The many faces of protease-protein inhibitor interaction

Proteases and their natural protein inhibitors are among the most intensively studied protein-protein complexes. There are about 30 structurally distinct inhibitor families that are able to block serine, cysteine, metallo- and aspartyl proteases. The mechanisms of inhibition can be related to the catalytic mechanism of protease action or include a mechanism-unrelated steric blockage of the active site or its neighborhood. The structural elements that are responsible for the inhibition most often include the N- or the C-terminus or exposed loop(s) either separately or in combination of several such elements. During complex formation, no major conformational changes are usually observed, but sometimes structural transitions of the inhibitor and enzyme occur. In many cases, convergent evolution, with respect to the inhibitors' parts that are responsible for the inhibition, can be inferred from comparisons of their structures or sequences, strongly suggesting that there are only limited ways to inhibit proteases by proteins.     

GnRH systems of <Emphasis Type="Italic">Cichlasoma dimerus</Emphasis> (Perciformes,Cichlidae) revisited: a localization study with antibodies and riboprobes to GnRH-associated peptides

The distribution of cells that express three prepro-gonadotropin-releasing hormones (GnRH), corresponding to salmon GnRH, sea bream GnRH (sbGnRH), and chicken II GnRH, was studied in the brain and pituitary of the South American cichlid fish, Cichlasoma dimerus. Although the ontogeny and distribution of GnRH neuronal systems have previously been examined immunohistochemically with antibodies and antisera against the various GnRH decapeptides, we have used antisera against various perciform GnRH-associated peptides (GAPs) and riboprobes to various perciform GnRH+GAPs. The results demonstrate that: (1) the GnRH neuronal populations in the forebrain (salmon and sea bream GAPs; sGAP and sbGAP, respectively) show an overlapping pattern along the olfactory bulbs, nucleus olfacto-retinalis, ventral telencephalon, and preoptic area; (2) projections with sGAP are mainly located in the forebrain and contribute to the pituitary innervation, with projections containing chicken GAP II being mainly distributed along the mid and hindbrain and not contributing to pituitary innervation, whereas sbGAP projections are restricted to the ventral forebrain, being the most important molecular form in relation to pituitary innervation; (3) sbGnRH (GnRH I) neurons have an olfactory origin; (4) GAP antibodies and GAP riboprobes are valuable tools for the study of various GnRH systems, by avoiding the cross-reactivity problems that occur when using GnRH antibodies and GnRH riboprobes alone.This work was supported by grants UBACYT X-217, Conicet PIP 0539/R188; NIH-HD-29186 and Fogarty International Fellowship TW00086.     

Preincubation with sodium ascorbate potentiates insulin-dependent PKB/Akt and c-Jun phosphorylation in L6 rat myoblasts challenged with reactive oxygen/nitrogen species

Previously, we reported that mitogenicity in L6 muscle cells was stimulated by insulin but inhibited by reactive oxygen/nitrogen species (ROS/RNS; []) and that preincubation with sodium ascorbate (ASC) protected from either the impaired DNA synthesis and/or loss of cell viability. Now, we addressed the question how ascorbate (AA) rescued DNA synthesis in L6 muscle cells being challenged with ROS/RNS. We assumed that AA might be able to influence insulin signaling. We found that insulin elevated the protein levels of both PKB/Akt kinase phosphorylated at Serine(473) (pS473-Akt), and c-Jun phosphorylated at Serine63, Serine73 (pS63, pS73-c-Jun) residues, respectively. A short-term treatment experiment (0 - 45 min) revealed that either insulin (0.1 muM) or hydrogen peroxide (0.1, 0.5 mM; H2O2) increased the pS473-Akt and pS63, pS73-c-Jun protein levels, although the effect of ROS/RNS peaked earlier (5 min) than that of insulin (45 min). Astonishingly, the elevated levels of both pS473-Akt and pS63, pS73-c-Jun in response to insulin were reduced by the concomitant treatment with H2O2 in a dose-dependent fashion. In contrast, a 4-hour preincubation with ASC (1 mM) augmented the signal from pS473-Akt and pS63, pS73-c-Jun, when both insulin and H2O2 were added. Moreover, a 24 h preincubation with ASC also elevated the pS473-Akt and pS63, pS73-c-Jun levels in response to insulin irrespective to ROS/RNS co-treatment. During chronic treatment studies, ROS/RNS stimulated neither phosphorylation of Akt nor c-Jun, indicating that ROS/RNS-dependent activation of the above-mentioned proteins was short-term and transient. Furthermore, higher levels of pS473 Akt and pS63, pS73-c-Jun after preincubation with ASC suggest that by this route AA could protect insulin-induced mitogenicity. Basal levels of Akt and its target p70(S6K) remained constant regardless of treatment. These results suggest that AA defends the insulin-stimulated mitogenicity hampered by ROS/RNS most likely by the amplification of insulin signal at the level of pS473-Akt and pS63, pS73-c-Jun, respectively.     

Stromelysin-2 (matrix metalloproteinase 10) is inducible in lymphoma cells and accelerates the growth of lymphoid tumors in vivo

Matrix metalloproteinase (MMP) 10 (stromelysin-2) is known to degrade various components of the extracellular matrix; however, the signals that regulate its expression and its role in lymphoma growth remain unknown. In the present work, we report the up-regulated expression of MMP10 in T lymphoma cells following contact with endothelial cells. The induction of MMP10 was found to be dependent on the specific interaction between LFA-1 and ICAM-1, which play a central role in regulating the expression of genes involved in the rate-limiting steps of lymphoma development. MMP10, but not MMP3 (stromelysin-1), was also up-regulated in human B lymphoma cells following exposure to IL-4, IL-6, and IL-13, but not to IL-1. To gain further insight into the role of MMP10 in lymphoma development, we generated lymphoma cell lines constitutively expressing high levels of MMP10 and studied these cells for their ability to form thymic lymphoma in vivo. Mice injected with lymphoma cells constitutively expressing MMP10 developed thymic lymphoma more rapidly than those injected with control lymphoma cells. These results provide the first in vivo evidence that overexpression of MMP10 promotes tumor development, and indicate that MMP10 induction is an important pathway activated not only upon ICAM-1/LFA-1-mediated intercellular contact, but also following activation of tumor cells with inflammatory cytokines.     

The long‐term immunological effects of alloferon and its analogues in the mealworm Tenebrio molitor

The subject of this article is a search for the long‐term immunological effects of alloferon and 3 structural analogues of alloferon, which were earlier characterized by the highest pro‐apoptotic activity in Tenebrio molitor. The differences in the actions of these peptides on immune response were observed. Alloferon increased nodulation and significantly phenoloxidase activity in the hemolymph of experimentally infected T. molitor. However, [Phe(p‐NH₂)¹]‐ and [Phe(p‐OMe)¹]‐alloferon strongly inhibited cellular and humoral defense of the mealworm against Staphylococcus aureus infection. One day after injection of these peptides, the specific biochemical and morphological hallmarks of apoptosis in bacteria‐challenged hemocytes were visible; in contrast, 3 days after peptides injection in all hemocytes, caspase activation was not observed. However, these new, circulating hemocytes differed from the control and the peptide‐untreated bacteria‐challenged hemocytes. They had an increased adhesion that led to a separation of viable, anucleated fragments of hemocytes that retain the ability to adhere and to form long filopodia. The peptide‐induced separation of hemocyte fragments may resemble the formation of platelets in mammals and perhaps play a role in sealing wounds in insects. The results of in vivo studies may suggest a long half‐life of studied peptides in the hemolymph of mealworm. Moreover, we showed the importance of the N‐terminal histidine residues at position one of the alloferon molecule for its immunological properties in insects. The results obtained here show that alloferon plays pleiotropic functions in insects.     

Tuning of Hemes b Equilibrium Redox Potential Is Not Required for Cross-Membrane Electron Transfer

In biological energy conversion, cross-membrane electron transfer often involves an assembly of two hemes b. The hemes display a large difference in redox midpoint potentials (ΔE_{m_}b), which in several proteins is assumed to facilitate cross-membrane electron transfer and overcome a barrier of membrane potential. Here we challenge this assumption reporting on heme b ligand mutants of cytochrome bc₁ in which, for the first time in transmembrane cytochrome, one natural histidine has been replaced by lysine without loss of the native low spin type of heme iron. With these mutants we show that ΔE_{m_}b can be markedly increased, and the redox potential of one of the hemes can stay above the level of quinone pool, or ΔE_{m_}b can be markedly decreased to the point that two hemes are almost isopotential, yet the enzyme retains catalytically competent electron transfer between quinone binding sites and remains functional in vivo. This reveals that cytochrome bc₁ can accommodate large changes in ΔE_{m_}b without hampering catalysis, as long as these changes do not impose overly endergonic steps on downhill electron transfer from substrate to product. We propose that hemes b in this cytochrome and in other membranous cytochromes b act as electronic connectors for the catalytic sites with no fine tuning in ΔE_{m_}b required for efficient cross-membrane electron transfer. We link this concept with a natural flexibility in occurrence of several thermodynamic configurations of the direction of electron flow and the direction of the gradient of potential in relation to the vector of the electric membrane potential.     

Noise pollution and decreased size of wooded areas reduces the probability of occurrence of Tawny Owl Strix aluco

At present, urban areas cover almost 3% of the Earth's land surface, and this proportion is constantly increasing along with human population growth. Although urbanization leads to biodiversity decline, at the same time it creates a novel and extensive environment that is exploited by whole assemblages of organisms. These include predators, which use the matrix of different habitat types within the urban environment for breeding and/or foraging. This study investigated how attributes of the urban landscape influence the distribution pattern of a nocturnal acoustic predator, the Tawny Owl Strix aluco. The probability of occurrence of this species was correlated with the presence of natural forests, and the increasing size of wooded habitat patches within the urban landscape; however, Tawny Owls were less likely to occur at sites with high noise levels at night. Our study suggests that the distribution pattern of acoustic predators is shaped by the availability of primary habitat but reduced by noise intensity (which may decrease hunting efficiency). The Tawny Owl is a top predator in the urban environment and its presence/absence may therefore affect populations of other species; this provides clear evidence of the indirect effect of noise pollution on animal populations inhabiting urban environments.     

Microbial Communities of Low Temperature,Saline Groundwater Used for Therapeutical Purposes in North Poland

Knowledge on microbial community composition and ecology in highly mineralized (TDS up to 71?g l^?1), cold waters (T?<?12?°C) from the zones of restricted water exchange is scarce. Therefore, the aim of our study was to reveal how the particular physicochemical water conditions of these zones affect the community composition. The community members, which could be involved in the sulfur cycling were of particular interest since they may strongly influence wells operation. The pristine, mineral waters of North Poland were extracted from boreholes reaching Cenozoic, Jurassic, and Triassic aquifers at the depth of 46–1248?m below ground surface. Both culture-dependent (culturing on R2A medium) and culture-independent techniques (microscopic methods and high-throughput 16S rDNA amplicon sequencing) were applied. The bacterial communities were characterized by low complexity and strongly varied across the sampling locations. Bacteria potentially involved in the sulfur cycle (sulfate reduction) were common in all mineral waters and were dominant (especially Desulfovibrio) in the deepest waters. The most important geochemical drivers of the observed microbial community composition were TDS including Cl^?, Na⁺, Mg²⁺, and NO₃^?. Bacterial isolates belonged mostly to the genus Bacillus. Sequences assigned to Archaea (Methanobacterium) were detected only in the deepest borehole water.     

The level of 8-oxo-7,8-dihydro-2′-deoxyguanosine is positively correlated with the size of the labile iron pool in human lymphocytes

It appears that the labile iron pool (LIP, low molecular weight iron) presence in cells can result in the production of reactive oxygen species (ROS). ROS may be responsible for the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in cellular DNA. In the present study we report on the relationship between LIP and the endogenous level of 8-oxodGuo in human lymphocytes. Good correlation has been determined between LIP and the oxidatively modified nucleoside. This in turn points out the possibility that under physiological condition there is the availability of LIP for catalyzing Fenton-type reactions in close proximity to cellular DNA. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0335-x.