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.     

Immunochemically detectable phytochrome is present at normal levels but is photochemically nonfunctional in the hy 1 and hy 2 long hypocotyl mutants of Arabidopsis

The hy 1 and hy 2 long hypocotyl mutants of Arabidopsis thaliana contain less than 20% (the detection limit) of the phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. In contrast, spectral measurements for the hy 3, hy 4, and hy 5 long hypocotyl mutants indicate that they each contain levels of phytochrome equivalent to the wild-type parent. Immunoblot analysis using a monoclonal antibody directed against the chromophore-bearing region of etiolated-oat phytochrome demonstrates that extracts of all mutant and wild-type Arabidopsis tissues, prepared by extraction of proteins into hot SDS-containing buffer, have identical levels of one major immunodetectable protein (116 kDa). An assay involving controlled in vitro proteolysis, known to produce distinctive fragmentation patterns for Pr and Pfr (Vierstra RD, Quail PH, Planta 156: 158–165, 1982), indicates that the 116 kDa polypeptide from the wild-type parent represents Arabidopsis phytochrome. The 116 kDa protein from either hy 3, hy 4, or hy 5 displays the same fragmentation pattern found for the wild type. Together with the spectral data, these results indicate that the mutant phenotype of these variants does not involve lesions in the polypeptide sequence that lead to gross conformational aberrations, and suggest that the genetic lesions may affect steps in the transduction chain downstream of the photoreceptor. In contrast, this same analysis for hy 1 and hy 2 has revealed that the 116 kDa protein from either of these mutants is not degraded differently in response to the different wavelengths of irradiation given in vitro. Moreover, whereas immunoblot analysis of tissue extracts from light-grown wild-type seedlings show that the 116 kDa phytochrome protein level is greatly reduced relative to dark-grown tissue as expected, similar extracts of light-grown hy 1 and hy 2 seedlings contain the 116 kDa polypeptide in amounts equivalent to those of dark-grown tissue. Combined, these data indicate that the hy 1 and hy 2 mutants both produce normal levels of immunochemically detectable phytochrome that is photochemically nonfunctional.     

Photophysiology and phytochrome content of long-hypocotyl mutant and wild-type cucumber seedlings

Photomorphogenetic responses have been studied in a cucumber (Cucumis sativus L.) mutant (lh), which has long hypocotyls in white light (WL). While etiolated seedlings of this mutant have a similar phytochrome content and control of hypocotyl elongation as wild type, deetiolation is retarded and WL-grown seedlings show reduced phytochrome control. Spectrophotometric measurements exhibit that WL-grown tissues of the lh mutant (flower petals and Norflurazon-bleached leaves) contain 35 to 50% of the phytochrome level in the wild type. We propose that this is a consequence of a lack of light-stable phytochrome, in agreement with our hypothesis proposed on the basis of physiological experiments. The lh mutant lacks an end-of-day far-red light response of hypocotyl elongation. This enables the end-of-day far-red light response, clearly shown by the wild type, to be ascribed to the phytochrome, deficient in the lh mutant. Growth experiments in continuous blue light (BL) and continuous BL + red light (RL) show that when RL is added to BL, hypocotyl growth remains inhibited in the wild type, whereas the lh mutant exhibits significant growth promotion compared to BL alone. It is proposed that the hypocotyls fail to grow long in low fluence rate BL because photosynthesis is insufficient to sustain growth.     

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     

Cloning of DNA involved in sporulation of Streptomyces griseus

Twenty-two bald mutants of Streptomyces griseus were isolated and classified into four phenotypic groups, two of which showed conditional sporulation. A 3-kilobase fragment of DNA was cloned in a high-copy-number vector and detected by its ability to restore sporulation to one class of conditionally bald mutants. Analysis of subclones demonstrated that the sporulation property was contained within a 2.5-kilobase fragment. Hybridization studies and restriction analysis indicated that this DNA fragment was present in several Streptomyces species and was distinct from DNA that has been shown to complement afsA mutants of S. bikiniensis and bldA mutants of S. coelicolor.     

Specific labeling and partial inactivation of cytochrome oxidase by fluorescein mercuric acetate

Addition of 1 eq of fluorescein mercuric acetate (FMA) to beef heart cytochrome oxidase was found to inhibit the steady-state electron transfer activity by 50%, but further additions up to 10 eq had no additional effect on activity. The partial inhibition caused by FMA is thus similar to that observed with other mercury compounds (Mann, A. J., and Auer, H. E. (1980) J. Biol. Chem. 255, 454-458). The fluorescence of FMA was quenched by a factor of 10 upon binding to cytochrome oxidase, consistent with the involvement of a sulfhydryl group. However, addition of mercuric chloride to FMA-cytochrome oxidase resulted in an increase in fluorescence, suggesting that FMA was displaced from the high affinity binding site. Cytochrome c binding to FMA-cytochrome oxidase resulted in a 10% decrease in the fluorescence, possibly caused by Forster energy transfer from FMA to the cytochrome c heme. The binding site for FMA in cytochrome oxidase was investigated by carrying out sodium dodecyl sulfate gel electrophoresis under progressively milder dissociation conditions. When FMA-cytochrome oxidase was dissociated with 3% sodium dodecyl sulfate and 6 M urea, FMA was predominantly bound to subunit II following electrophoresis. However, when the dissociation was carried out at 4 degrees C in the absence of urea with progressively smaller amounts of lithium dodecyl sulfate, the labeling of subunit II decreased and that of subunit I increased. These experiments demonstrate that mercury compounds bind to a high affinity site on cytochrome oxidase, possibly located in subunit I, but then migrate to subunit II under the normal sodium dodecyl sulfate gel electrophoresis conditions. A definitive assignment of the high affinity binding site in the native enzyme cannot be made, however, because it is possible that mercury compounds can migrate from one sulfhydryl to another under even the mildest electrophoresis conditions.