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Biomedical subjects

M A Fernando

Publications and source records attributed to M A Fernando.

At least 55 records · Page 3Linked to original sources

The effects of single and multiple doses of thiabendazole on growing and arrested stages of the rabbit stomach worm Obeliscoides cuniculi.

Preliminary observations, using 5 rabbits carrying long-standing infections, indicated that single doses of thiabendazole were effective against adult O. cuniculi and that repeated doses may have had some effect on arrested larvae. A multiple dosing regime, consisting of 1 dose at 110 mg kg-1 body weight followed by 8 doses at 70 mg kg-1 body weight at 4-h intervals, was successful in removing 99.12% of developing worms and 86.04% of arrested larvae.

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Eimeria necatrix: induced proteolytic sensitivity of infected chick crypt cells.

While devising a protocol for the isolation of chick crypt cells infected with Eimeria necatrix, it was observed that infected cells were readily lysed by 0.25% trypsin. Time-course studies at 17 C with 5.5 x 10(5) cells at 96 hr postinfection revealed that 0.001% trypsin effectively lysed greater than 90% of infected cells within 10 min. Uninfected crypt cells were not lysed under these conditions. To determine the site of action of trypsin, the plasma membrane proteins from trypsin-treated and untreated infected cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the major proteins were unaffected by the trypsin treatment, some minor changes were noted: (1) three components (congruent to 60, congruent to 52, and congruent to 20 KDa) were trypsin sensitive and (2) a new band (congruent to 42 KDa) appeared in the membrane of trypsin-treated infected cells. Previously, it was found that the plasma membrane of infected cells, in contrast to uninfected cells, accumulated gel-phase lipid (J. E. Thompson, M. A. Fernando, and J. Pasternak, Biochimica et Biophysica Acta 555, 472-487, 1979). Here, it was examined whether trypsin would perturb the physical state of the plasma membrane of infected cells. Both X-ray diffraction patterns and transition temperature studies revealed no difference between membranes from untreated and trypsin-treated infected cells. Thus, "trypsin sensitivity" may be a secondary phenomenon that is due primarily to the cellular leakiness that accompanies the accumulation of gel-phase lipid in the plasma membrane of infected cells. The uptake of trypsin may stimulate the release of catabolic enzymes that, consequently, lyse an infected cell.

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Invasion of chicken caecal and intestinal lamina propria by crypt epithelial cells infected with coccidia.

The development of second generation schizonts of Eimeria necatrix and E. tenella was studied with the electron microscope. Invasion of the crypt epithelial cells by merozoites of the first generation schizonts caused changes in the morphology of the infected cells and stimulated their migration into the lamina propria through breaks which appeared in the basement membrane of the crypts. Second generation schizonts developed in the lamina propria within these crypt cells whose epithelial origin was confirmed by their interconnection by desmosomes and tight junctions and by their possession of characteristic microvilli. A comparison is made between this invasion of the lamina propria by parasitized cells and invasion of connective tissue by malignant epithelial cells; the possible mechanisms involved are discussed.

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Ribosomal and translatable messenger RNA of Eimeria tenella.

RNA from oocysts of Eimeria tenella was purified into poly(A)--RNA and poly(A)+-RNA fractions with affinity chromatography on oligo(dT)-cellulose. Gel electrophoresis, under denaturing conditions of the poly(A)--RNA fraction revealed two major RNAs with molecular weights of 1.27 and 0.65 X 10(6). The values for these components not only represent the ribosomal RNAs of this species; but they also resemble the molecular sizes of the non-mRNAs of many other lower eukaryotes. In vitro translation assays with a cell-free wheat germ system showed that the level of translatable mRNA in the poly(A)--RNA fraction was negligible. Virtually all the translatable mRNA in E. tenella was polyadenylated, that is, it contained poly(A)-tracts with more than 15 adenylate residues. Consequently some of the key components of the protein translational machinery of this organism are eukaryotic in nature.

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Induction of gel-phase lipid in plasma membrane of chick intestinal cells after coccidial infection.

When chickens are infected with the coccidial parasite Eimeria necatrix, the plasma membrane of intestinal cells harbouring second-generation schizonts becomes refractory to mechanical shearing, hypotonic shock and ultrasonication. Plasma membrane from these infected cells was isolated to high purity as judged by enriched levels of ouabain-sensitive (Na+ + K+)-stimulated Mg2-dependent ATPase activity and sialic acid content, the lack of detectable cytochrome oxidase and glucose-6-phosphatase activities and electron microscopic analysis of the final preparation. Wide-angle X-ray diffraction patterns recorded from the isolated membranes revealed that during the later stages of parasite maturation the host cell plasma membrane acquires increasing proportions of gel-phase lipid. By contrast, purified membrane from isolated parasites is in a liquid-crystalline state. The transition temperature of host cell plasmalemma at 100 h postinfection is 61 degrees C, about 20 degrees C above physiological temperature. By contrast, liposomes of plasma membranes from infected cells undergo a thermal transition at about 28 degrees C. The accumulation of gel-phase lipid in the host cell plasma membrane is not attributable either to an increase in the constituent ratio of saturated to unsaturated fatty acids or to a significant change in the cholesterol to phospholipid ratio. During the late stages of infection, the cells become stainable with trypan blue which suggests that the acquisition of crystalline phase lipid disrupts the permeability of the host cell plasmalemma.

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Immunogenicity of a single sporocyst of Eimeria maxima.

Eight out of a total 40 chickens infected with single sporocysts of Eimeria maxima produced infective oocysts. These 8 chickens, when challenged with 4 X 10(5) oocysts per bird at 28 days postinfection, showed at least twice the percent average weight gain of the previously unexposed chickens, indicating that partial immunity was probably conferred by these infections. Also, these successful single sporocyst infections suggest that like E. tenella, sporozolles of E. maxima are probably sexually undifferentiated.

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Changes in the generation cycle of duodenal crypt cells in chickens infected with Eimeria acervulina.

Changes in the duration of the progenitor cycle and its four phases were determined for duodenal crypt cells in chickens infected with Eimeria acervulina. Metaphase curves were constructed using percent labelled metaphase nuclei in duodenal crypt cells at short intervals after the injection of [3H]thymidine. The duration of the progenitor cycle and its four phases were calculated using the synthetic index and data obtained from the metaphase curves. The cycle time was reduced from 14 h in control birds to 10.2 h at 2 days and 10.6 h at 4 days postinfection. The change was attributable entirely to a reduction in G1 or the presynthetic phase. In addition, the population of dividing cells within each duodenal crypt was almost doubled in infected birds. These increases in cell production precedes all the histological changes observed earlier in the intestines of E. acervulina infected chickens. At least in this instance, changes in crypt morphology seems, therefore, to result from an induced change in the functional activity of the crypt.

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Immuno-ultracentrifugation: a combination of density gradient centrifugation and immunodiffusion.

By incorporating acrylamide in a sucrose gradient and photopolymerizing the tubes at the end of the run, direct immunochemical and other analysis can be performed on the separated material in situ without recovering fractions from the centrifuge tube. Immunodiffusion performed on the sliced gel can be employed to locate antibody activity in the gel slice or determine the distribution of antigens in a sucrose gradient

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Isolation of chick intestinal cells infected with second-generation schizonts of Eimeria necatrix.

After infection with Eimeria necatrix, chick intestinal cells undergo a profound increase in size. Herein, we describe methods for isolating and purifying infected cells containing second-genration schizonts at various times post-infection. Our purification procedures depends upon three fundamental properties of infected cells: namely (1) large size, (2) resistance to shearing and (3) decreased susceptibility to osmotic shock in hypotonic solutions. The availability of simple techniques for isolating infected whole cells should prove valuable in elucidating various intracellular aspects of infection during schizogony.

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