Delayed Postconditionig Initiates Additive Mechanism Necessary for Survival of Selectively Vulnerable Neurons After Transient Ischemia in Rat Brain

1. The aim of this study was to validate the role of postconditioning, used 2 days after lethal ischemia, for protection of selectively vulnerable brain neurons against delayed neuronal death.2. Eight, 10, or 15 min of transient forebrain ischemia in rat (four-vessel occlusion model) was used as initial lethal ischemia. Fluoro Jade B, the marker of neurodegeneration, and NeuN, a specific neuronal marker were used for visualization of changes 7 or 28 days after ischemia without and with delayed postconditioning.3. Our results confirm that postconditioning if used at right time and with optimal intensity can prevent process of delayed neuronal death. At least three techniques, known as preconditioners, can be used as postconditioning: short ischemia, 3-nitropropionic acid and norepinephrine. A cardinal role for the prevention of death in selectively vulnerable neurons comprises synthesis of proteins during the first 5 h after postconditioning. Ten minutes of ischemia alone is lethal for 70% of pyramidal CA1 neurons in hippocampus. Injection of inhibitor of protein synthesis (Cycloheximide), if administered simultaneously with postconditioning, suppressed beneficial effect of postconditioning and resulted in 50% of CA1 neurons succumbing to neurodegeneration. Although, when Cycloheximide was injected 5 h after postconditioning, this treatment resulted in survival of 90% of CA1 neurons.4. Though postconditioning significantly protects hippocampal CA1 neurons up to 10 min of ischemia, its efficacy at 15 min ischemia is exhausted. However, protective impact of postconditioning in less-sensitive neuronal populations (cortex and striatum) is very good after such a damaging insult like 15 min ischemia. This statement also means that up to 15 min of ischemia, postconditioning does not induce cumulation of injuries produced by the first and the second stress.     

Distribution of cortical endoplasmic reticulum determines positioning of endocytic events in yeast plasma membrane

In many eukaryotes, a significant part of the plasma membrane is closely associated with the dynamic meshwork of cortical endoplasmic reticulum (cortical ER). We mapped temporal variations in the local coverage of the yeast plasma membrane with cortical ER pattern and identified micron-sized plasma membrane domains clearly different in cortical ER persistence. We show that clathrin-mediated endocytosis is initiated outside the cortical ER-covered plasma membrane zones. These cortical ER-covered zones are highly dynamic but do not overlap with the immobile and also endocytosis-inactive membrane compartment of Can1 (MCC) and the subjacent eisosomes. The eisosomal component Pil1 is shown to regulate the distribution of cortical ER and thus the accessibility of the plasma membrane for endocytosis.     

Helicobacter pylori and Nonmalignant Diseases

The incidence of peptic ulcer disease has declined over the last few decades, particularly in Western populations, most likely as a result of the decrease in Helicobacter pylori infection and the widespread use of proton-pump inhibitors (PPI) in patients with dyspepsia. The hospital admission rate for uncomplicated duodenal and gastric ulcers has significantly decreased worldwide. In contrast, admissions for complicated ulcer disease, such as bleeding peptic ulcers and perforation, remained relatively stable. Prophylactic H.?pylori eradication was found to be associated with a reduced risk of both gastric and duodenal ulcers and their complications, including bleeding in chronic users of nonsteroidal anti-inflammatory drugs. The recent Helicobacter Eradication Relief of Dyspeptic Symptoms trial presented important data relating to symptoms and quality of life of H.?pylori-positive patients with functional dyspepsia (FD) and also demonstrated significant benefits from eradication compared with the control group. The new Asian consensus report on FD recommended that dyspepsia accompanied by H.?pylori infection should be considered a separate disease entity from FD and that H.?pylori infection should be eradicated before diagnosing FD. The association of H.?pylori with gastroesophageal reflux disease (GERD) is still controversial. Treatment for H.?pylori does not seem to increase GERD symptoms or reflux esophagitis. However, documented eradication of H.?pylori appears to significantly improve GERD symptoms. Additional long-term intervention studies are needed to provide more information on which to base clinical decisions.     

Differential Mobility of Pigment-Protein Complexes in Granal and Agranal Thylakoid Membranes of C3 and C4 Plants

The photosynthetic performance of plants is crucially dependent on the mobility of the molecular complexes that catalyze the conversion of sunlight to metabolic energy equivalents in the thylakoid membrane network inside chloroplasts. The role of the extensive folding of thylakoid membranes leading to structural differentiation into stacked grana regions and unstacked stroma lamellae for diffusion-based processes of the photosynthetic machinery is poorly understood. This study examines, to our knowledge for the first time, the mobility of photosynthetic pigment-protein complexes in unstacked thylakoid regions in the C₃ plant Arabidopsis (Arabidopsis thaliana) and agranal bundle sheath chloroplasts of the C₄ plants sorghum (Sorghum bicolor) and maize (Zea mays) by the fluorescence recovery after photobleaching technique. In unstacked thylakoid membranes, more than 50% of the protein complexes are mobile, whereas this number drops to about 20% in stacked grana regions. The higher molecular mobility in unstacked thylakoid regions is explained by a lower protein-packing density compared with stacked grana regions. It is postulated that thylakoid membrane stacking to form grana leads to protein crowding that impedes lateral diffusion processes but is required for efficient light harvesting of the modularly organized photosystem II and its light-harvesting antenna system. In contrast, the arrangement of the photosystem I light-harvesting complex I in separate units in unstacked thylakoid membranes does not require dense protein packing, which is advantageous for protein diffusion.In higher plants, the photosynthetic apparatus is compartmentalized in the specialized chloroplast organelle. The molecular machinery for the primary photosynthetic processes, the sunlight-driven generation of metabolic energy equivalents, is harbored in an intricate thylakoid membrane system within the chloroplasts. Recent improvements in electron tomography have led to three-dimensional models of the complex architecture of thylakoid membranes (Mustárdy and Garab, 2003; Nevo et al., 2009; Austin and Staehelin, 2011; Daum et al., 2010; Kouřil et al., 2011). Although important details about the thylakoid structure are still highly controversial, consensus exists about the overall design of this membrane system. The thylakoid membrane consists of two morphologically distinct domains: strictly stacked cylindrical grana regions with a diameter of 300 to 600 nm are interconnected by unstacked stroma lamellae, thus forming a continuous membrane system. The molecular complexes that catalyze energy transformation are distributed heterogeneously between the stacked and unstacked membrane regions. The majority of the PSII complex and light-harvesting complex II (LHCII) are localized in stacked thylakoid regions, whereas PSI and the ATP-synthase complex are lacking from stacked grana (Staehelin and van der Staay, 1996; Albertsson, 2001; Dekker and Boekema, 2005). It is assumed that the fifth photosynthetic protein complex (cytochrome b₆f complex) is homogenously distributed.An essential feature of the thylakoid membrane system is its high flexibility, which is required for adaptability and maintenance of the photosynthetic machinery in plants. Highly responsive to environmental conditions, both the overall thylakoid architecture (e.g. number of grana discs) and the molecular membrane composition can change remarkably to optimize, protect, and maintain the photosynthetic apparatus (Walters, 2005; Anderson et al., 2008; Chuartzman et al., 2008; Dietzel et al., 2008; Betterle et al., 2009; Johnson et al., 2011). The underlying molecular processes require brisk protein traffic between stacked and unstacked thylakoid domains (Kirchhoff, 2008). The role of grana in these transport-based processes is poorly understood.Although photosynthetic energy conversion is possible without grana (Anderson et al., 2008), the fact that stacked thylakoids are ubiquitous in almost all land plants (with the exception of chloroplasts in bundle sheath [BS] cells in some C₄ plants; see below) highlights the evolutionary pressure to preserve this complex structural feature. Recently, the importance of grana formation was highlighted in Arabidopsis (Arabidopsis thaliana) mutants that lack the GRANA-DEFICIENT CHLOROPLAST1 gene; they grow much slower than the wild type and exhibit seed lethargy due to missing grana formation (Cui et al., 2011). The functional advantages of grana formation have been discussed extensively (Trissl and Wilhelm, 1993; Mullineaux, 2005; Anderson et al., 2008). It was hypothesized that grana could (1) increase the thylakoid membrane area, and the pigment concentration, in chloroplasts, (2) avoid energy spillover from PSII to PSI, (3) regulate the balance of energy distribution between PSII and PSI by state transition, and (4) enable transversal exciton energy transfer between adjacent grana discs. Although there are good arguments that these possibilities are important for photosynthetic energy conversion, the basis for the evolutionary development of grana has not been determined (Mullineaux, 2005; Anderson et al., 2008).A less considered aspect of grana formation is that it leads to a concentration of protein complexes (Murphy, 1986; Kirchhoff, 2008). The membrane area fraction that belongs to integral photosynthetic protein complexes is about 70%, making grana discs one of the most crowded biomembranes (Kirchhoff, 2008). Light harvesting by PSII benefits from a high protein-packing density for two reasons. First, a concentration of PSII and LHCII in grana ensures a high concentration of light-absorbing pigments that increase the probability of capturing sunlight, which is a “dilute” energy source on the molecular scale (Blankenship, 2002). Second, it has been demonstrated that a high protein-packing density in grana thylakoids is required for efficient intermolecular exciton energy transfer between LHCII and PSII (Haferkamp et al., 2010). Macromolecular crowding ensures that weakly interacting LHCII and PSII complexes come in close contact, allowing efficient Förster-type energy transfer.Besides these advantages, lateral protein traffic is challenged by macromolecular crowding (Mullineaux, 2005; Kirchhoff, 2008). The molecular mobility of proteins in grana thylakoids is reduced by numerous collisions of the diffusing object in the two-dimensional reaction space of the membrane with obstacles, integral membrane proteins, that increase the apparent diffusion path and, consequently, the diffusion time. The strong impairment of a high protein density in grana thylakoids on protein mobility was demonstrated by computer simulations (Tremmel et al., 2003; Kirchhoff et al., 2004) and by diffusion measurement on isolated grana membranes (Kirchhoff et al., 2008) and intact chloroplasts (Goral et al., 2010) using the fluorescence recovery after photobleaching (FRAP) technique. Processes that are expected to be affected by restricted protein mobility are a regulation of energy distributed between PSII and PSI by state transitions (Lemeille and Rochaix, 2010), the repair of photodamaged PSII (Mulo et al., 2008), membrane remodeling triggered by long-term environmental changes (Walters, 2005; Anderson et al., 2008), and the biogenesis of the thylakoid membrane network (Adam et al., 2011). Recently, evidence has accumulated that photoprotective high-energy quenching also requires large-scale diffusion-based structural reorganization within grana thylakoids (Betterle et al., 2009; Johnson et al., 2011).In contrast to our current understanding of diffusion-based processes in thylakoid membranes, knowledge about the factors that determine the mobility of photosynthetic protein complexes in different thylakoid domains is still fragmentary (Mullineaux, 2008). The protein-packing density is very likely a main element that determines protein mobility (Kirchhoff et al., 2008). However, other factors, like electrostatic interactions between proteins by membrane surface charges (Tremmel et al., 2005) or the size and molecular shape of protein complexes (Tremmel et al., 2003), can contribute significantly. However, data only exist about protein mobility for isolated grana thylakoids (Kirchhoff et al., 2008) and for chloroplasts from the grana-containing C₃ plant Arabidopsis (Goral et al., 2010). The diffusion characteristics of the latter are almost completely determined by granal proteins. Limiting information on protein diffusion exists for stroma lamellae of C₃ plants (Consoli et al., 2005; Vladimirou et al., 2009), and no data are available for agranal thylakoids, which occur in BS cells of some C₄ species.This study fills this gap in the knowledge base by studying lateral protein diffusion in unstacked thylakoid membranes in BS chloroplasts of two NADP-malate enzyme (ME)-type C₄ species, maize (Zea mays) and sorghum (Sorghum bicolor), in comparison with the grana-containing mesophyll (M) chloroplasts. The analysis was also complemented by studies on isolated thylakoid subfragments (grana core, grana, and stroma lamellae) from Arabidopsis. The protein mobility was measured by FRAP (Mullineaux and Kirchhoff, 2007), which has been shown to be a straightforward method to analyze protein diffusion in photosynthetic membranes by utilizing natural chlorophyll fluorescence (Kirchhoff et al., 2008; Goral et al., 2010). The comparison with diffusion characteristics in unstacked versus stacked membrane areas highlights the significance of grana formation on the lateral mobility of photosynthetic pigment-protein complexes.     

Fuzzy J-Means and VNS methods for clustering genes from microarray data

MOTIVATION: In the interpretation of gene expression data from a group of microarray experiments that include samples from either different patients or conditions, special consideration must be given to the pleiotropic and epistatic roles of genes, as observed in the variation of gene coexpression patterns. Crisp clustering methods assign each gene to one cluster, thereby omitting information about the multiple roles of genes. RESULTS: Here, we present the application of a local search heuristic, Fuzzy J-Means, embedded into the variable neighborhood search metaheuristic for the clustering of microarray gene expression data. We show that for all the datasets studied this algorithm outperforms the standard Fuzzy C-Means heuristic. Different methods for the utilization of cluster membership information in determining gene coregulation are presented. The clustering and data analyses were performed on simulated datasets as well as experimental cDNA microarray data for breast cancer and human blood from the Stanford Microarray Database. AVAILABILITY: The source code of the clustering software (C programming language) is freely available from Nabil.Belacel@nrc-cnrc.gc.ca     

Pyrrolizidine alkaloids from Lithospermum canescens Lehm

Seven pyrrolizidine alkaloids (PAs) have been isolated from Lithospermum canescens and their structures determined by spectroscopical methods. Besides the known lycopsamine, O7-acetyl-lycopsamine and O7-acetylintermedine four new PAs were found. Their structures are O7-(3-hydroxy-3-methyl-butanoyl)-O9-(+)-trachelanthoyl-heliotridine (= O7-(3-hydroxy-3-methyl-butanoyl)-rinderine = canescine), O7-(3-hydroxy-3-methyl-butanoyl)-O9-(-)-viridifloryl-heliotridine (= O7-(3-hydroxy-3-methyl-butanoyl)-echinatine = canescenine and their O13-acetyl-derivatives (= acetylcanescine; acetylcanescenine).     

Production of H2O2 in human embryos after long-lasting cryopreservation and subsequent culture

The production of hydrogen peroxide (H(2)O(2)) was investigated by means of cytochemical reaction with cerium chloride in human embryos cryostoraged for a long time period. The sites of H(2)O(2) generation were demonstrated at submicroscopic level in both freshly thawed embryos and in embryos and blastocysts formed after subsequent culture. The intact blastomeres as well as cells of well developed blastocysts did not produce any H(2)O(2). Two main intracellular sites of H(2)O(2) production were identified: mitochondria and plasma membranes. Some alterations and often destruction of plasma membrane integrity accompanied by massive H(2)O(2) generation are believed to be caused by the freezing and thawing.