The Ras superfamilies: regulatory proteins and post-translational modifications.

The Ras-like GTP-binding proteins comprise a large superfamily of proteins that play key roles in a wide variety of cellular activities, including cell growth, differentiation, secretion, and protein trafficking. During the past few years, it has become clear that these GTP-binding proteins are regulated by a variety of manners, including interactions with specific types of regulatory proteins and post-translational modification events.

IQGAP1, a Rac- and Cdc42-binding protein, directly binds and cross-links microfilaments.

Activated forms of the GTPases, Rac and Cdc42, are known to stimulate formation of microfilament-rich lamellipodia and filopodia, respectively, but the underlying mechanisms have remained obscure. We now report the purification and characterization of a protein, IQGAP1, which is likely to mediate effects of these GTPases on microfilaments. Native IQGAP1 purified from bovine adrenal comprises two approximately 190-kD subunits per molecule plus substoichiometric calmodulin. Purified IQGAP1 bound directly to F-actin and cross-linked the actin filaments into irregular, interconnected bundles that exhibited gel-like properties. Exogenous calmodulin partially inhibited binding of IQGAP1 to F-actin, and was more effective in the absence, than in the presence of calcium. Immunofluorescence microscopy demonstrated cytochalasin D-sensitive colocalization of IQGAP1 with cortical microfilaments. These results, in conjunction with prior evidence that IQGAP1 binds directly to activated Rac and Cdc42, suggest that IQGAP1 serves as a direct molecular link between these GTPases and the actin cytoskeleton, and that the actin-binding activity of IQGAP1 is regulated by calmodulin.

A GDP dissociation inhibitor that serves as a GTPase inhibitor for the Ras-like protein CDC42Hs.

Members of the family of Ras-related guanosine triphosphate (GTP) binding proteins appear to take part in the regulation of a number of biological processes, including cell growth and differentiation. Three different classes of proteins that regulate the GTP binding and GTP hydrolytic activities of the Ras family members have been identified. These different regulatory proteins inhibit guanosine diphosphate (GDP) dissociation (designated as GDIs), stimulate GDP dissociation and GDP-GTP exchange (designated as GDSs), or stimulate GTP hydrolysis (designated as GAPs). In the case of the Ras-like protein CDC42Hs, which is the human homolog of a Saccharomyces cerevisiae cell division cycle protein, the GDI protein also inhibited both the intrinsic and GAP-stimulated hydrolysis of GTP. These findings establish an additional role for the GDI protein--namely, as a guanosine triphosphatase (GTPase) inhibitory protein for a Ras-like GTP binding protein.

Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13.

Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho control cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide-binding proteins (G proteins) participate in several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein alpha subunits, Galpha12 and Galpha13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Galpha13 bound tightly to p115 RhoGEF and stimulated its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Galpha12 inhibited stimulation by Galpha13. Thus, p115 RhoGEF can directly link heterotrimeric G protein alpha subunits to regulation of Rho.

p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13.

Members of the regulators of G protein signaling (RGS) family stimulate the intrinsic guanosine triphosphatase (GTPase) activity of the alpha subunits of certain heterotrimeric guanine nucleotide-binding proteins (G proteins). The guanine nucleotide exchange factor (GEF) for Rho, p115 RhoGEF, has an amino-terminal region with similarity to RGS proteins. Recombinant p115 RhoGEF and a fusion protein containing the amino terminus of p115 had specific activity as GTPase activating proteins toward the alpha subunits of the G proteins G12 and G13, but not toward members of the Gs, Gi, or Gq subfamilies of Galpha proteins. This GEF may act as an intermediary in the regulation of Rho proteins by G13 and G12.

Downregulation of beta-catenin by human Axin and its association with the APC tumor suppressor, beta-catenin and GSK3 beta.

BACKGROUND: Inactivation of the adenomatous polyposis coli (APC) tumor suppressor protein is responsible for both inherited and sporadic forms of colon cancer. Growth control by APC may relate to its ability to downregulate beta-catenin post-translationally. In cancer, mutations in APC ablate its ability to regulate beta-catenin, and mutations in beta-catenin prevent its downregulation by wild-type APC. Moreover, signaling by the protein product of the wnt-1 proto-oncogene upregulates beta-catenin and promotes tumorigenesis in mice. In a Xenopus developmental system, Wnt-1 signaling was inhibited by Axin, the product of the murine fused gene. This suggests a possible link between Axin, the Wnt-1 signaling components beta-catenin and glycogen synthase kinase 3 beta (GSK3 beta), and APC. RESULTS: Human Axin (hAxin) binds directly to beta-catenin, GSK3 beta, and APC in vitro, and the endogenous proteins are found in a complex in cells. Binding sites for Axin were mapped to a region of APC that is typically deleted due to cancer-associated mutations in the APC gene. Overexpression of hAxin strongly promoted the downregulation of wild-type beta-catenin in colon cancer cells, whereas mutant oncogenic beta-catenin was unaffected. The downregulation was increased by deletion of the APC-binding domain from Axin, suggesting that APC may function to derepress Axin activity. In addition, hAxin dramatically facilitated the phosphorylation of APC and beta-catenin by GSK3 beta in vitro. CONCLUSIONS: Axin acts as a scaffold upon which APC, beta-catenin and GSK3 beta assemble to coordinate the regulation of beta-catenin signaling.

Functional interaction between the cytoplasmic leucine-zipper domain of HIV-1 gp41 and p115-RhoGEF.

The long cytoplasmic tail of the human immunodeficiency virus (HIV)-1 transmembrane protein gp41 (gp41C) is implicated in the replication and cytopathicity of HIV-1 [1]. Little is known about the specific functions of gp41C, however. HIV-1 or simian immunodeficiency virus (SIV) mutants with defective gp41C have cell-type- or species-dependent phenotypes [2] [3] [4] [5] [6]. Thus, host factors are implicated in mediating the functions of gp41C. We report here that gp41C interacted with the carboxy-terminal regulatory domain of p115-RhoGEF [7], a specific guanine nucleotide exchange factor (GEF) and activator of the RhoA GTPase, which regulates actin stress fiber formation, activation of serum response factor (SRF) and cell proliferation [8] [9]. We demonstrate that gp41C inhibited p115-mediated actin stress fiber formation and activation of SRF. An amphipathic helix region with a leucine-zipper motif in gp41C is involved in its interaction with p115. Mutations in gp41C leading to loss of interaction with p115 impaired HIV-1 replication in human T cells. These findings suggest that an important function of gp41C is to modulate the activity of p115-RhoGEF and they thus reveal a new potential anti-HIV-1 target.

Inter-rater agreement on assessment of outcome within a trauma registry.

INTRODUCTION: To better evaluate the degree of ongoing disability in trauma patients, it has been recommended that trauma registries introduce routine long-term outcome measurement. One of the measures recommended for use is the Extended Glasgow Outcome Scale (GOS-E). However, few registries have adopted this measure and further research is required to determine its reliability with trauma populations. This study aimed to evaluate the inter-rater agreement of GOS-E scoring between an expert rater and trauma registry follow-up staff with a sample of detailed trauma case scenarios. METHODS: Sixteen trauma registry telephone interviewers participated in the study. They were provided with a written summary of 15 theoretical adult trauma cases covering a spectrum of disability and asked to rate each case using the structured GOS-E interview. Their ratings were compared with those of an expert rater in order to calculate the inter-rater agreement for each individual rater-expert rater pair. Agreement was reported as the percentage of agreement, the kappa statistic, and weighted kappa. A multi-rater kappa value was also calculated for agreement between the 16 raters. RESULTS: Across the 15 cases, the percentage of agreement between individual raters and the expert ranged from 63% to 100%. Across the 16 raters, the percentage of agreement with the expert rater ranged from 73-100% (mean=90%). Kappa values ranged from 0.65 to 1.00 across raters (mean=0.86) and weighted kappa values ranged from 0.73 to 1.00 (mean=0.89) The multi-rater kappa value was 0.78 (95% CI: 0.66, 0.89). CONCLUSIONS: Sixteen follow-up staff achieved 'substantial' to 'almost perfect' agreement with an expert rater using the GOS-E outcome measure to score 15 sample trauma cases. The results of this study lend support to the use of the GOS-E within trauma populations and highlight the importance of ongoing training where multiple raters are involved to ensure reliable outcome reporting. It is also recommended that the structured GOS-E interview guide be used to achieve better agreement between raters. Ensuring the reliability of trauma outcome scores will enable more accurate evaluation of patient outcomes, and ultimately, more targeted trauma care.

Selected positions of acyl chains are affected differently by antibody binding which results in decreased membrane fluidity.

We have studied the interaction between monoclonal anti-trinitrophenyl antibodies (IgGl and IgG2a) and haptenated phospholipid vesicles using stopped-flow fluorometry. Conformational changes of the antibodies were induced very rapidly (within 0.1 s) after binding to lipid haptens (TNP-Cap-DPPE) on the membrane surfaces. Conversely, after that, the bound antibody molecules decreased the degree of molecular motion at different depths in the bilayer, ranging from the polar head group to the terminal methyl groups of the fatty acyl chains. Such an effect reaches all places of the bilayer within 40 s at 25 degrees C.

Importance of surgical resection in the successful management of soft tissue sarcoma.

HYPOTHESIS: A systemic disease-free state necessitates a local disease-free state. This cannot be accomplished without a properly performed resection by an experienced surgical team. Successful local management of soft tissue sarcoma (STS) may lead to improved disease-free survival. An STS treatment protocol using wide local excision followed by radiation therapy is effective in achieving local tumor control and survival similar to that of multiple-modality regimens, but with lower morbidity. DESIGN: Retrospective cohort review (August 1, 1987, to May 6, 1998). SETTING: Referral to a single musculoskeletal oncologic surgeon, with surgery performed at a tertiary care medical center in a large urban area. PATIENTS: Ninety patients with STS of the trunk or extremities. INTERVENTIONS: Preoperative evaluation included surveillance computed tomographic scan of the chest, magnetic resonance imaging of primary site to assess tumor extent and to plan the surgical approach, and angiography if vascular bypass was proposed. Wide local excision of tumor was performed, with concomitant vascular bypass and/or complex plastic reconstruction as needed. Postoperative radiation therapy was given in most patients. Adjuvant chemotherapy was used selectively. MAIN OUTCOME MEASURES: Morbidity, local recurrence rates, and survival. RESULTS: Histologically negative margins were obtained in 89 (99%) of 90 patients; 86 (96%) remained free of local disease at follow-up. Five patients died of systemic metastatic disease. CONCLUSION: Excellent local control obtained with aggressive, appropriate surgery followed by radiation therapy in most patients and chemotherapy in only selective high-risk patients leads to excellent survival, with low morbidity and good functional outcome.

Primary sclerosing cholangitis.

Primary sclerosing cholangitis was seen in only 7 of 6,494 biliary operations performed between 1948 and 1979. Fifteen more cases were seen in 1,467 biliary operations performed over a recent 5 year period. Surgery was directed primarily towards the differential diagnosis between cholangiocarcinoma and primary sclerosing cholangitis of the upper bile duct and the need for decompression (nine patients), accompanying gallstones (4 patients), and focal distal bile duct stricture with a question of cholangiocarcinoma (two patients). Invasive radiologic dilatation of the bile ducts was not possible in most instances. Two patients have had successful liver transplants and two more were pending at last follow-up.

Cholangiography and small duct injury.

Eighteen instances of bile duct injury over the past 25 years have been reported. Manipulation of a minute cystic duct where the common bile duct was 3 mm in diameter or less was responsible for the division of the duct in eight cases. Most of the commercial catheters are sharp, about 2 mm in diameter, and can easily divide the bile duct if pushed too far. Since manipulation of the duct with a mosquito or tonsil clamp to insert other smaller catheters can also divide a small bile duct, we have decided to discontinue obtaining cholangiograms under these circumstances. Longitudinal splitting of two additional 3 mm ducts with a 3 mm dilator was another major source of injury. Overclipping of the cystic artery to involve the hepatic duct and overmobilization of the bile duct were other sources of injury. We have ceased attempting to obtain cholangiograms in patients with small cystic ducts because of the availability of endoscopic sphincterotomy, the low yield of the films under these circumstances, and fear of damaging the ducts. We use hepaticojejunostomy with Silastic tube stenting for repair of bile duct injuries.

Radioimmunoguided surgery for colorectal cancer.

BACKGROUND: Improving the survival rate of surgical treatments of cancer depends upon accurate staging of disease and the subsequent ability to completely resect the lesions. Radioimmunoguided surgery (RIGS) has been employed in the treatment of colon and rectal cancer, and a multicenter trial is under way. METHODS: Twenty-eight patients with either primary or recurrent colorectal cancer were injected with the monoclonal antibody CC49 labeled with iodine 125. A hand-held gamma-detecting probe was used intraoperatively to detect radiolabeled antibody. At surgery the patients underwent both traditional and RIGS explorations. RESULTS: RIGS detected radiolabeled antibody in 73% (14 of 19) of patients with primary colorectal cancer and in 100% (7 of 7) of patients with recurrent disease. Based on RIGS, 26% (5 of 19) of patients with primary cancer were upstaged from stage I/II to stage III/IV. CONCLUSIONS: Although the final results of this phase III trial are incomplete, RIGS appears to provide immediate, valuable information that is not available from more conventional methods.

Choledochal cysts presenting in late childhood and adulthood.

Primary excision is well accepted in children. Although the length of follow-up is inadequate, our review of the literature and experience with five patients suggests primary excision as the procedure of choice in older children and adults. Although the number of cases is too small and the follow-up too sporadic to draw statistically valid conclusions, excision has a lower morbidity, mortality and reoperation rate than internal cyst drainage, and definitive removal of tissue at risk for malignant degeneration seems intuitively more appealing.

Pancreaticojejunostomy. Report of a 25 year experience.

Surgical therapy for 88 patients operated on between 1958 and 1982 has been reviewed. Ninety-three operative procedures were performed including pancreaticojejunostomy in 56, pancreaticocystojejunostomy in 12, pancreaticojejunostomy with resection of less than 10 percent of the pancreas in 16, and pancreaticojejunostomy with resection of more than 50 percent of the pancreas in 9. Operative mortality was 7.5 percent and operative morbidity was 25 percent. Overall, 63 percent of the patients had an excellent or good result in the postoperative follow-up period which averaged 4 years. In the nonalcohol-induced pancreatitis group, ductal diameter was a good predictor of postoperative success, whereas in the alcoholic patient population, abstinence from further alcohol intake was a more accurate predictor of the success of pancreatic drainage.

Prospective evaluation of formalin therapy for radiation proctitis.

BACKGROUND: Radiation proctitis is a troublesome complication of radiation therapy for as many as 75% of patients after pelvic irradiation. Five percent progress to chronic radiation proctitis complicated by telangiectasias and hemorrhage. The utility of formalin rectal instillation for treatment of bleeding is prospectively evaluated in this study. METHODS: Eleven patients (9 male, 2 female) with rectal bleeding after pelvic irradiation were treated with formalin therapy. In a single treatment, 4% formalin was instilled into the rectum in four separate 20-cc aliquots with total mucosal contact time of approximately 15 minutes. Patients were initially evaluated at 7 to 10 days and 1 month postoperatively and assessed for bleeding. RESULTS: All patients presented with rectal bleeding. Twenty-seven percent required transfusion. Thirty-six percent had failed other previous therapy. In follow-up of 3 to 64 months, 100% had initial success with cessation of bleeding. Three patients had recurrent bleeding; none required transfusion. One patient required repeat formalin instillation, with no further bleeding at 3 months follow-up. CONCLUSION: Local rectal instillation of 4% formalin is an efficacious therapy for treatment of radiation-induced lower gastrointestinal bleeding.

Ampullary disconnection during the course of biliary and duodenal surgery.

Ampullary disconnection is fortunately rare. As this experience indicates, the long-term salvage rate is good but demands immediate recognition, precise repair and frequently reoperation to obtain a satisfactory result. One should be aware that the ampulla may be located very proximally in patients with long-standing peptic ulceration. The common duct should be catheterized while reoperation is required in this area in cases of obscured anatomy of the distal bile and pancreatic ducts.

A prospective trial of computed tomography and ultrasonography for diagnosing appendicitis in the atypical patient.

BACKGROUND: The surgical diagnosis of acute appendicitis is customarily made on clinical grounds alone using history, physical examination, and white blood cell count. In the atypical patient, ie, the patient with prolonged symptoms, inconsistent history, or misleading physical examination, diagnostic studies should be helpful in establishing the appropriate diagnosis. Computed tomography (CT) scan and ultrasonography (US) have demonstrated utility in diagnosing appendicitis but have not been studied in the atypical patient population. METHODS: A retrospective review of 500 consecutive appendectomy patients, coupled with a review of the literature, resulted in the development of an algorithm to help the medical physician to differentiate the atypical patient requiring an imaging study from those requiring immediate surgical consultation. This patient population was entered into a prospective, randomized study of CT scan or US examination. RESULTS: Between May 1997 and May 1999, 106 patients were enrolled in the study; 17 were later excluded from the study because of typical presentation and direct admission to surgery without diagnostic imaging. Forty-nine patients were randomly assigned to CT scan and 40 to US examination. CT scan was 100% specific and 97% sensitive (P = 0.018). US was 90% specific and 76% sensitive. Based on the results of these imaging studies, 70 of the study patients underwent exploration for suspected appendicitis or other acute inflammatory process. CONCLUSION: A subset of patients presenting with possible acute appendicitis has been identified that should benefit from imaging of the appendix prior to surgical consultation. For this group, CT scan appears superior to abdominal and pelvic US in terms of diagnostic accuracy and reliability. With this approach, a low incidence of negative laparotomies was achieved.

Potentially curable masses in the pancreas.

Twenty patients with intraabdominal non-Hodgkin's lymphoma localized to the peripancreatic area were reviewed. Appropriate diagnosis and staging required exploratory laparotomy and biopsy. With appropriate chemotherapy and radiotherapy, 40 percent of these patients were alive 3 years after diagnosis without evidence of disease. It is for this reason that we have adopted an aggressive approach in obtaining histologic diagnoses for all pancreatic masses.

Structure of the core oligosaccharide in the lipopolysaccharide isolated from Aeromonas salmonicida ssp. salmonicida.

The core oligosaccharide isolated from the lipopolysaccharide of Aeromonas salmonicida ssp. salmonicida has been investigated by methylation analysis, NMR spectroscopy (13C and 1H), oxidation with periodate and chromium trioxide, and Smith degradation. The following structure is proposed: [Formula: see text]