Evaluation of ion gradient-dependent H+ transport systems in isolated enterocytes from the chick

Summary The experiments reported here evaluate the capability of isolated intestinal epithelial cells to accomplish net H⁺ transport in response to imposed ion gradients. In most cases, the membrane potential was kept constant by means of a K⁺ plus valinomycin voltage clamp in order to prevent electrical coupling of ion fluxes. Net H⁺ flux across the cellular membrane was examined at pH 6.0 (the physiological lumenal pH) and at pH 7.4 using methylamine distribution or recordings of changes in media pH. Results from both techniques suggest that the cells have an Na⁺/H⁺ exchange system in the plasma membrane that is capable of rapid and sustained changes in intracellular pH in response to an imposed Na⁺ gradient. The kinetics of the Na⁺/H⁺ exchange reaction at pH 6.0 [K _t for Na⁺=57mm,V _max=42 mmol H⁺/liter 3OMG (3-O-methylglucose) space/min] are dramatically different from those at pH 7.4 (K _t for Na⁺=15mm,V _max=1.7 mmol H⁺/liter 3OMG space/min). Experiments involving imposed K⁺ gradients suggest that these cells have negligible K⁺/H⁺ exchange capability. They exhibit limited but measurable H⁺ conductance. Anion exchange for base equivalents was not detected in experiments performed in media nominally free of bicarbonate.     

Transposon donor plasmids, based on ColIb-P9, for use in Pseudomonas putida and a variety of other gram negative bacteria

Summary The properties of pLG221, a derivative of the ColIb plasmid carrying the transposon Tn5 are described. This plasmid can be used to introduce Tn5 by conjugation from Escherichia coli into a variety of Gram negative bacteria outside the host range for maintenance of ColIb. Plasmid pLG221, and a similar plasmid pLG223 carrying Tn10 may be of general utility as vectors for transposon-mediated mutagenesis in a variety of Gram negative bacteria.     

Diterpenoids from Caesalpinia pulcherrima

Three new furanoditerpenoids of the caesalpin-type have been isolated from the roots of Caesalpinia pulcherrima. The structures of these compounds, vouacapen-5α-ol, 6β-cinnamoyl-7β-hydroxy-vouacapen-5α-ol and 8,9,11,14-didehydrovouacapen-5α-ol, were elucidated through interpretation of their spectral data. Sitosterol was also obtained.     

Stoichiometric translocation of adipocyte insulin receptors from the cell-surface to the cell-interior. Studies using a novel method to rapidly remove detergent and concentrate soluble receptors

A rapid one-step method was developed for harvesting and concentrating insulin receptors from solubilized adipocytes, which entails precipitating soluble receptors with polyethylene glycol and resuspending the receptor-containing pellet in a reduced volume of binding buffer. With this procedure 90-100% of receptors were recovered, while 80% of cellular protein was removed, thus resulting in a marked reduction of both ligand and receptor proteases and about a 5-fold purification of the receptor. More importantly, greater than 98% of the Triton X-100 detergent was removed during this procedure so that the reduced receptor affinity observed in solubilized extracts (due to detergent) was restored to normal. Reconstituted receptors exhibited normal binding characteristics similar to those observed for plasma membrane receptors. The general utility of our receptor precipitation-reconstitution method is highlighted by studies on insulin-induced translocation of receptors from the cell-surface to the cell-interior of adipocytes and studies on the assessment of the binding affinity of nascent intracellular receptors. The results of these studies are consistent with the following. 1) Insulin initiates endocytotic uptake of insulin receptors, which then recycle back to the cell-surface. 2) Chloroquine impairs the recycling of internalized receptors while preventing receptor degradation, resulting in the progressive trapping and accumulation of receptors within cells during insulin treatment. 3) Receptor translocation during acute insulin-induced down-regulation is stoichiometric in that receptors lost from the cell-surface can be quantitatively recovered within the cell-interior. 4) In the absence of ligand, these receptors within adipocytes are mainly newly synthesized receptors enroute to the cell-surface, and they possess an affinity similar, if not identical, to mature receptors on the plasma membrane.     

Experimental Methyl Mercury Neurotoxicity: Locus of Mercurial Inhibition of Brain Protein Synthesis In Vivo and In Vitro

Brain cell-free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration. In this report, we have identified the locus of mercurial inhibition of translation. Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the post-mitochondrial supernatant (PMS) harvested from the brain of young (10-20-day-old) rats. No mercurial-induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients. No difference in total RNA was found in the PMS. Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation. After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid-directed incorporation of [3H]phenylalanine. However, the in vivo inhibition was no longer observed when [3H]phenylalanyl-tRNAPhe replaced free [3H]phenylalanine in the incorporation assay. The formation of peptidyl[3H]puromycin revealed no difference from controls. There was significant mercurial inhibition of phenylalanyl-tRNA Phe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg-poisoned rats. These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.     

Multiline varieties and disease control

Summary A general model for the evolution of pathogen populations on mixtures or multilines is developed. This model is used to extend previous analyses of the effects of the widespread cultivation of multilines on the evolution of virulence in obligate parasites to mixtures of lines carrying different numbers of resistance genes. It is concluded that the composition of an equilibrium pathogen population growing on a multiline may vary within wide limits and the prinicipal determinant of its composition is the number of components in the multiline and the resistance genes they carry. Other factors of importance are (i) the relative contribution made by each host class (with different numbers of resistance genes) to the pathogen spore pool each generation; (ii) the levels of stabilizing selection against unnecessary virulence genes; and (iii) the way in which unnecessary genes for virulence combine to reduce pathogen fitness. Conditions for the fixation of avirulent biotypes in the pathogen population and the evolution of a pathogen superrace are given for multilines of various compositions.Paper No. 9246 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina. This investigation was supported in part by NIH Research Grant No. GM 11546 from the National Institute of General Medical Sciences     

The localization and rate of disappearance of a synaptic vesicle antigen following denervation

Summary A proteoglycan-specific antiserum has been used to monitor the effects of denervation in the electric organ of Torpedo marmorata. The antiserum was produced by injecting a highly purified synaptic vesicle fraction prepared from the electric organs of Torpedo marmorata. Following absorption the serum appears to be specific towards synaptic vesicles. The ultrastructural localization of the antigen determined by immuno-electron microscopy confirmed the specificity of the antiserum and showed that it did not crossreact with the proteoglycans of the basal lamina. The rate of disappearance of the vesicle proteoglycans following denervation was evaluated by means of the antiserum and was compared to the rate of disappearance of other vesicular and nerve terminal-associated markers. The results suggest that degeneration affects the vesicular constituents at varying rates resulting in a progressive disappearance of the entire functional capacity of the synaptic vesicles.     

Localization of Tamm-Horsfall-glycoprotein-like immunoreactivity in cultured baby-hamster kidney cells, shown by immunofluorescence and by light- and electron-microscopic immunoperoxidase techniques.

Tamm-Horsfall glycoprotein was isolated from hamster urine, and antiserum against it was produced in rabbits. IgG was isolated from the antiserum. Immunocytochemical methods were used to localize Tamm-Horsfall-like immunoreactivity in three substrains of baby-hamster kidney (BHK) cells. Indirect immunofluorescence techniques showed that, in two substrains (BHK-21/C13/2P and BHK-21/C13/3P), a proportion of the cells fluoresced brilliantly, whereas those of the third substrain (BHK-21/ICRF) were totally negative. Related findings were obtained by the immunoperoxidase optical-microscopic technique. From the results of immunoperoxidase techniques using the electron microscope, it was concluded that the substance was present in association with the plasma membranes of the reacting cells. Our data suggest that the line of baby-hamster kidney cells, BHK-21/C13, may contain cells of renal-tubular epithelial origin, and that the proportion of these may be variable from one subculture to another.     

Platelet immune complex interaction in pathogenesis of Kawasaki disease and childhood polyarteritis.

The role of platelets in the pathogenesis of vasculitis and the formation of coronary artery aneurysms was studied in 19 children with Kawasaki disease and five with polyarteritis. All patients with Kawasaki disease developed thrombocytosis in the third week of illness. The peak platelet count was significantly correlated (p less than 0.005) with the subsequent development of coronary artery aneurysms. The rise in platelet count was associated with the appearance in the circulation of a factor that induced aggregation and serotonin release in normal platelets. This factor was shown to be of high molecular weight, and its activity was lost at low pH--features suggestive of an immune complex. Immune complexes, detected by precipitation with polyethylene glycol, also appeared in the circulation as the platelet count increased. These complexes induced platelet aggregation, and there was a significant correlation (p less than 0.001) between the concentrations of IgG and IgA in the polyethylene glycol precipitated material and the platelet aggregating activity. Similar platelet aggregating activity was also detected in patients with polyarteritis but followed a different time course, persisting in the circulation for several months in association with continued disease activity. These findings imply that different mechanisms have a role in distinct phases of Kawasaki disease. The initial feverish phase (probably infective) is probably followed by an immune complex vasculitis that occurs when antibodies to the initiating agent appear in the circulation. The immune complexes aggregate platelets and induce release of serotonin. Platelet derived vasoactive mediators may increase vascular permeability and facilitate further deposition of complexes in the tissues.