Biochemical studies on the muscle microsomes of Ascaris lumbricoides var. suum. II. Purification and characterization of b-type cytochrome and NADH-ferricyanide reductase from Ascaris muscle microsomes.

A b-type cytochrome and NADH-ferricyanide (FC) reductase were solubilized from Ascaris muscle microsomes by detergents and purified by column chromatography. The purified b-type cytochrome displayed absorption bands at 560 (alpha-peak), 525 (beta-peak), and 424 nm (gamma-peak), with a marked shoulder at 555 nm in the reduced from, 415 nm (gamma-peak) in the oxidized form. This absorption spectrum was different from that of rat liver microsomal cytochrome b5. The molecular weight was estimated to be about 100,000 by SDS-polyacrylamide gel electrophoresis, and the absorption spectrum of alkaline pyridine ferrohemochrome suggested that the prosthetic group of this cytochrome is protoheme. The molecular weight of the purified NADH-FC reductase was estimated to be about 55,000 by SDS-polyacrylamide gel electrophoresis. The purified reductase required NADH as a specific electron donor. The reductase efficiently reduced some redox dyes with NADH, but the reduction of cytochrome c was much slower. The purified reductase, like the membrane-bound reductase, was not inhibited by thiol reagents.     

Ultrastructural analysis of sex determination in Ascaris lumbricoides var. suum

At metaphase of the first spermatocyte meiotic division in Ascaris lumbricoides var. suum the polymorphic bivalents are arranged at the equatorial plate peripheral to a central aggregate mass. The aggregate mass segregates to only one of the two daughter cells and thus behaves as a sex chromosome. The mass is comprised of two components: a granular chromatin that can be differentiated from the autosomes at metaphase I, and a condensed chromatin that has a similar morphology to the autosomes but is functionally differentiated at anaphase I. The condensed chromatin may be autosomes that have fused with the sex chromsomes and may be responsible for the segregation of the whole aggregate at anaphase I. Analysis of the origin of the mass is discussed and microtubular association in the movement of the mass is described.     

Chromatin diminution in early embryogenesis of Ascaris lumbricoides L. var. suum

The occurrence of chromatin diminution in early Ascaris lumbricoides L. embryos has been studied in detail, and it is shown that it is possible to preselect three characteristic types of mitoses: pre-diminution, diminution, and post-diminution mitosis. The first three embryonic mitotic divisions are of the pre-diminution type. Chromatin diminution occurs after the third mitosis, but there is a variation from embryo to embryo as to whether or not chromosomal diminution occurs during the fourth, fifth, and six divisions. However, the seventh embryonic division, which gives rise to an eight-cell embryo, always exhibits chromatin diminution. Subsequent mitoses of somatic cells already in the diminished state are of the post-diminution type of mitosis.     

Studies on the subunits of myosin from muscle layer of Ascaris lumbricoides suum.

1. A purified preparation of Ascaris myosin was obtained from the muscle layer of Ascaris lumbricoides suum, using gel filtration and ion-exchange chromatography. 2. Ascaris myosin whether purified or unpurified, had almost the same ability for ATP-splitting and superprecipitation. 3. Ascaris myosin and rabbit skeletal myosin were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A significant difference in the number of light chains between both myosins was found. Ascaris myosin was found to have one heavy chain and two distinct light chain components (LC1-A and LC2-A), having molecular weights of 18000 and 16000, respectively. These light chains correspond in molecular weight to the light chain 2 (LC2-S) and light chain 3 (LC3-S) in rabbit skeletal myosin. 4. LC1-A could be liberated from the Ascaris myosin molecule reacted with 5,5'-dithio-bis(2-nirobenzoic acid( Nbs2) with recovery of ATPase activity by addition of dithiothreitol. These properties are equivalent to those of the LC2-S in rabbit skeletal myosin, although Ascaris myosin when treated with Nbs2-urea lost its ATPase activity.     

Ascaris lumbricoides suum: morphological characterization of apparent cuticular pores by ionic permeability and electron microscopy

The cuticle of the parasitic roundworm Ascaris lumbricoides suum has been found to contain apparent pores, using scanning and transmission electron microscopy. However, X-ray spectral analysis and dot mapping analysis of diffusing divalent cations in the cuticle found these ions to be randomly distributed on the pseudocoelomic surface of the cuticle. This indicates that the pores seen with the electron microscope were not true structural pores and that the cuticle is homogeneously permeable to ions.     

Spermatogenesis and spermiogenesis in Ascaris lumbricoides Var. suum

Reorganization of the prophase I nucleus marks the beginning of the first meiotic division. A pair of centrioles is present at each pole at metaphase I and mitochondria are not observed in the spindle area. A chromosomal pellicle, which resembles a kinetochore plate but has no apparent association with microtubules, surrounds each autosome at metaphase I and II. The sex body lags behind the autosomes at anaphase I and segregates differentially to one daughter cell. Mitochondria and a pair of centrioles are present in the spindle during the second meiotic division. Localized condensation of chromatin and fusion of the condensed chromatin of the secondary spermatocyte telophase nucleus results in a compact spermatid nucleus. Loss of spermatid cytoplasm is effected by the ejection of a cytophore vesicle.     

Precocious formation of synaptonemal-like polycomplexes and their subsequent fate in female Ascaris lumbricoides var. suum

During meiotic interphase, before leptotene, synaptonemal-like polycomplexes are seen in the cytoplasm of the Ascaris lumbricoides oocytes and in the communal anucleate rachis. In some females short intranuclear synaptonemal complexes are present briefly at that early stage. The number of extranuclear complexes increases just before leptotene, some are attached to the pores of the nuclear membrane. During zygotene most polycomplexes disappear. At late pachytene they reappear in some females but not in others. The morphology, when first seen, is that of disorganized filamentous bodies, later lateral elements appear among the filaments. The dimensions of the lateral elements of the polycomplexes are variable. In the male the distribution of polycomplexes among the rachis, the cell cytoplasm, and at the nuclear envelope is similar to that observed in the female.These observations confirm the precocious occurrence of synaptonemal-like polycomplexes reported by Bogdanov (1977). Ascaris lumbricoides thus, uniquely, appears to manufacture synaptonemal complex-like material in the communal cytoplasm of the germ cells prior to the time that the full complement of synaptonemal complexes appears in the nucleus.     

Characterization and expression of a spliced leader RNA in the parasitic nematode Ascaris lumbricoides var. suum.

The parasitic nematode Ascaris spp. contains a 22-nucleotide spliced-leader (SL) sequence identical to the trans-SL previously described in Caenorhabditis elegans and other nematodes. The SL comprises the first 22 nucleotides of a approximately 110-base RNA and is transcribed by RNA polymerase II. The SL RNA contains a trimethylguanosine cap and a consensus Sm binding site. Furthermore, the Ascaris SL RNA has the potential to adopt a secondary structure which is nearly identical to potential secondary structures of similar SL RNAs in C. elegans and Brugia malayi.     

The fine structure of neuromuscular junctions and contact zones between body wall muscle cells of Ascaris lumbricoides (var. suum)

Summary Neuromuscular junctions and close membrane apposition between body wall muscle cells of Ascaris lumbricoides (var. suum) have been examined with the light and electron microscopes. It was found that the body wall muscle cells send out elongate processes from their basal, myofibril containing portion to terminate on dorsal and ventral nerves. When observed with the aid of the electron microscope the neuromuscular junctions were seen to consist of several muscle cell processes in apposition to a single axon. The intersynaptic cleft was approximately 350–500 Å wide. Both the axolemma and sarcolemma were triple layered membranes which were 75–80 Å thick. Electron dense patches were observed at intervals on the apposed membranes which were due to increased thickness of the inner membrane leaflets of axolemma and sarcolemma. Muscle cell membranes, at the level of the neuromuscular junction, were in close apposition resulting in an apparently five-layered membrane complex which was 170–210 Å thick. The sarcolemmata in these regions were separated by 10–50 Å. Presynaptic axons contained mitochondria, microtubules which were 180–270 Å in diameter, and two, morphologically distinct types and sizes of synaptic vesicles. One was 200–600 Å in diameter, with a single, triple-layered membrane bounding a center of low electron density. The other was 600–1200 Å in diameter, with a single, triple-layered membrane bounding a central, electron dense granule of 500–800 Å size.The functional significances of the close membrane appositions between body wall muscle cells and of the two types of synaptic vesicles found at the neuromuscular junctions of Ascaris lumbricoides were discussed with respect to their possible role in neuromuscular physiology.Supported by U.S.P.H.S. Grant No. NB-01528 and Research Career Development Award No. 9-K3-NB-15255. — The author wishes to express his grateful appreciation for the excellent technical assistance given by Miss Gabrielle Rouiller during the course of this investigation.