Cellular & molecular biology research最新文献

Collagenase, a prototypic matrix metalloproteinase, plays a major role in the degradation of the extracellular matrix. The essential amino acid L-tryptophan was recently shown to stimulate the expression of collagenase gene in human dermal fibroblast cultures. In this study, we focused our attention on the mechanisms responsible for activation of collagenase transcription by L-tryptophan. Incubation of fibroblasts with L-tryptophan resulted in a dose- and time-dependent elevation of collagenase and tissue inhibitor of metalloproteinase mRNA levels. The maximum enhancement in collagenae mRNA was approximately 50-fold. This effect was not abolished by cycloheximide, suggesting independence from ongoing protein synthesis. Transient cell transfections with a promoter/reporter gene construct containing 3.8 kb of 5' flanking DNA of the human collagenase gene linked to the chloramphenicol acetyl transferase (CAT) gene or a construct containing three phorbol ester-responsive AP-1 binding sequences (12-O-tetradecanoyl-phorbol-13-acetate-responsive element) in front of the thymidine kinase promoter linked to the CAT gene indicated enhancement of promoter activity by L-tryptophan. Furthermore, electrophoretic DNA mobility shift assays demonstrated enhanced DNA-protein complex formation specific for an AP-1 binding site probe with nuclear extracts prepared from cells incubated with L-tryptophan. These results collectively suggest that activation of collagenase gene expression in dermal fibroblasts by L-tryptophan is mediated through AP-1 binding elements in the collagenase gene promoter that are sufficient for gene response.

The transferrin receptor promoter is responsive to growth factors and mitogens. This induction is a delayed response and does not occur until several hours after stimulation of quiescent cells by mitogens. The results described here show that the transferrin receptor promoter is also activated by treatment with ultraviolet (UV) light. Activation of the promoter by UV light is dose dependent and requires the same cis-acting elements that are activated in response to serum and other mitogens. As with serum stimulation, activation of the promoter by UV light is a delayed event and is not initiated until 6 h after treatment. This coincides with the induction of nuclear factors that bind with specificity to the required cis-acting elements. A major GC-box binding factor induced by UV light is also induced by serum and has been shown to be supershifted by antibodies to the Sp1 transcription factor.

The present study was carried out to explore the mechanisms underlying hyaluronan-induced sperm motility. We demonstrate for the first time the enhanced cellular protein phosphorylation in hyaluronic acid stimulated cauda spermatozoa. Labeling of spermatozoa with [Pi] orthophosphoric acid yielded a wide range of labeled phosphoproteins in presence of hyaluronan. Under these experimental conditions, we further show tyrosine specific phosphorylation of proteins. In addition, this is also the first report showing enhanced phosphorylation of 34 kDa hyaluronan binding protein in response to hyaluronan. The role of 34 kDa hyaluronan binding protein in hyaluronan-induced spermatozoa is supported by elevated production of inositol triphosphate accompanied by increased phosphorylation in Triton X-100 insoluble cytoskeletal proteins. The evidence strongly suggests the biological importance of hyaluronan binding protein phosphorylation in the transduction of signals resulting from the interaction of hyaluronate with the sperm surface.

Early events of cardiovascular development have received renewed interest in recent years. The cardiovascular system is the first major organ system to become functional during early embryogenesis. Cells fated to form the cardiovascular system can be identified as early as during stages of gastrulation of avian and mammalian embryos. In the present brief summary, we describe the primitive-streak origin of the avian cardiovascular system and examine the state of commitment of prospective cardiogenic and vasculogenic areas of the primitive streak. In addition, we describe initial experiments aimed at elucidating the primitive-streak origin of the heart in mouse embryos. Finally, we consider the possible roles of Hensen's node and the "cardiac" endoderm in determination of cell fate and patterning of the avian developing heart tube. Although recent studies have shed considerable light on the origin, migration, and determination of the cardiovascular system, much still remains to be learned about mechanisms underlying cardiovascular patterning in the early embryo.

Understanding normal and abnormal cardiovascular development is of interest to basic scientists as well as to clinicians taking care of infants with heart defects. This article presents a visual overview of cardiac development. It provides a framework on which to understand how abnormal cardiac development leads to groups of cardiovascular defects requiring clinical care. Human heart development is presented schematically and is correlated with similar points in chick cardiac development. Studying both normal and abnormal cardiac development in neural crest-ablated embryos has highlighted two major themes of cardiac development: there is a mechanism of differential growth in the developing cardiovascular system that is not seen to a major extent after birth and cardiac defects can be pictured as arrested stages of normal development. At a particular stage of development, it is normal to have a certain relationship between developing structures. However, if the development is arrested and this relationship of structures is allowed to persist, it then becomes abnormal. Visualizing heart defects as arrested points in normal development is better used as a tool to categorize defects than as a causative mechanism. The exact mechanisms of how abnormal development results in cardiac defects is not well understood. Study of the neural crest model of cardiac defects suggests possible mechanisms.

As a marker of macrophage activation, IL-1 alpha was measured after stimulation of murine resident peritoneal macrophages (RPM) with endotoxin-associated protein (EP). Significant IL-1 alpha was produced by EP-stimulated RPM from both C3H/OuJ and C3H/HeJ mouse strains. This EP-mediated IL-1 alpha production was blocked by tyrosine kinase inhibitors including genistein and tyrphostin, suggesting the involvement of a protein tyrosine kinase in the activation of RPM by EP. Immunoblot analysis using antiphosphotyrosine antibody showed that EP induces the tyrosine phosphorylation of a 71 kD protein (p71). The p71 and the spleen tyrosine kinase p72syk found in other cell types share common features including: similar molecular weight, PKC independent tyrosine phosphorylation, and inhibition of phosphorylation by piceatannol. Furthermore, immunoblot analysis using anti-p72syk antibody detected the p72syk kinase in EP-activated RPM. These results suggest that the activation of RPM involves an early tyrosine phosphorylation of p72syk or a p72syk-like protein.

In this article, we survey the mechanisms involved in early embryonic angiogenesis. The first embryonic blood vessels are formed exclusively by endothelial cells. Therefore, the emergence and behavior of this cell type is the center of this article. We discuss both intra- and extraembryonic angiogenesis and the various modes of capillary formation. The high plasticity and migratory potential of endothelial cells and their precursors, the angioblists, are outlined. The promoting and inhibiting influences of the extracellular matrix on the behavior of angioblasts are a matter of concern, as is also the question of embryonic angiogenic factors. Cell-cell interactions that may lead to organ-specific differentiation of endothelial cells are mainly discussed in the context of blood-brain barrier formation and development of fenestrated capillaries. The last section deals with the development of the vascular wall.

AP1 is a heterodimeric complex containing products of the Jun and Fos oncogene families. The c-fos and c-jun protooncogenes act as transcriptional activator for numerous cellular genes, and the overexpression of these genes may cause malignant transformation. In this study, to show evidence of a possible inhibition of AP1 transcriptional activity in molecular mechanisms of foodborne molecules, known to be negative modulators of carcinogenesis, we established two rat liver epithelial (REL) cell lines overexpressing either c-fos (43C line) or c-jun (RELcJ1 line) oncoproteins. Contrary to the 43C line, which was spontaneously transformed, the c-jun-transfected REL cells were only transformed in vitro after 12-O-tetra-decanoylphorbol 13-acetate (TPA) exposure. All trans-retinoic acid (RA) abolished the transformation of the 43C line and TPA-treated RELcJ1 cells, suggesting that RA could decrease AP1 activity in these cells despite c-fos or c-jun overexpression. Furthermore, we show for the first time that a flavonoid, quercetin, which is a natural component of vegetables, inhibited only the transformation of the 43C line. The spontaneous transformation of the c-fos-transfected REL cells was associated with the appearance of c-fos/AP1 complexes binding TRE, suggesting that c-fos/AP1 complexes are involved in the antitransforming mechanism of quercetin.

Human or mouse epidermal keratinocytes NHEK or Pam212 was less susceptible to ultraviolet (UV)-B irradiation than mouse neuroblastoma NAs1 cells in culture, undergoing apoptosis-like cell death as shown by cell fragmentation and cell membrane integrity disruption. UV susceptibility was appreciably reduced by the reactive oxygen species (ROS)-scavenger L-ascorbic acid-2-phosphate (Asc2P) endowed with long-lasting functions but not by L-ascorbic acid (Asc) for each cell type. DehydroAsc reduced UV susceptibility of Pam212 or NAs1 established cell lines but not of normal diploid NHEK cells destined to be thereafter submitted to cellular senescence. The susceptibility reduction may not be ascribed to extracellular Asc2P or DehAsc, which was removed by aspirating and/or rinsing upon irradiation after the intracellular channelyzer analysis and dead cell-specific DNA-intercalator ethidium homodimer/fluorometry, respectively. Thus, the three cell types differed in UV susceptibility partly because of their different ROS-scavenging abilities, which may be potently promoted by Asc2P or dehydroAsc but not Asc.

We previously identified a transcriptional negative element (NE) present in the rabbit angiotensin-converting enzyme (ACE) gene. Here, we report that the NE can also repress transcription driven by the strong constitutive promoters of the human beta-actin gene and SV40 in both ACE-expressing and nonexpressing cell lines. The extent of repression was influenced by the relative positions of the NE and the SV40 promoter and enhancer. The NE could also repress transcription driven by interleukin-1 and interferon-alpha-inducible promoters. Finally, transcription from a TATA-less promoter was equally repressed by the NE. Taken together, these results suggest that the NE of the rabbit ACE gene can function as a universal transcriptional silencer element.