Nicastrin is an integral component of the high molecular weight presenilin complexes that control proteolytic processing of the amyloid precursor protein and Notch. We report here that nicastrin is most probably a type 1 transmembrane glycoprotein that is expressed at moderate levels in the brain and in cultured neurons. Immunofluorescence studies demonstrate that nicastrin is localized in the endoplasmic reticulum, Golgi, and a discrete population of vesicles. Glycosidase analyses reveal that endogenous nicastrin undergoes a conventional, trafficking-dependent maturation process. However, when highly expressed in transfected cells, there is a disproportionate accumulation of the endo-␤-N-acetylglucosaminidase H-sensitive, immature form, with no significant increase in the levels of the fully mature species. Immunoprecipitation revealed that presenilin-1 interacts preferentially with mature nicastrin, suggesting that correct trafficking and co-localization of the presenilin complex components are essential for activity. These findings demonstrate that trafficking and post-translational modifications of nicastrin are tightly regulated processes that accompany the assembly of the active presenilin complexes that execute ␥-secretase cleavage. These results also underscore the caveat that simple overexpression of nicastrin in transfected cells may result in the accumulation of large amounts of the immature protein, which is apparently unable to assemble into the active complexes capable of processing amyloid precursor protein and Notch.

Continuously growing cultures of E. coli B/r were irradiated with a fluence of broad-band near-ultraviolet radiation (315-405 nm) sufficient to cause extensive growth delay and complete cessation of net RNA synthesis. Chloramphenicol treatment was found to stimulate resumption of RNA synthesis, similar to that observed with chloramphenicol treatment after amino-acid starvation. E. coli strains in which amino-acid starvation does not result in cessation of RNA synthesis ("relaxed" or ret strains) show no cessation of growth and only a slight effect on the rate of growth or of RNA synthesis. These findings show that such near-UV fluences do not inactivate the RNA synthetic machinery but affect the regulation of RNA synthesis, in a manner similar to that produced by amino-acid starvation. Such regulation is believed to be mediated through alterations in concentration of guanosine tetraphosphate (ppGpp), and our estimations of ppGpp after near-UV irradiation are consistent with such an interpretation. These data, combined with earlier published data, strongly suggest that the mechanism of near-UV-induced growth delay in E. coil involves partial inactivation of certain tRNA species, which is interpreted by the cell in a manner similar to that of amino-acid starvation, causing a rise in ppGpp levels, a shut-off of net RNA synthesis, and the induction of a growth delay.Near-ultraviolet radiation (near-UV; 300-380 nm) is present in sunlight, but is absorbed only marginally, if at all, by proteins and nucleic acids. Yet the well-known deleterious effects of far-ultraviolet radiation (far-UV; below 300 nm), such as killing, induction of mutation, and inhibition of growth, are found to occur in bacteria throughout the near-UV region (1, 2).Of these effects, the one induced by the lowest fluences of near UV is the inhibition of growth*. Bacterial studies have shown that growth delay has a narrow action spectrum, peaking at 340 nm. Earlier suggestions that quinones might be the chromophores and oxidative phosphorylation the cellular target (1) have not been supported by recent work showing that coenzyme Q is resistant to near-UV radiation in vivo (3) and that ATP synthesis shows a threshold response (B. Lakchaura, T. Fossum, and J. Jagger, J. Bacteriol., 125, in press). In addition, it is unlikely that DNA is the target for near-UV-induced growth inhibition (4).Inhibition of growth clearly must involve inhibition of macromolecular synthesis. Swenson et al. (5) He has also shown that the action spectrum for induction of this inhibition of RNA synthesis is similar to the action spectrum for induction of growth delay in E. coli, and that the growth delay action spectrum fits very closely to the absorption spectrum of tRNAs that contain the unusual nucleoside 4-thiouridine (4Srd). It is known that near-UV irradiation in vitro of such tRNAs can induce an adduct between the 4Srd in the 8-position and a cytidine in the 13-position of the tRNA, and that this lowers the rate at which the tRNA accepts amino acids (7). The f...

Fluences (21-32 kJ/m2) of near-ultraviolet radiation that induce about a 1-h growth delay in continuously growing cultures of E. coli B/r are found to produce complete cessation of net RNA synthesis, while the effects on protein and DNA synthesis are considerably milder. The near-UV action spectrum for this inhibition of RNA synthesis is similar to the action spectrum for growth delay in E. coli B and to the absorption spectrum of E. coli valyl transfer RNA. In addition, the fluences required for inhibition of RNA synthesis and for growth delay are similar to those reported for formation of 4-thiouridine-cytidine adducts in transfer RNA. These findings suggest that the chromophore and target for near-UV-induced inhibition of both net RNA synthesis and growth in E. coli may be 4-thiouridine in transfer RNA.

Abstract— Near‐ultraviolet (near‐UV; 320–405 nm) irradiation of Escherichia coli B/r induces the formation in vivo of 4Srd‐Cyd adducts in transfer RNA, as evidenced by (1) fluorescence spectrum changes of tRNA extracted from irradiated cells and reduced with NaBH4, (2) thin‐layer chromatography on cellulose of hydrolysates of trichloroacetic acid‐precipitable extracts of irradiated cells, and (3) comparison of these findings with adduct formation induced by near‐UV irradiation of purified mixed tRNA from E. coli. The kinetics of induction of the 4Srd‐Cyd adduct in vivo, and the near‐UV fluences required, provide strong support for our earlier hypothesis that formation of these adducts is responsible for near‐UV‐induced growth delay in E. coli.

A mutant of Escherichia coli has been isolated that lacks 4-thiouridine, a rare base in transfer ribonucleic acid. The mutant grows at the same rate as wild-type cells. It shows little near-ultraviolet-induced growth delay, thus supporting earlier hypotheses that 4-thiouridine in transfer ribonucleic acid is the chromophore for this growth delay.