Spectroscopic and Scanning Electron Microscopy Studies of Bioaccumulation of Pollutants by Algae

The ability of Chlorella species and two other algal blooms collected locally to take up Cu⁺² and Ni⁺² was investigated using infrared and scanning electron microscopy (SEM) data. The percentage of metal uptake was determined with atomic absorption spectroscopy. The effects of pH and initial concentrations of metal ions on bioaccumulation were examined. The uptake of methyl orange dye by Chlorella species was also studied using Langmuir and Freundlich adsorption isotherms.     

Ageing-Induced Alterations in Lipid/Phospholipid Profiles of Rat Brain and Liver Mitochondria: Implications for Mitochondrial Energy-Linked Functions

Effects of ageing on the lipid/phospholipid profile of brain and liver mitochondria from rats were examined. In the brain mitochondria the contents of total phospholipid (TPL) and cholesterol (CHL) increased with simultaneous increase in the TPL/CHL (mole:mole) ratio. The proportion and contents of lysophospholipid (Lyso), sphingomyelin (SPM), phosphatidylinositol (PI), phosphatidylserine (PS) and diphosphatidylglycerol (DPG) components increased, with maximal increases seen for PS and PI; phosphatidylcholine (PC) and phosphatidylethanolamine (PE) components registered decrease. In the liver mitochondria contents of TPL and CHL increased. However, the TPL/CHL (mole:mole) ratio was not altered. Lyso, PI and PS increased. However, the magnitude of increase was competitively lower; PE and DPG decreased. SPM and PC did not change as a consequence of ageing. These changes altered the contents of individual phospholipids in the two membrane systems. Respiration with glutamate, pyruvate + malate, succinate and ascorbate + N,N,N’,N’-tetramethyl-p-phenylenediamine was significantly impaired in brain mitochondria from old animals. For liver mitochondria the respiratory activity declined with glutamate and succinate. Correlation studies by regression analysis revealed that the lipid/phospholipid classes regulate respiratory function differently in the mitochondria from the two tissues. The respiration-related parameters in the brain mitochondria were dependent on multiple lipid/phospholipid components, and the process of regulation was complex compared to the liver mitochondrial functions.     

Diabetic Modulation of the Temperature Kinetics Properties of Cytochrome Oxidase Activity in Rat Brain Mitochondria

The effects of alloxan-diabetes and subsequent treatment with insulin on temperature kinetics properties of cytochrome oxidase activity from rat brain mitochondria were examined. The enzyme activity decreased only at the late stage of diabetes which was not normalized by insulin treatment; however at early stage of diabetes hyper-stimulation occurred. In the control animals the Arrhenius plot was chair shaped with three energies of (E₁, E₂ and E₃) and two phase transition temperatures (T_t1 and T_t2). At early diabetic stage the Arrhenius plot became biphasic and E₁ and E₂ decreased_; insulin treatment reversed chair-shaped pattern with increase in E₂. These changes correlated with transient changes in the phospholipids profiles especially decreased acidic phospholipids. The temperature kinetics parameters were minimally affected at the late stage of diabetes or by insulin treatment. Thus at the late stage the brain tissue seems to have readjusted to its insulin homeostasis.     

Microbial fouling community analysis of the cooling water system of a nuclear test reactor with emphasis on sulphate reducing bacteria

Culture and molecular-based techniques were used to characterize bacterial diversity in the cooling water system of a fast breeder test reactor (FBTR). Techniques were selected for special emphasis on sulphate-reducing bacteria (SRB). Water samples from different locations of the FBTR cooling water system, in addition to biofilm scrapings from carbon steel coupons and a control SRB sample were characterized. Whole genome extraction of the water samples and SRB diversity by group specific primers were analysed using nested PCR and denaturing gradient gel electrophoresis (DGGE). The results of the bacterial assay in the cooling water showed that the total culturable bacteria (TCB) ranged from 10³ to 10⁵ cfu ml^?1; iron-reducing bacteria, 10³ to 10⁵ cfu ml^?1; iron oxidizing bacteria, 10² to 10³ cfu ml^?1 and SRB, 2–29 cfu ml^?1. However, the counts of the various bacterial types in the biofilm sample were 2–3 orders of magnitude higher. SRB diversity by the nested PCR-DGGE approach showed the presence of groups 1, 5 and 6 in the FBTR cooling water system; however, groups 2, 3 and 4 were not detected. The study demonstrated that the PCR protocol influenced the results of the diversity analysis. The paper further discusses the microbiota of the cooling water system and its relevance in biofouling.     

All-or-none membrane permeabilization by fengycin-type lipopeptides from Bacillus subtilis QST713

The fungicidal activity of Bacillus subtilis QST713 has been utilized for the highly effective and environmentally safe protection of crops against a variety of pathogens. It is based mainly on the production of cyclic lipopeptides of the fengycin (FEs), surfactin, and iturin families. The mixed population of native FEs forms micelles which solubilize individual FEs such as agrastatin 1 (AS1) that are otherwise rather insoluble on their own. Fluorescence lifetime-based calcein efflux measurements and cryo transmission electron microscopy show that these FEs show a unique scenario of membrane permeabilization. Poor miscibility of FEs with lipid probably promotes the formation of pores in 10% of the vesicles at only≈1μM free FE and in 15% of the vesicles at 10 μM. We explain why this limited, all-or-none leakage could nevertheless account for the killing of virtually all fungi whereas the same extent of graded vesicle leakage may be biologically irrelevant. Then, crystallization of AS1 and micellization of plipastatins cause a cut-off in leakage at 15% that might regulate the biological activity of FEs, protecting Bacillus and plant membranes. The fact that FE micelles solubilize only about 10 mol-% fluid lipid resembles the behavior of detergent resistance.     

Retracted: Aging is associated with altered inflammatory,arachidonic acid cascade,and synaptic markers,influenced by epigenetic modifications,in the human frontal cortex

Aging is a risk factor for Alzheimer's disease (AD) and is associated with cognitive decline. However, underlying molecular mechanisms of brain aging are not clear. Recent studies suggest epigenetic influences on gene expression in AD, as DNA methylation levels influence protein and mRNA expression in postmortem AD brain. We hypothesized that some of these changes occur with normal aging. To test this hypothesis, we measured markers of the arachidonic acid (AA) cascade, neuroinflammation, pro‐ and anti‐apoptosis factors, and gene specific epigenetic modifications in postmortem frontal cortex from nine middle‐aged [41 ± 1 (SEM) years] and 10 aged subjects (70 ± 3 years). The aged compared with middle‐aged brain showed elevated levels of neuroinflammatory and AA cascade markers, altered pro and anti‐apoptosis factors and loss of synaptophysin. Some of these changes correlated with promoter hypermethylation of brain derived neurotrophic factor (BDNF), cyclic AMP responsive element binding protein (CREB), and synaptophysin and hypomethylation of BCL‐2 associated X protein (BAX). These molecular alterations in aging are different from or more subtle than changes associated with AD pathology. The degree to which they are related to changes in cognition or behavior during normal aging remains to be evaluated.     

Thyroid Hormone-Induced Alterations in Membrane Structure-Function Relationships: Studies on Kinetic Properties of Rat Kidney Microsomal Na+,K+-ATPase and Lipid/Phospholipid Profiles

The effects of thyroidectomy (Tx) and subsequent treatment with 3,5,3′-triiodothyronine (T₃) or combined replacement therapy (T_R) with T₃ and thyroxine (T₄) on the substrate and temperature kinetics properties of Na⁺,K⁺-ATPase and lipid/phospholipid makeup of rat kidney microsomes were examined. Enzyme activity was somewhat high in the hypothyroid (Tx) animals and increased significantly following T₃ treatment, while T_R treatment caused a decrease. In the Tx and T₃ groups enzyme activity resolved in two kinetic components, while in the T_R group the enzyme showed allosteric behavior up to 0.5 mm ATP concentration. The K _m and V _max values of both the components decreased in Tx animals without affecting the catalytic efficiency. T₃ treatment caused a significant increase in the V _max of both the components, with a significant increase in the catalytic efficiency, while the K _m values were not upregulated. The T_R regimen lowered the K _m and V _max of component II but improved the catalytic efficiency. Thyroid status-dependent changes were also noted in the temperature kinetics of the enzyme. Regression analysis revealed that changes in the substrate and temperature kinetics parameters correlated with specific phospholipid components.     

Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation.     

A Strategy for O-Glycoproteomics of Enveloped Viruses—the O-Glycoproteome of Herpes Simplex Virus Type 1

Glycosylation of viral envelope proteins is important for infectivity and interaction with host immunity, however, our current knowledge of the functions of glycosylation is largely limited to N-glycosylation because it is difficult to predict and identify site-specific O-glycosylation. Here, we present a novel proteome-wide discovery strategy for O-glycosylation sites on viral envelope proteins using herpes simplex virus type 1 (HSV-1) as a model. We identified 74 O-linked glycosylation sites on 8 out of the 12 HSV-1 envelope proteins. Two of the identified glycosites found in glycoprotein B were previously implicated in virus attachment to immune cells. We show that HSV-1 infection distorts the secretory pathway and that infected cells accumulate glycoproteins with truncated O-glycans, nonetheless retaining the ability to elongate most of the surface glycans. With the use of precise gene editing, we further demonstrate that elongated O-glycans are essential for HSV-1 in human HaCaT keratinocytes, where HSV-1 produced markedly lower viral titers in HaCaT with abrogated O-glycans compared to the isogenic counterpart with normal O-glycans. The roles of O-linked glycosylation for viral entry, formation, secretion, and immune recognition are poorly understood, and the O-glycoproteomics strategy presented here now opens for unbiased discovery on all enveloped viruses.