A 150-kDa protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) was previously purified from rat brain. The sequences of peptides derived from the purified PLD inhibitor now identify it as synaptojanin, a nerve terminal protein that has been implicated in the endocytosis of fused synaptic vesicles and shown to be a member of the inositol polyphosphate 5-phosphatase family. Further characterization of the enzymatic properties of synaptojanin now shows that it hydrolyzes only the 5-phosphate from inositol 1,4,5-trisphosphate (I(1,4,5)P 3 ) and that it does not catalyze the dephosphorylation of either I(1,3,4)P 3 or inositol 1,4-bisphosphate. However, synaptojanin hydrolyzes both the 4-and 5-phosphates of PI(4,5)P 2 and the 4-phosphate of phosphatidylinositol 4-phosphate, converting both compounds to phosphatidylinositol. Magnesium is required for the hydrolysis of I(1,4,5)P 3 , but not for that of phosphoinositides, by synaptojanin. The inhibition of PLD by synaptojanin is attributable to its ability to hydrolyze PI(4,5)P 2 . Synaptojanin did not inhibit PLD in the absence of PI(4,5)P 2 , and the extent of PLD inhibition was related to the extent of PI(4,5)P 2 hydrolysis in substrate vesicles. It has been proposed that the biosynthesis of PI(4,5)P 2 and the activation of PLD by ADP-ribosylation factor constitute a positive loop to increase rapidly the concentrations of PI(4,5)P 2 and phosphatidic acid (PA) during membrane vesiculation. The PA thus produced, probably together with PI(4,5)P 2 , facilitates vesicle coat assembly. The hydrolysis of PI(4,5)P 2 , and consequent inhibition of PLD, by synaptojanin might therefore constitute a mechanism to halt the positive loop connecting PI(4,5)P 2 and PA during the endocytotic cycle of synaptic vesicles and serve as a signal for uncoating.

Rat brain cytosol contains proteins that markedly inhibit the activity of partially purified brain membrane phospholipase D (PLD) stimulated by ADP-ribosylation factor (Arf) and phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Sequential chromatography of the brain cytosol yielded four inhibitor fractions, which exhibited different kinetics to heat treatment at 70°C. Purification of the most heat-labile inhibitor to homogeneity yielded two preparations, which displayed apparent molecular masses of 150 kDa and 135 kDa, respectively, on SDSpolyacrylamide gels. Tryptic digests of the 150-and 135-kDa proteins yielded similar elution profiles on a C 18 reverse-phase column, suggesting that the 135-kDa form is a truncated form of the 150-kDa form. Sequences of two tryptic peptides were determined. A data base search revealed no proteins with these sequences.The purified 150-kDa inhibitor negated the PLD activity stimulated by Arf, RhoA, or Cdc42. The concentration required for half-maximal inhibition was 0.4 nM. Concentration dependence on the 150-kDa inhibitor was not affected by changes in the concentrations of Arf, PIP 2 , or phosphatidylcholine used in the assays, suggesting that the inhibition is not due to competition with the activators or substrate for PLD. The purified inhibitor did not affect the PIP 2 -hydrolyzing activity of a phospholipase C isozyme that was measured with substrate vesicles of lipid composition identical with that used for the PLD assay. Thus, the mechanism of inhibition appears to be a specific allosteric modification of PLD rather than disruption of substrate vesicles.

Effects of synthetic cytidine-phosphate-guanosine (CpG) oligodeoxynucleotide (ODN) on respiratory burst activity of olive flounder (Paralichthys olivaceus) head-kidney phagocytes and on protection against lethal infection with Edwardsiella tarda were investigated. Phagocytes precultured with a CpG ODN showed significantly higher chemiluminescence (CL) responses than phagocytes precultured with guanosine-phosphate-cytidine (GpC) ODN or culture medium alone (control) at all concentrations. Supernatants produced from leucocytes, which were pulsed with CpG ODN, induced significantly higher respiratory burst activity than supernatants produced by GpC ODN or culture medium alone. In an in vivo experiment, respiratory burst activities of the head kidney phagocytes in the groups injected either 0.25 or 0.5 µg fish -1 of CpG ODN were significantly higher than those in the groups injected with GpC ODN or HBSS (control) at 3, 5 and 7 d after injection. The groups of fish injected with 0.25 or 0.5 µg of CpG ODN showed higher survival rates (83.3%) than groups treated with GpC ODN (33.3%) and a control group (8.3%) after challenge with E. tarda. The present in vitro and in vivo experiments have demonstrated the ability of synthetic CpG ODN to increase phagocyte respiratory burst activity and disease resistance in olive flounder.

15980 -15985), synaptojanin is identified as a protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). Here, the purification from rat brain cytosol of another PLD-inhibitory protein that is immunologically distinct from synaptojanin is described, and this protein is identified as clathrin assembly protein 3 (AP3) by peptide sequencing and immunoblot analysis. AP3 binds both inositol hexakisphosphate and preassembled clathrin cages with high affinity. However, neither inositol hexakisphosphate binding nor clathrin cage binding affected the ability of AP3 to inhibit PLD. AP3 also binds to PI(4,5)P 2 with low affinity. But the PI(4,5)P 2 binding was not responsible for PLD inhibition, because the potency and efficacy of AP3 as an inhibitor of PLD were similar in the absence and presence of PI(4,5)P 2 . A bacterially expressed fusion protein, glutathione S-transferase-AP3 (GST-AP3), also inhibited PLD with a potency equal to that of brain AP3. The inhibitory effect of AP3 appeared to be the result of direct interaction between AP3 and PLD because PLD bound GST-AP3 in an in vitro binding assay. Using GST fusion proteins containing various AP3 sequences, we found that the sequence extending from residues Pro-290 to Lys-320 of AP3 is critical for both inhibition of and binding to PLD. The fact that AP3 is a synapse-specific protein indicates that the AP3-dependent inhibition of PLD might play a regulatory role that is restricted to the rapid cycling of synaptic vesicles.We recently showed that rat brain cytosol contains proteins that inhibit the activity of partially purified brain membrane phospholipase D (PLD) 1 stimulated by ADP-ribosylation factor (ARF) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) (1). Sequential chromatography of the brain cytosolic preparation yielded four inhibitory fractions, which exhibited differential sensitivity to heat. The most heat-labile inhibitor (inhibitor IA) was purified to yield a 150-kDa protein, which, in the accompanying paper (2), we showed to be synaptojanin, a nerve terminal-enriched protein with inositol polyphosphate phosphatase (IPPase) activity, and which inhibits PLD by hydrolyzing PI(4,5)P 2 .We now describe the purification of PLD inhibitor IB, which has an apparent molecular mass of 165 kDa and is less susceptible to heat treatment than synaptojanin. Sequences of tryptic peptides of inhibitor IB suggest that it is identical to clathrin assembly protein 3 (AP3). We further show that AP3 binds to PLD. EXPERIMENTAL PROCEDURESMaterials-The sources of rat brains, bovine brain phosphatidylcholine (PC), phosphatidylethanolamine (PE), GTP␥S, PI(4,5)P 2 , [cholinemethyl-3 H]dipalmitoylphosphatidylcholine ((pam) 2 PC) (50 Ci/mmol), [2-palmitoyl-9,10-3 H](pam) 2 PC (89 Ci/mmol), and n-octyl-␤-D-glucopyranoside were as described previously (2). Phytic acid (inositol hexakisphosphate, or IP 6 ) and monoclonal antibodies to AP3 (clone AP180-I) were obtained from Sigma. PLD, AR...

Of 24 tetracycline(Tc)-resistant Vibrio spp. isolated from different marine sources in Korea between 1993 and 2003, 23 were identified as carrying both tet(B) and tet(M), while 1 strain carried tet(B) only. In conjugation experiments, 3 strains appeared to be able to transfer both tet (B) and tet(M) to the recipient. Both discriminatory PCR and sequence analysis showed that tet(M) genes of Vibrio spp. appear to be a single allele containing a specific region of tet(M) in Tn1545. However, erm(B) and aphA3, known to be linked to Tn1545-like genes, were not detected in Tc-resistant Vibrio spp., even in 9 strains resistant to erythromycin. In analysis to examine the relative position of tet(B) and tet(M), it was shown that tet(M) was present at the 3'-end of the insertion sequence IS10 of Tn10 carrying tet (B). At the junctional region between Tn10 and tet(M), we found a 14 bp sequence of unknown function and the deletion of regulatory sequences reported to be needed for tet(M) expression in conjugative transposons. This is the first report of the simultaneous presence of tet (B) and tet(M), and of the tet(M) gene being linked to the 3'-end of Tn10 in Tc-resistant Vibrio spp. in Korea.

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