Phagocytes such as neutrophils play a vital role in host defense against microbial pathogens. The anti-microbial function of neutrophils is based on the production of superoxide anion (O 2 -), which generates other microbicidal reactive oxygen species (ROS) and release of antimicrobial peptides and proteins. The enzyme responsible for O 2 -production is called the NADPH oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two transmembrane proteins (p22phox and gp91phox, also called NOX2, which together form the cytochrome b558) and four cytosolic proteins (p47phox, p67phox, p40phox and a GTPase Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate agents. This process is dependent on the phosphorylation of the cytosolic protein p47phox. p47phox is a 390 amino acids protein with several functional domains: one phox homology (PX) domain, two src homology 3 (SH3) domains, an auto-inhibitory region (AIR), a proline rich domain (PRR) and has several phosphorylated sites located between Ser303 and Ser379. In this review, we will describe the structure of p47phox, its phosphorylation and discuss how these events regulate NADPH oxidase activation.

SummaryThe protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin. We investigated the expression of PAR-2 and the role of trypsin in cell proliferation in human colon cancer cell lines. A total of 10 cell lines were tested for expression of PAR-2 mRNA by Northern blot and RT-PCR. PAR-2 protein was detected by immunofluorescence. Trypsin and the peptide agonist SLIGKV (AP2) were tested for their ability to induce calcium mobilization and to promote cell proliferation on serum-deprived cells. PAR-2 mRNA was detected by Northern blot analysis in 6 out of 10 cell lines Cl.19A, SW480,. Other cell lines expressed low levels of transcripts, which were detected only by RT-PCR. Further results were obtained with HT-29 cells: (1) PAR-2 protein is expressed at the cell surface; (2) an increase in intracellular calcium concentration was observed upon trypsin (1-100 nM) or AP2 (10-100 µM) challenges; (3) cells grown in serum-deprived media supplemented with trypsin (0.1-1 nM) or AP2 (1-300 µM) exhibited important mitogenic responses (3-fold increase of cell number). Proliferative effects of trypsin or AP2 were also observed in other cell lines expressing PAR-2. These data show that subnanomolar concentrations of trypsin, acting at PAR-2, promoted the proliferation of human colon cancer cells. The results of this study indicate that trypsin could be considered as a growth factor and unravel a new mechanism whereby serine proteases control colon tumours.

The GK rat model of type 2 diabetes is especially convenient to dissect the pathogenic mechanism necessary for the emergence of overt diabetes because all adult rats obtained in our department (GK/Par colony) to date have stable basal mild hyperglycemia and because overt diabetes is preceded by a period of normoglycemia, ranging from birth to weaning. The purpose of this article is to sum up the information so far available related to the biology of the ␤-cell in the GK/Par rat. In terms of ␤-cell function, there is no major intrinsic secretory defect in the prediabetic GK/Par ␤-cell, and the lack of ␤-cell reactivity to glucose (which reflects multiple intracellular abnormalities), as seen during the adult period when the GK/Par rats are overtly diabetic, represents an acquired defect (perhaps glucotoxicity). In terms of ␤-cell population, the earliest alteration so far detected in the GK/Par rat targets the size of the ␤-cell population. Several convergent data suggest that the permanently reduced ␤-cell mass in the GK/Par rat reflects a limitation of ␤-cell neogenesis during early fetal life, and it is conceivable that some genes among the set involved in GK diabetes belong to the subset of genes controlling early ␤-cell development. Diabetes 50 (Suppl. 1):S89-S93, 2001T ype 2 diabetes develops as a consequence of interplay among ␤-cell dysfunction, peripheral insulin resistance, and elevated hepatic glucose production. However, it is not known which is the primary abnormality and which are abnormalities secondary to elevated plasma glucose, so-called glucose toxicity. To delineate the primary abnormalities, it is desirable to analyze individuals destined to become diabetic before the development of the disease. The advantage of using an animal model is that the development of diabetes can be predicted and thus it is possible to dissect the pathogenic mechanism necessary for the emergence of overt diabetes. The Goto-Kakizaki Wistar rat (GK rat) is especially useful because all adult animals of both sexes exhibit type 2 diabetes. This spontaneous diabetes model was produced by selective breeding (with glucose intolerance as a selection index) repeated over many generations, starting from a nondiabetic Wistar rat colony. The characteristics of GK animals bred in our colony in Paris (GK/Par) for more than 10 years (1) are very stable and remain close to those of the animals in the original Japanese colony (2): all of the rats have a basal mild hyperglycemia and impaired glucose tolerance. Males and females are similarly affected, and their diabetic state is stable over 72 weeks of follow-up (3). In adult GK rats, plasma insulin release in vivo in response to intravenous glucose is abolished (1,3). In vitro studies of insulin release with the isolated perfused pancreas (1) or with perifused islets (4) indicate that both early and late phases of glucoseinduced insulin release are markedly affected in the adult GK rat. Concerning insulin action in adult GK rats, we have reported decreased insulin sensitivity in the...

The aim of this study was to investigate, in markedly obese children, the effect of puberty on substrate oxidation during an acute bout of exercise. Two groups of markedly obese boys (7 pre pubertal, 8 post pubertal, matched for adiposity) performed an exercise-test designed for measuring carbohydrate and fat oxidation with indirect calorimetry, and consisting of five six-minute steady-state workloads at 20, 30, 40, 50, and 60 % of the theoretical maximal aerobic power. Fat oxidation (mg . min (-1)) is correlated to fat free mass (FFM) (r = 0.7, p = 0.02). When expressed in crude flow rate units, fat oxidation is slightly higher in PostP than PreP children (p < 0.05). However, when expressed per unit of FFM or as a percentage of total fuel oxidation, fat oxidation is lower in PostP than PreP children (p < 0.05). Multivariable analysis shows that the influence of age on the ability to oxidize fat at exercise is explained by the pubertal increase in FFM. In markedly obese children during puberty, the ability of each kg of FFM to oxidize fat at exercise decreases (- 28% at 20%Wmax th), but the pubertal increase in FFM overcomes this effect, resulting in an increase in whole body ability to oxidize fat at exercise (+ 17,3% at 20%Wmax th).

We examined to what extent the abnormal glucosedependent insulin secretion observed in NIDDM (noninsulin-dependent diabetes mellitus) is related to alterations in the handling of cytosolic ] i observed in response to high glucose and induced fast [Ca 2+ ] i oscillations with high amplitude in Wistar islets. The latter effect was not seen in GK and nSTZ islets. In these two NIDDM models, several common alterations in glucoseinduced Ca 2+ handling were revealed which may contribute to their poor glucose-induced insulin secretion.

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High affinity binding sites have been found in membrane preparations from hamster beta-cell tumors by using radiolabeled gastric inhibitory polypeptide (125I-GIP). HPLC of 125I-GIP resulted in two major peaks (A III and B III), with identical specific binding. It was verified that peaks A III and B III stimulate insulin release from the isolated perfused rat pancreas to an extent at least equal to that obtained with unlabeled GIP at 10(-9) M. Natural GIP competitively inhibited the binding of 125I-GIP in the range of 10(-10) -10(-6) M and half-maximal inhibition was observed at 1.9 +/- 0.19 X 10(-9) M GIP. The number of high affinity sites was 219 +/- 8 fmol/mg protein and the dissociation constant was 2.05 +/- 0.1 X 10(-9) M. None of 10 regulatory peptides tested exhibited any effect on the 125I-GIP binding at concentrations in the range of 10(-6) -10(-4) M. Consequently, saturable, high affinity and specific binding sites for the GIP have been found and characterized in the plasma membranes of beta-cells. This model can be of use in studying the interaction of GIP with its preponderant target tissue.