Superoxide dismutases of chestnut leaves, Castanea sativa: Characterization and study of their involvement in natural leaf senescence

Superoxide dismutases (SOD; EC 1.15.1.1) in chestnut ( Castanea sativa Mill., cv. 431) leaves were characterized by native polyacrylamide gel electrophoresis. The three molecular forms of SOD were distinguished from each other by their different sensitivity to cyanide and H₂O₂ Three CuZn-containing SODs were detected (CuZn-SOD I, II. and III), and all the isozymes had a molecular mass of 33 kDa. CuZn-SOD III was the most abundant isozyme. whereas CuZn-SOD II was present in a minor amount. In leaves showing typical symptoms of senescence increases of 2.5-. 7- and 4-fold in the specific activities of CuZn-SODs I, II, and III. respectively, were found. In addition, the pattern of the three isozymes was modified by the age of leaves, a rise in the CuZn-SOD II and a decrease in the CuZn-SOD 1 percentages being found in senescent leaves compared to green leaves. As to other activated oxygen-related enzymes, an increase in the superoxide-generating xanthine oxidase activity and a decline in both catalase and peroxidase activities during natural senescence of chestnut leaves were observed. Results obtained suggest that in natural senescence of chestnut leaves activated oxygen species are involved, and an overproduction of hydrogen peroxide and superoxide radicals probably takes place.     

Binding of [3H]-2-isobutyl-3-methoxypyrazine to cow olfactory mucosa

The odorant 2-isobutyl-3-methoxypyrazine binds to cow olfactorymucosa homogenate. The complex, which can be separated by gelfiltration on Sephadex G-100, appears to be made up of fourmacromolecular species. No significant binding has been measuredwith respiratory epithelium. The binding disappears after treatmentwith proteolytic enzymes, or in a SDS containing buffer, thusindicating that the receptors are proteins. Complete loss ofbinding capacity has been also observed as a consequence ofdialysis: this suggests the involvement of a low molecular weightcomponent.     

Liposome-entrapped superoxide dismutase reduces ischemia/reperfusion ‘oxidative stress’ in gerbil brain

Bilateral common carotid artery occlusion (15 min.) followed by two hours of recirculation reduced mitochondrial superoxide dismutase (SOD) and glutathione reductase (GR) activities, and increased susceptibility of mitochondrial membranes to in vitro lipid peroxidation in brain regions (i.e., cortex, striatum and hippocampus) of Mongolian gerbil. Intraperitoneal bolus injection (2 mg/kg b.w.) of liposome-entrapped CuZn superoxide dismutase (l-SOD) increased the endogenous SOD activity in normal brain tissue and, when given at the end of ischemia, counteracted both the ischemic reduction of endogenous SOD and the increased peroxidation of mitochondrial membranes. 1-SOD treatment was ineffective in reducing brain swelling, suggesting that superoxide radicals are not a main participant in the process of (post)ischemic brain edema formation.     

Crystal structure of RlmM, the 2��O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA

RlmM (YgdE) catalyzes the S-adenosyl methionine (AdoMet)-dependent 2′O methylation of C2498 in 23S ribosomal RNA (rRNA) of Escherichia coli. Previous experiments have shown that RlmM is active on 23S rRNA from an RlmM knockout strain but not on mature 50S subunits from the same strain. Here, we demonstrate RlmM methyltransferase (MTase) activity on in vitro transcribed 23S rRNA and its domain V. We have solved crystal structures of E. coli RlmM at 1.9 Å resolution and of an RlmM–AdoMet complex at 2.6 Å resolution. RlmM consists of an N-terminal THUMP domain and a C-terminal catalytic Rossmann-like fold MTase domain in a novel arrangement. The catalytic domain of RlmM is closely related to YiiB, TlyA and fibrillarins, with the second K of the catalytic tetrad KDKE shifted by two residues at the C-terminal end of a beta strand compared with most 2′O MTases. The AdoMet-binding site is open and shallow, suggesting that RNA substrate binding may be required to form a conformation needed for catalysis. A continuous surface of conserved positive charge indicates that RlmM uses one side of the two domains and the inter-domain linker to recognize its RNA substrate.     

The [2Fe-2S] cluster E(m) as an indicator of the iron-sulfur subunit position in the ubihydroquinone oxidation site of the cytochrome bc1 complex.

Recent crystallographic and kinetic data have revealed the crucial role of the large scale domain movement of the iron-sulfur subunit [2Fe-2S] cluster domain during the ubihydroquinone oxidation reaction catalyzed by the cytochrome bc(1) complex. Previously, the electron paramagnetic resonance signature of the [2Fe-2S] cluster and its redox midpoint potential (E(m)) value have been used extensively to characterize the interactions of the [2Fe-2S] cluster with the occupants of the ubihydroquinone oxidation (Q(o)) catalytic site. In this work we analyze these interactions in various iron-sulfur subunit mutants that carry mutations in its flexible hinge region. We show that the E(m) increases of the iron-sulfur subunit [2Fe-2S] cluster induced either by these mutations or by the addition of stigmatellin do not act synergistically. Moreover, the E(m) increases disappear in the presence of class I inhibitors like myxothiazol. Because various inhibitors are known to affect the location of the iron-sulfur subunit cluster domain, the measured E(m) value of the [2Fe-2S] cluster therefore reflects its equilibrium position in the Q(o) site. We also demonstrate the existence in this site of a location where the E(m) of the cluster is increased by about 150 mV and discuss its possible implications in term of Q(o) site catalysis and energetics.     

The 30-Base-Pair Deletion in Chinese Variants of the Epstein-Barr Virus LMP1 Gene Is Not the Major Effector of Functional Differences between Variant LMP1 Genes in Human Lymphocytes

One group of sequence variants of Epstein-Barr virus is characterized by a 10-amino-acid deletion within the CTAR-2 functional domain of the latent membrane protein, LMP1. A role for this deletion in enhancing the tumorigenicity of the viral oncogene in rodent fibroblasts was recently demonstrated. We examined the effect of this deletion upon LMP1 function in four human lymphoid cell lines by using three natural variants of LMP1: the prototype B95.8 gene and the CAO and AG876 genes, both of which have codons 343 to 352 of the B95.8-LMP1 deleted. These experiments revealed that LMP1-mediated upregulation of CD40 and CD54 was markedly impaired (by 60 to 90%) with CAO-LMP1 compared with B95.8-LMP1. In contrast, the function of AG876-LMP1 was indistinguishable from that of B95.8-LMP1 in two lines and was only slightly impaired in the other two lines. Activation of NF-κB by CAO-LMP1 was not impaired in any of the lines; rather, activation of an NF-κB reporter by CAO-LMP1 was consistently about twofold greater than the activation with B95.8- or AG876-LMP1. Therefore, while the CAO-LMP1 is functionally distinct from the prototype B95.8-LMP1 in human lymphocytes, the 10-amino-acid deletion appears not to be directly responsible. This conclusion was confirmed by using a B95.8-LMP1 mutant with codons 343 to 352 deleted and chimerae of CAO- and B95.8-LMP1 in which the CTAR-2 domains of these genes were exchanged. Sequences outside the CTAR-2 domain were implicated in the distinct functional characteristics of CAO-LMP1 in human lymphoid cells.     

A Simple Method for Isolating DNA of High Yield and Quality from Cotton (shape Gossypium hirsutum L.) Leaves

An easy, reproducible and fast procedure to isolate DNA from cotton leaves is described. The addition of 0.5 M glucose in the extraction buffer avoids browning by polyphenolic compounds and improves the quality of DNA for molecular analysis. The DNA yield ranged between 150–400 mg per gram of fresh tissue. The DNA was suitable for digestion by restriction enzymes and amplificatiion by Taq DNA polymerase.     

Influence of the composition and preparation method on the morphology and swelling behavior of alginate–chitosan hydrogels

In this study, a 2⁴ factorial experimental design was employed in order to evaluate the influence of the reaction conditions and preparation method on alginate–chitosan hydrogel properties. Alginate content, pH, chitosan molecular weight and the hydrogel preparation method were the independent variables and the reaction yield, particle size, swelling degree and point of zero surface charge were the dependent variables. The results showed that hydrogels were spherical with an average diameter of 5.0 ± 2.0 μm. Reaction yield varied according to the parameters, and chitosan molecular weight showed the greatest influence. Furthermore, the swelling degree and point of zero surface charge showed a linear dependence on the alginate content. In this regard, the study showed that hydrogels with a specific charge and swelling degree can be obtained by controlling the alginate content using the equation here provided to give an enhanced and site-specific controlled drug release.