Suboptimal growth temperatures enhance the biological activity of cultured cyanobacterium Gloeocapsa sp.

The cytotoxic, antibacterial, and antifungal activities of cyanobacterium Gloeocapsa sp. strain Gacheva 2007/R-06/1 were investigated and the possibility for an enhancement of these activities by changing the culture conditions evaluated. Fatty acids of this cyanobacterium were found to be active against Streptococcus pyogenes. Exopolysaccharides inhibited the growth of both Gram-positive and Gram-negative bacteria and the fungus Candida albicans. Both exopolysaccharides and fatty acid mixtures also significantly decreased the viability of human cervical carcinoma cells, HeLa. Greater biological activities were exhibited by Gloeocapsa sp., cultured at suboptimal temperatures (15–26°C) than at optimal and supraoptimal ones. In comparison with higher light intensity, the low-light cultivation stimulated the cytotoxicity of the fatty acids. In general, low temperatures decreased the growth of Gloeocapsa sp., but promoted its biological activity. Prolonged cultivation also had a beneficial impact on the bioactivity. Compared to 4 days, the 17-day cultivation resulted in fourfold higher antibacterial and antifungal activities of exopolysaccharides and more than twice increases in their cytotoxicity. The study revealed that this cyanobacterial isolate is a new source of natural products with potential for pharmacological and medical applications.     

Studies on the interactions of immunostimulated macrophages and Yersinia enterocolitica O:8

Immunological and electron microscopy investigations of the phagocytic and killing activities of peritoneal macrophages from rats and mice against Yersinia enterocolitica serotype O:8 cells were performed. The effect of in vivo application of cytoplasmic membranes (CM) from the stable Escherichia coli WF+ L-form on macrophage activity was also studied. It was established that rat macrophages more actively phagocytosed the plasmidless pYV(-) Y. enterocolitica cells, compared to the plasmid-bearing pYV(+) Y. enterocolitica cells. The killing ability against both variants of the Y. enterocolitica strain was significantly enhanced in macrophages from CM-treated rats after 2 h, 4 h, and 24 h incubation. The CM treatment enhanced the phagocytic activity of the macrophages. The in vitro interaction of normal and immunostimulated rat macrophages with both pYV(+) and pYV(-) variants of Y. enterocolitica did not lead to any additional apoptotic and necrotic changes in macrophages compared to control macrophages, which were cultivated without Y. enterocolitica. Electron-microscopic investigation showed that mouse macrophages eliminated Y. enterocolitica pYV(+) cells in vivo after 24 h. No engulfed or digested bacterial cells were observed. Activation of cell surfaces and vacuolization of macrophage cytoplasm, both of CM-treated non-infected and infected mice, were observed. The experimental results showed that Y. enterocolitica pYV(+) cells could be eliminated by peritoneal macrophages.     

Design and synthesis of new substrates of HtrA2 protease

HtrA2 belongs to the HtrA (high temperature requirement A) family of ATP-independent serine proteases. The primary function of HtrA2 includes maintaining the mitochondria homeostasis, cell death (by apoptosis, necrosis, or anoikis), and contribution to the cell signaling. Several recent reports have shown involvement of HtrA2 in development of cancer and neurodegenerative disorders. Here, we describe the profiling of HtrA2 protease substrate specificity via the combinatorial chemistry approach that led to the selection of novel intramolecularly quenched substrates. For all synthesized compounds, the highest HtrA2-mediated hydrolysis efficiency and selectivity among tested HtrA family members was observed for ABZ-Ile-Met-Thr-Abu-Tyr-Met-Phe-Tyr(3-NO₂)-NH₂, which displayed a specificity constant k_cat/K_M value of 14,535 M⁻¹ s⁻¹.     

Effect of a fungal Cu/Zn superoxide dismutase on the cell-mediated immune response in Graffi tumor bearing hamsters

The antibody-dependent cell cytotoxicity (ADCC) of spleen lymphocytes, isolated from hamsters with progressing myeloid Graffi tumor, was studied. The effect of the application of Cu/Zn superoxide dismutase, isolated from the fungal strain Humicola lutea (HL SOD), before and during tumor transplantation on the lymphocyte ADCC was examined. Myeloid Graffi tumor cells as target cells were used. Antibodies from a rabbit hyper-immune anti-tumor Graffi cells serum, or from tumor-bearing hamsters serum were used in the test. The leukocyte adherence inhibition (LAI) in the presence of tumor antigen was examined also during tumor progression. ADCC of the spleen lymphocytes, determined by both, rabbit and hamster anti-tumor antibodies, decreased during tumor progression. The optimum treatment of the animals by HL SOD induced a 20-30% increase of lymphocyte cytotoxicity against myeloid Graffi tumor cells. Cytotoxicity in presence of tumor bearing hamsters serum was twofold lower as compared to that one determined in the presence of rabbit hyper-immune anti-myeloid Graffi tumor cells serum. Leukocyte adherence inhibition (LAI) index in the presence of tumor antigen increased during tumor development in the groups of treated and untreated animals. The LAI indices of HL SOD-treated tumor-bearing hamsters were lower than that of untreated animals with tumors, what can be explained by a higher adherence ability of leukocytes induced by HL SOD treatment (in formula for calculation of LAI index the adherence value is in the denominator). The results show the beneficial effect of HL SOD on the cell-mediated immune response of myeloid Graffi tumor bearing hamsters, what is probably due to the participation of the enzyme in the host's oxidant-antioxidant balance.     

Effect of Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea 103 on antioxidant defense of Graffi tumor-bearing hamsters

A novel Cu/Zn-containing superoxide dismutase (SOD) was isolated from the fungal strain Humicola lutea 103. Previously, a protective effect of this enzyme (HLSOD) against tumor growth and also superoxide production in Graffi tumor-bearing hamsters (TBH) were established. The aim of the present study was to investigate the effect of HLSOD on the activity of endogenous SOD and catalase in the cells from TBH during tumor progression. Our results point out that transplantation of Graffi tumor causes a significant decrease in SOD activity in the cells from liver of the hosts (from 35 to 59% compared to the control). In the tumor cells relatively low levels of SOD (about 7 U mg protein(-1)) were found, and Cu/ZnSOD was the main isoenzyme in total SOD activity. Tumor growth resulted in a reduction of catalase activity, which correlated with the process of tumor progression. A single dose (65 U) treatment with HLSOD caused an increase in endogenous SOD and catalase activity in healthy animals and resulted in restoration of the antioxidant ability in liver cells of the hosts at the early stage of tumor progression. The results show the possible participation of HLSOD in the host oxidant-antioxidant balance, which is probably one of the factors of its immunoprotective action established earlier.     

The significance of nasal carriage of Staphylococcus aureus as risk factor for human skin infections

The effects of 2,2',5,5'-tetrachlorobiphenyl (TeCB), a PCB congener, and biphenyl on the cytoplasmic membranes of Ralstonia eutropha H850 were investigated by measuring fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as the probe, and determining the cellular fatty acid compositions. TeCB significantly affected the membrane of R. eutropha H850 cells grown on fructose by decreasing DPH fluorescence polarization. In contrast, the membrane of cells grown on biphenyl showed a considerably less significant effect of TeCB on membrane polarization than in fructose-grown cells. An increase in the ratio of total saturated to unsaturated fatty acids in cells grown on biphenyl suggested less of a fluidizing effect of TeCB on membranes in those cells. When biphenyl-grown cells were transferred back to a fructose medium, they required 25 generations for the membrane polarization and fatty acid compositions of these cells to revert back to those of the initial fructose-grown cells. The re-adaptation to a change in temperature required only five generations to return to normal. These results show that biphenyl affects cells in more ways than simply fluidizing the cytoplasmic membrane.     

Changes in extracellular action potential detect kainic acid and trimethyltin toxicity in hippocampal slice preparations earlier than do MAP2 density measurements

In vitro electrophysiological techniques for the assessment of neurotoxicity could have several advantages over other methods in current use, including the ability to detect damage at a very early stage, and could further assist in replacing animal experimentation in vivo. We investigated how an electrophysiological parameter, the extracellularly-recorded compound action potential ("population spike", PS) could be used as a marker of in vitro neurotoxicity in the case of two well-known toxic compounds, kainic acid (KA) and trimethyltin (TMT). We compared the use of this electrophysiological endpoint with changes in immunoreactivity for microtubule-associated protein 2 (MAP2), a standard histological test for neurotoxicity. We found that both toxic compounds reliably caused disappearance of the PS, and that such disappearance occurred after only 1 hour of exposure to the drug. By contrast, densitometric measurements of MAP2 immunoreactivity were unaffected by both KA and TMT after such a short exposure time. We conclude that, in the case of KA and TMT, the extracellular PS was abolished at a very early time-point, when MAP2 immunoreactivity levels were still comparable to those of the untreated controls. Electrophysiology could be a reliable and early indicator of neurotoxicity, which could improve our ability to test for neurotoxicity in vitro, thus further replacing the need for in vivo experimentation.     

Genotype‐environment interactions rule the response of a widespread butterfly to temperature variation

Understanding how organisms adapt to complex environments is a central goal of evolutionary biology and ecology. This issue is of special interest in the current era of rapidly changing climatic conditions. Here, we investigate clinal variation and plastic responses in life history, morphology and physiology in the butterfly Pieris napi along a pan‐European gradient by exposing butterflies raised in captivity to different temperatures. We found clinal variation in body size, growth rates and concomitant development time, wing aspect ratio, wing melanization and heat tolerance. Individuals from warmer environments were more heat‐tolerant and had less melanised wings and a shorter development, but still they were larger than individuals from cooler environments. These findings suggest selection for rapid growth in the warmth and for wing melanization in the cold, and thus fine‐tuned genetic adaptation to local climates. Irrespective of the origin of butterflies, the effects of higher developmental temperature were largely as expected, speeding up development; reducing body size, potential metabolic activity and wing melanization; while increasing heat tolerance. At least in part, these patterns likely reflect adaptive phenotypic plasticity. In summary, our study revealed pronounced plastic and genetic responses, which may indicate high adaptive capacities in our study organism. Whether this may help such species, though, to deal with current climate change needs further investigation, as clinal patterns have typically evolved over long periods.     

A plant polyphenol-rich extract restores the suppressed functions of phagocytes in influenza virus-infected mice

Influenza infection was induced in white ICR mice by intranasal (i.n.) inoculation of the virus A/Aichi/2/68 (H3N2). The number, migration and phagocyte indices of alveolar and peritoneal macrophages (pM?) and of blood polymorphonuclear leukocytes (PMNs), as well as the inhibition of the PMN adherence in the presence of a specific antigen were followed for 9 days after infection. The effect of the i.n. application of a polyphenol-rich extract, designated as polyphenolic complex (PC), isolated from the medicinal plant Geranium sanguineum L., on the inspected immune parameters was studied in parallel with the virological parameters of the infection, e.g. rate of mortality, mean survival time (MST), infectious lung virus titre and consolidation of the lungs. It was found that the application of PC induced a continuous 2- to 2.5-fold rise in the number of both peritoneal and alveolar macrophages (aM?) in the infected and healthy controls. The migration of both peritoneal and aM? increased 1.5- to 2-fold in the group of infected PC-treated animals and four to fivefold in the control group, the maximum being on day 9. PC stimulated phagocyte activities of blood PMNs in both infected and healthy mice. The leukocyte adherence inhibition (LAI) index decreased in the infected and PC-treated animals. The restoration of the suppressed functions of phagocytes in influenza virus-infected mice (VIM) was consistent with a prolongation of MST and reduction in mortality rate, infectious virus titre and lung consolidation. The immunoenhancing properties of PC apparently contribute to the overall protective effect of the plant preparation in the lethal murine experimental influenza A/Aichi infection.