Using a cDNA clone from the ovary of the frog, Xenopus laevis, we have identified the mRNA and determined the complete amino acid sequence of a major cytoskeletal protein expressed in the oocyte. A comparison with other cytoskeletal proteins of Xenopus and mammals identifies this polypeptide Mr 55,700 as a nonepidermal kind of cytokeratin of the basic (type II) subfamily, which represents the amphibian equivalent to cytokeratin no. 8 of simple epithelia of higher mammals. The sequence data demonstrate the high evolutionary stability of this protein. This cytokeratin and its mRNA are present in oocytes, eggs, embryos, liver, and intestinal mucosa of adult frogs, as well as cultured kidney epithelial cells. We suggest that epithelial cell differentiation in early stages of Xenopus embryogenesis differs from other known pathways of cell differentiation in that major cell-type-specific proteins--i.e., cytokeratins of the simple epithelial type--and their mRNAs are maternally provided and distributed to early epithelial cells by special sorting mechanisms.
The design and generation of an optimal expression construct is the first and essential step in in the characterization of a protein of interest. Besides evaluation and optimization of process parameters (e.g. selection of the best expression host or cell line and optimal induction conditions and time points), the design of the expression construct itself has a major impact. However, the path to this final expression construct is often not straight forward and includes multiple learning cycles accompanied by design variations and retesting of construct variants, since multiple, functional DNA sequences of the expression vector backbone, either coding or non-coding, can have a major impact on expression yields. To streamline the generation of defined expression constructs of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed. This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules with a minimal cloning burden. Combining these features with a standardized cloning strategy facilitates the identification of optimized DNA expression constructs in shorter time. The MoPET system currently consists of 53 defined DNA modules divided into eight functional classes and can be flexibly expanded. However, already with the initial set of modules, 792,000 different constructs can be rationally designed and assembled. Furthermore, this starting set was used to generate small and mid-sized combinatorial expression optimization libraries. Applying this screening approach, variants with up to 60-fold expression improvement have been identified by MoPET variant library screening.
Background and purpose: Rho‐kinase (ROCK) has been implicated in the pathophysiology of altered vasoregulation leading to hypertension. Here we describe the pharmacological characterization of a potent, highly selective and orally active ROCK inhibitor, the derivative of a class of azaindoles, azaindole 1 (6‐chloro‐ N 4 ‐{3,5‐difluoro‐4‐[(3‐methyl‐1 H ‐pyrrolo[2,3‐ b ]pyridin‐4‐yl)oxy]‐phenyl}pyrimidine‐2,4‐diamine). Experimental approach: Pharmacological characterization of azaindole 1 was performed with human recombinant ROCK in vitro . Vasodilator activity was determined using isolated vessels in vitro and different animal models in vivo . Key results: This compound inhibited the ROCK‐1 and ROCK‐2 isoenzymes with IC 50 s of 0.6 and 1.1 nM in an ATP‐competitive manner. Although ATP‐competitive, azaindole 1 was inactive against 89 kinases (IC 50 >10 μM) and showed only weak activity against an additional 21 different kinases (IC 50 =1 ‐ 10 μM). Only the kinases TRK und FLT3 were inhibited by azaindole 1 in the sub‐micromolar range, albeit with IC 50 values of 252 and 303 nM, respectively. In vivo , azaindole 1 lowered blood pressure dose‐dependently after i.v. administration in anaesthetized normotensive rats. In conscious normotensive and spontaneously hypertensive rats azaindole 1 induced a dose‐dependent decrease in blood pressure after oral administration without inducing a significant reflex increase in heart rate. In anaesthetized dogs, azaindole 1 induced vasodilatation with a moderately elevated heart rate. Conclusions and implications: Azaindole 1 is representative of a new class of selective and potent ROCK inhibitors and is a valuable tool for the elucidation of the role of ROCK in the cardiovascular system. British Journal of Pharmacology (2007) 152 , 1070–1080; doi: 10.1038/sj.bjp.0707484 ; published online 15 October 2007
Abstract A Xenopus laevis mRNA encoding a cytokeratin of the basic (type II) subfamily that is expressed in postgastrulation embryos was cDNA-cloned and sequenced. Comparison of the deduced amino acid sequence of this polypeptide (513 residues, calculated mol. wt 55454; Mr ∼ 58 000 on SDS–PAGE) with those of other cytokeratins revealed its relationship to certain type II cytokeratins of the same and other species, but also remarkable differences. Using a subclone representing the 3′-untranslated portion of the 2·4kb mRNA encoding this cytokeratin, designated XenCK55(5development of n blot experiments, we found that it differs from the only other Xenopus type II cytokeratin known, i.e. the simple epithelium-type component XenCKl(8), in that it is absent in unfertilized eggs and pregastrulation embryos. XenCK55(5/6) mRNA was first detected at gastrulation (stage 11) and found to rapidly increase during neurulation and further development. It was also identified in Xenopus laevis cultured kidney epithelial cells of the line A6 and in the adult animal where it is a major polypeptide in the oesophageal mucosa but absent in most other tissues examined. The pattern of XenCK55(5/6) expression during embryonic development was similar to that reported for the type I polypeptides of the ‘XK81 subfamily’ previously reported to be embryo-specific and absent in adult tissues. Therefore, we used a XK81 mRNA probe representing the 3′-untranslated region in Northern blots, SI nuclease and hybrid-selection-translation assays and found the ∼ 1·6kb XK81 mRNA and the resulting protein of Mr∼ 48 000 not only in postgastrula embryos and tadpoles but also in the oesophagus of adult animals. Our results show that both these type II and type I cytokeratins are synthesized only on gastrulation and are very actively produced in early development. However, their synthesis is not restricted to developmental stages but is continued in at least one epithelium of the adult organism. These observations raise doubts on the occurrence of Xenopus cytokeratins that are strictly specific for certain embryonic or larval stages and absent in the adult. They rather suggest that embryonically expressed cytokeratins are also produced in some adult tissues, although in a restricted pattern of tissue and cell type distribution.
