Uridine sugar nucleotides modified in their nucleoside moieties and their effect on lipid-linked saccharide formationin vitro

Uridine diphospho glucose (UDP-Glc) and uridine diphospho N-acetylglucosamine (UDP-GlcNAc), modified in the uridine moiety by either periodate oxidation of the ribose ring or substitution at position 5 of the uracil ring with fluorine, have been tested as potential inhibitors of glucosyl monophosphoryl dolichol (Glc-P-Dol) or N,N-diacetylchitobiosyl pyrophosphoryl dolichol [GlcNAc)2-PP-Dol) assembly in chick embryo cell membranes. The periodate oxidised sugar nucleotides inhibited glycosyl transfer from their respective natural counterparts by 50% at 230 micron periodate oxidised UDP-Glc and 70 micron periodate oxidised UDP-GlcNAc respectively. Inhibition in both cases was irreversible and addition of exogenous Dol-P stimulated only the residual non-inhibited reaction. Periodate oxidised UDP-GlcNAc preferentially inhibited the transfer of GlcNAc to GlcNac-PP-Dol. The sugar nucleotide containing 5-fluorouridine were, on the other hand, alternative substrates for Glc-P-Dol or (GlcNAc)2-PP-Dol synthesis. FUDP-Glc was a good substrate for Glc-P-Dol formation; having Km and Vmax values equal to those of UDP-Glc, whereas FUDP-GlcNAc was a less efficient substrate for the formation of (GlcNAc)2-PP-Dol; having Km and Vmax values one half and one third respectively of those of UDP-GlcNAc.     

Different effects of the glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine on the glycosylation of rat alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein.

The glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine were used to inhibit oligosaccharide processing in primary cultures of rat hepatocytes. Their effect on the glycosylation of alpha 1-proteinase inhibitor (alpha 1PI) and alpha 1-acid glycoprotein (alpha 1AGP) was studied. Of the three glucosidase inhibitors examined, 1-deoxynojirimycin inhibited not only oligosaccharide trimming but also glycosylation de novo of newly synthesized proteins, resulting in the formation of alpha 1PI with two and three (normally carrying three) and alpha 1AGP with two to five (normally carrying six) oligosaccharide side chains. In the presence of the glucosidase inhibitors, glucosylated high-mannose-type oligosaccharides accumulated. Whereas most of the endoglucosaminidase-H-sensitive oligosaccharides formed in the presence of 1-deoxynojirimycin contained only one glucose residue, N-methyl-1-deoxynojirimycin and castanospermine led mainly to the formation of oligosaccharides with three glucose residues. None of the three glucosidase inhibitors completely prevented the formation of complex-type oligosaccharides. Thus, in their presence, alpha 1PI and alpha 1AGP with a mixture of both high-mannose and complex-type oligosaccharides were secreted.     

UDP-glucosamine as a substrate for dolichyl monophosphate glucosamine synthesis.

The sugar nucleotide analogue UDP-glucosamine was found to function as a sugar donor in microsomal preparations of both chick-embryo cells and rat liver, yielding dolichyl monophosphate glucosamine (Dol-P-GlcN). This was characterized by t.l.c. and retention by DEAE-cellulose. Glucosamine was the only water-soluble product released on mild acid hydrolysis. Dol-P-GlcN did not serve as substrate by transferring its glucosamine moiety to dolichol-linked oligosaccharide. Competition experiments between UDP-[3H]glucose and UDP-glucosamine showed Dol-P-[3H]glucose synthesis to be depressed by 56 or 73% in microsomes from chick-embryo cells and rat liver respectively. The concentrations of the UDP-sugars in this experiment were comparable with those occurring in galactosamine-metabolizing liver. These findings suggest that Dol-P-GlcN, formed as a metabolite of D-galactosamine, may interfere with Dol-P-dependent reactions.     

Studies on lung surfactant replacement in respiratory distress syndrome. Rapid film formation from binary mixed liposomes

Binary mixed liposomes were prepared from dipalmitoylphosphatidylcholine (DPPC) and a minor compound, e.g., egg phosphatidylglycerol (PG) at a ratio of 9:1. Using different preparative techniques, large unilamellar vesicles (LUV), small unilamellar vesicles (SUV) or multilamellar vesicles (MLV) were obtained and were studied with an electron microscope for morphology, with a Wilhelmy balance for spreading and surface tension lowering potential, and in the surfactant-depleted isolated rat lung for their ability to restore expiratory lung capacity. Only the simultaneous investigation of phospholipids by negative staining and thin sectioning allows unequivocal classification of liposomes. The surface-active structures prepared with the technique of Bangham et al. (Bangham, A.D., Hill, M.W. and Miller, N.G.A. (1974) in Methods in Membrane Biology (Korn, E., ed.), Vol. 1, pp. 1-68, Plenum Press, New York) at room temperature are LUV. LUV containing DPPC:PG at a ratio of 9:1 rapidly spread to a film with high surface tension lowering potential. Within 5 min after injection into the subphase they rise to the surface and form a film at the air/liquid interface able to lower the surface tension to less than 1 mN/m at compression. SUV of the same chemical composition, however, are immediately surface-active only when spread directly onto the surface. MLV exhibit poor surface activity. LUV or pure DPPC, applied onto the surface, are weakly surface active within 5 min. DPPC vesicles injected into the subphase at 37 degrees C do not adsorb to any film with surface tension lowering potential in this time. The minor compounds PE, PI, PS, PA, lysoPC enable DPPC to form surface-active films after application on saline at 37 degrees C. Removal of surfactant decreases the expiratory lung capacity of the isolated rat lung from 49.7 to 12.4% at 4 cmH2O. After substitution with natural surfactant, the expiratory lung capacity is twice that of the washed lung (25.9%), but the original distensibility of the native lung is not restituted. The effect of LUV containing DPPC:PG at a ratio of 9:1 is also remarkable (21.2%).     

Rate theory models for ion transport through rigid pores. I. Time-dependent analysis in the case of vanishing interactions

In this first of a series of papers concerning the theoretical analysis of rate theory models for ion transport through rigid pores, the case of vanishing interactions is investigated. "Rigidity" means that ions crossing membranes through pores see a fixed structure of the pores, not changing in time. A single pore is considered to be a sequence of (n + 1) activation barriers separated by n energy minima. The explicit analytical treatment is restricted to pores with regular internal barrier structure, including the nonequilibrium situation of an applied electric field. In this case the connection with continuum diffusion models is demonstrated by performing in the limit n leads to infinity (n = number of binding sites within the pores) the transition to continuum. Thus, from diffusion equations describing a discrete number of jumps, the corresponding diffusion-like partial differential equations and boundary conditions are generated. For regular pores, from the time dependent solutions of the discrete equations, the corresponding solutions of the continuum equations are explicitly generated. The time-dependent relaxation behaviour of the discrete model is in good agreement with the continuum model if one assumes more than two binding sites in the pores.     

Inhibition of beta-1,4-Glucan Biosynthesis by Deoxyglucose : The Effect on the Glucosylation of Lipid Intermediates

GDP- and UDP-deoxyglucose inhibit the incorporation of glucose from UDP-glucose into dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides. GDP-deoxyglucose inhibits by competing with the physiological nucleotide sugars for dolichyl phosphate, and dolichyl phosphate deoxyglucose is formed. This inhibition is reversed by excess of dolichyl phosphate. UDP-deoxyglucose does not give rise to a lipid-linked derivative, and inhibition by this analog is not reversed by dolichyl phosphate. The UDP- and GDP-derivatives of deoxyglucose inhibit the incorporation of glucose into glucose-containing glycoproteins. This effect seems to be the result of the inhibition of lipid intermediates glucosylation and is comparable to the effect produced by coumarin. Cellulose synthetase activity is not affected by UDP- or GDP-deoxyglucose. On the other hand, deoxyglucose inhibits the formation of β-1,4-glucans in vivo.     

Disappearance and reformation of the nuclear lamina structure during specific stages of meiosis in oocytes

The nuclear lamina is a rigid, proteinaceous layer underlying the inner nuclear membrane of eucaryotic cells. It is present in somatic cell nuclei, disappears during mitosis, and is absent from male meiotic cells. We have investigated the disappearance and reformation of the nuclear lamina during meiosis in oocytes, using immunofluorescence and electron microscopy. We find that the status of the nuclear lamina during meiosis of oocytes differs from the reversible depolymerization seen in mitosis in two respects. First, the lamina disappears during meiotic prophase without affecting the structure of the nuclear membranes or the nuclear pores. Second, the proteins of the dissociated lamina are undetectable by immunological methods in pachytene oocytes, whereas they persist in the cytoplasm during mitosis.     

Oriented axon outgrowth from avian embryonic retinae in culture

The neural retina of avian embryos was spread on a membrane filter and cut in any desired orientation. Strips cut across the retina of 4- to 7-day chick or 3- to 6-day quail embryos were explanted onto collagen gels. Vigorous neurite outgrowth was seen for about 3 days, by which time many neurites were 3 mm long. Horseradish peroxidase (HRP) labeling showed that the cells producing the neurites were large and formed a layer near the inner limiting membrane, indicating that the neurites in vitro were axons of retinal ganglion cells. The size of the neurite population and the regions from which neurites emerged vaired with the donor age, while most neurites sprouted from the side of the explant formerly closest to the optic fissure. This pattern closely resembled that of axon growth in the normal retina, as revealed by SEM, silver staining, and HRP labeling. Mitotic inhibitors (Ara-C and FUdR) did not alter the neurite outgrowth. Pretreatment of retinae with trypsin or collagenase did not disorganize axons at the time of explantation, but tended to equalize neurite emergence on each side of the retinal strips. We suggest that microenvironmental factors, especially the enzyme-labile inner limiting membrane, are important for axon guidance in the retina.     

Steroid derivatives

Microbial transformation of some steroid 19-hydroxy compounds and 1(10),5-dienes, substituted by a hydroxyl group in position 3, by the action ofProactinomyces globerulus, yielded substances with aromatic A ring. Substrates with 17-keto group underwent simultaneously a partial reduction resulting in the formation of 17β-hydroxyl.     

Limestone gravel as growth medium in hydroponics

Summary Plants were grown in gravel beds of basalt or limestone. With the standard nutrient solution yields for the plants grown in the limestone gravel were very much reduced and the plants were chlorotic. This chlorosis and reduced yields remained even if the gravel was acid or water washed beforehand, or if it was pretreated in high phosphate concentrations.High yields, comparable to those obtained in basalt beds were obtained if phosphate and iron were added in small amounts (0.1mM P, 1 mg/1 Fe) at each irrigation. This technique to avoid lime induced chlorosis can be easily and economically accomplished in hydroponics. It proved successful here with lettuce, cucumber, tomato and eggplants.