Derenzini M, Sirri V, Pession A, Trerè D, Roussel P, Ochs R L, Hernandez-Verdun D
Centro di Patologia Cellulare, Dipartimento di Patologia Sperimentale, Bologna, Italy.
Exp Cell Res. 1995 Jul;219(1):276-82. doi: 10.1006/excr.1995.1228.
The relationship between the amount of the two major AgNOR proteins, nucleolin and protein B23, and the rate of ribosomal RNA (rRNA) synthesis was studied in cortisol-treated and regenerating rat hepatocytes after partial hepatectomy. In both experimental models the synthesis of rRNA was greatly stimulated, but only in regenerating hepatocytes was the increased synthesis associated with cells entering the mitotic cycle. Nucleolin and protein B23 were identified on SDS-polyacrylamide gels of nuclear proteins transferred to nitrocellulose and detected (1) by immunoreaction with specific monoclonal antibodies revealed by second antibodies coupled to peroxidase followed by chemiluminescence or (2) by the silver-staining procedure for AgNOR proteins. Nucleolin and protein B23 were then quantified by computerized densitometric analysis of the immunolabeling signals or the silver-stained bands at 105 kDa (nucleolin) and 39 kDa (protein B23). The synthesis of rRNA was measured by evaluating the amount of radioactivity incorporated into pre-rRNA after [3H]orotic acid injection. Densitometric analysis of silver-stained bands and immunolabeling signals showed no change in nucleolin and protein B23 amounts in cortisol-stimulated hepatocytes, whereas a moderate increase was found in regenerating hepatocytes at 12 h after partial hepatectomy. In both cortisol-stimulated and regenerating hepatocytes the synthesis of rRNA was highly increased (2.6-fold and 4.3-fold above the control level, respectively). To ascertain the relationship between quantitative changes in nucleolin and protein B23 and stimulation of rRNA transcriptional activity in regenerating hepatocytes, the quantitative distribution of these proteins was also investigated in the early times of regeneration using silver-stained nitrocellulose-transblotted nuclear proteins. The quantity of protein B23 was unchanged until 12 h after partial hepatectomy, whereas nucleolin appeared to be slightly increased at 9 h (1.15-fold above the control value) after partial hepatectomy. On the other hand, at just 6 h after partial hepatectomy, a significant increase of rRNA synthesis occurred in regenerating rat hepatocytes (1.8-fold above the control value). These data demonstrated that stimulation of rRNA transcriptional activity occurring in rat hepatocytes after cortisol treatment and in the early times after partial hepatectomy was not associated with quantitative changes in the amounts of nucleolin and protein B23.
在部分肝切除术后经皮质醇处理和再生的大鼠肝细胞中，研究了两种主要的AgNOR蛋白（核仁素和蛋白B23）的含量与核糖体RNA（rRNA）合成速率之间的关系。在这两种实验模型中，rRNA的合成均受到极大刺激，但只有在再生肝细胞中，合成增加才与进入有丝分裂周期的细胞相关。核仁素和蛋白B23通过转移至硝酸纤维素膜上的核蛋白的SDS-聚丙烯酰胺凝胶进行鉴定，并通过以下方法检测：（1）与特异性单克隆抗体进行免疫反应，随后用与过氧化物酶偶联的二抗进行化学发光检测；（2）采用AgNOR蛋白的银染程序。然后通过对免疫标记信号或105 kDa（核仁素）和39 kDa（蛋白B23）处的银染条带进行计算机密度分析，对核仁素和蛋白B23进行定量。通过评估注射[3H]乳清酸后掺入前体rRNA中的放射性量来测量rRNA的合成。银染条带和免疫标记信号的密度分析显示，皮质醇刺激的肝细胞中核仁素和蛋白B23的量没有变化，而在部分肝切除术后12小时的再生肝细胞中发现有适度增加。在皮质醇刺激的肝细胞和再生肝细胞中，rRNA的合成均显著增加（分别比对照水平高2.6倍和4.3倍）。为了确定再生肝细胞中核仁素和蛋白B23的定量变化与rRNA转录活性刺激之间的关系，还使用银染的硝酸纤维素转印核蛋白在再生早期研究了这些蛋白质的定量分布。蛋白B23的量在部分肝切除术后12小时之前没有变化，而核仁素在部分肝切除术后9小时似乎略有增加（比对照值高1.15倍）。另一方面，在部分肝切除术后仅6小时，再生大鼠肝细胞中的rRNA合成就发生了显著增加（比对照值高1.8倍）。这些数据表明，皮质醇处理后以及部分肝切除术后早期大鼠肝细胞中发生的rRNA转录活性刺激与核仁素和蛋白B23含量的定量变化无关。
