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W W Hauswirth

Publications and source records attributed to W W Hauswirth.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mitochondrial gene expression is regulated at the level of transcription during early embryogenesis of Xenopus laevis.

Mitochondrial transcription in the early Xenopus laevis embryo resumes several hours before active mtDNA replication, effectively decoupling mtDNA transcription and replication. This developmental feature makes Xenopus embryogenesis an appealing model system to investigate the regulation of mitochondrial transcription. Studies reported here refine our understanding of the timing, magnitude, and mechanism of this transcriptional induction event. Northern analyses of six mitochondrial mRNAs (normalized to mtDNA) reveal that transcript levels remain basal between fertilization and gastrulation and then undergo a coordinate induction, culminating in a 20-28-fold increase over egg levels by 48 h of development. Measurement of mitochondrial run-on transcription rates demonstrates a good correlation between transcription rates and transcript levels, showing that transcription itself is the primary determinant of transcript abundance. Experimental increases in mitochondrial ATP and energy charge also correlate with patterns of transcript levels and transcription rates, suggesting that developmental changes in the biochemical composition of the mitochondrial matrix could be regulating transcriptional activity. Consistent with this idea, transcriptional run-on rates in mitochondria of early embryos can be stimulated by the addition of tricarboxylic acid cycle intermediates to the run-on reaction. However, mitochondria of later stages do not show this response to the addition of metabolite. In combination, these data suggest that mitochondrial transcription is under metabolic regulation during early Xenopus embryogenesis.

Animals↗
PubMed1999

Adenoviral gene therapy with catalase suppresses experimental optic neuritis.

OBJECTIVE: To determine if adenoviral-mediated transfer of the gene for catalase (CAT), the reactive oxygen species scavenger, suppresses experimental optic neuritis. CLINICAL RELEVANCE: Gene therapy with CAT delivered by an adeno-associated viral vector was previously shown to suppress experimental optic neuritis. Because the transduction of protein expression with recombinant adeno-associated viral vector is relatively slow, taking weeks to reach full levels, we studied the effects of replication-deficient adenovirus containing CAT in suppressing experimental optic neuritis. Transduction with adenovirus occurs within days of inoculation, thus, it may be more applicable for the treatment of patients with acute optic neuritis. MATERIALS AND METHODS: Replication-deficient adenovirus containing CAT was injected above the right optic nerve heads of SJL/J mice that were simultaneously sensitized for experimental allergic encephalomyelitis. For controls, the left eyes were injected with the replication-deficient adenovirus without CAT or no virus. The histological effects of CAT on the lesions of experimental allergic encephalomyelitis were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), the extent of the cellular infiltrate, extravasated serum albumin labeled with immunogold (for disruption of the blood-brain barrier), and the in vivo hydrogen peroxide reaction product. RESULTS: After 1 month, cell-specific catalase activity, evaluated by the quantitation of catalase immunogold, was increased about 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the CAT-inoculated right optic nerves compared with the control left optic nerves. The increased cellular levels of catalase reduced demyelination by 30%, optic nerve head swelling by 25%, cellular infiltration by 26%, disruption of the blood-brain barrier by 61%, and in vivo levels of hydrogen peroxide by 81%. CONCLUSIONS: Adenoviral-mediated gene transfer increased catalase levels in all optic nerve cell types, and it persisted for 1 month after inoculation. The increased cellular levels of catalase suppressed demyelination and blood-brain barrier disruption at the foci in the optic nerve where prior magnetic resonance imaging and histopathologic studies have demonstrated the demyelinating inflammation of experimental and human optic neuritis. Together, they suggest that gene therapy with CAT may be helpful in the treatment of patients with optic neuritis.

Adenoviridae↗
PubMed1999

Reporter expression persists 1 year after adeno-associated virus-mediated gene transfer to the optic nerve.

OBJECTIVE: To determine the foci and duration of protein expression following virus-mediated gene transfer to the optic nerve. METHODS: A cytomegalovirus (CMV) promoter was linked to a lacZ-SV40 polyA reporter gene or a humanized green fluorescent protein (hgfp) reporter gene, then inserted into a bacterial plasmid containing adeno-associated virus (AAV) terminal repeat sequences. The CMV-lacZ or the CMV-hgfp construct were injected into the vitreous cavity of strain-13 guinea pigs. Controls consisted of eyes injected with AAV without the promoter and reporter elements or eyes that received no injections. The eyes and optic nerves were processed for beta-galactosidase immunohistochemistry and hgfp fluorescence analyses. Cellular transduction at the messenger RNA (mRNA) level was evaluated by in situ reverse transcription-polymerase chain reaction. RESULTS: Weekly fundus photography, done for 1 month, documented the absence of any ocular abnormality due to the viral injections. No in vivo hgfp fluorescence of the retina was visualized. Beta-galactosidase histochemical analysis of eye cups that received the lacZ gene construct showed blue lacZ staining of the optic nerve head at 2 weeks. Light microscopy revealed the blue beta-galactosidase reaction product in fibers, glial cells, and blood vessels of the optic nerve head and retrobulbar nerve. Histochemistry showed absence of beta-galactosidase in the optic nerve at 3 to 12 months, but immunochemistry showed the persistence of beta-galactosidase in fibers, glial cells, and blood vessels as late as 1 year after a single ocular injection. In the retina, histochemical staining showed evidence of lacZ at 3 months, but not later. In situ reverse transcription-polymerase chain reaction revealed brown lacZ mRNA reaction product in ganglion cells of the retina. Control eyes that received AAV without the promoter and reporter elements and the eyes that received no viral injections and were processed for beta-galactosidase showed no reporter gene expression in any ocular tissue or cell type. CONCLUSIONS: Viral-mediated gene transfer can be successfully accomplished in the optic nerve. Further evaluation is needed to determine whether the level of protein expression at 1 year after injection, which is clearly reduced relative to shorter postinjection time, is sufficient for therapeutic purposes. CLINICAL RELEVANCE: We have previously shown that gene therapy with catalase suppressed experimental optic neuritis at 1 month after injection. Viral-mediated gene transfer may be a powerful technique for the treatment of optic neuropathies, particularly for recurrences of optic neuritis, if long-term expression of transduced protein can be demonstrated in the optic nerve.

Animals↗
PubMed1998

Adeno-associated viral-mediated catalase expression suppresses optic neuritis in experimental allergic encephalomyelitis.

Suppression of oxidative injury by viral-mediated transfer of the human catalase gene was tested in the optic nerves of animals with experimental allergic encephalomyelitis (EAE). EAE is an inflammatory autoimmune disorder of primary central nervous system demyelination that has been frequently used as an animal model for the human disease multiple sclerosis (MS). The optic nerve is a frequent site of involvement common to both EAE and MS. Recombinant adeno-associated virus containing the human gene for catalase was injected over the right optic nerve heads of SJL/J mice that were simultaneously sensitized for EAE. After 1 month, cell-specific catalase activity, evaluated by quantitation of catalase immunogold, was increased approximately 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the optic nerve. Effects of catalase on the histologic lesions of EAE were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), extent of the cellular infiltrate, extravasated serum albumin labeled by immunogold (for blood-brain barrier disruption), and in vivo H2O2 reaction product. Relative to control, contralateral optic nerves injected with the recombinant virus without a therapeutic gene, catalase gene inoculation reduced demyelination by 38%, optic nerve head swelling by 29%, cellular infiltration by 34%, disruption of the blood-brain barrier by 64%, and in vivo levels of H2O2 by 61%. Because the efficacy of potential treatments for MS are usually initially tested in the EAE animal model, this study suggests that catalase gene delivery by using viral vectors may be a therapeutic strategy for suppression of MS.

Animals↗
PubMed1998

Retinal gene therapy 1998: summary of a workshop.

The 1998 Workshop on Retinal Gene Therapy evaluated the potential of gene therapy in treatment of retinal disease. Academic, industry, and private foundation representatives attended. Topics included: determing which retinal diseases are likely candidates for gene therapy, specific retinal degenerations and nonspecific neuronal survival mechanisms, design and use of viral and retroviral vectors in achieving regulated gene expression, animal models of retinal degeneration and associated therapies, human trials, and alternatives to gene therapy. The discussion of human trials explored the justification for moving from animal models to human testing, patient population concerns, lessons learned from previous human gene therapy trials, and the role of industry in support of basic and clinical research.

Adenoviridae↗
PubMed1998

Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa.

Ribozymes, catalytic RNA molecules that cleave a complementary mRNA sequence, have potential as therapeutics for dominantly inherited disease. Twelve percent of American patients with the blinding disease autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes can discriminate and catalyze the in vitro destruction of P23H mutant mRNAs from a transgenic rat model of ADRP. Here, we demonstrate that in vivo expression of either a hammerhead or hairpin ribozyme in this rat model considerably slows the rate of photoreceptor degeneration for at least three months. Catalytically inactive control ribozymes had less effect on the retinal degeneration. Intracellular production of ribozymes in photoreceptors was achieved by transduction with a recombinant adeno-associated virus (rAAV) incorporating a rod opsin promoter. Ribozyme-directed cleavage of mutant mRNAs, therefore, may be an effective therapy for ADRP and also may be applicable to other inherited diseases.

Animals↗
PubMed1998

Ribozyme-targeted destruction of RNA associated with autosomal-dominant retinitis pigmentosa.

PURPOSE: To design ribozymes--catalytic RNA molecules--to cleave the P23H and S334Ter mutant mRNA selectively and to test them in vitro to determine their potential as therapeutic agents in the prevention of autosomal dominant retinitis pigmentosa. METHODS: Synthetic RNA targets were used in cleavage assays to determine the catalytic efficiencies of the ribozymes in vitro. Cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis. Total retinal RNA was also used as a substrate, and opsin mRNA cleavage was assayed by reverse transcription-polymerase chain reaction. RESULTS: All three ribozymes cleaved the mutant target specifically. Substrate cleavage was seen in less than 5 mM magnesium and was detectable after 15 minutes of incubation. The most active ribozyme against the P23H target was the hammerhead (kcat:K(m) [Michaelis-Menton constant] ratio = 5 x 10(7) M/min), then the P23H hairpin ribozyme (kcat:K(m) ratio = 9 x 10(5) M/min) and the S334Ter hammerhead (kcat:K(m) ratio = 8 x 10(5) M/min). No cleavage activity was observed, when wild-type target sequences or inactive control ribozymes were used. The ribozymes bound and specifically digested the intact mutant opsin mRNA in the presence of all normal retinal RNA. CONCLUSIONS: Ribozymes can discriminate between the mutant and wild-type sequences of mRNA associated with autosomal dominant retinitis pigmentosa. The kinetics and specificity of ribozyme cleavage indicate that they should reduce the amount of aberrant rhodopsin in the rod cells and may have potential as therapeutic agents against genetic disease.

Animals↗
PubMed1997

Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus.

We describe a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina. Recombinant adeno-associated virus (rAAV) vectors were used to transfer the bacterial lacZ gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas after injection into the subretinal space. Using a proximal murine rod opsin promoter (+86 to -385) to drive expression, reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium. GFP-expressing photoreceptors typically encompassed 10-20% of the total retinal area after a single 2-microl injection. Photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site. We estimate approximately 2.5 million photoreceptors were transduced as a result of the single subretinal inoculation. This level of gene transfer and expression suggests the feasibility of genetic therapy for retinal disease. The gfp-containing rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infectious center assay, respectively. Thus, highly purified, helper virus-free rAAV vectors can achieve high-frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells.

Animals↗
PubMed1996

A "humanized" green fluorescent protein cDNA adapted for high-level expression in mammalian cells.

We constructed gfph, a synthetic version of the jellyfish Aequorea victoria green fluorescent protein (gfp) cDNA that is adapted for high-level expression in mammalian cells, especially those of human origin. A total of 92 base substitutions were made in 88 codons in order to change the codon usage within the gfp10 coding sequence so that it was more appropriate for expression in mammalian cells. We also describe a series of versatile recombinant adeno-associated virus and adenovirus vectors for delivery and expression of genes into mammalian cells and, using these vectors, demonstrate the efficient transduction and expression of the gfph gene in the human cell line 293 and also in vivo, within neurosensory cells of guinea pig eye. Cells infected with recombinant adeno-associated virus-GFPH can be readily sorted by fluorescence-activated cell sorting, suggesting that the newly designed gfph gene could be widely used as a reporter in many gene delivery technologies, including human gene therapy.

Amino Acid Sequence↗
PubMed1996

Developmentally important DNA elements within the bovine opsin upstream region.

PURPOSE: Nuclear run-on analysis has documented three categories of opsin gene expression during bovine fetal retinal development: basal levels of transcription occurring before 6 months gestation; opsin-specific enhanced levels of fetal transcription at 7-7.5 months gestation; and a switch from fetal to adult transcription, where transcriptional activity acquires a sarkosyl sensitive component in the adult. To begin determining the mechanism of these multiple levels of gene regulation, DNA regulatory elements within 2.1 kb of the rod opsin upstream region were identified in fetal and adult bovine retinal extracts that represented the transcriptional activities characteristic of each stage of opsin gene expression. METHODS: DNAse I footprint experiments were performed on fetal and adult bovine retinal extracts from each of the developmental stages of opsin expression. RESULTS: Ten regions of protection were mapped and the level of protection was quantitated as a function of the developmental stage. CONCLUSIONS: The 10 DNA elements identified fell into three categories of developmental protection: those elements that were protected similarly at each developmental stage; those elements that were more highly protected when opsin transcription was at basal levels (i.e., less than 6 months gestation); and those elements that were more highly protected in fetal ages as compared to the adult animal. These elements are likely to be important in the developmental regulation of rod opsin gene expression.

Animals↗
PubMed1995

Topographical regulation of cone and rod opsin genes: parallel, position dependent levels of transcription.

RNase protection assays were used to follow rhodopsin and red cone opsin mRNA levels during bovine fetal development as a function of retinal position. Following induction, an equivalent radial gradient of rod and cone opsin mRNA is present in the fetal retina. This gradient is maintained in the adult retina even though no corresponding gradient in rod or cone cell density is present. Since the mRNA expression gradient does not progress radially, position dependent levels of photoreceptor-specific transcription is suggested.

Animals↗
PubMed1995

Bovine opsin gene expression exhibits a late fetal to adult regulatory switch.

Rates of bovine photoreceptor gene transcription, as measured by nuclear run-on assays, exhibit gene-specific patterns of regulation. Here we investigate initiation and elongation in nuclear run-on assays with the use of sarkosyl to further understand the nature of these gene-specific elements. Opsin transcription, alone among several genes tested, proved sarkosyl-sensitive. This sensitivity is maximal in adult retinas, with inhibition first detected in mid-third trimester fetal retinas. Therefore, opsin transcription appears to involve different regulatory elements in adult and fetal retinas, implying a fetal to adult switch in the control of opsin gene expression. Although this regulatory switch is initially activated at a time when the fetal outer nuclear layer of the retina first achieves adult-like morphology, further maturation of opsin regulation takes place postpartum since levels of sarkosyl sensitivity are almost 5-fold greater in adult retinas compared to the 7.5 month fetus. We also show that the sarkosyl-induced reduction of opsin transcription is not due to prevention of de novo RNA polymerase II initiation in the run-on reaction, suggesting the detergent alters a positive-acting, postinitiation component of the transcriptional apparatus. Since levels of opsin transcription with sarkosyl are similar to those of the other visual transduction genes with or without sarkosyl, this detergent-sensitive transcriptional component appears to account for the singularly high, gene-specific rate of opsin transcription in retinal photoreceptor cells.

Aging↗
PubMed1995

Selective DNA amplification regulates transcript levels in plant mitochondria.

Most plant mitochondrial genomes exist as subgenomic-size fragments apparently due to recombination between repetitive sequences. This leads to the possibility that independently replicating subgenomic domains could result in mitochondrial gene copy number variation. We show, through Southern-blot analysis of both restricted and intact mtDNA, that there are gene-specific copy number differences in the monocot Zea mays. Comparison of two different maize genotypes, B37(N) and B37(T), a cytoplasmic male-sterile strain, reveal fewer gene copy number differences for B37(T) than for B37(N). In contrast to maize, significant gene copy number differences are not detected in the dicot Brassica hirta. We also demonstrate that mitochondrial transcriptional rates in both species are apparently dependent on gene copy number since relative rates determined by run-on analysis are proportional to relative gene copy numbers. Thus a direct relationship exists between plant mitochondrial gene copy number and transcriptional rate.

Blotting, Southern↗
PubMed1995

Fetal topography of bovine rhodopsin mRNA suggests retinotopographically determined gene expression.

PURPOSE: To understand the developmental processes in the differentiating bovine retina, topographic accumulation of rhodopsin mRNA in staged fetal and adult retinas was analyzed. METHODS: Isolated retinas were spread on a nylon membrane with the photoreceptor cells facing the membrane and dissected into 25-mm square tissue segments, sometimes with as many as 150 segments/eye. Subsequent to disruption of the tissue in each segment, rhodopsin and beta-actin mRNA levels were quantitated with a solution hybridization assay. Slight variations in RNA extraction efficiency and retinal segment size were corrected using beta-actin mRNA as an internal standard. RESULTS: Analysis of multiple fetal and adult bovine retinas revealed a relatively static central-to-peripheral gradient of rhodopsin mRNA level that appears at the time of transcriptional induction (6 to 6.5 months of gestation) and persists into adulthood. After induction of rhodopsin mRNA expression, increase of rhodopsin mRNA levels was detected simultaneously in all retinal segments. Furthermore, the rate of increase in rhodopsin mRNA levels in peripheral and central regions was identical. CONCLUSIONS: Fetal induction of rhodopsin mRNA expression occurs simultaneously in all photoreceptor cells across the retina, but the levels are set according to a topographically predetermined pattern. This suggests that regulation of accumulation of rhodopsin mRNA during development is determined according to spatial coordinates before gene induction, most likely in a nonphotoreceptor retinal cell type.

Actins↗
PubMed1994

Inter- and intrapopulation studies of ancient humans.

For a genetic analysis of ancient human populations to be useful, it must be demonstrated that the DNA samples under investigation represent a single human population. Toward that end, we have analyzed human DNA from the Windover site (7000-8000 BP). MHC-I analysis, using allele-specific oligonucleotide hybridization to PCR amplified Windover DNA, microsatellite analysis by PCR of the APO-A2 repeat and mtD-loop 3' region sequencing on multiple individuals spanning nearly the full range of estimated burial dates all confirm the hypothesis that there is a persistence of both nuclear and mitochondrial haplotypes at Windover throughout its entire period of use. Thus, Windover can be considered a single population. Neighbor-joining tree analysis of mtDNA sequences suggests that some mitochondrial types are clearly related to extant Amerind types, whereas others, more distantly related, may reflect genetically distinct origins. A more complete sequence analysis will be required to firmly resolve this issue. Calibrating genetic relationships deduced by tree analysis, radiocarbon dates and burial position, yields a human mtD-loop DNA rate of evolution of 3700 to 14,000 years per percent change. Both values are within the range of recent, independently calculated values using estimates of evolutionary divergence or theoretical population genetics. Thus we are beginning to realize the promise of ancient DNA analysis to experimentally answer heretofore unapproachable questions regarding human prehistory and genetic change.

Alleles↗