Inhibition of surfactant release from isolated type II cells by compound 4880

Release of [³H]phosphatidylcholine from pulmonary Type II epithelial cells was stimulated by terbutaline, forskolin and cytochalasin D. Compound $\text{48}\text{80}$ inhibited both basal and agonist-stimulated release of [³H]PC. The IC₅₀ for inhibition by compound $\text{48}\text{80}$ was 1–2 μg/ml, and was similar for inhibition of both basal and stimulated release of [³H]phosphatidylcholine. Inhibitory effects of $\text{48}\text{80}$ were noted following a 1 h exposure to compound $\text{48}\text{80}$ and persisted up to 3 h. The inhibitory effect of compound $\text{48}\text{80}$ was entirely reversed by removing compound $\text{48}\text{80}$ from the external milieu. Compound $\text{48}\text{80}$ had no effect on cytosolic cyclic AMP levels or lactate dehydrogenase release. Inhibition of surfactant release produced by compound $\text{48}\text{80}$ was unaffected by changes in extracellular calcium concentrations. Compound $\text{48}\text{80}$ is a non-toxic inhibitor of phosphatidylcholine release from Type II epithelial cells.     

Intrinsic factor secretion from isolated human gastric mucosal cells

Human gastric mucosal cells were isolated from the resected fundic mucosa of peptic ulcer patients. The intracellular content and secretion of intrinsic factor were estimated by binding to cyano[⁵⁷Co]cobalamin. The content was maximal in the enriched parietal cell fraction which also displayed the highest H⁺ production as measured by amino[¹⁴C]pyrine uptake. Secretagogues evoked full response after 15 min of incubation: pentagastrin (181% of basal secretion), carbachol (208%), histamine (250%) and dibutyryl cyclic adenosine monophosphate (304%). The phosphodiesterase inhibitor isobutylmethylxanthine was slightly more effective even than dibutyryl cAMP. The response to histamine was abolished by ranitidine, indicating activation of adenylate cyclase via histamine H₂ receptors, but remained unaffected by atropine, which in turn blocked the carbachol effect, whereas ranitidine was ineffective. The mean formation rate was 8.4 fmol intrinsic factor/10⁶ cells per h under basal conditions and 14.3 fmol in response to histamine.     

Evidence for intracellular formation of angiotensins: coexistence of renin and angiotensin-converting enzyme in Leydig cells of rat testis

Leydig cells were purified from rat testes by discontinuous metrizamide density gradient and were shown to contain renin (EC 3.4.99.1), angiotensin-converting enzyme (dipeptidyl carboxypeptidase, (EC 3.4.15.1), and the peptide hormone angiotensins I, II and III as determined by the combined HPLC and radioimmunoassay. In germinal cells only angiotensin II (AII) was found at a significant level. These findings provide evidence for intracellular formation of AII in testicular cells and demonstrate that an intracellular renin-angiotensin system exists in normal non-transformed cells.     

Brain CCK receptors are structurally distinct from pancreas CCK receptors

Brain and pancreas cholecystokinin (CCK) receptors differ markedly in their selectivity for CCK analogs. To determine the size and subunit structure of the brain CCK receptor and compare it to that of the pancreas, 125I-CCK33 was covalently cross-linked with ultraviolet light to its receptor on mouse brain particles and purified pancreatic plasma membranes. When CCK was crosslinked to brain membranes, a single consistent major labeled protein band of Mr = 55,000 was observed in both the presence and the absence of DTT. These data with brain receptors contrast to results with pancreatic receptors where two bands of Mr = 120,000 and 80,000 are labeled in the absence and presence of DTT, respectively. These studies indicate, therefore, that the brain and pancreas CCK receptors are structurally and functionally distinct.     

ATP and the processivity of Xenopus laevis DNA polymerase α

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase α from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase α to DNA and conclude that ATP increases the processivity of the enzyme.     

Resolution of multiple fluorescence lifetimes in heterogeneous systems by phase-modulation fluorometry

Procedures are described for the treatment of phase and modulation lifetime data in fluorescent systems having multiexponential decay. All computer procedures (called FIT programs) arise from the lifetime resolution theory for phase-modulation measurements (Weber, G (1981) J. Phys. Chem. 85, 949–953). The programs most successful in resolving heterogeneous lifetimes use a Monte Carlo approach in which phase and modulation lifetime data at three modulation frequencies are simultaneously utilized. These programs are shown to have more utility than the final closed form procedure presented by Weber (1981). The FIT routines are simple and require little computer time while yielding excellent results. To illustrate the applicability of these programs, defined binary (carbazole and pyrene) and ternary systems (carbazole, pyrene and POPOD) were examined. In most cases, the resolved lifetimes were within 5% of the independently measured value and the fractional fluorescence contributions were within 10% of that expected. These results demonstrate that phase-modulation measurements analyzed by appropriate computer programs are capable of solving for lifetimes in both binary and, in selected cases, ternary systems. An example is given from the recent literature (Dalbey, R., Weiel, J. and Yount, R.G. (1983) Biochemistry 22, 4696–4706) in which the above programs allowed the resolution of both binary and ternary lifetimes of a dansyl label on myosin, where Förster energy transfer was occuring. These lifetimes] were used to quantify changes in distances between two activity-related thiols on myosin upon the addition of Mg-ATP or its analogs.     

Field evaluation of symbiotic nitrogen fixation by rhizobial strains using15N methodology

Summary Small differences in N₂ fixation by nodulated soybeans (Glycine max. (L.) Merr.), inoculated with various strains ofRhizobium japonicum, were assessed in field experiments using¹⁵N methodology, and compared with yields of plant dry matter and total N. Percentage of plant-N derived from atmospheric N₂ and from fertilizer, and values of %¹⁵N atom excess had lower coefficients of variation than did total N and dry matter yield. Nevertheless the precision of estimates of kg N/ha fixed were sufficient to differentiate only the extremes of the range of strains tested, and there were discrepancies between ranking of strains based on % N derived from fertilizer and on total N yield.     

Endogenous proteinases modulate the function of the β-adrenergic receptor-adenylate cyclase system

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as p-azido-m-[^{125I]iodobenzylcarazolol}. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su− variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.     

An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on the cell growth status. Exponentially growing cells treated with 10^?3-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 × 10^?4. However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10^?3-0.6 LF/ml), the survival decreased to 2 × 10^?6 and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10^?3 mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.     

2-β-d-Glucopyranosyloxy-2-methylpropanol in Acacia sieberana var. woodii

A new diol glucoside, 2-β-d-glucopyranosyloxy-2-methylpropanol, the first reported naturally occurring monoglucoside of an aliphatic dihydric alcohol, was isolated from pods of Acacia sieberana var. woodii. Structure elucidation was based on ¹ H and ¹³C NMR spectroscopy, and enzymatic analyses. The compound was hydrolysed very slowly by almond β-glucosidase, but cleaved by a β-glucuronidase enzyme complex from Helix pomatia.