Isobutene production by Rhodotorula minuta

Summary Isobutene production by Rhodotorula minuta IFO 1102 was studied. It was confirmed that the gas species produced by this yeast was isobutene from the result of analysis with a gas chromatograph mass spectrometer. Oxygen supply was essential to the microbial production of isobutene. The optimum pH was found to be approximately pH 6.0 and optimum temperature 25°–27° C. Isobutene production rate was maximal when l-leucine and l-phenylalanine in the medium were being uptaken by the yeast.The results from an investigation of the role of l-leucine and l-phenylalanine suggested that l-leucine was the precursor of isobutene and l-phenylalanine the inducer for the enzyme concerned with isobutene production.     

Enhancement of γ-linolenic acid production by Mucor ambiguus with nonionic surfactants

Summary -Linolenic acid (GLA) production by Mucor ambiguus IFO 6742, immobilised in Biomass Support Particles (BSPs), has been investigated in a fluidized-bed fermenter in the presence of nonionic surfactants. In this system, repeated batch cultivation was achieved at higher yield and productivity than by conventional methods, since microbial lipids inlcuding GLA were significantly secreted into the culture broth and/or on the surface of the cell wall.     

The GDP-fucose:N-acetylglucosaminide 3-alpha-L-fucosyltransferases of LEC11 and LEC12 Chinese hamster ovary mutants exhibit novel specificities for glycolipid substrates

Previous studies have shown that the GDP-fucose:N-acetylglucosaminide 3-alpha-L-fucosyltransferase (alpha (1,3) fucosyltransferase (Fuc-T)) activities expressed by the Chinese hamster ovary cell mutants LEC11 (Fuc-TI) and LEC12 (Fuc-TII) are different enzymes and indicated that Fuc-TI might act on sialylated lactosamine sequences (Campbell, C., and Stanley, P. (1984) J. Biol. Chem. 259, 11208-11214). In this paper we show that CSLEX-1, a monoclonal antibody specific for NeuNac alpha (2,3)Gal beta (1,4)(Fuc alpha (1,3))GlcNAc beta 1 sequences, bound to LEC11 cells but not to LEC12 cells. Direct evidence that Fuc-TI could act on sialylated substrates was sought with a series of glycolipid acceptors. Optimal assay conditions in crude cell extracts were determined with nLc4, a glycolipid which accepted fucose with both Fuc-TI and Fuc-TII to generate the Lex antigenic determinant. The two enzymes differed in their detergent sensitivities, pH optima, Mn2+ requirements, and apparent Km values for nLc4. When sialylated glycolipids were examined as substrates, Fuc-TI added fucose to IV3NeuNAcnLc4 but not to IV6NeuNAcnLc4, whereas Fuc-TII was unable to utilize either glycolipid as a substrate. Further studies showed that Fuc-TI and Fuc-TII possess novel specificities for glycolipids containing two lactosamine sequences as potential fucose acceptors. Fuc-TI exhibited good activities with VI3NeuNAcnLc6 and VI6NeuNAcnLc6 whereas Fuc-TII had very low activity with both substrates. Glycosidase digestions of the labeled products showed that Fuc-TI added fucose primarily to the internal N-acetylglucosamine of both glycolipids. The same preference for the internal N-acetylglucosamine was shown by Fuc-TI when nLc6 was the acceptor. In contrast, Fuc-TII preferred to transfer fucose to the external acceptor site of nLc6, consistent with the low activities of Fuc-TII with sialylated nLc6 derivatives. Thus the two enzymes preferentially add fucose to different N-acetylglucosamines in the same substrate, nLc6. This indicates that the biosynthetic pathway for fucosylation of polylactosamine sequences in glycolipids and glycoproteins will vary depending upon the particular alpha (1,3)fucosyltransferase present.     

Kinetic studies of wheat carboxypeptidase-catalyzed reaction: differences in pressure and temperature dependence of peptidase and esterase activities

A kinetic study of hydrolytic catalysis by wheat bran carboxypeptidase (carboxypeptidase W) was carried out using 3-(2-furyl)acryloyl-acylated (Fua-) synthetic substrates. This enzyme showed high esterase activity in addition to the intrinsic carboxypeptidase activity. The optimum pH for the peptidase activity (kcat/Km) was at pH 3.3 and the kcat/Km value decreased with increasing pH with an apparent pKa of 4.50, while the esterase activity increased with pH up to pH 8 with an apparent pKa of 6.04. Optimum pH's for kcat for the peptidase and esterase reactions were also very different and their apparent pKa values were 3.80 and 6.15, respectively. From a measurement of the pressure dependences of kcat and Km, the activation volumes (delta V not equal to) and reaction volumes (delta V), respectively, were determined. delta V not equal to for kcat was -7 to -8 ml/mol for peptidase and -2 to -3 ml/mol for esterase. These results lead us to propose that the peptidase and esterase activities of carboxypeptidase W are different not in the rate-determining steps in a common reaction pathway, but in the binding modes and/or catalytic site(s).     

In vitro studies of the effects of naloxone on the root potentials in the frog spinal cord: enkephalin-like effect on the recurrent presynaptic inhibition

Specific binding of [3H]naloxone was demonstrated in the frog spinal cord. In isolated and perfused frog spinal cord, naloxone increased the spontaneous discharges of the ventral root. Naloxone decreased the ventral root-dorsal root potential (VR-DRP) in a dose-dependent manner, and inhibited presynaptic inhibition of the ventral root reflex. Methionine-enkephalin also decreased the VR-DRP, and naloxone partially antagonized this effect. These results suggest the existence of enkephalinergic control of spinal motor activities and that naloxone has a partial agonistic effect in the frog spinal cord.     

Stopped-flow investigation of the reaction between vitamin E radical and vitamin C in solution

Kinetic study of the reaction between vitamin E radical and vitamin C has been performed. The rates of reaction of vitamin C (ascorbic acid 1, 6-0-stearyl ascorbic acid 2, and 2,6-O-dipalmitoyl ascorbic acid 3) with vitamin E radical (5,7-diisopropyl-tocopheroxyl) in benzene-ethanol (2:1, v/v) solution have been determined spectrophotometrically, using stopped-flow technique. The second-order rate constants obtained are 549 +/- 30 M-1s-1 for 1, 626 +/- 53 M-1s-1 for 2, and 4.84 +/- 1.41 M-1s-1 for 3 at 25.0 degrees C. The result shows that the ascorbic acid ester 2 having a long-alkyl-chain at 6-position is 1.14 times as reactive as the ascorbic acid 1, whereas the ascorbic acid ester 3 substituted at 2-position is only 0.01 times as reactive as the ascorbic acid 1.     

Specific interaction of arabinose residue in ginsenoside with egg phosphatidylcholine vesicles

The interaction of the specific sugar residue in ginsenosides with egg phosphatidylcholine vesicles was investigated by ESR spectrometry using phosphatidic acid spin-labeled at the polar head groups. Ginsenoside-Rc, which has an alpha-L-arabinofuranose residue and agglutinability toward egg yolk phosphatidylcholine vesicles (Fukuda, K. et al. (1985) Biochim. Biophys. Acta 820, 199-206), caused the restriction of the segmental motion of spin-labeled phosphatidic acid in egg phosphatidylcholine vesicles, indicating that the saponin interacted with the polar head groups of vesicles. Other ginsenosides-Rb2, Rb1, Rd and p-nitrophenyl glycoside derivatives which have less or no agglutinability were also investigated in the same manner. Only ginsenoside-Rb2 and p-nitrophenyl alpha-L-arabinofuranoside which have the specific sugar residue (arabinose) showed a strong interaction with the polar head groups of vesicles. To gain an insight into the mechanism of agglutination by ginsenoside-Rc, the interaction with the fatty acyl groups was also studied by using phosphatidylcholine spin-labeled at the fatty acyl groups. Ginsenoside-Rc increased the order parameter of the spin-labeled phosphatidylcholine, indicating that the saponin was inserted into lipid bilayers. In other saponins investigated, only ginsenoside-Rb2 interacted with the fatty acyl part of vesicles. The process of expression of agglutination by ginsenoside-Rc was discussed on the basis of the ESR studies.     

Distribution and fine structure of ependymal cells possessing intracellular cysts in the aqueductal wall of the rat brain

Summary The wall of the cerebral aqueduct was examined in 20 male rats at the light- and electron-microscopic levels. Disorder in ciliary orientation was occasionally seen in ordinary ependymal cells. Ependymal cells possessing intracellular cysts of 5 to 30 urn in diameter were observed within and beneath the aqueductal ependyma in all animals examined. Light-microscopic reconstruction from serial, 10-m thick frontal sections revealed an extensive distribution of cystic ependymal cells (CECs), especially along the ependymal ridges in the rostral half of the aqueduct, and along the dorsal region of the aqueductal lining in the caudal half. Both cystic and surface membranes of CECs bore microvilli and cilia. Ectopic ependymal cells (EECs) characterized by densely packed microvilli, well-developed intermediate junctions and cilia, but without cysts, were situated in the subependymal region adjacent to a CEC or another EEC. The ependymal ridges were long, narrow and sporadically stratified ependymal linings extending rostrocaudally and bilaterally along the aqueductal surface. Tanycyte-like cells filled the surface region of the ridge, and CECs and EECs were frequently seen in the core. Intraventricularly injected microperoxidase was detected among densely packed microvilli but not in the cystic lumina of CECs, indicating that EECs and CECs are distinct entities.     

Effect of disuse on the ultrastructure of the achilles tendon in rats

We examined the influence exerted, through disuse of the hindlimb, on the collagen fibres of the achilles tendon in rats. With disuse the body mass decreased by 28%, and the mass of soleus muscle decreased by 20%. A decrease in the surface area and diameter was observed in the experimental group when compared to the control group. A histogram of the collagen fibres showed a decrease of the thick fibres in the experimental group. The maximum surface area of collagen fibres in the experimental group was seen to be only 43% of that of the control group. These results showed a decrease in the thickness of the collagen fibres of the achilles tendon through disuse. This seemed to suggest that resistance to tension is decreased by disuse.