Determination of Acetylcholine Release in the Striatum of Anesthetized Rats Using In Vivo Microdialysis and a Radioimmunoassay

A vertical-type in vivo microdialysis probe and a sensitive, specific radioimmunoassay (RIA) were used to study the mechanism of acetylcholine (ACh) release in the striatum of anesthetized rats. Without the use of physostigmine, a cholinesterase inhibitor, our RIA could still detect the amount of ACh present in the perfusate (5.6 +/- 0.6 fmol/min, n = 16). Tetrodotoxin (1 microM) produced a significant decrease in the amount of ACh collected in the perfusate, suggesting that basal ACh determined under the present experimental conditions was related to cholinergic neural activity. Atropine (0.1-1 microM) applied topically via the dialysis probe did not affect the amount of ACh recovered in the perfusate in the absence of physostigmine. Addition of physostigmine (10 microM) to the perfusion fluid produced about a 100-fold increase in the amount of ACh collected. In the presence of physostigmine, topical administration of atropine and pirenzepine (0.01-1 microM) through a dialysis probe produced a further three- to fourfold increase in ACh output, whereas a slight increase was produced by AF-DX 116 at the highest concentration (1 microM). These results indicate that presynaptic modulation of ACh release in the striatum does not occur under basal conditions, and that presynaptic M1 muscarinic receptors are involved in the modulation of ACh release when the ACh concentration is raised under certain conditions.     

Mobilization of newly synthesized RNAs into polysomes inXenopus laevis embryos

Summary The mobilization of newly synthesized 18S and 28S rRNAs, 4S RNA and poly(A)⁺ RNA into polysomes was studied in isolated cells ofXenopus laevis embryos between cleavage and neurula stages. Throughout these stages, 4S RNA and poly(A)⁺ RNA were mobilized immediately following their appearance in the cytoplasm. 18S rRNA however, stayed in the ribosomal subunit fraction for about 30 min until the 28S rRNA appeared, when the two rRNAs were mobilized together at an equimolar ratio. This mobilization, at a 1:1 molar ratio, appeared to be realized at initiation monome formation. Thus, the efficiency of the mobilization of two newly synthesized rRNAs, shortly after their arrival at the cytoplasm, differed considerably but difference disappeared once steady state was reached.The contribution of newly synthesized 18S and 28S rRNAs to polysomes remains small throughout early development. around 3% of newly synthesized 4S RNA is polysomal which is the same distribution observed for unlabeled 4S RNA. Less than 10% of the newly synthesized cytoplasmic poly(A)⁺ RNA was mobilized into polysomes during cleavage, but in later stages the proportion increased to around 20%–25%. These results show that newly synthesized RNAs are utilized for protein synthesis at characteristic rates soon after they are synthesized during early embryonic development. On the basis of the data presented here and elsewhere we discuss quantitative aspects of the utilization of newly synthesized and maternal RNAs during early embryogenesis.     

Polysaccharide Produced by the Genus Pullularia: I. Production of Polysaccharide by Growing Cells

A dextranlike polysaccharide was found to be produced on substrates of sucrose, maltose, glucose, and fructose by growing cells of various strains of the genus Pullularia. The polysaccharide, obtainable in amounts as large as 2 to 3 g per 100 ml of culture medium using various carbohydrates as the carbon source, was soluble in cold water but not in 50% alcohol. The polysaccharide obtained had a α_d = +197.5° (c = 0.2 in water), and its molecular weight, determined by the light-scattering method, was found to be approximately 250,000.     

Increase in the CO2 exchange rate of leaves of Ilex rotunda with elevated atmospheric CO2 concentration in an urban canyon

 It was found that the atmospheric carbon dioxide (CO₂) concentration in an urban canyon in Fukuoka city, Japan during August 1997 was about 30 μmol mol⁻¹ higher than that in the suburbs. When fully exposed to sunlight, in situ the rate of photosynthesis in single leaves of Ilex rotunda planted in the urban canyon was higher when the atmospheric CO₂ concentration was elevated. A biochemically based model was able to predict the in situ rate of photosynthesis well. The model also predicted an increase in the daily CO₂ exchange rate for leaves in the urban canyon with an increase in atmospheric CO₂ concentration. However, in situ such an increase in the daily CO₂ exchange rate may be offset by diminished sunlight, a higher air temperature and a lower relative humidity. Thus, the daily CO₂ exchange rate predicted using the model based soleley on the environmental conditions prevailing in the urban canyon was lower than that predicted based only on environmental factors found in the suburbs. Received: 24 October 1997; Received after revision: 25 March 1998; Accepted: 1 June 1998     

Effects of repeated chair restraint on physiological values in the rhesus monkey (Macaca mulatta)

The purpose of this study was to examine the effects of repeated chair restraint on some physiological values in the rhesus monkey. Although heart rate and blood pressure increased significantly at the onset of chair restraint, these increased values gradually decreased with repetition of restraint as well as with the lapse of time after restraining. Finally, no elevation of these levels was detected after repetitions lasting eight hours per day for five days. Plasma cortisol, CK and GOT values also showed similar trends. These elevated physiological values at the onset of chair restraint seemed to be due to high sympathetic tone and muscular activity used to try to avoid the restraint. It is concluded that repeated chair restraint has an accommodative component in monkeys resulting in the return of some elevated physiological values to the normal level.     

Differential scanning calorimetric studies on bovine serum albumin: III. Effect of sodium dodecyl sulphate

Measurements of differential scanning calorimetry (d.s.c.) have been made on the complex bovine serum albumin (BSA)--sodium dodecyl sulphate (SDS) under various conditions. There are two peaks P1 and P2 in the d.s.c. curve for BSA at pH 7 and in the absence of NaCl, indicating the presence of the heat-induced transition of BSA. There are three peaks P1, P2 and P3 in the curve for the system with the molar mixing ratio SDS/BSA = 1. With the increase in the amount of SDS, the peak P3 grows at the expense of P1 and P2. There is only a single peak P3 in the curve for the systems SDS/BSA > 7, and no peak at SDS/BSA = 50 and 100. There is a single peak P12 in the curve for BSA at pH 7 and in the presence of 0.05 M NaCl, indicating that the heat-induced transition is suppressed. There are two peaks P12 and P3 for the systems SDS/BSA = 1-5; the area ratio of the peak P3 to P12 increases with the increase in the amount of SDS. There is only a single peak P3 when SDS/BSA > 7, and no peak at SDS/BSA = 50. It is concluded that the peak P3 is a product of SDS regardless of the presence or absence of NaCl. Values of thermal denaturation temperature (Td) and enthalpy (delta H) of thermal denaturation indicate that the complex AD12 (A = BSA, D = SDS) is in the most thermostabilized state.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA analyses of XX and XX-hypospadiac males

Fourteen 46,XX males were analyzed by Southern blot hybridization with seventeen different Y chromosome-derived DNA probes and by the polymerase chain reaction for an additional two sites on the short arm of Y. Eight 46,XX males possessed various segments of the short arm of the Y chromosome, including the sex determining region. The detected segments ranged from the two most distal loci to nearly the entire length of the short arm, viz., 10 out of 11 loci. None of the eight patients had hypospadia. Five out of the six remaining cases had hypospadia and no Y sequence was detected, suggesting the presence of a causative difference between hypospadiac and non-hypospadiac groups.     

Discovery of a genetic polymorphism of human plasma protein C inhibitor (PCI): genetic survey utilizing isoelectric focusing followed by immunoblotting,immunological and biochemical characterization

Summary The objectives of this study were to determine the genetic basis of the electrophoretic differences of human plasma protein C inhibitors (PCI) from 977 individuals. Three discrete antibodies were produced against the PCI purified from human plasma and peptides that corresponded to the N-terminal 15 amino acid residues and the C-terminal 15 residues of human PCI, the chemical structures of which were determined by cDNA sequence analysis. The combined techniques of polyacrylamide gel isoelectric focusing and immunoblotting with these three different antibodies resolved the plasma PCI into several isoprotein bands, with a pH range of 6–7. These PCI isoproteins, however, were not stained by anti-human kallikrein, anti-human protein C or anti-human urokinase antibodies. Therefore, each of the PCI bands, which were detected by immunoblotting with the anti-PCI antibody and the two different anti-peptide antibodies, were derived from free PCI, and not an inactive PCI species. Two common phenotypes, designated PCI 1 and 1–2, were recognized, and family studies showed that they represented homozygosity or heterozygosity for two autosomal codominant alleles, PCI ^*1 and PCI ^*2. A population study of plasma samples collected from 977 Japanese individuals indicated that the frequencies of the PCI ^*1 and PCI ^*2 alleles were 0.988 and 0.012, respectively.     

Production of S-(+)-ibuprofen from a nitrile compound by Acinetobacter sp. strain AK226

S-(+)-2-(4'-Isobutylphenyl)propionic acid [S-(+)-ibuprofen] was produced from racemic 2-(4'-isobutylphenyl)propionitrile (Ibu-CN) by an isolated bacterial strain, Acinetobacter sp. strain AK226. Ammonium acetate, acetonitrile, or n-butyronitrile as a carbon source in the culture medium was effective for bacterial growth and induction of this activity. The optimum pH of the reaction was around 8.0. S-(+)-Ibuprofen formed from Ibu-CN by resting cells was present in a 95% enantiomeric excess. Acinetobacter sp. strain AK226 appeared to possess a nitrilase for Ibu-CN because 2-(4'-isobutylphenyl)propionamide was not detected in the reaction mixture and 2-(4'-isobutylphenyl)propionamide was not hydrolyzed to S-(+)-ibuprofen. Since S-(+)-ibuprofen was preferentially produced while the R enantiomer of Ibu-CN was left almost intact over the time course of the reaction, the putative nitrilase appeared to be highly specific for the S enantiomer of Ibu-CN.     

Lippia dulcis shoot cultures as a source of the sweet sesquiterpene hernandulcin

The axenic shoot culture of Lippia dulcis Trev., Verbenaceae, was established on hormone-free Murashige-Skoog solid medium containing 3% sucrose. Shoots were cultured in various liquid or solid media. Woody Plant liquid medium was best for shoot multiplication, but the production of hernandulcin was relatively low. The highest hernandulcin content (2.9% dry wt) was obtained after 28 days of culture on Murashige-Skoog solid medium containing 2% sucrose. The addition of chitosan to the culture media enhanced the growth of shoots as well as the production of hernandulcin, especially with the liquid medium.Abbreviations MS(2%) Murashige-Skoog medium containing 2 % sucrose - MS(3%) Murashige-Skoog medium containing 3 % sucrose - 1/2MS half strength Murashige-Skoog medium containing 2% sucrose - B5 Gamborg B5 medium containing 2% sucrose - WP Woody Plant medium containing 2% sucrose