Biomedical subjects

D W Russell

Publications and source records attributed to D W Russell.

At least 55 records · Page 3Linked to original sources

PubMed1998

Human gene targeting by viral vectors.

Stable transduction of mammalian cells typically involves random integration of viral vectors by non-homologous recombination. Here we report that vectors based on adeno-associated virus (AAV) can efficiently modify homologous human chromosomal target sequences. Both integrated neomycin phosphotransferase genes and the hypoxanthine phosphoribosyltransferase gene were targeted by AAV vectors. Site-specific genetic modifications could be introduced into approximately 1% of cells, with the highest targeting rates occurring in normal human fibroblasts. These results suggest that AAV vectors could be used to introduce specific genetic changes into the genomic DNA of a wide variety of mammalian cells, including therapeutic gene targeting applications.

Adenoviridae↗

PubMed1998

Two 7 alpha-hydroxylase enzymes in bile acid biosynthesis.

The addition of a 7-hydroxyl group is an early and often rate-limiting step in the synthesis of bile acids. This reaction is catalysed by two cytochrome P450 enzymes known as cholesterol 7 alpha-hydroxylase and oxysterol 7 alpha-hydroxylase. cDNAs encoding these proteins have been isolated and used to define two evolutionarily conserved pathways that produce 7 alpha-hydroxylated bile acids.

Amino Acid Sequence↗

PubMed1998

Infectious clones and vectors derived from adeno-associated virus (AAV) serotypes other than AAV type 2.

Adeno-associated viruses (AAVs) are single-stranded dependent parvoviruses being developed as transducing vectors. Although at least five serotypes exist (AAV types 1 to 5 [AAV1 to -5]), only AAV2, AAV3, and AAV4 have been sequenced, and the vectors in use were almost all derived from AAV2. Here we report the cloning and sequencing of a second AAV3 genome and a new AAV serotype designated AAV6 that is related to AAV1. AAV2, AAV3, and AAV6 were 82% identical at the nucleotide sequence level, and AAV4 was 75 to 78% identical to these AAVs. Significant sequence variation was noted in portions of the capsid proteins that presumably are responsible for serotype-specific functions. Vectors produced from AAV3 and AAV6 differed from AAV2 vectors in host range and serologic reactivity. The AAV3 and AAV6 vector serotypes were able to transduce cells in the presence of serum from animals previously exposed to AAV2 vectors. Our results suggest that vectors based on alternative AAV serotypes will have advantages over existing AAV2 vectors, including the transduction of different cell types, and resistance to neutralizing antibodies against AAV2. This could be especially important for gene therapy, as significant immunity against AAV2 exists in human populations and many protocols will likely require multiple vector doses.

Amino Acid Sequence↗

PubMed1998

Packaging cells based on inducible gene amplification for the production of adeno-associated virus vectors.

Although vectors based on adeno-associated virus (AAV) offer several unique advantages, their usage has been hampered by the difficulties encountered in vector production. In this report, we describe a new AAV packaging system based on inducible amplification of integrated helper and vector constructs containing the simian virus 40 (SV40) replication origin. The packaging and producer cell lines developed express SV40 T antigen under the control of the reverse tetracycline transactivator system, which allows inducible amplification of chromosomal loci linked to the SV40 origin. Culturing these cells in the presence of doxycycline followed by adenovirus infection resulted in helper and vector gene amplification as well as higher vector titers. Clonal producer cell lines generated vector titers that were 10 times higher than those obtained by standard methods, with approximately 10(4) vector particles produced per cell. These stocks were free of detectable replication-competent virus. The lack of a transfection step combined with the reproducibility of stable producer lines makes this packaging method ideally suited for the large-scale production of vector stocks for human gene therapy.

Cell Line, Transformed↗

PubMed1998

Marked reduction in bile acid synthesis in cholesterol 7alpha-hydroxylase-deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia.

These studies used mice that were deficient in cholesterol 7alpha-hydroxylase to determine the effects of reduced bile acid synthesis on cholesterol homeostasis. In mice lacking this enzyme, bile acid synthesis was reduced from 8.3 to 3.4 micromol/day per 100 g body weight, the intestinal bile acid pool was decreased from 62.5 to 13.2 micromol/100 g bw, and the proportion of hyodeoxycholate, relative to cholate, in this pool was significantly increased. Associated with these changes, intestinal cholesterol absorption decreased from 37% to <1% while triacylglycerol absorption and animal weight gain remained essentially unaffected. The very low rate of cholesterol absorption could be corrected by feeding the mutant mice cholate, but not hyodeoxycholate. The reduction in sterol uptake across the intestine was associated with a 2-fold increase in cholesterol synthesis in the small bowel and liver and an increase in fecal neutral sterol excretion from 15.2 to 35.7 micromol/day per 100 g bw. The size of the cholesterol pools in the plasma, various organs and whole animal remained constant. Thus, under circumstances where the excretion of sterol as bile acids was markedly reduced, total cholesterol turnover actually increased from 164 to 239 mg/day per kg bw. This study demonstrates the complex interactions between bile acid and cholesterol metabolism and the dramatic effects of eliminating a single gene product; however, even though a major catabolic pathway was deleted, cholesterol balance across the animal was maintained.

Animals↗

PubMed1997

Transfer of contaminants in adeno-associated virus vector stocks can mimic transduction and lead to artifactual results.

The potential of adeno-associated virus (AAV) vectors for gene transfer and gene therapy applications is currently being intensively investigated. Although much progress has been made in defining AAV vector biology, inconsistencies remain in the literature regarding the efficiency of AAV transduction in various cell types. In the course of exploring these differences, we have identified a problem associated with the use of AAV vector stocks that results in overestimation of gene transfer efficiencies. We show here that biologically active vector-encoded proteins can contaminate AAV vector stocks, especially cell lysate preparations that have not been further purified, and can be transferred in a virion-independent manner to target cells, a phenomenon called pseudotransduction. This observation is significant because impure cell lysate stocks have been widely employed in the AAV literature, and we demonstrate here that this phenomenon can occur with commonly used reporter proteins such as beta-galactosidase and alkaline phosphatase. We conclude that although there are many potential explanations for apparently conflicting results in the literature, the possibility of pseudotransduction must be considered, especially when cell lysate stocks of AAV vectors have been employed. This artifact can be avoided by further vector purification.

Alkaline Phosphatase↗

PubMed1997

Identification and characterization of a mouse oxysterol 7alpha-hydroxylase cDNA.

The synthesis of essential 7alpha-hydroxylated bile acids in the liver is mediated by two pathways that involve distinct 7alpha-hydroxylases. One pathway is initiated in the endoplasmic reticulum by cholesterol 7alpha-hydroxylase, a well studied cytochrome P450 enzyme. A second pathway is initiated by a less well defined oxysterol 7alpha-hydroxylase. Here, we show that a mouse hepatic oxysterol 7alpha-hydroxylase is encoded by Cyp7b1, a cytochrome P450 cDNA originally isolated from the hippocampus. Expression of a Cyp7b1 cDNA in cultured cells produces an enzyme with the same biochemical and pharmacological properties as those of the hepatic oxysterol 7alpha-hydroxylase. Cyp7b1 mRNA and protein are induced in the third week of life commensurate with an increase in hepatic oxysterol 7alpha-hydroxylase activity. In the adult mouse, dietary cholesterol or colestipol induce cholesterol 7alpha-hydroxylase mRNA levels but do not affect oxysterol 7alpha-hydroxylase enzyme activity, mRNA, or protein levels. Cholesterol 7alpha-hydroxylase mRNA is reduced to undetectable levels in response to bile acids, whereas expression of oxysterol 7alpha-hydroxylase is modestly decreased. The liver thus maintains the capacity to synthesize 7alpha-hydroxylated bile acids regardless of dietary composition, underscoring the central role of 7alpha-hydroxylated bile acids in lipid metabolism.

Animals↗

PubMed1997

Expression cloning and characterization of oxidative 17beta- and 3alpha-hydroxysteroid dehydrogenases from rat and human prostate.

Intracellular levels of active steroid hormones are determined by their relative rates of synthesis and breakdown. In the case of the potent androgen dihydrotestosterone, synthesis from the precursor testosterone is mediated by steroid 5alpha-reductase, whereas breakdown to the inactive androgens 5alpha-androstane-3alpha, 17beta-diol (3alpha-adiol), and androsterone is mediated by reductive 3alpha-hydroxysteroid dehydrogenases (3alpha-HSD) and oxidative 17beta-hydroxysteroid dehydrogenases (17beta-HSD), respectively. We report the isolation by expression cloning of a cDNA encoding a 17beta-HSD6 isozyme that oxidizes 3alpha-adiol to androsterone. 17beta-HSD6 is a member of the short chain dehydrogenase/reductase family and shares 65% sequence identity with retinol dehydrogenase 1 (RoDH1), which catalyzes the oxidation of retinol to retinal. Expression of rat and human RoDH cDNAs in mammalian cells is associated with the oxidative conversion of 3alpha-adiol to dihydrotestosterone. Thus, 17beta-HSD6 and RoDH play opposing roles in androgen action; 17beta-HSD6 inactivates 3alpha-adiol by conversion to androsterone and RoDH activates 3alpha-adiol by conversion to dihydrotestosterone. The synthesis of an active steroid hormone by back conversion of an inactive metabolite represents a potentially important mechanism by which the steady state level of a transcriptional effector can be regulated.

17-Hydroxysteroid Dehydrogenases↗

PubMed1997

Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7alpha-hydroxy dehydroepiandrosterone and 7alpha-hydroxy pregnenolone.

Steroids produced locally in brain (neurosteroids), including dehydroepiandrosterone (DHEA), influence cognition and behavior. We previously described a novel cytochrome P450, Cyp7b, strongly expressed in rat and mouse brain, particularly in hippocampus. Cyp7b is most similar to steroidogenic P450s and potentially could play a role in neurosteroid metabolism. To examine the catalytic activity of the enzyme mouse Cyp7b cDNA was introduced into a vaccinia virus vector. Extracts from cells infected with the recombinant showed NADPH-dependent conversion of DHEA (Km, 13.6 microM) and pregnenolone (Km, 4.0 microM) to slower migrating forms on thin layer chromatography. The expressed enzyme was less active against 25-hydroxycholesterol, 17beta-estradiol and 5alpha-androstane-3beta,17beta-diol, with low to undetectable activity against progesterone, corticosterone, and testosterone. On gas chromatography and mass spectrometry of the Cyp7b metabolite of DHEA the retention time and fragmentation patterns were identical to those obtained with authentic 7alpha-hydroxy DHEA. The reaction product also comigrated on thin layer chromatography with 7alpha-hydroxy DHEA but not with 7beta-hydroxy DHEA; when [7alpha-3H]pregnenolone was incubated with Cyp7b extracts the extent of release of radioactivity into the medium suggested that hydroxylation was preferentially at the 7alpha position. Brain extracts also efficiently liberated tritium from [7alpha-3H]pregnenolone and converted DHEA to a product with a chromatographic mobility indistinguishable from 7alpha-hydroxy DHEA. We conclude that Cyp7b is a 7alpha-hydroxylase participating in the synthesis, in brain, of neurosteroids 7alpha-hydroxy DHEA, and 7alpha-hydroxy pregnenolone.

17-alpha-Hydroxypregnenolone↗

PubMed1997

Conservation of function between mammalian and plant steroid 5alpha-reductases.

Arabidopsis det2 mutants are small dark-green dwarfs displaying pleiotropic defects in light-regulated development during multiple stages of the plant life cycle. The DET2 gene encodes a protein that shares approximately 40% sequence identity with mammalian steroid 5alpha-reductases and is implicated in the synthesis of a class of plant steroids, the brassinosteroids. Here we show that the DET2 protein, when expressed in human embryonic kidney 293 cells, catalyzes the 5alpha-reduction of several animal steroid substrates and has similar kinetic properties to the mammalian steroid 5alpha-reductase enzymes. Moreover, human steroid 5alpha-reductases expressed in det2 mutant plants can substitute for DET2 in brassinosteroid biosynthesis. These data indicate that DET2 is an ortholog of the mammalian steroid 5alpha-reductases and provide further evidence that brassinosteroids play an essential role in light-regulated plant development. The structural and functional conservation between DET2 and human steroid 5alpha-reductases raise interesting issues concerning the evolutionary origin of the steroid hormone signaling system.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1997

Persistent expression of human clotting factor IX from mouse liver after intravenous injection of adeno-associated virus vectors.

We previously found that gene transduction by adeno-associated virus (AAV) vectors in cell culture can be stimulated over 100-fold by treatment of the target cells with agents that affect DNA metabolism, such as irradiation or topoisomerase inhibitors. Here we show that previous gamma-irradiation increased the transduction rate in mouse liver by up to 900-fold, and the topoisomerase inhibitor etoposide increased transduction by about 20-fold. Similar rates of hepatic transduction were obtained by direct injection of the liver or by systemic delivery via tail vein injection. Hepatocytes were much more efficiently transduced than other cells after systemic delivery, and up to 3% of all hepatocytes could be transduced after one vector injection. The presence of wild-type AAV, which contaminates many AAV vector preparations, was required to observe a full response to gamma-irradiation. Injection of mice with AAV vectors encoding human clotting factor IX after gamma-irradiation resulted in synthesis of low levels of human clotting factor IX for the 5-month period of observation. These studies show the potential of targeted gene transduction of the liver by AAV vectors for treatment of various hematological or metabolic diseases.

Animals↗

PubMed1997

Molecular characterization of two mammalian bHLH-PAS domain proteins selectively expressed in the central nervous system.

Here we describe two mammalian transcription factors selectively expressed in the central nervous system. Both proteins, neuronal PAS domain protein (NPAS) 1 and NPAS2, are members of the basic helix-loop-helix-PAS family of transcription factors. cDNAs encoding mouse and human forms of NPAS1 and NPAS2 have been isolated and sequenced. RNA blotting assays demonstrated the selective presence of NPAS1 and NPAS2 mRNAs in brain and spinal cord tissues of adult mice. NPAS1 mRNA was first detected at embryonic day 15 of mouse development, shortly after early organogenesis of the brain. NPAS2 mRNA was first detected during early postnatal development of the mouse brain. In situ hybridization assays using brain tissue of postnatal mice revealed an exclusively neuronal pattern of expression for NPAS1 and NPAS2 mRNAs. The human NPAS1 gene was mapped to chromosome 19q13.2-q13.3, and the mouse Npas1 gene to chromosome 7 at 2 centimorgans. Similarly, the human NPAS2 gene was assigned to chromosome 2p11.2-2q13, and the mouse Npas2 gene to chromosome 1 at 21-22 centimorgans. The chromosomal regions to which human NPAS1 and NPAS2 map are syntenic with those containing the mouse Npas1 and Npas2 genes, indicating that the mouse and human genes are true homologs.

Amino Acid Sequence↗

PubMed1997

Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells.

Here we describe the cloning and characterization of a PAS domain transcription factor termed endothelial PAS-1 (EPAS1). This protein shares 48% sequence identity with hypoxia inducible factor (HIF-1alpha) and lesser similarity with other members of the basic helix-loop-helix/PAS domain family of transcription factors. Like HIF-1alpha, EPAS1 binds to and activates transcription from a DNA element originally isolated from the erythropoietin gene and containing the sequence 5'-GCCCTACGTGCTGTCTCA-3'. Activation by both HIF-1alpha and EPAS1 is stimulated by hypoxic conditions. EPAS1 forms a heterodimeric complex with the aryl hydrocarbon nuclear transporter prior to transcriptional activation of target genes. EPAS1 expression is limited to the endothelium of mouse embryos and, in agreement with its cell type-specific expression pattern, is capable of specifically activating the transcription of the endothelial tyrosine kinase gene Tie-2. These observations raise the possibility that EPAS1 may represent an important regulator of vascularization, perhaps involving the regulation of endothelial cell gene expression in response to hypoxia.

Amino Acid Sequence↗

PubMed1997

Expression and regulation of steroid 5 alpha-reductase in the genital tubercle of the fetal rat.

The genital tubercle is a bipotential anlage that can develop into a penis or a clitoris. Formation of the penis requires synthesis within the tubercle of the androgen dihydrotestosterone by steroid 5 alpha-reductase isozymes. Isozyme 1 mRNA and protein were present in epithelial cells, whereas isozyme 2 transcripts were present in mesenchymal cells of developing genital tubercles from day 17, 19, and 21 rat embryos. These expression patterns were observed in both uncommitted anlagen and in committed male or female structures. Androgens did not significantly influence levels of 5 alpha-reductase mRNAs or affect their cellular distributions. Together with genetic data, the results implicate the mesenchyme as the crucial cell type in formation of the male external genitalia.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1997

Loneliness and nursing home admission among rural older adults.

In this study, the authors tested the relation between loneliness and subsequent admission to a nursing home over a 4-year time period in a sample of approximately 3,000 rural older Iowans. Higher levels of loneliness were found to increase the likelihood of nursing home admission and to decrease the time until nursing home admission. The influence of extremely high loneliness on nursing home admission remained statistically significant after controlling for other variables, such as age, education, income, mental status, physical health, morale, and social contact, that were also predictive of nursing home admission. Several mechanisms are proposed to explain the link between extreme loneliness and nursing home admission. These include loneliness as a precipitant of declines in mental and physical health and nursing home placement as a strategy to gain social contact with others. Implications for preventative interventions are discussed.

Activities of Daily Living↗

PubMed1997

Personality, social networks, and perceived social support among alcoholics: a structural equation analysis.

In this study we tested relations among personality characteristics, social network properties, and perceived social support both concurrently and prospectively. A sample of 294 men in treatment at a Department of Veterans Affairs Alcohol Treatment Unit was assessed during treatment and 3 months after discharge. Results of the cross-sectional structural equation analyses indicated that the personality characteristics of extraversion and neuroticism were related to both social network properties and perceived social support. Characteristics of the alcoholic's social network were also related to perceived availability of support. Longitudinal analyses of perceived social support after treatment indicated that two social network properties (size of the network and the proportion of confidants) were predictive net of initial levels of social support. Extraversion and neuroticism were found to be indirectly related to perceived social support at Time 2 through their effects on social network properties and perceived social support during treatment. Implications of these findings for models of the nature and determinants of perceived social support are discussed.

Adaptation, Psychological↗

PubMed1997

Adeno-associated virus vector integration junctions.

Vectors derived from adeno-associated virus (AAV) have the potential to stably transduce mammalian cells by integrating into host chromosomes. Despite active research on the use of AAV vectors for gene therapy, the structure of integrated vector proviruses has not previously been analyzed at the DNA sequence level. Studies on the integration of wild-type AAV have identified a common site-specific integration locus on human chromosome 19; however, most AAV vectors do not appear to integrate at this locus. To improve our understanding of AAV vector integration, we analyzed the DNA sequences of several integrated vector proviruses. HeLa cells were transduced with an AAV shuttle vector, and integrated proviruses containing flanking human DNA were recovered as bacterial plasmids for further analysis. We found that AAV vectors integrated as single-copy proviruses at random chromosomal locations and that the flanking HeLa DNA at integration sites was not homologous to AAV or the site-specific integration locus of wild-type AAV. Recombination junctions were scattered throughout the vector terminal repeats with no apparent site specificity. None of the integrated vectors were fully intact. Vector proviruses with nearly intact terminal repeats were excised and amplified after infection with wild-type AAV and adenovirus. Our results suggest that AAV vectors integrate by nonhomologous recombination after partial degradation of entering vector genomes. These findings have important implications for the mechanism of AAV vector integration and the use of these vectors in human gene therapy.

Base Sequence↗

PubMed1997

Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration.

The ability of recombinant adeno-associated virus (AAV) vectors to integrate into the host genome and to transduce nondividing cells makes them attractive as vehicles for gene delivery. In this study, we assessed the ability of several AAV vectors to transduce airway cells in rabbits by measuring marker gene expression. AAV vectors that transferred either a beta-galactosidase (beta-gal) or a human placental alkaline phosphatase (AP) gene were delivered to one lobe of the rabbit lung by use of a balloon catheter placed under fluoroscopic guidance. We observed vector-encoded beta-gal or AP staining almost exclusively in the epithelial and smooth muscle cells in the bronchus at the region of balloon placement. The overall efficiency of transduction in the balloon-treated bronchial epithelium was low but reached 20% in some areas. The majority of the staining was in ciliated cells but was also observed in basal cells and airway smooth muscle cells. We observed an 80-fold decrease in marker-positive epithelial cells during the 60-day period after vector infusion, whereas the number of marker-positive smooth muscle cells stayed constant. Although treatment with the topoisomerase inhibitor etoposide dramatically enhanced AAV transduction in primary airway epithelial cells in culture, treatment of rabbits did not improve transduction rates in the airway. Vector readministration failed to produce additional transduction events, which correlated with the appearance of neutralizing antibodies. These results indicate that both readministration and immune modulation will be required in the use of AAV vectors for gene therapy to the airway epithelium.

Alkaline Phosphatase↗