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Biomedical subjects

J Pasternak

Publications and source records attributed to J Pasternak.

At least 37 records · Page 2Linked to original sources
PubMed1981

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.

Animals↗
PubMed1979

Isolation and characterization of the cuticle from the free-living nematode Panagrellus silusiae.

The cuticle (0.15 to 0.5 microns thick) of the microscopic free-living nematode Panagrellus silusiae was isolated intact by incubating worms with 1% sodium dodecyl sulfate at 37 degrees C overnight. After shearing and further treatment with detergent, electron microscopy revealed that the cuticular pieces were free of contaminating material and retained their characteristic in situ ultrastructure. From amino acid determinations, the cuticle is collagen-like with high levels of glycine (approximately equal to 31 residue %), proline (approximately equal to 20 residue %) and alanine (approximately equal to 21 residue %) although the hydroxyproline (2.6 residue %) content is low. Half-cystine (approximately equal to 1 residue %) is present in purified cuticles. Treatment with 8 M guanidine hydrochloride-2% beta-mercaptoethanol can solubilize more than 85% of the cuticular preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized cuticles from juvenile, adult and old dead worms revealed, at least, 18 discrete components. Estimated molecular weights ranged from about 26 000 (peak 1) to 250 000 (peak 18).

Amino Acids↗
PubMed1979

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.

Animals↗
PubMed1979

Characterization of the genome of the free-living nematode Panagrellus silusiae: absence of short period interspersion.

The complexity of the DNA of the free-living nematode Panagrellus silusiae has been examined. Reassociation kinetics of pressure-sheared fragments (approximately 290 nucleotides) in 0.18 M Na+ at 60 degrees C showed the presence of foldback, repetitive, and unique DNA sequence elements. The three classes comprise 9.3%, 26.1%, and 61.3% of the total DNA, respectively. The mean length of the foldback duplex DNA after digestion with S1 nuclease is about 185 nucleotides. There are about 1.8 x10(4) inverted repeats per genome. Sequence arrangement was deduced from (1) renaturation kinetic profiles of long and short fragments on hydroxylapatite; (2) the pattern of renaturation of tracer DNA, labeled in vitro with 125I, of various sizes after incubation with excess short fragments; and (3) thermal denaturation behavior of DNA that had been reassociated to various C0t values. It was found that DNA fragments of the repetitive fraction that are, at least, 2000 nucleotides in length are virtually free of unique sequences. Moreover, it is estimated that the repeated segments in this species could extend for 10,000 nucleotide pairs. Thus, Panagrellus DNA lacks the pattern of extensive short period interspersion that is typified by the DNA of Xenopus.

Animals↗
PubMed1979

Isolation and translation in vitro of poly(A)+RNA from the free-living nematode Panagrellus silusiae.

Polysomal RNA was isolated from the free-living nematode Panagrellus silusiae. Passage of this RNA through a cellulose column resulted in the fractionation of the input RNA into poly(A)-RNA (ca. 97.5% of the total) and poly(A)+ RNA (ca. 2.5% of the total). RNase digestion, followed by polyacrylamide gel electrophoresis, revealed that the poly(A)+ RNA contained poly(A) tracts that ranged from 75 to 104 nucleotides in length with a mean value of about 90 residues. There was no evidence of poly(A) sequences in the poly(A)- RNA fraction. Poly(A)+ RNA gave a 25- to 50-fold stimulation (over background) of amino acid incorporation in the wheat germ cell-free protein-synthesizing system. At least 26 proteins were evident after electrophoresis in cylindrical sodium dodecyl sulfate-polyacrylamide gels. Poly(A)-RNA was capable of stimulating protein synthesis in vitro with about five discrete proteins being produced. In summary, the properties of mRNA from a simple organism such as P. silusiae are very similar to those of more complex eukaryotes.

Animals↗
PubMed1978

In vitro translation of nematode cuticular collagens.

Phenanthroline treatment of growing cultures of the free-living nematode Panagrellus silusiae was used to lower the degree of hydroxylation of nascent collagen chains at the polysomal level. Under these conditions, the bound pentasome-hexasome fraction provided substrate for prolyl hydroxylase. When this polysomal fraction was subsequently tested in a cell-free wheat germ system, collagenase-susceptible translation products were observed after sodium dodecyl sulfate-acrylamide gel electrophoresis. The electrophoretic mobilities of each of these four major collagen products were similar to four collagens that are isolated from intact cuticles. In addition, purified polysomal RNA that adhered to unmodified cellulose directed the synthesis of four pepsin-resistant polypeptides that had molecular weights that coincided with four pepsin-resistant collagens that can be purified from the cuticle of this species. Thus, the polysomal site of the messenger RNAs for the cuticular collagens of P. silusiae was located. Although precursor forms of the cuticular collagens were not produced in the cell-free system, the question whether additional amino acid segments occur on the primary translational products of the cuticular collagens in vivo remains open.

Cell-Free System↗
PubMed1977

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.

Animals↗
PubMed1977

Isolation of host-cell nuclei from chick intestinal cells infected with second-generation schizonts of Eimeria necatrix.

A procedure is described for the preparation of purified nuclei of chick intestinal cells infected with Eimeria necatrix. During second-generation schizogony the host-cell membrane becomes resistant to homogenization, hypotonic shock and ultrasonication. However, brief treatment with a low concentration of trypsin (0.001%) will disrupt the cell membrane. Thereafter the nuclei can be selectively harvested from contaminating nuclear types and schizonts by a series of centrifugations. Electron microscopy and Feulgen-DNA microspectrophotometry showed that the nuclear preparations retained their integrity with little, if any, loss of DNA. Chemical determinations confirmed that host-cell nuclear DNA synthesis was stimulated by coccidial infection and further established that quantitatively there was a coincident increase in the amount of host nuclear RNA during schizogony. It is now feasible to examine directly the effects of coccidial infection upon the host's nuclear apparatus.

Animals↗
PubMed1977

Relationship between nuclear morphology and the phases of the cell cycle during cercarial development of the digenetic trematode Trichobilharzia ocellata.

During the proliferative phase of cercarial development in the digenetic trematode Trichobilharzia ocellata, nuclei varied in size, appearance of the chromatin, and intensity of Feulgen staining. On the basis of interphase nuclear morphology six nuclear classes were identified. Data from microspectrophotometric determinations and 3H-TdR labeling experiments were used to correlate each kind of interphase nucleus with a phase of the cell cycle. Marked variability in nuclear area developed between cells by time they reached late G1. Increases in nuclear area could not be correlated with the onset of DNA synthesis. Throughout all stages of development of T. ocellata cercariae, proliferating cells divide mitotically; meiotic divisions were never seen. Thus, the mode of reproduction appears to be asexual. No evidence was found to support previous suggestions of diploid parthenogenesis.

Animals↗
PubMed1977

Cell cycle analysis in developing cercariae of Trichobilharzia ocellata (Trematoda: Schistosomatidae).

Cellular proliferation has been analyzed during cercarial development of the digenetic trematode Trichobilharzia ocellata. Prior to the tail-bud stage (about 1,000 cells), cells were actively involved in cellular proliferation. The mean cell cycle was 15.2 hr. The time for mitosis was 1.6 hr; for G1, 5.6 hr; for G2, 3.2 hr, and for the S phase, 4.8 hr. Beginning with the tail-bud stage, an increasing proportion of cells accumulate in the G1 phase. Cytological evidence of changes in the amount of cytoplasm per cell revealed that these noncycling cells were differentiating. During organ development and differentiation, the proportion of proliferating cells decreases and by the 2,000-celled stage proliferation ceases. Our results do not support a germinal lineage theory of cercarial development since none of the observed nuclear types could be unequivocally identified as belonging to the germ line.

Animals↗
PubMed1976

Relative ribosomal RNA cistron multiplicity in oocytes and postembryonic stages of the eutelic nematode Panagrellus silusiae.

The number of ribosomal RNA cistrons has been measured in the total DNA extracted from L2 juvenile and adult stages of the free-living nematode Panagrellus silusiae. Saturation hybridization studies with homologous rRNA indicate that both stages have about 275 ribosomal genes per haploid equivalent. Using homologous 125I-labelled rRNA for in situ hybridization, the mean number of silver grains per DNA content for oocyte, hypodermis and gut nuclei was similar. The mean DNA contents of maturing oocyte, hypodermis and gut nuclei are about 20C, 2C, and 10C respectively. We conclude that rDNA amplication alone is insufficient to account for the variation in DNA content of oocytes and that postembryonic development in this eutelic organism occurs without a significant differential increase in the number of ribosomal cistrons per worm.

Animals↗
PubMed1976

Cytophotometric study of nuclear proteins during embryogenesis in two nematode species, Ascaris lumbricoides and Panagrellus silusiae.

Patterns of nuclear protein changes during embryogenesis in two nematode systems: Ascaris lumbricoides and Panagrellus silusiae were examined microspectrophotometrically. In both species, development is accompanied by an extensive loss of basic nuclear protein as noted by the diminished intranuclear binding of both the Sakaguchi reagent and fluorodinitrobenzene chromophore. The alkaline fast green (pH 8.1) procedure stained both nuclear and cytoplasmic regions of interphase cells to a similar extent throughout embryogenesis. In cells at metaphase the chromosomes bound the fast green dye intensely while the cytoplasm continued to give a positive reaction. Relative Feulgen stainability of nuclei during embryonic development varied between species. In Ascaris, the one-celled stage nucleus was Feulgen-negative and the LI juvenile somatic nuclei yielded a mean Feulgen-DNA value which was considerably less than a postdiminution diploid DNA equivilent. In Panagrellus, the nuclei of one-celled stage embryos contain DNA in excess of a 4C quantity which subsequently is reduced during embryonic divisions. As in Ascaris the nuclei of Panagrellus LI juveniles give hypodiploid Feulgen-DNA values. The significance of these changes remains to be determined.

Animals↗
PubMed1976

Cytophotometric analysis of basic nuclear proteins during gametogenesis in the free-living nematode Panagrellus silusiae.

The cytochemical staining characteristics of basic nuclear proteins during gametogenesis in both sexes of the free-living nematode Panagrellus silusiae were monitored by absorption microspectrophotometry. During spermatogenesis, the levels of both of the nuclear-bound Sakaguchi and 1-fluoro-2, 4-dinitrobenzene (FDNB) reagents per Feulgen-DNA content increase prior to meiosis and subsequently drop off during spermiogenesis. During oogenesis, the amount of Sakaguchi-protein to DNA ratio rises slightly whereas the FDNB-protein to DNA ratio undergoes a meagre decline. Since the oocyte of Panagrellus accumulates DNA in excess of a 4C DNA equivalent, the relative constancy of the ratio of basic nuclear protein content to DNA amount during oogenesis signifies that these proteins are being continously produced during oocyte maturation. After treatment with alkaline fast green (pH 8.1) the cytoplasmic and nuclear regions of the gonocytes of both sexes yield such an intense reaction that microspectrophotometric measurements could not be taken. The basis of the cytoplasmic staining reaction to the fast green (pH 8.1) dye was not determined. The rapid, highly deterministic programme of embryogenesis in Panagrellus may require a substantial stockpiling of basic nuclear proteins during oogenesis.

Animals↗