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a Gladstone Institute of Cardiovascular Disease and b Cardiovascular Research Institute, c Department of Medicine, University of California, San Francisco, CA 94141–9100
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONGENERATING AUTHENTIC MOUSE...USING LARGE GENOMIC CLONES...SECRETION OF APO...REFERENCES |
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KEY WORDS: • protein structure/function • yolk sac endoderm • heart • hypobetalipoproteinemia • metabolism
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONGENERATING AUTHENTIC MOUSE...USING LARGE GENOMIC CLONES...SECRETION OF APO...REFERENCES |
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). Apolipoprotein B100 is synthesized by the liver, where it is
necessary for the formation of VLDL, and by the yolk sac during
development, where it appears to play a role in the nutrition of the
embryo (Farese et al. 1995 and 1996a
). As discussed in this short
review, apoB100 is also made by cardiac myocytes. Apolipoprotein B48 is
synthesized in the intestine as a result of an apoB mRNA-editing
process that converts codon 2153 to a translational stop codon
(Davidson 1994
). Apolipoprotein B mRNA is extensively edited in the
intestines of all mammals (Greeve et al. 1993
). Thus, apoB48 is the
principal structural protein of intestinally derived lipoproteins.
Apolipoprotein B mRNA editing activity occurs in the livers of some
mammalian species, including mice and rats (Greeve et al. 1993
). Thus,
the liver of these species secretes both apoB48 and apoB100.
Over the past five years, several laboratories have used gene-targeted
mice as well as conventional transgenic mice to investigate various
aspects of apoB biology. For example, human apoB transgenic mice have
been used to create animal models to study atherogenesis (Callow et al. 1994
, Linton et al. 1993a
, Purcell-Huynh et al. 1995
) and to study the
DNA sequences that control the expression of the apoB gene (Nielsen et al. 1998a and 1998b
). Transgenic mice expressing mutant forms of human
apoB have also been used to study the structure/function of the apoB
molecule (Callow and Rubin 1995
, McCormick et al. 1995, 1996 and 1997
).
Gene knockout mice have been used to create animal models for the human
apoB deficiency syndromes (Farese et al. 1995
, Homanics et al. 1993
,
Huang et al. 1995
) and to study the "physiologic rationale" for the
existence of apoB48 and apoB100 in mammalian lipoprotein metabolism
(Farese et al. 1996b
). This review will not attempt to describe all of
these studies but rather will focus on three recent examples of how the
use of genetically modified mice has contributed to our understanding
of apoB.
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GENERATING AUTHENTIC MOUSE MODELS FOR THE HUMAN APO B DEFICIENCY SYNDROME, FAMILIAL HYPOBETALIPOPROTEINEMIA |
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TOPABSTRACTINTRODUCTIONGENERATING AUTHENTIC MOUSE...USING LARGE GENOMIC CLONES...SECRETION OF APO...REFERENCES |
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). Most of the
mutations causing hypobetalipoproteinemia have been point mutations,
either deletions of one or two nucleotides or single nucleotide
substitutions causing nonsense mutations (Linton et al. 1993b
). These
mutations often lead to the synthesis of a truncated form of apoB,
which can be detected in the plasma lipoproteins. In these cases, the
concentration of the truncated apoB in the plasma is invariably quite
low. For example, in heterozygotes, the concentration of the truncated
apoB is typically <5% of the concentration of apoB100 that is
produced by the wild-type apoB allele. One of the goals of our
laboratory has been to understand the basic mechanisms for the low
plasma levels of the truncated apoB proteins in these syndromes.
To create a mouse model of FHb, we used gene targeting to insert an
apoB83 mutation (Leu3798Stop, a mutation typical of those causing human
FHb) into the mouse apoB gene (Kim et al. 1998
). In addition to the
Leu3798Stop mutation, the apoB83-only (Apob83)
allele contained a mutation at codon 2153 that abrogated apoB48
formation. The latter mutation had no affect on apoB mRNA levels
(Farese et al. 1996b
). Unlike the apoB70 mutation described earlier by
the laboratory of Dr. Nobuyo Maeda (Homanics et al. 1993
), the
apoB83-only allele did not alter the structure of the apoB transcript.
Unlike the "apoB48-only" mutation generated earlier by our
laboratory (Farese et al. 1996b
), the apoB83 nonsense mutation was
inserted into an "unnatural" site within the apoB gene (i.e., one
that did not lead to the production of a physiologically normal apoB
protein).
In mice heterozygous for the Apob83 allele,
apoB83 was present only in trace levels in the plasma (~2% of the
level of apoB100 that was produced by the other allele), a phenotype
that is strikingly similar to that observed in human FHb heterozygotes
with an apoB83 mutation (Farese et al. 1992
). An analysis of the plasma
lipoproteins in the heterozygous apoB83-only mice revealed that apoB83
was present in low concentrations in the VLDL but was absent in the
LDL—a pattern identical to the distribution of apoB83 in human FHb
heterozygotes (Farese et al. 1992
).
An analysis of heterozygous apoB83-only mice uncovered dual
mechanisms for the low plasma levels of apoB83 (Kim et al. 1998
).
First, the Apob83 mRNA levels and apoB83
secretion by primary hepatocytes were reduced by ~75%. Thus, unlike
the nonsense mutation in the apoB48-only mice (Farese et al. 1996b
), a
nonsense mutation at this "unnatural" site within the apoB gene
caused low apoB mRNA levels and correspondingly low apoB secretion
rates. Of note, the Apob83 pre-mRNA levels were
not reduced (Kim et al. 1998
). Thus, we suspect that the
Apob83 nonsense mutation affects either the
stability or the processing of the Apob transcript. There
are a number of precedents for low mRNA levels associated with nonsense
mutations in other genetic diseases (Baserga and Benz 1988 and 1992
,
Kessler and Chasin 1996
, Maquat 1995
).
In addition to low synthesis rates, apoB83 was removed very rapidly
from the plasma, much more rapidly than apoB100 (Kim et al. 1998
). We
estimate that the absolute concentration of apoB83 in the plasma of
heterozygous apoB83-only mice is only ~2% of the concentration of
apoB100, despite the fact that apoB83 synthesis rates are ~25% as
high as the apoB100 synthetic rates. The rapid clearance of apoB83 was
documented by genetic experiments in which the apoB83 mutation was
placed on a background of apoE deficiency (Piedrahita et al. 1992
) or
LDL receptor deficiency (Ishibashi et al. 1993
) and by metabolic
experiments in which we blocked lipoprotein degradation with Triton
WR-1339 (Li et al. 1996
).
There are several explanations for the rapid clearance of apoB83.
First, apoB83-containing lipoproteins, like apoB48-containing
lipoproteins, might accommodate more apoE, which could be partially
responsible for the rapid clearance of the apoB83-containing
lipoproteins. However, this probably does not represent the entire
story. Apolipoprotein B83 contains the portion of the apoB molecule
that interacts with the LDL receptor, and we believe that apoB83 is
likely to be more effective than apoB100 in mediating the
uptake of LDL and VLDL by the LDL receptor. Enhanced uptake of
apoB83-containing VLDL would also explain the extremely low levels of
apoB83 in the plasma and why apoB83 is virtually absent from the LDL
fraction of Apob83/+mice (and human apoB83
heterozygotes). Extremely rapid apoB83 clearance by the LDL receptor
pathway would be consistent with data from Dr. G. Schonfeld's
laboratory (Parhofer et al. 1990
); they found that apoB89 (a truncated
apoB molecule in the plasma of a kindred with FHb) was cleared much
more rapidly than apoB100.
Homozygous Apob83/83 embryos manifested multiple
developmental abnormalities, including exencephalus and herniation of
the liver into the umbilical cord; none survived more than a few days
(Kim et al. 1998
). Drs. R. Farese, R. Hamilton and co-workers proposed
that apoB synthesis and secretion by the yolk sac plays a key role in
mouse development (Farese et al. 1996a
). The fact that
Apob83/83 mice display severe developmental
abnormalities indicates that the quarter-normal apoB synthesis rates
fall below the threshold required for normal mouse development.
Alternatively, the peculiar intrinsic metabolic properties of apoB83
(particularly the extremely rapid uptake of apoB83 by the LDL receptor
pathway) could "short-circuit" normal embryonic lipoprotein
delivery (e.g., prevent apoB83-containing lipoproteins from reaching
critical sites within the developing mouse embryo).
In humans, the FHb phenotype is somewhat variable, depending on the length of the truncated apoB. The plasma levels of apoB37 in human apoB37 heterozygotes are probably about 10-fold greater than the plasma levels of apoB83 in human apoB83 heterozygotes. To obtain a broader view of metabolic mechanisms in FHb, we have used gene-targeting techniques to introduce another apoB gene mutation (Asn1785Stop) into the mouse genome, thereby generating apoB39-only mice. Interestingly, the plasma levels of apoB39 in heterozygous apoB39-only mice (Apob39/+ mice) were 10- to 15-fold higher than the plasma levels of apoB83 in Apob83/+ mice (E. Kim and S. Young, unpublished observations). In addition, many of the homozygous apoB39-only mice were born and appeared to be completely healthy, in contrast to the homozygous apoB83-only mice. During the next year, we will compare the phenotypes of our apoB39-only and apoB83-only mice and attempt to understand why the plasma levels of the truncated apoB are higher in the apoB39-only mice and why they do not exhibit the severe neurodevelopmental abnormalities.
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USING LARGE GENOMIC CLONES TO GENERATE MUTANT FORMS OF HUMAN APO B |
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TOPABSTRACTINTRODUCTIONGENERATING AUTHENTIC MOUSE...USING LARGE GENOMIC CLONES...SECRETION OF APO...REFERENCES |
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, Linton et al. 1993a
).
In transgenic lines with >10 copies of the transgene, the plasma
levels of human apoB in hemizygous mice fed a standard laboratory diet
were 60–80 mg/100 mL, which are similar to those in normolipidemic
humans (Linton et al. 1993a
). The development of the p158-human apoB
transgenic mice represented a breakthrough as a "human apoB
expression system." One of our primary goals was to transform this "human apoB expression system" into a system that would be useful for studies of apoB structure and function. In particular, we were interested in understanding the LDL receptor–binding domain and in defining the features of the apoB molecule that are important for its interaction with apo(a) in the assembly of lipoprotein(a) [Lp(a)]. To analyze the structure of human apoB, we wanted to be able to insert mutations into clone p158, express the mutant p158 construct in transgenic mice and then analyze the properties of the mutant human apoB protein.
We began our studies of apoB structure with a discrete issue, i.e., to
define the structural features of the apoB molecule that are important
for Lp(a) assembly. To address this issue, we initially interrupted the
coding sequence of p158 with a transposon and then used the mutant p158
to create transgenic mice expressing a truncated form of apoB, apoB90.
An analysis of the human apoB90 revealed that it was completely
incapable of forming Lp(a), once again suggesting that the carboxyl
terminal 10% of the apoB molecule was essential for Lp(a) formation
(McCormick et al. 1994
).
The transposon mutagenesis approach allowed us to make many constructs
coding for truncated apoBs, but did not allow us to introduce point
mutations into p158. To overcome this obstacle, we sought to introduce
subtle mutations into the human apoB gene by using homologous
recombination in yeast artificial chromosome (YAC) (McCormick et al. 1995 and 1996
). For these studies, the human apoB gene (the insert from
p158) was cloned into a YAC, and "pop-in, pop-out" gene targeting
was used to introduce subtle mutations into the human apoB gene. The
YAC DNA was then used to generate transgenic mice expressing high
levels of the mutant human apoB (McCormick et al. 1995
).
To determine the utility of the YAC gene-targeting approach, we began
by testing the hypothesis that the last cysteine residue of apoB100,
cysteine-4326, was involved in the disulfide bond with apo(a). We used
the YAC gene-targeting system to change cysteine-4326 to a glycine and
then generated transgenic mice expressing the mutant human apoB. Our
studies with the mutant apoB protein demonstrated that it completely
lacked the capacity to bind to apo(a) and form Lp(a) (McCormick et al. 1995 and 1996
). These studies indicated that apoB cysteine-4326 is the
site of attachment for apo(a). Callow and Rubin (1995)
obtained the
same findings.
Recently, we performed additional experiments to define the apoB
sequences, aside from cysteine-4326, that are important for Lp(a)
assembly. To address this issue, we used the YAC gene-targeting
approach to introduce nonsense mutations into exon 29 of the apoB gene,
generating two truncated forms of human apoB, apoB95 (4330 amino acids)
and apoB97 (4397 amino acids). We then tested the ability of those
truncated apoB's to form Lp(a) (McCormick et al. 1997
). Our studies
revealed that Lp(a) was formed extremely slowly and inefficiently with
apoB95, even though that molecule contained the critical cysteine
residue (cysteine-4326). In contrast, Lp(a) was formed rapidly and
efficiently with apoB97. From these studies, we concluded that the
sequences carboxyl-terminal to cysteine-4326, particularly residues
4331–4397, play an important role in the initial interaction of apoB
with apo(a).
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SECRETION OF APO B–CONTAINING LIPOPROTEINS BY THE HEART |
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TOPABSTRACTINTRODUCTIONGENERATING AUTHENTIC MOUSE...USING LARGE GENOMIC CLONES...SECRETION OF APO...REFERENCES |
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, Kane and Havel 1989
, Young 1990
). During our initial characterization of the
p158-human apoB transgenic mice, we made a surprising and provocative
observation, i.e., the human apoB transgene was expressed in the heart.
This finding was confirmed by both RNA slot-blot studies and RNase
protection assays in many independent lines of the p158 transgenic
mice. The amount of apoB mRNA in the heart was ~4% of that in the
liver (Linton et al. 1993a
). Heart expression of the human apoB
transgene was confirmed in independent experiments by Callow et al. (1994)
.
Initially, we were extremely skeptical of the finding that the human
apoB gene was expressed in the heart. First, clone p158 clearly did not
contain all of the regulatory sequences required for the intestinal
expression of the human apoB gene (Nielsen et al. 1997
). Thus, p158
might have represented an "incomplete" genomic clone from the
perspective of the regulatory sequences controlling proper
tissue-specific gene expression (i.e., we hypothesized that p158 lacked
the regulatory sequences that would normally serve to silence the apoB
gene in the heart, in addition to lacking the regulatory sequences
required for expression of the apoB gene in the intestine). Second, the
heart seemed a very unlikely site for apoB gene expression. From the
perspective of lipoprotein metabolism, the heart has always been viewed
as a "consumer" of lipoprotein-derived lipids and plasma fatty
acids. Along with skeletal muscle and adipose tissue, the heart is one
of the principal sites for uptake of plasma free fatty acids (Goldberg 1996
). At first glance, it would seem extremely counterproductive for
the heart to express the apoB gene, inasmuch as apoB secretion would
serve to transport lipids away from the heart.
Subsequent studies have shown that apoB gene expression in the heart is
not simply a peculiarity of the transgenic mice generated with clone
p158. We have recently analyzed apoB gene expression in more than three
dozen transgenic lines developed with a large array of bacterial
artificial chromosome (BAC) clones (Nielsen et al. 1997
) modified by
RecA-assisted restriction endonuclease-cleavage (L. Nielsen and S.
Young, unpublished observations). In each of the BAC transgenic lines,
we documented human apoB gene expression in the heart. Like the p158
human apoB transgenic mice, the human apoB mRNA levels in the BAC
transgenic mice averaged 3–4% of those observed in the liver and
~10% of those in the intestine (L. Nielsen and S. Young, unpublished
observations). Interestingly, apoB gene expression in the hearts of
transgenic mice does not require distant gene-regulatory sequences.
Bacterial artificial chromosome constructs containing as little as 5 kb
of 5' flanking sequences and 1.5 kb of 3' flanking sequences were
expressed in the heart (L. Nielsen and S. Young, unpublished
observations).
We considered the possibility that the expression of human apoB in the
hearts of the p158- and BAC-transgenic mice might represent an artifact
resulting from the expression of a fragment of human genomic DNA in the
mouse. We therefore used RNase protection assays to assess the
expression of the endogenous apoB genes in human and mouse hearts. To
test this issue, we prepared RNA from nontransgenic mouse hearts and
from human hearts obtained from cardiac transplantation operations. Of
note, the apoB mRNA was easily detectable in RNA prepared from five
different human hearts and was present at levels similar to those
observed in the human apoB transgenic mice (Nielsen et al.1998
). In
addition, the mouse apoB gene was expressed in the hearts of
nontransgenic mice (Nielsen et al.1998
). Finding apoB gene expression
in the human heart led us to suspect that the human apoB cDNA would be
encountered in Expressed Sequence Tags (EST) databases. This suspicion
was borne out; the human apoB cDNA has been identified in two different
heart cDNA libraries (http://www.ncbi.nlm.nih.gov/dbEST). The only
other tissues in which the apoB cDNA was identified were liver and
intestine.
To localize the cell type responsible for cardiac expression of the
apoB gene, we performed initial immunohistochemical studies with both
human and mouse apoB-specific antibodies. These studies revealed very
intense staining of capillary endothelium and much less intense
staining of the cardiac myocytes (L. Nielsen and S. Young, unpublished
observations). These preliminary results led us to hypothesize that
apoB was synthesized and secreted by the capillary endothelium.
However, two lines of evidence suggested that this interpretation was
not correct. First, the laboratory of Dr. R. Hamilton performed
ultrastructural studies on the capillary endothelium of both human and
mouse heart tissue. They found that the caveolae of the endothelial
cells (the putative transcytotic vesicles) contained numerous
lipoproteins, whereas the Golgi apparatus from these cells contained
none (L. Nielsen, R. Hamilton and S. Young, unpublished observations).
Thus, the ultrastructural studies suggested that the staining of the
endothelial cells in the immunohistochemical studies was due to the
presence of plasma lipoproteins that were trapped within the
endothelial cells. Second, and probably more importantly, in situ
hybridization studies with a human apoB-specific riboprobe demonstrated
apoB gene expression in the cardiac myocytes (Nielsen et al. 1998
). In
those studies, surrounding "non-cardiac" tissues (including the
aorta, pulmonary artery and lungs) manifested no apoB gene expression,
whereas tissues known to secrete apoB (the liver and the absorptive
enterocytes of intestine) were intensely positive. Interestingly, the
signal was more intense in atrial myocytes than in ventricular
myocytes.
In the liver and intestine, the microsomal triglyceride transfer
protein (MTP) gene is expressed in association with the apoB gene and
is essential for lipoprotein assembly (Gordon et al. 1994
). We
suspected that the MTP gene might also be expressed in the heart. This
suspicion has been confirmed. A Western blot of human heart microsomes
revealed the presence of the 97-kDa fragment of MTP (Borén et al. 1998
), and Northern blots revealed the presence of the MTP mRNA in
mouse hearts as well as human hearts (Nielsen et al. 1998
). The finding
that the MTP gene is expressed in the heart is actually consistent with
a much earlier observation by Wetterau and Zilversmit (1986)
. Even
before the MTP cDNA was cloned, they had documented MTP activity in
microsomes prepared from rat heart (at levels that were ~5% as high
as those observed in liver microsomes).
The expression of both the apoB and MTP genes in the heart suggested
that the heart might actually be a lipoprotein-secreting organ. To test
this hypothesis, heart tissue from several different lines of human
apoB transgenic mice was metabolically labeled with
[35S]methionine/cysteine, and the media were fractionated
by sucrose density gradient ultracentrifugation. Apolipoprotein B was
immunoprecipitated from each fraction and examined by SDS-PAGE and
autoradiography. The human apoB transgenic mouse hearts secreted
apoB-containing lipoproteins, predominantly in the LDL fraction
(Borén et al. 1998
). Metabolic labeling experiments were also
performed on fresh human heart tissue (obtained from the explanted
diseased hearts at the time of cardiac transplantation) and on the
hearts of nontransgenic mice (Borén et al. 1998
). In both cases,
we documented the synthesis and secretion of apoB100-containing
lipoproteins. Further, metabolic labeling experiments performed in the
presence of an MTP inhibitor drug (Gordon et al. 1996
) revealed that
the secretion of apoB100-containing lipoproteins by the heart is
completely dependent on MTP (Borén et al. 1998
).
Recently, Dr. R. Hamilton and Ms. J. Wong performed ultrastructural
studies on human heart myocytes using special staining techniques that
permit the visualization of lipoproteins (Nielsen et al. 1998
). They
found examples of VLDL- and intermediate density lipoprotein
(IDL)-sized lipoproteins within the Golgi apparatus of heart myocytes
from two different human hearts. The lipoproteins were typically
observed singly or in very small groups in the Golgi cisternae or
vesicles. Lipoproteins were also identified within the Golgi vesicles
of ventricular myocytes from human apoB transgenic mice.
The conservation of apoB and MTP expression in the hearts of humans and mice, two species separated by 80 million years of evolution, suggests that heart lipoprotein secretion is important. But what is the precise purpose of heart lipoprotein secretion? Given the well-established role of apoB in assembling triglyceride-rich lipoproteins, we are attracted to a simple hypothesis, i.e., that lipoprotein secretion unloads surplus myocyte lipids, particularly triglycerides, into the extracellular space (and eventually into the bloodstream). Although the delivery of lipids to the heart is subject to metabolic regulation, it is easy to imagine that changing metabolic conditions (such as ischemia or a sudden change in physical activity) might occasionally lead to an accumulation of surplus fatty acids or triglycerides. Thus, the ability to secrete lipoproteins into the bloodstream might serve to limit both the accumulation of potentially toxic fatty acid intermediates and local storage of triglycerides. This hypothesis, which might reasonably be designated a "reverse triglyceride transport hypothesis," awaits testing.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Presented at the symposium "Assembly and
Physiology of Apolipoprotein B-Containing Lipoproteins It's Not Just
for Heart Disease Anymore!" as part of Experimental Biology 98, April
18–22, 1998, San Francisco, CA. The symposium was sponsored by the
Energy and Macronutrient Research Interest Section of the American
Society for Nutritional Sciences, the Egg Nutrition Center, the
American Heart Association-Western States Affiliate, Merck Research
Laboratories, Bristol-Meyers Squibb Pharmaceutical Research Institute
and Parke-Davis Pharmaceutical Research. Published as a supplement to
The Journal of Nutrition. Guest editors for this supplement
were Rosemary L. Walzem, University of California, Davis, and Robert L.
Hamilton, University of California, San Francisco, CA.
2 Supported by National Institutes of Health grant
HL41633.
3 Abbreviations used: Apo, apolipoprotein; BAC,
bacterial artificial chromosome; FHb, familial hypobetalipoproteinemia;
Lp, lipoprotein; MTP, microsomal triglyceride transfer protein; YAC,
yeast artificial chromosome.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONGENERATING AUTHENTIC MOUSE...USING LARGE GENOMIC CLONES...SECRETION OF APO...REFERENCES |
|---|
1.
Baserga S. J., Benz E. J., Jr. Nonsense mutations in the human ß-globin gene affect mRNA metabolism. Proc. Natl. Acad. Sci. U.S.A. 1988;85:2056-2060.
2.
Baserga S. J., Benz E. J., Jr. ß-Globin nonsense mutationdeficient accumulation of mRNA occurs despite normal cytoplasmic stability. Proc. Natl. Acad. Sci. U.S.A. 1992;89:2935-2939.
3. Borén J., Véniant M. M., Young S. G.. Apo B100–containing lipoproteins are secreted by the heart. J. Clin. Investig. 1998;101:1197-1202.[Medline]
4.
Callow M. J., Rubin E. M.. Site-specific mutagenesis demonstrates that cysteine 4326 of apolipoprotein B is required for covalent linkage with apolipoprotein(a) in vivo. J. Biol. Chem. 1995;270:23914-23917.
5.
Callow M. J., Stoltzfus L. J., Lawn R. M., Rubin E. M.. Expression of human apolipoprotein B and assembly of lipoprotein(a) in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 1994;91:2130-2134.
6. Davidson N. O.. RNA editing of the apolipoprotein B geneA mechanism to regulate the atherogenic potential of intestinal lipoproteins?. Trends Cardiovasc. Med. 1994;4:231-235.
7. Farese R. V., Jr, Cases S., Ruland S. L., Kayden H. J., Wong J. S., Young S. G., Hamilton R. L.. A novel function for apolipoprotein Blipoprotein synthesis in the yolk sac is critical for maternal-fetal lipid transport in mice. J. Lipid Res. 1996;37:347-360.[Abstract]
8. Farese R. V., Jr, Garg A., Pierotti V. R., Vega G. L., Young S. G.. A truncated species of apolipoprotein B, B-83, associated with hypobetalipoproteinemia. J. Lipid Res. 1992;33:569-577.[Abstract]
9.
Farese R. V., Jr, Ruland S. L., Flynn L. M., Stokowski R. P., Young S. G.. Knockout of the mouse apolipoprotein B gene results in embryonic lethality in homozygotes and protection against diet-induced hypercholesterolemia in heterozygotes. Proc. Natl. Acad. Sci. U.S.A. 1995;92:1774-1778.
10.
Farese R. V., Jr, Véniant M. M., Cham C. M., Flynn L. M., Pierotti V., Loring J. F., Traber M., Ruland S., Stokowski R. S., Huszar D., Young S. G.. Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100. Proc. Natl. Acad. Sci. U.S.A. 1996;93:6393-6398.
11. Goldberg I. J.. Lipoprotein lipase and lipolysiscentral roles in lipoprotein metabolism and atherogenesis. J. Lipid Res. 1996;37:693-707.[Abstract]
12.
Gordon D. A., Jamil H., Gregg R. E., Olofsson S.-O., Borén J.. Inhibition of the microsomal triglyceride transfer protein blocks the first step of apolipoprotein B lipoprotein assembly but not the addition of bulk core lipids in the second step. J. Biol. Chem. 1996;271:33047-33053.
13.
Gordon D. A., Jamil H., Sharp D., Mullaney D., Yao Z., Gregg R. E., Wetterau J.. Secretion of apolipoprotein B-containing lipoproteins from HeLa cells is dependent on expression of the microsomal triglyceride transfer protein and is regulated by lipid availability. Proc. Natl. Acad. Sci. U.S.A. 1994;91:7628-7632.
14. Greeve J., Altkemper I., Dieterich J.-H., Greten H., Windler E.. Apolipoprotein B mRNA editing in 12 different mammalian specieshepatic expression is reflected in low concentrations of apoB-containing plasma lipoproteins. J. Lipid Res. 1993;34:1367-1383.[Abstract]
15. Havel R. J., Kane J. P.. Introductionstructure and metabolism of plasma lipoproteins. Scriver C. R. Beaudet A. L. Sly W. S. Valle D. eds. The Metabolic and Molecular Bases of Inherited Disease 1995:1841-1851 McGraw-Hill New York, NY.. .
16.
Homanics G. E., Smith T. J., Zhang S. H., Lee D., Young S. G., Maeda N.. Targeted modification of the apolipoprotein B gene results in hypobetalipoproteinemia and developmental abnormalities in mice. Proc. Natl. Acad. Sci. U.S.A. 1993;90:2389-2393.
17. Huang L.-S., Voyiaziakis E., Markenson D. F., Sokol K. A., Hayek T., Breslow J. L.. Apo B gene knockout in mice results in embryonic lethality in homozygotes and neural tube defects, male infertility, and reduced HDL cholesterol ester and apo A-I transport rates in heterozygotes. J. Clin. Investig. 1995;96:2152-2161.
18. Ishibashi S., Brown M. S., Goldstein J. L., Gerard R. D., Hammer R. E., Herz J.. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J. Clin. Investig. 1993;92:883-893.
19. Kane J. P., Havel R. J.. Disorders of the biogenesis and secretion of lipoproteins containing the B apolipoproteins. Scriver C. R. Beaudet A. L. Sly W. S. Valle D. eds. The Metabolic Basis of Inherited Disease 1989:1139-1164 McGraw-Hill New York, NY.. .
20. Kessler O., Chasin L. A.. Effects of nonsense mutations on nuclear and cytoplasmic adenine phosphoribosyltransferase RNA. Mol. Cell. Biol. 1996;16:4426-4435.[Abstract]
21. Kim E., Cham C. M., Véniant M. M., Ambroziak P., Young S. G.. Dual mechanisms for the low plasma levels of truncated apolipoprotein B proteins in familial hypobetalipoproteinemiaAnalysis of a new mouse model with a nonsense mutation in the Apob gene. J. Clin. Investig. 1998;101:1468-1477.[Medline]
22. Li X., Catalina F., Grundy S. M., Patel S.. Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouseevidence for a very rapid turnover of VLDL and preferential removal of B-48- relative to B-100-containing lipoproteins. J. Lipid Res. 1996;37:210-220.[Abstract]
23. Linton M. F., Farese R. V., Jr, Chiesa G., Grass D. S., Chin P., Hammer R. E., Hobbs H. H., Young S. G.. Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a). J. Clin. Investig. 1993;92:3029-3037.
24. Linton M. F., Farese R. V., Jr, Young S. G.. Familial hypobetalipoproteinemia. J. Lipid Res. 1993;34:521-541.[Medline]
25. Maquat L. E.. When cells stop making senseeffects of nonsense codons on RNA metabolism in vertebrate cells. RNA 1995;1:453-465.[Abstract]
26.
McCormick S.P.A., Linton M. F., Hobbs H. H., Taylor S., Curtiss L. K., Young S. G.. Expression of human apolipoprotein B90 in transgenic miceDemonstration that apolipoprotein B90 lacks the structural requirements to form lipoprotein(a). J. Biol. Chem. 1994;269:24284-24289.
27.
McCormick S.P.A., Ng J. K., Cham C. M., Taylor S., Marcovina S. M., Segrest J. P., Hammer R. E., Young S. G.. Transgenic mice expressing human apoB95 and apoB97Evidence that sequences within the carboxyl-terminal portion of human apoB100 are important for the assembly of lipoprotein(a). J. Biol. Chem. 1997;272:23616-23622.
28.
McCormick S.P.A., Ng J. K., Taylor S., Flynn L. M., Hammer R. E., Young S. G.. Mutagenesis of the human apolipoprotein B gene in a yeast artificial chromosome reveals the site of attachment for apolipoprotein(a). Proc. Natl. Acad. Sci. U.S.A. 1995;92:10147-10151.
29. McCormick S.P.A., Peterson K. R., Hammer R. E., Clegg C. H., Young S. G.. Generation of transgenic mice from yeast artificial chromosome DNA that has been modified by gene targeting. Trends Cardiovasc. Med. 1996;6:16-24.
30.
Nielsen L. B., Kahn D., Duell T., Weier H.-U. G., Taylor S., Young S. G.. Apolipoprotein ß gene expression in a series of apoB transgenic mice generated with recA-assisted restriction endonuclease cleavage–modified bacterial artificial chromosomesan intestine-specific enhancer element is located between 54 and 62 kilobases 5' to the structural gene. J. Biol. Chem. 1998;273:21800-21807.
31.
Nielsen L. B., McCormick S.P.A., Pierotti V., Tam C., Gunn M. D., Shizuya H., Young S. G.. Human apolipoprotein B transgenic mice generated with 207- and 145-kilobase pair bacterial artificial chromosomesEvidence that a distant 5'-element confers appropriate transgene expression in the intestine. J. Biol. Chem. 1997;272:29752-29758.
32.
Nielsen L. B., Véniant M., Hamilton R. L., Borén J., Wong J. S., Tam C., Flynn L., Gunn M. D., Sanan D., Goldberg I., Young S. G.. The genes for apolipoprotein B and microsomal triglyceride transfer protein are expressed in the heartindications that the heart has capacity to synthesize and secrete lipoproteins. Circulation 1998;98:13-16.
33. Parhofer K. G., Daugherty A., Kinoshita M., Schonfeld G.. Enhanced clearance from plasma of low density lipoproteins containing a truncated apolipoprotein, apoB-89. J. Lipid Res. 1990;31:2001-2007.[Abstract]
34.
Piedrahita J. A., Zhang S. H., Hagaman J. R., Oliver P. M., Maeda N.. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 1992;89:4471-4475.
35. Purcell-Huynh D. A., Farese R. V., Jr, Johnson D. F., Flynn L. M., Pierotti V., Newland D. L., Linton M. F., Sanan D. A., Young S. G.. Transgenic mice expressing high levels of human apolipoprotein B develop severe atherosclerotic lesions in response to a high-fat diet. J. Clin. Investig. 1995;95:2246-2257.
36. Wetterau J. R., Zilversmit D. B.. Localization of intracellular triacylglycerol and cholesteryl ester transfer activity in rat tissues. Biochim. Biophys. Acta 1986;875:610-617.[Medline]
37.
Young S. G.. Recent progress in understanding apolipoprotein B. Circulation 1990;82:1574-1594.
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