Site-specific gene modification by oligodeoxynucleotides in mouse bone marrow-derived mesenchymal stem cells.

Synthetic oligodeoxynucleotides (ODNs) had been employed in gene modification and represent an alternative approach to 'cure' genetic disorders caused by mutations. To test the ability of ODN-mediated gene repair in bone marrow-derived mesenchymal stem cells (MSCs), we established MSCs cell lines with stably integrated mutant neomycin resistance and enhanced green fluorescent protein reporter genes. The established cultures showed morphologically homogenous population with phenotypic and functional features of mesenchymal progenitors. Transfection with gene-specific ODNs successfully repaired targeted cells resulting in the expression of functional proteins at relatively high frequency approaching 0.2%. Direct DNA sequencing confirmed that phenotype change resulted from the designated nucleotide correction at the target site. The position of the mismatch-forming nucleotide was shown to be important structural feature for ODN repair activity. The genetically corrected MSCs were healthy and maintained an undifferentiated state. Furthermore, the genetically modified MSCs were able to engraft into many tissues of unconditioned transgenic mice making them an attractive therapeutic tool in a wide range of clinical applications.

Delivery and mechanistic considerations for the production of knock-in mice by single-stranded oligonucleotide gene targeting.

Single-stranded oligodeoxynucleotide (ssODN) gene targeting may facilitate animal model creation and gene repair therapy. Lipofection of ssODN can introduce point mutations into target genes. However, typical efficiencies in mouse embryonic stem cells (ESC) are <10(-4), leaving corrections too rare to effectively identify. We developed ESC lines with an integrated mutant neomycin resistance gene (Tyr22Ter). After targeting with ssODN, repaired cells survive selection in G418. Correction efficiencies varied with different lipofection procedures, clonal lines, and ssODN designs, ranging from 1 to 100 corrections per million cells plated. Uptake studies using cell sorting of Cy5-labelled ssODN showed 40% of the corrections concentrated in the best transfected 22% of cells. Four different basepair mismatches were tested and results show that the base-specificity of the mismatch is critical. Dual mismatch ssODN also showed mismatch preferences. These ESC lines may facilitate development of improved ssODN targeting technologies for either animal production or ex vivo gene therapy.

Oligonucleotide-directed single-base DNA alterations in mouse embryonic stem cells.

We have investigated the use of single-stranded oligodeoxy-nucleotides (ssODN) to produce specific single-base alterations in episomal and chromosomal DNA in mouse embryonic stem (ES) cells. Two different reporter genes, EGFP and LacZ, each with a single point mutation that inactivates reporter activity, were used. ssODN homologous to the target sequence, except for a single mismatch at the mutant base, were used to correct the mutant reporter genes. When tested in CHO-K1 cells, the ssODN showed correction rates of 0.5-1.0%, consistent with prior reports. ssODN in the antisense orientation provided higher rates of gene conversion than those in the sense orientation for both reporter genes. Nuclear extracts from mouse ES cells exhibited nearly the same correction activity as extracts from CHO-K1 cells. ssODN corrected the mutant bases of both episomal and chromosomal mutant reporter genes in mouse ES cells. Although the efficiency of gene correction observed in ES cells is low, approximately 10(-4), these results demonstrate that ssODN can produce single-base alterations in the genomic DNA of mouse ES cells. As conversion efficiency is improved by the continued development of oligonucleotide structure and DNA delivery methods, ssODN could be used to produce ES cells with specific mutations in any gene in a single step. The targeted ES cells could in turn be used to create accurate mouse models of inherited diseases.

Necrotizing pancreatitis: contemporary analysis of 99 consecutive cases.

OBJECTIVE: To analyze the impact of a conservative strategy of management in patients with necrotizing pancreatitis, reserving intervention for patients with documented infection or the late complications of organized necrosis. SUMMARY BACKGROUND DATA: The role of surgery in patients with sterile pancreatic necrosis remains controversial. Although a conservative approach is being increasingly used, few studies have evaluated this strategy when applied to the entire spectrum of patients with necrotizing pancreatitis. METHODS: The authors reviewed 1,110 consecutive patients with acute pancreatitis managed at Brigham and Women's Hospital between January 1, 1995, and January 1, 2000, focusing on those with pancreatic necrosis documented by contrast-enhanced computed tomography. Fine-needle aspiration, the presence of extraintestinal gas on computed tomography, or both were used to identify infection. RESULTS: There were 99 (9%) patients with necrotizing pancreatitis treated, with an overall death rate of 14%. In three patients with underlying medical problems, the decision was made initially not to intervene. Of the other 62 patients without documented infection, all but 3 were managed conservatively; this group's death rate was 11%. Of these seven deaths, all were related to multiorgan failure. Five patients in this group eventually required surgery for organized necrosis, with no deaths. Of the 34 patients with infected necrosis, 31 underwent surgery and 3 underwent percutaneous drainage. Only four (12%) of these patients died, all of multiorgan failure. Of the total 11 patients who died, few if any would have been candidates for earlier surgical intervention. CONCLUSIONS: These results suggest that conservative strategies can be applied successfully to manage most patients with necrotizing pancreatitis, although some will eventually require surgery for symptomatic organized necrosis. Few if any patients seem likely to benefit from a more aggressive strategy.

Clinical features and mutations in patients with dominant retinitis pigmentosa-1 (RP1).

PURPOSE: To survey patients with dominant retinitis pigmentosa (RP) for mutations in the RP1 gene to determine the spectrum of dominant mutations in this gene, to estimate the proportion of dominant RP caused by this gene, and to determine whether the clinical features of patients with RP1 mutations differ from features of those with rhodopsin mutations. METHODS: A set of 241 patients who did not have mutations in the rhodopsin gene (based on previous work) formed the basis for the study. Of these patients, 117 had also been previously evaluated and were found not to carry mutations in the RDS gene. The single-strand conformation polymorphism (SSCP) method was used to search for sequence variants, which were then directly sequenced. The relatives of selected patients were recruited for segregation analyses. Clinical evaluations of patients included a measurement of Snellen visual acuity, final dark adaptation thresholds, visual fields, and ERGs. Clinical data were compared with those obtained earlier from a study of 128 patients with dominant rhodopsin mutations. RESULTS: Of the 241 patients, all were screened for the most common RP1 mutation (Arg677Ter), and 10 patients were found to have this mutation. In addition, an evaluation of a subset of 189 patients in whom the entire coding sequence was evaluated revealed the following mutations: Gln679Ter (1 case), Gly723Ter (2 cases), Glu729(1-bp del) (1 case), Leu762(5-bp del) (2 cases), and Asn763(4-bp del) (1 case). All of these mutations cosegregated with RP in the families of the index patients. Nine missense mutations that were each found in six or fewer patients were encountered. The segregation of eight of these was evaluated in the respective patients' families, and only one segregated with dominant RP. This cosegregating missense change was in cis with the nonsense mutation Gln679Ter. Although patients with RP1 mutations had, on average, slightly better visual acuity than patients with rhodopsin mutations, there was no statistically significant difference in final dark-adaptation thresholds, visual field diameters, or cone electroretinogram (ERG) amplitudes. Comparably aged patients with RP1 mutations had visual function that varied by approximately two orders of magnitude, based on visual fields and ERG amplitudes. CONCLUSIONS: Dominant RP1 alleles typically have premature nonsense codons occurring in the last exon of the gene and would be expected to encode mutant proteins that are only approximately one third the size of the wild-type protein, suggesting that a dominant negative effect rather than haploinsufficiency is the mechanism leading to RP caused by RP1 mutations. On average, patients with RP1 mutations have slightly better visual acuity than patients with dominant rhodopsin mutations; otherwise, they have similarly severe disease. The wide range in severity among patients with RP1 mutations indicates that other genetic or environmental factors modulate the effect of the primary mutation.

Pathways to photoreceptor cell death in inherited retinal degenerations.

The mutations that cause many forms of inherited retinal degenerations have been identified, yet the mechanisms by which these mutations lead to death of photoreceptor cells of the retina are not completely understood. Investigations of the pathways from mutation to retinal degeneration have focused on spontaneous and engineered animal models of disease. Based on the studies performed to date, four major categories of degeneration mechanism can be identified. These include disruption of photoreceptor outer segment morphogenesis, metabolic overload, dysfunction of retinal pigment epithelial cells, and chronic activation of phototransduction. Future investigations will likely identify additional mechanisms of photoreceptor damage. This review will summarize what has been learned from studying animal models of non-syndromic inherited retinal degenerations.

Retinal expression, regulation, and functional bioactivity of prostacyclin-stimulating factor.

Prostacyclin-stimulating factor (PSF) acts on vascular endothelial cells to stimulate the synthesis of the vasodilatory molecule prostacyclin (PGI2). We have examined the expression, regulation, and hemodynamic bioactivity of PSF both in whole retina and in cultured cells derived from this tissue. PSF was expressed in all retinal cell types examined in vitro, but immunohistochemical analysis revealed PSF mainly associated with retinal vessels. PSF expression was constitutive in retinal pericytes (RPCs) but could be modulated in bovine retinal capillary endothelial cells (RECs) by cell confluency, hypoxia, serum starvation, high glucose concentrations, or inversely by soluble factors present in early vs. late retinopathy, such as TGF-beta, VEGF, or bFGF. In addition, RPC-conditioned media dramatically increased REC PGI2 production, a response inhibited by blocking PSF with a specific antisense oligodeoxynucleotide (ODN). In vivo, PGI2 increased retinal blood flow (RBF) in control and diabetic animals. Furthermore, the early drop in RBF during the initial weeks after inducing diabetes in rats, as well as the later increase in RBF, both correlated with levels of retinal PSF. RBF also responded to treatment with RPC-conditioned media, and this effect could be partially blocked using the antisense PSF ODN. We conclude that PSF expressed by ocular cells can induce PGI2, retinal vascular dilation, and increased retinal blood flow, and that alterations in retinal PSF expression may explain the biphasic changes in RBF observed in diabetes.

Disease expression of RP1 mutations causing autosomal dominant retinitis pigmentosa.

PURPOSE: To determine the disease expression in heterozygotes for mutations in the RP1 gene, a newly identified cause of autosomal dominant retinitis pigmentosa (adRP). METHODS: Screening strategies were used to detect disease-causing mutations in the RP1 gene, and detailed studies of phenotype were performed in a subset of the detected RP1 heterozygotes using electroretinography (ERG), psychophysics, and optical coherence tomography (OCT). RESULTS: Seventeen adRP families had heterozygous RP1 changes. Thirteen families had the Arg677ter mutation, whereas four others had one of the following: Pro658 (1-bp del), Ser747 (1-bp del), Leu762-763 (5-bp del), and Tyr1053 (1-bp del). In Arg677ter RP1 heterozygotes, there was regional retinal variation in disease, with the far peripheral inferonasal retina being most vulnerable; central and superior temporal retinal regions were better preserved. The earliest manifestation of disease was rod dysfunction, detectable as reduced rod ERG photoresponse maximum amplitude, even in heterozygotes with otherwise normal clinical, functional, and OCT cross-sectional retinal imaging results. At disease stages when cone abnormalities were present, there was greater rod than cone dysfunction. Patients with the RP1 frameshift mutations showed similarities in phenotype to those with the Arg677ter mutation. CONCLUSIONS: Earliest disease expression of RP1 gene mutations causing adRP involves primarily rod photoreceptors, and there is a gradient of vulnerability of retinopathy with more pronounced effects in the inferonasal peripheral retina. At other disease stages, cone function is also affected, and severe retina-wide degeneration can occur. The nonpenetrance or minimal disease expression in some Arg677ter mutation-positive heterozygotes suggests important roles for modifier genes or environmental factors in RP1-related disease.

A pilot study of noninvasive methods to assess healed acute and chronic wounds.

BACKGROUND: A variety of instruments are available that can objectively assess physical parameters of the skin such as strength, firmness, elasticity, hydration, and color, often undetected by clinical assessment. OBJECTIVE: To assess the physical properties of healed acute and chronic wounds using several noninvasive instruments. METHODS: Four patients with healed acute wounds and four patients with healed chronic wounds were studied using ballistometric, impedance, levarometric, and spectrophotometric measurements. RESULTS: In general, scars were harder, less elastic, dryer, and more erythematous than control skin. These differences were more pronounced in healed chronic wounds. CONCLUSION: A scar from an acute surgical wound becomes softer, more elastic, dryer, less erythematous, and less pigmented as it ages. In contrast, chronic wound scars become harder as they age. These different properties of healed acute wounds and healed chronic wounds may be a result of the different healing processes in each wound type.

Mutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pigmentosa.

The autosomal dominant retinitis pigmentosa (RP) locus, designated RP1, has been mapped through linkage studies to a 4-cM interval at 8q11-13. Here we describe a new photoreceptor-specific gene that maps in this interval and whose expression is modulated by retinal oxygen levels in vivo. This gene consists of at least 4 exons that encode a predicted protein of 2,156 amino acids. A nonsense mutation at codon 677 of this gene is present in approximately 3% of cases of dominant RP in North America. We also detected two deletion mutations that cause frameshifts and introduce premature termination codons in three other families with dominant RP. Our data suggest that mutations in this gene cause dominant RP, and that the encoded protein has an important but unknown role in photoreceptor biology.

Regulation of vascular endothelial growth factor by oxygen in a model of retinopathy of prematurity.

OBJECTIVES: To investigate the role of vascular endothelial growth factor (VEGF) in the pathogenesis of the first phase of retinopathy of prematurity (ROP) and to examine the mechanism by which supplemental oxygen therapy might inhibit neovascularization in the second phase of ROP. METHODS: A novel combination of fluorescein-dextran perfusion and colorimetric whole-retina in situ hybridization was used to evaluate the expression of VEGF messenger RNA in relationship to the location of blood vessels in retinas from neonatal mice that were exposed to hyperoxia. Northern blot and immunoblot analyses were used to quantify the changes in VEGF messenger RNA and protein expression caused by hyperoxia. The ability of VEGF to prevent hyperoxia-induced vaso-obliteration was evaluated by injecting exogenous VEGF into the vitreous cavity prior to oxygen exposure. RESULTS: Vascular endothelial growth factor messenger RNA was produced in a reticular pattern just anterior to the developing blood vessels in normal retina on postnatal day 7. The expression of VEGF in the peripheral retina was down-regulated by hyperoxia in conjunction with the arrest of growth and the loss of some of the developing vasculature. Total VEGF messenger RNA and protein levels in retinas from animals on postnatal day 7 were decreased 55% and 85%, respectively, after 6 hours in 75% oxygen. Vaso-obliteration was inhibited 57% by pretreatment of animals with exogenous VEGF. In animals with retinal ischemia secondary to loss of vasculature, treatment with supplemental oxygen therapy decreased stimulated retinal VEGF levels by approximately 70%. CONCLUSIONS: Down-regulation of VEGF expression by hyperoxia may be partly responsible for the vaso-obliteration and cessation of normal retinal blood vessel growth observed in premature infants in whom ROP develops. Hyperoxia also has the potential to be used therapeutically to down-regulate VEGF expression in hypoxic retina in the hope of limiting the neovascular complications of ROP. Based on these findings about the regulation of VEGF expression in the retina, an explanation of the pathogenesis of ROP is proposed.

Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy.

Diseases characterized by retinal neovascularization are among the principal causes of visual loss worldwide. The hypoxia-stimulated expression of vascular endothelial growth factor (VEGF) has been implicated in the proliferation of new blood vessels. We have investigated the use of antisense phosphorothioate oligodeoxynucleotides against murine VEGF to inhibit retinal neovascularization and VEGF synthesis in a murine model of proliferative retinopathy. Intravitreal injections of two different antisense phosphorothioate oligodeoxynucleotides prior to the onset of proliferative retinopathy reduced new blood vessel growth a mean of 25 and 31% compared with controls. This inhibition was dependent on the concentration of antisense phosphorothioate oligodeoxynucleotides and resulted in a 40-66% reduction in the level of VEGF protein, as determined by Western blot analysis. Control (sense, nonspecific) phosphorothioate oligodeoxynucleotides did not cause a significant reduction in retinal neovascularization or VEGF protein levels. These data further establish a fundamental role for VEGF expression in ischemia-induced proliferative retinopathies and a potential therapeutic use for antisense phosphorothioate oligodeoxynucleotides.

Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins.

The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGF-neutralizing chimeric proteins were constructed by joining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGF-receptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P < 0.006) or hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-microns section averaged 47% +/- 4% (P < 0.001) and 37% +/- 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66%, respectively. No retinal toxicity was observed by light microscopy. These data demonstrate VEGF's causal role in retinal angiogenesis and prove the potential of VEGF inhibition as a specific therapy for ischemic retinal disease.

Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization.

Neovascular diseases of the retina are a major cause of blindness worldwide. Hypoxia is thought to be a common precursor to neovascularization in many retinal diseases, but the factors involved in the hypoxic neovascular response have not been fully identified. To investigate the role of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in retinal neovascularization, the expression of VEGF/VPF mRNA and protein were studied in a mouse model of proliferative retinopathy. RNA (Northern) blot analysis revealed that retinal VEGF/VPF mRNA expression increased 3-fold between 6 and 12 hr of relative retinal hypoxia and remained elevated during the development of neovascularization. In situ hybridization localized VEGF/VPF mRNA to cells bodies in the inner nuclear layer of the retina. Immunohistochemical confocal microscopy demonstrated that VEGF/VPF protein levels increase with a time course similar to that of the mRNA. The cells in the inner nuclear layer of the retina that produce VEGF/VPF were identified morphologically as Müller cells. These data suggest that VEGF/VPF expression in the retina plays a central role in the development of retinal ischemia-induced ocular neovascularization.

Point mutation in the cytoplasmic domain of the neutrophil p22-phox cytochrome b subunit is associated with a nonfunctional NADPH oxidase and chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a congenital disorder in which phagocytes cannot generate superoxide (O2-) and other microbial oxidants due to mutations in any one of four components of the O2(-)-generating complex, NADPH oxidase. We report here a female CGD patient in whom a missense mutation in one of these components, the p22-phox subunit of the neutrophil membrane cytochrome b [where phox indicates phagocyte oxidase (used to designate protein components of the phagocyte NADPH oxidase)] results in a nonfunctional oxidase and failure of neutrophils to produce O2- in response to phorbol 12-myristrate 13-acetate. Cytochrome b in the patient's neutrophils was normal in appearance and abundance as determined by visible spectroscopy and by immunoblots of the gp91 and p22 subunits. However, the neutrophil plasma membranes were devoid of activity in the cell-free oxidase activation system, whereas the cytosol functioned normally. We postulated that the patient was homozygous for a mutation in p22 that results in the synthesis of normal levels of a nonfunctional cytochrome b. A single-base substitution (C----A) was found in the patient's mononuclear cell p22-phox cDNA that predicts a nonconservative Pro----Gln substitution at residue 156. The same mutation was also identified in all clones sequenced from patient genomic DNA, demonstrating homozygosity for the mutant allele. An antipeptide antibody against p22 residues 153-164 was found to bind only to permeabilized neutrophils, indicating that the mutation occurs in a cytoplasmic domain. These studies establish that this domain of p22-phox is cytoplasmic and that mutations in this region can have profound effects on cytochrome b function.

Human neutrophil cytochrome b light chain (p22-phox). Gene structure, chromosomal location, and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease.

A membrane-bound cytochrome b, a heterodimer formed by a 91-kD glycoprotein (heavy chain) and a 22-kD polypeptide (light chain), is an essential component of the phagocyte NADPH-oxidase responsible for superoxide generation. Cytochrome b is absent in two subgroups of chronic granulomatous disease (CGD), an inherited disorder characterized by the lack of oxidase activity. Mutations in the cytochrome heavy chain gene, encoded by the CYBB locus in Xp21.1, result in the X-linked form of CGD. A rare subgroup of autosomal recessive CGD also lacks cytochrome b (A- CGD), but the genetic defect has not previously been identified. In order to search for possible mutations in the cytochrome light chain locus, CYBA, the structure of this gene was characterized. The CYBA locus was localized to 16q24, and the approximately 600-bp open reading frame determined to be encoded by six exons that span approximately 8.5 kb. Three unrelated patients with A- CGD were studied for evidence of mutations in the light chain gene. One patient, whose parents were first cousins, was homozygous for a large deletion that removed all but the extreme 5' coding sequence of the gene. The other two patients had a grossly normal light chain transcript on Northern blot of mononuclear cell RNA. The light chain transcript was amplified by the polymerase chain reaction and sequenced. One patient was a compound heterozygote for two alleles containing point mutations in the open reading frame that predict a frame shift and a nonconservative amino acid replacement, respectively. The second patient, whose parents were second cousins, was homozygous for a different single-base substitution resulting in another nonconservative amino acid change. These results indicate that A- CGD can results from defects in the gene encoding the 22-kD light chain of the phagocyte cytochrome b.

Regulation of the intestinal 1,25-dihydroxyvitamin D3 receptor during neonatal development in the rat.

Monoclonal antibodies have been used to examine the regulation of expression of the rat intestinal receptor for 1,25-dihydroxyvitamin D3 during neonatal development. 1,25-Dihydroxyvitamin D3 binding activity increases dramatically between Days 15 and 28 postpartum in normal neonatal rats. Immunoprecipitation and combined immunoprecipitation-immunoblotting experiments demonstrate that this increase in hormone binding activity is caused by an increase in the amount of receptor protein present in rat intestine between Days 18 and 28 of age. Very little receptor protein, if any, appears to be present in rat intestine prior to Day 18 of life. These results suggest that the expression of the gene for 1,25-dihydroxyvitamin D3 receptor is regulated during neonatal development in the rat.

Size and charge of the functional 1,25-dihydroxyvitamin D receptor in porcine intestine.

The 1,25-dihydroxyvitamin D3 receptor from porcine intestine has been characterized by immunoprecipitation and immunoblotting experiments. Immunoprecipitation studies demonstrate that monoclonal antibodies to the receptor that recognize two different epitopes on the receptor protein quantitatively precipitate the 1,25-dihydroxyvitamin D3 binding activity from nuclear and whole cell extracts of intestinal mucosa. These antibodies were then used to study the porcine receptor by both one- and two-dimensional immunoblotting techniques. One-dimensional immunoblots of nuclear extract and whole cell extract show the receptor to be a single polypeptide of Mr approximately 55,000. The migration of the receptor on one-dimensional gels was unchanged by preassociation in vitro with 1,25-dihydroxyvitamin D3. In two-dimensional immunoblots two receptor proteins were detected, both of molecular weight 55,000. In addition, a form of the receptor did not enter the isoelectric focusing gel from either the acidic or basic direction. It did, however, migrate in the Mr 55,000 region in the molecular separation. The major receptor protein has a pI of 6.1; the minor protein has a pI of 5.9. These studies represent the first determinations of the size and charge of a mammalian 1,25-dihydroxyvitamin D3 receptor in crude tissue preparations, and they indicate that the pig receptor is a Mr 55,000 protein that exhibits charge heterogeneity in vivo. The nature of this apparent charge heterogeneity and its significance in receptor function are under investigation.