Researches in our laboratory are focused on the molecular regulations of the phagocyte NADPH oxidase system that generates active oxygens essential for killing invading microbes and on a possible prevention of host DNA from active oxygens.

The phagocyte NADPH oxidase is composed of membrane-integrated flavocytochrome b558 containing gp91phox and p22phox, and cytosolic components such as p47phox and p67pox. Upon phagocytotic stimulation, cytosolic factors moves to membrane to make an active complex with the flavoocytochrome. Based on genetic analysis of CYBB, the gene encoding gp91phox, in a patient with chronic granulomatous disease, we found that PU.1, a transcriptional activator, bound to gp91phox promoter position centered to bp -53 was important for the expression of gp91phox in neutrophils, monocytes, and B lymphocytes, but not in eosinophils. On the other hand, GATA-1 bound to a position centered to bp -98 had an important role in the expression of the gene in eosinophils. We are now interested in the mechanism that can modulate the expression of CYBB in selected types of cells for improving primary defense system in inflammation and allergy.

In the analysis of a cis-element of gp91phox promoter, we found a novel GT-mismatched DNA binding protein. An addition of unlabelled competitor homoduplex with G/C at bp -177, generated, but not erased, a strong 'supershifted' band in EMSA using the labelled probe with A/T at bp -177. Newly paired heteroduplex with the unlabelled upper strand with G at bp -177 and the labelled lower strand with T at the same position recruited nGTBP. This protein strictly required TRTGNB (R=purine, N=any base, B=not adenine, G paired with T) and 14-mer or longer for binding. G can be replaced by deaminated A, namely, hypoxanthine, suggesting deaminated C-6 is essential for nGTBP binding. Deamination of nucleotide bases are increased by high temperature and the repair of deaminated portions of DNA would be more important in tropical area than other areas. Transitions appreciably occurred more at TRTRNB sites than at other sites in tumor supressor protein p53 exons, suggesting this particular sites were fragile in tumor-prone cells. Relative frequency of esophageal cancer due to transitions at p53 non-CpG sites in Brazilian mate-drinkers was relatively higher than that in world-wide patients. Cloning and purification of this nGTBP are now urgent issues in our laboraatory.

Chemotaxis of immune cells

Chemotaxis has mostly been assayed by Boyden chamber method that make low-to-high chemical gradients and cells movepartially dependent on gravity. The method requires many cells for an assay and therefore its application has been limited to popular cells. We want to develop a method capable of preparation of scanty cells and simultaneous aapply assay of their chemotxis in collaboration with Dr. S. Kanegasaki (Effector Cell Research Institute, Tokyo, Japan). This new approach will give us a chance to quantitatively analyse chemotactic activity of heman pripheral blood eosinophils and basophils and its correlation to other functions such as superoxide generations.