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Biomedical subjects

R E Hammer

Publications and source records attributed to R E Hammer.

At least 55 records · Page 3Linked to original sources
PubMed1998

Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants.

The multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) has been postulated to participate in a number of diverse physiological and pathological processes ranging from the homeostasis of plasma lipoproteins, atherosclerosis, and fibrinolysis to neuronal regeneration and survival. It has not been possible to demonstrate in vivo the physiological significance of LRP for each of these complex processes by a conventional gene knockout approach because LRP is essential for embryonic development. Here we have used the Cre/loxP recombination system to achieve inducible, tissue-specific and quantitative disruption of the LRP gene in adult mice. Inactivation of LRP in the livers of LDL receptor-deficient mice resulted in the accumulation of cholesterol-rich remnant lipoproteins in the circulation. In normal animals, this caused a compensatory upregulation of the LDL receptor in the liver. Conditional gene targeting has thus allowed us to isolate a specific physiological function of LRP for in vivo analysis and has provided unequivocal evidence for another LDL receptor-independent cholesterol clearance pathway in liver.

Animals↗
PubMed1998

Neurexin I alpha is a major alpha-latrotoxin receptor that cooperates in alpha-latrotoxin action.

alpha-Latrotoxin is a potent neurotoxin from black widow spider venom that binds to presynaptic receptors and causes massive neurotransmitter release. A surprising finding was the biochemical description of two distinct cell surface proteins that bind alpha-latrotoxin with nanomolar affinities; Neurexin I alpha binds alpha-latrotoxin in a Ca(2+)-dependent manner, and CIRL/latrophilin binds in a Ca(2+)-independent manner. We have now generated and analyzed mice that lack neurexin I alpha to test its importance in alpha-latrotoxin action. alpha-Latrotoxin binding to brain membranes from mutant mice was decreased by almost 50% compared with wild type membranes; the decrease was almost entirely due to a loss of Ca(2+)-dependent alpha-latrotoxin binding sites. In cultured hippocampal neurons, alpha-latrotoxin was still capable of activating neurotransmission in the absence of neurexin I alpha. Direct measurements of [3H]glutamate release from synaptosomes, however, showed a major decrease in the amount of release triggered by alpha-latrotoxin in the presence of Ca2+. Thus neurexin I alpha is not essential for alpha-latrotoxin action but contributes to alpha-latrotoxin action when Ca2+ is present. Viewed as a whole, our results show that mice contain two distinct types of alpha-latrotoxin receptors with similar affinities and abundance but different properties and functions. The action of alpha-latrotoxin may therefore be mediated by independent parallel pathways, of which the CIRL/latrophilin pathway is sufficient for neurotransmitter release, whereas the neurexin I alpha pathway contributes to the Ca(2+)-dependent action of alpha-latrotoxin.

Alternative Splicing↗
PubMed1998

Cranial and cardiac neural crest defects in endothelin-A receptor-deficient mice.

Neural crest cells arise in the dorsal aspect of the neural tube and migrate extensively to differentiate into a variety of neural and non-neural tissues. While interactions between neural crest cells and their local environments are required for the proper development of these tissues, little information is available about the molecular nature of the cell-cell interactions in cephalic neural crest development. Here we demonstrate that mice deficient for one type of endothelin receptor, ETA, mimic the human conditions collectively termed CATCH 22 or velocardiofacial syndrome, which include severe craniofacial deformities and defects in the cardiovascular outflow tract. We show that ETA receptor mRNA is expressed by the neural crest-derived ectomesenchymal cells of pharyngeal arches and cardiac outflow tissues, whereas ET-1 ligand mRNA is expressed by arch epithelium, paraxial mesoderm-derived arch core and the arch vessel endothelium. This suggests that paracrine interaction between neural crest-derived cells and both ectoderm and mesoderm is essential in forming the skeleton and connective tissue of the head. Further, we find that pharyngeal arch expression of goosecoid is absent in ETA receptor-deficient mice, placing the transcription factor as one of the possible downstream signals triggered by activation of the ETA receptor. These observations define a novel genetic pathway for inductive communication between cephalic neural crest cells and their environmental counterparts.

Animals↗
PubMed1998

Dual genetic pathways of endothelin-mediated intercellular signaling revealed by targeted disruption of endothelin converting enzyme-1 gene.

Recent gene targeting studies have revealed unexpected roles for endothelins in the development of neural crest-derived tissues. Endothelin converting enzyme-1 (ECE-1) catalyzes the proteolytic activation of big endothelin-1 to endothelin-1(ET-1) in vitro. However, the importance of ECE-1 cleavage in the multiple endothelin pathways in vivo is unknown. Here we generated a targeted null mutation in the mouse ECE-1 gene. ECE-1-/- term embryos exhibited craniofacial and cardiac abnormalities virtually identical to the defects seen in ET-1 and endothelin A receptor (ETA)-deficient embryos. Epidermal melanocytes as well as enteric neurons of the distal gut were also absent in ECE-1-/- embryos, reproducing the developmental phenotype seen in ET-3-/- and endothelin B receptor (ETB)-/- mice. Surprisingly, large amounts of mature ET-1 peptide are found in ECE-1-/- embryos, indicating that non-ECE-1 protease(s) can activate ET-1 at certain sites. However, these enzymes cannot produce sufficient mature endothelin at the locations crucial for normal embryonic development. These findings reveal that ECE-1 is a bona fide activating protease for both big ET-1 and big ET-3 in vivo, and that the cell-cell communication pathways represented by the ET-1/ECE-1/ETA axis and the ET-3/ECE-1/ETB axis are each involved in the development of distinct subsets of neural crest cell lineages. Mutations in ECE-1 may cause developmental defects in humans, such as Hirschsprung disease, velocardiofacial syndrome and related neurocristopathies.

Animals↗
PubMed1998

Egg culture: the foundation.

Ralph Brinster began his classic work on egg culture more than 35 years ago. His interest in mammalian egg culture had developed, in part, as a consequence of his experiences with animal breeding and reproduction that he gained while growing up on a farm. Ralph decided early in his career that an in vitro approach to culturing eggs would provide a powerful tool with which to study the development of these cells. Beginning at the close of the 19th century, a number of investigators had performed in vitro studies on egg culture and the related area of egg transfer; however, the ability to recover and transplant eggs had reached a much higher level of perfection than had culture. Eggs of many species could be successfully transferred, but there was no reliable technique for egg culture. In 1963, Ralph reported a method for culturing eggs in microdrops of medium under oil (Brinster, 1963), which has become universally used. Two years later, he identified pyruvate as the central and essential energy source for early stages of mouse eggs (Brinster, 1965b). These two developments revolutionized in vitro studies of mammalian eggs and issued in an era of intense research activity concerning egg culture and egg manipulation. Effective formulations of culture media could now be developed to allow routine in vitro maintenance of eggs, and important parameters for these recipes were soon determined. It was quickly established that the requirement for pyruvate as an energy source exists at ovulation in many species and is already present in germ cells of the mouse fetus. The metabolic activity of the fertilized mouse egg was shown to be low and comparable to bone; however, four days later, at the blastocyst stage of development, the metabolic activity was comparable to brain. Thus, a foundation of understanding about the biology of early mammalian eggs was established between 1960 and 1970, and subsequent studies have broadened this understanding. However, the greatest impact of a simple, reliable egg culture method has been to provide the ability to perform complicated manipulative procedures on preimplantation stages of mammalian embryos. In no area has this been more important than in development of transgenic animals. All methods for generating germ line genetic modifications rely on the ability to maintain and manipulate eggs and early developmental stages in vitro without loss of developmental competence. The importance of efficient egg culture to manipulation and transgenesis is fundamental and enabling.

Animals↗
PubMed1997

Hepatic fibrosis, glomerulosclerosis, and a lipodystrophy-like syndrome in PEPCK-TGF-beta1 transgenic mice.

Transgenic mice overexpressing a constitutively active human TGF-beta1 under control of the rat phosphoenolpyruvate carboxykinase regulatory sequences developed fibrosis of the liver, kidney, and adipose tissue, and exhibited a severe reduction in body fat. Expression of the transgene in hepatocytes resulted in increased collagen deposition, altered lobular organization, increased hepatocyte turnover, and in extreme cases, hemorrhage and thrombosis. Renal expression of the transgene was localized to the proximal tubule epithelium, and was associated with tubulointerstitial fibrosis, characterized by excessive collagen deposition and increased fibronectin and plasminogen activator inhibitor-1 immunoreactivity. Pronounced glomerulosclerosis was evident, and hydronephrosis developed with low penetrance. Expression of TGF-beta1 in white and brown adipose tissue resulted in a lipodystrophy-like syndrome. All white fat depots and brown fat pads were severely reduced in size, and exhibited prominent fibroplasia. This reduction in WAT was due to impaired adipose accretion. Introduction of the transgene into the ob/ob background suppressed the obesity characteristic of this mutation; however, transgenic mutant mice developed severe hepato- and splenomegaly. These studies strengthen the link between TGF-beta1 expression and fibrotic disease, and demonstrate the potency of TGF-beta1 in modulating mesenchymal cell differentiation in vivo.

Adipose Tissue↗
PubMed1997

Elevated levels of SREBP-2 and cholesterol synthesis in livers of mice homozygous for a targeted disruption of the SREBP-1 gene.

The synthesis of cholesterol and its uptake from plasma LDL are regulated by two membrane-bound transcription factors, designated sterol regulatory element binding protein-1 and -2 (SREBP-1 and SREBP-2). Here, we used the technique of homologous recombination to generate mice with disruptions in the gene encoding the two isoforms of SREBP-1, termed SREBP-1a and SREBP-1c. Heterozygous gene-disrupted mice were phenotypically normal, but 50- 85% of the homozygous (-/-) mice died in utero at embryonic day 11. The surviving -/- mice appeared normal at birth and throughout life. Their livers expressed no functional SREBP-1. There was a 1.5-fold upregulation of SREBP-2 at the level of mRNA and a two- to threefold increase in the amount of mature SREBP-2 in liver nuclei. Previous studies showed that SREBP-2 is much more potent than SREBP-1c, the predominant hepatic isoform of SREBP-1, in activating transcription of genes encoding enzymes of cholesterol synthesis. Consistent with this observation, the SREBP-1 -/- animals manifested elevated levels of mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase, farnesyl diphosphate synthase, and squalene synthase. Cholesterol synthesis, as measured by the incorporation of [3H]water, was elevated threefold in livers of the -/- mice, and hepatic cholesterol content was increased by 50%. Fatty acid synthesis was decreased in livers of the -/- mice. The amount of white adipose tissue was not significantly decreased, and the levels of mRNAs for lipogenic enzymes, adipocyte lipid binding protein, lipoprotein lipase, and leptin were normal in the -/- mice. We conclude from these studies that SREBP-2 can replace SREBP-1 in regulating cholesterol synthesis in livers of mice and that the higher potency of SREBP-2 relative to SREBP-1c leads to excessive hepatic cholesterol synthesis in these animals.

Adipose Tissue↗
PubMed1997

Transgenic mice expressing human ApoB95 and ApoB97. Evidence that sequences within the carboxyl-terminal portion of human apoB100 are important for the assembly of lipoprotein.

The structural features of apolipoprotein (apo) B that are important for its covalent linkage to apo(a) to form lipoprotein(a) (Lp(a)) are incompletely understood. Although apoB100 cysteine 4326 is required for the disulfide linkage with apo(a), other structural features, aside from a single free cysteine residue, must be important for apoB's initial interaction with apo(a) and for facilitating the formation of the disulfide bond. To determine if sequences carboxyl-terminal to cysteine 4326 affect the efficiency of Lp(a) formation, we used "pop-in, pop-out" gene targeting in a human apoB yeast artificial chromosome to introduce nonsense mutations into exon 29 of the apoB gene. The mutant yeast artificial chromosomes, which coded for the truncated versions of human apoB, apoB95, and apoB97, were then used to express these mutant forms of apoB in transgenic mice. As judged by in vitro assays of Lp(a) formation, apoB95 (4330 amino acids) formed a small amount of Lp(a) but did so slowly. In contrast, apoB97 (4397 amino acids) formed Lp(a) rapidly, although not quite as rapidly as the full-length apoB100 (4536 amino acids). These results were supported by an analysis of double-transgenic mice expressing both human apo(a) and either apoB95 or apoB97. In mice expressing both apoB95 and apo(a), there was only a trace amount of Lp(a) in the plasma, and most of the apo(a) was free, whereas in mice expressing both apoB97 and apo(a), virtually all of the apo(a) was bound to apoB97 in the form of Lp(a). These results show that sequences carboxyl-terminal to apoB95 (amino acids 4331-4536) are not absolutely required for Lp(a) formation, but this segment of the apoB molecule, particularly residues 4331-4397, is necessary for the efficient assembly of Lp(a).

Animals↗
PubMed1997

Novel HY peptide antigens presented by HLA-B27.

We have identified two peptides corresponding to the male-specific HY minor histocompatibility Ags presented by HLA-B27 in transgenic rodents, isolated from whole cell extracts and from immunoprecipitated B27 molecules of male B27 rat spleen cells. HPLC peptide fractions that sensitized female B27 targets for lysis by B27-restricted anti-HY CTL were analyzed by electrospray tandem mass spectrometry using a new highly sensitive quadrupole/time-of-flight instrument. Two peptide sequences were obtained, KQYQKSTER and AVLNKSNREVR. Synthetic peptides corresponding to these sequences bound B27 in vitro and were recognized by distinct B27-restricted anti-HY CTL populations. Neither peptide sequence entirely matches known protein sequences or shows a resemblance to known Y chromosome genes, but both show homology to known autosomally encoded proteins. Both peptides were shown to be controlled by the Sxr(b) segment of the short arm of the mouse Y chromosome, a segment known to contain all previously identified HY Ags. Taken together, these findings suggest that the two peptides arise as a result of Y chromosome-regulated control of one or more autosomal gene products. Although arginine at position 2 is a dominant anchor residue for peptides bound to B27, neither B27-presented HY sequence contains this residue. These studies, employing sensitive new methodology for identification of MHC-bound peptides, significantly extend the complexity of the genetic basis of HY Ags and expand the repertoire of antigenically active peptides bound to B27.

Animals↗
PubMed1997

Characterization of defective nucleotide excision repair in XPC mutant mice.

Nucleotide excision repair (NER) is a fundamental process required for maintaining the integrity of the genome in cells exposed to environmental DNA damage. Humans defective in NER suffer from the hereditary cancer-prone disease xeroderma pigmentosum. In order to model this disease in mice a mutation in the mouse XPC gene was generated and used to replace a wild-type XPC allele in mouse embryonic stem cells by homologous recombination. These cells were used to derive XPC mutant mice. Fibroblasts from mutant embryos were more sensitive to the cytotoxic effects of ultraviolet light than wild-type and heterozygous cells. Repair synthesis of DNA following irradiation with ultraviolet light was reduced in these cells, indicating a defect in NER. Additionally, XPC mutant embryo fibroblasts were specifically defective in the removal of pyrimidine (6-4) pyrimidone photoproducts from the non-transcribed strand of the transcriptionally active p53 gene. Mice defective in the XPC gene appear to be an excellent model for studying the role of NER and its interaction with other proteins in the molecular pathogenesis of cancer in mammals following exposure to environmental carcinogens.

Alleles↗
PubMed1997

Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells.

We have produced transgenic mice whose livers express a dominant positive NH2-terminal fragment of sterol regulatory element binding protein-1c (SREBP-1c). Unlike full-length SREBP-1c, the NH2-terminal fragment enters the nucleus without a requirement for proteolytic release from cell membranes, and hence it is immune to downregulation by sterols. We compared SREBP-1c transgenic mice with a line of transgenic mice that produces an equal amount of the NH2-terminal fragment of SREBP-1a. SREBP-1a and -1c are alternate transcripts from a single gene that differ in the first exon, which encodes part of an acidic activation domain. The 1a protein contains a long activation domain with 12 negatively charged amino acids, whereas the 1c protein contains a short activation domain with only 6 such amino acids. As previously reported, livers of the SREBP-1a transgenic mice were massively enlarged, owing to accumulation of triglycerides and cholesterol. SREBP-1c transgenic livers were only slightly enlarged with only a moderate increase in triglycerides, but not cholesterol. The mRNAs for the LDL receptor and several cholesterol biosynthetic enzymes were elevated in SREBP-la transgenic mice, but not in 1c transgenic mice. The mRNAs for fatty acid synthase and acetyl CoA carboxylase were elevated 9- and 16-fold in la animals, but only 2- and 4-fold in 1c animals. Experiments with transfected cells confirmed that SREBP-1c is a much weaker activator of transcription than SREBP-1a when both are expressed at levels approximating those found in nontransfected cells. SREBP-1c became a strong activator only when expressed at supraphysiologic levels. We conclude that SREBP-1a is the most active form of SREBP-1 and that SREBP-1c may be produced when cells require a lower rate of transcription of genes regulating cholesterol and fatty acid metabolism.

Acetyl-CoA Carboxylase↗
PubMed1997

Monoclonal antibody to the HLA class I alpha 3 domain inhibits T cell activation and prolongs cardiac allograft survival in HLA-transgenic mice.

Antibodies recognizing MHC class I molecules expressed on the surface of T cells have been shown to inhibit T cell responses in vitro. These findings suggested that therapy with such an antibody may prevent rejection and promote graft acceptance. We therefore tested the effect of an anti-HLA class I alpha 3 domain antibody (TP25.99) in vivo using transgenic C57BL/6 mice expressing HLA-B2705. Flow cytometric analysis confirmed the binding of TP25.99 to normal human peripheral blood lymphocytes and to mouse spleen cells, bone marrow cells and thymocytes isolated from hemizygous (+/-) transgenic littermates but not from homozygous (-/-) littermates. TP25.99 inhibited OKT-3-induced, but not PMA+ionomycin-induced, proliferation of human peripheral blood lymphocyte as well as anti-CD3 or Con A-induced proliferation of HLA+ mouse T cells. Both intact monoclonal antibody TP25.99 and TP25.99 Fab inhibited T cell proliferation. Reduced proliferation was associated with suppressed production of interleukin-2 as measured by ELISA. The efficacy of TP25.99 Fab in vivo was evaluated in a heart allograft model. Antibody therapy of (H-2h, B2705+) transgenic recipients of allogeneic Balb/c (H-2d) heart grafts prolonged graft survival significantly (MST = 19.8 +/- 6.4, p = 0.003) compared to treated (H-2b, B2705-) (MST = 9.17 +/- 2.2) or untreated (H-2b, B2705+) (MST = 10.0 +/- 2.8) transgenic recipients. This demonstrates that immunomodulation through anti-HLA class I antibody therapy can lead to prolongation of graft survival.

Animals↗
PubMed1997

A new MHC locus that influences class I peptide presentation.

We have investigated the HLA-B27-restricted CTL response to HY minor histocompatibility antigens in rats and mice transgenic for HLA-B27 and human beta2-microglobulin. A polymorphism was found at a locus within the H2 complex, producing two distinct but overlapping sets of B27-presented HY peptides. The locus, named Cim2, mapped between the K and Pb loci, and its product is therefore distinct from TAP, LMP, and tapasin. Identical findings in rats and mice, including identical HY peptide sequences and the failure of a rat Tap2A transgene to alter CTL recognition, suggest that a homologous locus with similar polymorphism exists in the rat. Cim2, or a closely linked locus, was found to exert a broad effect on peptide loading of both HLA-B27 and mouse class I alleles. The data thus establish a strong, previously unrecognized MHC-encoded influence on the class I antigen pathway.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Synergistic interactions between XPC and p53 mutations in double-mutant mice: neural tube abnormalities and accelerated UV radiation-induced skin cancer.

The significance of DNA repair to human health has been well documented by studies on xeroderma pigmentosum (XP) patients, who suffer a dramatically increased risk of cancer in sun-exposed areas of their skin [1,2]. This autosomal recessive disorder has been directly associated with a defect in nucleotide excision-repair (NER) [1,2]. Like human XP individuals, mice carrying homozygous mutations in XP genes manifest a predisposition to skin carcinogenesis following exposure to ultraviolet (UV) radiation [3-5]. Recent studies have suggested that, in addition to roles in apoptosis [6] and cell-cycle checkpoint control [7] in response to DNA damage, p53 protein may modulate NER [8]. Mutations in the p53 gene have been observed in 50% of all human tumors [9] and have been implicated in both the early [10] and late [11] stages of skin cancer. To examine the consequences of a combined deficiency of the XPC and the p53 proteins in mice, we generated double-mutant animals. We document a spectrum of neural tube defects in XPC p53 mutant embryos. Additionally, we show that, following exposure to UV-B radiation, XPC p53 mutant mice have more severe solar keratosis and suffer accelerated skin cancer compared with XPC mutant mice that are wild-type with respect to p53.

Animals↗
PubMed1996

Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a.

The NH2-terminal domain of sterol-regulatory element binding protein-1a (SREBP-1a) activates transcription of genes encoding enzymes of cholesterol and fatty acid biosynthesis in cultured cells. This domain is synthesized as part of a membrane-bound precursor that is attached to the nuclear envelope and endoplasmic reticulum. In sterol-depleted cells a two-step proteolytic process releases this NH2-terminal domain, which enters the nucleus and activates transcription. Proteolysis is suppressed by sterols, thereby suppressing transcription. In the current experiments we produce transgenic mice that overexpress a truncated version of human SREBP-1a that includes the NH2-terminal domain but lacks the membrane attachment site. This protein enters the nucleus without a requirement for proteolysis, and therefore it cannot be down-regulated. Expression was driven by the phosphoenolpyruvate carboxykinase (PEPCK) promoter, which gives high level expression in liver. When placed on a low carbohydrate/high protein diet to induce the PEPCK promoter, the transgenic mice developed progressive and massive enlargement of the liver, owing to the engorgement of hepatocytes with cholesterol and triglycerides. The mRNAs encoding 3-hydroxy-3-methylglutaryl CoA (HMG CoA) synthase, HMG CoA reductase, squalene synthase, acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase-1 were all elevated markedly, as was the LDL receptor mRNA. The rates of cholesterol and fatty acid synthesis in liver were elevated 5- and 25-fold, respectively. Remarkably, plasma lipid levels were not elevated. The amount of white adipose tissue decreased progressively as the liver enlarged. These studies indicate that the NH2-terminal domain of SREBP-1a can produce major effects on lipid synthesis and storage in the liver.

Adipose Tissue↗