Purification and physicochemical characterization of a cotyledonary lectin from Luetzelburgia auriculata

A lectin was purified from the cotyledons of Luetzelburgia auriculata (Fr. All) Ducke by affinity chromatography on agarose-N-acetyl-D-galactosamine. The lectin is a potent agglutinin for rabbit erythrocytes, reacts with human red cells, but is inactive against cow, sheep, and goat erythrocytes. Hemagglutination of rabbit erythrocytes was inhibited by either 0.39 mM N-acetyl-neuraminic acid or N-acetyl-D-galactosamin, 12.5 mM D-lactose or D-melibiose, 50 mM D-galactose or raffinose. Its hemagglutinating activity was lost at 80 degrees C, 5 min, and the activation energy required for denaturation was 104.75 kJ mol(-1). Chromatography on Sephadex G-100, at pH 7.6, showed that at this hydrogenic ionic concentration the native lectin was a homotetramer (123.5 kDa). By denaturing SDS-PAGE, LAA seemed to be composed of a mixture of 29 and 15 kDa polypeptide subunits. At acidic and basic pHs it assumed different conformations, as demonstrated by exclusion chromatography on Superdex 200 HR 10/30. The N-terminal sequence of the 29 kDa band was SEVVSFSFTKFNPNQKDII and the 15 kDa band contained a mixture of SEVVSFSFTKFNPNQKDII and KFNQIVAVEEDTDXESQPQ sequences, indicating that these bands may represent full-length and its endogenous fragments, respectively. The lectin is a glycoprotein having 3.2% neutral carbohydrate, with a pI of 5.8, containing high levels of Asp+Asn and Glu+Gln and hydroxy amino acids, and low amount or absence of sulfur amino acids. Its absorption spectrum showed a maximum at 280 nm and a epsilon (1%) x (1cm) of 5.2. Its CD spectrum was characterized by minima near 228 nm, maxima near 196 nm and a negative to positive crossover at 210 nm. The secondary structure content was 6% alpha-helix, 8% parallel beta-sheet, 38% antiparallel beta-sheet, 17% beta-turn, 31% unordered and others contribution, and 1% RMS (root mean square). In the fluorescence spectroscopy, excitation of the lectin solution at 280 nm gave an emission spectrum in the 285-445 nm range. The wavelength maximum emission was in 334.5 nm, typical for tryptophan residues buried inside the protein.     

Effect of the contents and form of rabies glycoprotein on the potency of rabies vaccination in cattle

One of the methods used for controlling cattle rabies in Brazil consists of vaccination. Sometimes, however, rabies occurs in cattle supposedly protected. Since rabies vaccine batches are officially controlled by tests performed on laboratory animals, it is questionable whether the minimal mandatory requirements really correspond to immunogenicity in the target species. We have analyzed the association among potencies of rabies vaccines tested by the NIH test, the contents and form (free-soluble or virus-attached) of rabies glycoprotein (G) in the vaccine batches, and the virus-neutralizing antibodies (VNA) titers elicited in cattle. No correlation was found between G contents in the vaccine batches and the NIH values, whatever the presentation of G. There was no correlation either between NIH values and VNA titers elicited in cattle. There was, however, a positive correlation (r = 0.8681; p = 0.0001) between the amounts of virion-attached G present in the vaccine batches and VNA elicited in cattle. This was not observed when the same analysis was performed with total-glycoprotein or free-soluble glycoprotein. The study demonstrated that NIH values can not predict the effect of the immunogen in cattle. On the other hand, the quantification of virus-attached rabies glycoprotein has a strong correlation with VNA elicited in cattle.     

Vasodilator effect of angiotensin-(1-7) in mature and sponge-induced neovasculature

Angiotensin-(1-7) (Ang-(1-7)), a peptide constituent of the renin-angiotensin system, has been shown to act as a vasodilator mediator in pre-existing (skin) and newly formed vasculatures (14-day-old sponge implants). Blood flow was determined by the outflow rate of sodium fluorescein applied intradermally or intraimplant and the results were expressed in t(1/2) values (time taken for the fluorescence to reach 50% of the peak in the systemic circulation). We showed that the t(1/2) value was significantly lower (4.1+/-0.46) in the implants compared with the cutaneous vasculature (5.7+/-0.5). Ang-(1-7) 20 ng was able to decrease t(1/2) values in both vasculatures. The specific receptor antagonist, D-Ala7-Ang-(1-7) (A-779), prevented Ang-(1-7)-induced vasodilation and altered the basal vascular tone of the implants. The vasodilator effect was also abolished by nitric oxide (NO) synthase inhibitors in both vasculatures and by indomethacin in the implant. Selective AT(1) and AT(2) receptor antagonists did not alter the vasodilation induced by the peptide. These results establish the vasodilator effect of Ang-(1-7) in the cutaneous and implant vasculature and that the peptide is produced endogenously by the fibrovascular tissue, and suggest that this peptide contributes for the vasodilation found in newly formed vascular beds (wound healing, chronic inflammatory processes and tumors).     

Secretory trichomes,a substitutive floral nectar source in Lundia A. DC. (Bignoniaceae), a genus lacking a functional disc

This is the first report of corolla-borne secretory trichomes that substitute in role for a non-functional disc in a species of the neotropical genus Lundia A. DC. (Bignoniaceae). The floral biology and flowering phenology of Lundia cordata were investigated at two remnants of tropical rainforest in northeastern Brazil. This species is a typically omithophilous liana, with reddish, tubular, scentless flowers. The flowers are resupinate, protandrous and last for 2 d. There is a vestigial non-functional perigynous disc and nectar is secreted by glandular trichomes distributed along the internal surface of the corolla. The nectar is stored at the base of the corolla tube, thus showing secondary nectar presentation. The nectariferous trichomes are multi-cellular, uniseriate, with a basal foot cell rooting in the epidermis, one neck cell, and a glandular head with 13 cells on average. Three species of hummingbirds (Amazilia fimbriata, Eupetomena macroura and Phaethornis pretrei) serve as pollinators. Phaethornis ruber, Xylocopa bees, wasps and diurnal moths are considered nectar thieves.     

Effect of prenatal stress on the hormonal response to acute and chronic stress and on immune parameters in the offspring

The effect of prenatal stress on the time course of the corticosterone response to acute and chronic stress and on hematological and immunological parameters in the offspring were analized in the present study. Pregnant Sprague-Dawley rats were stressed daily for 2 hours during the last week of gestation, and female and male off-spring were studied during adulthood. Corticosterone response to acute immobilization stress was not significantly different in either control or prenatally stressed rats. However, after 10 days of immobilization stress the corticosterone response completely disappeared in the control animals but not in the prenatally stressed group: high levels of corticosterone were found during the first hour of stress, although they were lower than those found in acutely stressed rats. Adrenal hypertrophy in response to prenatal stress was observed in females but not in male offspring, and chronic stress only increased adrenal weights in the male control group. Prenatal stress decreased the total peripheral leukocyte count, altered its diferential count decreasing lymphocytes and increasing neutrophil and eosinhophil counts, and significantly reduced the percentage of peripheral lymphocyte T CD8+ subset in male offspring. Chronic stress also reduced the percentage of the peripheral T CD8+ lymphocyte subset in the control group but not in the prenatally stressed group. These results suggest that the exposure to stress during pregnancy alters the adaptative response of the hypothalamus-pituitary-adrenocortical axis to chronic stress and presumably the immune competence in the offspring.     

13C-urea breath test to diagnose Helicobacter pylori infection in children aged up to 6 years

BACKGROUND: 13C-urea breath test (13C-UBT) is an accurate noninvasive tool for diagnosis of Helicobacter pylori infection. It is considered the best method for epidemiological studies, but there are few studies to evaluate the 13C-UBT in infants and toddlers. AIM: To evaluate the 13C-UBT performed with infrared spectroscopy in children aged up to 6 years. PATIENTS: Sixty-eight patients (6 months. to 5 years 11 months.) were evaluated prospectively and consecutively. METHODS: Helicobacter pylori infection was detected by positive culture, or rapid urease test and histological examination, both positive. 13C-UBT was performed with 50 mg of 13C-urea diluted in 100 ml of commercial orange juice. Two expired air samples were collected: before and 30 minutes after tracer ingestion. Cutoff of delta over baseline (DOB) was 4.0 per thousand and urea hydrolysis rate 10 microg/minute. RESULTS: Fifteen of 68 (22.1%) patients were H. pylori infected. Sensitivity was 93.3% (95% CI; 86.8%-99.7%) and specificity was 96.2% (95% CI; 93.6%-98.8%), and these values were equal for DOB and urea hydrolysis rate. Negative DOB values in noninfected patients ranged from -1.5 per thousand to 2.6 per thousand and positive DOB values ranged from 10.8 per thousand to 105.5 per thousand. There was no relationship between DOB values and age. Conclusion. 13C-UBT performed with infrared spectroscopy proved to be a reliable and accurate noninvasive diagnostic tool for H. pylori infection detection in children aged up to 6 years. Results far from cutoff value can clearly distinguish positive from negative 13C-UBT results in children up to 6 years old.     

Phenotypic evaluation to differentiate Candida albicans from Candida dubliniensis

This study evaluated the phenotypic tests used to differentiate Candida albicans from Candida dubliniensis. A total of 55 isolates from vaginal secretions, oral cavity and hemoculture were studied. They were originally identified as C. albicans, based on their morphological and physiological characteristics. These isolates were tested for colony color development on CHROMagar Candida medium, growth at 45 degrees C on Sabouraud Dextrose agar, lipolytic activity on Tween 80 Agar medium and colony morphology and chlamydoconidia formation on Staib agar medium. Of the 55 isolates studied, seven yielded one or more phenotypic characteristics suggestive of Candida dubliniensis. These isolates were tested by PCR with specific primers for Candida dubliniensis and API ID 32. The seven isolates were confirmed as Candida albicans. All of these finding indicate that DNA based tests should be used for definitive identification of Candida dubliniensis.     

Role of hydration in the closed-to-open transition involved in Ca2+ binding by troponin C

Troponin C (TnC) is the Ca(2+)-binding subunit of the troponin complex of vertebrate skeletal muscle. It consists of two structurally homologous domains, N and C, connected by an exposed alpha-helix. The C-domain has two high-affinity sites for Ca(2+) that also bind Mg(2+), whereas the N-domain has two low-affinity sites for Ca(2+). Previous studies using isolated N- and C-domains showed that the C-domain apo form was less stable than the N-domain. Here we analyzed the stability of isolated N-domain (F29W/N-domain) against urea and pressure denaturation in the absence and in the presence of glycerol using fluorescence spectroscopy. Increasing the glycerol concentration promoted an increase in the stability of the protein to urea (0-8 M) in the absence of Ca(2+). Furthermore, the ability to expose hydrophobic surfaces normally promoted by Ca(2+) binding or low temperature under pressure was partially lost in the presence of 20% (v/v) glycerol. Glycerol also led to a decrease in the Ca(2+) affinity of the N-domain in solution. From the ln K(obs) versus ln a(H)2(O), we obtained the number of water molecules (63.5 +/- 8.7) involved in the transition N <=>N:Ca(2) that corresponds to an increase in the exposed surface area of 571.5 +/- 78.3 A(2). In skinned fibers, the affinity for Ca(2+) was also reduced by glycerol, although the effect was much less pronounced than in solution. Our results demonstrate quantitatively that the stability of this protein and its affinity for Ca(2+) are critically dependent on protein hydration.     

Observing bacterial activity interferometrically

It is shown that bacterial activity, even of slowly growing species, can be detected by precise interferometric measurements of refractive index changes of the culture medium. The bacteria-containing sample is kept in an isothermal block together with a reference liquid without bacteria. The biological activity is obtained from the difference of the index changes of these samples. Experiments were performed with Bacilo Calmette-Guérin. The order of magnitude of the observed total refractive index change was compatible with theoretical estimates based on the amount of available oxygen. An unexpected positive index change during the lag phase was observed, which might permit fast diagnostics in medical applications. This technique may provide cheap and quick tests of bacterial susceptibility with respect to antibiotics.