Direct current ablation destroys multi-stage fibrosarcomas in rats.

INTRODUCTION: Direct current (DC) ablation offers the potential for precise targeting of tumors and stimulation of the immune system, but has not achieved widespread use. This study was conducted to evaluate the impact on tumor size and subject survival of combining a main ablation treatment with a low-current pretreatment, to assess potential immune system activation, and to assess stimulation-related parameters. METHODS AND RESULTS: Twenty-six female Fischer 344 rats were injected with methylcholanthrene-induced fibrosarcoma cells to create primary tumors and again in the contralateral flank when primary tumors reached 700 mm(3) (contralateral tumors were proxies for metastases). There were four treatment groups: two control animals received no treatment; six control plus placebo animals had electrodes implanted but received no stimulation; eight received high-current stimulation (80 coulombs (C) of charge at 20 milliamperes (mA), over 66 minutes); and eight received high-current treatment one day after a low-current pretreatment (10C, 10 mA, 16.6 min). Electrodes were inserted through the tumor base in a single plane, 4.0mm apart, alternating anode and cathode. Treatments commenced once primary tumors reached 700 mm(3). All control animals were sacrificed 55 days after primary tumor cell injection due to excessive tumor growth. Tumors disappeared from all 16 treated rats within eight days; retreatment was required in two animals. Pretreatment had no effect on tumor disappearance or survival. CONCLUSION: Direct current ablation provides highly effective tumor destruction in a rat model. Slower growth of contralateral tumors suggests a remote effect that may involve the immune system.

The vacuole system is a significant intracellular pathway for longitudinal solute transport in basidiomycete fungi.

Mycelial fungi have a growth form which is unique among multicellular organisms. The data presented here suggest that they have developed a unique solution to internal solute translocation involving a complex, extended vacuole. In all filamentous fungi examined, this extended vacuole forms an interconnected network, dynamically linked by tubules, which has been hypothesized to act as an internal distribution system. We have tested this hypothesis directly by quantifying solute movement within the organelle by photobleaching a fluorescent vacuolar marker. Predictive simulation models were then used to determine the transport characteristics over extended length scales. This modeling showed that the vacuolar organelle forms a functionally important, bidirectional diffusive transport pathway over distances of millimeters to centimeters. Flux through the pathway is regulated by the dynamic tubular connections involving homotypic fusion and fission. There is also a strongly predicted interaction among vacuolar organization, predicted diffusion transport distances, and the architecture of the branching colony margin.

Investigations into seed dormancy in Grevillea linearifolia, G. buxifolia and G. sericea: anatomy and histochemistry of the seed coat.

BACKGROUND AND AIMS: Seeds of east Australian Grevillea species generally recruit post-fire; previous work showed that the seed coat was the controller of dormancy in Grevillea linearifolia. Former studies on seed development in Grevillea have concentrated on embryology, with little information that would allow testing of hypotheses about the breaking of dormancy by fire-related cues. Our aim was to investigate structural and chemical characteristics of the seed coat that may be related to dormancy for three Grevillea species. METHODS: Seeds of Grevillea linearifolia, Grevillea buxifolia and Grevillea sericea were investigated using gross dissection, thin sectioning and histochemical staining. Water movement across the seed coat was tested for by determining the water content of embryos from imbibed and dry seeds of G. sericea. Penetration of intact seeds by Lucifer Yellow was used to test for internal barriers to diffusion of high-molecular-weight compounds. KEY RESULTS: Two integuments were present in the seed coat: an outer testa, with exo-, meso- and endotestal (palisade) layers, and an inner tegmen of unlignified sclerenchyma. A hypostase at the chalazal end was a region of structural difference in the seed coat, and differed slightly among the three species. An internal cuticle was found on each side of the sclerenchyma layer. The embryos of imbibed seeds had a water content six times that of dry seeds. Barriers to diffusion of Lucifer Yellow existed at the exotestal and the endotestal/hypostase layers. CONCLUSIONS: Several potential mechanisms of seed coat dormancy were identified. The embryo appeared to be completely surrounded by outer and inner barriers to diffusion of high-molecular-weight compounds. Phenolic compounds present in the exotesta could interfere with gas exchange. The sclerenchyma layer, together with strengthening in the endotestal and exotestal cells, could act as a mechanical constraint.

Retention of fluorescent probes during aldehyde-free anhydrous freeze-substitution.

Fluorescent probes are widely used for microscopy of live-cell processes, but few such probes can also be used with classically fixed or otherwise immobilized material, and none has been used without aldehyde fixation, which can introduce artefacts of structure and probe localization. Here we show that the fluorescence patterns in fungal hyphae loaded with chloromethyl aminocoumarin (CMAC), and then anhydrously freeze-substituted, without any aldehyde fixation, are similar to those seen in living hyphae. Probe loss into the mounting medium (Spurr's resin) with CMAC and five other probes tested indicated that some unwanted solubilization of probe occurred during embedding, but nevertheless vacuoles could be imaged by their retention of probe.

Motile tubular vacuoles in extramatrical mycelium and sheath hyphae of ectomycorrhizal systems.

Extramatrical mycelium and outer hyphae of the sheath of Eucalyptus pilularis-Pisolithus tinctorius mycorrhizas contain abundant motile tubular vacuoles which accumulate the carboxyfluorescein analogue Oregon Green 488 carboxylic acid. The fluorochrome accumulates in a system of small vacuoles, tubules, and larger vacuoles, which are interlinked, motile, and pleiomorphic, in external hyphae, cords, and hyphae of the outer sheath. There is often a difference in fluorescence between two neighbouring cells, indicating that the dolipore septum exercises control on the movement of material between cells. Generally the motile tubular vacuole system in mycorrhizas resembles that previously found in isolated mycelium. The majority of fungal cells in the sheath contain no fluorochrome even after long exposure of the mycorrhiza to the solution, but with differential interference optics the cells are clearly seen to be alive and to contain vacuoles resembling those in the outer hyphae. In translocation experiments, long-distance transport of the fluorochrome is slow and slight, or even nonexistent in some cases.

XMAP215 regulates microtubule dynamics through two distinct domains.

XMAP215 belongs to a family of proteins involved in the regulation of microtubule dynamics. In this study we analyze the function of different parts of XMAP215 in vivo and in Xenopus egg extracts. XMAP215 has been divided into three fragments, FrN, FrM and FrC (for N-terminal, middle and C-terminal, respectively). FrN co-localizes with microtubules in egg extracts but not in cells, FrC co- localizes with microtubules and centrosomes both in egg extracts and in cells, while FrM does not co- localize with either centrosomes or microtubules. In Xenopus egg extracts, FrN stimulates microtubule growth at plus-ends by inhibiting catastrophes, while FrM has no effect, and FrC suppresses microtubule growth by promoting catastrophes. Our results suggest that XMAP215 is targeted to centrosomes and microtubules mainly through its C-terminal domain, while the evolutionarily conserved N-terminal domain contains its microtubule-stabilizing activity.

Prostate carcinoma knowledge, attitudes, and screening behavior among African-American men in Central Harlem, New York City.

BACKGROUND: Although the benefits of prostate carcinoma screening in reducing mortality rates have not been proven or shown to be cost-effective, screening, particularly using prostate specific antigen (PSA) tests, is widespread. A better understanding of screening behavior, knowledge of prostate carcinoma risk, and attitudes toward screening among men at high risk, such as African-American men, would be valuable. METHODS: A prevalence survey was conducted using 2 samples of African-American men, aged 50-74 years: a clinic sample drawn from all clinics in Central Harlem (n = 404) and a random-digit dial sample from the same geographic region (n = 319). The prevalence of self-reported PSA screening was estimated using a cognitive survey methodology based on the internal consistency of answers to four different questions. Prevalence estimates were adjusted to take into account the high proportion of nontelephone residences. RESULTS: The clinic sample, representing a poorer, more ill population (as determined by MOS Physical Function Scores, was less likely to report PSA screening than the community sample (11.1% in clinic sample vs. 25.5% in community). The prevalence of PSA testing in Central Harlem overall in this age group by using two different techniques was estimated to be 24%. In multiple logistic models, self-reported PSA screening was associated with age, education, favorable attitudes toward screening, and knowing someone who had prostate carcinoma. However, the association between these factors and the likelihood of self-reported PSA screening differed between clinic and community samples. CONCLUSIONS: The prevalence of self-reported PSA screening in Central Harlem was lower than that reported for other populations. These findings may be useful in the design of health education campaigns and for counseling innercity, low-income African-American patients appropriately about the disease.

Structure and composition of the thick wall in hair root epidermal cells of Woollsia pungens.

• Hair roots of Woollsia pungens are shown to have thick-walled epidermal cells, a feature found in a small number of other species within the Epacridaceae. Hair roots otherwise had a structure typical of the Ericales. • Ultrastructural, immunocytochemical and histochemical techniques were used to investigate the structure and composition of these thick-walled epidermal cells. • The thick walls were multilamellate with a helicoidal arrangement of microfibrils typical of a secondary cellulosic wall. Staining techniques revealed a relatively high abundance of ß-glucans; these were not ß 1-3 linked and there was no detectable protein. Galactose side-chains were abundant but not mannose or glucose side-chains. The wall contained a pH-dependent net negative charge. Although apparently rich in COOH groups the thick wall did not react, or only minimally, with the monoclonal antibodies JIM5 and JIM7, testing for nonesterified and methyl-esterified pectins, respectively; this contrasted with the strong positive reaction in the cortical and stelar cells. In epidermal cells colonized by mycorrhizal fungi the thick wall had additional layers of spongy appearance with many interconnected, irregular patches containing dispersed material. Colonized cells retained their integrity longer than noncolonized cells. • The thick wall might be important in long-term survival of infected cells and the low levels of pectin might control mycorrhizal endophyte infection.

Brefeldin A affects growth, endoplasmic reticulum, Golgi bodies, tubular vacuole system, and secretory pathway in Pisolithus tinctorius.

Brefeldin A (BFA) reduced radial growth in Pisolithus tinctorius at a concentration as low as 2 microM. Use of endoplasmic reticulum (ER)-Tracker dye, unconjugated BFA, and fluorescent BFA (BODIPY-BFA) allowed comparison of the effects of BFA on the endomembrane system of P. tinctorius at the light and electron microscope levels. Both ER-Tracker dye and BODIPY-BFA have been shown previously to label the ER. Unconjugated BFA and BODIPY-BFA modified the ER network and disrupted the tubular vacuole system in the tip region. The ultrastructure in freeze-substituted hyphae showed that BFA treatment resulted in (i) disruption of the Spitzenkörper, (ii) reduction in number of apical vesicles, (iii) redistribution and mild dilation of ER, and (iv) persistence and increased size and complexity of Golgi bodies. The effects of BFA on the ER were only partially reversible in the time period examined. We conclude that in P. tinctorius, BFA as the free metabolite or BODIPY-BFA affects the tubular vacuole system as well as anterograde membrane flow between the ER and the Golgi bodies and post-Golgi transport.

Cancer education among primary care physicians in an underserved community.

INTRODUCTION: Urban minority groups, such as those living in north Manhattan, are generally underserved with regard to cancer prevention and screening practices. Primary care physicians are in a critical position to counsel their patients on these subjects and to order screening tests for their patients. METHODS: Eighty-four primary care physicians in two intervention communities who received educational visits about cancer screening and prevention were compared with 38 physicians in a nearby community who received no intervention. With pre- and post-test interviews over an 18-month period, the physicians were asked about their attitudes toward, knowledge of (relative to American Cancer Society guidelines), and likelihood of counseling and screening for breast, cervical, colorectal, and prostate cancers. RESULTS: Comparison of the two surveys of physicians indicated no statistically significant differences in knowledge of cancer prevention or screening. At post-test, however, intervention group physicians identified significantly fewer barriers to practice than control physicians (p<0.05). While overall, the educational visits to inner-city primary care physicians did not appear to significantly alter cancer prevention practices, there was a positive dose-response relationship among the subgroup of participants who received three or more project contacts. CONCLUSIONS: We uncovered significant changes in attitude due to academic detailing among urban primary care physicians practicing in north Manhattan. A significant pre-test sensitization effect and small numbers may have masked overall changes in cancer prevention and screening behaviors among physicians due to the intervention.

Cancer screening and prevention practices of inner-city physicians.

INTRODUCTION: Effective preventive services are needed most in underserved, inner-city settings that suffer disproportionately from morbidity and mortality. Primary care physicians can play an important role in the provision of efficacious cancer prevention and screening services to patients in these communities. METHOD: We surveyed 122 primary care physicians about their cancer prevention and screening knowledge, attitudes, and practices. RESULTS: Relative to the findings from national and local surveys, sample physicians were not as knowledgeable about national guidelines for preventive care, were less likely to counsel on smoking cessation, and were less likely to advise diet modification. Although physician practices reflected national cancer prevention and screening guidelines in general, a significant proportion of physicians suggested lung and prostate cancer screening tests that were inconsistent with national recommendations. CONCLUSIONS: Systematic efforts are needed to increase the knowledge and practices of inner-city physicians concerning cancer prevention and screening.

OOC-3, a novel putative transmembrane protein required for establishment of cortical domains and spindle orientation in the P(1) blastomere of C. elegans embryos.

Asymmetric cell divisions require the establishment of an axis of polarity, which is subsequently communicated to downstream events. During the asymmetric cell division of the P(1) blastomere in C. elegans, establishment of polarity depends on the establishment of anterior and posterior cortical domains, defined by the localization of the PAR proteins, followed by the orientation of the mitotic spindle along the previously established axis of polarity. To identify genes required for these events, we have screened a collection of maternal-effect lethal mutations on chromosome II of C. elegans. We have identified a mutation in one gene, ooc-3, with mis-oriented division axes at the two-cell stage. Here we describe the phenotypic and molecular characterization of ooc-3. ooc-3 is required for the correct localization of PAR-2 and PAR-3 cortical domains after the first cell division. OOC-3 is a novel putative transmembrane protein, which localizes to a reticular membrane compartment, probably the endoplasmic reticulum, that spans the whole cytoplasm and is enriched on the nuclear envelope and cell-cell boundaries. Our results show that ooc-3 is required to form the cortical domains essential for polarity after cell division.

ER-Tracker dye and BODIPY-brefeldin A differentiate the endoplasmic reticulum and golgi bodies from the tubular-vacuole system in living hyphae of Pisolithus tinctorius.

Two fluorochromes, ER-TrackerTM Blue-White DPX dye and the fluorescent brefeldin A (BFA) derivative, BODIPY-BFA, label the endoplasmic reticulum (ER) in hyphal tips of Pisolithus tinctorius and allow its differentiation from the tubular-vacuole system at the light microscope level in living cells. The ER-Tracker dye labels a reticulate network similar in distribution to ER as seen in electron micrographs of freeze-substituted hyphae. BODIPY-BFA stains a thicker axially aligned structure with an expanded region at the apex, which is similar to that seen when hyphae are stained with ER-Tracker dye in the presence of unconjugated BFA. This structure is considered to be ER modified by BFA, a view supported by ultrastructural observations of the effect of BFA on the fungal ER. Both fluorescent probes also stain punctate structures, which are most likely to be Golgi bodies. Neither probe labels the tubular-vacuole system.

Control of microtubule dynamics by the antagonistic activities of XMAP215 and XKCM1 in Xenopus egg extracts.

Microtubules are dynamic polymers that move stochastically between periods of growth and shrinkage, a property known as dynamic instability. Here, to investigate the mechanisms regulating microtubule dynamics in Xenopus egg extracts, we have cloned the complementary DNA encoding the microtubule-associated protein XMAP215 and investigated the function of the XMAP215 protein. Immunodepletion of XMAP215 indicated that it is a major microtubule-stabilizing factor in Xenopus egg extracts. During interphase, XMAP215 stabilizes microtubules primarily by opposing the activity of the destabilizing factor XKCM1, a member of the kinesin superfamily. These results indicate that microtubule dynamics in Xenopus egg extracts are regulated by a balance between a stabilizing factor, XMAP215, and a destabilizing factor, XKCM1.

Electron microscopy may reveal structure of docosahexaenoic acid-rich oil within Schizochytrium sp.

Schizochytrium sp. is an algae-like microorganism utilized for commercial production of docosahexaenoic acid (DHA)-rich oil and dried microalgae for use as a source of DHA in foods, feeds, and nutritional supplements. Electron microscopic analysis of whole cells of Schizochytrium sp. employing sample preparation by high-pressure freeze substitution suggests the presence of secondary and tertiary semicrystalline structures of triacylglycerols within the oil bodies in Schizochytrium sp. A fine secondary structure consisting of alternating light- and dark-staining bands was observed inside the oil bodies. Dark bands were 29 +/- 1 A in width, and light bands were 22 +/- 1 A in width. The tertiary (three-dimensional) structure may be a multilayered ribbon-like structure which appears coiled and interlaced within the oil body. In freeze-fracture photomicrographs, Schizochytrium oil bodies exhibited fracture planes with terraces averaging 52 +/- 7 A in height which could correspond to the combined width of two halves of two light bands and one dark band observed in the high-pressure freeze substitution photomicrographs. The results suggest that triacylglycerols within Schizochytrium sp. oil bodies may be organized in a triple chain-length structure. High-pressure freeze substitution electron micrographs of two other highly unsaturated oil-producing species of microalgae, Thraustochytrium sp. and Isochrysis galbana, also revealed this fine structure, whereas microalgae containing a higher proportion of saturated oil did not. The results suggest that the staining pattern is not an artifact of preparation and that the triple chain-length conformation of triacylglycerols in Schizochytrium sp. oil bodies may be caused by the unique fatty acid composition of the triacylglycerols.

Dispersed polyphosphate in fungal vacuoles in Eucalyptus pilularis/Pisolithus tinctorius ectomycorrhizas.

Ectomycorrhizas produced between Pisolithus tinctorius and Eucalyptus pilularis under axenic conditions were rapidly frozen, freeze-substituted in tetrahydrofuran and embedded anhydrously, and dry-sectioned for X-ray microanalysis. The vacuoles of the sheath and Hartig net hyphae were rich in phosphorus and potassium. They also contained sulfur and variable amounts of chlorine. In anhydrously processed freeze-substituted mycorrhizas, dispersed electron-opaque material filled the fungal vacuoles. X-ray maps indicated that P was distributed evenly throughout the entire vacuole profile and was not concentrated in spherical bodies or subregions of the vacuole. There were no electron-opaque granules surrounded by electron-lucent areas, such as are commonly seen in chemically fixed material. The fungal vacuoles were also rich in K, which similarly gave a signal from the entire vacuolar profile. Such P-rich vacuoles occurred in both the mycorrhizal sheath and Hartig net hyphae. Stained sections of ether-acrolein freeze-substituted mycorrhizas also showed only dispersed material in the fungal vacuoles as, in most cases, did acetone-osmium freeze-substituted material. Precipitation of metachromatic granules by ethanol suggested that large amounts of polyphosphate are stored in these regions under the conditions of our experiments, as well as in the tips of actively growing hyphae of the same fungus. The higher plant vacuoles of ectomycorrhizas gave a much lower signal for K, and P was barely detectable. Much more K was located in the vacuoles of the root exodermal cells than in epidermal cells. The analysis of element distribution between the vacuole and cytoplasm in root cells agrees well with that found for other plant species using other techniques. We conclude that polyphosphate is indeed present in the vacuoles of the fungal cells of these ectomycorrhizas, but that in vivo it is in a dispersed form, not in granules.

Microtubules, but not actin microfilaments, regulate vacuole motility and morphology in hyphae of Pisolithus tinctorius.

While it is now recognised that transport within the endomembrane system may occur via membranous tubules, spatial regulation of this process is poorly understood. We have investigated the role of the cytoskeleton in regulating the motility and morphology of the motile vacuole system in hyphae of the fungus Pisolithus tinctorius by studying (1) the effects of anti-microtubule (oryzalin, nocodazole) and anti-actin drugs (cytochalasins, latrunculin) on vacuolar activity, monitored by fluorescence microscopy of living cells; and (2) the ultrastructural relationship of microtubules, actin microfilaments, and vacuoles in hyphae prepared by rapid-freezing and freeze-substitution. Anti-microtubule drugs reduced the tubular component of the vacuole system in a dose-dependent and reversible manner, the extent of which correlated strongly with the degree of disruption of the microtubule network (monitored by immunofluorescence microscopy). The highest doses of anti-microtubule drugs completely eliminated tubular vacuoles, and only spherical vacuoles were observed. In contrast, anti-actin drugs did not reduce the frequency of tubular vacuoles or the motility of these vacuoles, even though immunofluorescence microscopy confirmed perturbation of microfilament organisation. Electron microscopy showed that vacuoles were always accompanied by microtubules. Bundles of microtubules were found running in parallel along the length of tubular vacuoles and individual microtubules were often within one microtubule diameter of a vacuole membrane. Our results strongly support a role for microtubules, but not actin microfilaments, in the spatial regulation of vacuole motility and morphology in fungal hyphae.

Regulation of Saccharomyces cerevisiae kinetochores by the type 1 phosphatase Glc7p.

We have investigated the role of protein phosphorylation in regulation of Saccharomyces cerevisiae kinetochores. By use of phosphatase inhibitors and a type 1 protein phosphatase mutant (glc7-10), we show that the microtubule binding activity, but not the centromeric DNA-binding activity, of the kinetochore complex is regulated by a balance between a protein kinase and the type 1 protein phosphatase (PP1) encoded by the GLC7 gene. glc7-10 mutant cells exhibit low kinetochore-microtubule binding activity in vitro and a high frequency of chromosome loss in vivo. Specifically, the Ndc10p component of the centromere DNA-binding CBF3 complex is altered by the glc7-10 mutation; Ndc10p is hyperphosphorylated in glc7-10 extracts. Furthermore, addition of recombinant Ndc10p reconstitutes the microtubule-binding activity of a glc7-10 extract to wild-type levels. Finally, the glc7-10-induced mitotic arrest is abolished in spindle checkpoint mutants, suggesting that defects in kinetochore-microtubule interactions caused by hyperphosphorylation of kinetochore proteins activate the spindle checkpoint.

Mitotic chromatin regulates phosphorylation of Stathmin/Op18.

Meiotic and mitotic spindles are required for the even segregation of duplicated chromosomes to the two daughter cells. The mechanism of spindle assembly is not fully understood, but two have been proposed that are not mutually exclusive. The 'search and capture' model suggests that dynamic microtubules become progressively captured and stabilized by the kinetochores on chromosomes, leading to spindle assembly. The 'local stabilization' model proposes that chromosomes change the state of the cytoplasm around them, making it more favourable to microtubule polymerization. It has been shown that Stathmin/Op18 inhibits microtubule polymerization in vitro by interaction with tubulin, and that overexpression in tissue culture cells of non-phosphorylatable mutants of Stathmin/Op18 prevents the assembly of mitotic spindles. We have used Xenopus egg extracts and magnetic chromatin beads to show that mitotic chromatin induces phosphorylation of Stathmin/Op18. We have also shown that Stathmin/Op18 is one of the factors regulated by mitotic chromatin that governs preferential microtubule growth around chromosomes during spindle assembly.