Study of O-glycan sialylation in C6 cultured glioma cells: evidence for post-translational regulation of a beta-galactoside alpha 2,3 sialyltransferase activity by N-glycosylation

We have studied the Gal beta 1-3GalNAc-R alpha 2,3 sialyltransferase from C6 glioma cells transferring Neu5Ac from CMP-Neu5Ac onto O-glycans of glycoproteins. Using synchronized C6 glioma cells, we showed that the alpha 2,3 sialyltransferase activity was inhibited by tunicamycin to a greater extend than DNA and protein biosynthesis suggesting inhibition of N-glycosylation of this enzyme. Additional demonstration of N-glycosylation of the alpha 2,3 sialytransferase was provided through ConA-Sepharose binding. Treatment of partially purified alpha 2,3 sialytransferase by peptide-N-glycosidase F showed a significative inhibition demonstrating that N-glycan moiety is required for complete activity of the C6 glioma cell alpha 2,3 sialyltransferase.     

Potential of ensiling for efficient management of spent residue from solid state fermentation system

Summary Studies on ensiling of spent solids from solid state fermentation process for production of cellulases byTrichoderma harzianum showed that good quality ensiled solids can be obtained by using about 43% initial substrate dry matter with 0.3% ensiling additive.     

Investigating the function of Gdt1p in yeast Golgi glycosylation

The Golgi ion homeostasis is tightly regulated to ensure essential cellular processes such as glycosylation, yet our understanding of this regulation remains incomplete. Gdt1p is a member of the conserved Uncharacterized Protein Family (UPF0016). Our previous work suggested that Gdt1p may function in the Golgi by regulating Golgi Ca^2 +/Mn^2 + homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdt1Δ mutant cultured in presence of high Ca^2 + concentration, as well as the pmr1Δ and gdt1Δ/pmr1Δ strains presented strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination. The addition of Mn^2 + confirmed the rescue of these defects. Interestingly, our structural data confirmed that the glycosylation defect in pmr1Δ could also completely be suppressed by the addition of Ca^2 +. The use of Pmr1p mutants either defective for Ca^2 + or Mn^2 + transport or both revealed that the suppression of the observed glycosylation defect in pmr1Δ strains by the intraluminal Golgi Ca^2 + requires the activity of Gdt1p. These data support the hypothesis that Gdt1p, in order to sustain the Golgi glycosylation process, imports Mn^2 + inside the Golgi lumen when Pmr1p exclusively transports Ca^2 +. Our results also reinforce the functional link between Gdt1p and Pmr1p as we highlighted that Gdt1p was a Mn^2 + sensitive protein whose abundance was directly dependent on the nature of the ion transported by Pmr1p. Finally, this study demonstrated that the aspartic residues of the two conserved motifs E-x-G-D-[KR], likely constituting the cation binding sites of Gdt1p, play a crucial role in Golgi glycosylation and hence in Mn^2 +/Ca^2 + transport.     

Spin-labelled sulfur containing neoglycolipids.

A neoglycolipid of structure beta-D-Glcp-S-(CH2)3N(OH)(CH2)4-O-cholest-5-en-3 beta-yl has been prepared in fair overall yield by reduction of the nitrone obtained by condensation of beta-D-Glcp-S-(CH2)3NHOH and OCH-(CH2)3-O-cholest-5-en-3 beta-yl. This synthetic procedure is very flexible, allowing a large range of lengths for the spacer arm, different positions for the NOH group along the spacer arm chain and the replacement of the sulfur by other bio-isosteric groups. The new neoglycolipid spontaneously oxidized to the corresponding nitroxide free radical whose EPR spectrum gave information on its conformational equilibrium which was further studied by molecular mechanics.     

Bicyclonucleosides and ring-chain interconversion.

Four types of bicyclonucleosides differing in the easiness of their ring-chain interconversion have been prepared, some exhibited anti-HIV activity and the ratio of their cyclic and open-chain forms could have some bearing on their biological activity.     

Lte1 contributes to Bfa1 localization rather than stimulating nucleotide exchange by Tem1

Lte1 is a mitotic regulator long envisaged as a guanosine nucleotide exchange factor (GEF) for Tem1, the small guanosine triphosphatase governing activity of the Saccharomyces cerevisiae mitotic exit network. We demonstrate that this model requires reevaluation. No GEF activity was detectable in vitro, and mutational analysis of Lte1’s putative GEF domain indicated that Lte1 activity relies on interaction with Ras for localization at the bud cortex rather than providing nucleotide exchange. Instead, we found that Lte1 can determine the subcellular localization of Bfa1 at spindle pole bodies (SPBs). Under conditions in which Lte1 is essential, Lte1 promoted the loss of Bfa1 from the maternal SPB. Moreover, in cells with a misaligned spindle, mislocalization of Lte1 in the mother cell promoted loss of Bfa1 from one SPB and allowed bypass of the spindle position checkpoint. We observed that lte1 mutants display aberrant localization of the polarity cap, which is the organizer of the actin cytoskeleton. We propose that Lte1’s role in cell polarization underlies its contribution to mitotic regulation.     

Isolation and characterization of a proteinaceous inhibitor of microbial proteinases induced during the hypersensitive reaction of tobacco to tobacco mosaic virus

A proteinase inhibitor is strongly induced in tobacco leaves reacting hypersensitively to tobacco mosaic virus. The tobacco inhibitor is highly active against four different serine endoproteinases of fungal and bacterial origin (EC 3.4.21.14) but inhibits poorly two serine endoproteinases of animal origin, trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1). The inhibitor has been purified to homogeneity by successive steps of conventional and high-performance liquid chromatography. When electrophoresed under denaturing conditions, it behaves as a small polypeptide with a molecular weight of about 6,000. From its amino acid composition and NH2-terminal amino acid sequence analysis, it appears that the inhibitor belongs to the potato inhibitor I family. A polyclonal antiserum was raised against the purified tobacco inhibitor and was used in immunoblotting experiments to follow inhibitor accumulation during the hypersensitive reaction of tobacco to tobacco mosaic virus. The inhibitor is highly efficient and might represent a potent fungicide and/or bactericide to be used in plant biotechnology.