Synthesis of Fructooligosaccharides in Banana `Prata` and Its Relation to Invertase Activity and Sucrose Accumulation

Levels of sucrose and total fructool igosaccha rides (FOS) were quantified in different phases of banana `Prata` ripening during storage at ambient (similar to 19 degrees C) and low (similar to 10 degrees C) temperature. Total FOS levels were detected in the first days after harvest, whereas 1-kestose remained undetectable until the sucrose levels reached approximately 200 mg/g (dry weight) in both groups. Sucrose levels increased slowly but constantly at low temperature, but they elevated rapidly when the temperature was raised to 19 degrees C. Total FOS and sucrose levels were higher in bananas stored at low temperature than in the control group. In both samples, total FOS levels were higher than those of 1-kestose. The carbohydrate profiles obtained by HPLC and TLC suggest the presence of neokestose, 6-kestose, and bifurcose. The enzymes putatively involved in banana fructosyltransferase activity were also evaluated. Results obtained indicate that the banana enzyme responsible for the synthesis of FOS by transfructosylation is an invertase rather than a sucrose-sucrosyl transferase-like enzyme.

Functional deficiencies of sulfite oxidase: Differential diagnoses in neonates presenting with intractable seizures and cystic encephalomalacia

Sulfite oxidase is a mitochondrial enzyme encoded by the SUOX gene and essential for the detoxification of sulfite which results mainly from the catabolism of sulfur-containing amino acids. Decreased activity of this enzyme can either be due to mutations in the SUOX gene or secondary to defects in the synthesis of its cofactor, the molybdenum cofactor. Defects in the synthesis of the molybdenum cofactor are caused by mutations in one of the genes MOCS1, MOCS2, MOCS3 and GEPH and result in combined deficiencies of the enzymes sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase. Although present in many ethnic groups, isolated sulfite oxidase deficiency and molybdenum cofactor deficiency are rare inborn errors of metabolism, which makes awareness of key clinical and laboratory features of affected individuals crucial for early diagnosis. We report clinical, radiologic, biochemical and genetic data on a Brazilian and on a Turkish child with sulfite oxidase deficiency due to the isolated defect and impaired synthesis of the molybdenum cofactor, respectively. Both patients presented with early onset seizures and neurological deterioration. They showed no sulfite oxidase activity in fibroblasts and were homozygous for the mutations c.1136A>G in the SUOX gene and c.667insCGA in the MOCS1 gene, respectively. Widely available routine laboratory tests such as assessment of total homocysteine and uric acid are indicated in children with a clinical presentation resembling that of hypoxic ischemic encephalopathy and may help in obtaining a tentative diagnosis locally, which requires confirmation by specialized laboratories. (C) 2009 Published by Elsevier B.V.

Functional and bioinformatics analyses reveal conservation of Cis-regulatory elements between sciaridae and Drosophilidae

The sciarid DNA puff C4 BhC4-1 gene is amplified and transcribed in salivary glands at the end of the larval stage. In transgenic Drosophila, the BhC4-1 promoter drives transcription in prepupal salivary glands and in the ring gland of late embryos. A bioinformatics analysis has identified 162 sequences similar to distinct regions of the BhC4-1 proximal promoter, which are predominantly located either in 5` or 3` regions or introns in the Drosophila melanogaster genome. A significant number of the identified sequences are found in the regulatory regions of Drosophila genes that are expressed in the salivary gland. Functional assays in Drosophila reveal that the BhC4-1 proximal promoter contains both a 129 bp (-186/-58) salivary gland enhancer and a 67 bp (-253/-187) ring gland enhancer that drive tissue specific patterns of developmentally regulated gene expression, irrespective of their orientation.

Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli

Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher`s disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

First isolation and molecular characterization of Ehrlichia canis in Costa Rica, Central America

The present study investigated Ehrlichia species in blood samples from dogs suspected of clinical ehrlichiosis, using molecular and isolation techniques in cell culture. From a total of 310 canine blood samples analyzed by 16S rRNA nested PCR, 148 (47.7%) were positive for Ehrlichia canis. DNA from Ehrlichia chaffeensis or Ehrlichia ewingii was not detected in any sample using species-specific primers in separated reactions. Leukocytes from five PCR-positive dogs were inoculated into DH82 cells; successful isolation of E. canis was obtained in four samples. Partial sequence of the dsb gene of eight canine blood samples (including the five samples for in vitro isolation) was obtained by PCR and their analyses through BLAST showed 100% of identity with the corresponding sequence of E. canis in GenBank. This study represents the first molecular diagnosis, isolation, and molecular characterization of E. canis in dogs from Costa Rica. (C) 2010 Elsevier Ltd. All rights reserved.

Carios mimon (Acari: Argasidae): description of adults and redescription of larva

Carios mimon is an argasid tick common on Chiroptera, originally described from larvae collected on bats Mimon crenulatum from Bolivia and Eptesicus brasiliensis from Uruguay. Later it was also registered from Argentina and recently included among the Brazilian tick fauna. In Brazil, this species is very aggressive to man, resulting in intense inflammatory response and pain. It is known only by the larval description and its morphology resembles that from other species currently included into the genus Carios, formerly classified into the subgenus Alectorobius, genus Ornithodoros. Here we describe adults and redescribe the larva of C. mimon, based on light and scanning electron microscopy. Remarks about its morphological similarity with other species of this genus are also discussed. Molecular analysis inferred from a portion of the 16S rRNA mitochondrial gene placed C. mimon in a cluster supported by maximal bootstrap value (100%) with other argasid species (mostly bat parasites in the New World), which have been classified into either the genus Ornithodoros or Carios, depending on the Argasidae classification adopted by different authors.

Detection and molecular characterization of a canine piroplasm from Brazil

In the beginning of the 20th century, a new canine disease was reported in Brazil under the name ""nambiuvu"", whose etiological agent was called Rangelia vitalii, a distinct piroplasm that was shown to parasitize not only erythrocytes, but also leucocytes and endothelial cells. In this new century, more publications on R. vitalii were reported from Brazil, including an extensive study on its ultrastructural analysis, in addition to clinical, pathological, and epidemiological data on nambiuvu. However, a molecular analysis of R. vitalii has not been performed to date. In the present study, we performed molecular phylogenetic analyses of R. vitalii based on fragments of the genes 18S rRNA and the heat shock protein 70 (hsp70), amplified by PCR performed on blood samples derived from five clinical cases of dogs presumably infected with R. vitalii in southern Brazil. In addition, we examined Giemsa-stained thin blood smears from these same dogs. DNA sequences (604-bp) of the 18S rRNA gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (95%) with Babesia sp. China-BQ1. DNA sequences (1056-bp) of the hsp70 gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (87%) with Babesia bigemina. Phylogenetic analyses inferred from either of the two genes resulted in the newly genotype being placed in the Babesia spp.sensu stricto clade with very high bootstrap support (95-100%) in three analyses (Neighbor-Joining, Maximum parsimony, and Maximum likelihood). Giemsa-stained thin blood smears from the dogs were shown to contain piroplasm organisms within erythrocytes, monocytes and neutrophils (individual forms), and schizont-like forms within neutrophils, in accordance with literature reports of R. vitalii. Based on these results, we conclude that R. vitalii, the etiological agent of ""nambiuvu"" in southern Brazil, is a valid species of piroplasm. Further studies are required to evaluate the validity of the genus Rangelia. (C) 2011 Elsevier B.V. All rights reserved.

Detection of Bartonella spp. in neotropical felids and evaluation of risk factors and hematological abnormalities associated with infection

Although antibodies to Bartonella henselae have been described in all neotropical felid species, DNA has been detected in only one species, Leopard us wiedii. The aim of this study was to determine whether DNA of Bartonella spp. could be detected in blood of other captive neotropical felids and evaluate risk factors and hematological findings associated with infection. Blood samples were collected from 57 small felids, including 1 Leopard us geoffroyi, 17 L wiedii, 22 Leopardus tigrinus, 14 Leopardus pardalis, and 3 Puma yagouaroundi; 10 blood samples from Panthera onca were retrieved from blood banks. Complete blood counts were performed on blood samples from small felids, while all samples were evaluated by PCR. DNA extraction was confirmed by amplification of the cat GAPDH gene. Bartonella spp. were assessed by amplifying a fragment of their 16S-23S rRNA intergenic spacer region; PCR products were purified and sequenced. For the small neotropical felids, risk factors [origin (wild-caught or zoo-born), gender, felid species, and flea exposure) were evaluated using exact multiple logistic regression. Hematological findings (anemia, polycythemia/hyperproteinemia, leukocytosis and leukopenia) were tested for association with infection using Fisher`s exact test. The 635 bp product amplified from 10 samples (10/67 = 14.92%) was identified as B. henselae by sequencing. Small neotropical felid males were more likely to be positive than females (95% CI = 0.00-0.451, p = 0.0028), however other analyzed variables were not considered risk factors (p > 0.05). Hematological abnormalities were not associated with infection (p > 0.05). This is the first report documenting B. henselae detection by PCR in several species of neotropical felids. (C) 2009 Elsevier B.V. All rights reserved.

Genotyping of potentially zoonotic Giardia duodenalis from exotic and wild animals kept in captivity in Brazil

We have studied the variability of glutamate dehydrogenase (gdh) and small subunit ribosomal (SSU) rRNA coding genes of Giardia species in fecal samples isolated from wild and exotic animals in Brazil, and compared with homologous sequences of isolates from human and domestic animals characterized in previous studies. Cysts of Giardia duodenalis were obtained from feces of naturally infected monkeys (Alouatta fusca) (n = 20), chinchillas (Chinchilla lanigera) (n = 3), ostriches (Struthio camelus) (n = 2) and jaguar (Panthera onca) (n = 1). Assemblage AI was assigned to the unique isolate of jaguar. All the samples from monkeys, chinchillas, and ostriches were assigned to Assemblage B. There was little evolutionary divergence between the referred isolates and isolates described elsewhere. The Assemblage B isolates identified in this study were closely related to Assemblage BIV isolated from humans. The molecular identification of Assemblages A and B of G. duodenalis isolates from exotic and wild animals demonstrates that such hosts may be a potential reservoir for zoonotic transmission of G. duodenalis. (C) 2011 Elsevier B.V. All rights reserved.

European 1: A globally important clonal complex of Mycobacterium bovis

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.

Diazepam decreases leukocyte-endothelium interactions in situ

High doses of diazepam reduce the inflammatory paw edema in rats. This effect was attributed to an action of diazepam on the Translocator Protein (TSPO). We evaluated the effects of diazepam (10 mg/kg, intraperitoneally) on leukocyte rolling and migration. In carrageenan-induced acute inflammation, diazepam decreased the interaction of leukocytes with endothelial cells (rolling) and the number of leukocytes in the mesentery (migration). RU486 (antagonist of glucocorticoid receptors) reduced the effects of diazepam on leukocyte rolling and migration, suggesting a participation of endogenous corticosteroids. We also showed that the effects of diazepam on leukocyte-endothelium interactions are mediated by nitric oxide (NO), since prior treatment with l-arginine (precursor of NO) partially precludes the inhibitory effects of diazepam; conversely, pretreatment with L-NAME (false substrate of the NO synthase) somewhat potentiates the effects of diazepam. The pathways that underlie the effects of diazepam remain to be further elucidated, but we believe that both local and systemic mechanisms may overlap to explain the influence of diazepam on leukocyte-endothelium interactions.

Physical, epidemiological, and molecular evaluation of infection by Cryptosporidium galli in Passeriformes

Due to the scarcity of information related to the epidemiology of Cryptosporidium infection in passerine birds, this study aimed to determine the periodicity of fecal shedding of Cryptosporidium spp. oocysts, after natural infection, and its clinical signs, mortality, and molecular characterization. Four hundred eighty fecal samples were collected from 40 birds, including 372 samples from 31 adult birds and 108 samples from nine young birds (up to 12 months old), housed in five aviaries, monthly from September 2007 to September 2008, with the exception of April. The birds originated from aviaries in which the following species were raised: great-billed seed-finch (Oryzoborus maximiliani), lesser seed-finch (Oryzoborus angolensis), ultramarine grosbeak (Cyanocompsa brissonii), and rusty-collared seedeater (Sporophila collaris). The samples were preserved in 2.5% potassium dichromate at 4A degrees C until processing. The oocysts were purified by centrifugal flotation in Sheather`s solution, followed by genomic DNA extraction and molecular characterization of oocysts using the nested polymerase chain reaction for amplification of fragments of the 18S subunit of rRNA gene. Intermittent shedding of oocysts was observed by positive amplification for Cryptosporidium spp. in 91 (24.5%) samples of adult birds and 14 (13%) of young birds. The sequencing of the amplified fragments enabled the identification of Cryptosporidium galli. Although all the aviaries had birds positive for C. galli, morbidity or mortality was observed in only one aviary and was associated with concomitant infection with Escherichia coli and Isospora sp.

Molecular characterization of Cryptosporidium spp. from fecal samples of birds kept in captivity in Brazil

The aim of this study was to determine the prevalence of Cryptosporidium species and genotypes in birds kept in captivity in Brazil. A total of 966 samples from 18 families of birds was collected and stored in 5% potassium dichromate solution at 4 degrees C until processing. Oocysts were purified in Sheather sugar solution following extraction of genomic DNA. Molecular analyses were performed using nested-PCR for amplification of fragments of the 18S subunit of rRNA gene and of the actin gene. Amplification of Cryptosporidium DNA fragments was obtained in 47 (4.86%) samples. Sequencing of amplified fragments and phylogenetic analyses allowed the identification of Cryptosporidium baileyi in a black vulture (Coragyps atratus), a domestic chicken (Gallus gallus domesticus) and a saffron finch (Sicalis flaveola); Cryptosporidium galli in canaries (Serinus canaria), a cockatiel (Nymphicus hollandicus) and lesser seed-finches (Oryzoborus angolensis); Cryptosporidium meleagridis in a domestic chicken (G. g. domesticus); Cryptosporidium parvum in a cockatiel (N. hollandicus); Cryptosporidium avian genotype I in a canary (S. canaria) and an Indian peafowl (Pavo cristatus); Cryptosporidium avian genotype II in ostriches (Struthio camelus) and Cryptosporidium avian genotype III in a cockatiel (N. hollandicurs) and a peach-faced lovebird (Agapornis roseicolis). (C) 2009 Elsevier B.V. All rights reserved.

Redescription of the female, description of the male, and several new records of Amblyomma parkeri (Acari: Ixodidae), a South American tick species

The tick Amblyomma parkeri Fonseca and Arago was described in 1952, based on female and immature ticks collected in the states of So Paulo and Santa Catarina, Brazil. Thereafter, there has been no further report of A. parkeri, and the male has remained unknown. Herein, we examined ticks collected on porcupines from a locality in the state of So Paulo. Some of the ticks were identified as Amblyomma longirostre (Koch, 1844), whereas others as A. parkeri, including male specimens, for which we provide the first description. We also provide additional reports of A. parkeri after examining collections of A. longirostre and Amblyomma geayi Neumann, 1899 from different tick collections. Morphological evidence to support the original description of A. parkeri is presented, supported by molecular analyses of portions of the 16S rRNA and 12S rRNA mitochondrial genes. Morphological particularities to separate A. parkeri, A. longirostre, and A. geayi are provided.

Detection of Herpesviruses and Periodontal Pathogens in Subgingival Plaque of Patients With Chronic Periodontitis, Generalized Aggressive Periodontitis, or Gingivitis

Background: Herpesviruses may be related to the etiology of aggressive periodontitis (AgP) and chronic periodontitis (CP) by triggering periodontal destruction or by increasing the risk for bacterial infection. This case-control study evaluated the presence of herpes simplex virus type 1 (HSV-1), Epstein-Barr virus type 1 (EBV-1), human cytomegalovirus (HCMV), Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia (previously T. forsythensis) in patients with generalized AgP (AgP group), CP (CP group), or gingivitis (G group) and in healthy individuals (C group). Methods: Subgingival plaque samples were collected with paper points from 30 patients in each group. The nested polymerase chain reaction (PCR) method was used to detect HSV-1, EBV-1, and HCMV. Bacteria were identified by 16S rRNA-based PCR. Results: HSV-1, HCMV, and EBV-1 were detected in 86.7%, 46.7%, and 33.3% of the AgP group, respectively; in 40.0%, 50.0%, and 46.7% of the CP group, respectively; in 53.3%, 40.0%, and 20.0% of the G group, respectively; and in 20.0%, 56.7%, and 0.0% of the C group, respectively. A. actinomycetemcomitans was detected significantly more often in the AgP group compared to the other groups (P<0.005). P. gingivalis and T. forsythia were identified more frequently in AgP and CP groups, and AgP, CP, and G groups had higher frequencies of P. intermedia compared to the C group. Conclusion: In Brazilian patients, HSV-1 and EBV-1, rather than HCMV, were more frequently associated with CP and AgP. J Periodontol 2008;79:2313-2321.