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Ultrastructure of the Alnus crispa var. mollis Fern. root nodule endophyte.

Nitrogen-fixing, field-obtained root nodules of the silky green alder were studied by transmission electron microscopy. The nodule endophyte exhibited a prokaryotic cytology and was present in two forms: the hypha(0.3-1.0mum), which was branched and septate, and the vesicle (3-5mum), which was also septate and developed at the parental hypha tip. Bacteria-like cells, previously observed in light microscopy studies, were not seen in the present work. The actinomycete-like endophyte penetrated through the host cell wall and becane enveloped by a capsular material (0.1mum), the whole being enclosed by host membranes. In some host cells, the endophyte appeared to lyse and become a mass of shrunken debris. The fine structure of the Alnus crispa var. mollis root nodule endophyte was found to be similar to that of other nonleguminous root nodule endophytes.

Bacteria

Observations on the male nucleus during fertilization in the fern Pteridium aquilinum.

Fertilization of eggs of Pteridium under controlled conditions has revealed that archegonia containing viable eggs require more than 30 min flooding before opening. Syngamy appears to take place by an engulfing action, and no evidence was found of a 'membrane-fusion' kind of conjugation. Although the gametic nucleus lacks a recognizable envelope, as soon as it comes into contact with the female cytoplasm an envelope is reconstituted from an opaque layer at the surface of its chromatin. This membrane also undergoes growth, the outer layer extending into the cytoplasm and surrounding spermatozoid mitochondria. The male envelope remains distinct from the female, and lacks evaginations and well defined pores. The male nucleus lies in irregular loops within a funnel, filled with egg cytoplasm, formed by the microtubular ribbon of the gamete. The mouth of the funnel is bordered by the multi-layered structure. Karyogamy is initiated at the anterior end of the male nucleus, the orifice connecting the 2 nuclei widening from about 0-1 to 2 mum in diameter during fusion. The contents of the male nucleus flow through the mouth of the funnel into the female, the chromatin relaxing locally, but retaining a rod-like form. The envelope of the male nucleus is probably resorbed at the site of junction. There is no evidence that it contributes significantly to the envelope of the zygotic nucleus.

Cell Nucleus

An ultrastructural study of the spermatozoid of the fern, Marsilea vestita.

The ultrastructure of the mature spermatozoid of Marsilea vestita was studied after its release from the microspore and prior to its penetration of the egg. The psermatozoid is a pear-shaped cell with a complex spiral structure coiled around the edge in the narrow anterior end. This coil is composed of a large mitochondrion, elongated nucleus with highly condensed chromatin, a ribbon of microtubules, and a dense band of material (flagellated band) into which the flagella are inserted. There are over a hundred flagella protruding from each spermatozoid along the length of the coil. At the anterior tip of the coil is a short multilayered structure. It is not known what maintains the helical shape of the coil. The microtubular ribbon could be involved, but it is also possible that either the flagellated band, the condensed chromatin, or both, are sufficiently rigid to retain their shpaes unaided. When the spermatozoid is first released from the microspore it includes a cytoplasmic vesicle in the posterior region containing plastids, mitochondria, and other organelles. This vesicle is shed, taking the nuclear envelope with it, before the spermatozoid reaches the egg.

Cell Nucleus

Developmental mechanisms in heterospory: cytochemical demonstration of spore-wall enzymes associated with beta-lectins, polysaccharides and lipids in water ferns.

Cytochemical methods are used to examine the distribution and localization of acid phosphatase, non-specific esterase, ribonuclease and peroxidase activity in the walls of the spores of the heterosporous Marsileaceae before and during germination. In the quiescent spore, the principal activity is associated with the perine layer of the wall and the intine, with some activity in the outer, gelatinous wall layer, but none in the exine. The microspores of Marsilea and Pilularia have non-specific esterase activity concentrated in the intine inthe immediate vicinity of the germinal site; that is, above the position of the future male gametangia. The enzymes are not leached from the wall during hydration of the spores, although ribonuclease is redistributed during imbibition with a high concentration of activity remaining in or around the germinal site. The wall enzymes occur together with PAS-reactive and acidic carbohydrates, lipids, and in the microspore perine, beta-lectins. Following the enzyme pattern, the beta-lectins are found to be concentrated in the region of the germinal site. beta-Lectin activity is absent from the megaspore wall. Acidic carbohydrates are confined to the gelatinous wall layer and this layer also binds concanavalin A. In contrast to what has been found for other plant cells, the spore-wall beta-lectins are not water-labile; the activity is not significantly diminished after hydration. This surprising stability suggests that these molecules, together with the enzymes, may be retained in position in the wall by the waterproof overlay of lipid. From the evidence of preliminary developmental studies, it appears that the enzymes associated with the perine layer of the wall originate in the sporophytic tapetal periplasmodium and inclusion of the activity is concurrent with wall differentiation, while the activity associated with the intine is derived from the gametophyte. It is possible, however, in the megaspore at least, that the distribution of the activity may to some extent be influenced by a system of exine channels which communicates between the two domains of the wall during sporogenesis. No definite information is obtained concerning the utility of the enzymes and associated molecules in the life of the spore. Acting separately or in co-operation, their role could conceivably be connected with one or more of four processes; wall differentiation, gametophyte nutrition, gametophyte protection or reproduction. Each of these possibilities is discussed.

Acid Phosphatase

Abnormal structures in the flagellar apparatus of the spermatozoa of two ferns.

The flagellar structure of spermatozoa is generally of the 9 + 2 pattern. However, in various animal species abnormalities have been found which some authors have recently suggested as being of value in systematics. Little information has, as yet, been published in this field on plants. In studying spermatogenesis, we found some abnormalities in connection with central and peripheral tubules. These abnormal structures were uncommon and affected only a few of the numerous flagella borne by a single spermatozoid. Such abnormalities do not seem to affect radically the motility of the spermatozoa, but they may have lost part of their fertilizing ability.

Male

Effect of drying with hot forced draft and of mincing bracken fern on its carcinogenic activity.

The carcinogenic activity of young bracken dried at a high temperature or minced by a mechanical chopper was compared with that of unprocessed bracken dried at room temperature. Inbred strain ACI rats of both sexes were divided into 4 groups; Group 1 received a diet containing the unprocessed bracken dried at a room temperature of below 30 degrees, Group 2 received a diet containing bracken dried at 70 approximately 90 degrees with a hot, forced draft, and Group 3 received the diet containing processed bracken that had been minced to a paste-like consistency using a mechanical chopper. The control group was fed a normal diet. Rats fed a bracken-containing diet developed tumors most frequently in the ileum. Comparison of Group 1 with Groups 2 and 3 showed no significant difference in the latent period, incidence, histologic types, or multiplicity of the intestinal tumors found in each animal.

Adenocarcinoma

Bovine enzootic haematuria in Queensland.

Bovine enzootic haematuria was diagnosed in Queensland in coastal areas in association with bracken fern (Pteridium esculentum) on 3 properties or with mulga fern or rock fern (Cheilanthes sieberi) on 4 properties, and in inland areas in association with C. sieberi on 3 properties. In the absence of bracken fern, long-term ingestion of C. sieberi is suggested as a cause of bovine enzootic haematuria. Haemangiomas, haemangiosarcomas, transitional cell carcinomas, papillomas, fibromas and an adenoma were detected in the urinary bladders of 19 affected cattle and were accompanied by chronic cystitis.

Animals

Studies on DNA sequences in the Osmundaceae.

Phylogenetic relationships of Osmunda cinnamomea, O. claytoniana, and O. regalis were explored by means of DNA sequence comparisons. Hydroxyapatite thermal elution profiles of self-reassociated repetitive DNA fragments were very similar, indicating the absence of gross differences in the amount of recent amplification or addition of repetitive DNA in any of these three genomes. Interspecific DNA sequence comparisons showed, in contrast to our earlier interpretation, that repeated DNA sequences of O. claytoniana are nearly equally diverged from those of O. cinnamomea and O. regalis. Differences between repetitive sequences of the three species can be interpreted as reflecting amplification events which occurred subsequent to speciation. The data obtained suggest that the three Osmunda species most likely arose more or less simultaneously from a common ancestor. These findings were verified in experiments with tracer DNA preparations enriched for single copy sequences. On the basis of the hydridization data presented here and of the fossil record, the rate of single copy sequence divergence in the ferns is comparable to that in the primates, although slower than that observed in other animal taxa. From this first evaluation of rates of DNA evolution in plants it would seem that the rates for plants and animals are roughly comparable. The evidence suggests that species divergence is accompanied by further reiteration of preexisting repeat sequences. The rate of addition of repetitive sequences probably is slower in ferns than in angiosperms. This difference might be attributable to the much larger effective generation time in ferns.

Base Sequence

Mutagens in a river heavily polluted with paper recycling wastes: results of field and laboratory mutagen assays.

Paper recycling industries generate considerable quantities of waterborne wastes, and thus water pollution constitutes the greatest environmental problem associated with this industrial activity [Hunt and Franklin, 1973]. Generally the impact of this water pollution is considered in terms of aesthetic blight and deterioration of water quality. We present data that document another aspect of this pollution, the environmental contamination of an aquatic ecosystem with mutagenic materials. A natural population of the fern Osmunda regalis growing in a river heavily polluted with paper recycling wastes had a high incidence of chromosome mutations. This population was monitored for four years for the frequency of two-break chromosome mutations. These mutations were postzygotic in origin and suggested the presence of mutagens in the river water. The fern population is downstream from the outfalls of a paper recycling mill, which was discharging 13.3 X 10(6) liters of untreated paper recycling waste per day. In 1977 a waste-water-treatment facility was constructed to remove the solid waste previously discharged into the river. This facility generates 69,300 kg of solid waste daily, which is taken to a landfill. Periodic samples of this solid waste were collected from the waste-treatment facility in the summer of 1978, extracted with various solvents, and the extracts tested for mutagenic activity with the Salmonella/mammalian microsome mutagenicity test [Ames et al, 1975]. A majority of the solid waste samples contained mutagenic materials, but in all cases S-9 activation was required for mutagenic activity. The samples also were assayed with the soybean mitotic crossing-over assay [Vig, 1975]. Four out of six samples were positive. These results document the presence of mutagens in the solid waste generated by a paper recycling industry and the genetic impact of these mutagens on the local biota.

Chromosome Aberrations