Correlations between cyclic AMP binding and chemoreception in Paramecium

Paramecium tetraurelia is attracted to cyclic AMP, which probably, as other attractants, signifies the presence of food. Attraction to cyclic AMP was specific, saturable, and, therefore, likely to be receptor-mediated. In these studies, we measured the binding of cyclic [3H]AMP to whole cells and found it to be saturable, reversible, and displaying specificity similar to that of attraction. An HPLC method of separating nucleotides was devised and used to determine that external cyclic AMP was degraded in the absence of IBMX, a phosphodiesterase inhibitor, and that cyclic AMP was taken into the cells in small amounts. Since binding and attraction were subsequently measured in the presence of IMBX, it was cyclic AMP and not a degradation product that served as the attractant stimulus for Paramecium.     

The dose rate dependence of the relative biological effectiveness of 241Am versus 226Ra gamma rays

The survival of Chinese hamster cells exposed to 59.5 keV 241Am gamma rays was compared with that obtained after exposure to 226Ra gamma rays. The Fricke dosimeter in conjunction with the calculational techniques of transition-zone dosimetry was employed to determine the dose rates to the cells at the petri dish/growth medium interface. The dose rates to the cells ranged from 11 to 133 cGy/h. In all cases, cell survival versus dose was best described by a simple exponential function of dose. For both radiations, graphs of D0 versus dose rate show complex but similar patterns of peaks and valleys. As the curve for 241Am is displaced toward lower dose rates compared with that for 226Ra, the relative biological effectiveness of 241Am vs 226Ra varies considerably with dose rate, ranging from 1.7 at 20 cGy/h to 1.1 at 40 cGy/h to 1.6 at 50 cGy/h. This phenomenon may be due to the LET-dependent accumulation of cells at the G2 + M interface in the cell cycle. The mean unrestricted track-average LET of 241Am (3.7 keV/microns) is 12 times higher than that for 226Ra (0.31 keV/microns) but only one-fifth that of carbon ions (18 keV/microns) for which G2 + M pile-up is observed. Application of the in vitro data derived from this study to the clinical situation, where the dose rate decreases rapidly with distance from the source, suggests that, dose for dose, 241Am will produce results little different from those obtained with 226Ra.     

Synthesis of trisaccharides containing 3-deoxy-D-manno-2-octulosonic acid residues related to the KDO-region of enterobacterial lipopolysaccharides

Glycosylation of methyl (allyl 7,8-O-carbonyl-3-deoxy-alpha-D-manno-2-octulo-pyranosid)o nate with an alpha-(2----4) linked per-O-acetylated KDO-disaccharide bromide derivative under Helferich conditions afforded a 2:1 mixture of the alpha- and beta-linked trisaccharide derivatives in 50% yield. Removal of the protecting groups gave sodium O-[sodium (3-deoxy-alpha-D-manno-2-octulopyranosyl)onate]-(2----4)-O-[ sodium (3-deoxy-alpha- and -beta-D-manno-2-octulopyranosyl)onate]-(2----4)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate. Radical copolymerization of the allyl glycosides afforded artificial antigens, suitable for defining antibody specificities directed against the KDO-region of enterobacterial lipopolysaccharides.     

A crystallographic study of the glutathione binding site of glutathione reductase at 0.3-nm resolution

The binding of glutathione, some related molecules and two redox compounds to crystals of glutathione reductase has been investigated by X-ray crystallography at 0.3-nm resolution. Models for several bound ligands have been built and subjected to crystallographic refinement. The results clearly show the residues involved in glutathione binding as well as the geometry of the disulfide exchange. Glutathione-I is bound in a V-shaped conformation, while glutathione-II is extended. The zwitterionic glutamyl end of glutathione-II appears to be the most tightly bound part of the substrate. All glutathione conjugates and derivatives studied show binding dominated by the interactions at this site. In the reduced enzyme, glutathione-I forms a mixed disulfide intermediate with Cys58. Other structural changes are observed on reduction of the enzyme, and it is demonstrated that the carboxamidomethylated enzyme is a good model for the reduced species. Lipoate, a weak substrate of the enzyme, assumes a defined binding site where its disulfide is available for being attacked by Cys58-S gamma. A second region with affinity for a number of compounds has been found in a large cavity at the dimer interface of the enzyme. No functional role of this site is known.     

Assay of L-3-hydroxyacyl-coenzyme A dehydrogenase with substrates of different chain lengths

A method for assaying L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) which permits rate measurements with L-3-hydroxyacyl-CoA substrates of various chain lengths at physiological pH is described. The method is based on a coupled assay system in which 3-ketoacyl-CoA compounds formed by the dehydrogenase are cleaved by 3-ketoacyl-CoA thiolase (EC 2.3.1.16) in the presence of CoASH. The advantages of this assay method are its irreversibility and elimination of product inhibition. The assay procedure was used to determine the kinetic parameters (Km, Vmax) of pig heart L-3-hydroxyacyl-CoA dehydrogenase with several substrates of various chain lengths. The data obtained show the enzyme to be most active with medium-chain substrates whereas Km values for medium-chain and long-chain substrates are almost equal but much lower than those previously reported.     

Atrial natriuretic peptide in lymphoid organs of various species

1. Evidence for the occurrence of atrial natriuretic peptide (ANP) in various lymphoid organs of different species (rat, mouse, pig, chicken) is provided. 2. ANP precursor material (1-126) as well the physiologically active ANP (99-126), were identified by chromatographic analysis and RIA in extracts of thymus, spleen and lymph nodes of rat, mouse and pig. 3. mRNA coding for ANP was demonstrated both in the thymus and in isolated thymocytes of these species. Furthermore, mRNA for ANP was detected in spleen and lymph nodes (rat and pig). 4. The bursa of Fabricius, thymus glands and spleen of chickens were also shown to express mRNA coding for ANP. 5. These findings provide a firm basis for a link of ANP to the immune system, a novel aspect of possible biological functions of this peptide.     

Soluble and membrane-bound ferrisiderophore reductases of Escherichia coli K-12

After uptake of microbial ferrisiderophores, iron is assumed to be released by reduction. Two ferrisiderophore-reductase activities were identified in Escherichia coli K-12. They differed in cellular location, susceptibility to amytal, and competition between oxygen and ferrichrome-iron(III) reduction. The ferrisiderophore reductase associated with the 40,000×g sediment (membrane-bound enzyme) was inhibited by 10 mM amytal in contrast to the ferrisiderophore reductase present in the 100,000×g supernatant (soluble enzyme). Reduction by the membrane-bound enzyme followed sigmoid kinetics, but was biphasic in the case of the soluble enzyme. The soluble reductase could be assigned to a protein consisting of a single polypeptide of M _r 26000. Reduction of iron(III) by the purified enzyme depended on the addition of NADH or NADPH which were equally active reductants. The cofactor FMN and to a lesser degree FAD stimulated the reaction. Substrate specificity of the soluble reductase was low. In addition to the hydroxamate siderophores arthrobactin, schizokinen, fusigen, aerobactin, ferrichrome, ferrioxamine B, coprogen, and ferrichrome A, the iron(III) complexes of synthetic catecholates, dihydroxy benzoic acid, and dicitrate, as well as carrier-free iron(III) were accepted as substrates. Both ferrisiderophore reductases were not controlled by the fur regulatory system and were not suppressed by anaerobic growth.Abbreviations DHB dihydroxybenzoic acid - MECAM 1,3,5-N,N,N-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene - MECAMS 2,3-dihydroxy-5-sulfonyl-derivative of MECAM     

Heterozygous expression of X-linked chondrodysplasia punctata

Summary Two females showing partial expression of X-linked chondrodysplasia punctata were identified in a family. Bone dysplasia was caused by an aberrant X chromosome that had an inverse duplication of the segment Xp21.2–Xp22.2 and a deletion of Xp22.3-Xpter. To characterise the aberrant X chromosome, dosage blots were performed on genomic DNA from a carrier using a number of X-linked probes. Anonymous sequences from Xp21.2–Xp22.2 to which probes D2, 99.61, C7, pERT87-15, and 754 bind were duplicated on the aberrant X chromosome. The proposita was heterozygous for all these markers. Dosage blots also showed that the loci for steroid sulfatase and the cell surface antigen 12E7 (MIC2) were deleted as expected from the cytogenetic results. Mouse human cell hybrids were constructed that retained the normal X in the active state. Analysis of these hybrid clones for the markers from Xp21.2–Xp22.2 revealed that all the alleles of the informative markers, present in a single dosage in the genomic DNA, were carried on the normal X chromosome of the proposita. The duplicated X chromosome therefore had two identical alleles, indicating that the aberration resulted from an intrachromosomal rearrangement.     

Probing FhuA''-''PhoA fusion proteins for the study of FhuA export into the cell envelope of Escherichia coli K12

Summary The FhuA protein (formerly TonA) is located in the outer membrane of Escherichia coli K12. Fusions between fhuA and phoA genes were constructed. They determined proteins containing a truncated but still active alkaline phosphatase of constant size and a variable FhuA portion which ranged from 11%–90% of the mature FhuA protein. The fusion sites were nearly randomly distributed along the FhuA protein. The FhuA segments directed the secretion of the truncated alkaline phosphatase across the cytoplasmic membrane. The fusion proteins were proteolytically degraded up to the size of alkaline phosphatase and no longer reacted with anti-FhuA antibodies. The fusion proteins were more stable in lon and pep mutants lacking cytoplasmic protease and peptidases, respectively. The larger fusion proteins above a molecular weight of 64000 dalton were predominantly found in the outer membrane fraction. They were degraded by trypsin when cells were converted to spheroplasts so that trypsin gained access to the periplasm. In contrast, FhuA protein in the outer membrane was largely resistant to trypsin. It is concluded that the larger FhuA-PhoA fusion proteins were associated with, but not properly integrated into, the outer membrane.     

Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.