Escherichia coli heat-labile enterotoxin B subunit is a more potent mucosal adjuvant than its vlosely related homologue, the B subunit of cholera toxin.

Although cholera toxin (Ctx) and Escherichia coli heat-labile enterotoxin (Etx) are known to be potent mucosal adjuvants, it remains controversial whether the adjuvanticity of the holotoxins extends to their nontoxic, receptor-binding B subunits. Here, we have systematically evaluated the comparative adjuvant properties of highly purified recombinant EtxB and CtxB. EtxB was found to be a more potent adjuvant than CtxB, stimulating responses to hen egg lysozyme when the two were coadministered to mice intranasally, as assessed by enhanced serum and secretory antibody titers as well as by stimulation of lymphocyte proliferation in spleen and draining lymph nodes. These results indicate that, although structurally very similar, EtxB and CtxB have strikingly different immunostimulatory properties and should not be considered equivalent as prospective vaccine adjuvants.

Cholera toxin and Escherichia coli enterotoxin B-subunits inhibit macrophage-mediated antigen processing and presentation: evidence for antigen persistence in non-acidic recycling endosomal compartments.

Cholera toxin (Ctx) and the closely related Escherichia coli heat-labile enterotoxin (Etx) not only act as mediators of diarrhoeal disease but also exert potent immunomodulatory properties on mammalian immune systems. The toxins normally exert their diarrhoeagenic effects by initiating receptor-mediated uptake into vesicles that enter a retrograde trafficking pathway, circumventing degradative compartments and targeting them to the trans-Golgi network (TGN) and endoplasmic reticulum. Here, we examine whether receptor-mediated binding and cellular entry by the toxin B-subunits also lead to concomitant changes in uptake and trafficking of exogenous antigens that could contribute to the potent immunomodulatory properties of these toxins. Treatment of the macrophage (J774.2) cell line with Etx B-subunit (EtxB) resulted in EtxB transport to the TGN and also led to the formation of large, translucent, non-acidic, EtxB-devoid vacuoles. When exogenous antigens were added, EtxB-treated cells were found to be proficient in both internalization of ovalbumin (OVA) and phagocytosis of bacterial particles. However, the internalized OVA, instead of trafficking along a lysosome-directed endocytic pathway via acidified endosomes, persisted in a non-acidic, light-density compartment that was distinct from the translucent vacuoles. The rerouted OVA did not co-localize with the endosomal markers rab5 or rab11, nor with EtxB, but was retained in a transferrin receptor-positive compartment. The failure of OVA to enter the late endosomal/lysosomal compartments correlated with a striking inhibition of OVA peptide processing and presentation to OVA-responsive CD4+ T-cells. CtxB also modulated OVA trafficking and inhibited antigen presentation. These findings demonstrate that the B-subunits of Ctx and Etx alter the progression of exogenous antigens along the endocytic processing pathway, and prevent or delay efficient epitope presentation and T-cell stimulation. The formation of such 'antigen depots' could contribute to the immunomodulatory properties of these bacterial virulence determinants.

Bcl10 is a positive regulator of antigen receptor-induced activation of NF-kappaB and neural tube closure.

Bcl10, a CARD-containing protein identified from the t(1;14)(p22;q32) breakpoint in MALT lymphomas, has been shown to induce apoptosis and activate NF-kappaB in vitro. We show that one-third of bcl10-/- embryos developed exencephaly, leading to embryonic lethality. Surprisingly, bcl10-/- cells retained susceptibility to various apoptotic stimuli in vivo and in vitro. However, surviving bcl10-/- mice were severely immunodeficient and bcl10-/- lymphocytes are defective in antigen receptor or PMA/Ionomycin-induced activation. Early tyrosine phosphorylation, MAPK and AP-1 activation, and Ca2+ signaling were normal in mutant lymphocytes, but antigen receptor-induced NF-kappaB activation was absent. Thus, Bcl10 functions as a positive regulator of lymphocyte proliferation that specifically connects antigen receptor signaling in B and T cells to NF-kappaB activation.

Palliative care at home: an audit of cancer deaths in Grampian region.

BACKGROUND: Ninety per cent of the last year of life of cancer patients is spent at home. Some studies have suggested that care in this setting is often suboptimal. Information on the standard of palliative care delivered at home by general practitioners (GPs) and their teams is limited, and clarification of the problems faced is needed. AIM: To audit the home-based palliative care of patients dying of cancer. METHOD: Matched postal questionnaires were sent to the GPs and nurses of 1086 successive patients dying of cancer in whatever setting in the Grampian region of Scotland some six weeks after the death to establish the professionals' perception of symptom control, communication problems, use of services, and information given to patients and relatives. RESULTS: Response rates were 88.8% for GPs (964 out of 1086) and 87.1% for nurses (325 out of 375 that were passed on to nurses). Two-thirds of patients received palliative care at home. Pain was poorly controlled in 15.7%, and poor control of other symptoms ranged from 13.8% (nausea and vomiting) to 21% (depression and dyspnoea). Communication difficulties were present in 93.7% of cases, although only 5.2% of these were of a major nature. District nurses were involved in 76.7% of cases and Macmillan nurses in 28.0%. Twenty-six per cent of referrals to district nurses were assessed as being late in the course of the illness. Patients were fully informed about the diagnosis in 66.3% of cases and about the prognosis in 55.4%. General practitioners were more likely to report the presence of communication problems between themselves and the patient (when compared with nurses: 43.9% versus 28.0%), more likely to report that patients were 'not at all informed' about self-help groups (57.5% versus 36.3%), and were less likely to report the involvement of occupational therapists (21.8% versus 39.7%). CONCLUSIONS: Levels of reporting of poor symptom control by professionals was much lower than levels reported by relatives in other studies, but there was no difference between the reporting of GPs and nurses. However, a number of areas were identified where care could be enhanced by improved teamwork and further education and training in symptom control, as well as in communication, use of services, and information provision.

Signal anchor sequence insertion into the outer mitochondrial membrane. Comparison with porin and the matrix protein targeting pathway.

We have addressed the question of overlap between the pathways for protein insertion into the outer mitochondrial membrane and import to the matrix compartment, using competition studies in vitro. A synthetic peptide corresponding to the matrix-targeting signal of pre-ornithine carbamyl transferase competed for outer membrane insertion of porin but did not compete for membrane insertion of outer membrane signal anchor-containing proteins. Conversely, however, a synthetic peptide corresponding to the signal anchor sequence of Tom70 competed for import of all proteins examined. Both peptides competed for a step beyond receptor binding. Import of all precursors examined was inhibited by antibodies raised against the import receptor Tom20. Following binding to the surface of the organelle, outer membrane integration of porin was sensitive to depletion of nucleoside triphosphates by apyrase, whereas signal anchor protein insertion was not. The results demonstrate that outer membrane signal anchor insertion overlaps with a general insertion pathway. However, it exhibits both properties and steps that differ from the pathway followed by porin and matrix-targeted protein.

Palliative care experience and training of Scottish general practitioner trainees.

OBJECTIVES: (a) To establish the clinical experience of Scottish general practice trainees in palliative care. (b) To establish the extent of their training in the discipline. (c) To elicit their views on home care. DESIGN: Postal questionnaire survey. SUBJECTS AND SETTING: Forty-seven trainee general practitioners attending the Scottish National Trainees Conference in Aberdeen in February 1995. MAIN OUTCOME MEASURES: Experience of palliative care in hospital and in general practice and training received in each setting. Perception of need for further training. Views on home care.

Identification of the human mitochondrial protein import receptor, huMas20p. Complementation of delta mas20 in yeast.

The human homolog of the S. cerevisiaelN. crassa mitochondrial protein import receptor, Mas20p/MOM19, has been identified and characterized. Sequence similarities between these three proteins is most pronounced within the NH2-terminal third of the molecules. However, the mammalian protein exhibits only weak homology to the tetratricopeptide repeat B domain that is found in Mas20p/MOM19. huMas20p is targeted and inserted into the outer membrane of isolated rat heart mitochondria, in the Nin-Ccyto orientation. Antibodies directed against the soluble portion of huMas20p inhibited in vitro mitochondrial import of a diverse set of precursor proteins (including inner membrane uncoupling protein), but failed to block import of a fusion protein bearing the signal-anchor sequence of Mas20p itself. Finally, expression of huMAS20 complemented the respiratory defect of delta mas20 yeast cells. Together, these results demonstrate that huMAS20p is a component of the mammalian import apparatus.

Import and insertion of proteins into the mitochondrial outer membrane.

Nuclear-encoded proteins destined for insertion into the mitochondrial outer membrane, follow the same general pathway for import as proteins that are translocated to interior compartments within the organelle. This observation is true both for beta-barrel-type proteins and for proteins that contain hydrophobic alpha-helical transmembrane segments. In this review, we describe what is known about the various steps leading to protein insertion into the outer membrane, and discuss the energetics that favor vectorial translocation into and across this membrane. The selection of the outer membrane during import may involve a lateral release of the translocating polypeptide from the import machinery so that the appropriate domains of the protein become embedded in the lipid bilayer. One type of topogenic domain that can guarantee such selection of the outer membrane is a signal-anchor sequence of the type characterized for the bitopic protein Mas70p. It is suggested that a signal-anchor sequence selective for the mitochondrial outer membrane causes abrogation of polypeptide translocation and triggers the release of the transmembrane segment into the surrounding lipid bilayer, prior to any possibility for the commitment of translocation to the interior of the organelle. Specific structural features of the signal-anchor sequence specify its orientation in the membrane, and can confer on this sequence the ability to form homo-oligomers and hetero-oligomers. Strategies other than a signal-anchor sequence may be employed by other classes of proteins for selection of the outer-membrane. Of note is the ability of the outer-membrane import machinery to catalyze integration of the correct set of proteins into the outer-membrane bilayer, while allowing proteins that are destined for integration into the bilayer of the inner membrane to pass through unimpeded. Again, however, different proteins may employ different strategies. One model proposes that this can be accomplished by a combination of a matrix-targeting signal and a distal stop-transfer sequence. In this model, the formation of contact sites, which is triggered when the matrix-targeting signal engages the import machinery of the inner membrane, may prevent the outer-membrane translocon from recognizing and responding to the downstream stop-transfer domain. This allows the transmembrane segment to pass across the outer-membrane, and subsequently integrate into the inner membrane.

Mitochondrial Mas70p signal anchor sequence. Mutations in the transmembrane domain that disrupt dimerization but not targeting or membrane insertion.

Mas70p is an integral membrane protein in Saccharomyces cerevisiae that is targeted and inserted into the mitochondrial outer membrane in an N(in)-Ccyto orientation by its NH2-terminal 29-amino acid signal anchor sequence. Recently, we demonstrated that the signal anchor was capable of mediating homo-oligomerization of a fusion protein, pOMD29, in the outer membrane in vitro (Millar, D. G., and Shore, G. C. (1992) J. Biol. Chem. 268, 18403-18406). Consistent with this finding, we show here that a synthetic peptide corresponding to the Mas70p signal anchor is capable of independent membrane insertion and dimerization with pOMD29. To further map the oligomerization domain in the signal anchor sequence, a deletion mutant of pOMD29 that lacks amino acids 2-10 was constructed. This protein, pOMD29 delta 2-10, efficiently participated in dimer formation following import, indicating that dimerization was mediated by the putative membrane spanning segment (amino acids 11-29). This segment is predicted to form an alpha-helix that has an alanine-rich face and contains multiple copies of a pentapeptide dimerization motif that is widespread among members of the receptor tyrosine kinase family. Substitution of the alanine residues in one of these copies with isoleucine, producing a potentially bulkier contact surface, resulted in a protein which was targeted and inserted into the outer membrane but failed to assemble into dimers. Taken together, these results identify a structural feature of the signal anchor transmembrane domain that is important for oligomerization but is not required for targeting and membrane insertion.

Targeting of Bcl-2 to the mitochondrial outer membrane by a COOH-terminal signal anchor sequence.

The protooncogene product Bcl-2 is an integral membrane protein that functions as a suppressor of programmed cell death. It contains a single predicted transmembrane segment located at its COOH terminus. Here, we show that the transmembrane domain of human Bcl-2 functions as a mitochondrial signal anchor sequence that targets and inserts the protein into the outer membrane in an Ncyto-C(in) orientation, leaving the bulk of the polypeptide facing the cytosol. Deletion of the COOH-terminal 22 amino acids of Bcl-2 abrogated protein targeting, whereas fusion of this domain to the COOH terminus of dihydrofolate reductase resulted in targeting and insertion of the hybrid protein into the outer membrane in a manner similar to that of Bcl-2. The sequence of the hydrophobic core of the Bcl-2 signal anchor is similar to the corresponding region of the NH2-terminal signal anchor of the mitochondrial outer membrane protein in yeast, Mas70p. A synthetic peptide comprising the Mas70p signal anchor sequence effectively competed for insertion of Bcl-2 into the outer membrane but had no effect on the comparatively low association that Bcl-2 makes with endoplasmic reticulum microsomes. Insertion of Bcl-2 into the mitochondrial outer membrane is mechanistically different than its association with microsomes.

The signal anchor sequence of mitochondrial Mas70p contains an oligomerization domain.

pOMD29 is a mitochondrial precursor protein that contains the NH2-terminal signal anchor sequence of Mas70p fused to dihydrofolate reductase. The signal anchor mediates insertion of pOMD29 into the outer mitochondrial membrane in the Nin-Ccyto orientation. Following import in vitro, pOMD29 was chemically cross-linked, via a unique cysteine residue adjacent to the signal anchor (residue 34), to form a product that was approximately twice the size of pOMD29. The cross-linked product was a dimer of pOMD29, as judged by the following. 1) It exhibited the same charge:mass ratio as pOMD29. 2) Formation of radioactive cross-linked product containing 35S-labeled pOMD29 was stimulated by co-import with unlabeled pOMD29. 3) Co-import of pOMD29 with a modified pOMD29 that contains two copies of dihydrofolate reductase resulted in formation of the predicted homo- and heterodimers. Cross-linking of pOMD29 was unaffected by concentrations of methotrexate that lock the dihydrofolate reductase moiety into its native monomeric conformation, indicating that oligomerization was mediated by the signal anchor rather than by the cytosolic domain of pOMD29. The predicted transmembrane core of the signal anchor sequence contains structural motifs similar to those found in a variety of signal-transducing cell surface receptors that dimerize through their transmembrane segments.

A signal-anchor sequence selective for the mitochondrial outer membrane.

pOMD29 is a hybrid protein containing the NH2-terminal topogenic sequence of a bitopic, integral protein of the outer mitochondrial membrane in yeast, OMM70, fused to dihydrofolate reductase. The topogenic sequence consists of two structural domains: an NH2-terminal basic region (amino acids 1-10) and an apolar region which is the predicted transmembrane segment (amino acids 11-29). The transmembrane segment alone was capable of targeting and inserting the hybrid protein into the outer membrane of intact mitochondria from rat heart in vitro. The presence of amino acids 1-10 enhanced the rate of import, and this increased rate depended, in part, on the basic amino acids located at positions 2, 7, and 9. Deletion of a large portion of the transmembrane segment (amino acids 16-29) resulted in a protein that exhibited negligible import in vitro. Insertion of pOMD29 into the outer membrane was not competed by import of excess precursor protein destined for the mitochondrial matrix, indicating that the two proteins may have different rate-limiting steps during import. We propose that the structural domains within amino acids 1-29 of pOMD29 cooperate to form a signal-anchor sequence, the characteristics of which suggest a model for proper sorting to the mitochondrial outer membrane.

Hostile emotion and obsessional neurosis.

The literature on the place of hostile emotion in obsessional neurosis is reviewed. In the main study 11 obsessional neurotic and 11 depressive patients matched for severity of illness were given questionnaires to measure experienced hostility and anger, and a repertory grid of standard form. The obsessional group showed a qualitatively distinct correlational pattern of measured aspects of hostility-anger, including a high correlation between outwardly directed aspects of hostility and anger; this pattern was replicated in a validation study. In contrast, the obsessional group was not differentiated by a negative cognitive set. Treatment implications for obsessional neurosis are briefly discussed.

The long term behaviour of autogeneou vein by-pass grafts.

The results of the follow-up of 50 consecutive atuogeneous vein bypass grafts during 1958-65 are described. Arteriography was performed at regular intervals throughout the follow-up lasting from 8-15 years. 11 grafts failed in less than 2 years and were considered technical failures. Thereafter, of the 37 cases on whom arteriographic information is available, a further 5 grafts failed. Deterioration of the graft itself occurred in 19 percent, proximal anastomosis dilatation it 16 percent, and distal anastomosis narrowing in 25 percent. Improvement of flow to the lower leg vessels did not accelerate progression of the long saphenous vein at the time of operation is the limiting factor in longterm graft deterioration. The majority of grafts remained patient without degeneration for up to 15 years.