The minimal number of genes required for the formation of gas vesicles in halophilic archaea has been determined. Single genes of the 14 gvp genes present in the p-vac region on plasmid pHH1 of Halobacterium salinarum (p-gvpACNO and p-gvpDEFGHIJKLM) were deleted, and the remaining genes were tested for the formation of gas vesicles in Haloferax volcanii transformants. The deletion of six gvp genes (p-gvpCN, p-gvpDE, and p-gvpHI) still enabled the production of gas vesicles in H. volcanii. The gas vesicles formed in some of these gvp gene deletion transformants were altered in shape (⌬I, ⌬C) or strength (⌬H) but still functioned as flotation devices. A minimal p-vac region (minvac) containing the eight remaining genes (gvpFGJKLM-gvpAO) was constructed and tested for gas vesicle formation in H. volcanii. The minvac transformants did not form gas vesicles; however, minvac/gvpJKLM double transformants contained gas vesicles seen as light refractile bodies by phase-contrast microscopy. Transcript analyses demonstrated that minvac transformants synthesized regular amounts of gvpA mRNA, but the transcripts derived from gvpFGJKLM were mainly short and encompassed only gvpFG(J), suggesting that the gvpJKLM genes were not sufficiently expressed. Since gvpAO and gvpFGJKLM are the only gvp genes present in minvac/JKLM transformants containing gas vesicles, these gvp genes represent the minimal set required for gas vesicle formation in halophilic archaea. Homologs of six of these gvp genes are found in Anabaena flos-aquae, and homologs of all eight minimal halobacterial gvp genes are present in Bacillus megaterium and in the genome of Streptomyces coelicolor.

Prostaglandin E2 (PGE2) produced by cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) plays an important role in the pathophysiology of inflammation, pain, and fever. We investigated the actions of TNFalpha toward stimulation of PGE2 synthesis in primary spinal cord neurons. TNFalpha induced COX-2 and mPGES-1 expression in neurons, followed by formation of PGE2, which was blocked by a selective COX-2 inhibitor. Surprisingly, the "selective COX-1" inhibitor SC-560 completely inhibited TNFalpha-induced PGE2 synthesis in neurons at nanomolar concentrations. Moreover, SC-560 inhibited PGE2 and thromboxane A2 synthesis in human monocytes and platelets with IC50 of 1.8 nM and 2.5 nM, respectively. SC-560 treatment neither altered TNFalpha-induced COX-2 or mPGES-1 expression nor did the addition of the calcium ionophore A23187 or arachidonic acid reverse the inhibition by SC-560. Moreover, no influence of SC-560 on PGE2 synthase activities or PGE2 transport was seen. Most importantly, SC-560 blocked TNFalpha-induced PGE2 synthesis in COX-1-deficient spinal cord neurons, demonstrating a COX-1-independent inhibition of PGE2 synthesis. Although SC-560 inhibited LPS-induced PGE2 synthesis in neurons and RAW264.7 macrophages in whole cell assays, no inhibition was observed in lysates of the same cells. Taken together our data demonstrate that SC-560 acts at least in some cell types as an unselective COX inhibitor despite its selectivity toward COX-1 under cell-free conditions.

The transcription of the 14 p-gvp genes involved in gas vesicle formation of Halobacterium salinarum PHH1 is driven by the four promoters pA, pD, pF and pO. The regulation of these promoters was investigated in Haloferax volcanii transformants with respect to the endogenous regulatory proteins GvpE and GvpD. Northern analyses demonstrated that the transcription derived from the pA and pD promoters was enhanced by GvpE, whereas the activities of the pF and pO promoters were not affected. Similar results were obtained using promoter fusions with the bgaH reporter gene encoding an enzyme with b-galactosidase activity. The largest amount of specific b-galactosidase activity was determined for pA-bgaH transformants, followed by pF-bgaH and pD-bgaH transformants. The presence of GvpE resulted in a severalfold induction of the pA and pD promoter, whereas the pF promoter was not affected. A lower GvpE-induced pA promoter activity was seen in the presence of GvpD in the pA-bgaH/DE ex transformants, suggesting a function of GvpD in repression. To determine the DNA sequences involved in the GvpE-mediated activation, a 50-nucleotide region of the pA promoter was investigated by 4-nucleotide scanning mutagenesis. Some of these mutations affected the basal transcription, especially mutations in the region of the TATA box and the putative BRE sequence element, and also around position "10. Mutant E, harbouring a sequence with greater identity to the consensus BRE element, showed a significantly enhanced basal promoter activity compared to wild-type.Mutations not affecting basal transcription, but yielding a reduced GvpE-mediated activation, were located immediately upstream of BRE. These results suggested that the transcription activation by GvpE is in close contact with the core transcription machinery.

Nociception-evoked prostaglandin E 2 (PGE 2 ) release in the spinal cord contributes considerably to the development of hyperalgesia and allodynia. Biosynthesis of PGE 2 involves the conversion of arachidonic acid to PGH 2 by cyclooxygenases (COXs), followed by an isomerization of PGH 2 to PGE 2 by PGE 2 synthases (PGESs). The roles of COX-1, COX-2, and the inducible microsomal PGES-1 have been studied in models of pain and inflammation. In contrast, in nociceptive processes, very little is known about the role of cytosolic PGES (cPGES), which has been described as being functionally coupled to COX-1. Here we show by in situ hybridization and immunohistological analysis that COX-1 and cPGES are constitutively expressed in neuronal and non-neuronal cells of the dorsal and ventral horns in the spinal cord of adult rats. The protein levels of both enzymes were not regulated by nociceptive stimuli; however, reduction of cPGES in rat spinal cord with intrathecal application of cPGES antisense oligonucleotides reduced the nociceptive behavior in zymosan-evoked thermal hyperalgesia and in the formalin assay. The data indicate that cPGES plays an important role in mediating early responses during spinal nociceptive processing.