We describe the isolation of a novel protein from the conditioned medium of human articular cartilage chondrocytes in primary culture. This 39-kDa protein has the N-terminal sequence YKL, which we have termed YKL-39. The 1434-nucleotide sequence of the YKL-39 cDNA predicts a 385-residue initial translation product and a 364-residue mature YKL-39. The amino acid sequence of YKL-39 is most closely related to YKL-40, followed by macrophage chitotriosidase, oviductal glycoprotein, and macrophage YM-1. All five proteins share significant sequence identity with bacterial chitinases and have the probable structure of an (␣␤) 8 barrel. YKL-39 lacks the active site glutamate, which is essential for the activity of chitinases, and as expected has no chitinase activity. The highest level of YKL-39 mRNA expression is seen in chondrocytes, followed by synoviocytes, lung, and heart. YKL-39 accounts for 4% of the protein in chondrocyte-conditioned medium, prostromelysin accounts for 17%, and YKL-40 accounts for 33%. In contrast to YKL-40, YKL-39 is not a glycoprotein and does not bind to heparin.In this study we report the discovery of a new member of the mammalian protein family related in sequence to bacterial chitinases. This protein family has an (␣␤) 8 barrel structure (1, 2) and includes a protein secreted from human macrophages that does have chitinase activity, which is termed chitotriosidase (3), and three proteins with no presently known enzymatic activity, YKL-40 (4), YM-1 (GenBank TM /EBI accession number M94584), and oviductal glycoprotein (6). Because the new member of this family, which we have termed YKL-39, is more closely related in size and sequence to YKL-40 than to other members of this family, it is useful to summarize research on YKL-40 as a background for the present investigation.YKL-40 is a 40-kDa glycoprotein that was first discovered as a heparin-binding protein secreted from bovine breast tissue during the massive tissue involution that follows the cessation of lactation (7). YKL-40 was subsequently discovered as a heparin-binding protein in the conditioned medium of human synoviocytes (8), chondrocytes (4, 9), and the MG-63 osteosarcoma cell line (10). YKL-40 has also been discovered as a heparin-binding protein expressed by porcine vascular smooth muscle cells undergoing a differentiation transition (11) and as a protein expressed selectively by murine mammary tumors initiated by neu/ras oncogenes (12). The present studies were initiated to further examine the expression of YKL-40 by human articular cartilage chondrocytes in culture. In the course of these studies YKL-39 was found as a protein that copurified with YKL-40.We report here the discovery, purification, characterization, and sequence of human YKL-39. MATERIALS AND METHODSIsolation and Culture of Chondrocytes and Synoviocytes-Chondrocytes and synoviocytes were obtained from Martin Lotz, director of the University of California, San Diego osteoarthritis cell culture facility and were isolated and cultured essentially as described (13). C...

We describe here the isolation of a novel non-collagenous protein from the acid demineralization extract of bovine cortical bone. This 24-kDa protein is multiply phosphorylated at serine residues in Ser-X-Glu/Ser(P) sequences, a recognition motif for phosphorylation by the secretory pathway protein kinase, and we have termed this protein secreted phosphoprotein 24 (spp24). The cDNA structure of spp24 was determined by sequencing cDNA fragments obtained by reverse transcription-polymerase chain reaction, 3'-rapid amplification of cDNA ends, and screening a lambda gt11 cDNA library. This cDNA sequence predicts a 200-residue initial translation product which consists of a 20-residue signal sequence and the 180-residue mature spp24. Northern blot analysis using the spp24 cDNA showed that spp24 mRNA is in liver and bone but not in heart, lung, kidney, or spleen. A search of existing protein sequences revealed that the N-terminal 107 residues of mature spp24 are related in sequence to the cystatin family of thiol protease inhibitors, which suggests that spp24 could function to modulate the thiol protease activities that are known to be involved in bone turnover. Several of the proteins in the cystatin family that are most closely related to spp24 are not only thiol protease inhibitors but are also precursors to peptides with potent biological activity, peptides such as bradykinin and the neutrophil antibiotic peptides. It is therefore possible that the intact form of spp24 found in bone could also be a precursor to a biologically active peptide, a peptide which could coordinate an aspect of bone turnover.

Studies have attributed several functions to the Eaf family, including tumor suppression and eye development. Given the potential association between cancer and development, we set forth to explore Eaf1 and Eaf2/U19 activity in vertebrate embryogenesis, using zebrafish. In situ hybridization revealed similar eaf1 and eaf2/u19 expression patterns. Morpholino-mediated knockdown of either eaf1 or eaf2/u19 expression produced similar morphological changes that could be reversed by ectopic expression of target or reciprocal-target mRNA. However, combination of Eaf1 and Eaf2/U19 (Eafs)-morpholinos increased the severity of defects, suggesting that Eaf1 and Eaf2/U19 only share some functional redundancy. The Eafs knockdown phenotype resembled that of embryos with defects in convergence and extension movements. Indeed, knockdown caused expression pattern changes for convergence and extension movement markers, whereas cell tracing experiments using kaeda mRNA showed a correlation between Eafs knockdown and cell migration defects. Cardiac and pancreatic differentiation markers revealed that Eafs knockdown also disrupted midline convergence of heart and pancreatic organ precursors. Noncanonical Wnt signaling plays a key role in both convergence and extension movements and midline convergence of organ precursors. We found that Eaf1 and Eaf2/U19 maintained expression levels of wnt11 and wnt5. Moreover, wnt11 or wnt5 mRNA partially rescued the convergence and extension movement defects occurring in eafs morphants. Wnt11 and Wnt5 converge on rhoA, so not surprisingly, rhoA mRNA more effectively rescued defects than either wnt11 or wnt5 mRNA alone. However, the ectopic expression of wnt11 and wnt5 did not affect eaf1 and eaf2/u19 expression. These data indicate that eaf1 and eaf2/u19 act upstream of noncanonical Wnt signaling to mediate convergence and extension movements. EAF1 (ELL-associated factor 1) was first discovered through its ability to associate with the protein ELL (eleven-nineteen lysine-rich leukemia), a fusion partner of MLL in the t(11; 19)(q23;p13.1) chromosomal translocation associated with acute myeloid leukemia (1, 2). Subsequent studies found a second binding partner for ELL, EAF2, which was independently identified as an androgen up-regulated gene in the rat prostate and named human up-regulated 19 (U19) (3, 4). ELL binds to RNA polymerase II and acts as a transcriptional elongation factor whose targeted deletion leads to embryonic lethality in mice (5, 6). Both EAF1 and EAF2, which share significant sequence homology, stimulate ELL elongation activity (7). Studies by Luo et al. (8) argued that EAF proteins are important in MLL-ELL leukemogenesis, whereas our previous studies showed that EAF2/U19 inhibits xenograft prostate tumor growth and undergoes down-regulation in prostate cancer cell lines (9). These findings link the EAF2/U19 gene with two major human cancers: prostate cancer and acute myeloid leukemia. To investigate the function EAF2/U19 in vivo, we constructed a murine knock-out model. The EAF2...

Background: FBXO32 is an E3 ubiquitin ligase that plays important roles in tumorigenesis and muscle atrophy. Results: c-Myc was found to be a target of FBXO32 for proteasomal degradation. Conclusion: FBXO32 targets Lys-326 of c-Myc to form polyubiquitin chains, resulting in inhibition of cell proliferation. Significance: FBXO32 may mediate c-Myc proteasomal degradation.

Abstract. The characteristics of ozone variations and the impacts of synoptic and local meteorological factors in northern China were quantitatively analyzed during the warm season from 2013 to 2017 based on multi-city in situ ozone and meteorological data as well as meteorological reanalysis. The domain-averaged maximum daily 8 h running average O3 (MDA8 O3) concentration was 122±11 µg m−3, with an increase rate of 7.88 µg m−3 yr−1, and the three most polluted months were closely related to the variations in the synoptic circulation patterns, which occurred in June (149 µg m−3), May (138 µg m−3) and July (132 µg m−3). A total of 26 weather types (merged into five weather categories) were objectively identified using the Lamb–Jenkinson method. The highly polluted weather categories included the S–W–N directions (geostrophic wind direction diverts from south to north), low-pressure-related weather types (LP) and cyclone type, which the study area controlled by a low-pressure center (C), and the corresponding domain-averaged MDA8 O3 concentrations were 122, 126 and 128 µg m−3, respectively. Based on the frequency and intensity changes of the synoptic circulation patterns, 39.2 % of the interannual increase in the domain-averaged O3 from 2013 to 2017 was attributed to synoptic changes, and the intensity of the synoptic circulation patterns was the dominant factor. Using synoptic classification and local meteorological factors, the segmented synoptic-regression approach was established to evaluate and forecast daily ozone variability on an urban scale. The results showed that this method is practical in most cities, and the dominant factors are the maximum temperature, southerly winds, relative humidity on the previous day and on the same day, and total cloud cover. Overall, 41 %–63 % of the day-to-day variability in the MDA8 O3 concentrations was due to local meteorological variations in most cities over northern China, except for two cities: QHD (Qinhuangdao) at 34 % and ZZ (Zhengzhou) at 20 %. Our quantitative exploration of the influence of both synoptic and local meteorological factors on interannual and day-to-day ozone variability will provide a scientific basis for evaluating emission reduction measures that have been implemented by the national and local governments to mitigate air pollution in northern China.

Life-sustaining responses to low oxygen, or hypoxia, depend on signal transduction by HIFs, but the underlying mechanisms by which cells sense hypoxia are not completely understood. Based on prior studies suggesting a link between the β-adrenergic receptor (β-AR) and hypoxia responses, we hypothesized that the β-AR mediates hypoxia sensing and is necessary for HIF-1α accumulation. Beta blocker treatment of mice suppressed hypoxia induction of renal HIF-1α accumulation, erythropoietin production, and erythropoiesis in vivo. Likewise, beta blocker treatment of primary human endothelial cells in vitro decreased hypoxia-mediated HIF-1α accumulation and binding to target genes and the downstream hypoxia-inducible gene expression. In mechanistic studies, cAMP-activated PKA and/or GPCR kinases (GRK), which both participate in β-AR signal transduction, were investigated. Direct activation of cAMP/PKA pathways did not induce HIF-1α accumulation, and inhibition of PKA did not blunt HIF-1α induction by hypoxia. In contrast, pharmacological inhibition of GRK, or expression of a GRK phosphorylation-deficient β-AR mutant in cells, blocked hypoxia-mediated HIF-1α accumulation. Mass spectrometry-based quantitative analyses revealed a hypoxia-mediated β-AR phosphorylation barcode that was different from the classical agonist phosphorylation barcode. These findings indicate that the β-AR is fundamental to the molecular and physiological responses to hypoxia.

Coordination between the endoderm and adjacent cardiac mesoderm is crucial for heart development. We previously showed that myocardial migration is promoted by convergent movement of the endoderm, which itself is controlled by the S1pr2/Gα 13 signaling pathway, but it remains unclear how the movements of the two tissues is coordinated. Here, we image live and fixed embryos to follow these movements, revealing previously unappreciated details of strikingly complex and dynamic associations between the endoderm and myocardial precursors. We found that during segmentation the endoderm underwent three distinct phases of movement relative to the midline: rapid convergence, little convergence and slight expansion. During these periods, the myocardial cells exhibited different stage-dependent migratory modes: co-migration with the endoderm, movement from the dorsal to the ventral side of the endoderm (subduction) and migration independent of endoderm convergence. We also found that defects in S1pr2/Gα 13 -mediated endodermal convergence affected all three modes of myocardial cell migration, probably due to the disruption of fibronectin assembly around the myocardial cells and consequent disorganization of the myocardial epithelium. Moreover, we found that additional cell types within the anterior lateral plate mesoderm (ALPM) also underwent subduction, and that this movement likewise depended on endoderm convergence. Our study delineates for the first time the details of the intricate interplay between the endoderm and ALPM during embryogenesis, highlighting why endoderm movement is essential for heart development, and thus potential underpinnings of congenital heart disease.

ics, Chinese Academy of Sciences. The goals of the network were to monitor and provide warnings of the atmospheric quality in Beijing and its surrounding area during the Beijing 2008 Olympic Games. The results showed that the atmospheric complex pollution exhibited high concentrations of ozone and fine particles and oxidation in summer, with a ubiquitous regional source. The regional mean concentrations of SO 2 , PM 2.5 , NO 2 , and O 3_8h max (the maximum daily 8 h mean) and O x were 22±11, 90±40, 25±5, 136±35 and 112±21 μg/m 3 in summer, respectively. During the Olympic Games, the mean concentration of SO 2 , PM 2.5 , NO 2 , O 3_8h max , and O x were 12.5±4, 56±28, 23±4, 114±29, 95±17 μg/m 3 in the region, respectively, and fell by 51.0%, 43.7%, 13%, 20.2%, and 18.9%, respectively, compared to the prophase mean before the Olympic Games. The concentration of atmospheric pollutants declined significantly and achieved the "Green Olympics" control goal of air quality. After the Olympic Games, SO 2 , PM 2.5 and NO x increased significantly as the temporary atmospheric pollution control measures were terminated.