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Publications and source records attributed to A J Conley.
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The expression of cytochromes P450 17 alpha-hydroxylase (P450c17) and aromatase (P450arom) was compared between preimplantation Chinese Meishan and domestic Yorkshire conceptuses during the period encompassing maternal recognition of pregnancy. Individual conceptuses were recovered on days 10.5, 11.0, 11.5, 12.0, and 14.0 of gestation. Diameter (spherical blastocysts), length (elongated blastocysts), DNA, protein and oestradiol content, as well as the amounts of P450c17 and P450arom (western analysis) were determined in individual conceptuses. Comparisons were made only between conceptuses of similar diameters on each day which restricted analyses to blastocysts 6 mm or less in diameter on days 10.5-12.0. Nonetheless, both DNA and protein content were greater in Yorkshire than in Meishan conceptuses. Oestradiol content also tended to be greater in Yorkshire than in Meishan conceptuses across days. A significant effect of breed and breed by day interaction was detected for P450c17. Expression of P450c17 in Yorkshire conceptuses increased markedly above that in Meishan conceptuses by day 11, remained high until day 11.5 and returned to values similar to those of Meishans by day 12. The expression of P450arom was also greater in Yorkshire than in Meishan conceptuses, but no breed by day interaction was detected. These data suggest that differences in development between Meishan and Yorkshire conceptuses include trophoblastic differentiation during preattachment stages. The significance and impact of this divergence in development on subsequent growth and survival remains to be determined.
We have used a panel of anti-gp160 MAbs to construct anti-HIV immunotoxins by coupling antibodies to ricin A chain (RAC). The ability of the immunotoxins to kill HIV-1-infected cells and halt the spread of infection was tested in tissue culture on persistently and acutely infected cell lines and primary lymphocyte cultures stimulated with phytohemagglutinin (PHA blasts). Laboratory strains and clinical isolates of HIV both were tested. The constitution and antigen-binding capacity of the immunotoxins were confirmed by ELISA and indirect immunofluorescence. Immunotoxins that bind epitopes exposed on the cell surface effectively killed persistently infected cells, although killing was not directly proportional to binding of immunotoxin to cell. The activity of anti-gp41, but not anti-gp120, immunotoxins was markedly enhanced in the presence of soluble CD4 or peptides corresponding to the CDR3 region of CD4. CD4-mediated enhancement of anti-gp41 immunotoxin activity was observed for laboratory strains neutralized by sCD4 and for clinical isolates that were resistant to neutralization by sCD4. Immunotoxin action was potentiated by brefeldin A, bafilomycin A1, cortisone, and an amphipathic fusion peptide, but not by cytochalasin D, nocodazol, monodansyl cadaverine, or trans-retinoic acid. Anti-HIV immunotoxins are useful tool with which to study the functional expression of gp120/gp41 antigens on the surface of HIV-infected cells, as well as potential AIDS therapeutics. Because these studies relate to the accessibility of viral antigens to antibody-mediated attack, these studies also have relevance for vaccine development.
Implantation is initiated on day 4 in the mouse and on day 13 in the pig. The preimplantation pig blastocyst synthesizes steroid hormones, but whether preimplantation rodent embryos also have this ability has remained unresolved for the last two decades. In this study, the mRNAs encoding NADPH-cytochrome P450 reductase (P450-reductase), adrenodoxin, lanosterol 14-demethylase P450 (CYP51), 17 alpha-hydroxylase P450 (CYP17), cholesterol side-chain cleavage P450 (CYP11A1), sterol 27-hydroxylase P450 (CYP27), and aromatase P450 (CYP19) were examined in day 4 mouse blastocysts (day 1 = vaginal plug) and in day 13 and 16 pig blastocysts using reverse transcription-polymerase chain reaction (RT-PCR). In mouse blastocysts, mRNAs of P450-reductase, adrenodoxin, and CYP51, but not CYP17, CYP11A1, CYP27, and CYP19, were detected. In agreement with this finding, no aromatase protein could be detected by immunohistochemistry. By contrast, all these mRNAs were detected in the pig blastocyst. Furthermore, both the ovarian and placental types of aromatase (CYP19) mRNAs were detected in the pig blastocyst on days 13 and 16 of pregnancy, although the ovarian form was more abundant. Both forms of aromatase were much higher in day 13 than in day 16 pig blastocysts. The results provide definitive evidence that the preimplantation mouse blastocyst, as opposed to the pig blastocyst, has no ability to synthesize estrogen and no steroidogenic capacity. Maternal estrogen synthesis is essential for implantation of the mouse blastocyst.
Previous studies in ovine adrenocortical cells in vitro have shown angiotensin II (AII) receptors are expressed on the zona fasciculata (ZF) cells and are functionally coupled to phosphoinositidase C and increased [Ca2+]i, but AII stimulation does not cause an acute change in cortisol biosynthesis. AII can, however, chronically regulate differential expression of P450c17 and 3 beta HSD in ovine adrenocortical cells in vitro. We have stained ovine adrenal sections with specific antisera to the angiotensin II Type-1 receptor (AT1-R), as well as P450c17 and 3 beta HSD in order to further test the hypothesis that changes in AT1-R expression underlie changes in zonal expression of P450c17 and 3 beta HSD in vivo. AT1-R expression was found to be highest in the outermost layer of cells (zona glomerulosa, ZG) which stained negatively for P450c17 and only faintly positive for 3 beta HSD, as expected. The adjacent layer of cells (ZF) stained much less strongly for AT1-R but stronger for P450c17 and 3 beta HSD. These findings are consistent with our previously reported in vitro expression data, and suggest that the transition from ZG to ZF phenotype, i.e. increased P450c17 and 3 beta HSD expression, may require reduced expression of AT1-R, but maintenance of reduced levels of AT1-R expression in the ovine ZF still allows for differential control of the P450c17: 3 beta HSD ratio. Thus, even though there is no acute cortisol response to AII alone in these cells, AII stimulation can oppose C19 steroid production in the face of cortisol biosynthesis by the ZF in response to agonists such as ACTH.
The expression of aromatase cytochrome P450 (P450arom) in the adrenal glands, testes, and placentas of fetal and newborn pigs was investigated. Western immunoblot analysis detected a single 48-50-kDa protein band in these tissues as well as in other porcine tissues known to express P450arom including Day 12 tubular conceptuses, theca interna, and granulosa. Slight differences in migration suggested that the P450arom protein expressed in the testis was larger than that in the adrenal gland, which was, in turn, larger than that in placenta, theca, and granulosa. Consistent with P450arom expression in these tissues, a cDNA encoding porcine P450arom hybridized to a 2.3-kb transcripts in Northern analyses of porcine blastocysts, placentas, and fetal and newborn adrenal glands and testes, as well as in theca and granulosa tissues from preovulatory follicles. No differences in transcript size were detectable among tissues. The identity of P450arom transcripts was confirmed by sequence analysis of partial cDNA clones amplified from porcine fetal adrenal glands, testes, and placentas according to the RACE procedure. The sequences of the adrenal and testis clones were identical but differed from the placental sequence, which represented the first 85 amino acids of porcine P450arom. Specifically, the adrenal and testis clones expressed transcripts that resembled the ovarian isoform of porcine P450arom, rather than the porcine placental isoform, predicting a two-amino acid deletion and 12 predicted amino acid substitutions. P450arom activity was examined to further define expression in these tissues. Activity in adrenal, testis, and placental homogenates was inhibited by 4-hydroxyandrostenedione (4OH-A4) whereas inhibition by etomidate was demonstrated in the adrenal and testis homogenates but not in the placental homogenates. The sensitivity of activity in the newborn porcine adrenal glands and testes to inhibition by etomidate was similar to that of ovarian P450arom activity. The level of P450arom activity was highest in the placenta and lowest in the adrenal gland, and no effect of fetal sex was noted in either tissue. Immunocytochemical studies localized the expression of P450arom in the adrenal gland of newborns to cells at the corticomedullary junction and, with greater intensity, to cells around the developing medullary lobules. The same cells expressed cytochrome P450 17 alpha-hydroxylase, the expression of which also extended throughout the zona fasciculata. The interstitial cells were the site of P450arom expression in the testis, but no expression could be detected in any cells with the spermatic tubules. These data demonstrate that fetal and newborn porcine adrenal glands and testes express an active P450arom that resembles the isoform expressed in the ovary. The localization of adrenal expression suggests a possible role in medullary maturation and function in the fetal and newborn pig.
The anti-gp41 virus neutralizing monoclonal antibody 2F5 was infused into chimpanzees, which were then given an intravenous challenge with a primary human immunodeficiency virus type I (HIV-1) isolate. In two control animals, the infection was established immediately, as evidenced by positive cell-associated DNA PCR and serum RNA PCR tests within 1 week, seroconversion by 4 weeks, and development of lymphadenopathy in this acute phase. Serum RNA PCR tests were negative in one of the two antibody-infused animals until week 8 and in the other antibody-infused animal until week 12; both animals seroconverted at week 14. The peak of measurable virus-specific serum RNA was delayed until week 16 in one antibody-infused animal. Virus-specific RNA in the other animal did not reach levels comparable to those in the other animals through 1 year of follow-up studies. Virus was isolated from the week 16 blood sample from one infused animal. Virus was not isolated from peripheral blood of the second animal but was isolated from lymph node cells taken at week 36. The infection of untreated chimpanzees with this primary isolate appears robust. Use of this isolate should widen the scope of possible experiments in the chimpanzee model. This antibody infusion study indicates that neutralizing antibody, when present at the time of challenge, affects the timing and level of infection and remains influential after it can no longer be detected in the peripheral circulation. It is possible that preexisting, neutralizing antibodies (passively administered or actively elicited) affect the course of acute-phase virus replication in humans. It remains to be established whether these immunologically mediated early effects will influence the course of HIV-1 disease.
The effect of sex on pig conceptus development to day 12 of gestation was investigated. On day 2 of gestation, reciprocal embryo transfers were performed resulting in four groups (Yorkshire-Yorkshire, Yorkshire-Meishan, Meishan-Yorkshire and Meishan-Meishan). Conceptuses at day 12 were recovered from each recipient and diameter, as well as DNA, protein and oestradiol content were determined for individual conceptuses. The sex of individual conceptuses at day 12 was determined by amplification of a fragment of the pig SRY gene, using the polymerase chain reaction. Embryos developed more rapidly to day 12 in Yorkshire recipients, but there was no detectable effect of sex on the diameter, DNA, protein or oestradiol content of conceptuses from any transfer group. Thus, no sex effect was apparent under conditions either promoting or retarding the rate of early pig blastocyst growth. These results provide strong evidence that pig embryonic development occurs at a rate determined by uterine environment and not by sex of the conceptus.
Functional isoforms of porcine aromatase cytochrome P-450 were cloned from placenta, and ovarian theca interna and granulosa tissues, and full length cDNAs were expressed in vitro. Porcine theca and granulosa expressed an identical form of P-450arom. This ovarian cDNA encoded for a protein of 501 amino acids, two amino acids shorter at the N-terminal end than placental P-450arom isoform (503 residues). Overall, the two isoforms exhibited 93% nucleotide and 87% amino acid identity with each other, and both were highly homologous, at the nucleotide and amino acid levels, to human and bovine P-450arom, also 503 amino acid proteins. Analysis of the predicted amino acid sequence further suggested that the regions of the cDNAs, corresponding to presumed exons III, V and IX, assuming conservation of intron-exon boundaries with the human P-450arom gene, were conserved in the porcine placental and ovarian enzymes, while sequence variance occurred in all other putative exons. In vitro expression indicated that the cDNA encoding porcine placental P-450arom was almost 10-fold more active in the synthesis of estrone from androstenedione than was the ovarian isoform which synthesized more 19OH-androstenedione than estrone. Western analysis of transfected Cos1 cells suggested that the differences in activity were not due to levels of expression of the cDNAs since similar levels of immunodetectable protein were observed in cells transfected with each construct. Both isoforms were sensitive to inhibition of activity by the specific aromatase inhibitors, 4OH-androstenedione and CGS16949A. In addition, activity of the enzyme encoded by the ovarian P-450arom cDNA was suppressed by etomidate, an inhibitor of cytochrome P-450 11beta-hydroxylase, but the placental P-450arom isoform was not. These functional differences were consistent with observations made in similar experiments involving P-450arom activity in freshly homogenized tissues. These data provide evidence of the existence of distinct, intraspecies isoforms of P-450arom, the first described in any species and suggest that pigs possess a unique mechanism for regulating androgen metabolism.
The oligomerization by chemical cross-linking of a recombinant human antiviral monoclonal antibody (MAb), r447-1, and its characterization are described. This MAb binds to an epitope residing in the hypervariable V3 region of the envelope protein (gp120/160) of HIV-1. A dimeric form of this MAb displays enhanced avidity and was found to be capable of neutralizing a greater variety of lymphoid cell culture-adapted HIV-1 variants and HIV-1 primary isolates than its monomeric form. The superior binding and breadth of reactivity of this antibody suggests it may have utility as a therapeutic and/or prophylactic agent, if it possesses an appropriate safety and immunogenicity profile.
An HIV-1-seropositive volunteer was infused with an expanded autologous cytotoxic T lymphocyte (CTL) clone directed against the HIV-1 nef protein. This clone was adoptively transferred to determine whether supplementing CTL activity could reduce viral load or improve clinical course. Unexpectedly, infusion was followed by a decline in circulating CD4+ T cells and a rise in viral load. Some of the HIV isolates obtained from the plasma or CD4+ cells of the patient were lacking the nef epitope. These results suggest that active CTL selection of viral variants could contribute to the pathogenesis of AIDS and that clinical progression can occur despite high levels of circulating HIV-1-specific CTLs.
Follicular and luteal morphology and steroidogenic function were investigated by immunohistochemistry for cytochrome P450 17 alpha-hydroxylase (P450c17) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) during the estrous cycle in pigs, sheep, and cows. The theca interna of all species expressed P450c17 during follicular development. In the pig, this constituted a continuous layer of cells around the follicle, but a sheath of cells lining the basement membrane appeared not to express P450c17. Neither was expression of P450c17 in ovine and bovine follicles uniform throughout the theca interna. In these two species, a beaded appearance was given by P450c17, since it was expressed in some regions but not in others. Therefore, staining for P450c17 defined functional sub-populations of cells within the theca interna of pigs, sheep, and cows. Ovulation was associated with a decrease in P450c17 in all species, but some expression persisted in theca-derived cells of developing and mature porcine CL. Expression of 3 beta-HSD in the preovulatory follicle was confined to the theca of the pig and sheep; in contrast, in the cow, it was highest in the granulosa. In general, 3 beta-HSD expression appeared to be greater in porcine than ovine or bovine follicles, the physiological relevance of which is discussed. Porcine and ovine theca continued to express 3 beta-HSD after ovulation, and granulosa-derived cells increased their 3 beta-HSD expression markedly as they luteinized in all three species. During early luteal development in pigs and sheep, theca-derived cells with high 3 beta-HSD encircled luteal lobules, but these cells appeared throughout the parenchyma of the mature CL. Luteal regression in sheep and cows was typified by the loss of many cells expressing 3 beta-HSD, whereas others, adjacent to them, appeared to be intact without loss of enzyme expression. These data further define differences in steroidogenesis during follicular and luteal development among the pig, sheep, and cow.
Conversion of androgens to estrogens is catalyzed by aromatase P450 (P450arom; the product of the CYP19 gene). Regulation of tissue-specific expression of P450arom in humans is due, in part, to alternative transcriptional start sites that arise as a consequence of the use of granulosa cells and placental tissue from cows, horses, and pigs (ungulates) in order to determine whether these species, like the human, utilize tissue-specific promoters to drive P450arom expression. The majority of transcripts in the placenta have 5'-termini that differ from those in the ovary upstream of a common site of divergence, indicative of a splice junction. The use of tissue-specific promoters by the bovine CYP19 gene would produce these results, as it does in the case of the human CYP19 gene. A bovine genomic library was then screened with probes that hybridize to ovary- or placenta-specific transcripts. Two clones of approximately 15 kb each in length were isolated; one hybridized with the ovary-specific sequence and the other hybridized with the placenta-specific sequence. Whereas the former sequence was contiguous with the downstream sequence containing the translational start site, the latter was identical only with the sequence of the placental transcripts upstream of the putative splice junction, indicating that this was the distal sequence. Bovine and human ovary-specific genomic sequences share 77% bp identity, while bovine and human placenta-specific sequences demonstrated only 39% bp identity. These results mirror those obtained in comparisons of human, bovine, equine, and porcine ovarian and placental RACE cDNA 5'-termini.(ABSTRACT TRUNCATED AT 250 WORDS)
We have used virus neutralization and antibody-binding techniques to define the epitope for a human monoclonal antibody, designated 19b, within the V3 region of the gp120 surface glycoprotein of human immunodeficiency virus type 1. Unusually, the 19b epitope encompasses residues on both flanks of the V3 loop. However, 19b binding to gp120 is independent of sequences at the crown of the V3 loop, provided that they are compatible with the formation of a type II beta turn that is presumably necessary to juxtapose the antigenic residues on the V3 flanks. By comparing the V3 sequences of virus gp120s able and unable to bind 19b, we were able to define the canonical 19b epitope as -I----G--FY-T, where residues at the positions indicated by the gaps do not contribute directly to the 19b-binding site. A few conservative substitutions at the more critical residues are also compatible with 19b binding. Inspection of V3 sequences in the human immunodeficiency virus database indicated that the canonical 19b epitope is well conserved among isolates from the North American-European clade B and also among clade E isolates from Thailand and clade F isolates from Brazil. A minority of gp120s from clades A and C also possess the 19b epitope. Consistent with the theoretical predictions of its cross-clade reactivity, 19b was found to bind to gp120s from clades A, B, C, E, and F in immunoassays. However, 19b was not able to reduce the infectivity of primary viruses from clades A, E, and F that were predicted to possess the 19b epitope and only modestly reduced the infectivity of a clade C virus at low input virus concentrations. Cross-clade neutralization via V3-directed antibodies may, therefore, be difficult, even if the antibodies show broad reactivities in binding assays and the viruses theoretically possess the relevant binding site.
The objectives of this study were to develop a rapid method for sex determination for several mammalian species using polymerase chain reaction (PCR) and to use this method to determine whether there is a significant developmental difference in spherical diameter between male and female d-10 or -11 porcine embryos. The PCR system was developed and verified using genomic DNA from pigs of known sex, then it was tested with genomic DNA from several other mammalian species. Sex is determined by amplification of two genes in a single reaction. The presence or absence of a region of the Sry (sex-determining region Y) gene determines sex, and amplification of the Zfy (male) or Zfx (female) genes acts as a positive control for PCR. Sex determination was successful for all animals tested, including pigs, cattle, sheep, goats, llamas, horses, humans, baboons, dogs, cats, rats, and mice. A total of 209 embryos were collected from 21 crossbred gilts on d 10 or 11 of gestation, and their diameters were measured. No significant difference in embryo diameter was detected between male and female embryos, indicating that sexual dimorphism in embryonic growth in pigs does not occur before the period of rapid embryo elongation. The present sexing technique using PCR is rapid (approximately 6 h from receipt of embryos to results), and it may be useful for examining the effects of sex on any trait of interest in early porcine embryos and embryos from several other mammals.
The antiviral characteristics of monoclonal antibody IAM-41-2F5 (2F5) were determined in cell culture. The antibody had been previously shown to bind a specific sequence, ELDKWA, within the external domain of the gp41 envelope glycoprotein human immunodeficiency virus type 1 (HIV-1). Selection by 2F5 of recombinant phage from an epitope library confirmed the identification of the antibody's binding determinant. The antibody was found to be capable of neutralizing a broad range of lymphoid cell culture-adapted HIV-1 variants as well as HIV-1 primary isolates. Sequence analysis of the latter showed that neutralization was related to the presence of the antibody binding site. From kinetic measurements using an epitope-containing peptide or gp41, the half-time of dissociation for 2F5 was determined to be 122 min for the peptide and 156 min for gp41. The region of gp41 expressing this sequence exhibits greater conservation among HIV-1 isolates than do the variable domains of gp120.
A successful prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine must elicit an immune response that will prevent establishment of the persistent viral infection. The only response shown to be effective in this regard is virus-neutralizing antibody directed against the viral gp120 hypervariable V3-loop region. Conjugate immunogens, containing cyclic peptides representing the V3 determinant covalently bound to a carrier protein, were capable of eliciting virus-neutralizing antibodies. The consistency of the response was related to peptide size. The smaller cyclic peptides, expressing relatively conserved sequences from the V3-loop apex, were poor inducers of neutralizing activity. In contrast, the largest cyclic peptides mediated neutralizing responses that were similar to those observed and previously reported for intact gp120 immunogens. A cyclic synthetic peptide expressing most of the prototypic HIV-1 MN variant V3 determinant warrants further study as a potentially effective vaccine immunogen.
The expression of four major steroidogenic enzymes in porcine theca and granulosa cell layers of preovulatory follicles was related to the levels of steroids in follicular fluid and gonadotropin concentrations in peripheral serum at slaughter. Ovaries were collected during proestrus, early estrus, and late estrus as evidenced by behavioral signs. Follicles were dissected from the ovaries, and theca, granulosa, and follicular fluids were pooled for each of 24 sows. Cytochromes P450 17 alpha-hydroxylase/17-20 lyase (P450c17), aromatase (P450arom) and side-chain cleavage (P450scc), as well as 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), were subjected to Northern and Western immunoblot analyses. The concentrations of estradiol-17 beta, testosterone, androstenedione, and progesterone were determined in follicular fluid, and peripheral serum was assayed for estradiol-17 beta, LH, and FSH. Stages of preovulatory development were verified by plasma levels of LH, FSH, and estradiol-17 beta. Thecca expressed P450c17, P450arom, P450scc, and 3 beta HSD whereas granulosa expressed only P450arom and low levels of P450scc. Thecal P450c17, thecal P450arom, and granulosa P450arom expression decreased coincidentally as serum estradiol-17 beta and follicular fluid estradiol-17 beta, testosterone, and androstenedione levels declined after the presumed gonadotropin surge. Unlike P450c17 and P450arom P450scc and 3 beta HSD remained relatively constant in theca and granulosa. From these data, we suggest that the theca interna may be the primary steroidogenic compartment of the porcine follicle during its final stages of preovulatory development. Moreover, preovulatory estrogen secretion appears to be controlled by the coordinated expression of a triad of enzymes in the porcine follicle that includes theca P450c17, theca P450arom and granulosa P450arom.