Elevation of Proteasomal Substrate Levels Sensitizes Cells to Apoptosis Induced by Inhibition of Proteasomal Deubiquitinases

Inhibitors of the catalytic activity of the 20S proteasome are cytotoxic to tumor cells and are currently in clinical use for treatment of multiple myeloma, whilst the deubiquitinase activity associated with the 19S regulatory subunit of the proteasome is also a valid target for anti-cancer drugs. The mechanisms underlying the therapeutic efficacy of these drugs and their selective toxicity towards cancer cells are not known. Here, we show that increasing the cellular levels of proteasome substrates using an inhibitor of Sec61-mediated protein translocation significantly increases the extent of apoptosis that is induced by inhibition of proteasomal deubiquitinase activity in both cancer derived and non-transformed cell lines. Our results suggest that increased generation of misfolded proteasome substrates may contribute to the mechanism(s) underlying the increased sensitivity of tumor cells to inhibitors of the ubiquitin-proteasome system.     

Hypoxia-inducible factor and vascular endothelial growth factor in the neuroretina and retinal blood vessels after retinal ischemia

Retinal ischemia arises from circulatory failure. As the retinal blood vessels are key organs in circulatory failure, our aim was to study the retinal vasculature separately from the neuroretina to elucidate the role of hypoxia-inducible factor (HIF) 1α and 1β and vascular endothelial growth factor (VEGF) in retinal ischemia. Retinal ischemia was induced in porcine eyes by applying an intraocular pressure, followed by 12 h of reperfusion. HIF-1α mRNA expression was not affected by ischemia, while immunofluorescence staining was higher after ischemia in the neuroretina. HIF-1β immunoreactivity and mRNA expression were unaffected. VEGF protein levels in the vitreous humor and VEGF staining in the neuroretina were more pronounced in eyes subjected to ischemia than in the sham eyes. VEGF may be activated downstream of HIF-1 and is known to stimulate retinal neovascularization, which causes sight-threatening complications. These results emphasize the need for pharmacological treatment to block the HIF and VEGF signaling pathways in retinal ischemia.     

The terminal basal mitosis of chicken retinal Lim1 horizontal cells is not sensitive to cisplatin-induced cell cycle arrest

For proper development, cells need to coordinate proliferation and cell cycle-exit. This is mediated by a cascade of proteins making sure that each phase of the cell cycle is controlled before the initiation of the next. Retinal progenitor cells divide during the process of interkinetic nuclear migration, where they undergo S-phase on the basal side, followed by mitoses on the apical side of the neuroepithelium. The final cell cycle of chicken retinal horizontal cells (HCs) is an exception to this general cell cycle behavior. Lim1 expressing (+) horizontal progenitor cells (HPCs) have a heterogenic final cell cycle, with some cells undergoing a terminal mitosis on the basal side of the retina. The results in this study show that this terminal basal mitosis of Lim1+ HPCs is not dependent on Chk1/2 for its regulation compared to retinal cells undergoing interkinetic nuclear migration. Neither activating nor blocking Chk1 had an effect on the basal mitosis of Lim1+ HPCs. Furthermore, the Lim1+ HPCs were not sensitive to cisplatin-induced DNA damage and were able to continue into mitosis in the presence of γ-H2AX without activation of caspase-3. However, Nutlin3a-induced expression of p21 did reduce the mitoses, suggesting the presence of a functional p53/p21 response in HPCs. In contrast, the apical mitoses were blocked upon activation of either Chk1/2 or p21, indicating the importance of these proteins during the process of interkinetic nuclear migration. Inhibiting Cdk1 blocked M-phase transition both for apical and basal mitoses. This confirmed that the cyclin B1-Cdk1 complex was active and functional during the basal mitosis of Lim1+ HPCs. The regulation of the final cell cycle of Lim1+ HPCs is of particular interest since it has been shown that the HCs are able to sustain persistent DNA damage, remain in the cell cycle for an extended period of time and, consequently, survive for months.     

Changes in autumn zooplankton in the pelagic zone of Lake Sevan (Armenia) during the increase in fish abundance

The qualitative composition and structure of the autumn zooplankton in the pelagic zone of Lake Sevan in years that were characterized by different abundancies of fish are described. In October 2013, upon the increase in whitefish abundance, the species richness of zooplankton increased insignificantly; the values of the trophic coefficient and the Shannon index, calculated by the number, increased; and the specific number of Rotifera and Cladocera decreased. At the same time, atypical changes were recorded in the development parameters of zooplankton invertebrates. They were expressed as an increase in the total biomass due to Cladocera, among which large Daphnia (Ctenodaphnia) magna Straus occupied the leading position. The possible reasons for the particular changes in zooplankton are discussed.     

Oxidative stress decreases extracellular homocysteine concentration in human hepatoma (HepG2) cell cultures

BACKGROUND: Mild hyperhomocysteinemia is associated with premature vascular disease. The mechanism behind the vascular injuries is, however, still unknown. Homocysteine may be catabolized in the trans-sulfuration pathway to cysteine. Cystathionine beta-synthase, which catalyses the first step in the trans-sulfuration pathway is redox-sensitive. We have therefore investigated total extracellular homocysteine turnover in the presence of oxidative stress in human cell lines. METHODS: The turnover of total extracellular homocysteine in HeLa and hepatoma cell cultures has been investigated in the presence of hydrogen peroxide. Furthermore, the effect of hydrogen peroxide on the removal of high amounts of exogenously added homocysteine was also studied. RESULTS: Total extracellular homocysteine concentration in hepatoma cell cultures decreased in the presence of hydrogen peroxide, whereas the extracellular homocysteine concentration in HeLa cell cultures was not influenced. There was no significant change of intracellular homocysteine in any type of cell cultures. Furthermore, the presence of hydrogen peroxide did not increase the removal of exogenously added homocysteine. CONCLUSION: The presence of hydrogen peroxide probably increases the activity of the trans-sulfuration pathway in hepatoma cell cultures, which increases the intracellular use of homocysteine and lowers its extracellular release. Consequently this mechanism might tend to lower total plasma homocysteine concentration in oxidative stress.     

Nitrogen cycling and storage in <Emphasis Type="Italic">Gagea spathacea</Emphasis> (Liliaceae): ecological insights for protecting a rare woodland species

Strategies to globally protect biological diversity are often hampered by an insufficient ecological knowledge about target species. This also applies to Gagea spathacea (Liliaceae), a ‘vulnerable’ woodland spring geophyte with a distribution largely restricted to the lowlands of Central Europe. We studied whether the species’ linkage to highly fertile forest soils is related to its high nitrogen (N) demands during its short developmental cycle. We hypothesized that the species exhibits a highly efficient N (re)cycling strategy, characterized by efficient resorption of N from the leaves and reallocation to bulbs at the end of the growing season. To test this assumption, we conducted a ¹⁵N tracer experiment and quantified ¹⁵N flows between soil, leaves, bulbs, and roots. Our findings support our hypothesis that G. spathacea is exceptionally efficient in recycling N, shown by the resorption of 68% of leaf N and its reallocation to bulbs at the end of the growing season. After 6 weeks of growth the plant showed a distinct shift in its N metabolism: The C:N ratio of leaves strongly increased and those of bulbs decreased, leaf ¹⁵N enrichment and recovery started to decrease, while total plant ¹⁵N recovery remained constant, indicating no further N uptake from the soil. Leaf N reallocation to bulbs was accompanied by a twofold increase of the bulbs’ biomass. Because of the stenoecious behaviour of G. spathacea, a careful protection and sustainable management of G. spathacea forest habitats is necessary, particularly in its Central European core area.     

Induction of synthesis of tetrahydropyrimidine derivatives in Streptomyces strains and their effect on Escherichia coli in response to osmotic and heat stress.

The metabolic responses of a number of Streptomyces strains to osmotic and heat stress were studied by 13C nuclear magnetic resonance spectroscopy. During cell growth in a chemically defined medium supplemented with 0.5 M NaCl, tetrahydropyrimidine derivatives (THPs), 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] and, to a lesser extent, 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)], were found to accumulate in a significant amount in all bacteria examined. In addition, when the growth temperature was shifted from 30 to 39 degrees C, the intracellular concentration of THP(A) increased significantly. Moreover, exogenously provided THP(A) or THP(B) or both reversed inhibition of Escherichia coli growth caused by osmotic stress and increased temperature. Although the ability of Streptomyces strains to tolerate high concentrations of NaCl is well known, very little is known about the osmoregulatory strategy in Streptomyces strains. Similarly, the mechanism by which compatible solutes accumulate in a variety of microorganisms is not understood. Our findings suggest the possibility of a novel mechanism of protection of DNA against salt and heat stresses involving the THPs.