Incidence and growth of methylcholanthrene-induced tumors in mice with altered immunological status

Summary BALB/c mice were treated s.c. with 3-methyl-cholanthrene (MCA), and tumor incidence and growth were followed for 9 months. Immunological status of mice was altered by various treatments. Thymectomized, lethally irradiated, bone marrow reconstituted mice served as T-cell deficient recipients. In order to suppres natural killer (NK)-cell/macrophage functions some mice were injected with silica particles; to enhance these functions some mice were given Corynebacterium parvum (CP). Silica and CP were given simultaneously with MCA to test their influence on the presumed function of surveillance of tumor incidence, and also 2 months after MCA to test their influence on the growth of greater numbers of transformed host cells. Almost all mice developed tumors at the inoculation site and at the end of the observation period there was no difference in tumor incidence among 9 experimental groups. However, in T-cell deficient mice we observed shorter tumor duration and earlier death than in normal mice. Silica particles appeared to enhance tumor growth but the differences compared to normal controls were not significant. A single injection of CP simultaneously with MCA caused earlier tumor appearance but also slowed its growth. In contrast, CP given 2 months after MCA significantly delayed the appearance of the tumors. In regard to the tumor growth immunosuppression had stronger effects in males than in females; the opposite was true for immunostimulation treatments. We concluded that immunological status does not influence long-term tumor incidence, but that both T-cell and NK-cell/macrophage compartments strongly influence the parameters of growth of chemically induced tumors, i.e., the immune and natural resistance mechanisms do not influence the frequency of de novo arising tumors but both can slow down tumor growth.Postgraduate and pregraduate students, University of Zagreb, Faculty of Medicine, Zagreb, Yugoslavia     

Generating thermostabilized agonist-bound GPR40/FFAR1 using virus-like particles and a label-free binding assay

Elucidating the detailed mechanism of activation of membrane protein receptors and their ligand binding is essential for structure-based drug design. Membrane protein crystal structure analysis successfully aids in understanding these fundamental molecular interactions. However, protein crystal structure analysis of the G-protein-coupled receptor (GPCR) remains challenging, even for the class of GPCRs which have been included in the majority of structure analysis reports among membrane proteins, due to the substantial instability of these receptors when extracted from lipid bilayer membranes. It is known that increased thermostability tends to decrease conformational flexibility, which contributes to the generation of diffraction quality crystals. However, this is still not straightforward, and significant effort is required to identify thermostabilized mutants that are optimal for crystallography. To address this issue, a versatile screening platform based on a label-free ligand binding assay combined with transient overexpression in virus-like particles was developed. This platform was used to generate thermostabilized GPR40 [also known as free fatty acid receptor 1 (FFAR1)] for fasiglifam (TAK-875). This demonstrated that the thermostabilized mutant GPR40 (L42A/F88A/G103A/Y202F) was successfully used for crystal structure analysis.     

Manganese requirement for optimum photosynthesis and growth in NAD-malic enzyme C-4 species

Despite the evidence for a critical role of Mn in malate decarboxylation and CO₂ release for carbon fixation reactions in C-4 plants, there is a lack of information on their Mn requirement. The objective of this study was to establish Mn levels needed for optimum growth and photosynthesis of four agriculturally important C-4 species, NAD-ME C-4 pearl millet and purple amaranth, and NADP-ME C-4 corn and sorghum, as compared to two C-3 species, wheat and squash. Plants were grown hydroponically in a complete nutrient solution with Mn concentrations ranging from 0 to 100 μM. We report that under these conditions, C-3 and NADP-ME C-4 plants reached their maximum biomass production with 2–5 μM Mn, the concentration commonly used in plant nutrient media. In contrast, Mn concentrations supporting maximum performance of NAD-ME C-4 plants were up to 20-fold higher and ranged between 50 and 100 μM. Although leaf tissue Mn concentrations increased in parallel with Mn nutrition in all plants, the higher leaf Mn had no effect on NADP-ME C-4 or C-3 plants, but it caused a large, up to 100%, increase in net photosynthetic rate in NAD-ME C-4 species. The highest photosynthetic rates across the spectrum of photon flux density were recorded for C-3 and NADP-ME C-4 plants receiving 2–5 μM Mn, and for NAD-ME C-4 species millet and amaranth supplied with 50 or 100 μM Mn, respectively. Squash (C-3) plants were the most sensitive to Mn and their photosynthetic rate was severely depressed with more than 10 μM Mn. The increase in photosynthetic rates of NAD-ME C-4 species occurred without an increase in stomatal conductance, eliminating CO₂ uptake as the main cause. We propose that the higher photosynthetic rates in NAD-ME C-4 species supplied with higher Mn were a result of increased activation of the Mn-dependent NAD-ME in bundle sheath cells, producing greater CO₂ supply for Calvin cycle reactions. This is, to our knowledge, the first report on a significantly higher Mn requirement for optimum photosynthesis and biomass production of NAD-ME C-4 species.     

Mechanisms for the formation of membranous nanostructures in cell-to-cell communication

Cells interact by exchanging material and information. Two methods of cell-to-cell communication are by means of microvesicles and by means of nanotubes. Both microvesicles and nanotubes derive from the cell membrane and are able to transport the contents of the inner solution. In this review, we describe two physical mechanisms involved in the formation of microvesicles and nanotubes: curvature-mediated lateral redistribution of membrane components with the formation of membrane nanodomains; and plasmamediated attractive forces between membranes. These mechanisms are clinically relevant since they can be affected by drugs. In particular, the underlying mechanism of heparin’s role as an anticoagulant and tumor suppressor is the suppression of microvesicluation due to plasma-mediated attractive interaction between membranes.     

Lysosomal pathways to cell death and their therapeutic applications

Lysosomes are the major cell digestive organelles that were discovered over 50 years ago. They contain a number of hydrolases that help them to degrade intracellular and extracellular material delivered. Among the hydrolases, the cathepsins, a group of proteases enclosed in the lysosomes, have a major role. About a decade ago, the cathepsins were found to participate in apoptosis. Following their release into the cytosol, they cleave Bid and degrade antiapoptotic Bcl-2 proteins, thereby triggering the mitochondrial pathway of apoptosis, with the lysosomal membrane permeabilization being the critical step in this pathway. Lysosomal dysfunction is linked with several diseases, including cancer and neurodegenerative disorders, thereby providing a potential for therapeutic applications. In this review lysosomes and lysosomal proteases involvement in apoptosis and their possible pharmaceutical targeting are discussed.     

Recognition of human tumor necrosis factor α (TNF-α) by therapeutic antibody fragment: energetics and structural features

Human tumor necrosis factor α (TNF-α) exists in its functional state as a homotrimeric protein and is involved in inflammation processes and immune response of a human organism. Overproduction of TNF-α results in the development of chronic autoimmune diseases that can be successfully treated by inhibitors such as monoclonal antibodies. However, the nature of antibody-TNF-α recognition remains elusive due to insufficient understanding of its molecular driving forces. Therefore, we studied the energetics of binding of a therapeutic antibody fragment (Fab) to the native and non-native forms of TNF-α by employing calorimetric and spectroscopic methods. Global thermodynamic analysis of data obtained from the corresponding binding and urea-induced denaturation experiments has been supported by structural modeling. We demonstrate that the observed high affinity binding of Fab to TNF-α is an enthalpy-driven process due mainly to specific noncovalent interactions taking place at the TNF-α-Fab binding interface. It is coupled to entropically unfavorable conformational changes and accompanied by entropically favorable solvation contributions. Moreover, the three-state model analysis of TNF-α unfolding shows that at physiological concentrations, TNF-α may exist not only as a biologically active trimer but also as an inactive monomer. It further suggests that even small changes of TNF-α concentration could have a considerable effect on the TNF-α activity. We believe that this study sets the energetic basis for understanding of TNF-α inhibition by antibodies and its unfolding linked with the concentration-dependent activity regulation.     

Interaction between Oligomers of Stefin B and Amyloid-�� in Vitro and in Cells

To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid-β-(1–40) peptide (Aβ). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to Aβ is oligomer specific. The dimers and tetramers of stefin B, which bind Aβ, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit Aβ fibril formation. When expressed in cultured cells, stefin B co-localizes with Aβ intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the Aβ epitope. Thus, stefin B is another APP/Aβ-binding protein in vitro and likely in cells.