Metalloproteinase-mediated Shedding of Integrin β2 promotes macrophage efflux from inflammatory sites

Macrophage exiting from inflammatory sites is critical to limit the local innate immune response. With tissue insult, resident tissue macrophages rapidly efflux to lymph nodes where they modulate the adaptive immune response, and inflammatory macrophages attracted to the site of injury then exit during the resolution phase. However, the mechanisms that regulate macrophage efflux are poorly understood. This study has investigated soluble forms of integrin β2 whose levels are elevated in experimental peritonitis at times when macrophages are exiting the peritoneum, suggesting that its proteolytic shedding may be involved in macrophage efflux. Both constitutive and inducible metalloproteinase-dependent shedding of integrin β2 from mouse macrophages are demonstrated. Soluble integrin β2 is primarily released as a heterodimeric complex with αM that retains its ability to bind its ligands intracellular adhesion molecule-1, fibrin, and collagen and thus may serve as a soluble antagonist. In a model of accelerated exiting, administration of a metalloproteinase inhibitor prevents macrophage efflux by 50% and impedes loss of macrophage integrin β2 from the cell surface. Exiting of peritoneal macrophages in mice lacking integrin β2 is accelerated, and antibody disruption of integrin β2-substrate interactions can reverse 50% of the metalloprotease inhibitor blockade of macrophage exiting. Thus, our study demonstrates the ability of metalloproteinase-mediated shedding of integrin β2 to promote macrophage efflux from inflammatory sites, and the release of soluble integrin heterodimers may also limit local inflammation.     

20th International Symposium on Shiftwork and Working Time: biological mechanisms, recovery, and risk management in the 24-h society

This dedicated issue of Chronobiology International is devoted to the selected proceedings of the 20th International Symposium on Shift Work and Working Time held in Stockholm, Sweden, 28 June to 1 July 2011. It constitutes the fifth such issue of the journal since 2004 dedicated to the selected proceedings to the meetings of the Working Time Society. The key theme of the 20th Symposium was "Biological Mechanisms, Recovery, and Risk Management in the 24-h Society." The collection of papers of this dedicated issue represents the best of contemporary research on the effects of night and rotating shift schedules on worker health and safety. The contents cover such topics as sleep restriction, injuries, health, and performance of night work and rotating shiftwork, plus light treatment as a countermeasure against the circadian disruption of shiftwork. The majority of the papers are observational field studies, including some of large sample size, and three studies are well-designed laboratory experiments.     

Familial secondary erythrocytosis due to increased oxygen affinity is caused by destabilization of the T state of hemoglobin Brigham (α(2) β(2) (Pro100Leu) )

Hemoglobin Brigham (β Pro100 to Leu) was first reported in a patient with familial erythrocytosis. Erythrocytes of an affected individual from the same family contain both HbA and Hb Brigham and exhibit elevated O(2) affinity compared with normal cells (P(50) = 23 mm Hg vs. 31 mmHg at pH 7.4 at 37°C). O(2) affinities measured for hemolysates were sensitive to changes in pH or chloride concentrations, indicating little change in the Bohr and Chloride effects. Hb Brigham was separated from normal HbA by nondenaturing cation exchange liquid chromatography, and the amino acid substitution was verified by mass spectrometry. The properties of Hb Brigham isolated from the patient's blood were then compared with those of recombinant Hb Brigham expressed in Escherichia coli. Kinetic experiments suggest that the rate constants for ligand binding and release in the high (R) and low (T) affinity quaternary states of Hb Brigham are similar to those of native hemoglobin. However, the Brigham mutation decreases the T to R equilibrium constant (L) which accelerates the switch to the R state during ligand binding to deoxy-Hb, increasing the rate of association by approximately twofold, and decelerates the switch during ligand dissociation from HbO(2) , decreasing the rate approximately twofold. These kinetic data help explain the high O(2) affinity characteristics of Hb Brigham and provide further evidence for the importance of the contribution of Pro100 to intersubunit contacts and stabilization of the T quaternary structure.     

Benzylideneoxymorphone: A new lead for development of bifunctional mu/delta opioid receptor ligands

Opioid analgesic tolerance remains a considerable drawback to chronic pain management. The finding that concomitant administration of delta opioid receptor (DOR) antagonists attenuates the development of tolerance to mu opioid receptor (MOR) agonists has led to interest in producing bifunctional MOR agonist/DOR antagonist ligands. Herein, we present 7-benzylideneoxymorphone (6, UMB 246) displaying MOR partial agonist/DOR antagonist activity, representing a new lead for designing bifunctional MOR/DOR ligands.     

Synthesis and in vitro evaluation of SG3227, a pyrrolobenzodiazepine dimer antibody-drug conjugate payload based on sibiromycin

A novel pyrrolobenzodiazepine dimer payload, SG3227, was rationally designed based on the naturally occurring antitumour compound sibiromycin. SG3227 was synthesized from a dimeric core in an efficient fashion. An unexpected room temperature Diels-Alder reaction occurred during the final step of the synthesis and was circumvented by use of an iodoacetamide conjugation moiety in place of a maleimide. The payload was successfully conjugated to trastuzumab and the resulting ADC exhibited potent activity against a HER2-expressing human cancer cell line in vitro.     

The hepcidin-binding site on ferroportin is evolutionarily conserved

Mammalian iron homeostasis is regulated by the interaction of the liver-produced peptide hepcidin and its receptor, the iron transporter ferroportin. Hepcidin binds to ferroportin resulting in degradation of ferroportin and decreased cellular iron export. We identify the hepcidin-binding domain (HBD) on ferroportin and show that a synthetic 19 amino acid peptide corresponding to the HBD recapitulates the characteristics and specificity of hepcidin binding to cell-surface ferroportin. The binding of mammalian hepcidin to ferroportin or the HBD shows an unusual temperature dependency with an increased rate of dissociation at temperatures below 15°C. The increased rate of dissociation is due to temperature- dependent changes in hepcidin structure. In contrast, hepcidin from poikilothermic vertebrates, such as fish or frogs, binds the HBD in a temperature-independent fashion. The affinity of hepcidin for the HBD permits a rapid, sensitive assay of hepcidin from all species and yields insights into the evolution of hepcidin.     

Impaired reproduction in three-spined sticklebacks exposed to ethinyl estradiol as juveniles

To investigate the population-level effects of exposure to environmental endocrine disrupters, a mesocosm-scale study was carried out in which the reproductive performance of groups of free-spawning three-spined sticklebacks, Gasterosteus aculeatus, exposed as juveniles to a model estrogen, was assessed. Juvenile sticklebacks were exposed to ethinyl estradiol (EE(2)) at measured concentrations of (mean +/- SEM) 1.75 +/- 0.37 ng L(-1) and 27.7 +/- 1.08 ng L(-1) for 4 wk posthatch and then reared thereafter in pristine lake water until they reached adulthood. Exposure to the higher EE(2) concentration resulted in the occurrence of ovotestis among males, whereas no gonadal abnormalities were evident among males exposed to the lower concentration of EE(2). In addition, when spawning was allowed in the mesocosm environment, fewer nests were built per male, and fewer eggs were deposited per nest, in the group exposed to 27.7 ng L(-1). Males from this group also exhibited a less intense nuptial coloration than control males. In the group exposed to 1.75 ng L(-1) EE(2) posthatch, significantly fewer nests were built than in the control group. These results demonstrate that the timing of exposure to estrogenic contaminants, in developmental terms, is critically important. Short-term exposure to estrogens as juveniles can clearly influence reproductive performance as adults, despite all growth and development subsequent to the exposure period taking place in an estrogen-free environment. In addition, these results suggest that reproductive dysfunction can occur even in fish with no gross abnormalities in gonadal structure. This suggests that the absence of gonadal intersex is not a reliable indicator of the reproductive potential, or estrogen-exposure history, of fish populations or the only important factor involved in compromising the reproduction of estrogen-exposed fish.     

Activation of ROCK by RhoA is regulated by cell adhesion, shape, and cytoskeletal tension

Adhesion to the extracellular matrix regulates numerous changes in the actin cytoskeleton by regulating the activity of the Rho family of small GTPases. Here, we report that adhesion and the associated changes in cell shape and cytoskeletal tension are all required for GTP-bound RhoA to activate its downstream effector, ROCK. Using an in vitro kinase assay for endogenous ROCK, we found that cells in suspension, attached on substrates coated with low density fibronectin, or on spreading-restrictive micropatterned islands all exhibited low ROCK activity and correspondingly low myosin light chain phosphorylation, in the face of high levels of GTP-bound RhoA. In contrast, allowing cells to spread against substrates rescued ROCK and myosin activity. Interestingly, inhibition of tension with cytochalasin D or blebbistatin also inhibited ROCK activity within 20 min. The abrogation of ROCK activity by cell detachment or inhibition of tension could not be rescued by constitutively active RhoA-V14. These results suggest the existence of a feedback loop between cytoskeletal tension, adhesion maturation, and ROCK signaling that likely contributes to numerous mechanochemical processes.     

Break-induced loss of heterozygosity in fission yeast: dual roles for homologous recombination in promoting translocations and preventing de novo telomere addition

Loss of heterozygosity (LOH), a causal event in tumorigenesis, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms leading to such extensive LOH are poorly understood. We investigated the mechanisms of DNA double-strand break (DSB)-induced extensive LOH by screening for auxotrophic marker loss approximately 25 kb distal to an HO endonuclease break site within a nonessential minichromosome in Schizosaccharomyces pombe. Extensive break-induced LOH was infrequent, resulting from large translocations through both allelic crossovers and break-induced replication. These events required the homologous recombination (HR) genes rad32(+), rad50(+), nbs1(+), rhp51(+), rad22(+), rhp55(+), rhp54(+), and mus81(+). Surprisingly, LOH was still observed in HR mutants, which resulted predominantly from de novo telomere addition at the break site. De novo telomere addition was most frequently observed in rad22Delta and rhp55Delta backgrounds, which disrupt HR following end resection. Further, levels of de novo telomere addition, while increased in ku70Delta rhp55Delta strains, were reduced in exo1Delta rhp55Delta and an rhp55Delta strain overexpressing rhp51. These findings support a model in which HR prevents de novo telomere addition at DSBs by competing for resected ends. Together, these results suggest that the mechanisms of break-induced LOH may be predicted from the functional status of the HR machinery.