High-affinity binding of bioactive glycosylation-inhibiting factor to antigen-primed T cells and natural killer cells

High-affinity binding was demonstrated between suppressor-T-cell-derived bioactive glycosylation-inhibiting factor (GIF) and helper T hybridomas and natural killer cell line cells. Inactive GIF present in cytosol of suppressor T cells and Escherichia coli-derived recombinant human GIF (rhGIF) failed to bind to these cells. However, affinity of rhGIF for the target cells was generated by replacement of Cys-57 in the sequence with Ala or of Asn-106 with Ser or binding of 5-thio-2-nitrobenzoic acid to Cys-60 in the molecule. Such mutations and the chemical modification of rhGIF synergistically increased the affinity of GIF molecules for the target cells. The results indicated that receptors on the target cells recognize conformational structures of bioactive GIF. Equilibrium dissociation constant (Kd) of the specific binding between bioactive rGIF derivatives and high-affinity receptors was 10–100 pM. Receptors for bioactive GIF derivatives were detected on Th1 and Th2 T helper clones and natural killer NK1.1+ cells in normal spleen but not on naive T or B cells. Neither the inactive rGIF nor bioactive rGIF derivatives bound to macrophage and monocyte lines or induced macrophages for tumor necrosis factor α production.

Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF

Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF/MIF molecules in human suppressor T (Ts) cell hybridomas. Peptide mapping and MS analysis of the affinity-purified GIF from the Ts cells revealed that one modification is cysteinylation at Cys-60, and the other is phosphorylation at Ser-91. Cysteinylated GIF, but not the wild-type GIF/MIF, possessed immunosuppressive effects on the in vitro IgE antibody response and had high affinity for GIF receptors on the T helper hybridoma cells. In vitro treatment of wild-type recombinant human GIF/MIF with cystine resulted in preferential cysteinylation of Cys-60 in the molecules. The cysteinylated recombinant human GIF and the Ts hybridoma-derived cysteinylated GIF were comparable both in the affinity for the receptors and in the immunosuppressive activity. Polyclonal antibodies specific for a stretch of the amino acid sequence in α2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wild-type GIF/MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF/MIF molecules are responsible for the generation of GIF bioactivity.

The crystal structure of human glycosylation-inhibiting factor is a trimeric barrel with three 6-stranded beta-sheets.

Glycosylation-inhibiting factor (GIF) is a cytokine that is involved in the regulation of IgE synthesis. The crystal structure of recombinant human GIF was determined by the multiple isomorphous replacement method. The structure was refined to an R factor of 0.168 at 1.9 angstrom resolution. The overall structure is seen to consist of three interconnected subunits forming a barrel with three 6-stranded beta-sheets on the inside and six alpha-helices on the outside. There is a 5-angstrom-diameter "hole" through the middle of the barrel. The barrel structure of GIF in part resembles other "trefoil" cytokines such as interleukin 1 and fibroblast growth factor. Each subunit has a new class of alpha + beta sandwich structure consisting of two beta-alpha-beta motifs. These beta-alpha-beta motifs are related by a pseudo-twofold axis and resemble both interleukin 8 and the peptide binding domain of major histocompatibility complex protein, although the topology of the polypeptide chain is quite different.

Association of the "major histocompatibility complex subregion" I-J determinant with bioactive glycosylation-inhibiting factor.

Murine suppressor T-cell hybridoma cells (231F1) secrete not only bioactive glycosylation-inhibiting factor (GIF) but also an inactive peptide comparable to bioactive GIF peptide in its molecular size and reactivity with anti-GIF; the amino acid sequence of the inactive peptide is identical to that of the bioactive homologue. The inactive GIF peptide in culture supernatant of both the 231F1 cells and a stable transfectant of human GIF cDNA in the murine suppressor T hybridoma selectively bound to Affi-Gel 10, whereas bioactive GIF peptides from the same sources failed to bind to the gel. The inactive cytosolic human GIF from the stable transfectant and Escherichia coli-derived recombinant human GIF also had affinity for Affi-Gel 10. Both the bioactive murine GIF peptide from the suppressor T hybridoma and bioactive recombinant human GIF from the stable transfectant bound to the anti-I-J monoclonal antibody H6 coupled to Affi-Gel. However, bioactive hGIF produced by a stable transfectant of human GIF cDNA in BMT10 cells failed to be retained in H6-coupled Affi-Gel. These results indicate that the I-J specificity is determined by the cell source of the GIF peptide and that the I-J determinant recognized by monoclonal antibody H6 does not represent a part of the primary amino acid sequence of GIF. It appears that the epitope is generated by a posttranslational modification of the peptide.

Diagnosis of 5α-reductase type 2 deficiency: Contribution of anti-Müllerian hormone evaluation

Aim: To evaluate anti-Müllerian hormone (AMH) levels in patients with clinical and molecular diagnosis of 5α-reductase 2 deficiency. Patients and methods: Data from 14 patients whose age ranged from 21 days to 29 years were analyzed according to age and pubertal stage. Sexual ambiguity was rated as Prader III in 11 patients. LH, FSH, testosterone (T), dihydrotestosterone (DHT) and AMH serum levels were measured in all but two patients, who had been previously submitted to gonadectomy; T and DHT were also measured in 20 age-matched controls. Results: Gonadotropin levels were normal in all but one patient who retained gonads (six of whom had reached puberty) and T/DHT ratio was elevated in all patients when compared to controls. All prepubertal patients had AMH levels < -1 SD for age, while most pubertal patients had AMH levels compatible with pubertal stage. Conclusions: Prepubertal patients with 5α-reductase 2 deficiency have AMH values in the lower part of the normal range. These data indicate that T does not need to be converted to DHT to inhibit AMH secretion by Sertoli cells. © Freund Publishing House Ltd., London.

Adjuvant therapy with GnRH agonists/tamoxifen in breast cancer should be a good council for patients with hormone receptor-positive tumours and wish to preserve fertility

Infertility represents one of the main long-term consequences of combination chemotherapy used for the treatment of breast cancer. Approximately 60%-65% of breast cancers express the nuclear hormone receptor in premenopausal women. Adjuvant endocrine therapy is an integral component of care for patients with hormone receptor-positive (HR+) tumours. The GnRH agonist (GnRHa) alone or in combination with tamoxifen produces results at least similar to those obtained with the different chemotherapy protocols in patients with HR+ tumors with respect to recurrence-free survival and overall survival, Presentation of the hypothesis: It is time to indicate adjuvant therapy with GnRHa associated with tamoxifen for patients with breast cancer (HR+ tumours) if they want to preserve their reproductive function. Testing the hypothesis: Assessment of ovarian reserve tests: follicle stimulating hormone (FSH), anti-Mullerian hormone (AMH), inhibin B, antral follicle count (AFC) and ovarian volume 6 months, and 1 year after the end of therapy with GnRHa/tamoxifen. The recurrence-free survival and overall survival should be analysed. Implications of the hypothesis: The major implication will be to avoid adjuvant chemotherapy for patients with breast cancer (HR+ tumours) that request fertility preservation. It is expected that ovarian function should not be altered in almost all cases. © Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida.

Índice preditor de reserva ovariana (ORPI) para controle de estimulação ovariana individualizado

Objective: to expand the evaluation of a new ovarian response prediction index (ORPI), which was based on the AMH, AFC and age, and to verify its reability as a predictor of ovarian response to stimulation in assisted reproductive technology (ART) cycles. Methods: A total of 129 patients enrolled in the ICSI programme were included. The ORPI values were calculated by multiplying the AMH level (ng/ml) by the number of antral follicles (2-9 mm), and the result was divided by the age (years) of the patient (ORPI=(AMH × AFC)/Patient age). Results: Spearman's test revealed significant correlations (P<0.0001) between the ORPI and the number of oocytes collected and the number of follicles. Logistic regression revealed that ORPI values were significantly associated with the likelihood of collecting ≥4 oocytes (OR=45.56), ≥4 MII oocytes (OR=6.01) and ≥15 oocytes (OR=6.15; P<0.0001). Based on the ROC curves, the ORPI accurately predicted a low ovarian response (<4 oocytes retrieved; area under the curve (AUC):0.91), collection of ≥4 MII oocytes (AUC:0.85) and an excessive ovarian response (≥15 oocytes retrieved; AUC:0.89). Conclusions: The ORPI exhibited an excellent ability to predict a low ovarian response and a good ability to predict a collection of ≥ 4 MII oocytes, an excessive ovarian response. The ORPI might be used to improve the cost-benefit ratio of ovarian stimulation regimens by guiding the selection of medications and by modulating the doses and regimens according to the actual needs of the patients. © Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida.

Associação genômica ampla para características reprodutivas em bovinos da raça Nelore

Pós-graduação em Medicina Veterinária - FCAV

Intermolecular interactions and characterization of the novel factor Xa exosite involved in macromolecular recognition and inhibition: Crystal structure of human Gla-domainless factor Xa complexed with the anticoagulant protein NAPc2 from the hematophagous nematode Ancylostoma caninum

NAPc2, an anticoagulant protein from the hematophagous nematode Ancylostoma caninum evaluated in phase-II/IIa clinical trials, inhibits the extrinsic blood coagulation pathway by a two step mechanism, initially interacting with the hitherto uncharacterized factor Xa exosite involved in macromolecular recognition and subsequently inhibiting factor VIIa (K-i = 8.4 pM) of the factor VIIa/tissue factor complex. NAPc2 is highly flexible, becoming partially ordered and undergoing significant structural changes in the C terminus upon binding to the factor Xa exosite. In the crystal structure of the ternary factor Xa/NAPc2/selectide complex, the binding interface consists of an intermolecular antiparallel beta-sheet formed by the segment of the polypeptide chain consisting of residues 74-80 of NAPc2 with the residues 86-93 of factor Xa that is additional maintained by contacts between the short helical segment (residues 67-73) and a turn (residues 26-29) of NAPc2 with the short C-terminal helix of factor Xa (residues 233-243). This exosite is physiologically highly relevant for the recognition and inhibition of factor X/Xa by macromolecular substrates and provides a structural motif for the development of a new class of inhibitors for the treatment of deep vein thrombosis and angioplasty. (c) 2006 Elsevier Ltd. All rights reserved.

Characterization and detection of naturally occurring antibodies against IL-1 alpha and IL-1 beta in normal human plasma.

During the development and testing of a radioreceptor assay (RRA) for human IL-1, we have detected and identified the presence of auto-antibodies to IL-1 in normal human plasma (NHP). The RRA is based on the competition between human 125I-labeled rIL-1 alpha and standard or unknown quantities of IL-1 alpha or IL-1 beta for binding to a limited amounts of IL-1 receptor (IL-1R) isolated from the EL4 mouse thymoma cell line. NHP from 20 out of 100 unselected blood donors were found to completely inhibit the binding of 125I-labeled IL-1 alpha to its receptor, suggesting the presence in these NHP samples of either abnormal amounts of IL-1 or of a factor binding to the 125I-labeled IL-1 alpha. Special care was taken to ascertain that the inhibitory factors were antibodies and not soluble IL-1 receptor antagonist. When plasma samples with inhibiting activity were incubated with labeled IL-1 alpha and chromatographed on a Sephadex G200 column, they were found to contain 125I-labeled complexes with an apparent molecular weight of 150-200kD. The IL-1 binding factor could be eliminated from plasma by incubation with protein A-Sepharose, suggesting that it consisted in IgG antibodies directed against IL-1. Furthermore, the antibody nature of the inhibiting factor was confirmed by its binding to purified rIL-1 coupled to Sepharose. Screening of 200 NHP samples by incubation with 100 pg of 125I-labeled IL-1 followed by precipitation with 12% of polyethylene glycol (PEG) confirmed that about 25% of NHP contain detectable IgG antibodies to IL-1 alpha, while only 2% of NHP contain antibodies to IL-1 beta. No correlation between the presence of these anti-IL-1 antibodies and any particular major histocompatibility complex or any pathological conditions was detected. We suggest that all serum samples assayed for IL-1 alpha or IL-1 beta content should be pretested with the PEG precipitation assay described here.

Role of the hypothalamic pituitary adrenal axis in the control of the response to stress and infection

The release of adrenocorticotropin (ACTH) from the corticotrophs is controlled principally by vasopressin and corticotropin-releasing hormone (CRH). Oxytocin may augment the release of ACTH under certain conditions, whereas atrial natriuretic peptide acts as a corticotropin release-inhibiting factor to inhibit ACTH release by direct action on the pituitary. Glucocorticoids act on their receptors within the hypothalamus and anterior pituitary gland to suppress the release of vasopressin and CRH and the release of ACTH in response to these neuropeptides. CRH neurons in the paraventricular nucleus also project to the cerebral cortex and subcortical regions and to the locus ceruleus (LC) in the brain stem. Cortical influences via the limbic system and possibly the LC augment CRH release during emotional stress, whereas peripheral input by pain and other sensory impulses to the LC causes stimulation of the noradrenergic neurons located there that project their axons to the CRH neurons stimulating them by alpha-adrenergic receptors. A muscarinic cholinergic receptor is interposed between the alpha-receptors and nitric oxidergic interneurons which release nitric oxide that activates CRH release by activation of cyclic guanosine monophosphate, cyclooxygenase, lipoxygenase and epoxygenase. Vasopressin release during stress may be similarly mediated. Vasopressin augments the release of CRH from the hypothalamus and also augments the action of CRH on the pituitary. CRH exerts a positive ultrashort loop feedback to stimulate its own release during stress, possibly by stimulating the LC noradrenergic neurons whose axons project to the paraventricular nucleus to augment the release of CRH.

Steinernema feltiae: ammonia triggers the emergence of their infective juveniles

Entomopathogenic nematodes complete their life cycles inside dead insects. The emergence of new infective juveniles from the cadaver has been attributed (but never demonstrated) to food depletion or to the accumulation of metabolites from the breakdown of the host's tissues. Here we give evidence that emergence is triggered by ammonia, a product of nematode defecation. We found that the emergence of Steinernema feltiae infective juveniles from Galleria mellonella cadavers was stimulated by a particular level of ammonia. Emergence was delayed when ammonia in the cadaver was decreased and was prompted when increased. These findings will further improve the understanding of the nematode life cycle. Here we speculate that production of infective juveniles can be mediated by ammonia and work in a manner analogous to that of the clatter recovery inhibiting factor (DRIF) in Caenorhabditis elegans. (C) 2008 Elsevier Inc. All rights reserved.

Computer-mediated communication in distance MA programmes: the student's perspective

This paper reports on a study of computer-mediated communication within the context of a distance MA in TEFL programme which used an e-mail discussion list and then a discussion board. The study focused on the computer/Internet access and skills of the target population and their CMC needs and wants. Data were collected from 63 questionnaires and 6 in-depth interviews with students. Findings indicate that computer use and access to the Internet are widespread within the target population. In addition, most respondents indicated some competence in Internet use. No single factor emerged as an overriding inhibiting factor for lack of personal use. There was limited use of the CMC tools provided on the course for student–student interaction, mainly attributable to time constraints. However, most respondents said that they would like more CMC interaction with tutors. The main factor which would contribute to greater Internet use was training. The paper concludes with recommendations and suggestions for learner training in this area.

Low cost technology applied to thermometry of grains

Modern agriculture demands investments in technology that allows the farmers to improve productivity and quality of their products, aiming to establish themselves in a competitive market. However, the high costs of acquiring and maintaining such technology may be an inhibiting factor to its spread and acceptance, mainly to a large number of small grain Brazilian farmers, who need low cost innovative technological solutions, suitable for their financial reality. Starting from this premise, this paper presents the development of a low cost prototype for monitoring the temperature and humidity of grains stored in silos, and the economic implications of cost/benefit ratio of innovative applications of low cost technology in the process of thermometry of grains. The prototype was made of two electronic units, one for acquisition and another one for data reception, as well as software, which offered the farmers more precise information for the control of aeration. The data communication between the electronic units and the software was reliable and both were developed using low cost electronic components and free software tools. The developed system was considered as potentially viable to small grain Brazilian farmers; it can be used in any type of small silos. It provided reduction of costs of installation and maintenance and also offered an easy expansion system; besides the low cost of development when compared to similar products available in the Brazilian market.

Genetische Analyse des Somatostatin-Systems

Somatostatin ist ein Molekül mit multifunktinonellem Charakter, dem Neurotransmitter-, Neuromodulator- und (Neuro)-Hormoneigenschaften zugeschrieben werden. Gemäß seiner ubiquitären Verteilung in Geweben beeinflusst es Stoffwechsel- und Entwicklungsprozesse, bis hin zu Lern-und Gedächtnisleistungen. Diese Wirkungen resultieren aus dem lokalen und zeitlichen Zusammenspiel eines Liganden und fünf G-Protein gekoppelter Rezeptoren (SSTR1-5). Zur Charakterisierung der biologischen Bedeutung des Somatostatin-Systems im Gesamtorganismus wurde eine Mutationsanalyse einzelner Systemkomponenten durchgeführt. Sie umfaßte die Inaktivierung der Gene für das Somatostatin-Präpropeptid und die der Rezeptoren SSTR3 und SSTR4 durch Gene Targeting. Die entsprechenden Ausfallmutationen belegen: Weder die Rezeptoren 3 und 4, noch Somatostatin sind für das Überleben des Organismus unter Standardhaltungsbedingungen notwendig. Die entsprechenden Mauslinien zeigen keine unmittelbar auffälligen Einschränkungen ihrer Biologie. Die Somatostatin-Nullmaus wurde zum Hauptgegenstand einer detaillierten Untersuchung aufgrund der übergeordneten Position des Liganden in der Signalkaskade und verfügbaren Hinweisen zu seiner Funktion. Folgende Schlußfolgerungen konnten nach eingehender Analyse gezogen werden: Der Ausfall des Somatostatin-Gens hat erhöhte Plasmakonzentrationen an Wachstumshormon (GH) zur Konsequenz. Dies steht im Einklang mit der Rolle Somatostatins als hemmender Faktor der Wachstumshormon-Freisetzung, die in der Mutante aufgehoben ist. Durch die Somatostatin-Nullmaus wurde zudem deutlich: Somatostatin interagiert als wesentliches Bindeglied zwischen der Wachstums- und Streßachse. Permanent erhöhte Corticosteron-Werte in den Mutanten implizieren einen negativen tonischen Einfluß für die Sekretion von Glukocorticoiden in vivo. Damit zeigt die Knockout-Maus, daß Somatostatin normalerweise als ein entscheidendes inhibierendes Kontrollelement der Steroidfreisetzung fungiert. Verhaltensversuche offenbarten ein Defizit im motorischen Lernen. Somatostatin-Nullmäuse bleiben im Lernparadigma “Rotierender Stabtest” hinter ihren Artgenossen zurück ohne aber generell in Motorik oder Koordination eingeschränkt zu sein. Diese motorischen Lernvorgänge sind von einem funktionierenden Kleinhirn abhängig. Da Somatostatin und seine Rezeptoren kaum im adulten, wohl aber im sich entwickelnden Kleinhirn auftreten, belegt dieses Ergebnis die Funktion transient in der Entwicklung exprimierter Neuropeptide – eine lang bestehende, aber bislang experimentell nicht nachgewiesene Hypothese. Die Überprüfung weiterer physiologischer Parameter und Verhaltenskategorien unter Standard-Laborbedingunggen ergab keine sichtbaren Abweichungen im Vergleich zu Wildtyp-Mäusen. Damit steht nun ein Tiermodell zur weiterführenden Analyse für die Somatostatin-Forschung bereit: In endokrinologischen, elektrophysiologischen und verhaltens-biologischen Experimenten ist nun eine unmittelbare Korrelation selektiv mit dem Somatostatin-Peptid bzw. mit den Rezeptoren 3 und 4 aber auch in Kombination der Ausfallmutationen nach entsprechenden Kreuzungen möglich.