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Biomedical subjects

R E Hammer

Publications and source records attributed to R E Hammer.

At least 109 records · Page 6Linked to original sources
PubMed1993

An endocrine-specific element is an integral component of an exocrine-specific pancreatic enhancer.

We have analyzed the function of individual elements of the elastase I transcriptional enhancer in transgenic animals. This pancreas-specific enhancer comprises three functional elements, one of which (the B element) plays a dual role. Within the context of the enhancer, the B element contributes to appropriate acinar cell expression. However, when separated from the other enhancer components, the B element selectively directs transcription in islet cells of transgenic animals. This islet-specific activity is normally suppressed by an upstream repressor domain. The B element binds a novel islet-specific factor, and similar B-like elements are present in other pancreatic genes, both exocrine and endocrine specific. We suggest that a principal role of this transcriptional element and its associated factors is to activate many pancreatic genes as part of the program of pancreatic determination prior to the divergence of the acinar and islet cell lineages.

Amylases↗
PubMed1993

Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.

We employed homologous recombination in embryonic stem cells to produce mice lacking functional LDL receptor genes. Homozygous male and female mice lacking LDL receptors (LDLR-/- mice) were viable and fertile. Total plasma cholesterol levels were twofold higher than those of wild-type litter-mates, owing to a seven- to ninefold increase in intermediate density lipoproteins (IDL) and LDL without a significant change in HDL. Plasma triglyceride levels were normal. The half-lives for intravenously administered 125I-VLDL and 125I-LDL were prolonged by 30-fold and 2.5-fold, respectively, but the clearance of 125I-HDL was normal in the LDLR-/- mice. Unlike wild-type mice, LDLR-/- mice responded to moderate amounts of dietary cholesterol (0.2% cholesterol/10% coconut oil) with a major increase in the cholesterol content of IDL and LDL particles. The elevated IDL/LDL level of LDLR-/- mice was reduced to normal 4 d after the intravenous injection of a recombinant replication-defective adenovirus encoding the human LDL receptor driven by the cytomegalovirus promoter. The virus restored expression of LDL receptor protein in the liver and increased the clearance of 125I-VLDL. We conclude that the LDL receptor is responsible in part for the low levels of VLDL, IDL, and LDL in wild-type mice and that adenovirus-encoded LDL receptors can acutely reverse the hypercholesterolemic effects of LDL receptor deficiency.

Alleles↗
PubMed1993

Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a).

The B apolipoproteins, apo-B48 and apo-B100, are key structural proteins in those classes of lipoproteins considered to be atherogenic [e.g., chylomicron remnants, beta-VLDL, LDL, oxidized LDL, and Lp(a)]. Here we describe the development of transgenic mice expressing high levels of human apo-B48 and apo-B100. A 79.5-kb human genomic DNA fragment containing the entire human apo-B gene was isolated from a P1 bacteriophage library and microinjected into fertilized mouse eggs. 16 transgenic founders expressing human apo-B were generated, and the animals with the highest expression had plasma apo-B100 levels nearly as high as those of normolipidemic humans (approximately 50 mg/dl). The human apo-B100 in transgenic mouse plasma was present largely in lipoproteins of the LDL class as shown by agarose gel electrophoresis, chromatography on a Superose 6 column, and density gradient ultracentrifugation. When the human apo-B transgenic founders were crossed with transgenic mice expressing human apo(a), the offspring that expressed both transgenes had high plasma levels of human Lp(a). Both the human apo-B and Lp(a) transgenic mice will be valuable resources for studying apo-B metabolism and the role of apo-B and Lp(a) in atherosclerosis.

Animals↗
PubMed1992

Selective expression of trypsin fusion genes in acinar cells of the pancreas and stomach of transgenic mice.

Fusion genes combining the 5'-transcriptional regulatory region of the rat trypsin I gene and the structural gene of human growth hormone as a reporter were expressed to the high levels characteristic of the endogenous trypsin I gene selectively in the acinar cells of the pancreas of transgenic mice. As little as 232 base pairs of trypsin gene sequences containing the transcriptional start site and upstream promoter elements were sufficient to direct pancreatic expression. The tissue-specific expression was controlled transcriptionally. Trypsin-human growth hormone fusion transgenes also were expressed, although at low levels, in the stomach, an unexpected site for the expression of pancreatic digestive enzymes. Expression in the stomach of endogenous trypsin, elastase, and amylase genes in both normal and transgenic mice verified that transgene expression was consistent with normal expression of pancreatic genes. Endogenous amylase colocalizes with pepsinogen in the acinar cell-like Chief cells of the glandular portion of the mouse stomach. The expression of pancreatic genes in stomach cells is probably the consequence of similar developmental origins of pancreatic and gastric acinar cells from the primordial gut.

Animals↗
PubMed1992

Atherogenesis in transgenic mice expressing human apolipoprotein(a)

Elevated plasma levels of the lipoprotein Lp(a) are associated with increased risk for atherosclerosis and its manifestations, myocardial infarction, stroke and restenosis (for reviews, see refs 1-3). Lp(a) differs from low-density lipoprotein by the addition of the glycoprotein apolipoprotein(a), a homologue of plasminogen that contains many tandemly repeated units which resemble the fourth kringle domain of plasminogen, and single homologues of its kringle-5 and protease domain. As plasma Lp(a) concentration is strongly influenced by heritable factors and is refractory to most drug and dietary manipulation, the effects of modulating it are difficult to mimic experimentally. In addition, the absence of apolipoprotein(a) from virtually all species other than primates precludes the use of convenient animal models. Here we show that transgenic mice expressing human apolipoprotein(a) are more susceptible than control mice to the development of lipid-staining lesions in the aorta, and that apolipoprotein(a) co-localizes with lipid deposition in the artery walls.

Animals↗
PubMed1992

Reconstitution of lipoprotein(a) by infusion of human low density lipoprotein into transgenic mice expressing human apolipoprotein(a).

Lipoprotein(a) (Lp(a)) is an atherosclerosis-causing lipoprotein that circulates in human plasma as a complex of low density lipoprotein (LDL) and apolipoprotein(a) (apo(a)). It is not known whether apo(a) attaches to LDL within hepatocytes prior to secretion or in plasma subsequent to secretion. Here we describe the development of a line of mice expressing the human apo(a) transgene under the control of the murine transferrin promoter. The apo(a) was secreted into the plasma, but circulated free of lipoproteins. When human (h)-LDL was injected intravenously, the circulating apo(a) rapidly associated with the lipoproteins, as determined by nondenaturing gel electrophoresis. Human HDL and mouse LDL had no such effect. When h-VLDL was injected, there was a delayed association of apo(a) with the lipoprotein fraction which suggests that apo(a) preferentially associated with a metabolic product of VLDL. The complex of apo(a) with LDL formed both in vivo and in vitro was resistant to boiling in the presence of detergents and denaturants, but was resolved upon disulfide reduction. These studies suggest that apo(a) fails to associate with mouse lipoproteins due to structural differences between human and mouse LDL, and that Lp(a) formation can occur in plasma through the association of apo(a) with circulating LDL.

Aminocaproates↗
PubMed1992

LDL receptor-related protein internalizes and degrades uPA-PAI-1 complexes and is essential for embryo implantation.

The low density lipoprotein receptor-related protein (LRP) is a large multifunctional clearance receptor that has been implicated in the hepatic uptake of chylomicron remnants and in the removal of protease-inhibitor complexes from the circulation and from the extracellular space. Disruption of the LRP gene in mice blocks development of LRP-/- embryos around the implantation stage. The expression pattern of LRP in the postimplantation stage embryo is identical to that of urokinase, a plasminogen activator that confers invasive properties to migrating cells. We demonstrate that LRP mediates uptake and degradation of urokinase-type plasminogen activator-plasminogen activator inhibitor 1 complexes and propose that the inability of the giant cells to remove the inactive protease complexes from their surfaces interferes with implantation of the embryo.

Animals↗
PubMed1992

Cytoplasmic sequence required for basolateral targeting of LDL receptor in livers of transgenic mice.

When expressed in livers of transgenic mice, the human low density lipoprotein (LDL) receptor is specifically targeted to the basolateral (sinusoidal) surface of hepatocytes as determined by immunofluorescence and immunoelectron microscopy. The COOH-terminal cytoplasmic domain of the receptor (residues 790-839) contains a signal for this targeting. A mutant receptor truncated at residue 812 was localized exclusively to the apical (bile canalicular) surface. A mutant receptor terminating at residue 829 showed the normal basolateral distribution, as did a receptor in which alanine was substituted for serine 833, which was previously shown to be a site for phosphorylation in vitro. These data localize the basolateral targeting signal to the 17-residue segment between residues 812 and 828. A 10-amino acid stretch within this segment shows a 4/10 match with a sequence within a previously identified basolateral sorting motif for the receptor for polymeric IgA/IgM in MDCK cells. The four shared residues are spaced at intervals of three, raising the possibility that they all face the same side of an alpha-helix. We conclude that this 10-amino acid stretch may contain a signal that directs certain proteins, including the LDL receptor and the polymeric IgG/IgM receptor, to the basolateral surface of polarized epithelia.

Amino Acid Sequence↗
PubMed1992

Tissue-specific expression of kallikrein family transgenes in mice and rats.

To define the regulatory strategy for the transcriptional control of the kallikrein multigene family, we analyzed the expression of several kallikrein/SV40 T-antigen (TAg) fusion genes in transgenic mice and rats. Kallikrein family members are normally expressed at a high level in the submandibular gland and are expressed in a wide range of tissues that vary among individual family members. A total of 1.7 kb of proximal 5'-flanking DNA from the tissue kallikrein gene (rKlk1) was sufficient to confer much of the correct tissue-specific pattern on a TAg reporter gene. TAg mRNA was detectable in tissues that normally express rKlk1 and TAg-induced tumors arose in brain and pancreas. However, absolute levels of transgene mRNA were very low relative to the expression of the normal endogenous tissue kallikrein gene. In particular, expression in the salivary glands, normally very high for endogenous rKlk1, was either low or absent. An intact rKlk1 transgene with extensive flanking DNA (4.5 kb 5' and 4.7 kb 3') and complete intragenic (4 kb) sequences was expressed similarly to the fusion transgene, demonstrating that regulatory elements necessary for comprehensively correct expression are not contained within these additional gene regions. Two additional kallikrein/SV40 fusion transgenes were derived from other family members, one from the rKlk2 gene, which encodes tonin, and another from the rKlk8 gene, which encodes a prostate kallikrein. Whereas the endogenous rKlk2 and rKlk8 genes normally are expressed at high levels in rat salivary glands, they were not expressed in the salivary glands as transgenes. The results for these transgenes of three different family members indicate that control elements that direct the particular nonsalivary gland expression pattern characteristic of each family member may be present within the proximal 5'-flanking region of each gene, whereas regulatory sequences necessary for normal levels of expression in these tissues and for maximal salivary gland expression are not. We propose that the gene-associated regulatory sequences are complemented by a dominant control region that imposes salivary gland expression on the extended kallikrein family locus.

Animals↗
PubMed1992

Cell-specific expression of cytosolic phosphoenolpyruvate carboxykinase in transgenic mice.

The gene encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK) is expressed in multiple cell types in diverse tissues including liver, kidney, intestine, and white and brown adipose tissues. It can thus be considered a model system for examining the regulation of cell-specific transcription. The PEPCK gene is transcribed from a single start site, but studies of transgenic mice have revealed that distinct cis-acting elements (and thus different trans-acting factors) regulate PEPCK expression in hepatocytes, renal proximal tubule epithelial cells, and adipocytes. Hepatocytes require elements between -457 and +69 bp; renal proximal tubule epithelia require elements between -363 and +69 bp; and adipocytes require elements between -2086 and -888 bp. An additional element downstream of +69 bp is required to either attenuate PEPCK mRNA levels in liver and fat or increase renal PEPCK mRNA. We hypothesize that the transcription factors C/EBP and DBP are the principal tissue-specific regulators in liver, and that HNF-1 and perhaps C/EBP are important for kidney-specific PEPCK expression. We propose that the putative downstream element is involved in regulating PEPCK mRNA turnover in liver and fat. Finally, we suggest that the fat-specific element is an enhancer that requires a novel adipogenic regulatory factor, ARF6, to function. The long-term objective will be to fine map the cis-acting elements and identify the cognate trans-acting factors that regulate PEPCK in liver, kidney and fat. This information will help elucidate the combinatorial mechanisms that control the cell-specific expression of this complex gene.

Adipose Tissue↗
PubMed1992

Tissue-specific, developmental, hormonal, and dietary regulation of rat phosphoenolpyruvate carboxykinase-human growth hormone fusion genes in transgenic mice.

The cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene is expressed in multiple tissues and is regulated in a complex tissue-specific manner. To map the cis-acting DNA elements that direct this tissue-specific expression, we made transgenic mice containing truncated PEPCK-human growth hormone (hGH) fusion genes. The transgenes contained PEPCK promoter fragments with 5' endpoints at -2088, -888, -600, -402, and -207 bp, while the 3' endpoint was at +69 bp. Immunohistochemical analysis showed that the -2088 transgene was expressed in the correct cell types (hepatocytes, proximal tubular epithelium of the kidney, villar epithelium of the small intestine, epithelium of the colon, smooth muscle of the vagina and lungs, ductal epithelium of the sublingual gland, and white and brown adipocytes). Solution hybridization of hGH mRNA expressed from the transgenes indicated that white and brown fat-specific elements are located distally (-2088 to -888 bp) and that liver-, gut-, and kidney-specific elements are located proximally (-600 to +69 bp). However, elements outside of the region tested are necessary for the correct developmental pattern and level of PEPCK expression in kidney. Both the -2088 and -402 transgenes responded in a tissue-specific manner to dietary stimuli, and the -2088 transgene responded to glucocorticoid stimuli. Thus, different tissues utilize distinct cell-specific cis-acting elements to direct and regulate the PEPCK gene.

Adipose Tissue↗
PubMed1991

Negative and positive selection of antigen-specific cytotoxic T lymphocytes affected by the alpha 3 domain of MHC I molecules.

The alpha 1 and alpha 2 domains of major histocompatibility complex (MHC) class I molecules function in the binding and presentation of foreign peptides to the T-cell antigen receptor and control both negative and positive selection of the T-cell repertoire. Although the alpha 3 domain of class I is not involved in peptide binding, it does interact with the T-cell accessory molecule, CD8. CD8 is important in the selection of T cells as anti-CD8 antibody injected into perinatal mice interferes with this process. We previously used a hybrid class I molecule with the alpha 1/alpha 2 domains from Ld and the alpha 3 domain from Q7b and showed that this molecule binds an Ld-restricted peptide but does not interact with CD8-dependent cytotoxic T lymphocytes. Expression of this molecule in transgenic mice fails to negatively select a subpopulation of anti-Ld cytotoxic T lymphocytes. In addition, positive selection of virus-specific Ld-restricted cytotoxic T lymphocytes does not occur. We conclude that besides the alpha 1/alpha 2 domains of class I, the alpha 3 domain plays an important part in both positive and negative selection of antigen-specific cells.

Animals↗
PubMed1991

Germ-line intrachromosomal recombination restores fertility in transgenic MyK-103 male mice.

Males of the MyK-103 line of transgenic mice are fertile and sire litters of normal size, but they never transmit the transgene, whereas females transmit the transgene with normal frequency. The chromosome originally bearing the transgene can be transmitted through the male germ line, but only after the transgene is deleted or rearranged by intrachromosomal recombination. The transgene encodes a functional herpes simplex virus (HSV) thymidine kinase gene that causes sperm infertility when expressed in postmeiotic germ cells. Immunocytochemistry revealed clones of germ cells that fail to express HSV thymidine kinase. We postulate that these cells arose by transgene deletion in embryonic germ cells and postnatal spermatogonial stem cells and that they are responsible for the normal fertility of MyK-103 males. The frequency of recombination events at the integration locus suggests that it contains a hotspot for mitotic recombination.

Animals↗
PubMed1991

Tolerance to liver-specific antigens.

We have described a TG model for peripheral tolerance of alloreactive CTL. Expression of Q10/L on hepatocytes renders mice functionally tolerant, although in vitro we observe that TG animals have normal numbers of CTL.Pf directed against this antigen. The basis for the tolerance presumably resides in the fact that the TG mice are lacking a subpopulation, either through deletion or anergy, that is responsible for recognition of the antigen on hepatocytes in vivo. The data are consistent with a tolerance model where cells with high affinity receptors are silenced. The presumed low affinity antigen-specific cells remaining in TG mice cannot be primed in vivo when immunized with antigen on spleen cells. Further, these CTL generate poor lytic activity in vitro. This failure to prime TG CTL in vivo could be attributed to primed cells traveling to the liver where they become tolerized when exposed to antigen on hepatocytes. However, we show that TG cells, after transfer to non-TG recipients, cannot be primed in vivo, indicating that the presumed low-affinity cells remaining in TG mice are not readily activable in this milieu. These data also indicate that this tolerance is not readily reversible during a 10- to 17-d time interval.

Animals↗
PubMed1990

Spontaneous inflammatory disease in transgenic rats expressing HLA-B27 and human beta 2m: an animal model of HLA-B27-associated human disorders.

Humans who have inherited the human class I major histocompatibility allele HLA-B27 have a markedly increased risk of developing the multi-organ system diseases termed spondyloarthropathies. To investigate the role of B27 in these disorders, we introduced the B27 and human beta 2-microglobulin genes into rats, a species known to be quite susceptible to experimentally induced inflammatory disease. Rats from one transgenic line spontaneously developed inflammatory disease involving the gastrointestinal tract, peripheral and vertebral joints, male genital tract, skin, nails, and heart. This pattern of organ system involvement showed a striking resemblance to the B27-associated human disorders. These results establish that B27 plays a central role in the pathogenesis of the multi-organ system processes of the spondyloarthropathies. Elucidation of the role of B27 should be facilitated by this transgenic model.

Animals↗
PubMed1990

Diet-induced hypercholesterolemia in mice: prevention by overexpression of LDL receptors.

The current studies were designed to determine whether chronic overexpression of low density lipoprotein (LDL) receptors in the liver would protect mice from the increase in plasma LDL-cholesterol that is induced by high-fat diets. A line of transgenic mice was studied that express the human LDL receptor gene in the liver under control of the transferrin promoter. When fed a diet containing cholesterol, saturated fat, and bile acids for 3 weeks, the transgenic mice, in contrast to normal mice, did not develop a detectable increase in plasma LDL. The current data indicate that unregulated overexpression of LDL receptors can protect against diet-induced hypercholesterolemia in mice.

Animals↗