Free cortisol is likely to be a better guide to cortisolemia in systemic infection because it corresponds more closely to illness severity. The attenuated cortisol increment after tetracosactrin in RAI is not due to low cortisol-binding proteins. Free cortisol levels can be determined reliably using total cortisol and CBG levels.

The process of ligand binding leading to receptor activation is an ordered and sequential one. Highaffinity binding of GM-CSF, interleukin 3 (IL-3), and IL-5 to their receptors induces a number of key events at the cell surface and within the cytoplasm that are necessary for receptor activation. These include receptor oligomerization, activation of tyrosine kinase activity, phosphorylation of the receptor, and the recruitment of SH2 (src-homology) and PTB (phosphotyrosine binding) domain proteins to the receptor. Such a sequence of events represents a recurrent theme among cytokine, growth factor, and hormone receptors; however, a number of very recent and interesting findings have identified unique features in this receptor system in terms of: A) how GM-CSF/IL-3/IL-5 bind, oligomerize, and activate their cognate receptors; B) how multiple biological responses such as proliferation, survival, and differentiation can be transduced from activated GM-CSF, IL-3, or IL-5 receptors, and C) how the presence of novel phosphotyrosine-independent signaling motifs within a specific cytoplasmic domain of β C may be important for mediating survival and differentiation by these cytokines. This review does not attempt to be all-encompassing but rather to focus on the most recent and significant discoveries that distinguish the GM-CSF/IL-3/IL-5 receptor subfamily from other cytokine receptors.

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Sphingosine kinase catalyzes the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in a number of agonist-driven cellular responses including mitogenesis, anti-apoptosis, and expression of inflammatory molecules. Despite the importance of sphingosine kinase, very little is known regarding its structure or mechanism of catalysis. Moreover, sphingosine kinase does not contain recognizable catalytic or substrate-binding sites, based on sequence motifs found in other kinases. Here we have elucidated the nucleotide-binding site of human sphingosine kinase 1 (hSK1) through a combination of site-directed mutagenesis and affinity labeling with the ATP analogue, FSBA. We have shown that Gly 82 of hSK1 is involved in ATP binding since mutation of this residue to alanine resulted in an enzyme with an ϳ45-fold higher K m(ATP) . We have also shown that Lys 103 is important in catalysis since an alanine substitution of this residue ablates catalytic activity. Furthermore, we have shown that this residue is covalently modified by FSBA. Our data, combined with amino acid sequence comparison, suggest a motif of SGDGX 17-21 K is involved in nucleotide binding in the sphingosine kinases. This motif differs in primary sequence from all previously identified nucleotide-binding sites. It does, however, share some sequence and likely structural similarity with the highly conserved glycine-rich loop, which is known to be involved in anchoring and positioning the nucleotide in the catalytic site of many protein kinases.

Background-C-reactive protein (CRP), a well-recognized marker of atherosclerosis, has recently been suggested to have a direct proinflammatory effect. The constitutive expression of low levels of CRP in normal plasma suggests the likelihood that a natural factor exists to neutralize the effect of CRP. This factor(s) has not yet been identified. Method and Results-The proinflammatory effect of CRP was measured by the induction of inflammatory adhesion molecules in human umbilical vein endothelial cells (HUVECs). We show that CRP significantly induced upregulation of adhesion molecules in both protein and mRNA levels. The CRP-induced expression of these inflammatory adhesion molecules was completely suppressed when the cells were preincubated with a physiological concentration (1 mg/mL apolipoprotein A-I) of HDLs derived from human plasma (native HDL) or reconstituted HDL (rHDL) at a very low concentration (0.01 mg/mL apolipoprotein A-I). A novel mechanism of HDL inhibition is likely to operate, because (1) rHDL was 100 times more potent than native HDL, (2) preincubation with HDL and its sustained presence were obligatory, and (3) oxidized 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine was the fundamental active component. Conclusions-The

The beta subunit (beta c) of the receptors for human granulocyte macrophage colony stimulating factor (GM‐CSF), interleukin‐3 (IL‐3) and interleukin‐5 (IL‐5) is essential for high affinity ligand‐binding and signal transduction. An important feature of this subunit is its common nature, being able to interact with GM‐CSF, IL‐3 and IL‐5. Analogous common subunits have also been identified in other receptor systems including gp130 and the IL‐2 receptor gamma subunit. It is not clear how common receptor subunits bind multiple ligands. We have used site‐directed mutagenesis and binding assays with radiolabelled GM‐CSF, IL‐3 and IL‐5 to identify residues in the beta c subunit involved in affinity conversion for each ligand. Alanine substitutions in the region Tyr365‐Ile368 in beta c showed that Tyr365, His367 and Ile368 were required for GM‐CSF and IL‐5 high affinity binding, whereas Glu366 was unimportant. In contrast, alanine substitutions of these residues only marginally reduced the conversion of IL‐3 binding to high affinity by beta c. To identify likely contact points in GM‐CSF involved in binding to the 365‐368 beta c region we used the GM‐CSF mutant eco E21R which is unable to interact with wild‐type beta c whilst retaining full GM‐CSF receptor alpha chain binding. Eco E21R exhibited greater binding affinity to receptor alpha beta complexes composed of mutant beta chains Y365A, H367A and I368A than to those composed of wild‐type beta c or mutant E366A. These results (i) identify the residues Tyr365, His367 and Ile368 as critical for affinity conversion by beta c, (ii) show that high affinity binding of GM‐CSF and IL‐5 can be dissociated from IL‐3 and (iii) suggest that Tyr365, His367 and Ile368 in beta c interact with Glu21 of GM‐CSF.

The importance of Mn 2؉ for pneumococcal physiology and virulence has been studied extensively. However, the specific cellular role(s) for which Mn 2؉ is required are yet to be fully elucidated. Here, we analyzed the effect of Mn 2؉ limitation on the transcriptome and proteome of Streptococcus pneumoniae D39. This was carried out by comparing a deletion mutant lacking the solute binding protein of the high-affinity Mn 2؉ transporter, pneumococcal surface antigen A (PsaA), with its isogenic wild-type counterpart. We provide clear evidence for the Mn 2؉ -dependent regulation of the expression of oxidative-stress-response enzymes SpxB and Mn 2؉ -SodA and virulence-associated genes pcpA and prtA. We also demonstrate the upregulation of at least one oxidativeand nitrosative-stress-response gene cluster, comprising adhC, nmlR, and czcD, in response to Mn 2؉ stress. A significant increase in 6-phosphogluconate dehydrogenase activity in the psaA mutant grown under Mn 2؉ -replete conditions and upregulation of an oligopeptide ABC permease (AppDCBA) were also observed. Together, the results of transcriptomic and proteomic analyses provided evidence for Mn 2؉ having a central role in activating or stimulating enzymes involved in central carbon and general metabolism. Our results also highlight the importance of high-affinity Mn 2؉ transport by PsaA in pneumococcal competence, physiology, and metabolism and elucidate mechanisms underlying the response to Mn 2؉ stress.

Human granulocyte-macrophage colonystimulating factor (GM-CSF) is a pleiotropic hemopoietic growth factor and activator of mature myeloid cell function. We have previously shown that residue 21 in the first helix of GM-CSF plays a critical role in both biological activity and high-affinity receptor binding. We have now generated analogues of GM-CSF mutated at residue 21, expressed them in Escherichia coli, and examined them for binding, agonistic, and antagonistic activitles. Binding experiments showed that GM E21A, E21Q, E21F, E21H, E21R, and E21K bound to the GM-CSF receptor a chain with a similar affinity to wild-type GM-CSF and had lost high-affinity binding to the GM-CSF receptor a-chain-common 13-chain complex. From these mutants, only the charge reversal mutants E21R and E21K were completely devoid of agonistic activity. Significantly we found that E21R and E21K antagonized the proliferative effect of GM-CSF on the erythroleukemic cell line TF-1 and primary acute myeloid leukemias, as well as GM-CSF-mediated stimulation of neutrophil superoxide production. This antagonism was specific for GM-CSF in that no antagonism of interleukin 3-mediated TF-1 cell proliferation or tumor necrosis factor a-mediated stimulation of neutrophil superoxide production was observed. E. coli-derived GM E21R and E21K were effective antagonists of both nonglycosylated and glycosylated wild-type GM-CSF. These results show that low-affinity GM-CSF binding can be dissociated from receptor activation and have potential clinical significance for the management of inflammatory diseases and certain leukemias where GM-CSF plays a pathogenic role.