Accuracy and predictability of intraocular lens power calculation after photorefractive keratectomy.

PURPOSE: To investigate the accuracy and predictability of intraocular lens (IOL) power calculation in postoperative photorefractive keratectomy (PRK) eyes. SETTING: Gimbel Eye Centre, Calgary, Alberta, Canada. METHODS: The results in 5 cataract surgery eyes that had had PRK were analyzed retrospectively. Target refractions based on actual and refraction-derived keratometric values were compared with postoperative achieved refractions. The target refractions calculated using 5 IOL formulas and 2 A-constants were also compared with the achieved refractions. RESULTS: In postoperative PRK eyes, the power calculation was more accurate and predictable when the smaller of either the actual or refraction-derived keratometric value was used to calculate the IOL power. The difference between target and achieved refractions appeared smaller when the Binkhorst formula was used. No significant hyperopic shift was observed after cataract surgery. CONCLUSION: The smaller of the actual or the refraction-derived keratometric value is recommended for calculating IOL power in post-PRK eyes.

Rare activation of the TCF/beta-catenin pathway in ovarian cancer.

OBJECTIVE: The activation of the T cell factor/beta-catenin pathway is a crucial event in colon cancer initiation. A recent report describing the presence of beta-catenin mutations in endometrioid ovarian cancer suggested that the TCF/beta-catenin pathway may be generally activated in ovarian cancer. We therefore undertook to determine the frequency of activation of this pathway in ovarian cancer cell lines using a functional screen. METHODS: We functionally screened a series of ovarian cancer cell lines for the presence of constitutive TCF/beta-catenin-mediated transcriptional activity using a reporter assay. Lines possessing such activity were subjected to mutational and gel-shift analysis, as well as sensitivity to the introduction of dominant-negative TCF or APC alleles. A cDNA harboring a beta-catenin point mutation found in an ovarian cancer line was incorporated into an expression plasmid for functional analysis. RESULTS: Constitutive TCF/beta-catenin transcriptional activity was detected in 21% (4 of 19) of ovarian lines studied, while 32% (6 of 19) exhibited greater than twofold repression. One of the constitutively active lines, UCI107, harbored an activating beta-catenin point mutation, which was shown to be capable of inducing TCF/beta-catenin transcriptional activity in transiently transfected 293 cells. A second active line, SW626, was shown to harbor an inactivating APC mutation and may in fact be of colonic origin. The third and fourth lines harbored neither an APC nor a beta-catenin mutation. Gel-shift analysis, together with the absence of sensitivity to dominant-negative TCF, indicated that the reporter activity exhibited by the latter two cell lines may not be due to a TCF/beta-catenin transcriptional complex. CONCLUSIONS: These results indicate that genuine constitutive activation of the TCF/beta-catenin pathway is infrequent in ovarian cancer, but that constitutive transcriptional repression from TCF sites is more common in this tumor type.

Phosphorylation- and activation-independent association of the tyrosine kinase Syk and the tyrosine kinase substrates Cbl and Vav with tubulin in B-cells.

Aggregation of the B-cell antigen receptor leads to the activation of the 72-kDa Syk protein-tyrosine kinase and the phosphorylation of tubulin on tyrosine. To explore the requirement of Syk catalytic activity for tubulin phosphorylation, tubulin was isolated from cytosolic fractions from anti-IgM-activated B-cells (DT40) that lacked endogenous Syk and immunoblotted with anti-phosphotyrosine antibodies. Tubulin was not tyrosine-phosphorylated in Syk- B-cells. Phosphorylation could be restored by the expression of wild-type, but not catalytically inactive, Syk. However, both catalytically inactive and wild-type Syk were capable of constitutive association with tubulin, indicating that tubulin phosphorylation is not required for this interaction. Anti-phosphotyrosine antibody immunoblotting of proteins adsorbed to colchicine-agarose revealed the presence of three major tubulin-associated phosphoproteins of 110, 90, and 74 kDa, the phosphorylation of which was dependent on Syk expression. The proteins of 110 and 90 kDa were identified as Cbl and Vav, two proto-oncogene products known to become prominently phosphorylated following receptor engagement. Both proteins were shown to be constitutively associated with tubulin.

Incidence and management of intraoperative and early postoperative complications in 1000 consecutive laser in situ keratomileusis cases.

OBJECTIVE: To identify intraoperative and early postoperative complications of laser in situ keratomileusis (LASIK) surgery learning curve and to offer recommendations on prevention and management. DESIGN: Retrospective noncomparative case series. PARTICIPANTS: The first 1000 consecutive myopic LASIK eyes (April 1995-February 1997) operated on by one surgeon (HVG) were examined. INTERVENTION: Myopic LASIK surgery was performed with the Chiron Corneal Shaper and NIDEK EC-5000 excimer laser. MAIN OUTCOME MEASURES: The preoperative and 6-month postoperative spherical equivalent, best-corrected visual acuity, and corneal status were recorded, as was the incidence of intraoperative and early postoperative complications. The rate of retreatments was also recorded. RESULTS: There were 32 (3.2%) intraoperative complications and surgical events recorded, including 19 (1.9%) microkeratome-related flap complications and 13 (1.3%) nonmicrokeratome-related surgical events such as inability to obtain sufficient suction. There were 18 (1.8%) postoperative complications requiring repositioning of microwrinkled or shifted flaps. Six-month spherical equivalent was -0.52 diopter [D] +/- 1.19 D for eyes with microkeratome complications, -0.56 D +/- 1.07 D for the group with nonmicrokeratome-related intraoperative events, and -0.78 D +/- 0.92 for eyes requiring postoperative flap repositioning. None of the 32 eyes with intraoperative complications and surgical events lost 2 or more lines of vision. One eye in the postoperative complications group lost two lines of vision. The rate of microkeratome complications related to surgical technique and the overall surgery times decreased over the course of the first 1000 myopic LASIK cases. CONCLUSION: The complications encountered during the early learning curve of LASIK surgery have not in this series resulted in a significant loss of best-corrected visual acuity. With increasing surgical experience, the incidence of complications, along with surgical times, has decreased.

Identification of the major sites of autophosphorylation of the murine protein-tyrosine kinase Syk.

The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1.

Syk, activated by cross-linking the B-cell antigen receptor, localizes to the cytosol where it interacts with and phosphorylates alpha-tubulin on tyrosine.

Syk (p72syk) is a 72-kDa, nonreceptor, protein-tyrosine kinase that becomes tyrosine-phosphorylated and activated in B lymphocytes following aggregation of the B-cell antigen receptor. To explore the subcellular location of activated Syk, anti-IgM-activated B-cells were fractionated into soluble and particulate fractions by ultracentrifugation. Activated and tyrosine-phosphorylated Syk was found predominantly in the soluble fraction and was not associated with components of the antigen receptor. Similarly, the activated forms of Syk and its homolog, ZAP-70, were found in soluble fractions prepared from pervanadate-treated Jurkat T-cells. A 54-kDa protein that co-immunoprecipitated with Syk from the soluble fraction of activated B-cells was identified by peptide mapping as alpha-tubulin. alpha-Tubulin was an excellent in vitro substrate for Syk and was phosphorylated on a single tyrosine present within an acidic stretch of amino acids located near the carboxyl terminus. alpha-Tubulin was phosphorylated on tyrosine in intact cells following aggregation of the B-cell antigen receptor in a reaction that was inhibited by the Syk-selective inhibitor, piceatannol. Thus, once activated, Syk releases from the aggregated antigen receptor complex and is free to associate with and phosphorylate soluble proteins including alpha-tubulin.

Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk.

The tyrosine kinase Syk (relative molecular mass 72,000), which is widely expressed in haematopoietic cells, becomes associated with and activated by engagement of the B-cell antigen receptor. Furthermore, it has been implicated in signalling through the receptors for interleukin-2 (IL-2), granulocyte colony-stimulating factor (G-CSF) and Fc, the T cell receptor, as well as through receptors for several platelet agonists. A homologous kinase, ZAP-70, is crucial in signalling through the T-cell receptor and in T-cell development. Using homologous recombination in embryonic stem cells, we created mice null for the syk gene which showed petechiae in utero and died shortly after birth. Irradiated mice reconstituted with Syk-deficient fetal liver showed a block in B-cell development at the pro-B to pre-B cell transition, consistent with a key role for Syk in pre-B-cell receptor signalling. Despite the production of small numbers of immature B cells, Syk-deficient radiation chimaeras failed to accumulate mature B cells, indicating a possible role for this protein in the production or maintenance of mature B cells. In addition, whereas the development of alpha beta T cells proceeded normally, Syk-deficient mice showed impaired development of thymocytes using the V gamma 3 variable region gene (V gamma 3+ thymocytes). Finally, we show that Syk is not required for signalling through the IL-2 and G-CSF receptors.

Signaling-induced association of a tyrosine-phosphorylated 36-kDa protein with p50csk.

The protein-tyrosine kinase, p50csk, is thought to participate in the regulation of signal transduction pathways by catalyzing the phosphorylation of the Src-related protein-tyrosine kinases on a negative regulatory tyrosine residue located near the COOH terminus. To study possible mechanisms by which the activity of p50csk might be regulated, we searched for p50csk-interacting proteins in human erythroleukemia cells. We found that in response to the treatment of cells with pervanadate, a potent inhibitor of protein tyrosine phosphatases, or to the cross-linking of Fc gamma RIIA receptors, p50csk becomes tightly associated with a 36-kDa protein (p36). This association is dependent on the tyrosine phosphorylation of p36 and involves its interaction with the SH2 domain of p50csk.p36 can be phosphorylated in vitro by p50csk or by a full-length GST-Csk fusion protein expressed in Escherichia coli. Tyrosine-phosphorylated p36 is found exclusively in the particulate membrane fraction of the cell. Conditions that induce the formation of the p50csk.p36 complex promote the appearance of p50csk in the particulate fraction. These data suggest that the association between p50csk and p36 serves to translocate the normally cytosolic p50csk to the membrane, where it presumably interacts with its physiologically relevant substrates.

Interactions of Lyn with the antigen receptor during B cell activation.

Signaling through the B cell antigen receptor requires a complex set of interactions involving transmembrane components of the IgM receptor complex and cytosolic protein-tyrosine kinases. We have focused on the nature of these protein-protein interactions, the requirements for their occurrence, as well as the temporal sequence of events during the activation process. We found that cross-linking B cell antigen receptors at 0 degree C resulted in the rapid association of the Src-family protein-tyrosine kinase, Lyn, with the antigen receptor complex as judged by the presence of Lyn in anti-IgM and anti-phosphotyrosine immune complexes and the presence of MB-1 in anti-Lyn immune complexes. Receptor engagement also resulted in the rapid association of Lyn with the phosphotyrosine phosphatase, CD45. This association of Lyn with receptor components was stable in the detergent Brij 96, but was readily disrupted by Nonidet P-40, suggesting the involvement of hydrophobic interactions in stabilizing formation of the Lyn-receptor complex. The protein-tyrosine kinase, Syk, was also found associated with activated receptor complexes. This association of Syk with components of the antigen receptor complex was stable to Nonidet P-40. Antibodies directed against the carboxyl teminus of Syk, but not against the amino-terminal SH2 domain, co-immunoprecipitated MB-1 from activated cells, consistent with the binding of Syk through an SH2 domain-phosphotyrosine interaction.