The Gram-negative bacterium Acetobacter xylinum assembles a cellulose ribbon composed of a number of microfibrils in the longitudinal axis of its envelope. The zone of ribbon assembly was investigated by freeze-etch electron microscopy. Freeze-etching revealed, beneath the cellulose ribbons, a linear array of pores on the lipopolysaccharide membrane. These pores have a rim diameter of 120-150 ]k and a central hole or deepening of ~35 A. The axes of pore arrays closely coincide with linear arrays of 100 A particles on the E-and P-faces of the fractured lipopolysaccharide membranes. Pores and particles in the lipopolysaccharide membrane are probably congruent. The pores are hypothesized to be the export sites (penetration sites) for cellulose.

The unusual amino acid D-aspartate is present in significant amounts in brain and endocrine glands and is supposed to be involved in neurotransmission and neurosecretion (Wolosker et al. [2000] Neuroscience 100:183-189). D-aspartate oxidase is the only enzyme known to metabolize D-aspartate and could regulate its level in different regions of the brain. We examined the cellular and subcellular distribution of this enzyme and its mRNA in human and rat brain by immunohistochemistry, in situ hybridization, and immunoelectron microscopy. D-aspartate oxidase protein and mRNA are ubiquitous. The protein shows a granular pattern, particularly within neurons and to a significantly lesser extent in astrocytes and oligodendrocytes. No evidence for a synaptic association was observed. Whereas between most positive neurons only gradual differences were observed, in the hypothalamic paraventricular nucleus, neurons with high enzyme content were found next to others with no labeling. cDNA cloning of D-aspartate oxidase corroborates an inherent targeting signal sequence for protein import into peroxisomes. Immunoelectron microscopy showed that the protein is localized in single membrane-bound organelles, apparently peroxisomes.

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D-Aspartate oxidase (EC 1.4.3.1) was assayed in subcellular fractions and in highly purified peroxisomes from rat, bovine and sheep kidney cortex as well as from rat liver. During all steps of subcellular-fractionation procedures, D-aspartate oxidase co-fractionated with peroxisomal marker enzymes. In highly purified preparations of peroxisomes, the enrichment of D-aspartate oxidase activity over the homogenate is about 32-fold, being comparable with that of the peroxisomal marker enzymes catalase and D-amino acid oxidase. Disruption of the peroxisomes by freezing and thawing released more than 90% of the enzyme activity, which is typical for soluble peroxisomal-matrix proteins. Our findings provide strong evidence that in these tissues D-aspartate oxidase is a peroxisomal-matrix protein and should be added as an additional flavoprotein oxidase to the known set of peroxisomal oxidases.

Background/aims-Ultraviolet (UV) irradiation of mammalian cells in culture evokes the transcriptional activation of diVerent proto-oncogenes, among them members of the fos/jun family which are known to play an important role in cell proliferation and diVerentiation. To investigate in vivo UV induced proto-oncogene expression of irradiated ocular cells, the expression of JunB, JunD, and Egr-1 was analysed in the cornea, lens, and retina. Furthermore, UV radiation is known to induce pleiotrophic events in irradiated cells which include growth arrest, inflammation, and even cell death. In order to determine the type of cell death-for example, apoptosis versus necrosis, sections of UV irradiated rat eyes were further examined for distinct ultrastructural morphology of cell death and DNA fragmentation. Methods-Eyes of anaesthetised rats were exposed to 1.5 J/cm 2 of ultraviolet radiation (280-380 nm). Animals were perfused 6 and 16 hours after irradiation and tissue sections of enucleated bulbi were processed for light and electron microscopy. Results-Under control conditions, Jun B was constitutively expressed in numerous superficial cells but also in scattered basal cells of the corneal epithelium. After UV exposure JunB expression was massively upregulated in many cells of the basal cell layers of the corneal epithelium, although during the entire experiment, both the corneal stroma and endothelium were JunB negative. In contrast, Egr-1 was expressed exclusively in lens epithelium showing only a faint expression pattern under control conditions. However, Egr-1 expression increased after UV exposure, so that many Egr-1 positive cells of the lens epithelium could be found several hours after UV illumination. JunD was expressed in single cells of both the ganglion cell layer and the inner nuclear layer of the retina, a pattern of expression which did not change after UV exposure. Regarding the type of cell death, features of apoptosis were only occasionally present in scattered superficial cells of the corneal epithelium of control eyes. After UV exposure, however, morphological signs of apoptosis and TUNEL positive cells were visible both in the stroma and epithelium of the rat cornea. In contrast, UV irradiated lens epithelial cells exhibited features typical of necrosis. The corneal endothelium and the retina did not show any indications of morphological changes indicative of cell death after UV irradiation. Conclusion-Each proto-oncogene encoded protein was found to be expressed in a tissue specific manner and UV irradiation diVerentially modulates the expression pattern of these transcriptional regulatory proteins. This temporospatial expression pattern of these proteins is accompanied by two morphologically distinct types of cell death in the cornea and lens after UV irradiation. (Br J Ophthalmol 1999;83:225-230)