Biological consequences of photoproducts in mammalian cell DNA partially substituted with 5-bromouracil

The response of Chinese hamster ovary cells in which 10 per cent of the thymine of one DNA strand was substituted with bromouracil (BU) was compared with normal cells following u.v. irradiation. The bromouracil substitution resulted in a 21/2 fold enhancement of both u.v.-induced killing and mutation induction at the HGPRT locus. These BU-photoproducts do not, however, result in any further inhibition of DNA replication or inhibition of the repair of u.v.-induced DNA photoproducts identified as antibody-binding sites.     

Growth response of barley and tomato to nitrogen stress and its control by abscisic acid,water relations and photosynthesis

Barley (Hordeum vulgare L.) and tomato Lycopersicon esculentum Mill.) were grown hydroponically and examined 2, 5, and 10 d after being deprived of nitrogen (N) supply. Leaf elongation rate declined in both species in response to N stress before there was any reduction in rate of dryweight accumulation. Changes in water transport to the shoot could not explain reduced leaf elongation in tomato because leaf water content and water potential were unaffected by N stress at the time leaf elongation began to decline. Tomato maintained its shoot water status in N-stressed plants, despite reduced water absorption per gram root, because the decline in root hydraulic conductance with N stress was matched by a decline in stomatal conductance. In barley the decline in leaf elongation coincided with a small (8%) decline in water content per unit area of young leaves; this decline occurred because root hydraulic conductance was reduced more strongly by N stress than was stomatal conductance. Nitrogen stress caused a rapid decline in tissue NO ₃ ^- pools and in NO ₃ ^- flux to the xylem, particularly in tomato which had smaller tissue NO ₃ ^- reserves. Even in barley, tissue NO ₃ ^- reserves were too small and were mobilized too slowly (60% in 2 d) to support maximal growth for more than a few hours. Organic N mobilized from old leaves provided an additional N source to support continued growth of N-stressed plants. Abscisic acid (ABA) levels increased in leaves of both species within 2 d in response to N stress. Addition of ABA to roots caused an increase in volume of xylem exudate but had no effect upon NO ₃ ^- flux to the xylem. After leaf-elongation rate had been reduced by N stress, photosynthesis declined in both barley and tomato. This decline was associated with increased leaf ABA content, reduced stomatal conductance and a decrease in organic N content. We suggest that N stress reduces growth by several mechanisms operating on different time scales: (1) increased leaf ABA content causing reduced cell-wall extensibility and leaf elongation and (2) a more gradual decline in photosynthesis caused by ABA-induced stomatal closure and by a decrease in leaf organic N.Abbreviation and symbols ABA abscisic acid - c_i leaf internal CO₂ concentration - L_p root hydraulic conductance     

The role of antibiotic production by a strain of Pseudomonas fluorescens in the suppression of Pseudocercosporella herpotrichoides, the causal agent of eyespot disease of cereals

Pseudomonas fluorescens strain 220 is an effective antagonist of Pseudocercosporella herpotrichoides , the eyespot pathogen of cereals. Culture filtrates of Ps. fluorescens 220 were inhibitory to spore germination and hyphal growth of P. herpotrichoides and at least two compounds with antifungal and antibacterial activity were identified in cultures grown in nutrient broth. In plant tests, both a culture broth of Ps. fluorescens 220 and a crude antibiotic extract reduced eyespot disease, whereas a mutant strain of 220 deficient in antibiotic production had no effect. Production of antibiotics would therefore appear to be a major factor in the suppression of P. herpotrichoides infection. A loss of disease control when Ps. fluorescens 220 was applied to plants in water was not due to lack of survival, as populations of a marked strain of Ps. fluorescens 220 applied to the stem base of wheat plants were similar whether applied in water or culture broth.     

Trypanosoma brucei: host parasite interaction in parasite destruction by salicylhydroxamic acid and glycerol in mice

Mouse blood and serum contain a synergistic factor which affects both the speed and completeness of destruction of Trypanosoma brucei in the presence of salicylhydroxamic acid (SHAM) and glycerol. The action of this factor is dose dependent producing complete killing in infected whole blood with 2 mM SHAM and 12 mM glycerol but not in a mixture of 20% infected whole blood and 80% buffer containing the same final concentration of SHAM and glycerol. This factor may account for the discrepancy in reports showing that SHAM-glycerol does not kill 100% of the exposed parasites in vitro yet is able to cure infected animals. The factor is not due to an acquired immune response, complement action, nor lipoproteins. Should the level of the factor be able to be increased, this could greatly increase the effectiveness of SHAM-glycerol.     

Protein export from the mitochondrial matrix

Assembly of a functional mitochondrion requires import of proteins from the cytosol and export of proteins from the matrix. Most previous studies have focused on the import pathway followed by nucleus-encoded proteins. However, it is now clear that proteins encoded in the nucleus as well as those encoded in the mitochondrion also move from the matrix into and across the inner membrane, a process defined here as export. These exported proteins are found in at least three cellular locations: the inner mitochondrial membrane, the intermembrane space and the cell surface. Here, we consider the pathways for export and the relationships between import and export.     

Structural Dynamics of the Activation of Elongation Factor 2 Kinase by Ca2 +-Calmodulin

Eukaryotic elongation factor 2 kinase (eEF-2K), the only known calmodulin (CaM)-activated α-kinase, phosphorylates eukaryotic elongation factor 2 (eEF-2) on a specific threonine (Thr-56) diminishing its affinity for the ribosome and reducing the rate of nascent chain elongation during translation. Despite its critical cellular role, the precise mechanisms underlying the CaM-mediated activation of eEF-2K remain poorly defined. Here, employing a minimal eEF-2K construct (TR) that exhibits activity comparable to the wild-type enzyme and is fully activated by CaM in vitro and in cells, and using a variety of complimentary biophysical techniques in combination with computational modeling, we provide a structural mechanism by which CaM activates eEF-2K. Native mass analysis reveals that CaM, with two bound Ca^2 + ions, forms a stoichiometric 1:1 complex with TR. Chemical crosslinking mass spectrometry and small-angle X-ray scattering measurements localize CaM near the N-lobe of the TR kinase domain and the spatially proximal C-terminal helical repeat. Hydrogen/deuterium exchange mass spectrometry and methyl NMR indicate that the conformational changes induced on TR by the engagement of CaM are not localized but are transmitted to remote regions that include the catalytic site and the functionally important phosphate binding pocket. The structural insights obtained from the present analyses, together with our previously published kinetics data, suggest that TR, and by inference, wild-type eEF-2K, upon engaging CaM undergoes a conformational transition resulting in a state that is primed to efficiently auto-phosphorylate on the primary activating T348 en route to full activation.     

Isolation of a cDNA from Saccharomyces cerevisiae that encodes a high affinity sulphate transporter at the plasma membrane

Resistance to selenate and chromate, toxic analogues of sulphate, was used to isolate a mutant of Saccharomyces cerevisiae deficient in the capacity to transport sulphate into the cells. A clone which complements this mutation was isolated from a cDNA library prepared from S. cerevisiae poly(A)⁺ RNA. This clone contains an insert which is 2775 by in length and has a single open reading frame that encodes a 859 amino acid polypeptide with a molecular mass of 96 kDa. Sequence motifs within the deduced amino acid sequence of this cDNA (SUL1) show homology with conserved areas of sulphate transport proteins from other organisms. Sequence analysis predicts the position of 12 putative membrane spanning domains in SUL1. When the cDNA for SUL1 was expressed in S. cerevisiae, a high affinity sulphate uptake activity (K_m = 7.5 ± 0.6 μM for SO ₄ ^2? ) was observed. A genomic mutant of S. cerevisiae in which 1096 by were deleted from the SUL1 coding region was constructed. This mutant was unable to grow on media containing less than 5 mM sulphate unless complemented with a plasmid containing the SUL1 cDNA. We conclude that the SUL1 cDNA encodes a S. cerevisiae high affinity sulphate transporter that is responsible for the transfer of sulphate across the plasma membrane from the external medium.     

Identification of pathogenicity‐related genes in Fusarium oxysporum f. sp. cepae

Pathogenic isolates of Fusarium oxysporum, distinguished as formae speciales (f. spp.) on the basis of their host specificity, cause crown rots, root rots and vascular wilts on many important crops worldwide. Fusarium oxysporum f. sp. cepae (FOC) is particularly problematic to onion growers worldwide and is increasing in prevalence in the UK. We characterized 31 F. oxysporum isolates collected from UK onions using pathogenicity tests, sequencing of housekeeping genes and identification of effectors. In onion seedling and bulb tests, 21 isolates were pathogenic and 10 were non‐pathogenic. The molecular characterization of these isolates, and 21 additional isolates comprising other f. spp. and different Fusarium species, was carried out by sequencing three housekeeping genes. A concatenated tree separated the F. oxysporum isolates into six clades, but did not distinguish between pathogenic and non‐pathogenic isolates. Ten putative effectors were identified within FOC, including seven Secreted In Xylem (SIX) genes first reported in F. oxysporum f. sp. lycopersici. Two highly homologous proteins with signal peptides and RxLR motifs (CRX1/CRX2) and a gene with no previously characterized domains (C5) were also identified. The presence/absence of nine of these genes was strongly related to pathogenicity against onion and all were shown to be expressed in planta. Different SIX gene complements were identified in other f. spp., but none were identified in three other Fusarium species from onion. Although the FOC SIX genes had a high level of homology with other f. spp., there were clear differences in sequences which were unique to FOC, whereas CRX1 and C5 genes appear to be largely FOC specific.