Differential opening of the brain endothelial barrier following neutralization of the endothelial luminal anionic charge in vitro.

This study was undertaken to determine the effect of neutralization of brain endothelial cell luminal membrane anionic charge on endothelial permeability properties. Mouse brain microvessel endothelial cells were grown to confluence on a nitrocellulose filter. The permeability of the endothelium to Evan's blue dye (EBD) (molecular weight 960) and fluoresceinated dextran (FITC-D) (molecular weight 20,000), both polar molecules, was assessed before and after the exposure of the endothelium to cationic ferritin (CF) or native ferritin (NF). The use of CF resulted in a significant increase in permeability of the endothelium to EBD compared to NF. This result indicates that ablation of endothelial surface anionic charge enhances endothelial transport of a small, polar-charged molecule. Cationic ferritin did not increase the permeability of FITC-D compared to NF. This negative result is not surprising because FITC-D differs from EBD in terms of charge and solubility as well as in size. The electrical resistance of the endothelial cell layer after the application of CF was unchanged from baseline values suggesting a transcellular rather than a paracellular route of the EBD leakage.

Immunoglobulin D(H) recombination signal sequence targeting: effect of D(H) coding and flanking regions and recombination partner.

V(D)J recombination is targeted by recombination signal sequences (RSS) located immediately adjacent to immune receptor gene segments. While the RSS flanking D(H) segments appear to be equivalent, they are not randomly utilized. During D(H) to J(H) rearrangement, the 3' D(H) RSS is virtually exclusively utilized, suggesting that the 3' D(H) RSS could simply be a better target for the recombinase. However, when we examined V(H) to D(H) (without J(H)) rearrangements, we found that the preference for D(H) RSS use changes, so that the 5' D(H) RSS are preferred. This suggests that the 3' D(H) RSS are not simply superior targets for the V(D)J recombinase, but instead that certain 12/23-bp spacer RSS combinations work better together to target recombination than do others. We have analyzed a series of artificial recombination substrates to delineate cis sequences that affect D(H) RSS selection. Our data suggest that coding sequences adjacent to the D(H) RSS, flanking sequences outside the D(H) gene segment itself, and recombination partner all affect D(H) RSS targeting.

Molecular basis for the lack of T cell proliferation induced by an altered peptide ligand.

In this report, we explore the mechanisms underlying cell cycle progression in T cells stimulated with an altered peptide ligand (APL) versus wild-type peptide. APL stimulation did not induce proliferation compared to wild-type peptide stimulation. To determine the point at which cell cycle progression is blocked, we have examined molecules responsible for regulating the retinoblastoma tumor suppressor gene product, pRb, which in its active state prevents G1/S progression. The majority of cells stimulated with an APL did not progress beyond G1; however, a small population did make the G1/S transition. These few cells passed the late G1 restriction point, divided and subsequently arrested at the next G1 phase. The lack of sustained signaling events following stimulation with an APL failed to induce cyclin E:cdk2 activity, a regulator which hyper-phosphorylates and inactivates pRb. Exogenous IL-2 addition did not compensate for the lack of proliferation following APL stimulation. Furthermore, the inability of the cells to enter S phase during partial T cell activation cannot be accounted for by p27Kip1 inhibition of cyclin E:cdk2 complexes. Upon APL stimulation, an increase in association of p27Kip1 with cyclin E:cdk2 complex was not observed, suggesting that instead, decreased cyclin E:cdk complex formation might contribute to the failure to progress from G1/S. Therefore, while for a majority of cells, wild-type stimulation results in cell cycle progression, APL stimulation is not sufficient to drive cells beyond G1.

Equine severe combined immunodeficiency: a defect in V(D)J recombination and DNA-dependent protein kinase activity.

V(D)J rearrangement is the molecular mechanism by which an almost infinite array of specific immune receptors are generated. Defects in this process result in profound immunodeficiency as is the case in the C.B-17 SCID mouse or in RAG-1 (recombination-activating gene 1) or RAG-2 deficient mice. It has recently become clear that the V(D)J recombinase most likely consists of both lymphoid-specific factors and ubiquitously expressed components of the DNA double-strand break repair pathway. The deficit in SCID mice is in a factor that is required for both of these pathways. In this report, we show that the factor defective in the autosomal recessive severe combined immunodeficiency of Arabian foals is required for (i) V(D)J recombination, (ii) resistance to ionizing radiation, and (iii) DNA-dependent protein kinase activity.