Arginine auxotrophs of Candida albicans deficient in argininosuccinate lyase

Auxotrophic mutants of Candida albicans FC18 were induced by a combination of treatments with nitrous acid and UV irradiation. Arginine (Arg-), histidine (His-) and methionine/cysteine (MetA-) auxotrophs were recovered by this means. The Arg- auxotrophs lacked active argininosuccinate lyase (EC 4.3.2.1), the enzyme catalysing the final step in arginine biosynthesis. Thus the locus may be designated arg-4. The mutant strains bearing this mutation did not form germ tubes unless the germination medium contained arginine.     

Polyphyly of Asian Tree Toads,Genus Pedostibes Günther, 1876 (Anura: Bufonidae), and the Description of a New Genus from Southeast Asia

The Asian Tree Toad genus Pedostibes, as currently understood, exhibits a conspicuously disjunct distribution, posing several immediate questions relating to the biogeography and taxonomy of this poorly known group. The type species, P. tuberculosus and P. kempi, are known only from India, whereas P. hosii, P. rugosus, and P. everetti are restricted to Southeast Asia. Several studies have shown that these allopatric groups are polyphyletic, with the Indian Pedostibes embedded within a primarily South Asian clade of toads, containing the genera Adenomus, Xanthophryne, and Duttaphrynus. Southeast Asian Pedostibes on the other hand, are nested within a Southeast Asian clade, which is the sister lineage to the Southeast Asian river toad genus Phrynoidis. We demonstrate that Indian and Southeast Asian Pedostibes are not only allopatric and polyphyletic, but also exhibit significant differences in morphology and reproductive mode, indicating that the Southeast Asian species’ are not congeneric with the true Pedostibes of India. As a taxonomic solution, we describe a new genus, Rentapia gen. nov. to accommodate the Southeast Asian species.     

A Novel Flow Cytometric Hemozoin Detection Assay for Real-Time Sensitivity Testing of Plasmodium falciparum

Resistance of Plasmodium falciparum to almost all antimalarial drugs, including the first-line treatment with artemisinins, has been described, representing an obvious threat to malaria control. In vitro antimalarial sensitivity testing is crucial to detect and monitor drug resistance. Current assays have been successfully used to detect drug effects on parasites. However, they have some limitations, such as the use of radioactive or expensive reagents or long incubation times. Here we describe a novel assay to detect antimalarial drug effects, based on flow cytometric detection of hemozoin (Hz), which is rapid and does not require any additional reagents. Hz is an optimal parasite maturation indicator since its amount increases as the parasite matures. Due to its physical property of birefringence, Hz depolarizes light, hence it can be detected using optical methods such as flow cytometry. A common flow cytometer was adapted to detect light depolarization caused by Hz. Synchronized in vitro cultures of P. falciparum were incubated for 48 hours with several antimalarial drugs. Analysis of depolarizing events, corresponding to parasitized red blood cells containing Hz, allowed the detection of parasite maturation. Moreover, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 to 24 hours of incubation. At 24 hours incubation, 50% inhibitory concentrations (IC50) were comparable to previously reported values. These results indicate that the reagent-free, real-time Hz detection assay could become a novel assay for the detection of drug effects on Plasmodium falciparum.     

Analysis of lymphocytes reactive to histocompatibility antigens. III. Detection of inclusion among allo-reactive lymphocyte populations by specific depletion of reactive cells.

We have used a quantitative titration assay for mixed lymphocyte interactions to determine the additivity of responses of rat lymphocyte populations to simultaneous stimulation with major histocompatibility complex alloantigens from pairs of apparently unrelated rat strains. Our inability to detect strict additive stimulation led us to investigate inclusion among allo-reactive lymphocyte populations. After specific depletion of reactivity to PVG by negative selection through an AO × PVG F₁ hybrid rat, AO thoracic duct lymphocytes showed an asymmetrical loss of reactivity to the unrelated strains DA and F344. We suggest that this differential loss of reactivity probably reflects alloantigen sharing among rat strains which had been previously undetected using other less sensitive techniques for the quantitation of lymphocyte reactivity after specific depletion of allo-reactive cells.     

MYELOGRAPHY—Diagnostic Value in Lesions of the Lumbar Intervertebral Discs with a Variation in Technique

Myelography using pantopaque in greater than usual amount with a variation in technique, which is described, is believed to provide increased accuracy in differential diagnosis and precise localization of lesions in the lumbar spine. The need for multiple space exploration is eliminated and more detailed information concerning the size and shape of lesions is provided as compared to that secured by the use of 3 or 6 cc. of opaque medium and fluoroscopic examination alone. In 53 cases in which lumbar myelography was performed and the diagnosis verified or disproved at operation, there was a 5 per cent diagnostic error in 41 instances in which the method outlined was used, as compared with 17 per cent error in 12 cases in which only 3 or 6 cc. of radiopaque material and fluoroscopy alone were used. The accuracy of the procedure would appear to warrant its use in the evaluation of patients suspected of having abnormalities of the lumbar discs associated with nerve root compression.     

Calcium binding mode of gamma-carboxyglutamic acids in conantokins.

Conantokin-T (con-T) and conantokin-G (con-G) are two highly homologous peptide toxins found in Conus venom. The former is a 21-residue peptide with four gamma-carboxyglutamic acid (Gla) residues (at positions 3, 4, 10 and 14), while the latter is a 17-residue peptide with five gamma-carboxyglutamic acid residues (at positions 3, 4, 7, 10 and 14). Despite the apparent similarity in number and relative positions of the gamma-carboxyglutamic acid residues, (113)Cd-NMR studies indicated a distinct metal binding behavior for con-G and con-T. There appears to be four binding sites in con-G in contrast to one metal binding site in con-T. To elucidate the mode of calcium binding by the gamma-carboxyglutamic acid residues in these conantokins, we designed various analogous peptides with their gamma-carboxyglutamic acid replaced by other amino acid residues. (113)Cd-NMR experiments on conantokin analogues reveal that the major difference in the number of metal binding sites between con-G and con-T is due to the residue at position 7. We also performed molecular simulations to calculate the relative binding free energies of several potential binding sites. Based on our theoretical and experimental results, we propose a 'four-site' binding model for conantokin-G and a 'single-site' binding model for conantokin-T.