Controlled and modulated release of basic fibroblast growth factor.

Basic fibroblast growth factor has multivariate effects in stimulating cell growth and the processes that surround tissue repair. Pathophysiologic studies have been hampered by the stability of the compound. Though very potent, basic fibroblast growth factor is rapidly degraded when injected or ingested. Controlled release of basic fibroblast growth factor would allow for examination of the chronic effects of this compound. Conventional matrix polymer-based release devices were fabricated and basic fibroblast growth factor released in a sustained fashion, but 99% of basic fibroblast growth factor mitogenic activity was lost. The source of these losses was identified and preventative measures examined. Preservation and stabilization of basic fibroblast growth factor was accomplished by binding the factor to heparin-Sepharose beads. This permitted prolonged storage, repeated handling, and the encapsulation of basic fibroblast growth factor within a microspherical controlled-release device using a naturally occurring polymer material, alginate. Encapsulation was accomplished with 77% efficiency and 87.5 +/- 12% of the basic fibroblast growth factor was released in a biologically active form. Release activation and regulation was achieved when cleavage of the basic fibroblast growth factor-heparin bonds was enhanced (e.g. by enzymatic bond cleavage with heparinase). Kinetic profiles were identified for a variety of experimental conditions and the effects of the controlled release of basic fibroblast growth factor on BALBc/3T3 fibroblasts examined.     

Characterization of the heat shock response in M-14 human melanoma cells continuously exposed to supranormal temperatures

The heat shock response elicited in a human melanoma cell line (M-14) by continuous exposures to supranormal temperatures has been characterized. The electrophoretic patterns of polypeptides labeled in vivo at different time-intervals during a continuous heating at 42 degrees C show that the hyperthermic stress induces the synthesis of three HSPs, with molecular weights, respectively, of 86 kDa, 70-72 kDa and 26 kDa. The relative rate of synthesis of the 70-72 kDa HSP--the preeminent HSP--increases during the first hours of treatment, reaching the maximum value after about 9 hr. Later on, the rate of synthesis of this protein progressively decreases, finally attaining a steady state level only slightly exceeding the constitutive one. On the contrary, the smaller molecular weight HSP is synthesized at an apparently constant rate in the course of 21 hr of heating treatment. A continuous exposure at 40 degrees C induces the synthesis of the same three HSPs observed in cells heated at 42 degrees C, but the rate of synthesis of all these HSPs is not so greatly enhanced over the control values as in the 42 degrees C-heated cells. Moreover, the repression of the 70-72 kDa HSP synthesis is faster, taking place within 4-6 hr of treatment. Coomassie blue stained gels show that a polypeptide, coincident with the 70-72 kDa HSP, accumulates in the course of a continuous heating either at 42 degrees C and at 40 degrees C. The final intracellular level attained by this protein species results higher in 42 degrees C-treated cells than in 40 degrees C-treated ones. Hybridization experiments between total RNAs obtained from cells heated at 42 degrees C and a radioactive DNA probe (containing sequences complementary to the mRNA coding for the human 70 kDa HSP) demonstrate that the kinetics of accumulation and decay of the 70 kDa HSP-mRNAs correlate with the kinetics of induction and repression of the corresponding protein.     

Root growth is modulated by differential hormonal sensitivity in neighboring cells

Coherent plant growth requires spatial integration of hormonal pathways and cell wall remodeling activities. However, the mechanisms governing sensitivity to hormones and how cell wall structure integrates with hormonal effects are poorly understood. We found that coordination between two types of epidermal root cells, hair and nonhair cells, establishes root sensitivity to the plant hormones brassinosteroids (BRs). While expression of the BR receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) in hair cells promotes cell elongation in all tissues, its high relative expression in nonhair cells is inhibitory. Elevated ethylene and deposition of crystalline cellulose underlie the inhibitory effect of BRI1. We propose that the relative spatial distribution of BRI1, and not its absolute level, fine-tunes growth.     

Developmental effects of chemicals and the heat shock response in Drosophila cells

Exposure of prokaryotic and eukaryotic cells to heat shock (hyperthermia) or to a number of diverse environmental stresses such as teratogens, anoxia, and inhibitors of oxidative phosphorylation results in the enhanced synthesis of a number of proteins which have been previously referred to as heat shock proteins (hsps). More recently, in view of the diverse types of agents that can induce these proteins, they have also been referred to as stress proteins. This phenomenon is one of the most basic regulatory mechanisms in living organisms. Exposure of Drosophila embryos, larvae, or pupae to these types of stresses also results in a variety of developmental abnormalities in the ensuing adult. Although the function(s) of these heat shock proteins has yet to be determined, they are widely thought to play an important role in cell survival and protection following some types of environmental stress. In our laboratory, we have developed an in vitro assay for detecting agents that act as teratogens, utilizing Drosophila embryonic cultures. Drosophila embryonic cells differentiate in vitro to a number of functional cell types including myotubes and ganglia. A number of drugs that have been shown to act as teratogens in mammals have also been found to inhibit muscle and/or neuron differentiation in Drosophila embryonic cultures. We have examined, by two-dimensional gel electrophoresis, the effects of such teratogens on protein synthesis in Drosophila embryonic cells. Inhibition of muscle and/or neuron differentiation correlates well with the induction of two proteins of about 20 kilodaltons. These are identical to two of the heat shock proteins (hsp 23, 22) as shown by electrophoretic mobilities and peptide mapping by partial proteolysis. Heat shock and other treatments such as exposure to some of the metal ions and ether induces the entire set of seven major heat shock proteins in the Drosophila embryonic cells. Dose-response studies of several teratogens show a correlation between the degree of inhibition of differentiation and the level of induction of hsps. Since heat shock proteins have been suggested as possibly serving a protecting role, our present studies are aimed at identifying the role of hsps in teratogenesis and investigating the differential regulation of heat shock genes in response to different external stimuli.     

转录因子环腺苷酸反应元件结合蛋白介导的细胞凋亡及其调控机制

细胞凋亡是一个精细而复杂的过程,需要一系列的蛋白参与.其中环腺苷酸反应元件结合蛋白(CREB)作为一个核转录因子,通过自身磷酸化实现调节转录功能,改变细胞内的信号转导途径,从而调控细胞凋亡过程.我们结合研究结果对CREB介导的细胞凋亡及其调控机制作一综述.     

The growth factor midkine is modulated by both glucocorticoid and retinoid in fetal lung development

The glucocorticoids (GC) and retinoids (RA) modulate branching morphogenesis and cytodifferentiation in the developing lung. We investigated downstream target genes that link glucocorticoid stimulation to the achievement of a mature lung in glucocorticoid receptor (GR) knockout mice. All GR(null) mice and approximately 80% of mice homozygous for a hypomorphic allele (GR(hypo)) die shortly after birth of respiratory failure. cDNA microarray analysis showed organ-specific upregulation of the retinoic acid responsive gene midkine (MK) and its chondroitin-sulfate binding partner PG-M/versican at fetal day 18 and at neonatal day 1 in lungs of GR(hypo) mice, and at neonatal day 1 in lungs of GR(null) mice. By contrast, lung MK and PG-M/versican were downregulated in these mice at fetal day 16.5. In situ hybridization studies showed a dramatic decrease in MK and PG-M/versican RNA between days 16.5 and 17.5 in GR(WT) but not in GR(null) mice. Continued diffuse and robust expression of MK protein was observed in GR(null) mice at neonatal day 1. These findings suggest that MK may contribute to the dysmature lung phenotype in GR-deficient mice. Exposure of cultured day 21 fetal rat lung cells to GC downregulated MK, whereas RA enhanced MK expression. Our findings demonstrate the coincident modulation of expression of MK at the same developmental time point by both GC and RA, providing a potential mechanism for the integration of GC and RA effects on fetal lung development.     

Induction of heat shock protein 72 synthesis by endogenous tumor necrosis factor via enhancement of the heat shock element-binding activity of heat shock factor 1

Endogenous tumor necrosis factor (enTNF) acts as a resistance factor against cytotoxicity caused by heat by inducing manganous superoxide dismutase (MnSOD), thereby scavenging reactive oxygen free radicals. On the other hand, it is also well known that heat shock proteins (HSP) which are induced by heat stress behave as cytoprotective factor against this stress. However, the relationship of these two resistance factors is not elucidated yet. In the present study, we therefore proposed the possibility that enTNF enhances HSP72 expression. Heat-sensitive L-M (mouse tumorigenic fibroblast) cells, which normally do not express enTNF, were transfected with a nonsecretory-type human TNF-α expression vector to produce enTNF. Stable transfectants showed resistance to heat treatment and an increase of HSP72 expression. Conversely, when HeLa (human uterine cervical cancer) cells, which normally produce an appreciable amount of enTNF, were transfected with an antisense TNF-α mRNA expression vector to inhibit enTNF synthesis, their heat sensitivity was enhanced and HSP72 expression was reduced by half. Although enTNF caused no difference in the level of heat shock factor (HSF) 1 in these cells, enTNF expression correlated well with the binding activity of HSF-1 to a ³²P-labeled synthetic oligonucleotide containing the human heat shock element (HSE). These results indicate that enTNF participates not only in intrinsic resistance against heat via induction of MnSOD but also via enhancement of the HSE-binding activity of HSF 1 followed by augmentation of HSP72 expression.     

Natural cytotoxic activity is not necessarily mediated by the release of tumour necrosis factor.

Natural cell-mediated cytotoxicity is mediated by a family of effector cells that express cytolytic activities distinct from those generally attributed to B cells, T cells and macrophages; it includes both natural killer (NK) and natural cytotoxic (NC) activities. There is now convincing evidence to show that NC activity, but not NK activity, is mediated by tumour necrosis factor (TNF). Further, it has been argued that it is the release of TNF, as a freely diffusible factor, that causes NC-mediated target lysis. Here, we present evidence that the admixture of NC-sensitive target cells and spleen cells, under conditions that result in NC-mediated target cell lysis, does not necessarily result in the release of freely diffusible TNF into the culture medium. Also, it is demonstrated that the procedures used do not result in inactivation or loss of significant amounts of TNF during the assay period, which might account for our failure to detect free TNF. These results suggest that NC activity is mediated by either a membrane-associated TNF activity, similar to that described for some of the lytic activity of activated macrophages, or by the release of TNF that is capable of acting only over a very short distance.     

Cellular and molecular effects of bleomycin are modulated by heat shock in Saccharomyces cerevisiae

To study some mechanisms underlying the stress responses in eukaryotic cells, we investigated the effect of heat shock (HS) on the induction of DNA double strand breaks as well as on potentially lethal and mutagenic events induced by the radiomimetic antibiotic bleomycin (BLM) in Saccharomyces cerevisiae. Haploid wild-type yeast cells in the logarithmic phase of growth were exposed to different concentrations of BLM (0–30 μg/ml, 1.5 h) without and with a previous HS (38°C, 1 h). Immediately after treatments, survival as well as mutation frequency were determined, and quantitative analysis of chromosomal DNA by laser densitometry were performed both immediately after treatments and after incubation of cells during different time intervals in liquid nutrient medium free of BLM. Our results indicate that HS induces resistance to potentially lethal and mutagenic effects of BLM. Quantitative analysis of chromosomal DNA performed immediately after treatments showed the same DNA fragmentation, either upon BLM as single agent or preceded by HS. However, HS pretreated cells incubated during 4 h in liquid nutrient medium free of BLM repaired DNA double strand breaks more efficiently as compared to non-pretreated cells. On this basis, we propose that the observed HS-induced resistance to BLM depends on a regulatory network acting after DNA-induced damage, which includes genes involved in DNA repair, HS response and DNA metabolism.