Cellular & molecular biology research最新文献

Differentiation of leukemic cells is frequently associated with downregulation of expression of genes that are important for cell proliferation and differentiation. The p18 gene encodes a major cytosolic phosphoprotein that appears to play a role in transducing signals that control the proliferation and differentiation of normal and leukemic cells. Recent reports have shown that p18 expression and phosporylation by p34cdc2 kinase is essential for progression through the cell cycle. It was previously shown that the level of p18 gene expression is markedly reduced when several different leukemic cell lines are induced to differentiate by exposer to a variety of chemical inducers. The mechanism of this downregulation of p18 mRNA expression has not been elucidated. We have explored the mechanism(s) of p18 mRNA downregulation in U937 promonocytic leukemia cells that are induced with phorbol esters to differentiate along a monocyte/macrophage pathway. We find that the half-life of p18 mRNA that is exceptionally stable in uninduced U937 cells does not change significantly with induced differentiation. We also determined that the stability of the p18 mRNA in these cells does not depend on the synthesis of a labile protein. Direct comparison of the transcription of this gene in induced and uninduced U937 cells showed that transcription is the predominant level of regulation of the activity of this gene in leukemic cells.

beta-Galactosidase (beta-gal) expressing vectors are commonly used to standardize the transfection efficiency in transient expression experiments. In the Chinese hamster ovary (CHO) cell line, we transfected beta-gal expressing vectors in combination with different plasmid DNAs. We reported here that the presence of specific DNAs led to statistically significant variations in the beta-gal expression level. Therefore, the measure of beta-gal activity is not necessarily an accurate method to monitor transfection efficiency, and its use to normalize the expression from reporter genes could be questionable.

Recessive mutant gene c in axolotls causes a failure of the hearts of affected embryos to function. The mutant hearts (c/c) lack organized sarcomeric myofibrils. The present study was undertaken to determine the overall pattern of in vivo protein synthesis and subsequent accumulation of the newly synthesized proteins for a 24-h period in normal (+/+ or +/c) and cardiac mutant (c/c) axolotl hearts at various stages of development. Additionally, selected cytoskeletal/myofibrillar proteins were analyzed in detail for their synthesis during heart development. For such analyses, the hearts were radiolabeled with 35S-methionine for 24 h and subjected to SDS-PAGE and autoradiography. Quantitative densitometric analyses of the bands show that, even though the overall protein pattern is similar in normal and mutant heart tissues, a general reduction in the synthesis of the proteins in mutant hearts is observed even at the earlier stages of development (stages 35-36 and 37-38). Synthesis and accumulation of most of the proteins is significantly inhibited in mutant hearts at later stages (stages 41-42). Tropomyosin synthesis in mutant hearts is at a level of only 72.6% of that in normal embryonic hearts at stage 35. The synthesis and the accumulation of the tropomyosin in mutant hearts decreases further with increasing age until the protein essentially stops being synthesized by stage 41.

We describe our studies on the characterization of the cell phenotypes in the wall of the aortic-arch-derived arteries from "late" chicken embryos. Using immunocytochemistry with antibodies against smooth muscle cytoskeletal and basement membrane proteins, we show that the smooth muscle of the aortic-arch-derived arteries from 13- to 19-d-old embryos contains two cell phenotypes organized in interchanging lamellae. One cell phenotype (lamellar cells), but not the other cell phenotype (interlamellar cells), expresses the cytoskeletal proteins desmin and alpha smooth muscle actin (alpha SMactin). Both cell phenotypes express the cytoskeletal protein vimentin. Furthermore, the lamellar cells but not the interlamellar cells are surrounded by the basement membrane proteins laminin and collagen type IV. Performing quail-chick transplantation experiments and using a quail specific antibody, we show that both lamellar and interlamellar cells in the "aortic arches" of a 15-d-old chimeric embryo are derived from neural crest cells. We conclude that the aortic smooth muscle cells from "late" chicken embryos consist of two distinct cell phenotypes which are derived from a common lineage.

Pathology of the prostate gland in rats and humans is associated with aging. Our objective was to examine the effects of aging on the activities of plasminogen activators and metalloproteases in the prostatic complex of rats. Plasminogen activator activities (very low in the anterior, lateral, and dorsal lobes, in contrast to higher activities in the ventral lobe of 4-month-old adult rats) increased with aging in the dorsal and anterior prostate lobes of 31-month-old rats; these activities also increased in the dorsal and lateral lobes upon castration of 18-month-old rats. The plasminogen activator activities in the ventral lobe did not increase with aging to 18 months but did increase 3-5-fold after castration of either young or old rats. Metalloprotease activities of 70 and 76 kDa were observed in the anterior and lateral lobes of 4 month untreated adult rats, whereas the dorsal lobe showed MP of 70 and 92 kDa. Castration of young adult rats increased activities of all three molecular forms of metalloprotease in these three lobes. Increased expression of metalloprotease activities was also found with aging to 31 months in the anterior, lateral, and dorsal lobes. However, changes in metalloprotease activities associated with age were most striking in the lateral lobe and included activities of 52, 55, 81, 93, 113, and 117 kDa at 18 months of age. Castration for 30 days at this age resulted in a decline in the 52, 55, 113, and 117 kDa activities and an increase in activities of the 70, 81, and 93 kDa forms. These latter metalloprotease activities were also increased in the dorsal lobe after castration. Our results suggest that some metalloprotease activities increased in the dorsal lobe after castration. Our results suggest that some metalloprotease activities increased in the lateral lobe with age possibly result from an increased accumulation of secretory proteins (i.e., 52, 55, 113, and 117 kDa), whereas the 70, 81, and 93 kDa metalloprotease activities may be related to possible prostatitis and/or involved in changes in tissue organization. The increased expression of metalloprotease activities in the lateral and dorsal prostate lobes with aging, and castration upon aging, may be indicative of altered hormonal regulation of these proteases in these lobes.

One form of a group of enzymes known as aspartate kinases, primarily reported in prokaryotes and plants, might also exist in animal cells. Here we report the immunodetection of an aspartate kinase-like activity in human platelets using antibodies against the pure form of the enzyme purified from Escherichia coli. Moreover, the enrichment of platelet extracts with the bacterial kinase results in the phosphorylation of discrete forms mainly of membrane-bound endogenous polypeptides.

ES/130 is a novel 130-kDa protein that has been linked previously to the transformation of endocardial endothelium into cushion mesenchyme. In the present study we report the localization of protein and mRNA for ES/130 in stages 7-plus through 20 chick embryos and present functional data related to a potential mechanism for ES/130. The temporal and spatial regulation of ES/130 expression suggests that this epithelial-to-mesenchymal transformation is a result of homogenetic induction. Functional studies indicate that myocardially derived ES/130 elicits expression of this protein by target AV endothelial cells, which is linked to a signal transduction cascade. The localization of ES/130 to other sites of inductive interactions (e.g., limb bud ectoderm, gut, and notochord) implies that this protein may have a more widespread importance to embryogenesis beyond its involvement in cardiac cushion tissue formation.

The effects of recombinant human tumor necrosis factor-alpha (TNF-alpha) on the expression of fibronectin and types (IV), (III), and (I) procollagen genes in cultured bovine pulmonary artery endothelial cells were examined in this study. Findings indicate that TNF-alpha increases steady-state levels of alpha l (IV) and alpha l (III) procollagen mRNAs while it decreases levels of fibronectin and alpha 2 (1) procollagen mRNA and leaves cytoskeletal actin mRNA levels unchanged. Both dose and exposure time moderated these effects. Treatment with TNF-alpha increased the stability of alpha l (IV) procollagen mRNA. The half-life of this mRNA, previously unreported in the literature, was increased by 118%, while the stability of fibronectin mRNA decreased by 44%. The stability of mRNAs for procollagens alpha l (III) and alpha 2 (I) were unchanged. Except in the case of procollagen alpha l (III), these effects were blocked by cycloheximide. Protein production induced by TNF-alpha was evaluated by immunoprecipitating proteins from the media and cell lysates. Our data indicate that TNF-alpha has a strong pretranslational regulatory role in cultured endothelial cell's expression of extracellular matrix protein genes.

Carboxyl-containing amino acids in the transmembrane segments appear to be important for sodium- and potassium-activated adenosinetriphosphatase (Na,K-ATPase) activity. Substitution of Glu779 with Leu in a ouabain-resistant isoform inactivates the overall enzyme activity (Jewell-Motz & Lingrel, 1993). Chemical modification of this residue results in inactivation of Na,K-ATPase in a Na+ and K+ protectable manner (Arguello and Kaplan, 1991, 1994). These experiments suggest that this residue is important in cation binding. To further understand the role of Glu779 in Na,K-ATPase function, we have substituted this with four amino acids (Gln, Asp, Ala, and Leu) using site-directed mutagenesis coupled with expression and characterized the expressed enzyme. The amino acid substitutions were introduced into a modified sheep RD alpha 1 isoform that is relatively resistant to this drug. Enzyme carrying the E779Q and E779A replacements conferred ouabain resistance to the sensitive HeLa cells, while expression of enzyme carrying the E779D and E779L substitutions did not. Further analysis of isolated plasma membranes containing altered enzymes E779Q and E779A confirmed that they retain Na,K-ATPase activity. Analysis of cation stimulation of Na,K-ATPase activity revealed that the E779Q substituted enzyme exhibited a similar apparent affinity for K+ and a 2.6-fold decrease in affinity for Na+ compared with control enzyme. The E779A replacement caused a 6.6-fold and 5-fold decrease in apparent affinity for K+ and Na+, respectively. There is no difference in apparent affinity for ATP at the low affinity site for either E779Q or E779A.(ABSTRACT TRUNCATED AT 250 WORDS)