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1.  High-Density Lipoprotein Maintains Skeletal Muscle Function by Modulating Cellular Respiration in Mice 
Circulation  2013;128(22):2364-2371.
Background
Abnormal glucose metabolism is a central feature of disorders with increased rates of cardio-vascular disease (CVD). Low levels of high density lipoprotein (HDL) are a key predictor for CVD. We used genetic mouse models with increased HDL levels (apoA-I tg) and reduced HDL levels (apoA-I ko) to investigate whether HDL modulates mitochondrial bioenergetics in skeletal muscle.
Methods and Results
ApoA-I ko mice exhibited fasting hyperglycemia and impaired glucose tolerance test (GTT) compared to wild type (wt) mice. Mitochondria isolated from gastrocnemius muscle of apoA-I ko mice displayed markedly blunted ATP synthesis. Endurance capacity (EC) during exercise exhaustion test was impaired in apoA-I ko mice. HDL directly enhanced glucose oxidation by increasing glycolysis and mitochondrial respiration rate (OCR) in C2C12 muscle cells. ApoA-I tg mice exhibited lower fasting glucose levels, improved GTT, increased lactate levels, reduced fat mass, associated with protection against age-induced decline of EC compared to wt mice. Circulating levels of fibroblast growth factor 21 (FGF21), a novel biomarker for mitochondrial respiratory chain deficiencies and inhibitor of white adipose lipolysis, were significantly reduced in apoA-I tg mice. Consistent with an increase in glucose utilization of skeletal muscle, genetically increased HDL and apoA-I levels in mice prevented high fat diet-induced impairment of glucose homeostasis.
Conclusions
In view of impaired mitochondrial function and decreased HDL levels in T2D, our findings indicate that HDL-raising therapies may preserve muscle mitochondrial function and address key aspects of T2D beyond CVD.
doi:10.1161/CIRCULATIONAHA.113.001551
PMCID: PMC3957345  PMID: 24170386
HDL; obesity; exercise; muscular glucose oxidation; mitochondrial bioenergetics
2.  Cholesterol efflux and atheroprotection: Advancing the concept of reverse cholesterol transport 
Circulation  2012;125(15):1905-1919.
doi:10.1161/CIRCULATIONAHA.111.066589
PMCID: PMC4159082  PMID: 22508840
High density lipoprotein; cholesterol efflux; reverse cholesterol transport; atherosclerosis; cardiovascular disease; vascular imaging
3.  The Effects of Type 2 Diabetes on Lipoprotein Composition and Arterial Stiffness in Male Youth 
Diabetes  2013;62(8):2958-2967.
Recent studies suggest HDL exists as numerous subpopulations with distinct protein/lipid compositions that are not reflected in the HDL cholesterol (HDL-C) number. In this study, we sought to evaluate HDL subpopulations in adolescents with type 2 diabetes (T2D) to determine if changes in HDL composition are associated with early vascular disease. T2D (n = 10), lean (n = 9), and obese (n = 11) youth were recruited. Plasma was fractionated using gel-filtration chromatography, and lipid-associated proteins were identified using mass spectrometry. Concurrently, vascular stiffness was assessed using pulse wave velocity (PWV). We found youth with T2D exhibited decreased phospholipid content in fractions containing large HDL particles that was inversely associated with PWV (P < 0.001). No association was noted between HDL-C and PWV. Proteomic analysis revealed changes in 7 of 45 identified proteins in the T2D group, including apolipoprotein (apo) A-II, apoE, and paraoxonase-1 (P < 0.05). Our data demonstrate early changes in the lipid and protein compositions of specific HDL subspecies in adolescents with T2D that are related to early markers of arterial disease. These findings suggest that analyzing the composition of HDL, rather than HDL-C, may be useful in assessing cardiovascular risk in this population.
doi:10.2337/db12-1753
PMCID: PMC3717874  PMID: 23835332
4.  Specific sequences in N termini of apolipoprotein A-IV modulate its anorectic effect 
Physiology & behavior  2013;120:136-142.
Rodent apoA-IV is expressed predominantly in small intestine and also expressed to a small extent in liver and hypothalamus. ApoA-IV has been shown to inhibit food intake in rats when injected centrally. In the current study, we hypothesize that a specific sequence within rat apoA-IV is responsible for mediating the anorectic effect. We use a bacterial expression system to generate truncation mutants (Δ249–371, Δ117–371 and Δ1–61) of rat apoA-IV and assess the ability of various regions of the molecule to inhibit food intake. The results indicate that a responsible sequence exists within the N-terminal 61 amino acids of rat apoA-IV. Synthetic peptides (1–30 EVTSDQVANVMWDYFTQLSNNAKEAVEQLQ, 1–15 EVTSDQVANVMWDYF and 17–30 QLSNNAKEAVEQLQ) were used to specify the region in between residues 1 and 30. A 14-mer peptide (17–30) encompassing this sequence was capable of reducing food intake in a dose-dependent manner whereas a peptide designed on a more C-terminal region (211–232) of apoA-IV (QEKLNHQMEGLAFQMKKNAEEL) failed to exhibit the dose-dependent anorectic effect. The isolation of this sequence provides a valuable tool for future work directed at identifying apoA-IV binding proteins and is a key step for exploring the potential of therapeutic manipulation of food intake via this pathway.
doi:10.1016/j.physbeh.2013.07.010
PMCID: PMC4054808  PMID: 23911688
Apolipoprotein A-IV; Food intake; Truncation mutation
5.  Eye movement recordings to investigate a supranuclear component in chronic progressive external ophthalmoplegia: a cross-sectional study 
The British journal of ophthalmology  2010;94(9):1165-1168.
Background
It has been postulated that eye movement disorders in chronic progressive external ophthalmoplegia (CPEO) have a neurological as well as a myopathic component to them.
Aim
To investigate whether there is a supranuclear component to eye movement disorders in CPEO using eye movement recordings.
Methods
Saccade and smooth pursuit (SP) characteristics together with vestibulo-ocular reflex (VOR) gain and VOR suppression (VORS) gain in 18 patients with CPEO and 34 normal patients were measured using Eyelink II video-oculography.
Results
The asymptotic values of the peak velocity main sequence curves were reduced in the CPEO group compared to those of normal patients, with a mean of 161°/s (95% CI 126°/s to 197°/s) compared with 453°/s (95% CI 430 to 475°/s), respectively. Saccadic latency was longer in CPEO (263 ms; 95% CI 250 to 278), compared to controls (185 ms; 95% CI 181 to 189). Smooth pursuit and VOR gains were impaired in CPEO, although this could be explained by non-supranuclear causes. VORS gain was identical in the two groups.
Conclusions
This study does not support a supranuclear component to the ophthalmoplegia of CPEO, although the increased latencies observed may warrant further investigation.
doi:10.1136/bjo.2009.165639
PMCID: PMC4038492  PMID: 20576783
6.  Improving the diffraction of apoA-IV crystals through extreme dehydration 
Apolipoprotein A-IV crystals consisted of a long unit-cell edge (540 Å) with a high mosaic spread, making them intractable for X-ray diffraction analysis. Extreme dehydration in 60% PEG 3350 was utilized as a post-crystallization treatment as well a screening method to significantly sharpen the mosaic spread and increase the overall resolution of diffraction.
Apolipoproteins are the protein component of high-density lipoproteins (HDL), which are necessary for mobilizing lipid-like molecules throughout the body. Apolipoproteins undergo self-association, especially at higher concentrations, making them difficult to crystallize. Here, the crystallization and diffraction of the core fragment of apolipoprotein A-IV (apoA-IV), consisting of residues 64–335, is presented. ApoA-IV64–335 crystallized readily in a variety of hexagonal (P6) morphologies with similar unit-cell parameters, all containing a long axis of nearly 550 Å in length. Preliminary diffraction experiments with the different crystal morphologies all resulted in limited streaky diffraction to 3.5 Å resolution. Crystal dehydration was applied to the different morphologies with variable success and was also used as a quality indicator of crystal-growth conditions. The results show that the morphologies that withstood the most extreme dehydration conditions showed the greatest improvement in diffraction. One morphology in particular was able to withstand dehydration in 60% PEG 3350 for over 12 h, which resulted in well defined intensities to 2.7 Å resolution. These results suggest that the approach of integrating dehydration with variation in crystal-growth conditions might be a general technique to optimize diffraction.
doi:10.1107/S1744309111048706
PMCID: PMC3253848  PMID: 22232185
apolipoproteins; dehydration; long unit cell; reflection elongation
7.  Apolipoprotein A-I structural organization in high density lipoproteins isolated from human plasma 
High density lipoproteins (HDL) mediate cholesterol transport and protection from cardiovascular disease. Although synthetic HDLs have been studied for 30 years, the structure of human plasma-derived HDL, and its major protein apolipoprotein (apo)A-I, is unknown. We separated normal human HDL into 5 density subfractions and then further isolated those containing predominantly apoA-I (LpA-I). Using cross-linking chemistry and mass spectrometry, we found that apoA-I adopts a structural framework in these particles that closely mirrors that in synthetic HDL. We adapted established structural models for synthetic HDL to generate the first detailed models of authentic human plasma HDL in which apoA-I adopts a symmetrical cage-like structure. The models suggest that HDL particle size is modulated via a twisting motion of the resident apoA-I molecules. This understanding offers insights into how apoA-I structure modulates HDL function and its interactions with other apolipoproteins.
doi:10.1038/nsmb.2028
PMCID: PMC3079355  PMID: 21399642
8.  High density lipoprotein: it’s not just about lipid transport anymore 
Plasma levels of high density lipoprotein cholesterol (HDL-C) have long been associated with protection against cardiovascular disease (CVD) in large populations. However, HDL-C has been significantly less useful for predicting CVD risk in individual patients. This has ignited a new debate on the merits of measuring HDL quantity versus quality in terms of protective potential. In addition, numerous recent studies have begun to uncover HDL functions that vary surprisingly from traditional lipid transport roles. In this paper, we review recent findings that point to important functions for HDL that go well beyond lipid transport. These discoveries suggest that HDL might be a platform that mediates protection from a host of disease states ranging from CVD to diabetes to infectious disease.
doi:10.1016/j.tem.2010.10.001
PMCID: PMC3036841  PMID: 21067941
9.  Proteomic Characterization of Human Plasma High Density Lipoprotein Fractionated by Gel Filtration Chromatography 
Journal of proteome research  2010;9(10):5239-5249.
Plasma levels of high density lipoprotein cholesterol (HDL-C) are inversely proportional to the incidence of cardiovascular disease. Recent applications of modern proteomic technologies have identified upward of 50 distinct proteins associated with HDL particles with many of these newly discovered proteins implicating HDL in nonlipid transport processes including complement activation, acute phase response and innate immunity. However, almost all MS-based proteomic studies on HDL to date have utilized density gradient ultracentrifugation techniques for HDL isolation prior to analysis. These involve high shear forces and salt concentrations that can disrupt HDL protein interactions and alter particle function. Here, we used high-resolution size exclusion chromatography to fractionate normal human plasma to 17 phospholipid-containing subfractions. Then, using a phospholipid binding resin, we identified proteins that associate with lipoproteins of various sizes by electrospray ionization mass spectrometry. We identified 14 new phospholipid-associated proteins that migrate with traditionally defined HDL, several of which further support roles for HDL in complement regulation and protease inhibition. The increased fractionation inherent to this method allowed us to visualize HDL protein distribution across particle size with unprecedented resolution. The observed heterogeneity across subfractions suggests the presence of HDL particle subpopulations each with distinct protein components that may prove to impart distinct physiological functions.
doi:10.1021/pr100520x
PMCID: PMC3100175  PMID: 20718489
high density lipoprotein; proteomics; lipoprotein; apolipoprotein; mass spectrometry
10.  Enhanced placental cholesterol efflux by fetal HDL in Smith–Lemli–Opitz syndrome 
Molecular genetics and metabolism  2008;94(2):240-247.
Previous studies from this laboratory have shown that maternal-derived cholesterol can be effluxed from trophoblasts to fetal HDL and plasma. We had the opportunity to study for the first time the ability of HDL and plasma from a fetus with the Smith–Lemli–Opitz syndrome (SLOS) to efflux cholesterol from trophoblasts. It was unclear whether cholesterol could be effluxed to fetuses with SLOS since lipoprotein levels are often very low. To answer this question, cord blood was collected from the placentas of an SLOS fetus and unaffected fetuses just after delivery. Plasma cholesterol concentrations were very low in the affected fetus; cholesterol, 7-dehydrocholesterol, and 8-dehydocholesterol concentrations were 14.1, 4.5, and 5.2 mg/dl, respectively. The HDL from the fetal SLOS effluxed ≈50% more cholesterol from a trophoblast cell line, were smaller in size, and had a lower cholesterol to phospholipid ratio as compared to HDL from unaffected fetuses or adults. Plasma from the SLOS fetus effluxed cholesterol to a similar percentage as unaffected fetal plasma or adult plasma, possibly due to fewer HDL particles as demonstrated in previous SLOS patients. These novel data demonstrate that the cholesterol-deficient SLOS fetus is able to obtain cholesterol from trophoblasts at a time when cholesterol is playing a critical role in development, and has implications for design of treatments for cholesterol deficiency syndromes as well as understanding of prenatal cholesterol transport in humans.
doi:10.1016/j.ymgme.2008.01.015
PMCID: PMC3037116  PMID: 18346920
Fetus; Trophoblast; BeWo cells; Pregnancy; Cholesterol transport
11.  The Role of Hydrophobic and Negatively Charged Surface Patches of Lipid-Free Apolipoprotein A-I in Lipid Binding and ABCA1-Mediated Cholesterol Efflux 
Biochimica et biophysica acta  2009;1801(1):64-69.
Summary
Recent models of lipid-free apolipoprotein A-I, including a cross-link/homology model and an x-ray crystal structure have identified two potential functionally relevant “patches” on the protein surface. The first is a hydrophobic surface patch composed of leucine residues 42, 44, 46, and 47 and the second a negatively charged patch composed of glutamic acid residues 179, 191, and 198. To determine if these domains play a functional role, these surface patches were disrupted by site-directed mutagenesis and the bacterially expressed mutants were compared with respect to their ability to bind lipid and stimulate ABCA1-mediated cholesterol efflux. It was found that neither patch plays a significant functional role in the ability of apoA-I to accept cholesterol in an ABCA1-dependent manner, but that the hydrophobic patch did affect the ability of apoA-I to clear DMPC liposomes. Interestingly, contrary to previous predictions, disruption of the hydrophobic surface patch enhanced the lipid binding ability of apoA-I. The hydrophobic surface patch may be important to the structural stability of the lipid binding regions of apoA-I, or may be a necessary permissive structural element for lipid binding.
doi:10.1016/j.bbalip.2009.09.012
PMCID: PMC2787660  PMID: 19782154
apolipoprotein A-I; ATP binding cassette transporter A1; functionality; surface patch; lipid-free model; lipid-binding
12.  High-Density Lipoprotein Proteomics: Identifying New Drug Targets and Biomarkers by Understanding Functionality 
Recent proteomics studies on human plasma high-density lipoprotein (HDL) have discovered up to 50 individual protein constituents. Many of these have known functions that vary surprisingly from the lipid transport roles commonly thought to mediate HDL’s ability to protect from coronary artery disease. Given newly discovered roles in inflammation, protease inhibition, complement regulation, and innate immunity, many have begun to view HDL as a broad collection of distinct particle subfamilies, each distinguished by unique protein compositions and functions. Herein we review recent applications of high-resolution proteomics to HDL and summarize evidence supporting the idea of HDL functional subspeciation. These studies have set the stage for a more complete understanding of the molecular basis of HDL functional heterogeneity and hold promise for the identification of new biomarkers that can predict disease or evaluate the success of clinical interventions.
doi:10.1007/s12170-009-0069-9
PMCID: PMC2901108  PMID: 20625533
High density lipoprotein; Proteomics; Mass spectrometry; Lipoprotein; Apolipoprotein; Reverse cholesterol transport; Cardiovascular disease; Protein
13.  Up-regulation of apolipoprotein E by leptin in the hypothalamus of mice and rats 
Physiology & behavior  2009;98(1-2):223-228.
Apolipoprotein E (apoE) is a satiation factor, playing an important role in the regulation of food intake and body weight. We previously reported that apoE was present in the hypothalamus, but it is unclear which type of the cells in this brain area expressing apoE. In addition, hypothalamic apoE mRNA levels were significantly reduced in both genetically obese ob/ob (leptin deficient) mice and high-fat diet-induced obese (leptin resistant) rats, raising the possibility that deficient leptin signaling might be related to the change in apoE gene expression. In the present studies, using double-staining immunohistochemistry, we demonstrated that apoE is mainly present in astrocytes. To characterize the effect of leptin on apoE gene expression, ob/ob and db/db mice were treated with recombinant mouse leptin (3 μg/g daily, i.p.) or vehicle for 5 days. We found that the increased hypothalamic apoE mRNA levels occurred only in leptin-treated ob/ob, but not in pair-fed ob/ob, or db/db, mice, indicating that leptin up-regulated hypothalamic apoE gene expression depends upon an intact leptin receptor, and this effect is not related to the changes in food intake and body weight. The reduced apoE gene expression caused by fasting, which also results in relatively lower leptin level, is restored by intracerebroventricular administration of leptin. In addition, leptin was significantly less efficacious in apoE KO mice because these animals consumed more food and lost less weight following leptin treatment, compared with wild-type controls. These observations imply that apoE signaling, at least partially, mediates the inhibitory effects of leptin on feeding.
doi:10.1016/j.physbeh.2009.05.013
PMCID: PMC2757054  PMID: 19481557
apolipoprotein; food intake; obesity
14.  Autonomic dysfunction in dementia 
Background
There are no studies of autonomic function comparing Alzheimer's disease (AD), vascular dementia (VAD), dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD).
Aims
To assess cardiovascular autonomic function in 39 patients with AD, 30 with VAD, 30 with DLB, 40 with PDD and 38 elderly controls by Ewing's battery of autonomic function tests and power spectral analysis of heart rate variability. To determine the prevalence of orthostatic hypotension and autonomic neuropathies by Ewing's classification.
Results
There were significant differences in severity of cardiovascular autonomic dysfunction between the four types of dementia. PDD and DLB had considerable dysfunction. VAD showed limited evidence of autonomic dysfunction and in AD, apart from orthostatic hypotension, autonomic functions were relatively unimpaired. PDD showed consistent impairment of both parasympathetic and sympathetic function tests in comparison with controls (all p<0.001) and AD (all p<0.03). DLB showed impairment of parasympathetic function (all p<0.05) and one of the sympathetic tests in comparison with controls (orthostasis; p = 0.02). PDD had significantly more impairment than DLB in some autonomic parameters (Valsalva ratio: p = 0.024; response to isometric exercise: p = 0.002). Patients with VAD showed impairment in two parasympathetic tests (orthostasis: p = 0.02; Valsalva ratio: p = 0.08) and one sympathetic test (orthostasis: p = 0.04). These results were in contrast with AD patients who only showed impairment in one sympathetic response (orthostasis: p = 0.004). The prevalence of orthostatic hypotension and autonomic neuropathies was higher in all dementias than in controls (all p<0.05).
Conclusion
Autonomic dysfunction occurs in all common dementias but is especially prominent in PDD with important treatment implications.
doi:10.1136/jnnp.2006.102343
PMCID: PMC2117678  PMID: 17178816
15.  Proteomic analysis of defined HDL subpopulations reveals particle-specific protein clusters: Relevance to antioxidative function 
Objective
Recent proteomic studies have identified multiple proteins that co-isolate with human HDL. We hypothesized that distinct clusters of protein components may distinguish between physicochemically-defined subpopulations of HDL particles, and that such clusters may exert specific biological function(s).
Methods and Results
We investigated the distribution of proteins across five physicochemically-defined particle subpopulations of normolipidemic human HDL (HDL2b, 2a, 3a, 3b, 3c) fractionated by isopycnic density gradient ultracentrifugation. Liquid chromatography/electrospray mass spectrometry identified a total of 28 distinct HDL-associated proteins. Using an abundance pattern analysis of peptide counts across the HDL subfractions, these proteins could be grouped into 5 distinct classes. A more indepth correlational network analysis suggested the existence of distinct protein clusters, particularly in the dense HDL3 particles. Levels of specific HDL proteins, primarily apoL-I, PON1 and PON3, correlated with the potent capacity of HDL3 to protect LDL from oxidation.
Conclusions
These findings suggest that HDL is composed of distinct particles containing unique (apolipo)protein complements. Such subspeciation forms a potential basis for understanding the numerous observed functions of HDL. Further work using additional separation techniques will be required to define these species in more detail.
doi:10.1161/ATVBAHA.109.186031
PMCID: PMC2845307  PMID: 19325143
high density lipoprotein; mass spectrometry; compositional heterogeneity; proteome; oxidation
16.  Breast cancer risk in elderly women with systemic autoimmune rheumatic diseases: a population-based case–control study 
British Journal of Cancer  2009;100(5):817-821.
Systemic autoimmune rheumatic diseases (SARDs) are chronic inflammatory and immuno-modulatory conditions that have been suggested to affect cancer risk. Using the Surveillance, Epidemiology and End Results–Medicare-linked database, women aged 67–99 years and diagnosed with incident breast cancer in 1993–2002 (n=84 778) were compared with an equal number of age-matched cancer-free female controls. Diagnoses of SARDs, including rheumatoid arthritis (RA, n=5238), systemic lupus erythematosus (SLE, n=340), Sjogren's syndrome (n=374), systemic sclerosis (n=128), and dermatomyositis (n=31), were determined from claim files for individuals from age 65 years to 1 year before selection. Associations of SARD diagnoses with breast cancer, overall and by oestrogen receptor (ER) expression, were assessed using odds ratio (OR) estimates from multivariable logistic regression models. The women diagnosed with RA were less likely to develop breast cancer (OR=0.87, 95% confidence interval (CI)=0.82–0.93). The risk reduction did not differ by tumour ER-status (OR=0.83, 95% CI=0.78–0.89 for ER-positive vs OR=0.91, 95% CI=0.81–1.04 for ER-negative, P for heterogeneity=0.14). The breast cancer risk was not associated with any of the other SARDs, except for a risk reduction of ER-negative cases (OR=0.49, 95% CI=0.26–0.93) among women with SLE. These findings suggest that systemic inflammation may affect breast epithelial neoplasia.
doi:10.1038/sj.bjc.6604906
PMCID: PMC2651404  PMID: 19190628
rheumatic diseases; autoimmune diseases; breast cancer
17.  ApoA-I Induced CD31 in Bone Marrow-derived Vascular Progenitor Cells Increases Adhesion: Implications for Vascular Repair 
Biochimica et biophysica acta  2008;1781(11-12):703-709.
Transgenic over expression of apolipoprotein A-I (ApoA-I) the major structural apolipoprotein of HDL appears to convey the most consistent and strongest anti atherogenic effect observed in animal models so far. We tested the hypothesis that ApoA-I mediates its cardio protective effects additionally through ApoA-I induced differentiation of bone marrow derived progenitor cells in vitro. This study demonstrates that lineage negative bone marrow cells (lin−BMCs) alter and differentiate in response to free ApoA-I. We find that lin−BMCs in culture treated with recombinant free ApoA-I at a concentration of 0.4µM are twice as large in size and have altered cell morphology compared to untreated cells; untreated cells retain the original spheroid morphology. Further, the total number of CD31 positive cells in the ApoA-I treated population consistently increased by two fold. This phenotype was significantly reduced in untreated cells and points towards a novel ApoA-I dependent differentiation. A protein lacking its best lipid-binding region (ApoA-IΔ10) did not stimulate any changes in the lin−BMCs cells indicating that ApoA-I may mediate its effects by regulating cholesterol efflux. The increased CD31 correlates with an increased ability of the lin−BMCs to adhere to both fibronectin and Mouse Brain Endothelial Cells. Our results provide the first evidence that exogenous free ApoA-I has the capacity to change the characteristics of progenitor cell populations and suggests a novel mechanism by which HDL may mediate its cardiovascular benefits.
doi:10.1016/j.bbalip.2008.08.002
PMCID: PMC2752855  PMID: 18775511
ApoA-I; bone marrow cells (BMCs); CD31; lineage minus; vascular progenitor cell; adhesion
18.  Characterization of Apolipoprotein A-IV in Brain Areas Involved in Energy Homeostasis 
Physiology & behavior  2008;95(1-2):161-167.
Apolipoprotein A-IV (apo A-IV) is a satiation protein synthesized in the small intestine and hypothalamus. To further understand its anorectic mechanisms, we used immunohistochemical techniques to characterize the distribution of apo A-IV in brain areas involved in energy homeostasis. Dense apo A-IV staining was detected in the arcuate (ARC) and ventromedial hypothalamic nuclei with less staining in cells in the paraventricular and dorsomedial nuclei. In the brainstem, apo A-IV staining was found in the nucleus of the solitary tract. Double staining immunohistochemistry revealed co-existence of apo A-IV with Neuronal Nuclei (a neuronal marker), but less with glial fibrillary acidic protein (a glial marker), in ARC, suggesting that apo A-IV is largely present in neurons. In the ARC, apo A-IV was co-localized with pro-opiomelanocortin (POMC), and apo A-IV administration stimulated hypothalamic POMC gene expression, suggesting that the brain apo A-IV system suppresses food intake by stimulating the ARC POMC system. To ascertain whether the apo A-IV detected in the brain is derived from the circulation, 125I-labeled recombinant rat apo A-IV was intravenously injected into mice. No increase of radioactive apo A-IV was found in the brain, consistent with a lack of uptake of co-injected 99mTc-labeled albumin, indicating that circulating apo A-IV is unable to cross the blood brain barrier. These data collectively support the hypothesis that apo A-IV, produced by neuronal cells, may exert its anorectic action by interacting with catabolic regulatory neuropeptides.
doi:10.1016/j.physbeh.2008.05.022
PMCID: PMC2574991  PMID: 18577393
Apolipoproteins; immunohistochemistry; blood brain barrier; neuropeptides
19.  Rainbow Smelt (Osmerus mordax) Genomic Library and EST Resources 
Genomic resources in rainbow smelt (Osmerus mordax) enable us to examine the genome duplication process in salmonids and test hypotheses relating to the fate of duplicated genes. They further enable us to pursue physiological and ecological studies in smelt. A bacterial artificial chromosome library containing 52,410 clones with an average insert size of 146 kb was constructed. This library represents an 11-fold average coverage of the rainbow smelt (O. mordax) genome. In addition, several complementary deoxyribonucleic acid libraries were constructed, and 36,758 sequences were obtained and combined into 12,159 transcripts. Over half of these transcripts have been identified, several of which have been associated with cold adaptation. These basic resources show high levels of similarity (86%) to salmonid genes and provide initial support for genome duplication in the salmonid ancestor. They also facilitate identification of genes important to fish and direct us toward new technologies for other studies in fish biology.
doi:10.1007/s10126-008-9089-6
PMCID: PMC2516299  PMID: 18386095
cDNA; EST database; BAC library; Rainbow smelt
20.  Apolipoprotein A-IV inhibits experimental colitis 
Journal of Clinical Investigation  2004;114(2):260-269.
The antiatherogenic properties of apoA-IV suggest that this protein may act as an anti-inflammatory agent. We examined this possibility in a mouse model of acute colitis. Mice consumed 3% dextran sulfate sodium (DSS) in their drinking water for 7 days, with or without daily intraperitoneal injections of recombinant human apoA-IV. apoA-IV significantly and specifically delayed the onset, and reduced the severity and extent of, DSS-induced inflammation, as assessed by clinical disease activity score, macroscopic appearance and histology of the colon, and tissue myeloperoxidase activity. Intravital fluorescence microscopy of colonic microvasculature revealed that apoA-IV significantly inhibited DSS-induced leukocyte and platelet adhesive interactions. Furthermore, apoA-IV dramatically reduced the upregulation of P-selectin on colonic endothelium during DSS-colitis. apoA-IV knockout mice exhibited a significantly greater inflammatory response to DSS than did their WT littermates; this greater susceptibility to DSS-induced inflammation was reversed upon exogenous administration of apoA-IV to knockout mice. These results provide the first direct support for the hypothesis that apoA-IV is an endogenous anti-inflammatory protein. This anti-inflammatory effect likely involves the inhibition of P-selectin–mediated leukocyte and platelet adhesive interactions.
doi:10.1172/JCI200421233
PMCID: PMC450164  PMID: 15254593
21.  Isolation of Ehrlichia chaffeensis from wild white-tailed deer (Odocoileus virginianus) confirms their role as natural reservoir hosts. 
Journal of Clinical Microbiology  1997;35(7):1681-1686.
Field and experimental studies have implicated white-tailed deer (Odocoileus virginianus) as probable reservoir hosts for Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis, but natural infection in deer has not been confirmed through isolation of E. chaffeensis. Thirty-five white-tailed deer collected from three Amblyomma americanum-infested populations in Georgia were examined for evidence of E. chaffeensis infection by serologic, molecular, cell culture, and xenodiagnostic methods. Twenty-seven deer (77%) had E. chaffeensis-reactive indirect fluorescent-antibody assay titers of > or = 1:64; and the blood, spleens, or lymph nodes of seven (20%) deer were positive in a nested PCR assay with E. chaffeensis-specific primers. E. chaffeensis was isolated in DH82 cell cultures from the blood of five (14%) deer, including two deer that were PCR negative. Combination of culture and PCR results indicated that six (17%) deer were probably rickettsemic and that nine (26%) were probably infected. Restriction digestion of PCR products amplified from deer tissues and cell culture isolates resulted in a banding pattern consistent with the E. chaffeensis 16S rRNA gene sequence. The sequences of all PCR products from deer tissues or cell culture isolates were identical to the sequence of the Arkansas type strain of E. chaffeensis. Xenodiagnosis with C3H mice inoculated intraperitoneally with deer blood, spleen, or lymph node suspensions was unsuccessful. When viewed in the context of previous studies, these findings provide strong evidence that E. chaffeensis is maintained in nature primarily by a tick vector-vertebrate reservoir system consisting of lone star ticks and white-tailed deer.
PMCID: PMC229821  PMID: 9196173
22.  Physician characteristics and prescribing for elderly people in New Brunswick: relation to patient outcomes. 
OBJECTIVE: To examine the relation between physician characteristics, prescribing behaviour and patient outcomes. DESIGN: Descriptive study linking four provincial databases. SETTING: New Brunswick. PARTICIPANTS: All 366 general practitioners (GPs) (accounting for 40% of all physicians with a general licence in New Brunswick) who ordered at least 200 prescriptions for elderly beneficiaries of the New Brunswick Prescription Drug Program and saw at least 20 elderly patients in an office setting between Apr. 1, 1990, and Mar. 31, 1991. Physicians with palliative care practices were excluded. OUTCOME MEASURES: GPs' personal, professional and practice characteristics, their prescribing patterns, and mortality, morbidity (number of days in hospital per patient) and hip-fracture rates among their elderly patients. RESULTS: Compared with the GPs who had a lower mortality rate, those with a higher mortality rate prescribed more drugs overall (p < 0.001), specifically antidepressants, bronchodilators, cholesterol-lowering agents, gastrointestinal drugs, neuroleptics and nonsteroidal anti-inflammatory drugs (NSAIDs). They also were more likely to be male (p < 0.01), had larger practices (p < 0.001), saw more patients per day (p < 0.05) and billed more per year (p < 0.001). Compared with the GPs who had a lower morbidity rate, those with a higher morbidity rate prescribed more drugs overall (p < 0.005), specifically bronchodilators, gastrointestinal drugs and NSAIDs. They also were more likely to be younger (p < 0.005) and male (p < 0.01), had fewer years in practice (p < 0.001), saw more patients per day (p < 0.05) and billed more per patient (p < 0.01). The GPs who had a higher hip-fracture rate prescribed more drugs overall (p < 0.001), notably antihypertensives, bronchodilators, cholesterol-lowering agents, gastrointestinal drugs and NSAIDs, than those who had a lower hip-fracture rate. They also had a larger practice (p < 0.001), practised more days per year (p < 0.005), had more patient visits per year (p < 0.05) and billed more per year (p < 0.001). Younger male GPs who practised with relatively more intensity and prescribed more drugs per patient had practices with higher morbidity, mortality and hip-fracture rates among their elderly patients than the other GPs. CONCLUSIONS: There is a significant relation between certain physician characteristics, their prescribing behaviour and patient outcomes. Further study is required to determine what physician characteristics and prescribing behaviours for specific illnesses contribute to patient outcomes. Regional differences should also be examined, as should incentives in this fee-for-service system. Linkage of these types of provincial databases may help in the evaluation of physicians' performance and in the development of strategic interventions and practice guidelines.
PMCID: PMC1337810  PMID: 7736373
23.  Susceptibility of white-tailed deer (Odocoileus virginianus) to infection with Ehrlichia chaffeensis, the etiologic agent of human ehrlichiosis. 
Journal of Clinical Microbiology  1994;32(11):2725-2728.
Although more than 320 cases of human ehrlichiosis have been diagnosed in 27 states since 1986, the reservoir host or hosts remain unknown. Since antibodies reactive to Ehrlichia chaffeensis, the etiologic agent of human ehrlichiosis, have been found in white-tailed deer (Odocoileus virginianus), we experimentally evaluated the susceptibilities of four white-tailed deer to infection with E. chaffeensis and Ehrlichia canis, a closely related species. A fifth deer served as a negative control. Isolation and nested PCR amplification results from peripheral blood indicated that E. chaffeensis circulated for at least 2 weeks. The deer developed antibodies to E. chaffeensis by day 10 after inoculation, but there was no indication of clinical disease. Immunohistochemical staining identified E. chaffeensis within macrophage-type cells in lymph nodes. The deer inoculated with E. canis did not become infected and did not seroconvert. These results indicate that white-tailed deer can support an E. chaffeensis infection with resulting rickettsemia of at least 2 weeks. The resistance to infection and the absence of seroconversion upon exposure to E. canis indicate that antibody responses previously detected among wild deer are not E. canis cross-reactions. The role of deer as competent reservoirs in the life cycle of E. chaffeensis remains to be explored with suspected tick vectors.
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PMCID: PMC264150  PMID: 7852563
24.  Relation between physician characteristics and prescribing for elderly people in New Brunswick. 
OBJECTIVE: To determine whether there is a relation between physician characteristics and prescribing for elderly patients. DESIGN: Descriptive study linking two provincial databases. SETTING: New Brunswick. PARTICIPANTS: All general practitioners (GPs) in New Brunswick who ordered at least 200 prescriptions for elderly beneficiaries of the New Brunswick Prescription Drug Program between Apr. 1, 1990, and Mar. 31, 1991; eligible GPs accounted for 376 (40%) of all physicians with a general licence in New Brunswick. MAIN OUTCOME MEASURES: GPs' personal and professional characteristics (age, sex, family practice accreditation, country of training and number of years in practice), practice characteristics (number of practice days, number of patients seen and medical services provided per day, average amount of billing per patient, total number of patients seen and their average age, and total amount of billings) and number of prescriptions by category of drug. RESULTS: High prescribers and low prescribers did not differ significantly in age, number of years in practice, mean practice size or patient age. Compared with the low prescribers the high prescribers were more likely to be male, have been trained in Canada and be qualified by the Canadian College of Family Physicians. Also, they had more practice days, saw more patients per day, performed more services per day, billed more per patient and billed on average 30% more during the study period. Overall, the high prescribers ordered on average 45% more prescriptions than the low prescribers. CONCLUSION: There is a significant relation between certain physician characteristics and prescribing behaviour. Further study is required to examine the relation between these variables and patient outcomes.
PMCID: PMC1486694  PMID: 8131124
25.  Addition of constitutive c-myc expression to Abelson murine leukemia virus changes the phenotype of the cells transformed by the virus from pre-B-cell lymphomas to plasmacytomas. 
Molecular and Cellular Biology  1993;13(4):2578-2585.
Abelson murine leukemia virus (A-MuLV), a retrovirus that expresses the v-abl oncogene, characteristically induces pre-B-cell lymphomas following in vivo infection of BALB/c mice or in vitro infection of suspensions of fetal liver or bone marrow cells. ABL-MYC, a retrovirus that expresses both v-abl and c-myc, induces solely plasmacytomas in BALB/c mice. To investigate how the addition of overexpression of c-myc to that of v-abl accomplishes this dramatic change in the phenotype of the cells transformed by these closely related retroviruses, we utilized helper-free A-MuLV (psi 2) and ABL-MYC (psi 2) in vitro to infect suspensions of cells from different lymphoid tissues and purified immature and purified mature B cells. As expected, A-MuLV(psi 2) induced only pre-B-cell lymphomas in vivo and in vitro when immature B cells were present. ABL-MYC(psi 2), on the other hand, produced only plasmacytomas, even when purified immature B lymphocytes were infected in vitro. Although the A-MuLV(psi 2)-induced pre-B-cell lymphomas express easily detectable levels of c-myc mRNA, maturation into more-mature forms of B lymphocytes is blocked. The constitutively overexpressed c-myc in the ABL-MYC retrovirus abrogates this block, permits maturation of infected immature B cells, and yields transformed plasma cells.
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PMCID: PMC359590  PMID: 8455630

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