Molecular and physiological evaluation of subtropical environmental isolates of Acanthamoeba spp., causal agent of Acanthamoeba keratitis

Previous molecular examination of Acanthamoeba spp. has resulted in the determination of distinct genotypes in this genus (designated T1-T12, T14). Genotype T4 has been responsible for the majority of cases of Acanthamoeba keratitis. Here we examine the relative abundance of environmental T4 isolates on beaches and ask whether they have temperature and salinity tolerances that could enhance pathogenicity. Twenty-four Acanthamoeba strains were isolated from beach sand (n = 20), soil (n = 3), and tap water (n = 1) in south Florida. Phylogenetic analysis identified 19 of 24 isolates as T4, the Acanthamoeba keratitis-associated genotype. The remaining isolates were genotype T5 (4) and T11 (1). Nearly all beach isolates were genotype T4, whereas the tap water and soil isolates were mostly T5. All amoebae grew at 0, 1.0, and 2.0% salt and 19 of 20 beach isolates also grew at 3.2%. No soil or tap-water acanthamoebae reproduced at 3.2%. All isolates grew at 37 degrees C and two (T5) at 42 degrees C. Little correlation existed between beach location, salt-tolerance, and genetic relatedness. Overall, the large majority of environmental isolates obtained were genotype T4, suggesting it may be the most common genotype in this environment and could be a potential source of Acanthamoeba keratitis infections.     

Remarkable Salinity Tolerance of Seven Species of Naked Amoebae (gymnamoebae)

The salinity tolerance of naked amoebae collected from sites ranging from ca. 0‰ to 160‰ were compared in laboratory experiments. Amoebae were collected from hypersaline ponds around the perimeter of the Salton Sea, California, where salinities averaged 160‰, and directly from the shoreline waters of the Sea where salinities were generally between 44 and 48‰. Naked amoebae were also collected from the intertidal zone of a Florida beach, a habitat subject (on occasion) to salinity fluctuations within the range 6–85‰. From these combined sites, 6 clones of amoebae were isolated for salinity tolerance experiments (2 marine beach isolates, 2 Salton Sea isolates, and 2 hypersaline pond isolates). A seventh clone, Acanthamoeba polyphaga, a common freshwater/soil amoeba, was obtained from a Culture Collection. Laboratory experiments compared the effects of gradually changing culture salinity versus no salinity acclimatization. Growth rate and culture yield were used as indices of effect. Generally, amoebae were tolerant over a wide range of salinity conditions (in terms of growth and yield) and were not markedly influenced by pre-conditioning to salinity changes throughout the experiments. Overall, the freshwater amoeba Acanthamoeba grew between 0 and 12‰, the marine clones grew in the range of 2–120‰, and the Salton Sea clones reproduced between 0 and 138 ‰. The hypersaline clones were the most resilient and grew between 0 and 270‰ salt. The survival and activity of large populations of naked amoebae in sites subject to salinity fluctuations suggest that they should be considered in future studies to better understand their, as yet, undefined ecological role.     

Tissue distribution of lipogenesis in vivo in the common murre (Uria aalge) and the domestic chicken (Gallus domesticus)

1. Total body lipogenesis was similar in the murre and the chicken. 2. The liver contributes 10.4% to whole body lipogenesis in fed murres when measured in vivo using 3H2O. 3. The liver contributes 28.0% to whole body lipogenesis in the fed chicken. 4. The lower contribution of the liver in the murre may be a consequence of the high fat diet of the murre relative to the chicken.     

Androgen receptor in rat liver: characterization and separation from a male-specific estrogen-binding protein

Many liver processes are sexually dimorphic. In particular, the microsomal content of specific enzymes and the synthesis of specific proteins are under sex steroid hormone control. Because the liver of male rats is strikingly androgen responsive, we sought evidence for an androgen receptor in this tissue. We detected and characterized both cytosolic and nuclear androgen-binding proteins. Both forms bind [3H]R1881 (methyltrienolone, 17 beta-hydroxy-17 alpha-methyl-4,9,11-estratriene-3-one) with the high affinity, low capacity, and specificity for androgens and antiandrogens characteristic of androgen receptors. No high-affinity binding of [3H]DHT could be detected in unfractionated cytosol because of the rapid metabolism of this ligand; however, binding of a DHT metabolite to the high-capacity male-specific estrogen binder (MEB) of cytosol was observed. Both gel filtration and heparin-Sepharose affinity chromatography separate the cytosolic androgen receptor from MEB. Incubation of cytosol in the absence of sodium molybdate resulted in androgen-binding activity which was retained by DNA-cellulose. Castration of male rats results in a time-dependent loss of both cytosolic and nuclear androgen binding, as well as a loss in MEB activity. Androgen-binding activity is low in livers from female rats, but can be induced by testosterone treatment. An intact pituitary is necessary for maintenance of androgen-binding activity, as hypophysectomy results in complete loss of activity.     

Use of alcohol oxidase to measure the methanol produced during the hydrolysis of D- and L-methyl-3-hydroxybutyric acid

An enzymatic assay for the measurement of methanol has been developed. The assay uses alcohol oxidase and peroxidase coupled to the oxidation of 2,2'-azino-di-(3-ethyl)-benzthiazoline-6-sulfonic acid as the chromogen. The assay is linear up to 50 nmol of methanol in a 200-microliters sample and sensitive; 1.25 nmol of methanol in a 200-microliters sample can be measured. The assay is rapid and measurements can be made at any convenient time between 15 min and 4 h after initiation of the reaction. The assay shows highest activity with methanol but significant activity with other primary alcohols up to 1-butanol. Little activity is shown with secondary alcohols and diols. We have used this assay to follow the hydrolysis of the two isomers of the methyl ester of 3-hydroxybutyric acid.     

Production and Purification of the Extracellular Ribonuclease of Bacillus Amyloliquefaciens (Barnase) and its Intracellular Inhibitor (Barstar)

Procedures are presented for the large-scale growth of Bacillus amylo-liquefaciens and the concentration and purification of its extracellular ribo-nuclease (barnase). Improvements over earlier methods include economy, the reduction of labor, and Improved purity of product, especially in the elimination of traces of protease. The bacterial cells obtained as a by-product may be used as a starting point for the isolation of barstar, the intracellular inhibitor of barnase¹. Experiments relating to the purity, amino-acid composition and molecular weight of the product are also reported.     

Relevant assay to study the adhesion of Plasmodium falciparum-infected erythrocytes to the placental epithelium

In placental malaria, Plasmodium falciparum-infected erythrocytes adhere to the apical plasma membrane of the placental epithelium, triggering an impairment of placental function detrimental to the fetus. The design of anti-adhesion intervention strategies requires a detailed understanding of the mechanisms involved. However, most adhesion assays lack in vivo relevance and are hardly quantitative. Here, we describe a flow cytometry-based adhesion assay that is fully relevant by using apical epithelial plasma membrane vesicles as the adhesion matrix, and being applicable to infected erythrocytes directly isolated from patients. Adhesion is measured both as the percentage of pathogens bound to epithelial membrane vesicles as well as the mean number of vesicles bound per infected erythrocytes. We show that adhesins alternative to those currently identified could be involved. This demonstrates the power of this assay to advance our understanding of epithelial adhesion of infected erythrocytes and in the design of intervention strategies.     

Effects of lipid environment on the conformational changes of an ABC importer

In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.     

Antibody to P. falciparum in pregnancy varies with intermittent preventive treatment regime and bed net use

BACKGROUND: Antibodies towards placental-binding P. falciparum are thought to protect against pregnancy malaria; however, environmental factors may affect antibody development. Methods and Findings: Using plasma from pregnant Malawian women, we measured IgG against placental-binding P. falciparum parasites by flow cytometry, and related results to intermittent preventive treatment (IPTp) regime, and bed net use. Bed net use was associated with decreased antibody levels at mid-pregnancy but not at 1 month post partum (1 mpp). At 1 mpp a more intensive IPTp regime was associated with decreased antibody levels in primigravidae, but not multigravidae. CONCLUSIONS/SIGNIFICANCE: Results suggest bed nets and IPTp regime influence acquisition of pregnancy-specific P. falciparum immunity.