NIMH Editor's Consortium on development and psychopathology.
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Adolescent↗
Biomedical subjects
W S Davidson
Publications and source records attributed to W S Davidson.
Canadian Bardet-Biedl syndrome family reduces the critical region of BBS3 (3p) and presents with a variable phenotype.
There are at least five distinct Bardet-Biedl syndrome (BBS) loci, four of which have been mapped: 11q (BBS1), 16q (BBS2), 3p (BBS3), and 15q (BBS4). A comparative study of the three Arab-Bedouin kindreds used to map the BBS2, BBS3, and BBS4 loci suggests that the variability in the number and severity of clinical manifestations, particularly the pattern of polydactyly, reflects chromosome-specific subtypes of BBS [Carmi et al., 1995a; Am J Med Genet 59:199-203]. We describe a Newfoundland kindred of northern European descent and confirm the initial finding of a BBS locus on chromosome 3. However, the "BBS3 phenotype," which includes polydactyly of all four limbs and a progression to morbid obesity, was not observed. Rather, four of the five BBS patients in this family had polydactyly restricted to their feet. The obesity in these patients was reversible with caloric restriction and/or exercise. Mental retardation has been considered a major symptom of BBS. However, formal IQ testing shows that these patients are of average intelligence. Haplotype analysis reduces the BBS3 critical region to a 6-cM interval between D3S1595-D3S1753.
Adult↗
Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes.
alpha-Synuclein is a highly conserved presynaptic protein of unknown function. A mutation in the protein has been causally linked to Parkinson's disease in humans, and the normal protein is an abundant component of the intraneuronal inclusions (Lewy bodies) characteristic of the disease. alpha-Synuclein is also the precursor to an intrinsic component of extracellular plaques in Alzheimer's disease. The alpha-synuclein sequence is largely composed of degenerate 11-residue repeats reminiscent of the amphipathic alpha-helical domains of the exchangeable apolipoproteins. We hypothesized that alpha-synuclein should associate with phospholipid bilayers and that this lipid association should stabilize an alpha-helical secondary structure in the protein. We report that alpha-synuclein binds to small unilamellar phospholipid vesicles containing acidic phospholipids, but not to vesicles with a net neutral charge. We further show that the protein associates preferentially with vesicles of smaller diameter (20-25 nm) as opposed to larger (approximately 125 nm) vesicles. Lipid binding is accompanied by an increase in alpha-helicity from 3% to approximately 80%. These observations are consistent with a role in vesicle function at the presynaptic terminal.
Amino Acid Sequence↗
Characterization of novel minisatellite repeat loci in Atlantic salmon (Salmo salar) and their phylogenetic distribution.
We present here the sequence and characterization of various minisatellite-like tandem repeat loci isolated from the genome of Atlantic salmon (Salmo salar). Their diversity of sequence and lack of core motifs common to minisatellites of other species suggest the presence of numerous and previously unidentified simple sequence repeat families in this salmonid. Evidence for their ubiquity was provided by screening of a salmon genomic library. Southern blot analysis of the phylogenetic distribution of a subset of the minisatellites found one sequence to be pervasive among vertebrates, others present only in Salmoninae or Salmonidae species, and one amplified only in Atlantic salmon. There is evidence for the positioning of microsatellite and minisatellite arrays in close proximity at many loci. Furthermore, one tandem repeat appears to have been inserted into the transposase coding region of a copy of the Tc1 transposon-like element recently identified in salmonids.
Animals↗
Methods of ensuring high follow-up rates: lessons from a longitudinal study of dual diagnosed participants.
Despite growing research interest in co-occurring psychiatric and substance misuse disorders, relatively few longitudinal studies have been conducted with dual diagnosis populations. Many of the longitudinal studies that have been done have experienced excessive attrition. Thus, investigators have noted that one of the primary difficulties of conducting longitudinal studies with this population is successfully minimizing attrition during follow-up. This study, conducted in 1991 1993, describes retention and tracking methods employed in a longitudinal study of 485 dual diagnosis participants. Follow-up interview completion rates at the 2-, 6-, 10-, 14-, and 18-month interviews were 86.7, 85.6, 85.1, 84.5, and 88.4%, respectively. Several case studies are provided that document some of the challenges facing project staff and successful strategies for handling them. A notable finding from this study was that participants reported that the relationship established with the interviewer was more important than material incentives in preventing study attrition.
Diagnosis, Dual (Psychiatry)↗
"Why don't they just call the cops?": Reasons for differential police contact among women with abusive partners.
Incidents of domestic violence are frequently not reported to police (e.g., Johnson, 1990; Langan & Innes, 1986; Roy, 1977), and people commonly assume that women's reasons for not calling about violence by a current or former partner are intrapersonal (e.g., shame, embarrassment, love). However, few researchers have asked battered women themselves about the frequency of their police contacts and their reasons for not calling the police. In this study, participants were recruited from a battered women's shelter and asked about their experiences with the police over the prior 6 months. Two thirds of the sample had had contact with the police during that time, but most did not have as much contact with the police as they had needed. Women gave multiple reasons for not calling the police; these reasons frequently included situational barriers, such as being physically prevented from using the telephone and/or being threatened with more violence. Only 3% of the sample reported that shame, embarrassment, or love were their sole reasons for not calling the police. Underreporting was related to previous (negative) experience with the police, as well as to the level of violence experienced. The practical implications of these findings are discussed.
Adult↗
Removal of cellular cholesterol by pre-beta-HDL involves plasma membrane microsolubilization.
High density lipoprotein (HDL) is able to remove unesterified cholesterol from peripheral cells in the process of reverse cholesterol transport by an aqueous diffusion mechanism as well as by an apolipoprotein (apo)-mediated process. The aqueous diffusion mechanism is understood but the molecular mechanism of lipid-poor pre-beta-HDL-(apo-) mediated cholesterol removal is not known. Measurements of the initial rates of efflux of unesterified cholesterol and phospholipid from human fibroblasts to lipid-free, human apoA-I showed that both lipids are released from the cells during a 10-min incubation with apoA-I. The concentration-dependence of efflux of the lipids is the same (Km = 0.4 and 0.6 microg apoA-I/ml for cholesterol and phospholipid flux, respectively), suggesting a membrane microsolubilization process. A finite pool of about 1% of the plasma membrane cholesterol is accessible for release by solubilization; the limited size of this cholesterol pool is not due to a lack of availability of apoA-I, but rather to the restricted amount of phospholipid that is removed from the plasma membrane. Plasma membrane domains may be involved in membrane microsolubilization, but caveolar cholesterol seems not to be specifically accessed in this process. Membrane microsolubilization is the process by which pre-beta1-HDL removes cell cholesterol in the first step of reverse cholesterol transport. When apoA-I is present in the extracellular space, the relative contributions of cholesterol efflux by membrane microsolubilization and by aqueous diffusion are determined by the degree of lipidation of the apoA-I molecules.
Apolipoprotein A-I↗
Purification and characterization of a regiospecific lipase from Aspergillus terreus.
Aspergillus terreus lipase was purified to homogeneity with 18.0% yield. The specific activity of the enzyme increased from 20.80 to 250 U/mg of protein. Ion exchange on Q-Sepharose was highly effective in the purification process. The molecular mass of the purified enzyme was 41+/-1 kDa as determined by SDS/PAGE. The purified lipase showed excellent temperature tolerance (15-90 degreesC) and was highly thermostable, retaining 100% activity at 60 degreesC for 24 h. It showed good pH tolerance (3.0-12.0) and was stable over a pH range of 4.0-10.0 for 24 h. The activity of the enzyme was inhibited by ionic detergents, whereas non-ionic detergents stimulated enzyme activity. Mg2+ and Ca2+ ions stimulated lipase activity, whereas Co2+, Cu2+, Ni2+ and Fe3+ ions caused inhibition. The enzyme was unaffected by the metal chelator EDTA or by 2-mercaptoethanol and potassium ferrocyanide. At a concentration of 100 microM, 3,4-dichloroisocoumarin caused weak inhibition with 40% loss of activity, but diethyl p-nitrophenyl phosphate at the same concentration strongly inhibited enzyme activity (98.12% loss of activity), confirming that the A. terreus lipase is a serine hydrolase. The lipase was highly active on pig fat (151% relative activity) and groundnut oil (103% relative activity) and least active on kusum oil (18% relative activity). Extensive dialysis did not affect enzyme activity up to 168 h, suggesting the absence of any dialysable cofactor in the enzyme. The A. terreus lipase retained significant activity on freeze-drying and had a shelf-life of more than 6 months at room temperature. The A. terreus lipase exhibited 1,3-regiospecificity and was stable in various organic solvents.
Aspergillus↗
The role of apolipoprotein AI domains in lipid binding.
Apolipoprotein AI (apoAI) is the principal protein constituent of high density lipoproteins and it plays a key role in human cholesterol homeostasis; however, the structure of apoAI is not clearly understood. To test the hypothesis that apoAI is organized into domains, three deletion mutants of human apo AI expressed in Escherichia coli were studied in solution and in reconstituted high density lipoprotein particles. Each mutant lacked one of three specific regions that together encompass almost the entire 243 aa sequence of native apoAI (apoAI delta 44-126, apoAI delta 139-170, and apoAI delta 190-243). Circular dichroism spectroscopy showed that the alpha-helical content of lipid-free apoAI delta 44-126 was 27% while the other mutants and native apoAI averaged 55 +/- 2%, suggesting that the missing N-terminal portion contains most of the alpha-helical structure of lipid-free apoAI. ApoAI delta 44-126 exhibited the largest increase in alpha-helix upon lipid binding (125% increase versus an average of 25% for the others), confirming the importance of the C-terminal half of apoAI in lipid binding. Denaturation studies showed that the N-terminal half of apoAI is primarily responsible for alpha-helix stability in the lipid-free state, whereas the C terminus is required for alpha-helix stability when lipid-bound. We conclude that the N-terminal half (aa 44-126) of apoAI is responsible for most of the alpha-helical structure and the marginal stability of lipid-free apoAI while the C terminus (aa 139-243) is less organized. The increase in alpha-helical content observed when native apoAI binds lipid results from the formation of alpha-helix primarily in the C-terminal half of the molecule.
Apolipoprotein A-I↗
Apolipoprotein A-I structural modification and the functionality of reconstituted high density lipoprotein particles in cellular cholesterol efflux.
The role of HDL and its major protein constituent, apolipoprotein (apo) A-I, in promoting the removal of excess cholesterol from cultured cells has been well established; however, the mechanisms by which this occurs are not completely understood. To address the effects of apoA-I modification on cellular unesterified (free) cholesterol (FC) efflux, three recombinant human apoA-I deletion mutants and plasma apoA-I were combined with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and FC to make reconstituted high density lipoprotein (rHDL) discoidal complexes. These particles were characterized structurally and for their efficiency as acceptors of mouse L-cell fibroblast cholesterol. The deletion mutant proteins lacked NH2-terminal (apoA-I (Delta44-126)), central (apoA-I (Delta139-170)), or COOH-terminal (apoA-I (Delta190-243)) domains of apoA-I. The three deletion mutants all displayed lipid-binding abilities and formed discoidal complexes that were similar in major diameter (13.2 +/- 1.5 nm) to those formed by human apoA-I when reconstituted at a 100:5:1 (POPC:FC:protein) mole ratio. Gel filtration profiles indicated unreacted protein in the preparation made with apoA-I (Delta190-243), which is consistent with the COOH terminus portion of apoA-I being an important determinant of lipid binding. Measurements of the percent alpha-helix content of the proteins, as well as the number of protein molecules per rHDL particle, gave an indication of the arrangement of the deletion mutant proteins in the discoidal complexes. The rHDL particles containing the deletion mutants had more molecules of protein present than particles containing intact apoA-I, to the extent that a similar number of helical segments was incorporated into each of the discoidal species. Comparison of the experimentally determined number of helical segments with an estimate of the available space indicated that the deletion mutant proteins are probably more loosely arranged than apoA-I around the edge of the rHDL. The abilities of the complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts. All four discoidal complexes displayed similar abilities to remove FC from the plasma membrane of L-cells when compared at an acceptor concentration of 50 microg of phospholipid/ml. Thus, none of the deletions imposed in this study notably altered the ability of the rHDL particles to participate in cellular FC efflux. These results suggest that efficient apoA-I-mediated FC efflux requires the presence of amphipathic alpha-helical segments but is not dependent on specific helical segments.
Animals↗
Correlates of functioning in a population with dual diagnoses: an examination of diagnosis and problem history.
This study examines the relationship between diagnosis and life functioning using the Addiction Severity Index (ASI) with 467 hospitalized individuals with mental illness and substance abuse problems. Persons diagnosed with schizophrenia were the best functioning group across most of the ASI domains except employment and psychiatric functioning. More robust relationships were found between problem history (i.e., prior symptomatology or treatment) and current functioning. Respondents with histories of drug treatment, prior experience of anxiety and depression, self-injurious behavior, or violence control problems experienced more severe medical, drug, alcohol, psychiatric, legal, and family/social problems at the time of hospitalization. Violence control problems were related to drug use and criminal involvement, whereas self-injurious behavior was more often related to alcohol use and psychiatric distress. These findings suggest that problem history may be a stronger predictor of treatment need at the time of hospital entry than are more commonly used indexes, such as diagnosis.
Adult↗
Comparison of the structural and functional effects of monomeric and dimeric human apolipoprotein A-II in high density lipoprotein particles.
High density lipoprotein (HDL) is thought to play a significant role in the process of reverse cholesterol transport. It has become clear that the apolipoprotein (apo) composition of HDL is important in determining the metabolic fate of this particle. The major proteins of human HDL are apoAI and APOAII; the latter protein is a disulfide-linked dimer in humans and higher primates but monomeric in the other species. The consequences of the apo Cys6-Cys6 disulfide bridge in apoAII for human HDL structure and function are not known. To address this issue, the influence of the Cys6-Cys6 disulfide bridge on the interaction of human apoAII with palmitoyl-oleoyl phosphatidylcholine has been studied. The size and valence of a series of homogeneous discoidal complexes containing either monomeric (reduced and carboxymethylated) or dimeric apoAII have been determined, and their ability to remove cholesterol from rat Fu5AH hepatoma cells grown in culture has been compared. The apoAII dimer and monomer form discoidal complexes of similar size, with twice as many of the latter molecule required per disc. Removal of the disulfide bond influences the stability of the helical segments around the edge of the disc as seen by a decrease in alpha-helix content of the monomeric protein. The discoidal particles containing the monomeric form of apoAII are somewhat more effective than particles containing either dimeric apoAII or apoAI in removing cellular cholesterol. Overall, reduction of the disulfide bridge of apoAII probably does not have a major effect in the determination of HDL particle size in vivo. It follows that the evolution of the Cys6-Cys6 disulfide bond in higher primates probably has not had a major effect on the function of the apoAII molecule.
Animals↗
Population differentiation and evolution in the common guillemot Uria aalge.
Common (Uria aalge) and Brünnich's guillemots (U. lomvia) are colonial seabirds that nest in temperate to arctic oceans throughout the Northern hemisphere. They are very similar in the characteristics of ecology, demography and life history that are thought to determine the extent of differentiation among populations, yet geographic variation in morphology is notably greater in common guillemots. Despite evidence of strong natal philopatry, previous analyses of allozymes and the mitochondrial cytochrome b gene revealed little genetic differentiation among North Atlantic colonies of Brünnich's guillemots. To determine if the more extensive morphological variability in common guillemots reflects greater genetic variability, we sequenced part of the cytochrome b gene for 160 common guillemots from 10 colonies distributed throughout the Northern hemisphere. Genotype frequencies and phylogenetic relationships among genotypes both indicated that Atlantic and Pacific populations are genetically distinct. Genetic divergence among genotypes suggested that differentiation of these populations has resulted from separation by Pleistocene glaciers and the Bering Landbridge, as well as by currently unsuitable breeding habitat in the Arctic Ocean. Cytochrome b genotype frequencies also differed among Atlantic colonies, and appeared to define a cline similar to that described for morphological characters. Analyses of sequence variation suggested that this variation probably results from secondary contact between two refugial populations from the Pleistocene glaciations, rather than from isolation by distance or selection. In contrast, the Atlantic population of Brünnich's guillemots appears to have arisen through recent expansion of a single homogeneous refugial population.
Animals↗
High yield overexpression and characterization of human recombinant proapolipoprotein A-I.
Human apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) where it defines the particle structure and stability and functions as the main activator of the enzyme lecithin:cholesterol acyltransferase (LCAT). ApoA-I is expressed in the liver as a preproprotein that is targeted to the endoplasmic reticulum for secretion; in plasma, an unknown protease removes the six amino acid long propeptide. In this study, the cDNA coding the human proapoA-I was cloned into an Escherichia coli vector; the overexpressed protein was purified to 99% homogeneity and was extensively characterized together with mature apoA-I purified from plasma. SDS-PAGE, mass spectrometry, and Edman sequence analysis showed that the initial Met residue needed for translation in E. coli is posttranslationally removed from the N-terminal sequence of the proapoA-I. The structural and functional analyses were carried out on the lipid-free and the lipid-bound proteins. ProapoA-I self associated, interacted with dimyristoyl phosphatidylcholine vesicles, and formed secondary structures very similar to the lipid-free apoA-I. Reconstituted HDL particles made with two initial molar ratios of palmitoyloleoyl phosphatidylcholine/cholesterol/apolipoprotein/Na-cholate had identical particle sizes and distributions when apoA-I or proapoA-I were used. Particles having diameters of 79 A and 98 A, containing two apoA-I or proapoA-I molecules per particle, were isolated and characterized. The particles contained the same amounts of alpha-helical structure, had very similar fluorescence properties, and activated LCAT equally well. We conclude that proapoA-I expressed and purified from E. coli is functionally and structurally indistinguishable from mature apoA-I purified from plasma when analyzed in vitro. Therefore, this recombinant proapoA-I and mutants derived from it will be important sources of protein for analyzing apoA-I structure and function, as well as for studies of proapoA-I processing.
Apolipoprotein A-I↗
Effects of the neutral lipid content of high density lipoprotein on apolipoprotein A-I structure and particle stability.
Alterations in high density lipoprotein (HDL) composition that occur in dyslipidemic states may modulate a number of events involved in cholesterol homeostasis. To elucidate the details of how HDL-core composition can affect the molecular structure of different kinds of HDL particles, the conformation and stability of apoA-I have been investigated in homogeneous recombinant HDL particles (LpA-I) containing palmitoyloleoyl phosphatidylcholine (POPC), triolein (TG), and/or cholesteryl linoleate (CE). In a discoidal particle containing two molecules of apoA-I and 85 molecules of POPC, apoA-I exhibits an alpha-helix content of 70% and a free energy of stability of its alpha-helical segments (delta G0D) of 2.2 kcal/mol. Inclusion of eight molecules of TG into the complex significantly reduces the alpha-helix content and stability of apoA-I, whereas inclusion of four molecules of CE into the complex has an opposite effect in that the alpha-helix content is significantly reduced and the stability of the remaining alpha-helical structure of apoA-I is increased. Neutral lipids have a different effect on apoA-I conformation in spherical LpA-I particles. In a sonicated-spherical LpA-I particle containing two molecules of apoA-I and 70 molecules of POPC, apoA-I exhibits an alpha-helix content of about 60% and a delta G0D of 1.2 kcal/mol apoA-I. Inclusion of either 10 molecules of TG or six molecules of CE into such a particle increases both the alpha-helix content and stability of apoA-I. Increasing the CE/TG ratio in LpA-I particles that contain both neutral lipids enhances the stability of the alpha-helical segments. ApoA-I molecules tend to dissociate and cause particle instability when delta G0D for the lipid-bound alpha-helices is less than that for helices in the lipid-free state. The stabilities of both discoidal and spherical LpA-I particles are relatively low when the only neutral lipid present is TG but the particle stability is enhanced by the presence of CE molecules. Such dissociation of apoA-I molecules from LpA-I particles that have a low CE/TG ratio would be promoted in the hypertriglyceridemic state in vivo.
Apolipoprotein A-I↗
Characterisation of fast, slow and cardiac muscle tropomyosins from salmonid fish.
Tropomyosin (TM) has been isolated from the cardiac muscle, and fast and slow trunk (myotomal) muscles of the mature salmonid fish Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri). When examined electrophoretically, isoforms of TM were detected which were specific, and exclusive, to each type of muscle. Cardiac and fast muscles contained single and distinct isoforms, while slow muscle contained two distinct isoforms, closely related in terms of apparent M(r), and pI. There was no detectable difference between the same TM type from either salmon or trout. On a variety of gel systems, the cardiac and slow isoforms migrated in close proximity to each other and to rabbit alpha-TM. The fast isoform comigrated with rabbit beta-TM. In developing salmon fry, a more acidic (unphosphorylated) variant of TM was present in addition to, and of similar M(r) to, the fast adult isoform. This TM declined in steady-state level during maturation and was virtually undetected in adult muscle. All of the isolated TMs contained little or no covalently bound phosphate and were blocked at the N-terminus. The amino acids released by carboxypeptidase A, when ordered to give maximal similarity to other muscle TMs, were consistent with the following sequences: fast (LDNALNDMTSI) and cardiac (LDHALNDMTSL). The C-terminal region of the slow TM contained His but was heterogeneous. In viscosity measurements, performed as a function of increasing protein concentration, at low ionic strength (t = 5 degrees C, pH 7.00), fast TM exhibited the highest relative viscosity values. Lower and equivalent levels of polymerisation occurred with the cardiac and slow TMs. Polymerisation of all three isoforms was temperature-dependent, with cardiac TM being least sensitive and fast TM being most sensitive. Determination of the complete coding sequence of adult fast TM confirmed the findings of the carboxypeptidase analysis, but the remainder of the sequence more closely resembled alpha-type TMs than beta-type TMs. Overall, salmon fast TM contains 20 (mostly conservative) substitutions compared to rabbit striated muscle alpha-TM and 40 (mostly conservative) substitutions compared to rabbit striated muscle beta-TM. This demonstrates that electrophoretic mobility is not, in all instances, a suitable method to assess the isomorphic nature of striated muscle TMs.
Amino Acid Sequence↗
Effects of acceptor particle size on the efflux of cellular free cholesterol.
Several subspecies of human high density lipoprotein (HDL) have been shown to exist, and particle size is one parameter that can be used to distinguish them. Recently, a small HDL subspecies has been described that may be a particularly efficient acceptor of peripheral cell unesterified (free) cholesterol (FC). To address the effects of particle size on the ability of HDL to remove FC from cells, homogeneous, well defined HDL particles were reconstituted (rHDL) that varied in particle diameter within the size range of human HDL particles (7-13 nm). The abilities of each of these particles to remove cellular FC from mouse L-cells and rat Fu5AH hepatoma cells were compared on the basis of their phospholipid (PL) content as well as on a per particle basis. The effect of particle size was also examined using small unilamellar vesicles (SUV) of 25 nm in diameter and large unilamellar vesicles (LUVs) of 70-180 nm in diameter. The SUV were prepared by sonication, and the LUVs were prepared by extrusion techniques. The FC efflux efficiency of these particles (in order of decreasing efficiency) was: rHDL > SUV > LUV when compared on the basis of acceptor PL content across a range of concentrations (i.e. at a given PL concentration for these three acceptor classes, smaller particles were more efficient). The FC efflux differences between the rHDL and the vesicles were not due to the absence of apolipoprotein in the vesicles. No difference was detected among the rHDL of varying size, nor was a difference detected among the LUVs of varying size when compared on the basis of PL content. When the FC efflux data for rHDL and LUVs were normalized on the basis of the number of acceptor particles present at a given PL concentration, a correlation was found between acceptor particle radius and the ability to accept cellular FC with larger particles being the most efficient. However, the dependence of the rate of FC efflux on acceptor particle size was not quantitatively the same within the rHDL and LUV classes of acceptor particles. The dependence of FC efflux on acceptor particle size may reflect differing abilities of the variously sized acceptor particles to access the region very close to the cell plasma membrane where most of the FC removal is expected to occur.
Animals↗