Constitutive and transport-related endocytotic pathways in turtle bladder epithelium

Summary Proton secretion in the urinary bladder of the fresh-water turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo-and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15°C. In addition, serosal application of 0.5mm acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15°C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1mm serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.     

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     

Peptide-N 4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducingN-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man₃(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and¹H- and¹³C-NMR assignments for the oligosaccharide Man₃(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).Abbreviations Endo endo--N-acetylglucosaminidase - Fuc fucose - GlcNAc N-acetylglucosamine - Man mannose - NMR nuclear magnetic resonance - PNGase peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase - Xyl xylose     

Differentiation of somatic embryos from protoplasts isolated from embryogenic suspension cultures of Abies alba L.

Embryogenic suspension cultures of Abies alba were established using an embryogenic suspensor mass culture originating from the zygotic embryo in immature seed explants (Schuller et al. 1989). Protoplasts were isolated from the suspension material. The protoplasts were immobilized in alginate layers in order to follow the development of single protoplasts. During the first days of protoplast culture a modified Kao and Michayluk (1975) medium proved to be necessary for subsequent divisions. The formation of proembryos succeeded within 2–3 weeks when subcultured with a modified Schenk and Hildebrandt (1972) liquid medium. Light, enhanced sugar concentration, and the addition of abscisic acid led to the formation of slightly green torpedo-shaped somatic embryos after 6–8 weeks from protoplast isolation.Abbreviations ABA abscisic acid - BAP N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - ESM Embryonal suspensor mass (Gupta and Durzan 1986) - KM Kao and Michayluk (1975) - LP (von Arnold and Eriksson 1977) - MES 2-(N-morpholino)ethane-sulfonic acid - NAA 1-naphthalene-acetic acid (sodium salt) - PVP polyvinylpyrrolidone - SH Schenk and Hildebrandt (1972) - Tween 80 polyoxyethylene-sorbitan-monooleate     

Extinction of populations by random influences

A unifying approach described by a random birth and death process which includes both environmental and demographic noise is introduced. It is shown that both of these noise sources play an essential role in extinction processes in general. The probability distribution of the lifetime of a population is determined and its dependence on the parameters of the model is discussed. Finally a population divided into subpopulations is modeled. The lifetime of this ensemble of subpopulations is compared to the lifetime of one large population.     

Uptake and distribution in rat brain of organic and inorganic selenium

Polyunsaturated fatty acids (PUFAs) occur in relatively high amounts in phospholipids of the synapses. PUFAs may thus determine the fluidity of the synaptosomal membrane and, hereby, they may regulate the neuronal transmission. It was therefore tempting to suggest a system in the brain, that inhibits autooxidation of PUFAs. In order to trace such a protection system, Wistar rats were equally loaded with 4500 kBq of 75-Se either as selenite or as L-Se-methionine. By means of gradient ultracentrifugation, particulate fractions of the brains were isolated, and the radioactivity as well as the glutathione-transferase and -peroxidase activities were estimated. The distribution of the two selenium components among the particulate fractions was different. Thus, selenite gave higher radioactivity in myelin, then followed by the light synaptosomal and the vesicular fraction. L-Se-methionine was more equally incorporated in all particulate fractions, although highest activity was found in the mitochondrial fraction. Myelin and synaptic vesicles were devoid of transferase activity. On the other hand, the synaptosomal fraction showed highest specific transferase activity. The glutathione peroxidase activity was highest in the myelin fraction, followed by the vesicular and the synaptosomal fractions. The data obtained thus support the idea that the PUFAs of the synaptic compartment are protected against peroxidation, at least in part, by the selenium containing glutathione peroxidase.     

A detailed study of the regulation and evolution of the two classes of patatin genes in Solanum tuberosum L.

The class-specific expression of patatin genes was investigated by analysing four new patatin genes. A class I patatin gene from cv. Berolina as well as a class I and two class II patatin genes from the monohaploid cultivar AM 80/5793 were isolated and partially sequenced. Sequence comparison indicates rearrangements as the major source for the generation of diversity between the different members of the classes. The expression of single genes was studied in potato plants transformed with chimaeric genes where the putative patatin promoters were fused to the GUS reporter gene. A detailed histochemical analysis reveals that both class I genes are expressed as the previously described class I patatin gene B33 from cv. Berolina [1], i.e. in the starch-containing cells of potato tubers and in sucrose-induced leaves. The class II gene pgT12 shows the same pattern as the previously described class II gene pgT2 [2], i.e. expression in root tips and in the vascular tissue of tubers, whereas no activity was detectable for pgT4. Thus the expression pattern of both classes of genes seems to be stable at least within or even between different cultivars.     

Thermogenic effect of adrenaline: interaction with insulin

The contribution of insulin (3.6 pmol.kg body mass-1.min-1) to adrenaline-induced (0.164 nmol.kg fat free mass-1.min-1) thermogenesis was studied in ten postabsorptive healthy volunteers using two sequential protocols. Variables considered were oxygen consumption as well as carbon dioxide production, heart rate, blood pressure, plasma concentrations of glucose, insulin, glycerol, free fatty acids, beta-HO-butyrate and lactate. Adrenaline increased plasma concentrations of glucose, glycerol, free fatty acids, and beta-HO-butyrate, and heart rate and metabolic rate during normo-insulinaemia [61.3 (SEM 6.6) pmol.l-1]. Similar effects were observed during hyperinsulinaemia [167.9 (SEM 18.7) pmol.l-1], but the effect of adrenaline on oxygen consumption was reduced. On average, metabolic rate increased by 12.9% during normo-insulinaemia and by 8.9% during hyperinsulinaemia. We concluded that relative hyperinsulinaemia resulted in decreased adrenaline-induced thermogenesis and therefore increased whole body anabolism.     

No evidence for linkage of autosomal dominant proximal spinal muscular atrophies to chromosome 5q markers

Summary Two recent articles have reported the linkage of a gene for recessive spinal muscular atrophy (SMA) on the chromosome region 5q11.2–13.3. Our data show no linkage of the dominantly inherited forms of SMA to this chromosome region.