Early cytokine responses after percutaneous magnetic resonance imaging guided laser thermoablation of malignant liver tumors

An early systemic response induced by magnetic resonance imaging (MRI)-guided interstitial percutaneous laser thermoablation was analyzed in 13 consecutive patients with malignant liver tumors by serum interleukin (IL)-1beta, IL-6, IL-10, tumor necrosis factor (TNF)-alpha, its receptor TNFRI, and C-reactive protein (CRP) levels up to 72h after the procedures. Only IL-6 (p=0.033) and TNFRI (p<0.001) increased statistically significantly after ablation, while the TNF-alpha, IL-1 beta, and IL-10 levels remained unchanged. The peak median CRP response was 92mg/l. There was a dose-dependent correlation between the energy used and the maximum CRP values (tau=0.68, p=0.013). MRI-guided laser thermoablation induced an early systemic inflammatory reaction with statistically significantly elevated IL-6, TNFRI, and CRP levels but not TNF-alpha or IL-10 levels and without procedure-related complications, favoring this procedure as a safe therapeutic alternative for well-selected patients with liver tumors.     

An ectophosphatase activity in Cryptococcus neoformans

There is increasing evidence in the literature showing that fungal pathogens express biologically active ectoenzymes. The expression of surface phosphatases at the cell surface of Cryptococcus neoformans, the etiologic agent of cryptococcosis, was evaluated in the present study. Different isolates of C. neoformans express ectophosphatase activity, which is not influenced by capsule size or serotype. The cryptococcal enzyme is an acid phosphatase, inhibited by classic inhibitors of ectophosphatases, including ammonium molybdate and sodium salts of fluoride and orthovanadate. Only the inhibition of enzyme activity caused by sodium orthovanadate has been shown to be irreversible. The cryptococcal ectoenzyme is also inhibited by Zn2+ and inorganic phosphate, the final product of reactions catalyzed by phosphatases. The ectophosphatase from C. neoformans efficiently releases phosphate groups from different phosphorylated amino acids, giving a higher rate of phosphate removal when phosphothreonine is used as a substrate. Yeast cells with irreversibly inhibited ectophosphatases are less capable of adhering to animal epithelial cells than fungi fully expressing enzyme activity, suggesting that ectoenzyme expression can contribute to the pathogenesis of C. neoformans.     

Protein carbonylation, cellular dysfunction, and disease progression

Carbonylation of proteins is an irreversible oxidative damage, often leading to a loss of protein function, which is considered a widespread indicator of severe oxidative damage and disease-derived protein dysfunction. Whereas moderately carbonylated proteins are degraded by the proteasomal system, heavily carbonylated proteins tend to form high-molecular-weight aggregates that are resistant to degradation and accumulate as damaged or unfolded proteins. Such aggregates of carbonylated proteins can inhibit proteasome activity. Alarge number of neurodegenerative diseases are directly associated with the accumulation of proteolysis-resistant aggregates of carbonylated proteins in tissues. Identification of specific carbonylated protein(s) functionally impaired and development of selective carbonyl blockers should lead to the definitive assessment of the causative, correlative or consequential role of protein carbonylation in disease onset and/or progression, possibly providing new therapeutic approaches.     

Analysis of the cerebellar proteome in a transgenic mouse model of inherited prion disease reveals preclinical alteration of calcineurin activity

Inherited prion diseases are linked to insertional and point mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. Transgenic (Tg) (PG14) mice express a mouse PrP homolog of a nine-octapeptide insertion associated with an inherited prion disorder. These mice develop a progressive neurological syndrome characterized by ataxia and cerebellar atrophy due to synaptic degeneration in the molecular layer and massive apoptosis of granule neurons. To investigate the molecular events that may contribute to neurological dysfunction, we carried out a differential proteomic analysis of cerebella from Tg(PG14) mice at the preclinical, onset, and symptomatic phases of their neurological illness. 2-D maps of cerebellar proteins from Tg(PG14) mice were compared to those obtained from age-matched Tg(WT) mice that express wild-type PrP and remain healthy. Proteins whose levels were significantly modified in at least one stage of the Tg(PG14) disease were identified by PMF. Analysis detected a preclinical decrease of the calcium/calmodulin-dependent phosphatase calcineurin (CaN) in granule neurons, suggesting that dysregulation of CaN activity induced by mutant PrP may be responsible for the cerebellar dysfunction in Tg(PG14) mice.     

Myosin binding protein C is differentially phosphorylated upon myocardial stunning in canine and rat hearts-- evidence for novel phosphorylation sites

Myocardial stunning is the transient cardiac dysfunction that follows brief episodes of ischemia and reperfusion without associated myocardial necrosis. Currently, there is limited knowledge about its cellular and biochemical mechanisms. In order to better understand the underlying mechanisms of contractile dysfunction associated with the stunning, comprehensive proteomic studies using 2-D DIGE were performed using a regional stunning model in canine heart. Cardiac myosin binding protein C (cMyBP-C), a regulatory myofilament protein associated with the thick filament, and nebulette, a thin filament associated protein, were differentially expressed. Phosphoprotein specific staining indicated both protein changes were due to phosphorylation. Subsequent phosphorylation mapping of canine cMyBP-C using IMAC and MS/MS identified five phosphorylation sites, including three novel sites. In order to further evaluate this finding in a different model, cMyBP-C phosphorylation was examined in a rat model of global stunning. In the rat model, stunning was associated with increased phosphorylation of cMyBP-C at a critical calcium/calmodulin-dependent kinase II site, and the increased phosphorylation was largely inhibited when stunning was prevented by either ischemic preconditioning or reperfusion in the presence of low-calcium buffer. These data indicate cMyBP-C phosphorylation plays an important role in myocardial stunning.     

Simple purification of immunoglobulins from whey proteins concentrate

We have developed a new protocol with only two steps for purification of immunoglobulins (Ig) from a protein concentrate of whey. Following this protocol, we have an 80% recovery of immunoglobulins, fairly pure. The purification was achieved by eliminating the BSA, via a strong adsorption on DEAE-agarose. Full desoprtion of the other serum proteins could be achieved without contamination with BSA. Thus, a protein solution containing only Ig and very small proteins (e.g., beta-lactoglobulins and alpha-lactalbumin) was obtained. Offering this protein mixture to a lowly activated aminated support, only Ig adsorbed on the support. It has been shown that BSA is able to interact with other proteins (including Ig and lactalbumins). This ability to form complexes with other proteins prevented the success of the direct adsorption of Ig on this mildly activated support, even although Ig should be the largest protein presented in dairy whey.     

Salt-induced formation of the A-state of ferricytochrome c--effect of the anion charge on protein structure

Structural information on partially folded forms is important for a deeper understanding of the folding mechanism(s) and the factors affecting protein stabilization. The non-native compact state of equine cytochrome c stabilized by salts in an acidic environment (pH 2.0-2.2), called the A-state, is considered a suitable model for the molten globule of cytochrome c, as it possesses a native-like alpha-helix conformation but a fluctuating tertiary structure. In this article, we extend our knowledge on anion-induced protein stabilization by determining the effect of anions carrying a double negative charge; unlike monovalent anions (which are thought to exert an 'ionic atmosphere' effect on the macromolecule), divalent anions are thought to bind to the protein at specific surface sites. Our data indicate that divalent anions, in comparison to monovalent ions, have a greater tendency to stabilize the native-like M-Fe(III)-H coordinated state of the protein. The possibility that divalent anions may bind to the protein at the same sites previously identified for polyvalent anions was evaluated. To investigate this issue, the behavior of the K88E, K88E/T89K and K13N mutants was investigated. The data obtained indicate that the mutated residues, which contribute to form the binding sites of polyanions, are important for stabilization of the native conformation; the mutants investigated, in fact, all show an increased amount of the misligated H-Fe(III)-H state and, with respect to wild-type cytochrome c, appear to be less sensitive to the presence of the anion. These residues also modulate the conformation of unfolded cytochrome c, influencing its spin state and the coordination to the prosthetic group.