The molybdenum and iron-sulphur centres of Escherichia coli nitrate reductase are non-randomly oriented in the membrane

Oriented membrane multilayers, prepared from Escherichiacoli grown anaerobically with nitrate, allowed the spatial organisation of electron paramagnetic resonance (e.p.r.)-detectable signals from the respiratory nitrate reductase to be examined. At low temperatures (7 K), two signals (g = 2.02, g = 1.98) have been assigned to an iron-sulphur cluster and their magnitudes shown to be dependent on the angle that the multilayer makes with the magnetic field of the e.p.r. spectrometer. Signals seen at 45 K (g = 1.985, g = 1.96) have been attributed to an anisotropic molybdenum centre. These redox components of the nitrate reductase are thus non-randomly oriented in the cytoplasmic membrane.     

Immobilization of whole Escherichia coli containing penicillin amidase using cross-linking agents and fillers

To obtain a catalyst with good mechanical stress stability and other operational characteristics, cross-linked aggregates of whole Escherichia coli containing penicillin amidase were reinforced with surface modified precipitated silica and chitosan. The immobilized cells plus mixed fillers possess better performance characteristics i.e. higher stability at 4°C and 30°C, least protein leaching capacity and good settling characteristics. Most suitable conditions to prepare catalyst with mixed fillers were: chitosan, 0.3: silica, 0.2 g/g d.wt cell; and minimum moisture content in catalyst, 30% (w/w).     

Comparison of the performance of a hollow-fiber microbe reactor with a reactor containing alginate entrapped cells

Summary Alginate entrapped Pseudomonas denitrificans have been compared with cells confined in the outer space of a hollow-fiber membrane unit with respect to continuous denitrification of water. The hollow-fiber unit had a higher productivity as well as a stability similar to that of the alginate unit. A reduction of cell-leakage in the eluate was found in the hollow-fiber unit. The nitrogen gas produced could be removed by circulating the cell containing fluid over a hydrophobic membrane.     

Effect of NaCl on lipid content of plasma membranes isolated from roots and cell suspension cultures of the dicot halophyte Kosteletzkya virginica (L.) Presl.

Lipid concent was examined in plasma membrane fractions isolated by discontinuous sucrose density-gradient centrifugation from both salinized and unsalinized roots and cell suspension cultures of Kosteletzkva virginica (L.) Presl., seashore mallow, a halophytic dicot. The distribution of marker enzymes along the gradient indicated that plasma membranes of roots and cell cultures accumulated primarily at the 34%/45% interface. Total sterol and phospholipid content increased significantly in plants and cell suspensions grown on salinized nutrient media. In addition, K. virginica plasma membranes were constitutively rich in sterols, and a high sterol-to-phospholipid ratio was maintained or elevated under saline conditions. These results are discussed in relation to membrane composition as a mechanism involved in the cellularly based salt tolerance of K. virginica.     

A proteomic screen with Drosophila Opa1-like identifies Hsc70-5/Mortalin as a regulator of mitochondrial morphology and cellular homeostasis

Mitochondrial morphology is regulated by conserved proteins involved in fusion and fission processes. The mammalian Optic atrophy 1 (OPA1) that functions in mitochondrial fusion is associated with Optic Atrophy and has been implicated in inner membrane cristae remodeling during cell death. Here, we show Drosophila Optic atrophy 1-like (Opa1-like) influences mitochondrial morphology through interaction with ‘mitochondria-shaping’ proteins like Mitochondrial assembly regulatory factor (Marf) and Drosophila Mitofilin (dMitofilin). To gain an insight into Opa1-like's network, we delineated bonafide interactors like dMitofilin, Marf, Serine protease High temperature requirement protein A2 (HTRA2), Rhomboid-7 (Rho-7) along with novel interactors such as Mortalin ortholog (Hsc70-5) from Drosophila mitochondrial extract. Interestingly, RNAi mediated down-regulation of hsc70-5 in Drosophila wing imaginal disc's peripodial cells resulted in fragmented mitochondria with reduced membrane potential leading to proteolysis of Opa1-like. Increased ecdysone activity induced dysfunctional fragmented mitochondria for clearance through lysosomes, an effect enhanced in hsc70-5 RNAi leading to increased cell death. Over-expression of Opa1-like rescues mitochondrial morphology and cell death in prepupal tissues expressing hsc70-5 RNAi. Taken together, we have identified a novel interaction between Hsc70-5/Mortalin and Opa1-like that influences cellular homeostasis through mitochondrial fusion.     

Validation of inhibitors of an ABC transporter required to transport lipopolysaccharide to the cell surface in Escherichia coli

The presence of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) of Gram-negative bacteria creates a permeability barrier that prevents the entry of most currently available antibiotics. The seven lipopolysaccharide transport (Lpt) proteins involved in transporting and assembling this glycolipid are essential for growth and division in Escherichia coli; therefore, inhibiting their functions leads to cell death. LptB, the ATPase that provides energy for LPS transport and assembly, forms a complex with three other inner membrane (IM) components, LptC, F, and G. We demonstrate that inhibitors of pure LptB can also inhibit the full IM complex, LptBFGC, purified in detergent. We also compare inhibition of LptB and the LptBFGC complex with the antibiotic activity of these compounds. Our long-term goal is to develop tools to study inhibitors of LPS biogenesis that could serve as potentiators by disrupting the OM permeability barrier, facilitating entry of clinically used antibiotics not normally used to treat Gram-negative infections, or that can serve as antibiotics themselves.     

Kinetic studies on alac-containing strain ofZymomonas mobilis

Summary Batch and continuous culture studies have been carried out on a strain ofZ.mobilis (ZM6306) which can convert lactose directly to ethanol. Previous strain development has established that thelac operon encoded on the transposon Tn951 can be expressed inZ.mobilis. Using a medium containing 80 g/l glucose and 40 g/l lactose, it was found that strain ZM6306 could convert about 13 g/l lactose to 4 g/l ethanol and 6 g/l galactose in continuous culture. Further lactose conversion is likely with increased cell concentration using a cell recycle system.     

Stereospecific oxidation of aliphatic alcohols catalyzed by galactose oxidase

The stereospecificity of galactose oxidase (EC 1.1.3.9) from Dactylium dendroides in the oxidation of simple three-carbon alcohols has been examined. The enzyme oxidizes glycerol to optically pure S(?)glyceraldehyde. In addition to this prochiral stereospecificity, galactose oxidase also exhibits enantiomeric stereospecificity in the oxidation of 3-halo-1,2-propanediols: the R isomer appears to be a better substrate than its S counterpart. The above stereochemistry of galactose oxidase-catalyzed oxidation of “unnatural” substrates, non-sugar alcohols, can be predicted on the basis of the conformation of the natural substrate of the enzyme, D-galactose.     

Effect of ethylene on sanguinarine production from Papaver somniferum cell cultures

A Papaver somniferum cell line capable of producing sanguinarine equivalent to 3% of cell dry weight was used to determine if ethylene was involved in signalling the biosynthesis of this alkaloid. A 3.3-fold increase in ethylene emanation from these cell suspension cultures was observed 7 h after elicitation with a Botrytis fungal homogenate. The rate of ethylene release then decreased to near zero after 48 h, suggesting that a pulse of ethylene production may be involved in sanguinarine production. However, sanguinarine biosynthesis was not promoted when either the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), or the ethylene releasing agent, 2-chloroethylphosphonic acid (ethephon), was added to the culture. These results strongly suggest that ethylene is not intimately involved in the production of sanguinarine from Papaver somniferum cell cultures or in the transduction of the elicitation event.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid     

The SNARE complex from yeast is partially unstructured on the membrane

Molecular recognition between cognate SNAREs leads to the formation of a four-helix bundle, which facilitates vesicle docking and membrane fusion. For a SNARE system involved in trafficking in yeast, target membrane (t-) SNARE Sso1p and vesicle associated (v-) SNARE Snc2p contribute one SNARE motif each, whereas another t-SNARE (Sec9) donates two N-terminal and C-terminal SNARE motifs (SN1 and SN2) to the helical bundle. By use of EPR, it is found that SN2 has a tendency to be uncoiled, leaving a significant population of the SNARE complexes to be partially unstructured on the membrane. In sharp contrast, SN2 is fully engaged in the four-helix bundle when removed from the membrane, showing that the membrane is the main destabilizing factor. Helix-breaking proline mutations in SN2 did not affect the rate of docking but reduced the rate of lipid mixing significantly, indicating that SN2 plays an essential role in activating the transition from docking to fusion.     

Effect of temperature on plasmid stability and expression of cloned cellulase gene in a recombinantBacillus megaterium

Summary The expression of carboxymethyl cellulase gene inBacillus megaterium (pCK108) was investigated with respect to temperature in batch culture. The suboptimal temperature supporting maximal cell growth rate was determined to be 30 °C at which stability of the plasmid pCK108 could be maintained stable. However, cellular plasmid contents, production rate of cellulase of the cell, and efficiency of the gene expression increased significantly with increase of the temperature from 30 °C to 44 °C, even though the plasmid stability decreased up to 60% level at the end of the culture.     

Hydroxylation of geraniol and nerol by a monooxygenase from Vinca rosea

A microsomal mixed function oxidase isolated from V. rosea seedlings was shown to catalyze the hydroxylation of the monoterpene alcohols, geraniol and nerol, to their corresponding 10-hydroxy derivatives. Hydroxylase activity was dependent upon NADPH and oxygen and was associated with the 100,000 X g pellet which exhibited a characteristic reduced P-450-CO binding spectra. Light reversible inhibition by CO as well as differential sensitivity to other inhibitors established the hydroxylase as a cytochrome P-450 type. Cis-trans isomerase activity was not observed in this preparation. Both geraniol and nerol were shown to be hydroxylated almost exclusively at the C-10 methyl group.     

Membrane effects of ethanol: bulk lipid versus lipid domains

It has been well-established that ethanol fluidizes the bulk lipid of membranes and that this effect may alter cell function and be involved in ethanol sensitivity and tolerance. This hypothesis has been supported in several studies, however, there is also a considerable amount of data that do not support such an explanation, e.g., direct effect of ethanol on proteins, other membrane acting drugs, temperature effects, effects of ethanol on aged membranes and inconsistent effects of chronic ethanol consumption on lipid content. This review examined the bulk membrane fluidization hypothesis in light of those data and proposed a modification of the bulk membrane hypothesis that is based on recent data that show that ethanol and other alcohols have a specific effect on the structural properties of different membrane domains. This specific effect of ethanol is discussed within the context of how changes in fluidity of domains may alter membrane function.     

Growth of baculovirus-infected insect cells in microcapsules to a high cell and virus density

Summary Insect cells (Spodoptera Frugiperda), infected with a temperature-sensitive mutant (TS10) of theAutographa Californica nuclear polyhedrosis virus (AcNPV), were cultured in multiple membrane alginate-polylysine (PLL) microcapsules. It was possible to obtain intracapsular cell densities of 8× 10⁷ cells/mL of capsules and virus concentrations of up to 10⁹ IFU/mL of capsules. This was higher by a factor of 10 than that which could be achieved by conventional cell suspension culture.     

How Epigallocatechin Gallate Can Inhibit α-Synuclein Oligomer Toxicity in Vitro

Oligomeric species of various proteins are linked to the pathogenesis of different neurodegenerative disorders. Consequently, there is intense focus on the discovery of novel inhibitors, e.g. small molecules and antibodies, to inhibit the formation and block the toxicity of oligomers. In Parkinson disease, the protein α-synuclein (αSN) forms cytotoxic oligomers. The flavonoid epigallocatechin gallate (EGCG) has previously been shown to redirect the aggregation of αSN monomers and remodel αSN amyloid fibrils into disordered oligomers. Here, we dissect EGCG''s mechanism of action. EGCG inhibits the ability of preformed oligomers to permeabilize vesicles and induce cytotoxicity in a rat brain cell line. However, EGCG does not affect oligomer size distribution or secondary structure. Rather, EGCG immobilizes the C-terminal region and moderately reduces the degree of binding of oligomers to membranes. We interpret our data to mean that the oligomer acts by destabilizing the membrane rather than by direct pore formation. This suggests that reduction (but not complete abolition) of the membrane affinity of the oligomer is sufficient to prevent cytotoxicity.     

Cytopathological features of cell suffering and death: Role of plasma membrane and cytoskeleton

Morphological and ultrastructural modifications related to the cell injury and leading to cell death have been investigated by using different compounds. Data obtained by treating various cultured cells with a quinone (menadione), a polar solvent (NMF) and a bacterial protein toxin (toxin B fromClostridium difficile) are here reported Differences seem to exist between such injuries, but changes in plasma membrane structure, called surface blebbing phenomenon, represent a common feature which can be in any case detected. Our results also allow to hypothesize an important role of cytoskeleton in such a process.     

Syntheses et Activites Biologiques de Nouvelles (E)-Alcenyl-5 Desoxy-2′ Uridines

Abstract

(E) 5-Alkenyl 2′-deoxyuridines were synthesized with moderate to high yields by the palladium catalyzed coupling of alkenyl-zirconium reagents with 0–3′, 5′-his (trimethylsilyl) deoxyuridine in T H F. Some of these 5-alkenyl-dUrd analogues, i.e. the 1-decenyl (5g) and 2- (1-hydroxycyclopentyl) ethenyl (5f) derivatives, inhibited murine L1210 cell growth at a concentration of about 4 μg/ml, whereas the 5-chloro-1-pentenyl (5c), 5-cyano-1-pentenyl (5d), 5-hexyn-1-enyl (5e) and 2-(1-hydroxycyclopentyl) ethenyl (5f) were inhibitory towards herpes simplex and vaccinia virus within the concentration range of 2–60 μg/ml. However, none of the newly synthesized 5-alkenyl-dUrd analogues proved selective in its antiviral action.     

Human kanadaptin and kidney anion exchanger 1 (kAE1) do not interact in transfected HEK 293 cells

Kanadaptin (k¯idney anion exchanger adaptor protein) is a widely expressed protein, shown previously to interact with the cytosolic domain of mouse Cl^?/HCO₃^? anion exchanger 1 (kAE1) but not erythroid AE1 (eAE1) by a yeast-two hybrid assay. Kanadaptin was co-localized with kAE1 in intracellular membranes but not at the plasma membrane in α-intercalated cells of rabbit kidney. It was suggested that kanadaptin is an adaptor protein or chaperone involved in targeting kAE1 to the plasma membrane. To test this hypothesis, the interaction of human kanadaptin with human kAE1 was studied in co-transfected HEK293 cells. Human kanadaptin contains 796 amino acids and was immuno-detected as a 90 kDa protein in transfected cells. Pulse-chase experiments showed that it has a half-life (t_1/2) of 7 h. Human kanadaptin was localized predominantly to the nucleus, whereas kAE1 was present intracellularly and at the plasma membrane. Trafficking of kAE1 from its site of synthesis in the endoplasmic reticulum to the plasma membrane was unaffected by co-expression of human kanadaptin. Moreover, we found that no interaction between human kanadaptin and kAE1 or eAE1 could be detected in co-transfected cells either by co-immunoprecipitation or by histidine6-tagged co-purification. Taken together, we found that human kanadaptin did not interact with kAE1 and had no effect on trafficking of kAE1 to the plasma membrane in transfected cells. Kanadaptin may not be involved in the biosynthesis and targeting of kAE1. As such, defects in kanadaptin and its interaction with kAE1 are unlikely to be involved in the pathogenesis of the inherited kidney disease, distal renal tubular acidosis (dRTA).