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The objective of this study was to determine if dietary gangliosides induce changes in the ganglioside content of intestinal mucosa, plasma and brain and to identify where GM3 and GD3 are localized in the enterocyte membrane. Male 18‐day‐old Sprague‐Dawley rats were fed a semipurified diet containing 20% (w/w) fat. The control diet contained triglyceride, reflecting the fat formulation of an existing infant formula. Two experimental diets were formulated by adding sphingomyelin (1% w/w of total fat) or a ganglioside‐enriched lipid (0.1% w/w of total fat) to the control diet fat. The ganglioside fraction of ganglioside‐enriched lipid diet contained more than 80% GD3. After 2 weeks of feeding, the total and individual ganglioside and cholesterol content was measured in small intestinal mucosa, plasma and brain. The ganglioside‐enriched lipid diet significantly increased total gangliosides in the intestinal mucosa, plasma and brain compared with the control diet. The ganglioside‐enriched lipid diet significantly increased the level of GD3 (7.5% w/w) in the intestine compared with control (3.2% w/w) while decreasing the level of GM3, the major ganglioside in the intestine. The ratio of cholesterol to ganglioside in the intestinal mucosa, plasma and brain decreased significantly in rats fed the ganglioside‐enriched lipid diet compared with controls. Confocal microscopy showed that GM3 is exclusively localized in the apical membrane of the enterocyte whereas GD3 is primarily localized in the basolateral membrane. The authors conclude that dietary ganglioside is absorbed in the small intestine and transported to different membrane sites, altering ganglioside levels in the intestinal mucosa, plasma and brain and thus possibly having the potential to change developing enterocyte function (and possibly that of other cell lines).

The objective of this study was to determine if dietary gangliosides induce changes in the ganglioside content of intestinal mucosa, plasma and brain and to identify where GM3 and GD3 are localized in the enterocyte membrane.

We have been experimenting with the use of animations to teach histology as part of an interactive multimedia program we are developing to replace the traditional lecture/laboratory‐based histology course in our medical and dental curricula. This program, called HistoQuest, uses animations to illustrate basic histologic principles, explain dynamic processes, integrate histologic structure with physiological function, and assist students in forming mental models with which to organize and integrate new information into their learning. With this article, we first briefly discuss the theory of mental modeling, principles of visual presentation, and how mental modeling and visual presentation can be integrated to create effective animations. We then discuss the major Web‐based animation technologies that are currently available and their suitability for different visual styles and navigational structures. Finally, we describe the process we use to produce animations for our program. The approach described in this study can be used by other developers to create animations for delivery over the Internet for the teaching of histology. Anat Rec (New Anat) 269:11–19, 2002. © 2002 Wiley‐Liss, Inc.

We have been experimenting with the use of animations to teach histology as part of an interactive multimedia program we are developing to replace the traditional lecture/laboratory-based histology course in our medical and dental curricula. This program, called HistoQuest, uses animations to illustrate basic histologic principles, explain dynamic processes, integrate histologic structure with physiological function, and assist students in forming mental models with which to organize and integrate new information into their learning. With this article, we first briefly discuss the theory of mental modeling, principles of visual presentation, and how mental modeling and visual presentation can be integrated to create effective animations. We then discuss the major Web-based animation technologies that are currently available and their suitability for different visual styles and navigational structures. Finally, we describe the process we use to produce animations for our program. The approach described in this study can be used by other developers to create animations for delivery over the Internet for the teaching of histology. Anat Rec (New Anat) 269:11–19, 2002. © 2002 Wiley-Liss, Inc.

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF-α-induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-α, sphingomyelinase, or C2-ceramide induced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, and stress fiber formation. Ly 294002, a phosphatidylinositol 3-kinase (PI 3-K) inhibitor, or expression of dominant/negative Ras (N17) completely blocked C2-ceramide- and sphingomyelinase-induced tyrosine phosphorylation of FAK and paxillin and severely decreased stress fiber formation. The TNF-α effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-α and C2-ceramide. TNF-α, sphingomyelinase, and C2-ceramide translocated Cdc42, Rac, and RhoA to membranes, and stimulated p21-activated protein kinase downstream of Ras-GTP, PI 3-K, and SK. Transfection with inactive RhoA inhibited the TNF-α- and C2-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-α, which increases sphingomyelinase activity and ceramide formation, activates sphingosine kinase, Rho family GTPases, focal adhesion kinase, and paxillin. This novel pathway of ceramide signaling can account for ∼70% of TNF-α-induced stress fiber formation and cytoskeletal reorganization.

Spectrin has been implicated in a variety of different processes during late embryogenesis, after transcription of the zygotic genome has been activated. However, relatively little is known about the role of maternally derived spectrin during the early cleavage divisions that give rise to a multicellular embryo. To investigate the role of spectrin in early development, we have microinjected anti-spectrin antibodies intoPatiria miniatastarfish embryos to inhibit the activity of the maternal pool of spectrin. Microinjection of affinity-purified anti-spectrin antibody, or low to moderate doses of Fabfragments, into one blastomere of a two-cell-stage embryo caused a dose-dependent, progressive increase in the length of the cell cycle compared to the uninjected control blastomere. The progeny of injected blastomeres were unable to participate in the formation of a blastula epithelium, instead forming a loose aggregate of cells that eventually stopped dividing. When division stopped, the cells formed surface protrusions and became motile. At high doses of either whole antibody or Fabfragments, cells initiated, but failed to complete, cytokinesis. Blastomeres injected with high doses of Fabfragments also failed to reform nuclei and underwent variable periods of cell cycle arrest up to 12 hr. Injected embryos stained with BODIPY-phallacidin exhibited extensive disruption of the cortical actin cytoskeleton. These results support previous studies implicating spectrin in stabilizing the cell surface and maintaining the organization of the cortical cytoskeleton. They further suggest that spectrin is not required for the initiation or contraction of the cleavage furrow, but functions in the completion of cytokinesis. Most surprisingly, however, the results demonstrate that inhibition of spectrin function alters cell cycle timing, suggesting that disruption of the actin cytoskeleton inhibits progression through the cell cycle.

Hypocrellins are naturally occurring compounds with photosensitizing properties in biological systems. We have prepared synthetic derivatives of hypocrellin B, which have promise as photosensitizers in the clinical application of photodynamic therapy. The inherent fluorescence of four selected compounds has provided a means of determining their uptake and distribution in monolayer cultures of EMT6/Ed murine tumor cells, via fluorescence confocal microscopy.

This paper investigates the relationship between the assembly of the cortical cytoskeleton and the reorganization of the egg surface following fertilization in the sea urchin. The surface of the unfertilized egg is covered with short microvilli, with dimensions of ~0.15 × 0.3 μm. Between 30 and 60 sec after insemination, slender finger-like processes, ~2 μm in length and 0.05 μm in diameter, grow from the tips of these short microvilli. Over the next 30 sec, clusters of these processes transform into broad protrusions of the egg surface, containing networks of actin filaments. By 2 min postinsemination, bundles of filaments start to form within these networks, beginning at the tips of the protrusions and growing inward. As the filament bundles develop, the plasma membrane encases them to form typical microvilli with average dimensions of 0.15 × 2 μm. The formation of the network of cortical actin filaments occurs during the transient increase in free cytoplasmic Ca⁺⁺ concentration, while the bundling of actin filaments occurs during the period of cytoplasmic alkalization. When cytoplasmic alkalization is inhibited, actin filament networks fail to reorganize into bundles, but microvilli develop by an identical series of surface shape changes. Eggs fertilized in the presence of cytochalasin B develop irregular surface protrusions that gradually transform into microvillar-like structures over a period of ~30 min without undergoing any definable series of shape changes or assembly of a normal cortical cytoskeleton. These results demonstrate that, while the formation of the large surface protrusions requires an intact network of cortical actin filaments, partitioning of these protrusions into microvilli does not depend on the formation of actin filament bundles. Further, microvillar-like structures are still able to form in eggs in which the normal sequence of surface shape changes has been blocked by inhibiting the assembly of the cortical actin filament network. J. Exp. Zool. 277:230–244, 1997. © 1997 Wiley-Liss, Inc.

We have investigated the functional properties of the mechanisms that control the reproduction of spindle poles in fertilized sea-urchin eggs. By prolonging mitosis by three independent means, we show that a spindle pole can split during mitosis into two functional poles of normal appearance. However, these poles have only half the normal reproductive capacity; each daughter cell that receives a split pole, always forms a monopolar spindle at the next division. Each monopolar spindle appears to be exactly half of a spindle because two of them can come together to form a functional bipolar spindle of normal appearance. The poles of such spindles show normal reproduction in subsequent divisions. By following the development of individual cells with monopolar spindles, we show that such a cell can stay in mitosis longer than normal, and the single pole splits into two asters, which move apart to give a functional bipolar spindle. The poles of such a spindle have only half the normal reproductive capacity, because the two daughters of the cell always form monopolar spindles at the next mitosis. This novel cycle of development is often repeated. The occurrence of such phenomena does not depend upon the method used to induce monopolar spindles. These results show that each normal pole has two polar determinants. The results also demonstrate that the reproduction of spindle poles consists of three distinct events: splitting of the polar determinants, physical separation of the two determinants, and duplication of the determinants to return the pole to a duplex state. Splitting and duplication are distinct events because they can be experimentally put out of phase with each other for several cell cycles.

The effects of different concentrations of the actin-disrupting drug cytochalasin D on tight junction permeability and distribution of actin filaments in MDCK epithelial cells were examined. Consistent with previous studies, 2 μg/ml cytochalasin D caused a significant decrease in transepithelial resistance, indicative of an increase in tight junction permeability. Surprisingly, increasing concentrations of cytochalasin D caused progressively smaller decreases in transepithelial resistance. The effects of cytochalasin D were reversible. Light microscopic analysis utilizing rhodamine-conjugated phalloidin demonstrated two distinct populations of actin filaments in MDCK cells: an apical peripheral ring of actin, presumably associated with the zonula adherens, and larger actin bundles more basally situated. When treated with 2 μg/ml cytochalasin D, both actin populations were severely disrupted and cells became flattened. Actin in the apical ring aggregated along cell boundaries, and these aggregates co-localized with similarly disrupted focal accumulations of the tight junction-associated protein ZO-1. The basal actin filament bundles also reorganized into focal aggregates. Increasing concentrations of cytochalasin D caused gradually less perturbation of the apical actin ring, consistent with the transepithelial resistance observations. However, the basal actin bundles were disrupted at all concentrations of cytochalasin D tested, demonstrating that the two actin populations are differentially sensitive to cytochalasin D and that apical actin filaments are more important in the regulation of tight junction permeability. Finally, treatment of cells with cytochalasin D inhibited the decrease in transepithelial resistance induced by the chelation of extracellular Ca2+. This indicates that the opening of tight junctions caused by removal of Ca2+ requires a functional actin cytoskeleton.

Objective. The purpose of this study was to determine whether patient ratings of general practice Registrars consulting skills are associated with ‘expert’ scoring using the MRCGP video assessment protocol. Methods. A cross-sectional observational study of general practice Registrars consultation skills was carried out in 23 practices in South East Scotland using two types of patient assessment compared with expert assessment of video consultation. The main outcome measures were rank correlation of Registrars overall level of attainment on the Royal College of General Practitioner (RCGP) video assessment with mean score on the Patient Enablement Instrument (PEI) and mean score on the Consultation Satisfaction Questionnaire (CSQ). Results. The rank correlation of Registrars mean PEI scores with marks on the RCGP video component was 0.01 (P = 0.97, n = 19) and mean CSQ score 0.05 (P = 0.83, n = 19). There were no adverse comments from patients, but Registrars and trainers found the process onerous. Conclusion. No meaningful association was identified between Registrars score on the RCGP video examination and patient assessment via either the PEI or the CSQ. This suggests that, with regard to measuring quality in the consultation, one or more of the assessments are invalid or that they are measuring different attributes. Further research to elucidate the reasons for the lack of correlation is required.