The formation of chylomicrons by the intestine is important for the absorption of dietary fats and fat-soluble vitamins (e.g., retinol, alpha-tocopherol). Apo B plays an essential structural role in ...the formation of chylomicrons in the intestine as well as the VLDL in the liver. We have developed genetically modified mice that express apo B in the liver but not in the intestine. By electron microscopy, the enterocytes of these mice lacked nascent chylomicrons in the endoplasmic reticulum and Golgi apparatus. Because these mice could not form chylomicrons, the intestinal villus enterocytes were massively engorged with fat, which was contained in cytosolic lipid droplets. These mice absorbed D-xylose normally, but there was virtually no absorption of retinol palmitate or cholesterol. The levels of alpha-tocopherol in the plasma were extremely low. Of note, the absence of chylomicron synthesis in the intestine did not appear to have a significant effect on the plasma levels of the apo B-containing lipoproteins produced by the liver. The mice lacking intestinal apo B expression represent the first genetic model of defective absorption of fats and fat-soluble vitamins and provide a useful animal model for studying nutrition and lipoprotein metabolism.
Familial hypobetalipoproteinemia is an autosomal codominant disorder resulting in a dramatic reduction in plasma concentrations of apolipoprotein (apo) B, cholesterol, and β-migrating lipoproteins. A ...benefit of hypobetalipoproteinemia is that mildly affected individuals may be protected from coronary vascular disease. We have used gene targeting to generate mice with a modified Apob allele. Mice containing this allele display all of the hallmarks of human hypobetalipoproteinemia: they produce a truncated apoB protein, apoB70, and have markedly decreased plasma concentrations of apoB, β-lipoproteins, and total cholesterol. In addition, the mice manifest several characteristics that are occasionally observed in human hypobetalipoproteinemia, including reduced plasma triglyceride concentrations, fasting chylomicronemia, and reduced high density lipoprotein cholesterol. An unexpected finding is that the modified Apob allele is strongly associated with exencephalus and hydrocephalus. These mice should help increase our understanding of hypobetalipoproteinemia, atherogenesis, and the eitiology of exencephalus and hydrocephalus.
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Conflict is an essential element of interesting stories. In previous work, we proposed a formal model of narrative conflict along with 4 quantitative dimensions which can be used to distinguish one ...conflict from another based on context: balance, directness, intensity, and resolution. This paper presents the results of an experiment designed to measure how well these metrics predict the responses of human readers when asked to measure these same values in a set of four stories. We conclude that our metrics are able to rank stories similarly to human readers for each of these four dimensions.
Familial hypobetalipoproteinemia (FHbeta), a syndrome characterized by low plasma cholesterol levels, is caused by mutations in the apo-B gene that interfere with the synthesis of apo-B100. FHbeta ...mutations frequently lead to the synthesis of a truncated form of apo-B, which typically is present in plasma at < 5% of the levels of apo-B100. Although many FHbeta mutations have been characterized, the basic mechanisms causing the low plasma levels of truncated apo-B variants have not been defined. We used gene targeting to create a mutant allele that exclusively yields a truncated apo-B, apo-B83. In mice heterozygous for the Apob83 allele, plasma levels and the size and density distribution of apo-B83-containing lipoproteins were strikingly similar to those observed in humans with FHbeta and an apo-B83 mutation. Analysis of mice carrying the Apob83 mutation revealed two mechanisms for the low plasma levels of apo-B83. First, Apob83 mRNA levels and apo-B83 secretion were reduced 76 and 72%, respectively. Second, apo-B83 was removed rapidly from the plasma, compared with apo-B100. This mouse model provides a new level of understanding of FHbeta and adds new insights into apo-B metabolism.
HYPOTHESIS A high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in soft tissue infections presents a treatment challenge. DESIGN Retrospective analysis. SETTING The San Francisco ...General Hospital Integrated Soft Tissue Infection (ISIS) Clinic. PATIENTS Patients treated at the ISIS Clinic from July 1, 2000, to June 30, 2003. MAIN OUTCOME MEASURES Information on patient demographics, surgical procedures, microbiologic studies, and antibiotic treatments was obtained for all patients treated in the ISIS Clinic. Microbial data and antibiotic susceptibility pattern of S aureus, treatment outcome, and antibiotic prescribed were analyzed for all evaluable patients. RESULTS The ISIS Clinic treated 6156 unique patients for 12 012 episodes of infection. In this cohort, 5164 (84%) were either homeless or had no health insurance. More than half of the patients (58%) were injection drug users, but most had only 1 prior visit to the clinic (62%). Patients underwent a surgical procedure 7707 times (64%). Of the 837 positive cultures obtained, S aureus was recovered 695 times (83%), and 525 (63%) of the cultures contained MRSA. Therefore, a full 76% of all S aureus isolated was MRSA. In a subset analysis of 622 cultures collected prospectively from consecutive patients, 282 (45%) grew organisms, of which 256 (91%) were S aureus. MRSA represented 59% of all S aureus isolated. Homelessness and injection drug use were risk factors for infection by S aureus and MRSA. In another subgroup of patients with soft tissue infections that required admission to the hospital, MRSA was recovered from the cultures in 149 patients. In these patients with MRSA, 44 (30%) only received a β-lactam antibiotic, inactive against MRSA, and had full resolution of their infection. CONCLUSIONS The prevalence of MRSA soft tissue infections in the medically underserved ISIS Clinic cohort is extremely high. The transmission of the MRSA seems to be in the community. Antibiotic therapy directed at MRSA may not be needed in a large number of patients with these soft tissue infections. Studies to identify the source and cause of this MRSA outbreak are urgently needed. Clinical trials to examine the need for antibiotic therapy in soft tissue infections should be conducted.Arch Surg. 2004;139:947-953-->
Posttranslational processing of Ras proteins has attracted considerable interest as a potential target for anticancer drug discovery. Rce1 encodes an endoprotease that facilitates membrane targeting ...of Ras and other prenylated proteins by releasing the carboxyl-terminal 3 amino acids (ie, the -AAX of the CAAX motif). Homozygous Rce1 mutant embryos(Rce1−/−)die late in gestation. To characterize the role of Rce1 in hematopoiesis, we performed adoptive transfers and investigated cells from the recipients. Rce1−/− fetal liver cells rescued lethally irradiated recipients and manifested normal long-term repopulating potential in competitive repopulation assays. The recipients of Rce1−/− cells developed modest elevations in mature myeloid cells (neutrophils + monocytes), but remained well. Bone marrow cells from mice that received transplants of Rce1−/− activated extracellular signal-related kinase (ERK) normally in response to granulocyte-macrophage colony-stimulating factor. These data suggest that pharmacologic inhibitors of Rce1 will have minimal effects on normal hematopoietic cells.
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Lipoprotein(a) Lp(a) is a lipoprotein formed by the disulfide linkage of apolipoprotein (apo) B100 of a low density lipoprotein particle to apolipoprotein(a). Prior studies have suggested that one of ...the C-terminal Cys residues of apo-B100 is involved in the disulfide linkage of apo-B100 to apo(a). To identify the apo-B100 Cys residue involved in the formation of Lp(a), we constructed a yeast artificial chromosome (YAC) spanning the human apo-B gene and used gene-targeting techniques to change Cys-4326 to Gly. The mutated YAC DNA was used to generate transgenic mice expressing the mutant human apo-B100 (Cys4326Gly). Unlike the wild-type human apo-B100, the mutant human apo-B100 completely lacked the ability to bind to apo(a) and form Lp(a). This study demonstrates that apo-B100 Cys-4326 is required for the assembly of Lp(a) and shows that gene targeting in YACs, followed by the generation of transgenic mice, is a useful approach for analyzing the structure of large proteins coded for by large genes.
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We recently reported that an 8-kilobase (kb) region, spanning from -54 to -62 kb 5' of the human apolipoprotein B (apoB) gene, contains intestine-specific regulatory elements that control apoB ...expression in the intestines of transgenic mice. In this study, we further localized the apoB intestinal control region to a 3-kb segment (-54 to -57 kb). DNaseI hypersensitivity studies uncovered a prominent DNaseI hypersensitivity site, located within a 315-base pair (bp) fragment at the 5'-end of the 3-kb segment, in transcriptionally active CaCo-2 cells but not in transcriptionally inactive HeLa cells. Transient transfection experiments with CaCo-2 and HepG2 cells indicated that the 315-bp fragment contained an intestine-specific enhancer, and analysis of the DNA sequence revealed putative binding sites for the tissue-specific transcription factors hepatocyte nuclear factor 3beta, hepatocyte nuclear factor 4, and CAAT enhancer-binding protein beta. Binding of these factors to the 315-bp enhancer was demonstrated in gel retardation experiments. Transfection of deletion mutants of the 315-bp enhancer revealed the relative contributions of these transcription factors in the activity of the apoB intestinal enhancer. The corresponding segment of the mouse apoB gene (located -40 to -83 kb 5' of the structural gene) exhibited a high degree of sequence conservation in the binding sites for the key transcriptional activators and also exhibited enhancer activity in transient transfection assays with CaCo-2 cells. In transgenic mouse expression studies, the 315-bp enhancer conferred intestinal expression to human apoB transgenes.
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We examined the relationship between the size of human apolipoprotein (apo) B and the formation and secretion of apoB-containing
lipoprotein particles. Stable transformants of the rat hepatoma cell ...line McA-RH7777 harboring a variety of human apoB cDNA
constructs were established, and these produced carboxyl-terminally truncated apoB proteins (apoB18, -B23, -B28, -B31, -B48,
and -B53). Immunoblotting of apoB proteins secreted into the culture medium and fractionated by equilibrium density ultracentrifugation
revealed that each of the truncated apoB species was secreted from the cells. The peak densities of the apoB-containing particles
decreased as the length of the apoB proteins increased. Apolipoproteins B18 and B23 appeared at the bottom of the salt gradient
(d = 1.23 g/ml), whereas particles containing apoB28, -B31, -B37, -B48, and -B53 exhibited progressive decreases in density.
The density distribution of secreted apolipoproteins was not affected by the expression or secretion of these recombinant
apoB species. As determined by nondenaturing gel electrophoresis, apoB28, -B31, -B37, -B48, and -B53 formed their own discrete
particles, and there was a direct correlation between the size of the particles and the length of the apoB species. The efficiency
and rate of secretion of these truncated forms of apoB were studied by measuring the decrease of immunoprecipitated 35S-labeled
apoB proteins in the cells and their accumulation in the medium. Proteins corresponding to apoB28 or larger were rapidly and
efficiently secreted, whereas apoB18 and apoB23 were secreted much more slowly. These data imply that the size of these truncated
apoB forms governs the lipid content of the apoB-containing lipoproteins formed as well as the kinetics of secretion.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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