Stress and Age Related Spots with Immunoreactivity to Ubiquitin-Antibody at Protoplast Surfaces

Mesophyll protoplasts from primary leaves of 2, 3, and 4 weekold Viciafaba L. plants and from not expanded leaves of 2 weekold plants were incubated with rabbit anti-ubiquitin antibodyand FITC labeled goat anti-rabbit IgG. Dependent on age of theplant material, an increase in size and number of immunoreactivespots at protoplast surfaces were observed, when incubationswere performed after 16 h storage to allow protoplast to recover.A relationship between isolation stress and the intensity ofimmunolabeling was demonstrated for protoplasts from not expandedleaves. Furthermore, the surface of isolation stressed protoplastsshowed an increasing number of immunoreactive spots when plantswere previously exposed to water deficiency conditions for 1,2 or 4 days. Water deficiency conditions and isolation stressare therefore thought to induce ubiquitination of surface locatedproteins. A phenomenon, which seemed to be normally correlatedwith early events of senescence. (Received October 28, 1993; Accepted February 21, 1994)     

Differential Dichrome Staining of Tissue Culture Monolayers: Alternate Dyes and Possible Mechanism

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.     

Purification of thymidine kinase by affinity chromatography with an enzyme inhibitor as the ligand

An affinity column for the purification of thymidine kinase is described. The ligand in this column is a glycoprotein isolated from rat kidney. This glycoprotein inhibits phosphorylation of thymidine in cultured cells and in a cell-free assay system. With an affinity column containing the glycoprotein as a ligand, a 24-fold purification of thymidine kinase from an ammonium sulfate fraction of a crude tissue extract can be obtained. Thymidine kinase eluted from the affinity column migrates as one major band on polyacrylamide and as one diffuse major band on sodium dodecyl sulfate-polyacrylamide. The affinity column, with thymidine kinase bound to the inhibitor, can also be used as an assay system. When the glycoprotein is covalently attached to Sepharose, it retains its binding capacity for thymidine kinase but has apparently lost its ability to inhibit the enzyme. Thymidine kinase eluted from the affinity column is again sensitive to the glycoprotein. It seems to be a carbohydrate moiety of the glycoprotein that is responsible for the inhibition.     

Activated-sludge process dynamics with continuous total organic carbon and oxygen uptake measurements

The dynamic responses of a bench-scale activated-sludge process to step changes and square-wave inputs in the feed flow and concentration were measured. Instrumentation permitted the continuous measurement of the oxygen uptake rate and dissolved organic carbon responses. Notable were the sensitivity of the oxygen uptake rate to process changes and the reliability of the dynamic oxygen electrode method. The responses were found to be greatly influenced by the organic loading, FS₀/XV, which was incorporated into a load-dependent kinetics model. Simulations showd good agreement with experiment in the case of the square-wave disturbances. Because of the changing and complex nature of the activated sludge it was necessary to reestimate the parameter set for each run.     

The return of an experimentally N-saturated boreal forest to an N-limited state: observations on the soil microbial community structure,biotic N retention capacity and gross N mineralisation

Background and aims

To find out how N-saturated forests can return to an N-limited state, we examined the recovery of biotic N sinks under decreasing N supply.

Methods

. We studied a 40-year-old experiment in Pinus sylvestris forest, with control plots, N0, three N treatments, N1-N3, of which N3 was stopped after 20 years, allowing observation of recovery.

Results

In N3, the N concentration in foliage was still slightly elevated, but the N uptake capacity of ectomycorrhizal (ECM) roots in N3 was no longer lower than in N0. Per area the amount of a biomarker for fungi, here mainly attributed ECM, was higher in N3 and N0 than in N1 and N2. Retention of labeled ¹⁵NH₄ ⁺ by the soil was greater in the control (99 %) and N3 (86 %), than in N1 (45 %) and N2 (29 %); we ascribe these differences to biotic retention because cation exchange capacity did not vary. Gross N mineralisation and retention of N correlated, negatively and positively, respectively, with abundance of ECM fungal biomarker.

Conclusions

. The results suggest a key role for ECM fungi in regulating the N cycle. We propose, in accordance with plant C allocation theory, that recovery is driven by increased tree below-ground C allocation to ECM roots and fungi.     

Studying genetics of adaptive variation in model organisms: flowering time variation in Arabidopsis lyrata

Arabidopsis thaliana has emerged as a model organism for plant developmental genetics, but it is also now being widely used for population genetic studies. Outcrossing relatives of A. thaliana are likely to provide suitable additional or alternative species for studies of evolutionary and population genetics. We have examined patterns of adaptive flowering time variation in the outcrossing, perennial A. lyrata. In addition, we examine the distribution of variation at marker genes in populations form North America and Europe. The probability of flowering in this species differs between southern and northern populations. Northern populations are much less likely to flower in short than in long days. A significant daylength by region interaction shows that the northern and southern populations respond differently to the daylength. The timing of flowering also differs between populations, and is made shorter by long days, and in some populations, by vernalization. North American and European populations show consistent genetic differentiation over microsatellite and isozyme loci and alcohol dehydrogenase sequences. Thus, the patterns of variation are quite different from those in A. thaliana, where flowering time differences show little relationship to latitude of origin and the genealogical trees of accessions vary depending on the genomic region studied. The genetic architecture of adaptation can be compared in these species with different life histories.     

Noncanonical regulation of alkylation damage resistance by the OTUD4 deubiquitinase

Repair of DNA alkylation damage is critical for genomic stability and involves multiple conserved enzymatic pathways. Alkylation damage resistance, which is critical in cancer chemotherapy, depends on the overexpression of alkylation repair proteins. However, the mechanisms responsible for this upregulation are unknown. Here, we show that an OTU domain deubiquitinase, OTUD4, is a positive regulator of ALKBH2 and ALKBH3, two DNA demethylases critical for alkylation repair. Remarkably, we find that OTUD4 catalytic activity is completely dispensable for this function. Rather, OTUD4 is a scaffold for USP7 and USP9X, two deubiquitinases that act directly on the AlkB proteins. Moreover, we show that loss of OTUD4, USP7, or USP9X in tumor cells makes them significantly more sensitive to alkylating agents. Taken together, this work reveals a novel, noncanonical mechanism by which an OTU family deubiquitinase regulates its substrates, and provides multiple new targets for alkylation chemotherapy sensitization of tumors.