Biomedical subjects

D J Zack

Publications and source records attributed to D J Zack.

At least 19 recordsLinked to original sources

PubMed2006

Sustained expression after nonviral ocular gene transfer using mammalian promoters.

The CMV promoter drives high transgene expression and is one of the most commonly used promoters for gene transfer. Tissue-specific mammalian promoters provide an alternative, and it would be useful to have a system to directly compare them to viral promoters free from potential confounding vector-related effects. In this study, we describe how electroporation after subretinal injection of plasmid DNA can be used to perform comparative quantitative analysis of promoter activities. Luciferase assay of eyecup homogenates was carried out after coinjection/electroporation of pGL2, a plasmid containing the promoter fragment of interest coupled to the firefly luciferase gene, and pRL-CMV, a plasmid containing the CMV promoter coupled to the Renilla luciferase gene for normalization. This technique was used to compare activity of different fragments of the 5'-upstream region of the vitelliform macular dystrophy 2 (VMD2) gene, which is selectively expressed in the retinal pigmented epithelial (RPE) cells, and results indicated positive regulatory elements between -104 and -154 bp and between -424 and -585 bp. Addition of a fragment from intron 1 reduced the activity of the -585/+38 bp fragment by 75%. Deletion analysis implicated a 342 bp region near the 5'-end of intron 1 in the repression. Results of transient transfections in two cell lines that constitutively express VMD2 were similar, and results in transgenic mice were consistent, providing validation for promoter analysis by in vivo electroporation. We then explored the time course of expression of the -585/+38 VMD2 promoter fragment and found that compared to cassettes driven by CMV or SV40 promoters, which showed peak luciferase activity on day 2 followed by a rapid decrease in activity, the VMD2 promoter fragment showed lower activity initially, but the activity was sustained for up to 56 days (longest time point measured). A promoter fragment from another RPE-specific gene, Rpe65, showed a similar pattern of sustained expression for at least 112 days. These data indicate that nonviral gene transfer can be used to quantitatively evaluate the activity of promoter fragments independent of influence from viral vectors. A potentially important finding using this new technique is the demonstration that relatively sustained passenger gene expression can be achieved with nonviral gene transfer using mammalian rather than viral promoters.

Animals↗

PubMed2005

Nonviral ocular gene transfer.

In this study, we explored the use of electroporation or media that promote lipoplex formation for nonviral gene transfer in the eye. There was no detectable staining for LacZ after subretinal, intravitreous, or periocular injection of a plasmid containing a CMV promoter expression cassette for LacZ, but when plasmid injection in each of the three sites was combined with electroporation, there was efficient transduction. Specific staining for LacZ was seen in retinal pigmented epithelial (RPE) cells after subretinal injection of a plasmid containing a vitelliform macular dystrophy 2 (VMD2) promoter expression cassette, demonstrating that this approach can be used to evaluate purported tissue-specific promoters in vivo. Electroporation with 10 V/mm resulted in strong LacZ staining, but was damaging to photoreceptors; substantial transduction with no evidence of retinal damage was seen using 3.4 V/mm. Staining for LacZ was also seen after subretinal or periocular, but not intravitreous, injection of plasmid DNA in medium containing 40% Lipofectamine2000 (Lf). Injection of 40% Lf into the subretinal space caused damage to photoreceptors, but subretinal injection of plasmid DNA in medium containing 10% NeuroPorter resulted in transduction of RPE cells with no adverse effects on retinal morphology or function as assessed by electroretinograms (ERGs). After either electroporation or lipofection, LacZ staining was detectable for at least 14 days, and could be reinduced by a second procedure. These data suggest that electroporation or lipofection can be used as experimental tools for ocular gene transfer to evaluate tissue-specific promoter fragments or to evaluate the effects of transgene expression in the retina. Also, with additional optimization, nonviral gene transfer may prove to be a valuable approach for the treatment of retinal and choroidal diseases.

Animals↗

PubMed2004

Angiopoietin 1 inhibits ocular neovascularization and breakdown of the blood-retinal barrier.

Several retinal and choroidal diseases are potentially treatable by intraocular delivery of genes whose products may counter or neutralize abnormal gene expression that occurs as part of the diseases. However, prior to considering a transgene, it is necessary to thoroughly investigate the effects of its expression in normal and diseased eyes. An efficient way to do this is to combine tissue-specific promoters with inducible promoter systems in transgenic mice. In this study, we used this approach to evaluate the effects of ectopic expression of angiopoietin-1 (Ang1) in normal eyes and those with ocular neovascularization. Adult mice with induced expression of Ang1 ubiquitously, or specifically in the retina, appeared normal and had no identifiable changes in retinal or choroidal blood vessels or in retinal function as assessed by electroretinography. Increased expression of Ang1 in eyes with severe retinal ischemia or in eyes with rupture of Bruch's membrane significantly suppressed the development of retinal or choroidal neovascularization, respectively. This inhibition of ocular neovascularization is particularly interesting and noteworthy, because overexpression of Ang1 in skin stimulates neovascularization. Ang1 also significantly reduced VEGF-induced retinal vascular permeability. These data suggest that intraocular delivery of ang1 has potential for treatment of ocular neovascularization and macular edema.

Angiopoietin-1↗

PubMed2002

Cross-reactivity and pathogenicity of anti-DNA autoantibodies in systemic lupus erythematosus.

Autoantibodies to DNA were discovered over 40 years ago following the discovery a few years earlier of the 'LE' cell phenomenon by Hargraves and colleagues in 1948. These investigators noted that, when leucocytes were incubated with serum from lupus patients, changes in the nucleus could be seen together with phagocytosis of nuclear remnants by polymorphonuclear leucocytes. Since that time numerous studies in many laboratories have investigated almost every aspect of anti-DNA antibodies, partly to identify what determines their pathology. Whilst a subset of anti-DNA antibodies, especially anti-native, or double-stranded DNA (dsDNA) antibodies constitutes a hallmark of lupus disease and a diagnostic criterion, it is now clear that not all anti-DNA autoantibodies are of pathogenic relevance. Moreover, anti-DNA autoantibodies may also be found in other connective tissue disorders. Here we briefly review studies presented at the fifth international workshop on anti-DNA autoantibodies held in London to highlight relevant properties of pathogenic anti-DNA antibodies.

Actinin↗

PubMed2001

Identification and functional consequences of a new mutation (E155G) in the gene for GCAP1 that causes autosomal dominant cone dystrophy.

Mutations in the gene for guanylate cyclase-activating protein-1 (GCAP1) (GUCA1A) have been associated with autosomal dominant cone dystrophy (COD3). In the present study, a severe disease phenotype in a large white family was initially shown to map to chromosome 6p21.1, the location of GUCA1A. Subsequent single-stranded conformation polymorphism analysis and direct sequencing revealed an A464G transition, causing an E155G substitution within the EF4 domain of GCAP1. Modeling of the protein structure shows that the mutation eliminates a bidentate amino acid side chain essential for Ca2+ binding. This represents the first disease-associated mutation in GCAP1, or any neuron-specific calcium-binding protein within an EF-hand domain, that directly coordinates Ca2+. The functional consequences of this substitution were investigated in an in vitro assay of retinal guanylate cyclase activation. The mutant protein activates the cyclase at low Ca2+ concentrations but fails to inactivate at high Ca2+ concentrations. The overall effect of this would be the constitutive activation of guanylate cyclase in photoreceptors, even at the high Ca2+ concentrations of the dark-adapted state, which may explain the dominant disease phenotype.

Amino Acid Sequence↗

PubMed2001

Cloning and characterization of a human beta,beta-carotene-15,15'-dioxygenase that is highly expressed in the retinal pigment epithelium.

Retinoids play a critical role in vision, as well as in development and cellular differentiation. beta,beta-Carotene-15,15'-dioxygenase (Bcdo), the enzyme that catalyzes the oxidative cleavage of beta,beta-carotene into two retinal molecules, plays an important role in retinoid synthesis. We report here the first cloning of a mammalian Bcdo. Human BCDO encodes a protein of 547 amino acid residues that demonstrates 68% identity with chicken Bcdo. It is expressed highly in the retinal pigment epithelium (RPE) and also in kidney, intestine, liver, brain, stomach, and testis. The gene spans approximately 20 kb, is composed of 11 exons and 10 introns, and maps to chromosome 16q21-q23. A mouse orthologue was also identified, and its predicted amino acid sequence is 83% identical with human BCDO. Biochemical analysis of baculovirus expressed human BCDO demonstrates the predicted beta,beta-carotene-15,15'-dioxygenase activity. The expression pattern of BCDO suggests that it may provide a local supplement to the retinoids available to photoreceptors, as well as a supplement to the retinoid pools utilized elsewhere in the body. In addition, the finding that many of the enzymes involved in retinoid metabolism are mutated in retinal degenerations suggests that BCDO may also be a candidate gene for retinal degenerative disease.

Amino Acid Sequence↗

PubMed2001

Expression and permeation properties of the K(+) channel Kir7.1 in the retinal pigment epithelium.

Bovine Kir7.1 clones were obtained from a retinal pigment epithelium (RPE)-subtracted cDNA library. Human RPE cDNA library screening resulted in clones encoding full-length human Kir7.1. Northern blot analysis indicated that bovine Kir7.1 is highly expressed in the RPE. Human Kir7.1 channels were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. The macroscopic Kir7.1 conductance exhibited mild inward rectification and an inverse dependence on extracellular K+ concentration ([K+]o). The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.013) > Na+ (0.003) approximately Li+ (0.001) and the sequence based on conductance ratios was Rb+ (9.5) >> K+ (1.0) > Na+ (0.458) > Cs+ (0.331) > Li+ (0.139). Non-stationary noise analysis of Rb+ currents in cell-attached patches yielded a unitary conductance for Kir7.1 of approximately 2 pS. In whole-cell recordings from freshly isolated bovine RPE cells, the predominant current was a mild inwardly rectifying K+ current that exhibited an inverse dependence of conductance on [K+]o. The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.021) > Na+ (0.003) approximately Li+ (0.002) and the sequence based on conductance ratios was Rb+ (8.9) >> K+ (1.0) > Na+ (0.59) > Cs+ (0.23) > Li+ (0.08). In cell-attached recordings with Rb+ in the pipette, inwardly rectifying currents were observed in nine of 12 patches of RPE apical membrane but in only one of 13 basolateral membrane patches. Non-stationary noise analysis of Rb+ currents in cell-attached apical membrane patches yielded a unitary conductance for RPE Kir of approximately 2 pS. On the basis of this molecular and electrophysiological evidence, we conclude that Kir7.1 channel subunits comprise the K+ conductance of the RPE apical membrane.

Animals↗

PubMed2001

Pigment epithelium-derived factor inhibits retinal and choroidal neovascularization.

In this study, we investigated whether overexpression of pigment epithelium-derived factor (PEDF) by gene transfer can inhibit neovascularization by testing its effect in three different models of ocular neovascularization. Intravitreous injection of an adenoviral vector encoding PEDF resulted in expression of PEDF mRNA in the eye measured by RT-PCR and increased immunohistochemical staining for PEDF protein throughout the retina. In mice with laser-induced rupture of Bruch's membrane, choroidal neovascularization was significantly reduced after intravitreous injection of PEDF vector compared to injection of null vector or no injection. Subretinal injection of the PEDF vector resulted in prominent staining for PEDF in retinal pigmented epithelial cells and strong inhibition of choroidal neovascularization. In two models of retinal neovascularization (transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors and mice with oxygen-induced ischemic retinopathy), intravitreous injection of null vector resulted in decreased neovascularization compared to no injection, but intravitreous injection of PEDF vector resulted in further inhibition of neovascularization that was statistically significant. These data suggest that sustained increased intraocular expression of PEDF by gene therapy might provide a promising approach for treatment of ocular neovascularization.

Adenoviridae↗

PubMed2001

Neurotrophic signaling in normal and degenerating rodent retinas.

Several types of insult cause up-regulation of neurotrophic factors and their receptors in the retina resulting in decreased photoreceptor cell death from subsequent injury. This phenomenon is more prominent in rats than in mice and neurotrophic factors are more efficacious in rats than mice. If up-regulation of neurotrophic factor receptors on photoreceptor cells early in the course of degenerations contributes to neurotrophic factor survival-promoting activity, it may also increase the ability to detect neurotrophic factor-induced signaling in photoreceptors, particularly in rats. In this study, these hypotheses were investigated by performing immunohistochemical staining for the phosphorylated form of extracellular receptor kinase (pERK) or c-fos after intravitreous injection of neurotrophic factors in wild type rats or mice, or those with inherited retinal degenerations. In both rats and mice either early or late in the course of degeneration, or in wild type animals, intravitreous injection of brain-derived neurotrophic factor, ciliary neurotrophic factor, or fibroblast growth factor-2 caused immunostaining for pERK and c-fos in cells of the inner retina, particularly Müller cells, but not in photoreceptors. These data add to the mounting evidence suggesting that neurotrophic factors act indirectly through Müller cells to promote photoreceptor survival.

Animals↗

PubMed2001

Fibroblast growth factor-2 decreases hyperoxia-induced photoreceptor cell death in mice.

Fibroblast growth factor-2 (FGF2) has neurotrophic effects in vitro and in vivo. It has been demonstrated to decrease photoreceptor cell death in rats exposed to constant light and in rats with an inherited defect in retinal pigmented epithelium (RPE) phagocytosis, but the effects of intravitreous injections of FGF2 in mice are equivocal. In this study, we used transgenic mice with increased expression of FGF2 in photoreceptors (rhodopsin promoter/FGF2 transgenics) to investigate the effects of sustained increased expression of FGF2 in mice with various types of photoreceptor degeneration, including rd mice that are homozygous for mutated phosphodiesterase beta subunit, Q344ter mice that undergo photoreceptor degeneration because of expression of mutated rhodopsin, and mice exposed to 75% oxygen for 1 or 2 weeks. At P21, the outer nuclear layer was markedly reduced in rd mice or Q344ter mice regardless of whether they inherited the rhodopsin promoter/FGF2 transgene. However, after 2 weeks of exposure to 75% oxygen, outer nuclear layer thickness was significantly reduced in littermate control mice compared to FGF2 transgenic mice (P = 0.0001). These data indicate that increased expression of FGF2 in photoreceptors protects them from hyperoxia-induced damage, but does not decrease cell death related to expression of mutated proteins involved in the phototransduction pathway. This suggests that FGF2 protects photoreceptors from oxidative damage, which may play a role in complex genetic diseases such as age-related macular degeneration.

Aging↗

PubMed2001

A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.

Stargardt-like macular dystrophy (STGD3, MIM 600110) and autosomal dominant macular dystrophy (adMD) are inherited forms of macular degeneration characterized by decreased visual acuity, macular atrophy and extensive fundus flecks. Genetic mapping data suggest that mutations in a single gene may be responsible for both conditions, already known to bear clinical resemblance. Here we limit the minimum genetic region for STGD3 and adMD to a 0.6-cM interval by recombination breakpoint mapping and identify a single 5-bp deletion within the protein-coding region of a new retinal photoreceptor-specific gene, ELOVL4, in all affected members of STGD3 and adMD families. Bioinformatic analysis of ELOVL4 revealed that it has homology to a group of yeast proteins that function in the biosynthesis of very long chain fatty acids. Our results are therefore the first to implicate the biosynthesis of fatty acids in the pathogenesis of inherited macular degeneration.

Amino Acid Sequence↗

PubMed2001

TIMP-1 promotes VEGF-induced neovascularization in the retina.

Proteolysis of vascular basement membranes and surrounding extracellular matrix is a critical early step in neovascularization. It requires alteration of the balance between matrix metalloproteinases (MMPs) and proteins that bind to and inactivate MMPs, tissue inhibitors of metalloproteinases (TIMPs). TIMP-1 has been demonstrated to inhibit neovascularization in chick chorioallantoic membranes. However, TIMP-1 has also been shown to either promote or inhibit cell proliferation and migration in different settings. To determine whether genetic alteration of the MMP/TIMP-1 ratio would alter retinal neovascularization, we crossed mice that express vascular endothelial growth factor (VEGF) in photoreceptors with TIMP-1-deficient mice or mice that overexpress TIMP-1. Compared to VEGF transgene-positive/TIMP-1-sufficient mice, VEGF transgene-positive/TIMP-1-deficient mice showed smaller neovascular lesions. There was also no difference between the two groups of mice in the appearance of the neovascularization by light or electron microscopy. Compound VEGF/TIMP-1 transgenic mice had increased expression of both VEGF and TIMP-1 in the retina, and had more neovascularization than mice that had increased expression of VEGF alone. These gain- and loss-of-function data suggest that alteration of the TIMP-1/MMP ratio modulates retinal neovascularization in a complex manner and not simply by altering the proteolytic activity and thereby invasiveness of endothelial cells.

Actins↗

PubMed2001

Isolation and characterization of a zebrafish homologue of the cone rod homeobox gene.

PURPOSE: To isolate and characterize a zebrafish CRX: homologue. Mammalian CRX: genes are expressed specifically in photoreceptors and pinealocytes, regulate photoreceptor gene expression, are necessary for normal photoreceptor differentiation, and when mutated cause a variety of photoreceptor degenerations. METHODS: A zebrafish retinal cDNA library was screened with a human CRX cDNA probe. Radiation hybrid mapping, Northern blot analysis, in situ hybridization, and transient transfection studies were performed using standard methods. RESULTS: Based on amino acid sequence comparisons, zebrafish crx shows 50% identity with human CRX, and 85% identity in the homeodomain. A phylogenetic analysis indicates that zebrafish crx is most closely related to the mammalian Crx proteins, and more distantly related to the Otx proteins. Zebrafish crx maps between 49.6 and 54.5 cm from the top of linkage group LG05C, a map position consistent with the location of the mouse and human CRX genes. Northern blot analysis and in situ hybridization indicate that zebrafish crx is expressed in the retina and pineal gland. In adult zebrafish, crx is expressed by both rods and cones in the outer nuclear layer, and in cells in the outer zone of the inner nuclear layer, in the region occupied by bipolar cells. Similar to mammalian Crx, zebrafish crx interacts with neural retinal leucine zipper (Nrl) to activate, although weakly, rhodopsin promoter activity. CONCLUSIONS: Based on molecular phylogeny, chromosomal location, expression pattern, and ability to activate rhodopsin promoter activity in transient transfection assays, zebrafish crx appears to be an orthologue and functional homologue of mammalian CRX:

Amino Acid Sequence↗

PubMed2001

Autosomal dominant retinal degeneration and bone loss in patients with a 12-bp deletion in the CRX gene.

PURPOSE: To define the phenotypic expression of a deletion in the gene encoding the transcription factor CRX in a large, seven-generation, white family. METHODS: Fourteen affected individuals, all heterozygous for the Leu146del12 mutation in the cone-rod homeobox gene (CRX), and four nonaffected relatives from the same family were examined with visual function tests, and 10 underwent bone mineral density (BMD) measurement. RESULTS: The ability of the mutated CRX protein to transactivate rhodopsin promoter was decreased by approximately 25%, and its ability to react synergistically with neural retinal leucine zipper (NRL) was reduced by more than 30%. The affected members of the family had an autosomal dominant ocular condition most closely resembling Leber congenital amaurosis (LCA) with severe visual impairment at an early age. Depending on age, affected members showed varying degrees of significant visual acuity loss, elevated dark-adaptation thresholds, significantly reduced cone and rod electroretinogram (ERG) amplitudes, and progressive constriction of the visual fields, in most cases leading to complete blindness. Six affected members had reduced levels of BMD in the spine and the hip (osteopenia). Four affected female members who were receiving long-term hormonal replacement therapy (HRT) demonstrated normal values of BMD. CONCLUSIONS: This large deletion of the CRX gene is associated with a severe form of autosomal dominant retinal degeneration. Affected members not receiving HRT showed reduced BMD (osteopenia). This phenotype may reflect the abnormal influence of mutant CRX on both retinal and pineal development.

Adult↗

PubMed2000

Functional domains of the cone-rod homeobox (CRX) transcription factor.

The paired-like homeodomain transcription factor CRX (cone-rod homeobox) is involved in regulating photoreceptor gene expression and rod outer segment development. Mutations in CRX have been associated with several retinal degenerative diseases. These conditions range from Leber congenital amaurosis (a severe cone and rod degeneration of childhood onset) to adult onset cone-rod dystrophy and retinitis pigmentosa (an adult onset condition that primarily affects rods). The goal of this study is to better understand the molecular basis of CRX function and to provide insight into how mutations in CRX cause such a variety of clinical phenotypes. We performed deletion analysis in conjunction with DNA binding and transient transfection-based transactivation studies to identify the functional domains within CRX. DNA binding requires a complete homeodomain. Furthermore, truncated proteins that did not contain an intact homeodomain failed to demonstrate detectable expression in tissue culture upon transfection. Transactivation analysis indicated that both the OTX tail and the WSP domain are important for controlling positive regulatory activity of CRX. Interestingly, the mapped CRX transactivation domains were also critical when coexpressed with NRL. Specifically, the synergy between CRX and NRL was constant regardless of which CRX variant was used.

Amino Acid Sequence↗

PubMed2000

The architectural transcription factor high mobility group I(Y) participates in photoreceptor-specific gene expression.

The nonhistone chromosomal proteins high mobility group I(Y) [HMG I(Y)] have been shown to function as architectural transcription factors facilitating enhanceosome formation on a variety of mammalian promoters. Specifically, they have been shown to act as a "molecular glue" mediating protein-protein and protein-DNA contacts within the enhanceosome complex. HMG I(Y) proteins are expressed at high levels in embryonic and transformed cells and have been implicated in transcriptional regulation in these cells. Terminally differentiated cells, however, have been reported to express only minimal, if any, HMG I(Y). In contrast to these observations, we show here that adult mouse retinal photoreceptors, which are terminally differentiated cells, express high levels of these proteins. Using retinoblastoma cells as an approximate model, we further demonstrate in transiently transfected cells that inhibition of HMG I(Y) expression and mutation of HMG I(Y) binding sites significantly reduce rhodopsin promoter activity. DNase I footprint analysis indicates that HMG I protein interacts with a discrete site within the rhodopsin proximal promoter. This site overlaps with the binding site for Crx, a paired-like homeodomain transcription factor that is essential for photoreceptor functioning and that when mutated causes several forms of human photoreceptor degeneration. Both biochemical and functional experiments demonstrate that HMG I(Y) physically associate with Crx and that their interaction with DNA is required for high-level transcription of the rhodopsin gene. These data provide the first demonstration that HMG I(Y) can be important for gene activation in terminally differentiated cells.

Animals↗

PubMed2000

The leucine zipper of NRL interacts with the CRX homeodomain. A possible mechanism of transcriptional synergy in rhodopsin regulation.

Photoreceptor-specific expression of rhodopsin is mediated by multiple cis-acting elements in the proximal promoter region. NRL (neural retina leucine zipper) and CRX (cone rod homeobox) proteins bind to the adjacent NRE and Ret-4 sites, respectively, within this region. Although NRL and CRX are each individually able to induce rhodopsin promoter activity, when expressed together they exhibit transcriptional synergy in rhodopsin promoter activation. Using the yeast two-hybrid method and glutathione S-transferase pull-down assays, we demonstrate that the leucine zipper of NRL can physically interact with CRX. Deletion analysis revealed that the CRX homeodomain (CRX-HD) plays an important role in the interaction with the NRL leucine zipper. Although binding with the CRX-HD alone was weak, a strong interaction was detected when flanking regions including the glutamine-rich and the basic regions that follow the HD were included. A reciprocal deletion analysis showed that the leucine zipper of NRL is required for interaction with CRX-HD. Two disease-causing mutations in CRX-HD (R41W and R90W) that exhibit reduced DNA binding and transcriptional synergy also decrease its interaction with NRL. These studies suggest novel possibilities for protein-protein interaction between two conserved DNA-binding motifs and imply that cross-talk among distinct regulatory pathways contributes to the establishment and maintenance of photoreceptor function.

Amino Acid Sequence↗

PubMed2000

Novel protein transfection of primary rat cortical neurons using an antibody that penetrates living cells.

An Ab-based system to deliver functional proteins into neurons was developed using the murine mAb, mAb 3E10. This was achieved by covalently conjugating catalase to the Ab so that the conjugate retained high activity for the degradation of hydrogen peroxide. Three-dimensional fluorescence microscopy was used to demonstrate penetration of the Ab into the nucleus of living primary cortical neurons. The Ab conjugate localized in both the cytoplasm and nucleus. Retention of catalase activity after penetration and distribution of conjugate was demonstrated by reduction in cell death following exposure of treated neurons to hydrogen peroxide. These studies illustrate the potential of this method for the intracellular delivery of therapeutic proteins.

Animals↗