Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system

Mycobacterium avium and Mycobacterium tuberculosis are human pathogens that infect and replicate within macrophages. Both organisms live in phagosomes that fail to fuse with lysosomes and have adapted their lifestyle to accommodate the changing environment within the endosomal system. Among the many environmental factors that could influence expression of bacterial genes are the concentrations of single elements within the phagosomes. We used a novel hard x-ray microprobe with suboptical spatial resolution to analyze characteristic x-ray fluorescence of 10 single elements inside phagosomes of macrophages infected with M. tuberculosis and M. avium or with avirulent M. smegmatis. The iron concentration decreased over time in phagosomes of macrophages infected with Mycobacterium smegmatis but increased in those infected with pathogenic mycobacteria. Autoradiography of infected macrophages incubated with (59)Fe-loaded transferrin demonstrated that the bacteria could acquire iron delivered via the endocytic route, confirming the results obtained in the x-ray microscopy. In addition, the concentrations of chlorine, calcium, potassium, manganese, copper, and zinc were shown to differ between the vacuole of pathogenic mycobacteria and M. smegmatis. Differences in the concentration of several elements between M. avium and M. tuberculosis vacuoles were also observed. Activation of macrophages with recombinant IFN-gamma or TNF-alpha before infection altered the concentrations of elements in the phagosome, which was not observed in cells activated following infection. Siderophore knockout M. tuberculosis vacuoles exhibited retarded acquisition of iron compared with phagosomes with wild-type M. tuberculosis. This is a unique approach to define the environmental conditions within the pathogen-containing compartment.     

Neuronal repellent Slit2 inhibits dendritic cell migration and the development of immune responses

One of the essential functions of dendritic cells is to take up Ags in peripheral tissues and migrate into secondary lymphoid organs to present Ags to lymphocytes for the induction of immune responses. Although many studies have demonstrated that the migration of dendritic cells is closely associated with the development of immune responses, little is known about factors that inhibit dendritic cell migration and control the extent of immune responses to Ag stimulation. We show that Slit2, a neuronal repellent factor, is up-regulated in the skin by allergen sensitization and down-regulates the migration of Langerhans cells. The effect is mediated by direct interaction of Slit2 with cells that express a Slit-specific receptor, Robo1. Slit2-mediated inhibition of Langerhans cell migration results in suppression of contact hypersensitivity responses. These findings provide insights into a novel mechanism by which Slit2 functions as an anti-inflammatory factor for the initiation of immune responses.     

Purification and characterization of Moschatin, a novel type I ribosomeinactivating protein from the mature seeds of pumpkin （Cucurbita moschata）, and preparation of its immunotoxin against human melanoma cells

A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of-29 kD. It is a rRNA Nglycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a ICs0 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.     

Hyper-Rayleigh scattering of protein-modified gold nanoparticles

The nonlinear optical properties of protein-modified gold nanoparticles has been studied by the hyper-Rayleigh scattering (HRS) technique. HRS signals from the nanoparticles coated with goat-anti-human IgG have been obtained when pumped with a laser pulse with a wavelength of 1064 nm. The HRS signals of gold nanoparticles with IgG were larger than those of bare gold nanoparticles. This can be explained by a noncentrosymmetric effect. It was also found that the HRS signals from the IgG-coated gold nanoparticles could be greatly increased when the antigen was added due to gold nanoparticle aggregation. Our experiment found that the HRS method could produce a measurable signal with 10 microg/ml antigen added, while the colorimetric method using UV spectrum detection required 100 microg/ml of added antigen. The results show that the HRS measurement of immunogold nanoparticles could become a potential immunoassay in determining small levels of antigen in aqueous samples.     

Autocrine and paracrine regulation of lymphocyte CB2 receptor expression by TGF-beta.

The marijuana-derived cannabinoid Delta(9)-tetrahydrocannabinol (THC) has been shown to be immunosuppressive. We report that THC induces the immunosuppressive cytokine TGF-beta by human peripheral blood lymphocytes (PBL). The ability of THC to stimulate TGF-beta production was blocked by the CB2 receptor specific antagonist SR144528 but not by the CB1 specific antagonist AM251. Furthermore, our data suggest that TGF-beta actively regulates lymphocyte CB2 receptor expression in an autocrine and paracrine manner. Whereas the addition of recombinant TGF-beta to PBL cultures downregulated CB2 receptor expression, anti-TGF-beta antibody treatment increased CB2 receptor expression. We conclude that one mechanism by which THC contributes to immune suppression is by stimulating an enhanced production of lymphocyte TGF-beta.     

Oligomeric behavior of Bordetella pertussis adenylate cyclase toxin in solution

Adenylate cyclase (AC) toxin from Bordetella pertussis inserts into eukaryotic cells, producing intracellular cAMP, as well as hemolysis and cytotoxicity. Concentration dependence of hemolysis suggests oligomers as the functional unit and inactive deletion mutants permit partial restoration of intoxication and/or hemolysis, when added in pairs [M. Iwaki, A. Ullmann, P. Sebo, Mol. Microbiol. 17 (1995) 1015-1024], suggesting dimerization/oligomerization. Using affinity co-precipitation and fluorescence resonance energy transfer (FRET), we demonstrate specific self-association of AC toxin molecules in solution. Flag-tagged AC toxin mixed with biotinylated-AC toxin, followed by streptavidin beads, yields both forms of the toxin. FRET measurements of toxin, labeled with different fluorophores, demonstrate association in solution, requiring post-translational acylation, but not calcium. AC toxin mixed with DeltaR, an inactive mutant, results in enhancement of hemolysis over that with wild type alone, suggesting that oligomers are functional. Dimers and perhaps higher molecular mass forms of AC toxin occur in solution in a manner that is relevant to toxin action.     

The stoichiometry of the chloroplast ATP synthase oligomer III in Chlamydomonas reinhardtii is not affected by the metabolic state

The chloroplast H(+)-ATP synthase is a key component for the energy supply of higher plants and green algae. An oligomer of identical protein subunits III is responsible for the conversion of an electrochemical proton gradient into rotational motion. It is highly controversial if the oligomer III stoichiometry is affected by the metabolic state of any organism. Here, the intact oligomer III of the ATP synthase from Chlamydomonas reinhardtii has been isolated for the first time. Due to the importance of the subunit III stoichiometry for energy conversion, a gradient gel system was established to distinguish oligomers with different stoichiometries. With this methodology, a possible alterability of the stoichiometry in respect to the metabolic state of the cells was examined. Several growth parameters, i.e., light intensity, pH value, carbon source, and CO(2) concentration, were varied to determine their effects on the stoichiometry. Contrary to previous suggestions for E. coli, the oligomer III of the chloroplast H(+)-ATP synthase always consists of a constant number of monomers over a wide range of metabolic states. Furthermore, mass spectrometry indicates that subunit III from C. reinhardtii is not modified posttranslationally. Data suggest a subunit III stoichiometry of the algae ATP synthase divergent from higher plants.     

Cancer metabolism: facts, fantasy, and fiction

The concept of a glycolytic cancer cell was introduced by Warburg over 70 years ago. This perception has since become the rationale that drives a considerable proportion of basic research on cancer, and it influences the current strategies for the diagnosis, monitoring, and treatment of cancer. Here we review the data from the last 40 years on this issue. We conclude that there is no evidence that cancer cells are inherently glycolytic, but that some tumours might indeed be glycolytic in vivo as a result of their hypoxic environment.     

低氧预适应增加小鼠脑组织内nPKCε膜转位

目的:初步探讨新奇型蛋白激酶C(novel protein kinases C,nPKCs)在脑低氧预适应发生发展过程中的作用.方法:利用蛋白电泳(SDS-PAGE)和蛋白印迹(Western blot)等生化技术,并结合本室已建立的小鼠低氧预适应模型,观察低氧预适应对小鼠海马和大脑皮层组织内nPKCs(nPKCε、δ、η、μ和θ亚型)膜转位(激活)的影响.结果:随低氧次数(H0-H4)或低氧耐受时间的增加,小鼠海马(H0:41.6%±1.4%vs H1-H4:46.9%±4.5%,52.7±3.9%,58.8%±2.7%,61.3%±3.7%)和大脑皮层(H0:38.4%±4.5%vsH1-H4:42.4%±5.0%,48.7%±6.5%,55.3%±8.9%,61.2%±10.2%)组织内nPKCε膜转位明显增加,且海马和大脑皮层分别在H2、H3、H4和H3、H4具有统计学的显著意义(P＜0.01);而nPKCδ、η、μ和θ亚型在海马和大脑皮层组织内的膜转位变化均无统计学意义.结论:nPKCε可能在脑低氧预适应的发生发展过程中发挥着重要作用,但需进一步的研究证实.     

Progressive Soil Drought Promotes the Export Function in the Birch (Betula platyphylla) Leaf

A greenhouse experiment, which imitated a short (4-day-long) and progressive (3-week-long) soil drought, was employed to assess, with an IR gas analyzer, leaf CO₂ exchange rate (CER) in intact one-year-old seedlings of Betula platyphylla as related to the flux of photosynthetically active radiation ranging from 0 to 1400 E/(m² s). The registered indices comprised leaf temperature, leaf transpiration conductivity, and the average daily increment of the leaf area. Within a week period following the transition from the short severe soil drought (20% H₂O per soil weight) to the conditions of sufficient water content (35–40%), the plants completely regained the initial leaf CER. Under the progressive soil drought, leaf CER was reduced by 30–35%, as compared to the conditions of sufficient water content, evidently due to a 3.7-fold drop in the transpiration conductivity as compared to the control plants. The apparent constant of Rubisco carboxylation and leaf respiration in the light were not affected by the drought period. The rate of leaf growth under the progressive drought was reduced by 64% as compared to the sufficient moisture conditions. Thus, under the progressive drought, the diminished stomatal conductivity reduced CO₂ concentration inside the leaf and lowered carbon photosynthetic assimilation. Meanwhile, the leaf source activity considerably increased in spite of diminished photosynthesis.