Two short peptides including segments of subunit A of Escherichia coli DNA gyrase as potential probes to evaluate the antibacterial activity of quinolones

Quinolones constitute a family of compounds with a potent antibiotic activity. The enzyme DNA gyrase, responsible for the replication and transcription processes in DNA of bacteria, is involved in the mechanism of action of these drugs. In this sense, it is believed that quinolones stabilize the so-called 'cleavable complex' formed by DNA and gyrase, but the whole process is still far from being understood at the molecular level. This information is crucial in order to design new biological active products. As an approach to the problem, we have designed and synthesized low molecular weight peptide mimics of DNA gyrase. These peptides correspond to sequences of the subunit A of the enzyme from Escherichia coli, that include the quinolone resistance-determining region (positions 75-92) and a segment containing the catalytic Tyr-122 (positions 116-130). The peptide mimic of the non-mutated enzyme binds to ciprofloxin (CFX) only when DNA and Mg2+ were present (Kd = 1.6 × 10 -6 m), a result previously found with DNA gyrase. On the other hand, binding was reduced when mutations of Ser-83 to Leu-83 and Asp-87 to Asn-87 were introduced, a double change previously found in the subunit A of DNA gyrase from several CFX-resistant clinical isolates of E. coli. These results suggest that synthetic peptides designed in a similar way to that described here can be used as mimics of gyrases (topoisomerases) in order to study the binding of the quinolone to the enzyme-DNA complex as well as the mechanism of action of these antibiotics. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.

Dynamics of handling time and functional response by larvae of Chrysomya albiceps (Dipt., Calliphoridae) on different prey species

One way to understand the behavioural patterns exhibited by a predator in response to prey density is to evaluate its functional response. Such evaluation yields information about basic mechanisms of prey-predator dynamics, and is an essential component of prey-predator models. In this paper we analysed experimentally the functional response and the handling time spent by Chrysomya albiceps on different prey species and larval instars of blowflies. The type II functional response was observed when second instar larvae of Chrysomya megacephala and Chrysomya macellaria were consumed. The handling time spent by the predator was significantly different between instars and species. The implications of the functional response and handling time for the interaction dynamics of Brazilian Chrysomyinae species are discussed.

Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: Activation of CD8+ cells, interferon-γ recovery and reduction of lung injury

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon-γ recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-α. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-γ and to restrict the growth of bacilli.

Avaliação de carcaças de suínos da raça large white utilizando medidas convencionais

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A specific short dextrin-hydrolyzing extracellular glucosidase from the thermophilic fungus Thermoascus aurantiacus 179-5

The thermophilic fungus Thermoascus aurantiacus 179-5 produced large quantities of a glucosidase which preferentially hydrolyzed maltose over starch. Enzyme production was high in submerged fermentation, with a maximal activity of 30 U/ml after 336 h of fermentation. In solid-state fermentation, the activity of the enzyme was 22 U/ml at 144 h in medium containing wheat bran and 5.8 U/ml at 48 h when cassava pulp was used as the culture medium. The enzyme was specific for maltose, very slowly hydrolyzed starch, dextrins (2-7G) and the synthetic substrate (α-PNPG), and did not hydrolyze sucrose. These properties suggest that the enzyme is a type II α-glucosidase. The optimum temperature of the enzyme was 70°C. In addition, the enzyme was highly thermostable (100% stability for 10 h at 60°C and a half-life of 15 min at 80°C), and stable within a wide pH range. Copyright © 2006, The Microbiological Society of Korea.

Cytokine profile and clinical metabolic alterations in HIV-1 infected individuals with and without lipodistrophy

The extensive use of Highly Active Antiretroviral Therapy (HAART) has transformed HIV infection into a chronic condition. Thus, metabolic alterations including lipodystrophy and dyslipidemia have been associated with the use of such medications. The objective of the present study was to analyze clinical metabolic alterations and the profile of TNF-α, IFN-γ, IL-2, IL-10, and TNF-α type II soluble receptor in serum of HIV-1 individuals with and without lipodystrophy. Eighty-four adults were evaluated, 42 males and 42 females, mean age 37 years, and HAART time of at least 15 months. Two groups were formed, G1: 42 individuals with lipodystrophy, and G2: 42 without lipodistropy. From the HAART used, stavudine was more associated with the lipodystrophy group and zidovudine with the non-lipodystrophy group. CD4 and CD8 values, viral load, glucose, albumin, and lipids were not different between groups, except for triglycerides, which were high in the lipodystrophy group, and HDL, whose concentration was reduced in G1. TNF-α, TNF-RII, and IL-10 profiles were high and had positive correlation; IL-2 and IFN-γ had reduced levels in the lipodystrophy group. High TNF-α and its receptor levels seem to be associated with lipodystrophy development in individuals under HAART therapy.

Pattern of macrophage activation in yersinia-resistant and yersinia-susceptible strains of mice

Th1 cells, in cooperation with activated macrophages, are required to overcome Yersinia enterocolitica infection in mice. The pathway macrophages utilize to metabolize arginine can alter the outcome of inflammation in different ways. The objective of this study was to verify the pattern of macrophages activation in Y. enterocolitica infection of BALB/c (Yersinia-susceptible) and C57BL/6 (Yersinia-resistant) mice. Both strains of mice were infected with Y. enterocolitica O:8 WA 2707. Peritoneal macrophages and spleen cells were obtained on the 1st, 3rd and 5th day post-infection. The iNOS and the arginase activities were assayed in supernatants of macrophage cultures, by measuring their NO/citrulline and ornithine products, respectively. TGFβ-1 production was also assayed. The Th1 and Th2 responses were evaluated in supernatants of lymphocyte cultures, by IFN-γ and IL-4 production. Our results showed that in the early phase of Y. enterocolitica infection (1st and 3rd day), the macrophages from C57BL/6 mice produced higher levels of NO/citrulline and lower levels of ornithine than macrophages from BALB/c mice. The infection with Y. enterocolitica leads to an increase in the TGF-β1 and IL-4 production by BALB/c mice and to an increase in the IFN-γ levels produced by C57BL/6 mice. These results suggest that Y. enterocolitica infection leads to the modulation of M1 macrophages in C57Bl/6 mice, and M2 macrophages in BALB/c mice. The predominant macrophage population (M1 or M2) at the 1st and 3rd day of infection thus seems to be important in determining Y. enterocolitica susceptibility or resistance.

Morfologia e morfometria dos tipos de fibras do músculo reto abdominal de cadelas (canis familiaris) durante a gestação

The present paper deals with the evaluation of morphological and morphometric alterations of slow-twist (I) and fast-twitch (II) fibers of rectus abdominis muscle of adult female dog during pregestational phase, at 30 and 60 days of pregnancy and at 30 days after the parturition. At the every phases, using the open biopsy technique, muscle samples were collected. The samples were plunged. Histological sections were cut in a microtome. For general morphology, some sections were stained with HE. Subsequent sections were reacted for myofibrillar ATPase (m-ATPase), after alkaline (pH 10.4) and acid (pH 4.4) pre-incubations, in order to identificate type I and II fibers. In the pre-gestational phase, muscle tissue revealed to be composed by fibers with different diameters, presenting polygonal outlines and one or more periphery nuclei. At 30 days of pregnancy, muscle fiber characteristics were similar. At 60 days, in addition to the existence of normal fibers, polymorphic and small diameter fibers were frequent. At 30 days after the parturation, the morphology of muscle fiber were similar to that observed in the pre-gestational phase. In the four phases, type II fiber diameters were lager than type I. The diameters of both fiber types showed a significant reduction in the 30 days phase and a significative increasing at 60 days. The expansion of the abdominal wall during the pregnancy represents a chronic stimulus, induced changes in the morphology and in the fiber type diameters.

Characterization of Zn0. 95Mn0.05O synthesized by polymeric precursors

This paper discusses the preparation and characterization of Zn 0.95Mn0.05O phase obtained by the polymeric precursor method for DMS applications. The as-obtained powders were calcined between 500 to 800°C and characterized by XRD, SEM and BET. The XRD analysis of the powder showed a crystalline material containing second phase. The crystallite sizes ranged from 20 to 51 nm. The micrographs showed that the powders consisted of soft and homogeneous agglomerations. The nitrogen adsorption/desorption curves of the Zn0.95Mn0.05O phases were type II curves, which is characteristic of mesoporous materials.

Scanning electron microscopic evaluation of the root apex of mandibular premolars.

This aim of this study was to evaluate the root apex of mandibular premolars regarding the presence of main and accessory foramina. The root apexes from fifty extracted mandibular single-rooted premolars were examined by scanning electron microscopy (SEM). The apical openings had their diameter measured and were identified as main or accessory foramina. Double blinded and calibrated examiners analyzed the SEM photographs and classified the premolar roots into three types, based on the presence and size of the apical openings. Type I: roots with a single main apical foramen and no accessory foramina; type II: roots with a main foramen and one or more accessory foramina; type III: roots with accessory foramina only. For the first premolar, 16 roots were classified as type I (48.48%), 4 as type II (12.12%) and 13 as type III (39.40%). For the second premolars, 10 roots were classified as type I (58.83%), 3 as type II (17.65%) and 4 as type III (23.52%). The high incidence of roots with accessory foramina only (type III), mainly in the first premolar, warns of the need for caution during working length determination and apical debridement.

Avaliação clínica e microbiológica do tratamento da estomatite protética com tintura de Schinus terebinthifolius Raddi (Aroeira)

Objective: To evaluate the clinical efficacy of Schinus terebinthifolius Raddi (aroeira) tincture in the treatment of denture stomatitis. Method: Eighteen removable denture wearers with clinical diagnosis of type II denture stomatts and presence of candidosis associated to the denture use, as confirmed by clinical and mycological examinations, were selected for the study. The patients were allocated to two groups: TG (test group) - treatment with Schinus terebinthifolius Raddi (aroeira) tincture; CG (control group), treatment with nystatin. All patients were instructed to clean the dentures with toothbrush and dentifrice, and then apply the product on the palatal mucosa and on denture surface 3 times a day, during 15 consecutive days, removing the denture at bedtime and keeping it in a receptacle with water. At the 15th day of use, the clinical and mycological examinations were redone to evaluate treatment efficacy. Data were analyzed statistically by Wilcoxon and Mann-Whitney tests at 5% significance level. Results: The inflammatory process and Candida spp. infect on were eliminated in 66.7% and 77.8% of the cases, respectively, in TG. In CG, elimination of the inflammatory process and fungal infect on occurred in 77.8% and 88.9% of the cases, respectively. These results were statistically significant (p=0.01). There was no statistically significant difference between the treatments (p>0.05). In all cases, fungal infect on was detected only on the denture, and C. albicans was the most prevalent microorganism, being present in 94.4% of the cases. Conclusion: The treatment with Schinus terebinthifolius Raddi (aroeira) tincture was effective in the treatment of denture stomatitis, promoting remission of the inflammatory process and Candida spp infection.

Capacidade predatória de chrysoperla externa sobre bemisia tabaci biótipo b expostas ao fungo metarhizium anisopliae

The objective of this study was to determine the influence of Metarhizium anisopliae on the predatory capacity and functional response of Chrysoperla externa fed Bemisia tabaci B biotype rearing cotton. The stock rearings of B. tabaci B biotype and C. externa were maintained in cages according to preexisting methodologies. Upon hatching, predator larvae were fed on eggs of Ephestia küehniella until their 3rd instar and then fed on whitefly nymphs in density exceeding their intake capacity (150 nymphs/individual). The nymphs were offered on cotton leaf disks (5 cm diameter), 24 hours after the treatments application following: T1: Control (water); T2: M. anisopliae at the concentration of 105 conidia cm-3; T3: 10 8 conidia cm-3 origined from Metharril® formulation of the 109 viable conidia cm-3 M. anisopliae active principle. The predatory capacity was obtained by the difference between the numbers of nymphs supplied and nymphs left. Five different nymphal densities (130, 160, 190, 220, and 250) were selected for the functional response study. The number of nymphs preyed was recorded after 24, 48, 72, and 96 hours. The M. anisopliae entomopathogenic fungi did not influence predatory capacity of C. externa which ranged from 70.6 individuals at the highest concentration to 87.1 in the control. Conclued that the C. externa predatory capacity was not affected by fungi M. anisopliae concentrations. The predator showed a type-II functional response after 24 hours, and a type-I response after 48, 72s, and 96 hours independent from fungi concentrations.

Sequential Sampling Plan for Tenuipalpus heveae Baker (Acari: Tenuipalpidae) on Rubber Tree

The objective of the present study was to develop a sequential sampling plan for the decision-making process to control Tenuipalpus heveae Baker (Acari: Tenuipalpidae), an important pest of the rubber tree crop. The experimental area was represented by 1,000 plants of the RRIM 600 clone divided in 100 plots with 10 plants each. Leaves were collected and the number of mites determined under laboratory conditions. The sequential sampling plan was developed in accordance with the Sequential Test Likelihood Ratio. The value 0.10 was pre-established for α and β representing type I and type II errors, respectively. The level of control adopted was six mites per 12 cm2. The operating characteristic curve and the curve of maximum expected sample were determined. Two lines were generated: the upper one, when the condition for chemical control is recommended (S1 = 23.3080 + 2.1972); and the lower, when chemical control is not recommended (S0 = -23.3080 + 2.1972). Sample size for the decision-making process to control T. heveae requires 6 to 18 plants. © 2013 Sociedade Entomológica do Brasil.

The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses

Tuberculosis remains as one of the main cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. The aroK-encoded M. tuberculosis Shikimate Kinase (MtSK), shown to be essential for survival of bacilli, catalyzes the phosphoryl transfer from ATP to the carbon-3 hydroxyl group of shikimate (SKH), yielding shikimate-3-phosphate and ADP. Here we present purification to homogeneity, and oligomeric state determination of recombinant MtSK. Biochemical and biophysical data suggest that the chemical reaction catalyzed by monomeric MtSK follows a rapid-equilibrium random order of substrate binding, and ordered product release. Isothermal titration calorimetry (ITC) for binding of ligands to MtSK provided thermodynamic signatures of non-covalent interactions to each process. A comparison of steady-state kinetics parameters and equilibrium dissociation constant value determined by ITC showed that ATP binding does not increase the affinity of MtSK for SKH. We suggest that MtSK would more appropriately be described as an aroL-encoded type II shikimate kinase. Our manuscript also gives thermodynamic description of SKH binding to MtSK and data for the number of protons exchanged during this bimolecular interaction. The negative value for the change in constant pressure heat capacity (ΔCp) and molecular homology model building suggest a pronounced contribution of desolvation of non-polar groups upon binary complex formation. Thermodynamic parameters were deconvoluted into hydrophobic and vibrational contributions upon MtSK:SKH binary complex formation. Data for the number of protons exchanged during this bimolecular interaction are interpreted in light of a structural model to try to propose the likely amino acid side chains that are the proton donors to bulk solvent following MtSK:SKH complex formation. © 2013 Rosado et al.

Search for methylation-sensitive amplification polymorphisms in mutant figs

Fig (Ficus carica) breeding programs that use conventional approaches to develop new cultivars are rare, owing to limited genetic variability and the difficulty in obtaining plants via gamete fusion. Cytosine methylation in plants leads to gene repression, thereby affecting transcription without changing the DNA sequence. Previous studies using random amplification of polymorphic DNA and amplified fragment length polymorphism markers revealed no polymorphisms among select fig mutants that originated from gamma-irradiated buds. Therefore, we conducted methylation-sensitive amplified polymorphism analysis to verify the existence of variability due to epigenetic DNA methylation among these mutant selections compared to the main cultivar 'Roxo-de-Valinhos'. Samples of genomic DNA were double-digested with either HpaII (methylation sensitive) or MspI (methylation insensitive) and with EcoRI. Fourteen primer combinations were tested, and on an average, non-methylated CCGG, symmetrically methylated CmCGG, and hemimethylated hmCCGG sites accounted for 87.9, 10.1, and 2.0%, respectively. MSAP analysis was effective in detecting differentially methylated sites in the genomic DNA of fig mutants, and methylation may be responsible for the phenotypic variation between treatments. Further analyses such as polymorphic DNA sequencing are necessary to validate these differences, standardize the regions of methylation, and analyze reads using bioinformatic tools. © FUNPEC-RP.