The N-terminal domain of human GABA receptor rho1 subunits contains signals for homooligomeric and heterooligomeric interaction

gamma-Aminobutyric acid type C (GABAC) receptors identified in retina appear to be composed of GABA rho subunits. The purpose of this study was to localize signals for homooligomeric assembly of rho1 subunits and to investigate whether the same region contained signals for heterooligomeric interaction with rho2 subunits. In vitro translated human rho1 was shown to be membrane-associated, and proteinase K susceptibility studies indicated that the N terminus was oriented in the lumen of ER-derived microsomal vesicles. This orientation suggested the involvement of the N terminus of rho1 in the initial steps of subunit assembly. To test this hypothesis, mutants were created containing only N-terminal sequences (N-rho1) or C-terminal sequences (C-rho1) of rho1. Co-immunoprecipitation studies revealed that N-rho1, but not C-rho1, interacted with rho1 in vitro. When coexpressed in Xenopus oocytes, N-rho1 interfered with rho1 receptor formation. Together, these data suggested that signals for rho1 homooligomeric assembly reside in the N-terminal half of the subunit. Sequential immunoprecipitations were then performed upon cotranslated rho1 and rho2 subunits which demonstrated that rho1 and rho2 interacted in vitro. Co-immunoprecipitation indicated that N-rho1 specifically associated with rho2. Therefore, the N-terminal regions of rho subunits contain the initial signals for both homooligomeric and heterooligomeric assembly into receptors with GABAC properties.     

Autologous lymphokine-activated killer cell therapy of Epstein-Barr virus-positive and -negative lymphoproliferative disorders arising in organ transplant recipients

Lymphoreticular malignancies, collectively called posttransplant lymphoproliferative disorders (PTLD), eventually develop in 2-5% of organ transplant recipients. They frequently undergo regression when immunosuppression is reduced or stopped. This feature has been associated with a previous or de novo Epstein-Barr virus (EBV) infection. We herein describe immunotherapy with autologous lymphokine-activated killer (LAK) cells in seven patients with PTLD (four EBV-positive patients and three EBV-negative patients). Autologous peripheral blood mononuclear cells were obtained by leukapheresis, depleted of monocytes, and cultured in the presence of interleukin 2 for 10 to 11 days. A single dose of 5.2 x 10(9) to 5.6 x 10(10) LAK cells was given intravenously. Systemic interleukin 2 was not administered. The four patients with EBV+ PTLD had complete tumor regression; two of them developed controllable rejection. Three patients are well 13-16 months after treatment; the fourth patient died of pneumonia 41 days after infusion. Three patients with EBV- lymphomas had no response despite prior evidence that their tumors also were subject to immune surveillance. Two of these three patients died after being given other treatment, and the third patient has persistent tumor. In conclusion, autologous LAK cell infusion was effective for treatment of four EBV+ organ transplant recipients. LAK cell efficacy for three patients with EBV- PTLD was not evaluable under the management circumstances in which this treatment was utilized.     

Novel high-performance liquid chromatographic assay using fluorescence detection for the quantitation of plasma gamma-methylene-10-deazaaminopterin and its major metabolite, 7-hydroxy-gamma-methylene-10-deazaaminopterin, in patients with solid cancers in a phase I trial

Gamma-methylene-10-deazaaminopterin (MDAM), a unique dihydrofolate reductase inhibitor, has demonstrated antitumor activity against a broad spectrum of human solid tumors in preclinical studies. A novel reversed-phase, ion-pair high-performance liquid chromatography (HPLC) assay that uses fluorescence detection has been developed to quantitate levels of MDAM and its major metabolite, 7-hydroxy-gamma-methylene-10-deazaaminopterin (7-OH-MDAM), in human plasma. The recovery of MDAM and 7-OH-MDAM from plasma was >97% by a simple one-step deproteinization process using tetrabutylammonium bromide (TBABr) and methanol. MDAM and 7-OH-MDAM remained stable in plasma over a 28-day test period at ambient temperatures, and neither compound was light-sensitive. The limit of quantitation was 0.005 microM for both MDAM and 7-OH-MDAM. This assay has been found to be simple, sensitive and reproducible in determining plasma concentrations of MDAM and 7-OH-MDAM in patients with solid cancers in a phase I trial.     

Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent tissues

A sensitive method was developed and applied to examine the distribution of K-ras gene mutations in histologically differing areas of lung tissues obtained from lung cancer patients. This method, which combines polymerase chain reaction (PCR), mutation allele enrichment (MAE), and denaturing gradient gel electrophoresis (DGGE), allows detection of one K-ras mutant allele present in 10(4) to 10(5) wild-type alleles. It was applied to analyze mutations in codon 12 of the K-ras gene in 43 tissue sites microdissected from paraffin-embedded sections obtained from 8 archival cases of lung cancer, all previously shown to have codon 12 K-ras mutations by direct sequencing. In four cases, mutations were detected only in the tumor, while in the other four cases, the same mutations were also found in tissues adjacent to tumors, using the MAE + DGGE method. No mutations were detected among normal-appearing cells in areas distant from the tumors in any of the cases studied. These findings demonstrate that K-ras mutations can be detected at low frequencies in normal-appearing cells from tissues adjacent to the tumor in some lung cancer cases. In addition, this approach also allowed detection of multiple mutations in colorectal tissues obtained from colorectal cancer patients. Thus, the MAE + DGGE method may be applicable to study of K-ras mutations in premalignant or morphologically suspicious lesions in bronchial mucosa or other types of human cancer.     

Heart rate variability in evaluation of the autonomic nervous system. A review

Analysis of heart rate variability is increasingly used for testing the function of the autonomic nervous system in cardiovascular disease and for the diagnosis of autonomic neuropathy. In cardiovascular disease, long-term data collection (several hours) is primarily used to describe the static activity in the different branches of the autonomic nervous system. In the diagnosis of autonomic neuropathy, short-term forced variations in heart rate are employed in order to describe the dynamic capacity of the parasympathetic nervous system. In the subsequent data-analysis several different principles may be used, and the purpose of this review is to describe these principles and their use in clinical and experimental settings.     

Concurrent primary carcinoma of the gallbladder and acute cholecystitis

BACKGROUND/AIMS: Primary carcinoma of the gallbladder is rare and associated with a late diagnosis and poor prognosis. Concurrent acute cholecystitis frequently obscures the presence of carcinoma. The information regarding gallbladder carcinoma with acute cholecystitis is limited. In order to better understand the presentation of gallbladder carcinoma with acute cholecystitis, we retrospectively reviewed the data of patients with primary carcinoma of the gallbladder. METHODOLOGY: The data of 86 patients with primary carcinoma of the gallbladder treated between 1979 and 1994 were compiled and reviewed. The patients were divided into 2 groups: Group 1 (with acute cholecystitis, 21 patients) and Group 2 (without cholecystitis, 65 patients). Clinicopathological comparisons were made and evaluated between these two groups RESULTS: The average age of Group 1 patients was older than that of Group 2 patients (75+/-2 years vs. 63+/-2 years; p<0.05). Three Group 1 patients presented with sepsis. The interval between the onset of symptoms and hospital admission in Group 2 patients was significantly (p<0.05) longer than that in Group 1 patients (243+/-95 days vs. 20+/-11 days). Leukocytosis (>11,000/mm3) was more common in Group 1 patients than in Group 2 patients (47.6% vs. 15.4%). Jaundice was more common in Group 2, and fever was common in Group 1. The majority of Group 2 gallbladder cancers were stage V (75.4%). In contrast, 52.4% of Group 1 gallbladder cancers were stage III and 38.1% were stage V. The 30-day postoperative mortality rate in Group 1 and Group 2 patients was 9.5% and 7.7%, respectively. The cumulative survival of Group 1 patients was not different from that of Group 2 patients (log-rank test, p>0.05). CONCLUSIONS: Age, the interval of symptoms prior to admission, the location of abdominal pain, fever, leukocytosis, and the absence of jaundice suggested the presence of acute cholecystitis in gallbladder carcinoma. A high index of suspicion of the disease, intraoperative examination of gallbladder specimens, and more aggressive surgical treatment may improve patient survival.