Cellular mechanisms of implantation in domestic farm animals.

In order for pregnancy to be established, the conceptus of domestic animals must signal its presence upon arrival in the uterus, a process known as maternal recognition of pregnancy. The conceptus derived signal(s) prevent(s) the structural and functional demise of the corpus luteum to ensure the maintenance of a uterine environment that supports implantation and embryonic development. Implantation is a remarkable event that has been described as a biological paradox because an adhesive interaction is formed between two apical surfaces of epithelial cell types that are typically covered by non-adhesive glycoproteins. In domestic animals (such as pigs, horses, sheep, does and cows), the implantation process is not invasive as it is in most other mammalian species. This review describes the interaction between the conceptus (embryo and surrounding membranes) and the uterine epithelial surface in domestic farm animals and ability of the conceptus to control the lifespan of the corpus luteum to maintain pregnancy.

The role of integrins in reproduction.

Fertilization, implantation, and placentation are dynamic cellular events that require not only synchrony between the maternal environment and the embryo, but also complex cell-to-cell communication. This communication involves integrins, a large family of proteins involved in the attachment, migration, invasion, and control of cellular function. Over the past decade, investigators have learned that integrins participate in multiple reproductive events including fertilization, implantation, and placentation in many species. This review will describe: (i) the expression of integrins on gametes and during the establishment and development of the placenta; (ii) regulatory pathways for controlling expression of integrins in the uterus and developing placenta; (iii) the function of integrins as determined by null-mutations; and (iv) reproductive dysfunction in women related to inappropriate integrin expression in the uterus and/or placenta.

The CTLA-4 gene is expressed in placental fibroblasts.

In order to elucidate the mechanisms that ensure survival of the allogeneic fetus, we are investigating the expression pattern of genes that are involved in peripheral self-tolerance in tissues at the maternal-fetal interface. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a negative regulator of T cell activation and may modulate peripheral self-tolerance. Previously, we reported the preferential transmission of maternally-inherited shorter alleles at a 3'-UTR microsatellite locus to liveborn children, but random transmission of paternally-inherited alleles, suggesting that CTLA-4 may be involved in the maintenance of tolerance at the maternal-fetal interface. In this report, we demonstrate that CTLA-4 mRNA and protein are indeed expressed in fetal tissues at the maternal-fetal interface throughout gestation.

Expression of cell adhesion molecules in murine placentas and a placental cell line.

Integrins and vascular cell adhesion molecule-1 (VCAM-1) are required for normal placental development. In this study, integrin subunits alpha4, alphav, beta1, and beta3, and VCAM-1 were investigated for expression in uteroplacental units (gestation day [g.d.] 6 and 8) and placentas (g.d. 10, 12, 14, 16, and 18) of Swiss-Webster mice. All subunits and VCAM-1 mRNA (identified by reverse transcriptase polymerase chain reaction [RT-PCR]) and protein (detected by immunofluorescence) were present in all tissues throughout gestation. VCAM-1 was expressed strongly in the ectoplacental cone and trophoblast giant cells, alpha4 was expressed strongly by trophoblast giant cells and moderately by spongiotrophoblast and labyrinthine trophoblast, and alphav was expressed more strongly in the spongiotrophoblast than in the labyrinthine zone. The beta1 was more strongly expressed in the labyrinthine than the spongiotrophoblast zone, while beta3 and VCAM-1 were essentially equal in the two zones. Trophoblast-like SM9-1 cells were positive for all of the adhesion molecules when tested by RT-PCR and immunocytochemistry. Adhesion molecule expression in SM9-1 cells was consistent with expression in the labyrinthine zone. Collectively, the results of this study demonstrate that murine placentas contain mRNA and protein for alpha4, alphav, beta1, beta3, and VCAM-1, and that expression is cell-specific. These results and the identification of an adhesion molecule-expressing trophoblastic cell line should facilitate future studies on the function of adhesion molecules in placental development.

Regulation of protein and prostaglandin secretion in polarized primary cultures of caprine uterine epithelial cells.

Caprine uterine epithelial (UE) cells were cultured on Matrigel-coated filters. Transmission electron microscopy revealed polarized UE cells characterized by basally located nuclei, apical microvilli, convoluted lateral membranes, and junctional complexes. Domain-specific secretion of prostaglandins and radiolabeled proteins provide further evidence of functional epithelial cell polarity. Two experiments were conducted to evaluate factors controlling prostaglandin E2 (PGE) and prostaglandin F2alpha (PGF) secretion. In experiment one, steroid-treated (estradiol, progesterone, or estradiol + progesterone) polarized UE cells were treated with interferon tau (IFNtau) and/or oxytocin (OT). Steroid treatment did not influence PGE or PGF secretion. However, analysis of variance revealed an IFNtau by OT interaction (P < .01) for both PGE and PGF This interaction was caused by a reduction in PGE and PGF secretion by cultures receiving only IFNtau and the inability of IFNtau to block OT-induced release of PGE or PGF. In experiment 2, polarized UE cells were cultured in progesterone, with or without IFNtau, and sequentially challenged with estradiol and OT. Oxytocin stimulated the release of both PGE and PGF by polarized cUE cells (P < .01) and resulted in an increased accumulation of PGE (OT*domain; P < .01) in the basal compartment. Interferon tau did not influence PGE (P < .1) secretion. However, further analysis revealed that IFNtau reduced PGF secretion and was unable to block OT-induced PGF secretion (IFNtau*OT; P < .05) by polarized UE cells. Therefore, caprine UE cells form polarized monolayers and retain responsiveness to IFNtau and OT in vitro.

Endometrial connexin expression in the mare and pig: evidence for the suppression of cell-cell communication in uterine luminal epithelium.

This investigation examines the relationship between implantation strategy and gap junction protein expression in uterine endometrium. The pattern of gap junction and connexin protein expression was analyzed in porcine and equine endometrium from cycling and pregnant animals using electron microscopy and immunocytochemistry. Functional analysis of cell-cell communication was also monitored by laser cytometry in primary cultures of endometrial epithelial cells. Gap junctions were detected in endometrial stroma of cycling and pregnant animals, which was correlated with immunoreactive Cx43 within stromal fibroblasts and vascular elements. No Cx26, Cx32, or Cx43 immunostaining was detected in luminal endometrial epithelium in either the mare or the pig at any stage of the estrous cycle or pregnancy. In contrast, endometrial glands of the mare exhibited a spatiotemporal pattern of Cx43 expression in the apicolateral plasma membrane which, when present, colocalized with the tight junction-associated protein, ZO-1. Uterine glandular Cx43 expression in mares was present from day 3 postovulation through day 14 of diestrus and until day 23 of pregnancy, whereas Cx43 was absent within uterine glands during seasonal anestrus, estrus, and after day 30 of pregnancy. Primary cultures of equine endometrial epithelial cells expressed both immunoreactive Cx43 and significant gap junction-mediated intercellular communication (GJIC) which was rapidly upregulated by 1.0 mM 8-bromo-cAMP or blocked with 1.0 mM octanol. No GJIC or connexin protein was detected in cultured porcine epithelial cells despite incubation with a variety of agents, including 8-bromo-cAMP, steroid hormones, retinoic acid, and/or prolactin. Junctional communication in endometrial epithelium of domestic farm animals is different than that reported for species exhibiting invasive implantation. The absence of GJIC in uterine luminal epithelium of the gilt and mare may be involved in limiting trophoblast invasiveness.

Spatial and temporal analyses of integrin and Muc-1 expression in porcine uterine epithelium and trophectoderm in vitro.

The expression of the glycoproteins Muc-1 and integrin subunits (alpha1, alpha3, alpha4, alpha5, alpha(v), beta1, and beta3), all of which may be involved in the control of uterine receptivity, was examined in cultured porcine uterine epithelial (pUE) cells. Integrin subunit expression was also determined in trophoblastic vesicles generated from Day 12 and Day 15 pig conceptuses. Immunocytochemistry was performed on pUE cells cultured on several substrates (glass, serum, fibronectin, or Matrigel) in DME/F12 medium with 5% charcoal/dextran-stripped fetal bovine serum either containing no steroids or supplemented with estrogen, progesterone, or estrogen plus progesterone, or in medium with 5% complete fetal bovine serum. In addition, pUE cells were grown on Matrigel-coated Millicell filter inserts to facilitate development of structurally and functionally distinct apical and basal domains (i.e., polarized pUE cells) and were maintained in the same medium with supplements. The major findings in this investigation are that 1) Muc-1 and alpha1, alpha3, alpha4, alpha5, alpha(v), beta1, and beta3 integrin subunits are expressed on pUE cells in vitro; 2) Muc-1 and several integrin subunits are modulated by steroid hormones if culture conditions are provided that permit development of cell polarity; 3) the expression and steroid-induced modulation of Muc-1 and several integrin subunits in polarized pUE cells are similar to those detected in vivo; and 4) the expression of alpha1, alpha3, alpha4, alpha5, alpha(v), beta1, and beta3 integrin subunits by trophoblastic vesicles is identical to their expression on trophectoderm of intact conceptuses in vivo. These results suggest that the properties of polarized pUE cells and trophoblastic vesicles are comparable to those of their counterparts in vivo and are, therefore, useful as in vitro model systems to study conceptus-endometrial interactions during the periimplantation period and the control of uterine receptivity to implantation.

Extracellular matrix and the implantation cascade in pigs.

The structural and functional alterations of uterine epithelial cells that permit the apical-apical union of conceptus and uterine epithelium are complex and are likely to involve many different adhesion molecules with distinct but inter-related functions. A number of changes in the molecular composition at the apical surface of uterine epithelial cells associated with the transition from the pre-receptive to the receptive state in the pig uterus are reviewed. Molecules that function in the adhesion cascade resulting in implantation are represented by a variety of adhesion systems. However, integrins are probably the dominant adhesion systems because their capacity to mediate adhesion is linked to their activation by engaging other surface molecules.

Spatial and temporal analyses of integrin and Muc-1 expression in porcine uterine epithelium and trophectoderm in vivo.

The spatial and temporal expressions of Muc-1, selected integrin subunits, and extracellular matrix components in porcine uterine epithelium from estrous (Days 0, 4, 8, 10-15) and early pregnant (Days 10-15 of pregnancy) gilts and from steroid-treated ovariectomized gilts were analyzed using indirect immunofluorescence analyses on cryosectioned tissues to identify potential components of uterine receptivity. Integrin subunit and extracellular matrix expressions were also examined in Day 11-15 conceptuses. Intense Muc-1 staining was detected on apical uterine epithelium on Day 0 but was absent by Day 10 in both cyclic and pregnant gilts. The result of estrogen treatment (E2; 100 micrograms/day for 10 days) was similar to that of the corn oil vehicle control, while treatment with progesterone (P4; 200 mg/day for 10 days) or E2 + P4 decreased Muc-1 staining in ovariectomized gilts. Immunostaining performed with antibodies directed against integrin subunits (alpha 1, alpha 3, alpha 4, alpha 5, alpha v, beta 1, and beta 3) in uterine epithelium revealed low (integrin subunits alpha 1 alpha 3), high (integrin subunits alpha v and beta 3), or modulated (integrin subunits alpha 4, alpha 5, and beta 1) expression, with the lowest expression on Day 0 and maximum expression by Days 10-15. Additionally, no differences due to pregnancy status were detected in staining of uterine epithelium on Days 10-15. Uterine epithelium from steroid-treated ovariectomized gilts had low expression of alpha 4, alpha 5, and beta 1 subunits in the presence of E2 that increased in response to P4 and E2 + P4 treatments. The expression of integrin subunits alpha 3, alpha v and beta 3 was not affected by sex steroids. Trophectoderm also expressed alpha 1, alpha 4, alpha 5, alpha v, beta 1, and beta 3, integrin subunits. Extracellular matrix constituents (fibronectin, vitronectin, laminin, and collagen type IV) were also examined. Fibronectin and vitronectin were present on trophectoderm, but only vitronectin was detected on uterine epithelium. The alpha 4, alpha 5, alpha v, beta 1, and beta 3 integrin subunits, vitronectin, and fibronectin were detected at sites of attachment between uterine epithelial cells and trophectoderm on Days 12-15 of pregnancy. These studies indicate that down-regulation of Muc-1 coincides with the transition of the prereceptive uterus to the receptive uterus. Additionally, the expression of alpha 4, alpha 5, alpha v, beta 1, and beta 3 integrin subunits along with the extracellular matrix components of fibronectin and vitronectin correlates with the time of implantation in swine.

Characterization of a polarized porcine uterine epithelial model system.

A porcine uterine epithelial cell (pUE) culture system that retains structural and functional properties of the surface epithelium in vivo was developed. Uterine luminal epithelial cells were isolated after pancreatin-dispase enzymatic release of epithelium from hysterectomized gilts. Cells were seeded on Millicell filters precoated with Matrigel in 24-well plates and subsequently allowed to proliferate to confluence. Purity of the isolation was confirmed by the presence of > 99% cytokeratin-positive cells. Epithelial cells became polarized in vitro and compared favorably in morphology to uterine epithelial cells in situ once a transepithelial resistance of > 600 omega cm2 was established. Microscopic analysis confirmed the presence of a simple columnar epithelium with prominent microvilli on the apical cell surface and a well-developed junctional complex containing tight junctions, belt and spot desmosomes, and interdigitating lateral cell processes. Indirect immunofluorescence of the tight junction-associated protein, ZO-1, indicated the formation of tight junctional complexes in the subapical region of the polarized cells. Functional polarity of epithelial cultures was also verified by 1) electrical resistance measurements, 2) basal preference for the secretion of prostaglandins F2 alpha and E2, 3) apical preference for the release of 35S-methionine-incorporated secretory proteins, and 4) apically and basally distinct secretory protein profiles. Steroid treatment (estrogen, progesterone, or estrogen plus progesterone) of the polarized pUE cells affected the release of radiolabeled methionine-incorporated secretory proteins. In addition, the protein profiles as compared to samples treated with fetal bovine serum or charcoal/dextran-stripped fetal bovine serum were altered. Steroid treatments did not alter the electrical resistance or the basal preference for prostaglandin secretion. This culture system may be useful for in vitro analysis of maternal recognition of pregnancy paradigms as well as the study of the direct actions of hormones, prostaglandin secretion, and epithelial-stromal interactions.

Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43.

The rapid effects of cAMP on gap junction-mediated intercellular communication were examined in several cell types which express different levels of the gap junction protein, connexin43 (Cx43), including immortalized rat hepatocyte and granulosa cells, bovine coronary venular endothelial cells, primary rat myometrial and equine uterine epithelial cells. Functional analysis of changes in junctional communication induced by 8-bromo-cAMP was monitored by a fluorescence recovery after photobleaching assay in subconfluent cultures in the presence or absence of 1.0 mM 1-octanol (an agent which uncouples cells by closing gap junction channels). Communicating cells treated with 1.0 mM 8-bromo-cAMP alone exhibited significant increases in the percent of fluorescence recovery which were detected within 1-3 min depending on cell type, and junctional communication remained significantly elevated for up to 24 hr. Addition of 1.0 mM 8-bromo-cAMP to cultured cells, which were uncoupled with 1.0 mM octanol for 1 min, exhibited partial restoration of gap junctional permeability beginning within 3-5 min. Identical treatments were performed on cultures that were subsequently processed for indirect immunofluorescence to monitor Cx43 distribution. The changes in junctional permeability of cells correlated with changes in the distribution of immunoreactive Cx43. Cells treated for 2 hr with 10 microM monensin exhibited a reduced communication rate which was accompanied by increased vesicular cytoplasmic Cx43 staining and reduced punctate surface staining of junctional plaques. Addition of 1.0 mM 8-bromo-cAMP to these cultures had no effect on the rate of communication or the distribution of Cx43 compared to cultures treated with monensin alone. These data suggest that an effect of cyclic AMP on Cx43 gap junctions is to promote increases in gap junctional permeability by increasing trafficking and/or assembly of Cx43 to plasma membrane gap junctional plaques.

Enhancement of melphalan toxicity by octanol in ovarian adenocarcinoma cell lines: effects of altered cell-cell communication, glutathione levels, and plasma membrane fluidity.

A2780 and COLO-316 ovarian adenocarcinoma cell lines were exposed to 1.0 mM 1-octanol for 12 hr in order to evaluate the potential effects of inhibition of gap junction-mediated intercellular communication (GJIC) on cellular responses to the chemotherapeutic drug melphalan. Other cellular endpoints relevant to drug-resistance mechanisms which were monitored after treatments included cellular glutathione levels, glutathione S-transferase activity, mitochondrial membrane potential, and plasma membrane lipid mobility. In cells which were sensitive to melphalan, octanol enhanced melphalan toxicity in the GJIC-competent (A2780/S) but not GJIC-incompetent (COLO-316/S) sensitive cells. Although octanol increases plasma membrane lipid mobility in A2780/S and COLO-316/S, it appears that enhancement of A2780/S sensitivity to melphalan may be due to inhibition of GJIC. In melphalan-resistant cells (A2780/R and COLO-316/R), 1.0 mM octanol treatment for 12 hr combined with melphalan reversed the resistance of the cells to the drug. Therefore, alterations in cellular glutathione metabolism and effects on the plasma membrane in addition to uncoupling of GJIC may be involved in sensitizing communication-competent and communication-incompetent resistant cells because COLO-316/R lacks gap junction-mediated intercellular communication. Further, analysis of mitochondrial membrane potential provided an index of acquired drug resistance and the efficacy of melphalan and combined octanol/melphalan toxicity.

Growth characteristics in vitro of myeloid leukaemic cells with t(6;9).

We report an analysis of in vitro growth characteristics of leukaemic cells from five patients with t(6;9)(p23;q34). Consistent with other reports of this abnormality, our patients were comparatively young (median age at diagnosis, 29 years), and responded poorly to conventional treatment (median survival from diagnosis, 10 months). During active disease the CFU-GM growth patterns were characterized by an abundance of granulocytic aggregates (mostly 20-100 cells in size) whose leukaemic origin was confirmed by cytogenetic analysis. During remission induction, colonies derived from regenerating normal progenitor cell colonies could be distinguished from those derived from persisting leukaemic cells on the basis of differences in size, morphology, in situ staining characteristics, and karyotype. Remission growth patterns were those of a normal bone marrow. Our findings add to a growing recognition that the t(6;9) identifies a subset of leukaemic patients with distinctive clinical, haematologic, molecular, and in vitro growth characteristics for whom conventional treatment offers little hope of cure or long survival.

Effect of heparin on capacitation/acrosome reaction of equine sperm.

The onset of sperm capacitation/acrosome reaction was evaluated using heparin. Equine semen was incubated at 38 degrees C for 4.5 h in culture medium with and without 10 micrograms/mL heparin and with and without 0.1 microM of Ca2+ ionophore. Sperm acrosome reaction was detected using chlortetracycline fluorescence (CTC) and transmission electron microscopy (TEM). The CTC assay provided staining patterns that corresponded with the capacitation/acrosome reaction in other mammalian species (man, mouse, guinea pig). The percentages of incapacitated sperm (PUC), capacitated acrosome-intact sperm (PC), and acrosome-reacted sperm (PAR) were evaluated following incubation times of 0.5 and 4.5 h in heparin-free and heparinized medium, and at 4.5 h only in sperm exposed to Ca2+ ionophore. The CTC assay was highly correlated with TEM for estimation of PAR. At 4.5 h, heparinized medium reduced PUC and increased PC and PAR, in comparison with heparin-free medium. Addition of Ca2+ ionophore to the medium reduced PUC and increased PC and PAR at 4.5 h, as compared with sperm in ionophore-free medium. Incubation time also affected PUC, PC, and PAR in heparin-free and heparinized medium without ionophore. The PUC was greater at 0.5 h than at 4.5 h, and PC and PAR were less at 0.5 h than at 4.5 h. It would appear that the initiation of capacitation/acrosome reaction of equine sperm in vitro is accelerated by heparin.

Concurrent analysis of intracellular glutathione content and gap junctional intercellular communication.

The potential for performing dual analysis of intracellular glutathione levels and assessment of gap junctional intercellular communication with thiol-specific fluorescent probes in anchored cells was evaluated. Gap junction-mediated diffusion of monochlorobimane and 5-chloromethylfluorescein diacetate following intracellular loading and conjugation with glutathione was compared with 5-carboxyfluorescein diacetate (which is routinely used in laser cytometry to monitor intercellular communication) by means of fluorescence recovery after photobleaching using a variety of communication-competent and communication-incompetent cells. The rate of diffusion of fluorescence among communication-competent cells was inversely proportional to the size of the fluorescent probe employed. The thiol-specific probes were also employed to monitor depletion and synthesis of glutathione following treatments to inhibit glutathione synthesis or consume glutathione by adduct formation. Analysis of gap junctional intercellular communication following glutathione depletion revealed a direct correlation between glutathione levels and intercellular communication. These studies support the utility of the thiol-specific probes to monitor the respective role of cellular glutathione and intercellular communication in the mechanisms of cellular injury.

Abnormal behaviors, with a special focus on rocking, and reproductive competence in a large sample of captive chimpanzees (Pan troglodytes).

Chimpanzees (Pan troglodytes) are endangered in the wild and may no longer be imported into the United States. Of those animals presently in captivity, candidates for breeding programs must be identified to insure a self-sustaining captive population. Some have suggested that poor reproductive performance might be linked to the performance of abnormal behaviors. In Study 1, three institutions housing breeding colonies of chimpanzees (86 males, 103 females) surveyed their animals for abnormal behaviors, copulatory performance, and, for females, maternal competence. In neither sex was there a positive association between absence of copulation and the presence of any of 18 forms of abnormal behavior. No one abnormal behavior was positively associated with inadequate maternal performance. Contrary to expectations, in both sexes (significantly for females), copulators exhibited more forms of abnormal behaviors than did noncopulators. In contrast, good mothers did show slightly fewer different forms of abnormal behaviors than did inadequate mothers. No specific combination of abnormal behaviors was associated with lack of copulatory performance in either sex or with inadequacy of maternal behavior. Significant sex differences occurred only in 2 of the 18 abnormal behaviors (coprophagy and self-clinging), both with females showing the higher prevalence. In Study 2, the rate of rocking in 5 male and 14 female chimpanzees at the Primate Foundation of Arizona was found to be relatively high among some reproductively competent subjects. Some rocking had forms and contexts indicative of aggressive displays. We conclude that chimpanzees should not be disregarded as potential breeders or as candidates for resocialization and breeding programs solely because they exhibit abnormal behaviors. Anyone involved in assessing well-being needs to be aware of individual differences among animals in the stimuli that may elicit rocking behavior. If they are strangers, they may elicit display rocking which does not indicate a lack of well-being. © 1992 Wiley-Liss, Inc.

Preparation and anti-HIV activities of aurintricarboxylic acid fractions and analogues: direct correlation of antiviral potency with molecular weight.

Aurintricarboxylic acid (ATA) was fractionated by a combination of dialysis, ultrafiltration, and gel permeation chromatography. The number average and weight average molecular weights of the ATA fractions were determined by the universal calibration method. The sulfonic acid analogue of ATA was prepared and separated in high and low molecular weight fractions. The phosphonic acid analogue of ATA was also synthesized. All of the ATA fractions were tested for prevention of the cytopathic effect of HIV-1 and HIV-2 in MT-4 cell culture as well as against HIV-1 in CEM cell culture. The abilities of the fractions and analogues to inhibit syncytium formation between HIV-1- and HIV-2-infected HUT-78 cells and uninfected MOLT-4 cells were evaluated. In addition, the fractions and analogues were tested for cytotoxicity in mock-infected MT-4 cells, prevention of the binding of the OKT4A monoclonal antibody to the CD4 receptor, inhibition of the binding of anti-gp120 monoclonal antibody to gp120, inhibition of attachment of HIV-1 virions to MT-4 cells, and inhibition of HIV-1 reverse transcriptase. In all of these assays except cytotoxicity, there was a correlation of potency with molecular weight. The higher the molecular weight, the higher the activity. Several of the lower molecular weight fractions of ATA, which bound to gp120 but not to CD4, prevented HIV-1 and HIV-2 cytopathicity. A similar profile was observed for the phosphonic acid analogue of ATA and the lower molecular weight fraction of the sulfonic acid analogue. The results on the ATA fractions indicate that the binding of ATA to gp120 in the absence of CD4 binding is sufficient for anti-HIV activity. The active compounds bind more avidly to gp120 than to CD4. The anti-HIV activity of the ATA fractions is due to inhibition of virus binding due to an interference with the gp120-CD4 interaction.

Synthesis and anti-HIV activities of low molecular weight aurintricarboxylic acid fragments and related compounds.

Several compounds corresponding to fragments of the schematic representation of the polymeric structure of aurintricarboxylic acid (ATA) have been prepared and tested for prevention of the cytopathic effect of HIV-1 and HIV-2 in MT-4 cell culture and HIV-1 in CEM cell culture. Both the triphenylcarbinol 3 as well as the triphenylmethane 5 were found to afford protection against the cytopathogenicity of HIV-2 in MT-4 cells and HIV-1 in CEM cells, but they were inactive against HIV-1 in MT-4 cells. Both substances were also found to inhibit syncytium formation when MOLT-4 cells were cocultured with HIV-2-infected HUT-78 cells, but were inactive in this assay against HIV-1-infected cells. When observed, the activity is generally moderate in degree of protection and requires concentrations in the 10(-4) molar range. In contrast to ATA, both of these substances were inactive when tested for prevention of the binding of the OKT4A monoclonal antibody to the CD4 receptor and also for inhibition of HIV-1 reverse transcriptase. These substances therefore appear act by a mechanism that is distinct from that of polymeric ATA. Several active and inactive structural analogues of 3 and 5 were also synthesized. The anti-HIV activity in this series seems to depend on the presence of anionic carboxylate groups, since the methyl esters 4, 6, and 12 were uniformly inactive. The diphenylmethanes 8, 14, 18, and 19 also reproducibly inhibited the cytopathic effect of HIV-1 in CEM cell culture.

Inhibition of human immunodeficiency virus-1 reverse transcriptase activity by rubromycins: competitive interaction at the template.primer site.

Rubromycins, a class of quinone antibacterials, were discovered to selectively inhibit human immunodeficiency virus-1 (HIV-1) RNA-directed DNA polymerase (reverse transcriptase) (RT) activity more potently than cellular DNA polymerase alpha. beta- and gamma-rubromycin each inhibited equipotently HIV-1 RT and avian myeloblastosis virus RT, in a concentration-dependent manner, and were significantly weaker as inhibitors of calf thymus DNA polymerase alpha. These agents inhibited HIV-1 RT reversibly, were competitive with respect to template.primer, and were noncompetitive with respect to TTP. Dixon analyses yielded HIV RT Ki values of 0.27 +/- 0.014 and 0.13 +/- 0.012 microM for beta- and gamma-rubromycin, respectively. Similarly, using DNA polymerase alpha, the Ki values were 25.1 +/- 4.3 and 3.9 +/- 0.6 microM for beta- and gamma-rubromycin, respectively. Because these agents were toxic to noninfected human T lymphoid cells using concentrations at or above 6 microM, HIV-1 infectivity studies were carried out at 0.8-6 microM. At these concentrations, which are below the range expected to provide protection, no significant antiviral activity was observed. Although beta- and gamma-rubromycins did not possess sufficient HIV RT inhibitory potency or selectivity versus mammalian DNA polymerase to demonstrate antiviral activities, these studies support the hypothesis that specific molecules containing quinone functional groups can selectively inhibit viral polymerase activities over cellular polymerase activities. In addition, these studies suggest that rubromycins may be lead structures for the development of more potent and selective agents.