Monoclonal antibodies with defined specificities for Torpedo nicotinic acetylcholine receptor cross-react with Drosophila neural tissue.

A panel of monoclonal antibodies with known specificity for the well-characterized nicotinic acetylcholine receptor from the electroplax of Torpedo californica, many of which cross-react with the mammalian muscle acetylcholine receptor, were examined for cross-reactivity in the fly, Drosophila melanogaster. Monoclonal antibodies with specificities for different epitopes on the transmembrane receptor complex from Torpedo cross-react with different regional subsets of neural tissue in Drosophila. Axonal tracts, neuropil, mechano-sensory bristle elements and photoreceptors, each are detected by separate monoclonal antibody classes corresponding to different epitope domains. A preliminary characterization of an antigenic determinant in Drosophila heads recognized by one of the cross-reacting monoclonal antibodies is presented. Monoclonal antibodies such as these may be useful in identifying molecules of homologous structure or function, possibly including a neuronal acetylcholine receptor.

Stimulation of acetylcholine release from guinea-pig ileal synaptosomes by cyclic nucleotides and forskolin.

Various agents which are known to affect intracellular levels of cAMP have been assessed for their ability to induce the release of [3H]acetylcholine ([3H]ACH) from a synaptosomal preparation derived from the guinea-pig ileum myenteric plexus. 8-Bromo-cAMP increased the release of [3H]ACh above basal levels. While 8-bromo-cGMP also increased the release, this nucleotide was far less potent than 8-bromo-cAMP. Comparison of the release caused by the cyclic nucleotides to the release induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP) suggested that there is some relationship, as yet undefined, between the 8-bromo-cAMP-induced and the DMPP-induced release, while no relationship was evident between the release induced by 8-bromo-cGMP and that caused by DMPP. The 8-bromo-cAMP-induced release was Ca2+-dependent. Neither adenosine, clonidine, nor oxotremorine (all of which modulate the nicotinically-induced release) affected the 8-bromo-cAMP-induced release. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine stimulated the release of [3H]ACh as did the adenylate cyclase activator forskolin. The forskolin-induced release was not affected by adenosine, clonidine or oxotremorine. The ability of the modulators to block the nicotinically-induced release but not the release caused by the cyclic nucleotides indicates that the modulation of release evoked by nicotinic activity does not occur at a step involving protein phosphorylation.

Short report: a comparative study of the interaction between antacid and H2-receptor antagonists.

The influence of concomitant antacid administration on the relative bioavailability of the H2-receptor antagonists cimetidine, famotidine, nizatidine and ranitidine, was investigated in a panel of 21 healthy, adult male volunteers in an eight-way crossover trial. Administration with antacid reduced the bioavailability of all agents tested. The reduction in area under the serum concentration-time curve (AUC) was greatest for cimetidine (23%) and ranitidine (26%) and least for nizatidine (12%) and famotidine (19%). Reductions in peak serum concentration (Cmax) followed a similar pattern. The times of peak serum concentrations were not affected by antacid. Comparison of the relative bioavailability among all drugs tested showed no statistically significant differences in the effect of antacid administration on these agents. However, a high degree of intersubject variability was observed.

Controlled study of the putative interaction between famotidine and theophylline in patients with chronic obstructive pulmonary disease.

The effects of famotidine (80 mg per day), cimetidine (1600 mg per day), and placebo on theophylline pharmacokinetic parameters in chronic obstructive pulmonary disease (COPD) patients were compared. This was an open-label, randomized, three-period cross-over study, in which each subject first underwent a seven-day theophylline washout period, and thereafter received three single intravenous doses of theophylline (5 mg/kg infused over 30 minutes) during the study. Each of the experimental treatments was administered orally every 12 hours for a total of 9.5 days (19 doses). Theophylline was infused after the 17th dose of each treatment. Fourteen serial blood samples were collected before the start of each infusion, and for 30 hours after the end of each infusion. Plasma samples were assayed for theophylline, pharmacokinetic parameters were estimated, and treatment effects on each parameter were compared. Fourteen COPD patients completed all three periods of the investigation. Famotidine treatment had virtually no effect on any of theophylline's pharmacokinetic parameters. In contrast, cimetidine treatment significantly altered every pharmacokinetic parameter of theophylline as follows: Cimetidine decreased theophylline geometric mean CL from 2.74 L/h to 2.07 L/h (P < .001), and prolonged theophylline harmonic mean half-life from 6.6 to 9.6 hours (P < .001) and mean residence time from 10.8 to 15.0 hours (P < .001). Cimetidine treatment slightly increased theophylline volume of distribution by approximately 10%, and that change also was statistically significant (P = .032). The authors conclude that the treatment effects of cimetidine on theophylline pharmacokinetic parameters were in accord with those reported by others, and that famotidine treatment had no effect on any of theophylline's pharmacokinetic parameters in COPD patients.

Alpha-bungarotoxin binding to a high molecular weight component from lower vertebrate brain identified on dodecyl sulfate protein-blots.

The binding of [125I]iodo-alpha-bungarotoxin [( 125]alpha-BuTX) to the dissociated alpha-subunit of Torpedo acetylcholine receptor (AChR) can be readily demonstrated in a modified 'protein-blot' analysis utilizing electrophoretically transferred, dissociated subunits immobilized onto positively charged nylon membranes which are then incubated directly with [125I]alpha-BuTX. We report here the use of the protein-blotting technique to detect the alpha-BuTX binding site present in the central nervous system of lower vertebrates and to characterize some of the physicochemical properties of the toxin binding site. High molecular weight (Mr greater than or equal to 200,000 and greater than or equal to 120,000) alpha-BuTX-binding components can be readily demonstrated in avian and fish brain extracts upon protein-blotting with [125I]alpha-BuTX following lithium dodecyl sulfate PAGE. Neither extensive reduction with dithiothreitol nor prior reduction followed by alkylation with iodoacetamide alter the mobility of the CNS-derived BuTX-binding sites. In contrast to our findings with Torpedo AChR or muscle AChR derived from a number of different species, no binding is observed in the molecular weight range of the alpha-subunit (Mr = 40,000) nor is any binding at any molecular weight observed in similar fractions prepared from adult, mammalian (rat, guinea pig) brain using this technique. These results demonstrate the existence in lower vertebrate brain of a BuTX binding site comparable in size to the AChR oligomeric complex of electric organ and muscle. They also suggest, however, striking structural differences between muscle AChR and the central neuronal BuTX-binding complex as well as a considerable difference between the neuronal BuTX-binding sites derived from lower and higher vertebrate brain.

Opiate binding to subcellular fractions from guinea pig ileum.

Binding of 3H-etorphine and 3H-D-Ala2-D-Leu5-enkephalin to opiate receptors in synaptosomal and microsomal fractions prepared from guinea pig ileum homogenates has been studied. It is found that the dissociation constants for etorphine from all fractions are the same. The binding capacity for etorphine for the purified synaptosomal fraction is greater than for other fractions by a factor of 5. For the enkephalin derivative binding to the microsomal fraction the dissociation constant is greater than for etorphine while the binding capacity is a factor of 3 lower. These results are in contrast to the case for binding to central nervous system subcellular fractions.

Renal cell carcinoma.

The behavior of renal cell carcinoma remains one of the most unpredictable of the genitourinary neoplasms. Once this disease has spread beyond the confines of the kidney, it is extremely difficult to control. This year, emphasis has focused on the characteristic cytogenetic and chromosomal changes that are seen in this tumor that help to explain partially its enigmatic behavior. Immunotherapy remains the mainstay of nonsurgical therapy. Recent studies have examined the efficacy of using combinations of interferons, interleukin-2, or specific subpopulations of lymphoid cells to control metastatic renal cell carcinoma. The role of surgery in metastatic disease, tumor extending into the vena cava, and parenchyma-sparing operations continues to be examined. This review examines the most recent literature on each of these aspects in the treatment of this difficult and challenging tumor.

Renal cell carcinoma.

Renal cell carcinoma represents a significant challenge to surgeons and oncologists treating urologic malignancies. Diagnostically, it is critically important to identify the precise extent of the tumor prior to therapeutic intervention. Therapeutically, a number of controversies continue to be debated, including the role of renal-conserving surgery and the role of surgery in patients with metastatic disease. New research is beginning to identify factors involved in the multidrug-resistant properties of these tumors that may allow us, in the future, to treat these tumors more effectively with systemic chemotherapy. Utilizing immunotherapy in the form of autolymphocytes, interferon, interleukin-2, or combinations of these regimens, a number of exciting advances have been made in the treatment of metastatic renal cell carcinoma. This review examines the most recent literature on each of the above-mentioned aspects in the treatment of this difficult and challenging tumor.

Activation of fluoride-stimulated adenylate cyclase by phospholipase A2 in the caudate nucleus of the rat brain.

Phospholipase A2 (PLA2) increases adenylate cyclase (AC) activity in the rat caudate nucleus in a dose-dependent manner. After maximal stimulation by fluoride, PLA2 treatment further increases AC activity 2.4 fold. Adenylate cyclase activity is maximal after 45% hydrolysis of the phospholipids. Of the products of PLA2 treatment only lysophosphatidylcholine (LPC) produces such an increase in AC activity. In contrast to PLA2 treatment, LPC solubilizes the enzyme, decreases the Km value for ATP, and requires much larger amounts of LPC than that produced by lipase treatment. After maximal stimulation with fluoride and PLA2, removal of most of the LPC does not reduce the activity of adenylate cyclase. These findings suggest that removal of membrane lipid rather than generation of LPC is responsible for the activation of brain adenylate cyclase by phospholipase A2.

Modulation of the release of acetylcholine from ileal synaptosomes by adenosine and adenosine 5'-triphosphate.

Low concentrations of either adenosine or adenosine 5'-triphosphate (ATP) inhibited the nicotinically induced release of [3H]acetylcholine from synaptosomes derived from the guinea-pig ileum myenteric plexus. Adenosine and ATP were equipotent in their ability to inhibit the release and the inhibition was reversible by theophylline in both cases. The data suggest that ATP may have acted after initial hydrolysis to adenosine and that the receptor involved may be the P1 or similar R site receptor. High concentrations of ATP caused marked increases in the release of [3H]acetylcholine. This release was neither temperature- nor calcium-dependent. Because the concentrations required were similar, however, to those which have been reported to cause ATP-induced contractions in intact preparations, further studies of the phenomenon were carried out. Lactate dehydrogenase was not released with the [3H]acetylcholine, suggesting that indiscriminate lysis of the membranes had not occurred. The release was not affected by theophylline, indomethacin, tetrodotoxin or adenosine. The ATP analog, adenylyl (beta, gamma-methylene)-diphosphonate did not cause the increase in release, therefore phosphorylation may be required for the effect. The mechanism of increased release remains to be defined, but the data suggest that it is unlikely that a P2 receptor is involved.

Post-translational modification of proteins by 15-carbon and 20-carbon isoprenoids in three mammalian cell lines.

A number of cellular proteins, including p21ras, lamin B, and the G-protein gamma subunits, undergo post-translational modification by 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid moieties derived from pyrophosphate intermediates of the cholesterol biosynthetic pathway. In this study, isoprenylated proteins in three mammalian cell lines (Hela cells, Rat-6 fibroblasts and COS cells) were radiolabeled with an isoprenoid precursor, [3H]mevalonate, and resolved by SDS gel electrophoresis. Groups of proteins with different molecular masses were eluted from the gels and the chain-lengths of the radiolabeled isoprenyl groups, released from the proteins by Raney-nickel-catalyzed desulfurization, were established by gel permeation chromatography. 15-Carbon and 20-carbon isoprenyl groups were found in separate classes of proteins within each cell line. With the exception of p21ras, which incorporated a 15-carbon group when expressed in COS cells, the proteins in the region of the 21-28 kDa ras-related GTP binding proteins contained mostly 20-carbon isoprenyl chains. In contrast, proteins belonging to the 66-72 kDa nuclear lamin family, as well as unidentified proteins with molecular masses of 41-46 kDa and 53-55 kDa, contained predominantly 15-carbon isoprenyl chains. The chain-lengths of the isoprenoids associated with particular classes of proteins did not vary from one cell line to another, suggesting that the nature of the isoprenoid modification (farnesyl versus geranylgeranyl) is determined by intrinsic structural features of the proteins, rather than the cell type in which the proteins are expressed.

Splenic abscess arising by direct extension from a perinephric abscess.

A case of a perinephric abscess invading the spleen in a 25-year-old woman with bladder exstrophy is reported. Treatment utilized both percutaneous drainage and open surgery. Perinephric abscesses have not been previously reported to extend into the spleen.

Binding of apomorphine to neural membranes.

The intrinsic fluorescence of apomorphine has been used to measure its binding to neural membranes. A large number of relatively weak binding sites are concentrated in myelin and synaptic membrane fractions. Butyrophenones have the highest affinities for these sites--KD = 43 micrometer for haloperidol--while dopamine and dopamine releasers and reuptake blockers, as well as a variety of other alkaloids, have much lower affinities. The sites are hydrophobic and undergo a phase transition to a highly fluid state near 26 degrees C. Calcium is a noncompetitive inhibitor of apomorphine binding. Some of the actions of neuroleptic drugs may result from binding to these hydrophobic membrane sites in vivo, blocking conduction in small catecholamine axons.

Medical therapy alone for the treatment of gas forming intrarenal abscess.

Gas forming renal infections are severe, potentially lethal conditions. Gas formation in an intrarenal abscess is extremely rare. Formerly, these patients were uniformly treated by a combined medical and surgical approach. We describe 2 patients with intrarenal gas abscess who were successfully treated with antibiotics alone. We review the literature concerning intrarenal gas abscess and propose a pathophysiological mechanism for its formation as well as a treatment schema.

Phyllodes type of atypical prostatic hyperplasia: a report of 3 new cases.

We report 3 new cases of phyllodes type of atypical prostatic hyperplasia. This lesion is characterized by epithelial and stromal proliferation. Stromal changes are the most characteristic finding in phyllodes type of atypical prostatic hyperplasia, which show atypical cells with enlarged, hyperchromatic sarcomatoid nuclei. Mitotic figures are not present. Although the histological appearance may mimic that of cystosarcoma phyllodes of the breast, this pattern is present only focally or not at all in phyllodes type of atypical prostatic hyperplasia. On computerized tomographic imaging phyllodes type of atypical prostatic hyperplasia has a distinct appearance. These patients can be expected to have a benign clinical course and distant metastases have not been reported. Treatment is by surgical excision as in benign prostatic hyperplasia.

Identification and purification of a kidney membrane protein which specifically binds the amino-terminal domain of native parathyroid hormone.

Nitrocellulose blots of bovine kidney membrane proteins were prepared from denaturing polyacrylamide gels. Strips of the blots were incubated with parathyroid hormone (PTH), washed, and then incubated with antisera against the hormone. Exposure to horseradish peroxidase-linked second antibody led to staining of a 51-kDa protein. No staining was observed in blots not incubated with PTH. Fragments 35-84 and 19-84 of PTH reacted strongly with the antisera, but did not lead to staining of the 51-kDa protein on the blots. Staining was visible, but greatly reduced, when fragment 9-84 was used. Oxidation of the native hormone at positions 8 and 18 led to reductions in staining of the band which were quantitatively similar to the reductions in biological activity induced by such oxidations. These properties suggested that the 51-kDa protein recognizes the amino-terminal portions of PTH, which is the segment of the molecule required for its biological activities. Several micrograms of the 51-kDa protein were purified to homogeneity by selective extraction from the membranes with detergent and by elution from multiple two-dimensional gels. The purified protein retained its PTH-dependent staining and specificity. This protein may be a PTH receptor or a fragment of a PTH receptor from kidney.

Cisplatin, methotrexate and vinblastine plus surgical restaging for patients with advanced transitional cell carcinoma of the urothelium.

Chemotherapy with cisplatin, methotrexate and vinblastine (CMV) is active in advanced transitional cell carcinoma of the urothelium. Aggressive surgical resection of residual disease following responses produced by CMV was incorporated into a combined modality approach. Between 1982 and 1990, 64 patients were entered into the study. Of 55 patients evaluable for response 11 (20%) had a pathological complete response, 14 (25%) achieved a complete response following resection of residual disease and 5 (9%) whose disease was not surgically restaged had a clinical complete response. The overall complete response rate was 55%. Patients with liver, lung or bone involvement had significantly decreased survival compared to patients without visceral disease (p = 0.002). With a median followup exceeding 50 months, 14 patients (22% of all patients entered into the study) were free of disease at 23 to 98+ months. There were no deaths related to treatment. CMV produced high rates of response in patients with advanced disease, including those with distant metastases. Surgical resection of residual disease following responses produced by chemotherapy proved to be feasible, without treatment related mortality, and may have prolonged survival in selected cases.