Checkpoint-Independent Regulation of Origin Firing by Mrc1 through Interaction with Hsk1 Kinase.

Mrc1 is a conserved checkpoint mediator protein that transduces the replication stress signal to the downstream effector kinase. The loss of checkpoint activity results in the aberrant activation of late/dormant origins in the presence of hydroxyurea. Mrc1 was also suggested to regulate orders of early origin firing in a checkpoint-independent manner, but its mechanism was unknown.

Epitope analysis of Japanese cedar pollen allergen Cry j2 with a human IgM class monoclonal antibody 404-117.

Japanese cedar pollen allergen Cry j2 is a causal allergen of seasonal pollinosis in Japan. To analyze B cell epitopes of Cry j2, we established two human-mouse hybridomas secreting IgM class human monoclonal antibodies to Cry j2. A pin-peptide enzyme-linked immunosorbent assay with synthesized icosa peptides showed that 404-117 monoclonal antibody bound to peptides #11-13 with cry j2 amino acid sequence of 101F-L140.

Epitope analysis of Japanese cedar pollen allergen Cry j1 with the human monoclonal antibody 4701-1.

We obtained a stable human-mouse hybridoma clone 4701-1 secreting IgM class human monoclonal antibody to Japanese cedar pollen allergen Cry j1. A pin-peptide enzyme-linked immunosorbent assay (ELISA) with synthesized pentadeca peptides showed a peptide with an amino acid sequence of LYTVT NSDDD PVNPA was found to be positive. Detailed analysis with deca to tetra peptides indicated that an amino acid sequence of TVTN was an essential sequence for antibody binding.

Hsk1 kinase and Cdc45 regulate replication stress-induced checkpoint responses in fission yeast.

In fission yeast, replication fork arrest activates the replication checkpoint effector kinase Cds1(Chk2/Rad53) through the Rad3(ATR/Mec1)-Mrc1(Claspin) pathway. Hsk1, the Cdc7 homologue of fission yeast required for efficient initiation of DNA replication, is also required for Cds1 activation. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3.

Interactions between Swi1-Swi3, Mrc1 and S phase kinase, Hsk1 may regulate cellular responses to stalled replication forks in fission yeast.

The Swi1-Swi3 replication fork protection complex and Mrc1 protein are required for stabilization of stalled replication forks in fission yeast. Hsk1 kinase also plays roles in checkpoint responses elicited by arrested replication forks. We show that both Swi1 and Swi3, the abundance of which are interdependent, are required for chromatin association of Mrc1.

Generation of mouse-human hybridomas secreting antibodies against peanut allergen Ara h1.

Two clones of mouse-human hybridomas, secreting human monoclonal antibodies to a peanut allergen Ara h1, were generated from human peripheral blood lymphocytes transformed with Epstein--Barr virus, followed by cell fusion with mouse myeloma cells. Epitope analysis with overlapping peptides synthesized on a multi-pin apparatus revealed antibody-binding sequences of Ara h1 protein.

Epitope analysis of peanut allergen Ara h1 with oligoclonal IgM antibody from human B-lymphoblastoid cells.

To analyze epitopes of peanut allergen Ara h1, Epstein-Barr virus-transformed human peripheral oligoclonal B-cells were cultured to obtain antibodies to Ara h1. The combined reaction pattern with six oligoclonal antibodies showed there were six antibody binding areas named a to f in Ara h1. We found the novel antibody binding area named "area c" (171-230aa).

Generation of mouse-human hybridomas secreting human monoclonal antibodies to Japanese cedar pollen allergen Cry j1.

Peripheral blood lymphocytes from 13 donors were transformed with Epstein-Barr virus (EBV) to establish immortalized human B-cells secreting antibodies to Japanese cedar (Cryptomeria japonica) pollen allergens. EBV-transformed B-lymphocytes were then fused with mouse myeloma SP2/O3, and three clones of mouse-human hybridomas secreting human IgM class monoclonal antibodies to a cedar pollen allergen Cry j1 were established.

Expression of lysosomal protective protein/cathepsin A in a stably transformed human neuroblastoma cell line during bi-directional differentiation into neuronal and Schwannian cells.

Human neuroblastoma GOTO cell lines were established that stably express recombinant human lysosomal protective protein/cathepsin A (PPCA) cDNA by transfection. Intracellular cathepsin A (acid serine carboxypeptidase) activity increased four-fold compared with in those of the parent and mock-transfected cell lines. The immunoreactive 54 kDa precursor/zymogen and mature 32/20 kDa two-chain forms were produced in the cells.