Immunomodulatory activity of ß-glucan-containing exopolysaccharides from Auricularia auricular in phagocytes and mice infected with Cryptococcus neoformans.

Current antifungal drugs present poor effectiveness and there is no available vaccine for fungal infections. Thus, novel strategies to treat or prevent invasive mycosis, such as cryptococcosis, are highly desirable. One strategy is the use of immunomodulators of polysaccharide nature isolated from mushrooms. The purpose of the present work was to evaluate the immunostimulatory activity of ß-(1,3)-glucan-containing exopolysaccharides (EPS) from the edible mushrooms Auricularia auricula in phagocytes and mice infected with Cryptococcus neoformans. EPS triggered macrophages and dendritic cell activation upon binding to Dectin-1, a pattern recognition receptor of the C-type lectin receptor family. Engagement of Dectin-1 culminated in pro-inflammatory cytokine production and cell maturation via its canonical Syk-dependent pathway signaling. Furthermore, upon EPS treatment, M2-like phenotype macrophages, known to support intracellular survival and replication of C. neoformans, repolarize to M1 macrophage pattern associated with enhanced production of the microbicidal molecule nitric oxide that results in efficient killing of C. neoformans. Treatment with EPS also upregulated transcript levels of genes encoding products associated with host protection against C. neoformans and Dectin-1 mediated signaling in macrophages. Finally, orally administrated ß-glucan-containing EPS from A. auricular enhanced the survival of mice infected with C. neoformans. In conclusion, the results demonstrate that EPS from A. auricula exert immunostimulatory activity in phagocytes and induce host protection against C. neoformans, suggesting that polysaccharides from this mushroom may be promising as an adjuvant for vaccines or antifungal therapy.

In vitro activity of essential oils of free and nanostructured Melaleuca alternifolia and of terpinen-4-ol on eggs and larvae of Haemonchus contortus.

Haemonchus contortus is one of the major gastrointestinal nematodes responsible for significant economic and production losses of sheep. Diseases caused by this species lack effective anthelmintic products, and the search for new compounds to replace synthetic anthelmintics has been extensive. The present investigation assesses the in vitro activity of the essential oil of melaleuca (Melaleuca alternifolia), both free (TTO) and nanostructured (nanoTTO), and terpinen-4-ol (terp-4-ol) on eggs and larvae of H. contortus. Tests of egg hatching (EHT) and inhibition of larval migration (LMIT) were used to assess the in vitro efficacy of TTO, nanoTTO and terp-4-ol. Using EHT, at a concentration of 3.5 mg/ml, 100% inhibition occurred using TTO and terp-4-ol, with LC50 values of 0.43 and 0.63 mg/ml, and LC90 values of 1.75 mg/ml and 3.12 mg/ml, respectively. NanoTTO had lower activity, with 82.6% inhibition at the same concentration. Using LMIT, TTO and nanoTTO had a similar activity with 88.0% and 84.8% inhibition, respectively, at a concentration of 56 mg/ml. Terp-4-ol had a greater effect on larvae, with 85.7% inhibition at a concentration of 56 mg/ml and 82.4% at 3.5 mg/ml, demonstrating high activity at the lowest concentration tested. Therefore, the results indicate that all substances tested showed ovicidal and larvicidal activity against H. contortus. TTO, terp-4-ol and, mainly, nanoTTO may be targeted in in vivo studies, besides being a promising line of research into the control and treatment of veterinary important helminths.

Selection of optimized candidate reference genes for qRT-PCR normalization in rice (Oryza sativa L.) during Magnaporthe oryzae infection and drought.

Drought and rice blast disease caused by Magnaporthe oryzae are two of the most serious threats to global rice production. To explore the mechanisms underlying gene expression induced in rice by stresses, studies involving transcriptome analyses have been conducted over the past few years. Thus, it is crucial to have a reliable set of reference genes to normalize the expression levels of rice genes affected by different stresses. To identify potential reference genes for studies of the differential expression of target genes in rice under M. oryzae infection and drought conditions, the present study evaluated five housekeeping genes for the normalization of gene expression. The stability of the expression of these genes was assessed using the analytical software packages geNorm and NormFinder. For all samples analyzed, the stability rank was UBQ5 > GAPDH > eIF-4α> ß-TUB > 18S rRNA. The data showed that the UBQ5, GAPDH, and eIF-4αgenes are appropriate, high-performing reference genes and will be highly useful in future expression studies of fungal infections and drought in rice.

Effect of intracervical anesthesia on pain associated with the insertion of the levonorgestrel-releasing intrauterine system in women without previous vaginal delivery: a RCT.

STUDY QUESTION: Is the pain associated with levonorgestrel-releasing intrauterine system (LNG-IUS) insertion reduced by intracervical anesthesia in women without previous vaginal birth? SUMMARY ANSWER: Intracervical anesthesia was not associated with reduced pain in women without previous vaginal birth. WHAT IS KNOWN ALREADY: The pain associated with the insertion of intrauterine contraceptives (IUCs) is a limiting factor for the use of these contraceptives by some women. No prophylactic pharmacological intervention has proven efficacy in relieving pain during or after the insertion of IUCs. However, previous studies included women with previous vaginal delivery, and injectable intracervical anesthesia was not evaluated in any of these studies. STUDY DESIGN, SIZE, DURATION: This was a randomized, open, parallel-group clinical trial that evaluated 100 women without previous vaginal delivery who wished to use the LNG-IUS for the first time. These women were evaluated immediately after LNG-IUS insertion and then 2 h and 6 h later. PARTICIPANTS/MATERIALS, SETTING, METHODS: The 100 women were randomized into two groups: (i) use of a non-steroidal anti-inflammatory drug (NSAID) (ibuprofen, 400 mg) 1 h prior to LNG-IUS insertion; or (ii) 2% lidocaine intracervical injection 5 min prior to LNG-IUS insertion. The women were evaluated immediately after LNG-IUS insertion and then 2 h and 6 h after insertion. Two pain scales were used (the visual analogue scale and the facial pain scale) in addition to assessing the ease of insertion (as rated by the provider) and the level of discomfort during the procedure (as rated by the patient). Multivariate logistic regression was performed to analyze the predictors associated with moderate/severe pain. MAIN RESULTS AND THE ROLE OF CHANCE: The pain and discomfort associated with LNG-IUS insertion, and the ease of insertion of the LNG-IUS did not differ between the groups. Nulliparity was more associated with moderate/severe pain [adjusted odds ratio (OR): 3.1 (95% confidence interval (CI): 1.3-7.80]. Injectable intracervical anesthesia use reduced the risk of moderate/severe pain by 40% [adjusted OR: 0.6 (95% CI: 0.2-1.4)]. The difference between the mean pain score in the intracervical anesthesia group and the NSAID group was <10%; thus, the effect size of the intervention was not significant. LIMITATIONS, REASONS FOR CAUTION: Intracervical anesthesia was compared with an oral medication in this study. Intracervical injection of a saline solution or even a dry needling as the placebo for a double-blind study could be a more adequate control; however, this approach was not a protocol approved by the institutional review board. Considering that the majority of the insertions were easy (>80% in both groups), the results may not be extrapolated to difficult insertions with moderate/severe pain where local anesthesia may have a role. WIDER IMPLICATIONS OF THE FINDINGS: The findings can be generalized to most insertions in nulliparous women or in those without a previous vaginal delivery. There is currently no evidence to recommend the routine use of prophylactic intracervical anesthesia prior to LNG-IUS insertion; there is no evidence that this treatment reduces insertion-related pain. STUDY FUNDING/COMPETING INTERESTS: RAF and CSV give occasional lectures for Bayer Healthcare. This study received funding from the National Institute of Hormones and Women's Health, National Council for Scientific and Technological Development (CNPq). TRIAL REGISTRATION NUMBER: NCT02155166.

Molecular cloning and characterization of an α-amylase cDNA highly expressed in major feeding stages of the coffee berry borer, Hypothenemus hampei.

α-Amylases are common enzymes responsible for hydrolyzing starch. Insect-pests, whose larvae develop in seeds, rely obligatorily on α-amylase activity to digest starch, as their major food source. Considering the relevance of insect α-amylases and the natural α-amylase inhibitors present in seeds to protect from insect damage, we report here the molecular cloning and nucleotide sequence of the full-length AmyHha cDNA of the coffee berry borer, Hypothenemus hampei, a major insect-pest of coffee crops. The AmyHha sequence has 1879 bp, containing a 1458 bp open reading frame, which encodes a predicted protein with 485 amino acid residues, with a predicted molecular mass of 51.2 kDa. The deduced protein showed 55-79% identity to other insect α-amylases, including Anthonomus grandis, Ips typographus and Sitophilus oryzae α-amylases. In depth analysis revealed that the highly conserved three amino acid residues (Asp184, Glu220, and Asp285), which compose the catalytic site are also presented in AmyHha amylase. The AmyHha gene seems to be a single copy in the haploid genome and AmyHha transcription levels were found higher in L2 larvae and adult insects, both corresponding to major feeding phases. Modeling of the AmyHha predicted protein uncovered striking structural similarities to the Tenebrio molitor α-amylase also displaying the same amino acid residues involved in enzyme catalysis (Asp184, Glu220 and Asp285). Since AmyHha gene was mostly transcribed in the intestinal tract of H. hampei larvae, the cognate α-amylase could be considered a high valuable target to coffee bean insect control by biotechnological strategies.

Exposing bovine cumulus-oocyte complexes to aromatizable androgen restore serum-induced low estradiol production in vitro.

We aimed in this study to assess whether serum-decreased bovine cumulus-oocyte complex (COC) steroidogenesis during in vitro maturation (IVM) is caused by deficient androgen milieu. For this approach, bovine COCs were cultured in serum-supplemented IVM medium in the presence or absence of 1 µM androstenedione. After 24 h of culture, medium was collected and analyzed for its content of estradiol-17ß (E2) and progesterone (P4). Medium E2 content markedly increased after incubation of COCs with androstenedione (17.52 ± 1.86 ng/ml to the androgen group; 0.32 ± 0.05 ng/ml to the non-androgen group). No significant difference in the P4 content was detected despite the presence of androstenedione (21.83 ± 1.61 ng/ml to the androgen group; 21.73 ± 1.67 ng/ml to the non-androgen group). Our data provide compelling evidence that bovine COCs steroidogenesis remains functional during culture in serum-supplemented medium and suggest that serum-induced decreased COCs estradiol secretion is caused by deficiency of an aromatizable androgen source.

Morpho-anatomical characterization of mature embryo-derived callus of rice (Oryza sativa L.) suitable for transformation.

The objective of this study was to morpho-anatomically characterize embryogenic rice calli during early induction of somatic embryogenesis of three Brazilian rice cultivars. Herein, we explored embryogenic units (EUs) from 2-week-old cut proliferated calli to verify whether they were suitable for Agrobacterium tumefasciens-mediated transformation. Histological analysis and scanning electron microscopy (SEM) were used to analyze these types of calli during early rice callogenesis in the cultivars BRS Primavera, BRS Bonança, and BRS Caiapó. The characteristics of the embryogenic cells were preserved in the EUs, which showed a globular, compact structure that contained tightly packed cells and thus rendered the cells suitable for transformation. The EUs of BRS Caiapó also maintained the characteristics of the non-embryogenic callus, such as an elongated morphology and a lack of cellular organization. In general, the observations of the histological sections corresponded with those of the SEM images. The histological analysis suggested that all cultivars used in these experiments have morphogenic potential. The EUs from proliferated 2-week-old cut calli maintained their embryogenic features. The EUs were subjected to Agrobacterium-mediated transformation, which exhibited a regeneration frequency of 58 % for transformed hygromycin-resistant cell lines. These results show that EUs from proliferated 2-week-old cut calli are suitable for plant transformation.

Preventing Microbial Contamination during Long-Term In Vitro Culture of Human Granulosa-Lutein Cells: An Ultrastructural Analysis.

Purpose. To investigate whether the addition of antibiotic/antimycotic during human granulosa-lutein cells (GLCs) isolation and cell-plating procedures prevents microbial contamination after 144 h of culture and also evaluate the effects of contamination on GLCs ultrastructure and steroid secretion. Methods. GLCs obtained from five women submitted to assisted reproductive techniques (ARTs) were isolated with PBS supplemented with antibiotic/antimycotic or PBS nonsupplemented and cultured for 144 h. GLCs were evaluated by transmission electron microscopy (TEM), and estradiol (E2) and progesterone (P4) secretion was assayed by chemiluminescence. Results. Although no contaminating microorganisms were identified by light microscopy, TEM analyses revealed several bacterial colonies in culture dishes of GLCs isolated with only PBS. Bacterial contamination disrupted the adherence of the GLCs to the culture plate interfering with monolayer formation affecting the growth pattern of GLCs. Various cellular debris and bacteria were observed, and no organelles were found in the cytoplasm of infected cells. While bacterial contamination decreased estradiol media levels, it increased progesterone, as compared with noncontaminated group. Conclusion. Taken together, our data showed that the addition of a high dose of antibiotic/antimycotic during the isolation and cell-plating procedures prevents microbial contamination of long-term GLCs culture as its effects on cells growth and function in vitro.

VIP expression in vessels of the vaginal wall: relationship with estrogen status.

OBJECTIVE: Vasoactive intestinal peptide (VIP) is a neuropeptide with elevated expression in regions that control urogenital functions. Estrogen appears to modulate VIP expression in various organs, but this effect has not been demonstrated in the vaginal wall. The aim of this study was to evaluate the influence of estrogen status on VIP expression in vessels of the vaginal wall. METHODS: Surgical specimens were removed from the vaginal walls of 18 premenopausal women and 12 postmenopausal women who were given surgery for genital prolapse grade I or II. Vaginal specimens were stained with estrogen receptor-alpha (ER-α) and VIP antibodies. Levels of follicle stimulating hormone (FSH), estradiol, prolactin, fasting glucose and serum thyroxine stimulating hormone were also measured. Estrogen status was assessed on the basis of FSH and ER-α scores. RESULTS: The vaginal walls of premenopausal women had significantly higher ER-α scores than those of menopausal women (premenopausal group, 3.6 ± 2.2; menopausal group, 1.4 ± 1.8; p = 0.01). Premenopausal women also had significantly higher levels of VIP in the vaginal wall than menopausal women (p = 0.02). Increasing age was associated with lower level of VIP staining (odds ratio 0.88; 95% confidence interval 0.78-0.99). CONCLUSION: Levels of ER-α and VIP expression in the posterior vaginal wall were higher in premenopausal than in menopausal women, but VIP expression was not associated with estrogen status. Age was an independent predictor of VIP staining in vaginal wall biopsies.

Anti-müllerian hormone is the best predictor of poor response in ICSI cycles of patients with endometriosis.

PURPOSE: To correlate ovarian reserve (OR) markers with response in assisted reproduction techniques (ART) and determine their ability to predict poor response among patients with endometriosis (EDT). METHODS: We evaluated ART cycles of 27 women with EDT and 50 with exclusive male factor. Basal follicle stimulating hormone (FSH) and anti-müllerian hormone (AMH) levels were determined. Ovarian response to gonadotropin stimulation was assessed and correlation coefficients calculated between the variables and reserve markers. Areas under the curve (AUC) determined ability of tests to predict poor response. RESULTS: AMH was significantly correlated with response in both groups and it was the only marker with significant discriminative capacity to predict poor response among EDT (AUC = 0.842; 95% CI: 0.651-0.952) and control group (AUC = 0.869; 95% CI: 0.743-0.947). CONCLUSION: Infertile patients with endometriosis can benefit from the pre-therapeutic assessment of OR markers. However, regardless of disease presence, only AMH predicts poor response to stimulus.

In Vivo Effects of Cagaita (Eugenia dysenterica, DC.) Leaf Extracts on Diarrhea Treatment.

Eugenia dysenterica is a plant typically found in the Cerrado biome and commonly used in popular medicine due to its pharmacological properties, which include antidiarrheal, skin healing, and antimicrobial activities. The effects of ethanolic extract, aqueous extract and infusion of E. dysenterica leaves on intestinal motility and antidiarrheal activity were evaluated using ricin oil-induced diarrhea in rats. At doses of 400 and 800 mg·Kg(-1), the ethanolic extract decreased intestinal motility while the other extracts showed no significant effects. Moreover, serum levels of chloride, magnesium, and phosphorus were also measured in rats. Histopathologic and enzymatic analyses were also performed to investigate any toxic effect. Animals treated with infusion, ethanolic extract, ricin oil, and loperamide presented morphological alterations in the small intestine, such as mucosa lesion, epithelial layer damage, and partial loss and/or morphological change of villi. Furthermore, the liver showed congestion and hydropic degeneration. Serum levels of alanine aminotransferase increased significantly in all treatments, but none rose above reference values. In summary, our results suggest that compounds present in leaves of E. dysenterica may have therapeutic benefits on recovery from diarrhea despite their toxic effects.

High prevalence of polycystic ovary syndrome in women born small for gestational age.

BACKGROUND: There is evidence that intrauterine growth restriction, resulting in newborn girls that are small for gestational age (SGA), may be related to the onset of polycystic ovary syndrome (PCOS). Thus, we studied whether women born SGA have a higher prevalence of PCOS than women born appropriate for gestational age (AGA). METHODS: This was a prospective birth cohort study of 384 women born at term between June 1, 1978, and May 31, 1979, in Ribeirão Preto, Brazil. After exclusion, 165 women effectively participated in this study, of whom 43 were SGA and 122 were AGA. The prevalence of PCOS was analysed. At a mean age of 29 years, the women agreed to follow the study protocol, which included: anamnesis, physical examination, serum tests [follicle stimulating hormone, luteinizing hormone, total and free testosterone, dehydroepiandrostenedione sulphate, 17-OH-progesterone, fasting insulin, sex steroid-binding globulin (SHBG) and fasting glucose] and pelvic ultrasound. Data regarding gestational age, birthweight, age at menarche and maternal data were obtained from the files of the cohort. The adjusted relative risk (RR) values of the SGA, insulin resistance, body mass index, maternal smoking and parity variables were analysed using Poisson regression with robust adjustment of variance for the prediction of PCOS. RESULTS: The prevalence of PCOS was higher in the SGA group than in the AGA group [adjusted RR = 2.44, 95% CI (1.39-4.28)]. Hyperandrogenism was more prevalent in the SGA women than in the AGA women (P = 0.011). Circulating SHBG was lower in the SGA women than in the AGA women (P = 0.041), but fasting insulinemia was similar in both groups. CONCLUSIONS: The prevalence of PCOS in SGA women was twice as high as in AGA women in our study population.

Identification of E. dysenterica laxative peptide: a novel strategy in the treatment of chronic constipation and irritable bowel syndrome.

Plants have contributed over the years to the discovery of various pharmacological products. Amongst the enormous diversity of herbs with remarkable medicinal use and further pharmacological potential, here in this report we evaluated pulp extracts from Eugenia dysenterica fruits and further identified the active principle involved in such laxative activity in rats. For protein isolation, fruits were macerated with an extraction solution following precipitation with (NH(4))(2)SO(4) (100%). After dialysis, the peptide was applied onto a reversed-phase semi-preparative HPLC column, and the major fraction was eluted with 26% and 66% acetonitrile. The evaluation of molecular masses by MALDI-TOF and Tris/Tricine SDS-PAGE of HPLC fractions showed the presence of a major peptide with approximately 7 kDa. The N-terminal amino acid peptide sequence was determined and showed no similarity to other proteins deposited in the Data Bank. Peptide from E. dysenterica was able to enhance rats' intestinal motility by approximately 20.8%, probably being responsible for laxative activity. Moreover, these proteins were non-toxic to mammals, as observed in histopathology and hemolytic analyses. In conclusion, results here reported indicate that, in the near future, proteins synthesized by E. dysenterica fruits could be utilized in the development of novel biotechnological pharmaceutics with laxative properties for use in chronic constipation and irritable bowel syndrome treatment.

Purification and characterization of a liver-derived beta-N-Acetylhexosaminidase from marine mammal Sotalia fluviatilis.

A beta-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from hepatic extracts of Sotalia fluviatilis, order Cetacea. The protein was purified by using ammonium sulfate fractionation and four subsequent chromatographies (Biogel A 1.5 m, Chitin, Deae-Biogel and hydroxyapatite resins). After these purification steps, the enzyme was purified 380.5-fold with an 8.4% yield. The molecular mass (10 kDa) was estimated by SDS-PAGE and MALDI-TOF analysis. A Km of 2.72 mM and Vmax 9.5 x 10(-6) micromol/(min x mg) were found for this enzyme, determined by p-nitrophenyl-beta-D: -hexosaminide substrate digestion. Optimal pH and temperature for beta-N-Acetylhexosaminidase activity were 5.0 and 60 degrees C, respectively. Enzyme activity was inhibited by sodium selenate (Na(2)SeO(4)), mercuric chloride (HgCl(2)) and sodium dodecyl sulfate (C(12)H(25)SO(4)Na), and activated by zinc, calcium, barium and lithium ions. Characterization of the beta-N-Acetylhexosaminidase in Sotalia fluviatilis can be a basis for physiological studies in this species.

Metabolic and cardiovascular impact of oral contraceptives in polycystic ovary syndrome.

Chronic anovulation, polycystic ovarian morphology and hyperandrogenism are the diagnostic criteria for polycystic ovary syndrome (PCOS). Metabolic disturbances are more common in PCOS women who are prone to develop metabolic syndrome and to present higher levels of some cardiovascular disease risk marker. Oral contraceptives are widely used in PCOS, but conflicting data have been reported regarding their impact on carbohydrate and lipid metabolism on PCOS women. This paper presents a critical evaluation of combined oral contraceptives (COCs) metabolic effect - carbohydrate metabolism and insulin sensitivity, lipid metabolism, haemostasis, body weight, arterial pressure and cardiovascular impact - on PCOS women. Because of the paucity of data on the impact of COCs on cardiovascular and metabolic parameters in PCOS patients, most of there commendations are based on studies involving ovulatory women. The use of low-dose COCs is preferable in PCOS, especially among patients with glucose intolerance, insulin resistance and uncomplicated diabetes mellitus. Although reported as a side effect of COCs, marked weight gain has not been confirmed among users. However, when arterial hypertension or elevated risk for thromboembolism is present, progestogen-only hormonal contraceptives should be used instead of COCs. Regarding dyslipidaemia, COCs reduce low-density lipoprotein and total cholesterol and elevate high-density lipoprotein and triglycerides, and therefore are not recommended for women with high triglycerides levels. The choice of a COC, which alleviates the PCOS-induced hyperandrogenism without significant negative impact on cardiovascular risk, is one of the greatest challenges faced by gynaecologists nowadays.

Digestive alpha-amylases from Tecia solanivora larvae (Lepidoptera: Gelechiidae): response to pH, temperature and plant amylase inhibitors.

The biochemical properties of the digestive alpha-amylase from Tecia solanivora larvae, an important and invasive insect pest of potato (Solanum tuberosum), were studied. This insect has three major digestive alpha-amylases with isoelectric points 5.30, 5.70 and 5.98, respectively, which were separated using native and isoelectric focusing gels. The alpha-amylase activity has an optimum pH between 7.0 and 10.0 with a peak at pH 9.0. The enzymes are stable when heated to 50 degrees C and were inhibited by proteinaceous inhibitors from Phaseolus coccineus (70% inhibition) and P. vulgaris cv. Radical (87% inhibition) at pH 6.0. The inhibitors present in an amaranth hybrid inhibited 80% of the activity at pH 9.0. The results show that the alpha-amylase inhibitor from amaranth seeds may be a better candidate to make genetically-modified potatoes resistant to this insect than inhibitors from common bean seeds.

Jaburetox-2Ec: an insecticidal peptide derived from an isoform of urease from the plant Canavalia ensiformis.

Canatoxin, a urease isoform from Canavalia ensiformis seeds, shows insecticidal activity against different insect species. Its toxicity relies on an internal 10 kDa peptide (pepcanatox), released by hydrolysis of Canatoxin by cathepsins in the digestive system of susceptible insects. In the present work, based on the N-terminal sequence of pepcanatox, we have designed primers to amplify by PCR a 270-bp fragment corresponding to pepcanatox using JBURE-II cDNA (one of the urease isoforms cloned from C. ensiformis, with high identity to JBURE-I, the classical urease) as a template. This amplicon named jaburetox-2 was cloned into pET 101 vector to obtain heterologous expression in Escherichia coli of the recombinant protein in C-terminal fusion with V-5 epitope and 6-His tag. Jaburetox-2Ec was purified on Nickel-NTA resin and bioassayed in insect models. Dysdercus peruvianus larvae were fed on cotton seed meal diets containing 0.01% (w/w) Jaburetox-2Ec and, after 11 days, all individuals were dead. Jaburetox-2Ec was also tested against Spodoptera frugiperda larvae and caused 100% mortality. In contrast, high doses of Jaburetox-2Ec were innocuous when injected or ingested by mice and neonate rats. Modeling of Jaburetox-2Ec, in comparison with other peptide structures, revealed a prominent beta-hairpin motif consistent with an insecticidal activity based on either neurotoxicity or cell permeation.

Use of the etonogestrel-releasing implant is associated with hypoactivation of the coagulation cascade.

BACKGROUND: The role of progestogens in haemostasis is controversial. Our objective is to evaluate the haemostatic effects of an etonogestrel-releasing implant. METHODS: This open-label, self-controlled, longitudinal study involved 20 healthy women receiving subcutaneous etonogestrel-releasing implants. At baseline, 1, 3 and 6 months, we measured the following: activated partial thromboplastin time; prothrombin time; thrombin time; fibrinogen; coagulation factors II, V, VII, VIII, IX, X and XI; von Willebrand factor; activated protein C (APC); antithrombin; free protein S; plasminogen activator inhibitor type 1 (PAI-1); alpha2-antiplasmin; thrombin-antithrombin (TAT) complex; prothrombin fragment 1 + 2 (F1 + 2); D-dimers; APC resistance. Statistical analyses included the Friedman test and ANOVA. RESULTS: Levels of APC (P < 0.01), factor II (P = 0.02), factor VII (P = 0.006), factor X (P = 0.01) and F1 + 2 (P < 0.001) were reduced, whereas those of PAI-1 (P = 0.01) and factor XI (P = 0.006) were transitory increased. All of these values, however, remained within normal ranges. Surprisingly, TAT concentrations fell below the normal range (P < 0.001). CONCLUSIONS: Our findings suggest that the etonogestrel-releasing implant does not induce a prothrombotic pattern during the first six months of use, and that its use is associated with a reduction in thrombin generation.

Proregion of Acanthoscelides obtectus cysteine proteinase: a novel peptide with enhanced selectivity toward endogenous enzymes.

Acanthoscelides obtectus is a devastating storage insect pest capable of causing severe bean crop losses. In order to maintain their own development, insect pest larvae feed continuously, synthesizing efficient digestive enzymes. Among them, cysteine proteinases (CPs) are commonly produced as inactive precursors (procysteines), requiring a cleavage of the peptide proregion to become active. The proregion fits tightly into the active site of procysteines, efficiently preventing their activity. In this report, a CP cDNA (cpao) was isolated from A. obtectus midgut larvae. In silico studies indicated that the complete CP sequence contains a hydrophobic signal peptide, a prodomain and a conserved catalytic region. Moreover, the encoding cDNA contains 963bp translating into a 321 residue protein, CPAo, which was expressed in E. coli, fused with thioredoxin. Enzymatic assays using the recombinant protein revealed that the enzyme was catalytically active, being able to cleave the synthetic substrate Z-Phe-Arg-7-AMC. Additionally, this report also focuses the cpao propeptide (PCPAo) subcloning and expression. The expressed propeptide efficiently inhibited CPAo, as well as digestive CP of other bean bruchids. Little or no activity was found against proteolytic enzymes of two other coleopterans: Rhyzopertha dominica and Anthonomus grandis. The data reported here indicate the possibility of endogenous propeptides as a novel strategy on bruchids control, which could be applicable to bean improvement programs.

Autonomic modulation of heart rate of young and postmenopausal women undergoing estrogen therapy.

The aim of the present study was to determine whether estrogen therapy (ET) reduces alterations of the autonomic control of heart rate (HR) due to hypoestrogenism and aging. Thirteen young (24 +/- 2.6 years), 10 postmenopausal (53 +/- 4.6 years) undergoing ET (PM-ET), and 14 postmenopausal (56 +/- 2.6 years) women not undergoing ET (PM) were studied. ET consisted of 0.625 mg/day conjugated equine estrogen. HR was recorded continuously for 8 min at rest in the supine and sitting positions. HR variability (HRV) was analyzed by time (SDNN and rMSSD indices) and frequency domain methods. Power spectral components are reported as normalized units (nu) at low (LF) and high (HF) frequencies, and as LF/HF ratio. Intergroup comparisons: SDNN index was higher in young (median: supine, 47 ms; sitting, 42 ms) than in PM-ET (33; 29 ms) and PM (31; 29 ms) women (P < 0.05). PM showed lower HFnu, higher LFnu and higher LF/HF ratio (supine: 44, 56, 1.29; sitting: 38, 62, 1.60) than the young group in the supine position (61, 39, 0.63) and the PM-ET group in the sitting position (57, 43, 0.75; P < 0.05). Intragroup comparisons: HR was lower in the supine than in the sitting position for all groups (P < 0.05). The HRV decrease from the supine to the sitting position was significant only in the young group. These results suggest that HRV decreases during aging. ET seems to attenuate this process, promoting a reduction in sympathetic activity on the heart and contributing to the cardioprotective effect of estrogen hormones.