Inflammation plays a critical role in the pathology of obesity-linked insulin resistance and is mechanistically linked to the effects of macrophage-derived cytokines on adipocyte energy metabolism, particularly that of the mitochondrial branched-chain amino acid (BCAA) and tricarboxylic acid (TCA) pathways. To address the role of inflammation on energy metabolism in adipocytes, we used high fat-fed C57BL/6J mice and lean controls and measured the down-regulation of genes linked to BCAA and TCA cycle metabolism selectively in visceral but not in subcutaneous adipose tissue, brown fat, liver, or muscle. Using 3T3-L1 cells, TNFα, and other proinflammatory cytokine treatments reduced the expression of the genes linked to BCAA transport and oxidation. Consistent with this, [(14)C]-leucine uptake and conversion to triglycerides was markedly attenuated in TNFα-treated adipocytes, whereas the conversion to protein was relatively unaffected. Because inflammatory cytokines lead to the induction of endoplasmic reticulum stress, we evaluated the effects of tunicamycin or thapsigargin treatment of 3T3-L1 cells and measured a similar down-regulation in the BCAA/TCA cycle pathway. Moreover, transgenic mice overexpressing X-box binding protein 1 in adipocytes similarly down-regulated genes of BCAA and TCA metabolism in vivo. These results indicate that inflammation and endoplasmic reticulum stress attenuate lipogenesis in visceral adipose depots by down-regulating the BCAA/TCA metabolism pathway and are consistent with a model whereby the accumulation of serum BCAA in the obese insulin-resistant state is linked to adipose inflammation.

Our laboratories have actively published in this area for several years and the objective of this chapter is to present as comprehensive an overview as possible. Following a brief review of the basic principles associated with 113Cd NMR methods, we will present the results from a thorough literature search for 113Cd chemical shifts from metalloproteins. The updated 113Cd chemical shift figure in this chapter will further illustrate the excellent correlation of the 113Cd chemical shift with the nature of the coordinating ligands (N, O, S) and coordination number/geometry, reaffirming how this method can be used not only to identify the nature of the protein ligands in uncharacterized cases but also the dynamics at the metal binding site. Specific examples will be drawn from studies on alkaline phosphatase, Ca2+ binding proteins, and metallothioneins. In the case of Escherichia coli alkaline phosphatase, a dimeric zinc metalloenzyme where a total of six metal ions (three per monomer) are involved directly or indirectly in providing the enzyme with maximal catalytic activity and structural stability, 113Cd NMR, in conjunction with 13C and 31P NMR methods, were instrumental in separating out the function of each class of metal binding sites. Perhaps most importantly, these studies revealed the chemical basis for negative cooperativity that had been reported for this enzyme under metal deficient conditions. Also noteworthy was the fact that these NMR studies preceeded the availability of the X-ray crystal structure. In the case of the calcium binding proteins, we will focus on two proteins: calbindin D9k and calmodulin. For calbindin D9k and its mutants, 113Cd NMR has been useful both to follow actual changes in the metal binding sites and the cooperativity in the metal binding. Ligand binding to calmodulin has been studied extensively with 113Cd NMR showing that the metal binding sites are not directly involved in the ligand binding. The 113Cd chemical shifts are, however, exquisitely sensitive to minute changes in the metal ion environment. In the case of metallothionein, we will reflect upon how 113Cd substitution and the establishment of specific Cd to Cys residue connectivity by proton-detected heteronuclear 1H-113Cd multiple-quantum coherence methods (HMQC) was essential for the initial establishment of the 3D structure of metallothioneins, a protein family deficient in the regular secondary structural elements of α-helix and β-sheet and the first native protein identified with bound Cd. The 113Cd NMR studies also enabled the characterization of the affinity of the individual sites for 113Cd and, in competition experiments, for other divalent metal ions: Zn, Cu, and Hg.

SUMMARYComplement-dependent clearance of immune complexes in humans is dependent on the activation and binding of the early components of the classical complement cascade. This prevents immune complex precipitation and promotes binding of the complexes by the C4bK3b complement receptor CR1 (CD35) found on erythrocytes. The fourth component of human complement is encoded by two closely linked genes within the MHC. These genes give rise to the isotypic forms C4A and C4B, and recent studies suggest that CRI binds activated C4A (C4Ab) to a greater extent than activated C4B (C4Bb). To study this difference in a more quantitative way the binding reactions between CR1 and C4Ab-and C4Bb-coated immune complexes and between CR1 and soluble dimers of C4Ab (C4Ab2) and C4Bb (C4Bb2) were analysed using the native receptor on human erythrocytes. The binding reaction between immune complexes with equivalent amounts of covalently bound C4Ab or C4Bb and erythrocyte CRI showed a two-fold higher binding of complexes coated with C4A. Furthermore, erythrocyte CR1 bound C4Ab2 with an apparent four-fold higher affinity (Kd = 1 . 4~ low7 M) than C4Bb2 (Kd = 4.8 x M), indicating a preferential binding of CR 1 for C4A.

There is a renewed enthusiasm for therapeutic vaccination as a viable treatment for patients with cancer. Early tumor vaccines were comprised of whole tumor cells, fragments of tumor cells, or protein lysate from tumor cells. Limited results with these approaches led investigators to begin developing the next generation of cancer vaccines based on defined tumor-associated antigens (TAAs). Defining and characterizing TAAs for human cancer, development of new approaches for identifying TAAs, and novel strategies to deliver the antigens as potent therapeutic vaccines have all been the focus of intense research in the past decade and will continue to be the focus for decades to come.

Objective Assessing and treating pain in nonverbal children with developmental disabilities are a clinical challenge. Current assessment approaches rely on clinical impression and behavioral rating scales completed by proxy report. Given the growing health relevance of the salivary metabolome, we undertook a translational-oriented feasibility study using proton nuclear magnetic resonance (NMR) spectroscopy and neuropeptide/cytokine/hormone detection to compare a set of salivary biomarkers relevant to nociception. Design Within-group observational design. Setting Tertiary pediatric rehabilitation hospital. Subjects Ten nonverbal pediatric patients with cerebral palsy with and without pain. Methods Unstimulated (passively collected) saliva was collected using oral swabs followed by perchloric acid extraction and analyzed on a Bruker Avance 700 MHz NMR spectrometer. We also measured salivary levels of several cytokines, chemokines, hormones, and neuropeptides. Results Partial least squares discriminant analysis showed separation of those children with/without pain for a number of different biomarkers. The majority of the salivary metabolite, neuropeptide, cytokine, and hormone levels were higher in children with pain vs no pain. Conclusions The ease of collection and noninvasive manner in which the samples were collected and analyzed support the possibility of the regular predictive use of this novel biomarker-monitoring method in clinical practice.

Flow cytometry (FCM) is useful for measuring DNA content as related to cell cycle position. We have extended this technology by developing a method that measures cellular DNA content using propidium iodide after permeabilization with lysolecithin. This technique maintains cell integrity such that high quality mRNA can be isolated from a sorted population. Unfixed MDA-468 cells, a human breast cancer cell line, were temporarily permeabilized with lysolecithin. A range of lysolecithin concentrations were studied in order to optimize DNA staining but minimize alterations in cell size and integrity. Cells permeabilized with lysolecithin in PBS, were stained with propidium iodide (50 pg/ml) in the presence of 1% bovine serum albumin, 1 mM Na,EDTA in PBS. The optimal concentration (4 pg lysolecithin/ml) combined staining -90% of the cells for DNA, with minimal effects on cell size. Subsequently, the cells were assayed for DNA content as a measure of cell cycle position. MDA-468 cells identified as being in G1 were sorted and collected. From these, high quality mRNA could be isolated, as judged by its ability to be in vitro translated into protein of a wide range of molecular weights. This technique should be useful for molecular studies requiring discrete cell populations based on DNA content andlor cell Cycle position. 0 1993 Wiley-Liss, Inc.Key terms: Flow cytometry, MDA-468, lysolecithin permeabilization, DNA staining, cell integrity, cell cycle, in vitro translationThe examination of DNA content by flow cytometric analysis has been widely used to investigate the ploidy status, DNA index, and cell cycle phase percentages of cells and nuclei from tissue and cell samples. Several fluorescent staining methods have been developed for the analysis and sorting of individual cells according to their DNA content. However, many of the common nonvital dyes used to stain cellular DNA quantitatively are efficiently excluded from intact plasma membranes. In contrast, some more recently introduced vital dyes are taken up by living cells. However, most have excitation wavelengths below 460 nm, limiting their use to lasers with UV capability. Therefore, preparative techniques that enable one to identify and collect a particular cell cycle fraction from heterogeneous cell populations using the newer air-cooled argon lasers would be useful. This is particularly true for studying gene expression correlated with differentiation and cell cycle status.We report a permeabilization technique using lysolecithin that allows DNA to be stained with dyes normally excluded from viable cells while maintaining the integrity of the cell sufficiently to allow for the isolation of high quality mRNA. Human breast cancer cells (MDA-468) were permeabilized with low concentrations of lysolecithin, stained for DNA using propidium iodide (PI), and analyzed for cell cycle kinetics. Cells in G1 of the cell cycle were identified and sorted. From these G1 sorts, high quality mRNA that could be efficiently translated into proteins of a wide range of molecular ...

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