Receptor-mediated binding of IgE-sensitized rat basophilic leukemia cells to antigen-coated substrates under hydrodynamic flow.

The physiological function of many cells is dependent on their ability to adhere via receptors to ligand-coated surfaces under fluid flow. We have developed a model experimental system to measure cell adhesion as a function of cell and surface chemistry and fluid flow. Using a parallel-plate flow chamber, we measured the binding of rat basophilic leukemia cells preincubated with anti-dinitrophenol IgE antibody to polyacrylamide gels covalently derivatized with 2,4-dinitrophenol. The rat basophilic leukemia cells' binding behavior is binary: cells are either adherent or continue to travel at their hydrodynamic velocity, and the transition between these two states is abrupt. The spatial location of adherent cells shows cells can adhere many cell diameters down the length of the gel, suggesting that adhesion is a probabilistic process. The majority of experiments were performed in the excess ligand limit in which adhesion depends strongly on the number of receptors but weakly on ligand density. Only 5-fold changes in IgE surface density or in shear rate were necessary to change adhesion from complete to indistinguishable from negative control. Adhesion showed a hyperbolic dependence on shear rate. By performing experiments with two IgE-antigen configurations in which the kinetic rates of receptor-ligand binding are different, we demonstrate that the forward rate of reaction of the receptor-ligand pair is more important than its thermodynamic affinity in the regulation of binding under hydrodynamic flow. In fact, adhesion increases with increasing receptor-ligand reaction rate or decreasing shear rate, and scales with a single dimensionless parameter which compares the relative rates of reaction to fluid shear.     

Kinetics of cell detachment: peeling of discrete receptor clusters.

Clustering of cell surface adhesion receptors is an essential step in the development of focal contacts, specialized cell-substrate attachment sites where receptors are simultaneously linked to extracellular ligand and cytoskeletal proteins. Previously, we examined the effect of receptor clustering on attachment strength. Here, we employ the numerical methodology developed by Dembo and colleagues (Dembo, M., D.C. Torney, K. Saxman, and D. Hammer. 1988. Proc. R. Soc. Lond. B. 234:55-83) to investigate the kinetics of cell detachment when receptors are clustered into discrete patches. We show that the membrane peeling velocity decreases if receptors are clustered within a patch located inside the contact region. Peeling of clusters is influenced by the chemistry and mechanics of receptor-ligand bonds within the patch. Detachment is also prohibited if the applied tension equals the critical tension of the patch, unless the patch length is small compared with the boundary length over which membrane bending occurs, in which case the patch will peel. Peeling of these short patches only occurs when the mechanical stiffness of clustered bonds is within an optimal range. We compare our model predictions with experimental measurements of T lymphocyte detachment from ICAM-1 substrates. We demonstrate that if discrete patches of receptors are present, detachment occurs through intervals of slow and fast peeling, similar to the dynamics of T lymphocyte peeling, indicating that clustering of LFA-1 receptors is one possible explanation for the observed detachment kinetics in this system.     

Seed transmission of turnip yellow mosaic virus in winter turnip and winter oilseed rapes

This is the first record of seed transmission of turnip yellow mosaic virus (TYMV) in oilseed and turnip rapes. The seed transmission of TYMV in a naturally infected winter turnip rape (Brassica napus var. silvestris) cultivar Perko PVH was investigated. By ELISA 1.6%, 3.2% and 8.3% seed transmission of the virus was found in seed of plants from three localities. The proportion of infected seeds produced by artificially infected plants of winter oilseed rape (Brassica napus ssp. oleifera) and winter turnip rape cultivars was determined. The virus transmission rate, expressed as the proportion of virus-infected plants which germinated from the seed was for the oilseed rape cvs Jet Neuf 0.1%, Solida 0.4%, Silesia 0.8%, Darmor 1.2%, SL-507 0.2%, SL-509 0.0% and for the winter turnip rape cv. Perko 1.5%. ELISA cannot be used in direct tests on bulk seed lots to estimate proportion of infected seed, but must be used on germinated seedlings.     

A theoretical analysis for the effect of focal contact formation on cell-substrate attachment strength.

For many cell types, growth, differentiation, and motility are dependent on receptor-mediated adhesion to ligand-coated surfaces. Focal contacts are strong, specialized, adhesive connections between cell and substrate in which receptors aggregate and connect extracellular ligand to intracellular cytoskeletal molecules. In this paper, we present a mathematical model to examine how focal contact formation affects cellular adhesive strength. To calculate adhesive strength with and without focal contacts, we use a one-dimensional tape peeling analysis to determine the critical tension necessary to peel the membrane. Receptor-ligand bonds are modeled as adhesive springs. In the absence of focal contacts, we derive analytic expressions for the critical tension at low and high ligand densities and show how membrane morphology affects adhesion. Then, focal contacts are modeled as cytoplasmic nucleation centers which bind adhesion receptors. The extent of adhesive strengthening upon focal contact formation depends on the elastic rigidity of the cytoskeletal connections, which determines the structural integrity of the focal contact itself. We consider two limits to this elasticity, very weak and rigid. Rigid cytoskeletal connections give much greater attachment strengths. The dependence of attachment strength on measurable model parameters is quite different in these two limits, which suggests focal contact structure might be deduced from properly performed adhesion experiments. Finally, we compare our model to the adhesive strengthening response reported for glioma cell adhesion to fibronectin (Lotz et al., 1989. J. Cell Biol. 109:1795-1805). Our model successfully predicts the observed detachment forces at 4 degrees C and yields values for the number of fibronectin receptors per glioma cell and the density of cytoskeletal connection molecules (talin) involved in receptor clusters which are consistent with measurements for other cell types. Comparison of the model with data at 37 degrees C suggests that while cytoskeletal cross-linking and clustering of fibronectin receptors significantly increases adhesion strength, specific glioma cell-substratum attachment sites possess little mechanical rigidity and detach through a peeling mechanism, consistent with the view that these sites of < or = 15 nm cell-substrate separation are precursors to fully formed, elastically rigid focal contacts.     

Synergistic interactions between XPC and p53 mutations in double-mutant mice: neural tube abnormalities and accelerated UV radiation-induced skin cancer

The significance of DNA repair to human health has been well documented by studies on xeroderma pigmentosum (XP) patients, who suffer a dramatically increased risk of cancer in sun-exposed areas of their skin [1] and [2]. This autosomal recessive disorder has been directly associated with a defect in nucleotide excision–repair (NER) [1] and [2]. Like human XP individuals, mice carrying homozygous mutations in XP genes manifest a predisposition to skin carcinogenesis following exposure to ultraviolet (UV) radiation [3], [4] and [5]. Recent studies have suggested that, in addition to roles in apoptosis [6] and cell-cycle checkpoint control [7] in response to DNA damage, p53 protein may modulate NER [8]. Mutations in the p53 gene have been observed in 50% of all human tumors [9] and have been implicated in both the early [10] and late [11] stages of skin cancer. To examine the consequences of a combined deficiency of the XPC and the p53 proteins in mice, we generated double-mutant animals. We document a spectrum of neural tube defects in XPC p53 mutant embryos. Additionally, we show that, following exposure to UV-B radiation, XPC p53 mutant mice have more severe solar keratosis and suffer accelerated skin cancer compared with XPC mutant mice that are wild-type with respect to p53.     

Potent inhibition of human immunodeficiency virus type 1 in primary T cells and alveolar macrophages by a combination anti-Rev strategy delivered in an adeno-associated virus vector.

The rate of viral replication appears to play a pivotal role in human immunodeficiency virus type 1 (HIV-1) pathogenesis and disease progression as it outstrips the capacity of the immune system to respond. Important cellular sites for HIV-1 production include T lymphocytes and tissue macrophages. Antiviral strategies, including newer treatment modalities such as gene therapy of HIV-1-susceptible cell populations, must be capable of engendering durable inhibitory effects to HIV-1 replication in both of these primary cell types in order to be effective. Among the potential genetic targets for intervention in the HIV-1 life cycle, the Rev regulatory system, consisting of Rev and its binding site, the Rev-responsive element (RRE), stands out as particularly attractive. Rev is essential for maintaining the stability of the viral genomic RNA as well as viral mRNAs encoding key structural and regulatory proteins. Moreover, it exhibits favorable threshold kinetics, in that Rev concentrations must rise above a critical level to exert their effect. To disable Rev function, primary T cells or macrophages were transduced with anti-Rev single-chain immunoglobulin (SFv) or RRE decoy genes either singly or in combination by employing adeno-associated virus vectors and then challenged with HIV-1. By directing both a protein and a nucleic acid against the normal interaction between Rev and the RRE, this genetic antiviral strategy effectively inhibited infection by either clinical or laboratory virus isolates. These results provide a framework for novel interventions to reduce virus production in the infected host.     

The role of lipid components of the diet in the regulation of the fatty acid composition of the rat liver endoplasmic reticulum and lipid peroxidation

The fatty acid compositions of the lipids and the lipid peroxide concentrations and rates of lipid peroxidation were determined in suspensions of liver endoplasmic reticulum isolated from rats fed on synthetic diets in which the fatty acid composition had been varied but the remaining constituents (protein, carbohydrate, vitamins and minerals) kept constant. Stock diet and synthetic diets containing no fat, 10% corn oil, herring oil, coconut oil or lard were used. The fatty acid composition of the liver endoplasmic reticulum lipid was markedly dependent on the fatty acid composition of the dietary lipid. Feeding a herring-oil diet caused incorporation of 8.7% eicosapentaenoic acid (C_20:5) and 17% docosahexaenoic acid (C_22:6), but only 5.1% linoleic acid (C_18:2) and 6.4% arachidonic acid (C_20:4), feeding a corn-oil diet caused incorporation of 25.1% C_18:2, 17.8% C_20:4 and 2.5% C_22:6 fatty acids, and feeding a lard diet caused incorporation of 10.3% C_18:2, 13.5% C_20:4 and 4.3% C_22:6 fatty acids into the liver endoplasmic-reticulum lipids. Phenobarbitone injection (100mg/kg) decreased the incorporation of C_20:4 and C_22:6 fatty acids into the liver endoplasmic reticulum of rats fed on a lard, corn-oil or herring-oil diet. Microsomal lipid peroxide concentrations and rates of peroxidation in the presence of ascorbate depended on the nature and quantity of the polyunsaturated fatty acids in the diet. The lipid peroxide content was 1.82±0.30nmol of malonaldehyde/mg of protein and the rate of peroxidation was 0.60±0.08nmol of malonaldehyde/min per mg of protein after feeding a fat-free diet, and the values were increased to 20.80nmol of malonaldehyde/mg of protein and 3.73nmol of malonaldehyde/min per mg of protein after feeding a 10% herring-oil diet in which polyunsaturated fatty acids formed 24% of the total fatty acids. Addition of α-tocopherol to the diets (120mg/kg of diet) caused a very large decrease in the lipid peroxide concentration and rate of lipid peroxidation in the endoplasmic reticulum, but addition of the synthetic anti-oxidant 2,6-di-t-butyl-4-methylphenol to the diet (100mg/kg of diet) was ineffective. Treatment of the animals with phenobarbitone (1mg/ml of drinking water) caused a sharp fall in the rate of lipid peroxidation. It is concluded that the polyunsaturated fatty acid composition of the diet regulates the fatty acid composition of the liver endoplasmic reticulum, and this in turn is an important factor controlling the rate and extent of lipid peroxidation in vitro and possibly in vivo.     

Aspects of hadrosaurian cranial anatomy

Supraorbital bones in Saurolophus angustirostris are described, and their presence in all hadrosaurs is suggested. Frontal-nasal and premaxillar-nasal fontanellae are distinguished in hadrosaurs; their presence is explained as connected with growth and considered to he responsible for the variability of crest structures. New data indicating the presence of a cartilaginous diverticulum nasi within the circumnarial depression in Saurobphus ongustirostris are presented. A physiological (respiratory and/or thermoregulatory) function of the nasal diverticulum is proposed.