The alkene monooxygenase from Xanthobacter Py2 is a binuclear non-haem iron protein closely related to toluene 4-monooxygenase

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter Py2 have been sequenced. The predicted amino acid sequence of the first ORF shows homology with the iron binding subunits of binuclear non-haem iron containing monooxygenases including benzene monooxygenase, toluene 4-monooxygenase (>60% sequence similarity) and methane monooxygenase (>40% sequence similarity) and that the necessary sequence motifs associated with iron co-ordination are also present. Secondary structure prediction based on the amino acid sequence showed that the predominantly α-helical structure that surrounds the binuclear iron binding site was conserved allowing the sequence to be modelled on the co-ordinates of the methane monooxygenase α-subunit. Significant differences in the residues forming the hydrophobic cavity which forms the substrate binding site are discussed with reference to the differences in reaction specificity and stereospecificity of binuclear non-haem iron monooxygenases.     

DNA-Launched Alphavirus Replicons Encoding a Fusion of Mycobacterial Antigens Acr and Ag85B Are Immunogenic and Protective in a Murine Model of TB Infection

There is an urgent need for effective prophylactic measures against Mycobacterium tuberculosis (Mtb) infection, particularly given the highly variable efficacy of Bacille Calmette-Guerin (BCG), the only licensed vaccine against tuberculosis (TB). Most studies indicate that cell-mediated immune responses involving both CD4+ and CD8+ T cells are necessary for effective immunity against Mtb. Genetic vaccination induces humoral and cellular immune responses, including CD4+ and CD8+ T-cell responses, against a variety of bacterial, viral, parasitic and tumor antigens, and this strategy may therefore hold promise for the development of more effective TB vaccines. Novel formulations and delivery strategies to improve the immunogenicity of DNA-based vaccines have recently been evaluated, and have shown varying degrees of success. In the present study, we evaluated DNA-launched Venezuelan equine encephalitis replicons (Vrep) encoding a novel fusion of the mycobacterial antigens α-crystallin (Acr) and antigen 85B (Ag85B), termed Vrep-Acr/Ag85B, for their immunogenicity and protective efficacy in a murine model of pulmonary TB. Vrep-Acr/Ag85B generated antigen-specific CD4+ and CD8+ T cell responses that persisted for at least 10 wk post-immunization. Interestingly, parenterally administered Vrep-Acr/Ag85B also induced T cell responses in the lung tissues, the primary site of infection, and inhibited bacterial growth in both the lungs and spleens following aerosol challenge with Mtb. DNA-launched Vrep may, therefore, represent an effective approach to the development of gene-based vaccines against TB, particularly as components of heterologous prime-boost strategies or as BCG boosters.     

Seasonal changes in moult,body mass and reproductive condition in siskins Carduelis spinus exposed to daylength regimes simulating different latitudes

Birds use change in daylength during the year to time events during their annual cycles. Individual Eurasian siskins Carduelis spinus can breed and winter in widely separated areas in different years. Birds at different latitudes will experience different changes in photoperiod. So how does latitude affect photoperiodic control? Our aim in this study was to find whether Siskins caught from the wild in Britain and exposed to different photoperiodic regimes, typical of widely separated latitudes, would differ in the subsequent timing and duration of their moults and associated processes. Siskins were caught in late February and early March, and initially kept outside on natural photoperiods. From the spring equinox (21 March), they were divided into three groups kept under photoperiodic regimes that simulated latitudes 40°, 55° and 70°N respectively. All three groups showed highly significant subsequent changes in body mass, fat scores and cloacal protuberance size. Moult of the primary feathers started during June – August (mean 9 July), and lasted 61–99 days (mean 75 days). Birds that started to moult late in the season had shorter moult durations. All individuals showed lower mass and fat levels during moult than before or after moult. Crucially, there were no significant differences in the timing of these events between the three photoperiodic groups. Apparently these birds did not use prevailing absolute photoperiod or the prevailing rate of change in photoperiod to time moult‐related seasonal events, but used instead some other feature of the annual photoperiod cycle or some form of interval timer linked to photoperiod.     

Immunopathological features of human pulmonary tumors following low-dose interleukin-2

Summary We administered preoperative low-dose interleukin-2 (IL-2) to 10 patients undergoing thoracotomy for pulmonary tumors. The in vivo effect of IL-2 on tumor-associated lymphocyte activity was assessed in the resected specimens by immunohistochemistry and compared with observations in 45 patients who did not receive IL-2. H & E evaluation revealed an increase in intra- and peritumoral lymphocyte infiltration in the IL-2-treated patients. Immunopathological evaluation with monoclonal antibodies revealed that this lymphocyte infiltration was predominantly CD5-positive T cells. The amount of intra-and peritumoral lymphocyte activity correlated with the dose of IL-2 administered (6000–90 000 international units/kg every 8 h for 48 h. IL-2-treated patients showed increases in T-cell-associated activation markers (IL-2 -receptor, transferrin receptor and HLA-DR) on peritumoral lymphocytes, but not on intratumoral lymphocytes. We previously reported that low-dose IL-2 increases the intrinsic natural killer cell cytotoxicity of intratumoral lymphocytes and suggest that this lymphocyte infiltration is further evidence that low-dose IL-2 can augment in vivo lymphocyte activity at the tumor site.This work was supported in part by USPHS grants CA 44 352 (S. H. G.) and 43 658 (A. J. C.). S. G. S. was supported by NIH Surgical Oncology Training Grant CA 09 010     

Velocity and displacement receptors in the skin of the hagfish Myxine glutinosa.

The skin of Myxine glutinosn contains both velocity-sensitive and displacement-sensitive mechanoreceptors. These can be distinguished from each other by the different time course of their response to skin indentation. Velocity receptors gave a brief response, consisting usually of three to four action potentials which could only be elicited by movement of the skin. No velocity receptor was spontaneously active. Displacement receptors, some of which were spontaneously active, gave a prolonged discharge which continued, with only slight adaptation, while the skin was indented. Velocity receptors code for velocity of indentation and continue to respond to repeated stimuli. Displacement receptor action potentials occur irregularly, with a Poisson-like distribution of interspike interval. The mean frequency of displacement receptor discharge increases with depth of skin indentation, while the interspike interval distribution becomes progressively biased towards shorter intervals. The threshold of displacement receptors is related to displacement velocity; sinusoidal indentations of the skin produce a minimum threshold around 60 Hz. These receptors fail to respond to oscillations in excess of 120 Hz. The structure of the skin is described. Stress-strain curves are given both for static and dynamic compression of the skin and these are discussed in relation to the properties of the skin mechanoreceptors.     

Regulation of spvR, the positive regulatory gene of Salmonella plasmid virulence genes

Abstract The regulation of the spvR promoter from the Salmonella dublin virulence plasmid was monitored using proter-reporter gene fusion constructs. Activity was dependent upon the presence of the spv region and was affected by the number of copies of the spv region present with the cell. Activity remained constant throughout exponential growth, and increased rapidly with the onset of stationary phase, under both aerobic and anaerobic conditions. Additionally, the level of spvR expression was controlled by the availability of iron, activity being greatest under low iron conditions in stationary phase. The spvA gene product negatively regulated spvR expression in a dose-dependent manner, indicating that SpvA provides a negative feedback mechanism for this operon.     

Intratumoral interleukin-2 immunotherapy: Activation of tumor-infiltrating and splenic lymphocytes in vivo

Direct intratumoral injection of interleukin-2 (IL-2) was evaluated in a murine model. Balb/c mice received 5 × 10⁴ Line 1 alveolar carcinoma cells (L1C2) by subcutaneous injection. On the third day following tumor implantation, mice received injections of IL-2 (5 × 10³–5 × 10⁴ units) or diluent twice daily, either by i. p. or intratumoral injection, 5 days/week for 3 weeks. Intratumoral injection of 5 × 10⁴ units IL-2 significantly reduced tumor volume (P <0.05 versus control), increased median survival time (P = 0.0001), and resulted in a 23.5% cure rate (P = 0.008). There were no long-term survivors in the other treatment groups. Both tumor-infiltrating lymphocytes (TIL) and splenic lymphocytes isolated directly from IL-2-treated mice demonstrated enhanced cytolytic activity compared to diluent-treated controls. To determine whether non-T-cell-mediated antitumor responses were active in our model, intratumoral immunotherapy was evaluated in athymic Balb/cnu/nu mice. In order to decrease the recruitment of lymphocyte precursors, nude mice were splenectomized and received cyclophosphamide prior to tumor injection and IL-2 therapy. Intratumoral IL-2 immunotherapy also significantly decreased tumor volume in these immunodeficient mice (P <0.02), but did not lead to long-term survival. We conclude that both TIL and splenic lymphocytes are activated in vivo in response to intratumoral IL-2 immunotherapy, suggesting that intratumoral therapy with IL-2 activates both local and systemic antitumor responses.Supported by the Tobacco-Related Disease Research Program of the University of California, the Cancer Research Coordinating Committee, the Jonsson Cancer Center Foundation, and Veterans Administration Medical Research Funds     

Identification of 3- and 4-phosphorylated phosphoinositides and inositol phosphates in stomatal guard cells

Components of the polyphosphoinositide signalling pathway have been identified in stomatal guard cells of Commelina communis L., one of the few plant systems shown unequivocally to be capable of responding to release of inositol 1,4,5-trisphosphate in the cytoplasm by increase in cytoplasmic Ca²⁺. 'Isolated' epidermal strips of C. communis (in which all cells other than guard cells have been killed by treatment at low pH) were radiolabelled with myo -[2n-³H]inositol or [³²P]orthophosphate for 17–18 h. The phosphoinositides and inositol phosphates were extracted. Phosphoinositides were deacylated and the head groups resolved by HPLC. The water-soluble products generated by mild periodate cleavage of HPLC-purified, deacylated lipid fractions were examined. The resulting biochemical analysis led to the identification of: PtdIns, PtdIns3 P , PtdIns4 P , PtdIns(3,4) P ₂ and PtdIns(4,5) P ₂. Thex inositol phosphates were resolved by HPLC. Preliminary analysis of HPLC-purified putative inositol phosphate fractions resulted in the identification of each inositol phosphate class, that is, Ins P , Ins P ₂, Ins P ₃, Ins P ₄, Ins P ₅ and Ins_P6. Many of these inositol phosphates occurred in different isomeric forms. The presence of 3-phosphorylated phosphoinositides suggests that they may have a role in signalling in stomatal guard cells.