Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing

Pernin P. 1984. Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing. International Journal for Parasitology14: 459–465. The isoenzymatic patterns of different strains of Naegleria were studied by isoelectric focusing (I.E.F.) on polyacrylamide gels for seven enzymatic activities (leucine amino peptidase; lactate dehydrogenase; glucose 6 phosphate dehydrogenase; propionyl esterase; glucose phosphate isomerase; malate dehydrogenase; acid phosphatase), two of which (lactate dehydrogenase and glucose 6 phosphate dehydrogenase) were being investigated for the first time. The three pathogenic N. fowleri strains share a common pattern for most of the enzymes tested except for glucose 6 phosphate dehydrogenase, and thus form a very homogeneous species, while thermophilic nonpathogenic strains show more heterogeneity particularly for leucine amino peptidase and glucose 6 phosphate dehydrogenase.I.E.F. must be considered as a supplementary and rapid method for the identification of N. fowleri and as a powerful tool to demonstrate the complexity of different genera of free-living amoebas.     

Use of an axenic medium for differentiation between pathogenic and nonpathogenic Naegleria fowleri isolates.

Growth in an axenic medium composed by Chang (3rd Int. Congr. Parasitol. Munich Abstr. ICPIII 1:187-188, 1974) allowed separation of pathogenic from nonpathogenic Naegleria fowleri strains, since only the former show luxuriant growth in this medium. On the basis of these results, this medium was used in early screening for virulent Naegleria isolates. During an extensive ecological study, data were obtained on 102 Naegleria strains. Twenty of these strains grew luxuriantly in this liquid medium. Seventeen of them were tested by intranasal instillation in mice, and all proved to be highly pathogenic. Strains showing only moderate growth or no growth at all in this axenic medium were found to be nonpathogenic for mice. Moreover, it was found that using this medium in the early stage of Naegleria sampling favors isolation of pathogenic strains in mixtures of Naegleria. During these experiments, further evidence was obtained that thermal polluted waters are the main origin of N. fowleri in the environment.     

Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells

The cytopathology for rat neuroblastoma cells (B-103) and the pathogenicity for B6C3F1 mice of four species of Naegleria have been compared. Both live amoebae and cell-free extracts of N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis added to 51Cr-labeled B-103 cells caused release of radiolabel. All four species of Naegleria exhibited surface extensions termed food cups. Only N. fowleri and N. australiensis were pathogenic for mice. Electron microscopic observations of cultures of either N. australiensis or N. lovaniensis with B-103 cells established that the cytopathology involved lysis of the B-103 target cells.     

Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells.

The cytopathology for rat neuroblastoma cells (B-103) and the pathogenicity for B6C3F1 mice of four species of Naegleria have been compared. Both live amoebae and cell-free extracts of N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis added to 51Cr-labeled B-103 cells caused release of radiolabel. All four species of Naegleria exhibited surface extensions termed food cups. Only N. fowleri and N. australiensis were pathogenic for mice. Electron microscopic observations of cultures of either N. australiensis or N. lovaniensis with B-103 cells established that the cytopathology involved lysis of the B-103 target cells.     

An agarose gel electrophoresis method for the separation of arabinogalactan proteins

A method for resolving plasma membrane associated arabinogalactan proteins (AGPs) has been developed. Plasma membranes purified by aqueous polymer two-phase partitioning were first subjected to Triton X-114 fractionation. The resulting water phase contained all detectable plasma membrane-bound AGPs. Plasma membrane AGPs were then resolved in an SDS-agarose gel electrophoresis system (SDS-AGE). For separating plasma membrane AGP species of the same apparent molecular weight but with different net charge, a two-dimensional electrophoresis system was used, utilizing isoelectric focusing in an immobilized pH gradient in the first dimension and SDS-AGE in the second dimension. These methods enabled the separation of individual plasma membrane AGPs. In comparison, SDS-PAGE methods left AGPs as unresolved high molecular-weight smears. The methods described here may help to establish some basic features of AGPs, such as the number, organization, and protein and carbohydrate characteristics of plasma membrane AGPs, as well as the relationship between plasma membrane and extracellular AGPs.     

Leishmania braziliensis: cell surface differences in promastigotes of pathogenic and nonpathogenic strains

A comparative study of cell surface characteristics of pathogenic and nonpathogenic promastigotes of Leishmania braziliensis, NR and LBY strains, respectively, was carried out by means of concanavalin A agglutination and labeling with concanavalin A-fluorescein isothiocyanate, concanavalin A-ferritin, and cationized ferritin. Cytochemical examination showed cell surface differences in lectin receptors and negative charge moieties in the two strains of L. braziliensis. The pathogenic NR strain agglutinated with low concentrations of concanavalin A and presented abundant lectin-binding and cationized ferritin-binding surface labeling. The nonpathogenic LBY strain neither agglutinated when incubated with concanavalin A, bound lectins, or cationized ferritin at the cell surface.     

Two-dimensional polyacrylamide gel electrophoresis: an improved method for ribosomal proteins

A two-dimensional electrophoresis system for analysis of ribosomal proteins with several advantages over previous systems is described. The general features of this system are: (1) first-dimension separation on the basis of mobility at pH 5.0 in 8 m urea and 4% polyacrylamide; (2) second-dimension separation on the basis of molecular weight using dodecyl sulfate detergent; (3) rapid electrophoretic shift between first- and second-dimension separation conditions; (4) high resolution separation can be obtained on 10-cm² slabs with proteins from approximately 100 μg of ribosomal subunits; (5) capacity for handling up to 10 samples at a time, with electrophoresis complete within about 10 hr; and (6) the apparatus is relatively simple and inexpensive to construct and use.     

Transformation in Naegleria gruberi: patterns of RNA synthesis examined by polyacrylamide gel electrophoresis

Naegleria gruberi were grown on bacteria and methods were devised to free the cellular RNA from bacterial RNA contamination. Use of actinomycin D and cycloheximide showed that the transformation of Naegleria from amoeba to flagellate required RNA synthesis for 30 min and protein synthesis for 40 min after the initial stimulus of distilled water. Comparison of the patterns of RNA synthesized during transformation with those during growth indicated a considerable amount of new RNA produced during the phenotypic change. Most marked was the increase in RNA co-migrating on polyacrylamide gels with the small ribosomal sub-unit RNA, together with RNAs between the latter and transfer RNA. These results were compared with other published results using axenically-grown cells cells and sucrose density gradient centrifugation. Cells placed in 80 mM NaCl instead of distilled water fail to transform but the pattern of newly-synthesized RNAs was not significantly different from that seen in transforming cells. This suggested that high salt concentrations inhibit transformation by inhibiting synthesis and/or assembly of certain proteins rather than RNA synthesis. Eluted material from various regions of polyacrylamide gels containing RNA extracted from transforming cells was used in a cell-free system. Incorporation of 3H-glutamic acid but not 3H-tryptophan was stimulated by material extracted from the 18S regions of the gels.     

Apparatus and method for preparative gel electrophoresis

A new apparatus for preparative gel electrophoresis with continuous elution which includes a miniaturized electrode and elution chamber system is described. The design provides high resolution, high yield, applicability for small and large amounts of peptide material, and easy operation. Furthermore, the apparatus enables a very accurate gel column or gel gradient to be formed. A method for preparative gel electrophoresis in sodium dodecyl sulfate which allows the purification of peptides and proteins without concurrently modifying tryptophane residues or blocking N-terminal α-amino groups is also described.     

Development of an effective sample preparation method for the proteome analysis of body fluids using 2-D gel electrophoresis

A sample preparation is still the most critical step in two-dimensional electrophoresis (2-DE) and should be optimized for each type of sample. In this study, a protein extraction method from body fluids was developed using a combined centrifugal filter device and a sample treating buffer. When plasma, amniotic fluid, urine, and tear were tested with this method, the recovery of protein reached almost 90% and high-quality separation of 2-DE gel was obtained.     

Improved gel electrophoresis matrix for hydrophobic protein separation and identification

We propose an improved acrylamide gel for the separation of hydrophobic proteins. The separation strategy is based on the incorporation of N-alkylated and N,N′-dialkylated acrylamide monomers in the gel composition in order to increase hydrophobic interactions between the gel matrix and the membrane proteins. Focusing on the most efficient monomer, N,N′-dimethylacrylamide, the potentiality of the new matrix was evaluated on membrane proteins of the human colon HCT-116 cell line. Protein analysis was performed using an adapted analytical strategy based on FT-ICR tandem mass spectrometry. As a result of this comparative study, including advanced reproducibility experiments, more hydrophobic proteins were identified in the new gel (average GRAVY: −0.085) than in the classical gel (average GRAVY: −0.411). Highly hydrophobic peptides were identified reaching a GRAVY value up to 1.450, therefore indicating their probable locations in the membrane. Focusing on predicted transmembrane domains, it can be pointed out that 27 proteins were identified in the hydrophobic gel containing up to 11 transmembrane domains; in the classical gel, only 5 proteins containing 1 transmembrane domain were successfully identified. For example, multiple ionic channels and receptors were characterized in the hydrophobic gel such as the sodium/potassium channel and the glutamate or the transferrin receptors whereas they are traditionally detected using specific enrichment techniques such as immunoprecipitation. In total, membrane proteins identified in the classical gel are well documented in the literature, while most of the membrane proteins only identified on the hydrophobic gel have rarely or never been described using a proteomic-based approach.     

Secondary pulsed field gel electrophoresis: a new method for faster separation of larger DNA molecules.

A novel technique, which we call secondary pulsed field gel electrophoresis (SPFG) has been developed. In SPFG, short pulses are applied in the direction of net migration of the DNA in addition to the reorienting pulses used in conventional pulsed field electrophoresis (PFG). Experimental results show that SPFG extends and improves the electrophoretic resolution of DNA for molecules from 0.5 megabase pairs to over 10 megabase pairs in size. This improved resolution is obtained with dramatically shorter run times. Thus SPFG appears to circumvent a number of the key limitations in previous PFG protocols.     

Agarose gel electrophoresis in isozyme separation and visualization

Isozyme analysis of rodent-human somatic cell hybrids has been used frequently to detect specific human chromosomes. The majority of these isozyme systems employs starch gels, the use of which can be laborious when screening large numbers of cell lines. We describe the development of two procedures to detect the long arms of human chromosomes 1 and 2 in Chinese hamster-human cell hybrids by a rapid and reproducible method using 1-mm-thick agarose gels. Detection of human chromosome 1q was accomplished by screening for human fumarate hydratase activity, whose gene has been mapped to 1q42.1. Detection of chromosome 2q was performed by screening for the isozyme isocitrate dehydrogenase 1, which has been localized to 2q32-qter. These systems provide a basis for the further development of procedures for detecting chromosome-specific isozyme markers in agarose gels.     

An improved method for separation of low-molecular-weight polypeptides by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel

An improved system for SDS-polyacrylamide gel electrophoresis, capable of analyzing polypeptides having molecular weights from 1500 to 100,000 (especially showing high resolving power in the 1500 to 25,000 molecular weight range) is described. The 10 to 18% linear gradient gel containing 7 M urea with an acrylamide:bisacrylamide ratio of 20:1 and the Laemmli discontinuous buffer was used. The use of the gel with a high crosslinkage ratio is shown to be effective in lowering the leakage of low-molecular-weight polypeptides from the gel. This method has facilitated rapid detection of small amounts of low-molecular-weight polypeptides in body fluids by the use of silver stain. A procedure is presented for the elimination of false bands on the gel frequently encountered during silver staining. The separation patterns of enzymatic cleavage products of proteins, uremic plasma, and urines from nephropathy patients are illustrated. This system is also applicable in the separation of lipopolysaccharides and also for the detection of phospholipids.     

Capillary gel electrophoresis: separation of major erythrocyte membrane proteins

A new separation method of human erythrocyte membrane proteins by sodium dodecyl sulfate capillary gel electrophoresis (SDS–CGE) is described. In this method, a replaceable gel matrix was used. Seven major erythrocyte membrane proteins, α-and β-spectrin, ankyrin 2.1, band 3 (anion-exchanger), 4.1a and b, and 4.2 (pallidin), were separated and identified by SDS–CGE method. High reproducible migration times of these proteins (inter-assay coefficients of variation less than 2%), as well as quantification (inter-assay coefficients of variation less than 11%) were obtained. This new SDS–CGE method may provide important diagnostic evidence for hereditary spherocytosis. It can be a powerful diagnostic tool in place of SDS polyacrylamide gel electrophoresis for erythrocyte membrane protein analysis.