During beneficial inflammation, potentially tissuedamaging granulocytes undergo apoptosis before being cleared by phagocytes in a non-phlogistic manner. Here we show that the rate of constitutive apoptosis in human neutrophils and eosinophils is greatly accelerated in both a rapid and concentration-dependent manner by the fungal metabolite gliotoxin, but not by its inactive analog methylthiogliotoxin. This induction of apoptosis was abolished by the caspase inhibitor zVAD-fmk, correlated with the inhibition of nuclear factor-kappa B (NF-B), and was mimicked by a cell permeable inhibitory peptide of NF-B, SN-50; other NF-B inhibitors, curcumin and pyrrolidine dithiocarbamate; and the proteasome inhibitor, MG-132. Gliotoxin also augmented dramatically the early (2-6 h) pro-apoptotic effects of tumor necrosis factor-␣ (TNF-␣) in neutrophils and unmasked the ability of TNF-␣ to induce eosinophil apoptosis. In neutrophils, TNF-␣ caused a gliotoxin-inhibitable activation of an inducible form of NF-B, a response that may underlie the ability of TNF-␣ to delay apoptosis at later times (12-24 h) and limit its early killing effect. Furthermore, cycloheximide displayed a similar capacity to enhance TNF-␣ induced neutrophil apoptosis even at time points when cycloheximide alone had no proapoptotic effect, suggesting that NF-B may regulate the production of protein(s) which protect neutrophils from the cytotoxic effects of TNF-␣. These data shed light on the biochemical and molecular mechanisms regulating human granulocyte apoptosis and, in particular, indicate that the transcription factor NF-B plays a crucial role in regulating the physiological cell death pathway in granulocytes.

he mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved T specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.

Full-length cDNA for the rat brain rolipram-sensitive cyclic AMP phosphodiesterase (PDE), RD1 was introduced into the expression vector pSVL. COS cells transfected with the recombinant vector pSVL-RD1 exhibited a 30-55% increase in homogenate PDE activity, which was abolished by rolipram (10 microM). Removal of the first 67 nucleotides of the RD1 cDNA yielded a truncated enzyme called Met26-RD1 which lacked the N-terminal first 25 amino acids. Whereas approx. 75% of RD1 activity was membrane-associated, Met26-RD1 activity was found exclusively in the cytosol fraction. Expression of RD1 nearly doubled membrane-associated PDE activity, while expression of Met26-RD1 increased cytosolic activity by approx. 30%. Membrane RD1 activity was found to be primarily associated with the plasma membrane, was not released by either high concentrations of NaCl or by a 'hypotonic shock' treatment, but was solubilized with low concentrations of Triton X-100. Phase separation of membrane components with Triton X-114 showed partition of RD1 into both the aqueous and detergent-rich phases, whereas Met26-RD1 partitioned exclusively into the aqueous phase. Both RD1 and Met26-RD1 specifically hydrolysed cyclic AMP; were unaffected by either Ca2+/calmodulin or by low cyclic GMP concentrations; exhibited linear Lineweaver-Burke plots with similar Km values for cyclic AMP (4 microM); both were potently and similarly inhibited by rolipram (Ki approx. 0.5 microM) and were similarly inhibited by cilostamide and 3-isobutyl-1-methylxanthine. Thermal inactivation, at 50 degrees C, showed that while the cytosolic-located fraction of RD1 (t0.5 approx. 3 min) and Met26-RD1 (t0.5 approx 3 min) were similarly thermolabile, membrane-bound RD1 was considerably more thermostable (t0.5 approx. 11 min). Treatment of both cytosolic RD1 and Met26-RD1 with Triton X-100 did not affect their thermostability, but solubilization of membrane RD1 activity with Triton X-100 markedly decreased its thermostability (t0.5 approx. 5 min). The N-terminal domain of RD1 appears not to influence either the substrate specificity or inhibitor sensitivity of this enzyme, but it does contain information which can allow RD1 to become plasma membrane-associated and thereby adopt a conformation which has enhanced thermostability.

After solubilization with the detergent Triton X-114, membrane proteins may be separated into three groups: if the membrane is sufficiently lipid-rich, one family of hydrophobic constituents separates spontaneously at low temperature; warming at 30 degrees C leads to separation of a detergent-rich phase and an aqueous phase. Using the chromaffin-granule membrane as a model, we found that many intrinsic membrane glycoproteins are found in the latter phase, probably maintained in solution by adherent detergent. They precipitate, however, when this is removed by dialysis, leaving in solution those truly hydrophilic proteins that were originally adhering to the membranes. We have used this method with mitochondria, and with Golgi- and rough-endoplasmic-reticulum-enriched microsomal fractions: it has proved to be a rapid and convenient method for effecting a partial separation of proteins from a variety of different membranes.

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