In vitro studies on the mechanisms of endospore release by Rhinosporidium seeberi

Studies of Rhinosporidium seeberi have demonstrated that this organism has a complex life cycle in infected tissues. Its in vivo life cycle is initiated with the release of endospores into a host's tissues from its spherical sporangia. However, little is known about the mechanisms of sporangium formation and endospore release since this pathogen is intractable to culture. We have studied the in vitro mechanisms of endospore release from viable R. seeberi's sporangia. It was found that watery substances visibly stimulates the mature sporangia of R. seeberi to the point of endospore discharge. The internal rearrangement of the endospores within the mature sporangia, the opening of an apical pore in R. seeberi's cell wall, and the active release of the endospores were the main features of this process. Only one pore per sporangium was observed. The finding of early stages of pore development in juvenile and intermediate sporangia suggested that its formation is genetically programed and that it is not a random process. The stimulation of R. seeberi's sporangia by water supports the epidemiological studies that had linked this pathogen with wet environments. It also explains, in part, its affinities for mucous membranes in infected hosts. The microscopic features of endospore discharge suggest a connection with organisms classified in the Kingdom Protoctista. This study strongly supports a recent finding that placed R. seeberi with organisms in the protoctistan Mesomycetozoa clade. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evidence for geographic clusters: Molecular genetic differences among strains of Pythium insidiosum from Asia, Australia and the Americas are explored

Twenty-eight isolates of Pythium insidiosum and P. destruens from Asia, Australia and the Americas were compared on the basis of restriction fragment-length polymorphisms of the amplified ribosomal intergenic spacer. Comparison of band profiles yielded three distinct clusters and an isolate that did not fall into any of the clusters. Cluster I consisted of 16 isolates, all from the Americas (Costa Rica, Brazil, Haiti, United States). Cluster II consisted of seven isolates from Asia (India, Thailand, Japan, Papua New Guinea) and Australia, including the two isolates of P. destruens. This cluster also included a United States isolate from a human who might have contracted an infection of P. insidiosum by contact with food from the Middle East. Cluster III was most distantly related to the other two clusters and consisted of two isolates from Thailand and one from the United States. The isolate excluded from all three clusters was from a spectacled bear in a zoo in the United States. These results indicate that all the isolates are more closely related to each other than to any other Pythium species and thus indeed might be one species, but they also point to geographical variants. Cluster III and Isolate M18 are so distant from the others that they might prove to be separate species. Knowledge of intraspecific variability in P. insidiosum might be important for the management of pythiosis in mammals.     

Uptake of water by lateral roots of small trees in an Amazonian Tropical Forest

A pulse chase technique was used to determine depth and breath of plant water uptake in an Amazonian evergreen forest. Two 2×2 m² plots were irrigated with deuterated water. The deuterium pulse, measured as D values of soil and plant sap water, was followed in the soil water profile and in stem water of small trees inside and up to 12 m away from the irrigated plots. The deuterium pulse percolation rate was measured to be approximately 0.25 m/month and similar to a previous study in central Amazon. There was little horizontal movement of label through the soil profile; allowing us to conclude that any evidence of label in plants away from the irrigation plots implies the presence of their roots inside the irrigation plots. The bulk of label uptake occurred in plants inside the irrigation plots. However, there were a few individuals as far as 10 m away picking up the label from the irrigation plots. This labeling pattern leads to the conclusion that small trees may have a core of water absorbing roots close to their main trunk, with some roots meandering far from their main trunk.     

Natural Schistosoma mansoni infection in Nectomys squamipes: histopathological and morphometric analysis in comparison to experimentally infected N. squamipes and C3H/He mice

Histopathologic and morphometric (area, perimeter, major and minor diameters) analysis of hepatic granulomas isolated from twelve naturally infected Nectomys squamipes were compared to four experimentally infected ones and six C3H/He mice. Liver paraffin sections were stained for cells and extracellular matrix. Both groups of N. squamipes presented peculiar granulomas consisting predominantly of large macrophages, full of schistosome pigment, characterizing an exudative-macrophage granuloma type, smaller than the equivalent granuloma type in mouse. Naturally infected animals exhibited granulomas in different stages of development, including large number of involutional types. Morphometric analysis showed that all measurements were smaller in naturally infected animals than in other groups. The results demonstrated that both N. squamipes groups reproduced, with small variations, the hepatic granuloma aspects already described in cricetidium (Calomys callosus), showing a genetic tendency to set up strong macrophage responses and small granulomas. Unexpectedly, natural infection did not engender distinguished histopathological characteristics distinct from those derived from experimental single infection, showing changes predominantly secondary to the duration of infection. It appears that the variability of the inocula (and the number of infections?) interfere more with the quantity than with the quality of the pathological changes, denoting some morpho-functional determinism in the response to schistosomal infection dependent on the animal species.     

Heterologous array analysis in Pinaceae: hybridization of Pinus taeda cDNA arrays with cDNA from needles and embryogenic cultures of P. Taeda, P. Sylvestris or Picea abies

Hybridization of labelled cDNA from various cell types with high-density arrays of expressed sequence tags is a powerful technique for investigating gene expression. Few conifer cDNA libraries have been sequenced. Because of the high level of sequence conservation between Pinus and Picea we have investigated the use of arrays from one genus for studies of gene expression in the other. The partial cDNAs from 384 identifiable genes expressed in differentiating xylem of Pinus taeda were printed on nylon membranes in randomized replicates. These were hybridized with labelled cDNA from needles or embryogenic cultures of Pinus taeda, P. sylvestris and Picea abies, and with labelled cDNA from leaves of Nicotiana tabacum. The Spearman correlation of gene expression for pairs of conifer species was high for needles (r(2) = 0.78 - 0.86), and somewhat lower for embryogenic cultures (r(2) = 0.68 - 0.83). The correlation of gene expression for tobacco leaves and needles of each of the three conifer species was lower but sufficiently high (r(2) = 0.52 - 0.63) to suggest that many partial gene sequences are conserved in angiosperms and gymnosperms. Heterologous probing was further used to identify tissue-specific gene expression over species boundaries. To evaluate the significance of differences in gene expression, conventional parametric tests were compared with permutation tests after four methods of normalization. Permutation tests after Z-normalization provide the highest degree of discrimination but may enhance the probability of type I errors. It is concluded that arrays of cDNA from loblolly pine are useful for studies of gene expression in other pines or spruces.     

Studying the functional genomics of stress responses in loblolly pine with the Expresso microarray experiment management system

Conception, design, and implementation of cDNA microarray experiments present a variety of bioinformatics challenges for biologists and computational scientists. The multiple stages of data acquisition and analysis have motivated the design of Expresso, a system for microarray experiment management. Salient aspects of Expresso include support for clone replication and randomized placement; automatic gridding, extraction of expression data from each spot, and quality monitoring; flexible methods of combining data from individual spots into information about clones and functional categories; and the use of inductive logic programming for higher-level data analysis and mining. The development of Expresso is occurring in parallel with several generations of microarray experiments aimed at elucidating genomic responses to drought stress in loblolly pine seedlings. The current experimental design incorporates 384 pine cDNAs replicated and randomly placed in two specific microarray layouts. We describe the design of Expresso as well as results of analysis with Expresso that suggest the importance of molecular chaperones and membrane transport proteins in mechanisms conferring successful adaptation to long-term drought stress.     

Carrageenophytes of occidental Portuguese coast: 1-spectroscopic analysis in eight carrageenophytes from Buarcos bay

Infrared and Raman spectroscopic analysis of the carrageenan (alkaline extraction) in eight species (representing seven genera and four families) of Gigartinales, in different reproductive phases from Buarcos bay (Figueira da Foz, Portugal), were studied. Female gametophytes and non-fertile thalli samples of Chondrus crispus, Mastocarpus stellatus, Chondracanthus teedei var. lusitanicus, Gigartina pistillata and Chondracanthus acicularis present a kappa-carrageenan profile or varying degrees of a kappa-iota hybrid. The presence of kappa-iota hybrid carrageenan in C. teedei var. lusitanicus was confirmed by 13C NMR. The carrageenans extracted from Gymnogongrus crenulatus and Ahnfeltiopsis devoniensis are constituted mainly by iota-carrageenan but seasonal variations in the nature of carrageenans are present. lambda-Family carrageenans were found in tetrasporophytes of C. crispus, M. stellatus, C. teedei var. lusitanicus, C. acicularis and G. pistillata. Calliblepharis jubata presents carrageenans of iota-type in all reproductive stages.