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.     

Mechanism of inhibition of net ion transport across frog corneal epithelium by calcium channel antagonists

Summary In the isolated bullfrog cornea, three calcium channel antagonists had dose-dependent inhibitory effects on the Cl-originated short-circuit current (SCC). Their order of decreasing potency was bepridil, verapamil and diltiazem. One millimolar diltiazem inhibited the SCC by 98% and subsequent incubation with the calcium ionophore A23187 had no restorative effect. Increasing the bathing solution Ca concentration from 0.05 to 15mm, however, decreased diltiazem's inhibitory efficacy. This antagonist depolarized the intracellular potential differenceV _m from –54 to –18 mV (tear: reference) and the voltage divider ratioFR ₀ decreased from 0.58 to 0.30, suggesting an increase in basolateral membrane electrical resistance. Additional indication of a basolateral membrane effect by the drug was that preincubation with 10⁵ m amphotericin B in Cl-free Ringer's did not eliminate the inhibitory effect of the drug on the Na- and K-elicited SCC. In the absence of amphotericin B in Cl-free Ringer's (SCC=0), 1 ×10³ m diltiazem depolarized theV _m from –78 to –9 mV suggesting that the increase in basolateral membrane resistance was due to K channel blockade. Diltiazem (1×10³ m) significantly decreased cyclic AMP content; however, isoproterenol in the presence of the drug increased cyclic AMP fourfold without having any restorative effect on the inhibited SCC. Therefore, the inhibition of the Cl-originated SCC resulting from an increase in basolateral membrane K resistance is not caused by a decline in cyclic AMP content. In plasma membrane-enriched fractions prepared from broken cell preparations of bovine corneal epithelium, 1×10³ m diltiazem had no inhibitory effects on either Na,K-ATPase or Ca,Mg-ATPase activities. These latter effects further point to the selectivity of diltiazem as an inhibitor of K-channel activity, but do not preclude a Ca-channel blocker effect by the drug in the micromolar range.     

The mechanistic nature of the membrane potential dependence of sodium-sugar cotransport in small intestine

Summary Methods are described which demonstrate the use of unidirectional influx of¹⁴C-tetraphenylphosphonium (¹⁴C-TPP⁺) into isolated intestinal epithelial cells as a quantitative sensor of the magnitude of membrane potentials created by experimentally imposed ion gradients. Using this technique the quantitative relationship between membrane potential () and Na⁺-dependent sugar influx was determined for these cells at various Na⁺ and -methylglucoside (-MG) concentrations. The results show a high degree of dependence for the transport Michaelis constant but a maximum velocity for transport which is independent of . No transinhibition by intracellular sugar (40mm) can be detected. Sugar influx in the absence of Na⁺ is insensitive to 1.3mm phlorizin and independent of . The mechanistic implications of these results were evaluated using the quality of fit between calculated and experimentally observed kinetic constants for rate equations derived from several transport models. The analysis shows that for models in which translocation is the potential-dependent step the free carrier cannot be neutral. If it is anionic, the transporter must be functionally asymmetric. A model in which Na⁺ binding is the potential-dependent step (Na⁺ well concept) also provides an appropriate kinetic fit to the experimental data, and must be considered as a possible mechanistic basis for function of the system.     

Modulation of carrier-mediated uptake of abscisic acid by methyl jasmonate in Phaseolus coccineus L

The effects of methyl jasmonate and jasmonic acid on uptake of abscisic acid (ABA) by suspension-cultured runner-bean cells and subapical runner-bean root segments have been investigated. Increasing concentrations of methyl jasmonate inhibit ABA uptake by the cultured cells with a K _i of 22±3 M. This is not due to cytoplasmic acidification or to effects on metabolism of ABA, and is not additive with inhibition of radioactive ABA uptake by nonradioactive ABA. Uptake of indol-3-yl acetic acid (IAA) is unaffected by methyl jasmonate. The maximum effect of nonradioactive ABA in inhibiting uptake of radioactive ABA, previously shown to reflect saturation of an ABA carrier, is generally greater than the effect of maximally inhibitory concentrations of methyl jasmonate. Similar results were obtained with root segments, but longer incubation times were necessary to observe inhibitory effects of methyl jasmonate. Demethylation of methyl jasmonate to jasmonic acid does not appear to be required since similar concentrations of jasmonic acid had no observable direct effect on ABA uptake other than that attributable to cytoplasmic acidification. Histidine reagents, a proton ionophore and acidic external pH all affect in parallel the inhibition by methyl jasmonate and nonradioactive ABA of uptake of radioactive ABA by the cultured cells. There is no effect of ABA or nonradioactive methyl jasmonate on uptake of radioactive methyl jasmonate by the cultured cells. It is proposed that methyl jasmonate interacts with the ABA carrier. Various models for this interaction are discussed.Abbreviations ABA abscisic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - IAA indol-3-yl acetic acid     

Trypanosoma brucei: analysis of relapsing populations in sensitive and resistant breeds of cattle

The clone DiTat 1.1 of Trypanosoma brucei brucei was injected into four bovids, and clones obtained from successive waves of parasitemia were used to study the expressed variant-specific surface glycoprotein repertoire. Twenty-four clones were obtained which could be classified into 12 different variable antigen types, in addition to the clone injected, using agglutination or immunofluorescence with monospecific antisera. The variable surface glycoproteins of the 25 clones were extracted using the detergent octyl-beta-D-glucopyranoside in the presence of the protease inhibitor, N-cbz-L-phenylalaninechloromethylketone. The molecular weights varied from 52,000 to 69,000 and the pI from 5.0 to 8.8. The virulence of 14 clones representing 13 variable antigen types was ascertained in mice. The mean survival time ranged from 20.5 to 43.0 days. Clones isolated from early peaks of parasitemia in the bovid were the most virulent while clones derived from later peaks were less virulent. It seems that organisms of diminishing virulence appear in bovids, leading to self-cure of the disease. All clones were sensitive to human serum in a blood infectivity inhibition test. Antibody against all virulent clones appeared in 20 cattle (10 Zebus, 10 Baoulés) which had been injected with T. brucei DiTat 1.1. There was no evidence for parasites of high or low virulence being preferentially expressed in resistant or sensitive hosts.     

Identification of the peptide bond cleaved during activation of human Clr

CNBr cleavage of unreduced proenzyme Clr yielded fragment CP2b, isolated by gel filtration and highpressure gel permeation chromatography. This fragment (˜ M_τ 55000) comprised at least 4 disulphidelinked peptides, which were separated by gel filtration after reduction and alkylation. Peptide CP2bRA4, overlapping the A- and B-chain regions in proenzyme Clr was digested by V8 staphylococcal protease, and the digest separated by reversed-phase HPLC. N-terminal sequence analysis of peptide CP2bRA4SP9 established that Clr activation involves the cleavage of a single Arg-Ile bond, located in the sequence: Gln-Arg-Gln-Arg-Ile-Ile-Gly-Gly     

Efficient separation of subfossil Chironomidae from lake sediments

Electron transport system (ETS) activity, CO₂ evolution, O₂ consumption, N₂-fixation (C₂H₂ reduction) and methanogenesis were appropriately measured in aerobic and anaerobically incubated sediment at 4, 10 and 20 ° C to better characterize these activities under different incubation conditions. ETS activity was always higher in the aerobically incubated sediment at all three incubation temperatures, whereas (C₂H₂ reduction was always greater in the anaerobic sediment. Carbon dioxide evolution was detected only in the aerobic sediment at 10 and 20 ° C but not at 4 ° C. Methane evolution in anaerobic sediment increased gradually with an increase in the incubation temperature.     

Uptake of free amino acids by the diatom,Melosira mediocris

Individual ¹⁴C-labelled amino acids are rapidly removed from dilute solution in artificial sea water (0.2 mol 1^–1) by suspensions of Meliosira medocris. The rate of disappearance of radioactivity corresponds closely to removal of primary amines as determined by measurement of the rate of decrease of fluorescamine-positive material. Net removal of naturally occurring free amino acids from the sea water habitat from which the alga was isolated is demonstrated using high performance liquid chromatography. Removal of amino acids from natural sources makes a significant contribution to the carbon requirements of the alga as well as supplying significant amounts of amino nitrogen.     

Axonal Transport of Glycoconjugates in the Rat Visual System

Long-Evans rats at 45 days of age were injected intraocularly with 25 mu Ci of [3H]glucosamine. Incorporation of radioactivity into retinal gangliosides, glycoproteins, and glycosaminoglycans (GAGs) was determined at various times after injection. Portions of all three classes of radioactive macromolecules were committed to rapid axonal transport in the retinal ganglion cells. With respect to gangliosides about 60% of those synthesized in the retina were retained in that structure, 30% were committed to transport to regions containing the nerve terminal structures (lateral geniculate body and superior colliculus), and about 10% were deposited in stationary structures of the axons (optic nerve and tract). With the exception of ganglioside GD3 the molecular species distribution of gangliosides synthesized in the retina matched that committed to transport. In contrast to gangliosides a smaller fraction of newly synthesized retinal glycoprotein (less than 12% of that synthesized in the retina) was committed to rapid transport to nerve ending regions and only about 0.5% was retained in the nerve and tract. The molecular-weight distribution of glycoproteins committed to transport differed quantitatively from that of the retina. With respect to GAGs an even smaller portion (1-2%) of that synthesized in the retina was committed to rapid transport; of this portion almost all was recovered in nerve terminal-containing structures. A constant proportion of each retinal GAG species was transported to the superior colliculus. We suggest that most of the retinal gangliosides are synthesized in neurons and preferentially in ganglion cells (possibly a function of the large surface membrane area supported by these cells). Subcellular fractionation experiments indicated that transported gangliosides, glycoproteins, and GAGs may be preferentially distributed into different subcellular compartments.