Effects of platelet activating factor and related lipids on phase transition of dipalmitoylphosphatidylcholine

Recent evidence localizing the inflammatory mediator, platelet activating factor, (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to the membranes of stimulated neutrophils raises the possibility that PAF may, in addition to its activities as a mediator, alter the physical properties of membranes. Accordingly, the effects of PAF and related alkyl ether and acyl analogs on phase transition thermodynamics of dipalmitoylphosphatidylcholine (DPPC) were studied using fluorescence polarization of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). PAF, its ester analog (1-palmitoyl-2-acetylphosphatidylcholine) and both the corresponding alkyl and acyl lysophospholipid analogs (each at a concentration of 10 mol%) significantly decreased the phase transition temperature and broadened the phase transition of DPPC (P less than 0.05). The relative potency of the lipids in causing this effect was ester-PAF greater than or equal to PAF greater than or equal to lyso-PAF greater than lyso-PC suggesting that the fluidization of the synthetic membranes was attributable to both the 2-position acetyl group and the 1-position alkyl linkage. Furthermore, using various related compounds, increases in chain length and degree of unsaturation in the 2-position were shown to enhance the depression in transition temperature and broadening of the phase transition. Phase transition thermodynamics were also assessed using differential scanning calorimetry. Similar depression in the phase transition temperature was measured for PAF and both the alkyl and acyl lysophospholipids. Broadening of the phase transition for DPPC by the various analogs was assessed by calculation of transition peak width and cooperative unit. Data from fluorescence polarization and differential scanning calorimetry provide similar though not identical results and support the hypothesis that the unique features of PAF may alter membrane physical properties and could ultimately explain some of its biologic actions.     

Vesicular trafficking permits evasion of cGAS/STING surveillance during initial human papillomavirus infection

Oncogenic human papillomaviruses (HPVs) replicate in differentiating epithelium, causing 5% of cancers worldwide. Like most other DNA viruses, HPV infection initiates after trafficking viral genome (vDNA) to host cell nuclei. Cells possess innate surveillance pathways to detect microbial components or physiological stresses often associated with microbial infections. One of these pathways, cGAS/STING, induces IRF3-dependent antiviral interferon (IFN) responses upon detection of cytosolic DNA. Virion-associated vDNA can activate cGAS/STING during initial viral entry and uncoating/trafficking, and thus cGAS/STING is an obstacle to many DNA viruses. HPV has a unique vesicular trafficking pathway compared to many other DNA viruses. As the capsid uncoats within acidic endosomal compartments, minor capsid protein L2 protrudes across vesicular membranes to facilitate transport of vDNA to the Golgi. L2/vDNA resides within the Golgi lumen until G2/M, whereupon vesicular L2/vDNA traffics along spindle microtubules, tethering to chromosomes to access daughter cell nuclei. L2/vDNA-containing vesicles likely remain intact until G1, following nuclear envelope reformation. We hypothesize that this unique vesicular trafficking protects HPV from cGAS/STING surveillance. Here, we investigate cGAS/STING responses to HPV infection. DNA transfection resulted in acute cGAS/STING activation and downstream IFN responses. In contrast, HPV infection elicited minimal cGAS/STING and IFN responses. To determine the role of vesicular trafficking in cGAS/STING evasion, we forced premature viral penetration of vesicular membranes with membrane-perturbing cationic lipids. Such treatment renders a non-infectious trafficking-defective mutant HPV infectious, yet susceptible to cGAS/STING detection. Overall, HPV evades cGAS/STING by its unique subcellular trafficking, a property that may contribute to establishment of infection.     

Induction of TGF-β1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

Background/Objective

Phosphatidylserine (PS) exposed on apoptotic cells has been shown to stimulate production of transforming growth factor-β (TGF-β) and promote anti-inflammatory responses. However, the PS receptor(s) responsible for this induction has not been clearly determined.

Methodology/Principal Findings

In the present study, using RAWTβRII cells in which a truncated dominant negative TGF-β receptor II was stably transfected in order to avoid auto-feedback induction of TGF-β, we show that TGF-β1 synthesis is initiated via activation of the scavenger receptor, CD36. The response requires exposure of PS on the apoptotic cell surface and was absent in macrophages lacking CD36. Direct activation of CD36 with an anti-CD36 antibody initiated TGF-β1 production, and signaling pathways involving both Lyn kinase and ERK1/2 were shown to participate in CD36-driven TGF-β1 expression.

Conclusion/Significance

Since CD36 has been previously implicated in activation of secreted latent TGF-β, the present study indicates its role in the multiple steps to generation of this important biological mediator.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase Cdelta

Phospholipid scramblase induces nonspecific bidirectional movement of phospholipids across the membrane during cell activation and has been proposed to mediate the appearance of phosphatidylserine (PS) in the plasma membrane outer leaflet during apoptosis, a cell surface change that is critical for apoptotic cell removal. We report here that protein kinase C (PKC) delta plays an important role in activated transbilayer movement of phospholipids and surface PS exposure by directly enhancing the activity of phospholipid scramblase. Specific inhibition of PKCdelta by rottlerin prevented both apoptosis- and activation-induced scramblase activity. PKCdelta was either selectively cleaved and activated in a caspase 3-dependent manner (during apoptosis) or translocated to the plasma membrane (in stimulated cells) and could directly phosphorylate scramblase immunoprecipitated from Jurkat cells. Furthermore, reconstitution of PKCdelta and scramblase, but not scramblase or PKCdelta alone in Chinese hamster ovary cells demonstrated enhanced scramblase activity.     

The role of phosphatidylserine in recognition of apoptotic cells by phagocytes

Exposure of phosphatidylserine on the outer leaflet of the plasma membrane is a surface change common to many apoptotic cells. Normally restricted to the inner leaflet, phosphatidylserine appears as a result of decreased aminophospholipid translocase activity and activation of a calcium-dependent scramblase. Phosphatidylserine exposure has several potential biological consequences, one of which is recognition and removal of the apoptotic cell by phagocytes. It is still not clear which receptors mediate PS recognition on apoptotic cells; however, several interesting candidates have been proposed. These include the Class B scavenger and thrombospondin receptor CD36, an oxLDL receptor (CD68), CD14, annexins, beta2 glycoprotein I, gas-6 and a novel activity expressed on macrophages stimulated with digestible particles such as beta-glucan. Whether PS is the sole ligand recognized by phagocytes or whether it associated with other molecules to form a complex ligand remains to be determined.     

Effect of chronic treatment with Rosiglitazone on Leydig cell steroidogenesis in rats: <Emphasis Type="Italic">In vivo</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> studies

Background  

The present study was designed to examine the effect of chronic treatment with rosiglitazone - thiazolidinedione used in the treatment of type 2 diabetes mellitus for its insulin sensitizing effects - on the Leydig cell steroidogenic capacity and expression of the steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (P450scc) in normal adult rats.     

Polyandry and multiple paternities in the threatened Agassiz’s desert tortoise, <Emphasis Type="Italic">Gopherus agassizii</Emphasis>

We used data from 17 to 20 microsatellite markers to investigate the incidence of multiple paternities in wild Agassiz’s desert tortoises, Gopherus agassizii. Neonates were sampled from clutches of eggs laid by wild mothers in nesting enclosures at Edwards Air Force Base and at the Marine Corps Air Ground Combat Center, California. We genotyped 28 clutches from 26 females sampling an average of six neonates per clutch. The number of paternal alleles was used to determine the minimum number of sires for each clutch. Based on conservative criteria requiring evidence from at least two loci to determine multiple paternity, a minimum of 64% of females were polyandrous, while a minimum of 57% of clutches were sired by multiple males. This formed one of the highest incidences of multiple paternities recorded to date in any species of tortoise. The high number of microsatellite loci involved in the analyses allowed detection of multiple paternities in clutches where this may have been missed if fewer loci were used. Our results highlighted the potential pitfalls of quantitatively comparing paternity studies based on differing sampling strategies. Finally, we summarized the conservation implications of the high rate of multiple paternities in this threatened species.     

Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte

Apoptotic-cell removal is critical for development, tissue homeostasis, and resolution of inflammation. Although many candidate systems exist, only phosphatidylserine has been identified as a general recognition ligand on apoptotic cells. We demonstrate here that calreticulin acts as a second general recognition ligand by binding and activating LDL-receptor-related protein (LRP) on the engulfing cell. Since surface calreticulin is also found on viable cells, a mechanism preventing inadvertent uptake was sought. Disruption of interactions between CD47 (integrin-associated protein) on the target cell and SIRPalpha (SHPS-1), a heavily glycosylated transmembrane protein on the engulfing cell, permitted uptake of viable cells in a calreticulin/LRP-dependent manner. On apoptotic cells, CD47 was altered and/or lost and no longer activated SIRPalpha. These changes on the apoptotic cell create an environment where "don't eat me" signals are rendered inactive and "eat me" signals, including calreticulin and phosphatidylserine, congregate together and signal for removal.