Michael Tilley

A simple modification of Blum's silver stain method allows for 30 minite detection of proteins in polyacrylamide gels

A simple and rapid protocol for silver staining of proteins following electrophoresis in polyacrylamide gels (PAGE) is described. We have reduced the number of steps in the procedure of Blum et al. (Electrophoresis (1987) 8, 93-99), and shortened fixation and washing times so that efficient detection of proteins can be achieved within 30 min. In common with more time-consuming silver-staining methods, the present protocol is capable of detecting nanogram quantities of proteins on a colorless background and is suitable for rapid screening of large numbers of samples.

Cloning and Analysis of a Cryptosporidium parvum Gene Encoding a Protein with Homology to Cytoplasmic Form Hsp70

ABSTRACT. An intronless gene encoding a protein of 674 amino acid residues with a molecular mass of 73,403 Da showing homology to the cytoplasmic form of the 70 kDa heat shock proteins has been cloned and sequenced from the intestinal pathogen Cryptosporidium parvum. Monospecific polyclonal antibodies obtained to recombinant protein recognized a single band with an approximate molecular mass of 70 kDa on a Western blot of C. parvum proteins, as well as the 70 kDa heat shock protein from bovine brain. Southern blot analysis suggested the gene was single copy in the C. parvum genome. Eleven perfect repeats of the sequence GGMP were found in the predicted protein near the carboxyl terminus.

Major antigens of Cryptosporidium parvum recognised by serum antibodies from different infected animal species and man

Serum humoral immune response to Cryptosporidium parvum was evaluated in six species: mouse, rabbit, lamb, calf, pig and man. Electrophoretic and immunoblot analysis showed that specific animal antibody response appeared between Day 4 and Day 15 post inoculation. The two main target antigens had apparent molecular weights of 15-17 and 23 kDa. They were recognised by each species studied. Serum IgA intensively recognised the 15-17 kDa antigen, except in rabbit. This study demonstrates that these two antigens are consistent targets of humoral immune response and can therefore be of great interest in studies of therapy/prophylaxis.

Comparison of Methods for Extracting Kafirin Proteins from Sorghum Distillers Dried Grains with Solubles

Use of coproducts generated during fermentation is important to the overall economics of biofuel production. The main coproduct from grain-based ethanol production is distillers dried grains with solubles (DDGS). High in protein, DDGS is a potential source of protein for many bioindustrial applications such as adhesives and resins. The objective of this research was to characterize the composition as well as chemical and physical properties of kafirin proteins from sorghum DDGS with various extraction methods including use of acetic acid, HCl-ethanol and NaOH-ethanol under reducing conditions. Extraction conditions affected purity and thermal properties of the extracted kafirin proteins. Extraction yields of 44.2, 24.2, and 56.8% were achieved by using acetic acid, HCl-ethanol and NaOH-ethanol, respectively. Acetic acid and NaOH-ethanol produced protein with higher purity than kafirins extracted with the HCl-ethanol protocol. The acetic acid extraction protocol produced protein with the highest purity, 98.9%. Several techniques were used to evaluate structural, molecular and thermal properties of kairin extracts. FTIR showed alpha-helix dominated in all three samples, with only a small portion of beta-sheet present. Electrophoresis results showed alpha(1), alpha(2) band and beta kafirins were present in all three extracts. Glass transition peaks of the extracts were shown by DSC to be approximately 230 degrees C. Kafirin degraded at 270-290 degrees C. Size exclusion chromatography revealed that the acetic acid and HCl-ethanol based extraction methods tended to extract more high molecular weight protein than the NaOH-ethanol based method. Reversed phase high-performance liquid chromatography showed that the gamma kafirins were found only in extracts from the NaOH-ethanol extraction method.

Sporozoites of Toxoplasma gondii lack dense-granule protein GRA3 and form a unique parasitophorous vacuole

The invasion of host cells by sporozoites of Toxoplasma gondii leads to the formation of parasitophorous vacuoles that are distinctly different from those surrounding tachyzoites. In sporozoite-infected cells, the fluid-filled space surrounding the sporozoite is many times larger in volume than the sporozoite, essentially lacks granular or tubular structures, and has no detectable continuous parasitophorous vacuolar membrane when prepared by conventional electron microscopic methods. Consistent with the ultrastructural differences, dense-granule protein GRA3, which associates with the parasitophorous vacuolar membrane of tachyzoites, was not detected by indirect immunofluorescence in sporozoite-infected cells 2-12 h post-inoculation or by Western blot analysis of sporozoite extracts. Western blots incubated with the alpha ROP/DG antiserum, which recognizes tachyzoite rhoptry and dense-granule proteins, revealed numerous other antigenic differences between sporozoites and tachyzoites. Cell cultures inoculated with sporozoites were monitored at various intervals for the expression of GRA3 and the developmentally-regulated tachyzoite surface protein SAG1. Expression of SAG1 and GRA3 was first observed in 30% of the sporozoite-infected cells at 12 and 15 h post-inoculation, respectively, and in all intracellular parasites at 24 h. Parasite replication was only observed in sporozoite-infected cells that were positive for GRA3 and SAG1. Thus, these data indicate that sporozoites and their interaction with host cells differ substantially from tachyzoites and the expression of tachyzoite-specific proteins is likely required for parasite replication.

A Comparative Study of the Development of Eimeria nieschulzi In Vitro Under Aerobic and Reducing Conditions

Sporozoites of the rat coccidian, Eimeria nieschulzi Dieben, 1924 (Apicomplexa: Eimeriidae), were inoculated onto monolayers of normal rat kidney (NRK) fibroblasts and cultured either under aerobic (5% CO2/95% air) or reducing (desiccator jars modified into candle jars) conditions in RPMI-1640 supplemented with 5% fetal bovine serum, sodium bicarbonate, and antibiotics. Under aerobic conditions, first-generation meronts were observed at 2 days postinoculation (DPI) and, except for individual third-generation meronts that were seen at 5 and 6 DPI, no further development was noted. Under reducing conditions, however, first-generation meronts observed at 2-5 DPI underwent additional development to form second-generation meronts (3-5 DPI), third-generation meronts (3-7 DPI), and a small number of fourth-generation meronts (5-8 DPI). Both second- and third-generation meronts were abnormal, exhibiting gigantism although the merozoites produced appeared normal. The gradual degeneration of cell monolayers under reducing conditions prevented further observations beyond 8 DPI. These results suggest that atmospheric conditions play an important role in the development of E. nieschulzi and maintenance of reducing conditions may be one key to achieving enhanced development of some species of coccidia in vitro.

EFFECT OF SELECT MEDIA SUPPLEMENTS ON MOTILITY AND DEVELOPMENT OF EIMERIA NIESCHULZI IN VITRO

A technique was developed to examine the effects of exogenous substances on apicomplexan sporozoite motility in vitro. Sporozoites of Eimeria nieschulzi were placed in the top compartments of blind well chemotactic chambers and separated from potential chemoattractant/chemokinetic agents by 8.0-microns pore size Millipore filters. After 3-hr incubations, the number of sporozoites migrating through the filters was assessed using a hemacytometer. Results revealed that several substances, especially albumin and fetuin, enhanced motility of coccidial sporozoites. Cell culture assays supported these data, with higher numbers of parasites found in cultures supplemented with albumin and/or fetuin.

Effects of overexpression of high molecular weight glutenin subunit 1Dy10 on wheat tortilla properties.

Wheat (Triticum aestivum L.) flour properties necessary for optimal tortilla production have not been identified. Transgenic wheats (Triticum aestivum L.) overexpressing high molecular weight glutenin subunit (HMW-GS) 1Dy10 were used to make tortilla and their quality was evaluated. The level of HMW-GS 1Dy10 in flours derived from transgenic wheats was 2.5-5.8-fold greater than in controls. Polymeric proteins in the transgenic samples had a molecular weight distribution shifted toward larger polymers as indicated by increased levels of polymeric proteins present and greater M(w) averages of the largest fractions in the insoluble polymeric proteins. Dough derived from transgenic wheats had greater resistance to extension and lower extensibility than controls. Tortilla quality evaluation revealed that tortillas originated from transgenic wheats had decreased diameter, greater thickness and rupture force, and lower rollability scores and stretchability than controls. The presence of 1RS chromosomal translocations from rye (Secale cereale L.) in transgenic wheat decreased the negative effects of overexpression of HMW-GS 1Dy10, as tortillas made with this flour mostly exhibited quality properties similar to those made from control flour. Results suggested that the negative effects of overexpression of HMW-GS 1Dy10 on tortilla properties were derived from a nonideal gluten matrix formation.

Functionality of Gliadin Proteins in Wheat Flour Tortillas

Gliadins are monomeric proteins that are encoded by the genes at the loci Gli 1 and Gli 2 present on the short arm of homologous wheat chromosomes 1 and 6, respectively. Studies have suggested that gliadins may play an important role in determining the functional properties of wheat flour. The main objective of this study was to understand the functionality of gliadins with respect to tortilla quality. The important tortilla quality attributes are diameter, opacity, and shelf stability, designated here as rollability or the ability to roll or fold the tortilla without cracking. In this study gliadin functionality in tortilla quality was studied using near-isogenic wheat lines that have deletions in either Gli A1, Gli D1, Gli A2, or Gli D2 gliadin loci. The deletion lines are designated by the same abbreviations. Dough and tortillas were prepared from the parent line used to derive these deletion lines, each individual deletion line, and a control commercial tortilla flour. Quantitative and qualitative evaluations were performed on the dough and tortillas derived from the flour from each of these lines. None of the deletions in the gliadin loci altered the shelf stability versus that found for the parent to the deletion lines or control tortilla flour. However, deletions in the Gli 2 loci, in particular Gli A2 reduced the relative proportion of alpha- and beta-gliadins with a greater cysteine amino acid content and gluten cross-link function versus the chain-terminating omega-gliadins in Gli 1, which were still present. As such, the dough and gluten matrix appeared to have greater extensibility, which improved the diameter and overall quality of the tortillas while not altering the rollability. Deletions in the Gli 1 loci had the opposite result with increased cross-linking of alpha- and beta-gliadins, polymeric protein content, and a stronger dough that decreased the diameter and overall quality of the tortillas. The data suggest that altering certain Gli 2 loci through null alleles could be a viable strategy to develop cultivars improved for the specific functionality requirements needed for the rapidly growing tortilla market.

Efficacy of a pentaiodide resin disinfectant on Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae) oocysts in vitro

The resin-I5 column developed at Kansas State University was tested for efficacy against oocysts of Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae). Cesium chloride gradient-purified oocysts were passed through 1.0-cm-diameter columns with lengths of 2.5, 5.0, and 10.0 cm at 23 C. Following column passage, oocyst viability was determined both in vitro by excystation and in vivo by the ability to establish infections in suckling mice. Oocysts were found to be retained by the pentaiodide resin in a linear fashion, probably by electrostatic interactions. Linear regression analysis revealed 100% of the oocysts should be removed in such a manner using a column length of greater than or equal to 25.7 cm. When compared to untreated control oocysts, less than 12% of the oocysts that passed through the columns appeared to be affected by the resin, as assessed by excystation. Inoculation of suckling mice with these column-treated oocysts supported the excystation data and revealed the coccidian to be viable. These results indicate that oocysts of C. parvum are retained on the pentaiodide column in a 1-hit manner and that, although killing of parasites may occur within the column, the greatest effect that the column may have on the parasite is as an electrostatic retention device.