SU-E-T-315: Planning and Verification of CT-Based HDR Intraluminal Brachytherapy Treatment for Malignant Obstructive Jaundice.

PURPOSE: To present our experience using CT to plan and verify intraluminal HDR treatment for a patient with obstructive jaundice. Due to the obstruction's proximity to the small bowel, along with small bowel adhesions from past surgical history, it was imperative to verify source position relative to the bowel before each treatment. METHODS: Treatment was administered to a total dose of 2000cGy in 5 fractions via a 6F intraluminal catheter inserted into the patient's 14F percutaneous drainage catheter. Graduations on the intraluminal catheter were used to measure the exact length of catheter inserted in to the patient's drainage tube allowing reproducibility. Dummy seeds inserted during CT were identified by iteratively aligning the planning system's 3D reconstruction axis to the catheter at multiple points as it snaked through the liver. Taking in to account the known offset between actual dwell positions and dummy source positions, we determined what dwell positions to activate for planning. CT verification was performed prior to each treatment to insure that the drainage catheter had not moved and that the distance from treatment site to small bowel was adequate. Dummy seeds and anatomical landmarks were identified on the scout image and correlated to the CT. RESULTS: Verification CTs showed remarkable consistency in the day-to-day drainage catheter position. The physician was able to easily identify the small bowel of concern on the CT and determine if a safe distance existed for treatment. CONCLUSIONS: The method outlined in this work provides a safe means by which to treat bile duct obstructions using HDR when critical structures are nearby. We were prepared to make real-time adjustments to our treatment plan to account for significant variation, but found it unnecessary to do so in this particular case.

The cytoskeleton in the unique cell reproduction by conidiogenesis of the long-neck yeast Fellomyces (Sterigmatomyces) fuzhouensis.

The morphology of conidiogenesis and associated changes in microtubules, actin distribution and ultrastructure were studied in the basidiomycetous yeast Fellomyces fuzhouensis by phase-contrast, fluorescence, and electron microscopy. The interphase cell showed a central nucleus with randomly distributed bundles of microtubules and actin, and actin patches in the cortex. The conidiogenous mother cell developed a slender projection, or stalk, that contained cytoplasmic microtubules and actin cables stretched parallel to the longitudinal axis and actin patches accumulated in the tip. The conidium was produced on this stalk. It contained dispersed cytoplasmic microtubules, actin cables, and patches concentrated in the cortex. Before mitosis, the nucleus migrated through the stalk into the conidium and cytoplasmic microtubules were replaced by a spindle. Mitosis started in the conidium, and one daughter nucleus then returned to the mother via an eccentrically elongated spindle. The cytoplasmic microtubules reappeared after mitosis. A strong fluorescence indicating accumulated actin appeared at the base of the conidium, where the cytoplasm cleaved eccentrically. Actin patches then moved from the stalk together with the retracting cytoplasm to the mother and conidium. No septum was detected in the long neck by electron microscopy, only a small amount of fine "wall material" between the conidium and mother cell. Both cells developed a new wall layer, separating them from the empty neck. The mature conidium disconnected from the empty neck at the end-break, which remained on the mother as a tubular outgrowth. Asexual reproduction by conidiogenesis in the long-neck yeast F. fuzhouensis has unique features distinguishing it from known asexual forms of reproduction in the budding and fission yeasts. Fellomyces fuzhouensis develops a unique long and narrow neck during conidiogenesis, through which the nucleus must migrate into the conidium for eccentric mitosis. This is followed by eccentric cytokinesis. We found neither an actin cytokinetic ring nor a septum in the long neck, from which cytoplasm retracted back to mother cell after cytokinesis. Both the conidium and mother were separated from the empty neck by the development of a new lateral wall (initiated as a wall plug). The cytoskeleton is clearly involved in all these processes.

Cytoskeleton in regenerating protoplasts and restoration of cell polarity in the yeast Saccharomyces cerevisiae.

The actin cables and microtubules were disrupted during protoplasting in Saccharomyces cerevisiae cells. In the process of protoplast regeneration, the cytoplasmic microtubules were the first to be restored; the actin patches remained regularly distributed under the surface of the growing protoplast. After the cell wall had been regenerated in a gelatine medium, the actin patches aggregated into clusters, which marked the site of bud development. An incomplete cell wall was the apparent cause for uncoupling between karyokinesis and cytokinesis. The presence of several nuclei in the cell gave rise to several buds emerging simultaneously and was probably related to their random positions. In haploids, however, the axial type of budding was also obvious in the reverting protoplasts. These observations suggest that actin is a structure involved in the restoration of polar growth during protoplast regeneration, and that the cell wall plays an important role in this process: in its absence, actin fails to polarise.

Microtubules and actin cytoskeleton in Cryptococcus neoformans compared with ascomycetous budding and fission yeasts.

Actin cytoskeleton and microtubules were studied in a human fungal pathogen, the basidiomycetous yeast Cryptococcus neoformans (haploid phase of Filobasidiella neoformans), during its asexual reproduction by budding using fluorescence and electron microscopy. Staining with rhodamine-conjugated phalloidin revealed an F-actin cytoskeleton consisting of cortical patches, cables and cytokinetic ring. F-actin patches accumulated at the regions of cell wall growth, i. e. in sterigma, bud and septum. In mother cells evenly distributed F-actin patches were joined to F-actin cables, which were directed to the growing sterigma and bud. Some F-actin cables were associated with the cell nucleus. The F-actin cytokinetic ring was located in the bud neck, where the septum originated. Antitubulin TAT1 antibody revealed a microtubular cytoskeleton consisting of cytoplasmic and spindle microtubules. In interphase cells cytoplasmic microtubules pointed to the growing sterigma and bud. As the nucleus was translocated to the bud for mitosis, the cytoplasmic microtubules disassembled and were replaced by a short intranuclear spindle. Astral microtubules then emanated from the spindle poles. Elongation of the mitotic spindle from bud to mother cell preceded nuclear division, followed by cytokinesis (septum formation in the bud neck). Electron microscopy of ultrathin sections of chemically fixed and freeze-substituted cells revealed filamentous bundles directed to the cell cortex. The bundles corresponded in width to the actin microfilament cables. At the bud neck numerous ribosomes accumulated before septum synthesis. We conclude: (i) the topology of F-actin patches, cables and rings in C. neoformans resembles ascomycetous budding yeast Saccharomyces, while the arrangement of interphase and mitotic microtubules resembles ascomycetous fission yeast Schizosaccharomyces. The organization of the cytoskeleton of the mitotic nucleus, however, is characteristic of basidiomycetous yeasts. (ii) A specific feature of C. neoformans was the formation of a cylindrical sterigma, characterized by invasion of F-actin cables and microtubules, followed by accumulation of F-actin patches around its terminal region resulting in development of an isodiametrical bud.

Cell wall and cytoskeleton reorganization as the response to hyperosmotic shock in Saccharomyces cerevisiae.

Transfer of exponentially growing cells of the yeast Saccharomyces cerevisiae to hyperosmotic growth medium containing 0.7-1 M KCl, 1 M mannitol, and/or 1 M glycerol caused cessation of yeast growth for about 2 h; thereafter, growth resumed at almost the original rate. During this time, formation of fluorescent patches on the inner surface of cell walls stained with Primulin or Calcofluor white was observed. The fluorescent patches also formed in solutions of KCl or when synthesis of the cell wall was blocked with cycloheximide and/or 2-deoxyglucose. The patches gradually disappeared as the cells resumed growth, and the new buds had smooth cell walls. Electron microscopy of freeze-etched replicas of osmotically stressed cells revealed deep plasma membrane invaginations filled from the periplasmic side with an amorphous cell wall material that appeared to correspond to the fluorescent patches on the cell surface. The rate of incorporation of D-[U-14C]glucose from the growth medium into the individual cell wall polysaccharides during osmotic shock followed the growth kinetics. No differences in cell wall composition between osmotically stressed yeast and control cells were found. Hyperosmotic shock caused changes in cytoskeletal elements, as demonstrated by the disappearance of microtubules and actin microfilaments. After 2-3 h in hyperosmotic medium, both microtubules and microfilaments regenerated to their original polarized forms and the actin patches resumed their positions at the apices of growing buds. The response of S. cerevisiae strains with mutations in the osmosensing pathway genes hog1 and pbs2 to hyperosmotic shock was similar to that of the wild-type strain. We conclude that, besides causing a temporary disassembling of the cytoskeleton, hyperosmotic shock induces a change in the organization of the cell wall, apparently resulting from the displacement of periplasmic and cell wall matrix material into invaginations of the plasma membrane created by the plasmolysis.

Reproducibility of temporomandibular joint clicking.

AIMS: To investigate the stability of temporomandibular joint (TMJ) clicking over a 10-day period and the effect of different open/close velocities on sound amplitude and power spectra in a group of subjects with subjectively stable unilateral clicking during the 3 months preceding the recordings. METHODS: Ten volunteers were recorded with a self-developed microcomputer-based system used in a previous study on asymptomatic subjects. The recordings were performed during 4 different sessions at 3 different open/close rates in each session. The subjective sound intensity was measured with a 100-mm visual analog scale (VAS). The VAS scores, the maximum amplitudes, and the power spectra of the signals were tested for statistical differences among the different open/close rates and over the sessions. The reliability of measurements was also calculated. RESULTS: The maximum amplitude and the power spectra of the TMJ clicking varied between subjects in a broad range that differed from those reported for asymptomatic subjects. No statistically significant differences were found within subjects for the subjective VAS scores for the maximum signal amplitudes or for the power spectrum parameters among the open/close rates and over the 4 sessions. For all 3 open/close rates and for the 4 sessions, a good to excellent reliability of measurements was determined, the values of r being mostly over 0.75. CONCLUSION: Within the limits of the experiment, TMJ clicking was subjectively and objectively stable over a period of 10 days. Therefore, the constant subjective perception of sound intensity was supported by the objective measurements.

Topology of microtubules and actin in the life cycle of Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

The morphology of budding and conjugating cells and associated changes in microtubules and actin distribution were studied in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) by phase-contrast and fluorescence microscopy. The non-budding interphase cell showed a nucleus situated in the central position and bundles of cytoplasmic microtubules either stretching parallel to the longitudinal cell axis or randomly distributed in the cell; none of these, however, had a character of astral microtubules. During mitosis, the nucleus divided in the daughter cell, cytoplasmic microtubules disappeared and were replaced by a spindle. The cytoplasmic microtubules reappeared after mitosis had finished. Actin patches were present both in the bud and the mother cell. Cells were induced to mate by transfer to ribitol-containing medium without nitrogen. Partner cells fused by conjugation projections where actin patches had been accumulated. Cell fusion resulted in a zygote that produced a basidium with parallel bundles of microtubules extended along its axis and with actin patches concentrated at the apex. The fused nucleus moved towards the tip of the basidium. During this movement, nuclear division was taking place; the nuclei were eventually distributed to basidiospores. Mitochondria appeared as vesicles of various sizes; their large amounts were found, often lying adjacent to microtubules, in the subcortical cytoplasm of both vegetative cells and zygotes.

Colocalization of microtubules and mitochondria in the yeast Schizosaccharomyces japonicus var. versatilis.

Both living and fixed cells of Schizosaccharomyces japonicus var. versatilis showed thread-like mitochondria when studied by phase-contrast and fluorescence microscopy. In the interphase cells, mitochondria extended from pole to pole and converged towards the growing tips. The mitochondrial threads did not disrupt but persisted during mitosis and, subsequently, their bundle was split between the two daughter cells by a concentrically growing septum. Mitochondria in the interphase cells were accompanied by cytoplasmic microtubules. These disappeared during mitosis and, instead, spindle microtubules were formed in the nucleus. The cytoplasmic microtubules reappeared after anaphase B, again in coaligment with mitochondria. Protoplasting as well as the action of microtubule inhibitors methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate (benomyl) and 2-methylbenzimidazole (MBC) resulted in rapid disintegration of microtubules and, suprisingly, also in disruption of mitochondria into small bodies. Removal of the inhibitors or a short regeneration of protoplasts allowed both the cytoplasmic microtubules and the thread-like mitochondria to reaggregate into the original pattern. Cytochalasin D treatment caused a complete disintegration of actin filaments, while the cytoplasmic microtubules and mitochondria remained intact. These findings of a transient close association of mitochondria and microtubules and their relative independence of the arrangement of actin filaments suggest that microtubules, but not actin cables, form supports for positioning or movement of mitochondria along the cylindrical cells. The persistence of mitochondria in the cell centre during mitosis may be accounted for by the fact that disrupted microtubules fail to provide support for mitochondrial movement towards the cell poles.

Microtubule-driven nuclear movements and linear elements as meiosis-specific characteristics of the fission yeasts Schizosaccharomyces versatilis and Schizosaccharomyces pombe.

Meiotic prophase in Schizosaccharomyces pombe is characterized by striking nuclear movements and the formation of linear elements along chromosomes instead of tripartite synaptonemal complexes. We analysed the organization of nuclei and microtubules in cells of fission yeasts undergoing sexual differentiation. S. japonicus var. versatilis and S. pombe cells were studied in parallel, taking advantage of the better cytology in S. versatilis. During conjugation, microtubules were directed towards the mating projection. These microtubules seem to lead the haploid nuclei together in the zygote by interaction with the spindle pole bodies at the nuclear periphery. After karyogamy, arrays of microtubules emanating from the spindle pole body of the diploid nucleus extended to both cell poles. The same differentiated microtubule configuration was elaborated upon induction of azygotic meiosis in S. pombe. The cyclic movements of the elongated nuclei between the cell poles is reflected by a dynamic and coordinated shortening and lengthening of the two microtubule arrays. When the nucleus was at a cell end, one array was short while the other bridged the whole cell length. Experiments with inhibitors showed that microtubules are required for karyogamy and for the elongated shape and movement of nuclei during meiotic prophase. In both fission yeasts the SPBs and nucleoli are at the leading ends of the moving nuclei. Astral and cytoplasmic microtubules were also prominent during meiotic divisions and sporulation. We further show that in S. versatilis the linear elements formed during meiotic prophase are similar to those in S. pombe. Tripartite synaptonemal complexes were never detected. Taken together, these findings suggest that S. pombe and S. versatilis share basic characteristics in the organization of microtubules and the structure and behaviour of nuclei during their meiotic cell cycle. The prominent differentiations of microtubules and nuclei may be involved in the pairing, recombination, and segregation of meiotic chromosomes.

Response of yeast protoplasts to their mating partners.

The mating process between two protoplasts or between a protoplast and a cell in the yeast Saccharomyces cerevisiae was manifested by a specific morphological response of only the cell partner. The cells produced projections, up to 5 micrometers long, to meet their protoplast partners. The protoplasts responded, after a period of nonspecific hernia-like growth, by ceasing to grow and assuming oval or spherical shapes. They never formed mating projections, apparently due to the absence of complete cell walls. Similarly to the cells, nuclear division in protoplasts was arrested and the nucleus migrated towards the plasma membrane at the site of protoplast-cell contact. Cytoplasmic microtubules were directed to this site, indicating the position of the spindle pole body (SPB) on the nucleus adjacent to the plasma membrane. Actin patches accumulated also in this region. These cytological features of the protoplasts were reminiscent of the reorganization of the cytoskeleton and nucleus characteristic of mating cells. This implies that the ability of protoplasts to produce and receive mating signals was unaffected by protoplasting. Fusion, however, was not initiated due to the absence of the complete cell wall in one of the partners. Thus, the cell wall appeared to be necessary for the expression of polarized growth during mating and for cell fusion.

Cell surface structures in osmotically fragile mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae characterized by osmotic fragility showed a marked fibrillar structure on the inner wall surface when studied by two electron microscopic techniques, i.e. freeze-etching of whole native cells and metal shadowing of isolated cell walls. The walls of the mutant cells were more permeable to macromolecules than were those of the wild-type parental strain. The synthesis and assembly of (1----3)-beta-D-glucan wall microfibrils studied in protoplasts of mutant cells were not impaired. It is suggested that the osmotic fragility of the mutant cells is related to the deficiency of the wall structure as a consequence of the srb1 mutation affecting biogenesis of the amorphous (glucan) component.

Sporothrix schenckii--a freeze-fracture study.

Freeze-fracture electron microscopy of a pathogenic dimorphic fungus Sporothrix schenckii revealed planar views of cell structures corresponding to those described already on thin sections. In addition to the characteristic differences in cell wall thickness between conidia, yeast forms and filaments, variations in plasma membrane invaginations were found. In conidia the invaginations were short and abundant, while in yeast forms they were scarce and longer. The plasma membrane of the filaments was smooth without invaginations. No differences were found in the frequency of intramembrane particles among the three forms. In the region of the septal pore the particles were circularly arranged with a characteristic partitioning on the P and E fracture faces.

Ultrastructure of Saccharomyces cerevisiae cells accumulating Golgi organelles.

Restrictive phenotype of sec 7 mutant of Saccharomyces cerevisiae was examined by freezefracture electron microscopy. In accordance with previous findings (NOVICK et al. 1981) dictyosomes and middle-size (200-600 nm) vesicles (Berkeley bodies) were found to accumulate. Dictyosomes are formed by aggregated flattened or dilated cisternae without associated secretory vesicles. After transfer to permissive conditions the dictyosomes disappear and are not detectable, just like in the wild type or in the permissive phenotype. Associated with the restrictive phenotype is an extended plasma membrane and tonoplasts with particle-free impressions.

Ultrastructure of two secretory mutants of Saccharomyces cerevisiae as revealed by freeze-fracture technique.

Permissive and restrictive phenotypes of two secretory mutants of Saccharomyces cerevisiae, sec 1 and sec 18, were studied by freeze-fracture technique. The sec 1 mutant, in addition to accumulating secretory vesicles, was characterized by a disappearance of the plasma membrane invaginations and by an aggregation of intra-membrane particles in vacuolar membranes. A prolonged incubation of the cells at 37 degrees C led to pathological fusion of some vesicles with the plasma membrane. After the cells were transferred back to the permissive temperature the invaginations reappeared rapidly while the accumulated vesicles disappeared only after budding had been resumed. The sec 18 mutant, apart from having distended endoplasmic reticulum membranes, also lost the plasma membrane invaginations at 37 degrees C and regained them at 24 degrees C. The described ultrastructural changes are typical for the restrictive phenotypes and represent further manifestations of the pleiotropic effect of the respective sec mutations.

Intramitochondrial ATP and cell functions: yeast cells depleted of intramitochondrial ATP lose the ability to grow and multiply.

Cells of the yeast Saccharomyces cerevisiae could be depleted of their intramitochondrial ATP bu culturing on glucose in the presence of antimycin A, which prevents production of ATP in mitochondria, along with bongkrekic acid, which prevents transport of ATP from the cytosol into mitochondria. Alternatively, the depletion could be achieved by culturing respiration-deficient mutants in the presence of bongkrekic acid. The depleted cells of the respiration-deficient mutant did not grow on glucose in a synthetic medium and growth for a few generations was made possible by adding peptone, yeast extract or some amino acids into the medium. The depleted cells did not differ from control cells in their content of amino acids, proteins, nucleic acids and major phospholipids and had preserved the ability to carry on protein and nucleic acid syntheses and to mate to other cells. No conspicuous cytological differences were found between the control and depleted cells. After culturing in a semi-synthetic medium in the presence of bongkrekic acid the cells of the respiration-deficient mutant exhibited almost no cytochrome c in their spectra and their azide-sensitive ATPase activity was drastically reduced. The results suggest that intramitochondrial syntheses of some low-molecular compounds as well as import and/or assembly of some cytoplasmically synthesized mitochondrial proteins into mitochondria may be impaired in cells lacking intramitochondrial ATP and this may be responsible for their inability to grow and multiply.

Identification of homologous ribosomal proteins in HeLa cells and in Tetrahymena pyriformis. A study of proteins binding 5-S RNA and acidic proteins released from 40-S subunits by EDTA.

Tetrahymena pyriformis 60-S ribosomal subunits treated with EDTA release a 7-S particle containing 5-S RNA and a 36000-Mr protein that is similar to mammalian 5-S-RNA-binding protein L5 in molecular weight, in two-dimensional acrylamide gel mobility, and in peptide pattern as generated by a simple, one-dimensional acrylamide gel technique. Human and T. pyriformis 40-S ribosomal subunits, treated with buffers lacking magnesium or containing EDTA, release varying amounts of two large acidic proteins. We have identified these released proteins by two-dimensional gel electrophoresis.