The ras-like GTP binding proteins cdc42, rac, and rho regulate diverse cellular processes including cell growth and actin remodeling associated with changes in cell morphology, growth, adhesion, and motility (1-4). In fibroblasts, cdc42 regulates actin polymerization and focal complexes necessary for filopodia formation, rac mediates actin polymerization and focal complex assembly within lamellipodia and membrane ruffles, and rho induces actin stress fiber and focal adhesion (FA) complex formation (5). A hierarchical relationship exists among cdc42, rac, and rho, whereby cdc42 regulates rac activity and rac regulates rho activity, suggesting that these proteins may orchestrate the spatial and temporal changes in the actin cytoskeleton necessary for cell movement (5, 6). cdc42 and rac also regulate activation of the c-Jun N-terminal kinase/stressactivated kinase via a mitogen-activated protein (MAP) kinase pathway (7-9), and rac and rho are essential for ras transformation (10, 11). cdc42, rho, and rac all appear to stimulate c-fos transcription (12), as well as cell cycle progression through GI and subsequent DNA synthesis (9). The activation state of ras-like GTP binding proteins is positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP, and negatively by GTPase activating proteins (2). A number of putative GEFs for rho-like GTPases have been identified by sequence comparison (2), and several of these demonstrate GEF activity in vitro. The dbl and ost oncogene products have cdc42 and rho GEF activity (13-15); the lbc oncogene product has rho GEF activity (16); the invasion-inducing Tiaml gene product has cdc42, rho, and rac GEF activity (17); and the yeast CDC24 gene product has cdc42 GEF activity (18). LAR is a broadly expressed transmembrane protein tyrosine phosphatase (PTPase) comprised of a cell adhesion-like extracellular region and two intracellular PTPase domains (19)(20)(21)(22). A role for LAR in regulating cell-matrix interactions was proposed, as LAR colocalizes with a coiled-coil protein, termed LAR interacting protein 1 (LIP.1) at the ends of FAs (23), and LAR expression was observed at regions of association between cells and basement membrane in various tissues (19). To identify putative substrates and other proteins involved in LAR-mediated signal transduction, we screened for proteins that bind the LAR PTPase domains using the interaction-trap assay and coimmunoprecipitation studies. A protein thus isolated is a novel multidomain GEF we have named Trio because it contains three enzyme domains: two GEF domains, one of which has racl GEF activity and the other has rhoA GEF activity, and a serine/threonine kinase (PSK) domain. In addition, Trio contains four N-terminal spectrinlike domains, two pleckstrin-like domains, and an Ig-like domain. Because proteins with cdc42, rac, or rho GEF activity are generally involved in regulating cytoskeletal organization (1), it is likely that Trio in conjunction with LAR plays a key role in coordinating the ...

Background-Biventricular pacing has been proposed to improve symptoms and exercise capacity in patients with advanced heart failure and wide electrocardiographic wave complexes. This study investigated the effect of biventricular pacing on reverse remodeling and the underlying mechanisms. Methods and Results-Twenty-five patients with NYHA class III to IV heart failure and electrocardiographic wave complex duration Ͼ140 ms receiving biventricular pacing therapy were assessed serially up to 3 months after pacing and when pacing was withheld for 4 weeks. Tissue Doppler echocardiography was performed using a 6-basal, 6-mid segmental model to assess the time to peak sustained systolic contraction (T S ). There was significant improvement of ejection fraction, dP/dt, and myocardial performance index; decrease in mitral regurgitation, left ventricular (LV) end-diastolic (205Ϯ68 versus 168Ϯ67 mL, PϽ0.01) and end-systolic volume (162Ϯ54 versus 122Ϯ42 mL, PϽ0.01); and improved 6-minute hall-walk distance and quality of life score after pacing for 3 months. The mechanisms of benefits were as follows: (1)

No abstract

Protein tyrosine phosphorylation is regulated by both protein tyrosine kinases and protein tyrosine phosphatases (PTPases). Recently, the structures of a family of PTPases have been described. In order to study the structure‐function relationships of receptor‐linked PTPases, we analyzed the effects of deletion and point mutations within the cytoplasmic region of the receptor‐linked PTPases, LCA and LAR. We show that the first of the two domains has enzyme activity by itself, and that one cysteine residue in the first domain of both LCA and LAR is absolutely required for activity. The second PTPase like domains do not have detectable catalytic activity using a variety of substrates, but sequences within the second domains influence substrate specificity. The functional significance of a stretch of 10 highly conserved amino acid residues surrounding the critical cysteine residue located in the first domain of LAR was assessed. At most positions, any substitution severely reduced enzyme activity, while missense mutations at the other positions tested could be tolerated to varying degrees depending on the amino acid substitution. It is suggested that this stretch of amino acids may be part of the catalytic center of PTPases.

Background-Patients with heart failure are frequently hospitalized for fluid overload. A reliable method for chronic monitoring of fluid status is therefore desirable. We evaluated an implantable system capable of measuring intrathoracic impedance to identify potential fluid overload before heart failure hospitalization and to determine the correlation between intrathoracic impedance and standard measures of fluid status during hospitalization. Methods and Results-Thirty-three patients with NYHA class III and IV heart failure were implanted with a special pacemaker in the left pectoral region and a defibrillation lead in the right ventricle. Intrathoracic impedance was regularly measured and recorded between the lead and the pacemaker case. During hospitalizations, pulmonary capillary wedge pressure and fluid status were monitored. Ten patients were hospitalized for fluid overload 25 times over 20.7Ϯ8.4 months. Intrathoracic impedance decreased before each admission by an average of 12.3Ϯ5.3% (PϽ0.001) over an average of 18.3Ϯ10.1 days. Impedance reduction began 15.3Ϯ10.6 days (PϽ0.001) before the onset of worsening symptoms. There was an inverse correlation between intrathoracic impedance and pulmonary capillary wedge pressure (

The transmembrane protein-tyrosinephosphatases (PTPases) LAR

The human transmembrane molecule LAR is a protein tyrosine phosphatase (PTPase) with a cell adhesion molecule‐like extracellular receptor region. The structure of LAR hinted at its involvement in the regulation of tyrosine phosphorylation through cell‐cell or cell‐matrix interactions. We show here that LAR is expressed on the cell surface as a complex of two non‐covalently associated subunits derived from a proprotein. The LAR E‐subunit contains the cell adhesion molecule‐like receptor region, while the LAR P‐subunit contains a short segment of the extracellular region, the transmembrane peptide and the cytoplasmic PTPase domains. Proprotein processing occurs intracellularly. Analysis of LAR mutants suggested that cleavage occurs in the LAR extracellular region at a paired basic amino acid site by a subtilisin‐like endoprotease. A single amino acid substitution at this site blocked LAR proprotein cleavage. The LAR E‐subunit is shed during cell growth, suggesting that LAR receptor shedding may be a mechanism for regulating PTPase function. The use of immunohistochemistry techniques on human tissues demonstrated the expression of LAR by various cell lineages, including epithelial cells, smooth muscle cells and cardiac myocytes. The LAR gene is mapped to chromosome 1, region p32–33, which contains candidate tumor suppressor genes.

Despite our knowledge of the protective role of antibodies passed to infants through breast milk, our understanding of immunity transfer via maternal leukocytes is still limited. To emulate the immunological interface between the mother and her infant while breast-feeding, we used murine pups fostered after birth onto MHC-matched and MHC-mismatched dams. Overall, data revealed that: 1) Survival of breast milk leukocytes in suckling infants is possible, but not significant after the foster-nursing ceases; 2) Most breast milk lymphocytes establish themselves in specific areas of the intestine termed Peyer’s patches (PPs); 3) While most leukocytes in the milk bolus were myeloid cells, the majority of breast milk leukocytes localized to PPs were T lymphocytes, and cytotoxic T cells (CTLs) in particular; 4) These CTLs exhibit high levels of the gut-homing molecules α4β7 and CCR9, but a reduced expression of the systemic homing marker CD62L; 5) Under the same activation conditions, transferred CD8 T cells through breast milk have a superior capacity to produce potent cytolytic and inflammatory mediators when compared to those generated by the breastfed infant. It is therefore possible that maternal CTLs found in breast milk are directed to the PPs to compensate for the immature adaptive immune system of the infant in order to protect it against constant oral infectious risks during the postnatal phase.