Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.     

Nucleotide Content Gradients in Maternally and Paternally Inherited Mitochondrial Genomes of the Mussel <Emphasis Type="Italic">Mytilus</Emphasis>

Several studies have shown that in vertebrate mtDNAs the nucleotide content at fourfold degenerate sites is well correlated with the site’s time of exposure to the single-strand state, as predicted from the asymmetrical model of mtDNA replication. Here we examine whether the same explanation may hold for the regional variation in nucleotide content in the maternal and paternal mtDNAs of the mussel Mytilus galloprovincialis. The origin of replication of the heavy strand (O_H) of these genomes has been previously established. A systematic search of the two genomes for sequences that are likely to act as the origin of replication of the light strand (O_L) suggested that the most probable site lies within the ND3 gene. By adopting this O_L position we calculated times of exposure for 0_FD (nondegenerate), 2_FD (twofold degenerate), and 4_FD (fourfold degenerate) sites of the protein-coding part of the genome and for the rRNA, tRNA and noncoding parts. The presence of thymine and absence of guanine at 4_FD sites was highly correlated with the presumed time of exposure. Such an effect was not found for the 2_FD sites, the rRNA, the tRNA, or the noncoding parts. There was a trend for a small increase in cytosine at 0_FD sites with exposure time, which is explicable as the result of biased usage of 4_FD codons. The same analysis was applied to a recently sequenced mitochondrial genome of Mytilus trossulus and produced similar results. These results are consistent with the asymmetrical model of replication and suggest that guanine oxidation due to single-strand exposure is the main cause of regional variation of nucleotide content in Mytilus mitochondrial genomes. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. David Pollock]     

The nuclear localization of SET mediated by impalpha3/impbeta attenuates its cytosolic toxicity in neurons

SET is a multi-functional protein in proliferating cells. Some of the proposed functions of SET suggest an important nuclear role. However, the nuclear import pathway of SET is also unknown and the function of SET in neurons is unclear. Presently, using cortical neurons, we report that the nuclear import of SET is mediated by an impalpha/impbeta-dependent pathway. Nuclear localization signal, (168)KRSSQTQNKASRKR(181), in SET interacts with impalpha3, which recruits impbeta to form a ternary complex, resulting in efficient transportation of SET into nucleus. By in vitro nuclear import assay based on digitonin-permeabilized neurons, we further demonstrated that the nuclear import of SET relies on Ran GTPase. We provide evidence that this nuclear localization of SET is important in neuronal survival. Under basal conditions, SET is predominately nuclear. However, upon death induced by genotoxic stress, endogenous SET decreases in the nucleus and increases in the cytoplasm. Consistent with a toxic role of SET in the cytoplasm, targeted expression of SET to the cytoplasm exacerbates death compared to wild type SET expression which is protective following DNA damage. Taken together, our results indicate that SET is imported into the nucleus through its association with impalpha3/impbeta, and that localization of SET is important in regulation of neuronal death.     

Mitochondrial reactive oxygen species signal hepatocyte steatosis by regulating the phosphatidylinositol 3-kinase cell survival pathway

Abnormal dietary intake of macronutrients is implicated in the development of obesity and fatty liver disease. Steatosis develops in cultured hepatocytes exposed to medium containing either a high concentration of long chain free fatty acids (HFFA) or medium deficient in methionine and choline (MCD). This study examined the mitochondrial reactive oxygen species (ROS)-dependent regulation of the phosphoinositol (PI) 3-kinase pathway in steatosis induced by exposure of AML-12 mouse hepatocytes to MCD or HFFA medium. Exposure to either MCD or HFFA medium resulted in increased production of superoxide anions and H(2)O(2), transduction of the PI 3-kinase pathway and steatosis. Inhibition of PI 3-kinase with LY294002 prevented steatosis. Pharmacologically inhibiting electron transport chain complex III production of ROS prevented activation of PI 3-kinase during macronutrient perturbation, whereas pharmacologically promoting electron transport chain complex III ROS production activated PI 3-kinase independent of nutrient input. The data suggest that H(2)O(2) is the ROS species involved in signal transduction; promoting the rapid conversion of superoxide to H(2)O(2) does not inhibit PI 3-kinase pathway activation during nutrient perturbation, and exogenous H(2)O(2) activates it independent of nutrient input. In addition to transducing PI 3-kinase, the ROS-dependent signal cascade amplifies the PI 3-kinase signal by maintaining phosphatase and tensin homolog in its inactive phosphorylated state. Knockdown of phosphatase and tensin homolog by small interfering RNA independently activated the PI 3-kinase pathway. Our findings suggest a common path for response to altered nutrition involving mitochondrial ROS-dependent PI 3-kinase pathway regulation, leading to steatosis.     

Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1

Crisponi syndrome is a severe autosomal recessive condition that is phenotypically characterized by abnormal, paroxysmal muscular contractions resembling neonatal tetanus, large face, broad nose, anteverted nares, camptodactyly, hyperthermia, and sudden death in most cases. We performed homozygosity mapping in five Sardinian and three Turkish families with Crisponi syndrome, using high-density single-nucleotide polymorphism arrays, and identified a critical region on chromosome 19p12-13.1. The most prominent candidate gene was CRLF1, recently found to be involved in the pathogenesis of cold-induced sweating syndrome type 1 (CISS1). CISS1 belongs to a group of conditions with overlapping phenotypes, also including cold-induced sweating syndrome type 2 and Stuve-Wiedemann syndrome. All these syndromes are caused by mutations of genes of the ciliary neurotrophic factor (CNTF)-receptor pathway. Here, we describe the identification of four different CRLF1 mutations in eight different Crisponi-affected families, including a missense mutation, a single-nucleotide insertion, and a nonsense and an insertion/deletion (indel) mutation, all segregating with the disease trait in the families. Comparison of the mutation spectra of Crisponi syndrome and CISS1 suggests that neither the type nor the location of the CRLF1 mutations points to a phenotype/genotype correlation that would account for the most severe phenotype in Crisponi syndrome. Other, still-unknown molecular factors may be responsible for the variable phenotypic expression of the CRLF1 mutations. We suggest that the syndromes can comprise a family of "CNTF-receptor-related disorders," of which Crisponi syndrome would be the newest member and allelic to CISS1.     

The myocardial response to adrenomedullin involves increased cAMP generation as well as augmented Akt phosphorylation

In this work we aimed to observe (1) the changes in adrenomedullin (AM) and its receptor system - calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) - in myocardial ischemic injury and (2) the response of injuried myocardia to AM and the phosphorylation of Akt to illustrate the protective mechanism of AM in ischemic myocardia. Male SD rats were subcutaneously injected with isoproterenol (ISO) to induce myocardial ischemia. The mRNA levels of AM, CRLR, RAMP1, RAMP2 and RAMP3 were determined by RT-PCR. Protein levels of Akt, phosphor-Akt, CRLR, RAMP1, RAMP2 and RAMP3 were assayed by Western blot. Results showed that, compared with that of the controls, ISO-treated rats showed lower cardiac function and myocardial injury. The mRNA relative amount of AM, CRLR, RAMP1, RAMP2 and RAMP3 in the myocardia of ISO-treated rats was increased. The elevated mRNA levels of CRLR, RAMP1, RAMP2 and RAMP3 were positively correlated with AM content in injured myocardia. The protein levels of CRLR, RAMP1, RAMP2 and RAMP3 in injured myocardia were increased compared with that of control myocardia. AM-stimulated cAMP generation in myocardia was elevated in the ISO group, and was antagonized by AM(22-52) and CGRP(8-37). Western blot analyses revealed that AM significantly enhanced Akt phosphorylation in injured myocardia, which was blocked by pretreatment with AM(22-52) or CGRP(8-37). Ischemia-injured myocardia hyper-expressed AM and its receptors - CRLR, RAMP1, RAMP2 and RAMP3 - and the response of ischemic myocardia to AM was potentiated, and the level of Akt phosphorylation was also increased, which suggests that changes in cardiac AM/AM receptor might play an important role in the pathogenesis of myocardial ischemic injury.     

四川山鹧鸪繁殖期的鸣声行为特征

2005年4—12月在四川老君山自然保护区,通过计算机声谱分析技术,对四川山鹧鸪(Arborophilarufipectus)雄性的鸣声行为特征进行了研究,其结果表明,雄性单音节鸣声出现在啼叫、保护领域和求偶中,这些鸣声持续时间差异明显,主峰值差异不明显;雄性双音节鸣声出现在啼叫、保护领域、竞争雌体和求偶中,这些鸣声第一音节时间、第二音节时间、音节间隔时间和主峰值差异极显著,全句时间差异不明显;雄性多音节鸣声出现在保护领域、警戒和惊吓中。雄性亚成体多音节鸣声出现在惊吓和警戒中;雄性成体和亚成体警戒鸣叫的音节持续时间、音节间隔时间和主峰值差异均不明显,全句持续时间差异明显;雄性亚成体和雄性成体的惊吓鸣叫音节持续时间、音节间隔时间、全句持续时间和主峰值差异均不明显。四川山鹧鸪表现出的各种鸣声行为是为了保护配偶和防止天敌,使该种群更好地繁衍。     

Culture media conditioned by heat-shocked osteoblasts enhances the osteogenesis of bone marrow-derived mesenchymal stromal cells

Osteogenic cells differentiated from bone marrow-derived mesenchymal stromal cells (MSC) hold much promise in bone tissue engineering and reconstructive surgery. There is a dire need for well-defined and efficient protocols to promote the osteogenesis of ex vivo cultured MSC. Hence, this study investigated whether a combination of chemical stimuli (ascorbic acid, beta-glycerophosphate and dexamethasone) and culture media conditioned by a human foetal osteoblast cell line (hFOB) had any synergistic effect on the osteogenesis of MSC. Conditioned media with or without prior heat shock treatment (42 degrees C for 1 h) of the hFOB cell line, were collected and tested on rabbit MSC cultures, in the presence and absence of chemical stimuli. Osteogenic differentiation of MSC was assessed on both day 14 and 21 of ex vivo culture. The results showed conclusively that conditioned media promoted osteogenesis of MSC, which was further enhanced by prior heat shock-treatment of the hFOB cells, as well as by the presence of chemical stimuli. Among all experimental groups, the combination of culture medium conditioned by heat shocked hFOB cells together with chemical stimuli, exhibited the highest level of calcium mineralization, as assessed by Von Kossa staining. This provides clear evidence of a synergistic effect of conditioned media, heat shock and chemical stimuli. It is hoped that the data may contribute to the development of a more well-defined and efficient in vitro culture protocol to promote the osteogenesis of MSC for both clinical and non-clinical applications.