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Genomic clones encompassing the entire coding region of the mouse lymphocyte pore-forming protein gene (Pfp) have been isolated and used to determine its intron-exon organization. In contrast to C9, Pfp has a simple structure, consisting of only three exons (two of which encode polypeptide), a large 5' intron, and a single, smaller intron that is situated approximately one-third of the way through the protein-coding portions of the gene. The regions encoding the homologous domains of PFP and C9 are encoded on exons 7, 8, 9, and 10 of C9, but form only approximately half of the open reading frame of exon III in Pfp. Although encoding polypeptides with related functions, the two genes possess such sharply contrasting structures as to suggest that their analogous regions may have risen independently, by a process of convergent evolution. Using a panel of somatic cell hybrid cell lines, Pfp has been mapped to chromosome 10.

The 65 kDa C8-binding protein or homologous restriction factor ( C8bp HRF ) protects cells from complement (C)-mediated lysis by binding to C8 and abrogating lytic channel formation. Human C8bp HRF is shown here to be immunologically related to human C8 and C9 and to murine lymphocyte poreforming protein (PFP, perforin). Polyclonal antibodies raised against purified C8, C9 and perforin react with C8bp HRF . The antigenic epitopes shared by these four proteins are limited to cysteine-rich or disulfide bridge-masked domains. Only complement proteins or perforin that have been disulfide-reduced elicit the production of cross-reactive antibodies when used as immunogens. Analogously, only C8bp HRF that has been disulfide-reduced reacts with these antibodies. These results suggest that C8bp HRF may belong to the complement/perforin supergene family. The function of homologous domains shared by these four proteins remains to be elucidated.

Perforin is one of the important cytolytic factors in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this paper, we report rat mAbs against mouse perforin established by immunization with a recombinant mouse perforin fragment. These mAbs reacted with purified mouse perforin prepared from cytoplasmic granules of an NK-like cell line in ELISA and Western blot analysis. However, none of these mAbs blocked the hemolytic activity of mouse perforin or absorbed it when fixed in the solid phase. These results indicate that all of these mAbs react with denatured but not with native mouse perforin. By using a combination of the mAbs, we established a sandwich ELISA, for quantitating the cellular contents of perforin. These mAbs were also useful for immunohistochemical staining analysis, and perforin was detected in the cytoplasmic granules of CTL and NK cell lines. Perforin was also detected in a minor population of lymphocytes of the spleen, liver, and lymph node. In normal spleen cells of 5- to 8-week-old mice, 12-15% of asialo GM1+ cells and 7-21% of CD8+ T cells were perforin-positive, but CD4+ T cells, B cells, and macrophages were totally negative. These data clearly show that perforin is expressed in cells of a cytotoxic character in normal mice, in the same way as in primed mice.

Pore-forming proteins (PFPs) are water-soluble proteins able to integrate into target membranes to form transmembrane pores. They are common determinants of bacterial pathogenicity and are often found in animal venoms. We recently isolated and characterized Hydralysins (Hlns), paralytic PFPs from the venomous green hydra Chlorohydra viridissima that are not found within the nematocytes, suggesting they are not involved in prey capture. The present study aimed to decipher the biological role of Hlns. Using in situ hybridization and immunohistochemistry, we show that Hlns are expressed by digestive cells surrounding the gastrovascular cavity (GVC) of Chlorohydra and secreted onto the prey during feeding. At biologically relevant concentrations, Hlns bind prey membranes and form pores, lysing the cells and disintegrating the prey tissue. Hlns are unable to bind Chlorohydra membranes, thus protecting the producing animal from the destructive effect of its own cytolytic protein. We suggest that osmotic disintegration of the prey within the GVC by Hlns, followed by phagocytosis and intracellular digestion, allows the soft-bodied green hydra to feed on hard, cuticle-covered prey while lacking the physical means to mechanically disintegrate it. Our results extend the biological significance of PFPs beyond the commonly expected offensive or defensive roles.

Chloride intracellular channel (CLIC) proteins possess the remarkable property of being able to convert from a water-soluble state to a membrane channel state. We determined the three-dimensional structure of human CLIC2 in its water-soluble form by X-ray crystallography at 1.8-A resolution from two crystal forms. In contrast to the previously characterized CLIC1 protein, which forms a possibly functionally important disulfide-induced dimer under oxidizing conditions, we show that CLIC2 possesses an intramolecular disulfide and that the protein remains monomeric irrespective of redox conditions. Site-directed mutagenesis studies show that removal of the intramolecular disulfide or introduction of cysteine residues in CLIC2, equivalent to those that form the intramolecular disulfide in CLIC1, does not cause dimer formation under oxidizing conditions. We also show that CLIC2 forms pH-dependent chloride channels in vitro with higher channel activity at low pH levels and that the channels are subject to redox regulation. In both crystal forms, we observed an extended loop region from the C-terminal domain, called the foot loop, inserting itself into an interdomain crevice of a neighboring molecule. The equivalent region in the structurally related glutathione transferase superfamily corresponds to the active site. This so-called foot-in-mouth interaction suggests that CLIC2 might recognize other proteins such as the ryanodine receptor through a similar interaction.

Omp85 proteins form a ubiquitous protein family, members of which are found in all Gram-negative bacteria. Omp85 of Neisseria meningitidis and YaeT of Escherichia coli are shown to be essential for outer membrane biogenesis. Interestingly, there exists a homologue to YaeT in E. coli and many proteobacteria, denoted YtfM, the function of which has not been described yet. Like YaeT, YtfM is predicted to consist of an amino-terminal periplasmic domain and a membrane-located carboxy-terminal domain. In this study, we present a first characterisation of YtfM by comparison to YaeT concerning structural, biochemical and electrophysiological properties. Furthermore, a knockout strain revealed that ytfM is a non-essential gene and lack of the protein had no effect on outer membrane composition and integrity. The only observable phenotype was strongly reduced growth, indicating an important role of YtfM in vivo.

Degenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases are believed to be causally related to the accumulation of amyloid oligomers that exhibit a common structure and may be toxic by a common mechanism involving permeabilization of membranes. We discovered that amyloid oligomers and the pore-forming bacterial toxin, alpha-hemolysin (alpha HL), as well as human perforin from cytotoxic T lymphocytes, share a structural and functional homology at the level of their common reactivity with a conformation-dependent antibody that is specific for amyloid oligomers, A11. The alpha HL oligomeric pores and partially folded alpha HL protomer, but not the monomer alpha HL precursor reacts with A11 antibody. A11 antibody inhibits the hemolytic activity of alpha HL, indicating that the structural homology is functionally significant. Perforin oligomers were also recognized by A11. Amyloidogenic properties of alpha HL and perforin were confirmed spectroscopically and morphologically. These results indicate that pore forming proteins (PFP) and amyloid oligomers share structural homology and suggest that PFPs and amyloid oligomers share the same mechanism of membrane permeabilization.

Cholesterol-dependent cytolysins (CDCs), a large family of bacterial toxins, are secreted as water-soluble monomers and yet are capable of generating oligomeric pores in membranes. Previous work has demonstrated that large scale structural rearrangements occur during this transition but the detailed mechanism by which these changes take place remains a puzzle. Despite evidence of structural and functional couplings between domains 3 and 4, the crystal structure of the CDC, perfringolysin O (PFO), shows the two domains do not make direct contact. Here, we present crystal structures of PFO that demonstrate movements of domain 4 are sufficient to trigger conformational changes that are transmitted through the molecule to the distant domain 3. These coupled movements result in a loss of many contacts between domain 3 and rest of the molecule that would eventually lead to the exposure of transmembrane regions in preparation for membrane insertion. The structures reveal a detailed molecular pathway that may be the basis for the allosteric transition that occurs on initial membrane binding leading to the exposure of membrane-spanning regions in a domain distant from the initial site of interaction.

The body fluid of earthworms is known to contain a variety of cytolytic and antibacterial activities to combat potential pathogens that may migrate from the environment into the body cavity. In the annelid Eisenia fetida, a multi-gene family exists that give rise to several isoforms, which display hemolytic and antibacterial activity. The hemolytic activity of lysenin, a major isoform, is known to be strictly dependend on sphingomyelin. As bacteria are devoid of sphingomyelin, the nature of the antibacterial activity of lysenin-like proteins appeared obscure. Here, we report the recombinant expression of lysenin, a defined single entity, which exerted hemolytic, antibacterial and membrane-permeabilizing activity comparable to that of the natural counterpart. Experiments using fluorescence resonance energy transfer spectroscopy with liposomes and planar lipid bilayers demonstrated membrane insertion and single channel fluctuations in the presence of sphingomyelin. By monitoring the lipid specificity of lysenin and its molecular organization on different target cell membranes, it became evident that oligomerization to stable pore-like structures occurs on erythrocytes but does not occur on bacterial membranes. However, bacterial membranes became permeabilized by lysenin, albeit much slower. Accordingly, lysenin appears to display sphingomyelin-dependent and sphingomyelin-independent activities to kill various foreign intruders of the earthworm's coelomic cavity.

The reactivity of immobilized glucose oxidase-containing liposomes (IGOL) prepared in our previous work (Wang et al. [2003] Biotechnol Bioeng 83:444-453) was considerably improved here by incorporating the channel protein OmpF from Escherichia coli into the liposome membrane as well as by entrapping inside the liposome's aqueous interior not only glucose oxidase (GO), but also catalase (CA), both from Aspergillus niger. CA was used for decomposing the hydrogen peroxide produced in the glucose oxidation reaction inside the liposomes. The presence of OmpF enhanced the transport of glucose molecules from the exterior of the liposomes to the interior. In a first step of the work, liposomes containing GO and CA (GOCAL) were prepared and characterized. A remarkable protection effect of the liposome membrane on CA inside the liposomes at 40 degrees C was found; the remaining CA activity at 72 h incubation was more than 60% for GOCAL, while less than 20% for free CA. In a second step, OmpF was incorporated into GOCAL membranes, leading to the formation of OmpF-embedded GOCAL (abbreviated GOCAL-OmpF). The activity of GO inside GOCAL-OmpF increased up to 17 times in comparison with that inside GOCAL due to an increased glucose permeation across the liposome bilayer, without any leakage of GO or CA from the liposomes. The optimal system was estimated to contain on average five OmpF molecules per liposome. Finally, GOCAL-OmpF were covalently immobilized into chitosan gel beads. The performance of this novel biocatalyst (IGOCAL-OmpF) was examined by following the change in glucose conversion, as well as by following the remaining GO activity in successive 15-h air oxidations for repeated use at 40 degrees C in an airlift bioreactor. IGOCAL-OmpF showed higher reactivity and reusability than IGOL, as well as IGOL containing OmpF (IGOL-OmpF). The IGOCAL-OmpF gave about 80% of glucose conversion even when the catalyst was used repeatedly four times, while the corresponding conversions were about 60% and 20% for the IGOL and IGOL-OmpF, respectively. Due to the absence of CA, IGOL-OmpF was less stable and resulted in drastically inhibited GO.

The first two authors equally contributed.Voltage Dependent Anion Channel (VDAC) is a pore forming protein located in outer mitochondrial membrane (1). Its lack is lethal in human (2) and it is involved in cellular cross-talk, important in the apoptotic cascade (3). Its structure is a transmembrane β-barrel (4) organized in 19 β-strands and a N-terminal α-helix probably important for gating (5). However the sidedness of VDAC inside the membrane still remains unresolved (4). This issue is essential in order to define the pore structural determinants interacting with soluble proteins. We expressed, in HeLa cells, a recombinant hVDAC1 carrying C-terminal tag including the hemagglutinin-tag (HA), the specific caspase 3/7 cleavage site (DEVD) and 7 histidine residues (7xHis). DEVD amino acidic sequence can be cleaved upon apoptosis induction by staurosporine. A complementary Fluorescence Protease Protection assay was performed where cell protein are exposed to protease digestion after plasma membrane permeabilization. If the C-terminus of VDAC1 is exposed to the cytosolic side, we expect to lose the His tag upon DEVD cleavage. As result only the HA can be detected at mitochondrial level. Conversely if the C-terminus is oriented towards the IMS, the DEVD is not cleavable and both tags will be detected in mitochondria. Our results by confocal microscopy and appropriate controls with other membrane-oriented constructs showed that this strategy is able to define the sidedness of the VDAC pore.1) Shoshan-Barmatz, V. et al (2010) Mol Aspects Med 31, 227-285.2) Huizing, M. et al (1994) 8924, 762-762.3) Tomasello M.F. et al (2009) Cell Res. 19, 1363-1376.4) De Pinto V. et al (2008) J. Bioenerg Biomemb 40, 139-147.5) De Pinto V. et al (2007) Chembiochem 8, 744-756.Acknowledgements to FIRB_PRIN Grants.

A perforin cDNA of Japanese flounder, Paralichthys olivaceus, was cloned from a cDNA library of kidney stimulated with ConA/PMA. The full-length cDNA is 2,157 bp, which encodes 587 amino acids. The Japanese flounder perforin gene consists of five exons and four introns, with a length of approximately 3 kb. The amino acid sequence of the Japanese flounder perforin is 36% identical to that of rat perforin and 37% identical to amino acid sequences of mouse and human perforin. The Japanese flounder perforin also showed low homology to human and mouse complement components (C6, C7, C8 and C9), ranging from 19% to 24%. However, the membrane attack complex/perforin domain is conserved. A phylogenetic analysis placed the Japanese flounder perforin in the same cluster with other known mammalian perforins. RT-PCR analysis revealed that the perforin gene was expressed in the peripheral blood leukocytes, head kidney, trunk kidney, spleen, heart, gill and intestine of healthy fish. Recombinant perforin produced in insect cells using the baculovirus expression system showed calcium-dependent hemolytic activity.

Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74–282)) and the conserved C-terminal region (p32 (1–221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.

Amoebapore A is a 77-residue protein from the protozoan parasite and human pathogen Entamoeba histolytica. Amoebapores lyse both bacteria and eukaryotic cells by pore formation and play a pivotal role in the destruction of host tissues during amoebiasis, one of the most life-threatening parasitic diseases. Amoebapore A belongs to the superfamily of saposin-like proteins that are characterized by a conserved disulfide bond pattern and a fold consisting of five helices. Membrane-permeabilizing effector molecules of mammalian lymphocytes such as porcine NK-lysin and the human granulysin share these structural attributes. Several mechanisms have been proposed to explain how saposin-like proteins form membrane pores. All mechanisms indicate that the surface charge distribution of these proteins is the basis of their membrane binding capacity and pore formation. Here, we have solved the structure of amoebapore A by NMR spectroscopy. We demonstrate that the specific activation step of amoebapore A depends on a pH-dependent dimerization event and is modulated by a surface-exposed histidine residue. Thus, histidine-mediated dimerization is the molecular switch for pore formation and reveals a novel activation mechanism of pore-forming toxins.

The protozoan parasite and human pathogen Entamoeba histolytica is protected against killing by its own lytic effector proteins. Amoebae withstand doses of amoebapores, their pore-forming polypeptides, that readily kill human Jurkat T cells. Moreover, the polypeptides do not bind to the amoebic surface membrane as evidenced by using fluorescently labelled amoebapores and confocal laser microscopy. Experiments employing liposomes as a minimalistic membrane system and the major isoform amoebapore A revealed that the lipid composition of amoebic membranes prevents binding of the cytolytic molecule and that both the phospholipid ingredients and the high content of cholesterol contributes to the protection of the toxin-producing cell.

The major outer membrane protein of Acinetobacter baumannii is the heat-modifiable protein HMP-AB, a porin with a large pore size allowing the penetration of solutes having a molecular weight of up to approximately 800 Da. Cross-linking experiments with glutardialdehyde failed to show any cross-linking between the monomers, a fact that proves again that this porin protein functions as a monomeric porin. The specific activity of this porin was found to be similar to that of other monomeric porins. Tryptic digestion of the outer membrane yielded a 23-kDa fragment of the HMP-AB protein that was resistant to further trypsin treatment. This observation indicates that HMP-AB is assembled in the membrane in a manner similar to monomeric porins. Cloning of the HMP-AB gene revealed an open reading frame of 1038 bp encoding a protein of 346 amino acids and a calculated molecular mass of 35,636 Da. The amino acid sequence and composition were typical of Gram-negative bacterial porins: a highly negative hydropathy index, absence of hydrophobic residue stretches, a slightly negative total charge, low instability index, high glycine content, and an absence of cysteine residues. Sequence comparison of HMP-AB with other outer membrane proteins revealed a clear homology with the monomeric outer membrane proteins, outer membrane protein A (OmpA) of Enterobacteria, and outer membrane protein F (OprF) of Pseudomonas sp. Secondary structure analysis indicated that HMP-AB has a 172-amino acid N-terminal domain that spans the outer membrane by eight amphiphilic beta strands and a C-terminal domain that apparently serves as an anchoring protein to the peptidoglycan layer. The results also indicate that HMP-AB belongs to the eight transmembrane beta-strand family of outer membrane proteins.

Pore-forming proteins are employed by many pathogens to achieve successful host colonization. Intracellular pathogens use pore-forming proteins to invade host cells, survive within and productively interact with host cells, and finally egress from host cells to infect new ones. The malaria-causing parasites of the genus Plasmodium evolved a number of life cycle stages that enter and replicate in distinct cell types within the mosquito vector and vertebrate host. Despite the fact that interaction with host-cell membranes is a central theme in the Plasmodium life cycle, little is known about parasite proteins that mediate such interactions. We identified a family of five related genes in the genome of the rodent malaria parasite Plasmodium yoelii encoding secreted proteins all bearing a single membrane-attack complex/perforin (MACPF)-like domain. Each protein is highly conserved among Plasmodium species. Gene expression analysis in P. yoelii and the human malaria parasite Plasmodium falciparum indicated that the family is not expressed in the parasites blood stages. However, one of the genes was significantly expressed in P. yoelii sporozoites, the stage transmitted by mosquito bite. The protein localized to the micronemes of sporozoites, organelles of the secretory invasion apparatus intimately involved in host-cell infection. MACPF-like proteins may play important roles in parasite interactions with the mosquito vector and transmission to the vertebrate host.

This project involved the use of supported phospholipid bilayers to design a new generation of highly rugged single molecule sensors devices. The devices employed alpha-hemolysin, a pore forming protein, as a single molecule transducer element. Our studies demonstrated that this ion channel could be inserted into supported bilayers while retaining two-dimensional mobility. Furthermore, we designed a variety of microfluidic and array-based platforms with concentration and temperature control for displaying this stochastic sensor element.

Sticholysin I (StI), a potent cytolysin isolated from the sea anemone Stichodactyla helianthus, was linked to the monoclonal antibody (mAb) ior C5. StI acts by forming hydrophilic pores in the membrane of the attacked cells leading to osmotic lysis. ior C5 is a murine IgG1, which recognizes the tumor associated antigen (TAA) ior C2. The cytolysin and the mAb were coupled by using the heterobifunctional cross-linking reagent sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). Two hybrid molecules composed by one ior C5 and one or two StI molecules were obtained (named conjugated I and II, respectively). The purified conjugates were evaluated by a binding affinity assay against an ior C2-positive colon cancer cell line (SW948). Both molecules were able to recognize the antigen (Ag) in the same way that unconjugated ior C5 does. The activity of both conjugates against human erythrocytes and SW948 cells was assessed. They lost most of their hemolytic activity but their residual activity was very similar. Nevertheless, when their cytotoxicity was studied on the SW948 cell line, only conjugate II killed efficiently the cells, indicating a specific mAb-Ag interaction. In this chimeric molecule the ratio between the cytotoxic and the hemolytic activity was larger than that of the free cytolysin. This fact indicates an increase of the specificity of the toxic effect toward the SW948 cell line and consequently an increase of the difference between its hemolytic and cytotoxic doses. The results herein support the feasibility of directing StI to the surface of cancer cells expressing ior C2 Ag via the mAb ior C5.

A large number of transmembrane proteins form aqueous pores or channels in the phospholipid bilayer, but the structural bases of pore formation and assembly have been determined experimentally for only a few of the proteins and protein complexes. The polypeptide segments that form the transmembrane pore and the secondary structure that creates the aqueous-lipid interface can be identified using multiple independent fluorescence techniques (MIFT). The information obtained from several different, but complementary, fluorescence analyses, including measurements of emission intensity, fluorescence lifetime, accessibility to aqueous and to lipophilic quenching agents, and fluorescence resonance energy transfer (FRET) can be combined to characterize the nature of the protein-membrane interaction directly and unambiguously. The assembly pathway can also be determined by measuring the kinetics of the spectral changes that occur upon pore formation. The MIFT approach therefore allows one to obtain structural information that cannot be obtained easily using alternative techniques such as crystallography. This review briefly outlines how MIFT can reveal the identity, location, conformation, and topography of the polypeptide sequences that interact with the membrane.

Introduction. Granzyme B is a serine protease commonly found in the granules of cytotoxic lymphocytes and natural killer cells. It is secreted with the pore forming protein perforin and mediates apoptosis in target cells. Granzyme B – mediated cytolysis is one of the regulatory mechanisms (together with IL-2 receptors (CD25)) by which T-regulatory cells (Tregs) influence on T-effectors cells. It is a well-known fact that Tregs participate in a pathogenesis of aGVHD and their amount after allo-HSCT inversely correlates with the probability of aGVHD incidence. No clinical data exist regarding the Granzyme B positive Tregs in this setting. Patients and methods. Peripheral blood samples were collected in EDTA-tubes at day +30, +60, +90 after allo-HSCT and at day of acute GVHD onset from 27 patients with hematological malignancies (AML=12, ALL n=12, LPD=2, MDS =1; 6 with active disease, 21 - in CR) after allo-HSCT (from matched unrelated donor n=23, from matched related donor n=4; MAC = 10, RIC=17). The anti-CD4-APC-Cy7, anti-CD25-FITC and anti-Granzyme B-PE (Becton Dickinson, USA) antibodies were used to determine T-regulatory cells population as CD4+CD25high. We did not include anti-FoxP3 antibodies as FoxP3 is not so specific in humans and particularly after allo-HSCT due to technical difficulties, several isoforms, FoxP3-positivity on activated nonregulatory CD4+ cells. CD4- lymphocytes (CD8+ and NK-cells certainly containing granzyme B) were used as internal positive control to define the percent of CD4+CD25highGranzymeB+ cells and gMFI (geometric mean fluorescence intensity) among the mononuclear cell fraction. 50000 of CD4+ cells were analyzed on a BD FACSCanto II (Becton Dickinson, USA). Results. 11 patients (42%) have developed aGVHD (II-IV) at a median time of +26 day (8 - 88) after HSCT. The percent of CD4+CD25highGranzymeB+ cells among CD4+cells at day +30 after allo-HSCT in patients who never developed aGVHD was statistically higher (9,49±2,79; p=0.003) than in patients at day of aGVHD onset (3,8±1,78). It’s worth to note that in 4 patients who did not have signs of a GVHD on day +30 but developed it later (on day +40, +45, +74, +88), the percent of CD4+CD25highGranzymeB+ cells at day +30 was as low (3,38±1,47) as at day of aGVHD onset and significantly lower (p=0.003) than in patients who never developed aGVHD, thus predicting the occurrence of aGVHD in the future. Conclusion Despite the fact that the analyzed group is small, we suggest that low relative amount of CD4+CD25highGranzymeB+ cells after allo-HSCT may contribute to the development aGVHD and even predict it onset. This fact, of course, needs further investigation. Disclosures No relevant conflicts of interest to declare.

We have characterized a pore-forming lytic protein from the saliva of the hematophagous insect Triatoma infestans, a vector of Chagas disease. This protein, named trialysin, has 22 kDa and is present in the saliva at about 200 microg/ml. Purified trialysin forms voltage-dependent channels in planar lipid bilayers with conductance of 880 +/- 40 pS. It lyses protozoan parasites and bacteria indicating that it has a role in the control of microorganism growth in the salivary glands. At higher concentrations, but below those found in saliva, trialysin can also permeabilize and lyse mammalian cells, suggesting that it might also facilitate insect blood feeding by interfering with the cell response of the host. The translated cDNA sequence of trialysin shows a basic, lysine-rich protein in which the N-terminal region is predicted to form an amphipathic alpha-helical structure with positive charges on one side and hydrophobic amino acids on the opposite side. A synthetic peptide corresponding to this cationic amphipathic alpha-helix induces protozoan lysis and mammalian cell permeabilization, showing that this region is involved in lytic activity. However, the lytic peptide G6V32 is 10-fold less efficient than trialysin in lysing parasites and 100-fold less efficient in permeabilizing mammalian cells. Trialysin activity is about 10-fold reduced in salivary gland homogenates prepared in the presence of an irreversible serine-protease inhibitor. Since trialysin precursor contains an anionic pro-sequence of 33 amino acids contiguous to the cationic amphipathic putative alpha-helix, we propose that removal of the acidic pro-sequence by limited proteolysis activates trialysin by exposing this lytic basic amphipathic motif.

Crude extracts of Leishmania amazonensis, but not of L. guyanensis, are lytic to erythrocytes and nucleated cells, including macrophages. L. amazonensis-mediated lysis is caused by a membrane-associated pore-forming protein, named a-leishporin. Here we show that L. amazonensis, but not L. guyanensis, promastigote extracts increase their hemolytic activity when kept at 4 degrees C for a few days or at 37 degrees C for a few hours. We show that the activation in the extracts is mediated by a cytosolic serine-protease. Although L. guyanensis extracts are hemolytically inactive and unable to generate hemolytic activity, their membrane fraction becomes hemolytic in the presence of the cytosolic fraction of L. amazonensis, also by the action of a serine-protease. This suggests that L. guyanensis contains a potential lytic molecule, named here g-leishporin. The cytosolic fraction of L. guyanensis is unable to activate either a- or g-leishporin, indicating that this species does not possess the protease(s) that activate(s) the cytolysin. Trypsin, chymotrypsin, collagenase, Pronase and proteinase K, are also effective in activating a-leishporin but not g-leishporin. This suggests that the inactive forms of a-leishporin and g-leishporin are distinct in structure and/or are activated by different mechanisms. We are considering two hypotheses for the activation of leishporins: (1) proteolysis of an inactive precursor and (2) dissociation and/or proteolytic degradation of an inhibitory oligopeptide. The present data and preliminary results argue for the second hypothesis. We speculate that leishporin could be activated in the protease-rich, low pH, and dissociating environment of parasitophorous vacuole contributing for the release of the parasites from the macrophage.

Cytotoxic T lymphocytes and natural killer cells kill virus-infected or transformed cells through the combined actions of a pore forming protein (perforin) and a family of serine proteases (granzymes). 1 Upon the formation of a stable conjugate between the two cells, the exocytosis of granule toxins from the lymphocyte results in the induction of specific pathways to apoptosis in the target cell. Perforin is both permissive and indispensable for apoptosis, as it enables the granzymes to access their key substrates in the target cell cytoplasm to bring about caspase-dependent and -independent cell death. While considerable progress has been made in our understanding of pro-apoptotic pathways activated by the granzymes, the precise molecular role played by perforin remains poorly understood. Over recent years, we have developed a number of robust expression modalities for mouse and human perforin, in order to characterise its molecular and cellular functions. These have included the capacity to express wild-type and mutated perforin molecules in a cellular context where cytoxicity can be measured, and in baculovirus-based systems where purified protein can be analysed for its cytotoxic activity. By combining these approaches, we have been able to map some of perforin’s functional domains, including a determination of the key residues responsible for calcium binding. 2 In addition, a repository of missense mutations identified in patients with the rare familial immune deficiency syndrome, familial haemophagocytic lymphohistiocytosis (FHL) has proven invaluable in identifying perforin residues that result in either presynaptic protein instability or post-synaptic dysfunction at the level of the target cell membrane. 3 Mutagenesis and modelling have recently allowed us to define the mechanism through which perforin forms transmembrane olgomers. 4 The functional implications of certain common human perforin polymorphisms such as Ala91Val and much rarer mutations will also be discussed, particularly with respect to perforin’s role in human cancer susceptibility. 5

It has been suggested that leptospiral hemolysins are important in the virulence and pathogenesis of leptospirosis. We have isolated an Escherichia coli clone carrying the 7.8 kb DNA insert from a genomic library of Leptospira interrogans serovar lai by plaque hybridization using a sequence derived from the sphingomyelinase C gene (sphA) of L. borgpetersenii. The clone showed a clear β-hemolytic zone on sheep blood agar and high hemolytic activities on both human and sheep erythrocytes in liquid assays. The clone carried at least two genes responsible for the hemolytic activities, encoded by two open reading frames of 1662 and 816 nucleotides, which are named sphH and hap-1 (hemolysis associated protein-1), respectively. The SphH showed 75% homology to the SphA at the amino acid level, and the Hap-1 showed no significant homology in major databases. Interestingly, however, E. coli cells harboring sphH did not show sphingomyelinase or phospholipase activities. Moreover, SphH-mediated hemolysis was osmotically protected by polyethylene glycol 5000, suggesting that the hemolysis is likely to be caused by pore formation on the membrane. The SphH was successfully expressed in E. coli as a histidine (His)–SphH fusion protein. Both sphH and hap-1 were highly conserved among the Leptospira species, except for the absence of sphH in non-pathogenic L. biflexa serovar patoc. We concluded that the SphH is a novel hemolysin of a pathogenic Leptospira species, which may be a putative pore-forming protein.

Bcl-2 family protein including anti-apoptotic (Bcl-2) or pro-apoptotic (Bax) members can form ion channels when incorporated into synthetic lipid bilayers. This contrasts with the observation that Bcl-2 stabilizes the mitochondrial membrane barrier function and inhibits the permeability transition pore complex (PTPC). Here we provide experimental data which may explain this apparent paradox. Bax and adenine nucleotide translocator (ANT), the most abundant inner mitochondrial membrane protein, can interact in artificial lipid bilayers to yield an efficient composite channel whose electrophysiological properties differ quantitatively and qualitatively from the channels formed by Bax or ANT alone. The formation of this composite channel can be observed in conditions in which Bax protein alone has no detectable channel activity. Cooperative channel formation by Bax and ANT is stimulated by the ANT ligand atractyloside (Atr) but inhibited by ATP, indicating that it depends on the conformation of ANT. In contrast to the combination of Bax and ANT, ANT does not form active channels when incorporated into membranes with Bcl-2. Rather, ANT and Bcl-2 exhibit mutual inhibition of channel formation. Bcl-2 prevents channel formation by Atr-treated ANT and neutralizes the cooperation between Bax and ANT. Our data are compatible with a menage a trois model of mitochondrial apoptosis regulation in which ANT, the likely pore forming protein within the PTPC, interacts with Bax or Bcl-2 which influence its pore forming potential in opposing manners.

Our knowledge concerning cytopathogenicity of Trichomonas vaginalis has been enriched in the past by numerous findings. In this paper, we review the latest advances in the field and discuss the different mechanisms and molecules responsible for the parasite's virulence.

The interactions of equinatoxin II (EqTxII) with zwitterionic (DPPC) and anionic (DPPG) phospholipids and an equimolar mixture of the two phospholipids (DPPC/DPPG) have been investigated by differential scanning calorimetry (DSC), CD-spectropolarimetry, intrinsic emission fluorescence spectroscopy, and ultrasonic velocimetry. EqTxII binds to small unilamellar vesicles formed from negatively charged DPPG lipids, causing a marked reduction in the cooperativity and enthalpy of their gel/liquid-crystalline phase transition. This transition is completely abolished at a lipid-to-protein ratio, L/P, of 10. For the mixed DPPC/DPPG vesicles, a 2-fold greater lipid-to-protein ratio (L/P = 20) is required to abolish the phase transition, which corresponds to the same negative charge (-10) of lipid molecules per EqTxII molecule. The disappearance of the phase transition of the lipids apparently corresponds to the precipitation of the lipid-protein complex, as suggested by our sound velocity measurements. Based on the far-UV CD spectra, EqTxII undergoes two structural transitions in the presence of negatively charged vesicles (DPPG). The first transition coincides with the gel/liquid-crystalline phase transition of the lipids, which suggests that the liquid-crystalline form of negatively charged lipids triggers structural changes in EqTxII. The second transition involves the formation of alpha-helical structure. Based on these observations, we propose that, in addition to electrostatic interactions, hydrophobic interactions play an important role in EqTxII-membrane association.

Equinatoxin II is a cysteineless pore-forming protein from the sea anemone Actinia equina. It readily creates pores in membranes containing sphingomyelin. Its topology when bound in lipid membranes has been studied using cysteine-scanning mutagenesis. At approximately every tenth residue, a cysteine was introduced. Nineteen single cysteine mutants were produced in Escherichia coli and purified. The accessibility of the thiol groups in lipid-embedded cysteine mutants was studied by reaction with biotin maleimide. Most of the mutants were modified, except those with cysteines at positions 105 and 114. Mutants R144C and S160C were modified only at high concentrations of the probe. Similar results were obtained if membrane-bound biotinylated mutants were tested for avidin binding, but in this case three more mutants gave a negative result: S1C, S13C and K43C. Furthermore, mutants S1C, S13C, K20C, K43C and S95C reacted with biotin only after insertion into the lipid, suggesting that they were involved in major conformational changes occurring upon membrane binding. These results were further confirmed by labeling the mutants with acrylodan, a polarity-sensitive fluorescent probe. When labeled mutants were combined with vesicles, the following mutants exhibited blue-shifts, indicating the transfer of acrylodan into a hydrophobic environment: S13C, K20C, S105C, S114C, R120C, R144C and S160C. The overall results suggest that at least two regions are embedded within the lipid membrane: the N-terminal 13‐20 region, probably forming an amphiphilic helix, and the tryptophan-rich 105‐120 region. Arg144, Ser160 and residues nearby could be involved in making contacts with lipid headgroups. The association with the membrane appears to be unique and different from that of bacterial pore-forming proteins and therefore equinatoxin II may serve as a model for eukaryotic channel-forming toxins.

The detection of organic molecules is important in many areas, including medicine, environmental monitoring and defence. Stochastic sensing is an approach that relies on the observation of individual binding events between analyte molecules and a single receptor. Engineered transmembrane protein pores are promising sensor elements for stochastic detection, and in their simplest manifestation they produce a fluctuating binary ('on/off') response in the transmembrane electrical current. The frequency of occurrence of the fluctuations reveals the concentration of the analyte, and its identity can be deduced from the characteristic magnitude and/or duration of the fluctuations. Genetically engineered versions of the bacterial pore-forming protein alpha-haemolysin have been used to identify and quantify divalent metal ions in solution. But it is not immediately obvious how versatile binding sites for organic ligands might be obtained by engineering of the pore structure. Here we show that stochastic sensing of organic molecules can be procured from alpha-haemolysin by equipping the channel with an internal, non-covalently bound molecular 'adapter' which mediates channel blocking by the analyte. We use cyclodextrins as the adapters because these fit comfortably inside the pore and present a hydrophobic cavity suitable for binding a variety of organic analytes. Moreover, a single sensing element of this sort can be used to analyse a mixture of organic molecules with different binding characteristics. We envisage the use of other adapters, so that the pore could be 'programmed' for a range of sensing functions.

The pathogenically important cholesterol-binding pore-forming bacterial “thiol-activated” toxins (TATs) are commonly believed to be monomeric in solution and to undergo a transition on membrane binding mediated by cholesterol to an oligomeric pore. We present evidence, gained through the application of a number of biochemical and biophysical techniques with associated modelling, that the TAT from Streptococcus pneumoniae, pneumolysin, is in fact able to self-associate in solution to form the same oligomeric structures. The weak interaction leading to solution oligomerization is manifested at low concentrations in a dimeric toxin form. The inhibition of toxin self-interaction by derivatization of the single cysteine residue in pneumolysin with the thiol-active agent dithio (bis)nitrobenzoic acid indicates that self-interaction is mediated by the fourth domain of the protein, which has a fold similar to other proteins known to self-associate. This interaction is thought to have implications for the understanding of mechanisms of pore formation and complement activation by pneumolysin.

Cytotoxic T lymphocytes and natural killer cells together comprise the means by which the immune system detects and rids higher organisms of virus-infected or transformed cells. Although differing considerably in the way they detect foreign or mutated antigens, these cells utilize highly analogous mechanisms for inducing target cell death. Both types of effector lymphocytes utilize two principal contact-dependent cytolytic mechanisms. The first of these, the granule exocytosis mechanism, depends on the synergy of a calcium-dependent pore-forming protein, perforin, and a battery of proteases (granzymes), and it results in penetration by effector molecules into the target cell cytoplasm and nucleus. The second, which requires binding of FasL (CD95L) on the effector cell with trimeric Fas (CD95) molecules on receptive target cells, is calcium independent and functions by generating a death signal at the inner leaflet of the target cell membrane. Exciting recent developments have indicated that both cytolytic mechanisms impinge on an endogenous signaling pathway that is strongly conserved in species as diverse as helminths and humans and dictates the death or survival of all cells.

The pore-forming protein perforin is one of the main effector molecules which cytotoxic lymphocytes utilize to kill their targets both in vivo and in vitro. Natural killer cells and cytotoxic T lymphocytes play an important role in host defense against a number of intracellular microorganisms such as virus and protozoan, but the exact way they help control infection is unknown. On the other hand, many microorganisms have evolved successful escape strategies to avoid immune-cell-mediated attack. It is thus necessary to investigate the direct interaction of infectious microorganisms with the lytic machinery of cytotoxic lymphocytes and other cells. In the present work we report the effect of perforin on both a protozoan, Trypanosoma cruzi, and the infected host cell. Epimastigote, amastigote, and trypomastigote forms of T. cruzi, as well as infected macrophages, were assayed for their susceptibility to perforin based on three different criteria. T. cruzi in all three differentiation stages were resistant to purified perforin at doses up to 100-fold larger than that sufficient to kill susceptible tumor cells. No morphological change was observed under electron microscopy. Survival rates and infectivities of the treated parasites in vitro were similar to those of control parasites. Moreover, the measurement of calcium influx using Fura-2 to assess membrane damage revealed that T. cruzi resist perforin attack by avoiding transmembrane pore formation. Resistance to perforin was not transferred to host cells since infected macrophages could be easily destroyed by perforin while intracellular amastigotes remained intact.

We investigated the cytotoxicity of CEL-III, one of four Ca2+-dependent galactose/N-acetylgalactosamine (GalNAc)-binding lectins from the marine invertebrate Cucumaria echinata. Among six cell lines tested, MDCK cells showed the highest susceptibility to CEL-III cytotoxicity and its LD50 was estimated to be 53 ng/ml, while no significant cytotoxicity of CEL-III was observed in CHO cells up to 10,000 ng/ml. In the presence of 0.1 M lactose, the cytotoxicity of CEL-III was strongly inhibited. The binding studies using FITC-labeled CEL-III revealed that the amount of CEL-III bound to MDCK cells was about 2-fold greater than that in the case of CHO cells. The cytotoxicity of CEL-III increased with decreasing temperature. The surviving fractions of Vero cells exposed to CEL-III at 4 degrees C were immediately decreased, and more than 90% of exposed cells were killed within 20 min, whereas at 37 degrees C much longer exposure period (more than 10 h) was required to kill 50% of the cells. CEL-III induced the release of carboxyfluorescein (CF) from CF-loaded MDCK cells and this activity was markedly increased at alkaline pH (pH 10) and at lower temperature (4 degrees C). Even in CHO cells, considerable CF release was induced by CEL-III at 4 degrees C and at pH 10 but not at pH 7.5 at both temperatures. In agreement with these results, CHO cells exposed to CEL-III at 4 degrees C and at pH 10 were killed in a dose-dependent manner. These results suggest that CEL-III exhibits cytotoxicity through damaging the plasma membrane by pore-formation in a temperature- and pH-dependent manner. Different susceptibility of each cell line to CEL-III cytotoxicity may be due to differences in the processes leading to pore-formation after binding to cell-surface carbohydrates.

The objective of our research is to develop a liposomal gene delivery system that specifically targets potent therapeutic genes to breast cancers which are positive for the human epidermal growth receptor 2 (Her-2). Current liposomal gene delivery systems predominately utilize cationic lipids, which efficiently bind and deliver plasmid DNA, but also result in nonspecific gene expression in the lungs and liver tissue. We attempt to improve on specificity, by using a two-component delivery system. The first component is a cationic polyethylene glycol (PEG) grafted polylysine which carries the plasmid DNA. The polylysine/DNA particle binds nonspecifically to cells but has limited ability to bypass the endosomal membrane. The second component is an anionic Her-2 targeting liposome which is disulfide bound to the pore forming protein Listeriolysin O (LLO). These liposomes bind with high selectivity to Her-2 positive cells and once internalized into the reducing conditions of an endosome, release LLO which forms pores in the endosomal membrane. Neither component delivers DNA plasmid on its own, but if the polylysine/DNA and LLO/liposome co-localize within an endosome, the DNA is able to access the cytoplasm which leads to gene expression of the plasmid. Using fluorescent microscopy and a cell panel, we show that labeled polylysine/DNA and LLO/liposomes have a much greater chance of co-localizing in an endosome of Her-2 positive breast cancer cells compared to Her-2 negative cells. In addition, utilizing luciferase plasmid, we demonstrate that this two-component system results in gene expression that is over 1000-fold greater in Her-2 overexpressing cells than in Her-2 negative cells. This is a vast improvement over standard cationic liposome techniques which show no specificity for Her-2 positive cells. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-02-02.

Porin or VDAC, the pore forming protein in the outer mitochondrial membrane, was identified as a binding site for microtubule-associated protein 2 (MAP2). By affinity chromatography using covalently bound MAP2, two proteins were selectively purified with molecular weights 63 and 70 KD. The 63 KD protein was identified to be the dimer of porin using affinity purified porin antibodies.

Publisher Summary This chapter discusses the theory of cell membrane damage in lymphocyte-mediated cytolysis—role of lymphocyte pore-forming protein perforin. Conjugation with the target cell triggers degranulation by the killer cell, which then results in the release of a number of putative cytotoxins into the intercellular space. A prominent member among these toxins is the pore-forming protein A; a large number of pore-forming toxins are known to create channels through a “barrel stave” mechanism. Three discrete steps have been defined for the pore formation in this model: (1) binding of the water-soluble monomers to the membrane, (2) insertion of the monomers into the membrane, and (3) aggregation of the monomers, such as barrel staves, surrounding a central pore such that the hydrophobic side of the proteins is exposed to the membrane acyl chains and the hydrophilic side lines up the pore. As a result, stable transmembrane pores are formed that allow ions and small molecules to flow passively across the bilayer. As many of the homopolymeric channels formed by toxins are not large enough to allow the passage of cytoplasmic proteins, this model predicts that the channels would produce an ionic imbalance in the cell, which then results in colloid osmotic lysis. Perforin is likely to lyse target cells by this mechanism.

CytB, a membrane pore-forming toxin from Bacillus thuringiensis subspecies kyushuensis, is specifically toxic to dipteran insect larvae but broadly cytolytic in vitro. It has been purified in the protoxin form from a recombinant Escherichia coli source and crystals have been obtained which diffract X-rays to at least 2.6 A resolution. The tendency for CytB to aggregate in solution was overcome by including 50 mM of urea or 8 mM of ethanolamine during crystallization. Mutants designed to add or subtract single cysteine residues for the purpose of heavy atom derivative preparation were similarly purified and crystallized. The crystals are hexagonal bipyramids. They belong to space group P6(1)22 (or P6(5)22) with lattice constants a = b = 67.34 A, c = 170.96 A, and contain one molecule of the CytB protoxin (MW 29235) per asymmetric unit and 27% solvent by volume.

The ability of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells to kill susceptible targets has been known for over 20 years. This observation led to an intensive if not somewhat prolonged search for the molecular mediators of cell death. With the knowledge that is now available to us, and which will be discussed here and in the various chapters of this volume, it is clear that killing involves at least two different pathways. The first is initiated by the content of the cytoplasmic granules which are exocytosed towards the target cell (Henkart 1985; Golstein et al. 1991). The second results from activation of the Fas receptor on the target by its ligand on the effector cell (Rouvier et al. 1993; Itoh et al. 1991; Suda et al. 1993; Vignaux and Golstein 1994). If it were not sufficiently complex that a cell uses two killing pathways, it also appears that cell death induced by granule exocytosis may utilize several molecular mediators: the pore forming protein perforin (Henkart et al. 1984; Podack et al. 1985, 1991), a group of CTL/NK-specific granule serine proteases known as granzymes (Hayes et al. 1989; Shiet al. 1992 a, b, 1994; Shiver et al. 1992) and perhaps a poly(A)-binding protein TIA-1 (Tian et al. 1991; Kawakami et al. 1992). Thus, it is not surprising that the mechanisms of CTL killing have revealed themselves only slowly.

Protein engineering is being used to produce a collection of pore-forming proteins with applications in biotechnology. Knowledge provided by investigations of the mechanism of self-assembly of staphylococcal alpha-hemolysin has allowed the design of genetically and chemically modified variants of the protein with pore-forming activities that can be triggered or switched on-and-off by chemical, biochemical and physical inputs. Examples include alpha-hemolysins that are activated by specific proteases and alpha a-hemolysins whose activity is controlled by divalent metal ions. These proteins have potential value in drug delivery as components of immunotoxins that can be activated at the surfaces of target cells. Further applications are likely in improved encapsulation techniques for drugs, enzymes and cells.

alpha-Hemolysin (alpha HL) is a 293 amino acid pore-forming toxin, which is secreted as a water-soluble monomer by Staphylococcus aureus. By forming a hexameric pore, alpha HL damages the plasma membranes of target cells. Previous studies established that alpha HL proteins with nicks near the midpoint of a central glycine-rich loop are held together by a domain-domain interaction and are hemolytically active. In contrast, alpha HL proteins comprising two alpha HL truncation mutants that overlap in the central loop have no or greatly reduced pore-forming activity, even though the two chains again form a tight complex. Based on these findings, overlap mutants have now been designed that are activated when redundant amino acids in the loop are removed by proteases. Further, the identity of the activating enzyme can be specified by additional mutagenesis of the protease recognition site in the overlap sequence. Mutants of alpha HL that are activated by tumor-associated proteases might be useful components of immunotoxins.

The decline in immunologic vigor that accompanies advancing age has been recognized for many years, and immunologic senescence has been the focus of two recent and extensive reviews (Thoman and Weigle, 1989; Miller, 1990). Although all immunologically active cells, including T cells, B cells, macrophages, and natural killer (NK) cells, appear to exhibit age-related alterations in one or more functions, T cells are widely considered to be the most vulnerable to the potentially deleterious effects of aging. Thymic involution, morphological evidence for which can be observed soon after the immune system functionally matures, is thought to be intimately connected with the subsequent decline in T-cell function (Hirokawa et al., 1990). Although evidence suggests that extrathymic T-cell development is also altered with advanced age (Gorczynski and Chang, 1984), the mechanisms are not yet understood.

Protease-activated triggers have been introduced Into a pore-forming protein, staphylococcal a-hemolysin (αHL). The hemolysin was remodeled by genetic engineering to form two-chain constructs with redundant polypeptide sequences at the central loop, the Integrity of which Is crucial for efficient pore formation. The new hemolysins are activated when the polypeptide extensions are removed by proteases. By alterating the protease recognition sequence in the loop, selective activation by specified proteases can be obtained. Protease-triggered pore-forming proteins might be used for the selective destruction of cancer cells that bear tumor-associated proteases. When certain two-chain constructs are treated with proteases, a full-length polypeptide chain forms as the result of a protease-mediated transpeptidation reaction. This reaction might be used to produce chimeric hemolysins that are Inaccessible by conventional routes.

In the present study, we have examined the exocytosis model for cellular lysis. Using monoclonal antibodies reactive with human pore-forming protein (PFP), we examined the localization of PFP at the interaction site of natural killer (NK) cells and the NK tumor targets K562 and Molt-4 as well as during antibody- dependent cellular cytotoxicity. Following the interaction of effector-target cell contact, an increased frequency of PFP was detected on the effector surface, in the micro- environment, and on the target surface of the interaction site. This temporal deposition of PFP was paralleled by loss of target cell integrity and release of chromium. In addition, selective deposition of PFP was seen at the interaction site of the target cell compared to other target cell regions. Collectively, these results are consistent with the exocytosis model and further support the hypothesis that PFP is one of the secreted moieties involved in NK cellular cytotoxicity

The senescent decline of cytolytic T lymphocyte (CTL) activity was examined (a) to learn more about the effect of aging on the immune system, and (b) to probe the mechanism of cell-mediated cytolysis. The effect of age on the generation of pore-forming protein (Pfp) was examined at the cellular level in a murine model using CTL stimulated in allogeneic mixed lymphocyte culture (MLC). Pfp expression was analyzed by immunocytochemistry and enzyme-linked immunosorbent assay (ELISA). Immunocytochemical analyses of Pfp in MLC-stimulated splenic T cells from a large number of mice revealed that although stimulated cells from aged mice exhibited fewer Pfp-producing cells than those from young, the diminution in the proportion of Pfp+ cells was small compared to the age-related decrease in lytic activities (approximately 2-fold vs. approximately 7.4-fold, respectively). Time-course analysis disclosed similar kinetics for the generation of Pfp+ cells among responding cells from young and aged mice. No significant age-related difference in the proportion of Pfp+ cells was observed in MLC-stimulated lymph node cells despite a large and significant difference in lytic activity (approximately 6.5-fold). Purified CD8+ T cells demonstrated a large age-related difference in CTL activity (approximately 3-11-fold) and accounted for virtually all the Pfp. Although little difference in the proportion of Pfp+ CD8+ T cells could be detected between age groups, stimulated CD8+ cells or whole splenic T cells from old mice consistently exhibited a striking reduction in both the intensity of Pfp staining and the apparent numbers of granules per cell. This difference in Pfp was examined by ELISA and total Pfp levels were found to be approximately 12-fold greater in CTL generated from splenic T cells of young compared to aged mice. The results demonstrate that Pfp levels are reduced in CTL from aged compared to young mice at the level of the individual cells and suggest the possibility that a threshold level of Pfp may be required for potency of effector cell function.

A toxin that causes a massive presynaptic activation of transmitter release from nerve terminals is alpha-latrotoxin, isolated from Latrodectus tredecimguttatus spider venom. This toxin has been highly purified, utilizing as a biological assay a toxin-dependent increase in 45Ca(2+)-accumulation by PC12 cells. The purification protocol includes an ion-exchange step and a gel-filtration column, by fast-flow liquid chromatography. The resulting toxin is a polypeptide of about 125 kDa in molecular mass. At nmol concentrations it specifically activates calcium influx and transmitter secretion after interacting with neuronal acceptors of the presynaptic membrane. The inhibitory effect of trivalent ions (which may develop as degradation product of 45Ca2+) on toxin-dependent calcium accumulation by PC12 cells is described. The results obtained suggest that calcium fluxes directly involved in the neurosecretory event, may occur through newly formed toxin-dependent channels.

A cluster of at lest six interferon-γ (IFNγ)-inducible genes designated Ifi201-204 and located on mouse chromosome 1 has recently been described. Here , we report a human IFN-γ-inducible gene, IFI 16, which has nucleotide sequence similarity with portions of two of the mouse genes, Ifi202 and Ifi204. A full-length cDNA clone derived from IFI 16 [2.709 kilobases (kb)] contained a single open reading frame of 2.187 kb which encoded a putative polypeptide of 729 amino acids and a predicted non-glycosylated M r of 80020. IFI 16 mRNA was found to be constitutively expressed in lymphoid cells and in cell lines of both the T and B lineages. By contrast, the mRNA was not expresed by the cell lines HL-60, U937, and K562, which represent early stages of myeloid development, but was strongly inducible in HL-60 and U937 with IFN-γ. The IFI 16 protein demonstrated a putative domain structure with patchy similarity to the proteins expressed from gene Ifi202 and Ifi204. The mouse and human proteins each contain two analogous ≈200 amino acid domains which are imperfect copies, but IFI 16 demonstrated additional unique regions, including a Lys-rich N-terminal portion and a “spacer” region between the reiterated domains, analogous to spacer regions in the CD5 and CD8α molecules. Using a panel of inter-species somatic cell hybrid cell lines, IFI 16 was localized to the chromosomal region 1q12→1qter, a region systenic between mouse an man. DNA blotting indicated that, in contrast to the mouse, IFI 16 is present as a single copy gene in the human genome. The authors are pleased to make the cDNA clones described in this paper available to interested investigators.

The domains of the pore-forming protein responsible for the gate and channel formations were separated and identified in the outer membrane of Pseudomonas aeruginosa. The proteolytic cleavage of the 46K channel protein, protein D2, yielded two major domains with apparent Mr of 27K and 19K. We identified the 27K polypeptide to be the channel-forming domain by an in vitro permeability assay. The channel size of purified 27K domain was indistinguishable from that of native protein D2. Degradation of the 19K domain into small subfragments increases the channel activity about ten times suggesting that the 19K polypeptide forms the gate or cap.