gaddl53 encodes a CCAAT/enhancer-binding protein (C/EBP)-related protein that lacks a functional DNA-binding domain. Since the gadd153 protein is capable of heterodimerizing with other C/EBPs, gadd153 may function as a negative regulator of these transcription factors. Here we examined the role of glucose in regulating gadd153 expression. We found that glucose deprivation markedly induces gaddl53 mRNA levels in both HeLa and 3T3-L1 cells and that addition of D-(+)-glucose resulted in a rapid decrease ofgaddl53 mRNA. Similar induction and reversal of gadd153 expression were observed at the protein level. Because C/EBPat appears to play an important role in regulating genes involved in adipogenesis and energy metabolism, we examined gaddl53 expression during the differentiation of 3T3-L1 preadipocytes and as a function of glucose utilization in differentiated adipocytes. Using a standard differentiation protocol that consisted of hormonal stimulation for 2 days followed by medium changes every 2 days thereafter, we observed that both C/EBPoa and gaddl53 mRNAs were elevated. However, C/EBPa induction occurred on day 3, while gaddl53 expression was not seen until day 4, when the cells were fully differentiated. Frequent addition of fresh medium to the cells during the differentiation process, as well as supplementation of medium with glucose, reduced gaddl53 expression without preventing C/EBPa expression or interfering with cellular differentiation. Thus, gaddl53 expression is not essential for the process of adipocyte differentiation but is significantly influenced by the availability of glucose to the cell. gaddl53 is a mammalian gene whose expression is induced by a wide variety of stresses, including growth arrest and DNA damage (16,20,21). gaddlS3 encodes a 19-kDa protein related to the CCAAT/enhancer-binding protein (C/ EBP) family of transcription factors (28). The C/EBPs are bZip proteins characterized by a conserved leucine zipper domain, through which various C/EBP monomers dimerize, adjacent to a basic domain involved in DNA sequence recognition and binding. Although the gaddl53 protein forms stable heterodimers with other C/EBP proteins, it lacks a functional DNA-binding domain (28). Thus, the gaddlS3 protein-C/EBP heterodimer is unable to bind known C/EBP-binding sites, suggesting that gaddlS3 may function as a negative regulator of C/EBP transcription factors.It has been hypothesized that C/EBPa is a central regulator of genes encoding proteins involved in energy metabolism (22). This proposal is based on the following observations: levels of C/EBPa expression are high in tissues with high rates of metabolism, such as liver, adipose, and placenta; the developmental pattern of C/EBPot expression in the liver coincides with the onset of gluconeogenic activity; C/EBPa can trans activate the promoters of genes involved in energy metabolism; and livers of cl4CoS/clCS mice show reduced levels of C/EBPa expression. These mutant mice, containing radiation-induced deletions involving the albino locus on chromo...

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The endoplasmic reticulum is the site of synthesis and folding of secretory proteins and is sensitive to changes in the internal and external environment of the cell. Both physiological and pathological conditions may perturb the function of the endoplasmic reticulum, resulting in endoplasmic reticulum stress. The chondrocyte is the only resident cell found in cartilage and is responsible for synthesis and turnover of the abundant extracellular matrix and may be sensitive to endoplasmic reticulum stress. Here we report that glucose withdrawal, tunicamycin, and thapsigargin induce up-regulation of GADD153 and caspase-12, two markers of endoplasmic reticulum stress, in both primary chondrocytes and a chondrocyte cell line. Other agents such as interleukin-1␤ or tumor necrosis factor ␣ induced a minimal or no induction of GADD153, respectively. The endoplasmic reticulum stress resulted in decreased chondrocyte growth based on cell counts, up-regulation of p21, and decreased PCNA expression. In addition, perturbation of endoplasmic reticulum function resulted in decreased accumulation of an Alcian Blue positive matrix by chondrocytes and decreased expression of type II collagen at the protein level. Further, quantitative realtime PCR was used to demonstrate a down-regulation of steady state mRNA levels coding for aggrecan, collagen II, and link protein in chondrocytes exposed to endoplasmic reticulum stress-inducing conditions. Ultimately, endoplasmic reticulum stress resulted in chondrocyte apoptosis, as evidenced by DNA fragmentation and annexin V staining. These findings have potentially important implications regarding consequences of endoplasmic reticulum stress in cartilage biology.

Summary:We evaluated the effects of early posttrau matic hypoxia on neurologic function, magnetic reso nance images (MRI), brain tissue specific gravities, and cerebral blood flow (CBF) in head-injured rats. By itself, an hypoxic insult (PaOZ 40 mm Hg for 30 min) had little effect on any measure of cerebral function. After tem poral fluid-percussion impact injury, however, hypoxia significantly increased morbidity. Of rats subjected to im pact (4.9 ± 0.3 atm) plus hypoxia, 71% had motor weak ness contralateral to the impact side 24 h after injury, while only 29% of rats subjected to impact alone had de monstrable weakness (p < 0.05). Lesions observed on MR images 24 h after injury were restricted to the impact site in rats with impact injury alone, but extensive areas with longer Tl relaxation times were observed throughout the ipsilateral cortex in rats with impact in-

Understanding how a subset of expressed genes dictates cellular phenotype is a considerable challenge owing to the large numbers of molecules involved, their combinatorics and the plethora of cellular behaviours that they determine1,2. Here we reduced this complexity by focusing on cellular organization—a key readout and driver of cell behaviour3,4—at the level of major cellular structures that represent distinct organelles and functional machines, and generated the WTC-11 hiPSC Single-Cell Image Dataset v1, which contains more than 200,000 live cells in 3D, spanning 25 key cellular structures. The scale and quality of this dataset permitted the creation of a generalizable analysis framework to convert raw image data of cells and their structures into dimensionally reduced, quantitative measurements that can be interpreted by humans, and to facilitate data exploration. This framework embraces the vast cell-to-cell variability that is observed within a normal population, facilitates the integration of cell-by-cell structural data and allows quantitative analyses of distinct, separable aspects of organization within and across different cell populations. We found that the integrated intracellular organization of interphase cells was robust to the wide range of variation in cell shape in the population; that the average locations of some structures became polarized in cells at the edges of colonies while maintaining the ‘wiring’ of their interactions with other structures; and that, by contrast, changes in the location of structures during early mitotic reorganization were accompanied by changes in their wiring.

Prostaglandins (PG) of the A series are potent inhibitors of cell proliferation. Recently, it was shown that PGA2-induced growth arrest was associated with the increased synthesis of stress proteins encoded by the HSP70 gene family. In this study, we have examined the molecular basis for this increased HSP70 expression. Northern (RNA) blot analysis and nuclear run-on assays demonstrated that induction of high levels of HSP70 mRNA results from an increase in the rate of transcription. High-level induction is specific to the HSP70 family of heat shock proteins and is rapid, reversible, dose dependent, and specific for PGs capable of growtharresting HeLa cells. In addition, the response was found to be highly dependent on the growth state of the cells, as induction occurs in growing but not in confluent nongrowing cell populations. Induction is dependent on the activation of heat shock factor. Cycloheximide pretreatment, which inhibits the antiproliferative effects of PGA2, prevents activation of the heat shock factor and induction of HSP70 mRNA by PGA2. These results support a role for HSP70 in mediating the antiproliferative effects of PGA2.The cyclopentenone prostaglandins (PG) PGA1 and PGA2 have been shown to be potent inhibitors of proliferation in a variety of cultured cells (2-4, 15, 37). These metabolites of PGE1 and PGE2, respectively, are characterized by the presence of an a,I-unsaturated carbonyl group in the cyclopentenone ring which is believed to be the active moiety in suppressing growth (3,19,24,33). They have been found to inhibit the growth of B16 melanoma and Friend erythroleukemia virus-infected cells in vivo as well as in vitro, suggesting their utility as effective antineoplastic agents (15,26,39). They are actively taken up by cells via a specific carrier on the cell membrane and are transported to the nucleus, where they associate with nuclear proteins (30-32). They affect cell cycle progression in two ways. They act on cells at any stage of the cell cycle to cause a general decrease in the rate of progression, and they act specifically on cells in the G1 phase to arrest progression (2, 35). Growth arrest has been associated with a decline in both c-myc and N-myc expression (22,27). However, the exact mechanism by which PGs control proliferation is largely unknown.The heat shock protein 70 (HSP70) gene family is part of a larger set of proteins whose syntheses are increased in response to a variety of chemical and biological stresses (25,40). The expression of these genes follows the stress-induced activation of one or more heat shock transcription factors (HSF) which bind to a specific DNA sequence, the heat shock element (HSE), in the promoter regions of the HSP genes to increase their rates of transcription (36,41,42). There is also evidence to suggest that there are both positive and negative roles for HSPs in normal cell growth and differentiation. For example, certain HSPs have been shown to be induced in response to proliferative stimuli (12,44 (20,21,23).Recently, two groups have re...

SummaryDespite the intimate link between cell organization and function, the principles underlying intracellular organization and the relation between organization, gene expression and phenotype are not well understood. We address this by creating a benchmark for mean cell organization and the natural range of cell-to-cell variation. This benchmark can be used for comparison to other normal or abnormal cell states. To do this, we developed a reproducible microscope imaging pipeline to generate a high-quality dataset of 3D, high-resolution images of over 200,000 live cells from 25 isogenic human induced pluripotent stem cell (hiPSC) lines from the Allen Cell Collection. Each line contains one fluorescently tagged protein, created via endogenous CRISPR/Cas9 gene editing, representing a key cellular structure or organelle. We used these images to develop a new multi-part and generalizable analysis approach of the locations, amounts, and variation of these 25 cellular structures. Taking an integrated approach, we found that both the extent to which a structure’s individual location varied (“stereotypy”) and the extent to which the structure localized relative to all the other cellular structures (“concordance”) were robust to a wide range of cell shape variation, from flatter to taller, smaller to larger, or less to more polarized cells. We also found that these cellular structures varied greatly in how their volumes scaled with cell and nuclear size. These analyses create a data-driven set of quantitative rules for the locations, amounts, and variation of 25 cellular structures within the hiPSC as a normal baseline for cell organization.

BAG-1 (Bcl-2 associated athanogene-1) is a multifunctional protein, linking cell proliferation, cell death, protein folding, and cell stress. In vivo, BAG-1 is expressed in growth plate and articular cartilage, and the expression of BAG-1 is decreased with aging. Chondrocytes respond to endoplasmic reticulum (ER) stress with decreased expression of extracellular matrix proteins, and prolonged ER stress leads to chondrocyte apoptosis. Here we demonstrate for the first time that BAG-1 is involved in ER stress-induced apoptosis in chondrocytes. Induction of ER stress through multiple mechanisms all resulted in downregulation of BAG-1 expression. In addition, direct suppression of BAG-1 expression resulted in chondrocyte growth arrest and apoptosis, while stable overexpression of BAG-1 delayed the onset of ER stress-mediated apoptosis. In addition to regulating apoptosis, we also observed decreased expression of collagen type II in BAG-1 deficient chondrocytes. In contrast, overexpression of BAG-1 resulted in increased expression of collagen type II. Moreover, under ER stress conditions, the reduced expression of collagen type II was delayed in chondrocytes overexpressing BAG-1. These results suggest a novel role for BAG-1 in supporting viability and matrix expression of chondrocytes.