A comparison of membrane vs. intracellular estrogen receptor-alpha in GH(3)/B6 pituitary tumor cells using a quantitative plate immunoassay.

The plasma membrane form of the estrogen receptor-alpha (mER-alpha) is involved in rapid estrogen-induced prolactin release from GH(3)/B6 rat pituitary tumor cells and can be detected immunocytochemically using several estrogen receptor-alpha (ER-alpha) antibodies. We recently described staining of fixed cells via a biotin-avidin-alkaline phosphatase sandwich assay. From this protocol, we have developed a rapid, quantifiable 96-well plate immunoassay for mER-alpha, using a different alkaline phosphatase substrate, para-nitrophenylphosphate, which generates a soluble yellow product, para-nitrophenol. We also permeabilized cells with detergent during fixation to measure intracellular ER-alpha (iER-alpha) with the same assay and then compared intracellular versus membrane ER-alpha levels in two GH(3)/B6 cell subclones originally selected for high and absent mER-alpha expression by immunocytochemistry. While the F10 subclone expresses plentiful amounts of the mER-alpha, the D9 subclone has undetectable levels of mER-alpha using this assay. In addition, there is a seven-fold difference in iER-alpha expression between the high (F10) and no (D9) mER-alpha expressing subclones. In the high mER-alpha expressing cell line, the mER-alpha totals approximately one third of total cellular ER-alpha. Neither membrane or intracellular forms of ER-beta were detected with this assay. The pNp assay allows convenient and quantitative comparison of multiple parameters of mER-alpha and iER-alpha regulation and should be applicable to other antigens that are expressed on the cell surface as well as intracellularly.

Membrane oestrogen receptors on rat pituitary tumour cells: immuno-identification and responses to oestradiol and xenoestrogens.

Our laboratory has identified plasma membrane oestrogen receptors on a GH3/B6 rat pituitary tumour cell line and several sublines which produce rapid (within minutes), non-genomic responses to oestrogens. Oestrogen receptors have been identified by their binding to nine different antibodies (Abs) which together recognize at least seven epitopes on the oestrogen receptor-alpha. GH3/B6/F10 cells, a membrane oestrogen receptor-enriched subline, elevate intracellular calcium levels in response to 10 nM oestradiol. Prolactin release in these cells is triggered by both 1 pM and 1 nM oestradiol and diethylstilbestrol (DES). A membrane oestrogen receptor-alpha immunocytochemical signal rapidly disappears (at 3 min) and reappears (at 12-15 min) when 1 nM oestradiol, 10 nM diethylstilbestrol, or 10 nM nonylphenol is applied to the cells. This suggests that both oestrogens and xenoestrogens can utilize this alternative pathway for oestrogenic action. Xenoestrogens, which have so far shown weak effects in genomic assay systems, should now be retested for activity in eliciting membrane-initiated oestrogenic responses.

The role of HIV counseling and testing in the developing world.

The role of voluntary HIV counseling and testing is still under debate, especially in the developing world. HIV counseling-and-testing (HIV CT) services are a major component of HIV and AIDS control programs in the industrialized world and are increasingly being advocated in the developing world. In the United States, voluntary HIV CT has been a major component of HIV prevention efforts since the HIV antibody test became available in 1985. Yet even in the United States, questions about the management, cost, and effectiveness of voluntary HIV CT services continue to be raised. Because HIV CT has multiple goals, the evaluation of its effectiveness is a complicated task. Worldwide, a broad range of ethical, social, policy, technical, and economic issues encompass this HIV prevention activity. This article identifies the substantial barriers and serious concerns that are raised about HIV CT services and attempts to highlight the potential advantages of providing HIV CT as part of a developing country's comprehensive HIV prevention strategy.

Annexins I, II and III are specific choline binding proteins.

We have isolated choline binding proteins from the plasma membrane fraction fraction of human lung epithelium-derived cell line (A549) by means of detergent solubilization, anion exchange and affinity chromatography. One of the affinity purified proteins had a specific choline binding activity of 44-57 pmol/mg, representing a two to three hundredfold enrichment relative to the specific activity of freshly prepared plasma membranes. The purified protein has a molecular mass of 38 kDa by SDS PAGE analysis and was identified as annexin II by N-terminal microsequencing. Annexin II, however, had not previously been known for choline binding activity. We therefore prepared a mixture of authentic annexins (I-V) from A549 cells. The mixture had a choline binding activity of 15 to 18 pmol/mg. The annexin mixture was subsequently affinity chromatographed on the choline-conjugated Sepharose 6B column. Analyses by SDS PAGE and immunoblot revealed that annexins I, II, and III are bound to the choline column while annexins IV and V did not. These results indicate that some of the annexins have specific choline binding activities.

Clients without health insurance at publicly funded HIV counseling and testing sites: implications for early intervention.

The characteristics of clients reporting no health insurance were compared with those reporting any health insurance at publicly funded human immunodeficiency virus (HIV) counseling and testing sites in the United States during 1992. Thirty of 65 funded health departments collect data on self-reported health insurance status. Data were dichotomized into two groups, clients reporting any health insurance versus those reporting none, and multivariate logistic models were developed to explore independent associations. Of the 885,046 clients studied, 440,416 reported that they lacked health insurance. Clients without health insurance were more likely to be male, members of racial or ethnic minorities, adolescent, and HIV seropositive. Prisoners (odds ratio = 0.26), clients of Hispanic ethnicity (odds ratio = 0.52), and clients receiving testing during field visits (odds ratio = 0.53) in drug treatment centers (odds ratio = 0.55) and in tuberculosis clinics (odds ratio = 0.55) were less likely to have health insurance. Injecting drug users, whether heterosexual (odds ratio = 0.65) or homosexual (odds ratio = 0.67), were less likely to have health insurance compared with other behavioral risk groups. Large numbers of clients receiving publicly funded HIV counseling and testing lack health insurance. Lack of health insurance may interfere with subsequent receipt of needed primary care services among high-risk clients, especially HIV seropositive clients in need of early intervention services.

Characterization of patients accepting and refusing routine, voluntary HIV antibody testing in public sexually transmitted disease clinics.

BACKGROUND AND OBJECTIVES: To determine the proportion of HIV-infected sexually transmitted disease (STD) clinic patients identified during routine, voluntary HIV counseling and testing and to characterize patients accepting and refusing counseling and testing, we linked data from a blinded HIV seroprevalence survey to data from the HIV counseling and testing program. GOAL OF THIS STUDY: This study characterizes patients accepting and refusing routine HIV counseling and testing in two public STD clinics. STUDY DESIGN: A cross-sectional, blinded HIV seroprevalence survey was conducted of 1,232 STD clinic patients offered HIV counseling and testing. RESULTS: HIV seroprevalence was higher among patients who refused voluntary testing (7.8% versus 3.6%, P = 0.001). Patients who refused testing were more likely to report a prior HIV test (45.6% versus 27.2%; P < 0.001). Among patients reporting a prior HIV test, differences were noted between reported prior results, both positive and negative, and blinded results. CONCLUSIONS: HIV-infected STD patients may not be detected by routine HIV testing, and self-reported HIV results should be confirmed.

Purification of a novel glycosylated ferritin from horse heart.

We have previously shown that mRNA coding for ferritin L subunit is present on both cytosolic ribosomes and endoplasmic reticulum-bound ribosomes in rat heart tissue [Campbell et al. (1989) Arch Biochem Biophys 273:89-98]; from this we infer that heart tissue is capable of making a secreted ferritin. We now report the purification from horse heart, of a ferritin that specifically binds to Concanavalin A-Sepharose and is immunologically cross-reactive with antibodies raised against both horse cellular ferritin and horse serum ferritin. Where cellular ferritin is 10 nm in diameter and contains primarily 21-kDa subunits (as determined by gel exclusion chromatography and electron microscopy), the glycosylated heart ferritin is smaller with diameters of 3-5 nm. Antisera raised against serum ferritin cross-reacted with the glycosylated heart ferritin did but did not show significant cross-reactivity with cellular ferritin thus indicating that serum ferritin and glycosylated heart ferritin have antigenic determinants which may not be present on cellular ferritin. The glycosylated ferritin also differs from cellular ferritin in subunit composition, with subunits of 66, 60.5, 53.5, 43.5, and 29.5 kDa, as shown by SDS-PAGE and Western blot analysis. Interestingly, ferritin purified from horse serum contains subunits of similar size.

Where injecting drug users receive HIV counseling and testing.

In 1990, nearly 1.5 million human immunodeficiency virus (HIV) antibody tests were performed at publicly funded sites. Eight percent of those tests were performed for self-identified illegal injecting drug users (IDU). The authors examined data from 28 project areas using a client record data base that permitted an analysis of self-reported risk behavior by type of service delivery site. Among self-identified IDUs, 68 percent of those tested and 82 percent of those found to be seropositive had obtained HIV counseling and testing services in settings other than drug treatment centers. The findings indicate that HIV-prevention programs for IDUs need to be available in various service delivery settings, not just in drug treatment programs. Strong links and cooperation between sites offering HIV counseling and testing and sites providing drug treatment programs are important to preventing HIV transmission to and from IDUs.

A study of clients returning for counseling after HIV testing: implications for improving rates of return.

Pretest and posttest counseling have become standard components of prevention-oriented human immunodeficiency virus (HIV) antibody testing programs. However, not all persons who receive pretest counseling and testing return for posttest counseling. Records of 557,967 clients from January through December 1990, representing more than 40 percent of all publicly funded HIV counseling and testing, were analyzed to determine variables independently associated with returning for HIV posttest counseling. On average, 63 percent of clients returned for posttest counseling. The rate varied by self-reported risk behavior, sex, race or ethnicity, age, site of counseling and testing, reason for visit, and HIV serostatus. In multivariate logistic models, persons who were young, African American, and pretest counseled in sexually transmitted disease (STD) clinics or family planning clinics were least likely to return for posttest counseling. Those clients who consider themselves to be at risk for HIV infection may be more likely to act on that perception and to follow through with posttest counseling than those who do not perceive risk. Counselors should make special efforts during pretest counseling to encourage adolescents, members of racial or ethnic minorities, and persons seen in STD and family planning clinics to return for posttest counseling by helping them understand and accept their own personal risk of HIV infection. Counselors need to establish, with the client's participation, a specific plan for receiving test results and posttest counseling.

Herpes simplex virus infection as a risk factor for human immunodeficiency virus infection in heterosexuals.

To determine if infection with herpes simplex virus (HSV) type 2 is associated with human immunodeficiency virus (HIV) type 1 infection among patients attending sexually transmitted diseases clinics, a case-control study was done on coded sera from 179 HIV-1-infected patients and 367 age-, race-, and gender-matched HIV-1-seronegative patients. Although only 13 (2.3%) of 546 patients had a history of genital herpes treatment, 72% and 56.6%, respectively, had serologic evidence of prior infection with HSV-1 and -2. HSV-1 antibody prevalence was similar among both patient groups; however, HSV-2 antibodies were more common among those infected with HIV-1. Among heterosexual men, 62.7% of those infected with HIV-1 had HSV-2 antibodies compared with 46.7% of those not infected (P less than .01). The HSV-2 seroprevalence among women with or without HIV infection was 78.1% and 57.7%, respectively (P less than .02). A history of intravenous drug use and a reactive serologic test for syphilis were each independently associated with HIV-1 infection in heterosexuals. These data suggest that the two most common causes of genital ulcerative disease in the United States, genital herpes and syphilis, may contribute to increased risk for HIV-1 infection among heterosexuals.

Ferritin mRNA is found on bound as well as on free polyribosomes in rat heart.

Free and endoplasmic reticulum-bound polyribosomes from rat heart were examined for their ferritin mRNA content. A procedure for separation and purification of the two ribosome populations that produced good yields of homogeneous mono- and polyribosomes with no contaminating ultrastructures and gave distinctive sedimentation profiles in 15-50% sucrose gradients was developed. 14C-labeled free and bound polyribosomes added to heart preparations indicated that only 3% of free and 5.5% of bound polyribosomes cross-contaminated the bound and free fractions, respectively. RNA from both polyribosome populations hybridized with [32P]cDNA for rat ferritin. The extent of hybridization with mRNA from endoplasmic reticulum (ER)-derived polyribosomes was much greater than what could be accounted for by cross-contamination with free polyribosomes. This indicates that heart ferritin is synthesized not only on free polyribosomes for internal use in iron storage but also on ER-bound polyribosomes, where it may be destined for secretion into the plasma.

Translational regulation of ferritin synthesis in rat spleen: effects of iron and inflammation.

Translational control of ferritin synthesis was studied in rat spleen, and compared with that for liver, heart and brain, in response to iron and inflammation. Spleen concentrations of total RNA in the ribonucleoprotein (mRNP) fraction was comparable to that for liver, while polyribosomal RNA was less. Both fractions were ten-fold lower in heart and brain. In untreated animals, the mRNP fraction of all tissues had the largest portion of the ferritin mRNA, as determined by slot blot hybridization with 32P-labeled cDNA for the L subunit. Acute treatment with ferric ammonium citrate shifted the spleen ferritin mRNA to the polyribosome fraction. This was also so in liver but not in the heart and brain which took up much less iron. The findings were confirmed by hybridization studies of mRNPs and polyribosomes separated in sucrose gradients. Turpentine-induced inflammation also caused a shift in ferritin mRNA from the mRNP to the polyribosome fraction of spleen and liver, over 12 h. We conclude that as in liver, spleen ferritin synthesis is under translational control by iron, and that both tissues also respond to inflammation by shifting of ferritin mRNA to the polyribosomes.

Human immunodeficiency virus infection among patients attending clinics for sexually transmitted diseases.

To assess the prevalence and associated risk factors for human immunodeficiency virus (HIV) infection in patients attending inner-city clinics for sexually transmitted diseases in Baltimore, we screened 4028 patients anonymously, of whom 209 (5.2 percent) were seropositive for HIV. HIV-seropositivity rates were higher among men (6.3 percent) than women (3.0 percent) (P less than 0.001) and among blacks (5.0 percent) than whites (1.2 percent) (P less than 0.02). Among men, but not women, HIV seroprevalence increased markedly and steadily up to the age of 40. In men, HIV seropositivity was independently associated with increased age, black race, a history of homosexual contact, and the use of parenteral drugs. In women, a history of parenteral drug use or of being a sexual partner of a bisexual man or parenteral drug user were independently predictive of HIV seropositivity. In men, HIV seropositivity was also associated with a history of syphilis or a reactive serologic test for syphilis, and in women, with a history of genital warts. Since these associations were independent of the type and number of reported sexual partners, they raise the possibility that sexually transmitted diseases that disrupt epithelial surfaces may be important in the transmissibility of HIV. In addition, on a self-administered questionnaire, one third of HIV-infected men and one half of infected women did not acknowledge previous high-risk behavior for HIV exposure. These data suggest that patients at clinics for sexually transmitted diseases represent a group at high risk for HIV infection, and that screening, counseling, and intensive education should be offered to all patients attending such clinics.

Binding sites for elastase on cultured human fibroblasts that do not mediate internalization.

The proteolytic actions of elastases have been implicated in extracellular matrix damage, which is characteristic of a variety of pathological conditions including emphysema and rheumatoid arthritis. In order to elucidate the molecular events involved in elastase interaction with connective tissue cells, the present study was designed to investigate the association of elastase with human fibroblasts at 4 degrees C. Elastase bound saturably to binding sites that were present on the surface of these cells. Analysis of cell-bound elastase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a high molecular weight complex (Mr 54,000) that was not formed with elastase whose catalytic site serine was derivatized with a diisopropylphosphate group. The complex did not represent elastase bound to either protease nexin or contaminating serum. The cellular component with which elastase formed a complex could not be detected in the cell culture medium. Unexpectedly, elastase that had been pre-bound at 4 degrees C was not internalized after cells were warmed to 37 degrees C. The elastase binding site described in this report is therefore distinct from high affinity binding sites involved in receptor-mediated endocytosis and intracellular degradation.

Interactions of serine proteases with cultured fibroblasts.

This review summarizes the mechanisms by which several serine proteases, particularly urokinase, thrombin, and elastase, interact with cultured fibroblasts. Many of these studies were prompted by findings that interactions of these proteases with cells and the extracellular matrix are important in a number of physiologic and pathologic processes. Two main pathways have been identified for specific interactions of these proteases with fibroblasts. One involves surface binding sites for the free protease that appear to bind only one particular protease. An unusual feature collectively shared by the binding sites for urokinase, thrombin, and elastase is that the bound protease is not detectably internalized by the fibroblasts. The other pathway by which serine proteases interact with fibroblasts involves proteins named protease nexins (PNs). Three PNs have been identified. They are secreted by fibroblasts and inhibit certain serine proteases by forming a covalent complex with the protease catalytic site serine. The complexes then bind back to the fibroblasts via the PN portion of the complex and are internalized and degraded. Recent studies showing that the fibroblast surface and extracellular matrix accelerate the inactivation of thrombin by PN-1 support the hypothesis that the PNs control protease activity at and near the cell surface. The PNs differ from plasma protease inhibitors in their molecular properties, absence in plasma, site of synthesis, and site of clearance of the inhibitor:protease complexes.