The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The K _m of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.Abbreviations MPO N-methylputrescine oxidase - PVDF polyvinylidene difluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We would like to thank members of the Plant Cell Biotechnology Group, Institute of Food Research, Norwich Laboratory, for their helpful discussions during the preparation of this paper.     

Synthesis and X-ray crystal structure of [Ag(phendio)<Subscript>2</Subscript>]ClO<Subscript>4</Subscript> (phendio = 1,10-phenanthroline-5,6-dione) and its effects on fungal and mammalian cells

The Cu(II) and Ag(I) complexes, [Cu(phendio)₃](ClO₄)₂4H₂O and [Ag(phendio)₂]ClO₄ (phendio = 1,10-phenanthroline-5,6-dione), are prepared in good yield by reacting phendio with the appropriate metal perchlorate salt. The X-ray crystal structure of the Ag(I) complex shows it to have a pseudo tetrahedral structure. `Metal-free' phendio and the Cu(II) and Ag(I) phendio complexes strongly inhibit the growth of the fungal pathogen Candida albicans, and are more active than their 1,10-phenanthroline analogues. The simple Ag(I) salts, AgCH<sub>3</sub>CO<sub>2</sub>, AgNO<sub>3</sub> and AgClO<sub>4</sub>.H<sub>2</sub>O display superior anti-fungal properties compared to analogous simple Cu(II) and Mn(II) salts, suggesting that the nature of the metal ion strongly influences activity. Exposing C. albicans to `metal-free' phendio, simple Ag(I) salts and [Ag(phendio)₂]ClO₄ causes extensive, non-specific DNA cleavage. `Metal-free' phendio and [Ag(phendio)₂]ClO₄ induce gross distortions in fungal cell morphology and there is evidence for disruption of cell division. Both drugs also exhibit high anti-cancer activity when tested against cultured mammalian cells.     

Aquaporin 9 expression along the male reproductive tract

Fluid movement across epithelia lining portions of the male reproductive tract is important for modulating the luminal environment in which sperm mature and reside, and for increasing sperm concentration. Some regions of the male reproductive tract express aquaporin (AQP) 1 and/or AQP2, but these transmembrane water channels are not detectable in the epididymis. Therefore, we used a specific antibody to map the cellular distribution of another AQP, AQP9 (which is permeable to water and to some solutes), in the male reproductive tract. AQP9 is enriched on the apical (but not basolateral) membrane of nonciliated cells in the efferent duct and principal cells of the epididymis (rat and human) and vas deferens, where it could play a role in fluid reabsorption. Western blotting revealed a strong 30-kDa band in brush-border membrane vesicles isolated from the epididymis. AQP9 is also expressed in epithelial cells of the prostate and coagulating gland where fluid transport across the epithelium is important for secretory activity. However, it was undetectable in the seminal vesicle, suggesting that an alternative fluid transport pathway may be present in this tissue. Intracellular vesicles in epithelial cells along the reproductive tract were generally poorly stained for AQP9. Furthermore, the apical membrane distribution of AQP9 was unaffected by microtubule disruption. These data suggest that AQP9 is a constitutively inserted apical membrane protein and that its cell-surface expression is not acutely regulated by vesicular trafficking. AQP9 was detectable in the epididymis and vas deferens of 1-wk postnatal rats, but its expression was comparable with adult rats only after 3--4 wk. AQP9 could provide a route via which apical fluid and solute transport occurs in several regions of the male reproductive tract. The heterogeneous and segment-specific expression of AQP9 and other aquaporins along the male reproductive tract shown in this and in our previous studies suggests that fluid reabsorption and secretion in these tissues could be locally modulated by physiological regulation of AQP expression and/or function.     

Phenylcoumaran Benzylic Ether Reductase Prevents Accumulation of Compounds Formed under Oxidative Conditions in Poplar Xylem

Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8–5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form. This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. Altogether, our data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against oxidative damage. In addition to describing the catalytic activity of one of the most abundant enzymes in wood, we provide experimental evidence for the antioxidant role of a phenylpropanoid coupling product in planta.     

Effect of leaf surface waxes on leaf colonization by Pantoea agglomerans and Clavibacter michiganensis

To evaluate the influence of leaf cuticular waxes on bacterial colonization of leaves, bacterial colonization patterns were examined on four glossy maize (Zea mays L.) mutants that were altered in their cuticular wax biosynthesis. Mutant gl3 was indistinguishable from the wild-type maize in its ability to foster colonization by the two bacterial species, Pantoea agglomerans and Clavibacter michiganensis subsp. nebraskensis. In contrast, the other three mutants supported the development of populations that significantly differed in size from those on the wild type. Mutant gl5 gl20 supported smaller populations of P. agglomerans, but not C. michiganensis, while mutant gl1 supported larger populations of C. michiganensis but not P. agglomerans. Mutant gl4 supported larger populations of both bacterial species. The exceptional ability of mutant gl4 to support bacterial colonization was hypothesized to result from the lower density of the crystalline waxes on gl4 than on the wild type, because a reduced crystal density could promote capillary water movement and water trapping among the wax crystals. This hypothesis was supported by the demonstration that the mechanical introduction of gaps among the wax crystals of the wild-type leaves resulted in the establishment of larger P. agglomerans populations on the altered leaves. These results provide the first direct evidence that leaf surface waxes affect bacterial leaf colonization at various stages of colonization and in a bacterial species-dependent manner.     

Bim and Bcl-2 mutually affect the expression of the other in T cells

The life and death of T cells is controlled to a large extent by the relative amounts of Bcl-2-related proteins they contain. The antiapoptotic protein Bcl-2 and the proapoptotic protein Bim are particularly important in this process with the amount of Bcl-2 per cell dropping by about one-half when T cells prepare to die. In this study we show that Bcl-2 and Bim each control the expression of the other. Absence of Bim leads to a drop in the amount of intracellular Bcl-2 protein, while having no effect on the amounts of mRNA for Bcl-2. Conversely, high amounts of Bcl-2 per cell allow high amounts of Bim, although in this case the effect involves increases in Bim mRNA. These mutual effects occur even if Bcl-2 is induced acutely. Thus these two proteins control the expression of the other, at either the protein or mRNA level.     

Vaccination to prevent persistent viral infection.

Persistent virus infections are increasingly being recognized as a significant cause of human morbidity and mortality. To establish persistence, a virus must establish infection and evade eradication by the host immune response, in particular by cytotoxic T lymphocytes (CTL). We have studied a virus that establishes persistence in part by suppressing the CTL response of the infected host. The virus persists in many cell types, including lymphocytes and macrophages. We show that prior immunization with a vaccine designed to induce CTL (in the absence of antiviral antibody) confers complete protection against subsequent establishment of persistence in all tissues analyzed. The vaccine can be designed to express as few as 10 amino acids of a viral protein that comprise the CTL epitope. Further, two CTL epitopes for two discrete MHC haplotypes can be successfully used in a single vaccine that protects both strains of mice. Hence, a "string of CTL epitopes" (beads) concept for vaccination is feasible. Finally, the CTL vaccine provided protection against the establishment of persistence by an immunosuppressive virus.