Effect of acid sphingomyelinase on nonalcoholic fatty liver disease via PPARα- PGC-1α pathway / 中国药理学通报

Aim To investigate the effects of acid sphingomyelinase(ASMase)on high-fat induced nonalcoholic fatty liver disease in mice and its regulation of PPARα- PGC-1α pathway. Methods ASMase knockout mice based on C57BL/6 background were constructed. Closed group heterozygotes were obtained through hybridized with wild-type mice(ASMase+/-),together with the littermate WT mice were prepared for NAFLD model in this study. The experiment was divided into four groups:WT+Chow:the WT mice were fed with normal diet for 12 weeks; WT+HFD:the WT mice were fed with high-fat diet for 12 weeks; ASMase+/-+Chow:the ASMase+/- mice were fed with normal diet for 12 weeks; ASMase+/- +HFD:the ASMase+/- mice were fed with high fat diet for 12 weeks. Biochemical method was used to detect serum TC,TG and liver TC,TG contents and liver function such as ALT and AST. Oil red staining,HE staining,Masson staining and Sirius red staining were performed to detect liver lipid accumulation,hepatocyte morphology and liver fibrosis. AmplexTM red sphingomyelinase kit was applied to detect ASMase activity. Western blot was performed to detect protein expressions of ASMase,PPARα,PGC-1α and CPT1. Results WT+HFD group displayed hypercholesterolemia and liver dysfunction. Levels of liver triglyceride(TG)were significantly higher than those in WT+Chow group(P<0.05 or P<0.01). Meanwhile,the hepatocytes showed marked steatosis,balloon-like changes,and fibrosis. Protein expression and activity of ASMase in liver increased significantly(P<0.01 or P<0.001),whereas CPT1,PPARα and PGC-1α expressions were not statistically significant compared with matched control group. Heterozygously ASMase-deficient mice reduced the elevated liver TG induced by HFD,as well as improving balloon-like changes and liver fibrosis. Furthermore,the expressions of PPARα,PGC-1α and CPT1 were up-regulated in ASMase+/- +HFD mice compared with WT+Chow group.Conclusions ASMase promotes hepatic steatosis and fibrosis,which may be related to its inhibition of PPARα-PGC-1α pathway.

Advances in the diagnosis of acid sphingomyelinase deficiency / 中国医师杂志

Acid sphingomyelinase deficiency (ASMD), also known as type A and B Niemann-Pick disease, is a group of intra-lysosomal lipid storage diseases caused by mutations in the SMPD1 gene that decrease acid sphingomyelinase activity or even cause deletion, resulting in abnormal deposition of sphingolipids. This disease can be diagnosed by bone marrow aspiration, pathological biopsy, acid sphingomyelinase activity measurement and SMPD1 gene testing. In recent years, with the rapid progress of molecular diagnostic techniques, new insights have been gained in the laboratory diagnosis of ASMD by means of molecular genetic tests, biomarkers and acid sphingomyelinase activity assay. This article will review the diagnostic progress of ASMD, aiming to reduce the misdiagnosis and leakage of the disease and improve the clinicians′ understanding of the disease.

Loxoscelismo cutáneo-visceral en una zona rural con desenlace favorable / Viscerocutaneous loxoscelism with a favorable outcome in a rural area

En el Perú los accidentes por mordedura de araña representan un problema de salud colectiva; de estos, el producido por la araña Loxosceles ha sido causa de numerosas muertes sobre todo en la costa. Esta tiende a ocasionar, ya sea una lesión cutánea o un cuadro sistémico, que puede llevar a la muerte del individuo si no es tratado a tiempo. Hasta la fecha no se cuenta con un protocolo de diagnóstico, predicción ni manejo a nivel internacional, por lo que se utilizan opciones terapéuticas sin respaldo de evidencia. Sin embargo, el manejo de soporte oportuno y adecuado es crucial para los cuadros severos. Se presenta el caso inusual de un loxoscelismo cutáneo-visceral o también llamado sistémico ocurrido en una zona rural. Hubo un compromiso renal severo que requirió hemodiálisis con un desenlace favorable a pesar del no uso de suero antiloxoscélico, lo que evidencia la importancia del manejo oportuno con las medidas de soporte adecuadas(AU)

Accidents caused by spider bites are a public health problem in Peru. Of these, those related to Loxosceles spider bites have been the cause of numerous deaths, mainly on the coast. These bites generally result in a cutaneous lesion or systemic involvement, which may threaten the person's life if not treated timely. An international protocol is not yet available for the diagnosis, prediction or management of Loxosceles spider bites. Therefore, therapeutic options are applied which are not supported by evidence. Still, timely and appropriate support management is crucial in severe cases. An unusual case of viscerocutaneous loxoscelism is presented, also known as systemic loxoscelism, which occurred in a rural area. The case was characterized by severe renal involvement requiring hemodialysis, but its outcome was favorable, despite not using antiloxoscelic serum, which shows the importance of timely management based on appropriate support measures(AU)

Effect of desipramine on apoptosis and endoplasmic reticulum stress in atherosclerotic plaque of rabbits / 中国药理学通报

Aim: To investigate the effect of desipramine (DES) on apoptosis and endoplasmic reticulum stress(ERS) in atherosclerotic model of rabbits. Methods: The rabbit model of atherosclerosis (AS) was established through abdominal aorta balloon injury and high fat diets for 12 weeks. They were divided into high-fat diet(HFD) group and HFD + DES group randomly. The same numbers of healthy rabbits with chow diet were divided randomly into normal control (NC) group and DES group. All interventions were given for the last 4 weeks. At the end of week 12, serum lipid was tested by conventional method. Plasma ox-LDL was measured by ELISA. Plasma and arterial acid sphingomyelinase(ASM) activity and ceramide levels were detected by UPLC analysis. Cell apoptosis in abdominal aorta was measured by TUNEL staining. Expression of GRP78 and CHOP proteins were detected by Western blot. Results: On the one hand, DES had no effect on serum lipid profiles including TG, TC, HDL-C.LDL-C and ox-LDL levels compared with either healthy or atherosclerosis rabbits. On the other hand, DES inhibited ASM and ceramide levels both in plasma and aorta, and decreased apoptotic cells and proteins of GRP78 and CHOP expression in abdominal aorta. Conclusions: DES attenuates AS via inhibition of ASM, down-regulating ceramide, attenuating ERS and thus reducing the apoptosis of AS plaques.

nSMase2/ceramide Regulates High Calcium and Phosphate-Induced Osteogenic Differentiation and Calcification of Vascular Smooth Muscle Cells / 中山大学学报(医学科学版)

[Objective] Vascular calcification is a gene-regulated biological process similar to bone formation.It is very common in the patients with chronic kidney disease.Neutral sphingomyelinase 2 (nSMase2) is a key regulator of bone development,and is responsible for ceramide generation through sphingomyelin hydrolysis,but the role of nSMase2 in vascular calcification remains unclear.The aim of this study is to determine whether nSMase2 regulates high calcium and phosphate-induced calcification of vascular smooth muscle cells (VSMC).[Methods] In vitro model of human VSMC calcification was used in this study and high calcium and phosphate were used to induce calcification of VSMCs.GW4869 was used to inhibit nSMase2 activity and nSMase2 was knockdowned in cultured VSMC using nSMase2 siRNA.The expression of Runx2,BMP2 and Osterix was analyzed by qRT-PCR and calcification was assessed by alizarin red staining.[Results] We found that nSMase2 expression and ceramide levels were increased in the process of VSMC calcification (P＜0.05).Inhibition of nSMase2 activity by GW4869 and knockdown of nSMase2 attenuated high calcium and phosphate-induced VSMC calcification and down-regulated the expression level of Runx2,BMP2 and Osterix (P＜0.05).By contrast,ceramide accelerated rat VSMC calcification and increased ALP activity (P＜0.05).[Conclusion] We demonstrate that nSMase2/ceramide promotes high calcium and phosphate-induced VSMC calcification,suggesting that nSMase2/ceramide could participate in the progression of vascular calcification in patients with chronic kidney disease.

Caracterización clínica del loxoscelismo dermonecrótico en equinos de córdoba, colombia / Clinical characterization of dermonecrotic loxoscelism in equine of Córdoba, Colombia / Caracterização clínica da loxoscelismo dermonecrótica em equinos de Córdoba, Colômbia

Resumen El objetivo de este estudio fue evaluar las características clínicas del loxoscelismo dermonecrótico (LDN) en equinos del departamento de Córdoba, Colombia. El estudio, fue de tipo descriptivo, no probabilístico, las muestra fueron seleccionadas por conveniencia. Se utilizaron nueve caballos criollos (Equus ferus caballus) y dos burros (Equus asinus africanus) con LDN diagnosticados clínica e histopatológicamente en diferentes producciones del Departamento. Al describir las lesiones cutáneas, éstas se caracterizaron por la presencia de una severa dermatitis necrótica, con edema y un área eritematosa focal, al detallar el área lesionada, se observó un punto necrótico central y dos halos alrededor (un halo blanco medial y un halo violáceo más externo) en la mayoría de los casos estudiados; así como ausencia de signos neurológicos y sistémicos. Las lesiones se ubicaron en la mayoría de los casos a nivel dorsal, seguido de la región rostral, pecho y pene. Histopatológicamente en la coloración de Hematoxilina Eosina (H&E), se observó severa dermatitis piogranulomatosa, con marcada infiltración de polimorfonucleares especialmente neutrófilos, con vacuolización de la capa basal de la epidermis y edema en la unión dermo-epidérmica. El diagnóstico definitivo de LDN en los 11 animales estudiados, se fundamentó en las manifestaciones clínicas observadas, las características anatomopatológicas de las lesiones y los resultados histopatológicos. El presente informe constituye el primer reporte de LDN en el Departamento de Córdoba, ya que no se encontraron reportes en la literatura consultada.

Abstract The aim of this study was to characterize the clinical aspects of dermonecrotic loxoscelism (LDN) in horses department of Cordoba, Colombia. This study was descriptive, not probabilistic, in animals of convenience. Nine horses (Equus ferus caballus) and 2 donkeys (Equus asinus africanus) were used with LDN diagnosed clinically and histopathologically in different productions of the Department. When describing the skin lesions, these were characterized by the presence of severe necrotic dermatitis, with edema and a focal erythematous area, when detailing the injured area, a central necrotic spot and two halos around it (a medial white halo and a halo Violet) in most of the cases studied; As well as absence of neurological and systemic signs. The lesions were located in most cases at the dorsal level, followed by the rostral region, chest and penis. Histopathologically in the staining of Hematoxylin Eosin (H & E), severe piogranulomatous dermatitis was observed, with marked infiltration of polymorphonuclear cells especially neutrophils, with vacuolization of the basal layer of the epidermis and edema in the dermo-epidermal junction. The definitive diagnosis of LDN in the 11 animals studied was based on the clinical manifestations observed, the anatomopathological characteristics of the lesions and the histopathological results. The present report constitutes the first LDN report in the Department of Córdoba, since no reports were found in the consulted literature.

Resumo O objetivo deste estudo foi caracterizar os aspectos clínicos da loxoscelismo dermonecrótica (LDN) em equinos de Córdoba, Colômbia. Este estudo foi descritivo, não probabilístico, em animais de conveniência. Foram usados 9 cavalos (Equus ferus caballus) e 2 jumentos (Equus asinus africanus) com LDN clinicamente e histologicamente diagnosticado no Departamento diferentes produções. Ao descrever as lesões da pele, que foram caracterizados pela presença de dermatite necrotizante grave, edema e focal área eritematosa, detalhando a área lesionada, um ponto necrótico central e dois halos foram observados em torno de (halo branco medial e halo mais exterior) violáceo, na maioria dos casos estudados; e ausência de sinais neurológicos e sistémicas. As lesões foram localizados na maioria dos casos ao nível dorsal, seguida da rostral, peito e do pénis. Foi observada histologicamente em hematoxilina-eosina (H & E), dermatite piogranulomatosa grave, com infiltração acentuada de neutrófilos polimorfonucleares especialmente com vacuolização da camada basal da epiderme e edema na junção dermo-epidérmica. O diagnóstico definitivo da LDN nos 11 animais estudados foi baseado nas manifestações clínicas observadas, características histopatológicas de lesões e os resultados histopatológicos. O presente relatório constitui o primeiro relatório LDN no Departamento de Córdoba, uma vez que não foram encontrados relatórios na literatura consultada.

Evolución clínica de pacientes con loxoscelismo sistémico y dermonecrótico en un hospital de tercer nivel / Clinical evolution of patients with systemic and dermonecrotic loxoscelism in a third level hospital

Resumen ANTECEDENTES: el loxoscelismo es una intoxicación por la mordedura de la araña Loxosceles reclusa, cuyo veneno contiene esfingomielinasa-D, causante de hemólisis y necrosis. Se reporta una serie de casos que describen su evolución clínica y respuesta al tratamiento. OBJETIVO: describir la evolución y características clínicas de pacientes con loxoscelismo sistémico y dermonecrótico, su respuesta al tratamiento y las complicaciones. PACIENTES Y MÉTODO: estudio que incluyó el análisis descriptivo de pacientes tratados en el servicio de Medicina Interna, Unidad Médica de Alta Especialidad Dr. Antonio Fraga Mouret, Centro Médico Nacional La Raza, de 2010 a 2015. RESULTADOS: se atendieron ocho hombres (67%) y cuatro mujeres (33%), con edad media de 39.4 años (16-83 años). El sitio de mordedura en cinco casos (42%) fue el miembro pélvico izquierdo; cuatro casos en el miembro torácico derecho (33%), dos en la cara (17%) y uno en el miembro torácico izquierdo (9%). Nueve casos provenían del Estado de México, dos de la Ciudad de México y uno de Hidalgo. Manifestación clínica: flictenas (cinco casos), edema y eritema (tres), placa liveloide (tres) y necrosis (un caso). Nueve (75%) pacientes se trataron inicialmente en la unidad de cuidados intensivos. Se reportaron las siguientes complicaciones sistémicas: renales (67%), pulmonares con administración de aminas (33%) y hematológicas (8%). Diez casos recibieron faboterápico, con media de 1.5 viales (0 a 4); cinco casos (42%) recibieron dapsona y cuatro de ellos (33%) padecieron metahemoglobinemia; once (92%) pacientes requirieron lavado-desbridación y 7 (58%) injerto cutáneo; cuatro pacientes (33%) tuvieron infección agregada de la herida. El promedio de estancia hospitalaria fue 16.2 días (3 a 40 días). CONCLUSIÓN: la evolución y pronóstico de esta afección depende de una sospecha inicial, diagnóstico y tratamiento oportunos. El loxoscelismo debe incluirse en los diagnósticos diferenciales de lesiones necróticas y progresivas, con o sin afección sistémica.

Abstract BACKGROUND: Loxoscelism is a poisoning caused by the bite of Loxosceles recluse spider, whose venom contains sphingomyelinaseD, causing hemolysis and necrosis. We report a case series describing their clinical course and response to treatment. OBJECTIVE: To describe the evolution and clinical characteristics of patients with systemic and dermonecrotic loxoscelism, their response to treatment and complications. PATIENTS AND METHOD: A descriptive analysis of patients treated in the Internal Medicine Service, Centro Médico Nacional La Raza, from 2010 to 2015. RESULTS: A total of 8 men (67%) and 4 women (33%) were included. Mean age was 39.4 years (16-83 years). Bite site was left pelvic limb in 5 cases (42%), 4 in the right forelimb (33%), 2 in the face (17%) and 1 in left forelimb (9%). Nine cases came from Estado de México, 2 from Mexico City and 1 from Hidalgo. Initial manifestations included blisters (five cases), edema and erythema (three cases), liveloide plate (three cases) and necrosis (one case). Nine (75%) patients were initially managed in ICU. Systemic complications were renal (67%), lung with use of amines (33%) and hematological (8%). Ten cases were treated with fabotherapy, with an average of 1.5 vials (0-4). Five cases (42%) received dapsone and 4 of them (33%) developed methemoglobinemia. Eleven (92%) required surgical washing and debridement and 7 (58%) skin graft; four patients (33%) had secondary wound infection. Average hospital stay was 16.2 days (3-40 days). CONCLUSIONS: The evolution and prognosis depends on initial suspicion early diagnosis and treatment. Loxoscelism should be included in the differential diagnosis of progressive necrotic lesions, with or without systemic involvement.

Actividad de una esfingomielinasa ácida tipo C producida por Mycobacterium tuberculosis / An acidic sphingomyelinase Type C activity from Mycobacterium tuberculosis

Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Sphingolipids are recognized as diverse and dynamic regulators of a multitude of cellular processes mediating cell cycle control, differentiation, stress response, cell migration, adhesion, and apoptosis. Bacterial SMases are virulence factors for several species of pathogens. Whole cell extracts of Mycobacterium tuberculosis strains H37Rv and CDC1551 were assayed using [N-methyl-14C]-sphingomyelin as substrate. Acidic Zn2+-dependent SMase activity was identified in both strains. Peak SMase activity was observed at pH 5.5. Interestingly, overall SMase activity levels from CDC1551 extracts are approximately 1/3 of those of H37Rv. The presence of exogenous SMase produced by M. tuberculosis during infection may interfere with the normal host inflammatory response thus allowing the establishment of infection and disease development. This Type C activity is different from previously identified M. tuberculosis SMases. Defining the biochemical characteristics of M. tuberculosis SMases helps to elucidate the roles that these enzymes play during infection and disease.

Las esfingomielinasas (SMasas) catalizan la hidrólisis de esfingomielina a ceramida y fosforilcolina. Los esfingolípidos son reconocidos como reguladores diversos y dinámicos de una multitud de procesos celulares que median en el control del ciclo celular, la diferenciación, la respuesta al estrés, la migración celular, la adhesión y la apoptosis. Las esfingomielinasas bacterianas son factores de virulencia reconocidos en varias especies de patógenos. En este trabajo se analizaron los extractos de células enteras de las cepas de Mycobacterium tuberculosis H37Rv y CDC1551 utilizando [N-metil-14C]-esfingomielina como sustrato. Se identificó actividad de SMasa-ácida dependiente de zinc en ambas cepas. La actividad máxima se observó a pH 5.5. Curiosamente, los niveles de actividad de SMasa generados a partir de extractos de la cepa CDC1551 son aproximadamente un tercio de los de la cepa H37Rv. La presencia de una SMasa exógena producida por M. tuberculosis durante la infección puede interferir con la respuesta inflamatoria del huésped, permitiendo así el establecimiento de la infección y el desarrollo de la enfermedad. Esta actividad tipo C es distinta de las actividades previamente reportadas para M. tuberculosis. Definir las características bioquímicas de las esfingomielinasas de M. tuberculosis ayudará a dilucidar el papel que desempeñan estas enzimas durante la infección y la enfermedad.

Analysis of the mutations of SMPD1 gene in Niemann-Pick disease / 临床儿科杂志

Objectives To study the molecular genetics of Niemann-Pick's disease (NPD), and its implication in the diagnosis of NPD. Methods The clinical data and blood samples of three unrelated families were collected. The genomic DNA was extracted from peripheral blood. The six coding exons and their lfanking intronic sequences of SMPD1 gene in all members of three pedigrees were ampliifed by polymerase chain reaction (PCR). The SMPD1 gene sequencing results were compared with the normal sequence from Genbank to identify possible causative mutations. The ampliifcation products of exons where mutations were located were cloned into TA vector for further conifrmation. Results Family 1 proband had homozygous T107C mutation and the parents had heterozygous T107C mutation. The homozygous delete mutation (c.108-113delGCTGGC) was detected and conifrmed by TA cloning in all members of family 2 and 3. The 20 normal control members did not have this delete mutation. Conclusions The genetic basis of NPD in the proband of family 1 is the homozygous T107C mutation in SMPD1 gene, while parents in family 1 are carriers of recessive T107C mutation. The homozygous mutation c.108-113delGCTGGC exists in SMPD1 gene in all members of the family 2 and 3. This delete mutation is considered to be genetic polymorphism.

First report of in vitro selection of RNA aptamers targeted to recombinant Loxosceles laeta spider toxins

BACKGROUND: Loxoscelism is the envenomation caused by the bite of Loxosceles spp. spiders. It entails severe necrotizing skin lesions, sometimes accompanied by systemic reactions and even death. There are no diagnostic means and treatment is mostly palliative. The main toxin, found in several isoforms in the venom, is sphingomyelinase D (SMD), a phospholipase that has been used to generate antibodies intended for medical applications. Nucleic acid aptamers are a promising alternative to antibodies. Aptamers may be isolated from a combinatorial mixture of oligonucleotides by iterative selection of those that bind to the target. In this work, two Loxosceles laeta SMD isoforms, Ll1 and Ll2, were produced in bacteria and used as targets with the aim of identifying RNA aptamers that inhibit sphingomyelinase activity. RESULTS: Six RNA aptamers capable of eliciting partial but statistically significant inhibitions of the sphingomyelinase activity of recombinant SMD-Ll1 and SMD-Ll2 were obtained: four aptamers exert ~17% inhibition of SMD-Ll1, while two aptamers result in ~25% inhibition of SMD-Ll2 and ~18% cross inhibition of SMD-Ll1. CONCLUSIONS: This work is the first attempt to obtain aptamers with therapeutic and diagnostic potential for loxoscelism and provides an initial platform to undertake the development of novel anti Loxoscelesvenom agents.

Loxoscelismo cutáneo: revisión narrativa de un problema actual / Cutaneous loxoscelism: narrative review of a current problem

The endemic presence of Loxosceles laeta species in Chile determines a constant validity of loxoscelism in clinical practice. Among their clinical scope, cutaneous loxoscelismis the most common presentation and it usually requires surgical management. The objective of this article is to review basic knowledge, epidemiologic data, clinical and therapeutic knowledge and advances in this disease based on the available evidence. Evidence does not support the routinary use of any pharmacological agent in cutaneous loxoscelism. General therapeutic measures, adequate analgesia, the delimitation of thedermonecrotic area and in cases if needed a conservative surgical approach are recommended. The use of antibiotics should be restricted to cases of concomitant infection. Emphasis should be on prevention of these injuries, stressing the importance of early consultation in the emergency service, and monitoring for signs and symptoms of the systemic compromise. A conservative surgical approach minimizes unnecessary morbidity.

Analysis of Acid Sphingomyelinase Activity in Dried Blood Spots Using Tandem Mass Spectrometry

BACKGROUND: Niemann Pick disease (NP) is a rare, lysosomal storage disorder due to deficiency of the intra-lysosomal enzyme acid sphingomyelinase (ASM) resulting in intracellular accumulation of sphingomyelin. We evaluated a tandem mass spectrometry (MS/MS) method to analyze ASM activity in dried blood spots (DBS) that may be suitable for laboratory diagnosis of NP including high throughput screening of at-risk populations and potentially for newborn screening. METHODS: ASM activity was measured in 3.2 mm punches from DBS. The eluate was incubated with the ASM substrate (N-Hexanoyl-D-erythro-sphingosylphosphorylcholine [C6-sphingomyelin (C29H59N2O6P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C22H43NO3)]). ASM product and IS were analyzed using MS/MS in multiple reaction monitoring mode for transitions m/z 370.6>264.3 (ASM internal standard) and m/z 398.6>264.3 (ASM product). RESULTS: ASM activities were stable for up to 2 months at or below 4degrees C. Position of the punch in the DBS and/or hematocrit of the DBS had a limited effect on ASM activities. Both intra- and inter-assay variability were below 10%. There was no carry-over. The median ASM activity in 2,085 newborn infants was 9.5 micromol/h/L (mean 10.6) with a SD of 5.06 micromol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 micromol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 micromol/h/L). CONCLUSIONS: This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP.

Analysis of Acid Sphingomyelinase Activity in Dried Blood Spots Using Tandem Mass Spectrometry

BACKGROUND: Niemann Pick disease (NP) is a rare, lysosomal storage disorder due to deficiency of the intra-lysosomal enzyme acid sphingomyelinase (ASM) resulting in intracellular accumulation of sphingomyelin. We evaluated a tandem mass spectrometry (MS/MS) method to analyze ASM activity in dried blood spots (DBS) that may be suitable for laboratory diagnosis of NP including high throughput screening of at-risk populations and potentially for newborn screening. METHODS: ASM activity was measured in 3.2 mm punches from DBS. The eluate was incubated with the ASM substrate (N-Hexanoyl-D-erythro-sphingosylphosphorylcholine [C6-sphingomyelin (C29H59N2O6P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C22H43NO3)]). ASM product and IS were analyzed using MS/MS in multiple reaction monitoring mode for transitions m/z 370.6>264.3 (ASM internal standard) and m/z 398.6>264.3 (ASM product). RESULTS: ASM activities were stable for up to 2 months at or below 4degrees C. Position of the punch in the DBS and/or hematocrit of the DBS had a limited effect on ASM activities. Both intra- and inter-assay variability were below 10%. There was no carry-over. The median ASM activity in 2,085 newborn infants was 9.5 micromol/h/L (mean 10.6) with a SD of 5.06 micromol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 micromol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 micromol/h/L). CONCLUSIONS: This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP.

Potential role of acid sphingomyelinase in environmental health / 中南大学学报(医学版)

Acid sphingomyelinase (ASM) is one enzyme responsible for the production of ceramide via the hydrolysis of sphingomyelin.Recent findings have revealed the important role of ASM in the initiation of ceramide-induced cell apoptosis,as well as in the pathophysiology of many common diseases (e.g.cardiovascular diseases,diabetes,pulmonary diseases,and neurological diseases).Other studies have also shown that ASM activation may occur through the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS),as well as by inflammatory mechanisms that may be induced by environmental and occupational stresses.ASM activation,in turn,may create excess or abnormally distributed ceramides,which could lead to tissue and organ injuries,including to the pulmonary,liver,kidney,and nervous systems.This review will discuss the basic biology of ASM and focus on the role and regulation of ASM in environmental stress responses.We propose that ASM activation is an important factor in environmental health,and that ASM-based therapeutics may have a key role in preventing environmental-induced tissue injury.

The role of acid sphingomyelinase in the development and progression of tumors and its application value / 肿瘤

Acid sphingomyelinase (aSMase) is an important enzyme in sphingolipid metabolism. Studies concerning the relationship between aSMase and tumor are relatively rare. However, the relationship between aSMase and the development and progression of tumors has already been demonstrated in previously published research. In this review, the mechanism of aSMase activation and the role of aSMase in the development and progression of tumors as well as its application value are summarized. Copyright © 2012 by TUMOR.

Expression pattern and apoptosis-inducing activity to murine macrophages and hepatocytes of Leptospira interrogans Sph2 hemolysin / 中华微生物学和免疫学杂志

Objective To determine the change of expression level of Leptospira interrogans sph2 gene, and hemolytic and cell apoptosis-inducing activities of sphingomyelinase hemolysin Sph2. Methods Entire sph2 gene fragment was amplified by PCR from genomic DNA of L. Interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai, and sequenced after T-A cloning. Subsequently, a prokaryotic expression system of sph2 gene was constructed. The expression of target recombinant Sph2( rSph2 ) was examined by SDS-PAGE and the expressed rSph2 was extracted by Ni-NTA affinity chromatogaphy. The hemolytic activity of rSph2 was measured by hemolytic test in sheep blood agar plate and spectrophotometry-based hemoglobin measurement, and the apoptosis-inducing activity of rSph2 to murine mononuclear-macrophagelike cell line(J774A. 1) and hepatic cell line(IAR20) was determined by flow cytometry. A real-time fluorescence quantitative RT-PCR was applied to detect the change of sph2 mRNA levels before and after L. Interrogans strain Lai infecting J774A. 1 and IAR20 cells. Results The cloned sph2 gene had 100% sequence identity to the corresponding gene in GenBank. The constructed prokaryotic expression system was able to efficiently express rSph2. The rSph2 could lyse sheep erythrocytes in concentration-dependent pattern. 10μg/ml rSph2 could induce the apoptosis of J774A. 1 cells and IAR20 cells, and the peak apoptotic rates were 23.96% and 32.92%, respectively. The mRNA level of sph2 gene was significantly elevated within 0.5-2 h of L. Interrogans strain Lai infecting either J774A. 1 or IAR20 cells, and then the mRNA level was quickly descended. Conclusion The sph2 gene of L. Interrogans strain Lai has a transient expression when the microbe contacts host cells. rSph2 possesses activities of sheep erythrocyte lysis and inducing macrophage and hepatocyte apoptosis, indicating Sph2 as an important virulence factor during pathogenic process of Leptospira.

Functional analysis of Leptospira interrogans sphingomyelinase hemolysin genes and their transcriptional level alterations in the infected host cells / 中华微生物学和免疫学杂志

Objective To determine the hemolytic activity of products of sphingomyelinase hemolysin encoding genes of Leptospira interrogaas, and the transcriptional level alterations in the infected host cells. Methods By using genomic DNAs of pathogenic L. interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai and serogroup Pomona serovar Pomona strain Luo, and non-pathogenic L. biflexa serogroup Sama-ranga serovar Patoc strain Patoc Ⅰ as templates, PCRs were performed to amplify entire sph1-sph4 genes. The amplified products were sequenced after T-A cloning. Prokaryotic expression systems of sph1-sph4 genes were re-spectively constructed, and the expressions of target recombinant proteins rSph1-rSph4 were examined by SDS-PAGE. Ni-NTA affinity chromatographic column was used to extract the expressed rSph1-rSph4. Hemolytic ac-tivities of rSph1-rSph4 on sheep blood agar plate were identified. Transcription alterations of sphl-sph4 genes in L. interrogans strain Lai after infected J774A. 1 cells were measured by real-time fluorescence quantitative RT-PCR. Results From genomic DNAs of both L. interrogans strain Lai and Luo, but not from that of L. biflexa strain Patoc Ⅰ , the target fragments of sph1-sph4 genes could be amplified. All the cloned sph1-sph4 genes had 100% nucleotide sequence identities compared to the corresponding reported sequences. The constructed pro-karyotic expression systems were able to efficiently express the target recombinant proteins rSph1-rSph4, respec-tively. All the rSph1-rSph4 had hemolytic activities, and among the four products rSph2 displayed the strongest hemolytic activity. After L. interrogaas strain Lai infecting J774A. 1 cells, the transcriptional levels of sph1-sph4 genes were remarkably up-regulated, especially for mRNA levels of sph2 and sph4 genes. Conclusion sph1- sph4 genes exist only in pathogenic L. interrogans species, and their products have hemolytic activity. The up-regulation of sph1-sph4 gene transcriptional levels in L. interrogans strain Lai after infected cells implies that the sphingomyelinase hemolysins may play important roles in the process of L. interrogans infection in hosts.

Differential effects of Fas cross-linking on phospholipase D activation and related lipid metabolism in Fas-resistant A20 cells

A20 murine lymphoma cells undergoing Fas-mediated apoptosis showed increase in the activity of phospholipase D (PLD), which is involved in proliferative or mitogenic cellular responses. Using A20 cell lines that were resistant to Fas-induced apoptosis, we investigated the differential effects of Fas cross-linking on PLD activity and sphingolipid metabolism. The basal PLD activities in all of the selected three Fas-resistant clones (#5, #8, and #11) were about 2~4 folds higher than that of wild type A20 cells. Among the PLD isoforms, PLD2 expression was increased in all of the selected Fas-resistant clones. The Fas downstream signaling events triggered by Fas cross-linking, including the activations of PLD, phosphatidy-lcholine-specific phospholipase C (PC-PLC), sphingomyelinase (SMase), the increase in diacylglycerol (DAG) and protein phosphorylation levels, and the translocation of protein kinase C to membrane were not changed in both of Fas-resistant clone #5 and #8. In contrast, Fas cross-linking stimulated the activity of PLD, PC-PLC, and SMase, translocation of PKC, and protein phosphorylation in Fas-resistant clone #11, similar to that of wild type cells. We also found that clone #11 had a different Fas sequence encoding Fas B which has been known to inhibit Fas-induced apoptosis. These findings suggest that increased PLD2 expression resulting in increased basal PLD activity and the blockade of Fas downstream signaling cascades may be involved to limit apoptosis induced by Fas cross-linking.

Ceramide is Involved in MPP+ -induced Cytotoxicity in Human Neuroblastoma Cells

To understand the cytotoxic mechanism of MPP+, we examined the involvement of ceramide in MPP+ -induced cytotoxicity to human neuroblastoma SH-SY5Y cells. When SH-SY5Y cells were exposed to MPP+, MPP+ induced dose-dependent cytotoxicity accompanied by 2-fold elevation of intracellular ceramide levels in SH-SY5Y cells. Three methods were used to test the hypothesis that the elevated intracellular ceramide is related to MPP+ -induced cytotoxicity: C2-ceramide was directly applied to cells, sphingomyelinase (SMase) was exogenously added, and oleoylethanolamine (OE) was used to inhibit degradation of ceramide. Furthermore, inhibition of ceramide-activated protein phosphatase (CAPP), the effector of ceramide, using okadaic acid (OA) attenuated cell death but treatment of fumonisin B1, the ceramide synthase inhibitor, did not alter the cytotoxic effect of MPP+. Based on these, we suggest that the elevation of intracellular ceramide is one of the important mediators in MPP+ -induced cell death.