Expanding roles for beta-arrestins as scaffolds and adapters in GPCR signaling and trafficking.

beta-arrestins play previously unsuspected and important roles as adapters and scaffolds that localize signaling proteins to ligand-activated G-protein-coupled receptors. As with the paradigmatic role of the beta-arrestins in uncoupling receptors from G proteins (desensitization), these novel functions involve the interaction of beta-arrestin with phosphorylated heptahelical receptors. beta-arrestins interact with at least two main classes of signaling proteins. First, interaction with molecules such as clathrin, AP-2 and NSF directs the clathrin-mediated internalization of G-protein-coupled receptors. Second, interaction with molecules such as Src, Raf, Erk, ASK1 and JNK3 appears to regulate several pathways that result in the activation of MAP kinases. These recent discoveries indicate that the beta-arrestins play widespread roles as scaffolds and/or adapter molecules that organize a variety of complex signaling pathways emanating from heptahelical receptors. It is likely that additional roles for the beta-arrestins remain to be discovered.

Upper jurassic dinosaur egg from utah.

The Upper Jurassic egg described here is the first known egg from the 100-million-year gap in the fossil record between Lower Jurassic (South Africa) and upper Lower Cretaceous (Utah). The discovery of the egg, which was found mixed in with thousands of dinosaur bones rather than in a nest, the pathological multilayering of the eggshell as found in modern and fossil reptilians, and the pliable condition of the eggshell at the time of burial indicate an oviducal retention of the egg at the time of burial.

Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes.

The Ras-dependent activation of mitogen-activated protein (MAP) kinase pathways by many receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) requires the activation of Src family tyrosine kinases. Stimulation of beta2 adrenergic receptors resulted in the assembly of a protein complex containing activated c-Src and the receptor. Src recruitment was mediated by beta-arrestin, which functions as an adapter protein, binding both c-Src and the agonist-occupied receptor. beta-Arrestin 1 mutants, impaired either in c-Src binding or in the ability to target receptors to clathrin-coated pits, acted as dominant negative inhibitors of beta2 adrenergic receptor-mediated activation of the MAP kinases Erk1 and Erk2. These data suggest that beta-arrestin binding, which terminates receptor-G protein coupling, also initiates a second wave of signal transduction in which the "desensitized" receptor functions as a critical structural component of a mitogenic signaling complex.

Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3.

beta-Arrestins, originally discovered in the context of heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) desensitization, also function in internalization and signaling of these receptors. We identified c-Jun amino-terminal kinase 3 (JNK3) as a binding partner of beta-arrestin 2 using a yeast two-hybrid screen and by coimmunoprecipitation from mouse brain extracts or cotransfected COS-7 cells. The upstream JNK activators apoptosis signal-regulating kinase 1 (ASK1) and mitogen-activated protein kinase (MAPK) kinase 4 were also found in complex with beta-arrestin 2. Cellular transfection of beta-arrestin 2 caused cytosolic retention of JNK3 and enhanced JNK3 phosphorylation stimulated by ASK1. Moreover, stimulation of the angiotensin II type 1A receptor activated JNK3 and triggered the colocalization of beta-arrestin 2 and active JNK3 to intracellular vesicles. Thus, beta-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR.

Interaction of tumor necrosis factor receptor-associated factor signaling proteins with the latent membrane protein 1 PXQXT motif is essential for induction of epidermal growth factor receptor expression.

The Epstein-Barr virus latent membrane protein 1 (LMP1) oncoprotein causes multiple cellular changes, including induction of epidermal growth factor receptor (EGFR) expression and activation of the NF-kappaB transcription factor. LMP1 and the cellular protein CD40, which also induces EGFR expression, interact with the tumor necrosis factor receptor-associated factor (TRAF) proteins. The LMP1 carboxy-terminal activation region 1 signaling domain interacts specifically with the TRAFs and is essential for EGFR induction through a mechanism independent of NF-kappaB alone. LMP1 and CD40 share a common TRAF binding motif, PXQXT. In this study, the PXQXT motifs in both LMP1 and CD40 were altered and mutant proteins were analyzed for induction of EGFR expression. Replacement of the T residue with A in CD40 completely blocked induction of the EGFR, while the same mutation in LMP1 did not affect EGFR induction. Replacement of both P and Q residues with A's in LMP1 reduced EGFR induction by >75%, while deletion of PXQXT blocked EGFR induction. These results genetically link EGFR induction by LMP1 to the TRAF signaling pathway. Overexpression of TRAF2 potently activates NF-kappaB, although TRAF2 did not induce expression of the EGFR either alone or in combination with TRAF1 and TRAF3. In vivo analyses of the interaction of the TRAFs with LMP1 variants mutated in the PXQXT domain indicate that high-level induction of EGFR expression requires interaction with TRAF1, -2, and -3. However, exogenous expression of TRAF3 decreased EGFR induction mediated by either LMP1 or CD40. These data suggest that TRAF-mediated activation of EGFR expression requires assembly of a complex containing the appropriate stoichiometry of TRAF proteins clustered at the cell membrane with LMP1.

The NPC derived C15 LMP1 protein confers enhanced activation of NF-kappa B and induction of the EGFR in epithelial cells.

The Epstein-Barr Virus (EBV) LMP1 protein is frequently expressed in nasopharyngeal carcinoma and is essential for the transforming effects of EBV. Analysis of LMP1 genes isolated from tumor biopsies has revealed considerable sequence variation including deletion of amino acids 343-352. Several studies have suggested that this sequence variation could enhance the transforming potential of LMP1. LMP1 has profound effects on cellular gene expression mediated in part through activation of the NF-kappa B transcription factor. In addition, LMP1 engages the TRAF signaling pathway resulting in the induction of EGFR expression. In this study, the LMP1 proteins derived from the laboratory strain B95-8 and the NPC strain C15 were analysed for their ability to activate NF-kappa B and also to induce expression of the EGFR. The data suggest that the C15-LMP1 protein activates NF-kappa B more efficiently and induces higher levels of the EGFR. Analysis of chimeric LMP1 proteins indicates that the amino terminal 181 amino acids of C15-LMP1 confer this increased signaling capability, and that deletion of amino acids 343-352 does not affect these properties. Finally, these data provide evidence that five amino acid changes within the transmembrane domain in the C15-LMP1 protein lead to enhanced NF-kappa B activation and EGFR induction.

The EGFR as a target for viral oncoproteins.

The epidermal growth factor receptor (EGFR) is a potent stimulator of the mitogen-activated protein kinase (MAPK) signaling pathway. Chronic stimulation of the EGFR and of multiple steps in the MAPK signaling pathway is involved in the development of cancer. Several tumor viruses encode proteins that induce EGFR expression or stimulate EGFR-mediated signaling and are thus likely to play an important role in the transformation of virus-infected cells.

Pharmacokinetic study of oral and bolus intravenous 2-chlorodeoxyadenosine in patients with malignancy.

PURPOSE: This study was designed to evaluate the absolute bioavailability (F value) of 2-chlorodeoxyadenosine (cladribine; 2-CdA) after multiple oral administrations, and the intersubject variability after oral and 2-hour intravenous (IV) administration schedules in patients with malignancy. PATIENTS AND METHODS: Patients with advanced malignancies were eligible. There were two treatment cycles; during cycle 1, patients received 2-CdA solution at 0.28 mg/kg/d orally under fasting conditions for 5 consecutive days concomitantly with omeprazole, and 4 weeks later during cycle 2 patients received 2-CdA as a 2-hour IV infusion of 0.14 mg/kg/d for 5 consecutive days. Serial blood samples for 2-CdA plasma levels were obtained after drug administrations on days 1 and 5 during each treatment cycle. RESULTS: Ten patients completed cycles 1 and 2. The F value of oral 2-CdA measured on days 1 and 5 was 37.2% and 36.7%, respectively. For both oral and IV multiple administrations, there was no significant accumulation in maximum concentration (Cmax), and the intersubject variabilities (coefficient of variation [CV], approximately 40%) in Cmax and area under the concentration-time curve from 0 to 24 hours [AUC(0-24)] values were comparable for both routes on days 1 and 5. A three-compartment open model was applied to the plasma concentration data after oral and IV administrations and resulted in good agreement between observed and simulated concentration-time profiles. Neutropenia was the principal adverse event observed when 2-CdA was administered orally and IV. CONCLUSION: The F value of 2-CdA after oral administration was approximately 37% and there were no cumulative differences in bioavailability observed on multiple dosing of the drug. The absorption and disposition characteristics of oral 2-CdA were linear and predictable with this dosing regimen.

Outpatient high-dose chemotherapy with autologous stem-cell rescue for hematologic and nonhematologic malignancies.

PURPOSE: A prospective study to determine the feasibility of high-dose chemotherapy (HDC) and autologous stem-cell rescue (ASCR) in the outpatient setting. METHODS: One hundred thirteen consecutive patients underwent 165 cycles of HDC/ASCR for a variety of malignancies. HDC regimens were disease-specific. Initially, patients were hospitalized for HDC, discharged on completion, and maintained as outpatients unless toxicities required rehospitalization (subtotal outpatient transplantation [STOT]). Once this was established as safe, a total outpatient transplant (TOT) program was developed in which patients received all of the HDC, as well as supportive care, as outpatients. Patients who declined the outpatient programs received the same HDC and supportive care as inpatients. RESULTS: In 140 of 165 (85%) HDC cycles, patients agreed to participate in one of the outpatient transplant programs. Five patients in the STOT program could not be discharged from the hospital because of toxicities that developed during HDC; thus, 135 patients were monitored the outpatient setting, 95 (70%) of whom were never readmitted. The mean +/- SEM total hospital length of stay (LOS), including all readmissions and excess days after chemotherapy, was 18.33 +/- 5.06 days for patients who refused the outpatient program, 8.22 +/- 5.76 days for patients in the STOT program, and 2.81 +/- 7.66 days for those in the TOT program (P < .001). One treatment-related death occurred in each treatment setting: day 120 inpatient, day 17 STOT, and day 110 TOT. CONCLUSION: Outpatient management of HDC/ASCR is safe and acceptable for the vast majority of patients. The STOT program resulted in significant reduction in hospital LOS, while the TOT program appears equally safe and further reduces LOS. Hospitalization for HDC/ASCR is unnecessary in most patients.

Arrestins as signaling molecules involved in apoptotic pathways: a real eye opener.

Recent data suggest that internalized receptor and arrestin complexes are actively involved in signal transduction. Miller and Lefkowitz discuss evidence from the Drosophila visual system that suggests that intracellular rhodopsin and arrestin2 complexes induce apoptosis. Experiments with activated mammalian G protein-coupled receptor and arrestin complexes point to a mechanism by which proliferative or proapoptotic signals can be mediated largely independent from G protein activation.

Primary inferior vena cava thrombosis: report of nine cases.

All cases of inferior vena cava obstruction diagnosed at the Mayo Clinic between 1950 and 1973 were reviewed. Diagnosis was confirmed by surgery, phlebography, or postmortem examinations in 64 cases; the cause in 55 cases is described. Carcinoma of the kidney was the most common cause (31% pf cases). In nine cases, extensive laboratory investigation failed to reveal the cause of the process. These cases were considered to be primary inferior vena cava thrombosis and are reviewed in detail.

Spontaneous remission of Cushing's syndrome in a patient with an adrenal adenoma.

A 23-yr-old male student presented with clinical and biochemical evidence of Cushing's syndrome. One month later, his elevated plasma and urinary adrenal steroids had returned to normal. At surgery, an adrenal adenoma was removed from his right side. We postulate that he either underwent a temporary spontaneous remission of his disease without treatment, prior to surgery, or that his adenoma secreted glucocorticoids in a cyclical fashion.

Nadolol versus diltiazem and combination for preventing exercise-induced ischemia in severe angina pectoris.

Combination beta-blocker and calcium antagonist therapy has been shown to be superior to monotherapy with either class of drugs in the treatment of patients with severe angina pectoris. Some combinations of these agents have resulted in an increased frequency of adverse effects. Although nadolol and diltiazem have low incidences of side effects as monotherapy, little is known about their combination. Thus, 18 patients with angina pectoris despite medical therapy were randomly assigned to 3-week periods of nadolol (160 mg/day), diltiazem (240 mg/day) or their combination. At the end of each treatment period, treadmill exercise testing and rest and peak bicycle exercise 2-dimensional echocardiography were performed. The heart rate-systolic blood pressure product was decreased most at peak treadmill exercise with the combination therapy versus monotherapy with either nadolol or diltiazem (12 vs 14 vs 22 x 10(3), respectively, p less than 0.05). Exercise duration did not differ with any of the 3 regimens, but the number of patients without angina during exercise was lowest with the combination therapy versus nadolol or diltiazem alone (5, 10 and 11, respectively, p less than 0.05); similar results were noted with the number of patients developing 1-mm ST depression on the exercise electrocardiogram (6, 10 and 13, respectively, p less than 0.05). The left ventricular ejection fraction at rest and during peak exercise was similar among the 3 treatments. The therapeutic combination of nadolol and diltiazem is well tolerated and results in less evidence of myocardial ischemia during exercise than monotherapy with either agent.

Transthoracic needle biopsy: accuracy and complications in relation to location and type of lesion.

Transthoracic needle biopsy has become a frequently used method for obtaining tissue for diagnosis of pulmonary lesions. The procedure carries an inherent risk of pneumothorax and hemorrhage, the latter usually manifested by transient hemoptysis. Data on 430 patients who underwent transthoracic needle aspirations from 1968 through 1977 were studied to determine accuracy and complications of this procedure as related to lesion type and location. Malilgnant disease, most often metastatic, was present in all but 10 patients. Also, 86% of patients were more than 50 years of age and most were not candidates for operation. Sufficient tissue for diagnosis was obtained in 82% of cases. The diagnostic yield diminished significantly in central lesions less than 2 cm in size. Pneumothorax and hemoptysis or pulmonary hemorrhage were the most common complications. Biopsy of central lesions, especially those in the mediastinum, were more often associated with pneumothorax, whereas lesions near the hilar region were more susceptible to hemorrhagic complications. Two deaths occurred, both from hemorrhage after biopsy in cavitating lesions with air/fluid levels. The incidence of pneumothorax was also higher in cavitating lesions. Fewer complications occurred with biopsy of lesions along the pleural surface and lesions in the periphery of the lung.

Prolonged, complete remission after 2-chlorodeoxyadenosine therapy in a patient with refractory essential mixed cryoglobulinemia.

Essential mixed cryoglobulinemia is a systemic disorder in which cutaneous vasculitis and frequently glomerulonephritis are associated with cryoprecipitable serum immune complexes. Typically, the treatment regimen consists of plasmapheresis, high-dose corticosteroids, and cytotoxic chemotherapy, as well as interferon alfa for hepatitis C virus-related cryoglobulinemia. Herein we describe a man with progressive, symptomatic cryoglobulinemia and multisystem organ dysfunction in whom corticosteroid and alkylating therapy failed; however, he had a complete and long-lasting remission after administration of 2-chlorodeoxyadenosine (cladribine).

Percutaneous placement of the Greenfield vena caval filter.

During the period from August 1986 to August 1987, 50 patients underwent percutaneous placement of a Greenfield vena caval filter from the right femoral vein, left femoral vein, or right internal jugular vein at our institution. All 50 patients had a contraindication to anticoagulation therapy or had complications of anticoagulation for deep venous thrombosis or pulmonary emboli. The percutaneous placement was accomplished in the angiographic suite with use of local anesthesia and was well tolerated by all patients. Only three complications related to the percutaneous approach occurred during the short-term follow-up (3 months to 1 year). These complications were deep venous thrombosis of the leg in two patients and misplacement of the filter in one patient. The three patients tolerated these complications well. We conclude that placement of Greenfield vena caval filters can be readily accomplished by means of percutaneous entry. Our experience demonstrated minimal associated morbidity and no mortality.

Percutaneous transluminal angioplasty in the lower extremities: a 5-year experience.

From January 1979 to March 1984, percutaneous transluminal angioplasty (PTA) was used to treat 148 limbs of 135 Mayo Clinic patients with occlusive arterial disease of the lower extremities. The procedure was technically successful in more than 95% of the attempts. The outcome was clinical improvement in 89 limbs and no improvement in 40 limbs; in 19 limbs, PTA was technically successful but the patient was dismissed from the hospital and lost to follow-up before the extent of improvement could be determined. Mean ankle/brachial pressure indices increased after PTA in those with clinical improvement but not in those without improvement. Clinical improvement was less likely to follow PTA in patients with advanced age, diabetes, severe initial symptoms, low ankle/brachial indices, or distal occlusive disease. In patients with improvement after PTA, the mean follow-up period was 33 months; during that time, failure (defined as recurrence of the original symptoms or the need for repeat PTA or operation) occurred at a rate of 6.4% per year. Serious complications occurred after three procedures (2.0%). We conclude that PTA is technically feasible and generally safe for many patients with occlusive arterial disease of the lower limbs.

The Mayo Lung Project for early detection and localization of bronchogenic carcinoma: a status report.

The Mayo Lung Project (MLP) is a screening program designed to detect bronchogenic carcinoma at a curable stage. Screening tests include chest roentgenograms, three-day "pooled" sputum cytology studies, and lung-health questionnaires. These are being applied every four months to a study population of outpatients who have a high probability of developing lung cancer. Initial patient acceptance of the screening program has been excellent. Small asymptomatic lung cancers have been detected both roentgenographically and cytologically. The two procedures have complemented each other with little overlap. Chest roentgenography has proved most useful in diagnosing peripherally situated cancers, whereas sputum cytology studies have been most effective in identifying early squamous cancer involving major airways. At present, more cancers have been detected roentgenographically than cytologically, but the cytologically detected cases appear to have a better prognosis. Roentgenographically occult cancers have been localized with regularity, although the localization process is complicated. Theoretically, vigorous application of radiologic and cytologic screening, combined with optimum use of localizing procedures and treatment, could increase the five-year survival rate among lung cancer patients to nearly 50 percent. However, the actual survivorship attained will ultimately be determined by currently imponderable factors such as patient acceptance of longterm screening, frequency of multicentric respiratory cancers, and incidence of noncancerous smoking-related diseases, especially chronic obstructive pulmonary disease and ischemic heart disease.

Mobilized peripheral blood progenitor cells (PBPC) in support of tandem cycles of high-dose chemotherapy (HDC).

We evaluated the efficacy, toxicity and feasibility of two cycles of high-dose chemotherapy (HDC), each supported with mobilized peripheral blood progenitor cells (PBPC). Ninety-six patients with metastatic or high-risk cancers received disease-specific HDC regimens. The first cycle consisted of cyclophosphamide 6000 mg/m2, thiotepa 500 mg/m2 and carboplatin 1200 mg/m2. Alternatively, some patients received etoposide 1800 mg/m2 substituted for thiotepa. A second cycle was planned 6-8 weeks later and consisted of mitoxantrone 60 mg/m2 with either melphalan 140 mg/m2 or thiotepa 600 mg/m2. PBPC were mobilized with either growth factor alone or cyclophosphamide followed by growth factor(s). Thirty-four of 96 enrolled patients (35%) did not receive the second cycle. The reasons were: patient refusal (15); insurance refusal (three); toxicities of cycle 1 (seven); no response to cycle 1 (four); and inadequate mobilization or poor engraftment, (five). Of the 33 patients who entered cycle 2 with measurable disease, 28 demonstrated further response after cycle 2 (including 10 who entered CR). One patient died of toxicity after each cycle. Hematologic recovery was rapid and complete in most patients. Tandem cycles of high-dose chemotherapy supported by PBPC are feasible and safe, although many patients fail to receive the second treatment. Preliminary evaluation shows evidence of further antitumor efficacy following cycle 2.

Ciprofloxacin-associated acute renal failure in patients undergoing high-dose chemotherapy and autologous stem cell rescue.

The broad spectrum of activity of ciprofloxacin makes it an ideal drug for the prophylaxis of bacterial infections in patients undergoing high-dose chemotherapy (HDC) with autologous stem cell rescue. We present two cases of ciprofloxacin-associated acute renal failure (ARF) in patients undergoing HDC. Maintaining a high index of suspicion for this complication will allow a prompt diagnosis, with discontinuation of the drug usually resulting in a reversal of renal failure. Renal biopsy usually reveals changes compatible with interstitial nephritis, but is not always possible in these patients due to severe thrombocytopenia following HDC. A brief course of steroid therapy may be beneficial although the role of glucocorticoids is difficult to ascertain in the absence of data regarding its efficiency in this clinical setting.