Egidijus E. Uzgiris

Study of lymphocyte and erythrocyte electrophoretic mobility by laser doppler spectroscopy

Abstract Laser Doppler spectroscopy, a technique that permits rapid and precise measurement of the distribution of electrophoretic mobilities in a population of suspended particles, has been applied to blood cells by using suspending medium of low ionic strength made isoosmotic with sucrose. Data on the mobilities of erythrocytes and lymphocytes, and the pH dependencies of these mobilities, appear in good agreement with those determined by classical procedures.

Laser Doppler methods in electrophoresis

Abstract Electrophoretic motion of particles, molecules, and biological cells can be readily measured by laser Doppler techniques. Small frequency shifts associated with the motion of the scatterers are detected by heterodyne detection of the scattered laser light. The principles of laser light scattering and heterodyne detection are reviewed. The central experimental problems associated with the application of electric fields to conducting solutions are considered in detail. Various types of laser Doppler spectrometers and electrophoresis chambers are compared both from fundamental physical points of view as well as in terms of resolving power of standard marker cells. As applications of the laser Doppler technique, measurements on proteins, virtues, nucleic acids, bioparticles and biological cells are reviewed.

Comparative measurements of size and polydispersity of several insect viruses

Abstract Comparative measurements of the sizes and polydispersity of the Tipula iridescent virus and the nuclear polyhedrosis viruses of the gypsy moth and European pine sawfly have been made by laser light-scattering spectroscopy, resistivepulse analysis, and electron microscopy. Size measurements are in substantial agreement by the techniques. Polydisperse components in the various viral preparations are distinctly resolvable by resistive-pulse analysis, are measurable by electron microscopy, but are combined into a weighted average size by laser light-scattering spectroscopy.

Complex thermal desorption of PCBs from soil

Abstract PCBs desorb from heated soil in a complex fashion with a fast phase and a much slower desorbing recalcitrant fraction. This recalcitrance is associated with clay layers of the montmorillonite type, the expandable clays. The recalcitrance arises from the fraction of the PCBs binding in the intercrystalline water layers of these clays. These sites provide kinetic traps rather than deep energy barriers to desorption. Such sites may play a role in the bioavailability of the PCBs for biodegradation or in ambient desorption and dispersal.

A Multimodal Contrast Agent for Preoperative MR Imaging and Intraoperative Tumor Margin Delineation

We have constructed a multimodal contrast agent suitable for near-infrared, NIR, fluorescent imaging as well as magnetic resonance imaging, MRI. This class of agents may be useful for preoperative tumor localization and tumor functional evaluation and for intraoperative delineation of tumor margins. We have covalently attached dyes of the cyanine family to a previously described polymeric contrast agent, Gd-DTPA-polylysine, of an extended, uncoiled conformation. The dual modality agent is as effective in imaging tumors by MRI as the parent compound provided that the dye loading on the polymer is such that it does not eliminate all the available free-lysine groups on the parent Gd-DTPA-polylysine polymers. NIR fluorescence from preclinical subcutaneous and orthotopic mammary gland tumors could be detected with a signal to background ratio of as high as 4.5 at 12 hours post agent injection at a dye dose of 125 nmole/kg. For intraoperative delineation of tumor margins, a wide-field illumination camera system was devised giving high signal to background NIR fluorescent images of surgically exposed orthotopic mammary gland tumors. Histologic microscopy confirmed the location of the dual modality agent at the boundary of the tumor with a margin distance of about 0.3 mm from labeled tumor cells.

Tumor imaging with a macromolecular paramagnetic contrast agent: Gadopentetate dimeglumine-polylysine

RATIONALE AND OBJECTIVES We evaluated magnetic resonance (MR) contrast enhancement of tumor tissue following injection of the macromolecular conjugate, gadopentetate dimeglumine-polylysine. METHODS T1-weighted MR imaging scans were performed on female Fisher-344 rats with subcutaneously implanted mammary adenocarcinoma tumors. Following the baseline scan, gadopentetate dimeglumine-polylysine or gadopentetate dimeglumine was injected at a dose of 0.1 mmol gadolinium per kilogram. RESULTS Gadopentetate dimeglumine-polylysine injection resulted in a maximum enhancement of tumor contrast of 310 +/- 60% (n = 7). Tumor tissue remained enhanced and well defined for several days after gadopentetate dimeglumine-polylysine injection. Gadopentetate dimeglumine injection at the same dose resulted in a 70 +/- 25% (n = 4) maximal tumor enhancement and a corresponding 25 +/- 4% muscle enhancement. CONCLUSION Gadopentetate dimeglumine-polylysine provides higher, more sustained tumor contrast than does gadopentetate dimeglumine for the same dosage of gadolinium.

Analysis of T cell subpopulations by laser Doppler spectroscopy and the association of electrophoretic mobility differences with differences in rosette-forming affinity.

Abstract Rosetting procedures were used to identify and isolate human T lymphocytes having different affinities for sheep red blood cells (SRBC). High-affinity E rosette-forming cells (E-RFC) were obtained by rosetting at 29°C with limited numbers of SRBC. Low-affinity and total E-RFC were obtained by rosetting at 4°C with an excess of SRBC. The E-RFC isolated from these fractions on Ficoll-Hypaque gradients exhibited different electrophoretic mobilities as measured by laser Doppler spectroscopy. The high-affinity E-RFC mobility was centered at 2.35 μm/sec/V/cm (for 25°C, 0.28 M sucrose medium of 0.005 ionic strength), whereas the low-affinity E-RFC mobility was centered at 2.15 μm/ sec/V/cm. The total E-RFC fractions consisted of cells of both mobilities. When IgG-sensitized E monolayers were used to remove cells bearing Fc receptors from mononuclear cell preparations, the non-adherent and the adherent fraction were each found to contain a different subpopulation of T cells having mobilities centered at 2.35 and 2.15 μm/sec V/cm, respectively. Rosetting assays and mobility measurements were performed simultaneously with the mononuclear cell preparations and the non-adherent fraction from IgG-sensitized E monolayers. In both cases, there was quantitative agreement between the number of high-affinity E-RFC and the number of high-mobility cells. In contrast, there were consistently more low-mobility cells than could be accounted for by low-affinity E-RFC. The determination of cell electrophoretic mobility is a direct physical measurement and its association with such surface markers as different affinity receptors for SRBC and Fc receptors for IgG shows that it should be useful parameter in the characterization of lymphocytes.

Association of electrophoretic mobility with other cell surface markers of T cell subpopulations in normal individuals and cancer patients.

Abstract Two major electrophoretically distinct T cell components were observed in peripheral blood of normal donors. The cells comprising these components differed in their binding capacity for sheep red blood cells (SRBC). High-affinity E-rosette-forming cells (E-RFC) were obtained by rosetting at 29°C with limited numbers of SRBC and belonged to the high-mobility component. Low-affinity E-RFC belonged to the low-mobility component. In cancer patients the fraction of cells falling in the high-mobility class decreased by 20 ± 11% of total cells, but no significant decrease of high-affinity E-RFC was observed. Thus, the correspondence between high-affinity and high-mobility found for normal donor lymphocytes may not hold for a subset of lymphocytes found in individuals with cancer.

Length of glycoprotein spikes of vesicular stomatitis virus and sindbis virus, measured in Situ using quasi elastic light scattering and a resistive-pulse technique

Abstract Sindbis and vesicular stomatitis virus (VSV) were sized in aqueous medium using two nondestructive methods: quasi-elastic laser light scattering (LLS), which measures the hydrodynamic Stokes-Einstein diameter of particles in suspension, and the Nanopar resistive-pulse technique (RP), which measures the diameter corresponding to the nonconductive volume of the particles. The total length of the glycoprotein spikes on the external surface of viral particles contributes to the hydrodynamic diameter, but the spikes make only a marginal contribution to the nonconductive diameter. Spherical Sindbis virus has a hydrodynamic Stokes-Einstein diameter of 735 ± 20 A as measured by LLS, and a diameter of 625 ± 6 A as measured by RP. Sindbis particles made spikeless by protease treatment have an RP diameter of 595 ± 6 A. From these measurements, a spike length of 70 ± 20 A was calculated. Similar comparison between the equivalent sphere diameters of intact and spikeless VSV measured by LLS and RP led to a calculated spike length of 175 ± 25 A. Upon the adsorption of purified VSV glycoprotein to uniform spherical polystyrene beads, a corresponding value of 170 ± 20 A was obtained via LLS as the thickness of the protein layer. Thus, the glycoproteins of VSV extend almost twice as far out of the membrane as do those of Sindbis, suggesting that the glycoprotein of these viruses must be characterized by quite different protein conformations.

Evanescent wave immunoprobe with high bivalent antibody activity

Abstract We have determined the kinetic response of an evanescent wave optical fibre immunosensor and its absolute sensitivity. Using both kinetic methods and optical determinations of bound antigen, we infer that there are 2·4 × 10 11 active antibodies per cm 2 probe area. We estimate that 75% of the active antibodies are in the bivalent form, with both binding site capable of binding antigen. Using the kinetic data and optical measurements of the number of antigens bound to the fibre, we estimate that the unstirred dead layer thickness next to the fibre surface in the stirred solution, across which the antigens must diffuse, is 55 μm. Approximately 10 9 FITC labelled antigens are necessary to achieve a threshold signal, defined as 1% of the signal obtained when the fibre is saturated with antigen.