Evidence of the activity of tyrosine kinase(s) and of the presence of phosphotyrosine proteins in pea plantlets

Homogenate fractions from etiolated pea plantlets showed tyrosine kinase activity when incubated with [32P]ATP and substrates like polyamino acid polymer (Glu-Ala-Tyr)n or [Val5]angiotensin II. When these tyrosine kinase substrates were recovered by high voltage electrophoresis, and analyzed by high pressure liquid chromatography after alkaline hydrolysis, yielded radioactive phosphotyrosine. The same product was obtained after acid hydrolysis of either endogenous or exogenous substrates. Phosphorylated polypeptides were extracted after sodium dodecyl sulfate gel electrophoresis of a pellet fraction incubated with [32P]ATP. After acid hydrolysis and high voltage electrophoresis, [32P]phosphotyrosine was found in gel bands with polypeptides of about 92 and 57 kDa. These results suggest that tyrosine kinase(s) and phosphotyrosine proteins are also present in higher plants.     

Structure–activity relationship study of acridine analogs as haspin and DYRK2 kinase inhibitors

Haspin is a serine/threonine kinase required for completion of normal mitosis that is highly expressed during cell proliferation, including in a number of neoplasms. Consequently, it has emerged as a potential therapeutic target in oncology. A high throughput screen of approximately 140,000 compounds identified an acridine analog as a potent haspin kinase inhibitor. Profiling against a panel of 270 kinases revealed that the compound also exhibited potent inhibitory activity for DYRK2, another serine/threonine kinase. An optimization study of the acridine series revealed that the structure–activity relationship (SAR) of the acridine series for haspin and DYRK2 inhibition had many similarities. However, several structural differences were noted that allowed generation of a potent haspin kinase inhibitor (33, IC₅₀ <60 nM) with 180-fold selectivity over DYRK2. In addition, a moderately potent DYRK2 inhibitor (41, IC₅₀ <400 nM) with a 5.4-fold selectivity over haspin was also identified.     

Wheat Chloroplastic Glutathione Reductase Activity is Regulated by the Combined Effect of pH, NADPH and GSSG

To study the mechanism of regulation of chloroplastic glutathionereductase (GR) under photooxidative conditions, GR activity,and the levels of NADPH, GSH and GSSG were measured in wheatchloroplasts under photooxidative light. GR was extremely labile,and the concentrations of GSH and GSSG progressively diminishedin chloroplasts prepared without ascorbate. The NADPH leveldid not significantly change during photooxidative treatment.The addition of 10 mM ascorbate to the incubation medium preventedthe decrease of GSH and GSSG and strongly protected GR activity.However, ascorbate had no effect on NADPH-dependent inhibitionof the chloroplastic GR purified from wheat leaves. We studiedthe effect of NADPH, temperature, pH and GSSG on the purifiedenzyme. The inhibition by NADPH was greatly dependent on temperatureand pH. NADPH inhibited GR by around 93% up to pH 7.5, but withina range of 8.0 to 9.5 the inhibition was only marginal. ThepH dependence of the NADPH inhibitory effect could be due, atleast in part, to different rates in the generation of NADPH-X,a derivative of NADPH which inactivates several pyridin nucleotidedehydrogenases. Furthermore, the NADPH-dependent inhibitionwas almost completely prevented by GSSG, but not by GSH. (Received July 9, 1998; Accepted April 30, 1999)     

Phylogenetic analysis of the alpha-globin pseudogene-4 (Hba-ps4) locus in the house mouse species complex reveals a stepwise evolution of t haplotypes [published erratum appears in Mol Biol Evol 1994 Jan;11(1):158]

A parsimony analysis was performed on restriction sites at the Hba-ps4 pseudogene locus within one of four inversions associated with mouse t haplotypes. The results suggest that all t haplotypes form a monophyletic group and that the in (17)4 inversion originated before the radiation of the Mus musculus species complex but after the divergence of the lineages leading to M. spretus, M. abbotti, and M. hortulanus. A time frame based on the evolutionary rate of mouse pseudogenes places the origin of this t haplotype inversion at 1.5 Mya, or approximately 1.5 Myr after the origin of the more proximal t complex inversion, in (17)2. The accumulated evidence indicates that complete t haplotypes have been assembled in a stepwise manner, with each of these inversions occurring on separate chromosomal lineages and at different evolutionary times. In addition, the evolutionary relationships of pseudogene sequences resulting from genetic exchange between wild-type and t haplotype alleles were examined. Analysis of sequences from the 5' and 3' sides of a putative site of recombination resulted in cladograms with different topologies. The implications for hypotheses concerning the evolutionary forces acting on t haplotypes and their rapid propagation throughout worldwide populations of mice are discussed.      

Fnr (EtrA) acts as a fine-tuning regulator of anaerobic metabolism in <Emphasis Type="Italic">Shewanella oneidensis</Emphasis>MR-1

Background  

EtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood.