Gap between odor perception threshold and identification threshold: Calculation based on a graph of the Biolfa(®) olfactory test.

OBJECTIVES: To develop a graph of semi-quantitative Biolfa(®) olfactory test data and to assess a method of mathematical measurement of the gap between odor perception and identification thresholds. PATIENTS AND METHODS: The semi-quantitative Biolfa(®) olfaction test comprises eight smells, each diluted in four increasing concentrations. Perception and identification threshold data for 158 patients was displayed in a radar-like diagram. The gap between odor perception threshold and odor identification threshold was quantified as the area between the perception and identification threshold curves. RESULTS: The gap calculated between odor perception and identification thresholds differentiated between etiologic origins of olfactory disorders. In cm², the gap ranged from 0 to 82.73 cm². Low values indicated a peripheral origin, and high values a central origin for olfactory disorders (p>0.05). On the other hand, no olfactory profile specific to each of the main etiologies responsible for olfactory disorder was found to exist. CONCLUSION: This radar-like graphic display of semi-quantitative Biolfa(®) olfactory test data is a simple means of providing an overall view of the data. Studying the gap between perception and identification thresholds for smells is relevant for diagnosis of central olfactory disorders.

Surfing the insulin signaling web.

The diverse biological actions of insulin and insulin-like growth factor I (IGF-I) are initiated by binding of the polypeptides to their respective cell surface tyrosine kinase receptors. These activated receptors phosphorylate a series of endogenous substrates on tyrosine, amongst which the insulin receptor substrate (IRS) proteins are the best characterized. Their phosphotyrosine-containing motifs become binding sites for Src homology 2 (SH2) domains on proteins such as SH2 domain-containing protein-tyrosine-phosphatase (SHP)-2/Syp, growth factor receptor bound-2 protein, (Grb-2), and phosphatidyl inositol 3 kinase (PI3 kinase), which participate in activation of specific signaling cascades. However, the IRS molecules are not only platforms for signaling molecules, they also orchestrate the generation of signal specificity, integration of signals induced by several extracellular stimuli, and signal termination and modulation. An extensive review is beyond the scope of the present article, which will be centered on our own contribution and reflect our biases.

Characterization of BIS20x3, a bi-specific antibody activating and retargeting T-cells to CD20-positive B-cells.

This paper describes a bi-specific antibody, which was called BIS20x3. It retargets CD3varepsilon-positive cells (T-cells) to CD20-positive cells and was obtained by hybrid-hybridoma fusion. BIS20x3 could be isolated readily from quadroma culture supernatant and retained all the signalling characteristics associated with both of its chains. Cross-linking of BIS20x3 on Ramos cells leads to DNA fragmentation percentages similar to those obtained after Rituximab-cross-linking. Cross-linking of BIS20x3 on T-cells using cross-linking F(ab')2-fragments induced T-cell activation. Indirect cross-linking of T-cell-bound BIS20x3 via Ramos cells hyper-activated the T-cells. Furthermore, it was demonstrated that BIS20x3 effectively re-targets T-cells to B-cells, leading to high B-cell cytotoxicity. The results presented in this paper show that BIS20x3 is fully functional in retargeting T-cells to B-cells and suggest that B-cell lymphomas may represent ideal targets for T-cell retargeting bi-specific antibodies, because the retargeted T-cell is maximally stimulated in the presence of B-cells. Additionally, since B-cells may up-regulate CD95/ Fas expression upon binding of CD20-directed antibodies, B-cells will become even more sensitive for T-cell mediated killing via CD95L/ Fas L, and therefore supports the intention to use T-cell retargeting bi-specific antibodies recognizing CD20 on B-cell malignancies as a treatment modality for these diseases.

Potential involvement of FRS2 in insulin signaling.

Shp-2 is implicated in several tyrosine kinase receptor signaling pathways. This phosphotyrosine phosphatase is composed of a catalytic domain in its C-terminus and two SH2 domains in its N-terminus. Shp-2 becomes activated upon binding through one or both SH2 domains to tyrosine phosphorylated molecules such as Shc or insulin receptor substrates. We were interested in finding a new molecule(s), tyrosine phosphorylated by the insulin receptor (IR), that could interact with Shp-2. To do so, we screened a human placenta complementary DNA (cDNA) library with the SH2 domain-containing part of Shp-2 using a modified yeast two-hybrid system. In this system we induce or repress the expression of a constitutive active IR beta-subunit. When expressed, IR phosphorylates proteins produced from the library that can then associate with Shp-2. Using this approach, we isolated FRS2 as a potential target for tyrosine phosphorylation by the IR. After cloning the entire cDNA, we found that 1) in the yeast two-hybrid system, FRS2 interacts with Shp-2 in a fashion dependent on the presence of the IR; and 2) in the PC12/IR cell-line, insulin leads to an increase in FRS2 association with the phosphatase. We next wanted to determine whether FRS2 could be a direct substrate for IR. In an in vitro kinase assay we found that wheat-germ agglutinin-purified IR phosphorylates glutathione-S-transferase-FRS2 fusion protein. Finally, in intact cells we show that insulin stimulates tyrosine phosphorylation of endogenous FRS2. In summary, by screening a two-hybrid cDNA library, we have isolated FRS2 as a possible substrate for IR. We found that IR can directly phosphorylate FRS2. Moreover, in intact cells insulin stimulates tyrosine phosphorylation of FRS2 and its subsequent association with Shp-2. Taken together these results suggest that FRS2 could participate in insulin signaling by recruiting Shp-2 and, hence, could function as a docking molecule similar to insulin receptor substrate proteins.

Interaction of insulin receptor substrate-1 (IRS-1) with phosphatidylinositol 3-kinase: effect of substitution of serine for alanine in potential IRS-1 serine phosphorylation sites.

Serine and threonine phosphorylation has been shown to down-regulate insulin signaling at multiple steps, including the receptor and downstream molecules such as insulin receptor substrate-1 (IRS-1). To further address the mechanism of this regulation at the level of IRS-1, we constructed a double serine mutant of IRS-1: S662A/S731A-IRS-1. The serines 662 and 731 mutated to alanine are surrounding tyrosines Y658 and Y727, respectively. These tyrosines are comprised in YXXM motifs, which are potential binding sites for the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase. In a first series of experiments using the yeast two-hybrid system, we show that IRS-1 interacts with p85alpha, and this interaction depends on tyrosine phosphorylation, as shown with the IRS-1 mutant F18 and 3Y-IRS-1. F18-IRS-1 contains 18 potential tyrosine phosphorylation sites mutated to phenylalanine; three of them, i.e. Y608, 628, and 658, which are potential binding sites for p85alpha, have been added back in the 3Y-IRS-1 mutant. The tyrosine phosphorylation of IRS-1, which is required for the interaction with p85alpha, is thought to occur via endogenous yeast kinases that phosphorylate IRS-1 at least on these PI 3-kinase-binding sites. Next, we show that not only p85alpha but also p55PIK, another regulatory subunit of PI 3-kinase, interacts with IRS-1 in yeast. Interestingly, for both regulatory subunits their interaction with IRS-1 is up-regulated by mutating serines 662 and 731 on IRS-1. In a previous study we found that insulin-stimulated PI 3-kinase activity was increased not only in the presence of S662A/S731A-IRS-1 but also under resting conditions compared with the activity seen with WT-IRS-1. Here we demonstrate in 293-EBNA cells overexpressing S662A/S731A-IRS-1 that insulin-stimulated protein kinase B activity is not augmented, whereas without insulin treatment, basal activity is increased compared with that in cells overexpressing wild-type IRS-1. In conclusion, we have shown that 1) potential serine phosphorylation sites on IRS-1, which are adjacent to YXXM binding motifs for PI 3-kinase, negatively regulate binding of IRS-1 to PI 3-kinase regulatory subunits; and 2) these modulations affect protein kinase B activity.

Insulin receptor substrate-1 as a signaling molecule for focal adhesion kinase pp125(FAK) and pp60(src).

Insulin receptor substrate-1 (IRS-1) is a major substrate of insulin and insulin-like growth factor-I receptors, which upon phosphorylation on tyrosine docks several signaling molecules. Recently, IRS-1 was found to interact with alphav beta3 integrins upon insulin stimulation. Integrins are transmembrane proteins that play an important role in adhesion between cells and between cells and extracellular matrix. One of the major proteins implicated in integrin signaling is pp125(FAK), a cytosolic tyrosine kinase, which upon integrin engagement becomes tyrosine-phosphorylated and subsequently binds to c-Src. Here, we established a mammalian two-hybrid system to show that pp125(FAK) binds to IRS-1. This association depends largely on the C terminus of pp125(FAK) but not on pp125(FAK) tyrosine kinase activity. Furthermore, we observed co-immunoprecipitation of pp125(FAK) with IRS-1 in 293 cells, suggesting a possible biological function of this association. When IRS-1 was expressed in 293 cells together with pp125(FAK) or Src, we found extensive IRS-1 tyrosine phosphorylation. In pp125(FAK)-expressing cells, this was concomitant with increased association of IRS-1 with Src homology 2-containing proteins such as growth factor receptor-bound protein 2, phosphatidylinositol (PI) 3-kinase p85alpha subunit, and Src homology 2-containing protein-tyrosine phosphatase-2. In addition, pp125(FAK)-induced association of IRS-1 with PI 3-kinase resulted in increased PI 3-kinase activity. In contrast, no change in mitogen-activated protein kinase activity was observed, indicating that pp125(FAK)-induced association between IRS-1 and growth factor receptor-bound protein 2 does not affect the mitogen-activated protein kinase pathway. Moreover, we found that engagement of integrins induced IRS-1 tyrosine phosphorylation. Considering our results together, we suggest that integrins and insulin/insulin-like growth factor-I receptor signaling pathways converge at an early point in the signaling cascade, which is the IRS-1 protein.

Interaction of wild type and dominant-negative p55PIK regulatory subunit of phosphatidylinositol 3-kinase with insulin-like growth factor-1 signaling proteins.

In a first series of experiments done in the yeast two-hybrid system, we investigated the nature of protein-protein interaction between the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), p55PIK, and several of its potential signaling partners. The region between the Src homology 2 (SH2) domains of p55PIK bound to the NH2 terminus region of p110alpha, as previously shown for p85alpha. Moreover, we found that the insulin-like growth factor-1 receptor (IGF-IR) bound to p55PIK; the interaction occurred at the receptor tyrosine 1316 and involved both p55PIK SH2 domains. Interaction between p55PIK and IGF-IR was seen not only in the yeast two-hybrid system, but also using in vitro binding and coimmunoprecipitation of lysates from IGF-1 stimulated 293 cells overexpressing p55PIK. Further, IGF-I stimulation of these cells led to tyrosine phosphorylation of p55PIK. In 293 cells association of p55PIK with insulin receptor substrate-1 and with IGF-IR was dependent on PI 3-kinase, since it was increased by wortmannin, an inhibitor of PI 3-kinase. Further, by deleting amino acids 203-217 of p55PIK inter-SH2 domain, we engineered a p55PIK mutant unable to bind to the p110alpha catalytic subunit of PI 3-kinase. This mutant had a dominant-negative action on insulin-stimulated glucose transport, since insulin's effect on Glut 4 myc translocation was inhibited in adipocytes expressing mutant p55PIK. Importantly, this dominant-negative mutant was more efficient than wild type p55PIK in associating to IGF-IR and insulin receptor substrate-1 in 293 cells. Taken together, our results show that p55PIK interacts with key elements in the IGF-I signaling pathway, and that these interactions are negatively modulated by PI 3-kinase itself, providing circuitry for regulatory feedback control.