The Role of Chloride in Acetylcholine Metabolism

Abstract: The chloride dependence of acetylcholine (ACh) synthesis and release and of choline uptake was studied in synaptosomal preparations from rat brain. The substitution of propionate for chloride, in the presence of 35 m m -potassium, lowered the ACh content of the synaptosomes. However, in the presence of 5 m m -potassium, the ACh level in synaptosomes was reduced, but significantly less so. Propionate had no effect on choline acetyltransferase (EC 2.3.1.6) activity when measured in a standard chloride-containing medium. In the presence of propionate, the spontaneous release of ACh was unchanged, but potassium-stimulated release of ACh was markedly reduced as compared with a chloride-containing medium. The synthesis of ACh, as measured by the net increase in the amount of ACh in the synaptosomes and that released to the medium, was reduced with propionate at 5 m m -potassium and was totally inhibited when the potassium concentration was increased to 35 m m . Choline uptake studies revealed that with propionate only a low-affinity component of the choline transport system existed. Further, the V _max was markedly reduced when the potassium concentration was increased to 35 m m . The results suggest that under certain conditions choline transported by a low-affinity system might provide a substantial source of choline for ACh synthesis.     

Skin temperature biofeedback and migraine headaches

To assess the relative contribution of specific and nonspecific effects of skin temperature biofeedback upon migraine headache, 11 migraine patients were taught to increase the temperature of their hand. Training to decrease the skin temperature of the hand served as a control for 12 other migraine patients. An additional 11 control subjects were not trained but kept records of migraine activity. Under carefully controlled double-blind procedures, migraine patients who learned to raise finger temperatures showed statistically significant and clinically therapeutic improvement during a 6-week follow-up period. However, they were not significantly better than those trained to lower finger temperatures, those who did not meet a learning criterion, or those receiving no training. While these groups did show some significant improvement when compared to subjects who learned to decrease finger temperature, the results are most parsimoniously explained through nonspecific rather than specific factors. The necessity of using double-blind procedures in evaluating therapeutic effectiveness is again stressed.This article was presented as a Citation Award Paper at the Biofeedback Society of America meetings, San Diego, 1979. The work was supported in part by Rehabilitation Services Administration Grant No. 16-P-56810/5-17 to the University of Minnesota Medical Rehabilitation Research and Training Center and by a grant from the Division of Health Care Psychology, University of Minnesota. We would like to acknowledge the advice and support of Lee Willerman and John Belknap. We are grateful for the assistance of John Hendrickson, Dale Armin Miller, and Stephanie Waddingham in conducting the study and for the technical assistance of Robert Patterson, Steve Sheffield, P. Thain Marston, Kathy Guttormson, and Nancy Belknap.     

High-performance liquid chromatographic and gas—liquid chromatographic determination of diazepam and nordiazepam in plasma

A one-step method for extraction of diazepam, nordiazepam, and internal standard into toluene is followed by chromatographic separation and detection with either dual-wavelength high-performance liquid chromatography or electron-capture gas—liquid chromatography. Agreement between the two methods was excellent for diazepam (r = 0.99, n = 38) and good for nordiazepam (r = 0.96, n = 79) over a concentration range that included subtherapeutic, therapeutic, and toxic plasma levels.     

Inhibition of Photosystem II by formate. Possible evidence for a direct role of bicarbonate in photosynthetic oxygen evolution

In broken chloroplasts the presence of 100 mM sodium formate at pH 8.2 will specifically lengthen the Photosystem II relaxation times of the reactions S′₂ → S₃ and S′₃ → S₀. Rates of reactions S′₀ → S₁ and S′₁ → S₂ remain unaffected. Evidence is presented which indicates the discrimination among S-states by formate cannot be attributed to a block imposed on the reducing side of Photosystem II. The results are interpreted in context of the known interaction of formate and CO₂ which is bound to the Photosystem II reaction center complex. It is proposed that those S-state transitions which show extended relaxation times in the presence of formate must result in the momentary release and rebinding of CO₂. Furthermore since formate is acting on the oxygen-evolving side of Photosystem II, it would seem that CO₂ is released in reactions that occur there. A chemical model of oxygen evolution is presented. It is based on the hypothesis that hydrated CO₂ is the immediate source of photosynthetically evolved oxygen and explains why, under certain conditions formate slows only the S-state transitions S′₂ → S₃ and S′₃ → S₀.     

The synthesis and cytotoxic activity of 1,3,4-tri-O-acetyl-2,6-dideoxy-L-arabino- and -L-lyxo-hexopyranose

Methyl 2,6-dideoxy-α-L-arabino-hexopyranoside (6) was prepared from L-rhamnose in five steps. Hydrolysis of6 with 50% aqueous acetic acid gave 2,6-dideoxy-L-arabino-hexopyranose. Treatment of 3,4-di-O-acetyl-L-rhamnal with acetic acid in the presence of acetic anhydride and 2% sulfuric acid afforded 1,2,3-tri-O-acetyl-2,6-dideoxy-L-arabino-hexopyranose in 65% yield. Selective benzoylation and subsequent mesylation of 6 afforded methyl 3-O-benzoyl-2,6-dideoxy-4-O-mesyl-α-L-arabino-hexopyranoside, which was treated with sodium benzoate and sodium azide in hexamethylphosphoric triamide to give the corresponding 3,4-dibenzoyl 9 and 4-azido 11 analogs. Hydrogenation and N-acetylation of 11 afforded the 4-acetamido derivative 12. Deprotection of 9 and 12 gave 2,6-dideoxy-L-lyxo-hexopyranose and 4-acetamido-2,4,6-trideoxy-L-lyxo-hexopyranose, which were characterized as their peracetates. The free and corresponding peracetylated derivatives were assayed for their ability to inhibit the growth of P388 leukemia cells in culture. Although the free sugars did not inhibit the replication of these tumor cells under the conditions employed, their peracetylated derivatives demonstrated significant activity.     

Development of a closed-loop artificial pancreas.

A closed-loop glucose controlled insulin infusion system was developed, consisting of elements for continuous blood glucose analysis, a computer control system, and infusion systems. Improvements include decreased size, cost reduction and better performance. The algorithm used was a piecewise linear representation of the sigmoidal curve commonly employed. The apparatus has been applied to simulation of the healthy beta cell and glucose clamp studies.     

Isolation and partial purification of the major abundant class rat seminal vesicle poly(A+)-messenger RNA

Total poly(A(+))-RNA (poly(A(+))-RNA(tot)) was isolated from rat seminal vesicle and its size distribution determined by 70% formamide 5-25% sucrose density analysis. One major peak was resolved in the 10-13 S region and accounted for approximately 35% of the total poly(A(+))-RNA applied. Preparative 1% SDS, 5-20% linear sucrose density gradients also resolved a single major peak in the 11S region (poly(A(+))(11S). Analysis of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) under denaturing conditions on 2% agarose gel electrophoresis demonstrated two major components in both poly(A(+))-RNA populations. Size estimations for these components are 620 and 540 NT respectively. (3)H-cDNA was made to both poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S). Back-hybridization of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) to their respective (3)H-cDNA revealed a highly abundant class representing 41% and 85% of the sequences in their respective (3)H-cDNA's. The highly abundant class corresponded to 3-5 sequences present in 30,000-50,000 copies/cell. Invitro translation of poly(A(+))-RNA(11S) resulted in two major polypeptides coded for by the 620 NT long and 540 NT long poly(A(+))-RNA respectively.Images     

Initiation of ovalbumin synthesis in chicken oviduct magnum: Transient labeling of the NH2-terminus by methionine

The incorporation of [³⁵S]methionine into ovalbumin, a protein containing NH₂-terminal N-acetylglycine, has been studied in chicken oviduct magnum cells. The purification of [³⁵S]methionine-labeled ovalbumin from total oviduct proteins was accomplished by dialysis of a crude extract at pH 3.6 followed by chromatography on carboxymethyl cellulose. The radioactive ovalbumin eluted from the column in three peaks (P₀, P₁, and P₂-containing 0, 1, and 2 moles of phosphate, respectively, per mole of ovalbumin). The kinetics of labeling of peaks P₀ and P₁ showed that the ratio of radioactivity in NH₂-terminal methionine to total incorporation was greater at 2 min of labeling than at later times. The transient labeling of the NH₂-terminus of ovalbumin with methionine indicates that methionine is the initiator amino acid for the synthesis of this protein, which in its mature form contains NH₂-terminal N-acetylglycine.