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D W Russell

Publications and source records attributed to D W Russell.

At least 37 records · Page 2Linked to original sources
PubMed2000

Design and packaging of adeno-associated virus gene targeting vectors.

Adeno-associated virus (AAV) vectors can transduce cells by several mechanisms, including (i) gene addition by chromosomal integration or episomal transgene expression or (ii) gene targeting by modification of homologous chromosomal sequences. The latter process can be used to correct a variety of mutations in chromosomal genes with high fidelity and specificity. In this study, we used retroviral vectors to introduce mutant alkaline phosphatase reporter genes into normal human cells and subsequently corrected these mutations with AAV gene targeting vectors. We find that increasing the length of homology between the AAV vector and the target locus improves gene correction rates, as does positioning the mutation to be corrected in the center of the AAV vector genome. AAV-mediated gene targeting increases with time and multiplicity of infection, similar to AAV-mediated gene addition. However, in contrast to gene addition, genotoxic stress did not affect gene targeting rates, suggesting that different cellular factors are involved. In the course of these studies, we found that (i) vector genomes less than half of wild-type size could be packaged as monomers or dimers and (ii) packaged dimers consist of inverted repeats with covalently closed hairpins at either end. These studies should prove helpful in designing AAV gene targeting vectors for basic research or gene therapy.

Alkaline Phosphatase↗
PubMed2000

Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes.

Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors.

Alkaline Phosphatase↗
PubMed2000

The bile acid synthetic gene 3beta-hydroxy-Delta(5)-C(27)-steroid oxidoreductase is mutated in progressive intrahepatic cholestasis.

We used expression cloning to isolate cDNAs encoding a microsomal 3beta-hydroxy-Delta(5)-C(27)-steroid oxidoreductase (C(27) 3beta-HSD) that is expressed predominantly in the liver. The predicted product shares 34% sequence identity with the C(19) and C(21) 3beta-HSD enzymes, which participate in steroid hormone metabolism. When transfected into cultured cells, the cloned C(27) 3beta-HSD cDNA encodes an enzyme that is active against four 7alpha-hydroxylated sterols, indicating that a single C(27) 3beta-HSD enzyme can participate in all known pathways of bile acid synthesis. The expressed enzyme did not metabolize several different C(19/21) steroids as substrates. The levels of hepatic C(27) 3beta-HSD mRNA in the mouse are not sexually dimorphic and do not change in response to dietary cholesterol or to changes in bile acid pool size. The corresponding human gene on chromosome 16p11.2-12 contains six exons and spans 3 kb of DNA, and we identified a 2-bp deletion in the C27 3beta-HSD gene of a patient with neonatal progressive intrahepatic cholestasis. This mutation eliminates the activity of the enzyme in transfected cells. These findings establish the central role of C(27) 3beta-HSD in the biosynthesis of bile acids and provide molecular tools for the diagnosis of a third type of neonatal progressive intrahepatic cholestasis associated with impaired bile acid synthesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1999

cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain.

The turnover of cholesterol in the brain is thought to occur via conversion of excess cholesterol into 24S-hydroxycholesterol, an oxysterol that is readily secreted from the central nervous system into the plasma. To gain molecular insight into this pathway of cholesterol metabolism, we used expression cloning to isolate cDNAs that encode murine and human cholesterol 24-hydroxylases. DNA sequence analysis indicates that both proteins are localized to the endoplasmic reticulum, share 95% identity, and represent a new cytochrome P450 subfamily (CYP46). When transfected into cultured cells, the cDNAs produce an enzymatic activity that converts cholesterol into 24S-hydroxycholesterol, and to a lesser extent, 25-hydroxycholesterol. The cholesterol 24-hydroxylase gene contains 15 exons and is located on human chromosome 14q32.1. Cholesterol 24-hydroxylase is expressed predominantly in the brain as judged by RNA and protein blotting. In situ mRNA hybridization and immunohistochemistry localize the expression of this P450 to neurons in multiple subregions of the brain. The concentrations of 24S-hydroxycholesterol in serum are low in newborn mice, reach a peak between postnatal days 12 and 15, and thereafter decline to baseline levels. In contrast, cholesterol 24-hydroxylase protein is first detected in the brain of mice at birth and continues to accumulate with age. We conclude that the cloned cDNAs encode cholesterol 24-hydroxylases that synthesize oxysterols in neurons of the brain and that secretion of 24S-hydroxycholesterol from this tissue in the mouse is developmentally regulated.

Amino Acid Sequence↗
PubMed1999

5alpha-reductase type 2 mutations are present in some boys with isolated hypospadias.

PURPOSE: We determined whether 5a-reductase type 2 mutations are present in boys with isolated hypospadias. MATERIALS AND METHODS: Penile skin tissues obtained at surgery during hypospadias repair were examined for 5alpha-reductase type 2 mutations by single strand conformational polymorphism and deoxyribonucleic acid sequence analysis. Clinical data, including family history of hypospadias and preoperative position of the urethral meatus, were correlated with the genetic findings. RESULTS: Of the 81 specimens examined 7 (8.6%) involved a mutation in at least 1, 5alpha-reductase type 2 gene, while 2 patients had mutations in both alleles. The mutations identified were A49T, L113V and H231R. The A49T mutation in 5 patients was the most common (71%) and it was generally present in less severe forms of hypospadias. To our knowledge neither the A49T nor the L113V mutation has been previously reported in association with 5alpha-reductase type 2 deficiency and to date they have only been identified in cases of isolated hypospadias. Family history was negative in the 7 patients with 5a-reductase type 2 mutations but positive in 5 without mutations. CONCLUSIONS: Some boys with isolated hypospadias have a mutation in at least 1 gene for 5alpha-reductase type 2. This finding suggests that a partial deficiency of 5alpha-reductase activity and inadequate levels of dihydrotestosterone in the fetal urethra may be sufficient to cause the phenotype of hypospadias without other clinical features of 5alpha-reductase deficiency. Family history may not be reliable for determining which boys with hypospadias are likely to have such mutations.

Child↗
PubMed1999

Behavior problems of children with Down syndrome and life events.

Behavior problems of 44 children with Down syndrome between the ages of 6 and 15 and 44 controls without mental retardation matched for age, sex, and socioeconomic status were compared on the basis of mother and teacher ratings. Ratings from both sources indicated that children with Down syndrome had more behavior problems, in particular attention deficit, noncompliance, thought disorder, and social withdrawal. Life events from the past year were significantly associated with mother but not teacher ratings of Down syndrome behavior problems.

Adaptation, Psychological↗
PubMed1999

High-fidelity correction of mutations at multiple chromosomal positions by adeno-associated virus vectors.

The gene targeting techniques used to modify chromosomes in mouse embryonic stem cells have had limited success with many other cell types, especially normal primary cells with restricted growth capacity outside the organism. This is due in large part to the technical problems and/or inefficiency of conventional DNA transfer methods, as well as the low rates of homologous recombination obtained in unselected cell populations. We recently described an alternative approach in which adeno-associated virus (AAV) vectors were used to modify homologous chromosomal sequences, and targeting rates close to 1% were observed at the single copy hypoxanthine phosphoribosyl transferase (HPRT) locus in normal human cells (D. W. Russell and R. K. Hirata, Nat. Genet. 18:325-330, 1998). Here we report experiments in which we used a retroviral shuttle vector system to introduce and characterize target loci in human chromosomes, and demonstrate that AAV vectors can correct several types of mutations with high fidelity, independent of chromosomal position. The gene targeting rates varied depending on the type of mutation being corrected, implicating cellular mismatch recognition functions in the reaction. Since AAV vectors can efficiently deliver DNA to many cell types both in vivo and ex vivo, our results suggest that AAV-mediated gene targeting will have wide applicability, including therapeutic gene correction.

Animals↗
PubMed1999

17Beta-hydroxysteroid dehydrogenase 3 deficiency in women.

In genetic males, mutation of the 17beta-hydroxysteroid dehydrogenase 3 (17HSD3)gene that is normally expressed in the testes impairs testosterone formation and causes development of male pseudohermaphroditism. We have ascertained seven women who are sisters of men with 17HSD3 deficiency and who are either homozygotes or compound heterozygotes for the same mutations as their affected brothers. Our findings confirm the concept that women with such mutations are asymptomatic.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

The parturition defect in steroid 5alpha-reductase type 1 knockout mice is due to impaired cervical ripening.

Successful delivery of the fetus (parturition) depends on coordinate interactions between the uterus and cervix. A majority (70%) of mice deficient in the type 1 isozyme of steroid 5alpha-reductase fail to deliver their young at term and thus manifest a parturition defect. Using in vitro and in vivo measurements we show here that rhythmic contractions of the uterus occur normally in these mutant mice at the end of gestation. In contrast, the cervix of the mutant animal fails to ripen at term as judged by biomechanical, histological, and endocrinological assays. Impaired metabolism of progesterone in the cervix of the mutant mice in late gestation leads to an accumulation of this steroid in the tissue. We conclude that a failure of cervical ripening underlies the parturition defect in mice lacking steroid 5alpha-reductase type 1 and that this enzyme normally plays an essential role in cervical progesterone catabolism at the end of pregnancy.

20-alpha-Dihydroprogesterone↗
PubMed1999

The effect of peers' alcohol consumption on parental influence: a cognitive mediational model.

OBJECTIVE: The current study was designed to elucidate familial and social influences on adolescent alcohol consumption by testing three hypotheses derived from the prototype/willingness model of adolescent risk behavior: (1) parents' prototypes of adolescent drinkers affect adolescent consumption through their impact on adolescents' prototypes, (2) strong parent-child relationships are associated with acceptance of parental influence regarding drinking and thus with less adolescent drinking and (3) association with peers who drink dilutes parental influence over adolescents' alcohol consumption. METHOD: Two hundred sixty-six rural adolescents, ages 15 through 17 at Time 1, and their parents and siblings completed questionnaires about drinking behavior and drinking-related cognitions at 1-year intervals for 3 years. RESULTS: Structural equation models provided evidence of transmission of prototypes of adolescent drinkers from parents to adolescents and evidence that these prototypes mediate adolescent alcohol consumption. They also provide evidence that although parents' prototypes and parent-adolescent relationships are important in shaping the adolescents' drinking, association with peers who drink significantly attenuates this influence. CONCLUSIONS: These data suggest that parents can influence their children's drinking, but that this influence has more of an impact if the adolescent is not involved in a drinking-conducive peer environment. The current analyses also suggest that the process of becoming an adolescent drinker involves an active rejection of parents' influence rather than a passive movement away from parents' attitudes and beliefs-a process that is accelerated by association with peers who drink.

Adolescent↗
PubMed1999

Male and female isoenzymes of steroid 5alpha-reductase.

There are two steroid 5alpha-reductase isoenzymes, designated type 1 and type 2, in mammals and recent experiments show that each plays a unique physiological role. In this article, the hypothesis is developed that the type 1 gene specifies a female isoenzyme, whereas the type 2 gene specifies a male isoenzyme. This idea results from the following observations. First, mutation of the 5alpha-reductase type 1 gene in mice affects reproduction in females by decreasing fecundity and blocking parturition, but has no effect on reproduction in males. Second, mutation of the 5alpha-reductase type 2 gene in mice and men prevents proper virilization but does not affect development or reproductive function in females. Analyses of these diverse phenotypes indicate that the isoenzymes catalyse both anabolic and catabolic reactions in steroid hormone metabolism.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1998

cDNA cloning of mouse and human cholesterol 25-hydroxylases, polytopic membrane proteins that synthesize a potent oxysterol regulator of lipid metabolism.

Oxysterols regulate the expression of genes involved in cholesterol and lipid metabolism and serve as intermediates in cholesterol catabolism. Among the most potent of regulatory oxysterols is 25-hydroxycholesterol, whose biosynthetic enzyme has not yet been isolated. Here, we report the cloning of cholesterol 25-hydroxylase cDNAs from the mouse and human. The encoded enzymes are polytopic membrane proteins of 298 and 272 amino acids, respectively, which contain clusters of histidine residues that are essential for catalytic activity. Unlike most other sterol hydroxylases, cholesterol 25-hydroxylase is not a cytochrome P450, but rather it is a member of a small family of enzymes that utilize diiron cofactors to catalyze the hydroxylation of hydrophobic substrates. The cholesterol 25-hydroxylase gene lacks introns, and in the human it is located on chromosome 10q23. The murine gene is expressed at low levels in multiple tissues. Expression of cholesterol 25-hydroxylase in transfected cells reduces the biosynthesis of cholesterol from acetate and suppresses the cleavage of sterol regulatory element binding protein-1 and -2. The data suggest that cholesterol 25-hydroxylase has the capacity to play an important role in regulating lipid metabolism by synthesizing a co-repressor that blocks sterol regulatory element binding protein processing and ultimately leads to inhibition of gene transcription.

Amino Acid Sequence↗
PubMed1998

Helper-free foamy virus vectors.

Retroviral vectors based on human foamy virus (HFV) have been developed and show promise as gene therapy vehicles. Here we describe a method for the production of HFV vector stocks free of detectable helper virus. The helper and vector plasmid constructs used both lack the HFV bel genes, so recombination between these constructs cannot create a wild-type virus. A fusion promoter that combines portions of the cytomegalovirus (CMV) immediate-early and HFV long terminal repeat (LTR) promoters was used to drive expression of both the helper and vector constructs. The CMV-LTR fusion promoter allows for HFV vector production in the absence of the Bel-1 trans-activator protein, which would otherwise be necessary for efficient transcription from the HFV LTR. Vector stocks containing either neomycin phosphotransferase or alkaline phosphatase reporter genes were produced by transient transfection at titers greater than 10(5) transducing units/ml. G418-resistant BHK-21 cells obtained by transduction with neo vectors contained randomly integrated HFV vector proviruses without detectable deletions or rearrangements. The vector stocks generated were free of replication-competent retrovirus (RCR), as determined by assays for LTR trans-activation and a marker rescue assay developed here for the detection of Bel-independent RCR.

Base Sequence↗
PubMed1998

The hypoxia-responsive transcription factor EPAS1 is essential for catecholamine homeostasis and protection against heart failure during embryonic development.

Mice lacking the hypoxia-inducible transcription factor EPAS1 die at mid-gestation. Despite normal morphological development of the circulatory system, EPAS1-deficient mice display pronounced bradycardia. In addition to the vascular endothelium, EPAS1 is expressed intensively in the organ of Zuckerkandl (OZ), the principle source of catecholamine production in mammalian embryos. EPAS1-deficient embryos contained substantially reduced catecholamine levels. Mid-gestational lethality was rescued by administration of the catecholamine precursor DOPS to pregnant females. We hypothesize that EPAS1 expressed in the OZ senses hypoxia during mid-gestational development and translates this signal into an altered pattern of gene expression, leading to increases in circulating catecholamine levels and proper cardiac function.

Animals↗
PubMed1998

Identification of a new inborn error in bile acid synthesis: mutation of the oxysterol 7alpha-hydroxylase gene causes severe neonatal liver disease.

We describe a metabolic defect in bile acid synthesis involving a deficiency in 7alpha-hydroxylation due to a mutation in the gene for the microsomal oxysterol 7alpha-hydroxylase enzyme, active in the acidic pathway for bile acid synthesis. The defect, identified in a 10-wk-old boy presenting with severe cholestasis, cirrhosis, and liver synthetic failure, was established by fast atom bombardment ionization-mass spectrometry, which revealed elevated urinary bile acid excretion, a mass spectrum with intense ions at m/z 453 and m/z 510 corresponding to sulfate and glycosulfate conjugates of unsaturated monohydroxy-cholenoic acids, and an absence of primary bile acids. Gas chromatography-mass spectrometric analysis confirmed the major products of hepatic synthesis to be 3beta-hydroxy-5-cholenoic and 3beta-hydroxy-5-cholestenoic acids, which accounted for 96% of the total serum bile acids. Levels of 27-hydroxycholesterol were > 4,500 times normal. The biochemical findings were consistent with a deficiency in 7alpha-hydroxylation, leading to the accumulation of hepatotoxic unsaturated monohydroxy bile acids. Hepatic microsomal oxysterol 7alpha-hydroxylase activity was undetectable in the patient. Gene analysis revealed a cytosine to thymidine transition mutation in exon 5 that converts an arginine codon at position 388 to a stop codon. The truncated protein was inactive when expressed in 293 cells. These findings indicate the quantitative importance of the acidic pathway in early life in humans and define a further inborn error in bile acid synthesis as a metabolic cause of severe cholestatic liver disease.

Amino Acid Sequence↗
PubMed1998

Reasoned action and social reaction: willingness and intention as independent predictors of health risk.

Three studies are described that assess elements of a new model of adolescent health-risk behavior, the prototype/willingness (P/W) model (F. X. Gibbons & M. Gerrard, 1995, 1997). The 1st analysis examined whether a central element of the prototype model, behavioral willingness, adds significantly to behavioral expectation in predicting adolescents' smoking behavior. The 2nd set of analyses used structural-equation-modeling procedures to provide the 1st test of the complete model in predicting college students' pregnancy-risk behavior. Finally, the 3rd study used confirmatory factor analysis to assess the independence of elements of the model from similar elements in other health behavior models. Results of the 3 studies provided support for the prototype model and, in particular, for 2 of its primary contentions: (a) that much adolescent health-risk behavior is not planned and (b) that willingness and intention are related but independent constructs, each of which can be an antecedent to risk behavior.

Adolescent↗