ESI-MS quantitation of increased sphingomyelin in Niemann-Pick disease type B HDL

HDLs have been proposed to have antiatherogenic properties because of their role in reverse cholesterol transport as lipid acceptors. To elucidate the phospholipid profile of these particles, we used electrospray ionization mass spectrometry to examine the phosphatidylcholine (PC) and sphingomyelin (SM) composition of HDLs purified from plasma and nascently generated in vitro from fibroblasts. We also quantitatively compared the phospholipids present in these lipoproteins between normal and Niemann-Pick disease type B (NPD-B) subjects characterized by sphingomyelinase (SMase) deficiency. We demonstrated that plasma HDLs from NPD-B were significantly enriched in SM by an average of 28%, particularly the palmitoyl SM (with an increase of 95%), which accounted for approximately 25-44% of total SM molecular species. Similarly, we observed an increase of approximately 63% in total SM levels in nascent HDLs prepared from NPD-B fibroblasts. Although PC levels in nascent HDLs were comparable between control and NPD-B cells, there was a 95% increase in total PC levels similar to that of SM in plasma HDLs extracted from NPD-B subjects. These data provide insight into the structure of HDLs and identify potential new roles for SMase in lipoprotein metabolism.     

Rescue of K562 cells from MDM2-modulated p53-dependent apoptosis by growth factor-induced differentiation

The wild-type human MDM2 protooncogene was tested for its ability to modulate apoptotic activity of the de novo expressed p53 tumor suppressor gene in K562 cells. We also studied the role of some cytokines in this phenomenon. K562, a human myeloid leukemia cell line, does not express p53 at the mRNA or protein level. In this study, we stably transfected K562 with eukaryotic vectors containing either normal p53 cDNA (pC53-SN3) or mutated p53 (143Val-->Ala) cDNA (pC53-SCX3). Transfectants expressing WT p53 or those expressing mutant p53 are called K562 SN and K562 SM respectively. Many leukemic cell lines undergo apoptosis when de novo WT p53 is expressed alone. In contrast, while the resulting clones (K562 SN and K562 SM) expressed p53, they did not undergo apoptosis. However, when treated with MDM2 mRNA antisense (MDM2 AS) oligodeoxynucleotides (ODNs), K562 SN demonstrated apoptotic features at both molecular and morphological levels. No change was observed when the other clones (K562 and K562 SM) were treated with MDM2 AS. Apoptosis induced in this manner was associated with a relatively small increase in intracellular calcium [Ca2+]i. Cells cultured in medium previously supplemented with recombinant human (rh) interleukin (IL)-3 and rh-erythropoietin (Epo) did not undergo apoptosis. Moreover, K562 SN cells were induced to differentiate. This differentiation was evaluated by measuring hemoglobin (Hb) level in cellular extracted proteins and by analyzing erythroid colony number and morphology. High Hb synthesis was obtained when K562 SN cells were cultured with cytokines (IL-3 + Epo) combined with MDM2 AS. Our results are consistent with the hypothesis that the function of the proto-oncogene MDM2 is to provide a 'feedback' mechanism for the p53-dependent pathway of apoptosis that could be shunted toward differentiation.     

The gut microbiota-artery axis: A bridge between dietary lipids and atherosclerosis?

Atherosclerotic cardiovascular disease is one of the major leading global causes of death. Growing evidence has demonstrated that gut microbiota (GM) and its metabolites play a pivotal role in the onset and progression of atherosclerosis (AS), now known as GM-artery axis. There are interactions between dietary lipids and GM, which ultimately affect GM and its metabolites. Given these two aspects, the GM-artery axis may play a mediating role between dietary lipids and AS. Diets rich in saturated fatty acids (SFAs), omega-6 polyunsaturated fatty acids (n-6 PUFAs), industrial trans fatty acids (TFAs), and cholesterol can increase the levels of atherogenic microbes and metabolites, whereas monounsaturated fatty acids (MUFAs), ruminant TFAs, and phytosterols (PS) can increase the levels of antiatherogenic microbes and metabolites. Actually, dietary phosphatidylcholine (PC), sphingomyelin (SM), and omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been demonstrated to affect AS via the GM-artery axis. Therefore, that GM-artery axis acts as a communication bridge between dietary lipids and AS. Herein, we will describe the molecular mechanism of GM-artery axis in AS and discuss the complex interactions between dietary lipids and GM. In particular, we will highlight the evidence and potential mechanisms of dietary lipids affecting AS via GM-artery axis.     

Increased sphingomyelin content impairs HDL biogenesis and maturation in human Niemann-Pick disease type B

We previously reported that human Niemann-Pick Disease type B (NPD-B) is associated with low HDL. In this study, we investigated the pathophysiology of this HDL deficiency by examining both HDL samples from NPD-B patients and nascent high density lipoprotein (LpA-I) generated by incubation of lipid-free apolipoprotein A-I (apoA-I) with NPD-B fibroblasts. Interestingly, both LpA-I and HDL isolated from patient plasma had a significant increase in sphingomyelin (SM) mass ( approximately 50-100%). Analysis of LCAT kinetics parameters (V(max) and K(m)) revealed that either LpA-I or plasma HDL from NPD-B, as well as reconstituted HDL enriched with SM, exhibited severely decreased LCAT-mediated cholesterol esterification. Importantly, we documented that SM enrichment of NPD-B LpA-I was not attributable to increased cellular mass transfer of SM or unesterified cholesterol to lipid-free apoA-I. Finally, we obtained evidence that the conditioned medium from HUVEC, THP-1, and normal fibroblasts, but not NPD-B fibroblasts, contained active secretory sphingomyelinase (S-SMase) that mediated the hydrolysis of [(3)H]SM-labeled LpA-I and HDL(3). Furthermore, expression of mutant SMase (DeltaR608) in CHO cells revealed that DeltaR608 was synthesized normally but had defective secretion and activity. Our data suggest that defective S-SMase in NPD leads to SM enrichment of HDL that impairs LCAT-mediated nascent HDL maturation and contributes to HDL deficiency. Thus, S-SMase and LCAT may act in concert and play a crucial role in the biogenesis and maturation of nascent HDL particles.     

Identification of prognostic alternative splicing signatures in hepatitis B or/and C viruses related hepatocellular carcinoma

BackgroundAlternative splicing (AS) takes a crucial part in tumor process. We aim to analyze AS in Hepatitis B virus (HBV) or/and hepatitis C virus (HCV) related hepatocellular carcinoma (HCC).MethodsCox regression analysis was conducted to screen survival-associated AS events. The receiver operating characteristic curve used to evaluate the predictive accuracy. Splicing network was built to investigate the relationship between splicing factors and AS events.ResultsNinety-six survival-associated AS events were obtained by univariate Cox regression. Final prognostic model could significantly distinguish the prognosis. We identified RBFOX2 as the hub gene in splicing network based on differentially expressed splicing factors, and obtained MAP3K13_AT as the key AS event in survival-related splicing network.ConclusionOur results highlight the AS signatures in HCC patients with HBV or/and HCV infection. Meanwhile, AS events and splicing factors in different virus-infected HCC subgroups can provide novel perspectives as biomarkers and individualized therapeutic targets.     

Epidermal sphingomyelins are precursors for selected stratum corneum ceramides

Epidermal ceramides (Cer) comprise a heterogeneous family of seven species, including two unique omega-hydroxylated Cer, that are key components of the stratum corneum (SC) intercellular lamellar membranes responsible for the epidermal permeability barrier. Although both glucosylceramide (GlcCer) and the phospho-sphingolipid sphingomyelin (SM) are potential precursors of SC Cer, based on reported chemical structures of epidermal GlcCer and SC Cer, it is assumed that all major subfractions of SC Cer are generated from lamellar body-derived GlcCer. Yet, we and others have shown that SM-derived Cer are required for normal barrier homeostasis. Moreover, two pools of SM, one from plasma membrane, the other from lamellar body-derived contents, are potentially available for Cer production. To clarify the role of SM as a potential precursor of bulk or specific SC Cer, we compared Cer moieties in epidermal SM, Cer generated from epidermal SM by sphingomyelinase treatment, Cer within SC, and Cer that persist in Gaucher SC, where GlcCer cannot generate Cer due to an absence of beta-glucocerebrosidase. Using gas chromatography-mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance for Cer characterization, epidermal SM comprise three major subfractions with distinctive amide-linked (N-acyl) fatty acid (FA) compositions: that is, either long-chain FA (SM-1; C(22;-26)), short-chain FA (SM-2; primarily C(16)), and short-chain alpha-hydroxy FA (SM-3; C(16;-18)). In contrast, only trace quantities of omega-hydroxy FA were present. For each SM subfraction, the sphingoid base was either sphingosine or sphinganine, but phytosphingosine was not detected. Comparison of these SM with corresponding sphingomyelinase-generated epidermal Cer and SC Cer revealed that the Cer moieties of SM-1 and SM-3 are equivalent to Cer 2 (NS) and Cer 5 (AS), respectively. Moreover, both Cer 2 and Cer 5 occurred in Gaucher SC, whereas other Cer subfractions did not occur.These results indicate that two epidermal SM, that is, SM-1 and SM-3, are important precursors of two corresponding Cer in mammalian SC, that is, Cer 2 and Cer 5, but other Cer species, including the omega-hydroxy Cer species, do not derive from SM.     

Structural Basis for the Recognition of Human Cytomegalovirus Glycoprotein B by a Neutralizing Human Antibody

Human cytomegalovirus (HCMV) infections are life-threating to people with a compromised or immature immune system. Upon adhesion, fusion of the virus envelope with the host cell is initiated. In this step, the viral glycoprotein gB is considered to represent the major fusogen. Here, we present for the first time structural data on the binding of an anti-herpes virus antibody and describe the atomic interactions between the antigenic domain Dom-II of HCMV gB and the Fab fragment of the human antibody SM5-1. The crystal structure shows that SM5-1 binds Dom-II almost exclusively via only two CDRs, namely light chain CDR L1 and a 22-residue-long heavy chain CDR H3. Two contiguous segments of Dom-II are targeted by SM5-1, and the combining site includes a hydrophobic pocket on the Dom-II surface that is only partially filled by CDR H3 residues. SM5-1 belongs to a series of sequence-homologous anti-HCMV gB monoclonal antibodies that were isolated from the same donor at a single time point and that represent different maturation states. Analysis of amino acid substitutions in these antibodies in combination with molecular dynamics simulations show that key contributors to the picomolar affinity of SM5-1 do not directly interact with the antigen but significantly reduce the flexibility of CDR H3 in the bound and unbound state of SM5-1 through intramolecular side chain interactions. Thus, these residues most likely alleviate unfavorable binding entropies associated with extra-long CDR H3s, and this might represent a common strategy during antibody maturation. Models of entire HCMV gB in different conformational states hint that SM5-1 neutralizes HCMV either by blocking the pre- to postfusion transition of gB or by precluding the interaction with additional effectors such as the gH/gL complex.     

Differential effects of sphingomyelin hydrolysis and resynthesis on the activation of NF-kappa B in normal and SV40-transformed human fibroblasts

The precise role of ceramide in NF-kappaB signaling remains unclear. The recent observation of differential sphingomyelin synthase (SMS) activity in normal (low SMS) versus SV40-transformed (high SMS) WI38 human lung fibroblasts provides an opportunity to assess the involvement of ceramide and SMS in NF-kappaB activation. Treatment of normal WI38 fibroblasts with bacterial sphingomyelinase resulted in a 4-fold elevation of ceramide and blocked NF-kappaB activation by serum stimulation. Such inhibition was not observed in SV40-transformed fibroblasts. Under regular growth conditions, after sphingomyelinase was washed out, normal WI38 did not show SM re-synthesis nor NF-kappaB activation. In SV40-WI38, on the other hand, sphingomyelinase washout induced resynthesis of SM due to the action of SMS on ceramide generated at the plasma membrane. NF-kappaB activation correlated with SM resynthesis. This activation was abrogated by D609, which inhibited SM resynthesis but not the initial formation of ceramide. The differential activity of SMS may explain the effects of ceramide in NF-kappaB signaling: in the absence of significant SMS activity, ceramide inhibits NF-kappaB, whereas with high SMS, the conversion of the ceramide signal to a diacylglycerol signal by the action of SMS stimulates NF-kappaB. These results also suggest a role for SMS in regulating NF-kappaB.     

Sulfur mustard induced cytokine production and cell death: Investigating the potential roles of the p38, p53, and NF‐κB signaling pathways with RNA interference

Cutaneous and ocular injuries caused by sulfur mustard (SM; bis‐(2‐chloroethyl) sulfide) are characterized by severe inflammation and death of exposed cells. Given the known roles of p38MAPK and NF‐κB in inflammatory cytokine production, and the known roles of NF‐κB and p53 in cell fate, these pathways are of particular interest in the study of SM injury. In this study, we utilized inhibitory RNA (RNAi) targeted against p38α, the p50 subunit of NF‐κB, or p53 to characterize their role in SM‐induced inflammation and cell death in normal human epidermal keratinocytes (NHEK). Analysis of culture supernatant from 200 μM SM‐exposed cells showed that inflammatory cytokine production was inhibited by p38α RNAi but not by NF‐κB p50 RNAi. These findings further support a critical role for p38 in SM‐induced inflammatory cytokine production in NHEK and suggest that NF‐κB may not play a role in the SM‐induced inflammatory response of this cell type. Inhibition of NF‐κB by p50 RNAi did, however, partially inhibit SM‐induced cell death, suggesting a role for NF‐κB in SM‐induced apoptosis or necrosis. Interestingly, inhibition of p53 by RNAi potentiated SM‐induced cell death, suggesting that the role of p53 in SM injury, may be complex and not simply prodeath. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:155–164, 2010; Published online inWiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20321     

Platelet-neutrophil interaction aggravates vascular inflammation and promotes the progression of atherosclerosis by activating the TLR4/NF-κB pathway

Platelet-neutrophil interaction is well known for its role in inflammatory diseases; however, its biological role in atherosclerosis (AS) progression remains unclear. Human peripheral blood neutrophils were obtained to compare toll-like receptor 4 (TLR4), tumor necrosis factor α (TNF-α), interleukin (IL)-1β and myeloid-related proteins 8/14 (Mrp8/14) levels in 22 AS patients with those in 18 healthy controls using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Meanwhile, mouse marrow neutrophils subjected to different treatment were collected for the ELISA assay, cell apoptosis, and Western blot analysis. Normal diet or high-fat diet ApoE^−/− mice with or without administration of Mrp8/14 antagonist paquinimod were used for plasma collection to measure total cholesterol, triglycerides, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol, TNF-α, IL-1β, Mrp8/14, TLR4, and nuclear factor (NF)-κB p65 levels. The results showed that Mrp8/14 and TLR4-mediated inflammatory pathway was activated in neutrophils of AS patients. In vitro experiments demonstrated that platelet-neutrophil interaction promoted the Mrp8/14 release and inhibited neutrophil apoptosis via P-selectin. Furthermore, platelet-neutrophil interaction upregulated TLR4/myeloid differentiation factor 88/NF-κB pathway. Conversely, Mrp8/14/TLR4/NF-κB interference alleviated AS progression. In conclusion, Mrp8/14/TLR4/NF-κB activated by platelet-neutrophil interaction is an important inflammatory signaling pathway for AS pathogenesis.     

Fate of streptomycin in mycelium of the producer organism

Summary While the mycelium of streptomycin (SM) producer takes up extracellular SM, it eliminates the accumulated SM after phosphorylation with SM 6-kinase. The rate of uptake of SM, rather than the content of SM 6-kinase in mycelium, limits the capacity of mycelium to phosphorylate the extracellular SM.     

Silencing of ATP2B1‐AS1 contributes to protection against myocardial infarction in mouse via blocking NFKBIA‐mediated NF‐κB signalling pathway

Myocardial infarction (MI) is an acute coronary syndrome that refers to tissue infarction of the myocardium. This study aimed to investigate the effect of long intergenic non‐protein‐coding RNA (lincRNA) ATPase plasma membrane Ca²⁺ transporting 1 antisense RNA 1 (ATP2B1‐AS1) against MI by targeting nuclear factor‐kappa‐B inhibitor alpha (NFKBIA) and mediating the nuclear factor‐kappa‐B (NF‐κB) signalling pathway. An MI mouse model was established and idenepsied by cardiac function evaluation. It was determined that ATP2B1‐AS1 was highly expressed, while NFKBIA was poorly expressed and NF‐κB signalling pathway was activated in MI mice. Cardiomyocytes were extracted from mice and introduced with a series of mouse ATP2B1‐AS1 vector, NFKBIA vector, siRNA‐mouse ATP2B1‐AS1 and siRNA‐NFKBIA. The expression of NF‐κBp50, NF‐κBp65 and IKKβ was determined to idenepsy whether ATP2B1‐AS1 and NFKBIA affect the NF‐κB signalling pathway, the results of which suggested that ATP2B1‐AS1 down‐regulated the expression of NFKBIA and activated the NF‐κB signalling pathway in MI mice. Based on the data from assessment of cell viability, cell cycle, apoptosis and levels of inflammatory cytokines, either silencing of mouse ATP2B1‐AS1 or overexpression of NFKBIA was suggested to result in reduced cardiomyocyte apoptosis and expression of inflammatory cytokines, as well as enhanced cardiomyocyte viability. Our study provided evidence that mouse ATP2B1‐AS1 silencing may have the potency to protect against MI in mice through inhibiting cardiomyocyte apoptosis and inflammation, highlighting a great promise as a novel therapeutic target for MI.     

Type A and type B monoamine oxidase activities in rat brain and liver mitochondria: a comparison of their properties using the non-ionic detergent Triton X-100

1. The effects of the non-ionic detergent Triton X-100 on the heterogeneity of monoamine oxidase activities were studied and compared in synaptic (fractions SM and SM2) and non-synaptic (fraction M) brain mitochondria and liver mitochondria. 2. Triton X-100 inhibited type A and type B monoamine oxidase activities in all four mitochondrial fractions in a concentration-dependent manner. Liver mitochondrial enzymatic activities were much more sensitive to this inhibition than those of brain mitochondria. The activities in the SM fraction of synaptic brain mitochondria were the least susceptible. 3. In all four mitochondrial fractions, type A activities were more sensitive to inhibition than type B activities. 4. These results suggest that the membrane micro-environment around the enzyme molecules in situ may be important in the functional expression of the activity of the enzyme.     

Association of common variants in KIF21B and ankylosing spondylitis in a Chinese Han population: a replication study

KIF21B polymorphisms were found associated with susceptibility to multiple sclerosis and ankylosing spondylitis (AS) in populations of white European ancestry. We aimed to replicate the association of polymorphisms around KIF21B and AS in a Chinese Han population. This case–control study included 665 patients with AS and 1,042 healthy controls genotyped for seven single nucleotide polymorphisms (SNPs) of KIF21B—rs12118246, rs4915464, rs502658, rs10494829, rs12089839, rs6687260, and rs957957—by TaqMan genotyping assay; statistical analyses involved the use of PLINK. We also estimated the linkage disequilibrium and haplotypes of these SNPs. Two SNPs—rs502658 (allelic p?=?0.0002, odds ratio [OR] 0.60, 95 % confidence interval [95 % CI] 0.47–0.76) and rs10494829 (allelic p?=?0.003, OR 1.30, 95 % CI 1.12–1.52)—were significantly associated with AS in the Chinese Han population. In addition, a linear regression test showed that they have independent contribution to disease susceptibility. For both SNPs, haplotype AT was strongly associated with AS and increased the risk of the disease (p?=?0.045, OR 1.183, 95 % CI 1.004–1.395), and the genotype GC reduced the risk (p?=?0.011, OR 0.715, 95 % CI 0.55–0.928). This work identified a significant association of two SNPs in KIF21B and AS in the Chinese Han population. KIF21B may play an important role in the pathogenesis of AS in the Chinese population and might be a new therapeutic target for AS.     

Non-autonomous mobile elements in the crenarchaeon Sulfolobus solfataricus

The genome of the archaeon Sulfolobus solfataricus P2 contains at least four types of short sequence elements lacking open reading frames which are similar to eukaryal non-autonomous mobile elements. The most- conserved elements SM1 (79-80 bp) and SM2 (183-186 bp), with 95 % sequence identity, are present in 40 and 25 copies, respectively. The less-conserved elements SM3 (127-139 bp) and SM4 (160-168 bp), with 75-97 % identity, occur in 44 and 34 copies, respectively. In total, the 143 SM elements constitute about 0.6 % of the genome. The wide distribution of each class of conserved element throughout the genome, and their precise locations, indicate that they are mobile. Direct evidence arises from the presence of SM1 and SM2 in only a fraction of genomic copies of a given class of insertion element, and within copies of open reading frames that are conserved in sequence. SM1 to SM4 are likely to be mobilized by transposases encoded by insertion elements ISC1048, ISC1217, ISC1058 and ISC1173, respectively. Furthermore, the occurrence of clusters of interwoven SM and insertion elements, in potentially mobile units, suggests a mechanism for the transfer of SM elements to other organisms.     

Reassessment of the evolution of wheat chromosomes 4A, 5A,and 7B

Key message

Comparison of genome sequences of wild emmer wheat and Aegilops tauschii suggests a novel scenario of the evolution of rearranged wheat chromosomes 4A, 5A, and 7B.

Abstract

Past research suggested that wheat chromosome 4A was subjected to a reciprocal translocation T(4AL;5AL)1 that occurred in the diploid progenitor of the wheat A subgenome and to three major rearrangements that occurred in polyploid wheat: pericentric inversion Inv(4AS;4AL)1, paracentric inversion Inv(4AL;4AL)1, and reciprocal translocation T(4AL;7BS)1. Gene collinearity along the pseudomolecules of tetraploid wild emmer wheat (Triticum turgidum ssp. dicoccoides, subgenomes AABB) and diploid Aegilops tauschii (genomes DD) was employed to confirm these rearrangements and to analyze the breakpoints. The exchange of distal regions of chromosome arms 4AS and 4AL due to pericentric inversion Inv(4AS;4AL)1 was detected, and breakpoints were validated with an optical Bionano genome map. Both breakpoints contained satellite DNA. The breakpoints of reciprocal translocation T(4AL;7BS)1 were also found. However, the breakpoints that generated paracentric inversion Inv(4AL;4AL)1 appeared to be collocated with the 4AL breakpoints that had produced Inv(4AS;4AL)1 and T(4AL;7BS)1. Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 either originated sequentially, and Inv(4AL;4AL)1 was produced by recurrent chromosome breaks at the same breakpoints that generated Inv(4AS;4AL)1 and T(4AL;7BS)1, or Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 originated simultaneously. We prefer the latter hypothesis since it makes fewer assumptions about the sequence of events that produced these chromosome rearrangements.