Serum immunoassay of a small cell lung cancer associated ganglioside: development of a sensitive scintillation proximity assay

We here report an enzyme linked immunosorbent assay (ELISA) and a scintillation proximity assay (SPA) for detection of the ganglioside FucGM₁ in sera from small cell lung cancer (SCLC) patients. The SPA was more sensitive and reproducible than the ELISA. In this assay, monoclonal antibodies specific for FucGM₁ were bound to SPA particles and incubated with labelled FucGM₁ and 100 µl test-serum overnight, and counted in a -counter. The sensitivity was 0.2 ng. Seven out of twenty sera from SCLC patients were positive, whereas none of twenty sera from healthy individuals were positive for FucGM₁. The SPA was more sensitive than the previously reported HPTLC as well as a direct ELISA.Abbreviations MAb monoclonal antibody - SPA scintillation proximity assay - HPTLC high performance thin layer chromatography - SCLC small cell lung cancer - FucGM₁ Fuc1-2Gal1-3GalNAc1-4(NeuAc2-3)-Gal1-4Glc1-1Cer - ELISA enzyme linked immunosorbent assay - FCS foetal calf serum - PBS phosphate buffered saline     

Enterochromaffin-like cells in the rat stomach: effect of α-fluoromethylhistidine-evoked histamine depletion

Summary In the rat, gastric histamine is stored predominantly in the enterochromaffin-like (ECL) cells, which are located basally in the oxyntic mucosa. The functional significance of histamine in the ECL cells is a matter of speculation. In this study the effect of depletion of histamine on the properties and ultrastructure of the ECL cells was examined. Histamine synthesis was inhibited with -fluoromethylhistidine (3 mg·kg^-1·h^-1) given via osmotic minipumps over a period of 24 h. The treatment reduced the histidine decarboxylase activity (approximately 20% remaining) and histamine concentration (less than 20% remaining) in the oxyntic mucosa, as well as the intensity of histamine- and chromogranin A-immunostaining in the ECL cells, compared to control rats. The cytoplasmic (secretory) granules/vesicles were greatly reduced in number and size following -fluoromethylhistidine administration. The histamine immunostaining of the mast cells, which occurs at the mucosal surface and in the submucosa, appeared unaffected. We conclude that ECL cell histamine accounts for at least 80% of the total oxyntic mucosal histamine in the rat and that it represents a more mobile pool than mast cell histamine. The reduction in the number and size of the ECL cell granules/vesicles following histamine depletion is in accord with the idea that they represent the storage site for histamine.     

Drug distribution studies with microdialysis. II. Caffeine and theophylline in blood, brain and other tissues in rats

Microdialysis was applied to estimate the pharmacologically active concentration of caffeine and theophylline in blood, adipose tissue, muscle, liver and brain of rats. The concentration of the drugs in the extracellular space was estimated by perfusion with varying concentrations of the drug through the microdialysis probe (difference method). Caffeine (20 mg/kg) was found to be evenly distributed with a free concentration of approximately 120 microM. Theophylline concentration in the brain was 91 microM and in other tissues approximately 120 microM. The rate of penetration into brain extracellular space was higher for caffeine than for theophylline. It is suggested that the lower levels of theophylline attained in the brain may to some extent explain the differences in clinical action profile between caffeine and theophylline.     

Ovarian function and pregnancy rates in reindeer calves (Rangifer tarandus ) in Southern Norway

Reindeer calves (n = 632) were slaughtered in November/December (n = 476) or in January (n = 156). Dressed weights and amount of perirenal fat were recorded, and the reproductive organs were collected. A separate group of 130 reindeer calves were weighed at 7 months of age and were followed up with repeated weighings and pregnancy examinations up to 21 months. The onset of puberty and the pregnancy rate were significantly influenced by body weight and the amount of perirenal fat. Approximately 60 g of perirenal fat and 22 kg dressed weight were found at the lower limits for pregnancy. A total of 222 (35%) animals had reached puberty and 126 (20%) were pregnant when examined after slaughter. Animals which conceived during their first autumn showed only a moderate weight gain the following year, and the calf mortality rate in these animals was 47.4%. It was concluded that calf pregnancies are common among the reindeer of Southern Norway and that measures need to be taken to prevent them.     

Plant protoplasts and genetic engineering II

Book Review

Plant protoplasts and genetic engineering IIY.P.S. Bajaj (Ed.), (Biotechnology in Agriculture and Forestry, Vol. 9). Berlin: Springer-Verlag, 1989. 510 pages. DM398.00. ISBN 3-540-50789-2     

Complete enzymatic deglycosylation of native sex steroid-binding protein (SBP or SHBG) of human and rabbit plasma: effect on the steroid-binding activity.

An enzymatic procedure for the complete removal of the N-linked and O-linked oligosaccharide side chains of the sex steroid-binding proteins (SBP or SHBG) of human and rabbit plasma under native conditions is described. Deglycosylation was catalyzed by N-glycanase, neuraminidase, and O-glycanase and was monitored by SDS-PAGE, lectin blotting, and molecular weight analyses by electrospray mass spectrometry. Digestion of rabbit SBP with N-glycanase generated a major 39,777-Da protein and two minor ones of 39,389 and 39,545 Da. The molecular weight of the major protein agrees with the molecular weight calculated from the sequence of the sugar-free polypeptide monomer (39,769 Da: Griffin, P.R., Kumar, S., Shabanowitz, J., Charbonneau, H., Namkung, P.C., Walsh, K.A., Hunt, D.F., & Petra, P.H., 1989, J. Biol. Chem. 264, 19066-19075), whereas the other two are deglycosylated proteolytic cleavage products lacking the TQR and TQ sequences at the amino-terminus. The N- and O-linked side chains of human SBP were removed by sequential digestion with N-glycanase and neuraminidase/O-glycanase. A 38,771-Da protein was generated, which agrees well with the molecular weight of the sugar-free polypeptide monomer (Walsh, K.A., Titani, K., Kumar, S., Hayes, R., & Petra, P.H., 1986, Biochemistry 25, 7584-7590). N-deglycosylation of human and rabbit SBP has no effect on the steroid-binding activity, but removal of the O-linked side chains of N-deglycosylated human SBP results in an apparent 50% loss of steroid-binding activity and an increase in the Kd for the binding of 5 alpha-dihydrotestosterone from 0.3 mM to 0.9 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme patterns during substrate shifts between phenol,acetate and glucose in continuous culture of Trichosporon cutaneum

Summary The soil yeast Trichosporon cutaneum was grown in continuous culture on phenol, acetate or glucose as sole carbon source. The activities of enzymes participating in the tricarboxylic acid cycle, glyoxylate cycle, 3-oxoadipate pathway, pentose phosphate pathway and glycolysis were determined in situ during shifts of carbon sources. Cells grown on phenol or glucose contained basal activity of the glyoxylate-cycle-specific isocitrate lyase. The derepression of the glyoxylate cycle enzymes was partly hindered in the presence of phenol but not in the presence of low levels of glucose. Phenol and glucose caused repression of isocitrate lyase. In the presence of either phenol or glucose, acetate accumulation in the medium increased. However, part of the supplied acetate was utilized simultaneously with phenol or glucose, the utilization rate of either carbon source being reduced in the presence of the other carbon source. Acetate caused repression but not inactivation of the phenol-degrading enzymes, phenol hydroxylase and catechol 1,2-dioxygenase. The simultaneous utilization of phenol and other carbon sources in continuous culture as well as the observed repression-derepression patterns of the involved enzymes reveal T. cutaneum to be an organism of interest for possible use in decontamination processes. Offprint requests to: H. Y. Neujahr Offprint requests to: H. Y. Neujahr     

Different oligosaccharides accumulate in the brain and urine of a cat with α-mannosidosis: structure determination of five brain-derived and seventeen urinary oligosaccharides

Five brain-derived and 17 urinary oligomannose-type oligosaccharides were isolated by ion-exchange chromatography on Mono Q or Dowex, followed by HPLC on Lichrosorb-NH₂ from a Persian cat suffering from -mannosidosis. The structures ofthe carbohydrate chains were determined by 500- or 600-MHz¹H-NMR spectroscopy. Different oligosaccharide patterns were found in brain and urine. 99% of the urinary oligosaccharides possess an (1-6)-linked mannose residue attached to -mannose, whereas only 5% of the brain-derived oligosaccharides contain such a residue. Furthermore, of the urinary carbohydrate chains 71% end with Man1-4GlcNAc1-4GlcNAc and 29% end with Man1-4GlcNAc, whereas the corresponding amounts are 23% and 77%, respectively, for the brain-derived oligosaccharides.Abbreviations MLEV-17 composite pulse devised by M. Levitt - HOHAHA homonuclear Hartman-Hahn spectroscopy - TPPI time-proportional phase incrementation - 2D two dimensional - GlcNAc N-acetylglucosamine - Man mannose - Fuc fucose     

Biphasic changes in leukocytes induced by strenuous exercise

Seven healthy male volunteers participated in short- (STR, 1.7 km), middle- (MTR, 4.8 km) and long- (LTR, 10.5 km) term runs at a speed close to their maximum. A prompt mobilization of white cells, and lymphocytes in particular, appeared following the exercise. The initial increase in the number of lymphocytes was succeeded by a significant decrease [(P less than 0.03) lymphopenial], which on average was 32%-39% of the pre-exercise values in all groups. A close correlation was found between the initial increase in plasma cortisol concentration after exercise and the subsequent lymphopenia. A modest enhancement in the number of granulocytes immediately after the exercise was accompanied by a comprehensive increase in polymorphonuclear (PMN) elastase concentration accounting for 78.6%, SEM 16.3%, 140.7%, SEM 31.8% and 241.3%, SEM 48.1% in the STR, MTR and LTR groups. No correlation was found between granulocyte number and the plasma PMN elastase concentration. A delayed granulocytosis was noted in all subjects, reaching a peak between 2 and 4 h after the exercise. The magnitude of the granulocytosis varied among subjects and peak values of the number of circulating granulocytes were found to be 5.7 x 10(9) cells.l-1, SEM 0.5, 6.7 x 10(9) cells.l-1, SEM 0.6 and 8.8 x 10(9) cells.l-1, SEM 0.5 in STR, MTR and LTR respectively, whereas the mean baseline value was 3.6 x 10(9) cells.l-1, SEM 0.4. The neutrophilic granulocytosis was not accompanied by a corresponding enhancement in PMN elastase concentration. The plasma cortisol concentration reached a peak 30 min after exercise and declined below the control level in 4 h. Neither the initial increase, nor the subsequent decrease in plasma cortisol concentration were found to be essential for the magnitude of the delayed leukocytosis.