Induction of sialic acid 9-O-acetylation by diverse gene products: implications for the expression cloning of sialic acid O- acetyltransferases

Sialic acids can be modified by O-acetyl esters at the 7- and/or 9- position, altering recognition by antibodies, lectins and viruses. 9(7)- O-acetylation is mediated by a sialic acid-specific O- acetyltransferase, which has proven difficult to purify. Two groups have recently isolated cDNAs possibly encoding this enzyme, by expression cloning of human melanoma libraries in COS cells expressing the substrate ganglioside GD3. Pursuing a similar approach, we have isolated additional clones that can induce 9-O-acetylation. One clone present in a melanoma library encodes a fusion protein between a bacterial tetracycline resistance gene repressor and a sequence reported to be part of the P3 plasmid. Expression of the open reading frame is necessary for inducing 9-O-acetylation, indicating that this is not a reaction to the introduction of bacterial DNA. Another clone from a rat liver cDNA library induced 9-O-acetylation on COS cells expressing alpha2-6-linked sialic acids, and encodes an open reading frame identical to the Vitamin D binding protein. However, truncation at the 5' end eliminates the amino-terminal hydrophobic signal sequence, predicting cytosolic hyperexpression of a truncated protein. Thus, diverse types of cDNAs can indirectly induce sialic acid 9-O- acetylation in the COS cell system, raising the possibility that the real enzyme may be composed of multiple subunits which would not be amenable to expression cloning. Importantly, the cDNAs we isolated are highly specific in their ability to induce 9-O-acetylation either on alpha2-6-linked sialic acids of glycoproteins (truncated vitamin D binding protein) or on the alpha2-8-linked sialic acids of gangliosides (Tetrfusion protein). These data confirm our prior suggestion that a family of O-acetyltransferases with distinctive substrate specificities exists in mammalian systems.      

Effect of filipin on Rat-liver and yeast mitochondria

1. Under the appropriate conditions intact yeast and mammalian mitochondria exhibit a heretofore unobserved sensitivity to the polyene antibiotic, filipin. The activity of the “filipin complex” (Filipins I, II, III and IV) is shown to be primarily due to the component designated Filipin II.

2. Yeast mitochondria treated with filipin complex, or purified Filipin II, exhibit “uncoupled” succinate oxidation and inhibited -ketoglutarate oxidation. Maximum filipin effect is observed at a concentration of 4 mM Filipin II. Rat-liver mitochondria are more sensitive to filipin than yeast mitochondria, and respiratory inhibition is observed regardless of substrate.

3. In liver mitochondria filipin-inhibited respiration is not relieved by Mg²⁺, K⁺, Ca²⁺ or 2,4-dinitrophenol, but is reversed by cytochrome c.

4. It is proposed that filipin treatment leads to altered membrane permeability and that respiratory inhibition is due to a loss of endogenous respiratory cofactors or an inactivation of primary dehydrogenases. The filipin-uncoupled yeast respiration may likewise be attributed to an altered phosphate permeability of the yeast mitochondrial membranes.     

Energy metabolism in Saccharomyces cerevisiae. Effect of progressive starvation on respiration and pyridine nucleotide reduction linked to ethanol oxidation in intact cells

The progressive effects of aerobic starvation on endogenous and ethanol-linked respiration and pyridine nucleotide reduction have been studied in the yeast Saccharomyces cerevisiae. Three distinct phases of pyridine nucleotide reduction were observed when ethanol was added to unstarved yeast: an initial phase of rapid reduction and accelerating respiration (A); a steady-state phase of reduction with maximal respiration (B); a final phase of rapid reduction at anaerobiosis (C).During the first 5 hr of aeration, the steady-state Phase B was replaced by a phase of slow pyridine nucleotide reduction, while Phases A and C were unaffected. During this period, both endogenous pyridine nucleotide reduction and endogenous respiration decreased sharply.Between 5 and 22 hr of aeration, the endogenous level of reduced pyridine nucleotide declined further, while endogenous respiration remained unchanged. Concurrently, the extent of the Phase A reduction doubled.The addition of ethanol to aerobic, unstarved yeast stimulated a rapid pyridine nucleotide reduction, with further reduction occurring at anaerobiosis. Under anaerobic conditions, the addition of ethanol to unstarved yeast caused little further reduction of pyridine nucleotide. Two hours of starvation decreased the extent of the endogenously supported anaerobic reduction and correspondingly increased the ethanol-induced reduction. These results suggest that, in unstarved yeast, reducing equivalents derived from ethanol under aerobic conditions and those derived from endogenous carbohydrate under anaerobic conditions have access to the same pool of pyridine nucleotide. With starvation, this pool becomes accessible to ethanol-derived (or ethanol-mobilized) reducing equivalents under anaerobic conditions.     

Effect of ultraviolet B radiation and 100 Hz electromagnetic fields on proliferation and DNA synthesis of Jurkat cells

The use of ultraviolet B light (UVB) has been proven to be highly effective for treatment of various inflammatory skin diseases, but UVB phototherapy is limited by its carcinogenic side effects. It is necessary to uncover effectors that augment UVB so that similar or improved efficacy can be obtained with lower UVB doses. We found that low frequency, low intensity electromagnetic fields (EMFs) can act as such an effector and synergistically inhibit T lymphocyte proliferation. We first characterized the effects of UVB on Jurkat cells, a model for cutaneous T lymphocytes, and determined UVB's dose dependent inhibition of cell proliferation and induction of apoptosis. Cells exposed to a sublethal UVB dose retained their sensitivity to UVB, but repetitive irradiation seemed to cause accumulation of delayed DNA damage. We then exposed cells to combinations of UVB plus EMFs and found that 100 Hz, 1 mT EMFs decrease DNA synthesis of UVB-activated Jurkat cells by 34 +/- 13% compared to UVB alone. The decrease is, however, most effective when relatively high UVB doses are employed. Since EMFs alone had only a very weak inhibitory effect (10 +/- 2%), the data suggest that EMFs augment the cell killing effects of UVB in a synergistic way. These findings could provide the basis for development of new and improved clinical phototherapy protocols.