Laparoscopic anterior esophageal myotomy and toupet fundoplication for achalasia.

Achalasia is an esophageal motility disorder characterized by the failure of lower esophageal sphincter relaxation and the absence of esophageal peristalsis. The purpose of this study was to evaluate the clinical outcomes of patients undergoing laparoscopic esophageal myotomy and Toupet fundoplication for achalasia. A 9-cm myotomy was performed in most cases extending 7 cm above and 2 cm below the gastroesophageal junction. Severity of dysphagia, heartburn, chest pain, and regurgitation was graded preoperatively and postoperatively using a five-point symptomatic scale (0-4). Patients also graded their outcomes as excellent, good, fair, or poor. Between December 1995 and November 2000 a total of 49 patients (23 male, 26 female) with a mean age of 44.3 years (range 23-71 years) were diagnosed with achalasia. Mean duration of symptoms was 40.2 months (range 4-240 months). Thirty-seven patients (76%) had had a previous nonsurgical intervention or combinations of nonsurgical interventions [pneumatic dilation (23), bougie dilation (five), and botulinum toxin (19)], and two patients had failed esophageal myotomies. Forty-five patients underwent laparoscopic esophageal myotomy and Toupet fundoplication. Two patients received laparoscopic esophageal myotomies without an antireflux procedure, and two were converted to open surgery. One patient presented 10 hours after a pneumatically induced perforation and underwent a successful laparoscopic esophageal myotomy and partial fundoplication. Mean operative time was 180.5 minutes (range 145-264 minutes). Mean length of stay was 1.98 days (range 1-18 days). There were five (10%) perioperative complications but no esophageal leaks. There was a significant difference (P < 0.05) between the preoperative and postoperative dysphagia, chest pain, and regurgitation symptom scores. All patients stated that they were improved postoperatively. Eighty-six per cent rated their outcome as excellent, 10 per cent as good, and 4 per cent as fair. Laparoscopic anterior esophageal myotomy and Toupet fundoplication effectively alleviates dysphagia, regurgitation, and chest pain accompanying achalasia and is associated with high patient satisfaction, a rapid hospital discharge, and few complications.

Mechanisms of nuclear import and export that control the subcellular localization of class II transactivator.

The presence of the class II transactivator (CIITA) activates the transcription of all MHC class II genes. Previously, we reported that deletion of a carboxyl-terminal nuclear localization signal (NLS) results in the cytoplasmic localization of CIITA and one form of the type II bare lymphocyte syndrome. However, further sequential carboxyl-terminal deletions of CIITA resulted in mutant forms of the protein that localized predominantly to the nucleus, suggesting the presence of one or more additional NLS in the remaining sequence. We identified a 10-aa motif at residues 405-414 of CIITA that contains strong residue similarity to the classical SV40 NLS. Deletion of this region results in cytoplasmic localization of CIITA and loss of transactivation activity, both of which can be rescued by replacement with the SV40 NLS. Fusion of this sequence to a heterologous protein results in its nuclear translocation, confirming the identification of a NLS. In addition to nuclear localization sequences, CIITA is also controlled by nuclear export. Leptomycin B, an inhibitor of export, blocked the nuclear to cytoplasmic translocation of CIITA; however, leptomycin did not alter the localization of the NLS mutant, indicating that this region mediates only the rate of import and does not affect CIITA export. Several candidate nuclear export sequences were also found in CIITA and one affected the export of a heterologous protein. In summary, we have demonstrated that CIITA localization is balanced between the cytoplasm and nucleus due to the presence of NLS and nuclear export signal sequences in the CIITA protein.

CD45 function is regulated by an acidic 19-amino acid insert in domain II that serves as a binding and phosphoacceptor site for casein kinase 2.

In this study experiments were conducted to elucidate the physical/functional relationship between CD45 and casein kinase 2 (CK2). Immunoprecipitation experiments demonstrated that CK2 associates with CD45 and that this interaction is inducible upon Ag receptor cross-linking in B and T cell lines as well as murine thymocytes and splenic B cells. However, yeast two-hybrid analysis failed to demonstrate a physical interaction between the individual CK2 alpha, alpha', or beta subunits and CD45. In contrast, a yeast three-hybrid assay in which either CK2 alpha and beta or alpha' and beta subunits were coexpressed with the cytoplasmic domain of CD45, demonstrated that both CK2 subunits are necessary for the interaction with CD45. Experiments using the yeast three-hybrid assay also revealed that a 19-aa acidic insert in domain II of CD45 mediates the physical interaction between CK2 and CD45. Structure/function experiments in which wild-type or mutant CD45RA and CD45RO isoforms were expressed in CD45-deficient Jurkat cells revealed that the 19-aa insert is important for optimal CD45 function. The ability of both CD45RA and CD45RO to reconstitute CD3-mediated signaling based on measurement of calcium mobilization and mitogen-activated protein kinase activation was significantly decreased by deletion of the 19-aa insert. Mutation of four serine residues within the 19-aa insert to alanine affected CD45 function to a similar extent compared with that of the deletion mutants. These findings support the hypothesis that a physical interaction between the CD45 cytoplasmic domain and CK2 is important for post-translational modification of CD45, which, in turn, regulates its catalytic function.

CD45 regulates tyrosine phosphorylation of CD22 and its association with the protein tyrosine phosphatase SHP-1.

Cross-linking of CD45 induced capping and physical sequestration from CD22 leading to an increase in tyrosine phosphorylation of CD22 and SHP-1 recruitment. Additionally, CD22 isolated from a CD45-deficient B cell line exhibited increased basal/inducible tyrosine phosphorylation and enhanced recruitment of SHP-1 compared with CD22 isolated from CD45-positive parental cells. Subsequent experiments were performed to determine whether enhanced SHP-1 recruitment to CD22 is responsible for attenuation of receptor-mediated Ca2+ responses in CD45-deficient cells. Catalytically inactive SHP-1 expressed in CD45-deficient cells interacted with CD22 and decreased phosphatase activity in CD22 immunoprecipitates to levels that were comparable to those in CD45-positive cells. Expression of catalytically inactive SHP-1 restored intracellular mobilization of Ca2+ in response to MHC class II cross-linking, but did not affect B cell Ag receptor- or class II-mediated Ca2+ influx from the extracellular space. These results indicate that CD45 regulates tyrosine phosphorylation of CD22 and binding of SHP-1. The data further indicate that enhanced recruitment and activation of SHP-1 in CD45-deficient cells affect intracellular mobilization of Ca2+, but are not responsible for abrogation of receptor-mediated Ca2+ influx from the extracellular space.

Major histocompatibility class II-mediated signal transduction is regulated by the protein-tyrosine phosphatase CD45.

Major histocompatibility complex class II molecules and the B cell antigen receptor (BCR) transduce similar signals when cross-linked by ligand. Therefore, studies were conducted to determine whether the protein tyrosine phosphatase CD45 regulates signaling via these transmembrane receptors in an analogous manner. Cross-linking of either class II molecules or the BCR on CD45-positive K46-17micromlambda B lymphoma cells was observed to induce activation of the Src family protein- tyrosine kinase Lyn, tyrosine phosphorylation of Syk and phospholipase Cgamma, and the production of inositol 1,4,5-trisphosphate leading to intracellular mobilization as well as extracellular influx of Ca2+. In the absence of CD45, cross-linking of either class II molecules or the BCR failed to induce activation of Lyn. Syk was inducibly phosphorylated on tyrosine in a normal manner, whereas phospholipase Cgamma exhibited a high basal level of tyrosine phosphorylation that was not significantly increased upon stimulation. Nevertheless, phospholipase Cgamma appeared to be functional because CD45-negative cells produced elevated levels of inositol 1,4,5-trisphosphate following stimulation through class II or the BCR. Regardless of this, CD45-negative cells exhibited Ca2+ mobilization responses that were greatly diminished and transient in nature. Whereas little or no mobilization of Ca2+ was observed in response to class II cross-linking, CD45-deficient cells mobilized Ca2+ from intracellular stores but not the extracellular environment in response to BCR cross-linking. These results demonstrate that CD45 regulates both Src family kinase activation and Ca2+ mobilization associated with class II- and BCR-mediated signal transduction.