EPR properties of mixed-valent μ-oxo and μ-hydroxo dinuclear iron complexes produced by radiolytic reduction at 77 K

 Radiolytic reduction at 77 K of oxo-/hydroxo-bridged dinuclear iron(III) complexes in frozen solutions forms kinetically stabilized, mixed-valent species in high yields that model the mixed-valent sites of non-heme, diiron proteins. The mixed-valent species trapped at 77 K retain ligation geometry similar to the initial diferric clusters. The shapes of the mixed-valent EPR signals depend strongly on the bridging ligands. Spectra of the Fe(II)OFe(III) species reveal an S=1/2 ground state with small g-anisotropy as characterized by the uniaxial component (g _z –g _av /2<0.03) observable at temperatures as high as ∼100 K. In contrast, hydroxo-bridged mixed-valent species are characterized by large g-anisotropy (g _z –g _av /2>0.03) and are observable only below 30 K. Annealing at higher temperatures causes structural relaxation and changes in the EPR characteristics. EPR spectral properties allow the oxo- and hydroxo-bridged, mixed-valent diiron centers to be distinguished from each other and can help characterize the structure of mixed-valent centers in proteins. Received: 27 June 1998 / Accepted: 25 February 1999     

Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13

Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594–1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)_n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.     

Mesothelioma Tumor Cells Modulate Dendritic Cell Lipid Content,Phenotype and Function

Dendritic cells (DCs) play an important role in the generation of anti-cancer immune responses, however there is evidence that DCs in cancer patients are dysfunctional. Lipid accumulation driven by tumor-derived factors has recently been shown to contribute to DC dysfunction in several human cancers, but has not yet been examined in mesothelioma. This study investigated if mesothelioma tumor cells and/or their secreted factors promote increases in DC lipid content and modulate DC function. Human monocyte-derived DCs (MoDCs) were exposed to human mesothelioma tumor cells and tumor-derived factors in the presence or absence of lipoproteins. The data showed that immature MoDCs exposed to mesothelioma cells or factors contained increased lipid levels relative to control DCs. Lipid accumulation was associated with reduced antigen processing ability (measured using a DQ OVA assay), upregulation of the co-stimulatory molecule, CD86, and production of the tolerogenic cytokine, IL-10. Increases in DC lipid content were further enhanced by co-exposure to mesothelioma-derived factors and triglyceride-rich lipoproteins, but not low-density lipoproteins. In vivo studies using a murine mesothelioma model showed that the lipid content of tumor-infiltrating CD4⁺CD8α^- DCs, CD4^-CD8α^- DCs DCs and plasmacytoid DCs increased with tumor progression. Moreover, increasing tumor burden was associated with reduced proliferation of tumor-antigen-specific CD8⁺ T cells in tumor-draining lymph nodes. This study shows that mesothelioma promotes DC lipid acquisition, which is associated with altered activation status and reduced capacity to process and present antigens, which may impair the ability of DCs to generate effective anti mesothelioma T cell responses.     

Liver Accumulation of Plasmodium chabaudi-Infected Red Blood Cells and Modulation of Regulatory T Cell and Dendritic Cell Responses

It is postulated that accumulation of malaria-infected Red Blood Cells (iRBCs) in the liver could be a parasitic escape mechanism against full destruction by the host immune system. Therefore, we evaluated the in vivo mechanism of this accumulation and its potential immunological consequences. A massive liver accumulation of P. c. chabaudi AS-iRBCs (Pc-iRBCs) was observed by intravital microscopy along with an over expression of ICAM-1 on day 7 of the infection, as measured by qRT-PCR. Phenotypic changes were also observed in regulatory T cells (Tregs) and dendritic cells (DCs) that were isolated from infected livers, which indicate a functional role for Tregs in the regulation of the liver inflammatory immune response. In fact, the suppressive function of liver-Tregs was in vitro tested, which demonstrated the capacity of these cells to suppress naive T cell activation to the same extent as that observed for spleen-Tregs. On the other hand, it is already known that CD4+ T cells isolated from spleens of protozoan parasite-infected mice are refractory to proliferate in vivo. In our experiments, we observed a similar lack of in vitro proliferative capacity in liver CD4+ T cells that were isolated on day 7 of infection. It is also known that nitric oxide and IL-10 are partially involved in acute phase immunosuppression; we found high expression levels of IL-10 and iNOS mRNA in day 7-infected livers, which indicates a possible role for these molecules in the observed immune suppression. Taken together, these results indicate that malaria parasite accumulation within the liver could be an escape mechanism to avoid sterile immunity sponsored by a tolerogenic environment.     

Estimates of the Prevalence of Obesity in Canadian Children

Childhood obesity is becoming a topic of great concern due to the rising prevalence of this condition in North America. Studies conducted in the United States have indicated that the prevalence of obesity has increased dramatically over the past few decades. The purpose of this study was to estimate the prevalence of obesity in Canadian children between the ages of 5 and 12 years by examining data from two national and two regional surveys. The 85th percentiles of each of four anthropometric indices derived from large normative populations were used as diagnostic criteria for obesity. As expected, the resulting prevalences varied according to the criteria used. A significant increase in childhood obesity between the 1981 to 1988 national surveys was observed when the three indices which used skinfolds were applied. Weight-for-height percentiles did not indicate an increase in obesity in these samples. Regional samples showed a less than expected prevalence of obesity among the middle-class children and a higher than expected rate among the inner city boys. It can be concluded that there is a need for a defined criteria for identifying obesity in children in order to avoid confusion resulting from the wide variation in estimates of prevalence resulting from different standards and measurements. Using adiposity-based criteria for obesity it was clearly evident that the prevalence of obesity has increased in Canadian children.     

The Redox Biochemistry of Protein Sulfenylation and Sulfinylation

Controlled generation of reactive oxygen species orchestrates numerous physiological signaling events (Finkel, T. (2011) Signal transduction by reactive oxygen species. J. Cell Biol. 194, 7–15). A major cellular target of reactive oxygen species is the thiol side chain (RSH) of Cys, which may assume a wide range of oxidation states (i.e. −2 to +4). Within this context, Cys sulfenic (Cys-SOH) and sulfinic (Cys-SO₂H) acids have emerged as important mechanisms for regulation of protein function. Although this area has been under investigation for over a decade, the scope and biological role of sulfenic/sulfinic acid modifications have been recently expanded with the introduction of new tools for monitoring cysteine oxidation in vitro and directly in cells. This minireview discusses selected recent examples of protein sulfenylation and sulfinylation from the literature, highlighting the role of these post-translational modifications in cell signaling.     

S-100 protein in human lung neuroendocrine neoplasms. Immunohistochemical study of 14 cases and review of the literature

A group of lung neuroendocrine (NE) neoplasms are investigated in view of the possible presence of S-100 protein immunoreactivity in their cells. The selected tumours were classified according to Gould et al. (1983a) and Mosca et al. (1985). They comprise 5 carcinoids, 3 neuroendocrine carcinomas of the well-differentiated type, or peripheral carcinoids, 5 neuroendocrine carcinomas of the intermediate cell type, or intermediate-cell, poorly differentiated carcinomas, 3 neuroendocrine carcinomas of the microcytoma type, or small cell carcinomas-SCC and a nodal metastasis of microcytoma. All but 2 tumours were immunoreactive for neuron specific enolase (NSE). Few S-100 immunoreactive cells were detected in 4 out of 5 carcinoids, in 1 out of 3 peripheral carcinoids, in 4 out of 5 poorly differentiated carcinomas and in the 3 microcytomas examined. No S-100 positive cells were found in the SCC's nodal metastasis. The S-100 immunolabelled cells can be interpreted as dendritic reticulum cells migrating through the tumours. However, in one case of typical carcinoid, abundant S-100 positive cells were detected: their stellate morphology and their intimate relation with neoplastic cells suggest that they are part of the neoplasia as a sort of satellite cell.     

Specific and nonspecific effects of nucleotides on hormone-induced phosphoinositide turnover in permeabilized human pituitary tumour cells (Flow 9000)

Previous studies have shown that agonist-induced inositol phosphate formation in the human embryonic pituitary cell line Flow 9000 is regulated by guanine nucleotides, and it is likely that a guanine nucleotide-binding protein is involved in coupling receptors to phosphoinositidase C (PIC) [(1986) Biochem.Soc.Trans. 14, 1135-1136]. We have now tested the specificity of various nucleotides in regulating PIC activity in the absence or presence of the hormone cholecystokinin (CCK-8) in saponin-permeabilized [3H]inositol-labelled Flow 9000 cells. We found that all nucleotides tested (i.e. CTP, UTP, ITP, TTP, GTP, GppNHp, GTP[S], ATP, AppNHp and ATP[S]) stimulated total [3H]inositol phosphate ([3H]IP) formation in a dose-dependent manner with similar potency and efficacy. However, only guanine nucleotides significantly enhanced CCK-8 stimulation of [3H]IP production. These results indicate a physiological role for guanine nucleotides in regulating hormone-induced phosphoinositide turnover. In addition, the effects of nucleotides on calcium-dependent PIC activity are discussed.     

The actions of Ca2+ ionophores on rat basophilic (2H3) cells are dependent on cellular ATP and hydrolysis of inositol phospholipids. A comparison with antigen stimulation

Calcium-specific ionophores are used widely to stimulate Ca2+-dependent secretion from cells on the assumption that permeabilization of the cell membranes to Ca2+ ions leads to a rise in concentration of cytosolic Ca2+ ([Ca2+]i), which in turn serves as a signal for secretion. In this way, events that precede mobilization of Ca2+ ions via receptor stimulation are bypassed. One such event is thought to be the rapid hydrolysis of membrane inositol phospholipids to form inositol phosphates and diacylglycerol. Accordingly, rat leukemic basophil (2H3) cells can be stimulated to secrete histamine either with the ionophores or by aggregation of receptors for IgE in the plasma membrane. We find, however, that ionophore A23187 stimulates secretion of histamine only at concentrations (200-1000 nM) that stimulate hydrolysis of membrane inositol phospholipids. The extent of hydrolysis of inositol phospholipids was dependent on the concentration of ionophore and the presence of external Ca2+ ions and correlated with the magnitude of the secretory response. A similar correlation between secretion and hydrolysis of inositol phospholipids was observed in response to the Ca2+-specific ionophore, ionomycin. Although this hydrolysis (possibly a consequence of elevated [Ca2+]i) was less extensive than that induced by aggregation of receptors, it may govern the secretory response to A23187. The studies revealed one paradox. The rise in [Ca2+]i depended on intracellular ATP levels, when either an ionophore or antigen was used as a stimulant irrespective of whether hydrolysis of inositol phospholipids was stimulated or not. The concept of how the ionophores act, therefore, requires critical reevaluation.