Modeling the influence of ectodomain affinities on the spatial distribution of membrane receptors

We have previously shown that the glycosylphosphatidyl-inositol (GPI)-linked urokinase-type plasminogen activator receptor (uPAR) reversibly associates with the integrins complement receptor type 3 (CR3; alphaMbeta2) and CR4 (alphaxbeta2) during leukocyte motility. These receptor-to-receptor interactions could potentially be accounted for by diffusion-controlled reactions or by directed transport phenomena. To address these alternatives, we have used computer simulation techniques. Our results show that a diffusion-controlled interaction between uPAR and CR4 during accumulation at lamellipodia is not physically reasonable. This suggests that a directed transport mechanism participates in establishing uPAR-integrin association.     

TNF-alpha opens a paracellular route for HIV-1 invasion across the blood-brain barrier.

BACKGROUND: HIV-1 invades the central nervous system early after infection when macrophage infiltration of the brain is low but myelin pallor is suggestive of blood-brain-barrier damage. High-level plasma viremia is a likely source of brain infection. To understand the invasion route, we investigated virus penetration across in vitro models with contrasting paracellular permeability subjected to TNF-alpha. MATERIALS AND METHODS: Blood-brain-barrier models constructed with human brain microvascular endothelial cells, fetal astrocytes, and collagen I or fibronectin matrix responded in a dose-related fashion to cytokines and ligands modulating paracellular permeability and cell migration. Virus penetration was measured by infectious and quantitative HIV-1 RNA assays. Barrier permeability was determined using inulin or dextran. RESULTS: Cell-free HIV-1 was retained by the blood-brain barrier with close to 100% efficiency. TNF-alpha increased virus penetration by a paracellular route in a dose-dependent manner proportionately to basal permeability. Brain endothelial cells were the main barrier to HIV-1. HIV-1 with monocytes attracted monocyte migration into the brain chamber. CONCLUSIONS: Early after the infection, the blood-brain barrier protects the brain from HIV-1. Immune mediators, such as TNF-alpha, open a paracellular route for the virus into the brain. The virus and viral proteins stimulate brain microglia and macrophages to attract monocytes into the brain. Infiltrating macrophages cause progression of HIV-1 encephalitis.     

Cloning of double stranded DNAs derived from polysomal mRNA of maize endosperm: isolation and characterisation of zein clones.

Starting from polysomal RNA of developing maize endosperm and applying the cloning procedure of integrating dsDNA into the Pst I site of plasmid pBR 322, clones containing sequences complementary to endosperm mRNAs were obtained. 25 per cent of these clones were identified as containing zein specific DNA sequences which hybridized either with the zein mRNA coding for the 22 000 Mr protein or with the zein mRNA coding for the 19 000 Mr protein. The zein-specific DNA inserts of the recombinant plasmids were further characterized by restriction enzyme analysis.     

CXC and CC chemokine receptors on coronary and brain endothelia

BACKGROUND: Chemokine receptors on leukocytes play a key role in inflammation and HIV-1 infection. Chemokine receptors on endothelia may serve an important role in HIV-1 tissue invasion and angiogenesis. MATERIALS AND METHODS: The expression of chemokine receptors in human brain microvascular endothelial cells (BMVEC) and coronary artery endothelial cells (CAEC) in vitro and cryostat sections of the heart tissue was determined by light and confocal microscopy and flow cytometry with monoclonal antibodies. Chemotaxis of endothelia by CC chemokines was evaluated in a transmigration assay. RESULTS: In BMVEC, the chemokine receptors CCR3 and CXCR4 showed the strongest expression. CXCR4 was localized by confocal microscopy to both the cytoplasm and the plasma membrane of BMVEC. In CAEC, CXCR4 demonstrated a strong expression with predominantly periplasmic localization. CCR5 expression was detected both in BMVEC and CAEC but at a lower level. Human umbilical cord endothelial cells (HUVEC) expressed strongly CXCR4 but only weakly CCR3 and CCR5. Two additional CC chemokines, CCR2A and CCR4, were detected in BMVEC and CAEC by immunostaining. Immunocytochemistry of the heart tissues with monoclonal antibodies revealed a high expression of CXCR4 and CCR2A and a low expression of CCR3 and CCR5 on coronary vessel endothelia. Coronary endothelia showed in vitro a strong chemotactic response to the CC chemokines RANTES, MIP-1alpha, and MIP-1beta. CONCLUSIONS: The endothelia isolated from the brain display strongly both the CCR3 and CXCR4 HIV-1 coreceptors, whereas the coronary endothelia express strongly only the CXCR4 coreceptor. CCR5 is expressed at a lower level in both endothelia. The differential display of CCR3 on the brain and coronary endothelia could be significant with respect to the differential susceptibility of the heart and the brain to HIV-1 invasion. In addition, CCR2A is strongly expressed in the heart endothelium. All of the above chemokine receptors could play a role in endothelial migration and repair.     

Disturbances of Amino Acids from Temporal Lobe Synaptosomes in Human Complex Partial Epilepsy

We have studied the levels of neuroactive amino acids in synaptosomes (P₂ fraction) isolated from brain tissue of ten patients with medically intractable epilepsy who were undergoing temporal lobectomy. First, lateral temporal tissue (nonfocal) was removed followed by medial temporal tissue (focal). A synaptosomal fraction (P₂) was immediately prepared from each tissue and analyzed for free amino acid concentrations. Statistically significant reductions were seen in glutamine and GABA concentrations in focal tissue compared to nonfocal tissue. The ratio of excitatory amino acids (aspartate and glutamate) to inhibitory amino acids (taurine and GABA) was significantly higher in focal tissue compared to nonfocal. The glutamine/glutamate ratio was significantly reduced. These data support the hypothesis that alterations in the balance between excitatory and inhibitory amino acids may be involved in the expression of epilepsy.     

Somatic mutation, affinity maturation and the antibody repertoire: a computer model

Somatic mutation has been implicated as a significant and possibly primary factor in the maturation of antibody affinity in the humoral immune response. B cells stimulated by antigen experience a hyper-mutation in the gene segments that code for the antigen-binding site of the antibody, creating antibody specificities that did not exist at the time of immunization. Although most of the mutations are likely to be disadvantageous, new specificities with a higher affinity for the antigen are sometimes created. These higher-affinity cells are preferentially selected for proliferation and eventual antibody secretion, resulting in a progressively higher average affinity over time. In this paper we present the results of an investigation of somatic mutation through the use of a computer model. At the basis of the model is a large repertoire of discrete antibodies and antigens, having three-dimensional structures, that exhibit properties similar to those of the real populations. The key factor is that the binding strength between any antibody/antigen pair can be calculated as a function of the complementarity of the (a) size, (b) shape and (c) functional groups that comprise the two structures. The created repertoires are imbedded in a dynamical system model of the immune response to directly evaluate the affect of somatic mutation on affinity maturation. We also present an expanded hypothesis of clonal selection and development to explain how the mutational restrictions imposed by the genetic code and the structure of the antibody repertoire, along with antigen concentration, affinity, and probabilistic factors may interact and contribute to the expansion of specific clones as the response develops over time.