Endothelial and leukocyte forms of IL-8. Conversion by thrombin and interactions with neutrophils

We have recently shown that endothelial cell-derived IL-8 inhibits neutrophil adhesion to IL1-beta-activated human umbilical vein endothelial cell monolayers. IL-8 secreted by T lymphocytes or monocytes has been characterized as a promoter of neutrophil degranulation and chemotaxis. The IL-8 isolated from each of these cell types is a mixture of two IL-8 polypeptides, one consisting of 72 amino acids (herein called [ser-IL-8]72) and the other 77 amino acids (an N-terminal extended form herein called [ala-IL-8]77). IL-8 derived from T lymphocytes and monocytes is predominantly [ser-IL-8]72, whereas endothelial-derived IL-8 is highly enriched (greater than 80%) in [ala-IL-8]77. We address the relationship and activities of these two forms of IL-8 using recombinant proteins expressed by both mammalian cells and Escherichia coli. Thrombin was found to efficiently convert [ala-IL-8]77 to [ser-IL-8]72. In contrast, urokinase and tissue-type plasminogen activator were unable to cleave [ala-IL-8]77, and trypsin generated multiple IL-8 cleavage fragments. In competitive binding assays using 125I[ala-IL-8]77 neutrophils exhibited a twofold preference for [ser-IL-8]72 over [ala-IL-8]77. Both forms of IL-8 inhibited neutrophil adhesion to IL-1-beta-activated HUVEC monolayers by up to 90%. However, [ser-IL-8]72 was approximately 10-fold more potent than [ala-IL-8]77 in these assays (ED50 approximately 0.3 nM for [ser-IL-8]72 vs approximately 3 nM for [ala-IL-8]77. Both forms of IL-8 promoted degranulation of cytochalasin B-treated neutrophils [[ser-IL-8]72 (ED50 greater than 10 nM) was two- to three-fold more potent than [ala-IL-8]77], although in this regard they were less active than FMLP. Our data suggest that [ala-IL-8]77 and [ser-IL-8]72 have qualitatively similar and potentially complex biological activities, and that full activation of IL-8 requires cleavage to the [ser-IL-8]72 form. In the case of inflamed endothelial cells this activation could be mediated by thrombin generated in the procoagulant environment associated with these cells.     

Inverse relationship between age at onset of Huntington disease and paternal age suggests involvement of genetic imprinting.

It is well recognized that age at onset of Huntington disease (HD) is strongly influenced by the sex of the affected parent, and this has lead to suggestions that genetic imprinting or maternal specific factors may play a role in the expression of the disease. This study evaluated maternal and paternal ages, birth order, parental age at onset, and sex of the affected parent and grandparent in 1,764 patients in the National HD Roster by using linear-regression techniques which incorporated a weighted least-squares approach to accommodate the correlation among siblings. It was found that paternal age is negatively associated with age at onset of HD, particularly among subjects who inherit the mutant gene from grandfathers. Apparent associations between age at onset and birth order and between age at onset and maternal age were not significant after adjustment for paternal age. The paternal age effect is strongest among juvenile-onset cases and individuals with anticipation of greater than or equal to 10 years, although it is detectable across the entire age-at-onset distribution. The tendency for older fathers, including those not transmitting the HD gene, to have affected offspring with early-onset disease may be consistent with a gene imprinting mechanism involving DNA methylation. Because paternal age in unaffected fathers is also a significant determinant of age at onset, methylation in this context might involve HD modifier genes or the normal HD allele.     

Vasomotor instability preceding tilt-induced syncope: does respiration play a role?

Lipsitz, Lewis A., Raymond Morin, Margaret Gagnon, DanKiely, and Aharon Medina. Vasomotor instability precedingtilt-induced syncope: does respiration play a role? J. Appl. Physiol. 83(2): 383-390, 1997.This studyaimed to determine whether alterations in cardiovascular dynamicsbefore syncope are related to changes in spontaneous respiration.Fifty-two healthy subjects underwent continuous heart rate (HR),arterial blood pressure (BP), and respiratory measurements during10-min periods of spontaneous and paced breathing (0.25 Hz) in thesupine and 60° head-up tilt positions. Data were evaluated by powerspectrum and transfer function analyses. During tilt, 27 subjectsdeveloped syncope or presyncope and 25 remained asymptomatic. Subjectswith tilt-induced syncope had significantly greater increases inlow-frequency (0.04-0.15 Hz) systolic BP, diastolic BP, and HRpower during tilt than the asymptomatic subjects(P  0.01). This difference waspresent during spontaneous but not paced breathing. However, averagetidal volume, respiratory rate, minute ventilation, proportion ofbreaths below 0.15 Hz, and low-frequency respiratory power during tilt did not differ between syncopal and nonsyncopal subjects. Transfer magnitudes between low-frequency respiration and BP, and between BP andinterbeat interval, were also similar between groups. Thus vasomotorinstability before syncope is not related to alterations in respirationor the cardiovagal baroreflex but may reflect oscillating centralsympathetic outflow to the vasculature.

     

Site-Specific Phosphorylation of the DNA Damage Response Mediator Rad9 by Cyclin-Dependent Kinases Regulates Activation of Checkpoint Kinase 1

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR–specific protein kinases.     

Effects of RACK1 on cell migration and IGF-I signalling in cardiomyoctes are not dependent on an association with the IGF-IR

RACK1 can act as a scaffolding protein to integrate IGF-IR and integrin signalling in transformed cells but its actions in regulating IGF-IR signalling in non-transformed cells are less well understood. Here, we investigated the function of RACK1 in the non-transformed cardiomyocyte cell line H9c2. Overexpression of RACK1 in H9c2 cells was sufficient to increase cell size, increase adhesion to collagen 1, enhance protection from hydrogen peroxide-induced cell death, and increase cell migration. However, cell proliferation was decreased in these cells. Small interfering RNA (siRNA)-mediated suppression of RACK1 in H9c2 cells resulted in decreased cell adhesion and migration, but had no effect on cell proliferation or size. Increased basal and IGF-I-mediated Erk phosphorylation was observed in RACK1-overexpressing H9c2 cells. Interestingly, contrary to observations in transformed cells, RACK1 was not observed to interact with the IGF-IR in H9c2 cells. Also in contrast to observations in transformed cells, IGF-I promoted recruitment of Src to RACK1 as well as recruitment of PKC, and PKC to RACK1. Overall, the data indicate that in H9c2 cells RACK1 can influence cell size, cell survival, adhesion, migration, but its responses to IGF-I are independent of an association with the IGF-IR. Thus, the composition of the RACK1 scaffolding complex and its effects on IGF-I signalling may be different in transformed and non-transformed cells.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.     

The electric picnic: synergistic requirements for exoelectrogenic microbial communities

Characterization of the various microbial populations present in exoelectrogenic biofilms provides insight into the processes required to convert complex organic matter in wastewater streams into electrical current in bioelectrochemical systems (BESs). Analysis of the community profiles of exoelectrogenic microbial consortia in BESs fed different substrates gives a clearer picture of the different microbial populations present in these exoelectrogenic biofilms. Rapid utilization of fermentation end products by exoelectrogens (typically Geobacter species) relieves feedback inhibition for the fermentative consortia, allowing for rapid metabolism of organics. Identification of specific syntrophic processes and the communities characteristic of these anodic biofilms will be a valuable aid in improving the performance of BESs.     

Nitrous Oxide Emission from Grazed Grassland Under Different Management Systems

Nitrous oxide (N₂O) emissions from grazed grasslands are estimated to be approximately 28% of global anthropogenic N₂O emissions. Estimating the N₂O flux from grassland soils is difficult because of its episodic nature. This study aimed to quantify the N₂O emissions, the annual N₂O flux and the emission factor (EF), and also to investigate the influence of environmental and soil variables controlling N₂O emissions from grazed grassland. Nitrous oxide emissions were measured using static chambers at eight different grasslands in the South of Ireland from September 2007 to August 2009. The instantaneous N₂O flux values ranged from -186 to 885.6 μg N₂O-N m⁻² h⁻¹ and the annual sum ranged from 2 ± 3.51 to 12.55 ± 2.83 kg N₂O-N ha⁻¹ y⁻¹ for managed sites. The emission factor ranged from 1.3 to 3.4%. The overall EF of 1.81% is about 69% higher than the Intergovernmental Panel on Climate Change (IPCC) default EF value of 1.25% which is currently used by the Irish Environmental Protection Agency (EPA) to estimate N₂O emission in Ireland. At an N applied of approximately 300 kg ha⁻¹ y⁻¹, the N₂O emissions are approximately 5.0 kg N₂O-N ha⁻¹ y⁻¹, whereas the N₂O emissions double to approximately 10 kg N ha⁻¹ for an N applied of 400 kg N ha⁻¹ y⁻¹. The sites with higher fluxes were associated with intensive N-input and frequent cattle grazing. The N₂O flux at 17°C was five times greater than that at 5°C. Similarly, the N₂O emissions increased with increasing water filled pore space (WFPS) with maximum N₂O emissions occurring at 60–80% WFPS. We conclude that N application below 300 kg ha⁻¹ y⁻¹ and restricted grazing on seasonally wet soils will reduce N₂O emissions.     

Mystique is a new insulin-like growth factor-I-regulated PDZ-LIM domain protein that promotes cell attachment and migration and suppresses Anchorage-independent growth

By comparing differential gene expression in the insulin-like growth factor (IGF)-IR null cell fibroblast cell line (R- cells) with cells overexpressing the IGF-IR (R+ cells), we identified the Mystique gene expressed as alternatively spliced variants. The human homologue of Mystique is located on chromosome 8p21.2 and encodes a PDZ LIM domain protein (PDLIM2). GFP-Mystique was colocalized at cytoskeleton focal contacts with alpha-actinin and beta1-integrin. Only one isoform of endogenous human Mystique protein, Mystique 2, was detected in cell lines. Mystique 2 was more abundant in nontransformed MCF10A breast epithelial cells than in MCF-7 breast carcinoma cells and was induced by IGF-I and cell adhesion. Overexpression of Mystique 2 in MCF-7 cells suppressed colony formation in soft agarose and enhanced cell adhesion to collagen and fibronectin. Point mutation of either the PDZ or LIM domain was sufficient to reverse suppression of colony formation, but mutation of the PDZ domain alone was sufficient to abolish enhanced adhesion. Knockdown of Mystique 2 with small interfering RNA abrogated both adhesion and migration in MCF10A and MCF-7 cells. The data indicate that Mystique is an IGF-IR-regulated adapter protein located at the actin cytoskeleton that is necessary for the migratory capacity of epithelial cells.