Plant viability assay

There is no single method in the long list given in Table 1 which can be used as an unequivocal criterion of viability. Many of the present methods of assay do correlate to various degrees with the final performance of the cell, tissue, organ, or the plant as a whole. Use of parallel viability tests indicating different cell functions is highly recommended. Great care should be taken to interpret the results of these assays. With several parallel tests, the validity of the interpretation can be enhanced, and in many cases, the interpretation may change considerably, depending upon the results from other tests, Since active transport systems have been implicated as one of the primary sites of freezing injury, more effort needs to be devoted to standardize viability assays based on this cell property. In general, the most popular viability assays for plants are based on biophysical rather than biochemical or metabolic functions. A specific test may be suited to a certain material more than another, yet our goal should be to devise a unique assay which will reflect the threshold of vitality versus death.     

Biofilm Modifies Expression of Ribonucleotide Reductase Genes in Escherichia coli

Ribonucleotide reductase (RNR) is an essential enzyme for all living organisms since is the responsible for the last step in the synthesis of the four deoxyribonucleotides (dNTPs) necessary for DNA replication and repair. In this work, we have investigated the expression of the three-RNR classes (Ia, Ib and III) during Escherichia coli biofilm formation. We show the temporal and spatial importance of class Ib and III RNRs during this process in two different E. coli wild-type strains, the commensal MG1655 and the enteropathogenic and virulent E2348/69, the prototype for the enteropathogenic E. coli (EPEC). We have established that class Ib RNR, so far considered cryptic, play and important role during biofilm formation. The implication of this RNR class under the specific growth conditions of biofilm formation is discussed.     

Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L⁵³⁵X₃G⁵³⁹X₂A⁵⁴²X₃V⁵⁴⁶X₂L⁵⁴⁹ rather than through the alternative glycine zipper motif A⁵³⁶X₃G⁵⁴⁰X₃G⁵⁴⁴ (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr⁵⁸⁸ and/or Tyr⁵⁹⁴) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.