Oligomerization and regulated proteolytic processing of angiopoietin-like protein 4

Angiopoietin-like protein 4 (Angptl4) is a recently identified circulating protein expressed primarily in adipose tissue and liver. Also known as peroxisome proliferator-activated receptor (PPAR)-gamma angiopoietin-related, fasting induced adipose factor, and hepatic fibrinogen/angiopoietin-related protein, recombinant Angptl4 causes increase of plasma very low density lipoprotein levels by inhibition of lipoprotein lipase activity. Similar to angiopoietins and other angiopoietin-like proteins, Angptl4 contains an amino-terminal coiled-coil domain and a carboxyl-terminal fibrinogen-like domain. We report here that Angptl4 is evolutionarily conserved among several mammalian species and that full-length Angptl4 protein is an oligomer containing intermolecular disulfide bonds. Oligomerized Angptl4 undergoes proteolytic processing to release its carboxyl fibrinogen-like domain, which circulates as a monomer. Angptl4's N-terminal coiled-coil domain mediates its oligomerization, which by itself is sufficient to form higher order oligomeric structure. Adenovirus-mediated overexpression of Angptl4 in 293 cells shows that conversion of full-length, oligomerized Angptl4 is mediated by a cell-associated protease activity induced by serum. These findings demonstrate a novel property of angiopoietin-like proteins and suggest that oligomerization and proteolytic processing of Angptl4 may regulate its biological activities in vivo.     

High-resolution mapping of genotype-phenotype relationships in cri du chat syndrome using array comparative genomic hybridization

We have used array comparative genomic hybridization to map DNA copy-number changes in 94 patients with cri du chat syndrome who had been carefully evaluated for the presence of the characteristic cry, speech delay, facial dysmorphology, and level of mental retardation (MR). Most subjects had simple deletions involving 5p (67 terminal and 12 interstitial). Genotype-phenotype correlations localized the region associated with the cry to 1.5 Mb in distal 5p15.31, between bacterial artificial chromosomes (BACs) containing markers D5S2054 and D5S676; speech delay to 3.2 Mb in 5p15.32-15.33, between BACs containing D5S417 and D5S635; and the region associated with facial dysmorphology to 2.4 Mb in 5p15.2-15.31, between BACs containing D5S208 and D5S2887. These results overlap and refine those reported in previous publications. MR depended approximately on the 5p deletion size and location, but there were many cases in which the retardation was disproportionately severe, given the 5p deletion. All 15 of these cases, approximately two-thirds of the severely retarded patients, were found to have copy-number aberrations in addition to the 5p deletion. Restriction of consideration to patients with only 5p deletions clarified the effect of such deletions and suggested the presence of three regions, MRI-III, with differing effect on retardation. Deletions including MRI, a 1.2-Mb region overlapping the previously defined cri du chat critical region but not including MRII and MRIII, produced a moderate level of retardation. Deletions restricted to MRII, located just proximal to MRI, produced a milder level of retardation, whereas deletions restricted to the still-more proximal MRIII produced no discernible phenotype. However, MR increased as deletions that included MRI extended progressively into MRII and MRIII, and MR became profound when all three regions were deleted.     

Oligomerization state-dependent hyperlipidemic effect of angiopoietin-like protein 4

Angiopoietin-like protein 4 (Angptl4) is the second member of the angiopoietin-like family of proteins previously shown to increase plasma triglyceride (TG) levels in vivo. We recently reported that Angptl4 is a variable-sized oligomer formed by intermolecular disulfide bonds and undergoes regulated proteolytic processing upon secretion. We now show that adenoviral overexpression of Angptl4 potently increases plasma TG levels by a mechanism independent of food intake or hepatic VLDL secretion. We determined that cysteine residues at positions 76 and 80 of Angptl4, conserved among mouse, rat, and human, are required to form higher order structures. By generating adenoviral expression vectors of Angptl4 containing different epitope tags at both N and C termini, we show that loss of oligomerization results in decreased stability of the N-terminal coiled-coil domain of Angptl4 as well as decreased ability to increase plasma TG levels, suggesting that intermolecular disulfide bond formation plays important roles in determining the magnitude of the hyperlipidemic effect of Angptl4. Because Angptl4 is more potent than Angptl3 in increasing plasma TG levels in mice, inappropriate oligomerization of Angptl4 could be associated with disorders of lipid metabolism in vivo.     

Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.     

Novel potassium N-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxylhex-1-yl]-L-amino acid dichloroplatinates(II) with high anti-tumor activity and low side reaction

To discover whether novel anti-tumor platinum agents are capable of selectively accumulating in tumor tissue, three novel potassium N-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxylhex-1-yl]-L-amino acid dichloroplatinates(II) were prepared. At a dose of 1.67 μmol kg(-1) the in vivo anti-tumor potencies of two of the compounds were higher than that of oxaliplatin. The mortality analysis indicated that these compounds resulted in a 100% survival rate, whereas oxaliplatin lead to an 80% survival rate. The organ damage examination indicated that these compounds induced less damage than oxaliplatin. The platinum accumulation in the organs, blood and bone was significantly lower than that of oxaliplatin treated mice, while the platinum accumulation in the tumor tissue was significantly higher than that of the oxaliplatin treated mice.     

小鼠血管内膜增生模型的建立

该文主要介绍一种可以在小鼠中有效地诱导血管内膜增生的方法。该方法使用硅胶管嵌套小鼠股动脉,造成股动脉血管内膜增生。病理切片观察表明,实验组小鼠股动脉与假手术组相比出现了内膜层和中膜层不规则增厚,管腔狭窄,细胞排列紊乱,内膜炎症细胞浸润等。免疫组织化学染色显示内膜增生部分是由于血管平滑肌细胞（VsMCs）增生和基质积聚所致。该模型的建立对动脉粥样硬化病理机制研究和治疗药物的研发具有重要意义。     

Design of synthetic hexapeptide substrates for prostate-specific antigen using single-position minilibraries.

Prostate-specific antigen (PSA), a serine endoprotease with chymotrypsin-like substrate specificity, is a marker used widely for detection of prostate cancer and other prostate diseases, catalyzing hydrolysis of the gel-forming proteins semenogelins I and II, which are synthesized and secreted by the seminal vesicle. In this study we report the use of two single-position minilibraries and RP-HPLC selection to optimize a hexapeptide substrate for PSA, spanning substrate positions P3 to P3'. PSA has been shown previously to prefer tyrosine in position P1 [Denmeade et al. (1997) Cancer Research, 57, 4924-4930]. Here we demonstrate preference for serine in position P1' and strong preference for phenylalanine in position P2. Based on these results we have designed and demonstrated the utility of the optimized fluorogenic PSA substrate 7-methoxy-coumarin-4-acetylGlnPheTyrSerSerAsnLys(epsilon-2,4-dinit rophenyl)amide, 1, which permits continuous monitoring of PSA endopeptidase activity at high sensitivity.     

Effect of insulin-like growth factor (IGF)-I and Des (1-3) IGF-I on the level of IGF binding protein-3 and IGF binding protein-3 mRNA in cultured porcine embryonic muscle cells

Insulin-like growth factor binding protein (IGFBP)-3 effects proliferation and differentiation of numerous cell types by binding to insulin-like growth factors (IGF) and attenuating their activity or by directly affecting cells in an IGF-independent manner. Consequently, IGFBPs produced by specific cells may affect their differentiation and proliferation. In this study we show that embryonic porcine myogenic cells, unlike murine muscle cell lines, produce significant quantities of a binding protein immunologically identified as IGFBP-3. Nonfusing cells subcultured from highly fused porcine myogenic cell cultures do not produce detectable IGFBP-3 protein or mRNA, thus suggesting the IGFBP-3 is produced by muscle cells in the porcine myogenic cell cultures. Treatment of porcine myogenic cultures with 20 ng of IGF-I or 20 ng of Des (1-3) IGF-I/ml serum-free media for 24 h results in a threefold reduction in the level of IGFBP-3 in conditioned media. This reduction is not affected by cell density over a sixfold range. Additionally, treatment for 24 h with 20 ng of IGF-I/ml media results in a sevenfold decrease in the steady-state level of IGFBP-3 mRNA. This IGF-I-induced decrease in IGFBP-3 mRNA level appears to be relatively unique to myogenic cells. IGF-I treatment also causes a fourfold increase in the steady-state level of myogenin mRNA. This increase in myogenin mRNA suggests that, as expected, IGF-I treatment accelerates differentiation of myogenic cells. The simultaneous decrease in IGFBP-3 mRNA and protein that accompanies IGF-I-induced myogenin expression suggests that differentiation of myogenic cells may be preceded or accompanied by decreased production of IGFBP-3.     

Supreme™ laryngeal mask airway use in general Anesthesia for category 2 and 3 Cesarean delivery: a prospective cohort study

Background

The Supreme? laryngeal mask airway (SLMA) is a single-use LMA with double lumen design that allows separation of the respiratory and the alimentary tract, hence potentially reducing the gastric volume and risk of aspiration. The purpose of this prospective cohort study is to evaluate the the role of the SLMA as an airway technique for women undergoing category 2 and 3 Cesarean delivery under general anesthesia.

Methods

We recruited 584 parturients who underwent category 2 or 3 Cesarean delivery under general anesthesia, in which 193 parturients underwent category 2 and 391 parturients underwent category 3 Cesarean delivery. The primary outcome was insertion success rate at 1st attempt in SLMA insertion. The secondary outcomes included anaesthetic, obstetric outcomes and maternal side effects associated with airway device.

Results

The 1st attempt insertion success rate was 98.3%, while the overall insertion success rate was 100%. The mean (Standard deviation) time to effective ventilation was 15.6 (4.4) seconds. Orogastric tube insertion was successful at the 1st attempt in all parturients. There was no clinical evidence of aspiration or regurgitation. No episodes of hypoxemia, laryngospasm or bronchospasm were observed intra-operatively. The incidence of complications was low and with good maternal satisfaction reported.

Conclusions

The SLMA could be an alternative effective airway in category 2 and 3 parturients emergency Cesarean Delivery under general anesthesia in a carefully-selected obstetric population.

Trial registration

Clinical Trials Registration: Clinicaltrials.gov Registration NCT02026882. Registered on December 31, 2013.