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Vascular injury and remodeling are common pathological sequelae of hypertension. Previous studies have suggested that the renin-angiotensin system acting through the type 1 angiotensin II (AT(1)) receptor promotes vascular pathology in hypertension. To study the role of AT(1) receptors in this process, we generated mice with cell-specific deletion of AT(1) receptors in vascular smooth muscle cells using Cre/Loxp technology. We crossed the SM22α-Cre transgenic mouse line expressing Cre recombinase in smooth muscle cells with a mouse line bearing a conditional allele of the Agtr1a gene (Agtr1a (flox)), encoding the major murine AT(1) receptor isoform (AT(1A)). In SM22α-Cre(+)Agtr1a (flox/flox) (SMKO) mice, AT(1A) receptors were efficiently deleted from vascular smooth muscle cells in larger vessels but not from resistance vessels such as preglomerular arterioles. Thus, vasoconstrictor responses to angiotensin II were preserved in SMKO mice. To induce hypertensive vascular remodeling, mice were continuously infused with angiotensin II for 4 weeks. During infusion of angiotensin II, blood pressures increased significantly and to a similar extent in SMKO and control mice. In control mice, there was evidence of vascular oxidative stress indicated by enhanced nitrated tyrosine residues in segments of aorta; this was significantly attenuated in SMKO mice. Despite these differences in oxidative stress, the extent of aortic medial expansion induced by angiotensin II infusion was virtually identical in both groups. Thus, vascular AT(1A) receptors promote oxidative stress in the aortic wall but are not required for remodeling in angiotensin II-dependent hypertension.

Prostaglandin E(2) (PGE(2)) plays an important role in the normal physiology of many organ systems. Increased levels of this lipid mediator are associated with many disease states, and it potently regulates inflammatory responses. Three enzymes capable of in vitro synthesis of PGE(2) from the cyclooxygenase metabolite PGH(2) have been described. Here, we examine the contribution of one of these enzymes to PGE(2) production, mPges-2, which encodes microsomal prostaglandin synthase-2 (mPGES-2), by generating mice homozygous for the null allele of this gene. Loss of mPges-2 expression did not result in a measurable decrease in PGE(2) levels in any tissue or cell type examined from healthy mice. Taken together, analysis of the mPGES-2 deficient mouse lines does not substantiate the contention that mPGES-2 is a PGE(2) synthase.

A mouse line has been developed that expresses low levels of the NMDA R1 (NR1) subunit of the NMDA receptor [Cell 98 (1999) 427]. These NR1 hypomorphic mice represent an experimental model of reduced NMDA receptor function that may be relevant to the pathophysiology of schizophrenia. To further characterize the neurobiological phenotype resulting from developmental NMDA receptor hypofunction, regional brain metabolic activity was assessed by autoradiographic analysis of 14C-2-deoxyglucose (2-DG) uptake. In addition, ligand binding to NMDA, AMPA, and kainate receptors was measured by quantitative autoradiography. MK-801 binding to NMDA receptors was reduced markedly throughout the brain of the NR1 hypomorphic mice. However, no alteration in 3H-AMPA or 3H-kainate binding was apparent in any region examined. Neuroanatomically specific alterations in regional 2-DG uptake were observed in the NR1 hypomorphic animals. Reduced relative 2-DG uptake was observed in the medial prefrontal and anterior cingulate cortices. Altered patterns of 2-DG uptake were also found in neocortical regions, with selective reductions of uptake in layer 6 in frontal regions of somatosensory and motor cortices. These data indicate alterations in cortical circuitry in the NR1 hypomorphic animals and are consistent with functional imaging studies in chronic schizophrenia patients which typically show reduced frontal cortical metabolic activity. Reduced relative 2-DG uptake was also found in the caudate, accumbens, hippocampus, and select thalamic regions in the NR1-deficient mice. However, in many other brain regions no alteration in 2-DG uptake was observed. The alterations in 2-DG uptake in the NR1 hypomorphic mice were distinctly different compared to those observed after acute challenge with the selective NMDA antagonist MK-801 in wild-type mice. The altered patterns of brain 2-DG uptake in the NR1 hypomorphic mice found in the present work, together with the altered behavioral phenotypes previously described, suggest that the mice may provide a valuable model to study novel therapeutic strategies to counteract the neurobiological consequences of chronic developmental NMDA receptor hypofunction.

BRCA1 is a nuclear phosphoprotein that has been classified as a tumor suppressor based on the fact that women carrying a mutated copy of the BRCA1 gene are at increased risk of developing breast and ovarian cancer. The association of BRCA1 with RAD51 has led to the hypothesis that BRCA1 is involved in DNA repair. We describe here the generation and analysis of murine embryonic stem (ES) cell lines in which both copies of the murine homologue of the human BRCA1 gene have been disrupted by gene targeting. We show that exogenous DNA introduced into these BRCA1 deficient cells by electroporation is randomly integrated into the genome at a significantly higher rate than in wild type ES cells. In contrast, integration of exogenous DNA by homologous recombination occurs in BRCA1 deficient cells at a significantly lower rate than in wild type controls. When BRCA1 expression is re-established at 5-10% of normal levels by introduction of a Brca1 transgene into BRCA1 deficient ES cells, the frequency of random integration is reduced to wild type levels, although the frequency of homologous recombination is not significantly improved. These results suggest that BRCA1 plays a role in determining the response of cells to double stranded DNA breaks.

Arachidonic acid metabolism by 5-lipoxygenase leads to production of the potent inflammatory mediators, leukotriene (LT) B4 and the cysteinyl LT. Relative synthesis of these subclasses of LT, each with different proinflammatory properties, depends on the expression and subsequent activity of LTA4 hydrolase and LTC4 synthase, respectively. LTA4 hydrolase differs from other proteins required for LT synthesis because it is expressed ubiquitously. Also, in vitro studies indicate that it possesses an aminopeptidase activity. Introduction of cysteinyl LT and LTB4 into animals has shown LTB4 is a potent chemoattractant, while the cysteinyl LT alter vascular permeability and smooth muscle tone. It has been impossible to determine the relative contributions of these two classes of LT to inflammatory responses in vivo or to define possible synergy resulting from the synthesis of both classes of mediators. To address this question, we have generated LTA4 hydrolase-deficient mice. These mice develop normally and are healthy. Using these animals, we show that LTA4 hydrolase is required for the production of LTB4 in an in vivo inflammatory response. We show that LTB4 is responsible for the characteristic influx of neutrophils accompanying topical arachidonic acid and that it contributes to the vascular changes seen in this model. In contrast, LTB4 influences only the cellular component of zymosan A-induced peritonitis. Furthermore, LTA4 hydrolase-deficient mice are resistant to platelet-activating factor, identifying LTB4 as one mediator of the physiological changes seen in systemic shock. We do not identify an in vivo role for the aminopeptidase activity of LTA4 hydrolase.

BRCA1 is a nuclear phosphoprotein that is expressed in a cell cycle regulated manner in virtually all normal dividing cells. Inheritance of a mutated copy of the BRCA1 gene increases a woman's risk for developing breast and ovarian cancer (1-3). Since the tumors that arise in these individuals consistently fail to express the wild-type allele, BRCA1 is believed to encode a tumor suppressor. Loss of the remaining functional BRCA1 allele, therefore, is one of the steps leading to neoplastic transformation of some types of epithelial cells. The isolation of the murine homologue of the human BRCA1 gene opened up the possibility of using a powerful genetic approach to study the role of this gene in both normal development and tumor formation. This genetic approach involves in vitro manipulation of the genome of embryonic stem (ES) cells, stable tissue culture cell lines derived from mouse blastocysts. After introducing mutations into the murine homologue of the BRCA1 gene Brca1 in these cell lines, four groups have generated mouse lines carrying the same mutations (4-7). Surprisingly, mice carrying a single mutant Brca1 allele do not display the increased risk for breast tumors seen in humans carrying similar mutations. However, while loss of BRCA1 appears to be a one of the many events involved in tumorgenesis in humans, these mouse lines demonstrate that gene expression is essential for development; as homozygosity for each of the Brca1 mutations results in postimplantation embryonic lethality. The survival of Brca1 deficient embryos is extended by one or two days in the absence of p53 and p21 (7,8).

Survival of newborn placental mammals depends on closure of the ductus arteriosus (DA), an arterial connection in the fetus which directs blood away from the pulmonary circulation and towards the placenta where oxygenation occurs. Here we show that morphological changes resulting in closure of the DA in mice are virtually identical to those observed in larger mammals, including humans, and that maintenance of the DA in the open, or patent, state in fetal mice is dependent on prostaglandin synthesis. This requirement is absent in mice lacking the prostaglandin E2 EP4 receptor (EP4(-/-) mice). In EP4(-/-) mice of the 129 strain, remodelling of the DA fails to occur after birth, resulting in a left-to-right shunt of blood and subsequently in death. This suggests that the neonatal drop in prostaglandin E2 that triggers ductal closure is sensed through the EP4 receptor. In contrast, 5% of EP4(-/-) mice of mixed genetic background survive, and selective breeding of these mice leads to a 21% survival rate, suggesting that alleles at other loci can provide an alternative mechanism for ductal closure.

We have generated a mouse line in which the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been mutated by gene targeting. Like human cystic fibrosis (CF) patients, mice lacking a functional CFTR gene, referred to as CFTR(-/-) mice, show increased numbers of goblet cells and obstruction of glands with inspissated eosinophilic secretions. The obstruction of glands often results in the destruction of gland-containing tissues in these animals. However, unlike the case in human CF patients, the most severe pathological changes in these mice were found, on preliminary analysis, to be confined to the intestinal tract and gallbladder. Although respiratory failure is the primary cause of death among humans with CF, we found only minor pathological alterations in the lungs and upper airways of our CFTR(-/-) animals. Possible explanations for the apparent lack of respiratory disease are the young age at which the animals were examined and the pathogen-free environment in which they were housed. In this manuscript, we examine the respiratory and other organ systems of CFTR(-/-) mice that have survived to adulthood. We also report on initial experiments in which CFTR(-/-) mice have been exposed to bacterial pathogens, and we present data on a single animal that displayed severe respiratory disease.

The structure-function relationships of the biological activities of mutant varieties of the pleiotropic cytokine interleukin-6 (human) were measured by three assays: induction of immunoglobulin M (IgM) secretion from an Epstein-Barr virus-transformed human B cell line and induction of fibrinogen secretion from either a human hepatoma cell line or a rat hepatoma cell line. The biological effects of the cytokine were characterized by three parameters as determined by a novel analysis: effectiveness (the maximal response attainable), efficiency (the concentration yielding a half-maximal response), and complexity (a measure of heterogeneity and feedback control). Substitution of serine for cysteine was associated with a reduction in the effectiveness of interleukin-6 in both fibrinogen secretion assays. In the assay with human hepatoma cells, there was also a profound reduction in efficiency. Serine substitution in the human IgM synthesis assay appears mainly to reduce the efficiency. Deletion of amino acids 4 to 23 increased the efficiency in the rat hepatoma assay. The complexity parameter suggests the presence of multiple receptor classes or negative feedback in all three assays. Use of the proposed sequential approach to the analysis of dose-response relations in bioassays provides a more useful quantitative assessment of activities as well as more insight into the complexity of the reactions.

Cystic fibrosis results from defects in the gene encoding a cyclic adenosine monophosphate-dependent chloride ion channel known as the cystic fibrosis transmembrane conductance regulator (CFTR). To create an animal model for cystic fibrosis, mice were generated from embryonic stem cells in which the CFTR gene was disrupted by gene targeting. Mice homozygous for the disrupted gene display many features common to young human cystic fibrosis patients, including failure to thrive, meconium ileus, alteration of mucous and serous glands, and obstruction of glandlike structures with inspissated eosinophilic material. Death resulting from intestinal obstruction usually occurs before 40 days of age.

We have examined the functional importance of the two disulfide bonds formed by the four conserved cysteines of human interleukin (IL-6). Using a bacterial expression system, we have synthesized a series of recombinant IL-6 mutants in which the constituent cysteines of the first (Cys45-Cys51), second (Cys74-Cys84), or both disulfide bonds of recombinant human interleukin-6 were replaced by other amino acids. Each mutant was partially purified and tested in four representative bioassays. While mutants lacking Cys45 and Cys51 retained activity similar to nonmutated recombinant IL-6, the activity of mutants lacking Cys74 and Cys84 was significantly reduced, especially in assays involving human cell lines. These results indicate that the first disulfide bond of human interleukin-6 is not required for maintenance of normal biologic activity. However, the fact that mutants lacking Cys45 and Cys51 were more active than corresponding cysteine-free mutants indicates that the disulfide bond formed by these residues contributes to biologic activity in the absence of the second disulfide bond. Competition binding studies with representative mutants indicate that their affinity for the human IL-6 receptor parallels their biologic activities on human cells.

The low natural abundance of many proteins is a major factor in preventing their development as therapeutic or diagnostic tools. To circumvent this barrier, we have used synthetic oligonucleotide technology to construct a gene based on the sequence of a cDNA for human interleukin 6 (IL-6). The synthetic gene encodes a cysteine-free, bioengineered rIL-6 protein, which is expressed at high concentrations in Escherichia coli as a tripartite fusion protein. Cleavage of the fusion protein with collagenase (EC 3.4.24.8) releases a 23-kDa rIL-6 protein that can be easily purified to homogeneity. This rIL-6 protein displays a range of biological activities similar to those of natural human IL-6, as demonstrated by its ability to (a) protect cells from viral infection and (b) stimulate the synthesis of fibrinogen in rat FAZA cells.

We have isolated clones with well over 100 randomly dispersed point mutations distributed throughout the 5' half of chemically synthesized adenovirus type 2 VA I genes. In addition, we have isolated clusters of mutations targeted to the regions corresponding to the A and B block consensus sequences of eukaryotic tRNA and adenovirus VA genes. In vitro analyses of these constructs have allowed us to survey in detail the importance of DNA sequence to transcriptional efficiency. Our analyses demonstrate that certain constructs with radically substituted A block regions can be transcribed efficiently. In contrast, there is little tolerance for variation in the sequence within the B block region. We propose that the B block sequence should be R-G-A/T-T-C-R-A-N-N-C for optimal transcriptional efficiency of the VA I gene in mammalian cells.