Enzymatic isomerization and epimerization of D-erythrose 4-phosphate and its quantitative analysis by gas chromatography/mass spectrometry

An enzyme preparation from beef liver catalyzed the isomerization and epimerization of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate. The presence of D-erythrulose 4-phosphate and D-threose 4-phosphate was demonstrated by several analytical methods. After dephosphorylation, the presence of D-erythrulose and D-threose was confirmed by thin-layer chromatography, gas-liquid chromatography and an enzymatic method depending upon D-erythrulose reductase. The enzymatic products were also identified and simultaneously quantitated by a new procedure using gas chromatography/mass spectrometry. Each of three tetroses was distinguished by the combination of the reduction with sodium borodeuteride and the determination of relative intensities of the ion pairs m/z 379 and 380 of sugar tetritol trifluoroacetate. By gas chromatography/mass spectrometry, we observed that D-threose 4-phosphate was also converted into D-erythrulose 4-phosphate and D-erythrose 4-phosphate. At the equilibrium, about 90% of the tetrose 4-phosphate existed in the form of D-erythrulose 4-phosphate. On the basis of gas chromatography/mass spectrometric evidence together with gas chromatographic and thin-layer chromatographic patterns, it is suggested that the single enzyme of the beef liver catalyzed both reactions of isomerization and epimerization of aldotetrose 4-phosphate.     

On the Stability of Clathrate Hydrates Encaging Polar Guest Molecules: Contrast in the Hydrogen Bonds of Methylamine and Methanol Hydrates

Abstract

The stability of clathrate hydrates encaging highly polar guests has been investigated in order to explain the experimental observation that some amines form clathrate hydrates but alcohols act as inhibitor to hydrate formation. We choose methylamine and methanol as guest species and examine the stable structure, at which the total potential energy has a minimum value. At the local minima of those two hydrates, the potential energies of water-water and guest-water, and their hydrogen bonded networks are compared. It is found that methanol does not retain the host lattice structure, while the host-network structure is kept in the presence of methylamine. It is shown that the difference in the magnitude of the partial charge on the hydrogen atom between the hydroxyl and amino groups plays a much more significant role on the stability of both clathrate hydrates than the difference in molecular geometry. This is supported from the result of a methylamine-like model that has the same partial charges on the atoms in the hydrophilic site as methanol.     

Mechanisms of modulation of neuronal nicotinic receptors by substance P and OAG

Substance P is known to modulate neuronal nicotinicacetylcholine receptors (nAChRs) in the sympathetic nervous system.There are two conflicting proposals for the mechanism of this effect, an indirect action mediated by protein kinase C (PKC) and a direct interaction with receptor subunits. We studied the mechanisms of thiseffect in PC-12 cells. Substance P enhanced the decay of thenicotine-induced whole cell current. This effect was fast in its onsetand was not antagonized by guanosine5'-O-(2-thiodiphosphate), a G protein blocker, orstaurosporine, a nonselective PKC blocker. Staurosporine failed toreverse the inhibition by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic diacylglycerol analog known to activate PKC. Theinhibitory effects of the peptide and OAG were preserved in excisedpatches, but substance P applied to the extra patch membrane wasineffective in the cell-attached patch configuration. We conclude thatsubstance P modulates neuronal nAChRs most likely by direct interactions with the receptors but independently from activation ofPKC or G proteins and that PKC does not participate in modulation by OAG.

     

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.     

Highly frequent single amino acid substitution in mammalian elongation factor 2 (EF-2) results in expression of resistance to EF-2-ADP-ribosylating toxins

Toxin-resistant polypeptide chain elongation factor 2 cDNA has been cloned from a mutant hamster cell line with only non-ADP-ribosylatable elongation factor 2. The mutation conferring resistance to diphtheria toxin and Pseudomonas aeruginosa exotoxin A is a G-to-A transition in the first nucleotide of codon 717. Codon 715 encodes a histidine residue that is modified post-translationally to diphthamide, which is the target amino acid for ADP-ribosylation by both toxins. Transfection of mouse L cells with a recombinant elongation factor 2 cDNA differing from the wild-type only by this G-to-A transition confers resistance to P. aeruginosa exotoxin A. The degrees of toxin-resistant protein synthesis of stable transfectants are dependent on the ratio of non-ADP-ribosylated elongation factor 2 to wild-type elongation factor 2, not the amount of non-ADP-ribosylated elongation factor 2. The mutation creates a new Mbo II restriction site in the elongation factor 2 gene. Several independently isolated diphtheria toxin-resistant Chinese hamster ovary cell lines show the same alteration in the Mbo II restriction pattern.     

Decreased intracellular free magnesium in erythrocytes of spontaneously hypertensive rats

Using 31p-NMR (the phosphorus nuclear magnetic resonance) spectroscopy, we measured intracellular free Mg levels in the erythrocytes of untreated (n = 7) and diltiazem-treated spontaneously hypertensive rats (SHR) (n = 8), and compared them with age-matched Wistar-Kyoto rats (WKY) (n = 10). The intracellular free Mg levels were significantly (p less than 0.01) decreased in untreated SHR compared with those in control WKY. A successful antihypertensive treatment with diltiazem increased the intracellular free Mg levels compared with untreated SHR (p less than 0.05). Furthermore, an inverse correlation was observed between intracellular free Mg levels and blood pressure levels in all groups (r = -0.48, p less than 0.01, n = 25). These observations suggest that abnormalities of intracellular Mg metabolism may be, in part, related to the development or the maintenance of hypertension in SHR.     

Cauliflower mosaic virus gene VI causes growth suppression, development of necrotic spots and expression of defence-related genes in transgenic tobacco plants

Summary In order to study possible functions of the inclusion body matrix protein (IBMP) encoded by gene VI of cauliflower mosaic virus (CaMV), the XbaI fragment containing the gene VI of a Japanese strain of CaMV (CaMV S-Japan) was transferred to tobacco plants by Ti mediated transformation. Eight out of 18 kanamycin resistant plants (40%) expressed detectable levels of IBMP. Those transgenic plants expressing IBMP produced leaves with light green color, and their growth was suppressed as compared with control plants. Symptom-like necrotic spots also appeared on the leaves and stems of the mature transgenic plants. Furthermore, in these transgenic plants, pathogenesis-related proteins 1a, 1b and 1c were highly expressed and the activity of 1,3--glucanase was increased up to eightfold. From these results, we concluded that expression of the IBMP is associated with symptom development.     

Characterization of Vegetal Plate Cells Separated from Cytochalasin B-Treated Blastulae of the Sea Urchin, Clypeaster japonicus

Vegetal plate forming cells (VPCs) of the vegetal plate blastulae of the sea urchin, Clypeaster japonicus , had a layer of microfilaments on the basal side. The VPCs specifically protruded from the embryos after a treatment with 1 μg/ml of cytochalasin B (CB). Based on scanning electron microscopy, unlike other epithelial cells the protruded VPCs possessed neither cilium nor microvilli on their surface. The protruded VPCs were easily separated from the embryos by stirring the embryo suspension with pipette. An in vitro immunohistochemistry using a primary mesenchyme cell (PMC) surface-specific monoclonal antibody (MAb) raised against PMCs of Strongylocentrotus purpuratus showed that the MAb also specifically bound to the PMCs in mesenchyme blastulae of C. japonicus . The MAb bound in 81% of the separated VPCs in C. japonicus vegetal plate blastulae examined. However, the MAb binding occurred only after the separated VPCs were incubated in artificial sea water (ASW) for at least 1 hr. In the VPC-deprived embryos, gastrulation occurred after they were transferred to normal ASW. However, the PMCs and the spicules were not formed in these embryos.
In conclusion, a majority of the VPCs separated from the CB-treated blastulae were presumptive PMCs. These VPCs provide an excellent source of presumptive PMCs.     

The direct activation of human multidrug resistance gene (MDR1) by anticancer agents

Enhanced expression of a multidrug-resistance gene (MDR1) is observed in some cancer patient, but any regulatory mechanisms of MDR1 gene expression in this phenomenon is not yet known. In this study, the regulation of MDR1 gene was analysed by transient expression assays in the presence of anticancer agents. We found that MDR1 promoter could be activated directly on the addition of anticancer agents including vincristine, daunomycin, adriamycin and colchicine. The results suggest that the level of MDR1 mRNA expression is associated with previous chemotherapy, including drugs that select the multidrug resistance phenotype.