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Biomedical subjects

E M Rubin

Publications and source records attributed to E M Rubin.

At least 37 records · Page 2Linked to original sources
PubMed1999

Lower plasma levels and accelerated clearance of high density lipoprotein (HDL) and non-HDL cholesterol in scavenger receptor class B type I transgenic mice.

Recent studies have indicated that the scavenger receptor class B type I (SR-BI) may play an important role in the uptake of high density lipoprotein (HDL) cholesteryl ester in liver and steroidogenic tissues. To investigate the in vivo effects of liver-specific SR-BI overexpression on lipid metabolism, we created several lines of SR-BI transgenic mice with an SR-BI genomic construct where the SR-BI promoter region had been replaced by the apolipoprotein (apo)A-I promoter. The effect of constitutively increased SR-BI expression on plasma HDL and non-HDL lipoproteins and apolipoproteins was characterized. There was an inverse correlation between SR-BI expression and apoA-I and HDL cholesterol levels in transgenic mice fed either mouse chow or a diet high in fat and cholesterol. An unexpected finding in the SR-BI transgenic mice was the dramatic impact of the SR-BI transgene on non-HDL cholesterol and apoB whose levels were also inversely correlated with SR-BI expression. Consistent with the decrease in plasma HDL and non-HDL cholesterol was an accelerated clearance of HDL, non-HDL, and their major associated apolipoproteins in the transgenics compared with control animals. These in vivo studies of the effect of SR-BI overexpression on plasma lipoproteins support the previously proposed hypothesis that SR-BI accelerates the metabolism of HDL and also highlight the capacity of this receptor to participate in the metabolism of non-HDL lipoproteins.

Animals↗
PubMed1999

A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration.

We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) in a developmental and tissue-specific manner identical to that observed in Huntington's disease (HD). YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or microaggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration.

Adaptation, Physiological↗
PubMed1999

Functional screening of an asthma QTL in YAC transgenic mice.

Many quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, but few causative genes have been identified. This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions. Transgenic mice have been successfully used to analyse well-characterized genes suspected of contributing to quantitative traits. Although this approach is powerful for examining one gene at a time, it can be impractical for surveying the large genomic intervals containing many genes that are typically associated with QTLs. To screen for genes contributing to an asthma QTL mapped to human chromosome 5q3 (refs 6,7), we characterized a panel of large-insert 5q31 transgenics based on studies demonstrating that altering gene dosage frequently affects quantitative phenotypes normally influenced by that gene. This panel of human YAC transgenics, propagating a 1-Mb interval of chromosome 5q31 containing 6 cytokine genes and 17 partially characterized genes, was screened for quantitative changes in several asthma-associated phenotypes. Multiple independent transgenic lines with altered IgE response to antigen treatment shared a 180-kb region containing 5 genes, including those encoding human interleukin 4 (IL4) and interleukin 13 (IL13 ), which induce IgE class switching in B cells. Further analysis of these mice and mice transgenic for mouse Il4 and Il13 demonstrated that moderate changes in Il4 and Il13 expression affect asthma-associated phenotypes in vivo. This functional screen of large-insert transgenics enabled us to identify genes that influence the QTL phenotype in vivo.

Animals↗
PubMed1999

Targeted disruption of the murine lecithin:cholesterol acyltransferase gene is associated with reductions in plasma paraoxonase and platelet-activating factor acetylhydrolase activities but not in apolipoprotein J concentration.

Lecithin:cholesteryl acyltransferase (LCAT) deficiency resulting from targeted disruption of the Lcat gene in the mouse is associated with dramatic decreases in HDL concentration and the accumulation of nascent HDL in the plasma. We examined whether LCAT deficiency in mice is associated with a concomitant decrease in two antioxidative enzymes, paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH). In control Lcat (+/+) mice both these enzymes are transported on HDL. Compared to Lcat (+/+) mice, HDL-cholesterol is reduced 94% and apoA-I, 90%, in Lcat (-/-) mice; this reduction in HDL is paralleled by a 71% decrease in PAF-AH activity and in a 58% decrease in PON activity. Apolipoprotein J (apoJ) levels, rather than being decreased, were significantly (P = 0.01) higher (36%) in Lcat (-/-) than in Lcat (+/+) mice, and the apo J/PON ratio was 3-fold greater in Lcat (-/-) than in Lcat (+/+) animals. Even though apolipoprotein A-I (apoA-I) concentration and PON activity were drastically reduced, there was no reduction in apoA-I and PON liver mRNA levels suggesting that post-transcriptional events are responsible for the reduction of plasma PON and apoA-I levels. Fast protein liquid chromatography (FPLC) revealed that in Lcat (+/+) mice both PON and PAF-AH activity is associated with large, apoA-I-containing HDL particles (9.7 nm by non-denaturing gradient gel electrophoresis) while in Lcat (-/-) mice both enzymes are associated with small 8.2 nm particles. We conclude that the concomitant reduction in HDL and apoA-I concentrations and PON and PAF-AH activities is best explained by rapid clearance of the small HDL particles found in LCAT deficiency.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1998

Neurobehavioral deficits in mice lacking the erythrocyte membrane cytoskeletal protein 4.1.

The erythrocyte membrane cytoskeletal protein 4.1 (4.1R) is a structural protein that confers stability and flexibility to erythrocytes via interactions with the cytoskeletal proteins spectrin and F-actin and with the band 3 and glycophorin C membrane proteins. Mutations in 4.1R can cause hereditary elliptocytosis, a disease characterized by a loss of the normal discoid morphology of erythrocytes, resulting in hemolytic anemia [1]. Different isoforms of the 4.1 protein have been identified in a wide variety of nonerythroid tissues by immunological methods [2-5]. The variation in molecular weight of these different 4.1 isoforms, which range from 30 to 210 kDa [6], has been attributed to complex alternative splicing of the 4.1R gene [7]. We recently identified two new 4.1 genes: one is generally expressed throughout the body (4. 1G) [8] and the other is expressed in central and peripheral neurons (4.1N) [9]. Here, we examined 4.1R expression by in situ hybridization analysis and found that 4.1R was selectively expressed in hematopoietic tissues and in specific neuronal populations. In the brain, high levels of 4.1R were discretely localized to granule cells in the cerebellum and dentate gyrus. We generated mice that lacked 4.1R expression; these mice had deficits in movement, coordination, balance and learning, in addition to the predicted hematological abnormalities. The neurobehavioral findings are consistent with the distribution of 4.1R in the brain, suggesting that 4.1R performs specific functions in the central nervous system.

Animals↗
PubMed1998

Ts1Cje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities.

A mouse model for Down syndrome, Ts1Cje, has been developed. This model has made possible a step in the genetic dissection of the learning, behavioral, and neurological abnormalities associated with segmental trisomy for the region of mouse chromosome 16 homologous with the so-called "Down syndrome region" of human chromosome segment 21q22. Tests of learning in the Morris water maze and assessment of spontaneous locomotor activity reveal distinct learning and behavioral abnormalities, some of which are indicative of hippocampal dysfunction. The triplicated region in Ts1Cje, from Sod1 to Mx1, is smaller than that in Ts65Dn, another segmental trisomy 16 mouse, and the learning deficits in Ts1Cje are less severe than those in Ts65Dn. In addition, degeneration of basal forebrain cholinergic neurons, which was observed in Ts65Dn, was absent in Ts1Cje.

Animals↗
PubMed1998

Deletion of amino acids Glu146-->Arg160 in human apolipoprotein A-I (ApoA-ISeattle) alters lecithin:cholesterol acyltransferase activity and recruitment of cell phospholipid.

Human apolipoprotein A-I (apoA-I) has an important role in the efflux of cholesterol from peripheral cells, the first step in reverse cholesterol transport. Deletion of amino acids Glu146-->Arg160 in apoA-I (apoA-ISeattle) removes a large section of a lipid binding helix and is associated in vivo with an atherogenic lipoprotein profile characterized by a deficiency in high-density lipoproteins (HDL). In the present study, we asked whether apoA-ISeattle had normal ability to recruit lipids from cells and to form nascent high-density lipoprotein (HDL) particles and whether the altered secondary structure affected lecithin:cholesterol acyltransferase (LCAT) activity. Wild-type apoA-I and apoA-ISeattle expressed in transfected Chinese hamster ovary cells formed nascent HDL particles with similar density distribution and protein-to-lipid ratio. Phospholipid subclass distribution of apoA-ISeattle nascent HDL demonstrated a significant increase in sphingomyelin and phosphatidylethanolamine compared to wild type. ApoA-ISeattle nascent HDL had a unique size distribution compared to wild-type nascent HDL; large (9-20 nm) particles predominated while there were virtually no small (7.5 nm) particles. LCAT reactivity was impaired by apoA-ISeattle nascent HDL where cholesterol esterification was only half that of wild-type complexes. The apoA-ISeattle conformation on nascent HDL was studied with a panel of monoclonal antibodies (Mabs) specific for apoA-I. Mabs that recognize the putative LCAT activation site, residues 95-122, had normal reactivity. As expected, the Mabs that recognized residues 141-164 were unreactive because of the 146-160 deletion; in addition, there was low reactivity with a Mab that recognizes residues 220-242. The data suggest that apoA-I residues 146-160 and/or 220-242 partake in normal LCAT activation and that cooperative interactions between helices may be important for maximal cholesterol esterification.

Animals↗
PubMed1998

Apolipoprotein(a) yeast artificial chromosome transgenic rabbits. Lipoprotein(a) assembly with human and rabbit apolipoprotein B.

The in vivo analysis of lipoprotein(a) (Lp(a)), an independent atherosclerosis risk factor in humans, has been limited in part by its restricted distribution among mammals. Although transgenic mice have been created containing Lp(a), the relatively small size of the mouse has precluded some studies. To examine the properties of this molecule in a significantly larger mammal, we have used a 270-kilobase yeast artificial chromosome clone containing the human apolipoprotein(a) (apo(a)) gene and a 90-kilobase P1 phagemid clone containing the human apolipoprotein B (apoB) gene to create transgenic rabbits that express either or both transgenes. Expression of both transgenes was tissue specific and localized predominantly to the liver. Average apolipoprotein plasma levels in the rabbits were 2.5 mg/dl for apo(a) and 17.6 mg/dl for human apoB. In contrast to observations in apo(a) transgenic mice, we found that apo(a) plasma levels in the rabbits were stable throughout sexual maturity. Also, apo(a) formed a covalent association with the endogenous rabbit apoB albeit with a lower efficiency than its association with human apoB. The analysis of Lp(a) transgenic rabbits has provided new insights into apo(a) expression and Lp(a) assembly. In addition, these transgenic rabbits potentially will provide an improved experimental model for the in vivo analysis of Lp(a) and its role in promoting atherosclerosis and restenosis.

Amino Acid Sequence↗
PubMed1998

Elevated triglycerides and low HDL cholesterol in transgenic mice expressing human apolipoprotein A-I(Milano).

In general, plasma concentrations of high density lipoproteins (HDL) are inversely related to the incidence of coronary artery disease. One exception to this trend is individuals with apolipoprotein A-I(Milano) (apo A-IM), a molecular variant of apo A-I, which results in very low plasma apo A-I and HDL-cholesterol levels. Despite these low levels, and other lipoprotein defects, individuals with this mutation have no increased risk for cardiovascular disease. As a first step in proving why apo A-IM carriers appear to be protected from the pro-atherogenic effect of a low HDL, transgenic mice expressing apo A-IM were generated. Mice expressing either wild-type human apo A-I or apo A-IM, together with human apo A-II, were crossed into mice lacking murine apo A-I. Apo A-IM/A-II mice had lower cholesterol and HDL plasma levels compared to apo A-I/A-II mice. Moreover, as in human carriers, apo A-IM mice were characterized by elevated triglyceride plasma levels and by the presence of a population of very small HDL particles. These results indicate that the expression of apo A-IM in a mouse model reproduces the major lipid/lipoprotein abnormalities observed in human carriers. Thus, apo A-IM transgenic mice appear to be a suitable model in which to assess whether the mutation has an anti-atherogenic effect.

Animals↗
PubMed1998

Rapid genotyping of mice with hemoglobinopathies and globin transgenes.

The hematology of the laboratory mouse has been well characterized. Normal genetic differences at the alpha- and beta-globin gene loci serve as useful markers for a wide variety of types of experimental studies. There are a number of naturally occurring or induced mutations that disrupt globin expression and produce thalassemic phenotypes. In addition, much has been learned of the workings of the globin locus control region from studies of transgenic mice, including those with mutations induced by targeted site-specific modifications. After a new mutation or transgene has been created, it must be maintained in living mice, and the genotypes of the offspring must be ascertained. While it is possible to determine genotypes by DNA analyses, such assays are time consuming and relatively expensive. An osmotic challenge test--originally developed for the genotyping of large-deletion alpha-thalassemia mutations in mice--has proven useful in detecting both severe and milder alpha- and beta-thalassemias, as well as some transgenic genotypes in mice carrying human globin genes. Reliable genotyping can, in some cases, be completed within a few minutes with minimal expense. Quantification of red cell fragility for a variety of thalassemic and transgenic mice is described here, along with a simplified test suitable for rapid, routine genotyping. The osmotic challenge test is perfectly reliable for distinguishing genotypes that cause significantly decreased release of hemoglobin from the red cells, but it is also useful for some of the conditions in which overall erythrocyte osmotic fragility is essentially normal.

Animals↗
PubMed1998

Effect of high-frequency oral airway and chest wall oscillation and conventional chest physical therapy on expectoration in patients with stable cystic fibrosis.

STUDY OBJECTIVE: To compare the effect of high-frequency oral airway oscillation, high-frequency chest wall oscillation, and conventional chest physical therapy (CPT) on weight of expectorated sputum, pulmonary function, and oxygen saturation in outpatients with stable cystic fibrosis (CF). DESIGN: Prospective randomized trial. SETTING: Pediatric pulmonary division of a tertiary care center. PATIENTS: Fourteen outpatients with stable CF recruited from the CF center. INTERVENTIONS: Two modes of oral airway oscillation (1: frequency 8 Hz; inspiratory to expiratory [I:E] ratio 9:1; 2: frequency 14 Hz; I:E ratio 8:1), two modes of chest wall oscillation (1: frequency 3 Hz; I:E ratio 4:1; 2: frequency 16 Hz; I:E ratio 1:1, alternating with frequency 1.5 Hz, I:E ratio 6:1), and CPT (clapping, vibration, postural drainage, and encouraged coughing) were applied during the first 20 min of 4 consecutive hours. MEASUREMENTS AND RESULTS: Sputum was collected on an hourly basis for a total of 6 consecutive hours. During the first and the last hour, patients collected sputum without having any treatment and underwent pulmonary function tests (PFTs). Oxygen saturation was measured at 30-min intervals during hours 1 to 6. For the first 20 min of the second to the fifth hour, patients received one of the treatments. To assess the effect of the intervention, the weight of expectorated sputum during hours 2 to 6 was averaged and expressed as percentage of the weight expectorated during the first hour (baseline). For the five treatment modalities, mean sputum dry and wet weights ranged between 122% and 185% of baseline. There was no statistically significant difference among the treatment modalities. As measured by sputum wet weight, all oscillatory devices tended to be less effective than CPT (p=0.15). As measured by dry weight, oral airway oscillation at 8 Hz with an I:E ratio of 9:1 and CPT tended to be more effective than the other treatment modalities (p=0.57). None of the treatment modalities had an effect on PFTs and oxygen saturation and all were well tolerated. CONCLUSION: In outpatients with stable CF, high-frequency oscillation applied via the airway opening or via the chest wall and CPT have comparable augmenting effects on expectorated sputum weight without changing PFTs or oxygen saturation. In contrast to CPT, high-frequency oral airway and chest wall oscillations are self-administered, thereby containing health-care expenses.

Adolescent↗
PubMed1998

ApoA-I deficiency causes both hypertriglyceridemia and increased atherosclerosis in human apoB transgenic mice.

To study the role of low levels of high density lipoprotein (HDL) and apolipoprotein (apo) A-I in atherosclerosis risk, human apoB transgenic mice (HuBTg) were crossed with apoA-I-deficient (apoA-I-/-) mice. After a high fat challenge, total cholesterol levels increased drastically due to an increase in the non-HDL cholesterol as confirmed by FPLC analysis. In addition, total cholesterol levels in A-I-/- HuBTg mice were lower than the control HuBTg mice, due mainly to decreased HDL-C in A-I-/- HuBTg mice. Analysis of atherosclerosis in the proximal aorta in mice fed a high-fat Western-type diet for 27 weeks revealed a 200% greater lesion area in female apoA-I-/- HuBTg mice (49740+/-9751 microm2) compared to control HuBTg mice (23320+/-4981 microm2, P = 0.03). Lesion size (12380+/-3281 microm2) in male A-I-/- HuBTg mice was also about 200% greater than that in the control HuBTg mice (5849+/-1543 microm2), although not statistically significant. Very few and small lesions were observed in both apoA-I-/- HuBTg and control HuBTg animals fed a chow diet. Therefore, the adverse effect of low HDL on atherosclerosis in mice was only evident when LDL-cholesterol was markedly elevated by high-fat challenge. Male apoA-I-/- HuBTg mice exhibited hypertriglyceridemia when challenged with a high-fat diet. This correlated with both a reduction in lipoprotein lipase activity and a decrease in lipoprotein lipase activation by HDL. In summary, low high density lipoprotein levels due to apolipoprotein A-I deficiency exacerbated the development of atherosclerotic lesions in mice with elevated atherogenic lipoproteins. This mouse model mimics human conditions associated with low HDL levels and provides additional evidence for the anti-atherogenic role of apoA-I.

Animals↗
PubMed1997

A role for Ras signaling in coreceptor regulation during differentiation of a double-positive thymocyte cell line.

Evidence suggests that the Ras/mitogen-activated protein kinase signaling pathway is required for positive selection of thymocytes. We have asked whether Ras activation is also sufficient to mediate changes in gene expression that are associated with positive selection. To accomplish this, we expressed a constitutively active form of Ras in the immature CD4+ 8+ DPK thymocyte cell line. DPK cells that express active Ras have reduced levels of CD8alpha and CD8beta at the level of cell surface protein and mRNA. These data provide evidence of a direct link between Ras signaling pathways and coreceptor regulation during positive selection. They also suggest that a sustained or potent Ras signal may play a critical role in directing thymocytes into the CD4 lineage. DPK cells that express active Ras, however, were not fully differentiated, indicating that Ras signaling provides only a partial signal for double-positive thymocyte differentiation.

Animals↗
PubMed1997

Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease.

To create mice expressing exclusively human sickle hemoglobin (HbS), transgenic mice expressing human alpha-, gamma-, and betaS-globin were generated and bred with knockout mice that had deletions of the murine alpha- and beta-globin genes. These sickle cell mice have the major features (irreversibly sickled red cells, anemia, multiorgan pathology) found in humans with sickle cell disease and, as such, represent a useful in vivo system to accelerate the development of improved therapies for this common genetic disease.

Anemia, Sickle Cell↗
PubMed1997

Lipoprotein(a) vascular accumulation in mice. In vivo analysis of the role of lysine binding sites using recombinant adenovirus.

Although the mechanism by which lipoprotein(a) [Lp(a)] contributes to vascular disease remains unclear, consequences of its binding to the vessel surface are commonly cited in postulated atherogenic pathways. Because of the presence of plasminogen-like lysine binding sites (LBS) in apo(a), fibrin binding has been proposed to play an important role in Lp(a)'s vascular accumulation. Indeed, LBS are known to facilitate Lp(a) fibrin binding in vitro. To examine the importance of apo(a) LBS in Lp(a) vascular accumulation in vivo, we generated three different apo(a) cDNAs: (a) mini apo(a), based on wild-type human apo(a); (b) mini apo(a) containing a naturally occurring LBS defect associated with a point mutation in kringle 4-10; and (c) human- rhesus monkey chimeric mini apo(a), which contains the same LBS defect in the context of several additional changes. Recombinant adenovirus vectors were constructed with the various apo(a) cDNAs and injected into human apoB transgenic mice. At the viral dosage used in these experiments, all three forms of apo(a) were found exclusively within the lipoprotein fractions, and peak Lp(a) plasma levels were nearly identical (approximately 45 mg/dl). In vitro analysis of Lp(a) isolated from the various groups of mice confirmed that putative LBS defective apo(a) yielded Lp(a) unable to bind lysine-Sepharose. Quantitation of in vivo Lp(a) vascular accumulation in mice treated with the various adenovirus vectors revealed significantly less accumulation of both types of LBS defective Lp(a), relative to wild-type Lp(a). These results indicate a correlation between lysine binding properties of Lp(a) and vascular accumulation, supporting the postulated role of apo(a) LBS in this potentially atherogenic characteristic of Lp(a).

Adenoviridae↗
PubMed1997

Modification of apolipoprotein(a) lysine binding site reduces atherosclerosis in transgenic mice.

Lipoprotein(a) contributes to the development of atherosclerosis through the binding of its plasminogen-like apolipoprotein(a) component to fibrin and other plasminogen substrates. Apolipoprotein(a) contains a major lysine binding site in one of its kringle domains. Destruction of this site by mutagenesis greatly reduces the binding of apolipoprotein(a) to lysine and fibrin. Transgenic mice expressing this mutant form of apolipoprotein(a) as well as mice expressing wild-type apolipoprotein(a) have been created in an inbred mouse strain. The wild-type apolipoprotein(a) transgenic mice have a fivefold increase in the development of lipid lesions, as well as a large increase in the focal deposition of apolipoprotein(a) in the aorta, compared with the lysine binding site mutant strain and to nontransgenic littermates. The results demonstrate the key role of this lysine binding site in the pathogenic activity of apolipoprotein(a) in a murine model system.

Animals↗
PubMed1997

Disruption of the murine lecithin:cholesterol acyltransferase gene causes impairment of adrenal lipid delivery and up-regulation of scavenger receptor class B type I.

Lecithin:cholesterol acyltransferase (LCAT) is the major determinant of the cholesteryl ester (CE) content of high density lipoprotein (HDL) in plasma. The selective uptake of HDL-CE is postulated to participate in delivery of tissue-derived cholesterol both to the liver and steroidogenic tissues. Recent studies comparing mice with similarly low levels of HDL, due to the absence of either of the two major HDL-associated apolipoproteins apoA-I and apoA-II, suggest that apoA-I is crucial in modulating this process, possibly through interaction with scavenger receptor class B type I (SR-BI). Because of the central role of LCAT in determining the size, lipid composition, and plasma concentration of HDL, we have created LCAT-deficient mice by gene targeting to examine the effect of LCAT deficiency on HDL structure and composition and adrenal cholesterol delivery. The HDL in the LCAT-deficient mice was reduced in its plasma concentration (92%) and CE content (96%). The HDL particles were heterogeneous in size and morphology and included numerous discoidal particles, mimicking those observed in LCAT-deficient humans. The adrenals of the male Lcat (-/-) mice were severely depleted of lipid stores, which was associated with a 2-fold up-regulation of the adrenal SR-BI mRNA. These studies demonstrate that LCAT deficiency, similar to apoA-I deficiency, is associated with a marked decrease in adrenal cholesterol delivery and supports the hypothesis that adrenal SR-BI expression is regulated by the adrenal cholesterol.

Adrenal Glands↗