CD36 gene deletion reduces muscle insulin sensitivity in mice by up-regulating PTP1B expression / 南方医科大学学报

OBJECTIVE@#To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism.@*METHODS@#Wild-type (WT) mice and systemic CD36 knockout (CD36-/-) mice with normal feeding for 14 weeks (n=12) were subjected to insulin tolerance test (ITT) after intraperitoneal injection with insulin (1 U/kg). Real-time PCR was used to detect the mRNA expressions of insulin receptor (IR), insulin receptor substrate 1/2 (IRS1/2) and protein tyrosine phosphatase 1B (PTP1B), and Western blotting was performed to detect the protein expressions of AKT, IR, IRS1/2 and PTP1B in the muscle tissues of the mice. Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), respectively.@*RESULTS@#CD36-/- mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice (P < 0.05). CD36-/- mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice (P < 0.05). Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36-/- mice as compared with the WT mice (P < 0.01). No significant differences were found in mRNA and protein levels of IR, IRS1 and IRS2 in the muscle tissues between WT and CD36-/- mice (P>0.05). In the muscle tissue of CD36-/- mice, tyrosine phosphorylation levels of IR and IRS1 were significantly decreased (P < 0.05), and the mRNA and protein levels of PTP1B (P < 0.05) and histone acetylation level of PTP1B promoters (P < 0.01) were significantly increased as compared with those in the WT mice. Intraperitoneal injection of claramine, a PTP1B inhibitor, effectively improved the impairment of insulin sensitivity in CD36-/- mice.@*CONCLUSION@#CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions, and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression, which results in detyrosine phosphorylation of IR and IRS1.

The receptors and entry of measles virus: a review / 生物工程学报

Measles virus is an enveloped virus with a non-segmented negative-sense RNA genome. Two envelope glycoproteins on the viral surface, namely hemagglutinin (H) and membrane fusion protein (F), are responsible for the virus entry into susceptible host cells. The specific interaction between H and its cellular receptors is a key step in successful virus infection, determining the infectivity and tissue tropism of the measles virus. Thus far, three H receptors have been identified, including the complement regulatory molecule CD46, the signaling lymphocyte activation molecule (SLAM) and the cell adhesion molecule Nectin-4. Here, we reviewed our molecular understanding on the recognition mechanism of these receptors by the viral H protein, aiming to promote future studies on antiviral drug design and measles virus-based oncolytic therapy.

CD46 polymorphism: A probable risk factor for recurrent spontaneous abortion in a northern Indian population.

Background. CD46 is a complement regulatory glycoprotein. Certain polymorphic forms of the CD46 gene have been associated with recurrent pregnancy loss in the Caucasian population. We assessed the role of CD46 polymorphism in recurrent spontaneous abortion in our setting, as this has not been done on Indian subjects till date. Methods. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was carried out on 44 samples each from women with recurrent spontaneous abortion and normal pregnancy. Genotyping of the CD46 gene was done using 2.5% agarose gel. Statistical analysis was done using the TFPGA software. Results. The absence of CD46H*1 homozygosity was more pronounced in women with recurrent spontaneous abortion in the Indian population. Of recurrent aborters, 9% had the H*1/*1 genotype as compared to 30% of normal pregnant women. Conclusion. Although our data did not fit the Hardy– Weinberg equilibrium, this pilot study indicates that further increasing the sample size might clarify whether polymorphism in the first intron of the CD46 gene can be regarded as a risk factor for recurrent spontaneous abortion.

Inhibitive effects of chimeric oncolytic adenovirus SG235 on leukemia cells in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the inhibitive effects of chimeric oncolytic adenovirus SG235 on leukemia cells in vitro.</p><p><b>METHODS</b>The ability of SG235 to infect leukemia cells and the expression of CD46 on blasts from the patient with leukemia were detected by flow cytometry (FACS). The cytotoxicity of the virus was evaluated by MTT assay. Apoptosis induced by SG235 was detected with Annexin-V/PI staining and TUNEL assay followed by FACS analysis.</p><p><b>RESULT</b>The majority of leukemia cells from the patient with acute leukemia was CD46-positive. GFP-positive cells were 45.1%, 35.7%, 54.2%, 37.0%, 30.1%, %67.1, 17.2% and 33.1% in Mutz-1, Kasumi-1, K562, HL60, Molt- 4, RPMI8226, L428, and Jurkat cell lines treated with SG235-EGFP vector at MOI (multiplicity of infection) of 50 for 48 h.SG235 treatment resulted in marked growth inhibition and apoptosis of Kassumi-1 cells, and also significantly inhibited expression of p-Akt.</p><p><b>CONCLUSION</b>The chimeric oncolytic adenovirus SG235 can infect leukemia cell effectively and results in the growth inhibition and apoptosis of Kasumi-1 cells in vitro.</p>

A Case of Familial Atypical Hemolytic Uremic Syndrome Associated with Complement Factor H Mutation in Adults / 대한신장학회지

The hemolytic uremic syndrome is a clinical syndrome defined by the presence of thrombocytopenia, microangiopathic hemolytic anemia and acute renal failure. Atypical hemolytic uremic syndrome (aHUS) which is not usually associated with prodromal symptoms, especially diarrhea, has a higher mortality rate and a stronger tendency to progress to chronic renal failure. In approximately 30-50% of patients with aHUS, mutations have been detected in complement factor H, membrane cofactor protein or factor I. Mutations in the complement regulator factor H are the most frequent and have a very poor prognosis, with most patients developing ESRD. We have experienced a 33-year-old man with a family history of renal failure diagnosed as aHUS resulted from factor H mutation, for whom we carried out hemodialysis, plasmapheresis and other conservative management.

Atenuação do vírus vacinal do sarampo: infecção sub-ótima do tecido linfático e alteração do tropismo / Measles vaccine virus attenuation: suboptimal lymphatic tissue infection and tropism alteration

Os mecanismos de atenuação do vírus vacinal do sarampo ainda não foram suficientemente caracterizados. Uma vez que a linhagem vacinal consegue penetrar as células através do receptor CD46 além do receptor primário do vírus do sarampo CD150 / SLAM (molécula de ativação e de sinalização linfocitária) nós nos perguntamos se (e como) o seu tropismo está alterado. Em tecido amigdaliano humano, a linhagem vacinal infecta linfócitos T virgens (CD45RA+ CD62L+), que expressam SLAM em pequena quantidade, com muito mais eficiência que as linhagens selvagens. Por outro lado, a linhagem vacinal infecta significativamente menos linfócitos B, macrófagos e células NK que as linhagens selvagens. Os níveis de infecção das linhagens selvagens se correlacionam com a freqüência de expressão da SLAM, sendo mais elevados nos linfócitos B, que apresentam níveis de infecção entre 40-55%. As células T que expressam SLAM são mais facilmente infectáveis que as células B que expressam essa molécula. Desta forma, a atenuação da linhagem vacinal parece ser causada por uma alteração no seu tropismo, juntamente com uma replicação menos eficiente.

The mechanisms of measles virus (MV) vaccine attenuation are insufficiently characterized. Because the vaccine strain can enter cells through CD46 in addition to the primary MV receptor signaling lymphocyte activation molecule (SLAM, CD150), we asked whether and how its tropism is altered. In human tonsillar tissue, this vaccine strain infects naive (CD45RA+ CD62L+) T lymphocytes, which express SLAM very infrequently, with much higher efficiency than do wild-type strains. By contrast, it infects B-lymphocytes, macrophages, and NK cells with significantly lower efficiencies than those of wild-type strains. Infection levels by wild-type strains correlate with the frequency of SLAM expression, and are highest in B-cells, which are 40-55% infected. SLAM-expressing Tcells are more readily infected by all MV strains than are SLAM-expressing B-cells. Thus, vaccine attenuation may be caused by tropism alteration in combination with suboptimal replication.

Co-expression and synergic effect of human complement regulatory proteins DAF and MCP / 生物工程学报

Recombinant expression vector pcDNA3-DAFMCP-DP containing human membrane complement regulatory proteins (hCRPs) decay accelerating factor (DAF) and membrane cofactor protein (MCP) cDNA was constructed by using two independent promoters. After transfected into NIH3T3 cells by calcium phosphate-DNA precipitate method, NIH3T3 pcDNA3-DAFMCP-DP transfectants were obtained by G418 selection. Extraneous genes integration was identified by PCR. The co-expression of human DAF and MCP at both mRNA and protein levels was confirmed by using RT-PCR and Western blot analysis. Human DAF and MCP cDNA were integrated into NIH3T3 pcDNA3-DAFMCP-DP genomic DNA after continuous 30 times passages, indicating that NIH3T3 pcDNA3-DAFMCP-DP were stable cell lines. Human C-mediated cytolysis assays showed that NIH3T3 cells transfected stably with pcDNA3-DAF, pcDNA3-MCP, and pcDNA3-DAFMCP-DP were protected from C-mediated damage and co-expressed human DAF and MCP provided more excellent protection against C-mediated attack, which was compared with either DAF or MCP alone. These results suggest that the dicistronic vector could improve the efficiency of multi-gene delivery and benefit the synergic effect of human membrane complement regulatory proteins DAF and MCP.

Hemolytic uremic syndrome / 소아과

The hemolytic uremic syndrome (HUS) is a rare disease of microangiopathic hemolytic anemia, low platelet count and renal impairment. HUS usually occurs in young children after hemorrhagic colitis by shigatoxin-producing enterohemorrhagic E. coli (D+HUS). HUS is the most common cause of acute renal failure in infants and young children, and is a substantial cause of acute mortality and morbidity; however, renal function recovers in most of them. About 10% of children with HUS do not reveal preceding diarrheal illness, and is referred to as D- HUS or atypical HUS. Atypical HUS comprises a heterogeneous group of thrombomicroangiopathy (TMA) triggered by non-enteric infection, virus, drug, malignancies, transplantation, and other underlying medical condition. Emerging data indicate dysregulation of alternative complement pathway in atypical HUS, and genetic analyses have identified mutations of several regulatory genes; i.e. the fluid phase complement regulator Factor H (CFH), the integral membrane regulator membrane cofactor protein (MCP; CD46) and the serine protease Factor I (IF). The uncontrolled activation of the complement alternative pathway results in the excessive consumption of C3. Plasma exchange or plasma infusion is recommended for treatment of, and has dropped the mortality rate. However, overall prognosis is poor, and many patients succumb to end- stage renal disease. Clinical presentations, response to plasma therapy, and outcome after renal transplantation are influenced by the genotype of the complement regulators. Thrombotic thrombocytopenic purpura (TTP), another type of TMA, occurs mainly in adults as an acquired disease accompanied by fever, neurologic deficits and renal abnormalities. However, less frequent cases of congenital or hereditary TTP associated with ADAMTS-13 (a disintegrin and metalloprotease, with thrombospondin 1-like domains 13) gene mutations have been reported, also. Recent advances in molecular genetics better allow various HUS to be distinguished on the basis of their pathogenesis. The genetic analysis of HUS is important in defining the underlying etiology, predicting the genotype-related outcome and optimizing the management of the patients.

Activation of Intrarenal Complement System in Mouse Model for Chronic Cyclosporine Nephrotoxicity

PURPOSE: Local activation of the complement system plays a role in target organ damage. The aim of our study was to investigate the influence of cyclosporine (CsA)- induced renal injury on the complement system in the kidney. MATERIALS AND METHODS: Mice fed a low salt (0.01%) diet were treated with vehicle (VH, olive oil, 1mL/kg/day) or CsA (30mg/kg/day) for one or four weeks. Induction of chronic CsA nephrotoxicity was evaluated with renal function and histomorphology. Activation of the complement system was assessed through analysis of the expression of C3, C4d, and membrane attack complex (MAC), and the regulatory proteins, CD46 and CD55. CsA treatment induced renal dysfunction and typical morphology (tubulointerstitial inflammation and fibrosis) at four weeks. RESULTS: CsA-induced renal injury was associated with increased the expression of C3, C4d, and MAC (C9 and upregulation of complement regulatory proteins (CD 46 and CD55). Immunohistochemistry revealed that the activated complement components were mainly confined to the injured tubulointerstitium. CONCLUSION: CsA-induced renal injury is associated with activation of the intrarenal complement system.

Gene Expression Profile of Lung Cancer Cells Following Photodynamic Therapy / 결핵및호흡기질환

BACKGROUND: Photodynamic therapy is a viable option for lung cancer treatment, and many studies have shown that it is capable of inducing cell death in lung cancer cells. However, the precise mechanism of this cell death has not been fully elucidated. To investigate the early changes in cancer cell transcription, we treated A549 cells with the photosensitizer DH-I-180-3 and then we illuminated the cells. METHODS: We investigated the gene expression profiles of the the A549 lung cancer cell line, using a DEG kit, following photodynamic therapy and we evaluated the cell viability by performing flow cytometry. We identified the genes that were significantly changed following photodynamic therapy by performing DNA sequencing. RESULTS: The FACS data showed that the cell death of the lung cancer cells was mainly caused by necrosis. We found nine genes that were significantly changed and we identified eight of these genes. We evaluated the expression of two genes, 3-phosphoglycerate dehydrogenase and ribosomal protein S29. The expressed level of carbonic anhydrase XII, clusterin, MRP3s1 protein, complement 3, membrane cofactor protein and integrin beta 1 were decreased. CONCLUSION: Many of the gene products are membrane-associated proteins. The main mechanism of photodynamic therapy with using the photosensitizing agent DH-I-180-3 appears to be necrosis and this may be associated with the altered production of membrane proteins.

Expression of Complement Regulator Genes in Abeta1-42 Stimulated Human Neuroblastoma Cell

BACKGROUND: Endogenous complement inhibitors in the brain may protect against the neuroinflammation in Alzheimer's disease. Human neuroblastoma cells were stimulated by Abeta1 - 4 2 to investigate whether the expression of various complement regulator genes is upregulated. METHODS: SK-N-SH cells were incubated overnight with a single dose of 20 microM of Abeta1-42 or 0.5 ng/ml - 5 ng/ml of TNFalpha or both. Actinomycin D (2.5 microM) or cycloheximide (2.5 microM) was also added to the cell suspension. Messenger RNA expression of decay accelerating factor (DAF), membrane cofactor protein (MCP), CD59, complement-receptor 1(CR1), C1 inhibitor (C1-INH), C4-binding protein, factor H, factor I, clusterin and S-protein was measured by RT-PCR. RESULTS: Abeta1-42 and TNFalpha upregulated the expression of C1- INH significantly but increased expression of mRNA for factor H was not statistically significant. The expression of mRNAs for DAF and MCP was at low a level after stimulation. Factor I, CD59 and clusterin were not changed in their mRNA level. The mRNAs for S-protein, C4-binding protein and CR1 were not detected. Actinomycin D suppressed mRNA levels of C1-INH and CD59 significantly. Cycloheximide also inhibited the expression of both C1-INH and CD59. CONCLUSIONS: Early upregulated expression of C1-INH in Abeta1-42 stimulated neuroblastoma cell may contribute to a host defense mechanism against complement-mediated neuronal cell damage.

Expression of Complement Regulator Genes in Abeta1-42 Stimulated Human Neuroblastoma Cell

BACKGROUND: Endogenous complement inhibitors in the brain may protect against the neuroinflammation in Alzheimer's disease. Human neuroblastoma cells were stimulated by Abeta1 - 4 2 to investigate whether the expression of various complement regulator genes is upregulated. METHODS: SK-N-SH cells were incubated overnight with a single dose of 20 microM of Abeta1-42 or 0.5 ng/ml - 5 ng/ml of TNFalpha or both. Actinomycin D (2.5 microM) or cycloheximide (2.5 microM) was also added to the cell suspension. Messenger RNA expression of decay accelerating factor (DAF), membrane cofactor protein (MCP), CD59, complement-receptor 1(CR1), C1 inhibitor (C1-INH), C4-binding protein, factor H, factor I, clusterin and S-protein was measured by RT-PCR. RESULTS: Abeta1-42 and TNFalpha upregulated the expression of C1- INH significantly but increased expression of mRNA for factor H was not statistically significant. The expression of mRNAs for DAF and MCP was at low a level after stimulation. Factor I, CD59 and clusterin were not changed in their mRNA level. The mRNAs for S-protein, C4-binding protein and CR1 were not detected. Actinomycin D suppressed mRNA levels of C1-INH and CD59 significantly. Cycloheximide also inhibited the expression of both C1-INH and CD59. CONCLUSIONS: Early upregulated expression of C1-INH in Abeta1-42 stimulated neuroblastoma cell may contribute to a host defense mechanism against complement-mediated neuronal cell damage.

The Promoted Inhibition of Complement Aetivation by CR1 / MCP Hybrid Molecule

Hyperacute rejection (HAR) is the major obstacle to xenotransplantation. In HAR complement (C') cascade is activated following the binding of xenoreactive antibodies to the donor tissue. Complement receptor type 1 (CR1), the most efficient protein in inhibiting activated C's, was modified with membrane cofactor protein (MCP) to make a more efficient C'-inhibiting hybrid molecule. Modification was done by swapping the four active site short consensus repeats (SCRs) of MCP for the SCRs 8-11 of CR1. The hybrid molecule (CR1/MCP) was expressed on the surface of mouse L cells. When the complement inhibitory activity of the CR1/MCP protein was compared with that of the wild CR1 (wCR 1) protein, CR1/MCP's inhibitory activity was weaker than wCR1's. CR1/MCP protein's L cell protecting activity from complement's attack was more prominent in adherent state than in suspension state. From these results it was suggested that the conformational direction of MCP's inhibitory action on C' is different from that of CR1 and most of the MCP expression seems to be confined to the apical side but not to the basal side of the L cell in adherent state. The wCR1's expression seems to be present on all sides of the L cell. Finally, the inverted direction of SCRS-11 of CR1 or variable length of the serine-threoninrich structure of MCP could be tried to make other CR1/MCP variants with more powerful C' inhibitory activities.