β-adrenoceptors in human tracheal smooth muscle: characteristics of binding and relaxation

Specific binding of [¹²⁵I]-(−)-cyanopindolol to human tracheal smooth muscle membranes was saturable, stereo-selective and of high affinity (K_d=5.3±0.9 pmol/l and R_T=78±7fmol/g tissue). The β₁-selective antagonists atenolol and LK 203-030 inhibited specific [¹²⁵I]-(−)-cyanopindolol binding according to a one binding site model with low affinity in nearly all subjects, pointing to a homogeneous β₂-adrenoceptor population. In one subject using LK 203-030 a small β-adrenoceptor subpopulation could be demonstrated. The beta-mimetics isoprenaline, fenoterol, salbutamol and terbutaline recognized high and low affinity agonist binding sites. Isoprenaline's pK_H- and pK_L- values for the high and low affinity sites were 8.0±0.2 and 5.9±0.3 respectively. In functional experiments isoprenaline relaxed tracheal smooth muscle strips having intrinsic tone with a pD₂-value of 6.63±0.19.     

The binding of binuclear platinum(II)-terpyridine complexes to DNA.

The binding of platinum (II)-terpyridine complexes to DNA was studied by using equilibrium dialysis. Optical absorption methods were used to measure the ability of the ligands to aggregate in aqueous buffer. Scatchard plots for the binding of the monomeric [Pt(terpy)SC4H9]+ cation to DNA at I0.01 are curvilinear, concave upwards, suggesting two modes of binding. The association constant decreases at higher ionic strengths, consistent with polyelectrolyte theory, and 1.1 cations are released per bound ligand molecule. The association constants of the binuclear ligands [Pt(terpy)S[CH2]4S(terpy)Pt]2+ and [Pt(terpy)S[CH2]6S(terpy)Pt]2+ are 8 and 23 times larger respectively than the affinity of the monomer. For the latter binuclear derivative the increase may be ascribed to bifunctional reaction. Differential dialysis experiments with DNAs of differing base composition show that [Pt(terpy)SC4H9]+ has a requirement for a single G X C base-pair at the highest-affinity site. However, in the binuclear ligands chromophore specificity is severely compromised. Similar experiments indicate that 9-aminoacridine and selected methylene-linked diacridines show no significant sequence selectivity.     

Synergism between eosinophil cationic protein and oxygen metabolites in killing of schistosomula of Schistosoma mansoni

To study the cytotoxic reactions responsible for mediating eosinophil damage to schistosomula of Schistosoma mansoni, we have used cytoplasts (eosinophil or neutrophil vesicles devoid of granules and nuclei, with an intact oxidase in their plasma membrane) in combination with purified eosinophil cationic protein (ECP) or major basic protein (MBP) in a cytotoxicity test toward schistosomula. Suboptimal concentrations of ECP (10(-6) M) or MBP (10(-6) M) resulting in less than 10% killing were used in combination with cytoplasts. Cytoplasts alone in the presence of immune serum tested over a wide range of cytoplast:schistosomula ratios generated superoxide and hydrogen peroxide, but were unable to damage schistosomula. However, when a suboptimal ECP concentration (10(-6) M) was combined with neutroplasts or eosinoplasts, 43.9% +/- 8.5 (n = 7) and 24.7% +/- 9.8 (n = 3), respectively, of the schistosomula were killed. Oxygen metabolites were responsible for the synergism, because cytoplasts from a patient with chronic granulomatous disease were unable to act in synergy with ECP. In contrast to ECP, no synergism was found between cytoplasts and MBP (10(-6) to 2 X 10(-5)M). These results show that oxygen metabolites are important for the killing of schistosomula by lowering the concentration of ECP needed to inflict damage.     

Biological characterization of purified native 20-kDa human growth hormone

Because of the propensity of the 20-kDa variant of human growth hormone (GH) to aggregate with itself and with 22-kDa human GH, it has been difficult to prepare monomeric 20-kDa GH in highly purified form. This has been a major complicating factor in determining whether 20-kDa GH has a biological activity profile distinct from that of 22-kDa GH. In the present study, native 20-kDa GH was isolated from a human GH dimer concentrate and purified by a procedure that included column electrophoresis in agarose suspension as a final separation step. This procedure yielded highly purified monomeric 20-kDa GH, which was contaminated to an extent of less than 1% with 22-kDa GH, and which exhibited only a small degree of dimerization upon storage. The native 20-kDa GH was quite active in stimulating growth in hypophysectomized rats, when growth was assessed by body weight gain, longitudinal bone growth, the stimulation of sulfation of cartilage, and the elevation of serum IGF-1 level. However, in all of these growth assays, the 20-kDa GH was somewhat less active than the native 22-kDa GH to which it was compared; e.g., in the body weight gain and longitudinal bone growth assays, it had an estimated potency of 0.6 relative to the 22-kDa GH. The 20-kDa GH exhibited substantial diabetogenic activity when tested for the ability to raise fasting blood glucose concentration and to impair glucose tolerance in ob/ob mice. Also, the native 20-kDa GH had significant in vitro insulin-like activity, although its potency was approximately 20% that of the native 22-kDa GH to which it was compared. Thus, the biological activity profile of native 20-kDa GH differs from that of 22-kDa GH primarily in that insulin-like activity is markedly attenuated.     

Effects of adrenergic agonists and mitochondrial energy state on the Ca2+ transport systems of mitochondria

This study investigates the effects of adrenergic agonists and mitochondrial energy state on the activities of the Ca2+ transport systems of female rat liver mitochondria. Tissue perfusion with the alpha-adrenergic agonist phenylephrine and with adrenaline, but not with the beta-adrenergic agonist isoprenaline, induced significant activation of the uniporter and the respiratory chain. Uniporter activation was evident under two sets of experimental conditions that excluded influences of delta psi, i.e., at high delta psi, where uniporter activity was delta psi independent, and at low delta psi, where uniporter conductance was measured. Preincubation of mitochondria with extracts from phenylephrine-perfused tissue quantitatively reproduced uniporter activation when comparison was made with mitochondria treated similarly with extracts from tissue perfused without agonist. Similar, but more extensive, data were obtained with heart mitochondria pretreated with extracts from hearts perfused with the alpha-adrenergic agonist methoxamine. Phenylephrine did not affect Ca2+ efflux mediated by the Na+-Ca2+ carrier or the Na+-independent system. In contrast, the liver mitochondrial Na+-Ca2+ carrier was activated by tissue perfusion with isoprenaline; the Na+-independent system was unaffected. Na+-Ca2+ carrier activation was not associated with any change in a number of basic bioenergetic parameters. It is concluded that the Ca2+ transport systems of liver mitochondria may be controlled in an opposing manner by alpha-adrenergic agonists (promotion of Ca2+ influx) and beta-adrenergic agonists (promotion of Ca2+ efflux). At delta psi values greater than 110 mV, the Na+-independent system was activated by increase in delta psi; the uniporter and Na+-Ca2+ carrier activities were insensitive to delta psi changes in this range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sarcomere length uniformity determined from three-dimensional reconstructions of resting isolated heart cell striation patterns.

A- and I-band striation positions have been obtained, three-dimensionally reconstructed, and statistically analyzed from the volumes of resting isolated heart cells. Striation patterns from optically discrete subvolumes are imaged along the length of these myocytes with a computer-interfaced optical microscope imaging system. Planar striation maps are reconstructed by the computer from sequentially obtained striation pattern images displaced across the width or depth of the cell in controlled steps. Multiple planar maps are combined to form full three-dimensional (3-D) reconstructions that illustrate the sarcomeric structure and ordering throughout the volume of the cell. These reconstructions demonstrate a high degree of striation registration throughout most regions of cardiac cells. The striation registration is often slightly (less than 10 degrees) skewed across the width or depth of nearly every cell and is occasionally disrupted between adjacent groups of sarcomeres. These disruptions in registration are always associated with the locations of the nuclei. Rigorous statistical analyses indicate small volumetric regions of the cell delineated by these disruptions can have significantly (0.014-0.113 micron) shorter or longer average sarcomere length periodicities. Unlike skeletal muscle "fibrillenstruktur," these data from cardiac cells exhibit no evidence of helical packing schemes for sarcomere order. These observations suggest that the relatively large nuclei displace and disrupt the normal registration of the sarcomeres, which is probably mediated by internal cytoskeletal structures.     

Variables of myocardial backdiffusion,determined with 17-IODO-131 heptadecanoic acid in the normal dog heart

Summary Under normal and ischemic conditions backdiffusion of radiolabeled non-esterified fatty acids (NEFA) has been demonstrated. In the fasted normal canine heart the extraction fraction (EF) during interventions with glucose or lactate loading, vasodilation, and metabolic level augmentation was determined, and compared with the control EF. Backdiffusion alterations were deduced from the EF changes. After iv injection of 17-iodo-131 heptadecanoic acid (IHDA), 11 blood samples were drawn from aorta and coronary sinus in a time period of 60 minutes. In the control and vasodilation group the EF slowly decreased from 40 to 10%. In contrast, the EF in the noradrenaline group was constant. During glucose and lactate infusion the EF became negative within 10 min and remained negative. These results suggest that during physiological circumstances backdiffusion is determined by the metabolic level of the heart and its substrate availability.     

Single-stranded DNA used as an efficient new vehicle for transformation of plant protoplasts

In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3–10 fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts form recalcitrant plant species.Abbreviations ss single-stranded - ds double-stranded - CAT chloramphenicol acetyl transferase - NPTII neomycin phosphotransferase II - RT room temperature     

Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes

In this paper we describe the organization and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol-4-reductase (DFR) in Petunia hybrida. A nearly full-size DFR cDNA clone (1.5kb), isolated from a corolla-specific cDNA library was compared at the nucleotide level with the pallida gene from Antirrhinum majus and at the amino acid level with enzymes encoded by the pallida gene and the A1 gene from Zea mays.The P. hybrida and A. majus DFR genes transcribed in flowers contain 5 introns, at identical positions; the three introns of the A1 gene from Z. mays coincide with first three introns of the other two species. P. hybrida line V30 harbours three DFR genes (A, B, C) which were mapped by RFLP analysis on three different chromosomes (IV, II and VI respectively).Steady-state levels of DFR mRNA in the line V30 follow the same pattern during development as chalcone synthase (CHS) and chalcone flavanone isomerase (CHI) mRNA. Six mutants that accumulate dihydroflavonols in mature flowers were subjected to Northern blot analysis for the presence of DFR mRNA. Five of these mutants lack detectable levels of DFR mRNA. Four of these five also show drastically reduced levels of activity for the enzyme UDPG: flavonoid-3-O-glucosyltransferase (UFGT), which carries out the next step in flavonoid biosynthesis; these mutants might be considered as containing lesions in regulatory genes, controlling the expression of the structural genes in this part of the flavonoid biosynthetic pathway. Only the an6 mutant shows no detectable DFR mRNA but a wild-type level for UFGT activity. Since both an6 and DFR-A are located on chromosome IV and DFR-A is transcribed in floral tissues, it is postulated that the An6 locus contains the DFR structural gene. The an9 mutant shows a wild-type level of DFR mRNA and a wild-type UFGT activity.