Michio Nakamura

Helicobacter pylori-mediated transcriptional regulation of the human beta-defensin 2 gene requires NF-kappaB.

Human β‐defensin 2 (hBD‐2) is an antimicrobial peptide involved in host defence against bacterial infection in epithelial tissues. Its levels are dramatically increased after bacterial infection. The involvement of NF‐κB in Helicobacter pylori‐mediated induction of hBD‐2 promoter activity was examined. A luciferase reporter plasmid containing the hBD‐2 promoter extending from −2110 base pairs to −1 was transiently expressed in MKN45 cells, and promoter activity was determined after incubation with H. pylori for 6u2003h. Deletion or mutation of the NF‐κB site at −208 abolished activation of the hBD‐2 promoter. Only H. pylori strains carrying a cag pathogenicity island (PAI) induced activation of the NF‐κB site of the hBD‐2 promoter gene. By gel retardation analyses, H. pylori increased NF‐κB binding to hBD‐2 promoter gene sequences. Supershift analysis demonstrated that whereas H. pylori activated NF‐κB p65–p65 and p50–p50 homodimers, and the p65–p50 heterodimer of NF‐κB, only the p65–p65 homodimer bound to the NF‐κB site of the hBD‐2 promoter. Thus, specific NF‐κB proteins are important cis‐elements for induction of hBD‐2 gene transcription by H. pylori.

Location of the epitope for 7D5, a monoclonal antibody raised against human flavocytochrome b558, to the extracellular peptide portion of primate gp91phox.

Flavocytochrome b558 is the membrane component of the phagocyte NADPH oxidase, and is a heterodimer composed of gp91phox and p22phox subunits. Human flavocytochrome b558 is recognized by monoclonal antibody 7D5 at an unidentified extracellular domain, although our previous study suggested it might recognize p22phox. 7D5 has proven useful in rapid screening of individuals for X‐linked chronic granulomatous disease by flow‐cytometry. Therefore, we re‐evaluated the location of the 7D5 epitope using gene‐engineered cell lines expressing hybrid flavocytochromes composed of human and murine subunit homologues. The current study demonstrates that the 7D5 recognizes epitope only of primate gp91phox. Flow‐cytometric analyses showed that 7D5 consistently bound to cells expressing human gp91phox. In addition, 7D5 immunoprecipitated the ~58 kDa unglycosylated gp91phox protein from solubilized membrane fractions of tunicamycin‐treated PLB‐985 granulocytes, indicating that glycans were not required for 7D5 binding. Transgenic COS7 cells expressing human gp91phox but not p22phox were recognized by 7D5. These results localized the epitope of 7D5 to an extracellular peptide portion of primate gp91phox and indicate that the antibody will be useful for monitoring the efficiency of gene therapy in patients with flavocytochrome b558‐deficient chronic granulomatous disease and for elucidating structural characteristics of flavocytochrome b558.

SATB1 Makes a Complex with p300 and Represses gp91phox Promoter Activity

The expression of gp91phox, the key component of the phagocyte NADPH oxidase, is regulated by various factors binding to its proximal promoter. Two nuclear matrix attachment region (MAR)‐binding proteins, special AT‐rich binding protein 1 (SATB1) and CCAAT displacement protein (CDP), have been reported as rare examples of gp91phox gene repressors. However, their individual roles and interactions with other factors in the promoter have not been elucidated in detail. We have focused on these two repressive proteins recognizing the bp −115 to bp −106 segment of the gene and obtained the following results: 1. SATB1 makes a complex, mainly with p300, regardless of the presence of DNA. 2. SATB1/p300 complex binding to the 5′ upstream AT‐rich region in the bp −115 to bp −106 segment represses the gp91phox promoter activity, and the repressed activity is partially released by CDP binding to the CCAAT element directly downstream of the AT‐rich region. Our findings imply a novel role for p300 in SATB1‐associated global transcription regulation.

GATA‐3 represses gp91phox gene expression in eosinophil‐committed HL‐60‐C15 cells

To study the regulatory mechanism of gp91phox gene expression in eosinophils, we transiently transfected eosinophil‐committed HL‐60‐C15 cells with gp91phox promoter constructs, and identified a negative element from bp −267 to −246 of the gp91phox gene, the deletion of which caused an 83% increase in promoter activity. Electrophoresis mobility shift assays demonstrated GATA‐3 binds to the GATA consensus site from bp −256 to −250. An 81% increment in promoter activity was obtained when a mutation was introduced in the GATA‐3 binding site of the bp −267 to +12 construct, which is comparable to that of the bp −245 to +12 construct. We therefore conclude that GATA‐3 specifically binding to the GATA site negatively regulates the expression of the gene in HL‐60‐C15 cells.

FOXK2 transcription factor is a novel G/T-mismatch DNA binding protein

DNA mismatch repair is an important mechanism in the prevention of mutations. We reported the existence in the HL60 cell line of a novel G/T-mismatch DNA-binding protein (nGTBP) requiring strict DNA sequences. In this report, we identify the FOXK2 transcription factor as the nGTBP. FOXK2 fragments were obtained as the only clones with specifically binding activity to the G/T-mismatch DNA by screening of a human brain expression library. The recombinant forkhead domain of FOXK2 specifically recognized the G/T-mismatch DNA. The forkhead domain also recognized hypoxanthine/T and G/uracil, derived from the deamination of the exocyclic amino groups of A/T and G/C, respectively. An electrophoretic mobility shift assay (EMSA) analysis using HL60 cell nuclear extract and antibody raised against FOXK2 resulted in the exclusive binding of FOXK2 to G/T-mismatch DNA. Furthermore, FOXK2 bound to G/T-mismatch DNA with higher affinity than match FOXK2 consensus DNA. We therefore propose that FOXK2 is a G/T-mismatch DNA-binding protein and a deaminated DNA-binding protein.

Superoxide-dependent and -independent pathways are involved in the transmission blocking of malaria.

Abstract. Superoxide plays a crucial role in innate immunity to various pathogens. We examined the role of superoxides in the transmission of malaria using gp91phox knockout (X-CGD) mice that lack the ability to produce superoxide. Mosquitoes that fed on X-CGD mice infected intraperitoneally with Plasmodium berghei NK65 ANKA formed more oocysts than did those that fed on control mice at any day after infection. The number of oocysts peaked on dayxa05 post-infection in X-CGD and control mice and then decreased significantly after dayxa05 post-infection. However, on dayxa07 post-infection, the infectivity of gametocytes in X-CGD mice was significantly higher than that in control mice. These results show that two pathways, superoxide-dependent and -independent, are involved in the host systems regulating the transmission of malaria and inhibiting gametocyte development.

A 25-kb deletion in the 5′ region of the cytochrome b558 heavy chain gene (CYBB) in a patient with X-linked chronic granulomatous disease

Abstract We performed molecular genetic analyses of the family of a boy suffering from chronic granulomatous disease (CGD) after immunocytochemically confirming him and his mother to be an X-linked CGD patient and a mosaic carrier, respectively. Southern blot hybridization using cDNA for the cytochrome b558 heavy chain gene (CYBB) as a probe showed that the patient had a deletion in the 5′ region of the CYBB and his phenotypically normal mother was heterozygous for this deletion. Polymerase chain reaction analyses of all 13 exons of the patient’s CYBB gene demonstrated that the deletion extends from exon 7 or neighboring introns to 5′ upstream. The length of the deletion was determined by pulsed-field gel electrophoresis and Southern blotting of genomic DNA using CYBB cDNA and the genetic marker pERT55-5, centromeric to CYBB, as probes. Both probes recognized common SfiI-NotI fragments of 120 kb and 95 kb in normal individuals and the patient, respectively. These results revealed that the patient has a 25-kb deletion spanning from the middle of CYBB to 5′ upstream. This is the only report of a large 5′ deletion in CYBB and also the first observation that CYBB and pERT55-5 are within 120 kb in Xp21.

Cell adhesion markedly increases lucigenin-enhanced chemiluminescence of the phagocyte NADPH oxidase.

Lucigenin‐enhanced chemiluminescence (LECL) is widely used for the detection of reactive oxygen species released from various cells and mitochondria. However, the LECL response varies depending on cell species and assay conditions at least in part by unknown factors. Here we report that cell adhesion is an important factor for increasing LECL of tetradecanoylphorbol acetate (TPA)‐stimulated human neutrophils. More than 90% LECL remained even after complete removal of the cell suspension 10 min after TPA stimulation, and ~22.5% of neutrophils were adhered to the reaction tube. These results indicate that LECL by an adhering neutrophil is ~45× higher than that by a non‐adhering neutrophil. LECL by leukocyte adhesion deficiency neutrophils was one‐fifth of that by normal neutrophils and completely disappeared when the cell suspension was removed, confirming that LECL depends highly on cell adhesion. The oxidase activity of adhering neutrophils measured after permeabilization with Renex 30 together with NADPH addition was similar to that of non‐adhering neutrophils, indicating that lucigenin and cell adhesion do not enhance the oxidase activity. Based on these findings, we propose that a mixture of adhering and non‐adhering neutrophils can be used for simultaneous screenings of adhering activity and the oxidase activity of neutrophils.

Expression of Fimbriae and Hemagglutination Activity in Shigella boydii

This report describes the presence of type 1 fimbriae on Shigella boydii 5 which agglutinate guinea pig erythrocytes and feature mannose‐sensitive adherence. Morphologically, the fimbriae were thin, rigid cylinders 2–5 μm in length and 3–5 nm in diameter, and the organella retained axial holes. This is the first study to have revealed the existence of type 1 fimbriae on S. boydii.

In vitro reconstitution of an erythropoietin gene transcription system using its 5′-flanking sequence and a nuclear extract from anemic kidney

We have developed an in vitro transcription system for the erythropoietin (Epo) gene. This system uses a plasmid carrying 0.2 kb of 5-flanking sequence from the human Epo gene, rNTPs and a nuclear extract from mouse kidney. The transcribed RNA was assayed by primer extension with an end-labeled primer complementary to the sequence of the plasmid, dNTPs and reverse transcriptase. The primer extension product corresponding to the transcript was detected on a sequencing gel. The in vitro promoter activity of the Epo 5-flanking sequence was observed with a nuclear extract from anemic kidney but not with that from normal kidney.