Flip-flop-induced relaxation of bending energy: implications for membrane remodeling.

Cellular and organellar membranes are dynamic materials that underlie many aspects of cell biology. Biological membranes have long been thought of as elastic materials with respect to bending deformations. A wealth of theory and experimentation on pure phospholipid membranes provides abundant support for this idea. However, biological membranes are not composed solely of phospholipids--they also incorporate a variety of amphiphilic molecules that undergo rapid transbilayer flip-flop. Here we describe several experimental systems that demonstrate deformation-induced molecular flip-flop. First we use a fluorescence assay to track osmotically controlled membrane deformation in single component fatty acid vesicles, and show that the relaxation of the induced bending stress is mediated by fatty acid flip-flop. We then look at two-component phospholipid/cholesterol composite vesicles. We use NMR to show that the steady-state rate of interleaflet diffusion of cholesterol is fast relative to biological membrane remodeling. We then use a Förster resonance energy transfer assay to detect the transbilayer movement of cholesterol upon deformation. We suggest that our results can be interpreted by modifying the area difference elasticity model to account for the time-dependent relaxation of bending energy. Our findings suggest that rapid interleaflet diffusion of cholesterol may play a role in membrane remodeling in vivo. We suggest that the molecular characteristics of sterols make them evolutionarily preferred mediators of stress relaxation, and that the universal presence of sterols in the membranes of eukaryotes, even at low concentrations, reflects the importance of membrane remodeling in eukaryotic cells.

Reconstructing the emergence of cellular life through the synthesis of model protocells.

The complexity of modern biological life has long made it difficult to understand how life could emerge spontaneously from the chemistry of the early earth. The key to resolving this mystery lies in the simplicity of the earliest living cells, together with the ability of the appropriate molecular building blocks to spontaneously self-assemble into larger structures. In our view, the two key components of a primitive cell are not only self-assembling, but also self-replicating, structures: the nucleic acid genome and the cell membrane. Here, we summarize recent experimental progress toward the synthesis of efficient self-replicating nucleic acid and membrane vesicle systems and discuss some of the issues that arise during efforts to integrate these two subsystems into a coherent whole. We have shown that spontaneous nucleic-acid-copying chemistry can take place within membrane vesicles, using externally supplied activated nucleotides as substrates. Thus, membranes need not be a barrier to the uptake of environmentally supplied nutrients. We examine some of the remaining obstacles that must be overcome to enable the synthesis of a complete self-replicating protocell, and we discuss the implications of these experiments for our understanding of the emergence of Darwinian evolution and the origin and early evolution of cellular life.

Structure of a biological oxygen sensor: a new mechanism for heme-driven signal transduction.

The FixL proteins are biological oxygen sensors that restrict the expression of specific genes to hypoxic conditions. FixL's oxygen-detecting domain is a heme binding region that controls the activity of an attached histidine kinase. The FixL switch is regulated by binding of oxygen and other strong-field ligands. In the absence of bound ligand, the heme domain permits kinase activity. In the presence of bound ligand, this domain turns off kinase activity. Comparison of the structures of two forms of the Bradyrhizobium japonicum FixL heme domain, one in the "on" state without bound ligand and one in the "off" state with bound cyanide, reveals a mechanism of regulation by a heme that is distinct from the classical hemoglobin models. The close structural resemblance of the FixL heme domain to the photoactive yellow protein confirms the existence of a PAS structural motif but reveals the presence of an alternative regulatory gateway.

Imidazole is a sensitive probe of steric hindrance in the distal pockets of oxygen-binding heme proteins.

The FixL heme-based sensor, despite its low affinity for oxygen, is much more reactive than myoglobin toward the large polar ligand imidazole. To determine which features of a myoglobin heme pocket favor binding of imidazole, we have measured binding of this ligand to the FixL heme domain, elephant myoglobin, wild-type sperm whale myoglobin, and sperm whale myoglobins having alanine, valine, threonine, glutamine, leucine, phenylalanine, or tryptophan substitutions of the distal (E7) histidine residue. Except for histidine, the association rate constants dropped more than 3000-fold as the volume of the E7 side chain, at position 64, was expanded from alanine (10(6) M-1 s-1) to phenylalanine (10(3) M-1 s-1). There was inhibition of imidazole binding due to displacement of coordinated water from H64 and H64Q sperm whale myoglobins, where the E7 side chain hydrogen bonds directly to the bound ligand. The imidazole dissociation rate constants varied less dramatically and less consistently with any single factor, though they were measurably decreased by hydrogen bonding to an E7 glutamine or histidine. On the whole, the results for the sperm whale myoglobin E7 substitutions show that the rate constants for imidazole binding are useful and sensitive indicators of steric hindrance and polar interactions in the distal pockets of myoglobins. The combined effects of the glutamine 64 and phenylalanine 29 in elephant myoglobin largely account for its increased imidazole association and dissociation rate constants, respectively, compared to those of sperm whale myoglobin. An unhindered distal pocket not competent to stabilize positive poles is indicated by the large imidazole association (>/=10(4) M-1 s-1) and dissociation (>/=50 s-1) rate constants, parameters that are characteristic of FixL.

Effect of octreotide on the pathology of hepatic schistosomiasis.

In clinical practice, octreotide (CAS 83150-76-9) has its greatest impact in the management of bleeding varices. The present work is the first one which was undertaken to investigate the possible use of octreotide as an antifibrotic agent and to study its effect on hepatic vasculature in Schistosoma mansoni infection. The material of this investigation consisted of two groups of albino mice (A, B), subdivided each into normal control, infected control, subgroups treated with octreotide, praziquantel (CAS 55268-74-1), and a combination of octreotide and praziquantel. Groups A and B were sacrificed at the 8th week and the 18th week post infection, respectively. By analysis of the obtained results, octreotide induced a reduction of the portal pressure, the weight of the spleen and the liver, the liver egg load (number of eggs) granuloma size and cellularity, and of the degree of hepatic fibrosis quantified by serum N-terminal peptide of type III procollagen in serum, serum laminin and tissue collagen using a Picrosirius red dye assay. Moreover, the biochemical state of hepatocytes has been improved. The subgroups treated with octreotide in association with praziquantel revealed better results than the subgroups treated with praziquantel alone. These obtained data were analysed in terms of histological extent of liver fibrosis in sections stained with Masson trichrome and sirius red, hepatocytic and sinusoidal changes at an ultrastructural level and by immunohistochemical demarcation of endothelial cells of blood vessels through the determination of factor VIII-related antigen. The promising results detected in this study may encourage to further investigate the positive findings of this drug with the intention of its possible application on a clinical level.

Octreotide decreases connective tissue formation and improves vascular changes associated with hepatic schistosomiasis.

In clinical practice, Octreotide has its greatest impact in the management of bleeding varices. The present work is the first one which was undertaken to investigate the possible use of Octreotide as an antifibrotic agent and to study its effect on hepatic vasculature in Schistosoma mansoni infection. The material of this investigation consisted of two groups of albino mice (A&B) subdivided each, into normal control, infected control, Octreotide treated, Praziquantel treated and Octreotide with Praziquantel treated subgroups. Groups A & B were sacrificed at the 8th week and the 18th week post infection respectively. By analysis of the obtained results, Octreotide has induced reduction of the portal pressure, the weight of the spleen and the liver, the number of liver egg load, granuloma size and cellularity, and of the degree of hepatic fibrosis quantified by serum PIIINP, serum laminin and tissue collagen using sirius red dye assay. Moreover, the biochemical state of hepatocytes has been improved. The subgroups treated with Octreotide in association with Praziquantel revealed better results than the subgroups treated with Praziquantel alone. Data were analysed in terms of histological extent of liver fibrosis in sections stained with Masson trichrome and sirius red, hepatocytic and sinusoidal changes at an ultrastuctural level and by immunohistochemical demarcation of endothelial cells of blood vessels through the determination of factor VIII related antigen. The promising results detected in this study may encourage to further investigate the positive findings of this drug with the intention of its possible application on a clinical level.

Cellular constituent and intercellular adhesion in Schistosoma mansoni granuloma: an ultrastructural study.

The present work deals with the structural analysis of Schistosoma mansoni granuloma and the visualization of cellular interaction at an ultrastuctural level in the acute (8 weeks) and chronic (20 weeks) stages of infection, for more detailed understanding of pathophysiology of the disease. Although, S. mansoni granuloma is mediated by T-lymphocytes, yet in this work the macrophage cells and not the lymphocytes represented the main cell type in cellular and fibrocellular granulomas. The cellular and fibrocellular granulomas detected in the acute stage of infection elicited no difference in cellular constituent to those of the chronic stage respectively. Macrophage cells and fibrocytes were the only cell type detected in fibrotic granuloma. The monocytes may be considered the first cell reaching the site of the trapped egg as they formed the first row of cells around the egg. The cellular infiltrate forming the granuloma: monocytes, macrophages, lymphocytes, eosinophils, fibroblasts and plasma cells revealed direct contact or adherence between them and even between the individual cell type, through extending protrusion from the cell membrane of adjacent cells. They constituted an integrated network which encircled the egg. Similar adhesion between inflammatory cells in the blood vessels and between the inflammatory cells and the endothelial cells were displayed. These points of intercellular adhesion appeared as if, not only used for functional communication between the cells, but also for cellular deplacement either in the extracellular matrix or in the blood stream until extravasation. In conclusion, S. mansoni granuloma is a highly organized cellular lesion, in which cell to cell communication occurs through direct cell contact.

Evaluation of colchicine with or without praziquantel therapy in the control of hepatic fibrosis in murine schistosomiasis.

Colchicine alone or following praziquantel was given to mice infected with Schistosoma mansoni either 6 or 10 weeks post infection. Praziquantel increased body weight gain, histologically reduced number, diameter and cellularity of granuloma and improved liver function parameters. Early praziquantel therapy decreased hepatic collagen content as detected by the colorimetric method and the serum procollagen propeptide (PIIIP), while later treatment at the 10th week of infection increased hepatic collagen content and serum PIIIP. Colchicine therapy significantly decreased body weight gain with significant weight loss after early treatment. Colchicine did not change the histologic picture of schistosomal liver fibrosis; it induced a detectable hepatocytic injury recorded ultrastructurally and histologically with excitation of the inflammatory reaction in the granuloma and in portal tracts after early treatment. Excess pigmentation in macrophages and Kupffer cells, binucleation and large sized hepatocytic nuclei were evident after colchicine therapy. Colchicine increased hepatic collagen content microgram/mg protein, raised globulin and total serum protein and normalized the raised serum PIIIP of infected mice, but had no effect on PIIIP of normal mice. Early cessation of schistosomal infection evidently minimized the adverse effects of colchicine.