Temporal complexity in clinical manifestations of lung disease

In this review, we summarize results of recent research on the temporal variability of lung function, symptoms, and inflammatory biomarkers. Specifically, we demonstrate how fluctuation analysis borrowed from statistical physics can be used to gain insight into neurorespiratory control and complex chronic dynamic diseases such as asthma viewed as a system of interacting components (e.g., inflammatory, immunological, and mechanical). Fluctuation analysis tools are based on quantifying the distribution and the short- and long-term temporal history of tidal breathing and lung function parameters to assess neurorespiratory control and monitor chronic disease. The latter includes the assessment of severity and disease control, the impact of treatment and environmental triggers, the temporal characterization of disease phenotypes, and the individual risk of exacerbation. While in many cases specific mechanistic insight into the fluctuations still awaits further research, appropriate analyses of the fluctuations already impact on clinical science and practice.     

Enhancement of suboptimal CD8 cytotoxic T cell effector function in vivo using antigen-specific CD80 defective T cells

T cell upregulation of B7 molecules CD80 and CD86 limits T cell expansion in immunodeficient hosts; however, the relative roles of CD80 separate from CD86 on CD4 versus CD8 T cells in a normal immune system are not clear. To address this question, we used the parent-into-F1 (P→F1) murine model of graft-versus-host disease and transferred optimal and suboptimal doses of CD80 and/or CD86 knockout (KO) T cells into normal F1 hosts. Enhanced elimination of host B cells by KO T cells was observed only at suboptimal donor cell doses and was greatest for CD80 KO→F1 mice. Wild-type donor cells exhibited peak CD80 upregulation at day 10; CD80 KO donor cells exhibited greater peak (day 10) donor T cell proliferation and CD8 T cell effector CTL numbers versus wild-type→F1 mice. Fas or programmed cell death-1 upregulation was normal as was homeostatic contraction of CD80 KO donor cells from days 12-14. Mixing studies demonstrated that maximal host cell elimination was seen when both CD4 and CD8 T cells were CD80 deficient. These results indicate an important role for CD80 upregulation on Ag-activated CD4 and CD8 T cells in limiting expansion of CD8 CTL effectors as part of a normal immune response. Our results support further studies of therapeutic targeting of CD80 in conditions characterized by suboptimal CD8 effector responses.     

Agricultural recovery of a formerly radioactive area: I. Establishment of high-resolution quantitative protein map of mature flax seeds harvested from the remediated Chernobyl area

In recent years there has been an increasing tendency toward remediation of contaminated areas for agriculture purposes. The study described herein is part of a comprehensive, long-term characterization of crop plants grown in the area formerly contaminated with radioactivity. As a first step, we have established a quantitative map of proteins isolated from mature flax (Linum usitatissimum L.) seeds harvested from plants grown in a remediated plot localized directly in Chernobyl town. Flax was selected because it is a crop of economic and historical importance, despite the relative paucity of molecular resources. We used 2-dimensional electrophoresis followed by tandem mass spectrometry to establish a high-resolution seed proteome map. This approach yielded quantitative information for 318 protein spots. Genomic sequence resources for flax are very limited, leaving us with an “unknown function” annotation for 38% of the proteins analyzed including several that comprise very large spots. In addition to the seed storage proteins, we were able to reliably identify 82 proteins many of which are involved with central metabolism.     

Modeling stochastic and spatial heterogeneity in a human airway tree to determine variation in respiratory system resistance

Asthma is a variable disease with changes in symptoms and airway function over many time scales. Airway resistance (Raw) is variable and thought to reflect changes in airway smooth muscle activity, but just how variation throughout the airway tree and the influence of gas distribution abnormalities affect Raw is unclear. We used a multibranch airway lung model to evaluate variation in airway diameter size, the role of coherent regional variation, and the role of gas distribution abnormalities on mean Raw (Raw) and variation in Raw as described by the SD (SDRaw). We modified an anatomically correct airway tree, provided by Merryn Tawhai (The University of Auckland, New Zealand), consisting of nearly 4,000 airways, to produce temporal and spatial heterogeneity. As expected, we found that increasing the diameter variation by twofold, with no change in the mean diameter, increased SDRaw more than fourfold. Perhaps surprisingly, Raw was proportional to SDRaw under several conditions-when either mean diameter was fixed, and its SD varied or when mean diameter varied, and SD was fixed. Increasing the size of a regional absence in gas distribution (ventilation defect) also led to a proportionate increase in both Raw and SDRaw. However, introducing regional dependence of connected airways strongly increased SDRaw by as much as sixfold, with little change in Raw. The model was able to predict previously reported Raw distributions and correlation of SDRaw on Raw in healthy and asthmatic subjects. The ratio of SDRaw to Raw depended most strongly on interairway coherent variation and only had a slight dependence on ventilation defect size. These findings may explain the linear correlation between variation and mean values of Raw but also suggest that regional alterations in gas distribution and local coordination in ventilation amplify any underlying variation in airway diameters throughout the airway tree.     

Metabolic oligosaccharide engineering: perspectives, applications, and future directions

Many adhesion and signaling molecules critical for development, as well as surface markers implicated in diseases ranging from cancer to influenza, contain oligosaccharides that modify their functions. Inside a cell, complex glycosylation pathways assemble these oligosaccharides and attach them to proteins and lipids as they traffic to the cell surface. Until recently, practical technologies to manipulate glycosylation have lagged unlike the molecular biologic and genetic methods available to intervene in nucleic acid and protein biochemistry; now, metabolic oligosaccharide engineering shows promise for manipulating glycosylation. In this methodology, exogenously-supplied non-natural sugars intercept biosynthetic pathways and exploit the remarkable ability of many of the enzymes involved in glycosylation to process metabolites with slightly altered chemical structures. To date, non-natural forms of sialic acid, GalNAc, GlcNAc, and fucose have been incorporated into glycoconjugates that appear on the cell surface; in addition O-GlcNAc protein modification involved in intracellular signaling has been tagged with modified forms of this sugar. Reactive functional groups, including ketones, azides, and thiols, have been incorporated into glycoconjugates and thereby provide chemical 'tags' that can be used for diverse purposes ranging from drug delivery to new modes of carbohydrate-based cell adhesion that can be used to control stem cell destiny. Finally, strategies for further engineering non-natural sugars to improve their pharmacological properties and provide complementary biological activities, such as addition of short chain fatty acids, are discussed in this article.     

Kinase networks integrate profiles of N-methyl-D-aspartate receptor-mediated gene expression in hippocampus

The postsynaptic N-methyl-d-aspartate (NMDA) receptor activates multiple kinases and changes the phosphorylation of many postsynaptic proteins organized in signaling networks. Because the NMDA receptor is known to regulate gene expression, it is important to examine whether networks of kinases control signaling to gene expression. We examined the requirement of multiple kinases and NMDA receptor-interacting proteins for gene expression in mouse hippocampal slices. Protocols that induce long-term depression (LTD) and long-term potentiation (LTP) activated common kinases and overlapping gene expression profiles. Combinations of kinases were required for induction of each gene. Distinct combinations of kinases were required to up-regulate Arc, Npas4, Egr2, and Egr4 following either LTP or LTD protocols. Consistent with the combinatorial data, a mouse mutant model of the human cognition disease gene SAP102, which couples ERK kinase to the NMDA receptor, showed deregulated expression of specific genes. These data support a network model of postsynaptic integration where kinase signaling networks are recruited by differential synaptic activity and control both local synaptic events and activity-dependent gene expression.     

Airway smooth muscle cell tone amplifies contractile function in the presence of chronic cyclic strain

Chronic contractile activation, or tone, in asthma coupled with continuous stretching due to breathing may be involved in altering the contractile function of airway smooth muscle (ASM). Previously, we (11) showed that cytoskeletal remodeling and stiffening responses to acute (2 h) localized stresses were modulated by the level of contractile activation of ASM. Here, we investigated if altered contractility in response to chronic mechanical strain was dependent on repeated modulation of contractile tone. Cultured human ASM cells received 5% cyclic (0.3 Hz), predominantly uniaxial strain for 5 days, with once-daily dosing of either sham, forskolin, carbachol, or histamine to alter tone. Stiffness, contractility (KCl), and "relaxability" (forskolin) were then measured as was cell alignment, myosin light-chain phosphorylation (pMLC), and myosin light-chain kinase (MLCK) content. Cells became aligned and baseline stiffness increased with strain, but repeated lowering of tone inhibited both effects (P < 0.05). Strain also reversed a negative tone-modulation dependence of MLCK, observed in static conditions in agreement with previous reports, with strain and tone together increasing both MLCK and pMLC. Furthermore, contractility increased 176% (SE 59) with repeated tone elevation. These findings indicate that with strain, and not without, repeated tone elevation promoted contractile function through changes in cytoskeletal organization and increased contractile protein. The ability of repeated contractile activation to increase contractility, but only with mechanical stretching, suggests a novel mechanism for increased ASM contractility in asthma and for the role of continuous bronchodilator and corticosteroid therapy in reversing airway hyperresponsiveness.     

Genome-wide analysis of DNA methylation status of CpG islands in embryoid bodies, teratomas, and fetuses

Differentiation of embryonic stem (ES) cells into embryoid bodies (EBs) provides an in vitro system for the study of early lineage determination during mammalian development. We have previously reported that there are 247 CpG islands that potentially have tissue-dependent and differentially methylated regions (T-DMRs). This provided evidence that the formation of DNA methylation patterns at CpG islands is a crucial epigenetic event underlying mammalian development. Here we present an analysis by the restriction landmark genomic scanning (RLGS) using NotI as a landmark enzyme of the genome-wide methylation status of CpG islands of ES cells and EBs and of teratomas produced from ES cells. These results are considered in relation to the methylation status of CpG islands of genomic DNA from normal fetus (10.5 dpc) and adult tissues. We have prepared a DNA methylation panel that consists of 259 T-DMRs and includes novel T-DMRs that are distinctly methylated or unmethylated in the teratomas. The DNA methylation pattern was complex and differed for the ES cells, EBs, and teratomas, providing evidence that differentiation of cells involves both de novo DNA methylation as well as demethylation. Comparison of the numbers of T-DMRs, that were differentially methylated or unmethylated among the cells and tissue types studied, revealed that the teratomas were the most epigenetically different from ES cells. Thus, analysis of the DNA methylation profiles prepared in this study provides new insights into the differentiation of ES cells and development of fetus, EB, teratoma, and somatic tissues.     

GlcNAc 2-epimerase can serve a catabolic role in sialic acid metabolism

Sialic acid is a major determinant of carbohydrate-receptor interactions in many systems pertinent to human health and disease. N-Acetylmannosamine (ManNAc) is the first committed intermediate in the sialic acid biosynthetic pathway; thus, the mechanisms that control intracellular ManNAc levels are important regulators of sialic acid production. UDP-GlcNAc 2-epimerase and GlcNAc 2-epimerase are two enzymes capable of generating ManNAc from UDP-GlcNAc and GlcNAc, respectively. Whereas the former enzyme has been shown to direct metabolic flux toward sialic acid in vivo, the function of the latter enzyme is unclear. Here we study the effects of GlcNAc 2-epimerase expression on sialic acid production in cells. A key tool we developed for this study is a cell-permeable, small molecule inhibitor of GlcNAc 2-epimerase designed based on mechanistic principles. Our results indicate that, unlike UDP-GlcNAc 2-epimerase, which promotes biosynthesis of sialic acid, GlcNAc 2-epimerase can serve a catabolic role, diverting metabolic flux away from the sialic acid pathway.     

Characterization of the cellular uptake and metabolic conversion of acetylated N-acetylmannosamine (ManNAc) analogues to sialic acids

"Sialic acid engineering" refers to the strategy where cell surface carbohydrates are modified by the biosynthetic incorporation of metabolic intermediates, such as non-natural N-acetylmannosamine (ManNAc) analogues, into cellular glycoconjugates. While this technology has promising research, biomedical, and biotechnological applications due to its ability to endow the cell surface with novel physical and chemical properties, its adoption on a large scale is hindered by the inefficient metabolic utilization of ManNAc analogues. We address this limitation by proposing the use of acetylated ManNAc analogues for sialic acid engineering applications. In this paper, the metabolic flux of these "second-generation" compounds into a cell, and, subsequently, into the target sialic acid biosynthetic pathway is characterized in detail. We show that acetylated ManNAc analogues are metabolized up to 900-fold more efficiently than their natural counterparts. The acetylated compounds, however, decrease cell viability under certain culture conditions. To determine if these toxic side effects can be avoided, we developed an assay to measure the cellular uptake of acetylated ManNAc from the culture medium and its subsequent flux into sialic acid biosynthetic pathway. This assay shows that the majority ( > 80%) of acetylated ManNAc is stored in a cellular "reservoir" capable of safely sequestering this analogue. These results provide conditions that, from a practical perspective, enable the acetylated analogues to be used safely and efficaciously and therefore offer a general strategy to facilitate metabolic substrate-based carbohydrate engineering efforts. In addition, these results provide fundamental new insights into the metabolic processing of non-natural monosaccharides.