Sialic acid is an essential moiety of mucin as a hydroxyl radical scavenger

In this work, we examined the antioxidant role of mucin, a typical sialic acid containing high-molecular weight glycoprotein. The function of mucin as a hydroxyl radical (.OH) scavenger was characterized using bovine submaxillary gland mucin (BSM). Non-treated BSM effectively protected DNA from the attack of .OH; however, desialylated BSM lost this potential. Moreover, we estimated the scavenging effects of BSM against .OH generated by UV irradiation of hydrogen peroxide using ESR analysis. Our results indicate that BSM has .OH scavenging ability the and sialic acid in mucin is an essential moiety to scavenge .OH.     

Translocation and activation of sphingosine kinase 1 by ceramide-1-phosphate

Sphingosine kinases (SphKs) and ceramide kinase (CerK) phosphorylate sphingosine to sphingosine-1-phosphate (S1P) and ceramide to ceramide-1-phosphate (C1P), respectively. S1P and C1P are bioactive lipids that regulate cell fate/function and human health/diseases. The translocation and activity of SphK1 are regulated by its phosphorylation of Ser ²²⁵ and by anionic lipids such as phosphatidic acid and phosphatidylserine. However, the roles of another anionic lipid C1P on SphK1 functions have not yet been elucidated, thus, we here investigated the regulation of SphK1 by CerK/C1P. C1P concentration dependently bound with and activated recombinant human SphK1. The inhibition of CerK reduced the phorbol 12-myristate 13-acetate-induced translocation of SphK1 to the plasma membrane (PM) and activation of the enzyme in membrane fractions of cells. A treatment with C1P translocated wild-type SphK1, but not the SphK1-S225A mutant, to the PM without affecting phosphorylation signaling. A cationic RxRH sequence is proposed to be a C1P-binding motif in α-type cytosolic phospholipase A ₂ and tumor necrosis factor α-converting enzyme. The mutation of four cationic amino acids to Ala in the 56-RRNHAR-61 domain in SphK1 reduced the phorbol 12-myristate 13-acetate- and C1P-induced translocation of SphK1 to the PM, however, the capacity of C1P to bind with and activate SphK1 was not affected by this mutation. In conclusion, C1P modulates SphK1 functions by interacting with multiple sites in SphK1.     

Myristoylation of human immunodeficiency virus type 1 gag protein is required for efficient env protein transportation to the surface of cells

Highly conserved amino acids in the N-terminal region of the human immunodeficiency virus type 1 (HIV-1) Pr55(gag) are recognized to be critical for the attachment of myristic acid. We previously reported that the env protein was not detected on the cell surface by blocking of N-myristoylation of Pr55(gag) with N-myristoyl glycinal diethylacetal. Here, we constructed a mutant by substituting the N-terminal glycine of Pr55(gag) with alanine to demonstrate that N-myristoylation of Pr55(gag) is required for efficient env protein transportation to the cell surface. The expression level of the env protein on the surface of Jurkat cells transfected with the myristoylation-defective phenotype was observed to be significantly reduced by electron microscopic analyses with a gold-labeled monoclonal antibody against the env protein. In addition, Jurkat cells transfected with the myristoylation-defective phenotype lost the ability of envelope-mediated cell-to-cell fusion. The results suggest that N-myristoylation of the HIV-1 gag protein is necessary for efficient env protein transportation to the cell surface.     

A novel glucokinase regulator in pancreatic beta cells: precursor of propionyl-CoA carboxylase beta subunit interacts with glucokinase and augments its activity

A glucokinase regulatory protein has been reported to exist in the liver, which suppresses enzyme activity in a complex with fructose 6-phosphate, whereas no corresponding protein has been found in pancreatic beta cells. To search for such a protein in pancreatic beta cells, we screened for a cDNA library of the HIT-T15 cell line with the cDNA of glucokinase from rat islet by the yeast two hybrid system. We detected a cDNA encoding the precursor of propionyl-CoA carboxylase beta subunit (pbetaPCCase), and glutathione S-transferase pull-down assay illustrated that pbetaPCCase interacted with recombinant rat islet glucokinase and with glucokinase in rat liver and islet extracts. Functional analysis indicated that pbetaPCCase decreased the K(m) value of recombinant islet glucokinase for glucose by 18% and increased V(max) value by 23%. We concluded that pbetaPCCase might be a novel activator of glucokinase in pancreatic beta cells.     

The forkhead-associated domain of NBS1 is essential for nuclear foci formation after irradiation but not essential for hRAD50[middle dot]hMRE11[middle dot]NBS1 complex DNA repair activity

NBS1 (p95), the protein responsible for Nijmegen breakage syndrome, shows a weak homology to the yeast Xrs2 protein at the N terminus region, known as the forkhead-associated (FHA) domain and the BRCA1 C terminus domain. The protein interacts with hMRE11 to form a complex with a nuclease activity for initiation of both nonhomologous end joining and homologous recombination. Here, we show in vivo direct evidence that NBS1 recruits the hMRE11 nuclease complex into the cell nucleus and leads to the formation of foci by utilizing different functions from several domains. The amino acid sequence at 665-693 on the C terminus of NBS1, where a novel identical sequence with yeast Xrs2 protein was found, is essential for hMRE11 binding. The hMRE11-binding region is necessary for both nuclear localization of the complex and for cellular radiation resistance. On the other hand, the FHA domain regulates nuclear foci formation of the multiprotein complex in response to DNA damage but is not essential for nuclear transportation of the complex and radiation resistance. Because the FHA/BRCA1 C terminus domain is widely conserved in eukaryotic nuclear proteins related to the cell cycle, gene regulation, and DNA repair, the foci formation could be associated with many phenotypes of Nijmegen breakage syndrome other than radiation sensitivity.     

Alteration of mosaic methylation of the repeat unit of the human ribosomal RNA genes in lung cancer

We have investigated the methylation status of the repeat unit of the human ribosomal RNA genes in lung cancer. Using a Southern blot analysis approach we have determined that the non-transcribed region of these genes was generally heavily methylated, while the transcribed region was not methylated in either tumor or normal DNA. Our study also revealed that, in one tumor, the boundary of mosaic methylation of the repeat unit was not distinct. In the same tumor, both the non-transcribed ribosomal spacer region and the L1 interspersed repeat sequences became partially demethylated. In tumor cells, the methylation status of DNA can be altered, but the methylation of subtelomeric repeats was found to be maintained. These results suggest that the mosaic methylation of the repeat unit is not necessarily maintained in tumor DNA, while subtelomeric repeats escape tumor-specific wave of demethylation.