High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity

The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.      

Corynebacterium pseudodiphtheriticum isolated from relevant clinical sites of infection: a human pathogen overlooked in emerging countries

Aims:  To examine the occurrence of and to determine the antimicrobial susceptibility of Corynebacterium pseudodiphtheriticum among patients with bacterial infections at a teaching hospital.
Methods and Results:  A total of 113 Coryne. pseudodiphtheriticum strains identified by conventional biochemical methods and API-Coryne System were recovered from patients from different age groups: 65·48% adults (18 to ≤59 years old), 9·73% aged (≥60 years old); 14·15% infants (<18 years old); 4·42% newborns (0–7 days). Micro-organisms were mostly related to infections in the urinary (29·2%) and respiratory tracts (27·45%) and intravenous sites (18·6%). Clinical samples were obtained only from 32·7% patients (26 adults, four aged, four infants and three newborns) presenting at least one of the predisposing conditions: end-stage renal disease; renal transplant; AIDS and Mycobacterium tuberculosis infection; cancer, hepatic cirrhosis; haemodialysis and catheter use. Antimicrobial susceptibility tests identified multiresistant phenotypes. Most strains (>50%) were resistant to oxacillin, erythromycin and clindamycin.
Conclusions:  Despite significant differences in age and functional status of patients Coryne. pseudodiphtheriticum may be implicated as a cause of respiratory and nonrespiratory human infections.
Significance and Impact of the Study:  Data are valuable for practitioners indicating the occurrence of multiresistant phenotypes and the possibility of severe infections due to Coryne. pseudodiphtheriticum , a pathogen usually overlooked in emerging countries.     

The role of lateral roots in bypass flow in rice (Oryza sativa L.)

Although an apoplastic pathway (the so‐called bypass flow) is implicated in the uptake of Na⁺ by rice growing in saline conditions, the point of entry of this flow into roots remains to be elucidated. We investigated the role of lateral roots in bypass flow using the tracer trisodium‐8‐hydroxy‐1,3,6‐pyrenetrisulphonic acid (PTS) and the rice cv. IR36. PTS was identified in the vascular tissue of lateral roots using both epifluorescence microscopy and confocal laser scanning microscopy. Cryo‐scanning electron microscopy and epifluorescence microscopy of sections stained with berberine‐aniline blue revealed that the exodermis is absent in the lateral roots. We conclude that PTS can move freely through the cortical layers of lateral roots, enter the stele and be transported to the shoot via the transpiration stream.     

Soybean physiology and gene expression during drought

Soybean genotypes MG/BR46 (Conquista) and BR16, drought-tolerant and -sensitive, respectively, were compared in terms of morphophysiological and gene-expression responses to water stress during two stages of development. Gene-expression analysis showed differential responses in Gmdreb1a and Gmpip1b mRNA expression within 30 days of water-deficit initiation in MG/BR46 (Conquista) plants. Within 45 days of initiating stress, Gmp5cs and Gmpip1b had relatively higher expression. Initially, BR16 showed increased expression only for Gmdreb1a, and later (45 days) for Gmp5cs, Gmdefensin and Gmpip1b. Only BR16 presented down-regulated expression of genes, such as Gmp5cs and Gmpip1b, 30 days after the onset of moisture stress, and Gmgols after 45 days of stress. The faster perception of water stress in MG/BR46 (Conquista) and the better maintenance of up-regulated gene expression than in the sensitive BR16 genotype imply mechanisms by which the former is better adapted to tolerate moisture deficiency.     

Prion‐like Doppel gene polymorphisms and scrapie susceptibility in portuguese sheep breeds

The establishment of an association between prion protein gene (PRNP) polymorphisms and scrapie susceptibility in sheep has enabled the development of breeding programmes to increase scrapie resistance in the European Union. Intense selection for PRNP genotype may lead to correlated selection for genes linked to PRNP. We intended to investigate if any association exists between genetic variation in prion‐like protein Doppel gene (PRND) and scrapie susceptibility, determined through PRNP genotyping. Sampling included 460 sheep from eight Portuguese breeds and the PRND gene coding region was analysed by multiple restriction fragment‐single strand conformation polymorphism (MRF‐SSCP), whereas PRNP genotyping was carried out by primer extension. A synonymous substitution (c.78G>A) was detected in codon 26 of the PRND gene, in all breeds except Churra Mondegueira. Linkage disequilibrium was found between the PRND and PRNP loci (P = 0.000). Specifically, PRND was monomorphic in the 45 animals with the more resistant ARR/ARR PRNP genotype (P = 0.003), whereas a higher frequency of PRND heterozygotes (GA) was associated with ARQ/AHQ (P = 0.029). These results constitute preliminary evidence of an association between a polymorphism in the PRND gene and scrapie susceptibility, and indicate that the possibility of undesirable consequences from widespread selection for PRNP genotype on genetic diversity and reproduction traits needs to be further investigated.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.