Estrogen accelerates immune complex glomerulonephritis but ameliorates T cell-mediated vasculitis and sialadenitis in autoimmune MRL lpr/lpr mice.

Estrogen is known to influence immune responses in healthy subjects in a dichotomous fashion. Thus, in number of previous studies we and others have demonstrated that B cell activities are augmented after exposure to estrogen whereas T cell reactivity is suppressed. Furthermore, it has been shown that this hormone has significant impact on the course of certain human and experimental autoimmune diseases. In this study we report that treatment with physiological doses of estradiol exerts dichotomous effects on different manifestations of the lupus disease in MRL/l mice. On one hand immune complex-mediated glomerulonephritis was significantly accelerated. This outcome was due to polyclonal B cell activation with increased production of antibodies to double-stranded DNA and formation of circulating immune complexes. In contrast, T cell-mediated lesions such as focal sialadenitis, renal vasculitis, and periarticular inflammation were all significantly ameliorated in MRL/l mice exposed to estrogen. Thus, we were able to demonstrate that, within one subject and even within one organ, administration of estrogen leads to differential outcome of SLE morbidity. We propose that the differential effect of estrogen on the manifestations of the autoimmune disease of MRL/l mice is due to its dichotomous effects on B and T cell-mediated immune responses.     

Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs

Our objective was to examine the ability of nucleate and anucleate fragments of artificially activated mouse eggs to transform sperm nucleus into male pronucleus. To this end, zona-free oocytes in metaphase II were activated by ethanol and bisected into halves (one with the spindle, the other anucleate) either within 10 to 20 min (series A) or 3 or 5 hr later (series B). In series A, the fragments were inseminated 3,5, and 8 h after activation, and in series B. 3 and 5 h after activation. Both nucleate and anucleate fragments lose the capability of transforming sperm nucleus into fully formed pronucleus sometime between 3 and 5 h after activation. In 8 h old parthenogenetic fragments, the majority of sperm nuclei remain unchanged or begin decondensation but never reach the stage of an early pronucleus. In over 1/3 of anucleate fragments of this age group, sperm nuclei develop defectively: chromatin decondenses inside the persisting nuclear envelope. In other experimental groups, the incidence of these abnormal sperm nuclei varies between 0 and 10%. In general, the anuclcate fragments retain the capability to transform sperm nuclei (fully or partially) longer than their nuclear counterparts. This difference may be accounted for by a different level of substances required for pronuclcar growth (extrachromosomal constituents of the germinal vesicle and nuclear lamins): high and constant in the cytoplasm of anucleate egg halves and low and progressively decreasing in the nucleate halves because of their putative uptake by the female pronucleus. However, the cytoplasmic factors responsible for the initial stages of transformation (nuclear envelope breakdown, chromatin decondensation) become eventually inactivated both in the presence and in the absence of a female pronucleus.     

Lipoproteins in Drosophila melanogaster-Assembly, Function, and Influence on Tissue Lipid Composition

Interorgan lipid transport occurs via lipoproteins, and altered lipoprotein levels correlate with metabolic disease. However, precisely how lipoproteins affect tissue lipid composition has not been comprehensively analyzed. Here, we identify the major lipoproteins of Drosophila melanogaster and use genetics and mass spectrometry to study their assembly, interorgan trafficking, and influence on tissue lipids. The apoB-family lipoprotein Lipophorin (Lpp) is the major hemolymph lipid carrier. It is produced as a phospholipid-rich particle by the fat body, and its secretion requires Microsomal Triglyceride Transfer Protein (MTP). Lpp acquires sterols and most diacylglycerol (DAG) at the gut via Lipid Transfer Particle (LTP), another fat body-derived apoB-family lipoprotein. The gut, like the fat body, is a lipogenic organ, incorporating both de novo-synthesized and dietary fatty acids into DAG for export. We identify distinct requirements for LTP and Lpp-dependent lipid mobilization in contributing to the neutral and polar lipid composition of the brain and wing imaginal disc. These studies define major routes of interorgan lipid transport in Drosophila and uncover surprising tissue-specific differences in lipoprotein lipid utilization.     

In-gel digestion for mass spectrometric characterization of proteins and proteomes

In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.     

Clumping factor A-mediated virulence during Staphylococcus aureus infection is retained despite fibrinogen depletion

Clumping factor A (ClfA), a fibrinogen-binding protein expressed on the Staphylococcus aureus cell surface, has previously been shown to act as a virulence factor in experimental septic arthritis. Although the interaction between ClfA and fibrinogen is assumed to be of importance for the virulence of S. aureus, this has not been demonstrated in any in vivo model of infection. Therefore, the objective of this study was to investigate the contribution of this interaction to ClfA-mediated virulence in murine S. aureus-induced arthritis. Ancrod, a serine protease with thrombin-like activity, was used to induce in vivo depletion of fibrinogen in mice. Ancrod treatment significantly aggravated septic arthritis following inoculation with a ClfA-expressing strain (Newman) compared to control treatment. Also, ancrod treatment tended to enhance the arthritis induced by a clfA mutant strain (DU5876), indicating that fibrinogen depletion exacerbates septic arthritis in a ClfA-independent manner. Most importantly, the ClfA-expressing strain was much more arthritogenic than the isogenic clfA mutant, following inoculation of fibrinogen-depleted mice. This finding indicates that the interaction between ClfA and free fibrinogen is not required for ClfA-mediated functions contributing to S. aureus virulence. It is conceivable that ClfA contributes to the virulence of S. aureus through interactions with other host ligands than fibrinogen.     

Influence of spring and autumn phenological transitions on forest ecosystem productivity

We use eddy covariance measurements of net ecosystem productivity (NEP) from 21 FLUXNET sites (153 site-years of data) to investigate relationships between phenology and productivity (in terms of both NEP and gross ecosystem photosynthesis, GEP) in temperate and boreal forests. Results are used to evaluate the plausibility of four different conceptual models. Phenological indicators were derived from the eddy covariance time series, and from remote sensing and models. We examine spatial patterns (across sites) and temporal patterns (across years); an important conclusion is that it is likely that neither of these accurately represents how productivity will respond to future phenological shifts resulting from ongoing climate change. In spring and autumn, increased GEP resulting from an ‘extra’ day tends to be offset by concurrent, but smaller, increases in ecosystem respiration, and thus the effect on NEP is still positive. Spring productivity anomalies appear to have carry-over effects that translate to productivity anomalies in the following autumn, but it is not clear that these result directly from phenological anomalies. Finally, the productivity of evergreen needleleaf forests is less sensitive to phenology than is productivity of deciduous broadleaf forests. This has implications for how climate change may drive shifts in competition within mixed-species stands.     

Stimulation,Inhibition, or Stabilization of Na,K-ATPase Caused by Specific Lipid Interactions at Distinct Sites

The activity of membrane proteins such as Na,K-ATPase depends strongly on the surrounding lipid environment. Interactions can be annular, depending on the physical properties of the membrane, or specific with lipids bound in pockets between transmembrane domains. This paper describes three specific lipid-protein interactions using purified recombinant Na,K-ATPase. (a) Thermal stability of the Na,K-ATPase depends crucially on a specific interaction with 18:0/18:1 phosphatidylserine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-l-serine; SOPS) and cholesterol, which strongly amplifies stabilization. We show here that cholesterol associates with SOPS, FXYD1, and the α subunit between trans-membrane segments αTM8 and -10 to stabilize the protein. (b) Polyunsaturated neutral lipids stimulate Na,K-ATPase turnover by >60%. A screen of the lipid specificity showed that 18:0/20:4 and 18:0/22:6 phosphatidylethanolamine (PE) are the optimal phospholipids for this effect. (c) Saturated phosphatidylcholine and sphingomyelin, but not saturated phosphatidylserine or PE, inhibit Na,K-ATPase activity by 70–80%. This effect depends strongly on the presence of cholesterol. Analysis of the Na,K-ATPase activity and E₁-E₂ conformational transitions reveals the kinetic mechanisms of these effects. Both stimulatory and inhibitory lipids poise the conformational equilibrium toward E₂, but their detailed mechanisms of action are different. PE accelerates the rate of E₁ → E₂P but does not affect E₂(2K)ATP → E₁3NaATP, whereas sphingomyelin inhibits the rate of E₂(2K)ATP → E₁3NaATP, with very little effect on E₁ → E₂P. We discuss these lipid effects in relation to recent crystal structures of Na,K-ATPase and propose that there are three separate sites for the specific lipid interactions, with potential physiological roles to regulate activity and stability of the pump.     

Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels

Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.     

Fibrinogen binding sites P336 and Y338 of clumping factor A are crucial for Staphylococcus aureus virulence

We have earlier shown that clumping factor A (ClfA), a fibrinogen binding surface protein of Staphylococcus aureus, is an important virulence factor in septic arthritis. When two amino acids in the ClfA molecule, P(336) and Y(338), were changed to serine and alanine, respectively, the fibrinogen binding property was lost. ClfAP(336)Y(338) mutants have been constructed in two virulent S. aureus strains Newman and LS-1. The aim of this study was to analyze if these two amino acids which are vital for the fibrinogen binding of ClfA are of importance for the ability of S. aureus to generate disease. Septic arthritis or sepsis were induced in mice by intravenous inoculation of bacteria. The clfAP(336)Y(338) mutant induced significantly less arthritis than the wild type strain, both with respect to severity and frequency. The mutant infected mice developed also a much milder systemic inflammation, measured as lower mortality, weight loss, bacterial growth in kidneys and lower IL-6 levels. The data were verified with a second mutant where clfAP(336) and Y(338) were changed to alanine and serine respectively. When sepsis was induced by a larger bacterial inoculum, the clfAP(336)Y(338) mutants induced significantly less septic death. Importantly, immunization with the recombinant A domain of ClfAP(336)SY(338)A mutant but not with recombinant ClfA, protected against septic death. Our data strongly suggest that the fibrinogen binding activity of ClfA is crucial for the ability of S. aureus to provoke disease manifestations, and that the vaccine potential of recombinant ClfA is improved by removing its ability to bind fibrinogen.     

Using Integrative Modeling Platform to compute,validate, and archive a model of a protein complex structure

Biology is advanced by producing structural models of biological systems, such as protein complexes. Some systems are recalcitrant to traditional structure determination methods. In such cases, it may still be possible to produce useful models by integrative structure determination that depends on simultaneous use of multiple types of data. An ensemble of models that are sufficiently consistent with the data is produced by a structural sampling method guided by a data‐dependent scoring function. The variation in the ensemble of models quantified the uncertainty of the structure, generally resulting from the uncertainty in the input information and actual structural heterogeneity in the samples used to produce the data. Here, we describe how to generate, assess, and interpret ensembles of integrative structural models using our open source Integrative Modeling Platform program ( https://integrativemodeling.org ).