Listeria monocytogenes phosphatidylinositol (PI)-specific phospholipase C has low activity on glycosyl-PI-anchored proteins.

The ability of the phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes to hydrolyze glycosyl phosphatidylinositol (GPI)-anchored membrane proteins was compared with the ability of the PI-PLC from Bacillus thuringiensis to hydrolyze such proteins. The L. monocytogenes enzyme produced no detectable release of acetylcholinesterase from bovine, sheep, and human erythrocytes. The cleavage of the GPI anchors of alkaline phosphatase from rat and rabbit kidney slices was less than 10% of the cleavage seen with the PI-PLC from B. thuringiensis. Activity for release of Fc gamma receptor IIIB (CD16) on human granulocytes was also low. Variations in pH and salt concentration had little effect on the release of GPI-anchored proteins. Our data show that L. monocytogenes PI-PLC has low activity on GPI-anchored proteins.     

Structure and seasonal dynamics of humid tropical grasslands in Meghalaya,India

Abstract. Four humid grassland communities at three different locations in Meghalaya, India were analysed during 1988 and 1989 for species and life-form composition, diversity and dominance in relation to altitude, soil and prevailing disturbances. Due to the adverse interactive influences of exceptionally high annual rainfall (> 10 000 mm), topography and human interference on soil fertility, the grassland at Cherrapunji, at 1300 m altitude, had a low species diversity (H'= 1.74) and was dominated by three perennial grass species. Similar grasslands, at both higher and lower altitudes on fertile soil and with lower rainfall (ca. 2000 mm), showed higher diversity values (H'= 2.28 at Burnihat and 2.31 at Upper Shillong). The proportion of perennial species and chamaephytes increased with elevation. At the high altitude site a grassland under short-term protection from fires and grazing had a higher species richness, density and basal cover than an unprotected grassland. All grasslands show a clear seasonality, albeit with different patterns, with a maximum in density and basal cover in August. The differences in structure and seasonality are discussed in terms of different levels of stress.     

Production and potential applications of a xylanase from a new strain of Streptomyces cuspidosporus

Enzyme production by a new mesophilic Streptomyces isolate was investigated which grew optimally on 1% (w/v) xylan and 10% (w/v) wheat bran at pH 7 and 37 °C. Xylan induced only CMCase (0.29 U/ml) besides xylanase (22–35 U/ml, 40–49 U/mg protein). Wheat bran induced xylanase (105 U/ml, 17.5 U/mg protein), CMCase (0.74 U/ml), -xylosidase (0.009 U/ml), -glucosidase (0.026 U/ml), -L-arabinofuranosidase (0.049 U/ml), amylase (1.6 U/ml) and phytase (0.432 U/ml). The isolate was amenable to solid state cultivation and produced increased levels of xylanase (146 U/ml, 28 U/mg protein). The pH and temperature optima of the crude xylanase activity were 5.5 and 65 °C respectively. The pI was 6.0 as determined by PEG precipitation. The crude enzyme was applied in treatment of paper pulp and predigestion of poultry feed and was found to be effective in releasing sugars from both and soluble phosphorus from the latter.     

Considerations for a Pediatric Biopharmaceutics Classification System (BCS): Application to Five Drugs

It has been advocated that biopharmaceutic risk assessment should be conducted early in pediatric product development and synchronized with the adult product development program. However, we are unaware of efforts to classify drugs into a Biopharmaceutics Classification System (BCS) framework for pediatric patients. The objective was to classify five drugs into a potential BCS. These five drugs were selected since both oral and intravenous pharmacokinetic data were available for each drug, and covered the four BCS classes in adults. Literature searches for each drug were conducted using Medline and applied to classify drugs with respect to solubility and permeability in pediatric subpopulations. Four pediatric subpopulations were considered: neonates, infants, children, and adolescents. Regarding solubility, dose numbers were calculated using a volume for each subpopulation based on body surface area (BSA) relative to 250 ml for a 1.73 m² adult. Dose numbers spanned a range of values, depending upon the pediatric dose formula and subpopulation. Regarding permeability, pharmacokinetic literature data required assumptions and decisions about data collection. Using a devised pediatric BCS framework, there was agreement in adult and pediatric BCS class for two drugs, azithromycin (class 3) and ciprofloxacin (class 4). There was discordance for the three drugs that have high adult permeability since all pediatric permeabilities were low: dolasetron (class 3 in pediatric), ketoprofen (class 4 in pediatric), and voriconazole (class 4 in pediatric). A main contribution of this work is the identification of critical factors required for a pediatric BCS.     

Astrocytes are poised for lactate trafficking and release from activated brain and for supply of glucose to neurons

Brain is a highly-oxidative organ, but during activation, glycolytic flux is preferentially up-regulated even though oxygen supply is adequate. The biochemical and cellular basis of metabolic changes during brain activation and the fate of lactate produced within brain are important, unresolved issues central to understanding brain function, brain images, and spectroscopic data. Because in vivo brain imaging studies reveal rapid efflux of labeled glucose metabolites during activation, lactate trafficking among astrocytes and between astrocytes and neurons was examined after devising specific, real-time, sensitive enzymatic fluorescent assays to measure lactate and glucose levels in single cells in adult rat brain slices. Astrocytes have a 2- to 4-fold faster and higher capacity for lactate uptake from extracellular fluid and for lactate dispersal via the astrocytic syncytium compared to neuronal lactate uptake from extracellular fluid or shuttling of lactate to neurons from neighboring astrocytes. Astrocytes can also supply glucose to neurons as well as glucose can be taken up by neurons from extracellular fluid. Astrocytic networks can provide neuronal fuel and quickly remove lactate from activated glycolytic domains, and the lactate can be dispersed widely throughout the syncytium to endfeet along the vasculature for release to blood or other brain regions via perivascular fluid flow.     

Lack of Cytochrome c in Mouse Fibroblasts Disrupts Assembly/Stability of Respiratory Complexes I and IV

Cytochrome c (cyt c) is a heme-containing protein that participates in electron transport in the respiratory chain and as a signaling molecule in the apoptotic cascade. Here we addressed the effect of removing mammalian cyt c on the integrity of the respiratory complexes in mammalian cells. Mitochondria from cyt c knockout mouse cells lacked fully assembled complexes I and IV and had reduced levels of complex III. A redox-deficient mutant of cyt c was unable to rescue the levels of complexes I and IV. We found that cyt c is associated with both complex IV and respiratory supercomplexes, providing a potential mechanism for the requirement for cyt c in the assembly/stability of complex IV.The mitochondrial electron transport chain consists of four multisubunit complexes, namely, NADH-ubiquinone oxidoreductase (complex I),² succinate-ubiquinone oxidoreductase (complex II), ubiquinone-cytochrome c oxidoreductase (complex III), and cytochrome c oxidase (complex IV, COX). Cytochrome c (cyt c) shuttles electrons from oxidative phosphorylation complex III to complex IV. Electrons are transferred from reduced cyt c sequentially to the Cu_A site, heme a, heme a₃, and Cu_B binuclear center in the complex IV before being finally transferred to molecular oxygen to generate water (1). Respiratory complexes are assembled into supercomplexes (also called respirasomes). These contain complex I bound to dimeric complex III and a variable copy number of complex IV (2).In Saccharomyces cerevisiae, cyt c is encoded by two genes: CYC1 and CYC7. Mutagenesis studies in yeast have shown that cyt c is required for the assembly of COX (3, 4). In yeast lacking both the cyt c genes (CYC1 and CYC7), COX assembly was absent. It was also shown that cyt c is only structurally required for COX assembly, because a catalytic mutant of cyt c (W65S) was sufficient to bring about near normal levels of COX. However, because yeast lacks complex I, they could not analyze the role of cyt c in the assembly/stability of complex I. Mammals possess two different isoforms of cyt c encoded on different chromosomes: the somatic (cyt cS)- and testis (cyt cT)-specific isoforms. In mouse, the cDNAs bear 74% homology, whereas the proteins possess 86% identity with most dissimilarity in the C terminus.Cardiolipin (CL) is an anionic phospholipid present almost exclusively in the mitochondrial membranes and constitutes 25% of its total phospholipids (5). Work from several laboratories showed that CL is essential for the membrane anchorage of the respiratory supercomplexes. CL has two main roles in the mitochondrial structure and function, namely, stabilization of mitochondrial membranes and specific interactions with proteins. CL deficiency results in inefficient energy transformation by oxidative phosphorylation, swelling of mitochondria, decreased ATP/oxygen ratio, and reduced membrane potential (6, 7). In accordance, in S. cerevisiae lacking CL synthase, the supercomplex comprising complexes III and IV is unstable (8). Assembly mutants of COX had significantly reduced CL synthase activity, whereas assembly mutants of respiratory complex III and complex V showed less inhibition (9). Subsequently, the proton gradient across the inner mitochondrial membrane was found to be important for CL formation and that CL synthase was stimulated by alkaline pH at the matrix side (10). In this study, we investigated the role of cyt c depletion on CL levels by examining its content and composition in cyt c null cells.Here we aimed to answer the following questions: What is the role of cyt c in the assembly and maintenance of the different respiratory complexes in mammals? Are there changes in the content/composition of lipids in the cyt c-ablated cells? Analysis of mouse fibroblasts revealed that cyt c is essential for the assembly/stability of COX, and a catalytically mutant form of cyt c cannot rescue the COX defect in the cyt c null cells. CL and triacylglycerols showed significant differences in the cyt c null cells, both in content and composition.     

Kinetic and structural studies of the role of the active site residue Asp235 of human pyridoxal kinase

Pyridoxal kinase catalyzes the phosphorylation of pyridoxal (PL) to pyridoxal 5′-phosphate (PLP). A D235A variant shows 7-fold and 15-fold decreases in substrate affinity and activity, respectively. A D235N variant shows ∼2-fold decrease in both PL affinity and activity. The crystal structure of D235A (2.5 Å) shows bound ATP, PL and PLP, while D235N (2.3 Å) shows bound ATP and sulfate. These results document the role of Asp235 in PL kinase activity. The observation that the active site of PL kinase can accommodate both ATP and PLP suggests that formation of a ternary Enz·PLP·ATP complex could occur in the wild-type enzyme, consistent with severe MgATP substrate inhibition of PL kinase in the presence of PLP.     

Ly6Chigh Monocytes Become Alternatively Activated Macrophages in Schistosome Granulomas with Help from CD4+ Cells

Alternatively activated macrophages (AAM) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells. Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage. Here, we characterized monocyte and macrophage dynamics in the livers of infected CX3CR1^GFP/+ mice. CX₃CR1-GFP⁺ monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression, indicating differentiation into AAM. Intravital imaging of CX₃CR1-GFP⁺ Ly6C^low monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas. Differential labeling of CX₃CR1-GFP⁺ cells in the blood and the tissue showed CD4⁺ T cell dependent accumulation of PD-L2⁺ CX₃CR1-GFP⁺ AAM in the tissues as granulomas form. By adoptive transfer of Ly6C^high and Ly6C^low monocytes into infected mice, we found that AAM originate primarily from transferred Ly6C^high monocytes, but that these cells may transition through a Ly6C^low state and adopt patrolling behavior in the vasculature. Thus, during chronic helminth infection AAM can arise from recruited Ly6C^high monocytes via help from CD4⁺ T cells.