Apolipoprotein A-V (apoA-V) is postulated to modulate intra-hepatic triglyceride (TG) trafficking. Stably transfected McA-RH7777 hepatocarcinoma cells expressing human apoA-V displayed enhanced neutral lipid staining while conditioned media from these cells had 40 ± 8 % less TG than cells transfected with a control vector. To obtain homogeneous cell lines expressing different amounts of apoA-V, a strategy of clonal selection was pursued. Immunoblot analysis of two distinct apoA-V stable cell lines yielded one that expresses low amounts of apoA-V and another that expresses higher amounts. Confocal fluorescence microscopy of control cells and cells expressing low levels of apoA-V had similar numbers of lipid droplets while cells expressing higher amounts of apoA-V had twice as many lipid droplets, on average. Thus, apoA-V expression promotes lipid droplet accumulation in these cells.
triacylglycerol; lipid droplet; apolipoprotein A-V; flow cytometry; confocal fluorescence microscopy
Mechanisms to increase plasma high-density lipoprotein (HDL) or to promote egress of
cholesterol from cholesterol-loaded cells (e.g., foam cells from atherosclerotic lesions) remain an
important target to regress heart disease. Reconstituted HDL (rHDL) serves as a valuable vehicle to
promote cellular cholesterol efflux in vitro and in vivo. rHDL were prepared with wild type
apolipoprotein (apo) A-I and the rare variant, apoA-I Milano (M), and each apolipoprotein was
reconstituted with phosphatidylcholine (PC) or sphingomyelin (SM). The four distinct rHDL generated
were incubated with CHO cells, J774 macrophages, and BHK cells in cellular cholesterol efflux
assays. In each cell type, apoA-I(M) SM-rHDL promoted the greatest cholesterol efflux. In BHK cells,
the cholesterol efflux capacities of all four distinct rHDL were greatly enhanced by increased
expression of ABCG1. Efflux to PC-containing rHDL was stimulated by transfection of a nonfunctional
ABCA1 mutant (W590S), suggesting that binding to ABCA1 represents a competing interaction. This
interpretation was confirmed by binding experiments. The data show that cholesterol efflux activity
is dependent upon the apoA-I protein employed, as well as the phospholipid constituent of the rHDL.
Future studies designed to optimize the efflux capacity of therapeutic rHDL may improve the value of
this emerging intervention strategy.
apolipoprotein; ABCA1; ABCG1; reverse cholesterol transport; macrophage
Signal transduction pathways involving the second messenger cyclic di-GMP [bis-(3′-5′)-cyclic di-guanosine monophosphate] occur widely in bacteria where they act to link perception of environmental or intracellular cues and signals to specific alterations in cellular function. Such alterations can contribute to bacterial lifestyle transitions including biofilm formation and virulence. The cellular levels of the nucleotide are controlled through the opposing activities of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). The GGDEF domain of DGCs catalyses the synthesis of cyclic di-GMP from GTP, whereas EAL or HD-GYP domains in different classes of PDE catalyse cyclic di-GMP degradation to pGpG and GMP. We are now beginning to understand how alterations in cyclic di-GMP exert a regulatory action through binding to diverse receptors or effectors that include a small ‘adaptor’ protein domain called PilZ, transcription factors and riboswitches. The regulatory action of enzymically active cyclic di-GMP signalling proteins is, however, not restricted to an influence on the level of nucleotide. Here, I will discuss our recent findings that highlight the role that protein–protein interactions involving these signalling proteins have in regulating functions that contribute to bacterial virulence.
Expressed protein ligation (EPL) was performed to investigate sequence requirements for a variant human apolipoprotein A-I (apoA-I) to adopt a folded structure. A C-terminal truncated apoA-I, corresponding to residues 1–172, was expressed and isolated from E. coli. Compared to full length apoA-I (243 amino acids), apoA-I(1-172) displayed less α-helix secondary structure and lower stability in solution. To determine if extension of this polypeptide would confer secondary structure content and/or stability, 20 residues were added to the C-terminus of apoA-I(1-172) by EPL, creating apoA-IMilano(1-192). The EPL product displayed biophysical properties similar to full-length apoA-IMilano. The results provide a general protein engineering strategy to modify the length of a recombinant template polypeptide using synthetic peptides as well as a convenient, cost effective way to investigate the structure / function relations in apolipoprotein fragments or domains of different size.
Expressed protein ligation; apolipoprotein A-I; Milano; Protein stability; Intein
The Gender, Race And Clinical Experience (GRACE) study was conducted between October 2006 and December 2008 to evaluate sex- and race-based differences in outcomes after treatment with a darunavir/ritonavir-based antiretroviral regimen. Between June 2010 and June 2011, former participants of the GRACE trial at participating sites were asked to complete a 40-item questionnaire as part of the GRACE Participant Survey study, with a primary objective of assessing patients' characteristics, experiences, and opinions about participation in GRACE. Of 243 potential survey respondents, 151 (62%) completed the survey. Respondents were representative of the overall GRACE population and were predominantly female (64%); fewer were black, and more reported recreational drug use compared with nonrespondents (55% vs. 62% and 17% vs. 10%, respectively). Access to treatment (41%) and too many blood draws (26%) were reported as the best and worst part of GRACE, respectively. Support from study site staff was reported as the most important factor in completing the study (47%). Factors associated with nonadherence, study discontinuation, and poor virologic response in univariate analyses were being the primary caregiver for children, unemployment, and transportation difficulties, respectively. Patients with these characteristics may be at risk of poor study outcomes and may benefit from additional adherence and retention strategies in future studies and routine clinical care.
Apolipoprotein (apo) A-V is a low abundance protein with a profound influence on plasma triacylglycerol levels. In human populations, single nucleotide polymorphisms and mutations in APOA5 positively correlate with hypertriglyceridemia. As an approach to preventing the deleterious effects of chronic hypertriglyceridemia, apoA-V gene therapy has been pursued.
Methods and Results
Recombinant adeno-associated virus (AAV) 2/8 harboring the coding sequence for human apoA-V or a control AAV2/8 was transduced into hypertriglyceridemic apoa5 (−/−) mice. After injection of 1×1012 viral genome AAV2/8-apoA-V, maximal plasma levels of apoA-V protein were achieved at 3 to 4 weeks, after which the concentration slowly declined. Complementing the appearance of apoA-V was a decrease (50±6%) in plasma triacylglycerol content compared with apoa5 (−/−) mice treated with AAV2/8-β-galactosidase. After 8 weeks the mice were euthanized and plasma lipoproteins separated. AAV2/8-apoA-V–transduced mice displayed a dramatic reduction in very low–density lipoprotein triacylglycerol content. Vector generated apoA-V in plasma associated with both very low–density lipoprotein and high-density lipoprotein fractions.
Taken together, the data show that gene transfer of apoA-V improves the severe hypertriglyceridemia phenotype of apoa5 (−/−) mice. Given the prevalence of hypertriglyceridemia, apoA-V gene therapy offers a potential strategy for maintenance of plasma triacylglycerol homeostasis.
apolipoprotein A-V; gene therapy; lipoprotein; triacylglycerol
The discovery of apolipoprotein A-V (apoA-V) in 2001 has raised a number of intriguing questions about role in lipid transport and triglyceride (TG) homeostasis. Genome wide association studies (GWAS) have consistently identified APOA5 as a contributor to plasma TG levels. Single nucleotide polymorphisms (SNP) with-in the APOA5 gene locus have been shown to correlate with elevated plasma TG. Furthermore, transgenic and knockout mouse models support the view that apoA-V plays a critical role in maintenance of plasma TG levels. The present review describes recent concepts pertaining to apoA-V SNP analysis and their association with elevated plasma TG. The interaction of apoA-V with glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) is discussed relative to its postulated role in TG-rich lipoprotein catabolism. The potential role of intracellular apoA-V in regulation of TG homeostasis, as a function of its ability to associate with cytosolic lipid droplets, is reviewed. While some answers are emerging, numerous mysteries remain with regard to this low abundance, yet potent, modulator of TG homeostasis. Given the strong correlation between elevated plasma TG and heart disease, there is great scientific and public interest in deciphering the numerous biological riddles presented by apoA-V. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.
Apolipoprotein A-V; Triglyceride; Lipid droplet; Genome wide association study; Glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1; Nonalcoholic fatty liver disease
There is an increasing appreciation of the polymicrobial nature of many bacterial infections such as those associated with cystic fibrosis (CF) and of the potentially important role for interspecies interactions in influencing both bacterial virulence and response to therapy. Patients with CF are often co-infected with Pseudomonas aeruginosa and other pathogens including Burkholderia cenocepacia and Stenotrophomonas maltophilia. These latter bacteria produce signal molecules of the diffusible signal factor (DSF) family, which are cis-2-unsaturated fatty acids. We have previously shown by in vitro studies that DSF from S. maltophilia leads to altered biofilm formation and increased resistance to antibiotics by P. aeruginosa; these responses of P. aeruginosa require the sensor kinase PA1396. Here we show that DSF signals are present in sputum taken from patients with CF. Presence of these DSF signals was correlated with patient colonization by S. maltophilia and/or B. cenocepacia. Analysis of 50 clinical isolates of P. aeruginosa showed that each responded to the presence of synthetic DSF by increased antibiotic resistance and these strains demonstrated little sequence variation in the PA1396 gene. In animal experiments using CF transmembrane conductance regulator knockout mice, the presence of DSF promoted P. aeruginosa persistence. Furthermore, antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells was enhanced in the presence of DSF. Taken together, these data provide substantial evidence that interspecies DSF-mediated bacterial interactions occur in the CF lung and may influence the efficacy of antibiotic treatment, particularly for chronic infections involving persistence of bacteria.
interspecies signaling; diffusible signal factor; biofilm formation; virulence; cystic fibrosis; next-generation sequencing
In Burkholderia cenocepacia, the second messenger cyclic diguanosine monophosphate (c-di-GMP) has previously been shown to positively regulate biofilm formation and the expression of cellulose and type-I fimbriae genes through binding to the transcriptional regulator Bcam1349. Here, we provide evidence that cellulose and type-I fimbriae are not involved in B. cenocepacia biofilm formation in flow chambers, and we identify a novel Bcam1349/c-di-GMP-regulated exopolysaccharide gene cluster which is essential for B. cenocepacia biofilm formation. Overproduction of Bcam1349 in trans promotes wrinkly colony morphology, pellicle, and biofilm formation in B. cenocepacia. A screen for transposon mutants unable to respond to the overproduction of Bcam1349 led to the identification of a 12-gene cluster, Bcam1330–Bcam1341, the products of which appear to be involved in the production of a putative biofilm matrix exopolysaccharide and to be essential for flow-chamber biofilm formation. We demonstrate that Bcam1349 binds to the promoter region of genes in the Bcam1330–Bcam1341 cluster and that this binding is enhanced by the presence of c-di-GMP. Furthermore, we demonstrate that overproduction of both c-di-GMP and Bcam1349 leads to increased transcription of these genes, indicating that c-di-GMP and Bcam1349 functions together in regulating exopolysaccharide production from the Bcam1330–Bcam1341 gene cluster. Our results suggest that the product encoded by the Bcam1330–Bcam1341 gene cluster is a major exopolysaccharide that provides structural stability to the biofilms formed by B. cenocepacia, and that its production is regulated by c-di-GMP through binding to and promotion of the activity of the transcriptional regulator Bcam1349.
Biofilm; Burkholderia cenocepacia; c-di-GMP; exopolysaccharide
Biofilm formation and dispersal in the black rot pathogen Xanthomonas campestris pathovar campestris (Xcc) is influenced by a number of factors. The extracellular mannanase ManA has been implicated in biofilm dispersal whereas biofilm formation requires a putative glycosyl transferase encoded by the xag gene cluster. Previously we demonstrated that the post-transcriptional regulator RsmA exerts a negative regulatory influence on biofilm formation in Xcc. Here we address the mechanisms by which RsmA exerts this action. We show that RsmA binds to the transcripts of three genes encoding GGDEF domain diguanylate cyclases to influence their expression. Accordingly, mutation of rsmA leads to an increase in cellular levels of cyclic di-GMP. This effect is associated with a down-regulation of transcription of manA, but an upregulation of xag gene transcription. Mutation of clp, which encodes a cyclic di-GMP-responsive transcriptional regulator of the CRP-FNR family, has similar divergent effects on the expression of manA and xag. Nevertheless Clp binding to manA and xag promoters is inhibited by cyclic di-GMP. The data support the contention that, in common with other CRP-FNR family members, Clp can act as both an activator and repressor of transcription of different genes to influence biofilm formation as a response to cyclic di-GMP.
The Gram-negative bacteria Pseudomonas aeruginosa and Burkholderia cenocepacia are opportunistic human pathogens that are responsible for severe nosocomial infections in immunocompromised patients and those suffering from cystic fibrosis (CF). These two bacteria have been shown to form biofilms in the airways of CF patients that make such infections more difficult to treat. Only recently have scientists begun to appreciate the complicated interplay between microorganisms during polymicrobial infection of the CF airway and the implications they may have for disease prognosis and response to therapy.
To gain insight into the possible role that interaction between strains of P. aeruginosa and B. cenocepacia may play during infection, we characterised co-inoculations of in vivo and in vitro infection models. Co-inoculations were examined in an in vitro biofilm model and in a murine model of chronic infection. Assessment of biofilm formation showed that B. cenocepacia positively influenced P. aeruginosa biofilm development by increasing biomass. Interestingly, co-infection experiments in the mouse model revealed that P. aeruginosa did not change its ability to establish chronic infection in the presence of B. cenocepacia but co-infection did appear to increase host inflammatory response.
Taken together, these results indicate that the co-infection of P. aeruginosa and B. cenocepacia leads to increased biofilm formation and increased host inflammatory response in the mouse model of chronic infection. These observations suggest that alteration of bacterial behavior due to interspecies interactions may be important for disease progression and persistent infection.
We assessed metabolic changes for darunavir/ritonavir (DRV/r) once daily (qd) versus atazanavir/ritonavir (ATV/r) qd with fixed-dose tenofovir/emtricitabine. This was a phase 4, multicenter, open-label, randomized exploratory study. Treatment-naive, HIV-1-infected adults received DRV/r 800/100 mg qd or ATV/r 300/100 mg qd, both with emtricitabine/tenofovir 200/300 mg qd. Primary end point: change in triglyceride levels from baseline to week 12. Secondary end points: week 12 and week 48 changes in lipid parameters, insulin sensitivity, inflammatory/coagulation/bacterial translocation biomarkers, viral load, CD4+ cell count, and week 48 changes in adipose tissue distribution and subjects' perceptions of body changes. In the DRV/r arm, 32/34 and 29/34 subjects completed weeks 12 and 48, respectively; in the ATV/r arm, 30/31 and 25/31 subjects completed weeks 12 and 48, respectively. Small changes in lipid parameters from baseline to weeks 12 and 48 were observed in both arms. Differences were noted between arms in mean changes in total cholesterol (DRV/r, 20.3 mg/dl; ATV/r, 4.6 mg/dl) and apolipoprotein A1 (DRV/r, 10.7 mg/dl; ATV/r, –0.7 mg/dl) at week 12. At week 48, no clinically relevant differences between arms were noted for changes in any lipid parameter, fasting glucose, or insulin sensitivity. Biomarkers generally decreased and efficacy parameters improved in both arms over 48 weeks. Changes in adipose tissue were small and comparable between arms. Subjects' perceptions of body changes generally improved in both study arms. This first pilot comparison in HIV-1-infected subjects suggests that DRV/r has a metabolic profile similar to ATV/r over 48 weeks of treatment. Further randomized studies are warranted.
The synthesis of virulence factors by pathogenic bacteria is highly regulated and occurs in response to diverse environmental cues. An array of two component systems (TCSs) serves to link perception of different cues to specific changes in gene expression and/or bacterial behaviour. Those TCSs that regulate functions associated with virulence represent attractive targets for interference in anti-infective strategies for disease control. We have previously identified PA2572 as a putative response regulator required for full virulence of Pseudomonas aeruginosa, the opportunistic human pathogen, to Galleria mellonella (Wax moth) larvae. Here we have investigated the involvement of candidate sensors for signal transduction involving PA2572. Mutation of PA2573, encoding a probable methyl-accepting chemotaxis protein, gave rise to alterations in motility, virulence, and antibiotic resistance, functions which are also controlled by PA2572. Comparative transcriptome profiling of mutants revealed that PA2572 and PA2573 regulate expression of a common set of 49 genes that are involved in a range of biological functions including virulence and antibiotic resistance. Bacterial two-hybrid analysis indicated a REC-dependent interaction between PA2572 and PA2573 proteins. Finally expression of PA2572 in the PA2573 mutant background restored virulence to G. mellonella towards wild-type levels. The findings indicate a role for the orphan chemotaxis sensor PA2573 in the regulation of virulence and antibiotic tolerance in P. aeruginosa and indicate that these effects are exerted in part through signal transduction involving PA2572.
We sought to review the current literature with regards to future risks of hemorrhage following cerebral revascularization in Moyamoya disease (MMD).
We performed a comprehensive literature review using PubMed to inspect the available data on the risk of hemorrhage after revascularization in MMD.
In this review, we identify the risk factors associated with hemorrhage in MMD both before and after cerebral revascularization. We included proposed pathophysiology of the hemorrhagic risk, role of the type of bypass performed, treatment options, and future needs for investigation.
The published cases and series of MMD treatment do show a risk of hemorrhage after treatment with either direct or indirect bypass both in the immediate as well as long-term future. While there are no discernible patterns in the rate of these hemorrhages, there is Class III evidence for the predictive effect of multiple microbleeds on preoperative imaging. Also, whereas revascularization, both direct and indirect, has been shown to reduce ischemic complications from MMD, there is not an association with the risk of hemorrhage after the procedure. Further studies need to be performed to help evaluate what the risk factors are and how to counsel patients as to the long-term outlook of this disease process.
Angioarchitecture; cerebral hemorrhage; cerebral perfusion; moyamoya
Gender-based differences in lipids have been noted in antiretroviral clinical trials. We present the metabolic and anthropometric data from the GRACE (Gender, Race And Clinical Experience) study by gender. Treatment-experienced adults received darunavir/ritonavir (DRV/r) 600/100 mg twice daily, plus a background regimen, over 48 weeks. Fasting blood samples were obtained for lipid, glucose, and insulin measurements at baseline and at weeks 24 and 48/early discontinuation. Anthropometric measurements were taken at baseline and at weeks 12, 24, and 48/discontinuation. The Assessment of Body Change and Distress questionnaire was administered at baseline and regular intervals. Descriptive statistics as well as comparisons using a Wilcoxon rank-sum test are reported. Four hundred twenty-nine patients (women, n=287; men, n=142) enrolled in GRACE; 94 women (32.8%) and 33 men (23.2%) discontinued the trial. Median changes in triglycerides from baseline to week 48 were higher in men (25 mg/dL versus 8 mg/dL; p=0.006); the mean change in triglycerides was higher in men than in women in all racial subgroups. Other lipid and glucose level changes were similar between genders. Anthropometric parameters increased for both genders, with larger increases in women. Patients' perceptions of body changes concurred with physical measurements. The proportion of women who were “satisfied” or “very satisfied” with their bodies increased from 45.2% to 57.8% from baseline to week 48 (p=0.005), while the proportion of men who were “satisfied” or “very satisfied” with their bodies increased from 56.3% to 61.5% from baseline to week 48 (p=0.317). DRV/r-based therapy was associated with small to moderate changes in metabolic parameters, and few between-gender differences were observed. Levels of self-reported, body-related distress improved for women and men over 48 weeks.
Treatment of cerebral tumors and peritumoral brain edema remains a clinical challenge and is associated with high morbidity and mortality. Dexamethasone (DEX) is an effective drug to treat brain edema, but is associated with well-described side effects. Corticorelin acetate (Xerecept) or human corticotrophin releasing factor (hCRF) is a comparatively new drug and was evaluated in two orthotopic glioma models (U87 and C6), by a direct comparison with dexamethasone and temozolomide.
In vitro mono- and combination-treatments showed a variable response in 6 different glioma cell lines. In vivo studies showed a dose-dependent effect of hCRF (0.03 and 0.1 mg/kg/q12h) on survival of U87 intracranial xenograft-bearing animals [median survival: control 41 days (95% CI 25–61 d); “low-hCRF” 74.5 d (95% CI 41–88 d); “high-hCRF” >130 d (95% CI not reached)]. Dexamethasone treatment had no effect on survival, but significant toxicity was observed. A survival benefit was observed with TMZ and TMZ + hCRF - treated animals, but with significant TMZ toxicity. C6-bearing animals showed no survival benefit, but similar treatment toxicities. The difference in hCRF-treatment response between U87- and C6-intracranial gliomas can be explained by a difference in receptor expression. RT-PCR identified CRF2r mRNA in U87-xenografts; no CRF-receptors were identified in C6-xenografts.
HCRF was more effective than either dexamethasone or temozolomide in the treatment of U87 xenografts, with long-term survivors and only mild toxicity. HCRF therapeutic efficacy appears to be dependent on tumor hCRF-receptor expression. These results support further clinical assessment hCRF therapeutic efficacy and levels of CRFr expression in different human gliomas.
glioma; corticotropin-releasing factor; dexamethasone
Using a novel approach for analysis of TRPC channel activity, we report here that NSAIDs are involved into regulation of TRPC channels in the podocytes of the freshly isolated decapsulated glomeruli. Fluorescence and electron microscopy techniques confirmed the integrity of podocytes in the glomeruli. Western blotting showed that TRPC1,3 and 6 are highly expressed in the glomeruli. Single-channel patch clamp analysis revealed cation currents with distinct TRPC properties. This is the first report describing single TRPC-like currents in glomerular podocytes. Furthermore, our data provide a novel mechanism of NSAIDs regulation of TRPC channels, which might be implicated in maintaining the glomerular filtration barrier.
TRPC6; podocyte; FSGS; focal segmental glomerulosclerosis
Stenotrophomonas maltophilia encodes proteins related to the Rax proteins of Xanthomonas oryzae, which are required for the synthesis and secretion of the Ax21 protein. Here we show that Ax21 acts as a cell-cell signal to regulate a diverse range of functions, including virulence, in this nosocomial pathogen.
Mantle cell lymphoma (MCL) is a pre-germinal center neoplasm characterized by cyclin D1 overexpression resulting from t(11;14)(q13;q32). Since MCL is incurable with standard lymphoma therapies, new treatment approaches are needed that target specific biologic pathways. In the present study, we investigated a novel drug delivery nanovehicle enriched with the bioactive polyphenol, curcumin (curcumin nanodisks; curcumin-ND). Cells treated with curcumin-ND showed a dose-dependent increase in apoptosis. This was accompanied by enhanced generation of reactive oxygen species (ROS). The antioxidant, N-acetylcysteine, inhibited curcumin-ND induced apoptosis, suggesting that ROS generation plays a role in curcumin action on MCL cells. Curcumin-ND decreased cyclin D1, pAkt, pIκBα, and Bcl2 protein. In addition, enhanced FoxO3a and p27 expression as well as caspase-9, -3, and poly(ADP-ribose) polymerase (PARP) cleavage were observed. Curcumin-ND treatment led to enhanced G1 arrest in two cultured cell models of MCL.
Nanodisks; mantle cell lymphoma; curcumin; cell cycle; apoptosis
Xanthomonas is a large genus of bacteria that collectively cause disease on more than 300 plant species. The broad host range of the genus contrasts with stringent host and tissue specificity for individual species and pathovars. Whole-genome sequences of Xanthomonas campestris pv. raphani strain 756C and X. oryzae pv. oryzicola strain BLS256, pathogens that infect the mesophyll tissue of the leading models for plant biology, Arabidopsis thaliana and rice, respectively, were determined and provided insight into the genetic determinants of host and tissue specificity. Comparisons were made with genomes of closely related strains that infect the vascular tissue of the same hosts and across a larger collection of complete Xanthomonas genomes. The results suggest a model in which complex sets of adaptations at the level of gene content account for host specificity and subtler adaptations at the level of amino acid or noncoding regulatory nucleotide sequence determine tissue specificity.
Nanodisks (ND) are nanoscale, disk-shaped phospholipid bilayers whose edge is stabilized by apolipoproteins. In the present study, ND were formulated with the bioactive polyphenol, curcumin, at a 6:1 phospholipid:curcumin molar ratio. Atomic force microscopy revealed that curcumin-ND are particles with diameters <50 nm and thickness of a phospholipid bilayer. When formulated in ND, curcumin is water-soluble and gives rise to a characteristic absorbance spectrum with a peak centered at 420 nm. Fluorescence spectroscopy of curcumin-ND provided evidence of self-quenching. Incubation of curcumin-ND with empty-ND relieved the self-quenching, indicating redistribution of curcumin between curcumin loaded- and empty-ND. In HepG2 cells, curcumin-ND mediated enhanced cell growth inhibition compared to free curcumin. In a cell culture model of mantle cell lymphoma, curcumin-ND were a more potent inducer of apoptosis than free curcumin. The nanoscale size of the complexes, combined with their ability to solubilize curcumin, indicates ND may have in vivo therapeutic applications.
Curcumin; Nanodisk; Phospholipid; Apolipoprotein; Delivery
Objectives. Evaluation of pharmacokinetics and pharmacodynamics of darunavir and etravirine among HIV-1–infected, treatment-experienced adults from GRACE, by sex and race.
Methods. Patients received darunavir/ritonavir 600/100mg twice daily plus other antiretrovirals, which could include etravirine 200mg twice daily. Population pharmacokinetics for darunavir and etravirine were determined over 48 weeks and relationships assessed with virologic response and safety. Rich sampling for darunavir, etravirine, and ritonavir was collected in a substudy at weeks 4, 24, and 48.
Results. Pharmacokinetics were estimated in 376 patients for darunavir and 190 patients for etravirine. Median darunavir AUC12h and C0h were 60,642ng·h/mL and 3624ng/mL, respectively; and for etravirine were 4183ng · h/mL and 280ng/mL, respectively. There were no differences in darunavir or etravirine AUC12h or C0h by sex or race. Age, body weight, or use of etravirine did not affect darunavir exposure. No relationships were seen between darunavir pharmacokinetics and efficacy or safety. Patients with etravirine exposure in the lowest quartile generally had lower response rates. Rich sampling showed no time-dependent relationship for darunavir, etravirine, or ritonavir exposure over 48 weeks. Conclusions. Population pharmacokinetics showed no relevant differences in darunavir or etravirine exposure by assessed covariates. Lower etravirine exposures were associated with lower response rates.
GPIHBP1, a glycosylphosphatidylinositol-anchored Ly6 protein of capillary endothelial cells, binds lipoprotein lipase (LPL) avidly, but its ability to bind related lipase family members has never been evaluated. We sought to define the ability of GPIHBP1 to bind other lipase family members as well as other apolipoproteins and lipoproteins.
Methods and Results
As judged by cell-based and cell-free binding assays, LPL binds to GPIHBP1 but other members of the lipase family do not. We also examined the binding of apoAV–phospholipid disks to GPIHBP1. ApoAV binds avidly to GPIHBP1-transfected cells; this binding requires GPIHBP1’s amino-terminal acidic domain and is independent of its cysteine-rich Ly6 domain (the latter domain is essential for LPL binding). GPIHBP1-transfected cells did not bind HDL. Chylomicrons binds avidly to GPIHBP1-transfected CHO cells, but this binding is dependent on GPIHBP1’s ability to bind LPL within the cell culture medium.
GPIHBP1 binds LPL but does not bind other lipase family members. GPIHBP1 binds apoAV but did not bind apoAI or HDL. The ability of GPIHBP1-transfected CHO cells to bind chylomicrons is mediated by LPL; chylomicron binding does not occur unless GPIHBP1 first captures LPL from the cell culture medium.
lipoprotein lipase; chylomicronemia; hypertriglyceridemia; GPIHBP1
Apolipoprotein (apo) E has a storied history as a lipid transport protein. The integral association between cholesterol homeostasis and lipoprotein clearance from circulation are intimately related to apoE's function as a ligand for cell surface receptors of the low density lipoprotein receptor family. The receptor binding properties of apoE are strongly influenced by isoform specific amino acid differences as well as the lipidation state of the protein. As understanding of apoE as a structural component of circulating plasma lipoproteins has evolved, exciting developments in neurobiology have revitalized interest in apoE. The strong and enduring correlation between the apoE4 isoform and age of onset and increased risk of Alzheimer's disease has catapulted apoE to the forefront of neurobiology. Using genetic tools generated for study of apoE lipoprotein metabolism, transgenic “knock-in” and gene-disrupted mice are now favored models for study of its role in a variety of neurodegenerative diseases. Key structural knowledge of apoE and isoform specific differences is driving research activity designed to elucidate how a single amino acid change can manifest such profoundly significant pathological consequences. This review describes apoE through a lens of structure-based knowledge that leads to hypotheses that attempt to explain the functions of apoE and isoform specific effects relating to disease mechanism.
Apolipoprotein E; cholesterol; cardiovascular disease; neurobiology; Alzheimer's disease
Apolipoprotein A-V (apoA-V), a minor protein associated with lipoproteins, has a major effect on triacylglycerol (TG) metabolism. We investigated whether apoA-V complexed with phospholipid in the form of a reconstituted HDL (rHDL) has potential utility as a therapeutic agent for treatment of hypertriglyceridemia when delivered intravenously.
Methods and Results
Intravenous injection studies were performed in genetically engineered mouse models of severe hypertriglyceridemia including apoav-/- and gpihbp1-/- mice. Administration of apoA-V rHDL to hypertriglyceridemic apoav−/− mice resulted in a 60% reduction in plasma TG concentration after 4 h. This decline can be attributed to enhanced catabolism/clearance of VLDL where VLDL TG and cholesterol were reduced ∼60%. ApoA-V which associated with VLDL after injection was also rapidly cleared. Site-specific mutations in the heparin-binding region of apoA-V (amino acids 186-227) attenuated apoA-V rHDL TG-lowering activity by 50% suggesting this sequence element is required for optimal TG-lowering activity in vivo. Unlike apoav-/- mice, injection of apoA-V rHDL into gpihbp1-/- mice had no effect on plasma TG levels and apoA-V remained associated with plasma VLDL.
Intravenously injected apoA-V rHDL significantly lowers plasma TG in an apoA-V deficient mouse model. Its intravenous administration may have therapeutic benefit in human subjects with severe HTG, especially in cases involving apoA-V variants associated with HTG.
apoav-/- mice; gpihbp1-/- mice; very low density lipoproteins; apoA-V heparin binding mutant; lipoprotein lipase