Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H⁺-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A₁ or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.     

The impact of cell fixation on coherent anti‐stokes Raman scattering signal intensity in neuronal and glial cell lines

A number of studies require sample fixation, aimed to preserve cells in a physiological state with minimal changes of morphology and intracellular molecular content. Sample fixation may significantly distort experimental data, which makes the data interpretation process more challenging. It is particularly important for study of lipid‐related diseases, where the biochemical and morphological characteristics of the cells need to be well preserved for an accurate data analysis. This study investigates the effects of formaldehyde and ethanol (EtOH) fixatives on coherent anti‐stokes Raman scattering (CARS) signal of proteins and lipids in major cellular compartments of neuronal and glial cells. We found that both fixatives induce alteration of proteins and lipids signal in studied cell lines. Furthermore, the impact of sample preservation methods on CARS signal varies between cell lines. For instance, our data reveals that EtOH fixation induces ~45% increase of CARS signal of proteins in the nucleolus of neuronal cells and ~35% decrease of CARS signal in glial cells. The results indicate that aldehyde fixation is a preferable method for preservation of neuronal and glial cells prior to CARS imaging, as it less affects both CARS signal and intracellular distribution of proteins and lipids.      

人单拷贝及重复DNA序列的原位PCR

在培养的人小肠癌转移腹水细胞系细胞中进行了Y染色体特异的重复序列及单拷贝序列的原位扩增与检测.结果显示原位PCR法的灵敏度比直接的原位杂交法明显提高.     

The role of fibroblast growth factor 8 in cartilage development and disease

Fibroblast growth factor 8 (FGF‐8), also known as androgen‐induced growth factor (AIGF), is presumed to be a potent mitogenic cytokine that plays important roles in early embryonic development, brain formation and limb development. In the bone environment, FGF‐8 produced or received by chondrocyte precursor cells binds to fibroblast growth factor receptor (FGFR), causing different levels of activation of downstream signalling pathways, such as phospholipase C gamma (PLCγ)/Ca²⁺, RAS/mitogen‐activated protein kinase‐extracellular regulated protein kinases (RAS/MAPK‐MEK‐ERK), and Wnt‐β‐catenin‐Axin2 signalling, and ultimately controlling chondrocyte proliferation, differentiation, cell survival and migration. However, the molecular mechanism of FGF‐8 in normal or pathological cartilage remains unclear, and thus, FGF‐8 represents a novel exploratory target for studies of chondrocyte development and cartilage disease progression. In this review, studies assessing the relationship between FGF‐8 and chondrocytes that have been published in the past 5 years are systematically summarized to determine the probable mechanism and physiological effect of FGF‐8 on chondrocytes. Based on the existing research results, a therapeutic regimen targeting FGF‐8 is proposed to explore the possibility of treating chondrocyte‐related diseases.     

Baculovirus transduction of rat articular chondrocytes: roles of cell cycle

BACKGROUND: We have previously demonstrated highly efficient baculovirus transduction of primary rat articular chondrocytes, thus implicating the possible applications of baculovirus in gene-based cartilage tissue engineering. However, baculovirus-mediated gene expression in the chondrocytes is transient. METHODS: In this study, we attempted to prolong the expression by supertransduction, but uncovered that after long-term culture the chondrocytes became more refractory to baculovirus transduction. Therefore, the correlation between baculovirus-mediated enhanced green fluorescent protein (EGFP) expression and cell cycle was investigated by comparing the cycling chondrocytes and chondrocytes rich in quiescent cells, in terms of EGFP expression, virus uptake, cell cycle distribution, nuclear import and methylation of viral DNA. RESULTS: We demonstrated, for the first time, that baculovirus-mediated transduction of chondrocytes is correlated with the cell cycle. The chondrocytes predominantly in G2/M phase were approximately twice as efficient in EGFP expression as the cycling cells, while the cells in S and G1 phases expressed EGFP as efficiently as the cycling cells. Notably, the chondrocyte populations rich in quiescent cells resulted in efficient virus uptake, but less effective nuclear transport of baculoviral DNA and higher degree of methylation, and hence poorer transgene expression. CONCLUSIONS: These findings unravel the practical limitations when employing baculovirus in cartilage tissue engineering. The implications and possible solutions are discussed.     

Expression and regulation of Angiopoietins and their receptor Tie-2 in sika deer antler

The cartilage vascularization and chondrocyte survival are essential for endochondral ossification which occurs in the process of antler growth. Angiopoietins (Ang) is a family of major angiogenic growth factors and involved in regulating the vascularization. However, the expression and regulation of Angs in the antler are still unknown. The aim of this study is to localize the expression of Ang-1, Ang-2 and their receptor Tie-2 in sika deer antler using in situ hybridization and focused on analyzing the regulation of testosterone, estrogen, all-trans-retinoic acid (ATRA) and 9cRA on their expression in antler chondrocytes. The results showed that Ang-1, Ang-2 and Tie-2 were highly expressed in antler chondrocytes. Administration of testosterone to antler chondrocytes led to a notable increase in the expression of Ang-1 and Tie-2, and a reduction in the expression of Ang-2. The similar result was also observed after estrogen treatment. In contrast, ATRA and 9cRA could inhibit the expression of Ang-1 in antler chondrocytes and heighten the expression of Ang-2. Simultaneously, ATRA could downregulate the expression of Tie-2 in antler chondrocytes at 12 and 24?h, while 9cRA upregulate the expression of Tie-2 at 3 and 6?h. Collectively, Ang-1, Ang-2 and Tie-2 are expressed in antler chondrocytes and their expression can be affected by testosterone, estrogen, ATRA and 9cRA.     

Overexpression and mislocalization of a tail-anchored GFP redefines the identity of peroxisomal ER

Peroxisomal ascorbate peroxidase (APX) sorts indirectly via a subdomain of the ER (peroxisomal ER) to the boundary membrane of peroxisomes in tobacco Bright Yellow 2 cells. This novel subdomain characteristically appears as fluorescent reticular/circular compartments distributed variously in the cytoplasm. Further characterizations are presented herein. A peptide possessing the membrane targeting information for peroxisomal APX was fused to GFP (GFP-APX). Transiently expressed GFP-APX sorted to peroxisomes and to reticular/circular compartments; in both cases, the GFP moiety faced the cytosol. Of particular interest, both homotypic and heterotypic aggregates of peroxisomes, mitochondria, and/or plastids were formed. The latter two organelles comprised the circular portion of the reticular/circular compartments, apparently as a consequence of oligomerization (zippering) of the GFP moieties after insertion into the outer membranes of the affected organelles. These results, coupled with the accumulation of endogenous peroxisomal APX in cytoplasmic, noncircular compartment(s) following treatment with brefeldin A, indicate that authentic peroxisomal ER is composed only of a reticular compartment(s). Equally important, the data show that overexpressed, membrane-targeted GFP fusion proteins have a propensity to form organelle aggregates that may lead to misinterpretations of sorting pathways of trafficked proteins.     

Sclerostin Immunoreactivity Increases in Cortical Bone Osteocytes and Decreases in Articular Cartilage Chondrocytes in Aging Mice

Sclerostin is a 24-kDa secreted glycoprotein that has been identified as a negative modulator of new bone formation and may play a major role in age-related decline in skeletal function. Although serum levels of sclerostin markedly increase with age, relatively little is known about whether cells in the skeleton change their expression of sclerostin with aging. Using immunohistochemistry and confocal microscopy, we explored sclerostin immunoreactivity (sclerostin-IR) in the femurs of 4-, 9-, and 24-month-old adult C3H/HeJ male mice. In the femur, the only two cell types that expressed detectable levels of sclerostin-IR were bone osteocytes and articular cartilage chondrocytes. At three different sites along the diaphysis of the femur, only a subset of osteocytes expressed sclerostin-IR and the percentage of osteocytes that expressed sclerostin-IR increased from approximately 36% to 48% in 4- vs. 24-month-old mice. In marked contrast, in the same femurs, there were ~40% fewer hypertrophic chondrocytes of articular cartilage that expressed sclerostin-IR when comparing 24- vs. 4-month-old mice. Understanding the mechanism(s) that drive these divergent changes in sclerostin-IR may provide insight into understanding and treating the age-related decline of the skeleton.     

细胞外钙调素对烟草悬浮培养细胞蛋白质磷酸化作用的影响

用液体闪烁计数法研究了细胞外钙调素对烟草悬浮培养细胞质蛋白质磷酸化的作用。结果表明烟草细胞细胞质蛋白质磷酸化活性在细胞培养过程中逐渐增加,达到最高峰后又开始下降。在细胞质蛋白质磷酸化强度高峰时,加入抗CaM血清后,细胞质蛋白质磷酸化活性受到了部分抑制。加抗CaM血清后再补加CaM能够部分解除抗CaM血清对细胞质部分与细胞核部分蛋白质磷酸化的抑制作用。外加纯化钙调素可以引起烟草悬浮培养细胞细胞质蛋白质磷酸化的活性增强,并且这种增强作用具有时间（高峰为70min）与剂量（最适为CaM10-7mmol/L）依赖性。CaM引起的细胞质蛋白质磷酸化变化与红光所引起的细胞质蛋白质磷酸化变化在时间进程上是不相同的。     

杀螨剂对螨类不同螨态四种毒力室内测定标准的建议

针对当前杀螨剂毒力测定标准的适用局限性提出了适合所有杀螨剂类型毒力测定的新标准建议,并以水培花生叶培养二斑叶螨Tetranychus urticae Koch进行Potter喷雾法毒力测定为例,描述了测定其不同螨态的4种室内毒力的操作要点,阐明对螨类这种世代历期短、繁殖能力强、经济危害地位重要的特殊生物类群,采用新的杀螨剂毒力测定标准,不仅对正确评价杀螨剂的作用特性、指导科学合理用药非常必要,而且操作上是可行的。