Human α2-macroglobulin structure Location of Cys-949 residues within a half-molecule measured by fluorescence energy transfer

AbstractTo localize the pair of thiol-ester-derived cysteine-949 residues within a half-molecule of α2M and estimate the internal diameter of this α2-macroglobulin trap, we have measured the separation between these cysteines. We unexpectedly found that the two cysteines of intermediate form α2M had different reactivity, which permitted selective modification of one cysteine with dansyl, and the other with fluorescein fluorophores. From fluorescence energy transfer measurements, we calculated a separation of 41 ± 10 Å between these fluorophores. This indicates that the Cys-949 residues are probably located at the perimeter of the trap with an internal diameter at least as large as this separation

An approach to assessment of endocrine disruption in the National Children's Study.

In this article we consider the importance of assessing endocrine disruption in a large new cohort that has been proposed, the National Children's Study (NCS). We briefly review evidence that endocrine disruption is a potentially important hypothesis for human studies and weigh the need to assess endocrine disruption in the NCS. We note the salient features of earlier, similar cohort studies that serve as reference points for the design of the NCS. Finally, we discuss features of the NCS that would allow or enhance assessment of endocrine disruption, even if endocrine disruption were not a primary hypothesis motivating the study. At this time, the evidence supporting endocrine disruption in humans with background-level exposures is not strong. Thus, a compelling rationale for the NCS will probably need to be based on core hypotheses that focus on other issues. Nonetheless, if properly designed, the NCS could serve as an excellent resource for investigating future hypotheses regarding endocrine disruption

Thiol ester role in correct folding and conformation of human α2-macroglobulin Properties of recombinant C949S variant

AbstractTo determine the role of the thiol ester in the folding of human α2-macroglobulin (α2M) in the active conformation, we have characterized a recombinant variant of α2M, C949S, expressed in baby hamster kidney cells, that lacks the thiol ester-forming cysteine. C949S α2M behaves like methylamine-treated plasma α2M, with correctly formed inter-subunit disulfide bridges, non-covalent association of covalent dimers to form tetramers, and exposure of the receptor binding domain, but an inability to inhibit proteinases, and inaccessibility of the bait regions to proteolysis. We concluded that correct folding of monomers or their association to give tetrameric α2M does not require a pre-formed thiol ester. Active α2M may form in vivo by a two-step process involving initial folding to give a structure resembling that of C949S α2M followed by thiol ester formation and a conformational change that gives the native active state

The glyceryl ester of prostaglandin E(2) mobilizes calcium and activates signal transduction in RAW264.7 cells

Glyceryl prostaglandins (PG-Gs) are generated by the oxygenation of the endocannabinoid, 2-arachidonylglycerol, by cyclooxygenase 2. The biological consequences of this selective oxygenation are uncertain because the cellular activities of PG-Gs have yet to be defined. We report that the glyceryl ester of PGE(2), PGE(2)-G, triggers rapid, concentration-dependent Ca(2+) accumulation in a murine macrophage-like cell line, RAW264.7. Ca(2+) mobilization is not observed after addition of PGE(2), PGD(2)-G, or PGF(2α)-G but is observed after addition of PGF(2α). Moreover, PGE(2)-G, but not PGE(2), stimulates a rapid but transient increase in the levels of inositol 1,4,5-trisphosphate (IP(3)) as well as the membrane association and activation of PKC. PGE(2)-G induces a concentration-dependent increase in the levels of phosphorylated extracellular signal regulated kinases 1 and 2 through a pathway that requires the activities of PKC, IP(3) receptor, and phospholipase C β. The results indicate that PGE(2)-G triggers Ca(2+) mobilization, IP(3) synthesis, and activation of PKC in RAW264.7 macrophage cells at low concentrations. These responses are independent of the hydrolysis of PGE(2)-G to PGE(2)

[¹²³I]-Celecoxib Analogues as SPECT Tracers of Cyclooxygenase-2 in Inflammation

We report the synthesis and evaluation of a series of iodinated celecoxib analogues as cyclooxygenase-2 (COX-2)-targeted single photon emission computerized tomography (SPECT) imaging agents for the detection of inflammation. The structure−activity relationship identified 5-(4-iodophenyl)-1-{4-(methylsulfonyl)phenyl}-3-(trifluoromethyl)-1<i>H</i>-pyrazole (<b>8</b>) as a promising compound with IC₅₀ values of 0.05 μM against purified COX-2 and 0.03 μM against COX-2 in activated macrophages. The arylstannane of <b>8</b> undergoes facile radio-[¹²³I]-iodination upon treatment with Na¹²³I/NaI and chloramine T using an EtOAc/H₂O two-phase system. The [¹²³I]-<b>8</b> was produced in a radiochemical yield of 85% and a radiochemical purity of 99%. In vivo SPECT imaging demonstrated that the radiotracer was taken up by inflamed rat paws with an average 1.7-fold enrichment over contralateral noninflamed paws. This study suggests that conversion of celecoxib into its isomeric iodo-[¹²³I]-analogues is a useful approach for generating novel and efficacious agents for COX-2-targeted SPECT imaging of inflammation