Protein self-modification by heme-generated reactive species

In the presence of H(2)O(2), heme proteins form active intermediates, which are able to oxidize exogenous molecules. Often these products are not stable compounds but reactive species on their own, such as organic radicals. They can both diffuse to the bulk of the solution or react with the protein that generated them. Here, we describe the self-modification underwent by heme proteins with globin-type fold, that is, myoglobin, hemoglobin, and neuroglobin when treated with NO(2) (-) or catechols in the presence of H(2)O(2). The reactive nitrogen species generated by NO(2) (-) give rise to nitration, oxidation, and/or crosslinking reactions between the proteins or their subunits. The quinones formed upon reaction with catechols easily modify Cys and His residues and eventually cause protein aggregation, which induces precipitation. The pattern of modifications undergone by the protein strongly depends on the nature of the protein and the reaction conditions.     

Summer and spring trophic niche of larval and juvenile <Emphasis Type="Italic">Pleuragramma antarcticum</Emphasis> in the Western Ross Sea,Antarctica

The aim of this paper is to contribute to the knowledge on the feeding habits of larvae and juvenile Pleuragramma antarcticum in the western Ross Sea. In summer, the diet of P. antarcticum postlarvae (8–17 mm) was dominated by calanoid eggs (35.5%), Limacina (32.1%) and tintinnids (17.6%), while the principal food of juveniles consisted mainly of copepods (98.2%), with Oncaea curvata being the most abundant (85.1%) and the most frequently consumed prey. The food composition of P. antarcticum postlarvae (24–29 mm), collected in spring, suggest that they fed actively under the sea ice. Stephos longipes, Harpacticus furcifer and Paralabidocera antarctica sea ice copepods represent, in all their different developmental stages, the most abundant biomass food in Terra Nova Bay in this period. Our results therefore suggest that the diet of younger Pleuragramma specimens shifted in prey composition from the first summer to the following spring. This study draws attention to the key role of the copepod, P. antarctica, in the food web of Terra Nova Bay. This article belongs to a special topic: Five articles coordinated by L. Guglielmo and V. Saggiomo appear in this issue of Polar Biology and are a result of a workshop on Sea-ice communities in Terra Nova Bay (Ross Sea) held in August 2007 in Capo Calavà, Messina, Italy.     

Design and expression of peptides with antimicrobial activity against Salmonella typhimurium

We showed previously that insertion of Synechocystis Δ¹²‐desaturase in salmonella's membrane alters membrane physical state (MPS), followed by the expression of stress genes causing inability to survive within murine macrophages (MΦ). Recently, we showed that expression of one membrane lipid domain (MLD) of Δ¹²‐desaturase (ORF200) interferes with salmonella MPS, causing loss of virulence in mice and immunoprotection. Here, we postulate that an α‐antimicrobial peptide (α‐AMP) intercalates within membrane lipids, and depending on its amino acid sequence, it does so within specific key sensors of MLD. In this study, we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg²⁺ concentration. We synthesised a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in salmonella typhimurium. We showed that the pattern of gene expression controlled by PhoP/PhoQ highlights dysregulation of pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens. RNA‐Seq of strain expressing ORF200 showed that the pattern of those genes is also altered here. Accumulation of NUF conferred temporary immunoprotection. This represents a powerful procedure to address synthetic α‐AMPs to a specific MLD generating live non‐virulent bacterial strains.     

CSN complex controls the stability of selected synaptic proteins via a torsinA-dependent process

DYT1 dystonia is caused by an autosomal dominant mutation that leads to a glutamic acid deletion in torsinA (TA), a member of the AAA+ ATPase superfamily. In this study, we identified a novel-binding partner of TA, the subunit 4 (CSN4) of CSN signalosome. TA binds CSN4 and the synaptic regulator snapin in neuroblastoma cells and in brain synaptosomes. CSN4 and TA are required for the stability of both snapin and the synaptotagmin-specific endocytic adaptor stonin 2, as downregulation of CSN4 or TA reduces the levels of both proteins. Snapin is phosphorylated by the CSN-associated kinase protein kinase D (PKD) and its expression is decreased upon PKD inhibition. In contrast, the stability of stonin 2 is regulated by neddylation, another CSN-associated activity. Overexpression of the pathological TA mutant (ΔE-TA) reduces stonin 2 expression, causing the accumulation of the calcium sensor synaptotagmin 1 on the cell surface. Retrieval of surface-stranded synaptotagmin 1 is restored by overexpression of stonin 2 in ΔE-TA-expressing cells, suggesting that the DYT1 mutation compromises the role of TA in protein stabilisation and synaptic vesicle recycling.     

A novel anti-aldolase C antibody specifically interacts with residues 85–102 of the protein

Aldolase C is a brain-specific glycolytic isozyme whose complete repertoire of functions are obscure. This lack of knowledge can be addressed using molecular tools that discriminate the protein from the homologous, ubiquitous paralog aldolase A. The anti-aldolase C antibodies currently available are polyclonal and not highly specific. We obtained the novel monoclonal antibody 9F against human aldolase C, characterized its isoform specificity and tested its performance. First, we investigated the specificity of 9F for aldolase C. Then, using bioinformatic tools coupled to molecular cloning and chemical synthesis approaches, we produced truncated human aldolase C fragments, and assessed 9F binding to these fragments by western blot and ELISA assays. This strategy revealed that residues 85–102 harbor the epitope-containing region recognized by 9F. The efficiency of 9F was demonstrated also for immunoprecipitation assays. Finally, surface plasmon resonance revealed that the protein has a high affinity toward the epitope-containing peptide. Taken together, our findings show that epitope recognition is sequence-driven and is independent of the three-dimensional structure. In conclusion, given its specific molecular interaction, 9F is a novel and powerful tool to investigate aldolase C’s functions in the brain.     

Hydroxyamino compounds produced by the oxidation of diamines with diamine oxidase in the presence of alcohol dehydrogenase.

When the diamines putrescine, cadaverine, cystamine and lanthionamine are oxidized by purified pig kidney diamine oxidase in the presence of NADH and either liver or yeast crystalline alcohol dehydrogenase, NADH is oxidized. Chromatographic evidence obtained in the case of putrescine and cystamine indicates the production of the respective hydroxy-amino compound. In the case of cystamine, the product of the reaction is mercapto-ethanol-cysteamine mixed disulfide which may represent a biological source for the production of mercaptoethanol used for other reactions.     

Gas chromatography/mass spectrometry of catechol estrogens.

Catecholestrogens (CCEs), namely 2- or 4-hydroxyestradiol and hydroxyestrone, are highly polar, reactive, and extremely labile estrogen metabolites in many experimental conditions. For these reasons, indirect assay methods mainly have been used. Some experimental evidence suggests that CCEs are synthesized and biologically active mostly in target cells. At this level, unfortunately, the indirect assays cannot be used. We present a method of gas chromatographic/mass spectral (GC/MS) analysis for the identification of individual CCEs; the major fragmentation ions of authentic estrogen standards as trimethylsilylether derivatives, and the MS patterns of the major CCEs, namely, 2-hydroxyestradiol and hydroxyestrone, are included. Few examples of CCEs detected in human breast cancer tissues and in breast cyst fluids are reported. Sample extracts were submitted to reversed-phase, high-performance liquid chromatography (RP-HPLC) and were quantified by "on line" electrochemical (EC) detection; thereafter, either crude extracts or single eluted peaks were submitted to GC/MS, by which detection limits of less than 5 pmol were attained. As expected, the molecular ion was the most relevant molecule in all but one case. On the contrary, the other relative intensities of major fragmentation ions M -15, M -30, M -90, and M -15 + (-90) were unevenly distributed, although represented in the majority of cases. In all cases, the GC/MS of peak fractions, purified by RP-HPLC and UV detection, confirmed the results of liquid chromatographic analysis combined with EC detection. In contrast, GC/MS of crude extracts was not equally satisfactory. Comparison of a liquid chromatography system with EC detection and the GC/MS approach revealed some inconsistency in quantitation of individual CCEs.(ABSTRACT TRUNCATED AT 250 WORDS)     

The development of an EMG-assisted model to assess spine loading during whole-body free-dynamic lifting

Low back disorders (LBDs) are the most common and costly occupationally-related compensable conditions facing employers today. Over the years several biomechanical assessment models have been developed that intended to assess the load profile imposed upon the spine during lifting and, thus, intended to facilitate the control of LBD risk in the workplace. Many of these biomechanical models have evolved based upon assumptions about how the trunk musculature respond to loads imposed upon the body during lifting. However, few of these models have been able to accurately predict the co-contraction of the trunk musculature which has been shown to have a major influence on the development of spinal loads. Thus, our understanding of how the spine is loaded under realistic dynamic lifting conditions has been deficient. A biologically-assisted or EMG-assisted model has been developed in our laboratory over the past 15 years which endeavours to overcome these traditional problems. The model has been assessed in the sagittal, coronal, and torsional planes of the body. The model development and performance will be reviewed as well as the benefits for controlling occupationally-related LBDs.