Regulation of β-catenin stabilization in human platelets

The Wnt/β-catenin pathway controls developmental processes and homeostasis; however, abnormal activation of this pathway has been linked to several human diseases. Recent reports have demonstrated regulation of platelet function by canonical and non-canonical Wnt signalling. Platelet aggregation plays a crucial role in haemostasis and thrombosis. Here we report for the first time that, induction of sustained aggregation of platelets by a strong agonist in the presence of calcium was associated with nearly complete proteolysis of β-catenin, which was abrogated upon depletion of calcium from platelet suspension. β-catenin cleavage was disallowed in absence of aggregation, thus implicating integrin α_IIbβ₃ engagement in β-catenin proteolysis. Degradation of β-catenin was blocked partially by inhibitors of either proteasome or calpain and completely when cells were exposed to both the inhibitors. Protein kinase C inhibition, too, abolished β-catenin degradation. Thus activities of proteasome, calpain and protein kinase C regulate stabilization of β-catenin in aggregated human platelets.     

A substitution of cytosine for thymine in codon 110 of the human β-globin gene is a novel cause of β-thalassemia phenotypes

Summary We have described a novel human globin gene mutation that produced in a Japanese family the -thalassemia phenotype through a post-translational mechanism. Substitution of proline for leucine at position 110 in the G-helix of the -globin chain greatly reduced the molecular stability of the -globin subunit, leading to total destruction of the variant globin chains by proteolysis and hence to the -thalassemia phenotype. The mutation could be identified after MspI digestion. This detection of the mutation on the gene level is valuable for diagnostic purposes.     

Population study of a sequence polymorphism in intron 2 of the human β-globin gene

The distribution of a nucleotide polymorphism in intron 2 of the -globin gene (IVS-2 nt 666 C > T was examined in populations in southern Germany and Cameroon. The allelic frequencies were 0.86 for T and 0.14 for C in southern Germany and 0.87 for T and 0.13 for C in Cameroon, respectively.     

In silico analysis of single nucleotide polymorphism (SNPs) in human β-globin gene

Single amino acid substitutions in the globin chain are the most common forms of genetic variations that produce hemoglobinopathies--the most widespread inherited disorders worldwide. Several hemoglobinopathies result from homozygosity or compound heterozygosity to beta-globin (HBB) gene mutations, such as that producing sickle cell hemoglobin (HbS), HbC, HbD and HbE. Several of these mutations are deleterious and result in moderate to severe hemolytic anemia, with associated complications, requiring lifelong care and management. Even though many hemoglobinopathies result from single amino acid changes producing similar structural abnormalities, there are functional differences in the generated variants. Using in silico methods, we examined the genetic variations that can alter the expression and function of the HBB gene. Using a sequence homology-based Sorting Intolerant from Tolerant (SIFT) server we have searched for the SNPs, which showed that 200 (80%) non-synonymous polymorphism were found to be deleterious. The structure-based method via PolyPhen server indicated that 135 (40%) non-synonymous polymorphism may modify protein function and structure. The Pupa Suite software showed that the SNPs will have a phenotypic consequence on the structure and function of the altered protein. Structure analysis was performed on the key mutations that occur in the native protein coded by the HBB gene that causes hemoglobinopathies such as: HbC (E→K), HbD (E→Q), HbE (E→K) and HbS (E→V). Atomic Non-Local Environment Assessment (ANOLEA), Yet Another Scientific Artificial Reality Application (YASARA), CHARMM-GUI webserver for macromolecular dynamics and mechanics, and Normal Mode Analysis, Deformation and Refinement (NOMAD-Ref) of Gromacs server were used to perform molecular dynamics simulations and energy minimization calculations on β-Chain residue of the HBB gene before and after mutation. Furthermore, in the native and altered protein models, amino acid residues were determined and secondary structures were observed for solvent accessibility to confirm the protein stability. The functional study in this investigation may be a good model for additional future studies.     

Contribution of gene conversion in the evolution of the human β-like globin gene family

Gene conversion is referred to as one of two types of mechanisms known to act on gene families, mainly to maintain their sequence homogeneity or, in certain cases, to produce sequence diversity. The concept of gene conversion was established 20 years ago by researchers working with fungi. A few years later, gene conversion was also observed in the human genome, i.e. the γ-globin locus. The aim of this article is to emphasize the role of genetic recombination, particularly of gene conversion, in the evolution of the human β-like globin genes and further to summarize its contribution to the convergent evolution of the fetal globin genes. Finally, this article attempts to re-examine the origin and spread of specific mutations of the β-globin cluster, such as the sickle cell or β-thalassemia mutations, on the basis of repeated gene conversion events. Received: 13 February 1997 / Accepted: 15 May 1998     

Expression of human β-defensin-2 gene induced by CpG-DNA in human B cells

Defensins have a broad range of antimicrobial activity against bacteria, fungi, and viruses. The expression of human β-defensin-2 (hBD-2) is prevalently observed in epithelial cells and is induced by bacterial infection. Here, we have shown that the expression of the hBD-2 gene and release of hBD-2 protein into the medium is up-regulated in response to CpG-DNA in human B cell line RPMI 8226. The induction of hBD-2 was dependent on CG sequence and phosphorothioate backbone-modification. This was also confirmed in primary human lymphocytes. To shed light on the molecular mechanism involved in hBD-2 induction by CpG-DNA, we examined the contribution of the NF-κB signaling pathway in RPMI 8226 cells. Suppression of MyD88 function and inhibition of NF-κB nuclear localization blocked hBD-2 induction. The NF-κB pathway inhibitors also abolished hBD-2 induction. These results may contribute to a better understanding on the therapeutic effects of CpG-DNA against infectious diseases.