Radiosensitizing properties of novel hydroxydicarboxylatoplatinum(II) complexes with high or low reactivity with thiols: two modes of action

We examined radiosensitizing properties of two novel platinum complexes (ethylenediamine(L-malato)platinum(II)), Pt1 and bis(1-ethylimidazole(L-malato)platinum(II)), Pt4. Initial double strand break (DSB) level and DSB rejoining were measured, using pulse field gel electrophoresis (PFGE) in human G1 phase lymphocytes subjected to Pt complex treatment alone and in combination with 10Gy of X-rays. Effects of Pt complex pre-treatment followed by X-irradiation were examined on survival (clonogenic ability) and growth (48 h growth tests) in Chinese hamster ovary (CHO-K1), xrs6 and L5178Y (LY) cells (LY-R and LY-S sublines). Cell cycle distributions of CHO cells after drug treatment were determined with the use of flow cytometry. Pt1 slowed down rejoining of X-ray induced DSB. It exerted a more than additive lethal effect on CHO-K1 cells but not on L5178Y cells subjected to combined Pt complex treatment and X-irradiation. In xrs6 cells the effect of combined Pt1+X treatment was additive. We conclude that, as earlier proposed for other Pt complexes, the radiosensitizing effect of Pt1 is connected with converting repairable DNA damage into irrepairable one (mode (i) of action). The requirements for this mode of sensitization are functional DNA repair systems (nucleotide excision repair (NER) and non-homologous end-joining (NHEJ)). Pt4 does not slow down DSB rejoining. It shows a considerable ability to arrest cells in G2 phase. We assume that Pt4 pre-treatment arrests cells in G2 phase and thus sensitizes to X-rays these cells that have a radiosensitive G2 phase (mode (ii) of action).     

Mechanical strain regulation of the chicken glypican-4 gene expression in the avian eggshell gland

Comparison of RNA fingerprinting of the avian eggshell gland (ESG) without and with an egg revealed upregulation of a 382-bp cDNA fragment that showed high homology to the mammalian glypican 4 (GPC-4). The gene sequence revealed a conserved glypican signature, a glycosyl phosphatidyl inositol-anchorage site, and cystein residues, most of which were conserved. GPC-4 was expressed in the ESG in a circadian fashion only during the period of eggshell calcification, when maximal mechanical strain was imposed. Removal of the egg just before to its entry into the ESG, with consequent elimination of the mechanical strain, caused reduction in the gene expression. Artificial application of the mechanical strain induced expression of the GPC-4 gene that was related to the level of the strain. GPC-4 expression was strain dependent in other parts of the oviduct. In the ESG, GPC-4 was expressed exclusively by the glandular epithelium and not by the pseudostratified epithelium facing the lumen. In summary, we cloned the avian homologue of GPC-4, established its pattern of expression in the avian ESG, and demonstrated for the first time that this gene is regulated by mechanical strain.     

Changes in the thymus of peripubertal rats induced by centrally applied somatostatin-28

The aim of this study was to investigate the effects of centrally applied somatostatin-28 on morphometric characteristics of the thymus, the thymocyte subpopulations, as well as, on apoptosis and phases of cell cycle in thymocytes. For this purpose, peripubertal male rats were cannulated intracerebroventriculary and treated with repeated, nanomolar concentrations of somatostatin-28 (experimental group) or saline (control group). Animals were sacrificed and their thymuses were used for the analysis of thymocyte subpopulations, cell cycle and apoptosis by flow cytometry and for the evaluation of morphometric parameters by stereological analysis. Our results showed that somatostatin-28 caused decrease of the thymic mass and volume, as well as total thymocytes number. Stereological analysis revealed volume decrease of thymic cortex and medulla accompanied with cellularity decrease. Somatostatin in the deeper cortex decreased the number of thymocytes, per volume unit, while in outer cortex raised their number. A significant increase in the percentage of double-negative and both single-positive thymocyte subpopulations, in parallel with a diminished percentage of double-positive cells was found. The cellularity of double-positive and single-positive thymocyte subpopulations was decreased. Somatostatin-28 treatment augmented the percentage of apoptotic cells, while the percentage of the cells represented in phases of cell cycle was reduced. These results suggest that somatostatin-28 induce thymus hypotrophy as result of decreasing cortex and medulla volume and cellularity. Changes in the percentage and cellularity of thymocyte subpopulations and numerical density of thymocytes in outer and deeper cortex, indicate that somatostatin-28 evoked disturbance in transition of double-negative to double-positive thymocytes.     

Invasiveness of transformed human breast epithelial cell lines is related to cathepsin B and inhibited by cysteine proteinase inhibitors

The activities'of the lysosomal cysteine proteinases cathepsin B and L are regulated by their endogenous inhibitors, stefins A and B, and cystatin C, and their imbalance may be associated with increased invasiveness and development of the malignant cell phenotype. The aim of this study was to investigate mRNA, protein and activity levels of the above proteins in relation to in vitro invasiveness and to the reported in vivo tumorigenicity of four human breast tumor cell lines: the spontaneously immortalized cell line MCF10A, its c-Ha-ras transfectant MCF10AT, and two tumorigenic derivative cell lines, MCF10AT-Ca1a and MCF10AT-Ca1d. Invasiveness did not correlate with tumorigenicity, since the MCF10AT cell was the most invasive and the remaining three were at about half of its level. Cathepsin B expression paralleled the in vitro invasiveness through matrigel at all levels of expression, but cathepsin L did not. Stefin levels were elevated several-fold in the tumorigenic cell lines, but not in MCF10AT. The hypothesis that cathepsin B plays an active role in the invasion of breast cancer cell lines was confirmed by the fact that synthetic cysteine proteinase inhibitors, particularly those selective for cathepsin B, significantly reduced the invasion of the MCF10AT cells.     

An unusual halotolerant alpha-type carbonic anhydrase from the alga Dunaliella salina functionally expressed in Escherichia coli

A 60-kDa, salt-inducible, internally duplicated alpha-type carbonic anhydrase (Dca) is associated with the plasma membrane of the extremely salt-tolerant, unicellular, green alga Dunaliella salina. Unlike other carbonic anhydrases, Dca remains active over a very broad range of salinities (0-4M NaCl), thus representing a novel type of extremely halotolerant enzyme. To elucidate the structural principles of halotolerance, structure-function investigations of Dca have been initiated. Such studies require considerable amounts of the enzyme, and hence, large-scale algal cultivation. Furthermore, the purified enzyme is often contaminated with other, co-purifying algal carbonic anhydrases. Expression in heterologous systems offers a means to produce, and subsequently purify, sufficiently large amounts of Dca required for activity and structural studies. Attempts to over-express Dca in the Escherichia coli BL21(DE3)pLysS strain, after optimizing various expression parameters, produced soluble, but weakly active protein, composed of fully reduced and variably -S-S- cross-linked chains (each of the Dca repeats contains a pair of cysteine residues, presumably forming a disulfide bond). However, when the E. coli Origami B(DE3)pLysS strain was used as a host, a functionally active enzyme with proper disulfide bonds was formed in good yield. Affinity-purified recombinant Dca resembled the native enzyme from D. salina in activity and salt tolerance. Hence, this expression system offers a means of pursuing detailed studies of this extraordinary protein using biochemical, biophysical, and crystallographic approaches.     

The calanoid copepod Acartia italica Steuer, phenomenon in the small saline Lake Rogoznica (Eastern Adriatic coast)

Lake Rogoznica, near ibenik, Croatia, is a salt coastal lakewith a maximum depth of about 15 m. During most of the year,this small, naturally eutrophied lake is highly stratified,with hypoxia/anoxia occurring in the bottom layer. Total anoxia,with the presence of hydrogen sulphide, was recorded on September27, 1997. At that moment, massive mortality of all planktonicand benthic organisms was observed. After the anoxia, Acartiaitalica, the only planktonic copepod species of the lake, re-establishedquickly. Before anoxia, the population dynamics of the A.italicapopulation depended primarily on predator/prey relationships.However, in the post-anoxic period, nutrient–phytoplankton–copepodrelationships became more important, as there was no longerany predation pressure. From all accounts, it would appear thatonly A.italica is adapted to the extreme conditions which appearfrom time to time in the lake, and that this species has animportant role in the functioning of the lake ecosystem. Acartiaitalica was very important for the gradual normalization oflife in the lake after total anoxia.     

Virus Attenuation after Deletion of the Cytomegalovirus Fc Receptor Gene Is Not due to Antibody Control

The murine cytomegalovirus (MCMV) fcr-1 gene codes for a glycoprotein located at the surface of infected cells which strongly binds the Fc fragment of murine immunoglobulin G. To determine the biological significance of the fcr-1 gene during viral infection, we constructed MCMV fcr-1 deletion mutants and revertants. The fcr-1 gene was disrupted by insertion of the Escherichia coli lacZ gene. In another mutant, the marker gene was also deleted, by recombinase cre. As expected for its hypothetical role in immunoevasion, the infection of mice with fcr-1 deletion mutants resulted in significantly restricted replication in comparison with wild-type MCMV and revertant virus. In mutant mice lacking antibodies, however, the fcr-1 deletion mutants also replicated poorly. This demonstrated that the cell surface-expressed viral glycoprotein with FcR activity strongly modulates the virus-host interaction but that this biological function is not caused by the immunoglobulin binding property.