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J R Stanley

Publications and source records attributed to J R Stanley.

At least 19 recordsLinked to original sources
PubMed1997

Targeted disruption of the pemphigus vulgaris antigen (desmoglein 3) gene in mice causes loss of keratinocyte cell adhesion with a phenotype similar to pemphigus vulgaris.

In patients with pemphigus vulgaris (PV), autoantibodies against desmoglein 3 (Dsg3) cause loss of cell-cell adhesion of keratinocytes in the basal and immediate suprabasal layers of stratified squamous epithelia. The pathology, at least partially, may depend on protease release from keratinocytes, but might also result from antibodies interfering with an adhesion function of Dsg3. However, a direct role of desmogleins in cell adhesion has not been shown. To test whether Dsg3 mediates adhesion, we genetically engineered mice with a targeted disruption of the DSG3 gene. DSG3 -/- mice had no DSG3 mRNA by RNase protection assay and no Dsg3 protein by immunofluorescence (IF) and immunoblots. These mice were normal at birth, but by 8-10 d weighed less than DSG3 +/- or +/+ littermates, and at around day 18 were grossly runted. We speculated that oral lesions (typical in PV patients) might be inhibiting food intake, causing this runting. Indeed, oropharyngeal biopsies showed erosions with histology typical of PV, including suprabasilar acantholysis and "tombstoning" of basal cells. EM showed separation of desmosomes. Traumatized skin also had crusting and suprabasilar acantholysis. Runted mice showed hair loss at weaning. The runting and hair loss phenotype of DSG3 -/- mice is identical to that of a previously reported mouse mutant, balding (bal). Breeding indicated that bal is coallelic with the targeted mutation. We also showed that bal mice lack Dsg3 by IF, have typical PV oral lesions, and have a DSG3 gene mutation. These results demonstrate the critical importance of Dsg3 for adhesion in deep stratified squamous epithelia and suggest that pemphigus autoantibodies might interfere directly with such a function.

Animals↗
PubMed1997

Purification and characterization of secretory IgA from baboon colostrum.

In this report, we describe a method for purifying secretory immunoglobulin A (sIgA) from baboon (Papio anubis) colostrum. The colostrum was first clarified by centrifugation and then analyzed with various anti-human Ig-specific immunologic reagents. Cross-reactive IgA in the baboon colostrum was identified by ELISA. Western blot analysis also demonstrated cross-reactive epitopes associated with human IgA1, IgA2, secretory component (SC), and joining (J) chain. To purify the sIgA, colostrum was separated into 4 distinct fractions by gel filtration chromatography. Analysis of the individual fractions by ELISA indicated that the IgA elutes over one peak. The IgA fraction was compared with purified human sIgA on SDS-PAGE, and exhibited heavy (H) chains, light (L) chains, SC, and J chain. The baboon colostrum was also analyzed by ELISA for specific IgG H and L chain epitopes utilizing monoclonal antibodies (MAbs). No significant quantity of IgG was detected in the baboon colostrum or in the individual 4 fractions, while L chain reactivity was observed in the sIgA fraction. The sIgA fraction was pooled, concentrated, and was found to contain approximately 7 mg/ml sIgA. To determine if the baboon sIgA was dimeric like human sIgA, the purified sIgA was sized by molecular sieve chromatography. The molecular size of the sIgA preparation (350 kDa) was determined empirically by comparison to known molecular species used to calibrate the column. In addition, native SDS-PAGE indicated that baboon sIgA, like human sIgA, migrates between IgG and IgM, suggesting it has a dimeric form. The purified baboon sIgA preparation should prove useful in the future study of mucosal immune responses induced in non-human primate species and for the generation of sIgA-specific immunological reagents.

Animals↗
PubMed1997

Effects of chronic infusion of angiotensin II on renin and blood pressure in the late-gestation fetal sheep.

OBJECTIVES: Our purpose was to determine whether chronic physiologic elevations in plasma angiotensin II levels decrease plasma renin concentration, alter the relationship between active renin and prorenin in fetal plasma and kidney, and depress the expression of renal renin messenger ribonucleic acid in the fetus. STUDY DESIGN: Seventeen chronically catheterized ovine fetuses at approximately 130 days' gestation were infused with either angiotensin II (48.9 +/- 3.5 ng/kg x min) or vehicle (5% glucose in water) for 72 hours. RESULTS: Mean arterial pressure increased significantly by 1 hour of infusion and continued to increase throughout the infusion. The plasma active renin concentration was significantly decreased by 1 hour of the infusion, whereas the prorenin concentration was not decreased until 24 hours of the infusion. After 72 hours of angiotensin II infusion the renal tissue prorenin content decreased (21.5 +/- 5.1 ng/mg x hr angiotensin I vs 46.4 +/- 6.6 ng/mg - hr angiotensin I in the control animals, p = 0.01), whereas the active renin concentration did not change (26.6 +/- 5.1 ng/mg x hr angiotensin I vs 35.1 +/- 5.4 ng/mg x hr angiotensin I in the control animals, p = 0.28). The renal renin messenger ribonucleic acid expression tended to be lower in the angiotensin II-treated fetuses (p = 0.10). CONCLUSION: Chronic physiologic increases in fetal plasma angiotensin II suppress the secretion of active and prorenin and alter the relationship between processing and secretion of renin in the fetal kidney.

Angiotensin II↗
PubMed1997

The baboon as a nonhuman primate model for assessing the effects of maternal immunization with Haemophilus influenzae type b polysaccharide vaccines.

These studies were performed to assess the utility of the baboon as a nonhuman primate model to evaluate vaccines for use in humans. Specifically, we examined the antibody response of baboons immunized during the third trimester of pregnancy with Haemophilus influenzae type b (Hib) polyribosylribitol phosphate (PRP) conjugate and unconjugated polysaccharide vaccines. Some of the vaccinated mothers failed to respond to a single immunization with unconjugated Hib PRP. Specific Hib PRP immunoglobulin G (IgG) but not IgM antibodies cross the baboon placenta and are detected in the offspring. Higher-titer baboon anti-Hib PRP did not express two previously defined cross-reactive human anti-Hib PRP idiotypes and was biased towards lambda light-chain expression. Spectrotype analysis indicated that baboon anti-Hib PRP was restricted in heterogeneity and oligoclonal.

Animals↗
PubMed1996

Periconceptional folate intake and neural tube defects.

Approximately 50% of neural tube defects may be folate-preventable and perhaps even more in other countries where prevalence is high. The Public Health Service has issued the recommendation that all women of childbearing age in the United States who are capable of becoming pregnant should consume 400 micrograms of folic acid/day for the purpose of reducing this risk. Ways in which a reproductive age woman could achieve this goal include: 1) fortifying a food sample with folic acid, 2) consuming supplements containing at least 400 mcg of folic acid, or 3) increasing nutrient intake by eating foods rich in folate. Advantages of consuming foods high in folate content are that it is a natural behavior and consistent with other dietary recommendations. However, this method is dependent upon a proper diet, and equivalencies of conjugated (dietary form) vs. unconjugated (in supplements) folate are unknown. The benefit of a supplementation policy is that the appropriate group can be targeted as pregnancies are planned, whereas primary limitations to taking a supplement would be compliance and most cases in need would not be reached. The advantage of fortification is that it is likely to reach everyone before conception, while the major disadvantage is that nontargeted populations will also receive more folic acid. Adequate consumption of folic acid should begin before and continue during at least the first 4 weeks after conception when the fetal neural tube is being formed. Standard methods of screening for neural tube defects should continue during pregnancy. The risk of a recurrent neural tube defect is 2-3%, and a higher periconception daily intake of folic acid (4 mg per day) is recommended.

Adolescent↗
PubMed1996

Pemphigus vulgaris antigen (desmoglein 3) is localized in the lower epidermis, the site of blister formation in patients.

In Patients with pemphigus vulgaris, autoantibodies against the desmosomal glycoprotein desmoglein 3 (Dsg3) cause blisters due to loss of keratinocyte cell-cell adhesion in the basal and immediate suprabasal layer of the deeper epidermis, leaving the superficial epidermis intact. Autoantibodies from these patients, however, usually bind to the cell surface of keratinocytes throughout the entire epidermis, as determined by indirect immunofluorescence. To explain this apparent paradox, we immunoadsorbed pemphigus vulgaris sera with the extracellular domains of Dsg3 and desmoglein 1 (Dsg1) produced by insect cells infected with recombinant baculovirus. When adsorbed with extracellular domains of both Dsg3 and Dsg1, these sera no longer stained epidermis, demonstrating that most, if not all, of their cell surface reactivity can be attributed to antibodies against the extracellular domains of these desmogleins. Adsorption with only the Dsg1 extracellular domain left antibodies that stained only the basal and immediate suprabasal layers of the epidermis and immunoprecipitated only Dsg3, not Dsg1, from extracts of cultured cells synthesizing these molecules. In contrast, adsorption with only the Dsg3 extracellular domain left antibodies that stained only the more superficial epidermis and immunoprecipitated only Dsg1. These data localize Dsg3 exactly to the area in the epidermis where blisters occur in pemphigus vulgaris.

Autoantibodies↗
PubMed1995

Defective cell-cell adhesion in the epidermis.

The disastrous effects of loss of epidermal cell adhesion are epitomized by the life-threatening blistering skin diseases pemphigus foliaceus and pemphigus vulgaris. Clinical and experimental observations show that loss of cell adhesion is induced by these patients' autoantibodies. Pemphigus foliaceus antigen is desmoglein 1 (dsg-1), a desmosomal transmembrane glycoprotein limited in distribution to stratified squamous epithelia. It is linked to plakogoblin, a desmosomal plaque protein. Molecular cloning has shown that desmogleins are members of the cadherin gene superfamily. The originally described cadherins (e.g. E-cadherin) are transmembrane, calcium-dependent, homophilic adhesion molecules. Pemphigus vulgaris antigen is a 130 kDa glycoprotein also linked to plakoglobin. Molecular cloning has shown that pemphigus vulgaris antigen is also a desmoglein, dsg-3. Antibodies against pemphigus vulgaris antigen subdomains homologous to the binding subdomains of classical cadherins cause loss of epidermal cell adhesion, which suggests that desmogleins mediate adhesion, although direct evidence for this is lacking. The extracellular domain of pemphigus vulgaris antigen cannot substitute in function for that of E-cadherin. Future studies should address the cell biological function of desmogleins.

Animals↗
PubMed1995

Effects of intracervical prostaglandin E2 on fetal heart rate and uterine activity patterns in the presence of oligohydramnios.

OBJECTIVE: Our purpose was to compare fetal heart rate patterns and uterine activity before and after preinduction prostaglandin E2 administration in the presence or absence of oligohydramnios. STUDY DESIGN: In a retrospective case-controlled review we examined cases in which prostaglandin E2 (Prepidil) was inserted intracervically for gravid women requiring an induction of labor in the presence of either oligohydramnios (amniotic fluid index < or = 5.0) or adequate fluid (amniotic fluid index 5.1 to 23.9). Uterine activity and fetal heart rate tracings that were begun 1 hour before and continued for 6 hours after dosing were interpreted without knowledge of amniotic fluid volume. RESULTS: Cases in the oligohydramnios (n = 51) and adequate fluid (n = 49) groups were the same for maternal age, race, parity, gestational age, and predose Bishop score. Patients with oligohydramnios had more high-amplitude contractions in the first hour after dosing (9.0 +/- 1.2 vs 6.1 +/- 0.9, p < 0.05), but there were no significant differences in the frequency or duration of contractions during the subsequent 5 hours. Uterine hyperstimulation was not seen, and there were no differences in the frequency of variable or late fetal heart rate decelerations. CONCLUSION: For pregnancies undergoing preinduction cervical ripening with intracervical prostaglandin E2, the presence of oligohydramnios was not associated with a greater risk of fetal heart rate decelerations, although contractions were more common during the first hour after dosing.

Adult↗
PubMed1995

Intracellular domain of desmoglein 3 (pemphigus vulgaris antigen) confers adhesive function on the extracellular domain of E-cadherin without binding catenins.

For the extracellular (EC) domain of E-cadherin to function in homophilic adhesion it is thought that its intracytoplasmic (IC) domain must bind alpha- and beta-catenins, which link it to the actin cytoskeleton. However, the IC domain of pemphigus vulgaris antigen (PVA or Dsg3), which is in the desmoglein subfamily of the cadherin gene superfamily, does not bind alpha- or beta-catenins. Because desmogleins have also been predicted to function in the cell adhesion of desmosomes, we speculated that the PVA IC domain might be able to act in a novel way in conferring adhesive function on the EC domain of cadherins. To test this hypothesis we studied aggregation of mouse fibroblast L cell clones that expressed chimeric cDNAs encoding the EC domain of E-cadherin with various IC domains. We show here that the full IC domain of PVA as well as an IC subdomain containing only 40 amino acids of the PVA intracellular anchor (IA) region confer adhesive function on the E-cadherin EC domain without catenin-like associations with cytoplasmic molecules or fractionation with the cell cytoskeleton. This IA region subdomain is evolutionarily conserved in desmogleins, but not classical cadherins. These findings suggest an important cell biologic function for the IA region of desmogleins and demonstrate that strong cytoplasmic interactions are not absolutely necessary for E-cadherin-mediated adhesion.

Amino Acid Sequence↗
PubMed1995

Pemphigus sera recognize conformationally sensitive epitopes in the amino-terminal region of desmoglein-1.

To identify regions of the Desmoglein-1 (Dsg1) extracellular domain that are targeted by pemphigus antisera, cDNA sequences encoding various regions of the Dsg1 extracellular domain were ligated to sequences encoding the E-cadherin extracellular anchor and transmembrane and cytoplasmic domains. These constructs were then expressed in mammalian cells, and pemphigus sera were tested for the ability to recognize the Dsg1 extracellular domains. When analyzed by immunoblot, very few pemphigus foliaceus sera recognized the Dsg1 domains. To determine whether pemphigus sera recognize non-denatured Dsg1 domains, constructs were expressed in cultured cells and tested for reactivity with pemphigus sera using live-cell immunofluorescence. The pemphigus foliaceus sera reacted strongly with the Dsg1 extracellular domain by live-cell immunofluorescence and recognized predominantly the amino-terminal region of the Dsg1 extracellular domain. In addition, sera from patients with pemphigus vulgaris also demonstrated strong reactivity with the Dsg1 extracellular domain when tested using live-cell immunofluorescence. In contrast, sera from normal human controls and sera from bullous pemphigoid patients did not react with the Dsg1 extracellular domain. These data indicate that both pemphigus foliaceus and pemphigus vulgaris sera react with conformationally sensitive epitopes in the amino-terminal region of Dsg1.

Base Sequence↗
PubMed1995

Plakoglobin binding by human Dsg3 (pemphigus vulgaris antigen) in keratinocytes requires the cadherin-like intracytoplasmic segment.

Desmogleins are transmembrane desmosomal cadherins. Two desmogleins, Dsg3 and Dsg1, have been shown to bind plakoglobin, an intracytoplasmic (IC) desmosomal plaque protein. This binding may be critical for desmosome assembly or stability. The IC domain of desmogleins consists of subdomains that are either desmoglein specific or homologous with the IC region of classical cadherins. Here we identify the domains of human Dsg3 that are critical for plakoglobin binding in human keratinocytes. We constructed eukaryotic expression vectors containing chimeric cDNAs that encode the extracellular domain of mouse E-cadherin (Ecad) with the transmembrane and IC domains of Dsg3, with increasing truncations eliminating various IC subdomains from the carboxy-terminus. These constructs were used for transient transfection of HaCaT cells. Extracts were subjected to immunoprecipition with an anti-mouse Ecad antibody (that does not precipitate human Ecad), thus precipitating the chimeric protein and any tightly associated plakoglobin. Co-precipitation of plakoglobin was confirmed by immunoblotting. These data show that the desmoglein-specific IC subdomains are not necessary for plakoglobin binding, but the carboxy-terminal 87 amino acids of the IC-cadherin-like segment subdomain are critical. Finally, we confirmed these results outside cells with in vitro transcription and translation, which also demonstrates that the Dsg3-plakoglobin interaction is direct and does not depend on other cellular factors. These results underscore the importance of a region, highly conserved in all desmogleins, in the carboxy terminus of the IC-cadherin-like subdomain for the localization of plakoglobin to desmosomes.

Amino Acid Sequence↗
PubMed1995

A statewide, toll-free telephone service to improve obstetric care.

The University of Oklahoma Health Sciences Center began in 1993 to provide a statewide, toll-free telephone service for pregnancy counseling to primary care physicians. The service was available 24 hours each day, and responses were made by the on-call maternal-fetal medicine specialist. This report summarizes our first full year of operation. Inquiries came from 34 (63%) of the state's 54 counties having physicians who provide obstetric care. One hundred twenty-eight physicians made 523 inquiries (median 3, range 1-15). Information was sought about prenatal genetic disorders, risks from drugs, exposure to infection, environmental hazards, and active obstetric or medical complications. Funds from targeted ultrasounds, genetic amniocenteses, more detailed counselling, and maternal transfers provided support for this expanding educational resource.

Female↗
PubMed1994

Extracellular domain of pemphigus vulgaris antigen (desmoglein 3) mediates weak homophilic adhesion.

Pemphigus vulgaris antigen is in the cadherin supergene family. We hypothesized that the extracellular domain of pemphigus vulgaris antigen might mediate homophilic cell adhesion because 1) the originally described cadherins (e.g., E-cadherin) mediate this type of adhesion, 2) pemphigus vulgaris antigen is localized in desmosomes that are cell adhesion junctions, and 3) autoantibodies in pemphigus vulgaris patients cause loss of cell adhesion. To test this hypothesis we used a system developed for E-cadherin that, when transfected into L cells (mouse fibroblasts), has been shown to cause aggregation. Because this aggregation requires the cytoplasmic domain of E-cadherin to bind to catenins, we made a chimeric cDNA construct that encodes the extracellular domain of pemphigus vulgaris antigen and the cytoplasmic domain of E-cadherin. Analysis by immunofluorescence and flow cytometry with pemphigus vulgaris sera indicated that the pemphigus vulgaris antigen extracellular domain of this chimeric molecule (PVEC) was expressed on the cell surface of transiently transfected cells and permanently transfected L-cell clones. Immunoprecipitation of the chimeric molecule from extracts of these clones showed that the E-cadherin cytoplasmic domain bound catenins. Surprisingly, these L-cell clones displayed only slight aggregation compared to an L-cell clone transfected with E-cadherin. This weak aggregation was, however, specific and homophilic, as determined by cell sorting of only PVEC transfectants into aggregates from mixtures of PVEC and neomycin resistance gene transfectants, one of which was labeled with a fluorescent dye. We conclude that the extracellular domain of pemphigus vulgaris antigen mediates weak homophilic adhesion and is not interchangeable in function with the extracellular domain of E-cadherin.

Animals↗
PubMed1994

Pemphigus vulgaris antigen lacks biochemical properties characteristic of classical cadherins.

Pemphigus vulgaris antigen (PVA) is a member of the desmoglein subfamily of the cadherin supergene family. PVA has homology to the classical cadherins (e.g., E-cadherin), both in its extracellular and cytoplasmic domains. Classical cadherins possess certain well-defined and characteristic biochemical properties of both domains. The cytoplasmic domain binds alpha-, beta-, and gamma-catenins. The extracellular domain is protected by calcium from degradation by trypsin. In this study we show that PVA does not share these characteristic biochemical features. Immunoprecipitation of E-cadherin and PVA from human keratinocytes shows that under the same conditions in which the catenins co-precipitate with E-cadherin, only plakoglobin (which co-migrates with gamma-catenin) co-precipitates with PVA. Treatment of keratinocytes with 0.01% trypsin in 1 mM calcium (T/C) does not degrade the extracellular region of E-cadherin, but does partially degrade that of PVA. This increased T/C susceptibility of PVA is not due to its cytoplasmic domain, as the same sensitivity of the extracellular domain of PVA to T/C was observed in L cell clones transfected with a chimeric cDNA that encoded for the extracellular domain of PVA and the cytoplasmic domain of E-cadherin. These data demonstrate that although the desmogleins and classical cadherins share striking amino acid homologies in both the cytoplasmic and extracellular domains, they do not exhibit identical biochemical properties and, by extension, may not subserve identical cell biologic functions.

Amino Acid Sequence↗
PubMed1994

Extracellular domain of pemphigus vulgaris antigen (desmoglein 3) mediates weak homophilic adhesion.

Pemphigus vulgaris antigen is in the cadherin supergene family. We hypothesized that the extracellular domain of pemphigus vulgaris antigen might mediate homophilic cell adhesion because 1) the originally described cadherins (e.g., E-cadherin) mediate this type of adhesion, 2) pemphigus vulgaris antigen is localized in desmosomes that are cell adhesion junctions, and 3) autoantibodies in pemphigus vulgaris patients cause loss of cell adhesion. To test this hypothesis we used a system developed for E-cadherin that, when transfected into L cells (mouse fibroblasts), has been shown to cause aggregation. Because this aggregation requires the cytoplasmic domain of E-cadherin to bind to catenins, we made a chimeric cDNA construct that encodes the extracellular domain of pemphigus vulgaris antigen and the cytoplasmic domain of E-cadherin. Analysis by immunofluorescence and flow cytometry with pemphigus vulgaris sera indicated that the pemphigus vulgaris antigen extracellular domain of this chimeric molecule (PVEC) was expressed on the cell surface of transiently transfected cells and permanently transfected L-cell clones. Immunoprecipitation of the chimeric molecule from extracts of these clones showed that the E-cadherin cytoplasmic domain bound catenins. Surprisingly, these L-cell clones displayed only slight aggregation compared to an L-cell clone transfected with E-cadherin. This weak aggregation was, however, specific and homophilic, as determined by cell sorting of only PVEC transfectants into aggregates from mixtures of PVEC and neomycin resistance gene transfectants, one of which was labeled with a fluorescent dye. We conclude that the extracellular domain of pemphigus vulgaris antigen mediates weak homophilic adhesion and is not interchangeable in function with the extracellular domain of E-cadherin.

Animals↗