The effects of native and synthetic estrogenic compounds as well as vitamin D less-calcemic analogs on adipocytes content in rat bone marrow.

BACKGROUND: We demonstrated previously that phytoestrogens and vitamin D analogs like estradiol-17ß (E2) modulate bone morphology in rat female model. AIM: We now analyze the effects of phytoestrogens, E2, selective E2 re ceptor modulators, and the less-calcemic analogs of vitamin D: JKF1624F2-2 (JKF) or QW1624F2-2 (QW) on fat content in bone marrow (BM) from long bones in ovariectomized female rats (OVX). MATERIALS AND METHODS: OVX rats were injected with treatments known to affect bone formation, 5 days per week for 2.5 month for analysis of fat content in BM. RESULTS: In OVX young adults there is a decreased bone formation and a 10-fold increase in fat cells content in BM. Treatment with E2, raloxifene (Ral) or DT56a resulted in almost completely abolishment of fat cells content. Daidzein (D) decreased fat cells content by 80%, genistein (G) or biochainin A (BA) did not change fat cells content and carboxy BA (cBA) had a small but significant effect. JKF or QW did not affect fat cells content, whereas combined treatment of JKF or QW with E2 resulted in complete abolishment of fat cells content. These changes in fat cells content are inversely correlated with changes in bone formation. CONCLUSIONS: Our results demonstrate that adipogenesis induced by OVX is a reversible process which can be corrected by hormonal treatments. The awareness of a relationship between fat and bone at the marrow level might provide a better understanding of the pathophysiology of bone loss as well as a novel approach to diagnosis and treatment of postmenopausal osteoporosis.

The effects of peptides with estrogen-like activity on cell proliferation and energy metabolism in human derived vascular smooth muscle cells.

Hormone replacement therapy (HRT) for post-menopausal symptoms in diabetes is associated with increased risk of coronary heart disease and stroke. Therefore, there is a need for new HRT with no adverse effects on diabetic post-menopausal women. We developed peptides as potential estrogen mimetic compounds and now we evaluated the effects of the most efficacious peptide; hexapeptide estrogen-mimetic peptide 1 (EMP-1) (VSWFFE) in comparison to estrogen (E(2)) and peptides with weak activity A44 (KAWFFE) and A45 (KRAFFE) on modulation of cell proliferation of vascular smooth muscle cells (VSMC) growing in normal (ng) or high glucose (hg) concentrations. In ng EMP-1-like E(2) inhibited cell proliferation at high concentration, and stimulated at low concentration. EMP-1 did not affect E(2) stimulation of DNA, but inhibited E(2) inhibition of cell proliferation at high concentration. All effects by the combination of EMP-1 and E(2) were abolished at hg. A44-stimulated cell proliferation at all concentrations and A45 had no effect. When A44 was co-incubated with E(2) at both concentrations, DNA synthesis was stimulated, but abolished at hg. A45 abolished E(2) stimulation and inhibition of cell proliferation at both glucose concentrations. All peptides tested except A45-stimulated CK-specific activity at both glucose concentrations. In hg A44 stimulation of DNA was unaffected as well as its inhibition by EMP-1. EMP-1 and A44 similar to E(2)-stimulated MAPK activity in ng or hg, suggesting similar mechanism of action. The results presented here suggest that EMP-1 provided it acts similarly in vivo can replace E(2) for treatment of post-menopausal women in hyperglycemia due to diabetes.

Daidzein but not other phytoestrogens preserves bone architecture in ovariectomized female rats in vivo.

Ovariectomy of immature female rats, results in significant decrease of trabecular bone volume and in cortical bone thickness. Previously, we found that estradiol-17beta (E(2)) restored bone structure of ovariectomized (Ovx) female rats to values obtained in intact sham-operated female rats. E(2) also selectively stimulated creatine kinase (CK) specific activity a hormonal-genomic activity marker. In the present study, we compared the effects of E(2) and the phytoestrogens: daidzein (D), biochainin A (BA), genistein (G), carboxy-derivative of BA (cBA), and the SERM raloxifene (Ral) in Ovx, on both histological changes of bones and CK, when administered in multiple daily injections for 2.5 months. Bone from Ovx rats, showed significant disrupted architecture of the growth plate, with fewer proliferative cells and less chondroblasts. The metaphysis underneath the growth plate, contained less trabeculae but a significant increased number of adipocytes in the bone marrow. D like E(2) and Ral but not G, BA, or cBA, restored the morphology of the tibiae, similar to that of control sham-operated animals; the bony trabeculeae observed in the primary spongiosa was thicker, with almost no adipocytes in bone marrow. Ovariectomy resulted also in reduced CK, which in both epiphysis and diaphysis was stimulated by all estrogenic compounds tested. In summary, only D stimulated skeletal tissues growth and differentiation as effectively as E(2) or Ral, suggesting that under our experimental conditions, D is more effective in reversing menopausal changes than any of the other isolated phytoestrogens which cannot be considered as one entity.

Decreased ovarian hormones during a soya diet: implications for breast cancer prevention.

Ovarian hormones are biomarkers for breast cancer risk. Soybean consumption may be responsible in part for lower levels of ovarian hormones and decreased rates of breast cancer in women in Asia compared with Western populations. Soybeans contain a significant amount of the isoflavones daidzein and genistein, which are weak estrogens. The purpose of this study was to determine whether soya feeding decreases circulating levels of ovarian hormones and gonadotropins. Ten healthy, regularly cycling women consumed a constant soya-containing diet on a metabolic unit, starting on day 2 of a menstrual cycle until day 2 of the next cycle. Blood and urine samples were obtained daily for one menstrual cycle before and during soy feeding. The diet was calculated to maintain constant body weight, included 400 kilocalories from a 36-ounce portion of soymilk, and provided 113-207 mg/day (154.0+/-8.4 mg/day, mean +/- SE) of total isoflavones. For the group, the soya diet provided more carbohydrate and less protein than the home diets. Daily consumption of the soya diet reduced circulating levels of 17beta-estradiol by 25% (P<0.01, Wilcoxon signed rank test, two-tailed) and of progesterone by 45% (P<0.0001) compared with levels during the home diet period but had no effect on luteinizing hormone or follicle-stimulating hormone. Mean menstrual cycle length did not change during the soya diet; a slight decrease in mean luteal cycle length was marginally statistically significant (P = 0.06). Urinary excretion of isoflavones was 33.8+/-5.3 mg/day (mean +/- SE) and when expressed as percentage of intake, varied substantially (21.9+/-3.3% of intake; range, 9.1-36.7%) among the subjects. Mean daily serum levels of daidzein and genistein (free and conjugated forms) 15 h after soymilk were 2.89+/-0.53 microg/ml and 0.85+/-0.22 microg/ml, respectively, indicating systemic bioavailability of these substances. Secondary analyses by multiple regression showed that decreases in follicular and luteal phase 17beta-estradiol levels were positively associated with urinary isoflavone excretion, an association affected by age, and were inversely associated with decreases in protein intake. Decreases in progesterone levels during the soya diet were inversely associated with increases in intakes of genistein and were affected by the interaction of the intakes of daidzein with energy or with fiber. Consumption of an isoflavone-containing soya diet reduced levels of ovarian steroids in normal women over the entire menstrual cycle without affecting gonadotropins. This suggests that at least under the conditions of this study, soya-induced reductions of circulating ovarian steroids are not mediated by gonadotropins. Decreases in ovarian hormones are related to isoflavones contained in soy and also to energy intake and other components such as protein and fiber but not fat. Our results may explain decreased ovarian hormone levels and decreased risk of breast cancer in populations consuming soya diets and have implications for reducing breast cancer risk by dietary intervention.

Cloning of LL5, a novel protein encoding cDNA from a rat pituitary library.

While screening a rat pituitary cDNA library for a peptide hormone receptor, we identified a cDNA that represents a novel gene. The 3.8 kb cDNA has an open reading frame of 2.3 kb encoding a protein of 781 amino acids (M(r) = 87,507). Southern blot analysis of rat, mouse, bovine and human genomic DNAs revealed that a homologous gene is present in these species probably in a single copy. Northern blot analysis showed that in addition to the pituitary gland, the gene is also expressed in other rat tissues. Scanning of DNA and protein databanks revealed no significant homology to any other sequence. Thus, this gene encodes a heretofore unidentified protein.

Membranal effects of phytoestrogens and carboxy derivatives of phytoestrogens on human vascular and bone cells: new insights based on studies with carboxy-biochanin A.

Estradiol-17beta (E2) and some phytoestrogens induce a biphasic effect on DNA synthesis in cultured human vascular smooth muscle cells (VSMC), i.e., stimulation at low concentrations and inhibition at high concentrations. These compounds also increase the specific activity of creatine kinase (CK) as well as intracellular Ca2+ concentration in both VSMC and human female-derived cultured bone cells (OBs), and stimulate ERK1/2 phosphorylation in VSMC. At least some of these effects are exerted via membranal binding sites (mER), as would appear from observations that protein-bound, membrane impermeant estrogenic complexes can mimic the effect of E2 on DNA synthesis, intracellular Ca2+ concentration and MAPK, but not on CK activity. We now extend these studies by examining the effects of a novel carboxy-derivative of biochanin A, 6-carboxy-biochanin A (cBA) in VSMC and human osteoblasts in culture. cBA increased DNA synthesis in VSMC in a dose-dependent manner and was able to maintain this effect when linked to a cell membrane impermeable protein. In VSMC both cBA and estradiol, in their free or protein-bound forms induced a steep and immediate rise in intracellular calcium. Both the free and protein-bound conjugates of cBA and estradiol increased net MAPK-kinase activity. Neither the stimulatory effect of cBA nor the inhibitory effect of estradiol on DNA synthesis in VSMC could be shown in the presence of the MAPK-kinase inhibitor UO126. The presence of membrane binding sites for both estradiol and cBA was supported by direct visualization, using fluorescence labeling of their respective protein conjugates, E2-BSA and cBA-ovalbumin. Furthermore, these presumed membrane ER for estradiol and cBA were co-localized. In cultured human osteoblasts, cBA stimulated CK activity in a dose related fashion, which paralleled the increase in CK induced by estradiol per se, confirming the estrogenic properties of cBA in human bone cells. Both the free and protein-bound forms of cBA elicited immediate and substantial increments in intracellular Ca2+, similar to, but usually larger than the responses elicited by estradiol per se. cBA also increased ERalpha and suppressed ERbeta mRNA expression in human osteoblasts. Cultured human osteoblasts also harbor membrane binding sites for protein-bound form of cG, which are co-localized with the binding sites for protein-bound estradiol. The extent to which these properties of the novel synthetic phytoestrogen derivatives may be utilized to avert human vascular and/or bone disease requires further study.

Decrease in blood and ovarian 3 alpha- and 3 beta-androstanediol levels in rats induced to ovulate with pregnant mare serum gonadotropin.

Several hours before the first ovulation progesterone metabolism in the rat ovary, in vitro, is shifted from the production of 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) as the major metabolite toward the production of 4-ene-3-oxosteroids. In the present paper, changes in levels of 3 alpha-diol and its 3 beta-epimer as well as testosterone in blood and ovaries around the time of the first ovulation have been studied in immature PMSG-treated rats. Forty-eight hours after PMSG, a considerable increase in blood and ovarian testosterone concentration was observed, whereas the concentrations of both androstanediols in blood decreased sharply. At 52 h, the level of ovarian 3 beta-diol was only one third of the control level and continued to fall. The decrease in ovarian 3 alpha-diol was less pronounced, but reached about half, or less, of the control value. In PMSG-treated rats in which the LH surge was blocked by pentobarbitone, the decrease in blood diols was delayed but not prevented. It is concluded that the decrease in production of the androstanediols preceding the first ovulation observed previously in isolated ovaries, also occurs in the intact rat. The decrease in androstanediols occurs very shortly before an ovulation induced with an injection of PMSG and is dependent on the occurrence of an LH surge. Since it is assumed that the androstanediols have a prepubertal role in inhibiting uterine and ovarian growth and in preventing cyclic LH release, it is essential that their concentration decrease several hours before the first ovulation.

Characterization of an antiidiotypic antibody mimicking the actions of estradiol and its interaction with estrogen receptors.

The ability of a monoclonal antiidiotypic antibody (clone 1D5) directed against the binding site of a monoclonal antiestradiol antibody to interact with the estrogen receptor (ER) was investigated. The following lines of evidence indicate that clone 1D5 has the capacity of mimicking the actions of estradiol, and recognizes ER: 1) in binding experiments, clone 1D5 inhibited the binding of [3H]estradiol to porcine cytosolic 32-kilodalton ER fragment in a dose-dependent manner; irrelevant antibody had no effect; 2) in sucrose gradient density analysis, clone 1D5 abolished the specific peak of the [3H] estradiol-ER complex in the 4S region; 3) in immunoprecipitation experiments, clone 1D5 interacted with unoccupied ER, but not with estradiol-occupied ER; 4) in direct immunofluorescence studies clone 1D5 stained the nuclei of cultured rat epithelial cells and recognized estrogen binding sites in nuclear cryostat sections prepared from human, rat, and mouse estrogen-responsive tissues; and 5) When clone 1D5 was injected to immature female rats, it caused 46% increase in uterine creatine kinase activity, suggesting that clone 1D5 may possess estrogenic like activity. Under the same experimental conditions, estradiol caused 58% increase in creatine kinase activity. Collectively, these results suggest that clone 1D5 interacts with the steroid binding site of ER. Therefore, clone 1D5 can serve as a tool in the study of function and structure relationship of ER and to detect changes of ER levels in target cells of various species.

Regulation of rat granulosa cell differentiation by extracellular matrix produced by bovine corneal endothelial cells.

The effect of bovine corneal extracellular matrix (ECM) on gonadotropin-primed rat granulosa cells in vitro was studied by examining the following parameters: 1) rate of cell attachment to culture dishes; 2) modulation of cell morphology; 3) specific binding of [125I]human(h)CG to LH/hCG receptors; 4) cAMP response to hCG stimulation; and 5) basal and hCG stimulated progesterone production. Attachment of cells to culture dishes occurred significantly earlier on ECM, as compared with uncoated dishes (6 h vs. 24 h). Cells grown on ECM were epitheloid and organized in multilayer aggregates, closely resembling their organization in the intact wall of the ovarian follicle. In contrast, cultures on uncoated dishes grew as a monolayer of markedly flattened cells. A 2-fold increase in number of LH/hCG receptors occurred on ECM within 48 h, probably due to de novo synthesis. Scatchard analysis revealed no change in hormone affinity to the receptor during the culture period [association constant (Ka) = 2.5 X 10(10)M-1 for hCG]. Cells grown on ECM had a parallel increase in cAMP responsiveness to hCG stimulation. Cells grown in serum-free medium on ECM-coated dishes preserved only 50% of LH/hCG receptors and cAMP responsiveness after 48 h. Cells cultured on ECM showed a marked elevation in progesterone production even in the absence of gonadotropin stimulation, whereas cells grown on uncoated dishes almost completely lost their ability to produce progesterone both in the presence and absence of hCG. These results indicate that ECM plays a substantial role in the maintenance and further propagation of granulosa cell differentiation in vitro.

The antigenic epitopes of human grown hormone as mapped by monoclonal antibodies.

The antigenic epitopes of human GH (hGH) have been investigated using monoclonal antibodies (Mabs) to hGH. A panel of 12 Mabs was incorporated into two-site binding assays in order to investigate the antigenic surface of hGH. Nine reaction patterns were observed. The Mabs were further characterized in terms of their cross-reactivity with human placental lactogen, human PRL, with recombinant hGH molecules (MetLeu8hGH, Met14hGH), and with fragments derived from enzymatic or chemical digestion of hGH. These studies provided information on the antigenic sites that are shared with human placental lactogen and on the N-terminal and C-terminal regions of the hGH molecule. Based upon these findings, we tentatively suggest the epitope model for hGH comprising at least 10 antigenic sites (epitopes) which may be grouped into five antigenic regions.

Synaptic remodeling in the arcuate nucleus during the estrous cycle is induced by estrogen and precedes the preovulatory gonadotropin surge.

We have shown that the ovarian cycle is accompanied by a fall in the axosomatic synapses on randomly selected neurons of the arcuate nucleus by the morning of estrus, with a return to the preovulatory levels by the morning of metestrus, indicating a possible role in positive feedback. However, it remains to be proven that the circulating estradiol is the actual regulator of this physiological synaptic plasticity, or that estrogen-induced synaptic retraction precedes in the surge of gonadotropins at midcycle. To resolve these questions, we used an estradiol-immunoneutralization protocol and studied arcuate nucleus axosomatic synapses during the critical points of the estrous cycle. In addition to blocking positive feedback, estrogen immunoneutralization abolished synaptic retraction in the arcuate nucleus. As a positive control, the nonbinding estrogen diethylstilbestrol maintained the gonadotropin surge and synaptic retraction in the antiestradiol-treated animals. Furthermore, in the diluent-treated cycling control females, the synaptic retraction was found to precede the preovulatory LH surge. We demonstrated that the midcycle synaptic retraction of arcuate nucleus synapses is induced by the preovulatory estradiol surge, and that these morphological events precede the preovulatory gonadotropin surge. Taken together, these observations strongly suggest that the hypothalamic mechanism underlying the physiological disinhibition of gonadotropins at midcycle (positive feedback) requires estrogen-induced synaptic retraction in the arcuate nucleus.

Effects of gonadal steroids and their antagonists on DNA synthesis in human vascular cells.

The cardiovascular effect of estrogen is currently under intense investigation, but the role of androgens in vascular biology has attracted little attention. Because endothelial repair and vascular smooth muscle cell (VSMC) proliferation affect atherogenesis, we analyzed the effects of 17beta-estradiol (E2), dihydrotestosterone (DHT), and sex hormone antagonists on DNA synthesis in human umbilical VSMCs and in E304 cells (a human umbilical endothelial cell line). In VSMCs, both E2 and DHT had a biphasic effect on [3H]thymidine incorporation into DNA: low concentrations (0.3 nmol/L for E2, 3 nmol/L for DHT) stimulated [3H]thymidine incorporation (+35% and +41%, respectively), whereas high concentrations (30 nmol/L for E2, 300 nmol/L for DHT) inhibited [3H]thymidine incorporation (-40%). In contrast, E2 (0.3 to 300 nmol/L) and DHT (3 to 3000 nmol/L) dose-dependently enhanced [3H]thymidine incorporation in E304 cells (peak, +85% for both). In VSMCs, high concentrations of E2 and DHT inhibited platelet-derived growth factor (PDGF)-or insulin-like growth factor (IGF-1)-induced DNA synthesis (-50% to 80%), whereas PDGF- or IGF-1-dependent DNA synthesis in E304 cells was further increased by E2. The antiestrogens tamoxifen and raloxifene mimicked the effects of E2 on DNA synthesis in both VSMCs and E304 cells. However, when coincubated with a stimulatory concentration of E2 (0.3 nmol/L), tamoxifen and raloxifene blocked E2-induced [3H]thymidine incorporation in E304 cells but not in VSMCs. Finally, the androgen antagonist flutamide inhibited the biphasic effects of DHT on VSMCs and blocked the increase in DNA elicited by DHT in E304 cells. The results suggest complex, dose-dependent, and cell-specific interactions of estrogens, androgens, and their respective antagonists in the control of cellular proliferation in the vascular wall. Gonadal steroid-dependent inhibition of VSMC proliferation and stimulation of endothelial replication may contribute to vascular protection and remodeling responses to vascular injury.

Monoclonal anti-biotin antibodies simulate avidin in the recognition of biotin.

The sequence of the VH gene of a monoclonal anti-biotin antibody was determined. Biotin-binding motifs, similar to those in avidin and streptavidin, were identified in complementary determining regions 2 and 3, suggesting that natural selection of functional motifs may occur in unrelated protein types.

Direct time-resolved fluorescence immunoassay for serum oestradiol based on the idiotypic anti-idiotypic approach.

We report a novel competitive type immunoassay for oestradiol based on the idiotypic anti-idiotypic approach. This has been achieved by the production of an anti-idiotypic antibody (anti-Id) which is directed against the oestradiol binding site of the primary idiotypic antibody (Ab1). In this format the primary Ab1 was captured onto the surface of microtitre wells and oestradiol standards or serum samples were then allowed to compete with europium labelled anti-Id for the binding sites of Ab1. Fluorescence was proportional to the concentration of oestradiol over the range 0-8 ng/ml. The sensitivity of the assay was 80 +/- 20 pg/ml, whilst the intra-assay variation ranged from 3 to 10%, and the inter-assay variation from 7.3 to 15%. The results obtained by the fluorescence immunoassay correlated well with those obtained by an extraction radioimmunoassay using tritiated antigen and dextran-coated charcoal for separation of bound and free ligand (n = 60, r = 0.98). The idiotypic anti-idiotypic approach in hapten immunoassays enables antibodies to be labelled instead of haptens, and thus permits the development of robust and sensitive immunoassays.

Immunometric assay for lutropin (hLH) based on the use of universal reagents for enzymatic labelling and magnetic separation and monitored by enhanced chemiluminescence.

A solid-phase immunometric assay of human lutropin (hLH) is described. Two different anti-hLH antibodies were utilized as capture antibodies, and anti-IgG antibodies covalently coupled to magnetic particles and horseradish peroxidase, respectively, served as 'universal' detection reagents. An anti-hLH antibody raised in rabbits was incubated with a goat anti-rabbit IgG covalently bound to magnetic particles. The resulting complex was added to a separately incubated mixture of hLH and monoclonal anti-hLH antibody. Following incubation, the immunocomplex was sedimented in a magnetic field and the supernatant discarded. Finally a sheep anti-mouse antibody (F(ab')2 fragment) conjugated to horseradish peroxidase as label was added. Following a further incubation, the particles were sedimented in the magnetic field and washed. The hLH content of the sample was quantitated by measuring 'enhanced chemiluminescence'. The sensitivity of the assay was 2.5 +/- 0.9 IU/l (mean +/- SD), the within-run variation ranged from 7.9 to 11%, the between-run variation from 12.9 to 19.8%. Cross-reaction with hFSH or hTSH could not be detected, but was approximately 0.1% with hCG. The results correlated well with those obtained by radioimmunoassay (r = 0.84).

Synthesis of a novel biotin-estradiol conjugate and its use for the development of a direct, broad range enzyme immunoassay for plasma estradiol.

The synthesis and purification by high pressure liquid chromatography of a novel biotinoyl-diaminodioxaoctane-estradiol conjugate is described. The conjugate was used for the development of an enzyme immunoassay (EIA) for the direct determination of estradiol in human plasma. This assay was characterized by a low limit of detection (77 pM) and a broad working range. Estradiol concentrations ranging from 77 to 24300 pM, i.e. from the lower levels observed in the follicular phase of the normal menstrual cycle to the very high levels attained during hyperstimulation for in vitro fertilization, could be determined directly in undiluted plasma samples. Intra-assay variation ranged from 3.6 to 10.9% and interassay variation from 6.1 to 12%. The results obtained by the present EIA correlate well with target values obtained by isotope-dilution gas chromatography-mass spectrometry (r = 0.96) and with results obtained by a direct RIA (r = 0.97). The EIA requires no special apparatus, is simple, fast and robust, and could be applied in any clinical laboratory.

A direct non-competitive idiometric enzyme immunoassay for serum oestradiol.

We report a novel non-competitive enzyme immunoassay for oestradiol based on the use of two types of anti-idiotypic antibody that recognize different epitopes within the hypervariable region of the primary anti-oestradiol idiotypic antibody (Ab1). The first anti-idiotype, the betatype, competes with the analyte for an epitope of the primary antibody at the binding site. On the other hand, the second anti-idiotype, the alphatype, binds to the Ab1 in the presence of analyte but does not bind to the betatype/Ab1 complex because of steric hindrance. In the present format the biotinylated alphatype was captured onto anti-biotin IgG which was adsorbed on the surface of microtitre wells. Reaction mixtures containing the Ab1 complexed sequentially with an enzyme labelled second antibody reagent, with oestradiol standards or serum samples and with the betatype anti-idiotypic antibody were then allowed to react with the immobilized alphatype anti-idiotypic antibody. The enzyme activity of the bound fraction measured at 405 nm increased with increasing oestradiol concentrations over the range 0.06-2.5 ng/ml. The detection limit of the assay was 28 pg/ml. The intra-assay variation ranged from 3.5 to 12.4%, and inter-assay variation from 6 to 13.4%. The results obtained by the colorimetric idiometric immunoassay correlated well with those obtained by a direct radioimmunoassay (n = 85, r = 0.97). This non-competitive immunoassay, termed idiometric assay, for haptens permits the development of sensitive immunoassays with a wide working range, and a variety of end-point determinations depending on the label used (e.g., enzyme, chemiluminescent or fluorogenic compound).

Preliminary evaluation of a lateral flow immunoassay device for screening urine samples for the presence of sulphamethazine.

A lateral flow immunoassay (LFIA) device was developed and applied to testing urine samples for residues of the antimicrobial sulphamethazine (SMZ). This report describes the preparation of a rat monoclonal antibody to SMZ and its characterisation in an ELISA format. Apart from SMZ, the antibody showed high (> or =50%) cross-reactivity to N4-acetyl-sulphamethazine (55%), sulphamerazine (59%) and sulphisoxazole (50%) and lower cross-reactivity of 18% to sulphachlorpyridazine and sulphadiazine. The LFIA device consisted of a nitrocellulose membrane spotted with SMZ-ovalbumin and goat anti-mouse antibody as capture line and control line, respectively. Mouse anti-rat IgG F(ab')2 fragment specific antibody, adsorbed to colloidal carbon, was used as the detection ligand in the LFIA. The LFIA device had a cut-off value of 6.3 ng/ml in diluted (1/10) urine. Urine samples from SMZ-treated pigs, and bovine and porcine urine samples fortified with SMZ were used for a blind, four-laboratory evaluation of the performance of the LFIA device. Concentrations of SMZ in the test samples (n=29), as determined by LC-MS/MS, ranged from 0 (<3) to 1174 ng/ml. The evaluation of the LFIA device showed an overall sensitivity of 100%, a specificity of 71%, and positive and negative prediction values of 73% and 100%, respectively. The LFIA device has been fabricated as a test kit for determining SMZ residues in animals produced for slaughter.

Pediatric telephone advice in the emergency department: results of a mock scenario.

To assess the appropriateness of pediatric telephone advice given by emergency departments (EDs), a mock scenario simulating a 5-week-old with fever of 102 degrees F and signs compatible with meningitis was used to evaluate the responses of 61 randomly selected EDs, of which half were affiliated with pediatric residency training programs. All EDs were given the identical chief complaint: "My baby has been having a fever all day, and I can't seem to get it down." Calls were made by research technicians and monitored by one or more of the investigators by speakerphone. Fifty-three (87%) programs gave advice by telephone; in 42 (79%) of the 53 respondents the individual giving advice was a nurse. Fourteen programs (26%) gave advice without asking either the age of the child or height of the fever. Few of the respondents took historical information assessing irritability (4 programs), fluid intake (11 programs), urine output (8 programs), or breathing pattern (6 programs). Thirty-eight (71.7%) EDs advised the patient to see a physician, while only 32 (60.4%) suggested same-day evaluation. In several instances the caller was not advised to seek medical attention despite having given a history documenting the infant's fever, irritability, and lethargy. These findings show variability and inadequacies in pediatric telephone advice currently offered by the EDs that were studied.

Role of putative membrane receptors in the effect of androgens on human vascular cell growth.

We have reported previously that dihydrotestosterone (DHT) induces a biphasic effect on DNA synthesis in human vascular smooth muscle cells (VSMC), i.e. stimulation at low concentrations and inhibition at high concentrations. In contrast, DHT dose-dependently stimulated [(3)H]thymidine incorporation in a human endothelial cell line (ECV304). Additionally, DHT increased the specific activity of creatine kinase (CK) in both vascular cell types. In the present study, we have determined whether some of these effects are exerted via membrane-binding sites. We measured changes in DNA synthesis and CK after treatment with DHT and the membrane-impermeant testosterone-3-carboxymethyl oxime conjugated to bovine serum albumin (BSA) (T-BSA). High concentrations of either DHT or T-BSA inhibited VSMC proliferation (by 52+22% and 51+25% respectively). DHT as well as T-BSA increased DNA synthesis in ECV304 cells dose-dependently. In contrast, T-BSA did not affect CK in either cell type. In both cell types, DHT as well as T-BSA increased mitogen-activated protein kinase (MAPK) kinase activity as measured by total phosphorylated MAPK. Further, the inhibitory effect of either the free or protein-bound androgens on DNA synthesis was blocked by UO126, an inhibitor of MAPK kinase activity. T-BSA conjugate labeled with Europium showed binding to whole VSMC, which could be displaced by excess T-BSA, but not by estradiol-BSA or the free hormones. Finally, using T-BSA linked to the fluorescent dye Cy3.5, we directly demonstrated the presence of membrane-binding sites for androgen in VSMC. Hence, the inhibitory effects of testosterone on DNA synthesis in VSMC are apparently exerted by membrane-binding sites for androgen, do not require intracellular entry of the hormone and its binding to the classical nuclear receptors and are linked to MAPK activation.