Gastric inhibitory polypeptide receptor in hamster pancreatic beta cells. Direct cross-linking, solubilization and characterization as a glycoprotein

125I-labelled gastric inhibitory polypeptide (125I-GIP) is directly cross-linked to its specific receptor in hamster pancreatic beta cell membranes by using an ultraviolet irradiation procedure. This approach results in the identification of a GIP-protein complex of apparent Mr 64,000. The labelling of this protein species is specific since it is inhibited when incubating the membranes with increasing doses of native GIP (0.1 nM-1 microM) together with 125I-GIP, half-maximal inhibition being elicited by 5 nM peptide. Reduction of the GIP-protein complex by 100 mM dithiothreitol induces a decrease of the electrophoretic mobility of the complex. Alternatively pretreatment of membranes with dithiothreitol (up to 1 M) does not prevent the binding of 125I-GIP to its receptor. When prelabelled membranes are extracted by 0.5% Triton X-100 (v/v) and the extract is layered on a Sephadex G-50 column, a high peak of radioactivity is eluted with the void volume of the column. Treatment of this peak by 10 min ultraviolet irradiation followed by SDS-PAGE leads to identification of a major band of Mr 64,000. When the peak is further layered on Sephacryl S-200 it yields a single peak of radioactivity corresponding to a protein species with a Stokes radius of 3.2 nm and an apparent Mr of 65,000. The solubilized GIP-receptor complex is specifically adsorbed by Sepharose coupled to wheat germ agglutinin and concanavalin A and eluted from these lectins by their respective sugars. In conclusion the GIP receptor in pancreatic beta cells is a protein monomer of apparent Mr 59 000; its structure is maintained by intrachain disulfide bridges, these bonds being, however, not involved in the interaction of GIP with its receptor; the GIP receptor is a glycoprotein containing N-acetylglucosamine, mannose and probably sialic acid in its carbohydrate moiety.     

Hybridization of DNA from methanogenic bacteria with nitrogenase structural genes (nifHDK)

Summary Using the Southern hybridization technique, homologies were examined between restricted DNA of four methanogenic bacteria (Methanobacterium ivanovi, Methanobacterium thermoautotrophicum, Methanococcus voltae, Methanosarcina barkeri) and the nif (nitrogen fixation) genes of Klebsiella pneumoniae and Anabaena strain 7120. With K. pneumoniae probes, no hybridization was observed with nifA, nifNE, and nifJ but positive results were obtained with the nifHDK genes coding for nitrogenase. Homology was detected, in the four strains, with K. pneumoniae and Anabaena nifH probes. In M. voltae and M. ivanovi, the homology found with nifH was estimated to be about 70% and a weaker hybridization was observed also with nifD and nifK. In M. voltae, the sequence homologous to nifH was found on a 3.0 kbp HindIII fragment and sequences homologous to nifD and nifK on a 3.8 kbp HindIII fragment. The 3.0 kbp fragment was cloned and the region homologous to nifH was localized more precisely. When this fragment was used as a probe against other DNAs, it behaved as a K. pneumoniae and Anabaena nifH probe. The results suggest that the structural genes for nitrogenase may be present in archaebacteria and raise interesting questions regarding their evolution.     

Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction

Summary We investigated whether the efficiency of transformation of plant cells by Agrobacterium tumefaciens during cocultivation is limited by the properties of the plant cells or by the infecting bacteria.Therefore, tobacco protoplasts were infected by cocultivation with two different agrobacteria strains carrying Ti plasmids with distinguishable T-DNAs. These T-DNAs cotransform plant cells at a frequency equal to the product of their independent transformation frequencies, which indicates that all plant cells are equally competent. On the other hand, when these T-DNAs are located on the same Ti plasmid vector within one bacterial strain, the cotransformation frequency is significantly higher than the product of the single transformation frequencies. We interpret these results to indicate that transformation is limited more by the establishment of effective bacteria/plant cell interaction than by (i) the process of DNA integration and (ii) by the number of plant cells capable of being transformed by Agrobacterium. We found that most plant cells are transformed by only one or a few agrobacteria. Analysis of the number of T-DNA copies in these clonally transformed lines indicates amplification of the original, infecting T-region copy.     

N-Methylaspartate-Evoked Liberation of Taurine and Phosphoethanolamine In Vivo: Site of Release

The effect of N-methyl-D,L-aspartic acid (NMA) on extracellular amino acids was studied in the rabbit hippocampus with the brain dialysis technique. Administration of 0.5 or 5 mM NMA caused a concentration-dependent liberation of taurine and phosphoethanolamine (PEA). Taurine increased by 1,200% and PEA by 2,400% during perfusion with 5 mM NMA whereas most other amino acids rose by 20-100%. The effect of NMA appeared to be receptor-mediated, as coperfusion with D-2-amino-5-phosphonovaleric acid curtailed the NMA response by some 90%. The NMA-stimulated release of taurine and PEA was suppressed when Ca2+ was omitted and further inhibited when Co2+ was included in the perfusion medium. The effect of NMA was mimicked by the endogenous NMA agonist quinolinic acid and the partial NMA agonist D,L-cis-2,3-piperidine dicarboxylic acid. Although the NMA-evoked release of taurine and PEA was Ca2+-dependent in vivo, NMA had no effect on Ca2+ accumulation in hippocampal synaptosomes. The previously reported NMA-induced activation of dendritic Ca2+ spikes and the lack of effect on synaptosomal Ca2+ uptake suggest that taurine and PEA are released from sites other than nerve terminals, possibly from dendrosomatic sites. This notion was strengthened by the absence of an effect of NMA on the efflux of radiolabelled taurine from hippocampal synaptosomes. In contrast, high K+ stimulated synaptosomal uptake of Ca2+ and release of taurine.     

Zymomonas mobilis mutants blocked in fructose utilization

Summary A mutant ofZymomonas mobilis deficient in the utilization of fructose for growth and ethanol formation was shown to lack fructokinase activity. When grown in media which contained glucose+fructose or sucrose, both the mutant and wild type produced sorbitol in amounts up to 60 g·l^-1, depending on the initial concentrations of sugars. Sorbitol formation was accompanied by an accumulation of acetaldehyde, gluconate, and acetoin. A ferricyanide-dependent sorbitol dehydrogenase could be localized in the cell membrane; it thus resembles the sorbitol dehydrogenase ofGluconobacter suboxydans. Neither a NAD(P)H dependent reduction of fructose nor a NAD(P) dependent dehydrogenation of sorbitol could be detected in cell-free extracts. The use of fructose-negative mutants ofZ. mobilis for the enrichment of fructose in glucose+fructose mixtures is discussed.     

Effects of increased salinity on the diatom assemblage in Fonda Lake,Michigan

A salt storage facility has been located adjacent to Fonda Lake since 1953. In February 1981 a core was taken from the profundal sediments of the lake and analyzed to determine the effects of salt perturbation on the diatom community over a 32-year period. Diatom assemblages from different levels were compared using multivariate techniques including cluster analysis and principal component analysis. Shifts in diatom composition related to salinification were revealed most clearly by subdominant taxa. Five distinct groups of diatom taxa were found to correspond with 5 depth intervals. The diatom component of the lake up to 1960 included two groups of taxa which were alkaliphilous and chloride indifferent. A reduction in species diversity beginning in 1960 may indicate a salt effect. By 1968, when diversity reached a minimum, a variety of halophilic taxa (including Diatoma tenue, Navicula gregaria and Synedra fasciculata) attained their highest relative abundances. At the top of the core, diversity increased slightly and some halophilic taxa decreased in relative abundance, which suggests a possible decrease in salt loading to the lake.     

Effects of starvation on growth and reproductive apparatus of two immature freshwater snails Biomphalaria pfeifferi and Biomphalaria glabrata (Gastropoda: Planorbidae)

Starvation of immature snails of Biomphalaria pfeifferi and B. glabrata results in an arrest of growth and animals remain immature. Spermatogenesis is limited to spermatogonia (B.g.) or spermatocytes 1 (B.p.). The number and the size of oocytes remain inferior to that of controls. Animals show reduced genital tracts.Once feeding is restored, growth is resumed but wet weight and shell diameter do not reach the same level as in controls. Fecundity and gametogenesis do not differ from that in controls. Genital tracts weight is proportional to body weight.     

A kinetic model for the hydrolysis and synthesis of maltose, isomaltose, and maltotriose by glucoamylase

Kinetic results on the glucomylase-catalysed hydrolysis of maltose and maltotriose, and glucose polymerization into maltose and isomaltose up to 450 g/L total sugar concentration are presented. Whereas the enzyme has a faster hydrolytic and synthetic activity on alpha-(1-->4) than on alpha-(1-->6) linkages, at equilibrium, on the contrary, the isomaltose level which represents 15% (w/w) of the total sugar concentration at the highest investigated concentrations is much higher than the corresponding maltose level. Under a wide range of initial conditions, experimental results are adequately described by a new kinetic model with simple first- and second-order, or Michaelian-type, rate expressions for the reversible hydrolysis of maltotriose, maltose, and isomaltose. The model also accounts for the inhibition of hydrolysis by glucose, but does not consider the concentration of water which, under the present conditions, was not found kinetically limiting.     

Cellobiose metabolism in Erwinia: genetic study

Summary The study of mutants of Erwinia specifically unable to ferment cellobiose indicates that the mutations are clustered between arg and ile on the chromosome of this organism. In vivo cloning of the genes responsible for cellobiose utilization lead to a plasmid, pBEC2, which complements all Erwinia Clb^- specific mutants. When introduced into wild-type E. coli it allows this organism to use cellobiose, arbutin and salicin; it also complements bglB and bglC mutants of Escherichia coli indicating that arbutin and salicin utilization is due to the products of the pBEC2 cloned genes. From the characterization of mutants pleiotropically affected in the utilization of various carbon sources, including cellobiose, arbutin and salicin, it is proposed that the three--glucosides are substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS).