Steroid deficiencies are diseases affecting salt levels, sugar levels, and sexual differentiation. To study steroid deficiency in more detail, we used a gene-targeting technique to insert a neo gene into the first exon to disrupt Cyp11a1, the first gene in steroid biosynthetic pathways. Cyp11a1 null mice do not synthesize steroids. They die shortly after birth, but can be rescued by steroid injection. Due to the lack of feedback inhibition by glucocorticoid, their circulating ACTH levels are exceedingly high; this results in ectopic Cyp21 gene expression in the testis. Male Cyp11a1 null mice are feminized with female external genitalia and underdeveloped male accessory sex organs. Their testis, epididymis, and vas deferens are present, but undersized. In addition, their adrenals and gonads accumulate excessive amounts of lipid. The lack of steroid production, abnormal gene expression, and aberrant reproductive organ development resemble various steroid deficiency syndromes, making these mice good models for studies of steroid function and regulation.

SummaryLethal toxin (LT) from Clostridium sordellii (strain IP82) inactivates in glucosylating the small GTPases Ras, Rap, Ral and Rac. In the present study we show that LT-IP82 induces cell death via an intrinsic apoptotic pathway in the myeloid cell-line HL-60. LT-IP82 was found to disrupt mitochondrial homeostasis as characterized by a decrease in mitochondrial transmembrane potential and cardiolipin alterations, associated with the release of cytochrome c in the cytosol. Time-course studies of caspase activation revealed that caspase-9 and caspase-3 were activated before caspase-8. Moreover, although LT-IP82-induced cell death was abrogated by caspase-inhibitors, these inhibitors did not suppress mitochondrial alterations, indicating that caspase activation occurs downstream of mitochondria. Protection of mitochondria by Bcl-2 overexpression prevented mitochondrial changes as well as apoptosis induction. Furthermore, evidence is provided that LT-IP82-induced apoptosis is not a consequence of cortical actin disorganization, suggesting that Rac inactivation does not initiate the apoptotic process. Cell exposure to LT-IP82 leads to a co-localization of the toxin with a mitochondrial marker within 2 h. Therefore, we suggest that LT-IP82 could act at the mitochondrion level independently of its enzymatic effect on small GTPases.

Lethal toxin (LT) from Clostridium sordellii has been shown in HeLa cells to glucosylate and inactivate Ras and Rac and, hence, to disorganize the actin cytoskeleton. In the present work, we demonstrate that LT treatment provokes the same effects in HL-60 cells. We show that guanosine 5-O-(3-thiotriphosphate)-stimulated phospholipase D (PLD) activity is inhibited in a timeand dose-dependent manner after an overnight treatment with LT. A similar dose response to the toxin was found when PLD activity was stimulated by phorbol 12-myristate 13-acetate via the protein kinase C pathway. The toxin effect on actin organization seemed unlikely to account directly for PLD inhibition as cytochalasin D and iota toxin from Clostridium perfringens E disorganize the actin cytoskeleton without modifying PLD activity. However, the enzyme inhibition and actin cytoskeleton disorganization could both be related to a major decrease observed in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ). Likely in a relationship with this decrease, recombinant ADP-ribosylation factor, RhoA, Rac, and RalA were not able to reconstitute PLD activity in LT-treated cells permeabilized and depleted of cytosol. Studies of phosphoinositide kinase activities did not allow us to attribute the decrease in PtdIns(4,5)P 2 to inactivation of PtdIns4P 5-kinase. LT was also found to provoke a major inhibition in phosphatidylinositol 3-kinase that could not account for the inhibition of PLD activity because wortmannin, at doses that fully inhibit phosphatidylinositol 3-kinase, had no effect on the phospholipase activity. Among the three small G-proteins, Ras, Rac, and RalA, inactivated by LT and involved in PLD regulation, inactivation of Ral proteins appeared to be responsible for PLD inhibition as LT toxin (strain 9048) unable to glucosylate Ral proteins did not modify PLD activity. In HL-60 cells, LT treatment appeared also to modify cytosol components in relationship with PLD inhibition as a cytosol prepared from LT-treated cells was less efficient than one from control HL-60 cells in stimulating PLD activity. Phosphatidylinositol transfer proteins involved in the regulation of polyphosphoinositides and ADP-ribosylation factor, a major cytosolic PLD activator in HL-60 cells, were unchanged, whereas the level of cytosolic protein kinase C␣ was decreased after LT treatment. We conclude that in HL-60 cells, lethal toxin from C. sordellii, in inactivating small G-proteins involved in PLD regulation, provokes major modifications at the membrane and the cytosol levels that participate in the inhibition of PLD activity. Although Ral appeared to play an essential role in PLD activity, we discuss the role of other small G-proteins inactivated by LT in the different modifications observed in HL-60 cells. Phospholipase D (PLD)1 hydrolyzes phosphatidylcholine, the major membrane phospholipid, yielding choline and phosphatidic acid (PA). PA, a possible second messenger, is fusogenic, and its increase is associated with several important cellular modifications suc...

Mass spectrometry was applied to the identification of the destruxins (dtxs), cyclic peptides that are commonly produced by the fungal insect-pathogen, Metarhizium anisopliae. The aim of the study was to optimise a methodology in order to firstly determine whether these compounds were present in other species and to determine the effect of differing growth conditions upon the dtx content detected. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) was initially used to analyse the dtxs, but limitations were indicated. Nano-scale high-performance liquid chromatography/electrospray ionisation mass spectrometry (HPLC/ESI-MS) and automated 'data-dependent' tandem mass spectrometric (MS/MS) analysis were also applied, utilising characteristic neutral losses during fragmentation to confirm the presence of the dtxs. This latter approach distinguished the dtx E and B isoforms by retention time and diagnostic neutral losses during fragmentation allowing extraction of the destruxin data from a complex dataset. This process revealed the presence of a number of dtxs in the fungal species Lecanicillium longisporum, a species previously not known to produce dtxs, and dtx production in this species was shown to be significantly higher in aerated cultures compared with still cultures.