No abstract

The developmental cycle of the myxobactetium Myxococcus xanthus consists of three partially overlapping morphological stages referred to as rippling, fruiting body formation, and sporulation, all of which are absent in csgA null mutants. The CsgA gene product is an extracellular protein, referred to as the C signal, which is essential for developmental cell-cell interactions. csgA expression increases throughout development, reaching its peak during sporulation. CsgA was made limiting for development by constructing nested deletions upstream from the csgA gene, which resulted in reduced csgA expression. Successively larger deletions resulted in termination of development at earlier and earlier stages, with rippling requiring -20% maximum csgA expression, fruiting body formation requiring -30% expression, and sporulation requiring 82% expression. Conversely, artificial induction of csgA also induced development provided nutrients were limiting. These results suggest that steady increases in CsgA over the course of development entrain the natural sequence of morphological events. The csgA upstream region appears to process information concerning the levels of nutrients, peptidoglycan components, and the B signal. In the absence of nutrients, a region extending 400 bp upstream from the start site of transcription was necessary for development and maximal csgA expression. In the presence of low levels of nutrients, a region extending -930 bp upstream was essential for the same tasks. It appears that the upstream region extending from -400 to -930 stimulates csgA expression in the presence of excess carbon, nitrogen, and phosphate, thereby allowing development to go to completion.

We have identified the beta (beta) isoform of the 14‐3‐3 family of proteins as an activator of the Raf‐1 protein kinase. 14‐3‐3 was isolated in a yeast two‐hybrid screen for Raf‐1 kinase domain binding proteins. Purified bovine brain 14‐3‐3 interacted specifically with both c‐Raf‐1 and the isolated Raf‐1 kinase domain. Association was sensitive to the activation status of Raf‐1; 14‐3‐3 bound to unactivated Raf‐1, but not Raf‐1 activated by protein kinase C alpha or Ras and Lck. The significance of these interactions under physiological conditions was demonstrated by co‐immunoprecipitation of Raf‐1 and 14‐3‐3 from extracts of quiescent, but not mitogen‐stimulated, NIH 3T3 cells. 14‐3‐3 was not a preferred Raf‐1 substrate in vitro and did not significantly affect Raf‐1 kinase activity in a purified system. However, in cell‐free extracts 14‐3‐3 acted as a Ras‐independent activator of both c‐Raf‐1 and the Raf‐1 kinase domain. The same results were obtained in vivo using transfection assays; 14‐3‐3 enhanced both c‐Raf‐1‐ and Raf‐1 kinase domain‐stimulated expression of AP‐1‐ and NF‐kappa B‐dependent reporter genes and accelerated Raf‐1 kinase domain‐triggered differentiation of PC12 cells. We conclude that 14‐3‐3 is a latent co‐activator bound to unactivated Raf‐1 in quiescent cells and mediates mitogen‐triggered but Ras‐independent regulatory effects aimed directly at the kinase domain.

Addition of mitogenic growth factors to quiescent cells triggers complex signal transduction cascades that result in the reprogramming of gene expression and entry into the cell cycle. We have found that an oncogenic variant of the c-Raf-1 protein kinase stimulated the expression of promoters containing NF-,cB binding sites. In situ immunofluorescence analysis revealed elevated nuclear levels of the p65 subunit of NF-wB in v-raf-transformed NIH 3T3 cells. Incubation of HeLa cell cytoplasmic extracts with a purified recombinant glutathione S-transferase-raf fusion protein in the presence ofATP released active NF-wB that could be detected by electrophoretic gel mobility shift assay. Coincubation of purified recombinant I#cB and glutathione S-transferase-raf in the presence of ATP resulted in the phosphorylation of IdcB. Coexpression of GALA (activation domain)-I,B and GALA (DNA-binding domain)-raf fusion proteins in yeast resulted in stimulation of a GAL4-responsive reporter gene, indicating that IB and Raf interact physically in vivo. These results indicate that the Raf-l kinase functions in signal transduction in part by activating the NF-icB transcription factor by phosphorylating 1KB in the cytoplasmic IicB-NF-icB complex to release active NF-ucB.

No abstract

No abstract

A series of intercellular signals are involved in the regulation of gene expression during fruiting body formation of Myxococcus xanthus. Mutations which block cell interactions, such as csgA (formerly known as spoC), also prevent expression of certain developmentally regulated promoters. csgA+ cells containing TnS lac QiDK4435, a developmentally regulated promoter fused to lacZ, began synthesizing lacZ mRNA 12 to 18 h into the developmental cycle. j-Galactosidase specific activity increased about 12 h later. Neither lacZ mRNA nor ,B-galactosidase activity was detected in a developing csgA mutant containing flDK4435. The developmental promoter and its fused lacZ reporter gene were cloned into a pBR322-derived plasmid vector containing a portion of bacteriophage Mx8. These plasmids preferentially integrated into the M. xanthus chromosome by site-specific recombination at the bacteriophage Mx8 attachment site and maintained a copy number of 1 per chromosome. The integrated plasmids were relatively stable, segregating at a frequency of 0.0007% per generation in the absence of selection. The cloned and integrated promoter behaved like the native promoter, expressing I8-galactosidase at the proper time during wild-type development and failing to express the enzyme during development of a csgA mutant. The overall level of ,-galactosidase expression in merodiploid cells containing one native promoter and one promoter fused to lacZ was about half that of cells containing a single promoter fused to lacZ. These results suggest that the timing of developmentally regulated gene expression is largely independent of the location of this gene within the chromosome. Furthermore, they show that site-specific recombination can be a useful tool for establishing assays for promoter or gene function in M. xanthus.

The heart is an extremely sophisticated organ with nanoscale anisotropic structure, contractility and electro-conductivity; however, few studies have addressed the influence of cardiac anisotropy on cell transplantation for myocardial repair. Here, we hypothesized that a graft's anisotropy of myofiber orientation determines the mechano-electrical characteristics and the therapeutic efficacy. We developed aligned- and random-orientated nanofibrous electrospun patches (aEP and rEP, respectively) with or without seeding of cardiomyocytes (CMs) and endothelial cells (ECs) to test this hypothesis. Atomic force microscopy showed a better beating frequency and amplitude of CMs when cultured on aEP than that from cells cultured on rEP. For the in vivo test, a total of 66 rats were divided into six groups: sham, myocardial infarction (MI), MI + aEP, MI + rEP, MI + CM-EC/aEP and MI + CM-EC/rEP (n ≥ 10 for each group). Implantation of aEP or rEP provided mechanical support and thus retarded functional aggravation at 56 days after MI. Importantly, CM-EC/aEP implantation further improved therapeutic outcomes, while cardiac deterioration occurred on the CM-EC/rEP group. Similar results were shown by hemodynamic and infarct size examination. Another independent in vivo study was performed and electrocardiography and optical mapping demonstrated that there were more ectopic activities and defective electro-coupling after CM-EC/rEP implantation, which worsened cardiac functions. Together these results provide comprehensive functional characterizations and demonstrate the therapeutic efficacy of a nanopatterned anisotropic cardiac patch. Importantly, the study confirms the significance of cardiac anisotropy recapitulation in myocardial tissue engineering, which is valuable for the future development of translational nanomedicine.