Discrimination Amongst Leishmania by Polymerase Chain Reaction and Hybridization with Small Subunit Ribosomal DNA Derived Oligonucleotides

ABSTRACT. A method for discriminating among Leishmania is described, based upon small subunit ribosomal DNA sequence differences. The method was to amplify the entire 2.2 kb small subunit rDNA by polymerase chain reaction using conserved primers specific for the 5' and 3' termini of the small subunit ribosomal RNA, and then hybridize the product dotted onto nylon membranes with labeled oligonucleotides. The design of the hybridization probes was based upon complete small subunit rDNA sequences from L. amazonensis, L. major and L. guyanensis and partial sequences of L. mexicana, L. braziliensis, L. tropica and L. chagasi. A high degree of sequence similarity (> 99%) among species was found. However, sufficient sequence divergence occurred to permit the design of internal oligonucleotide probes specific for species complexes. This procedure successfully discriminated amongst a wide range of Leishmania isolates. The method detected as few as 10 cultured organisms and detected parasites in tissue samples from experimentally infected animals. Non-radioactive labeling showed the same specificity and sensitivity as radioactive probes.     

Cl- Fluxes and Cl- Content of Dunaliella acidophila--An Alga with a Positive Membrane Potential

The Cl^– fluxes across the plasma membrane and the Cl^–content of the acid–resistant alga Dunaliella acidophila(optimal growthat pH 1.0, positive membrane potential) werestudied in the presence of 0.01–300 mM Cl^–. Up to40 mM Cl^– in the medium, theinternal Cl^– concentrationis higher than that predicted by the electrochemical equilibrium,whereas at higher external Cl^– concentrations internalCl^– levels are lower than expected for the electrochemicalequilibrium. Growth in the absence of Cl^– is significantlylower than in the standard growth medium (2.2 mM Cl^–)and this reduction cannot be overcome by the addition ofothermonovalent anions such as Br^– or NO^–₃ The latterimplies a specific Cl^– requirement in addition to therole of Cl^– as apermeant anion during ion translocations.Growth and photosynthesis tolerate an excess of Cl^– upto 300 mM (without stepwiseadaptation to increasing salinity).The uptake of Cl^– (measured by tracer techniques) exhibitsMichaelis–Menten kinetics (K_M = 0.75 mM Cl^–) andis stimulated by light and high H⁺ concentrations. Internalacidification by acetic acid causes an inhibition of Cl^–uptake. The uptake of Cl^– is inhibited by the monovalentanions Br^–, I^–, and NO^–₃ with K¹, values notvery much different from the K_M. value for Cl^–. The aniontransport inhibitors SITS and DIDS do not affect photosynthesis,but strongly suppressthe uptake of Cl^–. The Cl^–channel blockers A–9–C and NPPB cause inhibitionsof Cl^– uptake as well as of photosynthesis andthe ATPpool. FCCP strongly depresses the internal ATP–pool withouta marked effect on Cl^– uptake. Cl^– efflux was inhibitedbyDIDS and SITS, but stimulated or inhibited by FCCP, dependingon the external Cl^– concentration. Results are in agreementwiththe hypothesis that Cl^– uptake into D. acidophila is dueto catalysed diffusion and is primarily independent of the hydrolysisofATP. Cl^– efflux is assumed to be coupled to an activepump. Data suggest tight co–operativity between the systemsresponsiblefor Cl^– uptake and Cl^– efflux, with thecytoplasmic pH and the membrane potential being important mediators. Key words: Acid resistance, chloride carrier, chloride channels, Dunaliella acidophila, membrane potential, plasma membrane     

Anatomy of extrafloral nectaries in Fabaceae from dry‐seasonal forest in Brazil

Extrafloral nectaries (EFNs) are found in many species of Fabaceae. The aim of this work is to describe the internal morphology of the EFNs from species of Fabaceae found in areas of dry‐seasonal forest in north‐eastern Brazil. All species of Fabaceae with EFNs found were collected and samples were submitted to conventional techniques for anatomical and scanning electronic microscopy analysis. EFNs were found in 35 species, of which 32 were examined anatomically. All types have epidermal cells, secretory tissues and vascular bundles in the EFNs. Sclerenchymatous cells were found between the secretory tissues and the vascular tissues, with a few exceptions. The function of these cells is not clear; however, a role in the transportation of the sap in the nectary or with the support of the secretory tissue is possible. The nectar is released through glandular trichomes, secretory pores or even by breaking the epidermal cells and cuticle. The internal patterns found in the EFNs from different species and genera can provide important information for taxonomic and evolutionary studies in the family. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163 , 87–98.