Hydroxyapatite affinity chromatography for the highly selective enrichment of mono‐ and multi‐phosphorylated peptides in phosphoproteome analysis

The most challenging analytical task facing phosphoproteome determination requires the isolation of phosphorylated peptides from the myriad of unphosphorylated species. In the past, several strategies for phosphopeptide isolation have been proposed in combination with subsequent mass spectrometric investigations. Among these techniques, immobilized metal affinity chromatography and titanium dioxide have been recognized as the most effective. Here, we present an alternative method for the enrichment of phosphopeptides based on hydroxyapatite (HAP) chromatography. By taking advantage of the strong interaction of HAP with phosphate and calcium ions, we developed an efficient method for the selective separation and fractionation of phosphorylated peptides. The effectiveness and efficiency of recovery for this procedure was assayed using tryptic digests of standard phosphorylated protein mixtures. Based on the higher affinity of multi‐phosphorylated peptides for HAP surfaces, the introduction of a phosphate buffer gradient for stepwise peptide elution resulted in the separation of mono‐, di‐, tri‐, and multi‐phosphorylated peptides. Thus, we demonstrated that this technique is highly selective and independent of the degree of peptide phosphorylation.     

A proteomic approach to the bilirubin‐induced toxicity in neuronal cells reveals a protective function of DJ‐1 protein

Unconjugated bilirubin (UCB) is a powerful antioxidant and a modulator of cell growth through the interaction with several signal transduction pathways. Although newborns develop a physiological jaundice, in case of severe hyperbilirubinemia UCB may become neurotoxic causing severe long‐term neuronal damages, also known as bilirubin encephalopathy. To investigate the mechanisms of UCB‐induced neuronal toxicity, we used the human neuroblastoma cell line SH‐SY5Y as an in vitro model system. We verified that UCB caused cell death, in part due to oxidative stress, which leads to DNA damage and cell growth reduction. The mechanisms of cytotoxicity and cell adaptation to UCB were studied through a proteomic approach that identified differentially expressed proteins involved in cell proliferation, intracellular trafficking, protein degradation and oxidative stress response. In particular, the results indicated that cells exposed to UCB undertake an adaptive response that involves DJ‐1, a multifunctional neuroprotective protein, crucial for cellular oxidative stress homeostasis. This study sheds light on the mechanisms of bilirubin‐induced neurotoxicity and might help to design a strategy to prevent or ameliorate the neuronal damages leading to bilirubin encephalopathy.     

Hypoxia and mitochondrial oxidative metabolism

It is now clear that mitochondrial defects are associated with a large variety of clinical phenotypes. This is the result of the mitochondria's central role in energy production, reactive oxygen species homeostasis, and cell death. These processes are interdependent and may occur under various stressing conditions, among which low oxygen levels (hypoxia) are certainly prominent. Cells exposed to hypoxia respond acutely with endogenous metabolites and proteins promptly regulating metabolic pathways, but if low oxygen levels are prolonged, cells activate adapting mechanisms, the master switch being the hypoxia-inducible factor 1 (HIF-1). Activation of this factor is strictly bound to the mitochondrial function, which in turn is related with the oxygen level. Therefore in hypoxia, mitochondria act as [O₂] sensors, convey signals to HIF-1directly or indirectly, and contribute to the cell redox potential, ion homeostasis, and energy production. Although over the last two decades cellular responses to low oxygen tension have been studied extensively, mechanisms underlying these functions are still indefinite. Here we review current knowledge of the mitochondrial role in hypoxia, focusing mainly on their role in cellular energy and reactive oxygen species homeostasis in relation with HIF-1 stabilization. In addition, we address the involvement of HIF-1 and the inhibitor protein of F₁F₀ ATPase in the hypoxia-induced mitochondrial autophagy.     

Mitochondrial respiratory chain super-complex I–III in physiology and pathology

Recent investigations by native gel electrophoresis showed the existence of supramolecular associations of the respiratory complexes, confirmed by electron microscopy analysis and single particle image processing. Flux control analysis demonstrated that Complex I and Complex III in mammalian mitochondria kinetically behave as a single unit with control coefficients approaching unity for each component, suggesting the existence of substrate channeling within the super-complex. The formation of this supramolecular unit largely depends on the lipid content and composition of the inner mitochondrial membrane. The function of the super-complexes appears not to be restricted to kinetic advantages in electron transfer: we discuss evidence on their role in the stability and assembly of the individual complexes, particularly Complex I, and in preventing excess oxygen radical formation. There is increasing evidence that disruption of the super-complex organization leads to functional derangements responsible for pathological changes, as we have found in K-ras-transformed fibroblasts.     

Proteomics and transcriptomics investigation on longissimus muscles in Large White and Casertana pig breeds

Consumer complaints against the blandness of modern lean meat and the frequent reference to the more strongly flavored meat that was available years ago have prompted reconsideration of high fat-depositing typical pig breeds. Casertana and Large White pig breeds are characterized by a different tendency toward fat accumulation as they exhibit opposite genetic and physiological traits with respect to the energy metabolism. These physiological differences were investigated in longissimus lumborum muscles through proteomics (2-DE, MS/MS) and microarray approaches. Data were analyzed for pathway and network analyses, as well as GO term enrichment of biological functions. As a result, Casertana showed a greater amount of proteins involved in glycolitic metabolism and mainly rely on fast-mobilizable energy sources. Large White overexpressed cell cycle and skeletal muscle growth related genes. Metabolic behavior and other implications are discussed.     

Cloning, functional identification and structural modelling of Vitis vinifera S-adenosylmethionine decarboxylase

In this paper we report the cloning and full sequencing of S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) cDNA from Vitis vinifera L. (VV) leaves, an enzyme belonging to the polyamine biosynthetic pathway, which appears to play an important role in the regulation of plant growth and development. The presence of two overlapping ORFs (tiny ORF and small ORF) upstream of the main ORF is reported in the Vitis cDNA. When the Vitis SAMDC cDNA was expressed in yeast without the two upstream ORFs, the resulting activity was about 50 times higher than the activity obtained with the full cDNA. These results demonstrated the strong regulatory activity of the tiny and small ORFs. RT-PCR expression analysis showed evidence of a similar mRNA level in all the tissues tested, with the exception of the petioles. The VV SAMDC was also modelled using its homologues from Solanum tuberosum and Homo sapiens as template. The present work confirmed, for the first time in a woody plant of worldwide economic interest such as grapevine, the presence of a regulatory mechanism of SAMDC, enzyme that has a well-established importance in the modulation of plant growth and development.     

Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1

Crisponi syndrome is a severe autosomal recessive condition that is phenotypically characterized by abnormal, paroxysmal muscular contractions resembling neonatal tetanus, large face, broad nose, anteverted nares, camptodactyly, hyperthermia, and sudden death in most cases. We performed homozygosity mapping in five Sardinian and three Turkish families with Crisponi syndrome, using high-density single-nucleotide polymorphism arrays, and identified a critical region on chromosome 19p12-13.1. The most prominent candidate gene was CRLF1, recently found to be involved in the pathogenesis of cold-induced sweating syndrome type 1 (CISS1). CISS1 belongs to a group of conditions with overlapping phenotypes, also including cold-induced sweating syndrome type 2 and Stuve-Wiedemann syndrome. All these syndromes are caused by mutations of genes of the ciliary neurotrophic factor (CNTF)-receptor pathway. Here, we describe the identification of four different CRLF1 mutations in eight different Crisponi-affected families, including a missense mutation, a single-nucleotide insertion, and a nonsense and an insertion/deletion (indel) mutation, all segregating with the disease trait in the families. Comparison of the mutation spectra of Crisponi syndrome and CISS1 suggests that neither the type nor the location of the CRLF1 mutations points to a phenotype/genotype correlation that would account for the most severe phenotype in Crisponi syndrome. Other, still-unknown molecular factors may be responsible for the variable phenotypic expression of the CRLF1 mutations. We suggest that the syndromes can comprise a family of "CNTF-receptor-related disorders," of which Crisponi syndrome would be the newest member and allelic to CISS1.     

Electromagnetic fields produced by GSM cellular phones and heart rate variability

In this study, 26 healthy young volunteers were submitted to 900 MHz (2 W) GSM cellular phone exposure and to sham exposure in separate sessions. The study was designed to assess cardiac regulatory mechanism in different autonomic nervous system (ANS) states during exposure to low-intensity EMF. Rest-to-stand protocol was applied to evaluate ANS in quiet condition (rest, vagal prevalence) and after a sympathetic activation (stand). The procedure is conducted twice in a double-blind design: once with a genuine EMF exposure and once with a sham exposure (at least 24 h apart). During each session three-leads electrocardiograms were recorded and RR series extracted off-line. Time domain and frequency domain HRV parameters were calculated in every phase of the protocol and during different exposures. The analysis of the data show there was no statistically significant effect due to EMF exposure both on main (i.e., RR mean) and most of the other HRV parameters. A weak interaction between some HRV parameters (i.e., SDNN, TINN, and triangular index in time domain and LF power in frequency domain analysis) and RF exposure was observed and this effect seems to be gathered around the sympathetic response to stand.     

Programmed cell death and stem cell differentiation are responsible for midgut replacement in <Emphasis Type="Italic">Heliothis virescens</Emphasis> during prepupal instar

We have analyzed midgut development during the fifth larval instar in the tobacco budworm Heliothis virescens. In prepupae, the midgut formed during larval instars undergoes a complete renewal process. This drastic remodeling of the alimentary canal involves the destruction of the old cells by programmed cell-death mechanisms (autophagy and apoptosis). Massive proliferation and differentiation of regenerative stem cells take place at the end of the fifth instar and give rise to a new fully functioning epithelium that is capable of digesting and absorbing nutrients and that is maintained throughout the subsequent pupal stage. Midgut replacement in H. virescens is achieved by a balance between this active proliferation process and cell-death mechanisms and is different from similar processes characterized in other insects. This work was supported by FAR 2006 (University of Insubria) to G.T., by a MIUR-FIRB-COFIN grant (no. RBNE01YXA8/2004077251), and by the Centro Grandi Attrezzature (University of Insubria).