Apoptosis repressor with caspase recruitment domain is regulated by MAPK/PI3K and confers drug resistance and survival advantage to AML.

The apoptosis repressor with caspase recruitment domain (ARC) protein is known to suppress both intrinsic and extrinsic apoptosis. We previously reported that ARC expression is a strong, independent adverse prognostic factor in acute myeloid leukemia (AML). Here, we investigated the regulation and role of ARC in AML. ARC expression is upregulated in AML cells co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) and suppressed by inhibition of MAPK and PI3K signaling. AML patient samples with RAS mutations (N = 64) expressed significantly higher levels of ARC than samples without RAS mutations (N = 371) (P = 0.016). ARC overexpression protected and ARC knockdown sensitized AML cells to cytarabine and to agents that selectively induce intrinsic (ABT-737) or extrinsic (TNF-related apoptosis inducing ligand) apoptosis. NOD-SCID mice harboring ARC-overexpressing KG-1 cells had significantly shorter survival than mice injected with control cells (median 84 vs 111 days) and significantly fewer leukemia cells were present when NOD/SCID IL2Rγ null mice were injected with ARC knockdown as compared to control Molm13 cells (P = 0.005 and 0.03 at 2 and 3 weeks, respectively). Together, these findings demonstrate that MSCs regulate ARC in AML through activation of MAPK and PI3K signaling pathways. ARC confers drug resistance and survival advantage to AML in vitro and in vivo, suggesting ARC as a novel target in AML therapy.

Age-based formulae to estimate children's weight in the emergency department.

OBJECTIVE: To develop an age-based weight estimation rule in a Chinese population and to compare its performance with existing formulae. DESIGN: Population-based observational study. SETTING: Schools and kindergartens in Hong Kong. SUBJECTS: Healthy Chinese children aged 1-10 years old on their last birthday. INTERVENTIONS: Weight was measured to the nearest 0.2 kg. MAIN OUTCOME MEASURES: Linear regression was used to derive a simple formula relating weight to the child's age on his or her last birthday. The accuracy and precision of different age-based weight formulae was compared using coefficient of variation, Bland Altman plots, and by determining the proportion of children with estimates >30% outside the actual weight. RESULTS: The Chinese Age Weight Rule is a simple linear formula that is more accurate than and at least as precise as any other age-based weight estimation rule: weight in kg=(3 × age last birthday)+5. It is accurate and precise in children <7 years old, but all age-based weight estimates are imprecise in older children. CONCLUSIONS: The Chinese Age Weight Rule should be used in a Chinese population in preference to any other age-based weight estimation rule. Caution should be taken when using it in older children in whom other weight-estimation tools may be more appropriate.

Disease severity prediction for nursing home-acquired pneumonia in the emergency department.

BACKGROUND: Prediction rules exist for the assessment of community-acquired pneumonia but their use in nursing home-acquired pneumonia (NHAP) remains undefined. The objectives of this study were to compare the prognostic ability for severe NHAP of five prediction rules (PSI, CURB-65, M-ATS, R-ATS, España rule), and to evaluate their usefulness to identify patients with less severe disease in the emergency department for outpatient care. METHODS: A prospective observational study of consecutive NHAP patients was conducted at a university teaching hospital emergency department in Hong Kong between January 2004 and June 2005. The primary outcome was severe pneumonia (defined as combined 30-day mortality and/or intensive care unit (ICU) admission). RESULTS: 767 consecutive NHAP patients were included. Mean (SD) age was 83.4 (9.0) years; 350 (45.6%) were male and 644 (84.0%) had coexisting illness. 95 patients died within 30 days (12.4%), five patients were admitted to the ICU (0.7%) and 98 patients had severe pneumonia (12.8%). Sensitivity and specificity of each decision rule ranged from 37.8% to 95.9% and 15.1% to 87.6% respectively. The overall predictive performance of each rule was between 0.627 and 0.712. The negative likelihood ratios of PSI (0.27) and CURB-65 (0.23) were lower than M-ATS (0.71), R-ATS (0.45) and España (0.39). After excluding 204 patients with either poor functional status or those >90 years of age, sensitivities of M-ATS (96.0%) and R-ATS (100%) improved greatly with negative likelihood ratios of <0.1. CONCLUSION: PSI and CURB-65 are useful for identification of patients with less severe NHAP.

Predictors of important neurological causes of dizziness among patients presenting to the emergency department.

OBJECTIVES: Dizziness is a common presenting complaint in the emergency department (ED). This prospective study describes the incidence, causes and outcome of ED patients presenting with dizziness and tries to identify predictors of central neurological causes of dizziness. METHODS: Single-centre prospective observational study in a university teaching hospital ED in Hong Kong. All ED patients (> or = 18 years old) presenting with dizziness were recruited for 1 month. Symptoms, previous health, physical findings, diagnosis and disposition were recorded. The outcome at 3 months was evaluated using hospital records and telephone interviews. Follow-up was also performed at 55 months using computerised hospital records to identify patients with subsequent stroke and those who had died. RESULTS: 413 adults (65% female, mean 57 years) were recruited. The incidence of dizziness was 3.6% (413/11 319). Nausea and/or vomiting (46%) and headache (20%) were the commonest associated findings. Hypertension (33%) was the commonest previous illness. Central neurological causes of dizziness were found in 6% (23/413) of patients. Age > or = 65 years (OR=6.13, 95% CI 1.97 to 19.09), ataxia symptoms (OR=11.39, 95% CI 2.404 to 53.95), focal neurological symptoms (OR=11.78, 95% CI 1.61 to 86.29), and history of previous stroke (OR=3.89, 95% CI 1.12 to 13.46) and diabetes mellitus (OR=3.57, 95% CI 1.04 to 12.28) predicted central causes of dizziness. CONCLUSIONS: Most dizzy patients had benign causes. Several clinical factors favoured a diagnosis of central neurological causes of dizziness.

Cost-consequence analysis comparing 2-octyl cyanoacrylate tissue adhesive and suture for closure of simple lacerations: a randomized controlled trial.

STUDY OBJECTIVE: We investigate the cost difference between conventional suture and tissue adhesive methods in simple wound closure. METHODS: A cost-consequence analysis was conducted alongside a nonblinded randomized controlled trial comparing 2-octyl cyanoacrylate tissue adhesive with conventional suture in simple lacerations closure in emergency departments (EDs) of a university teaching hospital and a major regional hospital in Hong Kong. One hundred eighty-six adult patients with simple lacerations of length within 8 cm were randomized to receive tissue adhesive (93 patients) or conventional suture (93 patients) for wound closure. The primary outcome measures were the costs to the Hospital Authority and the charges on participants incurred in each treatment method. The secondary outcome measures included the cosmetic visual analog scale, visual analog scale, Wound Evaluation Score, total time spent in each closure method, and the overall patients' satisfaction on the whole process of wound management. RESULTS: The 2 groups had similar baseline characteristics. The tissue adhesive method incurred a higher cost to the Hospital Authority (216.12 [US $27.70] versus 171.33 [US $21.96]; absolute difference 44.79 [US $5.74] [95% confidence interval (CI) 32.76 to 55.95 [US $4.20 to 7.14]]) but a lower charge to patients (109.68 [US $14.06] versus 156.96 [US $20.12]; absolute difference 47.28 [US $6.06] [95% CI, 35.58 to 58.98 [US $4.56 to 7.56]) than the conventional suture method. The mean cosmetic visual analog scale score, visual analog scale score, and Wound Evaluation Score of the 2 groups were similar at various intervals within 3 months after wound closure. Compared with the suture group, the tissue adhesive group had a shorter median procedure time, fewer patients had wound erythema or swelling after wound closure, fewer patients required analgesics on discharge at ED, and there was a higher overall patient satisfaction score. CONCLUSION: Simple wounds closed by tissue adhesives incur a higher cost to the Hospital Authority than the conventional suture but may be favored by patients because of lower personal charge.

A T-cell-selective interleukin 2 mutein exhibits potent antitumor activity and is well tolerated in vivo.

Human interleukin 2 (IL-2; Proleukin) is an approved therapeutic for advanced-stage metastatic cancer; however, its use is restricted because of severe systemic toxicity. Its function as a central mediator of T-cell activation may contribute to its efficacy for cancer therapy. However, activation of natural killer (NK) cells by therapeutically administered IL-2 may mediate toxicity. Here we have used targeted mutagenesis of human IL-2 to generate a mutein with approximately 3,000-fold in vitro selectivity for T cells over NK cells relative to wild-type IL-2. We compared the variant, termed BAY 50-4798, with human IL-2 (Proleukin) in a therapeutic dosing regimen in chimpanzees, and found that although the T-cell mobilization and activation properties of BAY 50-4798 were comparable to human IL-2, BAY 50-4798 was better tolerated in the chimpanzee. BAY 50-4798 was also shown to inhibit metastasis in a mouse tumor model. These results indicate that BAY 50-4798 may exhibit a greater therapeutic index than IL-2 in humans in the treatment of cancer and AIDS.

Combined inhibition of ß-catenin and Bcr-Abl synergistically targets tyrosine kinase inhibitor-resistant blast crisis chronic myeloid leukemia blasts and progenitors in vitro and in vivo.

Tyrosine kinase inhibitor (TKI) resistance and progression to blast crisis (BC), both related to persistent ß-catenin activation, remain formidable challenges for chronic myeloid leukemia (CML). We observed overexpression of ß-catenin in BC-CML stem/progenitor cells, particularly in granulocyte-macrophage progenitors, and highest among a novel CD34+CD38+CD123hiTim-3hi subset as determined by CyTOF analysis. Co-culture with mesenchymal stromal cells (MSCs) induced the expression of ß-catenin and its target CD44 in CML cells. A novel Wnt/ß-catenin signaling modulator, C82, and nilotinib synergistically killed KBM5T315I and TKI-resistant primary BC-CML cells with or without BCR-ABL kinase mutations even under leukemia/MSC co-culture conditions. Silencing of ß-catenin by short interfering RNA restored sensitivity of primary BCR-ABLT315I/E255V BC-CML cells to nilotinib. Combining the C82 pro-drug, PRI-724, with nilotinib significantly prolonged the survival of NOD/SCID/IL2Rγ null mice injected with primary BCR-ABLT315I/E255V BC-CML cells. The combined treatment selectively targeted CML progenitors and inhibited CD44, c-Myc, survivin, p-CRKL and p-STAT5 expression. In addition, pretreating primary BC-CML cells with C82, or the combination, but not with nilotinib alone, significantly impaired their engraftment potential in NOD/SCID/IL2Rγ-null-3/GM/SF mice and significantly prolonged survival. Our data suggest potential benefit of concomitant ß-catenin and Bcr-Abl inhibition to prevent or overcome Bcr-Abl kinase-dependent or -independent TKI resistance in BC-CML.

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621.

The role of pyruvate metabolism in the triggering of aerobic, alcoholic fermentation in Saccharomyces cerevisiae has been studied. Since Candida utilis does not exhibit a Crabtree effect. this yeast was used as a reference organism. The localization, activity and kinetic properties of pyruvate carboxylase (EC 6.4.1.1), the pyruvate dehydrogenase complex and pyruvate decarboxylase (EC 4.1.1.1) in cells of glucose-limited chemostat cultures of the two yeasts were compared. In contrast to the general situation in fungi, plants and animals, pyruvate carboxylase was found to be a cytosolic enzyme in both yeasts. This implies that for anabolic processes, transport of C4-dicarboxylic acids into the mitochondria is required. Isolated mitochondria from both yeasts exhibited the same kinetics with respect to oxidation of malate. Also, the affinity of isolated mitochondria for pyruvate oxidation and the in situ activity of the pyruvate dehydrogenase complex was similar in both types of mitochondria. The activity of the cytosolic enzyme pyruvate decarboxylase in S. cerevisiae from glucose-limited chemostat cultures was 8-fold that in C. utilis. The enzyme was purified from both organisms, and its kinetic properties were determined. Pyruvate decarboxylase of both yeasts was competitively inhibited by inorganic phosphate. The enzyme of S. cerevisiae was more sensitive to this inhibitor than the enzyme of C. utilis. The in vivo role of phosphate inhibition of pyruvate decarboxylase upon transition of cells from glucose limitation to glucose excess and the associated triggering of alcoholic fermentation was investigated with 31P-NMR. In both yeasts this transition resulted in a rapid drop of the cytosolic inorganic phosphate concentration. It is concluded that the relief from phosphate inhibition does stimulate alcoholic fermentation, but it is not a prerequisite for pyruvate decarboxylase to become active in vivo. Rather, a high glycolytic flux and a high level of this enzyme are decisive for the occurrence of alcoholic fermentation after transfer of cells from glucose limitation to glucose excess.

The new RGA locus encodes a negative regulator of gibberellin response in Arabidopsis thaliana.

We have identified a new locus involved in gibberellin (GA) signal transduction by screening for suppressors of the Arabidopsis thaliana GA biosynthetic mutant gal-3. The locus is named RGA for repressor of gal-3. Based on the recessive phenotype of the digenic rga/gal-3 mutant, the wild-type gene product of RGA is probably a negative regulator of GA responses. Our screen for suppressors of gal-3 identified 17 mutant alleles of RGA as well as 10 new mutant alleles at the previously identified SPY locus. The digenic (double homozygous) rga/gal-3 mutants are able to partially repress several defects of gal-3 including stem growth, leaf abaxial trichome initiation, flowering time, and apical dominance. The phenotype of the trigenic mutant (triple homozygous) rga/spy/gal-3 shows that rga and spy have additive effects regulating flowering time, abaxial leaf trichome initiation and apical dominance. This trigenic mutant is similar to wild type with respect to each of these developmental events. Because rga/spy/gal-3 is almost insensitive to GA for hypocotyl growth and its bolting stem is taller than the wild-type plant, the combined effects of the rga and spy mutations appear to allow GA-independent stem growth. Our studies indicate that RGA lies on a separate branch of the GA signal transduction pathway from SPY, which leads us to propose a modified model of the GA response pathway.

Stage-dependent changes in steroidogenic enzymes and estrogen receptors during spermatogenesis in the testis of the dogfish, Squalus acanthias.

In the spiny dogfish (Squalus acanthias), germ cells and adjacent steroidogenic elements are topographically segregated within the testis according to stage of development. In the experiments reported here, we have taken advantage of this favorable anatomical arrangement to demonstrate stage-specific variations in steroidogenic enzymes and estrogen receptor number. The testes of Squalus collected in July-November were sectioned transversely and further subdivided into three zones as follows: I) germinal bed plus immature lobules with spermatogonia; II) lobules with primary or secondary spermatocytes; III) lobules with spermatids or mature spermatozoa. The morphology of these zones was verified by light microscopy and, in a separate study, by electron microscopy. Through the course of spermatogenesis, Sertoli cells increased dramatically in size and in the abundance of steroidogenic organelles. By contrast, interstitial tissue was sparse in all stages of development, and only relatively undifferentiated Leydig-like cells were present. Microsomes prepared from each zone were incubated with [3H] progesterone, [3H]17 alpha-hydroxyprogesterone, or [3H]androstenedione to evaluate androgen and estrogen biosynthetic potentials. Based on product formation, 17 alpha-hydroxylase and C-17,20-lyase activities increased progressively from less mature to more mature regions (zone III greater than II greater than I), whereas aromatase was greatest in regions undergoing meiosis or early spermiogenesis (zone II). These enzymes were not detected in semen, although C21 substrates were converted to unidentified polar metabolites in high yield. Estrogen receptors were concentrated in immature zones (zone I greater than II greater than III), and the percentage of occupied receptors revealed the same distribution. Semen and epigonal tissues were receptor negative. We conclude that Sertoli cells are responsible for steroidogenesis in Squalus testis and that hormone production is keyed to the spermatogenic cycle. The data are consistent with an important role for androgens during spermatid maturation and/or after sperm release, but reveal that estrogen actions are expressed primarily during early spermatogenic stages. This report demonstrates the usefulness of unconventional animal models for obtaining new information of general relevance.

Tissue and species specificity of unmasked nuclear acceptor sites for the estrogen receptor of Squalus testes.

The testicular estrogen receptor of the shark Squalus acanthias is restricted to nuclear subfractions when tissue is homogenized in low salt buffers and adheres tightly to nuclei and DNA-cellulose even when exposed to high salt conditions. Therefore, we examined the binding characteristics of this receptor to chromatin subfractions from homologous and heterologous tissues. Squalus chromatin linked to cellulose and partially deproteinized by 0-8 M guanidine hydrochloride (GuHCl) gave extraction patterns similar to those obtained with mammalian and avian chromatin. Chromatin as prepared in our laboratory contained no bound estrogen receptor. The binding pattern of the [3H]estradiol-labeled nuclear estrogen receptor to chromatin fractions from Squalus testicular zones I/II (containing spermatogonia, spermatocytes, and high receptor levels) revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 2-4 M GuHCl (350% increase over unextracted chromatin), with a 40% decrease from maximal binding at 5-8 M GuHCl. By contrast, binding to chromatin from zone III (containing spermatozoa and little or no detectable receptor) showed no major peak and 4 times less binding at 3 M GuHCl-extracted chromatin. We have previously shown that zones I and II contain the majority of testicular receptors and, presumably, are the primary sites of estrogen action, whereas receptor activity in zone III is minimal (less than 5%), indicating a secondary or nontarget tissue. Squalus testicular [3H]estradiol-receptor complexes bound minimally to rabbit uterine chromatin. Likewise, [3H]estradiol-receptor complexes from rabbit uterus, Squalus oviduct, or mouse testis bound minimally to Squalus testicular chromatin. Thus, maximal binding occurred only with Squalus zones I/II chromatin and Squalus testicular receptor. The binding of [3H]estradiol-receptor complexes to testicular chromatin (zones I/II) was of high affinity (Kd = 1.9 X 10(-10) M) and low capacity and was optimal in the presence of 150 mM KCl, but was unaffected by the addition of urea in an amount similar to that of Squalus body fluids (300 mM). [3H]Estradiol binding to chromatin required undenatured receptor and was competitively inhibited by radioinert estradiol-receptor complexes, confirming saturability of acceptor sites. The affinity of the Squalus estrogen receptor for homologous chromatin was in the same range as that reported for other systems, despite the unusual nuclear extractability characteristics of Squalus receptor. This study provides new evidence for tissue and species specificity of receptor binding to chromatin acceptor sites.

Retinoid X receptor homodimers function as transcriptional activators in yeast.

The possibility that different retinoids activate transcription from a specific retinoic acid (RA)-responsive element known as site A via different homo and heterodimeric versions of RA receptors cannot be evaluated in mammalian cells because they contain endogenous RA receptors (RAR). However, this limitation can be overcome by using yeast cells, which do not contain endogenous RAR, to study retinoid signaling pathways. Here, we describe heterologous expression of the human retinoid X receptor (RXR alpha) in yeast and hormone-dependent activation of a reporter construct containing site A upstream from a yeast promoter fused to the lacZ gene of Escherichia coli. Western blot analysis of yeast extracts containing RXR alpha revealed a distinct immunoreactive polypeptide co-migrating with the mammalian-produced RXR alpha. Electrophoretic mobility shift assays demonstrated that RXR alpha produced in yeast binds efficiently to site A in the absence of 9-cis-RA. However, transcription activation experiments showed that RXR alpha transactivates a yeast basal promoter linked to site A only in the presence of 9-cis-RA. We conclude that RXR alpha homodimers bind to site A in the absence of 9-cis-RA, but function as ligand-dependent transactivators in yeast cells. This retinoid-responsive transcription unit created in yeast cells provides a powerful genetic tool for the systemic unraveling of the synergistic interactions between RXR alpha and its heterodimeric partners.

Identification of amino acid residues required for a specific interaction between Src-tyrosine kinase and proline-rich region of phosphatidylinositol-3' kinase.

The binding of ligand to B-cell antigen receptors (BCR) leads to the activation of receptor-associated Src-family kinases and phosphatidylinositol-3' kinase (PI-3 kinase). Although it has been demonstrated that SH3 domains of several Src-family kinases interact with PI-3 kinase by binding to a proline-rich region of PI-3 kinase in vitro, there is no direct evidence to support their interaction in vivo. Thus, we utilized the yeast two-hybrid assay to reconstitute this protein-protein interaction. This genetic screen clearly indicates that the interaction between SH3 domain of Fyn and the proline-rich region (residues: 80-104) of PI-3 kinase is highly specific. Mutational analysis revealed that amino acid residues Asp92, Tyr93, Arg96 and Thr97 of the SH3 domain of Fyn are essential for interacting with the proline-rich peptide of PI-3 kinase.

Reconstitution of hepatitis C virus protease activities in yeast.

The hepatitis C virus (HCV) protease genes (NS2/3 and NS3) were expressed in yeast with their natural substrates fused to a ligand-dependent transcriptional activator, the retinoic acid receptor (RARbeta). RARbeta can activate transcription in yeast cells in response to retinoic acids. We hypothesized that cis-cleavage at the NS2-3 or NS3-4A junctions by the appropriate HCV proteases would release RARbeta, thereby activating transcription of a reporter gene. Our results from Western blot analyses and reporter gene activation indicate that the wild-type NS2/3 and NS3 enzymes are catalytically active in yeast cells, whereas mutations in the catalytic domain of NS2(C993V) and NS3(S1165A) lead to inactive enzymes. We conclude that HCV NS2/3 and NS3 protease activities can be reconstituted in yeast.

A yeast screen system for aromatase inhibitors and ligands for androgen receptor: yeast cells transformed with aromatase and androgen receptor.

Endocrine disruptors are hormone mimics that modify hormonal action in humans and animals. It is thought that some endocrine disruptors modify estrogen and androgen action in humans and animals by suppressing aromatase activity. Aromatase cytochrome P450 is the key enzyme that converts C19 androgens to aromatic C18 estrogenic steroids. We have developed a novel aromatase inhibitor screening method that allows us to identify antiaromatase activity of various environmental chemicals. The screen was developed by coexpressing the human aromatase and the mouse androgen receptor in yeast cells, which carry the androgen-responsive ss-galactosidase reporter plasmid. Functional expression of aromatase in yeast has been demonstrated using the [3H]-water release assay with intact cells as well as with yeast microsomes. The aromatase activity could be blocked by known aromatase inhibitors such as aminoglutethimide (AG). Yeast-produced androgen receptors were able to transactivate a yeast basal promoter linked to an androgen-responsive element in response to androgens. The resultant triple yeast transformant responded to the treatment of testosterone, androstenedione, or 5 alpha-dihydrotestosterone (5 alpha-DHT). In the absence of the aromatase inhibitor AG, transcriptional activation was observed only for the nonaromatizable androgen 5 alpha-DHT. However, the two aromatizable androgens (testosterone and androstenedione) induced the reporter activity in the presence of AG. Using this yeast-based assay, we confirmed that two flavones, chrysin and alpha-naphtholflavone, are inhibitors of aromatase. Thus, this yeast system allows us to develop a high-throughput screening method, without using radioactive substrate, to identify aromatase inhibitors as well as new ligands (nonaromatizable androgen mimics) for the androgen receptors. In addition, this screening method also allows us to distinguish nonandrogenic aromatase inhibitors from inhibitors with androgenic activity. This yeast screening method will be useful to screen environmental chemicals for their antiaromatase activity and for their interaction with androgen receptor.

Drosophila ecdysone receptor functions as a constitutive activator in yeast.

Transcriptional activation of the Drosophila ecdysone receptor (EcR) was studied in yeast cells, which carry a reporter plasmid containing the ecdysone response element in the absence or presence of its heterodimeric partners, ultraspiracle protein (USP) or human retinoid X receptor (RXRalpha). High constitutive transcriptional activation was detected in the yeast strain expressing EcR, but not USP or RXRalpha in the absence of ponasterone or muristerone A. Incubation of these ligands with yeast cells coexpressing EcR and USP or RXRalpha did not enhance the constitutive transcriptional activity. However, specific ligand binding using [3H]ponasterone A as a radioactive ligand was detected only in yeast extracts prepared from the yeast strain coexpressing EcR and USP, but not from yeast strains expressing only EcR or USP. The ligand binding characteristics of the EcR/USP complexes were similar to those reported in an insect cell line with a Kd value of 1.8 nM for [3H]ponasterone A. These data are in contrast to mammalian cell transfection studies, and indicate that the EcR is the only member of the nuclear receptor superfamily of ligand-activated transcription factors which functions as a constitutive transcriptional activator in yeast, although the EcR/USP complexes exhibit normal ligand binding properties.

Estrogen receptors in the turtle brain.

Estrogen binding activity in the CNS of the freshwater turtle, Chrysemys picta, was investigated using DNA-cellulose affinity chromatography. An estrogen binding component (EBC) with the characteristics of an estrogen receptor species was demonstrated in the brain cytosol extracts from both sexes. This EBC exhibited high affinity (K d = 10 -10 M), low capacity (n = 0.8 to 6.0 fmol/mg cytosol protein) and binding specificity. The bound estradiol-17 beta which adhered to DNA-cellulose was sensitive to excess synthetic and natural estrogens (DES, E2, E1 and E3) but not to progesterone and androgens (5 alpha- DHT and T). Specific estradiol-17 beta binding was not detected in plasma or non-target tissues such as lung, kidney and muscle. The topographic distribution of cytoplasmic EBC was similar in males and females, with binding highest in the hypothalamus-preoptic areas (HPOA) followed by the remaining forebrain (RFB). The mid/hindbrain (HB), consisting of the optic lobes, cerebellum and underlying brain stem had significantly lower concentrations of EBC. Monthly data (from May to October) suggest that variations in EBC concentrations occur during the year.

Effect of antimicrobial apomyoglobin 56-131 peptide on liposomes and planar lipid bilayer membrane.

The horse apomyoglobin 56-131 peptide is a convenient object for studies on the recently discovered antimicrobial activities of haem-binding protein fragments called haemocidins. The purpose of this study was to determine the effect of this peptide on planar lipid bilayer membranes and on liposomes of different lipid compositions. Micromolar concentrations of the apomyoglobin 56-131 fragment disrupt phosphatidylserine/phosphatidylethanolamine planar lipid bilayers without discrete conductance changes. The observed detergent-like action is dependent on peptide concentration; the lower amount of peptide resulted in longer bilayer lifetime. The cholesterol has an inhibitory effect on peptide-induced liposome lysis as shown by calcein release from liposomes. Additionally, there was considerable lytic activity on liposomes formed from anionic lipids of the sort found in bacterial membranes. Circular dichroism (CD) experiments showed that the peptide had a disordered structure in aqueous solutions and folds gradually to form helices in both membrane-mimetic trifluoroethanol solutions as well as in liposome suspensions. The features of the apomyoglobin 56-131 fragment that are similar to the cationic antimicrobial peptides acting in a 'carpet-like' manner are discussed.

Evidence for the presence of different alpha-1-proteinase inhibitor genes products in mouse plasma.

The plasma alpha-1-proteinase inhibitor (API) of three mouse species Mus domesticus, M. caroli and M. pahori was isolated. Each of the species isoforms were then separated by chromatofocusing; however, no significant differences in association rate constants toward human neutrophil elastase and bovine chymotrypsin were observed. The amino-acid sequence of the P'1-P'15 C-terminal fragments of the API variants indicate that mouse plasma contains at least two different active API isoforms in the case of M. domesticus (five API genes) but only one active API isoform in M. pahori and M. caroli (one API gene).

Antibacterial peptides of the moth Galleria mellonella.

The work describes purification and biochemical characterization of two inducible antimicrobial peptides from the hemolymph of Galleria mellonella. The peptides were isolated by a sequence of reversed-phase chromatography steps from the hemolymph of larvae immunized with viable bacteria. The first peptide is a member of the cecropin family while the second one is rich in proline residues and has a unique sequence.