Nuclear DNA Amounts in Angiosperms and their Modern Uses--807 New Estimates

The DNA amount in the unreplicated haploid nucleus of an organismis known as its C-value. C-values differ about 1000-fold amongangiosperms and are characteristic of taxa. The data are usedin many biological fields, so they should be easily available.Values for 2802 angiosperm species (1%) were estimated during1950–1997, and five collected lists of C-values were publishedfor reference purposes during 1976–1997. Numbers of newangiosperm C-values published recently remained high, necessitatinga further supplementary list. This paper lists DNA C-valuesfor 807 angiosperm species from 70 original sources, including520 (75.2%) from sources published after 1996, and 691 for speciesnot included in any of the previous five lists. There is a continuingneed to estimate accurate DNA C-values for plant taxa, as shownin a workshop on this biodiversity topic sponsored by Annalsof Botany and held at Kew in 1997. Its key aim was to identifymajor gaps in our knowledge of plant DNA amounts and to recommendtargets and priorities for new work to fill them. A target ofestimating first C-values for the next 1% of angiosperm speciesin 5 years was set. The proportion of such C-values in the presentwork (85.6%) is very high; and the number being published (approx.220 per annum) has never been exceeded. In 1997, C-values werestill unknown for most (68%) families, so a target of completecoverage was set. This paper includes first C-values for 12families, but as less than 2% of such values listed here targetednew families, the need to improve familial representation remains.Copyright 2000 Annals of Botany Company Angiosperm DNA amounts, DNA C-values, nuclear genome sizes, plant DNA database     

Quantification of the Role of Acclimation Temperature in Temperature Tolerance of Fishes

The relative effect of acclimation temperature on temperature tolerance was estimated from a geometrical partitioning of the temperature tolerance polygon of a fish species into three distinct zones relative to four key tolerance temperatures. This approach yields a middle tolerance zone which is independent of acclimation temperature bounded by upper and lower acclimation dependent zones. Acclimation dependent and independent temperature tolerance zones can be quantified by either areal or linear methods. Both methods were applied to quantify the effect of acclimation temperature in 21 species of temperate fishes for which temperature tolerance polygons were available. Temperature tolerance polygon areas of these 21 species ranged from 468 to 1380°C² and are linearly related (r ²=0.93, p<0.001) to ultimate incipient upper lethal temperatures. Although areal and linear partitioning methods yielded similar acclimation independent and dependent tolerances, estimates from the areal method incorporates additional information concerning the shape of the temperature tolerance polygon, in particular lower and upper lethal temperature plateaus. Mean combined acclimation dependent and independent tolerance areas of these 21 species were not different, indicating that acclimation effectively doubles the temperature tolerance polygon. Mean lower acclimation dependent area was nearly three times greater than mean upper acclimation dependent area, suggesting that acclimation plays a larger role in tolerance of low rather than high temperatures. Among these 21 species, temperature tolerance of brook charr and sheepshead minnow were the least and most affected by acclimation temperature, respectively.     

P2X (purinergic) receptor redistribution in rabbit aorta following injury to endothelial cells and cholesterol feeding

The redistribution of purinergic P2X receptor subunits (P2X₁ to P2X₇) within the rabbit aorta wall three weeks after endothelial balloon injury/cholesterol feeding was examined. P2X₁ receptor cluster density was elevated in the media following balloon injury/cholesterol feeding by about 30% and these clusters appeared on smooth muscle cells throughout the greatly expanded neointima but they did not change significantly on the endothelial cells following balloon injury. P2X₄ clusters were found in high density throughout the media and in very high density in the enlarged neointima following balloon injury, particularly on the endothelial cells where the density increased about 10-fold after balloon injury. P2X₅ clusters were found in high density in the media of normal aorta but with little change following balloon injury. P2X₃, P2X₆ and P2X₇ cluster density was low in normal aorta and remained unchanged following balloon injury. All receptor subunits were found on endothelial cells. It is suggested that the release of ATP from damaged endothelial cells and from smooth muscle cells sufficient to activate P2X₄ receptors may contribute to neointimal proliferation.     

Changes in the distribution of different subtypes of P2X receptor clusters on smooth muscle cells in relation to nerve varicosities in the pregnant rat urinary bladder

Clusters of purinergic receptor subunits, about 1 μm diameter, are found on the smooth muscle cell membrane beneath junctional varicosities in the detrusor muscle of the rat urinary bladder. We have examined the extent of redistribution of the six different subunit clusters, P2X₁ to P2X₆, with respect to junctional varicosities during pregnancy, as it is known that the detrusor muscle undergoes changes in purinergic innervation during this period. Before pregnancy, clusters at junctional varicosities are principally composed of the subtypes P2X₁, P2X₂, P2X₃ and P2X₅. However this subtype distribution changes dramatically during pregnancy, such that by day 14 of pregnancy, the extent of P2X₁, P2X₂, P2X₃ and P2X₅ junctional clusters has decreased by more than 80% whereas the extent of P2X₄ and P2X₆ junctional clusters has increased by more than 80%. These changes were confirmed with Western blots for different subtypes. It is suggested that the changes in the purinergic innervation of the detrusor muscle during pregnancy reflect changes in the P2X subtypes found on the smooth muscle membrane beneath junctional varicosities.     

Effects of resin flow and monoterpene composition on susceptibility of lodgepole pine to attack by the Douglas-fir pitch moth, Synanthedon novaroensis (Lep., Sesiidae)

Resin flow differed significantly among three of six clones of lodgepole pine seed orchard trees, but did not differ among the clones categorized as susceptible or resistant to attack by the Douglas-fir pitch moth. A stepwise regression analysis identified δ-3-carene, cyclohexene, and α-terpinolene as significant compounds, explaining 49.9% of the variation in the number of attacks per tree. δ-3-Carene alone explained 41.8% of the variation in the regression, and analysis of variance showed that resistant clones consistently had high relative amounts (>17.9%) of this compound, whereas susceptible clones had low amounts (<10%). The significant effect by cyclohexene and α-terpinolene in the stepwise regression appeared to be due to a correlation between α-terpinolene and δ-3-carene in several clones, and that cyclohexene was only present in one clone, rather than any discernable biological relationship. Limonene co-eluted with β-phellandrene, so its role must be determined by additional study.     

Single molecule analysis indicates stimulation of MUTYH by UV-DDB through enzyme turnover

The oxidative base damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is a highly mutagenic lesion because replicative DNA polymerases insert adenine (A) opposite 8-oxoG. In mammalian cells, the removal of A incorporated across from 8-oxoG is mediated by the glycosylase MUTYH during base excision repair (BER). After A excision, MUTYH binds avidly to the abasic site and is thus product inhibited. We have previously reported that UV-DDB plays a non-canonical role in BER during the removal of 8-oxoG by 8-oxoG glycosylase, OGG1 and presented preliminary data that UV-DDB can also increase MUTYH activity. In this present study we examine the mechanism of how UV-DDB stimulates MUTYH. Bulk kinetic assays show that UV-DDB can stimulate the turnover rate of MUTYH excision of A across from 8-oxoG by 4–5-fold. Electrophoretic mobility shift assays and atomic force microscopy suggest transient complex formation between MUTYH and UV-DDB, which displaces MUTYH from abasic sites. Using single molecule fluorescence analysis of MUTYH bound to abasic sites, we show that UV-DDB interacts directly with MUTYH and increases the mobility and dissociation rate of MUTYH. UV-DDB decreases MUTYH half-life on abasic sites in DNA from 8800 to 590 seconds. Together these data suggest that UV-DDB facilitates productive turnover of MUTYH at abasic sites during 8-oxoG:A repair.     

Differential regulation of the accumulation of the light-harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves

The photoregulation of chloroplast development in pea leaves has been studied by reference to three polypeptides and their mRNAs. The polypeptides were the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO), and the light-harvesting chlorophyll a/b protein (LHCP). The polypeptides were assayed by a sensitive radioimmune assay, and the mRNAs were assayed by hybridization to cloned DNA probes. LSU, LSU mRNA, and LHCP mRNA were detectable in etiolated seedlings but LHCP, SSU, and SSU mRNA were at or below the limit of detection. During the first 48 hr of de-etiolation under continuous white light, the mRNAs for LSU, SSU, and LHCP increased in concentration per apical bud by about 40-fold, at least 200-fold, and about 25-fold, respectively, while the total RNA content per apical bud increased only 3.5-fold. In the same period, the LSU, SSU, and LHCP contents per bud increased at least 60-, 100-, and 200-fold, respectively. The LHCP increased steadily in concentration during de-etiolation, whereas the accumulation LSU, SSU, and SSU mRNA showed a 24-hr lag. The accumulation of SSU, SSU mRNA, and LHCP mRNA showed classical red/far-red reversibility, indicating the involvement of phytochrome in the regulatory mechanism. LSU and LSU mRNA were induced equally well by red and far-red light. The LHCP failed to accumulate except under continuous illumination. These results indicate that the accumulation of SSU is controlled largely through the steady-state level of its mRNA, which is in turn almost totally dependent on light as an inducer and on phytochrome as one of the photoreceptors. The accumulation of LSU is largely but not totally determined by the level of its mRNA, which appears to be under strong photoregulation, which has yet to be shown to involve phytochrome. Phytochrome is involved in the regulation of LHCP mRNA levels but substantial levels of the mRNA also occur in the dark. LHCP accumulation is not primarily governed by the levels of LHCP mRNA but by posttranslational stabilization in which chlorophyll synthesis plays a necessary but not sufficient role.     

Effect of different levels of NADH availability on metabolic fluxes of Escherichia coli chemostat cultures in defined medium

Escherichia coli overexpressing a NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii was grown in chemostat culture on various carbon sources at 0.05 h(-1) dilution rate, under anaerobic conditions using defined medium and compared to a control without the heterologous FDH pathway. Metabolic fluxes, NADH/NAD(+) ratios and NAD(H/(+)) levels were determined under a range of intracellular NADH availability. The effect of NADH manipulation on the distribution of metabolic fluxes in E. coli was assessed under steady-state conditions. The heterologous FDH pathway converts 1 mol of formate into 1 mol of NADH and carbon dioxide, in contrast with the native FDH where no cofactor involvement is present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed and reached 4.6 mol NADH/mol of substrate when sorbitol was used as a carbon source in a complex medium. In the current study, it was found that higher NADH yields and NADH/NAD(+) ratios were achieved with our in vivo NADH regeneration system compared to a control lacking the new FDH pathway in the three carbon sources (glucose, gluconate and sorbitol) examined suggesting a more reduced intracellular environment. The total NAD(H/(+)) amounts were very similar for all the combinations studied. It was also found that the ethanol to acetate ratio increased with increased NADH availability. This ratio increased from 1.05 for the control strain in glucose to 9.45 for the strain expressing the heterologous NAD(+)-dependent FDH in sorbitol.     

Perturbation of the mutated EGFR interactome identifies vulnerabilities and resistance mechanisms

We hypothesized that elucidating the interactome of epidermal growth factor receptor (EGFR) forms that are mutated in lung cancer, via global analysis of protein–protein interactions, phosphorylation, and systematically perturbing the ensuing network nodes, should offer a new, more systems‐level perspective of the molecular etiology. Here, we describe an EGFR interactome of 263 proteins and offer a 14‐protein core network critical to the viability of multiple EGFR‐mutated lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) had differential dependence of the core network proteins based on the underlying molecular mechanisms of resistance. Of the 14 proteins, 9 are shown to be specifically associated with survival of EGFR‐mutated lung cancer cell lines. This included EGFR, GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A. With the use of a drug network associated with the core network proteins, we identified two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib. Our results, enabled by interactome mapping, suggest new targets and combination therapies that could circumvent EGFR TKI resistance.