Reaction of bovine-liver copper-zinc superoxide dismutase with hydrogen peroxide. Evidence for reaction with H2O2 and HO2- leading to loss of copper

The reaction of hydrogen peroxide with the copper-zinc bovine-liver superoxide dismutase at low molar ratios (0.2-20.0) of H2O2/active site between pH 7.3-10.0 leads to the loss of native enzyme as a distinct form monitored by electrophoresis. The pH dependence of the loss of native enzyme between 7.3 and 9.0 indicates the involvement of a conjugate base on the enzyme of pKa of 8.7 +/- 0.1. The rate of loss of the native enzyme is first order with respect to the concentration of both enzyme and hydrogen peroxide between pH 7.3 and 9.0 with no evidence for binding of peroxide. A second-order rate constant of 3.0 +/- 1.0 M-1 s-1 is obtained from these data. At pH 10.0 the reaction is first order with respect to enzyme concentration but saturable in H2O2. All data are consistent with the interpretation that H2O2 reacts with the enzyme at the lower pH where the reaction is dependent upon the conjugate base of a functional group on the enzyme. At the higher pH, the data are consistent with the reaction of HO2- and H2O2 with the dismutase. The dissociation constant for HO2- calculated from the kinetic data at pH 10.0 is between 25-50 microM and the rate constant for the breakdown of the HO2- dismutase complex is 1.10 + 0.05 x 10(-2) s-1. The change in the electrophoretic pattern at all pH values is accompanied by the loss of the ability of the enzyme to bind copper. Weakly bound or free copper can be detected using bathocuproine disulfonate. Furthermore copper-defficient forms of the enzyme can be detected by staining gels of the peroxide-treated dismutase with diethyldithiocarbamate.     

Spontaneous cytotoxicity and tumor necrosis factor production by peripheral blood monocytes from AIDS patients

Peripheral blood monocytes (PBM) from AIDS patients have exhibited defects in some but not all of the immune functions yet tested. This study has examined the capacity of AIDS PBM to lyse tumor target cells as well as their ability to secrete TNF. Untreated PBM from AIDS patients were significantly cytotoxic to U937 target cells and responded to IFN-gamma pretreatment with augmented cytotoxicity. Both the spontaneous and IFN-gamma-stimulated cytotoxic activity was significantly (p less than 0.01) higher than that observed with normal PBM. The cytotoxic activity depended on the E:T ratio used and was higher in AIDS PBM at all ratios tested (10:1 to 40:1). Because TNF has been implicated in macrophage cell-mediated cytotoxicity, we examined whether the elevated cytotoxic activity of AIDS PBM was associated with an increase in TNF production. Supernatants from PBM cultured overnight with or without IFN-gamma were tested in a bioassay measuring cytotoxicity against U937 target cells as well as in an RIA specific for TNF. Supernatants derived from either unstimulated or IFN-gamma-treated AIDS PBM exhibited significantly higher levels of cytotoxicity than supernatants from normal macrophages. Both normal and AIDS PBM produced higher levels of cytotoxic factors in response to IFN-gamma. As determined by the RIA, AIDS PBM spontaneously released high levels of TNF whereas little TNF was produced by normal PBM. Treatment with IFN-gamma augmented the level of TNF production in both AIDS and normal PBM. These results demonstrate that PBM from AIDS patients have undergone in vivo activation as manifested by both cytotoxicity against tumor target cells and production of TNF. Target cell lysis by both AIDS PBM and their supernatants was inhibited by monoclonal anti-rTNF, suggesting that the increase in PBM cell-mediated cytotoxicity was caused by an increase in TNF production. The significance of these findings in the pathogenesis of the disease is discussed.     

Isoelectric focusing--polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities.

Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities.     

Cell Budding from Normal Appearing Epithelia: A Predictor of Colorectal Cancer Metastasis?

Background: Colorectal carcinogenesis is believed to be a multi-stage process that originates with a localized adenoma, which linearly progresses to an intra-mucosal carcinoma, to an invasive lesion, and finally to metastatic cancer. This progression model is supported by tissue culture and animal model studies, but it is difficult to reconcile with several well-established observations, principally among these are that up to 25% of early stage (Stage I/II), node-negative colorectal cancer (CRC) develop distant metastasis, and that circulating CRC cells are undetectable in peripheral blood samples of up to 50% of patients with confirmed metastasis, but more than 30% of patients with no detectable metastasis exhibit such cells. The mechanism responsible for this diverse behavior is unknown, and there are no effective means to identify patients with pending, or who are at high risk for, developing metastatic CRC.Novel findings: Our previous studies of human breast and prostate cancer have shown that cancer invasion arises from the convergence of a tissue injury, the innate immune response to that injury, and the presence of tumor stem cells within tumor capsules at the site of the injury. Focal degeneration of a capsule due to age or disease attracts lymphocyte infiltration that degrades the degenerating capsules resulting in the formation of a focal disruption in the capsule, which selectively favors proliferating or “budding” of the underlying tumor stem cells. Our recent studies suggest that lymphocyte infiltration also triggers metastasis by disrupting the intercellular junctions and surface adhesion molecules within the proliferating cell buds causing their dissociation. Then, lymphocytes and tumor cells are conjoined through membrane fusion to form tumor-lymphocyte chimeras (TLCs) that allows the tumor stem cell to avail itself of the lymphocyte''s natural ability to migrate and breach cell barriers in order to intravasate and to travel to distant organs. Our most recent studies of human CRC have detected nearly identical focal capsule disruptions, lymphocyte infiltration, budding cells, and the formation of TLCs. Our studies have further shown that age- and type-matched node-positive and -negative CRC have a significantly different morphological and immunohistochemical profile and that the majority of lymphatic ducts with disseminated cells are located within the mucosa adjacent to morphologically normal appearing epithelial structures that express a stem cell-related marker.New hypothesis: Based on these findings and the growth patterns of budding cells revealed by double immunohistochemistry, we further hypothesize that metastatic spread is an early event of carcinogenesis and that budding cells overlying focal capsule disruptions represent invasion- and metastasis-initiating cells that follow one of four pathways to progress: (1) to undergo extensive in situ proliferation leading to the formation of tumor nests that subsequently invade the submucosa, (2) to migrate with associated lymphocytes functioning as “seeds” to grow in new sites, (3) to migrate and intravasate into pre-existing vascular structures by forming TLCs, or (4) to intravasate into vascular structures that are generated by the budding cells themselves. We also propose that only node-positive cases harbor stem cells with the potential for multi-lineage differentiation and unique surface markers that permit intravasation.     

Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins

In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over polymerase chain reaction-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a double stranded DNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based protection of LET) method effectively increases superfolder green fluorescent protein (sfGFP) expression levels from LETs in Escherichia coli CFPS reactions by sixfold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from nonmodel organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.     

Exploring Sources of Emotional Distress among People Living with Scleroderma: A Focus Group Study

Background

Systemic sclerosis, or scleroderma, is a chronic and rare connective tissue disease with negative physical and psychological implications. Sources of emotional distress and the impact they have on the lives of people with scleroderma are not well understood.

Objectives

To gain an in-depth understanding of the emotional experiences and sources of emotional distress for women and men living with scleroderma through focus group discussions.

Methods

Three semi-structured focus group discussions were conducted (two in English, one in French) with a total of 22 people with scleroderma recruited through the Scleroderma Society of Ontario in Hamilton, Ontario and a scleroderma clinic in Montreal, Canada. Interviews were recorded, transcribed, and then coded for emerging themes using thematic inductive analysis.

Results

Core themes representing sources of emotional distress were identified, including: (a) facing a new reality; (b) the daily struggle of living with scleroderma; (c) handling work, employment and general financial burden; (d) changing family roles; (e) social interactions; and (f) navigating the health care system. Collectively, these themes refer to the stressful journey of living with scleroderma including the obstacles faced and the emotional experiences beginning prior to receiving a diagnosis and continuing throughout the participants’ lives.

Conclusion

Scleroderma was portrayed as being an unpredictable and overwhelming disease, resulting in many individuals experiencing multiple sources of emotional distress. Interventions and supportive resources need to be developed to help individuals with scleroderma and people close to them manage and cope with the emotional aspects of the disease.     

A simpler model of the human circadian pacemaker

Numerous studies have used the classic van der Pol oscillator, which contains a cubic nonlinearity, to model the effect of light on the human circadian pacemaker. Jewett and Kronauer demonstrated that Aschoff's rule could be incorporated into van der Pol type models and used a van der Pol type oscillator with higher order nonlinearities. Kronauer, Forger, and Jewett have proposed a model for light preprocessing, Process L, representing a biochemical process that converts a light signal into an effective drive on the circadian pacemaker. In the paper presented here, the authors use the classic van der Pol oscillator with Process L and Jewett and Kronauer's model of Aschoff's rule to model the human circadian pacemaker. This simpler cubic model predicts the results of a three-pulse human phase response curve experiment and a two-pulse amplitude reduction study with as much, or more, accuracy as the models of Jewett and Kronauer and Kronauer, Forger, and Jewett, which both employ a nonlinearity of degree 7. This suggests that this simpler cubic model should be considered as a potential alternative to other models of the human circadian system currently available.     

Cooperativity,smooth energy landscapes and the origins of topology-dependent protein folding rates

The relative folding rates of simple, single-domain proteins, proteins whose folding energy landscapes are smooth, are highly dispersed and strongly correlated with native-state topology. In contrast, the relative folding rates of small, Gō-potential lattice polymers, which also exhibit smooth energy landscapes, are poorly dispersed and insignificantly correlated with native-state topology. Here, we investigate this discrepancy in light of a recent, quantitative theory of two-state folding kinetics, the topomer search model. This model stipulates that the topology-dependence of two-state folding rates is a direct consequence of the extraordinarily cooperative equilibrium folding of simple proteins. We demonstrate that traditional Gō polymers lack the extreme cooperativity that characterizes the folding of naturally occurring, two-state proteins and confirm that the folding rates of a diverse set of Gō 27-mers are poorly dispersed and effectively uncorrelated with native state topology. Upon modestly increasing the cooperativity of the Gō-potential, however, significantly increased dispersion and strongly topology-dependent kinetics are observed. These results support previous arguments that the cooperative folding of simple, single-domain proteins gives rise to their topology-dependent folding rates. We speculate that this cooperativity, and thus, indirectly, the topology-rate relationship, may have arisen in order to generate the smooth energetic landscapes upon which rapid folding can occur.