Motile plant cell body: a "bug" within a "cage"

Analysis of the cytoskeleton in morphogenetically active plant cells allows us to propose a unified concept for the structural organization of eukaryotic cells. Their cytoarchitecture is determined by two principal structural complexes: nucleus-microtubule-based cell bodies ("bugs") and plasma-membrane-F-actin-based cell periphery complexes ("cages"). There are dynamic interactions between each of these entities in response to extracellular and intracellular signals. In the case of the cell body, these signals determine its polarization, rotation and migration. Interactions between cell body and cell periphery complexes determine cell growth polarity and morphogenesis throughout the eukaryotic kingdom.     

Interplay between activator-inhibitor coupling and cell-matrix adhesion in a cellular automaton model for chondrogenic patterning

We present a stochastic cellular automaton model for the behavior of limb bud precartilage mesenchymal cells undergoing chondrogenic patterning. This "agent-oriented" model represents cells by points on a lattice that obey rules motivated by experimental findings. The "cells" follow these rules as autonomous agents, interacting with other cells and with the microenvironments cell activities produce. The rules include random cell motion, production and lateral deposition of a substrate adhesion molecule (SAM, corresponding to fibronectin), production and release of a diffusible growth factor ("activator," corresponding to TGF-beta) that stimulates production of the SAM, and another diffusible factor ("inhibitor") that suppresses the activity of the activator. We implemented the cellular automaton on a two-dimensional (2D) square lattice to emulate the quasi-2D micromass culture extensively used to study patterning in avian limb bud precartilage cells. We identified parameters that produce nodular patterns that resemble, in size and distribution, cell condensations in leg-cell cultures, thus establishing a correspondence between in vitro and in silico results. We then studied the in vitro and in silico micromass cultures experimentally. We altered the standard in vitro micromass culture by diluting the initial cell density, transiently exposing it to exogenous activator, suppressing the inhibitor, and constitutively activating fibronectin production. We altered the standard in silico micromass culture in each case by changing the corresponding parameter. In vitro and in silico experiments agreed well. We also used the model to test hypotheses for differences in the in vitro patterns of cells derived from chick embryo forelimb and hindlimb. We discuss the applicability of this model to limb development in vivo and to other organ development.     

In vitro and in vivo activated T cells display increased sensitivity to PGE2.

In this study we report that in vitro activation of T cells increased the cyclic AMP response to subsequent prostaglandin E2 (PGE2) stimulation severalfold per cell. This sensitization of T cells to PGE2-induced cyclic AMP generation was observed when the T cells had been stimulated in vitro for 5 days with either the CD3 monoclonal antibody OKT3, phytohemagglutinin, or the combination phytohemagglutinin plus the phorbol ester PMA. Enhanced cyclic AMP generation following mitogenic activation was seen in response to both PGE2 and forskolin, direct activator of the adenylate cyclase, indicating that the amount of adenylate cyclase had increased during the in vitro activation course. In order to investigate whether various T cell subsets in general and in vivo activated T cells in particular would differ in their susceptibility to PGE2, we isolated CD4+, CD8+, CD4-CD8-, CD4+CD45RO+ ("memory"), and CD4+CD45RA+ ("virgin") T cells and studied PGE2-mediated inhibition of CD3-induced proliferation, as well as cyclic AMP generation in response to PGE2, respectively. We found that CD8+ T cells are more susceptible to PGE2 inhibition and produce more cyclic AMP than CD4+ T cells. Double-negative T cells (enriched for gamma delta T cell receptor positive cells) were found to be sensitive to PGE2 as well. Within the CD4+ T cell population, CD45RO+ ("memory") T cells were significantly more sensitive to PGE2-mediated suppression than CD45RA+ ("virgin") T cells. CD45RO+ cells required a 10-fold lower dose of PGE2 for half-maximum suppression of proliferation. However, no difference in cyclic AMP production could be demonstrated between these two subsets. We propose that substantial heterogeneity exists among peripheral blood T lymphocyte subsets regarding their sensitivity to the immunosuppressive action of PGE2 and that the sensitivity of individual cells changes in the course of an immune response.     

Hippocampal "time cells" bridge the gap in memory for discontiguous events

The hippocampus is critical to remembering the flow of events in distinct experiences and, in doing so, bridges temporal gaps between discontiguous events. Here, we report a robust hippocampal representation of sequence memories, highlighted by "time cells" that encode successive moments during an empty temporal gap between the key events, while also encoding location and ongoing behavior. Furthermore, just as most place cells "remap" when a salient spatial cue is altered, most time cells form qualitatively different representations ("retime") when the main temporal parameter is altered. Hippocampal neurons also differentially encode the key events and disambiguate different event sequences to compose unique, temporally organized representations of specific experiences. These findings suggest that hippocampal neural ensembles segment temporally organized memories much the same as they represent locations of important events in spatially defined environments.     

"Aequanimitas" Redux: William Osler on detached concern versus humanistic empathy

Recent critics make William Osler "the father of cool detachment" in medicine, largely because of his "Aequanimitas" address emphasizing objectivity and imperturbability. Closer analysis suggests that Osler's aequanimitas resembles more nearly the metriopatheia of later Stoic philosophy than the apatheia of the early Stoics. A previously unpublished memoir clarifies at least in part Osler's motive for teaching control of the "medullary centres" to minimize facial expression: he did not want to frighten patients, who typically had serious illnesses for which he lacked effective therapy. Twenty-first century challenges to medicine as a profession differ substantially from those of Osler's era. Physicians and educators must focus more closely on the tension between detached concern ("competence") and humanistic empathy ("caring") if medicine is to thrive as a learned profession as opposed to a technical service, a commodity to be bought and sold like any other.     

Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach

We present a comprehensive mass spectrometric approach that integrates intact protein molecular mass measurement ("top-down") and proteolytic fragment identification ("bottom-up") to characterize the 70S ribosome from Rhodopseudomonas palustris. Forty-two intact protein identifications were obtained by the top-down approach and 53 out of the 54 orthologs to Escherichia coli ribosomal proteins were identified from bottom-up analysis. This integrated approach simplified the assignment of post-translational modifications by increasing the confidence of identifications, distinguishing between isoforms, and identifying the amino acid positions at which particular post-translational modifications occurred. Our combined mass spectrometry data also allowed us to check and validate the gene annotations for three ribosomal proteins predicted to possess extended C-termini. In particular, we identified a highly repetitive C-terminal "alanine tail" on L25. This type of low complexity sequence, common to eukaryotic proteins, has previously not been reported in prokaryotic proteins. To our knowledge, this is the most comprehensive protein complex analysis to date that integrates two MS techniques.     

Cellular receptors for lymphotoxin: correlation of binding and cytotoxicity in sensitive and resistant target cells.

The binding of radioactively labeled lymphotoxin (LT) to both lymphotoxin-sensitive and -resistant cell clones was examined. The sensitive clone had a low- capacity, high-affinity ("specific") binding component, the curve of which closely followed the cytotoxicity curve of the lymphocyte mediator. The capacity of this binding component was calculated to be about 600 molecules of LT/cell. In addition, there was a low-affinity, high-capacity ("nonspecific") binding component. In striking contrast, the high-affinity, low-capacity ("specific") component was absent or greatly diminished from the resistant clone, whereas the low-affinity, high-capacity ("nonspecific") component was present at a similar level as in the sensitive cells.These binding characteristics closely resemble those observed by us and other investigators working with a variety of steroid hormones in steroid-sensitive and- resistant cell lines.     

Human peripheral blood cells differentially recognize and respond to two distinct CPG motifs

Oligodeoxynucleotides (ODN) that contain unmethylated CpG dinucleotides trigger a strong innate immune response in vertebrates. CpG ODN show promise as vaccine adjuvants, anti-allergens, and immunoprotective agents in animal models. Their transition to clinical use requires the identification of motifs that are optimally stimulatory in humans. Analysis of hundreds of novel ODN resulted in the identification and characterization of two structurally distinct "clusters" of immunostimulatory CpG ODN. One cluster ("D") preferentially stimulates IFN-gamma production by NK cells, whereas the other ("K") stimulates cell proliferation and the production of IL-6 and IgM by monocytes and B cells. The distinct immunostimulatory properties of K and D ODN can improve the design of CpG-based products to achieve specific therapeutic goals.     

Role of cell division in branching morphogenesis and differentiation of the embryonic pancreas

A new culture system for the embryonic pancreas enables the formation of a branched organ in vitro. In such cultures, each terminal branch originates as a small bud and the number of buds and of terminal branches increases progressively with the expansion of the culture. However buds can also be resorbed during growth. The normal labelling index of cells in incipient buds ("tips") is greater than between buds ("dips") suggesting that budding may be driven by a local increase of cell division. Consistent with this, treatments that reduce cell division repress the formation of buds and branches. It is not possible to initiate budding in isolated endodermal epithelium by treatment with fibroblast growth factor, although this does increase the degree of differentiation of exocrine cells. Cultures in which cell division is completely inhibited by aphidicolin treatment will produce more endocrine cells than usual and inhibit the differentiation of exocrine cells. Consistent with this it is found that in untreated cultures the division of endocrine precursors cannot be detected by BrdU labelling whereas the division of exocrine precursors is frequent. It is concluded that cell division is necessary for bud formation in the embryonic pancreas and that the growth factors required for this normally come from the mesenchyme. Cell division is also necessary for exocrine differentiation. Endocrine cells, however, can arise from undifferentiated progenitors without cell division.     

Mechanism of action of T cell factors regulating antibody formation]

While the induction of antibody synthesis depends on antigen specific T cell factors, its magnitude is under control of non antigen specific T cell factors. In this respect, TRF ("T cell Replacing Factor") amplifies antibody responses while IBF ("Immunoglobulin Binding Factor") acts as a suppressor factor. Using cultures of spleen cells from nude mice, we show that both factors act sequentially, influencing the final differenciation of B cells to antibody producing cells. We have no evidence of direct interaction between TRF and IBF.     

Relative contributions of "pressure pump" and "peristaltic pump" to gastric emptying

The relative contributions to gastric emptying from common cavity antroduodenal pressure difference ("pressure pump") vs. propagating high-pressure waves in the distal antrum ("peristaltic pump") were analyzed in humans by high-resolution manometry concurrently with time-resolved three-dimensional magnetic resonance imaging during intraduodenal nutrient infusion at 2 kcal/min. Gastric volume, space-time pressure, and contraction wave histories in the antropyloroduodenal region were measured in seven healthy subjects. The subjects fell into two distinct groups with an order of magnitude difference in levels of antral pressure activity. However, there was no significant difference in average rate of gastric emptying between the two groups. Antral pressure history was separated into "propagating high-pressure events" (HPE), "nonpropagating HPEs," and "quiescent periods." Quiescent periods dominated, and average pressure during quiescent periods remained unchanged with decreasing gastric volume, suggesting that common cavity pressure levels were maintained by increasing wall muscle tone with decreasing volume. When propagating HPEs moved to within 2-3 cm of the pylorus, pyloric resistance was found statistically to increase with decreasing distance between peristaltic waves and the pylorus. We conclude that transpyloric flow tends to be blocked when antral contraction waves are within a "zone of influence" proximal to the pylorus, suggesting physiological coordination between pyloric and antral contractile activity. We further conclude that gastric emptying of nutrient liquids is primarily through the "pressure pump" mechanism controlled by pyloric opening during periods of relative quiescence in antral contractile wave activity.     

Asymmetry and directionality in production of new cell types during clonal growth: the switching pattern of homothallic yeast.

Homothallic Saccharomyces yeasts efficiently interconvert between two cell types, the mating types a and alpha. These interconversions have been proposed to occur by genetic rearrangement ("cassette" insertion) at the locus controlling cell type (the mating type locus). The pattern of switching from one cell type to the other during growth of a clone of homothallic cells has been followed by direct microscopic observation, and the results have been summarized as "rules" of switching. First, when a cell divides, it produces either two cells with the same mating type as the original cell or two cells that have switched to the other mating type. This observation suggests that the mating type locus is changed early in the cell cycle, in late Gl or during S. Second, the ability to produce cells that have switched mating type is restricted to cells that have previously divided ("experienced cells"). Spores and buds ("inexperienced cells") rarely if ever give rise to cells with changed mating type. A homothallic yeast cell thus exhibits asymmetric segregation of the potential for mating type interconversion--at each cell division, the mother, but not the daughter, is capable of switching cell types in its next division. Homothallic cells also exhibit directionality in switching: experienced cells switch to the opposite cell type in more than 50% of cell divisions. These results show that the process of mating type interconversion is itself controlled during growth of a clone of homothallic cells. By analogy and extension of these results, we propose that multiple cell types can be produced in a specific pattern during development of a higher eucaryote in a model involving sequential cassette insertion.     

Frequency analysis of the antibody specificity repertoire of mitogen-reactive B cells and "spontaneously" occurring "background" plaque-forming cells in nude mice

The antibody specificity repertoire of lipopolysaccharide (LPS)-reactive B cells has been determined in the spleens and bone marrow (BM) of C57BL/Ka athymic nude mice using a limiting dilution culture system that allows the growth and development of every LPS-reactive B cell into a clone of IgM-secreting cells. In addition, the numbers of "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells as well as the "background" IgM antibody specificity repertoire has been assessed in spleens and BM. The frequencies of antigen-specific LPS-reactive B cells of C57BL/Ka nude and thymus-bearing mice showed a great similarity and ranged from 1 in 1000 to 1 in 2500 for sheep red blood cells (SRBC), horse red blood cells (HRBC), and goat red blood cells (GRBC), from 1 in 10 to 1 in 25 for 5-iodo-3-nitrophenyl-coupled (SRBC), from 1 in 15 to 1 in 150 for 4-hydroxy-3,5-dinitrophenyl-coupled SRBC, and from 1 in 70 to 1 in 140 for 2,4,6-trinitrophenyl-coupled SRBC. The specificity repertoire of the "background" IgM-secreting cells differed from that of age-matched thymus-bearing controls and was different in young and old C57BL/Ka nude mice. Within the limitations of having assessed only a minor fraction of the total B-cell antibody specificity repertoire and supposing that nude mice are largely devoid of functional T cells, the data presented suggest that the generation of the specificity repertoire of newly-formed B cells is hardly or not affected by T cells. On the other hand, T cells do affect the expression of the established repertoire, represented by "background" immunoglobulin-secreting cells.     

Integrating 'top-down" and "bottom-up" mass spectrometric approaches for proteomic analysis of Shewanella oneidensis

Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as well as fractionated via anion-exchange liquid chromatography for integrated studies. A portion of each fraction was proteolytically digested, with the resulting peptides characterized by on-line liquid chromatography/tandem mass spectrometry. The remaining portion of each fraction containing the intact proteins was examined by high-resolution Fourier transform mass spectrometry. This "top-down" technique provided direct measurement of the molecular masses for the intact proteins and thereby enabled confirmation of post-translational modifications, signal peptides, and gene start sites of proteins detected in the "bottom-up" experiments. A total of 868 proteins from virtually every functional class, including hypotheticals, were identified from this organism.     

Analysis of ligand-receptor interactions in cells by atomic force microscopy

Atomic force microscopy (AFM) increasingly has been used to analyse "receptor" function, either by using purified proteins ("molecular recognition microscopy") or, more recently, in situ in living cells. The latter approach has been enabled by the use of a modified commercial AFM, linked to a confocal microscope, which has allowed adhesion forces between ligands and receptors in cells to be measured and mapped, and downstream cellular responses analysed. We review the application of AFM to cell biology and, in particular, to the study of ligand-receptor interactions and draw examples from our own work and that of others to show the utility of AFM, including for the exploration of cell surface functionalities. We also identify shortcomings of AFM in comparison to "standard" methods, such as receptor auto-radiography or immuno-detection, that are widely applied in cell biology and pharmacological analysis.     

Inhibitory epidermal pentapeptide modulates proliferation and differentiation of transformed mouse epidermal cells in vitro.

A transformed mouse epidermal cell line ("308 cells") and nontransformed rat tongue squamous epithelial cells ("RT10 cells") were treated 3 times weekly for a period of two weeks with relatively large doses (150 micrograms/ml) of a synthetic inhibitory epidermal pentapeptide; pyroGlu-Glu-Asp-Ser-GlyOH. The peptide was recently isolated from mouse skin extracts and inhibits normal epidermal cells in vivo and in vitro at a restricted and low dose level. Repeated treatments with the large dose was followed by a 30-40% reduction in the number of 308 cells per well, starting as early as day 1. The number of RT10 cells was reduced about 20% only at termination of the experiment on day 14. In contrast to this, the number of unattached cornified envelopes on day 10 in the RT10 cells was increased by 85%, while the number of cornified, unattached 308 cells was similar to that in the controls. The effects of the pentapeptide thus seem to affect differentiation stronger than proliferation in the nontransformed cell line. Bivariate BrdUrd/DNA flow cytometry analysis on day 10 indicated that the reduced number of 308 cells was mainly due to a slower rate of cell proliferation and not to a increased sloughing off of keratinized cells. This analysis also demonstrated that an inhibition of DNA synthesis in the RT10 cells could be detected prior to a reduction of the cell number per well.     

Purpose-driven biomaterials research in liver-tissue engineering

Bottom-up engineering of microscale tissue ("microtissue") constructs to recapitulate partially the complex structure-function relationships of liver parenchyma has been realized through the development of sophisticated biomaterial scaffolds, liver-cell sources, and in vitro culture techniques. With regard to in vivo applications, the long-lived stem/progenitor cell constructs can improve cell engraftment, whereas the short-lived, but highly functional hepatocyte constructs stimulate host liver regeneration. With regard to in vitro applications, microtissue constructs are being adapted or custom-engineered into cell-based assays for testing acute, chronic and idiosyncratic toxicities of drugs or pathogens. Systems-level methods and computational models that represent quantitative relationships between biomaterial scaffolds, cells and microtissue constructs will further enable their rational design for optimal integration into specific biomedical applications.     

Increased accumulation of hybrid V(D)J joins in cells expressing truncated versus full-length RAGs.

RAG1 and RAG2 (RAGs) initiate V(D)J recombination by introducing breaks between two coding segments and flanking recombination signals (RSs). Nonhomologous end-joining (NHEJ) proteins then join the coding segments and join the RSs. In wild-type cells, both full-length and truncated ("core") RAGs lead to accumulation of "hybrid" V(D)J joins, in which an RS is appended to a different coding sequence. We now show that while hybrid joins do not accumulate in NHEJ-deficient cells that express full-length RAGs, they do accumulate in NHEJ-deficient cells that express the core RAGS; like those catalyzed by core RAGs in vitro, however, they are sealed on just one DNA strand. These results suggest a potential role for the non-core regions in repressing potentially harmful transposition events.