The role of N-glycosylation for functional expression of the human platelet-activating factor receptor. Glycosylation is required for efficient membrane trafficking

Streptococcus pneumoniae has been shown to utilize the platelet activating factor receptor for binding and invasion of host cells (Cundell, D. R., Gerard, N. P., Gerard, C., Idanpaan-Heikkila, I., and Tuomanen, E. I. (1995) Nature, in press). Because bacterial binding is in part carbohydrate dependent, and the human platelet-activating factor (PAF) receptor bears a single N-linked glycosylation sequence in the second extracellular loop, we undertook studies to determine the role of this epitope in PAF receptor function. Binding of pneumococci to COS cells transfected with the human PAF receptor is greatly reduced for a receptor mutant that bears no N-linked glycosylation site. Immunohistochemical and binding analyses show decreased expression of the non-glycosylated molecule on the cell membrane relative to the wild type receptor; however, metabolic labeling and immunopurification indicate it is synthesized intracellularly at a level similar to the native molecule. A mutant receptor encoding a functional glycosylation site at the NH2 terminus is better expressed at the cell surface compared with the non-glycosylated form, indicating that trafficking to the cell surface is facilitated by glycosylation, but its location is relatively unimportant. The binding affinity for PAF is not significantly effected by the presence or location of the carbohydrate, and variations in cell surface expression have little influence on signal transduction, as the non-glycosylated PAF receptor is equally effective for activation of phospholipase C as the native molecule. These data are supportive of pneumococcal binding on protein moiety(ies) of the PAF receptor and indicate that N-glycosylation facilitates expression of the protein on the cell membrane.     

Aristolochic acids, aristolactam alkaloids and amides from Aristolochia kankauensis

Fourteen aristolochic acid derivatives: ariskanins A-E, 3-hydroxy-4-methoxy-10-nitrophenanthrene-1-carboxylic acid methyl ester, aristolochic acid-II methyl ester, aristolochic acid-IV methyl ester, aristolochic acid-C, -I, -II, -III, -IV, -IVa; nine aristolactam alkaloids: aristolactam, aristolactam-AII, -BIII, -AIII, -III, cepharanone-A, 9-methoxy-aristolactam-I, aristolactam-N-beta-D-glucoside and aristolactam-C-N-beta-D-glucoside; a 4,5-dioxoaporphine alkaloid: cepharadione-A, together with 12 other compounds: methyl-p-coumarate, N-trans-feruloyltyramine, N-cis-feruloyltyramine, methyl vanillate, methyl paraben, allantoin, an octadecyl- and eicosyl ferulate mixture, cis- and trans-p-coumaric acid, N-p-coumaroyltyramine, N-p-cis-coumaroyltyramine, methyl ferulate and isorhamnetin-3-O-rutinoside were isolated and characterized from the fresh root and stem of Aristolochia kankauensis. Their structures were elucidated by spectral and chemical methods. Among them, ariskanins-A-E and N-p-cis-coumaroyltyramine are reported for the first time from a natural source. The cytotoxicity and antiplatelet activity of the compounds isolated are also discussed.     

Dosimetric analysis of chimeric monoclonal antibody cMOv18 IgG in ovarian carcinoma patients after intraperitoneal and intravenous administration

In this study the potential of intraperitoneal (i.p.) and intravenous (i.v.) administration of chimeric iodine-131-labelled MOv18 IgG for radioimmunotherapy was determined. The dosimetry associated with both routes of administration of cMOv18 IgG was studied in patients. Eight patients suspected of having ovarian carcinoma received 150 MBq 131I-cMOv18 IgG i.p. Blood and urine were collected and serial gamma camera images were acquired. Another group of four patients received 7.5 MBq 131I-cMOv18 IgG i.v. For all patients, tissue biopsies were obtained at surgery. Activity in the blood after i.p. administration was described by a bi-exponential curve with a mean uptake and elimination half-life of 6.9+/-3.2 h and 160+/-45 h, respectively. For i.v. infusion the mean half-life for the elimination phase was 103+/-12 h. Cumulative excretion in the urine was 17%+/-3% ID and 21%+/-7% ID in 96 h for i.p. and i.v. administration, respectively. Scintigraphic images after i.p. administration showed accumulation in ovarian cancer lesions, while all other tissues showed decreasing activity with time. Tumour uptake determined in the ovarian cancer tissue specimens ranged from 3.4% to 12.3% ID/kg for i.p. administration and from 3.6% to 5.4% ID/kg for i.v. administration. Dosimetric analysis of the data indicated that 1.7-4.3 mGy/MBq and 1.7-2.2 mGy/MBq can be guided to solid or ascites cells after i.p. and i.v. administration, respectively. Assuming that an absorbed dose to the bone marrow of 2 Gy will be dose limiting, a total activity of 4.1 GBq 131I-cMOv18 IgG can be administered safely via the i.p. route and 3.5 GBq via the i.v. route. At this maximal tolerated dose, a maximum absorbed dose to 1-g tumours in the peritoneal cavity of 18 and 8 Gy can be reached after i.p. and i.v. administration, respectively. For the i. p. route of administration, dose estimates for the tumour are even higher when the electron dose of the peritoneal activity is also taken into account: total doses to the tumour of 30 Gy and 22 Gy will be absorbed at the tumour surface and at 0.2 mm depth, respectively. In conclusion, therapeutic tumour doses can be achieved with 131I-cMOv18 IgG in patients with intraperitoneal ovarian cancer lesions with no normal organ toxicity. The i.p. route of administration seems to be preferable to i.v. administration.     

Combination of systemic acetazolamide and topical dorzolamide in reducing intraocular pressure and aqueous humor formation

BACKGROUND: Neuroendocrine tumors commonly metastasize to the liver. Although surgical resection is considered a treatment option for patients with localized metastases confined to the liver, the longterm survival benefit of liver resection has not been clearly demonstrated. We examined the survival of patients undergoing liver resection for this disease. STUDY DESIGN: Between 1984 and 1995, we evaluated 38 patients with liver-only metastases from neuroendocrine tumors, including 21 carcinoid, 13 islet cell, and 4 atypical neuroendocrine neoplasms. Data from a combined prospective and retrospective database and a tumor registry were analyzed. Of these patients, 15 underwent complete resection of all known disease. The remaining 23 patients, who also had disease confined to the liver, had comparable tumor burden but were believed to be unresectable. The longterm survival rates of these two groups were compared. RESULTS: Patients who underwent liver resection did not differ from those who were unresectable with regard to age, pathology, primary tumor site, serum alkaline phosphatase levels, or percentage of the liver involved. All resections were complete, leaving no residual disease, and consisted of lobectomy (n = 3), segmentectomy (n = 1), and wedge resections (n = 11). There were no operative deaths. Patients who underwent hepatic resection had a significantly longer survival than unresected patients. Although median survival had not been reached in resected patients, the median survival in the unresectable group was 27 months. Patients who underwent liver resection had a higher 5-year actuarial survival (73% versus 29%). CONCLUSIONS: Hepatic resection in selected patients with isolated liver metastases from neuroendocrine tumors may prolong survival. This conclusion was reached by comparing our resected group with an unresectable group with similar tumor burden.     

Decompressive laminectomies with clinical improvement but persistent myelographic blocks

Surgical decompression for spinal cord compression secondary to metastatic epidural neoplasms is a common procedure. Preoperative myelography is nearly always done, and clinical improvement postoperatively is assumed to be correlated with relief of the radiologically demonstrated subarachnoid block. Three cases with postoperative clinical improvement but persistent subarachnoid blocks are reported. The recognition of this is important to avoid unnecessary secondary surgery.     

Structure-activity relationships of four 11-hydroxyestrones isomeric at the C-9 and C-11 positions

The synthesis of 11 alpha-hydroxyestrone, 11 alpha-hydroxy-9 beta-estrone, and 11 beta-hydroxy-9 beta-estrone are presented. The reduction of 11-keto-9 beta-estrone 17-ethyleneketal by sodium in ethanol or sodium borohydride resulted in 11-hydroxy-9 beta-estrones. The 11-hydroxyl group configurations were opposite to expectations: sodium in boiling ethanol afforded the axial 11 beta-hydroxy-9 beta-estrone, while sodium borohydride in boiling tetrahydrofuran gave the equatorial 11 alpha-hydroxy-9 beta-estrone. In immature rat uterotropic bioassays using subcutaneous injections, 11 alpha-hydroxyestrone was 2 times as active as 11 alpha-hydroxy-9 beta-estrone, and 11 beta-hydroxyestrone was 10 times as active as 11 beta-hydroxy-9 beta-estrone.     

Detection and sequence analysis of hepatitis B virus integration in peripheral blood mononuclear cells

A PCR-based technique was used to detect hepatitis B virus (HBV) integration in peripheral blood mononuclear cells from patients with chronic hepatitis B. Integrated HBV DNA sequences, with virus-cell junctions located in the cohesive region between direct repeat 1 (DR1) and DR2, were found in 2 of 10 studied patients.     

A cruciform-dumbbell model for inverted dimer formation mediated by inverted repeats

Small inverted repeats (small palindromes) on plasmids have been shown to mediate a recombinational rearrangement event in Escherichia coli leading to the formation of inverted dimers (giant palindromes). This recombinational rearrangement event is efficient and independent of RecA and RecBCD. In this report, we propose a cruciform-dumbbell model to explain the inverted dimer formation mediated by inverted repeats. In this model, the inverted repeats promote the formation of a DNA cruciform which is processed by an endonuclease into a linear DNA with two hairpin loops at its ends. Upon DNA replication, this linear dumbbell-like DNA is then converted to the inverted dimer. In support of this model, linear dumbbell DNA molecules with unidirectional origin of DNA replication (ColE1 ori ) have been constructed and shown to transform E.coli efficiently resulting in the formation of the inverted dimer. The ability of linear dumbbell DNA to transform E.coli suggests that the terminal loops may be important in bypassing the requirement of DNA supercoiling for initiation of replication of the ColE1 ori.