A semianalytical model to study the effect of cortical tension on cell rolling

Cell rolling on the vascular endothelium plays an important role in trafficking of leukocytes, stem cells, and cancer cells. We describe a semianalytical model of cell rolling that focuses on the microvillus as the unit of cell-substrate interaction and integrates microvillus mechanics, receptor clustering, force-dependent receptor-ligand kinetics, and cortical tension that enables incorporation of cell body deformation. Using parameters obtained from independent experiments, the model showed excellent agreement with experimental studies of neutrophil rolling on P-selectin and predicted different regimes of cell rolling, including spreading of the cells on the substrate under high shear. The cortical tension affected the cell-surface contact area and influenced the rolling velocity, and modulated the dependence of rolling velocity on microvillus stiffness. Moreover, at the same shear stress, microvilli of cells with higher cortical tension carried a greater load compared to those with lower cortical tension. We also used the model to obtain a scaling dependence of the contact radius and cell rolling velocity under different conditions of shear stress, cortical tension, and ligand density. This model advances theoretical understanding of cell rolling by incorporating cortical tension and microvillus extension into a versatile, semianalytical framework.     

Extensive apoptotic death of rat colonic cells deprived of crypt habitat

Apoptosis in cells of different lineages is restrained by survival signals which depend upon cell-to-cell communication. The aim of this study was to determine whether colonic cells deprived of crypt ambient are doomed to die prior to their normal chronological demise. Apoptosis was studied in rat whole colonic tissue, in isolated intact crypts, and in colonic cell populations collected from the crypt axis at different stages of proliferation and differentiation. In a number of experiments, cell harvest was performed in the presence of either a tetrapeptide (YVAD-CMK) inhibitor of interleukin-1β-converting enzyme (ICE), or tyrphostin A25, a protein tyrosine kinase inhibitor, or sodium-orthovanadate, a phosphatase inhibitor. DNA fragmentation was assessed by electrophoretic and nonisotopic-labeling procedures. The ultrastructure of colonic tissue specimens and isolated cells was examined by transmission electron microscopy. Apoptosis in whole colonic tissue and in isolated crypts was confined predominantly to cells resident in the upper crypt regions. In contrast, extensive apoptotic death was observed in isolated colonic cells, irrespective of their developmental stage and positional hierarchy within the crypt continuum at harvest time. An apoptotic gradient, however, was evident. Exposure to YVAD-CMK resulted in a marked decrease in the number of apoptotic cells. Treatment with tyrphostin A25 caused a sharp rise in the apoptotic index; conversely, vanadate significantly impeded apoptosis. Cumulatively, these results indicate that disordered intercellular communication provokes unscheduled ICE-mediated apoptosis of colonocytes, and that local signals along the crypt continuum control both the reprieve from death and the timely demise of distinct colonic cell populations. Attenuation of tyrosine phosphorylation may be a contributory event in the acquisition of the apoptotic phenotype. J. Cell. Physiol. 177:377–386, 1998. © 1998 Wiley-Liss, Inc.     

ARTS, the unusual septin: structural and functional aspects

The human Septin 4 gene (Sept4) encodes two major protein isoforms; Sept4_i1 (H5/PNUTL2) and Sept4_i2/ARTS. Septins have been traditionally studied for their role in cytokinesis and their filament-forming abilities, but subsequently have been implicated in diverse functions, including membrane dynamics, cytoskeletal reorganization, vesicle trafficking, and tumorigenesis. ARTS is localized at mitochondria and promotes programmed cell death (apoptosis). These features distinguish ARTS from any other known human septin family member. This review compares the structural and functional properties of ARTS with other septins. In addition, it describes how a combination of two distinct promoters, differential splicing, and intron retention leads to the generation of two different Sept4 variants with diverse biological activity.     

Mannose binding lectin: a biomarker of systemic lupus erythematosus disease activity

Introduction

A role for mannose binding lectin (MBL) in autoimmune diseases has been demonstrated earlier and elevated level of MBL has been shown in systemic lupus erythematosus (SLE) patients. In the current study, we investigated MBL as a potential biomarker for disease activity in SLE.

Methods

In a case control study SLE patients (93 females) and 67 age, sex, ethnicity matched healthy controls were enrolled. Plasma MBL levels were quantified by enzyme linked immunosorbent assay (ELISA). Clinical, serological and other markers of disease activity (C3, C4 and anti-dsDNA) were measured by standard laboratory procedures.

Results

Plasma MBL levels were significantly high in SLE patients compared to healthy controls (P < 0.0001). MBL levels were variable in different clinical categories of SLE. Lower levels were associated with musculoskeletal and cutaneous manifestations (P = 0.002), while higher and intermediate MBL levels were significantly associated with nephritis in combination with other systemic manifestations (P = 0.01 and P = 0.04 respectively). Plasma MBL correlated with systemic lupus erythematosus disease activity index (SLEDAI) (P = 0.0003, r = 0.36), anti-dsDNA (P < 0.0001, r = 0.54), proteinuria (P < 0.0001, r = 0.42) and negatively correlated with C3 (P = 0.007, r = -0.27) and C4 (P = 0.01, r = -0.29).

Conclusions

Plasma MBL is a promising marker in the assessment of SLE disease activity.     

Hypothesis: Transgene establishment in wild relatives of wheat can be prevented by utilizing the Ph1 gene as a senso stricto chaperon to prevent homoeologous recombination

Durum and bread wheat need transgenic traits such as herbicide and disease resistance due to recent evolution of herbicide resistant grass weeds and an intractable new strain of stem rust. Transgenic wheat varieties have not been commercialized partly due to potential transgene movement to wild/weedy relatives, which occurs naturally to closely related Aegilops and other spp. Recombination does not occur in the F₁ hybrid between wheat and its relatives due to the presence of the Ph1 gene on wheat chromosome arm 5BL, which acts as a chaperone, preventing promiscuous homoeologous pairing to similar, but not homologous chromosomes of the wild/weedy species. Thus recombination must occur during backcrossing after the wheat Ph1 gene has been eliminated. Based on these findings, we speculate that Ph1 could be used to prevent gene introgression into weedy relatives. We propose two methods to prevent such transgene establishment: (1) link the transgene in proximity to the wheat Ph1 gene and (2) insert the transgene in tandem with the lethal barnase on any chromosome arm other than 5BL, and insert barstar, which suppresses barnase on chromosome arm 5BL in proximity to Ph1. The presence of Ph1 in backcross plants containing 5BL will prevent the homoeologous establishment of barnase coupled to the desired transgene in the wild population. 5BL itself will be eliminated during repeated backcrossing to the wild parent, and progeny bearing the desired transgene in tandem with barnase but without the Ph1-barstar complex will die.     

Modulation of membrane composition of swine vascular smooth muscle cells by homologous lipoproteins in culture

Swine vascular smooth muscle cells were exposed to homologous low-density or high-density lipoprotein fractions for 24 h. Total cell membranes were isolated from the post-nuclear supernatant of the cell homogenates, fractionated by sucrose density gradient centrifugation and characterized by enzyme assays. The membrane fraction with the lowest density was enriched in plasma membrane marker enzymes. Cholesterol analysis showed that cells exposed to low-density lipoprotein had higher cholesterol-to-protein ratios in total cells, total cell membranes and individual membrane fractions than had the cells exposed to high-density lipoproteins. Cholesterol-to-phospholipid ratios of the plasma membrane-enriched fraction from cells exposed to low-density lipoprotein were higher than the same membrane fraction of cells exposed to high-density lipoprotein. Studies with iodinated lipoproteins showed that these compositional changes could not be due to lipoprotein contamination. Membrane microviscosity was determined by fluorescence depolarization with diphenylhexatriene and the microviscosity of the plasma membrane-enriched fraction was different in the cells exposed to the two different lipoprotein fractions. This difference in membrane microviscosity was significant only when the medium cholesterol content was 40 μg per ml or greater; cells exposed to low-density lipoprotein gave membranes with higher microviscosity.These results demonstrate that the properties of vascular smooth muscle cell membranes are influenced by exposure of the cells to homologous lipoprotein fractions.     

Comparisons of the molecular evolutionary process at rbcL and ndhF in the grass family (Poaceae)

We examine rate heterogeneity among evolutionary lineages of the grass family at two plasmid loci, ndhF and rbcL, and we introduce a method to determine whether patterns of rate heterogeneity are correlated between loci. We show both that rates of synonymous evolution are heterogeneous among grass lineages and that are heterogeneity is correlated between loci at synonymous sites. At nonsynonymous sites, the pattern of rate heterogeneity is not correlated between loci, primarily due to an aberrant pattern of rate heterogeneity at nonsynonymous sites of rbcL. We compare patterns of synonymous rate heterogeneity to predictors based on the generation time effect and the speciation rate hypotheses. Although there is some evidence for generation time effects, neither generation time effects nor speciation rates appear to be sufficient to explain patterns of rate heterogeneity in the grass plastid sequences.      

The Rap1p-telomere complex does not determine the replicative capacity of telomerase-deficient yeast

Telomeres are nucleoprotein structures that cap the ends of chromosomes and thereby protect their stability and integrity. In the presence of telomerase, the enzyme that synthesizes telomeric repeats, telomere length is controlled primarily by Rap1p, the budding yeast telomeric DNA binding protein which, through its C-terminal domain, nucleates a protein complex that limits telomere lengthening. In the absence of telomerase, telomeres shorten with every cell division, and eventually, cells enter replicative senescence. We have set out to identify the telomeric property that determines the replicative capacity of telomerase-deficient budding yeast. We show that in cells deficient for both telomerase and homologous recombination, replicative capacity is dependent on telomere length but not on the binding of Rap1p to the telomeric repeats. Strikingly, inhibition of Rap1p binding or truncation of the C-terminal tail of Rap1p in Kluyveromyces lactis and deletion of the Rap1p-recruited complex in Saccharomyces cerevisiae lead to a dramatic increase in replicative capacity. The study of the role of telomere binding proteins and telomere length on replicative capacity in yeast may have significant implications for our understanding of cellular senescence in higher organisms.     

A Yersinia effector with enhanced inhibitory activity on the NF-κB pathway activates the NLRP3/ASC/caspase-1 inflammasome in macrophages

A type III secretion system (T3SS) in pathogenic Yersinia species functions to translocate Yop effectors, which modulate cytokine production and regulate cell death in macrophages. Distinct pathways of T3SS-dependent cell death and caspase-1 activation occur in Yersinia-infected macrophages. One pathway of cell death and caspase-1 activation in macrophages requires the effector YopJ. YopJ is an acetyltransferase that inactivates MAPK kinases and IKKβ to cause TLR4-dependent apoptosis in naïve macrophages. A YopJ isoform in Y. pestis KIM (YopJ^KIM) has two amino acid substitutions, F177L and K206E, not present in YopJ proteins of Y. pseudotuberculosis and Y. pestis CO92. As compared to other YopJ isoforms, YopJ^KIM causes increased apoptosis, caspase-1 activation, and secretion of IL-1β in Yersinia-infected macrophages. The molecular basis for increased apoptosis and activation of caspase-1 by YopJ^KIM in Yersinia-infected macrophages was studied. Site directed mutagenesis showed that the F177L and K206E substitutions in YopJ^KIM were important for enhanced apoptosis, caspase-1 activation, and IL-1β secretion. As compared to YopJ^CO92, YopJ^KIM displayed an enhanced capacity to inhibit phosphorylation of IκB-α in macrophages and to bind IKKβ in vitro. YopJ^KIM also showed a moderately increased ability to inhibit phosphorylation of MAPKs. Increased caspase-1 cleavage and IL-1β secretion occurred in IKKβ-deficient macrophages infected with Y. pestis expressing YopJ^CO92, confirming that the NF-κB pathway can negatively regulate inflammasome activation. K⁺ efflux, NLRP3 and ASC were important for secretion of IL-1β in response to Y. pestis KIM infection as shown using macrophages lacking inflammasome components or by the addition of exogenous KCl. These data show that caspase-1 is activated in naïve macrophages in response to infection with a pathogen that inhibits IKKβ and MAPK kinases and induces TLR4-dependent apoptosis. This pro-inflammatory form of apoptosis may represent an early innate immune response to highly virulent pathogens such as Y. pestis KIM that have evolved an enhanced ability to inhibit host signaling pathways.