Anatoli Ianoul

Steady-State and Transient Ultraviolet Resonance Raman Spectrometer for the 193–270 nm Spectral Region

We describe a state-of-the-art tunable ultraviolet (UV) Raman spectrometer for the 193–270 nm spectral region. This instrument allows for steady-state and transient UV Raman measurements. We utilize a 5 kHz Ti-sapphire continuously tunable laser (∼20 ns pulse width) between 193 nm and 240 nm for steady-state measurements. For transient Raman measurements we utilize one Coherent Infinity YAG laser to generate nanosecond infrared (IR) pump laser pulses to generate a temperature jump (T-jump) and a second Coherent Infinity YAG laser that is frequency tripled and Raman shifted into the deep UV (204 nm) for transient UV Raman excitation. Numerous other UV excitation frequencies can be utilized for selective excitation of chromophoric groups for transient Raman measurements. We constructed a subtractive dispersion double monochromator to minimize stray light. We utilize a new charge-coupled device (CCD) camera that responds efficiently to UV light, as opposed to the previous CCD and photodiode detectors, which required intensifiers for detecting UV light. For the T-jump measurements we use a second camera to simultaneously acquire the Raman spectra of the water stretching bands (2500–4000 cm−1) whose band-shape and frequency report the sample temperature.

Interactions of antimicrobial peptide from C-terminus of myotoxin II with phospholipid mono- and bilayers

Comparative studies of the effect of a short synthetic cationic peptide, pEM-2 (KKWRWWLKALAKK), derived from the C-terminus of myotoxin II from the venom of the snake Bothrops asper on phospholipid mono- and bilayers were performed by means of Langmuir Blodgett (LB) monolayer technique, atomic force microscopy and calcein leakage assay. Phospholipid mono- and bilayers composed of single zwitterionic or anionic phospholipids as well as lipid mixtures mimicking bacterial cell membrane were used. LB measurements indicate that the peptide binds to both anionic and zwitterionic phospholipid monolayers at low surface pressure but only to anionic at high surface pressure. Preferential interaction of the peptide with anionic phospholipid monolayer is also supported by a more pronounced change of the monolayer pressure/area isotherms induced by the peptide. AFM imaging reveals the presence of nanoscale aggregates in lipid/peptide mixture monolayers. At the same time, calcein leakage experiment demonstrated that pEM-2 induces stronger disruption of zwitterionic than anionic bilayers. Results of the study indicate that electrostatic interactions play a significant role in the initial recognition and binding of pEM-2 to the cell membrane. However, membrane rupturing activity of the peptide depends on interactions other than simple ionic attraction.

Photosensitivity and volume gratings in phosphate glasses for rare-earth-doped ion-exchanged optical waveguide lasers

Phosphate glasses containing enough sodium to allow the fabrication of optical channel waveguides by ion exchange are photosensitive to intense ultraviolet irradiation from excimer lasers at 193 and 248nm. Permanent submicrometer-period volume index gratings with modulation amplitudes larger than 10−4 were obtained even after annealing the irradiated samples for 2h at 230°C. Ultraviolet-visible absorption changes were measured in the irradiated areas, as well as structural modifications observed by surface profiling. Comparative studies were carried out for glasses that were also codoped with erbium and ytterbium to provide high gain at wavelengths near 1.5μm. Finally, we demonstrate narrowband gratings with reflectivities larger than 80% in silver ion-exchanged channel waveguides made in these phosphate glass substrates.

P-glycoprotein effect on the properties of its natural lipid environment probed by Raman spectroscopy and Langmuir-Blodgett technique

Behavior of P‐glycoprotein (Pgp) natural lipid environment within the membrane of CEM cells expressing Pgp in the quantities varying from 0% to 32% of the total amount of all membrane proteins is described for the first time. Observed cooperative effect of Pgp‐induced increase of membrane stability, decrease of the temperature of gel‐to‐crystal lipids transition and predominance of the lipid liquid crystalline phase at physiological temperatures should have an impact in development of multidrug resistance phenotype of tumor cells by favoring the Pgp intercellular transfer and Pgp ATPase activity.

Near-Field Scanning Optical Microscopy to Identify Membrane Microdomains

Near-field scanning optical microscopy (NSOM) allows optical imaging with a spatial resolution that is significantly better than the diffraction limited resolution achievable with conventional optical microscopy. NSOM has the potential to study the nanoscale organization of membrane surfaces and ultimately to resolve questions concerning lipid rafts in both model-supported membranes and cellular membranes. Supported phospholipid monolayers and bilayers of phase separated binary and ternary lipid mixtures that model the composition of lipid rafts in natural membranes have been studied by NSOM. The results illustrate the ability of NSOM measurements with 50-100 nm probe apertures to obtain detailed nanoscale information for small, closely spaced domains, and the utility of two-color experiments to probe localization of raft markers in supported membranes.

Raman, SERS, and induced circular dichroism techniques as a probe of pharmaceuticals in their interactions with the human serum albumin and p-glycoprotein

Camptothecin (CPT) derivatives are the well known inhibitors of the human DNA topoisomerase (topo) I. Two of them, irinotecan and topotecan, are just in the clinics; 9-amino- CPT is on the stage II of clinical trials, and the active search for new derivatives is now in progress. Stability of the CPT derivatives on their way to the target and resistance of cancer cells to these drugs present the crucial problem of the chemotherapy. Human serum albumin (HSA) is the mediator of transport and metabolism of numerous pharmaceuticals in the blood and P-glycoprotein (P- gp) plays a crucial role of the mediator of the multidrug resistance (MDR) of the cancer cells. This paper present the result of analysis of molecular interactions of some drugs of CPT family with the HSA and P-gp. Induced circular dichroism (CD) and Raman techniques have been applied for monitoring molecular interaction of drugs with HSA as well as to identify the conformational transition of the protein induced by the drug binding. Drug molecular determinants responsible for interaction have been identified and their binding sites within the HSA have been localized. New cancer cells lines exhibiting an extremely high level of MDR resistance have been established and were shown to contain the P-gp overproduced in the quantities of 35 percent from the all membrane proteins. The membrane fractions of these cells with the controls presented by the membranes of the parental membrane proteins. The membrane fractions of these cells with the controls presented by the membranes of the parental sensitive cells may be used as a model system for spectroscopic analysis of the specific pharmaceuticals/P-gp interactions.