Association of the interleukin-4 receptor alpha chain with p47phox, an activator of the phagocyte NADPH oxidase in B cells.

Interleukin (IL)-4 plays an important role in IgE synthesis in B cells and in Th2 differentiation in T cells. IL-4 conducts its biological activities through binding to the IL-4 receptor (IL-4R) on the surface of target cells. IL-4R are thought to be composed of the IL-4R alpha chain (IL-4R alpha) and either the IL-2R gamma chain or the IL-13R alpha chain. We have previously shown that the membrane-proximal portion in the cytoplasmic domain of the human IL-4R alpha (hIL-4R alpha) is critical for proliferation, generation of germline epsilon transcript, and activation of STAT6, based on analyses of truncated hIL-4R alphas. In this study, we found that p47phox, an activator of the phagocyte NADPH oxidase, binds to this portion by the two-hybrid system. Furthermore, we observed the association of p47phox with the hIL-4R alpha in B cells derived from a normal donor. These results suggest that p47phox is involved in the signal transduction of IL-4 in B cells. However, activation of STAT6, CD23 expression, and IgE synthesis induced by IL-4 were not affected in p47phox-deficient patients, which raises the possibility that p47phox may be important in other signaling activities as well in B cells.

A novel intracellular membrane-bound calcium-independent phospholipase A(2).

We have cloned human cDNA encoding a novel protein of 782 amino acids that contains the lipase consensus sequence Gly-Xaa-Ser-Xaa-Gly and several stretches surrounding the motif, which are homologous to those of the catalytic domain of cytosolic calcium-independent phospholipase A(2) (iPLA(2)). When expressed in COS-7 cells, the protein predominantly exists in the membrane fraction and exhibits a phospholipase A(2) activity in a calcium-independent manner. The transcript of the membrane-bound iPLA(2) gene is ubiquitously observed as a single band of approximately 3.3 kb on Northern blot, with the most abundant expression in the skeletal muscle and heart. By a search of the database, we have also identified its putative C. elegans homologue, which shows 47% identity with that of human in the iPLA(2) catalytic region. Thus the novel type of iPLA(2) is evolutionarily well conserved, suggestive of its biological significance.

Interaction of the PDZ domain of human PICK1 with class I ADP-ribosylation factors.

We have cloned the cDNA encoding human PICK1 (protein interacting with C kinase 1), a PDZ domain-containing protein of 415 amino acids, and also identified the Drosophila homologue by search of the databank. Northern blot analysis shows a single mRNA of about 2.0 kb ubiquitously expressed in human tissues. Although PICK1 proteins harbor a region homologous to arfaptin1 and arfaptin2, two proteins that bind to the ARF (ADP-ribosylation factor), this region of PICK1 does not interact with ARFs in the yeast two-hybrid system. On the other hand, the PDZ domain of PICK1 is capable of interacting with constitutively active, GTP-bound forms of ARF1 and ARF3, but neither with those of ARF5/6 nor with the GDP-bound ARFs. The PICK1-ARF interaction is abrogated by introduction of mutations in the PDZ domain or by deletion of the extreme C-terminus of ARF1. Thus, PICK1 specifically interacts with ARF1/3 in the GTP-bound state, suggesting that PICK1 participates in ARF1/3-mediated cellular processes.

An enzyme-linked immunosorbent assay for urinary albumin in patients with insulin-dependent diabetes mellitus.

Microalbuminuria is considered to be an early indicator of diabetic nephropathy. In this report, we developed an enzyme-linked immunosorbent assay (ELISA) for the measurement of urinary albumin (UA) and examined UA in 38 patients with insulin-dependent diabetes mellitus (IDDM). The assay range of ELISA for albumin was 5-1,000 ng/ml, and the albumin levels determined by ELISA were well correlated with those by immunoagglutination methods. The UA values of daytime single-void specimens in control subjects, which correlated significantly with UA excretion rates (micrograms/minute) in 24-hour urine, were 10.9 +/- 8.2 micrograms/mg creatinine. In 38 IDDM patients, there were four cases with microalbuminuria and one case with overt nephropathy. Their disease duration was longer than 8 years, and the diabetic control was fair to poor. On SDS-PAGE analysis. the urinary protein of the cases with microalbuminuria consisted mainly of albumin, and in the case of nephropathy, an IgG band was also detected. The measurements of UA in single-void specimens by ELISA is a satisfactory approach to detect impending nephropathy in IDDM patients.

The PX domain as a novel phosphoinositide- binding module.

The phox (phagocyte oxidase) homology (PX) domain occurs in the mammalian phox proteins p40(phox) and p47(phox), the polarity establishment protein Bem1p in budding yeast, and a variety of proteins involved in membrane trafficking. Here we show that the PX domains of p40(phox) and p47(phox) directly bind to phosphoinositides: p40(phox) prefers Ptdlns(3)P, while p47(phox) does Ptdlns(4)P and Ptdlns(3,4)P(2). In addition, the Bem1p PX domain also interacts with Ptdlns(4)P. When the p40(phox) PX domain is expressed as a fusion to green fluorescent protein in HeLa cells, it exists at early endosomes where Ptdlns(3)P is enriched. Furthermore, a mutant p40(phox) PX carrying the substitution of Lys for Arg105 only weakly binds to phosphoinositides in vitro, and fails to locate to early endosomes. Thus the PX domain functions as a novel phosphoinositide-binding module and likely participates in targeting of proteins to membranes.

Human homologues of the Caenorhabditis elegans cell polarity protein PAR6 as an adaptor that links the small GTPases Rac and Cdc42 to atypical protein kinase C.

BACKGROUND: Asymmetric cell division in the Caenorhabditis elegans embryos requires products of par (partitioning defective) genes 1-6 and atypical protein kinase C (aPKC), whereas Cdc42 and Rac, members of the Rho family GTPases, play an essential role in cell polarity establishment in yeast and mammalian cells. However, little is known about a link between PAR proteins and the GTPases in cell polarization. RESULTS: Here we have cloned cDNAs for three human homologues of PAR6, designated PAR6alpha, beta and gamma, comprising 345, 372 and 376 amino acids, respectively. The PAR6 proteins harbour a PDZ domain and a CRIB-like motif, and directly interact with GTP-bound Rac and Cdc42 via this motif and with the aPKC isoforms PKCiota/lambda and PKCzeta via the N-terminal head-to-head association. These interactions are not mutually exclusive, thereby allowing the PAR6 proteins to form a ternary complex with the GTPases and aPKC, both in vitro and in vivo. When PAR6 and aPKC are expressed with a constitutively active form of Rac in HeLa or COS-7 cells, these proteins co-localize to membrane ruffles, which are known to occur at the leading edge of polarized cells during cell movement. CONCLUSION: Human PAR6 homologues most likely play an important role in the cell polarization of mammalian cells, by functioning as an adaptor protein that links activated Rac and Cdc42 to aPKC signalling.

Epidemic of gastrointestinal tract infection including hemorrhagic colitis attributable to Shiga toxin 1-producing Escherichia coli O118:H2 at a junior high school in Japan.

BACKGROUND: An epidemic of gastrointestinal disturbances related to food ingestion occurred at a junior high school in Komatsu, Japan, and was caused by specifically Shiga toxin (Stx) 1-producing Escherichia coli O118:H2, which has not been reported previously in humans. No outbreak of E coli-producing Stx 1 alone had occurred. METHODS: A total of 526 students and 35 adult staff members who ate the same food at lunch in the school were investigated. Questionnaires about food consumption at lunch were given to all 561 subjects as well as to clinics and hospitals that had treated 79 patients. Stool specimens from 525 subjects, and food, water, and environmental specimens, including cooking utensils, were collected in an attempt to identify the pathogen. RESULTS: A total of 126 subjects (22.5%) developed a diarrheal illness. The pathogen was isolated from the stool in 131 subjects, 49 of which were asymptomatic, and from a dipper. Salads served over several days were identified as high-risk from food analysis. Gastrointestinal symptoms resembled those associated with previous infections of Stx-producing E coli, but were mild. No cases of the hemolytic-uremic syndrome developed. Headache was present in 87 patients. Three patients underwent surgery for acute appendicitis during this epidemic. Four of five carriers had received an antibiotic effective against the pathogen. CONCLUSIONS: This outbreak of E coli O118:H2 demonstrated the clinical and epidemiologic features of infection by E coli that produces Stx 1 alone. Infections with such organisms are being recognized increasingly, and the pattern of disease observed may differ from the pattern observed with E coli O157:H7.