A slow sustained increase in cytosolic Ca2+ levels mediates stalk gene induction by differentiation inducing factor in Dictyostelium.

During Dictyostelium stalk cell differentiation, cells vacuolate, synthesize a cellulose cell wall and die. This process of programmed cell death is accompanied by expression of the prestalk gene ecmB and induced by the differentiation inducing factor DIF. Using cell lines expressing the recombinant Ca2+-sensitive photoprotein apoaequorin, we found that 100 nM DIF increases cytosolic Ca2+ ([Ca2+]i) levels from approximately 50 to 150 nM over a period of 8 h. The Ca2+-ATPase inhibitor 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ) induced a similar increase in [Ca2+]i levels and induced expression of the prestalk gene ecmB to the same level as DIF. The [Ca2+]i increases induced by DIF and BHQ showed similar kinetics and preceded ecmB gene expression by approximately 1-2 h. The Ca2+ chelator 1,2-bis(o-aminophenoxy)-ethane-N,N,N'N'-tetra-acetic acid (BAPTA) efficiently inhibited the BHQ-induced [Ca2+]i increase and blocked DIF-induced expression of the ecmB gene. These data indicate that the effects of DIF on stalk gene expression are mediated by a sustained increase in [Ca2-]i. Sustained [Ca2+]i elevation mediates many forms of programmed cell death in vertebrates. The Dictyostelium system may be the earliest example of how this mechanism developed during early eukaryote evolution.     

The analysis of the NSW wheat variety database. I. Modelling trial error variance

The retrospective analysis of a large database on wheat variety testing in New South Wales (NSW) is considered. This analysis involved three key steps. Initially error variance heterogeneity is modelled, indicating significant differences in error variance due to trial location, year of trialling, sowing date and trial mean yield. The implication of this modelling for the estimaion of variance components is discussed.     

Inheritance of resistance to potyviruses in Phaseolus vulgaris L. IV. Inheritance, linkage relations, and environmental effects on systemic resistance to four potyviruses

We have examined the genetics of systemic resistance in Phaseolus vulgaris to azuki bean mosaic virus (AzMV) and cowpea aphid-borne mosaic virus (CABMV) and the relationship of this resistance to a phenotypically similar resistance to watermelon mosaic virus (WMV) and soybean mosaic virus (SMV). In P. vulgaris cv Great Northern 1140 (GN1140), resistance to SMV and WMV has been attributed to the genes Smv and Wmv, respectively, which have been shown to segregate as a unit. Systemic resistance to AzMV is conferred by two incompletely dominant alleles, Azm1 and Azm2, at unlinked loci. At least three resistance alleles must be present at these two loci for systemic resistance to be expressed in the plant. Systemic resistance to CABMV in GN 1140 is conditioned by a dominant allele that has been designated Cam2. Under some environmental conditions, a recessive allele at an unlinked locus, cam3, also controls a resistant response to CABMV. Resistance to AzMV and CABMV does not assort independently from Wmv/Smv, but also does not consistently cosegregate, suggesting that perhaps in each case one of the factors involved in resistance is associated with Smv/Wmv.     

Structure and dynamics of melittin in lysomyristoyl phosphatidylcholine micelles determined by nuclear magnetic resonance.

Mixed micelles of the 26-residue, lytic peptide melittin (MLT) and 1-myristoyl-2-hydroxyl-sn-glycero-3-phosphocholine (MMPC) in aqueous solution at 25 degrees C were investigated by (13)C- and (31)P-NMR spectroscopy. (13)C alpha chemical shifts of isotopically labeled synthetic MLT revealed that MLT in the micelle is predominantly alpha-helical and that the peptide secondary structure is stable from pH 4 to pH 11. Although the helical transformation of MLT as determined from NMR is evident at lipid:peptide molar ratios as low as 1:2, tryptophan fluorescence measurements demonstrate that well-defined micellar complexes do not predominate until lipid:peptide ratios exceed 30:1. (31)P linewidth measurements indicate that the interaction between phosphate ions in solution and cationic groups on MLT is pH dependent, and that the phosphoryl group of MMPC senses a constant charge, most likely +2, on MLT from pH 4 to pH 10. (13)C-NMR relaxation data, analyzed using the model-free formalism, show that the peptide backbone of MLT is partially, but not completely, immobilized in the mixed micelles. Specifically, order parameters (S(2)) of C alpha-H vectors averaged 0.7 and were somewhat larger for residues in the N-terminal half of the molecule. The amino terminal glycine had essentially the same range of motion as the backbone carbons. Likewise, order parameters for the trp side chain were similar to those found for the peptide C alpha moieties, as was verified by trp fluorescence anisotropy decay data. In contrast, the motion of the lysine side chains was less restricted, the average S(2) values for the C epsilon-H vectors being 0.19, 0.30, and 0.44 for lys-7, 21, and 23, respectively, for MLT in the mixed micelles. Values of the effective correlation time of the local motion tau e were in the motional narrowing limit and usually longer for side-chain atoms than for those in the backbone. The dynamics were independent of pH from pH 4 to pH 9, but at pH 11 the correlation time for the rotational motion of the mixed micelles as a whole increased from 10 ns to 16 ns, and S(2) for the lys side chains increased. Overall it appears that the MLT helix lies near the surface of the micelle at low to neutral pH, but at higher pH its orientation changes, accompanied by deeper penetration of the lysine side chains into the micelle interior. It is apparent, however, that the MLT-lipid interaction is not dependent on deprotonation of any of the titratable cationic groups in the peptide in the pH 4-10 range, and that there is substantial backbone and side-chain mobility in micelle-bound MLT.     

The status and ecology of a Juniperus excelsa subsp. polycarpos woodland in the northern mountains of Oman

Juniperus excelsa subsp. polycarpos (K. Koch) Takhtajan is found in mountain areas from Turkey through to India and as an isolated population on Jebel Akhadar in the northern mountains of Oman. Juniperus is one of the dominant plant species in these mountains and a major landscape feature of several proposed National Nature and Scenic Reserves and of Hayl Juwari, a wooded valley at 2250 m altitude proposed as a Botanical Site of Special Interest. Above 2400 m altitude the Juniperus woodlands generally appear to be regenerating and in good condition, both on exposed slopes and in wadis and sheltered gullies, whereas below 2400 m most stands are in poor condition and exhibit few signs of regeneration. If the apparently poor condition of the lower altitude woodlands is due to any long term change in climatic conditions, both tree status and regeneration would be poorer in relatively more xeric habitats. To test this prediction we have carried out a detailed survey of the status and ecology of a 32 ha area of Hayl Juwari, and analysed differences in tree status and regeneration between wadis (relatively more nesic sites) and non-wadi areas (relatively more xeric). Approximately one third of the trees are dead, and an analysis of the height, condition, regeneration, female cone production, preferred germination sites and spatial distribution of trees indicates the importance of topography, hydrology and microclimate for growth. However, although there are relatively greater numbers of dead and poor-condition trees in the more xeric non-wadi habitat, there is no unequivocal evidence that the present distribution of small, sexually immature trees in both habitats could not form a pattern of larger, sexually mature trees similar to that seen today. We speculate, however, that the climate at this altitude may be marginal for the survival of a J. excelsa subsp. polycarpos woodland and that even small increases in climatic stress could imperil the woodland's present status.     

Detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts,ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20–24 dyn/cm² were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells’ ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor-derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell’s ability to metastasize.     

Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats.

Fimbrial production by Porphyromonas gingivalis was inactivated by insertion-duplication mutagenesis, using the cloned gene for the P. gingivalis major fimbrial subunit protein, fimA. by several criteria, this insertion mutation rendered P. gingivalis unable to produce fimbrilin or an intact fimbrial structure. A nonfimbriated mutant, DPG3, hemagglutinated sheep erythrocytes normally and was unimpaired in the ability to coaggregate with Streptococcus gordonii G9B. The cell surface hydrophobicity of DPG3 was also unaffected by the loss of fimbriae. However, DPG3 was significantly less able to bind to saliva-coated hydroxyapatite than wild-type P. gingivalis 381. This suggested that P. gingivalis fimbriae are important for adherence of the organism to saliva-coated oral surfaces. Further, DPG3 was significantly less able to cause periodontal bone loss in a gnotobiotic rat model of periodontal disease. These observations are consistent with other data suggesting that P. gingivalis fimbriae play an important role in the pathogenesis of human periodontal disease.     

Dose-response of two cellular proliferation phenotypes produced by simian virus 40 large T antigen

Abstract. A set of cell lines was constructed by infection of established murine fibroblasts with recombinant retroviruses encoding the simian virus 40 large T antigen (Tag) gene. By immunofluorescence flow cytometry, it was shown that these cell lines expressed Tag over at least a 20-fold concentration range. Using these cells, the dose-response relationship between Tag concentration and a phenotype detected by flow cytometry that measures the rate at which proliferating cells transit the cell cycle (i.e. cell-cycling phenotype) was determined. This relationship between Tag concentration and phenotype was not linear. Instead, the cell-cycling phenotype became saturated at relatively low Tag concentrations, i.e. a further increase in Tag concentration did not change the phenotype. The dose-response relationship between Tag and a second phenotype, colony formation in soft agar, was also determined. Colony formation in soft agar is a measurement of cell transformation. In contrast to the cell-cycling phenotype, transformation was linearly related to Tag over the entire 20-fold Tag concentration range. This phenotype did not saturate at high Tag concentrations. Therefore, the dose-response relationship between Tag concentration and the cell-cycling phenotype was different from that between Tag concentration and cellular transformation. Since the Tag gene is comprised of multiple genetic domains that independently affect cellular proliferation, one possibility is that the differences in dose-response of the two phenotypes indicate that different genetic domains of the gene are necessary for production of each phenotype.     

Screening species and cultivars for their tolerance to acidic soil conditions.

The major phytotoxins in acid soils are aluminium and manganese. Tolerances to Al and to excessive Mn are independently inherited and Al and Mn solubilities in soils vary. In this work, the response of pasture grasses and legumes to soil acidity was studied on three soils with different Al and Mn concentrations. One provides moderate concentrations of Al with little Mn; one provides high concentrations of both Al and Mn and another provides a very high concentration of Mn at relatively low concentrations of Al. The response of a plant cultivar to changes in the soil acidity induced by lime or acid additions reflects the degree of Al and/or Mn stress provided by a particular soil, and the ability of the cultivar to tolerate those stresses. Examples are given of the way cultivars with different tolerances to Al and Mn toxicity respond to changes in acidity on the soils with different Al and Mn solubility characteristics. The utility of this screening technique to define the tolerance of cultivars to acidity on classically different soils is highlighted.     

Structural and dynamic studies of a non-self-complementary dodecamer DNA duplex.

A non-self-complementary dodecamer duplex d(CCTAAATTTGCC).d(GGCAAATTTAGG) has been investigated in solution by high resolution 1H NMR. Almost complete resonance assignment of both non-exchangeable and exchangeable protons, has been achieved. The duplex is essentially B-type, with distortions apparent at the AT and TA steps. These distortions and their affects on dynamics have been probed by the measurement of base-pair lifetimes, and observation of water of hydration. Base-pair opening rates were derived from measurements of T1's and effects on linewidths of the T and G imino protons on addition of an exchange catalyst. Our results are generally in line with observations reported for other systems, but we see only a slight drop in the A.T base-pair lifetime on moving out from the central region. This observation is reinforced by the detection of DNA-water nOe's for residues distributed throughout the dodecamer sequence.