Growth factor transgenes interactively regulate articular chondrocytes

Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin‐like growth factor I (IGF‐I), fibroblast growth factor‐2 (FGF‐2), transforming growth factor beta1 (TGF‐β1), bone morphogenetic protein‐2 (BMP‐2), and bone morphogenetic protien‐7 (BMP‐7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF‐I and FGF‐2 maximized cell proliferation. The three‐transgene group encoding IGF‐I, BMP‐2, and BMP‐7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell‐based articular cartilage repair. J. Cell. Biochem. 114: 908–919, 2013. © 2012 Wiley Periodicals, Inc.     

Contribution of NDH-dependent cyclic electron transport around photosystem I to the generation of proton motive force in the weak mutant allele of pgr5

In angiosperms, cyclic electron transport (CET) around photosystem I (PSI) consists of two pathways, depending on PGR5/PGRL1 proteins and the chloroplast NDH complex. In single mutants defective in chloroplast NDH, photosynthetic electron transport is only slightly affected at low light intensity, but in double mutants impaired in both CET pathways photosynthesis and plant growth are severely affected. The question is whether this strong mutant phenotype observed in double mutants can be simply explained by the additive effect of defects in both CET pathways. In this study, we used the weak mutant allele of pgr5-2 for the background of double mutants to avoid possible problems caused by the secondary effects due to the strong mutant phenotype. In two double mutants, crr2-2 pgr5-2 and ndhs-1 pgr5-2, the plant growth was unaffected and linear electron transport was only slightly affected. However, NPQ induction was more severely impaired in the double mutants than in the pgr5-2 single mutant. A similar trend was observed in the size of the proton motive force. Despite the slight reduction in photosystem II parameters, PSI parameters were severely affected in the pgr5-2 single mutant, the phenotype that was further enhanced by adding the NDH defects. Despite the lack of ?pH-dependent regulation at the cytochrome b₆f complex (donor-side regulation of PSI), the plastoquinone pool was more reduced in the double mutants than in the pgr5-2 single mutants. This phenotype suggests that both PGR5/PGRL1- and NDH-dependent CET contribute to supply sufficient acceptors from PSI by balancing the ATP/NADPH production ratio.     

Ethanol Tolerance and Alcohol Dehydrogenase (ADH; EC1.1.1.1) Activity in Species of the cardini Group of Drosophila

Ethanol tolerance, alcohol dehydrogenase (ADH;EC1.1.1.1) activity, and tissue-specific expression wereexamined in species of the cardini group ofDrosophila using D. melanogaster as astandard of comparison. In contrast to most fruit-breeding species, allcardini species examined, two from the cardini subgroupand five from the dunni subgroup, were ethanol sensitive(LC₅₀ 2.05%) and the mean ADH activityof males ranges from only 8 to 16% that of D.melanogaster Adh^FF. Among all sevencardini species, there were small but significantdifferences in ethanol tolerance and ADH activity.Differences in enzyme mobility were in accordance with the proposedphylogeny for the dunni-subgroup species. ADH isexpressed in the fat body and midgut. Males of D.acutilabella and of D. belladunni havesignificantly less ethanol tolerance and express less ADH activitythan females in zymograms and histologicalpreparations.     

Evaluation of physiological importance of metallothionein protein expressed by Tetrahymena cadmium metallothionein 1 (TMCd1) gene in Escherichia coli

TMCd1 is a cadmium inducible metallothionein (MT) gene. In the present study the TMCd1 gene of a ciliate protozoan has been expressed in E. coli and the function of the expressed TMCd1 protein as a metal-binding protein has been evaluated. The growth of E. coli cells expressing the GST fused TMCd1 proteins in the presence of cadmium metal clearly demonstrated the role of TMCd1 as a metal-binding protein. The metal accumulation experiments showed that the bacterial cells expressing the functional TMCd1 protein accumulated 19-fold more cadmium in contrast to control cells that lacked the TMCd1 protein expression. The results clearly demonstrate a physiological role of full length TMCd1 protein of a ciliate, expressed in E. coli, in cadmium metal sequestration and detoxification.     

Helicobacter pylori CagA inhibits the expression of Runx3 via Src/MEK/ERK and p38 MAPK pathways in gastric epithelial cell

Infection with CagA-positive Helicobacter pylori is the strongest risk factor for gastric carcinoma. Upon delivery into gastric epithelial cells, CagA disturbs cellular functions by physically interacting with and deregulating intracellular signaling molecules via both tyrosine phosphorylation-dependent and -independent mechanisms. Runx3 was suggested to be a tumor suppressor and closely associated with tumorigenesis and progression of gastric cancer. The aim of our study is to verify the effect of H. pylori virulence factor CagA on Runx3 expression level and investigate the corresponding molecular mechanisms and signaling pathways influencing Runx3 expression. Human gastric epithelial immortalized GES-1 cells were transfected with CagA-expression vector or control vector with FuGENE HD transfection reagent. Runx3 expression levels were determined by QRT-PCR and immunoblotting. Then we constructed a 1,150 bp Runx3 promoter luciferase reporter plasmid, pGL(3)-1150 bp, which was co-transfected into GES-1 cell with CagA-expression vector or control vector. Luciferase reporter assay was used to determine the effects of CagA on the 1,150 bp promoter activity of Runx3. Signal inhibitors were used to detect the signal pathway(s) through which CagA affects Runx3. Our results showed that CagA can reduce the expression level of Runx3 at both mRNA and protein levels significantly. Importantly, the 1,150 bp Runx3 promoter activity was decreased in cells transfected with CagA-expression vector comparing with cells transfected with control vector. And this inhibition is dependent on the phosphorylation of CagA. Signal pathways Src/MEK/ERK and p38 MAPK are involved in this regulation. Our findings provide new insights for understanding the mechanism of H. pylori carcinogenesis.