A newly identified isoform of Slp2a associates with Rab27a in cytotoxic T cells and participates to cytotoxic granule secretion.

Cytotoxic T lymphocytes (CTLs) and natural killer cells help control infections and tumors via a killing activity that is mediated by the release of cytotoxic granules. Granule secretion at the synapse formed between the CTL and the target cell leads to apoptosis of the latter. This process involves polarization of the CTL's secretory machinery and cytotoxic granules. The small GTPase Rab27a and the hMunc13-4 protein have been shown to be required for both granule maturation and granule docking and priming at the immunologic synapse. Using a tandem affinity purification technique, we identified a previously unknown hematopoietic form of Slp2a (Slp2a-hem) and determined that it is a specific effector of the active form of Rab27a. This interaction occurs in vivo in primary CTLs. We have shown that (1) Rab27a recruits Slp2a-hem on vesicular structures in peripheral CTLs and (2) following CTL-target cell conjugate formation, the Slp2a-hem/Rab27a complex colocalizes with perforin-containing granules at the immunologic synapse, where it binds to the plasma membrane through its C2 domains. The overexpression of a dominant-negative form of Slp2a-hem markedly impaired exocytosis of cytotoxic granules-indicating that Slp2a is required for cytotoxic granule docking at the immunologic synapse.

Role of protein kinase C delta in reactivation of Kaposi's sarcoma-associated herpesvirus.

TPA (12-O-tetradecanoylphorbol-13-acetate), a well-known activator of protein kinase C (PKC), can experimentally induce reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) in certain latently infected cells. We selectively blocked the activity of PKC isoforms by using GF 109203X or rottlerin and demonstrated that this inhibition largely decreased lytic KSHV reactivation by TPA. Translocation of the PKCdelta isoform was evident shortly after TPA stimulation. Overexpression of the dominant-negative PKCdelta mutant supported an essential role for the PKCdelta isoform in virus reactivation, yet overexpression of PKCdelta alone was not sufficient to induce lytic reactivation of KSHV, suggesting that additional signaling molecules participate in this pathway.

Diffusion of beta-lactam antibiotics through oligomeric or monomeric porin channels of some gram-negative bacteria.

The penetration of anionic beta-lactam antibiotics through porins was evaluated as a mechanism of drug resistance. The major proteins with porin activity were purified from the outer membranes of six bacteria. Three of the six porins were oligomeric porins. The molecular weights of their monomers were 37 kDa from Photobacterium damsela, 42 kDa from Serratia liquefaciens, and 36 kDa from E. coli B. The other three porins were heat-modifiable monomeric porins with molecular weights of 43 kDa from Porphyromonas asaccharolytica and Acinetobacter baumannii, and 37 kDa from Escherichia coli K12. Comparison of the six porin proteins revealed that, independent of their aggregation state, their amino acid content is similar but not identical. All have double the amount of negatively charged amino acids compared with positively charged amino acids. They have a similar polarity and polarity index. Two of the six tested bacteria do not produce beta-lactamase. These two bacteria were sensitive to the different beta-lactams tested. The other four bacteria were resistant to all or to several beta-lactams.A modified liposome swelling method was used for determining the rate of penetration of charged beta-lactam antibiotics. Zwitterionic beta-lactams were found to penetrate into liposomes at a rate that more or less fits their molecular weight, whether the porins are monomeric or oligomeric. The penetration rates of negatively charged beta-lactams are different for oligomeric and monomeric porins. Negatively charged beta-lactams penetrate through oligomeric porins better than estimated by their molecular weight, whereas monomeric porins are less penetrable to negatively charged beta-lactams than estimated by their molecular weight. The contribution of all types of porins to the susceptibility of bacteria to beta-lactam antibiotics (zwitterionic or negatively charged) is apparently doubtful. The porins may decrease or increase bacterial penetration rates to beta-lactams, and only the existence of a potential beta-lactamase that can destroy the penetrating drug will cause resistance.