The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.

Treatment of developmental defect of upper lip with carbon dioxide laser radiation (CO2): First surgical time

With the exception of the cleft lip, developmental defects (DD) of the lip are rare. The upper lip originates from the ectomesenchyme and is formed by the merging of the nasal medial and lateral processes with the maxillary process. Disturbances during this formation period can cause DD with functional and/or esthetic repercussions. We present a case of DD of the upper lip in a patient with a history of progressive growth of the left lateral portion of the upper lip that occurred from the time of birth until the age of 22 years. Clinical examination revealed hypertrophy of the area from the left philtral columns to the left commissure of the lip, extending the portion of the surface mucosa creating a flaccid and asymptomatic tissue mass. All other buccal structures appeared to be within normal limits and without any evidence of defects or deformities. In the surgical planning we decided to carry out corrective surgery in two phases. The first phase accomplished a conservative excision of the total abnormal labial tissue mass with a CO2 laser radiation (5 W in continuous mode, bunch diameter φ = 0.6 mm with a power density of 768 W/cm2 and fluency of 0.231 J/cm2) being careful to preserve the vermilion portion of the lip. Postsurgical clinical evaluations were done every three days until the skin sutures were removed and then every seven days until two months post surgery. While the entire mass of excessive tissue could not be completely removed, the removal of the excessive mucosal tissue produced a very good outcome relative to lip function, with a good esthetic result without scarring, and good tissue mobility. The results showed that the CO2 laser is an extremely useful instrument that can provide excellent control of the surgical field and allow for healing that produces excellent functional and esthetic results. © 2005 Taylor & Francis.

Effects of copper vapor laser radiation on the root canal wall of human teeth: A scanning electron microscope study

Objective: The aim of this study is to analyze the effects of copper vapor laser radiation on the radicular wall of human teeth. Materials and Methods: Immediately after the crowns of 10 human uniradicular teeth were cut along the cement-enamel junction, a chemical-surgical preparation of the radicular canals was completed. Then the roots were longitudinally sectioned to allow for irradiation of the surfaces of the dentin walls of the root canals. The hemi-roots were separated into two groups: one (control) with five hemi-roots that were not irradiated, and another (experimental) with 15 hemi-roots divided into three subgroups that were submitted to the following exposure times: 0.02,0.05, and 0.1 s. A copper vapor laser (510.6 nm) with a total average power of 6.5 W in green emission, frequency of 16.000 Hz, and pulse duration of 30 ns was used. Results: The results obtained by scanning electron microscope analysis showed the appearance of a cavity in the region of laser beam impact, with melting, recrystallization, and cracking on the edges of the dentin of the cavity due to heat diffusion. Conclusions: We determined that the copper vapor laser produces significant morphologic changes in the radicular wall of human teeth when using the parameters in this study. However, further research should be done to establish parameters that are compatible with dental structure in order to eliminate thermal damages. © Mary Ann Liebert, Inc.

Kinetics and crystal structure of human purine nucleoside phosphorylase in complex with 7-methyl-6-thio-guanosine

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and drugs that inhibit this enzyme may have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Here, we describe kinetics and crystal structure of human PNP in complex with 7-methyl-6-thio-guanosine, a synthetic substrate, which is largely used in activity assays. Analysis of the structure identifies different protein conformational changes upon ligand binding, and comparison of kinetic and structural data permits an understanding of the effects of atomic substitution on key positions of the synthetic substrate and their consequences to enzyme binding and catalysis. Such knowledge may be helpful in designing new PNP inhibitors. © 2005 Elsevier Inc. All rights reserved.

2,3,4,6-Tetra-O-(3-nitropropanoyl)-O-β-D-glucopyranoside, a new antimicrobial from the roots of Heteropteris aphrodisiaca

A new 2,3,4,6-tetra-O-(3-nitropropanoyl)-O-β-D-glucopyranoside anomer was isolated from the roots of Heteropteris aphrodisiaca and characterized by spectroscopic methods. Activity against Staphylococcus aureus, Bacillus subtilis, Candida albicans, C. parapsilosis, C. krusei, and C. tropicalis was demonstrated.

Estimation of variance components and prediction of breeding values in rubber tree breeding using the REML/BLUP procedure

The present paper deals with estimation of variance components, prediction of breeding values and selection in a population of rubber tree [Hevea brasiliensis (Willd. ex Adr. de Juss.) Müell.-Arg.] from Rio Branco, State of Acre, Brazil. The REML/BLUP (restricted maximum likelihood/best linear unbiased prediction) procedure was applied. For this purpose, 37 rubber tree families were obtained and assessed in a randomized complete block design, with three unbalanced replications. The field trial was carried out at the Experimental Station of UNESP, located in Selvíria, State of Mato Grosso do Sul, Brazil. The quantitative traits evaluated were: girth (G), bark thickness (BT), number of latex vessel rings (NR), and plant height (PH). Given the unbalanced condition of the progeny test, the REML/BLUP procedure was used for estimation. The narrow-sense individual heritability estimates were 0.43 for G, 0.18 for BT, 0.01 for NR, and 0.51 for PH. Two selection strategies were adopted: one short-term (ST - selection intensity of 8.85%) and the other long-term (LT - selection intensity of 26.56%). For G, the estimated genetic gains in relation to the population average were 26.80% and 17.94%, respectively, according to the ST and LT strategies. The effective population sizes were 22.35 and 46.03, respectively. The LT and ST strategies maintained 45.80% and 28.24%, respectively, of the original genetic diversity represented in the progeny test. So, it can be inferred that this population has potential for both breeding and ex situ genetic conservation as a supplier of genetic material for advanced rubber tree breeding programs. Copyright by the Brazilian Society of Genetics.

cDNA cloning and 1.75 Å crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 ± 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed β(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-β-d-glucopyranose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 Å resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (βα) 8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182. © 2006 The Authors.

Phylogenetic systematics of dart-poison frogs and their relatives (Amphibia: Athesphatanura: Dendrobatidae)

The known diversity of dart-poison frog species has grown from 70 in the 1960s to 247 at present, with no sign that the discovery of new species will wane in the foreseeable future. Although this growth in knowledge of the diversity of this group has been accompanied by detailed investigations of many aspects of the biology of dendrobatids, their phylogenetic relationships remain poorly understood. This study was designed to test hypotheses of dendrobatid diversification by combining new and prior genotypic and phenotypic evidence in a total evidence analysis. DNA sequences were sampled for five mitochondrial and six nuclear loci (approximately 6,100 base pairs [bp]; x=3,740 bp per terminal; total dataset composed of approximately 1.55 million bp), and 174 phenotypic characters were scored from adult and larval morphology, alkaloid profiles, and behavior. These data were combined with relevant published DNA sequences. Ingroup sampling targeted several previously unsampled species, including Aromobates nocturnus, which was hypothesized previously to be the sister of all other dendrobatids. Undescribed and problematic species were sampled from multiple localities when possible. The final dataset consisted of 414 terminals: 367 ingroup terminals of 156 species and 47 outgroup terminals of 46 species. Direct optimization parsimony analysis of the equally weighted evidence resulted in 25,872 optimal trees. Forty nodes collapse in the strict consensus, with all conflict restricted to conspecific terminals. Dendrobatids were recovered as monophyletic, and their sister group consisted of Crossodactylus, Hylodes, and Megaelosia, recognized herein as Hylodidae. Among outgroup taxa, Centrolenidae was found to be the sister group of all athesphatanurans except Hylidae, Leptodactyidae was polyphyletic, Thoropa was nested within Cycloramphidae, and Ceratophryinae was paraphyletic with respect to Telmatobiinae. Among dendrobatids, the monophyly and content of Mannophryne and Phyllobates were corroborated. Aromobates nocturnus and Colostethus saltuensis were found to be nested within Nephelobates, and Minyobates was paraphyletic and nested within Dendrobates. Colostethus was shown to be rampantly nonmonophyletic, with most species falling into two unrelated cis- and trans-Andean clades. A morphologically and behaviorally diverse clade of median lingual process-possessing species was discovered. In light of these findings and the growth in knowledge of the diversity of this large clade over the past 40 years, we propose a new, monophyletic taxonomy for dendrobatids, recognizing the inclusive clade as a superfamily (Dendrobatoidea) composed of two families (one of which is new), six subfamilies (three new), and 16 genera (four new). Although poisonous frogs did not form a monophyletic group, the three poisonous lineages are all confined to the revised family Dendrobatidae, in keeping with the traditional application of this name. We also propose changes to achieve a monophyletic higher-level taxonomy for the athesphatanuran outgroup taxa. Analysis of character evolution revealed multiple origins of phytotelm-breeding, parental provisioning of nutritive oocytes for larval consumption (larval oophagy), and endotrophy. Available evidence indicates that transport of tadpoles on the dorsum of parent nurse frogs-a dendrobatid synapomorphy-is carried out primitively by male nurse frogs, with three independent origins of female transport and five independent origins of biparental transport. Reproductive amplexus is optimally explained as having been lost in the most recent common ancestor of Dendrobatoidea, with cephalic amplexus arising independently three times. © American Museum of Natural History 2006.

Molecular modeling databases: A new way in the search of protein targets for drug development

DBMODELING is a relational database of annotated comparative protein structure models and their metabolic, pathway characterization. It is focused on enzymes identified in the genomes of Mycobacterium tuberculosis and Xylella fastidiosa. The main goal of the present database is to provide structural models to be used in docking simulations and drug design. However, since the accuracy of structural models is highly dependent on sequence identity between template and target, it is necessary to make clear to the user that only models which show high structural quality should be used in such efforts. Molecular modeling of these genomes generated a database, in which all structural models were built using alignments presenting more than 30% of sequence identity, generating models with medium and high accuracy. All models in the database are publicly accessible at http://www.biocristalografia.df.ibilce.unesp.br/tools. DBMODELING user interface provides users friendly menus, so that all information can be printed in one stop from any web browser. Furthermore, DBMODELING also provides a docking interface, which allows the user to carry out geometric docking simulation, against the molecular models available in the database. There are three other important homology model databases: MODBASE, SWISSMODEL, and GTOP. The main applications of these databases are described in the present article. © 2007 Bentham Science Publishers Ltd.

The inhibition of 5-enolpyruvylshikimate-3-phosphate synthase as a model for development of novel antimicrobials

EPSP synthase (EPSPS) is an essential enzyme in the shikimate pathway, transferring the enolpyruvyl group of phosphoenolpyruvate to shikimate-3-phosphate to form 5-enolpyruvyl-3-shikimate phosphate and inorganic phosphate. This enzyme is composed of two domains, which are formed by three copies of βαβαββ-folding units; in between there are two crossover chain segments hinging the nearly topologically symmetrical domains together and allowing conformational changes necessary for substrate conversion. The reaction is ordered with shikimate-3-phosphate binding first, followed by phosphoenolpyruvate, and then by the subsequent release of phosphate and EPSP. N-[phosphomethyl]glycine (glyphosate) is the commercial inhibitor of this enzyme. Apparently, the binding of shikimate-3-phosphate is necessary for glyphosate binding, since it induces the closure of the two domains to form the active site in the interdomain cleft. However, it is somehow controversial whether binding of shikimate-3-phosphate alone is enough to induce the complete conversion to the closed state. The phosphoenolpyruvate binding site seems to be located mainly on the C-terminal domain, while the binding site of shikimate-3-phosphate is located primarily in the N-terminal domain residues. However, recent results demonstrate that the active site of the enzyme undergoes structural changes upon inhibitor binding on a scale that cannot be predicted by conventional computational methods. Studies of molecular docking based on the interaction of known EPSPS structures with (R)- phosphonate TI analogue reveal that more experimental data on the structure and dynamics of various EPSPS-ligand complexes are needed to more effectively apply structure-based drug design of this enzyme in the future. © 2007 Bentham Science Publishers Ltd.

The reproductive biology and cycle of Rioraja agassizi (Müller and Henle, 1841) (Chondrichthyes: Rajidae) in southeastern Brazil, SW Atlantic Ocean

Specimens of Rioraja agassizi were collected at Guarujá pier, São Paulo State, Brazil, from March 2005 to March 2006. A total of 275 males were captured. Based on the analysis of the clasper length, gonad weight, clasper gland length, alar thorns and lobule diameter, the size-at-maturity was estimated to be 32 cm. There were 1049 females in the record. Size-at-maturity was calculated in 40 cm. Sexual resting females were observed. Gonadosomatic and hepatosomatic indexes varied significantly in both sexes throughout the year. However, ovulation, egg-laying and presence of sperm in the seminal vesicle were observed all year round. For this reason, an annual cycle with at least one peak in the sexual activity is proposed for this species.

Cryopreservation of dog spermatozoa: Effect of bovine serum albumin on acrosomal integrity and pregnancy rates after artificial insemination

The objective of the present study was to compare the in vitro and in vivo profile of frozen dog semen with Tris-bovine serum albumin (TB) and Tris-egg yolk (TE) extenders. Twenty dogs were used as donors. Each dog was stimulated by penile massage and only the sperm-rich fraction was collected weekly until 40 ejaculates were obtained. After macroscopic and microscopic analysis, equal parts of each ejaculate were diluted with TB and TE by the one-step method at 37 °C. The semen was added to 0.5-mL French straws which presented normal characteristics before freezing and after thawing. Acrosomal integrity was evaluated by double Trypan blue-Giemsa staining, in which alive intact (LI), alive reacted (LR), dead intact (DI) and dead reacted (DR) spermatozoa, were identified by the time of thawing and up to 4 h of incubation at 39 °C, the TE being significantly superior to TB (P<0,01) in the LI and LR variables. The TB being significantly superior to TE (P<0,01) in the DR variable. Female dogs in natural heat were submitted to artificial insemination, 20 receiving TE-semen and 20 receiving TB-semen with the Osiris probe (IMV, L'Aigle, France) and the numbers indicate that TE was significantly better than TB (P<0,01) to pregnancy rate and number of puppies/delivery. We concluded from this study, that TE was better than TB, because this, induced an eady acrossome reaction in dog's sperm.

Sexual development and reproductive cycle of the Eyespot skate Atlantoraja cyclophora (Regan, 1903) (Chondrichthyes: Rajidae: Arhynchobatinae), in southeastern Brazil

Specimens o/Atlantoraja cyclophora were collected monthly from commercial fishing landings at Guarujá, São Paulo State, Brazil, from March 2005 to April 2006 at depths between 10 and 146 m. Males ranged from 13.3 to 58.5 cm TL (n = 396). Both the smallest mature male and the largest immature male were 47.0 cm long. Males' size-at-50% maturity was calculated to be 46.3 cm. Females ranged from 11.5 to 68.0 cm (n = 401). The smallest mature and the largest immature female were 51.6 and 53.0 cm long respectively. For the females, size-at-50% maturity was calculated to be 53.2 cm. In the males the hepatosomatic andgonadosomatic indices varied between 0.48 (August) and 3.54 (November) and between 0.15 (November) and 1.45 (June) respectively, with no significant variation for the fourteen-month period. In the females the hepatosomatic and gonadosomatic indices varied from 1.55 and 6.30 3.54 (both for April 2006) and from 0.08 (December) to 4.41 (October) respectively, with no significant difference among months. Egg-bearing females were found in all months with proportions varying from 0.03 (March) to 0.67 (April). Both males and females undergo an annual cycle, with slight seasonal variations in reproductive activity and a peak in the proportion of egg bearing females between April and July.

Usefulness of Mycobacterium tuberculosis molecular typing in a tuberculosis low-endemic agro-industrial setting of Brazil

To highlight the transmission and major phylogenetic clades of Mycobacterium tuberculosis, a retrospective study was carried out at two health facilities in a small agro-industrial area in São Paulo, Brazil, that has a low tuberculosis incidence rate. IS6110-RFLP and spoligotyping were performed on the isolates, with the former revealing that 31.3% (35/112) of strains were clustered. Epidemiological links were found in 16 of the 35 clustered patients and were associated with transmission among patients living in public housing. Spoligotyping grouped 62.8% of the strains. The T genetic family predominated among the isolates. Of interest is that five strains had a pattern characteristic of African or Asian origin (ST535), and two others were of the rare localized type ST1888 (BRA, VEN). In addition, three new types-1889, 1890, and 1891-were identified. Spoligotyping showed that some ST may be circulating to or from Brazil, and RFLP revealed ongoing transmission in inadequately ventilated public-housing buildings. This may point to a failure in tuberculosis control policy.

A Bayesian approach to estimate the accuracy of in-house ELISA assay to measure rabies antibodies from compulsory vaccinated dogs and cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)