The Modified Heparin-Binding L-Asparaginase of Wolinella succinogenes.

The modified asparaginase Was79 was derived from the recombinant wild-type L-asparaginase of Wolinella succinogenes. The Was79 contains the amino acid substitutions V23Q and K24T responsible for the resistance to trypsinolysis and the N-terminal heparin-binding peptide KRKKKGKGLGKKR responsible for the binding to heparin and tumor K562 cells in vitro. When tested on a mouse model of Fischer lymphadenosis L5178Y, therapeutic efficacy of Was79 was significantly higher than that of reference enzymes at all single therapeutic doses used (125-8000 IU/kg). At Was79 single doses of 500-8000 IU/kg, the complete remission rate of 100 % was observed. The Was79 variant can be expressed intracellularly in E. coli as a less immunogenic formyl-methionine-free form at high per cell production levels.

[Modeling organic matter dynamics in conifer-broadleaf forests win different site types upon fires: a computational experiment].

The effect of forest fires differing in intensity on organic matter dynamics in forest soils has been assessed in different types of forest sites using the EFIMOD 2 system of models. Differences between the patterns of organic matter dynamics according to scenarios of forest ecosystem development under normal conditions and upon forest fires have been analyzed. Recovery rates of soil organic matter pools after fires depend on their intensity and frequency. The most profound changes take place upon high-intensity crown fires, which may even result in ecosystem destruction.

Effects of mistletoe lectin I and ionizing radiation on the glucose and thymidine uptake in tumour cells in vitro.

The increased uptake of hexose by mammalian cells is considered to be a general response to stress. Nowadays, mistletoe lectin separated from the extracts of the European mistletoe (Viscum album L.) is often used in adjuvant cancer therapy. The present work studies the effect of the lectin on unirradiated and x-irradiated tumour cells. The response of cultured human lung carcinoma cells (Calu-1) was followed by radioactive glucose uptake as well as by tritiated thymidine incorporation. The cells were maintained either in a complete or a so-called restrictive medium. Slight metabolic changes were found in the restrictive medium but not in the complete one. Mistletoe lectin I at a very low concentration (0.001 ng/mL) increased the glucose uptake and thymidine incorporation. Ionizing radiation (1 Gy) did not influence the hexose uptake but it enhanced the incorporation of thymidine. It seems that the actions of two different factors (mistletoe lectin I and radiation) proved to be rather provoking stress effects for the tumour cells as detected in the restrictive medium.

Cellular alterations upon IR-laser (890 nm) exposures, in vivo.

Exposure of cultured cells and small animals to ionizing radiation as well as irradiation of cultured cells with He-Ne laser can cause changes in the functional condition of plasma membranes. The ionizing radiation-induced cell membrane alterations have been determined after either partial or local exposures. The aim of the present study was to reveal whether the local laser treatments cause a general, distant, so called abscopal" effect measured at cellular level, when the laser treatment is intended as a stimulatory procedure. The biological effect of infrared laser (mean power of 5 Watts, 150 Hz frequency, 890 nm wavelength) was demonstrated through 3H-concanavalin A binding by blood cells of daily irradiated (altogether 10 exposures) oncological and non-oncological patients as well as by changes in the proliferation of bone marrow cells of whole body gamma-irradiated (4 Gy) rats, partially laser-treated. The lectin binding of lymphocytes of oncological, as well as ischaemic heart disease patients was increased immediately after the first laser treatment. However, it was decreased after completion of the full course. In cases of inflammatory diseases the test parameters were either unchanged or decreased as compared to their self-control values. The platelets and erythrocytes did not react in any group. Gamma irradiation caused a deep inhibition of proliferation of rat bone marrow cells. The number of fibroblast colony-forming units (CFU-F) could be increased again if the animals were partially exposed to laser. Laser irradiation of one of the femurs led to some recovery of CFU-F values in the exposed as well as unexposed femur. Thus, local infrared laser treatment induces abscopal effects on the cell membrane and cell proliferation characteristics.

Effect of visible light on some cellular and immune parameters.

The biological effect of visible light of low energy density was investigated in this study. The effects of diffuse (DL) and linearly polarized (LPL) light were compared on models in vitro and in vivo. Experiments in vitro were performed on human lymphocytes to study their blast-transformation and rosette-formation abilities. Both DL and LPL increased the number of blast-transformed cells even in a lymphocyte culture without PHA, and reduced rosette-formation of T lymphocytes. LPL had a more pronounced effect. In vivo exposure to DL and LPL of the spleens of tumour-bearing mice caused the appearance of factor(s) in their serum, inhibiting the incorporation in vitro of [3H]-thymidine into the tumour cells obtained from non-exposed animals. In the other series of experiments serum samples were taken from tumorous animals after the exposure of their spleens to LPL. Following the daily administration of these sera to another group of non-exposed tumorous mice a decreasing tendency of the mitotic kinetics of ascites tumour was observed. The application of visible (preferably linearly polarized) light for the stimulation of human immune competent cells, and clinical trials with extracorporeal irradiation of blood for the promotion of natural defences of an immune-repressed organism are suggested.

Detection of early membrane and nuclear alterations of thymocytes upon in vitro ionizing irradiation.

Some membrane and nuclear parameters of rat thymocytes were studied after in vitro X- or gamma-irradiation with doses of 0.5, 1, 2 and 4 Gy followed by incubation for 0.5 to 4 hours at 21-22 degrees C. Early (within the first 2 hours) distinct functional changes of plasma membranes, i.e. increase in Con A binding, autologous rosette-forming capacity, Alcian Blue-induced agglutination, and a decrease in amount of surface negative charges were observed. Meanwhile, the doses applied did not influence the DNA content, and the proportion of pyknotic nuclei did not grossly differ from that of the time-matched controls. However, an increase in AT-rich DNA component was noted. The radiation-induced changes proved to be transient and dose-dependent. In the whole cell populations no irreversible, death-associated events could be detected under the given experimental conditions.

Modifying effects of the parent and radio-detoxified endotoxins on cell-mediated cytotoxicity and cytokine release.

The cytotoxic effects of the parent and radio-detoxified lipopolysaccharides (LPS and RD-LPS, respectively) were studied at various concentrations (0-50 micrograms/ml) upon cultured human lung carcinoma target cells. There was no significant difference between the effects of the two endotoxins. LPS and RD-LPS at the same concentrations, however, modified the cytolytic activity of human leukocytes (effector cells) and their mediators in different extent. The most remarkable difference was found at 10 micrograms/ml. A much higher cytotoxic activity of effector cells was observed in the case of RD-LPS as compared to the parent LPS. This concentration used for the treatment of X-irradiated and unexposed effector cells at 24 h incubation resulted in elevated release of some cytokines as measured by ELISA. For increasing the natural defence, RD-LPS as immunomodulator may be of practical value.

Effects of modulated and continuous microwave irradiation on the morphology and cell surface negative charge of 3T3 fibroblasts.

Mouse embryo 3T3 cells were irradiated with 2450 MHz continuous and low frequency (16 Hz) square modulated waves of absorbed energy ranging from 0.0024 to 2.4 mW/g. The low frequency modulated microwave irradiation yielded more morphological cell changes than did the continuous microwave fields of the same intensity. The amount of free negative charges (cationized ferritin binding) on cell surfaces decreased following irradiation by modulated waves but remained unchanged under the effect of a continuous field of the same dose. Modulated waves of 0.024 mW/g dose increased the ruffling activity of the cells, and caused ultrastructural alteration in the cytoplasm. Similar effects were experienced by continuous waves at higher (0.24 and 2.4 mW/g) doses.

Effects of low energy beta-irradiation from tritiated water on the morphology of 3T3 fibroblasts.

Cellular alterations of cultured 3T3 cells irradiated with beta-rays from tritiated water were studied by scanning and transmission electron microscopy. We observed decreased negative surface charges, vacuolization of rough endoplasmic reticulum and Golgi-complex, degeneration of mitochondria, increase of lysosomal activity and changes in distribution and amount of microfilaments in the irradiated cells, that parallelled changes in cell shape.

Humoral leukocyte adherence inhibition (H-LAI) follow-up trials on malignant process in mice.

Humoral antitumor response of Lewis lung tumor-bearing mice was measured by humoral leukocyte adherence inhibition (H-LAI) test. The reactivity of serum against tumor antigens prepared from primary tumor and lung metastases could be revealed at the first day after injection of tumor cells. On the other hand, the macroscopical appearance of the primary tumors and lung metastases was observed after 5 and 11 days, respectively. These findings suggest that the immune reaction of the host could be detected by H-LAI test earlier than the tumor can manifest itself. The antigens prepared from primary tumors and metastases seemed to be very similar, however, the metastasis antigen had additional determinants as detected by the H-LAI technique. Comparison of two tumor lines, the original Lewis lung tumor (LLT) and its variant with high metastatic capacity (LLT-HH), showed high similarity between their antigens as tested in H-LAI system by cross-reaction probes.

Humoral leukocyte adherence inhibition (H-LAI) test in screening of high risk group for lung cancer.

In the screening examinations of 150 ore miners the positive humoral immune response against lung tumor antigen was measured in 30 serum samples. Repeated testing of positive sera (after 1-3 years) was possible only in 15 cases. Among them, the reaction of 12 serum samples was again positive, and 2 persons died of lung cancer. The results obtained in these follow-up investigations are discussed.

Electron microscopic studies on radiation-induced changes of cell surface negative charges.

The amount and distribution of negatively charged sites of different cells (human fibroblasts, B16 melanoma cells, a human lymphoid leukemic cell type) were investigated. In the non-irradiated fibroblasts and B16 melanoma cells the negatively charged sites were localized mainly on apical and lateral surfaces as well as in a polarized manner. However, negatively charged sites on the control human lymphoid leukemic cells often have patched distribution. It was demonstrated that the amount and distribution of negatively charged sites on primary human fibroblasts and B16 melanoma cells changed upon ionizing radiation. However, the topology of negative charges on investigated human leukemic cell membrane did not change.

Changes in arachidonic acid content of X-irradiated human lymphocytes and plasma membrane fractions.

The 14C-arachidonic acid content of pre-labelled human lymphocytes was followed during in vitro incubation in untreated and X-irradiated cells. Loss of about 35-40 per cent of the incorporated radioactivity was found already at 10 minutes after irradiation as detected in whole cells, phospholipid, free fatty acid and phosphatidylcholine fractions of plasma membranes. When pulse labeling with 14C-arachidonic acid was performed at various intervals after in vitro irradiation, the uptake of precursor by irradiated cells did not differ from untreated ones. At the same time the activity of phospholipid, free fatty acid and phosphatidylcholine fractions was approximately 50 per cent lower compared to the control, then it increased gradually reaching the control level.

Surface heterogeneity of tumor cells and changes upon ionizing radiation.

Heterogeneous distribution of surface domains is a characteristic feature of the tumor cell surface and the distribution differs from that of normal cells. During the malignant transformation the heterogeneity may change or disappear. Cell lines with various metastasizing capacities show different distributions of membrane domains or other differences in membrane or surface organization. We have demonstrated that the amount and distribution of negatively charged sites of B 16 melanoma membranes changed upon ionizing radiation (X-ray, 60Co-gamma). In the case of the P 388 lymphoma, however, only the amount of negatively charged sites change after irradiation, the distribution remains unaltered. Both features proved to be radioresistant in human lymphoid leukemic cells.

Polarized membrane domains of fibroblasts.

Data on the heterogeneous distribution of various membrane domains on the surface of fibroblasts are reviewed. Polarized localization of negative charges is demonstrated and new results on the development and maintenance of polarity in primary human fibroblasts are presented. Cell membrane heterogeneity in fibroblasts and in other cells is compared. Our results indicate that the regional localization of membrane domains of fibroblasts, and their dependence on cell movement and cell contacts is in several aspects similar to that described for epithelial cells.

Modified humoral leukocyte adherence inhibition test for detection of lung cancer.

The freshly separated indicator cells (suspension of leukocytes) used in humoral leukocyte adherence inhibition test were labeled either with 14C-amino acid mixture or 3H-concanavalin A (3H-ConA). Instead of counting the adherent cells, the amount of 'adherent' radioactivity was measured by a liquid scintillation spectrometer. By the modified method, sera from 25 lung-carcinoma-bearing patients as well as serum samples from 21 healthy persons were examined in the presence of crude antigens prepared from 'normal' lung tissue or lung tumors of various histologic types. Although the results demonstrated high specificity and reproducibility of both methods, the binding of 3H-ConA to the surface of adherent cells is more expressed and assures higher levels of radioactivity.

Effect of X-irradiation on adenylate cyclase activity and cyclic AMP content of primary human fibroblasts.

Ionizing radiation provokes an increase of the cAMP level in several organs and body fluids. After reviewing the relevant literature we present the results of our own experiments on primary human fibroblasts. X-irradiation at doses of 0.5 and 2.5 Gy in vitro evoked a rapid and reversible increase of adenylate cyclase enzyme activity. A significant increase in cAMP level of these cells was also observed. Adenylate cyclase was usually localized basolaterally on the surface of unirradiated cells, while irradiation resulted in a modification of distribution, i.e., the enzyme activity also appeared in apical localization.

The effects of low doses of ionizing radiation upon the micromorphology and functional state of cell surface.

The cellular membrane as one of the targets of ionizing radiation might play an important role in the development and modification of radiation-induced alterations after low doses. The present paper reviews the micromorphological and functional changes of plasma membranes of irradiated blood and cultured cells with special emphasis on the surface conditions: lectin binding, negative surface charges. The review is completed by our own studies on distribution of positive surface charges and the bindings of two lectins, the Concanavalin A and the wheat germ agglutinin. It was found that the decrease of negative surface charges is unconcomitant with appearance of domains exposing positive ones, particularly on the surfaces of rufflings. The distribution of Concanavalin A binding sites turned from a uniform distribution to a polarized one, especially on apical regions where it appeared in large aggregates. The polarity in localization of wheat germ agglutinin on untreated fibroblasts observed in our experiments ceased shortly after irradiation.

Radiation-induced changes of negative charge on the cell surface of primary human fibroblasts.

By binding cationized ferritin (CF) to the plasma membrane of primary human fibroblasts, the amount and topology of negatively charged sites on cell surfaces were studied after X-irradiation. The CF binding was tested both on fixed and unfixed cells. Using various enzymes, the chemical nature of sites carrying the negative charges on cell surfaces was investigated. The results suggest that in unirradiated fibroblasts the CF binding occurred in a polarized manner, i.e. the particles were localized mainly on the apical surface of cells and formed clusters. The thin cytoplasmic protrusions and cell-to-cell contact sites bound CF to a greater extent than the bleb-like formations. Enzymatic digestion of surface polysaccharides showed that the main carriers of negatively charged sites are the glycosaminoglycans associated with the cell surface. The fixation of cells with glutaraldehyde did not influence the topology of CF binding either before or after the enzymatic treatment. After X-irradiation with 2.5 Gy the topology of CF binding did not change but the CF coverage of cells as well as the amount of ferritin particles per unit of surface area decreased within 10 min. The changes proved to be reversible as the values reached the pre-irradiation level by 1 h after irradiation.