Physiochemical properties of rice starch for production of vermicelli with premium quality.

Rice vermicelli is a main food consumed in China and Southeast Asia. Quality of rice vermicelli varies with rice cultivars. Parameters including amylose content, amylopectin distribution, thermal and pasting characteristics, gel texture and starch granules of three rice cultivars "Zhongjiazao 17", "Xiangzaoxian 24" and "Thai Jasmine Rice", were studied for their impacts on vermicelli quality. Results showed significant differences for the measurements of the quality traits and indicated that a favorable quality of vermicelli was not determined by any single factor instead of a combination of multi-parameters. A vermicelli with a favorable quality could be produced from a rice variety with a high apparent amylose content (>25%), a protein content of 11%, an intermediate gelatinization temperature and gel consistency, and a gel hardness (~3 N for a Rapid Viscosity Analyzer pasting) and moderate retrogradation capacity (a setback viscosity of 30-100 RVU).

NSPc1 promotes cancer stem cell self-renewal by repressing the synthesis of all-trans retinoic acid via targeting RDH16 in malignant glioma.

Polycomb group (PcG) proteins play an important role in development and stem cell maintenance, and their dysregulation have been closely linked to oncogenesis and cancer stem cell phenotypes. Here, we found that nervous system polycomb 1 (NSPc1) was highly expressed in stem cell-like glioma cells (SLCs). Knockdown of NSPc1 in SLCs resulted in impaired neurosphere formation and self-renewal abilities, down-regulated expression of stemness markers such as NESTIN, CD133 and SOX2, and decreased capacity to propagate subcutaneous xenografts. In contrast, glioma cells overexpressing NSPc1 exhibited a stem cell-like phenotype, up-regulated expression of stemness markers NESTIN, CD133 and SOX2, and an enhanced capacity to propagate subcutaneous xenografts. Furthermore, we identified that NSPc1 epigenetically repressed the expression of retinol dehydrogenase 16 (RDH16) by directly binding to a region upstream (-1073 to -823) of the RDH16 promoter. Next, we confirmed that RDH16 is a stemness suppressor that partially rescues SLCs from the NSPc1-induced increase in neurosphere formation. Finally, we showed that ATRA partly reversed the NSPc1-induced stemness enhancement in SLCs, through mechanisms correlated with an ATRA-dependent decrease in the expression of NSPc1. Thus, our results demonstrate that NSPc1 promotes cancer stem cell self-renewal by repressing the synthesis of ATRA via targeting RDH16 and may provide novel targets for glioma treatment in the future.

The cadmium and lead content of the grain produced by leading Chinese rice cultivars.

The cadmium (Cd) and lead (Pb) content in both white and wholemeal flour milled from 110 leading rice cultivars was assessed. The white flour Cd content ranged from <0.0025 to 0.2530mg/kg (geometric mean (GM)=0.0150mg/kg), while its Pb content ranged from <0.0250 to 0.3830mg/kg (GM=0.0210mg/kg). The indica types took up higher amounts of Cd and Pb than did the japonica types. Although the heavy metal content of wholemeal flour tended to higher than that of white flour, nevertheless 84.5% (Cd) and 95.4% (Pb) of the entries were compliant with the national maximum allowable concentration of 0.2000mg/kg of each contaminant. An analysis of the Cd content in the white flour of three indica type cultivars grown in two consecutive years at two locations indicated that Cd content may be significantly affected by the conditions prevailing in the growing season.

Characterization and gene cloning of the rice (Oryza sativa L.) dwarf and narrow-leaf mutant dnl3.

The dwarf and narrow-leaf rice (Oryza sativa L.) mutant dnl3 was isolated from the Japonica cultivar Zhonghua 11 (wild-type). dnl3 exhibited pleiotropic developmental defects. The narrow-leaf phenotype resulted from a marked reduction in the number of vascular bundles, while the dwarf stature was caused by the formation of foreshortened internodes and a reduced number of parenchyma cells. The suggestion that cell division is impaired in the mutant was consistent with the transcriptional behavior of various genes associated with cell division. The mutant was less responsive to exogenously supplied gibberellic acid than the wild-type, and profiling the transcription of genes involved in gibberellin synthesis and response revealed that a lesion in the mutant affected gibberellin signal transduction. The dnl3 phenotype was inherited as a single-dominant gene, mapping within a 19.1-kb region of chromosome 12, which was found to harbor three open reading frames. Resequencing the open reading frames revealed that the mutant carried an allele at one of the three genes that differed from the wild-type sequence by 2-bp deletions; this gene encoded a cellulose synthase-like D4 (CSLD4) protein. Therefore, OsCSLD4 is a candidate gene for DNL3. DNL3 was expressed in all of the rice organs tested at the heading stage, particularly in the leaves, roots, and culms. These results suggest that DNL3 plays important roles in rice leaf morphogenesis and vegetative development.

Optimisation of near-infrared reflectance model in measuring protein and amylose content of rice flour.

Near-infrared reflectance spectroscopy (NIRS) has been used to predict the cooking quality parameters of rice, such as the protein (PC) and amylose content (AC). Using brown and milled flours from 519 rice samples representing a wide range of grain qualities, this study was to compare the calibration models generated by different mathematical, preprocessing treatments, and combinations of different regression algorithm. A modified partial least squares model (MPLS) with the mathematic treatment "2, 8, 8, 2" (2nd order derivative computed based on 8 data points, and 8 and 2 data points in the 1st and 2nd smoothing, respectively) and inverse multiplicative scattering correction preprocessing treatment was identified as the best model for simultaneously measurement of PC and AC in brown flours. MPLS/"2, 8, 8, 2"/detrend preprocessing was identified as the best model for milled flours. The results indicated that NIRS could be useful in estimation of PC and AC of breeding lines in early generations of the breeding programs, and for the purposes of quality control in the food industry.

Fine mapping and characterization of a novel dwarf and narrow-leaf mutant dnl1 in rice.

Plant height is one of the most important agronomic traits of rice (Oryza sativa). Dwarf mutants are ideal materials for research on the mechanisms of regulation of rice plant height. We examined a new dwarf and narrow-leaf mutant dnl1. Phenotypic analysis showed that the dnl1 mutant has a thinner culm and more tillers, but the number of grains per panicle, the seed setting rate and the grain weight of dnl1 mutant were found to be significantly lower than in the wild-type. Based on scanning electron microscopic observations, the number of cells in the y-axis in internodes was significantly lower than in the wild-type. In phytohormone induction experiments, dnl1 was gibberellic acid-insensitive. The expression of some genes involved in the gibberellins metabolic pathways was affected in the dnl1 mutant, based on the real-time PCR analysis, suggesting that the dnl1 gene likely plays a role in gibberellin metabolic pathways. Genetic analysis showed that the dwarf and narrow leaf phenotype is controlled by a novel single recessive gene, here referred to as the dwarf and narrow leaf 1 (dnl1), which is located within the region between markers Ind12-11 and RM8214 on the short arm of chromosome 12. By means of fine-mapping strategy, the dnl1 gene was localized within an interval of 285.75 kb physical distance. These results will be useful for dnl1 gene cloning and to improve our understanding of the molecular mechanisms involved in the regulation of growth and development of rice.

Risk factors of pulmonary hemorrhage in very-low-birth-weight infants: a two-year retrospective study.

Pulmonary hemorrhage is a serious complications in very-low-birth-weight (VLBW) infants with respiratory distress syndrome (RDS). We undertook a 2-year retrospective study to investigate the predisposing factors and the incidence of pulmonary hemorrhage in VLBW infants. From January 1997 through December 1998, twenty infants were diagnosed with massive pulmonary hemorrhage (MPH) according to the following criteria: active bleeding from the endotracheal tube, acute drop in hematocrit (> or = 10%), and the development of multilobar infiltration on chest radiograph. The mean gestational age was 26.9 +/- 2.5 weeks, the mean birth weight was 909 +/- 290 g. Twenty historic controls with similar gestational age and birth weight were retrospectively identified during the study period. The incidence of MPH in VLBW infants was 5.9%(20/340). A lack of prenatal corticosteroid administration, surfactant replacement therapy for RDS, and a patent ductus arteriosus (PDA) with cardiovascular dysfunction requiring dopamine support were the significantly predisposing factors of MPH in the acute stage (< or = 7th day of life). To avoid MPH and decrease mortality and morbidity in the acute stage, prenatal corticosteroid administration, evaluation of the necessity of surfactant therapy, and early recognition and aggressive treatment of hemodynamically significant PDA were necessary.

Partial liquid ventilation in normal rabbits: comparison of three kinds of perfluorocarbon.

Perfluorocarbon liquids have been used in liquid ventilation studies and considered an effective technique of gas exchange with less barotrauma when compared with gas ventilation. We compared the effects of partial liquid ventilation (PLV) using 3 kinds of perfluorocarbon liquids (Fluorinert FC 43, FC 77 and FC 84) available in Taiwan in normal rabbits. We were able to achieve adequate oxygenation and ventilation during a 2-hour-duration of PLV using FC 43, FC 77 or FC 84. There was no significant difference in hemodynamic status or laboratory findings between control group and PLV groups. There were also no significant differences before LV and after 2 hours of PLV among PLV groups. Histological study of lung tissue revealed intact and well expanded alveoli, and no significant pathological change after 2 hours of PLV. These results show that PLV using FC 43, FC 77 or FC 84 is an effective technique for maintaining adequate pulmonary gas exchange in normal rabbits.

Activity of phosphatidylethanolamine-N-methyltransferase in brain affected by Alzheimer's disease.

To determine whether phospholipid abnormality in Alzheimer's disease is associated with modification of phosphatidylethanolamine-N-methyltransferase, the activity of the enzyme was analysed in the frontal and occipital cortex of the brain from patients with Alzheimer's disease and from aged-matched control. The optimum pH for phosphatidylethanolamine-N-methyltransferase in human brain was 9.0. The enzyme activity was stimulated by detergent TWEEN 20 but inhibited by Triton X-100. Neither magnesium dependence nor chemical methylation was found. A decrease in activity of phosphatidylethanolamine-N-methyltransferase was observed in the frontal cortex of brain affected with Alzheimer's disease. The addition of exogenous phosphatidylethanolamine resulted in no modification in the methylation rate as compared with that of endogenous PE. The addition of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine resulted in significantly increased rates of methylation in brain tissues. However, the increased rate of phosphatidylethanolamine-N-methyltransferase activity stimulated by exogenous phospholipids was lower in the frontal cortex of brains with Alzheimer's disease when compared to the normals and there was no difference in the occipital cortex between Alzheimer's disease and the control. It is plausible that the decreased activity of phosphatidylethanolamine-N-methyltransferase and its low compensating ability could relate to the modification of phosphatidylcholine in brain tissues from Alzheimer's disease patients.

Glucose and insulin infusion versus kayexalate for the early treatment of non-oliguric hyperkalemia in very-low-birth-weight infants.

Forty very low birth weight (VLBW) infants with non-oliguric hyperkalemia in the first few days after birth were enrolled in this study. They were randomly divided into 2 groups, regular insulin (RI) infusion group and kayexalate resin enema group. Therapy was administered when serum potassium level was greater than 6 mEq/L. None of these infants received blood transfusion during this study course. In RI group (n = 20), the ratio of infusion glucose to regular insulin was 10-15 gm glucose to 1 unit RI, and the glucose infusion rate was maintained at least 6 mg/Kg/min. In Kayexalate group (n = 20), the dose of Kayexalate was 1 gm/Kg body weight given rectally every four hours. All treatment discontinued after the serum potassium level returned to normal for 6 hours. The mean gestational ages were 27.4 +/- 1.8 weeks in RI group and 28.4 +/- 2.4 weeks in Kayexalate group, respectively. Mean birth weights were 935 +/- 259 gm (RI) and 1065 +/- 214 gm (Kayexalate). The ages at onset of hyperkalemia were 24.6 +/- 8.2 (RI) and 22.2 +/- 8.1 (Kayexalate) hours after birth. The mean urine outputs during the 8-hour interval prior to development of hyperkalemia were 5.4 +/- 1.3 (RI) and 5.5 +/- 0.9 (Kayexalate) ml/kg/min. The durations of hyperkalemia were 26.4 +/- 14.9 (RI) and 38.6 +/- 13.3 (Kayexalate) hours. The peak serum potassium levels during therapy were 7.3 +/- 0.9 and 7.4 +/- 0.6 mEq/L. The incidences of grade II and above intraventricular hemorrhage (IVH) were 15% (3/20) and 50% (10/20). The incidences of cardiac dysrhythmia were 5% (1/20) and 10% (2/20). Significantly shorter duration of non-oliguric hyperkalemia and lower incidence of IVH were noted in RI group, but there were no differences in the peak potassium level or the incidence of cardiac dysrhythmia between these two groups. We conclude that to use early continuous regular insulin infusion therapy for the treatment of non-oliguric hyperkalemia in VLBW infants is more effective than kayexalate in decreasing the duration of hyperkalemia and reducing the incidence of intraventricular hemorrhage.

Two selective 5-HT1A receptor antagonists, WAY-100 635 and NDL-249, stimulate locomotion in rats acclimatised to their environment and alter their behaviour: a behavioural analysis.

The aim was to study firstly, the motor effects of a new 5-HT1A antagonist, NDL-249 [(R)-3-(N-cyclopentyl-N-propylamino)-8-fluoro-3,4-dihydro-2H-1-benzopyra n-5-carboxamide hydrochloride] and of the reference 5-HT1A antagonist WAY-100 635 [N-(2-(1-(4-(2-methoxyphenyl)piperazinyl))ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride], in comparison to the 5-HT1A agonist (+/-)-8-OH-DPAT [(8-hydroxy-2-(di-N-propylamino) tetralin, hereafter 8-OH-DPAT], in rats acclimatised to the automated activity cages; secondly, to study whether the behavioural effects of NDL-249 and 8-OH-DPAT are sensitive to the 5-HT depleting effects of p-chlorophenylalanine (PCPA); thirdly, to characterise the nature of the antagonist-induced activation seen in the automatic activity cages with the aid of a behavioural observation analysis; fourthly, to examine the interaction between the 5-HT1A receptors mediating the behavioural effects and dopamine (DA) receptors. NDL-249 was found to bind in vitro to rat hippocampal 5-HT1A receptors with high affinity and selectivity. In second messenger studies, it was devoid of agonist-like effects. In the locomotor activity studies, each antagonist significantly increased the incidence of horizontal activity, peripheral activity and rearing. 8-OH-DPAT, while significantly increasing peripheral and horizontal activities, decreased the incidence of rearing. PCPA blocked the motor effects of NDL-249 but did not affect those of 8-OH-DPAT. Observational analyses indicated that NDL-249 induced significant increases at one or more doses in sniffing, rearing and locomotion together with a significant reduction in stillness. WAY-100 635 significantly increased the incidence of rearing, intense grooming and vacuous chewing. The significant increases in sniffing, grooming and intense grooming and the significant decrease in stillness induced by the DA D1 agonist, SK&F 38393 [(+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride], were not altered by concomitant pre-treatment with NDL-249. Pre-treatment of rats with either the DA D1 antagonist SCH-23390 (2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol) or the DA D2 antagonist, raclopride, blocked the reduced stillness and increased sniffing and rearing induced by NDL-249. In conclusion, 5-HT1A antagonists including the new selective antagonist, NDL-249, induce mild behavioural activation in rats, which is mediated probably indirectly via DA systems.

Crash risks of older drivers: a panel data analysis.

Considerable progress has been made on understanding older drivers' safety issues. None the less, findings from previous research have been rather inconclusive. Differences in data and research methodology have been suggested as factors that contribute to the discrepancies in previous findings. One of the methodological limitations is the lack of considering temporal order between events (i.e. the time between onset of medical condition, symptom and crash). Without time-series data, a 'snap-shot' of medical conditions and driving patterns were often linked to more than 1 year--of crash data, hoping to accumulate enough data on crashes. The interpretation of the results from these studies is difficult in that one cannot explicitly attribute the increase in highway crash rates to medical conditions and/or physical limitations. This paper uses a panel data analysis to identify factors that place older drivers at greater crash risk. Our results show that factors that place female drivers at greater crash risk are different from those influencing male drivers. More risk factors were found to be significant in affecting older men's involvement in crashes than older women. When the analysis controlled for the amount of driving, women who live alone or who experience back pain were found to have a higher crash risk. Similarly, men who are employed, score low on word-recall tests, have a history of glaucoma, or use antidepressant drugs were found to have a higher crash risk. The most influential risk factors in men were the number of miles driven, and use of antidepressants.

A survey on the treatment strategy of patent ductus arteriosus in very low-birth-weight infants.

This study is a retrospective analysis of the clinical data of 67 very low birth weight infants (VLBWI) with symptomatic patent ductus arteriosus (PDA); all were admitted to four neonatal intensive care units (NICU) from January 1, 1996 through December 31, 1996. The mean gestational age was 27.9 +/- 2.4 weeks, the mean birth weight was 1078 +/- 193 g. Fifty-six infants (83.6%) had RDS, and 53 infants (79%) received artificial surfactant. The NICU at a regional hospital used CVD score > or =3 as the sole criteria and the remaining three NICUs used color Doppler echocardiogram to confirm a symptomatic PDA and to treat it; sometimes LA/AO > or = 1.3 was used as the criteria for indomethacin treatment. Two of these three hospitals sometimes used the pulsed Doppler echocardiogram as well as color Doppler examination as the treatment criteria. Seven infants (10.5%) had contraindication for indomethacin treatment; four of them closed after conservative treatment, and another three were subjected to surgical ligation. Of the remaining 60 infants, 3 were treated with oral indomethacin and 57 were treated with intravenous indomethacin. The mean age when initial treatment given was 3.8 +/- 1.5 days (range, 8 hours approximately 20 days). Among them 10 (16.7%) were within 24 hours after birth, 25 (41.7%) were between 24 and 48 hours, and 25 (41.7%) were beyond 48 hours. The dosage of indomethacin was 0.2 mg/kg per dose intravenously every 12 to 24 hours for three doses as a full course, if not contraindicated. The mean dose of indomethacin was 2.8 +/- 1.5; 10 infants (16.7%) received 1 dose, 15 (25%) received 2 doses, 27 (45%) received 3 doses, 3 (5%) received 4 doses and 5 (8.3%) received 6 doses. Among them, 51 infants (85%) PDA closed (including 2 treated with oral indomethacin), 9 (15%) failed to close and 6 of them received surgical ligation (including 1 treated with oral indomethacin). The complications associated with indomethacin treatment were hypoglycemia (52%), decreased urine output (42%) and gastrointestinal hemorrhage (32%). The infants with RDS had an earlier mean age of initial treatment than non-RDS infants (3.3 +/- 2.5 vs. 7.6 +/- 5.6 days; p < 0.05), and also had a higher closure rate (89% vs. 57%; p < 0.05). There was a closure rate of 85% in this multicenter retrospective analysis. Even though the infants received only one or two doses, they still had a good chance of ductal closure (21/25, 84%). To minimize the complications associated with indomethacin treatment in VLBWI, the protocol of indomethacin treatment should be re-evaluated.

The pharmacological characterization of a novel selective 5-hydroxytryptamine1A receptor antagonist, NAD-299.

The pharmacological properties of a novel selective 5-hydroxytryptamine1A (5-HT1A) receptor antagonist, NAD-299 [(R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R)-tartrate monohydrate] were examined in vitro and in vivo and compared with the reference 5-HT1A receptor antagonist, WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazin-yl))ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride]. The new compound had high affinity for 5-HT1A receptors in vitro with a Ki value of 0.6 nM. The only other receptors for which NAD-299 had affinity less than 1 microM were alpha-1 and beta adrenoceptors with Ki values of 260 and 340 nM, respectively. Thus, the selectivity of NAD-299 for 5-HT1A receptors was more than 400 times. WAY-100635 had considerably higher affinity than NAD-299 for alpha-1 adrenoceptors (Ki = 45 nM) and dopamine D2 and D3 receptors (Ki = 79 and 67 nM, respectively). Like WAY-100635, NAD-299 competitively blocked 5-HT-induced inhibition of vasoactive intestinal peptide-stimulated cAMP production in GH4ZD10 cells and had no intrinsic activity. Both compounds were therefore 5-HT1A receptor antagonists in vitro and also behaved as such in in vivo experiments. Thus, they competitively antagonized the 8-hydroxy-2-(di-n-propylamino)tetralin-induced 5-HT behavioral effects, hypothermia, corticosterone secretion and inhibition of passive avoidance behavior without causing any actions of their own. The effective dose of NAD-299 varied between 0.03 and 0.35 micromol/kg s.c., depending on the test and the dose of 8-hydroxy-2-(di-n-propylamino)tetralin.

NMDA receptor-mediated increase in cyclic GMP in the rat cerebellum in vivo is blocked by alaproclate and GEA-857.

The effects of alaproclate and GEA-857 (2-(4-chlorophenyl)-1,1-dimethylethyl 2-amino-3-methylbutanoate) on the production of cyclic GMP in the rat cerebellum in vivo induced by stimulation of N-methyl-D-aspartate (NMDA) receptors were studied. Alaproclate per se at a dose of 20 mg/kg subcutaneously, did not influence the basal cGMP level. The increase in cGMP induced by harmaline (20 mg/kg subcutaneously) was dose-dependently antagonized by alaproclate (5-40 mg/kg subcutaneously). S-(-)-Alaproclate was 2-5 times more potent than the R-(+)-enantiomer. GEA-857 which in contrast to alaproclate is a very weak 5-HT uptake inhibitor shared the ability of alaproclate to inhibit the effect of harmaline on cGMP accumulation with similar potency to S-(-)-alaproclate. Alaproclate at 15 mg/kg subcutaneously blocked the increase in cGMP in cerebellum caused by NMDA itself at 200 mg/kg subcutaneously. In contrast to alaproclate, the K+ channel antagonist, 4-aminopyridine, 5 mg/kg subcutaneously, produced per se an increase in cGMP levels in the rat cerebellum by 300% which was antagonized by the NMDA receptor antagonists, dizocilpine, phencyclidine and (+/-)-CCP, the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester and by alaproclate. Alaproclate. Alaproclate and GEA-857 antagonized seizures induced by NMDA, 200 mg/kg subcutaneously at doses similar to those antagonizing the harmaline- and NMDA-induced elevation of cerebellar cGMP. Neither alaproclate nor GEA-857 caused any behavioural effects typical for uncompetitive NMDA receptor antagonists except a slight increase in motor activity and sniffing. The effect of alaproclate on the NMDA receptor-mediated increase in cGMP in rat cerebellum in vivo might be due to blockade of the cation channel of the NMDA receptor complex previously observed in in vitro experiments and these compounds seems to belong to the group of low-affinity uncompetitive NMDA receptor antagonists that might have clinical interest.

On the usefulness of Fura-2 measurements of intrasynaptosomal calcium levels in rat cortical synaptosomes to study mechanisms of presynaptic function.

Levels of [Ca2+]i in rat cortex synaptosomes were measured using the Ca2+ indicator Fura-2. Ca2+ influx was induced by veratridine in a concentration-dependent manner (1-10 microM). The resulting increase in [Ca2+]i was inhibited by tetrodotoxin (TTX). K+ (18 mM) increased the [Ca2+]i which was not influenced by TTX. K(+)-channel blockers such as 4-aminopyridine, alpha- and delta-dendrotoxin pre se were ineffective. The veratridine-induced Ca2+ influx in synaptosomes was reduced by L-type Ca(2+)-channel blockers, such as felodipine, nifedipine and PN-200-110, verapamil and diltiazem. omega-Conotoxin, and N-type Ca(2+)-channel blocker, did not inhibit the veratridine-stimulated [Ca2+]i increase. Bay K 8644, and L-channel agonist, stimulated an increase of [Ca2+]i in synaptosomes which was not sensitive to TTX. R-N6-Phenyl-isopropyl-adenosine (R-PIA) and clonidine, agonists at adenosine A1-receptors and alpha 2-adrenoceptors, respectively, did not influence the veratridine-stimulated [Ca2+]i increase. R-PIA did not interact with Bay K 8644-stimulated [Ca2+]i increase in synaptosomes. The results for all the substances used show major differences between the effects on Ca2+ influx in synaptosomes and on the electrically evoked neurotransmitter release in slice preparations. Thus, the synaptosome preparation is not a generally applicable experimental model for the study of Ca2+ mechanisms of presynaptic neuromodulation.

Effect of an intracellular calcium chelator on the regulation of electrically evoked [3H]-noradrenaline release from rat hippocampal slices.

1. The electrically (3 Hz, 5 min) evoked [3H]-noradrenaline ([3H]-NA) release from rat hippocampal slices was reduced by prior treatment of the slices with 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetomethylester (BAPTA/AM) in a concentration-(10 to 500 microM) dependent manner (40% at 30 microM). Reduction of medium calcium from 1.3 to 0.5 mM caused a larger (70%) decrease. BAPTA free acid (100 mM), a non-permeable Ca(2+)-chelator had no significant effect. 2. Basal [3H]-noradrenaline release was reduced by BAPTA/AM in a concentration-dependent manner (50% at 30 microM), but reduction of external Ca2+ from 1.3 to 0.5 mM did not alter basal release. 3. About 10% of total [3H]-NA in the slices was released at 3 Hz stimulation in 1.3 mM Ca2+ buffer. Addition of the alpha 2-adrenoceptor antagonist, idazoxan (1 microM), increased electrically evoked [3H]-NA release to 26% but stimulated release was not altered by the adenosine A1-receptor antagonist, 8-cyclopentyl theophylline (8-CPT) (1 microM). 4. Evoked release was reduced by the alpha 2-receptor agonist, UK 14,304, in a concentration-dependent manner in the presence of 8-CPT (1 microM). The magnitude of this effect was not altered by the treatment of slices with 30 microM BAPTA/AM. 5. The adenosine A1 receptor agonist, N6-cyclohexyl adenosine (CHA) (1 microM) inhibited electrically evoked [3H]-NA release by about 40% in the presence of idazoxan (1 microM). The effect of CHA was not significantly altered by treatment of slices with BAPTA/AM. 7. The present results show that spontaneous [3H]-NA release is affected by reduction of intracellular Ca2+, but not by reduction of extracellular Ca2+ or by the presynaptic agonists or w-conotoxin. By contrast, electrically evoked release was affected more strongly by alterations of extracellular Ca2+ than by buffering intracellular Ca2+. The reduction of electrically evoked [3H]-NA release by agonists at the adenosine Al-receptor and a2-adrenoceptor is probably mediated through the control of Ca2+ entry via membrane ion channels or at a low affinity Ca2'-site governing evoked release.

Energy-linked transhydrogenase. Characterization of a nucleotide-binding sequence in nicotinamide nucleotide transhydrogenase from beef heart.

Purified nicotinamide nucleotide transhydrogenase from beef heart was investigated with respect to labeling and subsequent sequence analysis of a nicotinamide nucleotide-binding site. A photo-activated azide derivative, 8-azidoadenosine 5'-monophosphate, was used as an active-site-directed photoaffinity label, which was shown to be specific for the NAD(H)-binding site in the dark. Light-activated incorporation of the label in transhydrogenase was accompanied by an inactivation, which approached 100% at the incorporation of about 1 mol label/mol transhydrogenase monomer. As expected from the assumed site-specificity of the label. NADH prevented both labeling and inactivation to some extent. However, NADPH also prevented labeling and inactivation marginally. The oxidized substrates NAD+ and NADP+ were inhibitory by themselves under these conditions, and the substrate analogs 5'-AMP and 2'-AMP were also poor protectors. The NAD(H)-site specificity of the azido compound was thus largely lost upon illumination and covalent modification. Radioactive labeling of transhydrogenase with 8-azido-[2-3H]-adenosine 5'-monophosphate followed by protease digestion, isolation of labeled peptides and amino-acid sequence analysis showed that Tyr 1006 in the sequence 1001-1027 close to the C-terminus was labeled. This sequence shows homologies with nucleotide-binding sequences in, e.g., F1-ATPase. On the basis of sequence homologies with other NAD(P)-dependent enzymes it is proposed that transhydrogenase contains 4 nucleotide-binding sites, of which 2 constitute the adenine nucleotide-binding domains of the catalytic sites for NAD(H) and NADP(H) close to the N- and C-terminals, respectively. Each of these domains has an additional vicinal nucleotide-binding sequence which may constitute a non-catalytic nucleotide-binding site or the nicotinamide nucleotide-binding domain of the catalytic site. The present results indicate that 8-azidoadenosine 5'-monophosphate is kinetically specific for the catalytic NAD(H)-binding site, but reacts covalently with Tyr 1006 of the putative non-catalytic site or nicotinamide nucleotide-binding domain formed by the 1001-1027 amino acid sequence of the catalytic NADP(H)-binding site. Interactions between the catalytic NAD(H) and NADP(H) binding sites, and the assumed non-catalytic sites, may be facilitated by a ligand-triggered formation of a narrow pocket, which normally allows an efficient hydride ion transfer between the natural substrates.

Comparison of the effects of four dendrotoxin peptides, 4-aminopyridine and tetraethylammonium on the electrically evoked [3H]noradrenaline release from rat hippocampus.

We have examined the effects of four dendrotoxin (DaTX) peptides, alpha-, beta-, gamma- and delta-DaTX, separated from the venom of the green mamba (Dendroaspis angusticeps), on field stimulation-evoked [3H]noradrenaline (NA) release from rat hippocampus and compared their effects with those of two other inhibitors of K+ channels, 4-aminopyridine (4-AP) and tetraethylammonium (TEA). 4-AP (10-300 microM) and TEA (0.1-5 mM) facilitated the evoked [3H]NA release in a concentration-dependent manner. The evoked [3H]NA release was reduced to about half by alpha 2-adrenoceptor stimulation (UK 14,304; 100 nM) and this reduction was antagonized by 4-AP (10-100 microM), whereas TEA even at 5 mM was a poor inhibitor of alpha 2-effects. alpha-DaTX (10-200 nM) mimicked 4-AP in increasing the electrically evoked [3H]NA release and diminishing the inhibitory effects of UK 14,304 in a concentration-dependent manner. delta-DaTX did not itself alter the electrically evoked [3H]NA release, but at 200 nM, it reduced the effects of alpha 2-receptor stimulation. beta- and gamma-DaTX (up to 200 nM) had no significant effects. 4-AP, 3,4-diaminopyridine (3,4-DAP), TEA and the four dendrotoxins displaced the binding of [3H]p-aminoclonidine ([3H]PAC) from alpha 2-receptors. The IC50 values were 6.6 x 10(-4), 1.42 x 10(-3), 5.6 x 10(-2) for 4-AP, 3,4-DAP and TEA, respectively, and 3.19 x 10(-6) M for alpha-DaTX. Thus, their potency as inhibitors of alpha 2-receptors is apparently too low to account alone for the antagonism by K+ channel inhibitors of alpha 2-effects on NA release.(ABSTRACT TRUNCATED AT 250 WORDS)