Hydrolysis of fish oil by hyperactivated rhizomucor miehei lipase immobilized by multipoint anion exchange

Rhizomucor miehei lipase (RML) is greatly hyperactivated (around 20‐ to 25‐fold toward small substrates) in the presence of sucrose laurate. Hyperactivation appears to be an intramolecular process because it is very similar for soluble enzymes and covalently immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence of sucrose laurate) on cyanogen bromide‐activated Sepharose (very mild covalent immobilization through the amino terminal residue), on glyoxyl Sepharose (intense multipoint covalent immobilization through the region with the highest amount of Lys residues), and on different anion exchangers (by multipoint anionic exchange through the region with the highest density of negative charges). Covalent immobilization does not promote the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains the hyperactivated enzyme even in the absence of a detergent. The hydrolysis of fish oils by these hyperactivated enzyme derivatives was sevenfold faster than by covalently immobilized derivatives and three and a half times faster than by the enzyme hyperactivated on octyl‐Sepharose. The open structure of the hyperactivated lipase is fairly exposed to the medium, and no steric hindrance should interfere with the hydrolysis of large substrates. These new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML immobilized inside porous supports. In addition, the hyperactivated derivatives are fairly stable against heat and organic cosolvents. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

α-MELANOCYTE-STIMULATING HORMONE: A POSSIBLE NEURONAL MARKER OF THE AGEING BRAIN

Abstract— The amount of α-melanocyte-stimulating hormone (α-MSH) in the entire hypothalamus as well as the amount of α-MSH in free granule and synaptosome fractions of hypothalamic homogenates was investigated throughout the lifespan of female rats (1-24 months). A 900 g supernatant fluid was prepared from hypothalami following homogenization in an iso-osmotic sucrose solution, and free granules and synaptosomes containing α-MSH were fractionated by means of continuous sucrose density gradient centrifugation. α-MSH was quantified by radioimmunoassay. The total amount of α-MSH in the hypothalamus, as well as the amount in free granules and synaptosomes prepared from hypothalami increased progressively from the 1st to the 5th month of life, and this increase was more pronounced in the free granules than in the synaptosomes. On the other hand, the amount of α-MSH in the hypothalamus and the amount present in free granules and synaptosomes prepared from 5-24-month-old animals decreased with age, and this decrease appeared to proceed at similar rates in both subcellular compartments. Based on these results, it is suggested that ageing of α-MSH neurons in the hypothalamus is accompanied by a degeneration of the axons and/or an alteration in the biosynthetic and degradative activities of the neuron.     

Strain Dependent Variation of Immune Responses to A. fumigatus: Definition of Pathogenic Species

For over a century microbiologists and immunologist have categorized microorganisms as pathogenic or non-pathogenic species or genera. This definition, clearly relevant at the strain and species level for most bacteria, where differences in virulence between strains of a particular species are well known, has never been probed at the strain level in fungal species. Here, we tested the immune reactivity and the pathogenic potential of a collection of strains from Aspergillus spp, a fungus that is generally considered pathogenic in immuno-compromised hosts. Our results show a wide strain-dependent variation of the immune response elicited indicating that different isolates possess diverse virulence and infectivity. Thus, the definition of markers of inflammation or pathogenicity cannot be generalized. The profound understanding of the molecular mechanisms subtending the different immune responses will result solely from the comparative study of strains with extremely diverse properties.     

Reactive oxygen-mediated damage to murine mammary tumor cells

We have shown, in a preliminary report, that macrophages can induce strand breaks in the DNA of co-cultured tumor cells (Chong et al., 1988). The present study is designed to determine if oxygen-centered species generated by the cell-free enzyme-substrate combination of hypoxanthine and xanthine oxidase can induce similar lesions and to identify the specific mediator(s). We report that co-incubation of murine mammary tumor cell lines with hypoxanthine and xanthine oxidase leads to the induction of DNA-strand breaks as determined by fluorescence analysis of DNA unwinding (FADU) assay or alkaline elution techniques. This damage is preventable by catalase which removes hydrogen peroxide but no protection is provided by agents to remove or prevent the formation of superoxide anion (superoxide dismutase), or hydroxyl radical (mannitol or the iron chelator o-phenanthroline). Likewise, cyclooxygenase or lipoxygenase inhibitors of arachidonate metabolism (indomethacin, nordihydroguaiaretic acid, caffeic acid) or bromophenacyl bromide do not alter the degree of DNA scission. Treatment with higher doses of oxygen species leads to significant toxicity as determined by evaluation of cell growth potential or colony-forming ability. Again, toxicity is prevented only by the presence of catalase. Tumor cells are able to rejoin strand breaks at lower, less toxic doses. When comparing different tumor cell subpopulations at various stages of progression, i.e., metastatic vs. nonmetastatic, for sensitivity to hydrogen peroxide-induced strand breakage, we found that at lower concentrations (less than 5μM) metastatic populations are sensitive whereas nonmetastatic populations exhibit no significant breakage. At higher concentrations of hydrogen peroxide, all lines were sensitive, suggesting that a lower threshold of sensitivity may exist for more progressed tumour cell lines.     

Effect of Growth Conditions and Trehalose Content on Cryotolerance of Bakers' Yeast in Frozen Doughs

The cryotolerance in frozen doughs and in water suspensions of bakers' yeast (Saccharomyces cerevisiae) previously grown under various industrial conditions was evaluated on a laboratory scale. Fed-batch cultures were very superior to batch cultures, and strong aeration enhanced cryoresistance in both cases for freezing rates of 1 to 56°C min⁻¹. Loss of cell viability in frozen dough or water was related to the duration of the dissolved-oxygen deficit during fed-batch growth. Strongly aerobic fed-batch cultures grown at a reduced average specific rate (μ = 0.088 h⁻¹ compared with 0.117 h⁻¹) also showed greater trehalose synthesis and improved frozen-dough stability. Insufficient aeration (dissolved-oxygen deficit) and lower growth temperature (20°C instead of 30°C) decreased both fed-batch-grown yeast cryoresistance and trehalose content. Although trehalose had a cryoprotective effect in S. cerevisiae, its effect was neutralized by even a momentary lack of excess dissolved oxygen in the fed-batch growth medium.     

Inhibition ofCandida albicans andStaphylococcus aureus by phenolic compounds from the terrestrial cyanobacteriumNostoc muscorum

Phenolic compounds were determined in methanolic extract from the algal mass of aNostoc muscorum culture. Bioassays with two human pathogens,Candida albicans andStaphylococcus aureus indicated that algal phenolic compounds evoked significant growth inhibition for both species (89.1% and 88.2%, respectively). It is suggested that this strong inhibitory effect is of potential medicinal value.     

Voltammetric studies on the electrochemical behaviour of membrane-entrapped hemes

Summary The electrochemical behaviour of Fe(III)-protoporphyrin IX entrapped into a cellulose triacetate membrane has been investigated by cyclic voltammetry. The physical entrapment into a solid matrix does not modify the redox properties of the entrapped berries, which also act as efficient promoters in the electrochemistry of cytochromec. Such a system represents a promising example of a simple solid-state promoter, and stimulates further investigations in order to obtain more complex systems that may be of significance for basic and applied bioelectrochemistry.     

Isolation and characterization of a thermophilic Methanobacterium able to use formate,the strain FTF

A thermophilic anaerobic which produced methane from formate and H₂ and CO₂ was isolated from a bench-scale digester treating a mixture of solid wastes at 55°C, after enrichment cultures on sodium acetate. The cells were slightly crooked rods occurring singly or in filaments. The bacterium was not motile, and stained Gram positive. Colonies appearing after 1 week of incubation were white with filamentous edges and 1 mm in diameter. The organism used H₂:CO₂ or formate as an energy source. Yeast extract was not required but stimulated growth significantly. Casamino acids were stimulatory and could serve as a nitrogen source. Cysteine was used as a sulfur source. The optimum pH for growth was 7.5. Growth occurred from 35 to 70°C with an optimum at 55°C. The deoxyribonucleic acid base composition was 49.2 mol% guanine plus cytosine. Though this isolate conforms to Methanobacterium thermoformicium, its proper assignment awaits further studies. It has been deposited in the Deutsche Sammlung von Mikroorganismen as strain DSM 3012.This work was supported in part by the Conseil Régional Nord/Pas-de-Calais     

Chromatin reconstitution on small DNA rings. I

Chromatin was reconstituted using the four core histones on 359 base-pair nicked and closed rings by salt dialysis and/or at physiological ionic strength by means of polyglutamic acid. The products, which consisted of mono- and dinucleosomes, were characterized by gel electrophoresis, sedimentation in sucrose gradients and high-resolution electron microscopy. The results were as follows. (1) The efficiency of the reconstitution was found first to increase with the negative linking difference of the closed rings relative to their relaxed configuration to reach a maximum for -2 turns, and then to decrease for the largest difference of -3 turns. Discrepancies between topoisomers were also observed with regard to differential formation of mono- and dinucleosomes. Topoisomer -1 reconstituted monomers easily but reconstituted dimers with difficulty, whilst this discrimination was virtually absent in the case of topoisomers -2 and -3. Moreover, mononucleosomes on the nicked ring were, with respect to their electrophoretic mobility, similar to mononucleosomes formed on topoisomer -1 but not to those on the other topoisomers, whose mobilities were greater. These features were interpreted in terms of the linking number change associated with the formation of a nucleosome monomer and dimer, approximately -1 and -2 turns, respectively. (2) Two dinucleosome subtypes were found to form in a sequential manner. Their different electrophoretic mobilities and sedimentation coefficients suggested that the early subtype is lighter, probably because of an incomplete histone complement in the second nucleosome of that subtype as a result of an impaired co-operativity in octamer assembly due to the small ring size. (3) An electron microscopic examination of the chromatin reconstituted on topoisomer -2 revealed that both mono- and dinucleosomes adopt two different, salt-dependent, morphologies each: in type I, entering and exiting DNAs do not cross, whilst they do in type II. Type I configuration is favoured in lower salt, whereas type II is favoured in higher salt. Such behaviour explains why nucleosomes in dimers were found to be always diametrically opposed on the rings rather than sometimes apposed, as would have been expected from a random deposition of the histone cores.