Domain swapping reveals complement control protein modules critical for imparting cofactor and decay-accelerating activities in vaccinia virus complement control protein

Vaccinia virus encodes a structural and functional homolog of human complement regulators named vaccinia virus complement control protein (VCP). This four-complement control protein domain containing secretory protein is known to inhibit complement activation by supporting the factor I-mediated inactivation of complement proteins, proteolytically cleaved form of C3 (C3b) and proteolytically cleaved form of C4 (C4b) (termed cofactor activity), and by accelerating the irreversible decay of the classical and to a limited extent of the alternative pathway C3 convertases (termed decay-accelerating activity [DAA]). In this study, we have mapped the VCP domains important for its cofactor activity and DAA by swapping its individual domains with those of human decay-accelerating factor (CD55) and membrane cofactor protein (MCP; CD46). Our data indicate the following: 1) swapping of VCP domain 2 or 3, but not 1, with homologous domains of decay-accelerating factor results in loss in its C3b and C4b cofactor activities; 2) swapping of VCP domain 1, but not 2, 3, or 4 with corresponding domains of MCP results in abrogation in its classical pathway DAA; and 3) swapping of VCP domain 1, 2, or 3, but not 4, with homologous MCP domains have marked effect on its alternative pathway DAA. These functional data together with binding studies with C3b and C4b suggest that in VCP, domains 2 and 3 provide binding surface for factor I interaction, whereas domain 1 mediates dissociation of C2a and Bb from the classical and alternative pathway C3 convertases, respectively.     

Comparative effect of Ocimum sanctum, Commiphora mukul, folic acid and ramipril on lipid peroxidation in experimentally-induced hyperlipidemia

Treatment with C. mukul and O. sanctum, showed a significant decrease in cholesterol and triglyceride levels respectively. O. sanctum also significantly increased serum HDL-cholesterol compared to control. Serum MDA levels were significantly reduced in all the treated groups compared to control suggesting that each of the drugs under study were effective in their free radical scavenging action. Erythrocyte SOD activity was increased in all the treatment groups with C. mukul showing the maximum effect followed by O. sanctum, folic acid and ramipril. The erythrocyte CAT activity was significantly increased in all the drug treated groups with maximum increase seen in O. sanctum and ramipril treated groups, whereas lesser effects were observed with C. mukul and folic acid groups. Thus, the indigenous drugs, C. mukul and O. sanctum had beneficial effect on hypercholesterolemic rabbit model, both in terms of lipid profile as well as antioxidant potential. Ocimum sanctum was found to be the most promising of all the drugs. Moreover, it could be hypothesized that these plant products along with folic acid and ramipril can be explored for synergistic effect for treatment for hypercholesterolemic conditions.     

Cutting edge: CD4 is the receptor for the tick saliva immunosuppressor, Salp15

Salp15 is an Ixodes scapularis salivary protein that inhibits CD4+ T cell activation through the repression of TCR ligation-triggered calcium fluxes and IL-2 production. We show in this study that Salp15 binds specifically to the CD4 coreceptor on mammalian host T cells. Salp15 specifically associates through its C-terminal residues with the outermost two extracellular domains of CD4. Upon binding to CD4, Salp15 inhibits the subsequent TCR ligation-induced T cell signaling at the earliest steps including tyrosine phosphorylation of the Src kinase Lck, downstream effector proteins, and lipid raft reorganization. These results provide a molecular basis to understanding the immunosuppressive activity of Salp15 and its specificity for CD4+ T cells.     

Intervention in a family of Boolean networks

MOTIVATION: Intervention in a gene regulatory network is used to avoid undesirable states, such as those associated with a disease. Several types of intervention have been studied in the framework of a probabilistic Boolean network (PBN), which is a collection of Boolean networks in which the gene state vector transitions according to the rules of one of the constituent networks and where network choice is governed by a selection distribution. The theory of automatic control has been applied to find optimal strategies for manipulating external control variables that affect the transition probabilities to desirably affect dynamic evolution over a finite time horizon. In this paper we treat a case in which we lack the governing probability structure for Boolean network selection, so we simply have a family of Boolean networks, but where these networks possess a common attractor structure. This corresponds to the situation in which network construction is treated as an ill-posed inverse problem in which there are many Boolean networks created from the data under the constraint that they all possess attractor structures matching the data states, which are assumed to arise from sampling the steady state of the real biological network. RESULTS: Given a family of Boolean networks possessing a common attractor structure composed of singleton attractors, a control algorithm is derived by minimizing a composite finite-horizon cost function that is a weighted average over all the individual networks, the idea being that we desire a control policy that on average suits the networks because these are viewed as equivalent relative to the data. The weighting for each network at any time point is taken to be proportional to the instantaneous estimated probability of that network being the underlying network governing the state transition. The results are applied to a family of Boolean networks derived from gene-expression data collected in a study of metastatic melanoma, the intent being to devise a control strategy that reduces the WNT5A gene's action in affecting biological regulation. AVAILABILITY: The software is available on request. SUPPLEMENTARY INFORMATION: The supplementary Information is available at http://ee.tamu.edu/~edward/tree     

Lysosome membrane lipid microdomains: novel regulators of chaperone-mediated autophagy

Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins in lysosomes. The limiting step of this type of autophagy is the binding of substrates to the lysosome-associated membrane protein type 2A (LAMP-2A). In this work, we identify a dynamic subcompartmentalization of LAMP-2A in the lysosomal membrane, which underlies the molecular basis for the regulation of LAMP-2A function in CMA. A percentage of LAMP-2A localizes in discrete lysosomal membrane regions during resting conditions, but it exits these regions during CMA activation. Disruption of these regions by cholesterol-depleting agents or expression of a mutant LAMP-2A excluded from these regions enhances CMA activity, whereas loading of lysosomes with cholesterol significantly reduces CMA. Organization of LAMP-2A into multimeric complexes, required for translocation of substrates into lysosomes via CMA, only occurs outside the lipid-enriched membrane microdomains, whereas the LAMP-2A located within these regions is susceptible to proteolytic cleavage and degradation. Our results support that changes in the dynamic distribution of LAMP-2A into and out of discrete microdomains of the lysosomal membrane contribute to regulate CMA.     

Molecular epidemiology of ceftiofur-resistant Escherichia coli isolates from dairy calves

Healthy calves (n = 96, 1 to 9 weeks old) from a dairy herd in central Pennsylvania were examined each month over a five-month period for fecal shedding of ceftiofur-resistant gram-negative bacteria. Ceftiofur-resistant Escherichia coli isolates (n = 122) were characterized by antimicrobial resistance (disk diffusion and MIC), serotype, pulsed-field gel electrophoresis subtypes, beta-lactamase genes, and virulence genes. Antibiotic disk diffusion assays showed that the isolates were resistant to ampicillin (100%), ceftiofur (100%), chloramphenicol (94%), florfenicol (93%), gentamicin (89%), spectinomycin (72%), tetracycline (98%), ticarcillin (99%), and ticarcillin-clavulanic acid (99%). All isolates were multidrug resistant and displayed elevated MICs. The E. coli isolates belonged to 42 serotypes, of which O8:H25 was the predominant serotype (49.2%). Pulsed-field gel electrophoresis classified the E. coli isolates into 27 profiles. Cluster analysis showed that 77 isolates (63.1%) belonged to one unique group. The prevalence of pathogenic E. coli was low (8%). A total of 117 ceftiofur-resistant E. coli isolates (96%) possessed the bla(CMY2) gene. Based on phenotypic and genotypic characterization, the ceftiofur-resistant E. coli isolates belonged to 59 clonal types. There was no significant relationship between calf age and clonal type. The findings of this study revealed that healthy dairy calves were rapidly colonized by antibiotic-resistant strains of E. coli shortly after birth. The high prevalence of multidrug-resistant nonpathogenic E. coli in calves could be a significant source of resistance genes to other bacteria that share the same environment.     

A network diffusion model of disease progression in dementia

Patterns of dementia are known to fall into dissociated but dispersed brain networks, suggesting that the disease is transmitted along neuronal pathways rather than by proximity. This view is supported by neuropathological evidence for "prion-like" transsynaptic transmission of disease agents like misfolded tau and beta amyloid. We mathematically model this transmission by a diffusive mechanism mediated by?the brain's connectivity network obtained from tractography of 14 healthy-brain MRIs. Subsequent graph theoretic analysis provides a fully quantitative, testable, predictive model of dementia. Specifically, we predict spatially distinct "persistent modes," which, we found, recapitulate known patterns of dementia and match recent reports of selectively vulnerable dissociated brain networks. Model predictions also closely match T1-weighted MRI volumetrics of 18 Alzheimer's and 18 frontotemporal dementia subjects. Prevalence rates predicted by the model strongly agree with published data. This work has many important implications, including dimensionality reduction, differential diagnosis, and especially prediction of future atrophy using baseline MRI morphometrics.     

Damage to mitochondrial complex I during cardiac ischemia reperfusion injury is reduced indirectly by anti-anginal drug ranolazine

Ranolazine, an anti-anginal drug, is a late Na(+) channel current blocker that is also believed to attenuate fatty acid oxidation and mitochondrial respiratory complex I activity, especially during ischemia. In this study, we investigated if ranolazine's protective effect against cardiac ischemia/reperfusion (IR) injury is mediated at the mitochondrial level and specifically if respiratory complex I (NADH Ubiquinone oxidoreductase) function is protected. We treated isolated and perfused guinea pig hearts with ranolazine just before 30 min ischemia and then isolated cardiac mitochondria at the end of 30 min ischemia and/or 30 min ischemia followed by 10 min reperfusion. We utilized spectrophotometric and histochemical techniques to assay complex I activity, Western blot analysis for complex I subunit NDUFA9, electron paramagnetic resonance for activity of complex I Fe-S clusters, enzyme linked immuno sorbent assay (ELISA) for determination of protein acetylation, native gel histochemical staining for respiratory supercomplex assemblies, and high pressure liquid chromatography for cardiolipin integrity; cardiac function was measured during IR. Ranolazine treated hearts showed higher complex I activity and greater detectable complex I protein levels compared to untreated IR hearts. Ranolazine treatment also led to more normalized electron transfer via Fe-S centers, supercomplex assembly and cardiolipin integrity. These improvements in complex I structure and function with ranolazine were associated with improved cardiac function after IR. However, these protective effects of ranolazine are not mediated by a direct action on mitochondria, but rather indirectly via cytosolic mechanisms that lead to less oxidation and better structural integrity of complex I.     

Tyrosine nitration of voltage-dependent anion channels in cardiac ischemia-reperfusion: reduction by peroxynitrite scavenging

Excess superoxide (O(2)(-)) and nitric oxide (NO) forms peroxynitrite (ONOO(-)) during cardiac ischemia reperfusion (IR) injury, which in turn induces protein tyrosine nitration (tyr-N). Mitochondria are both a source of and target for ONOO(-). Our aim was to identify specific mitochondrial proteins that display enhanced tyr-N after cardiac IR injury, and to explore whether inhibiting O(2)(-)/ONOO(-) during IR decreases mitochondrial protein tyr-N and consequently improves cardiac function. We show here that IR increased tyr-N of 35 and 15kDa mitochondrial proteins using Western blot analysis with 3-nitrotyrosine antibody. Immunoprecipitation (IP) followed by LC-MS/MS identified 13 protein candidates for tyr-N. IP and Western blot identified and confirmed that the 35kDa tyr-N protein is the voltage-dependent anion channel (VDAC). Tyr-N of native cardiac VDAC with IR was verified on recombinant (r) VDAC with exogenous ONOO(-). We also found that ONOO(-) directly enhanced rVDAC channel activity, and rVDAC tyr-N induced by ONOO(-) formed oligomers. Resveratrol (RES), a scavenger of O(2)(-)/ONOO(-), reduced the tyr-N levels of both native and recombinant VDAC, while L-NAME, which inhibits NO generation, only reduced tyr-N levels of native VDAC. O(2)(-) and ONOO(-) levels were reduced in perfused hearts during IR by RES and L-NAME and this was accompanied by improved cardiac function. These results identify tyr-N of VDAC and show that reducing ONOO(-) during cardiac IR injury can attenuate tyr-N of VDAC and improve cardiac function.