Mitochondrial function in the yeast form of the pathogenic fungus <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis>

Differences between the respiratory chain of the fungus Paracoccidioides brasiliensis and its mammalian host are reported. Respiration, membrane potential, and oxidative phosphorylation in mitochondria from P. brasiliensis spheroplasts were evaluated in situ, and the presence of a complete (Complex I–V) functional respiratory chain was demonstrated. In succinate-energized mitochondria, ADP induced a transition from resting to phosphorylating respiration. The presence of an alternative NADH–ubiquinone oxidoreductase was indicated by: (i) the ability to oxidize exogenous NADH and (ii) the lack of sensitivity to rotenone and presence of sensitivity to flavone. Malate/NAD⁺-supported respiration suggested the presence of either a mitochondrial pyridine transporter or a glyoxylate pathway contributing to NADH and/or succinate production. Partial sensitivity of NADH/succinate-supported respiration to antimycin A and cyanide, as well as sensitivity to benzohydroxamic acids, suggested the presence of an alternative oxidase in the yeast form of the fungus. An increase in activity and gene expression of the alternative NADH dehydrogenase throughout the yeast’s exponential growth phase was observed. This increase was coupled with a decrease in Complex I activity and gene expression of its subunit 6. These results support the existence of alternative respiratory chain pathways in addition to Complex I, as well as the utilization of NADH-linked substrates by P. brasiliensis. These specific components of the respiratory chain could be useful for further research and development of pharmacological agents against the fungus.     

Epithelial cells treated with genistein inhibit adhesion and endocytosis of<Emphasis Type="Italic"> Paracoccidioides brasiliensis</Emphasis>

Paracoccidioidomycosis is caused by Paracoccidioides brasiliensis, which although not formally considered an intracellular pathogen, can be internalized by epithelial cells in vitro and in vivo. The mechanisms used by P. brasiliensis to adhere to and invade non-professional phagocytes have not been identified. The signal-transduction networks, involving protein tyrosine kinase (PTK) and protein phosphatase activities, can modulate crucial events during fungal infections. In this study, the involvement of PTK has been investigated in P. brasiliensis adherence and invasion in mammalian epithelial cells. A significant inhibition of the fungal invasion occurred after the pre-treatment of the epithelial cells with genistein, a specific tyrosine kinase inhibitor, indicating that the tyrosine kinase pathway is involved in P. brasiliensis internalization. In contrast, when the fungus was treated, a slight (not significant) inhibition of PTK was observed, suggesting that PTK might not be the fungus’ transduction signal pathway during the invasion process of epithelial cells. An intense PTK immunofluorescence labeling was observed in the periphery of the P. brasiliensis infected cells, little PTK labeling was found in both uninfected cells and yeast cells, at later infection times (8 and 24 h). Moreover, when the epithelial cells were treated with genistein and infected with P. brasiliensis, no labeling was observed, suggesting the importance of the PTK in the infectious process. These results suggest that PTK pathway participates in the transduction signal during the initial events of the adhesion and invasion processes of P. brasiliensis to mammalian epithelial cells.     

Prostaglandin E<Subscript>2 </Subscript>production by high and low virulent strains of <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis>

The production of prostaglandins (PGs) during fungal infections could be an important suppressor factor of host immune response. Host cells are one source of prostaglandin E₂ (PGE₂); however another potential source of PGE₂ is the fungal pathogen itself. Thus, both host and fungal PGE2 production is theorized to play a role in pathogenesis, being critical for growth of the fungus and to modulate the host immune response. The purpose of this work was to investigate if high and low virulent strains of Paracoccidioides brasiliensis have the capacity to produce PGE₂ in vitro, and if this production was related to the fungal growth. The results demonstrated that both strains of P. brasiliensis produce high levels of PGE₂ and the treatment with indomethacin, a cyclooxygenase inhibitor, significantly reduced the production of this mediator, as well as the viability of the fungus. Thus, our data indicate that PGE₂ is produced by P. brasiliensis by a cyclooxygenase–dependent metabolic pathway, and its production is required for fungal survival. This discovery reveals an important factor that has potentially great implications for understanding the mechanisms of immune deviation during infection.     

Protection induced in BALB/c mice by the high-molecular-mass (hMM) fraction of <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis>

Paracoccidioidomycosis (PCM) is a granulomatous disease caused by a dimorphic fungus, Paracoccidioides brasiliensis. The present study investigated the protective activity of the P. brasiliensis high-molecular-mass (hMM) fraction (~380 kDa) in experimental murine PCM. In the first step, lymphocyte proliferation and production of IFNγ (but not IL-4) were observed in “in vitro” spleen cells (from female BALB/c mice infected (i.v.) with P. brasiliensis) that were stimulated with hMM fractions. In the second step, female BALB/c mice were previously immunized (s.c.) with hMM fraction (25 μg/protein = F-25 and 50 μg/protein = F-50), and the colony-forming units (CFU) of the lung and spleen, the histopathological characteristics of the granulomatous lesions, and plasmatic gp43 soluble antigens and anti-hMM IgG levels were analyzed at 28 and 56 days after infection. The lung and liver CFU were lower in mice previously immunized with the hMM fraction (P < 0.05). The granulomatous lesions revealed a greater degree of compaction and organization, with no dissemination of the fungus to other organs. Lower soluble antigen levels (P < 0.05) and higher IgG anti-hMM fraction (P < 0.05) were observed in immunized groups. The results for CFU, histopathology and antigenemia suggest that the hMM fraction has a protective effect in experimental paracoccidioidomycosis in BALB/c mice.     

Structural comparison of the <Emphasis Type="Italic">hsp70</Emphasis> gene cluster in the <Emphasis Type="Italic">Drosophila virilis</Emphasis> species group

One of the Drosophila montana hsp70 genes was cloned and sequenced. Its 3′-flanking sequence proved to harbor a fragment of the SGM mobile element. The element was also found in the hsp70 3′-flanking region of and other species of the species group. A reorganization of the cluster with the involvement of full-length SGM was found in a strain. It was assumed that the presence of SGM in the cluster is conserved among species of the group and that SGM played a role in evolutionarily rearrangements of the cluster.     

Mice Immunization with Radioattenuated Yeast Cells of <Emphasis Type="Italic">Paracoccidiodes brasiliensis</Emphasis>: Influence of the Number of Immunizations

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America. Up to the moment no vaccine has been reported. The aim of this study was to evaluate the influence of the number of immunizations on the protection elicited by radioattenuated yeast cells of P. brasiliensis. BALB/c mice were divided into two groups that were immunized once (Group 1) or twice (Group 2), respectively. In each group, mice were divided into sub-groups that were challenged 30, 45, or 60 days after the second immunization. Organ colony-forming units (CFUs) was determined 90 days post-challenge. A significant reduction in CFUs recovery was verified in both groups, but it was higher in Group 2. Histologic alterations were observed only in Group 1. The cytokines IL-4, IL-10, and IFN-γ were produced in mice of Group 1. In Group 2, only IFN-γ was significantly detected. IgG2a predominance relative to IgG1 was also observed in Group 2. Altogether, our results indicated that mice immunized once developed a mixed Th1/Th2 response, which was less efficient in the infection control, while a trend to a Th1 pattern was obtained with two immunizations, promoting optimal elimination of P. brasiliensis yeast cells from mice tissues.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Characteristics of Environmental <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Isolates

The ecological niche or exact habitat of the fungus Paracoccidioides brasiliensis is not known, and few isolates have been obtained from the environment. In this study, ten isolates were analyzed with respect to antigenic composition, serology, pathogenicity, and molecular aspects. Gp43 is considered to be the molecular basis for the serodiagnosis of paracoccidioidomycosis; however, in this study only six of the environmental isolates secreted this molecule (four in great amounts and two in small amounts). Other molecules were also produced. When exoantigens from these isolates were tested using immunodiffusion, only four preparations were positive by ID tests. However, when these exoantigens were tested by ELISA, all of them except one were able to detect anti-P. brasiliensis antibodies. In Western blot assays, these exoantigens showed different reactivities. Isolates that secreted gp43 presented positive reactions for this molecule, and isolates that did not secrete gp43 gave positive reactions for other minor molecules. RAPD analysis revealed that there is great genetic variation between these environmental isolates. These isolates were non-pathogenic: no mortality was observed among the inoculated mice during an 18-month follow-up period.     

<Emphasis Type="Italic">Caragana aliensis</Emphasis>, a new species of <Emphasis Type="Italic">Caragana</Emphasis> (<Emphasis Type="Italic">Leguminosae</Emphasis>) from Tibet,China

Summary   Caragana aliensis a new species from Tibet, China, is described and illustrated.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Influence of Temperature on Virulence of Fungal Isolates of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and <Emphasis Type="Italic">Beauveria bassiana</Emphasis> to the Two-Spotted Spider Mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Twenty-three isolates of Metarhizium anisopliae (Metschnikoff) Sokorin and three isolates of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales: Clavicipitaceae) were assessed for their virulence against the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). Based on the screening results, nine isolates of M. anisopliae and two isolates of B. bassiana were tested for their virulence against young adult (1- to 2-day-old) female T. urticae at constant temperatures of 20, 25, 30 and 35°C. At all temperatures tested, all the fungal isolates were pathogenic to T. urticae but mortality varied with isolates and temperatures. Fungal isolates were more virulent at 25, 30 and 35°C than at 20°C. The lethal time to 50% mortality (LT₅₀) and lethal time to 90% mortality (LT₉₀) values decreased with increased temperature. There were no significant differences in virulence between fungal isolates at 30 and 35°C; however, significant differences were observed at 20 and 25°C.     

New species in <Emphasis Type="Italic">Barleria</Emphasis> sect. <Emphasis Type="Italic">Stellatohirta</Emphasis> (<Emphasis Type="Italic">Acanthaceae</Emphasis>) from Africa

Summary  Three new species are described in Barleria L. sect. Stellatohirta M. Balkwill from tropical Africa: B. aristata from south-central Tanzania, B. aenea from south-western Tanzania and northeast Zambia, and B. purpureotincta from south-western Zambia. Their affinities and conservation status are discussed.     

A novel cryptic plasmid pBMB175 from <Emphasis Type="Italic">Bacillus thuringiensis </Emphasis>subsp. <Emphasis Type="Italic">tenebrionis </Emphasis>YBT-1765

A new cryptic plasmid pBMB175 from Bacillus thuringiensis subsp. tenebrionis YBT-1765 was isolated and characterized. Sequence analysis showed that pBMB175 (14,841 bp and 31% GC content) contained at least eighteen putative open reading frames (ORFs), among which nine ORFs displayed the homology with the hypothetical proteins in rolling-circle replication plasmid pGI3. Deletion analysis revealed that the pBMB175 minireplicon located in a novel 1,151 bp fragment. This fragment contains ORF7 coding sequence, which encodes a protein (Rep175, 149 amino acids [aa]) indispensable for plasmid replication. Rep175 has no significant homology with known function proteins. Furthermore, a putative double-strand origin (dso), having no DNA similarity with characterized dso of other replicon so far, was identified in this minireplicon fragment. These features showed that pBMB175 could be placed into a new plasmid family.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan