Effect of nonanoic acid and other short chain fatty acids on exchange properties in embryonic axes of Cicer arietinum during germination

Nonanoic acid, which inhibits germination in several seeds, enhanced ion efflux from embryonic axes of Cicer arietinum L., especially at temperatures above 25°C. Other short chain fatty acids had little effect on germination and ion leakage. Nonanoic acid also decreased uptake of ⁸⁶Rb⁺ and ²²Na⁺ and increased efflux of both isotopes from the embryonic axes into the incubation solution. Fusicoccin, which stimulates early germination in C. arietinum , counteracted the effects of nonanoic acid at both 25 and 30°C. These results suggest that nonanoic acid affects the integrity of plasmalemma and other membrane systems. Nonanoic acid thus inhibits cell elongation during early germination by disturbing ion exchange and inhibiting water uptake.     

Effect of calcium ion on cytoplasmic streaming during turgor regulation in a brackish water charophyte Lamprothamnium

Abstract The brackish water charophyte Lamprothamnium succinctum regulates its turgor pressure against changes in the external osmotic pressure. Upon hypotonic treatment, the rate of cytoplasmic streaming in the internodal cells fell to almost zero, and then recovered to the original value within 20 min. The decrease could be inhibited by lowering the external Ca²⁺ concentration in the hypotonic medium. Also, cytoplasmic streaming in tonoplast-free cells of L. succintum was sensitive to Ca²⁺ like freshwater charophyte. Thus, the concentration of free Ca²⁺ in the cytoplasm seems to increase transiently upon hypotonic treatment.     

Purification of a delayed hypersensitivity-inducing protein from Listeria monocytogenes

Abstract Cell-envelope fragments were prepared from Listeria monocytogenes L242, serotype 4b. Delayed hypersensitivity (DH)-inducing proteins were extracted with deoxycholate and separated into two fractions by filtration through a Sephacryl S-200 column equilibrated with deoxycholate buffer. The second peak eluting from the Sephacryl column was fractionated using ion exchange chromatography on a DEAE Sepharose CL-6B column in the presence of 6 M urea. A purified 20 400-Da protein which induced DH against L. monocytogenes was obtained by isocratic elution. Three other DH-inducing fractions containing several protein bands were eluted by a gradient of potassium thiocyanate (KSCN) in urea buffer. Our results indicate that denaturing conditions can be employed for the fractionation and purification of DH inducing proteins from L. monocytogenes . In addition, it is suggested that the procedure described might also be useful for the purification of other antigens involved in cellular immune reactions.     

One- and two-dimensional NMR spectral analysis of the consequences of single amino acid replacements in proteins

The traditional approach of using homologous sequences to elucidate the role of specific amino acid residues in protein structure and function becomes more meaningful as the number of differences is minimized, with the limit being alteration of a single residue. For small proteins in solution, NMR spectroscopy offers a means of obtaining detailed information about each residue and its response to a given change in the protein sequence. Extraction of this information has been aided by recent progress in spectrometer technology (higher magnetic fields, more sensitive signal detection, more sophisticated computers) and experimental strategies (new NMR pulse sequences including multiple-quantum and two-dimensional NMR methods). The set of avian ovomucoid third domains, which consists of the third domain proper plus a short leader (connecting peptide) and has a maximum of 56 amino acid residues, offers an attractive system for developing experimental methods for investigating sequence-structure and structure-function relationships in proteins. Our NMR results provide examples of sequence effects on pKa' values, average conformation, and internal motion of amino acid side chains.     

Localized coupling in oxidative phosphorylation by mitochondria from Jerusalem artichoke (Helianthus tuberosus)

Delocalized chemiosmotic coupling of oxidative phosphorylation requires that a single-value correlation exists between the extent of and the kinetic parameters of respiration and ATP synthesis. This expectation was tested experimentally in nigericin-treated plant mitochondria in single combined experiments, in which simultaneously respiration (in State 3 and in State 4) was measured polarographically, FΔψ (which under these conditions was equivalent to ) was evaluated potentiometrically from the uptake of tetraphenylphosphonium⁺ and the rate of phosphorylation was estimated from the transient depolarization of mitochondria during State 4-State 3-State 4 transitions. The steady-state rates of the different biochemical reactions were progressively inhibited by specific inhibitors active with different modalities on various steps of the energy-transducing process: succinate respiration was inhibited competitively with malonate or noncompetitively with antimycin A, or by limiting the rate of transport into the mitochondria of the respiratory substrate with phenylsuccinate; was dissipated by uncoupling with increasing concentrations of valinomycin; ADP phosphorylation was limited with oligomycin. The results indicate generally that when the rate of respiratory electron flow is decreased, a parallel inhibition of the rate of phosphorylation is also observed, while very limited effects can be detected on the extent of . This behavior is in marked contrast to the effect of uncoupling where the decreased rate of ATP synthesis is clearly due to energy limitation. Extending previous observations in bacterial photosynthesis and in respiration by animal mitochondria and submitochondrial particles the results indicate, therefore, that respiration tightly controls the rate of ATP synthesis, with a mechanism largely independent of . These data cannot be reconciled with a delocalized chemiosmotic coupling model.     

Sulfate influx across the rabbit ileal brush border membrane: Sodium and proton dependence,and substrate specificities

Summary In intact ileal mucosa, uptake of SO₄ across the brush border membrane requires the presence of Na and is saturable, withK1/2=1.3mm at 140mm Na (P.L. Smith, S.A. Orellana & M. Field, 1981.J. Membrane Biol. 63:199–206). The present study examines the substrate specificities and transport stoichiometry of the Na-dependent SO₄ uptake process. The effects of variations in medium anion and cation composition on lumen-to-epithelium influx of SO₄ (J _me ^SO4 ) were determined under short-circuit conditions.J _me ^SO4 is inhibited by thiosulfate, but not by phosphate, methylsulfate, vanadate or taurocholate. Cl is weakly inhibitory. Uptake of SO₄ is poorly supported by Li, and is unaffected by K, indicating a specific dependence on Na. At low SO₄ concentration (0.22mm),J _me ^SO4 is a hyperbolic function of medium Na concentration; the corresponding Hill plot is linear with a slope of 1.0, suggesting a transport stoichiometry of 1 Na: 1 SO₄. At high SO₄ concentration (6.7mm), the Na-dependent SO₄ velocity curve is sigmoidal and yields a Hill plot which is again linear but has a slope of 1.56, suggesting transport of more than 1 Na per SO₄. SO₄ uptake in presence of Na exhibits a dependence on medium pH. At 0.22mm SO₄ and 140mm Na,J _me ^SO4 was doubled by lowering pH from 7.4 to 6.8. However, at 6.7mm SO₄ and 140mm Na, changing pH had no effect onJ _me ^SO4 over the range 6.8 to 8.5. The pH dependence ofJ _me ^SO4 at 6.7mm SO₄ was restored when medium Na was lowered to 3mm, suggesting that pH sensitivity is a function of the concentration of preformed NaSO ₄ ^– ion pair. The results suggest that SO₄ influx across the ileal brush border occurs by electroneutral Na⁺/NaSO ₄ ^– or Na⁺/H⁺/SO ₄ ^2– cotransport, the former being favored by high concentrations of Na and SO₄.     

Structural and developmental differences between three types of Na channels in dorsal root ganglion cells of newborn rats

Summary The changes in Na current during development were studied in the dorsal root ganglion (DRG) cells using the whole-cell patch-clamp technique. Cells obtained from rats 1–3 and 5–8 days after birth were cultured and their Na currents were compared. On top of the two types of Na currents reported in these cells (fast-FA current and slow-S current) a new fast current was found (FN). The main characteristics of the three currents are: (i) The voltages of activation are –37, –36, and –23 mV for the FN, FA and S currents, respectively. (ii) The activation and inactivation kinetics of FN and FA currents are about five times faster than those of the S current. (iii) The voltages at which inactivation reaches 50% are –139, –75 and –23 mV for the FN, FA and S currents, respectively.The kinetics and voltage-dependent parameters of the three currents and their density do not change during the first eight days after birth. However, their relative frequency in the cells changes. In the 1–3 day-old rats the precent of cells with S, FA, and mixed S+FN currents is 22, 18, and 60% of the cells, respectively. In the 5–8 day-old, the percent of cells with S, FA, and FN+S is 10, 66 and 22%. The relative increase in the frequency of cells with FA current during development can contribute to the ease of action potential generation compared with cells with FN currents, which are almost completely inactivated under physiological conditions. The predominance of FA cells also results in a significant decrease in the relative frequency of cells with the high-threshold, slow current.Antibodies directed against a part of the S₄ region of internal repeat I of the sodium channel (C ₁ ⁺ , amino acids 210–223, eel channel numbering) were found to shift the voltage dependence of FA current inactivation (but not of FN or S currents) to more negative potentials. The effect was found only when the antibodies were applied externally. The results suggest that FN, FA and S types of Na currents are generated by channels, which are different in the topography of the C ₁ ⁺ region in the membrane.     

Use of Microdialysis for Monitoring Tyrosine Hydroxylase Activity in the Brain of Conscious Rats

An on-line microdialysis system was developed which monitored the 3,4-dihydroxyphenylalanine (DOPA) formation in the striatum during infusion of a submicromolar concentration of an L-aromatic amino-acid decarboxylase inhibitor (NSD 1015). The absence of DOPA in dialysates of 6-hydroxydopamine-pretreated rats and the disappearance of DOPA after administration of alpha-methyl-p-tyrosine indicated that the dialyzed DOPA was derived from dopaminergic nerve terminals. Next we investigated whether the steady-state DOPA concentration in striatal dialysates could be considered as an index of tyrosine hydroxylase activity. The increase in DOPA output after intraperitoneal administration of haloperidol or gamma-butyrolactone and the decrease in DOPA output after intraperitoneal administration of apomorphine are in excellent agreement with results of postmortem studies, in which a decarboxylase inhibitor was used to measure the activity of tyrosine hydroxylase. The effect of haloperidol on DOPA formation was not visible when a U-shaped cannula (0.80 mm o.d.) was used. Some methodological problems related to microdialysis of the haloperidol-induced increase in DOPA formation are discussed. We concluded that the proposed model is a powerful and reliable in vivo method to monitor tyrosine hydroxylase activity in the brain. The method is of special interest for investigating the effect of compounds which are not able to pass the blood-brain barrier. As an application of the method in the latter situation, we report the effect of infusion the neurotoxin 1-methyl-4-phenylpyridinium ion (10 mmol/L infused over 20 min) on the activity of striatal tyrosine hydroxylase.     

[3H]1-Methyl-4-Phenyl-2,3-Dihydropyridinium Ion Binding Sites in Mouse Brain: Pharmacological and Biological Characterization

Because 1-methyl-4-phenyl-2,3-dihydropyridinium ion (MPP+) appears to damage the dopaminergic neuron and cause neuronal death, we characterized [3H]MPP+ binding sites in mouse brain membranes. Among several compounds tested, debrisoquin [3,4-dihydro-2(1H)-isoquinolinecarboxamidine] and some analogues were able to antagonize [3H]MPP+ binding. Debrisoquin is able to block adrenergic transmission and inhibit the activity of monoamine oxidase A (MAO-A). We found a certain correlation between the ability of these agents to displace [3H]MPP+ from its binding sites and their capacity to inhibit MAO-A activity. These data and the finding of a higher number of [3H]MPP+ binding sites in human placenta compared to mouse brain suggest that these sites may correspond to MAO-A enzymes. Recently it has been demonstrated in human brain that neurons in regions rich in catecholamines are positive for MAO-A. Accordingly, we suggest MAO-A as a possible accumulation site of MPP+ within the dopaminergic neuron. We also indicate the chemical structural requirement associated with the best binding of debrisoquin analogues with [3H]MPP+ sites. It would be reasonable to test the effects of debrisoquin-like drugs able to pass the blood-brain barrier on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity.