Generation of megakaryocytes and platelets from preadipocyte cell line 3T3-L1, but not the parent cell line 3T3, in vitro

Platelets are produced from megakaryocytes (MKs), although the pathway leading from stem cells to MK lineages are not yet fully understood. Recently, we reported to obtain abundant MKs and platelets from human subcutaneous adipose tissues. Adipose tissues contain various cell types, most of which are lineage cells from mesenchymal or adipocyte-derived stem cells, distinct from hematopoietic cells. To identify the cells responsible for the differentiation MK lineages in adipose tissues, this study examined whether the preadipocyte cell line 3T3-L1 and fibroblast cell line 3T3 differentiated into MK lineages in vitro. Cells were cultured in megakaryocyte lineage induction medium. By day 4, most of 3T3 cell-derived cells leaded to cell death. In contrast, 3T3-L1-derived cells on days 8 showed to have typical characterizations of MK lineages in analyses for specific marker, DNA ploidy, transmission electro micrograph. 3T3-L1-derived platelet-sized cells on day 12 could be stimulated by ADP and PAR4-activating peptide. This study clearly shows in vitro differentiation from 3T3-L1 cells, not from 3T3 cells, into MK lineages.     

PIK3C3/VPS34, the class III PtdIns 3-kinase,plays indispensable roles in the podocyte

The mammalian homolog of yeast Vps34 (PIK3C3/VPS34) is implicated in the regulation of autophagy, and recent studies have suggested that autophagy is a key mechanism in maintaining the integrity of renal glomerular podocytes. To date, however, the role of PIK3C3 in podocytes has remained unknown. We generated a line of podocyte-specific Pik3c3-knockout (Pik3c3^pdKO/mVps34^pdKO) mice and demonstrated an indispensable role for PIK3C3 in the regulation of intracellular vesicle trafficking and processing to protect the normal cellular metabolism, structure and function of podocytes.     

Production of 3-hydroxy-γ-decalactone, the precursor of two decenolides with flavouring properties, by the yeast Yarrowia lipolytica

3-Hydroxy-γ-decalactone is the precursor of dec-2 and dec-3-en-4-olides which are valuable aroma compounds not yet produced. To promote the accumulation of this lactone, the yeast Yarrowia lipolytica was placed in different environmental conditions aiming at altering β-oxidation fluxes. The concentration of substrate, pH, aeration and dissolved oxygen level were modified. We observed an important accumulation at low aeration (0.40 molar yields) and, to a lesser extent, at lower pH (0.15). As oxygen played a key-role, we evaluated its effect at fixed dissolved oxygen and at the pH which was the most favourable to the biotransformation (pH 4.5). At 5% and 30% dissolved oxygen, yields reached 0.50. β-Oxidation fluxes are very dependent on the presence of oxygen and conditions of accumulation of 3-hydroxy-γ-decalactone with very high yields were identified. These results are an important step in the production of the two decenolides. Moreover, they show the high dependence of β-oxidation fluxes on environmental conditions and relate these conditions to the accumulation of intermediates, results that are of interest to all the processes using yeast on lipids or alkanes.     

Biosynthesis of the Myelin 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterases

We have investigated the site of synthesis of the 2',3'-cyclic nucleotide 3'-phosphodiesterases (CNPs I and II) in rat brain. Rapid kinetics of incorporation of CNPs into oligodendrocyte plasma membrane in the intact brain are consistent with their synthesis on free polysomes. This hypothesis was confirmed by the translation in vitro of RNA isolated from free and bound polysomes, respectively. Unlike myelin basic protein (MBP) mRNAs, CNP mRNAs are not enriched in a myelin-associated pool of RNA. MBPs, but not CNPs, were found to readily associate in vitro with membrane vesicles derived from rough endoplasmic reticulum. The avidity of MBPs in binding to membranes is probably related to the previously observed spatial segregation of MBP mRNAs into actively myelinating cellular processes of the oligodendrocyte. Such a segregation would ensure that newly synthesized MBPs are immediately incorporated into myelin. In contrast, the CNPs probably associate with the cytoplasmic surface of the oligodendrocyte plasma membrane through interaction with a membrane-bound receptor.     

Involvement of MAP-kinase, PI3-kinase and EGF-receptor in the stimulatory effect of Neurotensin on DNA synthesis in PC3 cells

The mechanism by which neurotensin (NT) promotes the growth of prostate cancer epithelial cells is not yet defined. Here, androgen-independent PC3 cells, which express high levels of the type 1 NT-receptor (NTR1), are used to examine the involvement of epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (ERK, SAPK/JNK and p38), PI3 kinase and PKC in the mitogenic effect of NT. NT dose dependently (0.1–30 nM) enhanced phosphorylation of EGFR, ERK and Akt, reaching maximal levels within 3 min as measured by Western blotting. These effects were associated with an accumulation of EGF-like substance(s) in the medium (assayed by EGFR binding) and a 2-fold increase in DNA synthesis (assayed by [³H]thymidine incorporation). The DNA synthesis enhancement by NT was non-additive with that of EGF. The NT-induced stimulation of EGFR/ERK/Akt phosphorylation and DNA synthesis was inhibited by EGFR-tyrosine kinase inhibitors (AG1478, PD153035), metallo-endopeptidase inhibitor phosphoramidon and by heparin, but not by neutralizing anti-EGF antibody. Thus, transactivation of EGFR by NT involved heparin-binding EGF (HB-EGF or amphiregulin) rather than EGF. The effects of NT on EGFR/ERK/Akt activation and DNA synthesis were attenuated by PLC-inhibitor (U73122), PKC-inhibitors (bisindolylmaleimide, staurosporine, rottlerin), MEK inhibitor (U0126) and PI3 kinase inhibitors (wortmannin, LY 294002). We conclude that NT stimulated mitogenesis in PC3 cells by a PKC-dependent ligand-mediated transactivation of EGFR, which led to stimulation of the Raf–MEK–ERK pathway in a PI3 kinase-dependent manner.     

河南郑州老奶奶庙第3地点初步研究

老奶奶庙第3地点位于河南省郑州市,年代距今约4.5万年或稍早。2016年度的发掘,发现动物化石575件,石制品66件。从遗物的密度和分布情况来看,本遗址应为第1地点或其它中心营地外围的临时性地点,古人类曾在此活动,但并不频繁。动物化石的种属组合揭示出当时应为草原-疏林景观。石制品属于典型的华北地区小石片石器工业,与本区MIS3阶段其他遗址工业面貌相似,应属于同一技术体系。     

lncRNA HOXB-AS3 exacerbates proliferation,migration, and invasion of lung cancer via activating the PI3K-AKT pathway

Lung cancer remains the leading cause of cancer-related death all over the world. In spite of the great advances made in surgery and chemotherapy, the prognosis of lung cancer patients is poor. A substantial fraction of long noncoding RNAs (lncRNAs) can regulate various cancers. A recent study has reported that lncRNA HOXB-AS3 plays a critical role in cancers. However, its biological function remains unclear in lung cancer progression. In the current research, we found HOXB-AS3 was obviously elevated in NSCLC tissues and cells. Functional assays showed that inhibition of HOXB-AS3 was able to repress A549 and H1975 cell proliferation, cell colony formation ability and meanwhile, triggered cell apoptosis. Furthermore, the lung cancer cell cycle was mostly blocked in the G1 phase whereas the cell ratio in the S phase was reduced. Also, A549 and H1975 cell migration and invasion capacity were significantly repressed by the loss of HOXB-AS3. The PI3K/AKT pathway has been implicated in the carcinogenesis of multiple cancers. Here, we displayed that inhibition of HOXB-AS3 suppressed lung cancer cell progression via inactivating the PI3K/AKT pathway. Subsequently, in vivo experiments were utilized in our study and it was demonstrated that HOXB-AS3 contributed to lung cancer tumor growth via modulating the PI3K/AKT pathway. Overall, we implied that HOXB-AS3 might provide a new perspective for lung cancer treatment via targeting PI3K/AKT.     

Lovastatin-induced up-regulation of the BH3-only protein, Bim, and cell death in glioblastoma cells

The mechanism of lovastatin-induced cell death was examined in three established human glioblastoma cell lines; U87, U251, and U138. Changes in potential modifiers of apoptosis, including Bcl-2 family proteins and MAP kinase targets after such lovastatin treatment, were evaluated. Lovastatin (5 microm) treatment causes extensive cell death in two of the cell lines, U87 and U251; but only minimal in a third, U138. Lovastatin-induced death occurs in correlation with significantly increased levels of the BH3-only protein, Bim. The up-regulation of Bim levels was directly associated with an increased incidence of apoptosis. Lovastatin treatment in U87 cells results in activation of targets of three major mitogen-activating protein kinase cascades including Erk1/2, JNK and p38. Changes in levels of Bim, as well as increase phosphorylation of Erk1/2, c-jun, and p38 are all prevented by co-incubation of lovastatin and the isoprenylation metabolite, geranylgeranyl pyrophosphate. Inhibition of the MAP kinase pathways failed to block the increased expression of Bim expression or cell death. Further elucidation of the mechanisms of lovastatin-induced up-regulation of Bim and apoptosis in glioblastoma cells are important in determining a potential role for lovastatin as a chemotherapy agent.     

Metabolism of 3H-1α,25-dihydroxyvitamin D3 in cultured human keratinocytes

In the present investigation we studied the metabolism of 1α,25-dihydroxy-[1β-³H] vitamin D₃ (³H-1,25(OH)₂D₃) in culture-grown human keratinocytes (CHK). Our results showed that the cellular uptake of ³H-1,25(OH)₂D₃, upon incubation with CHK, occurred very rapidly; and it paralleled a decrease in the concentration of ³H-1,25(OH)₂D₃ in the medium. The amount of ³H-calcitroic acid, on the other hand, increased slowly in the medium, while the concentration of ³H-calcitroic acid in the cell remained undetectable during the whole period of incubation. When the cells were preincubated with 1,25(OH)₂D₃ (10^?8M), conversion of ³H-1,25(OH)₂D₃ to ³H-calcitroic acid increased almost twofold, indicating that 1,25(OH)₂D₃ catalyzed its own catabolism. © 1995 Wiley-Liss, Inc.     

High-performance liquid chromatographic method for the determination of the major quinine metabolite, 3-hydroxyquinine, in plasma and urine

The determination of 3-hydroxyquinine in urine and plasma samples is described. Extraction was performed using a mixture of toluene–butanol (75:25, v/v), followed by back-extraction into the mobile phase, which consisted of 0.1 M phosphate buffer, acetonitrile, tetrahydrofuran and triethylamine. A reversed-phase liquid chromatography system with fluorescence detection and a CT-sil C₁₈ column were used. The within-assay coefficient of variation of the method was 2% at the higher concentration values in plasma, 2.95 μM, 4% at 227 nM and 9% at the lower limit of quantitation, 4.5 nM. In urine, the coefficient of variation was 11% at the lower concentration, 227 nM and was 3% at 56.8 μM. The between-assay coefficient of variation was 4% at the low concentration (5.1 nM) in plasma, 2% at 276.8 nM and 3% at 1.97 μM. In urine, the between assay coefficient of variation was 4% at 204.6 nM, 3% at 5.12 μM and 2% at 56.8 μM.     

Enantiospecific enzymatic preparation of (2S,3S)-3-alkylaspartic acids of current interest in the synthesis of stereoregular poly[β-(2S,3S)-3-alkylmalic acids] as new optically active functional polyesters

(2S,3S)-3-methyl- and 3-isopropylaspartic acids were synthesized by bioconversion of the corresponding alkylfumarates (mesaconate and 3-isopropylfumarate) using β-methylaspartase from cell-free extracts of Clostridium tetanomorphum. Optically pure (2S,3S)-3-alkylaspartic acids were transformed in several steps to benzyl (3S,4R)-3-alkylmalolactonates without any racemization of the two chiral centers. These optically active α,β-substituted-β-lactones were polymerized by anionic ring opening polymerization yielding optically active semi-crystalline polyesters. ¹³C NMR analysis of poly[benzyl β-3-isopropylmalate] in CDCl₃ has shown that only the iso-type stereosequence is present in the polymer, indicating that the macromolecular chain is constituted by the only units of benzyl β-(2S,3S)-3-isopropylmalate monomer. The polymerization reaction was done without any racemization of the two stereogenic centers as in the case of benzyl (3S,4R)-3-methylmalolactonate. © 1996 Wiley-Liss, Inc.     

3H-31, A Non-structural Protein of Heliothis virescens ascovirus 3h,Inhibits the Host Larval Cathepsin and Chitinase Activities

Virologica Sinica - 3h-31 of Heliothis virescens ascovirus 3h (HvAV-3h) is a highly conserved gene of ascoviruses. As an early gene of HvAV-3h, 3h-31 codes for a non-structural protein (3H-31) of...     

LC3, a microtubule-associated protein1A/B light chain3, is involved in cytoplasmic lipid droplet formation

The cytoplasmic lipid droplet (LD) is one of organelles that has a neutral lipid core with a single phospholipid layer. LDs are believed to be generated between the two leaflets of the endoplasmic reticulum (ER) membrane and to play various roles, such as high effective energy storage. However, it remains largely unknown how LDs are generated and grow in the cytoplasm. We have previously shown that the Atg conjugation system that is essential for autophagosome formation is involved in LD formation in hepatocytes and cardiac myocytes. We show here that LC3 itself is involved in LD formation by using RNA interference (RNAi). All cultured cell lines examined, in which the expression of LC3 was suppressed by RNAi, showed reduced LD formation. Triacylglycerol, a major component of LDs, was synthesized and degraded in LC3 mRNA-knockdown cells as well as in control cells. Interestingly, potential of the bulk protein degradation in the knockdown-cells was also evident in the control cells. These findings indicate that LC3 is involved in the LD formation regardless of the bulk degradation, and that LC3 has two pivotal roles in cellular homeostasis mediated by autophagy and lipid metabolism.     

Phosphorylation of the stress-activated protein kinase,MEKK3, at serine 166

Much effort has focused on the identification of MAPK cascades that are activated by the MEKK family of protein kinases. However, direct phosphorylation and regulation of the MEKK proteins has not been shown. To address this question, we have expressed recombinant (His)6FLAG.MEKK3 in Sf9 insect cells and tethered the purified protein to Ni-Sepharose so that we could precipitate interacting proteins and then identify such proteins by liquid chromatography and mass spectrometry (LC-MS). We identified 14-3-3 proteins as interacting with MEKK3, which suggested that (His)6FLAG.MEKK3 was phosphorylated on serine since 14-3-3 proteins are known to associate with phosphorylated proteins. We identified two phosphorylated amino acids at Ser166 and Ser337 of tryptic peptides derived from (His)6FLAG.MEKK3 by using LC-MS. Antibodies were developed that recognize the specific phosphorylated amino acid and with these antibodies, we demonstrate that various stimuli (tumor necrosis factor, arsenite, forskolin, and serum) promote phosphorylation of Ser166 and Ser337. However, neither of these phosphorylated amino acids is required for association with 14-3-3 protein or regulation of MEKK3-dependent ERK and JNK activity. Nonetheless, these results suggest that MEKK3 is a convergence point of multiple upstream signaling pathways.     

Macrocyclic inhibitors of 3C and 3C-like proteases of picornavirus,norovirus, and coronavirus

The design, synthesis, and in vitro evaluation of the first macrocyclic inhibitor of 3C and 3C-like proteases of picornavirus, norovirus, and coronavirus are reported. The in vitro inhibitory activity (50% effective concentration) of the macrocyclic inhibitor toward enterovirus 3C protease (CVB3 Nancy strain), and coronavirus (SARS-CoV) and norovirus 3C-like proteases, was determined to be 1.8, 15.5 and 5.1 μM, respectively.     

Cellular and Subcellular Distribution of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase and Its mRNA in the Rat Central Nervous System

The 2',3'-cyclic nucleotide 3'-phosphodiesterases (CNPs) are closely related oligodendrocyte proteins whose in vivo function is unknown. To identify subcellular sites of CNP function, the distribution of CNP and CNP mRNA was determined in tissue sections from rats of various developmental ages. Our results indicate that CNP gene products were expressed exclusively by oligodendrocytes in the CNS. CNP mRNA was concentrated around oligodendrocyte perinuclear regions during all stages of myelination. Developmentally, initial detection of CNP mRNA closely paralleled initial detection of its translation products. In electron micrographs of immunostained ultrathin cryosections, CNP was associated with oligodendrocyte membranes during the earliest phase of axonal ensheathment. In more mature fibers, immunocytochemistry established that the CNPs are not major components of compact myelin but are concentrated within specific regions of the oligodendrocyte and myelin internode. These include (a) the plasma membrane of oligodendrocytes and their processes, (b) the periaxonal membrane and inner mesaxon, (c) the outer tongue process, (d) the paranodal myelin loops, and (e) the "incisure-like" membranes found in many larger CNS myelin sheaths. A cytoplasmic pool of CNP was also detected in oligodendrocyte perikarya and larger oligodendrocyte processes. CNP was also enriched in similar locations in myelinated fibers of the PNS.