Normalization of Red Cell Enolase Level Following Allogeneic Bone Marrow Transplantation in a Child with Diamond-Blackfan Anemia

We describe a girl with Diamond-Blackfan anemia with accompanying red cell enolase deficiency. At the age of 9 yr old, the patient received allogeneic bone marrow transplantation from her HLA-identical sister who had normal red cell enolase activity. While the post transplant DNA analysis with short tandem repeat has continuously demonstrated a stable mixed chimerism on follow-up, the patient remains transfusion independent and continues to show a steady increase in red cell enolase activity for over two and a half years following bone marrow transplantation.

Characterization of plasmin(ogen) binding to Streptococcus pneumoniae.

BACKGROUND & OBJECTIVES: The proteolytic activity of plasmin promotes migration of pathogenic bacteria through the human extracellular matrix. The human pathogen Streptococcus pneumoniae binds both human plasminogen and plasmin via the surface displayed alpha-enolase designated Eno. Electron microscopic studies verified the surface exposition of the glycolytic enzyme alpha-enolase and moreover, its ability to reassociate to the cell surface. Carboxyterminal lysine residues of recently described eukaryotic and prokaryotic plasminogen-binding proteins such as SEN of S. pyogenes are involved in interaction with lysine binding sites of kringle domains of plasminogen. In this study, the role of carboxy terminal lysyl residue of eno in plasminogen binding is further analysed. METHODS: Site-directed mutagenesis of eno gene was done using DNA primers with Hind III-restriction enzyme sites for cloning. Purified Eno fusion proteins were separated by SDS-PAGE and human plasminogen binding assay was performed. Radioiodinated ligand binding was done by competitive inhibition assay. RESULTS: Binding assays performed under reduced conditions indicated also a role of the C-terminal lysyl residues of Eno for plasmin(ogen) binding. Binding of pneumococci to radioiodinated plasminogen was competitively inhibited in the presence of plasminogen, kringle 1-3 (LBS 1) and the lysineanalogon epsilon-amino caproic acid indicating the crucial role of lysine-binding sites of plasminogen. However, binding analysis of plasminogen and LBS 1 to wild type Eno and carboxy terminal modified Eno proteins did not reveal any difference in plasminogen-binding activity under native conditions. INTERPRETATION & CONCLUSION: The present results suggested the presence of a further plasminogenbinding motif in Eno. This hypothesis was confirmed by plasminogen-binding activity of reassociated C-terminal modified enolase to the pneumococcal surface and indicated, therefore, the presence of a further binding motif in Eno for plasminogen binding.

Cloning and sequencing of complete tau-crystallin cDNA from embryonic lens of Crocodylus palustris.

tau-Crystallin is a taxon-specific structural protein found in eye lenses. We present here the cloning and sequencing of complete tau-crystallin cDNA from the embryonic lens of Crocodylus palustris and establish it to be identical to the a-enolase gene from non-lenticular tissues. Quantitatively, the tau-crystallin was found to be the least abundant crystallin of the crocodilian embryonic lenses. Crocodile tau-crystallin cDNA was isolated by RT-PCR using primers designed from the only other reported sequence from duck and completed by 5'- and 3'-rapid amplification of cDNA ends (RACE) using crocodile gene specific primers designed in the study. The complete tau-crystallin cDNA of crocodile comprises 1305 bp long ORF and 92 and 409 bp long untranslated 5'- and 3'-ends respectively. Further, it was found to be identical to its putative counterpart enzyme a-enolase, from brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of tau-crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the gene lineage reconstruction analysis helps our understanding of the evolution of crocodilians and avian species.