The action of adrenoceptor agonists and antagonists on the guinea pig and dog trachea

The action of beta- and alpha-adrenoceptor agonists (isoprenaline, orciprenaline, noradrenaline, phenylephrine and ephedrine) and antagonists (propranolol, metipranolol, exaprolol, BL 445 and phentolamine) on the resting tension and cAMP level of the guinea pig and the mechanical and electrical activities of the dog trachea were studied. By activating beta 2-adrenoceptors, isoprenaline and orciprenaline relaxed the smooth muscle, elevated the membrane potential and attenuated the excitatory effect of histamine on membrane potential and muscle tension. Noradrenaline and phenylephrine, acting on alpha 1-receptors, did not affect the membrane potential and increased the basal tension of the dog trachea only insignificantly. Ephedrine, in high concentrations, however, hyperpolarized the smooth muscle membrane and relaxed the dog trachea, while it did not alter the cAMP level in the guinea pig preparations. It is, therefore unlikely that alpha 1-adrenoceptors play a major role in the excitation of the dog trachea under resting conditions whereas the participation of alpha 2-receptors in the mechanisms of adrenergic relaxation could not be ruled out. All the beta-adrenoceptor antagonists studied enhanced the action of low isoprenaline concentrations and competitively antagonized it in high concentrations. The order of their antagonistic potency in the guinea pig trachea was as follows: metipranolol greater than propranolol = exaprolol greater than or equal to BL 445. It was suggested that metipranolol and exaprolol are nonselective beta-adrenoceptor antagonists, similarly as propranolol, whereas BL 445 shown some beta 1-selectivity. In contrast to their antagonistic effects on the membrane activities and muscle tension, both histamine and isoprenaline increased the level of cAMP in smooth muscle cells and, when present simultaneously, their effect was additive. The mechanism of histamine-induced cAMP level elevation and the possible involvement of different subcellular compartments in the action of isoprenaline and histamine in relation to the contraction-relaxation cycle is discussed.     

7C female sterile mutants fail to accumulate early eggshell proteins necessary for later chorion morphogenesis in Drosophila

Seven noncomplementing female sterile mutations that affect eggshell assembly in Drosophila have been mapped to the 7C1-3 region of the X-chromosome. TEM of the mature eggshell of one of the alleles, fs(1)410, shows a lack of organization within the endochorion and an accumulation of electron dense material in the vitelline membrane of stage 14 eggchambers. SDS-PAGE of radiolabeled eggshell proteins shows that two proteins, s67 and s85, fail to accumulate in the fs(1)410 eggshell. In wild-type flies s85 is produced during stage 10 of oogenesis and then processed to s67 in stages 13 and 14. Neither s85 nor an additional stage 10 specific follicle cell protein (s130) are detected in fs(1)410 or four of the mutant alleles. Short-term labeling studies, analyses of in vitro translation products, and the simultaneous occurrence of s85 and s130 as electrophoretic variants in geographic fly strains indicate s85 is derived from s130. Although major biochemical differences appear in stage 10, mutant and wild-type eggshells are morphologically indistinguishable until stages 13-14. These results suggest that follicle cell proteins synthesized during the time of vitelline membrane deposition (stage 10) are important for proper assembly of the chorion layers during stages 13 and 14.     

Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure.

Methionine aminopeptidase (MAP) catalyzes the removal of amino-terminal methionine from proteins. The Escherichia coli map gene encoding this enzyme was cloned; it consists of 264 codons and encodes a monomeric enzyme of 29,333 daltons. In vitro analyses with purified enzyme indicated that MAP is a metallo-oligopeptidase with absolute specificity for the amino-terminal methionine. The methionine residues from the amino-terminal end of the recombinant proteins interleukin-2 (Met-Ala-Pro-IL-2) and ricin A (Met-Ile-Phe-ricin A) could be removed either in vitro with purified MAP enzyme or in vivo in MAP-hyperproducing strains of E. coli. In vitro analyses of the substrate preference of the E. coli MAP indicated that the residues adjacent to the initiation methionine could significantly influence the methionine cleavage process. This conclusion is consistent, in general, with the deduced specificity of the enzyme based on the analysis of known amino-terminal sequences of intracellular proteins (S. Tsunasawa, J. W. Stewart, and F. Sherman, J. Biol. Chem. 260:5382-5391, 1985).     

Transposon Mutants of Bradyrhizobium japonicum Altered in Attachment to Host Roots

Transposon mutants of Bradyrhizobium japonicum 110 ARS were produced and screened for changes in attachment ability. Mutant CFK4 produced twice as many piliated cells, attached in 2.5-fold-higher numbers to soybean root segments, and colonized roots in about 2-fold-higher numbers than did the parental strain, 110 ARS. Mutants CFK35 and CFK38 were reduced in their attachment about 2-fold and 3.5-fold, respectively. This corresponded to reductions in piliated cells in their populations, reduced reaction with anti-pilus antiserum, and reduced hydrophobic attachment. Mutants CFK4 and CFK38 nodulated soybeans at about the same level as the parent strain, but CFK35 induced only pseudonodules. Two-dimensional gel analyses of the proteins from the mutants showed relatively few changes in proteins.     

Covalent binding of 4-carbamoylbenzenediazonium chloride to deoxyguanine bases of DNA resulting in apparent irreversible inhibition of poly(adenosine diphosphoribose) polymerase at the nicotinamide binding site

The poly(adenosine diphosphoribose) polymerase activity of isolated liver nuclei was inhibited by 4-carbamoylbenzenediazonium chloride, referred to as 4-diazoniobenzamide, an effect that was dependent on the time of incubation and the concentration of the diazonium compound, with inhibition following first-order kinetics. The inhibition was not reversed by reisolation of nuclei and centrifugal washing, whereas the inhibition by benzamide or 4-aminobenzamide was completely reversible under these conditions. Simultaneous incubation of 4-diazoniobenzamide with benzamide prevented enzyme inhibition. The 4-diazoniobenzoic acid analogue was not inhibitory. The mechanism of action of 4-diazoniobenzamide was traced to a specific covalent binding to dGMP of DNA to form N2-[(4-carbamoylphenyl)azo]-2'-deoxyguanosine 5'-monophosphate. Coenzymic DNA, by tight association with the polymerase protein, fixes the -C(O)NH2 moiety of the adduct at the nicotinamide-binding site of the enzyme.     

Flow cytometric analysis of the recruitment of G0 cells in human epidermis in vivo following tape stripping

Tape stripping of human skin elicits a proliferative response of a synchronously-dividing group of cells. The progress of this cohort of cells has been monitored using two windows in the cell cycle, one located in mid-S phase and the other centred around G2 + M. The cellular DNA is measured with flow cytometry, the windows are defined by two ranges in the DNA histogram. The cohort can be described as the recruitment of cells from a pre-existing G0 compartment which consists of 76% of all proliferative cells. The duration of the S phase is calculated to be 10.2 hr and G2 + M phase 5.1 hr. The cell cycle time of 39 hr for normal human keratinocytes derived from these figures is in line with recent values obtained by different techniques.     

Benzamide-DNA interactions: deductions from binding, enzyme kinetics and from X-ray structural analysis of a 9-ethyladenine-benzamide adduct

The interaction of benzamide with the isolated components of calf thymus poly(ADP-ribose) polymerase and with liver nuclei has been investigated. A benzamide-agarose affinity gel matrix was prepared by coupling o-aminobenzoic acid with Affi-Gel 10, followed by amidation. The benzamide-agarose matrix bound the DNA that is coenzymic with poly(ADP-ribose) polymerase; the matrix, however, did not bind the purified poly(ADP-ribose) polymerase protein. A highly radioactive derivative of benzamide, the 125I-labelled adduct of o-aminobenzamide and the Bolton-Hunter reagent, was prepared and its binding to liver nuclear DNA, calf thymus DNA and specific coenzymic DNA of poly(ADP-ribose) polymerase was compared. The binding of labelled benzamide to coenzymic DNA was several-fold higher than its binding to unfractionated calf thymus DNA. A DNA-related enzyme inhibitory site of benzamide was demonstrated in a reconstructed poly(ADP-ribose) polymerase system, made up from purified enzyme protein and varying concentrations of a synthetic octadeoxynucleotide that serves as coenzyme. As a model for benzamide binding to DNA, a crystalline complex of 9-ethyladenine and benzamide was prepared and its X-ray crystallographic structure was determined; this indicated a specific hydrogen bond between an amide hydrogen atom and N-3 of adenine. The benzamide also formed a hydrogen bond to another benzamide molecule. The aromatic ring of benzamide does not intercalate between ethyladenine molecules, but lies nearly perpendicular to the planes of stacking ethyladenine molecules in a manner reminiscent of the binding of ethidium bromide to polynucleotides. Thus we have identified DNA as a site of binding of benzamide; this binding is critically dependent on the nature of the DNA and is high for coenzymic DNA that is isolated with the purified enzyme as a tightly associated species. A possible model for such binding has been suggested from the structural analysis of a benzamide-ethyladenine complex.     

Crossovers in two German cystic fibrosis families determine probe order for MET, 7C22 and XV-2c/CS.7

Summary We have followed the segregation of the probes pJ3.11, 7C22, pB79a, and MET through cystic fibrosis families in the German Democratic Republic with two affected sibs. Two families with a crossover between MET and the CF phenotype were detected. In one of these families recombination was also observed between the DNA probe 7C22 and CF, and between the markers XV-2c and CF, which suggests that XV-2c, MET and 7C22 are all on the same side of CF. The other MET recombinant family is informative with XV-2c and does not recombine, which excludes the genetic order XC-2c-MET-CF if multiple recombinant events are disregarded. These two families together demonstrate that recombinations may occur in a very small genetic interval, which has important implications for prenatal diagnosis based on data from linked markers.     

Effects of N-immissions on nutrient status and vitality ofPinus sylvestris near a hen-house

Near a hen house (50–600 m), vitality ofPinus sylvestris, N-, P-, K-, Ca-, Mg-contents of the needles, N-, Mg-, K-, Ca- and Al-contents in soil extracts and NH₃/NH ₄ ⁺ -contents of the air were determined. Damage symptoms occurred when N-immissions hit the canopy directly. In contrast no visible decline of the above ground plant could be observed if N was mainly deposited on the soil.     

Chromatographic and immunological evidence that chloroplastic and cytosolic pea (Pisum sativum L.) NADP-isocitrate dehydrogenases are distinct isoenzymes

Two NADP-isocitrate dehydrogenase isoenzymes designated as NADP-IDH₁ and NADP-IDH₂ (EC 1.1.1.42) were identified in pea (Pisum sativum) leaf extracts by diethylaminoethylcellulose chromatography. The predominant form was found to be NADP-IDH₁ while NADP-IDH₂ represented only about 4% of the total leaf enzyme activity. These enzymes share few common epitopes as NADP-IDH₂ was poorly recognized by the specific polyclonal antibodies raised against NADP-IDH₁, and as a consequence NADP-IDH₂ does not result from a post-translational modification of NADP-IDH₁. Subcellular fractionation and isolation of chloroplasts through a Percoll gradient, followed by the identification of the associated enzymes, showed that NADP-IDH₁ is restricted to the cytosol and NADP-IDH₂ to the chloroplasts. Compared with the cytosolic isoenzyme, NADP-IDH₂ was more thermolabile and exhibited a lower optimum pH. The data reported in this paper constitute the first report that the chloroplastic NADP-IDH and the cytosolic NADP-IDH are two distinct isoenzymes. The possible functions of the two isoenzymes are discussed.Abbreviations BSA bovine serum albumin - DEAE diethylaminoethyl - NADP-IDH NADP-isocitrate dehydrogenase - NADP-IDH₁ cytosolic NADP-IDH - NADP-IDH₂ chloroplastic NADP-IDH