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Biomedical subjects

E M Rubin

Publications and source records attributed to E M Rubin.

At least 55 records · Page 3Linked to original sources
PubMed1997

A transgenic mouse model of hemoglobin S Antilles disease.

Hemoglobin (Hb) S Antilles is a naturally occurring form of sickling human Hb but causes a more severe phenotype than Hb S. Two homozygous viable Hb S Antilles transgene insertions from Tg58Ru and Tg98Ru mice were bred into MHOAH mice that express high oxygen affinity (P50 approximately 24.5 mm Hg) rather than normal (P50 approximately 40 mm Hg) mouse Hbs. The rationale was that the high oxygen affinity MHOAH Hb, the lower oxygen affinity of Hb S Antilles than Hb S (P50 approximately 40 v 26.5 mm Hg), and the lower solubility of deoxygenated Hb S Antilles than Hb S (approximately 11 v 18 g/dL) would favor deoxygenation and polymerization of human Hb S Antilles in MHOAH mouse red blood cells (RBCs). The Tg58 x Tg98 mice produced have a high and balanced expression (approximately 50% each) of h alpha and h beta(S Antilles) globins, 25% to 35% of their RBCs are misshapen in vivo, and in vitro deoxygenation of their blood induces 30% to 50% of the RBCs to form classical looking, elongated sickle cells with pointed ends. Tg58 x Tg98 mice exhibit reticulocytosis, an elevated white blood cell count and lung and kidney pathology commonly found in sickle cell patients, which should make these mice useful for experimental studies on possible therapeutic intervention of sickle cell disease.

Anemia, Sickle Cell↗
PubMed1997

Cholesterol efflux, lecithin-cholesterol acyltransferase activity, and pre-beta particle formation by serum from human apolipoprotein A-I and apolipoprotein A-I/apolipoprotein A-II transgenic mice consistent with the latter being less effective for reverse cholesterol transport.

Studies assessing fatty streak formation in mice have revealed that human apolipoprotein A-I (apoAI) transgenic mice (TgAI) have 15-fold less atherosclerosis susceptibility than combined human apolipoprotein A-I/human apolipoprotein A-II (apoAI:AII) transgenics (TgAI:AII) and 40-fold less than nontransgenic control mice. In order to examine the biochemical mechanisms underlying those in vivo observations, we have compared in vitro properties of serum from the different groups of animals for participation in cholesterol efflux, LCAT activation, and pre-beta particle formation. Analysis of cholesterol efflux from both Fu5AH hepatoma and Ob1771 adipose cells revealed serum from the TgAI to be the most efficient in promoting efflux. The two-dimensional electrophoresis of mouse serum shows that control mice have exclusively apoAI in alpha particles. TgAI and TgAI:AII mice have 30 and 38% of total apoAI in particles with pre-beta electrophoretic mobility, respectively. The distribution of cell-derived cholesterol between these apoAI-containing lipoprotein subspecies after 1 and 60 min of incubation with Fu5AH hepatoma cells was examined. This revealed after a 1 min incubation 66 +/- 8 and 83 +/- 9% of the counts in particles with pre-beta mobility for TgAI and TgAI:AII mice, respectively; while after 60 min of incubation, only 6 +/- 2% of counts remained in pre-beta particles from the TgAI and 30 +/- 3% for the TgAI:AII. This suggests faster movement of cholesterol from pre-beta to alpha particles in plasma from the TgAI. Consistent with this is the observation that LCAT activity with both exogenous and endogenous substrate increased in the TgAI versus the TgAI:AII mice. The previously observed decrease in fatty streak formation in the TgAI versus the TgAI:AII and control mice is consistent with the in vitro studies presented here and suggests that HDL containing human apoAI is a more effective participant in the postulated early steps in reverse cholesterol transport than HDL containing both human apoAI and human apoAII, and/or murine HDL.

Animals↗
PubMed1997

Induction of the early growth response (Egr) family of transcription factors during thymic selection.

There is little known about the regulation of gene expression during TCR-mediated differentiation of immature CD4+8+ (double positive) thymocytes into mature T cells. Using the DPK CD4+8+ thymocyte precursor cell line, we demonstrate that the early growth response-1 gene (Erg-1), encoding a zinc finger transcription factor, is rapidly upregulated after TCR stimulation. We also report that Egr-1 is expressed by a subset of normal double positive thymocytes in the thymic cortex, as well by a majority of medullary single positive thymocytes. Expression of Egr-1 is dramatically reduced in the thymus of major histocompatibility complex knockout mice, but can be induced by anti-CD3 antibody stimulation of isolated thymocytes from these animals. These and other data suggest that high level expression of Egr-1 in the thymus is a consequence of selection. A similar pattern of expression is found for family members Egr-2 and Egr-3. Using the DPK cell line, we also demonstrate that expression of Egr-1, 2, and 3 is dependent upon ras activation, as is the initiation of differentiation to a single positive cell. In contrast, the calcineurin inhibitor cyclosporin A, which inhibits DPK cell differentiation as well as positive selection, inhibits expression of Egr-2 and Egr-3, but not Egr-1. The identification of the Egr family in this context represents the first report of a link between the two known signaling pathways involved in positive selection and downstream transcriptional regulators.

Animals↗
PubMed1997

The vibrator mutation causes neurodegeneration via reduced expression of PITP alpha: positional complementation cloning and extragenic suppression.

The mouse vibrator mutation causes an early-onset progressive action tremor, degeneration of brain stem and spinal cord neurons, and juvenile death. We cloned the vibrator mutation using an in vivo positional complementation approach and complete resequencing of the resulting 76 kb critical region from vibrator and its parental chromosome. The mutation is an intracisternal A particle retroposon insertion in intron 4 of the phosphatidylinositol transfer protein alpha gene, causing a 5-fold reduction in RNA and protein levels. Expression of neurofilament light chain is also reduced in vibrator, suggesting one signaling pathway that may underlie vibrator pathology. The vibrator phenotype is suppressed in one intercross. We performed a complete genome scan and mapped a major suppressor locus (Mvb-1) to proximal chromosome 19.

Alleles↗
PubMed1997

Functional screening of 2 Mb of human chromosome 21q22.2 in transgenic mice implicates minibrain in learning defects associated with Down syndrome.

Using Down syndrome as a model for complex trait analysis, we sought to identify loci from chromosome 21q22.2 which, when present in an extra dose, contribute to learning abnormalities. We generated low-copy-number transgenic mice, containing four different yeast artificial chromosomes (YACs) that together cover approximately 2 megabases (Mb) of contiguous DNA from 21q22.2. We subjected independent lines derived from each of these YAC transgenes to a series of behavioural and learning assays. Two of the four YACs caused defects in learning and memory in the transgenic animals, while the other two YACs had no effect. The most severe defects were caused by a 570-kb YAC; the interval responsible for these defects was narrowed to a 180-kb critical region as a consequence of YAC fragmentation. This region contains the human homologue of a Drosophila gene, minibrain, and strongly implicates it in learning defects associated with Down syndrome.

Animals↗
PubMed1997

Functional screening and complex traits: human 21q22.2 sequences affecting learning in mice.

Libraries of the mammalian genome have generally been propagated in single cells and have been used for gene discovery through in vitro analyses. We have expanded upon this concept by the creation of panels of YAC transgenic mice propagating targeted megabase regions of the genome. Such a panel of mice can be called an 'in vivo library' and genes can be identified based on functional screens of members of the library. To test this approach, we created a 2 Mb in vivo library of human chromosome 21q22.2. Analysis of the library has revealed that one 570 kb YAC, in two separate founder lines, was associated with distinct learning deficits compared with the other 21q22 YAC transgenics and non-transgenic control animals. We have localized the gene on the YAC that causes the deficits by taking advantage of fragmentation of the YAC during the process of microinjection. The responsible gene is the human minibrain gene, and the homolog of the gene in Drosophila is also associated with learning defects. These results suggest that altered dosage of minibrain is associated with abnormal neural development in flies and mice and, in humans, may also be involved in the molecular pathology of Down syndrome.

Animals↗
PubMed1997

Computational and biological analysis of 680 kb of DNA sequence from the human 5q31 cytokine gene cluster region.

With the human genome project advancing into what will be a 7- to 10-year DNA sequencing phase, we are presented with the challenge of developing strategies to convert genomic sequence data, as they become available, into biologically meaningful information. We have analyzed 680 kb of noncontiguous DNA sequence from a 1-Mb region of human chromosome 5q31, coupling computational analysis with gene expression studies of tissues isolated from humans as well as from mice containing human YAC transgenes. This genomic interval has been noted previously for containing the cytokine gene cluster and a quantitative trait locus associated with inflammatory diseases. Our analysis identified and verified expression of 16 new genes, as well as 7 previously known genes. Of the total of 23 genes in this region, 78% had similarity matches to sequences in protein databases and 83% had exact expressed sequence tag (EST) database matches. Comparative mapping studies of eight of the new human genes discovered in the 5q31 region revealed that all are located in the syntenic region of mouse chromosome 11q. Our analysis demonstrates an approach for examining human sequence as it is made available from large sequencing programs and has resulted in the discovery of several biomedically important genes, including a cyclin, a transcription factor that is homologous to an oncogene, a protein involved in DNA repair, and several new members of a family of transporter proteins.

Animals↗
PubMed1997

Vascular accumulation of Lp(a): in vivo analysis of the role of lysine-binding sites using recombinant adenovirus.

Despite the importance of lipoprotein(a) [Lp(a)] as an atherogenic risk factor, very little information, especially from in vivo studies, is available concerning which structural features of apo(a) contribute to the interactions of Lp(a) with the vessel wall and its proatherogenic properties. Nearly all the proposed and proven activities of apolipoprotein(a) [apo(a)] focus on its high degree of sequence homology with plasminogen and the possibility that structural features shared by these two molecules contribute to the atherogenesis associated with high Lp(a) plasma levels in humans. In these studies, we examined the properties of three forms of Lp(a) differing at postulated lysine-binding domains contained in the constituent apo(a). We used the recombinant adenoviral gene delivery system to produce apo(a) in the plasma of human apoB transgenic mice, resulting in high levels of Lp(a) similar to those found in the plasma of humans. By comparison of in vitro lysine-binding properties of these forms of Lp(a) with measurements of Lp(a) vascular accumulation in the mice, we have demonstrated that lysine-binding defective forms of Lp(a) have a diminished capacity for vascular accumulation in vivo.

Adenoviridae↗
PubMed1997

Evidence that apolipoprotein A-IMilano has reduced capacity, compared with wild-type apolipoprotein A-I, to recruit membrane cholesterol.

Human carriers of apolipoprotein (apo) A-IMilano are heterozygous for an Arg173-->Cys substitution in the apoA-I primary sequence; despite severe reductions in HDL cholesterol concentrations, affected individuals do not develop coronary heart disease, suggesting that apoA-IMilano may possess antiatherogenic properties. As the beneficial effects of wild-type apoA-I are linked to its role in HDL cholesterol transport, we examined the capacity of apoA-IMilano to recruit cell cholesterol and activate lecithin:cholesterol acyltransferase (LCAT) (two key events in the antiatherogenic reverse cholesterol transport pathway). ApoA-IMilano and wild-type apoA-I were expressed in Chinese hamster ovary cells, and their ability to recruit membrane phospholipid and cholesterol for the assembly of nascent HDL was compared. Both clonal cell lines exhibited similar levels of apolipoprotein accumulation in serum-free medium (approximately 2 micrograms/mg cell protein per 24 hours), and 15% of each apolipoprotein was associated with membrane lipids to form nascent HDL (d = 1.063 to 1.21 g/mL). SDS-PAGE showed that a majority (66 +/- 12%) of the lipidated apoA-IMilano was in the homodimer form. Compositional analyses revealed that apoA-IMilano nascent HDL had a significantly lower (P < .001) unesterified cholesterol/phospholipid mole ratio (0.47 +/- 0.10) than wild-type apoA-I complexes (1.29 +/- 0.14), indicating that apoA-IMilano had a reduced capacity to recruit cell cholesterol. In addition to the reduced unesterified cholesterol/phospholipid ratio, apoA-IMilano nascent HDL consisted mostly of small 7.4-nm particles compared with wild-type apoA-I, in which 11- and 9-nm particles predominated. Despite these changes in nascent HDL particle size and composition, apoA-IMilano activated LCAT normally. We conclude that, even though apoA-IMilano is a normal activator of LCAT, it is less efficient that wild-type apoA-I in recruiting cell cholesterol, suggesting that the putative antiatherogenic properties attributed to apoA-IMilano may be unrelated to the initial stages of reverse cholesterol transport.

Animals↗
PubMed1997

HDL deficiency in genetically engineered mice requires elevated LDL to accelerate atherogenesis.

In humans, a low HDL concentration is one of the strongest indicators of increased risk for coronary heart disease. Apolipoprotein A-I (apo A-I) synthetic defects results in extremely low HDL levels and are frequently although not invariably associated with premature atherosclerosis. To investigate atherosclerosis susceptibility associated with HDL deficiency alone and in combination with other risk factors, such as high levels of LDL, we have quantified diet-induced atherogenesis in a series of genetically engineered mice, including mice with low HDL levels due to targeted disruption of both apo A-I alleles (AI KO mice), mice with high LDL levels due to expression of a human apolipoprotein B transgene (Btg mice), and mice with combined high LDL and low HDL levels due to the presence of the human apo B transgene and apo A-I knockout alleles, respectively (AI KO/Btg mice). After exposure to an atherogenic diet, AI KO and control mice had negligible lesions. All mice expressing the apo B transgene developed extensive lesions, but AI KO/Btg mice developed significantly larger lesions than Btg mice: 56, 260 +/- 4630 micron 2 for AI KO/Btg (n = 27) versus 38, 120 +/- 3350 micron 2 for Btg mice (n = 19) (P < .02). Results of this study, consistent with several human epidemiological studies, indicate that HDL deficiency in the mouse does not by itself lead to the development of atherosclerosis but does increase atherosclerosis susceptibility when accompanied by other risk factors, in this case elevated LDL.

Animals↗
PubMed1997

High density lipoprotein particle size restriction in apolipoprotein A-I(Milano) transgenic mice.

Human carriers of apolipoprotein A-I(Milano) (Arg173 --> Cys substitution in apolipoprotein A-I) are characterized by an HDL deficiency in which small, dense HDL accumulate in plasma. Because affected individuals are heterozygous for this mutation, the full impact of apolipoprotein A-I(Milano) (apoA-I(Milano)) on HDL-cholesterol metabolism is unknown. In this study, apoA-I(Milano) transgenic mice were used to evaluate the extent of apoA-I(Milano) dimerization and HDL particle size restriction in the absence of wild-type apoA-I. Murine apoA-I knockout mice were utilized to express apoA-I(Milano) and human apoA-II in the presence of wild-type, human apoA-I (apoA-IMilano/A-Iwt/A-II) and in its absence (apoA-IMilano/A-II). Plasma HDL-cholesterol concentrations were similar (30 mg/dl) in both lines of apoA-I(Milano) transgenic mice. In the apoA-IMilano/A-Iwt/A-II phenotype, 14% of the apoA-I(Milano) formed homodimers and 33% formed heterodimers with apoA-II. ApoA-I(Milano) homodimers increased by 71% in the apoA-IMilano/A-II transgenics and was associated with an abundance of small, 7.6-nm HDL3-sized particles compared to the 9.5, 8.3, and 7.6-nm-sized particles in apoA-IMilano/A-Iwt/A-II mice. The unesterified cholesterol/cholesteryl ester mole ratio of HDL was elevated by 45% in apoA-IMilano/A-Iwt/A-II mice and by 90% in apoA-IMilano/A-II transgenics compared to wild-type (human apoA-I/A-II). Both apoA-I(Milano) transgenics possessed normal levels of plasma LCAT activity, but endogenous cholesterol esterification rates were reduced by 50% compared to controls. Thus, HDL particle size restriction was not the result of impaired LCAT activation; rather, dimerization of apoA-I(Milano) limited the esterification of cholesterol on endogenous HDL. In the absence of wild-type apoA-I, the more extensive dimerization of apoA-I(Milano) severely limited cholesteryl ester accumulation on plasma HDL accounting for the abundance of small, 7.6-nm HDL3 particles in apoA-IMilano/A-II mice.

Animals↗
PubMed1996

Feedback mechanism of focal vascular lesion formation in transgenic apolipoprotein(a) mice.

Apolipoprotein(a) (apo(a)), the distinguishing protein of atherogenic lipoprotein(a), directs accumulation of the lipoprotein(a) particle to sites in the arterial wall where atherosclerotic lipid lesions develop in man and in transgenic mice expressing human apo(a). It has been proposed that focal apo(a) accumulation in the transgenic mouse vessel wall causes the observed severe local inhibition of transforming growth factor-beta (TGF-beta) activity and the consequent activation of the smooth muscle cells, which subsequently accumulate lipid to form lesions if the mice are fed a high fat diet. We show that blocking formation of these vascular lesions by two independent mechanisms, tamoxifen treatment and increasing high density lipoprotein, also abolishes apo(a) accumulation, inhibition of TGF-beta activity, and activation of smooth muscle cells. The data are consistent with a feedback mechanism in which an initial accumulation of apo(a) inhibits local TGF-beta activity, leading to further accumulation of apo(a). Breaking the feedback loop prevents smooth muscle cell activation and therefore lipid lesion development.

Animals↗
PubMed1996

Magnetic resonance evidence of hypoxia in a homozygous alpha-knockout of a transgenic mouse model for sickle cell disease.

All transgenic mouse models for sickle cell disease express residual levels of mouse globins which complicate the interpretation of experimental results. We now report on a mouse expressing high levels of human betaS and 100% human alpha-globin. These mice were created by breeding the alpha-knockout and the mouse beta(major)-deletion to homozygosity in mice expressing human alpha- and betaS-transgenes. These betaS-alpha-knockout mice have accelerated red cell destruction, altered hematological indices, ongoing organ damage, and pathology under ambient conditions which are comparable with those found in alphaH betaS-Ant[betaMDD] mice without introduction of additional mutations which convert betaS into a "super-betaS" such as the doubly mutated betaS-Antilles. This is of particular importance for testing strategies for gene therapy of sickle cell disease. Spin echo magnetic resonance imaging at room air and 100% oxygen demonstrated the presence of blood hypoxia (high levels of deoxygenated hemoglobin) in the liver and kidneys that was absent in control mice. We demonstrate here that transgenic mice can be useful to test new noninvasive diagnostic procedures, since the magnetic resonance imaging technique described here potentially can be applied to patients with sickle cell disease.

Anemia, Sickle Cell↗
PubMed1996

A novel functional role for apolipoprotein B in male infertility in heterozygous apolipoprotein B knockout mice.

Male infertility, affecting as many as 10% of the adult population, is an extremely prevalent disorder. In most cases, the cause of the condition is unknown, and genetic factors that might affect male fertility, other than some sequences on the Y chromosome, have not been identified. We report here that male mice heterozygous for a targeted mutation of the apolipoprotein B (apo B) gene exhibit severely compromised fertility. Sperm from these mice failed to fertilize eggs both in vivo and in vitro. However, these sperm were able to fertilize eggs once the zona pellucida was removed but displayed persistent abnormal binding to the egg after fertilization. In vitro fertilization-related and other experiments revealed reduced sperm motility, survival time, and sperm count also contributed to the infertility phenotype. Recognition of the infertility phenotype led to the identification of apo B mRNA in the testes and epididymides of normal mice, and these transcripts were substantially reduced in the affected animals. Moreover, when the genomic sequence encoding human apo B was introduced into these animals, normal fertility was restored. These findings suggest that this genetic locus may have an important impact on male fertility and identify a previously unrecognized function for apo B.

Animals↗
PubMed1996

Apolipoprotein B RNA editing enzyme-deficient mice are viable despite alterations in lipoprotein metabolism.

RNA editing in the nucleus of higher eukaryotes results in subtle changes to the RNA sequence, with the ability to effect dramatic changes in biological function. The first example to be described and among the best characterized, is the cytidine-to-uridine editing of apolipoprotein B (apo-B) RNA. The editing of apo-B RNA is mediated by a novel cytidine deaminase, apobec-1, which has acquired the ability to bind RNA. The stop translation codon generated by the editing of apo-B RNA truncates the full-length apo-B100 to form apo-B48. The recent observations of tumor formation in Apobec-1 transgenic animals, together with the fact that Apobec-1 is expressed in numerous tissues lacking apo-B, raises the issue of whether this enzyme is essential for a variety of posttranscriptional editing events. To directly test this, mice were created with a null mutation in Apobec-1 using homologous recombination in embryonic stem cells. Mice, homozygous for this mutation, were viable and made apo-B100 but not apo-B48. The null animals were fertile, and a variety of histological, behavioral, and morphological analyses revealed no phenotype other than abnormalities in lipoprotein metabolism, which included an increased low density lipoprotein fraction and a reduction in high density lipoprotein cholesterol. These studies demonstrate that neither apobec-1 nor apo-B48 is essential for viability and suggest that the major role of apobec-1 may be confined to the modulation of lipid transport.

APOBEC-1 Deaminase↗
PubMed1996

Contrasting in vivo effects of murine and human apolipoprotein A-II. Role of monomer versus dimer.

The role of apolipoprotein A-II (apoA-II) in high density lipoprotein (HDL) structure and metabolism has been studied previously in transgenic mice overexpressing either human or murine apoA-II. These studies have shown differences between these two groups of transgenic animals in the levels of very low density, low density, and high density lipoproteins, in the HDL particle size distribution, and in the relationship between apoA-II levels and lipoprotein levels. To determine whether these differences are due to the fact that human apoA-II is dimeric and murine apoA-II monomeric, we have examined the effects of monomeric human apoA-II (hA-IImon) in transgenic mice. Site-directed mutagenesis (Cys6 -> Ser) was used to generate 15 transgenic founder lines of hA-IImon mice, that contained plasma hA-IImon concentrations over a 10-fold range (11 mg/dl to 185 mg/dl). The hA-IImon floated in the d < or = 1.21 g/ml fraction and migrated as an apoA-II monomer by nonreducing SDS-polyacrylamide gel electrophoresis. HDL levels were not correlated with hA-IImon levels (r = -0.26); HDL particle size and size distribution, as well as very low density and low density lipoprotein levels and sizes, were unchanged compared to nontransgenic control mice. These results suggest that differences between mice overexpressing human dimeric apoA-II and those overexpressing murine apoA-II are the result of sequence differences between these two apoA-II molecules and are not solely due to the fact that human apoA-II exists as a dimer.

Amino Acid Sequence↗