Human plasma kininogens are identical with alpha-cysteine proteinase inhibitors. Evidence from immunological, enzymological and sequence data

Human high- and low-Mr kininogens were shown to be potent inhibitors of cysteine proteinases such as cathepsin L and papain (Ki = 17-48 pM). A strong immunological cross-reaction between the kininogens and low-Mr alpha-cysteine proteinase inhibitor from human plasma was found. Comparison of partial amino acid sequences from high- and low-Mr kininogen and low-Mr alpha-cysteine proteinase inhibitor demonstrated sequence identity for all segments analyzed. These findings suggest that the kininogens and the alpha-cysteine proteinase inhibitors from human plasma are identical proteins.     

Synthesis of (+)-(S)-ibuproxam and preparation of some new complexes of racemic and (+)-(S)-ibuproxam with β-cyclodextrin and its derivatives

(+)-(S)-Ibuproxam, a prodrug of (+)-(S)-ibuprofen, the pharmacologically active component of ibuprofen, was synthesized in order to minimize side effects (especially gastric irritation) and reduce effective dose. The low water solubility of (+)-(S)-ibuproxam, which prevents rapid dissolution and absorption from the gastrointestinal tract, was overcome by complexation with β-cyclodextrin and its derivatives. The inclusion complex formation was confirmed by differential scanning calorimetry (DSC), by ¹H-NMR spectroscopy, and X-ray powder diffractometry. The physicochemical characteristics of ibuproxam were significantly improved by the complexation. © 1995 Wiley-Liss, Inc.     

Induced Transmembrane Voltage and Its Correlation with Electroporation-Mediated Molecular Transport

Exposure of a cell to an electric field results in inducement of a voltage across its membrane (induced transmembrane voltage, ΔΨ _m) and, for sufficiently strong fields, in a transient increase of membrane permeability (electroporation). We review the analytical, numerical and experimental methods for determination of ΔΨ _m and a method for monitoring of transmembrane transport. We then combine these methods to investigate the correlation between ΔΨ _m and molecular transport through an electroporated membrane for isolated cells of regular and irregular shapes, for cells in dense suspensions as well as for cells in monolayer clusters. Our experiments on isolated cells of both regular and irregular shapes confirm the theoretical prediction that the highest absolute values of ΔΨ _m are found in the membrane regions facing the electrodes and that electroporation-mediated transport is confined to these same regions. For cells in clusters, the location of transport regions implies that, at the field strengths sufficient for electroporation, the cells behave as electrically insulated (i.e., as individual) cells. In contrast, with substantially weaker, nonelectroporating fields, potentiometric measurements show that the cells in these same clusters behave as electrically interconnected cells (i.e., as one large cell). These results suggest that sufficiently high electric fields affect the intercellular pathways and thus alter the electric behavior of the cells with respect to their normal physiological state.     

Effects of electrogenetherapy with p53wt combined with cisplatin on curvival of human tumor cell lines with different p53 status

The aim of our study was to evaluate electrogenetherapy with p53wt alone or combined with cisplatin on two colorectal (HT-29 and LoVo) and two prostatic (PC-3 and Du145) carcinoma cell lines with different p53 status. In addition, the feasibility of electrogenetherapy with p53wt was tested also in vivo on PC-3 prostatic cancer xenografts. Electrogenetherapy with p53wt was dependent on the p53 status of the cell lines used. Electrogenetherapy was the most effective on the PC-3 (p53 null) and Du145 (p53mt) cells, and to the much lesser extent in LoVo cells (p53wt). The exception was the HT-29 cell line with overexpressed mutated p53, where electrogenetherapy with p53wt was the least effective. Sensitivity of the cell lines to cisplatin was independent of the p53 status. Furthermore, the presence of exogenous p53 due to electrogenetherapy did not enhance cisplatin cytotoxicity, since the combination of these therapies resulted in additive cytotoxic effect. The effectiveness of electrogenetherapy with p53wt was also demonstrated in vivo by successful treatment of subcutaneous PC-3 tumors in mice. In conclusion, our study shows that electrogenetherapy with p53wt is feasible, and resulted in comparable cytotoxic and antitumor effectiveness to viral-mediated p53wt gene therapy. This therapy was effective and dependent on the p53 status of the tumor cell lines. Combination of electrogenetherapy and cisplatin resulted in additional cell kill by cisplatin, and was not dependent on the p53 status.     

Theoretical evaluation of the distributed power dissipation in biological cells exposed to electric fields

The paper deals with the power dissipation caused by exposure of biological cells to electric fields of various frequencies. With DC and sub-MHz AC frequencies, power dissipation in the cell membrane is of the same order of magnitude as in the external medium. At MHz and GHz frequencies, dielectric relaxation leads to dielectric power dissipation gradually increasing with frequency, and total power dissipation within the membrane rises significantly. Since such local increase can lead to considerable biochemical and biophysical changes within the membrane, especially at higher frequencies, the bulk treatment does not provide a complete picture of effects of an exposure. In this paper, we theoretically analyze the distribution of power dissipation as a function of field frequency. We first discuss conductive power dissipation generated by DC exposures. Then, we focus on AC fields; starting with the established first-order model, which includes only conductive power dissipation and is valid at sub-MHz frequencies, we enhance it in two steps. We first introduce the capacitive properties of the cytoplasm and the external medium to obtain a second-order model, which still includes only conductive power dissipation. Then we enhance this model further by accounting for dielectric relaxation effects, thereby introducing dielectric power dissipation. The calculations show that due to the latter component, in the MHz range the power dissipation within the membrane significantly exceeds the value in the external medium, while in the lower GHz range this effect is even more pronounced. This implies that even in exposures that do not cause a significant temperature rise at the macroscopic, whole-system level, the locally increased power dissipation in cell membranes could lead to various effects at the microscopic, single-cell level.     

Evaluation of cell membrane electropermeabilization by means of a nonpermeant cytotoxic agent

For the evaluation of cell membrane electropermeabilization, cells are usually exposed to electric pulses in the presence of propidium iodide, a fluorescent dye activated by binding to cellular DNA. The fraction of permeabilized cells is then determined using a flow cytometer. This widely established method has several drawbacks: (i) an arbitrary choice of minimum fluorescence intensity for characterization of permeabilized cells; (ii) the inability to detect cells disintegrated because of intense electropermeabilization; and (iii) false detection of cellular ghosts devoid of fluorescence because of leakage of DNA caused by electropermeabilization. Here, we present a simple and inexpensive method that eliminates these drawbacks. The method is based on the use of a cytotoxic agent that cannot permeate through an intact plasma membrane and thus leads to selective death of the electropermeabilized cells. The amount of nonpermeabilized cells is then determined by a suitable viability test. Bleomycin at a 5-nM concentration causes no statistically significant effect on cell survival in the absence of electric pulses, yet this concentration is sufficient for lethal toxicity in electropermeabilized cells. The amount of cells surviving the exposure relative to the control gives a reliable value of the fraction of nonpermeabilized cells.     

Increases in Endogenous or Exogenous Progestins Promote Virus-Target Cell Interactions within the Non-human Primate Female Reproductive Tract

Currently, there are mounting data suggesting that HIV-1 acquisition in women can be affected by the use of certain hormonal contraceptives. However, in non-human primate models, endogenous or exogenous progestin-dominant states are shown to increase acquisition. To gain mechanistic insights into this increased acquisition, we studied how mucosal barrier function and CD4+ T-cell and CD68+ macrophage density and localization changed in the presence of natural progestins or after injection with high-dose DMPA. The presence of natural or injected progestins increased virus penetration of the columnar epithelium and the infiltration of susceptible cells into a thinned squamous epithelium of the vaginal vault, increasing the likelihood of potential virus interactions with target cells. These data suggest that increasing either endogenous or exogenous progestin can alter female reproductive tract barrier properties and provide plausible mechanisms for increased HIV-1 acquisition risk in the presence of increased progestin levels.     

Estimating quantitative physiological and morphological tissue parameters of murine tumor models using hyperspectral imaging and optical profilometry

Understanding tumors and their microenvironment are essential for successful and accurate disease diagnosis. Tissue physiology and morphology are altered in tumors compared to healthy tissues, and there is a need to monitor tumors and their surrounding tissues, including blood vessels, non-invasively. This preliminary study utilizes a multimodal optical imaging system combining hyperspectral imaging (HSI) and three-dimensional (3D) optical profilometry (OP) to capture hyperspectral images and surface shapes of subcutaneously grown murine tumor models. Hyperspectral images are corrected with 3D OP data and analyzed using the inverse-adding doubling (IAD) method to extract tissue properties such as melanin volume fraction and oxygenation. Blood vessels are segmented using the B-COSFIRE algorithm from oxygenation maps. From 3D OP data, tumor volumes are calculated and compared to manual measurements using a vernier caliper. Results show that tumors can be distinguished from healthy tissue based on most extracted tissue parameters (). Furthermore, blood oxygenation is 50% higher within the blood vessels than in the surrounding tissue, and tumor volumes calculated using 3D OP agree within 26% with manual measurements using a vernier caliper. Results suggest that combining HSI and OP could provide relevant quantitative information about tumors and improve the disease diagnosis.