Several mixed-function oxidation systems catalyze the inactivation of Escherichia coli glutamine synthetase. Inactivation involves modification of a single histidine residue in each enzyme subunit and makes the enzyme susceptible to proteolytic degradation. We show here that 10 key enzymes in metabolism are inactivated by a bacterial NADH oxidase and by an oxidase system comprised of NADPH, cytochrome P450 reductase, and cytochrome P450 isozyme 2 from rabbit liver microsomes. Most of the inactivatable enzymes require a divalent cation for activity and all but one (enolase) possess a nucleotide binding site. Glutamine synthetase, pyruvate kinase, and phosphoglycerate kinase are protected from inactivation by their substrates; substrate protection of other enzymes was not tested. We propose that inactivation involves mixed-function oxidization system-catalyzed synthesis of H202 and reduction of Fe(EI) to Fe(ll) followed by oxidation of enzyme-bound Fe(II) by H202 to generate oxygen radicals that attack a histidine (or other oxidizable amino acid) at the metal binding site of the enzyme. This is supported by the following: (i) most of the inactivation reactions are inhibited by EDTA and by catalase, (ii) both mixed-function oxidation systems reduce Fe(S), and (iii) H202 together with Fe(II) catalyzes nonenzymic inactivation of glutamine synthetase. In view of the fact that inactivation of glutamine synthetase makes it susceptible to proteolytic degradation, it is possible that mixed-function oxidation system-catalyzed inactivation of enzymes is a regulatory step in enzyme turnover. In addition, the implication of oxidative inactivation reactions in ageing is suggested by the fact that many of the enzymes inactivated by mixed-function oxidation systems are known to accumulate as inactive forms during ageing. coli (1-3). Inactivation of glutamine synthetase by all enzyme systems is dependent on 02 and NAD(P)H (except in the case of xanthine oxidase, for which hypoxanthine serves as an electron donor). All systems are stimulated by Fe(III) and inhibited by catalase, Mn(II), EDTA, o-phenanthroline, and histidine (1-3). Inactivation of glutamine synthetase by either the ascorbate system or the NADH oxidase system (other systems not tested) is associated with the modification of a single histidine residue in each glutamine synthetase subunit (4). This inactivation renders the glutamine synthetase more susceptible to proteolytic degradation by subtilisin (5) as well as by proteases in E. coli (1, 2). Thus, the inactivation reaction appears to "mark" glutamine synthetase for proteolytic digestion.We report here that, in addition to the glutamine synthetase from E. coli, the glutamine synthetase from rat liver and a number of other key metabolic-enzymes, many of which possess ahistidine or another easily oxidizable residue at the catalytic site, are inactivated in vitro by the NADH oxidase system and also by the rabbit liver microsomal cytochrome P-450 system. During ageing, inactive or less active forms of severa...

Obesity is associated with an increased risk of breast cancer. A number of adipocytokines are increased in obesity causing low-level chronic inflammation associated with an increased risk of tumors. The adipocytokine leptin shows profound anti-obesity and pro-inflammatory activities. We have hypothesized that in common obesity, high circulating leptin levels might contribute to an increased risk of breast cancer by affecting mammary cell proliferation and survival. Leptin exerts its activity not only through leptin receptor (LepR), but also through crosstalk with other signaling systems implicated in tumorigenesis. In this study, we focused our attention on the relationship between the leptin/LepR axis and the estrogen receptor-a (ERa). To this aim, we utilized two human breast cancer cell lines, one ERa-positive cell line (MCF 7) and the other ERa-negative cell line (MDA-MB 231). We observed that the two cell lines had a different sensitivity to recombinant leptin (rleptin): on MCF 7 cells, rleptin induced a strong phosphorylation of the signal transducer and activator of transcription (STAT) 3 and of the extracellular related kinase 1/2 pathways with an increased cell viability and proliferation associated with an increased expression of ERa receptor. This response was not present in the MDA-MB 231 cells. The effects induced by leptin were lost when LepR was neutralized using either a monoclonal inhibitory antibody to LepR or LepR genesilencing siRNA. These data suggest that there is a bidirectional communication between LepR and ERa, and that neutralization and/or inactivation of LepR inhibits proliferation and viability of human breast cancer cell lines. This evidence was confirmed by ex vivo studies, in which we analyzed 33 patients with breast cancer at different stages of disease, and observed that there was a statistically significant correlation between the expression of LepR and ERa. In conclusion, this study suggests a crosstalk between LepR and ERa, and could envisage novel therapeutic settings aimed at targeting the LepR in breast cancers.

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We have analyzed Mytilus galloprovincialis' sperm chromatin, which consists of three protamine-like proteins, PL-II, PL-III, and PL-IV, in addition to a residual amount of the four core histones. We have probed the structure of this sperm chromatin through digestion with micrococcal nuclease (MNase) in combination with salt fractionation. Furthermore, we used the electrophoretic mobility shift assay to define DNA-binding mode of PL-II and PL-III and turbidimetric assays to determine their self-association ability in the presence of sodium phosphate. Although in literature it is reported that M. galloprovincialis' sperm chromatin lacks nucleosomal organization, our results obtained by MNase digestion suggest the existence of a likely unusual organization, in which there would be a more accessible location of PL-II/PL-IV when compared with PL-III and core histones. So, we hypothesize that in M. galloprovincialis' sperm chromatin organization DNA is wrapped around a PL-III protein core and core histones and PL-II and PL-IV are bound to the flanking DNA regions (similarly to somatic histone H1). Furthermore, we propose that PL's K/R ratio affects their DNA-binding mode and self-association ability as reported previously for somatic and sperm H1 histones.

The biology of transposable elements (TEs) is a fascinating and complex field of investigation. TEs represent a substantial fraction of many eukaryotic genomes and can influence many aspects of DNA function that range from the evolution of genetic information to duplication, stability, and gene expression. Their ability to move inside the genome has been largely recognized as a double-edged sword, as both useful and deleterious effects can result. A fundamental role has been played by the evolution of the molecular processes needed to properly control the expression of TEs. Today, we are far removed from the original reductive vision of TEs as “junk DNA”, and are more convinced that TEs represent an essential element in the regulation of gene expression. In this review, we summarize some of the more recent findings, mainly in the animal kingdom, concerning the active roles that TEs play at every level of gene expression regulation, including chromatin modification, splicing, and protein translation.

Methylation of specific lysine residues in histone tails has been proposed to function as a stable epigenetic marker that directs biological functions altering chromatin structure. Recent findings have implicated alteration in heterochromatin formation as a contributing factor in cancer development. In order to verify whether changes in the overall level of H3K4 histone methylation could be involved in oral squamous carcinoma, the levels of H3K4me1, me2 and me3 were measured in oral squamous carcinoma, leukoplakias and normal tissues. The levels of H3K4me2 and me3 were significantly different in oral squamous cell carcinoma in comparison with normal tissue: the level of H3K4me2 was increased while that of H3K4me3 decreased. No significant differences could be found between the two types of tissues in the level of H3K4me1. A similar trend was found in the leukoplakias that appeared more like the pathological than normal tissue. These results support the idea that alteration of chromatin structure could contribute to oncogenic potential.

Antibacterial effects of various arginine- and lysine-rich polycationic proteins and polymers were evaluated by broth and solid dilution assay on a range of foodborne pathogens, Gram-positive and Gram-negative bacteria. The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of α-poly-L-lysine (poly-lys), α-poly-L-arginine (poly-arg) and protamines from herring sperm (clupeine sulphate) and salmon sperm (salmine sulphate) were determined on Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Shigella sonnei, Escherichia coli O157:H7 and Pseudomonas aeruginosa. All these molecules showed antibacterial activity on all strains with different MIC and MBC values. The molecular mechanisms underlying the effect of α-poly-L-arginine might be related to the entrance of the molecule into the cell. In fact α-poly-L-arginine labelled with 7-Diethylamino coumarin-3-carboxylic acid, succinimidyl ester (DEAC,SE) showed ability to permeate the cell membrane of B. cereus and E. coli O157:H7.

The mode of binding of sperm and somatic H1 histones to DNA has been investigated by analyzing the effect of their addition on the electrophoretic mobility of linear and circular plasmid molecules. Low concentrations of sperm histones do not appear to alter the electrophoretic mobility of DNA, whereas at increasing concentrations, an additional DNA band is observed near the migration origin. This band then becomes the only component at higher values. In contrast, somatic histones cause a gradual retardation in the mobility of the DNA band at low concentrations and aggregated structures are observed only at higher values. Experiments on the H1 globular domain obtained by limited proteolysis indicate that the mode of binding to DNA depends on the H1 globular domain. The arginine residues appear to be relevant for the different effects as indicated by experiments on sperm histone and on protamine with arginines deguanidinated to ornithines. The modified molecules influence DNA mobility like somatic H1s, indicating that the positive guanidino groups of arginines cannot be substituted by the positive amino groups of ornithines. Modifications of the amino groups of lysines show that these residues are necessary for the binding of H1 histones to DNA but they have no influence on the binding mode.