Primary structure of an N-linked sugar chain derived from glucoamylase of Rhizopus niveus.

The primary structure of the N-linked sugar chain of Rhizopus niveus glucoamylase (major component) was investigated. The carbohydrate moiety was released from the polypeptide backbone by Flavobacterium sp. endo-beta-N-acetylglucosaminidase digestion. Studies using the method of exoglycosidase digestion of the fluorescent pyridylamino derivative, gel-permeation chromatography on Bio-Gel P-4 and 400-MHz 1H-NMR spectroscopy revealed that the most abundant structure is (Man)8-GlcNac-ol.

State-dependent learning produced by chlordiazepoxide and its transfer at different dose levels.

In Experiment I, the rat was given 15 mg/kg of chlordiazepoxide (CDP) and trained on a black-white discrimination task, motivated by electric shocks; the same animal, when given saline, was treated similarly with the reversed cue relationship on different and usually alternate sessions. Training was continued until a learning criterion under both drug and saline states and then tested with only one of five testing CDP doses (2.5, 5, 10, 20 and 25 mg/kg). During training, the rat made more errors under drug than under saline but there were little differences in starting and running times at least during the last sessions. In the transfer test, choice responses showed a bi-directional gradient around the training dose as in stimulus generalization gradient. Experiment II was identical to Experiment I, except that the same drug was tested at all five transfer doses and the resulting transfer gradient was a simple monotonic increasing function of dose levels. Discrepancy between the two experiments was discussed.

Differential effects of chlordiazepoxide on simultaneous and successive brightness discrimination learning in rats.

Chlordiazepoxide (CDP) at a dose of 20 mg/kg, i.p. was found to exert a marked disruptive effect on black-white successive discrimination learning in rats, while this effect failed to be obtained on black-gray simultaneous discrimination. Since trials to the learning criterion were found almost equal for saline rats on both successive and simultaneous discrimination tasks, the differential inhibitory effects of CDP could not be accounted for in terms of differences in difficulties of the two tasks. It is suggested that present results might be interpreted in terms of the hypotheses (a) that CDP produces a deficit in the integration of relevant stimulus cues when they were presented across trials or (b) that CDP has a disinhibitory effect in that inhibition of dominant response (positional preference) patterns is weakened by the drug, in a manner similar to effects observed in hippocampectomized animals.

Studies on the uronic acid-containing glycoproteins of Fusarium sp. M7-1: I. Isolation and some properties of the glycoproteins.

An acidic heteropolysaccharide preparation derived from the mycelium of Fusarium sp. M7-1 was fractionated into two fractions, precipitable and nonprecipitable, by treatment with cetyltrimethylammonium bromide (Cetavlon). These two fractions were further purified to apparent homogeneity on ultracentrifugation by treatment with charcoal and gel filtration chromatographies. Two glycoproteins, precipitable GP I and nonprecipitable GP II, were obtained. The molecular weights of GP I and GP II were estimated to be about 8.8 x 10(4) and 3.7 x 10(4), respectively, on gel filtration chromatography. Both GP I and GP II contained a high proportion of serine and threonine. Treatment of GP I and GP II with alkaline solution resulted in an increase in absorbance at 240 nm. Alkaline borohydride treatment markedly decreased the number of seryl and threonyl residues and resulted in an increase in alanine and the formation of 2-aminobutyric acid. It also resulted in release of low and high molecular weight carbohydrate chains. From these results, we conclude that both GP I and GP II are glycoproteins with carbohydrate chains attached to the protein moiety through O-glycosidic linkages to the hydroxyl group of serine and/or threonine.

Studies on the uronic acid-containing glycoproteins of Fusarium sp. M7-1: III. The primary structures of the acidic polysaccharides of the glycoproteins.

Two O-glycosidically linked acidic polysaccharides, AP I and AP II, were released, respectively, from glycoproteins GP I and GP II of Fusarium sp. M7-1 by alkaline borohydride treatment and purified by gel filtration chromatography. They were found to be apparently homogeneous on gel filtration chromatography and analytical ultracentrifugation. Their molecular weights were estimated to be 8.2 x 10(4) and 3.1 x 10(4), respectively. The various oligosaccharide fragments obtained from AP I and AP II by acetolysis and partial acid hydrolysis were purified by gel filtration chromatography and HPLC. Their primary structures were resolved mainly by NMR spectrometry in combination with methylation mass spectrometry. Analyses of the acetolysis and partial acid hydrolysis products and the 1H-NMR spectrum of AP I and AP II showed that they are analogues. Thus, we propose that the main parts of the acidic polysaccharides have the following structures. [formula: see text] *X, unidentified oligosaccharide chains. The numbers on the left and the numbers in parentheses outside the brackets indicate the approximate number of side chains of AP I and AP II, respectively, the saccharide sequences of which are not specified.

Studies on the uronic acid-containing glycoproteins of Fusarium sp. M7-1: II. The primary structures of the low molecular weight carbohydrate chains of the glycoproteins.

The primary structures of the O-glycosidically linked oligosaccharides isolated from glycoproteins GP I and GP II of Fusarium sp. M7-1 were established. The oligosaccharides released by alkaline borohydride treatment from the glycoproteins were purified by Bio-Gel P-4 and HPLC. This approach resulted in one monosaccharide and seven oligosaccharides. Their primary structures were resolved mainly by NMR spectrometry in combination with methylation mass spectrometry and fast atom bombardment mass spectrometry. The following structures have been determined. [formula: see text].

Identification of five embryonic hemoglobins of rat and ontogeny of their constituent globins during fetal development.

Hemoglobins of rats switch from an embryonic to an adult type during fetal development. However, very little is known about the structures and molecular species of hemoglobins occurring in the fetal life of rats. In the present study we isolated five embryonic hemoglobins, designated E1, E2, E3, E4, and E5, from the blood of rat fetuses on day 14 of gestation by ion exchange chromatography. Reverse-phase high performance liquid chromatography revealed that these hemoglobins each consist of two kinds of globins: E1(11 alpha:epsilon 1), E2(1 alpha: epsilon 1), E3(zetta: epsilon 1), E4(1 alpha: epsilon 3), and E5(zetta: epsilon 3), respectively. The complete amino acid sequences of the zetta, epsilon 1, and epsilon 3 globins were determined. The zetta globin showed characteristic features common in alpha-type embryonic globins of known species in that the N-terminus is blocked and the amino acid at position 38 is Gln. epsilon 1 and epsilon 3 are beta-type embryonic globins, sharing 73.7% amino acid homology. Interestingly, they are more similar to the corresponding mouse beta-type embryonic globins, y and z, respectively, than to each other, implying that these globins have evolved orthologously from common ancestral proteins. It was also shown that the zetta, epsilon 1, and epsilon 3 globins are almost completely replaced by the adult type alpha and beta globins in the blood of rat fetuses by day 18 of gestation.

Studies on the uronic acid-containing glycoproteins of Fusarium sp. M7-1: IV. Isolation and identification of four novel oligosaccharide units derived from the acidic polysaccharide chain.

Four novel oligosaccharide units were isolated from the acetolysis products of the acidic polysaccharide chain derived from the glycoproteins of Fusarium sp. M7-1. Their chemical structures were resolved mainly by 1H-NMR spectrometry in combination with methylation analysis and mass spectrometry. The results indicate that these oligosaccharide units originated from the side chains, GlcNAc alpha 1-->4GlcA alpha 1-->2(GlcNac alpha 1-->4)GlcA alpha 1-->2Gal, GlcNAc alpha 1-->4GlcA alpha 1-->2(GlcNAc alpha 1-->4)GlcA alpha 1-->2(GlcNac alpha 1-->4)GlcA alpha 1-->2Gal, ChN<--P--> 6Man beta 1-->4GlcA alpha 1-->2Gal, and Man beta 1-->2(ChN<--P-->6)Man beta 1-->4GlcA alpha 1-->2Gal linked together with the other units reported previously [Jikibara et al. (1992) J. Biochem. 111, 236-243] through beta 1-->6galactofuranoside linkages in the acidic polysaccharide chain.

Temporal sequence of frequency specific and nonspecific effects of flickering lights upon the occipital electrical activity in man.

Human subjects were exposed 10 times to light flicker for a period of 10 sec, being altered with equal non-flicker periods. Four flicker frequencies were studied: the resting alpha frequency, twice the frequency, and 14 and 6 c/sec, which are both unrelated to alpha rhythms. The effect of flicker stimulation upon the occipital EEG was quantified in terms of integrated values per band over an epoch of 10 sec using a Walter-type frequency analyzer.

Effects of median raphe nucleus lesions on hippocampal EEG in the freely moving rat.

The effects of median raphe lesions on the hippocampal EEG were examined in freely moving rats. First, median raphe lesions, including those restricted to the median raphe nucleus, unequivocally produced hippocampal low-frequency theta activity (5.8 Hz, SD = 0.47 Hz) during relaxed immobility which was not observed under normal conditions. This lesion-induced theta activity during immobiliy continued for at least 20 days, and was markedly suppressed by atropine sulfate (10 mg/kg, i.p.). On the other hand, reticular formation lesions had little effect on either hippocampal EEG patterns during immobility, movement or PS. Second, the mean frequency of theta activity was significantly reduced during movement and PS on the day following the median raphe lesion. These findings suggest a raphe-hippocampal pathway in which the median raphe mucleus plays a major role in hippocampal desynchronization (irregular pattern) by exerting an inhibitory influence on the hippocompal theta generating or facilitating mechanism. Thus the theta activity will be induced by the disinhibition following median raphe lesions.

Mutagenicity and dominant lethal test of kojic acid--Ames test, forward mutation test in cultured Chinese hamster cells and dominant lethal test in mice.

The mutagenicity of kojic acid was studied by means of reversion mutation test in bacteria (Ames test), forward mutation test in cultured Chinese hamster cells and dominant lethal test in mice. A positive result was obtained only in Ames test (TA 98, 1535 and 100) which was not modified by the presence of S-9 fraction. Thus, it is concluded that although kojic acid is a weak mutagen in bacteria, it is nonmutagenic in eukaryotic systems either in vivo or in vitro.