STAT3 activation in circulating immune cells is related to neovascular age-related macular degeneration

Purpose:The Signal Transducer and Activator of Transcription 3 (STAT3) pathway is known to play an important role in inflammation and angiogenesis. STAT3 can be activated by IL-6 family cytokines through the receptor IL-6R/gp130. Increased IL-6 has been detected in the plasma and vitreous in neovascular age-related macular degeneration (nAMD) patients. The aim of this study was to investigate the role of the STAT3 pathway in the pathogenesis of nAMD.

Methods:Blood cells from nAMD patients (n = 11) and age-, gender-matched healthy controls (n = 13) were stimulated with IL-6 for 20 minutes. The expression of the activated form of STAT3 (p-STAT3) was examined by flow cytometry. The mRNA levels of gp130, IL-6R and the suppressor of cytokine signalling 3 (SOCS3, a negative regulator of p-STAT3) were evaluated by real-time RT-PCR. Laser-induced choroidal neovascularisation (CNV) was performed in WT C57BL/6J mice as well as in the myeloid cell specific SOCS3 deficiency mice i.e., the LysMCre-SOCS3fl/fl mice. STAT3 activation in CNV lesions was examined by western blot. The size of CNV at different times after laser treatment was measured by confocal microscopy of RPE/choroidal flatmounts.

Results:The expression of p-STAT3 in CD11b+ monocytes was significantly increased in nAMD patients compared to healthy controls, although mRNA expression of gp130, IL-6R and SOCS3 did not differ between patients and controls. The expression of p-STAT3 in the retinal and RPE/choroidal tissues was increased at 1 and 3 days after laser treatment. The administration of a STAT3 inhibitor LLL12 significantly suppressed CNV. CD11b+ monocytes from LysMCre-SOCS3fl/fl mice expressed higher levels of p-STAT3 compared to the cells from WT mice. Laser induced CNV developed earlier and were larger in LysMCre-SOCS3fl/fl mice compared to WT C57BL/6J mice.

Conclusions:Our results suggest that STAT3 activation in circulating monocytes may contribute to the development of choroidal neovascularisation in AMD, and targeting the STAT3 pathway may have therapeutic potential in nAMD.

N-terminal glycation of cholecystokinin-8 abolishes its insulinotropic action on clonal pancreatic B-cells

Monoglycated cholecystokinin octapeptide (Asp(1)-glucitol CCK-X) was prepared under hyperglycaemic reducing conditions and purified by reverse phase-high performance liquid chromatography. Electrospray ionisation mass spectrometry and automated Edman degradation demonstrated that CCK-8 was glycated specifically at the amino-terminal Asp(1) residue. Effects of Asp(1)-glucitol CCK-8 and CCK-8 on insulin secretion were examined using glucose-responsive clonal BRIN-BD11 cells. In acute (20 min) incubations, 10(-10) mol/l CCK-8 enhanced insulin release by 1.2-1.5-fold at 5.6-11.1 mmol/l glucose. The stimulatory effect induced by 10(-10) mom CCK-8 was abolished following glycation. At 5.6 mmol/l glucose, CCK-8 at concentrations ranging from 10(-11) to 10(-7) mol/l induced a significant 1.6-1.9-fold increase in insulin secretion. Insulin output in the presence of Asp(1)-glucitol CCK-8 over the concentration range 10(-11)-10(-7) mol/l was decreased by 21-35% compared with CCK-8, and its insulinotropic action was effectively abolished. Asp(1)-glucitol CCK-8 at 10(-8) mol/l also completely blocked the stimulatory effects of 10(-11)-10(-8) mol/l CCK-8. These data indicate that structural modification by glycation at the amino-terminal Asp(1) residue effectively abolishes and/or antagonises the insulinotropic activity of CCK-8. (C) 1999 Elsevier Science B.V. All rights reserved.

Exposure to aflatoxin B1 in utero is associated with DNA methylation in white blood cells of infants in The Gambia

BACKGROUND: Exposure to environmental toxins during embryonic development may lead to epigenetic changes that influence disease risk in later life. Aflatoxin is a contaminant of staple foods in sub-Saharan Africa, is a known human liver carcinogen and has been associated with stunting in infants.

METHODS: We have measured aflatoxin exposure in 115 pregnant women in The Gambia and examined the DNA methylation status of white blood cells from their infants at 2-8 months old (mean 3.6 ± 0.9). Aflatoxin exposure in women was assessed using an ELISA method to measure aflatoxin albumin (AF-alb) adducts in plasma taken at 1-16 weeks of pregnancy. Genome-wide DNA methylation of infant white blood cells was measured using the Illumina Infinium HumanMethylation450beadchip.

RESULTS: AF-alb levels ranged from 3.9 to 458.4 pg/mg albumin. We found that aflatoxin exposure in the mothers was associated to DNA methylation in their infants for 71 CpG sites (false discovery rate < 0.05), with an average effect size of 1.7% change in methylation. Aflatoxin-associated differential methylation was observed in growth factor genes such as FGF12 and IGF1, and immune-related genes such as CCL28, TLR2 and TGFBI. Moreover, one aflatoxin-associated methylation region (corresponding to the miR-4520b locus) was identified.

CONCLUSIONS: This study shows that maternal exposure to aflatoxin during the early stages of pregnancy is associated with differential DNA methylation patterns of infants, including in genes related to growth and immune function. This reinforces the need for interventions to reduce aflatoxin exposure, especially during critical periods of fetal and infant development.

Suspension plasma sprayed coatings using dilute hydrothermally produced titania feedstocks for photocatalytic applications

Titanium dioxide coatings have potential applications including photocatalysts for solar assisted hydrogen production, solar water disinfection and self-cleaning windows. Herein, we report the use of suspension plasma spraying (SPS) for the deposition of conformal titanium dioxide coatings. The process utilises a nanoparticle slurry of TiO2 (ca. 6 and 12 nm respectively) in water, which is fed into a high temperature plasma jet (ca. 7000-20 000 K). This facilitated the deposition of adherent coatings of nanostructured titanium dioxide with predominantly anatase crystal structure. In this study, suspensions of nano-titanium dioxide, made via continuous hydrothermal flow synthesis (CHFS), were used directly as a feedstock for the SPS process. Coatings were produced by varying the feedstock crystallite size, spray distance and plasma conditions. The coatings produced exhibited ca. 90-100% anatase phase content with the remainder being rutile (demonstrated by XRD). Phase distribution was homogenous throughout the coatings as determined by micro-Raman spectroscopy. The coatings had a granular surface, with a high specific surface area and consisted of densely packed agglomerates interspersed with some melted material. All of the coatings were shown to be photoactive by means of a sacrificial hydrogen evolution test under UV radiation and compared favourably with reported values for CVD coatings and compressed discs of P25.

Specific role of neuronal nitric-oxide synthase when tethered to the plasma membrane calcium pump in regulating the beta-adrenergic signal in the myocardium

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates beta-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser(16) phospholamban (PLB) phosphorylation as well as Ser(22) and Ser(23) cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the beta-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1)

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.

Antimicrobial activity of prodigiosin is attributable to plasma-membrane damage

The bacterial pigment prodigiosin has various biological activities; it is, for instance, an effective antimicrobial. Here, we investigate the primary site targeted by prodigiosin, using the cells of microbial pathogens of humans as model systems: Candida albicans, Escherichia coli, Staphylococcus aureus. Inhibitory concentrations of prodigiosin; leakage of intracellular K+ ions, amino acids, proteins and sugars; impacts on activities of proteases, catalases and oxidases; and changes in surface appearance of pathogen cells were determined. Prodigiosin was highly inhibitory (30% growth rate reduction of C. albicans, E. coli, S. aureus at 0.3, 100 and 0.18 μg ml−1, respectively); caused leakage of intracellular substances (most severe in S. aureus); was highly inhibitory to each enzyme; and caused changes to S. aureus indicative of cell-surface damage. Collectively, these findings suggest that prodigiosin, log Poctanol–water 5.16, is not a toxin but is a hydrophobic stressor able to disrupt the plasma membrane via a chaotropicity-mediated mode-of-action.

Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane

Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30-300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging.