Gustatory responses of eel palatine receptors to amino acids and carboxylic acids

The gustatory receptors of the eel palate were found to be extremely sensitive to amino acids and carboxylic acids. The results obtained are as follows: (a) 11 amino acids which are among naturally occurring amino acids elicited responses in the palatine nerve, but 9 amino acids did not elicit a response even at a high concentration. The effect of D-amino acids was always much less than that of their corresponding L-isomers. There was no appreciable difference in the effectiveness of an alpha-amino acid (alpha-alanine) and beta-amino acid (beta-alanine). (b) The threshold concentrations of the most potent amino acids (arginine, glycine) were between 10(-8) and 10(-9) M. A linear relation between the magnitude of the response and log stimulus concentration held for a wide concentration range for all the amino acids examined. (c) The palatine receptors responded sensitively to various carboxylic acid solutions whose pH was adjusted to neutral. The threshold concentrations varied between 10(-4) and 10(-7) M. The magnitude of the response at 10(-2) M increased with an increase of carbon chain length. (d) The extent of cross-adaptation was examined with various combinations of amino acids. A variety of the response patterns showing complete cross-adaptation, no cross-adaptation, or synergetic interaction was observed. The synergetic interaction was also observed when one amino acid below its threshold concentration was added to the other amino acid below its threshold concentration was added to the other amino acid. No cross-adaptation was observed between amino acids and fatty acids. (e) The treatment of the palate with papain led to loss of the responses to arginine, glycine, and histidine without affecting those to proline and acetic acid. The treatment with pronase E eliminated selectively the response to proline. The possibility that the eel gustatory receptors are responsible for sensing food at a distance was discussed.     

Biosynthesis of Auxin-Induced Ethylene. Effects of Indole-3-Acetic Acid, Benzyladenine and Abscisic Acid on Endogenous Levels of 1-Aminocyclopropane-l-Carboxylic Acid (ACC) and ACC Synthase

Auxin-induced ethylene biosynthesis and its regulatory stepsin etiolated mung bean hypocotyl segments were examined. Theendogenous content of 1-aminocyclopropane- 1-carboxylic acid(ACC), an immediate precursor of ethylene, increased correspondingto the rate of ethylene production. Benzyladenine (BA), whichis a synergistic stimulator of auxin-induced ethylene production,increased the ACC content parallel to the rate of ethylene productionin the presence of IAA, but failed to increase the ACC contentin the absence of IAA while ethylene production was significantlystimulated by BA. Abscisic acid (ABA) inhibited the formationof ACC. The ACC synthase activity in the tissue was increasedby IAA, and the increase was further promoted by the presenceof BA. Cycloheximide severely inhibited the development of auxin-inducedACC synthase. The enzymatic properties of mung bean ACC synthasewere similar to those of the tomato fruit enzyme. Aminoethoxyvinylglycine(AVG) and aminooxyacetic acid, which inhibit the ACC synthasereaction, stimulated the development of ACC synthase. The regulatorymechanisms of the growth regulators are discussed in relationto ACC formation. (Received December 3, 1980; Accepted January 22, 1981)     

Exquisite Light Sensitivity of Drosophila melanogaster Cryptochrome

Drosophila melanogaster shows exquisite light sensitivity for modulation of circadian functions in vivo, yet the activities of the Drosophila circadian photopigment cryptochrome (CRY) have only been observed at high light levels. We studied intensity/duration parameters for light pulse induced circadian phase shifts under dim light conditions in vivo. Flies show far greater light sensitivity than previously appreciated, and show a surprising sensitivity increase with pulse duration, implying a process of photic integration active up to at least 6 hours. The CRY target timeless (TIM) shows dim light dependent degradation in circadian pacemaker neurons that parallels phase shift amplitude, indicating that integration occurs at this step, with the strongest effect in a single identified pacemaker neuron. Our findings indicate that CRY compensates for limited light sensitivity in vivo by photon integration over extraordinarily long times, and point to select circadian pacemaker neurons as having important roles.     

Gating and permeation properties of two types of calcium channels in neuroblastoma cells.

The gating and permeation properties of two types of calcium channels were studied in the neuroblastoma cell line N1E-115. Calcium channel currents as carried by Ba2+ (50 mM) were recorded using the whole-cell variation of the patch electrode voltage-clamp technique. The two types of calcium channels showed similar membrane potential dependence with respect to the steady-state activation and inactivation gating properties. However, the properties of the long-lasting type II channels were shifted approximately 30 mV in the depolarizing direction compared with those of the transient type I channels. Activation of type I channels developed with a sigmoidal time course which was described by m2 kinetics, whereas the activation of type II channels was described by a single exponential function. Tail current upon repolarization followed an exponential decay in either type of calcium channels. In comparison to type I channels, the activation process of type II channels was shifted approximately 30 mV in the positive direction, while the deactivation process showed a 60 mV shift in the positive direction. The rate constants of activation obtained from the activation and deactivation processes indicated that under comparable membrane potential conditions, type II channels close 2.4 times faster than type I channels upon repolarization. When external 50 mM Ba2+ was replaced with Ca2+ or Sr2+ on the equimolar basis, the amplitudes of transient and long-lasting currents were altered without a significant change in their time courses. The ion permeability ratios determined from the maximum amplitude of the inward current were as follows: Ba2+ (1.0) = Sr2+ (1.0) greater than Ca2+ (0.7) for type I channels, and Ba2+ (1.0) greater than Sr2+ (0.7) greater than Ca2+ (0.3) for type II channels. Replacement of Ba2+ with Ca2+ caused a 10-12 mV positive shift in the current-voltage relation for type II channels. However, the shift for type I channels was much less. This suggests that negative surface charges are present around type II channels. After correction for the surface charge effect on the ion permeation, there was no significant difference between the permeability ratios of these cations for the two channel types. It was concluded that the two types of calcium channels have many common properties in their gating and permeation mechanisms despite their differential voltage sensitivity and ion selectivity.     

Molecular characteristics and transcription of the gene encoding a multifunctional alcohol dehydrogenase in relation to the deactivation of pyruvate formate-lyase in the ruminal bacterium<Emphasis Type="Italic"> Streptococcus bovis</Emphasis>

To clarify the deactivation mechanism of pyruvate formate-lyase (PFL) and its role in the regulation of fermentation in Streptococcus bovis, the molecular properties and genetic expression of multifunctional alcohol dehydrogenase (ADHE) were investigated. S. bovis was found to have ADHE, which was deduced to consist of 872 amino acids with a molecular mass of 97.4 kDa. The ADHE was shown to harbor three enzyme activities: (1) alcohol dehydrogenase, (2) coenzyme-A-linked acetaldehyde dehydrogenase that catalyzes the conversion of acetyl-CoA to ethanol, and (3) PFL deactivase. Similar to Escherichia coli ADHE, S. bovis ADHE required Fe2+ for its activity. The gene encoding ADHE ( adhE) was shown to be monocistronic. The level of adhE mRNA changed in parallel with the mRNA levels of the genes encoding PFL (pfl) and PFL-activating enzyme (act) as the growth conditions changed, although these genes are independently transcribed. Synthesis of ADHE, PFL-activating enzyme, and PFL appears to be regulated concomitantly. Overexpression of ADHE did not cause a change in the formate-to-lactate ratio. It is conceivable that ADHE is not significantly involved in the reversible inactivation of active PFL under anoxic conditions. Partition of the flow from pyruvate appears to be mainly regulated by the activities of lactate dehydrogenase and PFL.     

Free fatty acid-induced hepatic insulin resistance: a potential role for protein kinase C-delta

The mechanisms of the impairment in hepatic glucose metabolism induced by free fatty acids (FFAs) and the importance of FFA oxidation in these mechanisms remain unclear. FFA-induced peripheral insulin resistance has been linked to membrane translocation of novel protein kinase C (PKC) isoforms, but the role of PKC in hepatic insulin resistance has not been assessed. To investigate the biochemical pathways that are induced by FFA in the liver and their relation to glucose metabolism in vivo, we determined endogenous glucose production (EGP), the hepatic content of citrate (product of acetyl-CoA derived from FFA oxidation and oxaloacetate), and hepatic PKC isoform translocation after 2 and 7 h Intralipid + heparin (IH) or SAL in rats. Experiments were performed in the basal state and during hyperinsulinemic clamps (insulin infusion rate, 5 mU. kg(-1). min(-1)). IH increased EGP in the basal state (P < 0.001) and during hyperinsulinemia (P < 0.001) at 2 and 7 h. Also, 7-h infusion of IH induced resistance to the suppressive effect of insulin on EGP (P < 0.05). Glycerol infusion (resulting in plasma glycerol levels similar to IH infusion) did not have any effect on EGP. IH increased hepatic citrate content by twofold, independent of the insulin levels and the duration of IH infusion. IH induced hepatic PKC-delta translocation from the cytosolic to membrane fraction in all groups. PKC-delta translocation was greater at 7 compared with 2 h (P < 0.05). In conclusion, 1) increased FFA oxidation may contribute to the FFA-induced increase in EGP in the basal state and during hyperinsulinemia but is not associated with FFA-induced hepatic insulin resistance, and 2) the progressive insulin resistance induced by FFA in the liver is associated with a progressive increase in hepatic PKC-delta translocation.     

A temperature-dependent timing mechanism is involved in the circadian system that drives locomotor rhythms in the fruit fly Drosophila melanogaster

The circadian clock of Drosophila melanogaster is thought to include rhythmic expression of period gene. Recent studies suggested, however, that a per-less oscillation is also involved in the regulation of circadian locomotor rhythms. In the present study, we examined the existence and the property of the possible per-less oscillation using arrhythmic clock mutant flies carrying per (01), tim(01), dClk(Jrk) or cyc(01), which lack rhythmic per expression. When temperature cycles consisting of 25 degrees C and 30 degrees C with various periods (T=8-32 hr) were given, wild-type (Canton-S) flies showed locomotor rhythms entrained to temperature cycles over a wide range of period (T=8-32 hr) in constant light (LL) while only to T=24 hr in constant darkness (DD). The mutant flies showed rhythms synchronizing with the given cycle both under LL and DD. In per(01) and tim(01) flies, the phase of a major peak slightly changed dependent on Ts in DD, while it did not in dClk(Jrk) and cyc(01) flies. When they were transferred from a constant temperature to a temperature cycle under DD, several cycles were necessary to establish a clear temperature entrainment in per(01) and tim (01) flies. These results suggest that per(01) and tim(01) flies have a temperature-entrainable weak oscillatory mechanism and that the per-less oscillatory mechanism may require dClk and cyc. In addition, per (01) and tim(01) flies changed from thermoactive in DD to cryoactive in LL, while dClk(Jrk) and cyc(01) flies did not. It is thus suggested that dClk and cyc are also involved in determining the light-associated temperature preference in per(01) and tim(01) flies.     

Balance of tumor necrosis factor alpha and interleukin-10 in a buccal infection in a streptozotocin-induced diabetic rat model

This study evaluates the local levels of proinflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), and anti-inflammatory cytokine, interleukin-10 (IL-10), in an experimental buccal abscess of a diabetic rat model. We prepared a buccal cavity induced by injection of carrageenin in a diabetic rat (blood glucose, 460.6 +/- 54.7 mg/dl, mean +/- SE) induced by streptozotocin (STZ). The buccal abscess was formed by the direct inoculation of Streptococcus pyogenes S-8 (2 x 10(7) cfu) into the buccal cavity at day 5 after carrageenin injection. Cytokine levels in the exudate of the buccal abscess were measured by enzyme-linked immunosorbent assay for 48 h after infection. Bacterial counts, weighing of exudate, and histological analysis were also performed. The mean TNF-alpha levels in the buccal abscess exudate of the diabetic group, which were generally higher than those of the control group, tended to increase over time until 48 h after infection, while the TNF-alpha levels in the control group peaked at 24 h after infection and then decreased. The IL-10 levels in the diabetic group remained almost unchanged until 48 h after infection, while the IL-10 levels in the control group were significantly higher than in the diabetic group at 12-24 h after infection. The mean ratio of TNF-alpha to IL-10 levels was 1.17-1.67 in the diabetic group, which was higher than the 0.26-0.69 of the control group. The bacterial counts in the buccal abscess and the weight of exudate were significantly higher in the diabetic group compared to the control group at 36-48 h. Histological findings showed that inflammatory cell infiltration was remarkable in the diabetic group compared to that of the control group. These results suggest that the elevated proinflammatory TNF-alpha levels and decreased anti-inflammatory IL-10 levels, which are produced at local infection sites, may at least in part be related to the severity of inflammation in this rat model with diabetes induced by STZ.     

Molecular characterization and physiological role of ascorbate peroxidase from halotolerant Chlamydomonas sp. W80 strain

A cDNA clone encoding an ascorbate peroxidase was isolated from the cDNA library from halotolerant Chlamydomonas W80 by a simple screening method based on the bacterial expression system. The cDNA clone contained an open reading frame encoding a mature protein of 282 amino acids with a calculated molecular mass of 30,031 Da, preceded by the chloroplast transit peptide consisting of 37 amino acids. In fact, ascorbate peroxidase was localized in the chloroplasts of Chlamydomonas W80 cells; the activity was detected in the stromal fraction but not in the thylakoid membrane. The deduced amino acid sequence of the cDNA showed 54 and 49% homology to chloroplastic and cytosolic ascorbate peroxidase isoenzymes of spinach leaves, respectively. The enzyme from Chlamydomonas W80 cells was purified to electrophoretic homogeneity. The molecular properties of the purified enzyme were similar to those of the other algal ascorbate peroxidases rather than those of ascorbate peroxidases from higher plants. The enzyme was relatively stable in ascorbate-depleted medium compared with the chloroplastic ascorbate peroxidase isoenzymes of higher plants. The presence of NaCl (3%) as well as of beta-d-thiogalactopyranoside was needed for the expression of Chlamydomonas W80 ascorbate peroxidase in Escherichia coli.