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Biomedical subjects

P K Hepler

Publications and source records attributed to P K Hepler.

66 records · Page 4Linked to original sources
PubMed1976

Production of guard cell protoplasts from onion and tobacco.

Guard cell protoplasts (GCP) from young cotyledons of onion and tobacco were isolated in culture microchambers where optimal isolating and culture conditions could be determined in situ. The digestion course was quantified by following under polarized light the loss of.retardation of the birefringent cellulose of the guard cells. The assay showed that driselase has a 5-fold higher cellulytic activity than cellulysin. Driselase is, however, harmful to the GCP. Calcofluor staining was less adequate for establishing digestion courses because it increases sharply after exposing guard cells to cellulysin.Osmotic conditions were crucial for GCP survival. Onion guard cells fragment in the presence of strong plasmolyticum (>0.45 m) indicating cytoplasmic connections between neighboring guard cells and/or cytoplasmic attachments to the wall. Tobacco guard cells plasmolyzed with 0.7 m mannitol revealed several areas of strong attachment to the wall which resulted in severe damage to the cells. Healthy tobacco GCP are obtained by an initial digestion with 4% (w/v) cellulysin in 0.23 m mannitol for 2 to 3 hours followed by an increase in the osmoticum to 0.7 m to stabilize the forming protoplasts.Onion GCP were obtained by digesting paradermal slices with 4% (w/v) cellulysin in 0.23 m mannitol. Protoplasts can be osmotically released by replacing the enzyme solution with 0.23 m mannitol at early stages of digestion. They are also available after prolonged digestion (6-12 hours). Paradermal slices also yield mesophyll and epidermal cell protoplasts but they can be selectively washed away if a pure preparation of GCP is desired. Onion GCP have been kept alive in a simple culture solution for up to 10 days.

Journal Article↗
PubMed1976

The blepharoplast of Marsilea: its de novo formation and spindle association.

Blepharoplast formation and function has been investigated by light and electron microscopy in developing microspores of the water fern Marsilea vestita. When single-celled microspores are hydrated they undergo a series of 9 cell divisions producing 7 sterile cells plus 32 spermatids. The blepharoplast, a densely staining, spherical inclusion (0-5-1-0 mum in diameter) interpenetrated by numerous lightly stained channels, is a precursor organelle for the production of the 100-150 basal bodies of the motile sperm. During development it arises twice in the dividing spermatogenous cells; first in telophase of the second to last or 7th division, and again in telophase of the next to last or 8th division. The blepharoplast first forms on the distal side of the nucleus during telophase of the 7th division but soon degenerates as the cell enters prophase and metaphase of the 8th division. The blepharoplast first forms on the distal side of the nucleus during telophase of the 7th division but soon degenerates as the cell enters prophase and metaphase of the 8th division. During telophase of the 8th division the blepharoplast again arises in the cytoplasm near a small indentation of the nuclear envelope. The forming blepharoplast consists of a sphere of lightly stained flocculent material within which 2 plaques separated by 40-50 nm arise. Each plaque possesses 2 dense layers (20 nm) separated by a light layer (10 nm). During subsequent development the distal layer of each plaque becomes progressively more densely stained. Light-staining channels appear within the accumulating dense material a-d 2 hemispherical blepharoplasts emerge. The blepharoplasts become spherical through continued growth and finally separate from each other as they move to the spindle poles during prophase of the last or 9th division. The blepharoplast appears to act as a microtubule organizing centre during formation of the spindle apparatus in prophase of the 9th division. While it remains at the pole throughout mitosis it does not continue to serve as the focal point for the spindle tubules. During metaphase-anaphase of the 9th division the blepharoplast swells, and the channels at its surface begin transforming into procentrioles and finally basal bodies. The results support the contention that basal bodies in Marsilea arise de novo, since no preexisting template such as a centriolar pinwheel is observed and sine the intermediates which initially occur are structurally dissimilar from a procentriole.

Microtubules↗
PubMed1975

Identification of actin in situ at the ectoplasm-endoplasm interface of Nitella. Microfilament-chloroplast association.

Using a glycerination procedure designed to avoid excessive plasmolysis or disruption of the ectoplasm, microfilaments in bundles at the ectoplasm-endoplasm interface of Nitella internode cell segments were found to bind rabbit heavy meromyosin (HMM) in situ. All HMM arrowheads in a bundle seem to have the same polarity and many lie in register as judged from the electron micrographs; the arrowhead periodicity is approximately 380 . The decorated microfilaments are thus similar to those seen in negatively stained cytoplasmic suspensions of internode cells. In glycerinated material, as well as in suspensions, the microfilaments are closely associated with chloroplasts. The microfilaments lie adjacent to or are attached to the chloroplast envelope. The results provide further evidence that the microfilaments thought to play a role in cytoplasmic streaming in vivo in Nitella consist of actin and suggest that they may be anchored to the chloroplasts.

Actins↗
PubMed1974

Actin in the green alga, Nitella.

Bundles of microfilaments very similar in appearance to actin are present in cytoplasmic suspensions obtained from Nitella flexilis. The microfilaments bind rabbit heavy meromyosin in arrowhead arrays similar to those produced on muscle actin. The arrowheads are removed with ATP. The results provide evidence that actin is present in green plants, probably in the form of microfilaments thought to be involved in cytoplasmic streaming.

Actins↗
PubMed1968

Microtubules and early stages of cell-plate formation in the endosperm of Haemanthus katherinae Baker.

A fine structure study of the phragmoplast and developing cell plate has been made on glutaraldehyde-osmium tetroxide-fixed, dividing, cultured cells of the liquid endosperm of Haemanthus katherinae Baker. The phragmoplast arises between the telophase nuclei, usually in association with a remnant strand of spindle elements, and consists of an accumulation of microtubules oriented at right angles to the plane of the future cell plate. The microtubules, which are 200-240 A in diameter, occur in small clusters spaced at approximately 0.2-0.3 micro intervals along the plate. Short interconnections interpreted as "cross-bridges" have been observed between individual microtubules. Within each cluster there is an electron-opaque zone about 0.3 micro in width which can be attributed in part to an overlap of microtubules from both sides of the plate and in part to a local accumulation of an amorphous electron-opaque material. During development these dense zones become aligned in a plane which itself defines the plane of the plate. Vesicles, commonly observed in long files, are derived from a cytoplasmic matrix rich in elements of the endoplasmic reticulum and sparse in dictyosomes. They aggregate between the clusters of microtubules and eventually coalesce to form the cell plate.

Cell Division↗
PubMed1968

An atypical crista resembling a "tight junction" in bean root mitochondria.

The conformation and structure of an atypical crista found in a small percentage of the mitochondria in root tip cells of Phaseolus vulgaris L. have been studied electron microscopically in material fixed in glutaraldehyde followed by osmium tetroxide. In its transformation into an atypical crista, a normal crista elongates, broadens, and flattens, and the inner leaflets of its apposed unit membranes appear to fuse in a manner analogous to the formation of "tight junctions" between certain animal cells. The result is a large platelike, quintuple-layered structure, 240-260 A thick, whose long axis parallels that of the mitochondrion. The outer layers of the "plate," bordering on the mitochondrial matrix, are thickened and exhibit striking patterns in the micrographs. The structure of the plate is compared with that previously described for tight junctions between animal cells.

Membranes↗
PubMed1966

The feeding mechanism of avian malarial parasites.

Electron microscope studies of the erythrocytic forms, including gametocytes and asexual schizonts, of the protozoa Plasmodium fallax, P. lophurae, and P. cathemerium, have revealed a "cytostome," a specialized organelle of the pellicular membrane which is active in the ingestion of host cell cytoplasm. In material fixed in glutaraldehyde and postfixed in OsO(4), the cytostome appears in face view as a pore limited by two dense circular membranes and having an inside diameter of approximately 190 mmicro. In cross-section, the cytostome is a cavity bounded on each side by two dense segments corresponding to the two dense circles observed in face view; its base consists of a single unit membrane. In the process of feeding, the cytostome cavity enlarges by expansion of its membrane, permitting a large quantity of red cell cytoplasm to come into contact with the cytostome wall. Subsequent digestion of erythrocyte cytoplasm occurs exclusively in food vacuoles which emanate from the cytostome invagination. As digestion progresses, the food vacuoles initially stain more densely and there is a marked build-up of hemozoin granules. In the final stage of digestion, a single membrane surrounds a cluster of residual pigment particles and very little of the original host cell cytoplasm remains. The cytostome in exoerythrocytic stages of P. fallax has been observed only in merozoites and does not seem to play the same role in the feeding mechanism.

Animals↗
PubMed1966

The fine structure of the exoerythrocytic stages of Plasmodium fallax.

The fine structure of the exoerythrocytic cycle of an avian malarial parasite, Plasmodium fallax, has been analyzed using preparations grown in a tissue culture system derived from embryonic turkey brain cells which were fixed in glutaraldehyde-OsO(4). The mature merozoite, an elongated cell 3- to 4-micro long and 1- to 2-micro wide, is ensheathed in a complex double-layered pellicle. The anterior end consists of a conoid, from which emanate two lobed paired organelles and several closely associated dense bodies. A nucleus is situated in the mid portion of the cell, while a single mitochondrion wrapped around a spherical body is found in the posterior end. On the pellicle of the merozoite near the nucleus a cytostomal cavity, 80 to 100 mmicro in diameter, is located. Based on changes in fine structure, the subsequent sequence of development is divided into three phases: first, the dedifferentiation phase, in which the merozoite loses many complex structures, i.e. the conoid, paired organelles, dense bodies, spherical body, and the thick inner layers of the pellicle, and transforms into a trophozoite; second, the growth phase, which consists of many nuclear divisions as well as parallel increases in mitochondria, endoplasmic reticulum, and ribosomes; and third, the redifferentiation and cytoplasmic schizogony phase, in which the specialized organelles reappear as the new merozoites bud off from the mother schizont.

Animals↗