Inhibition of L-lactate transport and band 3-mediated anion transport in erythrocytes by the novel stilbenedisulphonate N,N,N',N'-tetrabenzyl-4,4'-diaminostilbene-2,2'-disulpho nat e (TBenzDS).

(1) The synthesis of the novel stilbenedisulphonate N,N,N',N'-tetrabenzyl- 4,4'-diaminostilbene-2,2'-disulphonate (TBenzDS) is described, and its interaction with the lactate transporter and band 3 protein of erythrocytes investigated. At 10% haematocrit the IC50 (concn. required for 50% inhibition) for inhibition of transport of 0.5 mM L-lactate into rat erythrocytes at 7 degrees C was approx. 1.6 microM, as low as any other inhibitor of the transporter. In human erythrocytes at 10% haematocrit the IC50 value was increased from approx. 3 microM to 9 microM upon raising the temperature from 7 degrees C to 25 degrees C. (2) TBenzDS inhibited transport of L-lactate into rat erythrocytes in a manner that was competitive with the substrate, as is the case for some other stilbene disulphonate derivatives (Poole, R.C. and Halestrap, A.P. (1991) Biochem. J. 275, 307-312). (3) Increasing the haematocrit from 5 to 20% caused a 3-fold increase in the IC50 value for inhibition of L-lactate transport in rat erythrocytes. (4) TBenzDS was found to bind to erythrocyte membranes, with a partition coefficient (Pm) of 6000-7000 under all conditions tested. (5) TBenzDS also inhibited band 3-mediated sulphate transport in rat erythrocytes; 50% inhibition required approx. 2.5 microM TBenzDS for cells at 10% haematocrit. (6) TBenzDS is fluorescent, and an enhancement of this fluorescence occurs upon addition of BSA or erythrocyte membranes. The fluorescence enhancement caused by erythrocyte membranes is due to binding of the inhibitor to the band 3 protein at the same site as the stilbenedisulphonate 4,4'-diisothiocyanodihydrostilbene-2,2'-disulphonate (H2DIDS).     

Glycoprotein (GP) Ib-IX-transfected cells roll on a von Willebrand factor matrix under flow. Importance of the GPib/actin-binding protein (ABP-280) interaction in maintaining adhesion under high shear

Adhesion of platelets to sites of vascular injury is critical for hemostasis and thrombosis and is dependent on the binding of the vascular adhesive protein von Willebrand factor (vWf) to the glycoprotein (GP) Ib-V-IX complex on the platelet surface. A unique but poorly defined characteristic of this receptor/ligand interaction is its ability to support platelet adhesion under conditions of high shear stress. To examine the structural domains of the GPIb-V-IX complex involved in mediating cell adhesion under flow, we have expressed partial (GPIb-IX), complete (GPIb-V-IX), and mutant (GPIbalpha cytoplasmic tail mutants) receptor complexes on the surface of Chinese hamster ovary (CHO) cells and examined their ability to adhere to a vWf matrix in flow-based adhesion assays. Our studies demonstrate that the partial receptor complex (GPIb-IX) supports CHO cell tethering and rolling on a bovine or human vWf matrix under flow. The adhesion was specifically inhibited by an anti-GPIbalpha blocking antibody (AK2) and was not observed with CHO cells expressing GPIbbeta and GPIX alone. The velocity of rolling was dependent on the level of shear stress, receptor density, and matrix concentration and was not altered by the presence of GPV. In contrast to selectins, which mediate cell rolling under conditions of low shear (20-200 s-1), GPIb-IX was able to support cell rolling at both venous (150 s-1) and arterial (1500-10,500 s-1) shear rates. Studies with a mutant GPIbalpha receptor subunit lacking the binding domain for actin-binding protein demonstrated that the association of the receptor complex with the membrane skeleton is not essential for cell tethering or rolling under low shear conditions, but is critical for maintaining adhesion at high shear rates (3000-6000 s-1). These studies demonstrate that the GPIb-IX complex is sufficient to mediate cell rolling on a vWf matrix at both venous and arterial levels of shear independent of other platelet adhesion receptors. Furthermore, our results suggest that the association between GPIbalpha and actin-binding protein plays an important role in enabling cells to remain tethered to a vWf matrix under conditions of high shear stress.     

The von Willebrand factor-glycoprotein Ib/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and glycoprotein Ib/V/IX-transfected cells

Platelet adhesion to sites of vascular injury is initiated by the binding of the platelet glycoprotein (GP) Ib-V-IX complex to matrix-bound von Willebrand factor (vWf). This receptor-ligand interaction is characterized by a rapid on-off rate that enables efficient platelet tethering and rolling under conditions of rapid blood flow. We demonstrate here that platelets adhering to immobilized vWf under flow conditions undergo rapid morphological conversion from flat discs to spiny spheres during surface translocation. Studies of Glanzmann thrombasthenic platelets (lacking integrin alpha(IIb)beta(3)) and Chinese hamster ovary (CHO) cells transfected with GPIb/IX (CHO-Ib/IX) confirmed that vWf binding to GPIb/IX was sufficient to induce actin polymerization and cytoskeletal reorganization independent of integrin alpha(IIb)beta(3). vWf-induced cytoskeletal reorganization occurred independently of several well characterized signaling processes linked to platelet activation, including calcium influx, prostaglandin metabolism, protein tyrosine phosphorylation, activation of protein kinase C or phosphatidylinositol 3-kinase but was critically dependent on the mobilization of intracellular calcium. Studies of Oregon Green 488 1, 2-bis(o-amino-5-fluorophenoxy)ethane-N,N,N',N-tetraacetic acid tetraacetoxymethyl ester-loaded platelets and CHO-Ib/IX cells demonstrated that these cells mobilize intracellular calcium in a shear-dependent manner during surface translocation on vWf. Taken together, these studies suggest that the vWf-GPIb interaction stimulates actin polymerization and cytoskeletal reorganization in rolling platelets via a shear-sensitive signaling pathway linked to intracellular calcium mobilization.     

Synergistic adhesive interactions and signaling mechanisms operating between platelet glycoprotein Ib/IX and integrin alpha IIbbeta 3. Studies in human platelets ans transfected Chinese hamster ovary cells

This study investigates three aspects of the adhesive interaction operating between platelet glycoprotein Ib/IX and integrin alpha(IIb)beta(3). These include the following: 1) examining the sufficiency of GPIb/IX and integrin alpha(IIb)beta(3) to mediate irreversible cell adhesion on immobilized von Willebrand factor (vWf) under flow; 2) the ability of the vWf-GPIb interaction to induce integrin alpha(IIb)beta(3) activation independent of endogenous platelet stimuli; and 3) the identification of key second messengers linking the vWf-GPIb/IX interaction to integrin alpha(IIb)beta(3) activation. By using Chinese hamster ovary cells transfected with GPIb/IX and integrin alpha(IIb)beta(3), we demonstrate that these receptors are both necessary and sufficient to mediate irreversible cell adhesion under flow, wherein GPIb/IX mediates cell tethering and rolling on immobilized vWf, and integrin alpha(IIb)beta(3) mediates cell arrest. Moreover, we demonstrate direct signaling between GPIb/IX and integrin alpha(IIb)beta(3). Studies on human platelets demonstrated that vWf binding to GPIb/IX is able to induce integrin alpha(IIb)beta(3) activation independent of endogenous platelet stimuli under both static and physiological flow conditions (150-1800 s(-)(1)). Analysis of the key second messengers linking the vWf-GPIb interaction to integrin alpha(IIb)beta(3) activation demonstrated that the first step in the activation process involves calcium release from internal stores, whereas transmembrane calcium influx is a secondary event potentiating integrin alpha(IIb)beta(3) activation.     

Gas-liguid chromatography of parathion metabolites as a sensitive index of induction of liver microsomal mixed-function oxidases

A method for the gas-liquid chromatographic evaluation of induction of liver microsomal mixed-function oxidases is described. The sensitivity and reproducibility of this method allows statistically significant comparisons of inducers in small numbers of animals, or, by using larger treatment groups, should allow evaluation of minimal levels of inducer. The method is compared to several other indices of mixed-function oxidase induction in rats pretreated with DDT or DDE. The induction of squirrel monkey liver microsomal mixed-function oxidases by the drug diphenylhydantoin has also been demonstrated using this procedure.     

Design of a bovine low-density SNP array optimized for imputation

The Illumina BovineLD BeadChip was designed to support imputation to higher density genotypes in dairy and beef breeds by including single-nucleotide polymorphisms (SNPs) that had a high minor allele frequency as well as uniform spacing across the genome except at the ends of the chromosome where densities were increased. The chip also includes SNPs on the Y chromosome and mitochondrial DNA loci that are useful for determining subspecies classification and certain paternal and maternal breed lineages. The total number of SNPs was 6,909. Accuracy of imputation to Illumina BovineSNP50 genotypes using the BovineLD chip was over 97% for most dairy and beef populations. The BovineLD imputations were about 3 percentage points more accurate than those from the Illumina GoldenGate Bovine3K BeadChip across multiple populations. The improvement was greatest when neither parent was genotyped. The minor allele frequencies were similar across taurine beef and dairy breeds as was the proportion of SNPs that were polymorphic. The new BovineLD chip should facilitate low-cost genomic selection in taurine beef and dairy cattle.     

Leucine-rich repeats 2-4 (Leu60-Glu128) of platelet glycoprotein Ibalpha regulate shear-dependent cell adhesion to von Willebrand factor

Glycoprotein (GP) Ib-IX-V binds von Willebrand factor (VWF), initiating thrombosis at high shear stress. The VWF-A1 domain binds the N-terminal domain of GPIbalpha (His1-Glu282); this region contains seven leucine-rich repeats (LRR) plus N- and C-terminal flanking sequences and an anionic sequence containing three sulfated tyrosines. Our previous analysis of canine/human and human/canine chimeras of GPIbalpha expressed on Chinese hamster ovary (CHO) cells demonstrated that LRR2-4 (Leu60-Glu128) were crucial for GPIbalpha-dependent adhesion to VWF. Paradoxically, co-crystal structures of the GPIbalpha N-terminal domain and GPIbalpha-binding VWF-A1 under static conditions revealed that the LRR2-4 sequence made minimal contact with VWF-A1. To resolve the specific functional role of LRR2-4, we compared wild-type human GPIbalpha with human GPIbalpha containing a homology domain swap of canine for human sequence within Leu60-Glu128 and a reverse swap (canine GPIbalpha with human Leu60-Glu128) for the ability to support adhesion to VWF under flow. Binding of conformation-specific anti-GPIbalpha antibodies and VWF binding in the presence of botrocetin (which does not discriminate between species) confirmed equivalent expression of wild-type and mutant receptors in a functional form competent to bind ligand. Compared with CHO cells expressing wild-type GPIbalpha, cells expressing GPIbalpha, where human Leu60-Glu128 sequence was replaced by canine sequence, supported adhesion to VWF at low shear rates but became increasingly ineffective as shear increased from 50 to 2000 s(-1). Together, these data demonstrate that LRR2-4, encompassing a pronounced negative charge patch on human GPIbalpha, is essential for GPIbalpha.VWF-dependent adhesion as hydrodynamic shear increases.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.