding of biological signaling pathways responsible for epidemiology and clinical parameters of clear cell RCC (cRCC). Thirty-six pairs of paraffin-embedded clear cRCC and adjacent nontumoral tissues samples using immunohistochemistry (IHC) were analyzed and compared with corresponding clinicopathological parameters. Immunohistochemistry indicated statistically significant decreased SFRP3 expression in tumor tissues but no consistency in SFRP1 expression in analyzed normal and tumor tissue. The TCF1 expression level was significantly weaker in normal tissue compared to tumor samples while LEF1 protein levels were significantly weaker in tumor tissue. To our knowledge, this is the first report on analysis of the expression of transcription factors TCF1 and LEF1 in clear cell renal cell carcinoma and their comparison with Wnt signal pathway antagonists belonging to SFRP family.

Porous Silicon Covered with Silver Nanoparticles as Surface-Enhanced Raman Scattering (SERS) Substrate for Ultra-Low Concentration Detection

Microporous and macro-mesoporous silicon templates for surface-enhanced Raman scattering (SERS) substrates were produced by anodization of low doped p-type silicon wafers. By immersion plating in AgNO3, the templates were covered with silver metallic film consisting of different silver nanostructures. Scanning electron microscopy (SEM) micrographs of these SERS substrates showed diverse morphology with significant difference in an average size and size distribution of silver nanoparticles. Ultraviolet–visible–near-infrared (UV-Vis-NIR) reflection spectroscopy showed plasmonic absorption at 398 and 469 nm, which is in accordance with the SEM findings. The activity of the SERS substrates was tested using rhodamine 6G (R6G) dye molecules and 514.5 nm laser excitation. Contrary to the microporous silicon template, the SERS substrate prepared from macro-mesoporous silicon template showed significantly broader size distribution of irregular silver nanoparticles as well as localized surface plasmon resonance closer to excitation laser wavelength. Such silver morphology has high SERS sensitivity that enables ultralow concentration detection of R6G dye molecules up to 10−15 M. To our knowledge, this is the lowest concentration detected of R6G dye molecules on porous silicon-based SERS substrates, which might even indicate possible single molecule detection.

Macrophages and Leydig cells in testicular biopsies of azoospermic men.

A number of studies have indicated that testicular macrophages play an important role in regulating steroidogenesis of Leydig cells and maintain homeostasis within the testis. The current paper deals with macrophages (CD68 positive cells) and Leydig cells in patients with nonobstructive azoospermia (NOA). Methods employed included histological analysis on semi- and ultrathin sections, immunohistochemistry, morphometry, and hormone analysis in the blood serum. Histological analysis pointed out certain structural changes of macrophages and Leydig cells in NOA group of patients when compared to controls. In the testis interstitium, an increased presence of CD68 positive cells has been noted. Leydig cells in NOA patients displayed a kind of a mosaic picture across the same bioptic sample: both normal and damaged Leydig cells with pronounced vacuolisation and various intensity of expression of testosterone have been observed. Stereological analysis indicated a significant increase in volume density of both CD68 positive and vacuolated Leydig cells and a positive correlation between the volume densities of these cell types. The continuous gonadotropin overstimulation of Leydig cells, together with a negative paracrine action of macrophages, could result in the damage of steroidogenesis and deficit of testosterone in situ.

FTIR spectroscopy reveals lipid droplets in drug resistant laryngeal carcinoma cells through detection of increased ester vibrational bands intensity

The major obstacle to successful chemotherapy of cancer patients is drug resistance. Previously we explored the molecular mechanisms of curcumin cross-resistance in carboplatin resistant human laryngeal carcinoma 7T cells. Following curcumin treatment we found a reduction in curcumin accumulation, and reduced induction of reactive oxygen species (ROS) and their downstream effects, compared to parental HEp-2 cells. In order to shed more light on mechanisms involved in drug resistance of 7T cells, in the present study we applied Fourier transform infrared (FTIR) spectroscopy, a technique that provides information about the nature and quantities of all molecules present in the cell. By comparing the spectra from parental HEp-2 cells and their 7T subline, we found an increase in the intensity of ester vibrational bands in 7T cells. This implied an increase in the amount of cholesteryl esters in resistant cells, which we confirmed by an enzymatic assay. Since cholesteryl esters are localized in lipid droplets, we confirmed their higher quantity and serum dependency in 7T cells compared to HEp-2 cells. Moreover, treatment with oleic acid induced more lipid droplets in 7T when compared to HEp-2 cells, as shown by flow cytometry. We can conclude that along with previously determined molecular mechanisms of curcumin resistance in 7T cells, these cells exhibit an increased content of cholesteryl esters and lipid droplets, suggesting an alteration in cellular lipid metabolism as a possible additional mechanism of drug resistance. Furthermore, our results suggest the use of FTIR spectroscopy as a promising technique in drug resistance research.

Spectroscopic studies of alpha tocopherol interaction with a model liposome and its influence on oxidation dynamics

The influence of α-tocopherol on the surface conformation of liposome, as a model component of lipoproteins, and its role in oxidation process were studied. FT-IR spectra from suspensions of neat liposome, mixtures of liposome and α-tocopherol and liposome with incorporated α-tocopherol were analyzed. When α-tocopherol was incorporated into liposome, intensities of some bands were decreased or increased in comparison with the spectra of liposome and α-tocopherol mixture. These changes reflect the different localization of α-tocopherol in two types of liposome suspensions. The oxidation of liposome suspensions was initiated by addition of cupric ions. After prolonged oxidation, the differences in FT-IR spectra of oxidized samples were recorded. Differences were observed in comparison with spectra of native and oxidized liposomes were analyzed. The rate of oxidation was measured by EPR oximetry. Oxidation was generally very slow, but faster in liposome without α-tocopherol, indicating the protective role of α-tocopherol against liposome oxidation. On the other hand, liposome suspensions with EDTA in the buffer were not oxidized at all, while those with α-tocopherol and liposome mixture were only slightly oxidized. In this case the consumption of oxygen was the result of liposome oxidation supported by α-tocopherol. These results reflect the ambivalent role of α-tocopherol in liposome oxidation, similarly to findings in studies of lipoprotein oxidation.

Location of PRODAN in lipid layer of HDL particle: a Raman study

FT Raman spectroscopy has been applied to determine the location of PRODAN within HDL and to investigate its influence on the structure of the particle. The complex spectra of HDL and HDL labeled with PRODAN were divided into three regions according to the wave numbers, and adherent spectra were compared separately. Additionally, recorded spectra of protein and lipid fractions of HDL were used as a support for the assignment of particular vibrations in intact particles. In high frequency region, the shift in vibrational frequencies of CH3 groups but almost negligible shift of CH2 groups suggests that PRODAN is situated at the water/lipid interface in the vicinity of the protein. The statement is supported by the observed influence of PRODAN on particular lipid vibrations of phospholipids head-groups. In the fingerprint region, the influence of PRODAN is observed as the slight change in β-strand secondary structure of apolipoprotein and strongly reduced vibrations of the acyl chain in lipids. That additionally confirms that PRODAN mainly interacts with the lipid domain of the particle. In the low frequency region, the lack of change in Tyr Fermi resonance doublet and only slight differences in the pattern of CS and SS stretching vibrations in labeled HDL confirms that PRODAN has no influence on structure of apolipoprotein embedded in lipid domain. The main conclusions drawn from the vibrational spectra of HDL with and without PRODAN clearly confirm that PRODAN induces negligible changes in HDL structure and hence is reliable fluorescent label for the structural analysis.

Porous silicon by galvanostatic electrochemical anodisation of epitaxial silicon, polycrystalline silicon and silicon on insulator layers

Porous silicon (PSi) samples were prepared by galvanostatic electrochemical anodization of epitaxial silicon, polycrystalline silicon and silicon on insulator layers. Structural and optical properties of prepared samples were investigated by Raman and photoluminescence (PL) spectroscopy, field emission scanning electron microscopy (FE-SEM) and energy dispersive x-ray spectroscopy (EDS). Epitaxial silicon layers of n-type and {111} orientation grown on n-type {111} oriented silicon substrates were anodized as a function of concentration of 48 % HF in ethanol solution and anodization time. Electrical resistivities of the epitaxial layers and of the silicon substrate were ∼2 and ∼0.015 Ω cm, successively. Within the epitaxial layer and on the substrate surface, micro- and nano-pores of different sizes in dependence on HF concentration and anodization time were obtained. For anodization times longer than 30 min epitaxially grown layer detached from the substrate. A high density of micrometer sized pores with regions of three-dimensional photonic crystal expressed in an intersecting oriented macropore lattice on {111} oriented crystal was observed. After detaching the epitaxial layer, the black colored substrate consisted of fine nanometer sized cobweb-like silicon structures whose morphology and density depended on HF concentration and anodization time was found. The Raman spectra of such structures show broadened and red-shifted optical phonon band O(Γ), depending on the size of silicon nanostructures. The intensity of PL of such fine porous substrate shows the sensitivity on the level of the optical phonon confinement. Polycrystalline p-type silicon film were prepared by low pressure chemical vapor deposition (LPCVD) and anodized in aqueous hydrofluoric acid (HF)/ethanol electrolyte. The FE-SEM images showed preferential anodization and macro-PSi formation along grain boundaries. Weak PL signal was detected in all samples, while Raman measurements indicated minimal or no confinement effects. The anodization of silicon on insulator layers was performed by direct and alternating currents at 50 Hz. Raman spectra of DC samples showed no significant shift of c-Si peak while PL spectra showed intensive luminescence over the entire surface. When etched with AC, a very intensive PL was observed at the circular edge of the sample that exhibited micrometer sized island-like porous structures, while the center of the sample showed moderate PL signal similar to DC samples. The formation of such island-like structures was interpreted as stress due to difference of the piezoelectric effect of silicon and quartz layers (buried SiO2). Micro-Raman spectra of islands show strong phonon confinement in the range 1.4–3.5 nm.

W1244 The Role of Adenine and Guanine in Beneficial Effects of the Gastric Pentadecapeptide BPC 157 (PL 14736) On Ileoileal Anastomosis Healing in Rats Impaired with Systemic Corticosteroid Application. Raman Spectroscopy Study

Safe in inflammatory bowel disease (PL14736 Pliva, Croatia), no toxicity reported (Gastroenterology, 2005), gastric pentadecapeptide BPC 157 (BPC 157) heals the intestinal anastomosis (Surg Today, 2007). Raman spectroscopy was used to show the beneficial effect of BPC 157 on anastomosis healing in rats impaired with systemic corticosteroid application. Assessed were Raman spectroscopy, ileoileal anastomosis dehiscence, histology, biomechanical presentation (the volume (ml) (infunded through syringe-perfusion pump system (1ml /10 sec)) and the leakage pressure (mmHg) (catheter connected with chamber and monitor, at 10 cm proximal to anastomosis), on days 1, 2, 3, 4, 5 and 6. BPC 157 10µg, 6-alpha-methylprednisolone (MP) 1 mg, given alone or together /kg i.p. (or an equivolume of saline 5ml/kg). First application was after surgery and last was 24 h before sacrifice. Raman spectroscopy. Measurements used Perkin Elmer Spectrum GX FT Raman spectrometer with 4 cm-1 resolution. Excitation was NdYaG laser at 1064 nm wavelength and used laser power was 500 mW. Spectra were recorded in the area between 300 and 2300 cm-1, with 50 scans for better S/N ratio. Prior to mounting on a universal holder, the specimen was cut to a 0.3 x 0.3 cm piece. Presenting almost constant intensity for all investigated samples, all spectra were baseline corrected and normalized to band at 1450 cm-1. Raman spectroscopy. To point out an advanced healing rate the spectral areas from 510 to 560 cm-1, 800 to 1150 cm-1 and band at 1490 cm-1 were individually observed. We focused the band at 1490 cm-1, assigned to vibrations of adenine and guanine, to be indicative for increased tissue regeneration, since it fully correlates with the evidenced healing process. BPC 157 rats showed an increase throughout the 1-6 days following surgery ; MP-rats did not show before day 6. This healing delay is fully counteracted when rats received BPC 157 simultaneously with MP. Macroscopic and histology data support these findings: adhesion formation attenuated, blood vessels filled, mild passage obstruction only temporary ; anastomosis without leakage sustains markedly higher both volume and pressure, with consistent increase to healthy values ; decreased edema, granulocyte number and necrosis, along with granulation tissue, reticulin and collagen formation substantially increased, and finally epithelization increased (BPC 157) ; (MP)causing aggravation. These data signify the role of adenine and guanine in the anastomosis healing process, BPC 157 has a beneficial effect, MP negative effect, and counteracted corticosteroid impairment by BPC 157 application.