Sphingolipids, especially ceramide and S1P, are structural components of biological membranes and bioactive molecules which participate in diverse cellular activities such as cell division, differentiation, gene expression and apoptosis. Emerging evidence demonstrates the role of sphingolipids in hepatocellular death, which contributes to the progression of several liver diseases including ischaemia-reperfusion liver injury, steatohepatitis or hepatocarcinogenesis. Furthermore, some data indicate that the accumulation of some sphingolipids contributes to the hepatic dysfunctions. Hence, understanding of sphingolipid may open up a novel therapeutic avenue to liver diseases. This review focuses on the progress in the sphingolipid metabolic pathway with a focus on hepatic diseases and drugs targeting the sphingolipid pathway.
Apoptosis ; Ceramides ; metabolism ; Fatty Liver ; metabolism ; physiopathology ; Humans ; Liver Diseases ; metabolism ; physiopathology ; Lysophospholipids ; metabolism ; Reperfusion Injury ; metabolism ; physiopathology ; Sphingolipids ; metabolism ; Sphingosine ; analogs & derivatives ; metabolism

Objective To analysis the genomic sequence of a novel human leukocyte antigen (HLA)-B*3818 allele.Methods Full length genomic sequence of an unknown HLA-B allele was cloned,followed by bi-directional sequencing and the specificity of the antigen coded by this novel allele was defined by microcytotoxicity assay.The frequency and haplotype of this novel allele was acquired by population census and parentage analysis.Results The full length genomic sequence of this novel HLA-B*3818 allele with accession number FJ561482 differs from HLA-B*380201 by two nucleotide changes in exon 4 and intron 5,respectively.One change is located at nt 660 in exon 4 where C→A alternation,which results in an amino acid substitution from Asp(GAC)to Glu(GAA)at codon 196.This alternation is a new single nucleotide polymorphism compared with all other HLA-B alleles.Another is located at genomic position 2133 in intron 5(A→C).Except for this substitution,the intron sequences of HLA-B*3818 allele are identical to those of other HLA-B*38 alleles including HLA-B*380101,B*380201 and B*3814.The serological specificity of HLA-B*3818 is B38 and the frequency of this new allele is less than 0.000 5 in Chinese Han population.The parentage analysis showed the haplotype of novel allele is A*030101-Cw*010201-B*3818-DRB1*1312-DOB1*060101.Conclusion The simultaneous mutations in exon and intron were found in the Hovel HLA-B*3818 allele,and so it can present more sequence information for studies and applications associated with HIA genes by analyzing the genomic sequences of novel HLA alleles.

OBJECTIVESThe mutations of growth hormone receptor (GHR) gene results in growth hormone insensitivity (Laron syndrome) or partial growth hormone insensitivity. This study aimed to understand the relation between mutations of GHR gene and short stature with non-growth-hormone deficiency, and the clinical feature of the patients with the GHR gene mutations.

METHODS(1) Forty-seven patients with non-growth-hormone deficiency and short stature were enrolled in this study, 33 were male and 14 female. The age of the patients were at a range of 2 - 16 years. (2) The mutations of GHR gene were identified by PCR-SSCP and DNA sequencing. (3) The characteristics of the GHR mutation was assumed by screening for the same mutations in patients' family members and the control samples.

RESULTS(1) Four GHR mutations were identified in 5 patients with non-growth-hormone deficiency: H56R, G148E, IVS6-30, -31CA > TG and IVS8 + 10G > C. These mutations were located within the extracellular domain of GHR and not reported before. Five patients were the heterozygous of H56R, G148E, IVS6-30, -31CA > TG and IVS8 + 10G > C. The detection rate of mutant heterozygous individual accounted for 10.6% (5/47). The mutations were considered non-polymorphism by the GHR gene analysis in patients' family members and control samples. (2) Comparison of the amino acid sequence of different species and the position of the mutations H56R and G148E in the GHR protein structure suggested impact of the mutations on the protein function. (3) A polymorphism site was identified in exon 6 of GHR gene: G168G (GGA > GGG). The allelic frequency of G168G had no difference between the patients with non-growth-hormone deficiency and control samples but had significant difference between Chinese and Caucasian. It seems that the G168G was a polymorphism and has no relationship with the height stature. However, there was the allele diversity in different races.

CONCLUSIONThe mutations of GHR gene were detected in the patients with non-growth-hormone deficiency. Special attention should be paid clinically to its potential pathogenesis for short stature.

Adolescent ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Case-Control Studies ; Child ; Child, Preschool ; DNA Mutational Analysis ; European Continental Ancestry Group ; genetics ; Female ; Growth Disorders ; genetics ; Humans ; Male ; Molecular Sequence Data ; Mutation ; Polymorphism, Genetic ; Receptors, Somatotropin ; genetics

Objective To analyze the ultrasonographic features of retroperitoneal ifbrosis (RPF). Methods Totally 13 patients with retroperitoneal ifbrosis from February 2000 to October 2012 in the Long Gang central Hospital of Shenzhen were retrospectively analyzed. Results In all patients who underwent ultrasound examination, there were ten cases of idiopathic RPF and three cases of secondary RPF with abdominal tumors. In 11 cases, the masses were hypoechoic locating at retroperitoneum and surrounding the abdominal aorta without deifnitive margin. One case showed hypoechoic mass with clear boundary. In ten cases, the internal echogenicity of masses were uniform. In two cases, the internal echogenicity of masses were uneven with a small amount of ifbrous separator with slightly higher echogenicity. No blood was found in all masses. The encasement of inferior vena cava was found in three casesand the masses extended to iliac arteries in three cases. Hydronephrosis could be found in 11 patients (84.6%) and ureter dilatation was found in ten cases. Ureteral localized stenosis were found in two cases. Conclusion Ultrasonography is a preferred imaging method in diagnosing RPF.

To construct gene vaccine of PPV and to investigate the effects of interleukin 2 (IL-2) as an adjuvant on immune responses in mouse, the recombinant expression plasmid of pCIneo-IL2-VP2 was constructed and transfected into PK-15 cells by lipofectamine, the expressed product was detected by immunofluore assay. To study the immune effects of DNA vaccine in vitro and in vivo, mice were used as the animal model. The recombinant plasmid pCIneo-IL2-VP2, the control plasmid pCI-neo and the PPV live vaccine were immunized by intramuscular injection. Anti-PPV antibodies were measured by ELISA, lymphocyte proliferation activity was detected using MTT method, and the specific killing activities of CTL were assayed too. The results show that the immunized mice produced PPV antibody after one week, and reached to highest after four weeks. Compared with the control group, the pCIneo-IL2-VP2 immunized group produced significant differences in the antibody titers, the lymphocyte proliferation activity and the specific killing activities of CTL. The pCIneo-IL2-VP2 induced humoral and cellular immunity responses similarly to that the live vaccine induced. These results manifested that the PPV DNA vaccine successfully induced humoral and cellular immunity response in mice with the IL-2 gene as an adjuvant.
Adjuvants, Immunologic ; genetics ; Animals ; Antibodies, Viral ; blood ; Antigens, Viral ; genetics ; immunology ; Capsid Proteins ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Immunization ; Interleukin-2 ; genetics ; immunology ; Mice ; Parvovirus, Porcine ; genetics ; immunology ; Random Allocation ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Vaccines, DNA ; immunology ; Viral Vaccines ; immunology

The aim of this study was to gain more insight into the understanding of myelodysplastic syndrome in the clinical and laboratory features. The clinical data of 65 patients with MDS were reviewed and analysed. According to FAB criteria, 65 patients were classified as follows: 27 patients with RA, 1 patient with RAS, 33 patients with RAEB, 3 patients with RAEB-T, and 1 patient with CMML. The median age of them was 66 years old (range 19-89 years), and 6 patients had a history of toxic exposure (secondary MDS). The bone marrow smears, bone marrow biopsy and cytogenetic examinations were performed in this study. The results showed that dysplasia was found in 64 patients examined with bone marrow smears (98.5%), among them trilineage dysplasia in 21 patients (32.3%), bilineage dysplasia in 33 patients (50.8%), only erythroid dysplasia in 8 cases (12.3%) and 2 patients (3.1%) only with myeloid dysplasia. The bone marrow biopsy was performed in 38 patients, abnormal localization of immature precursor (ALIP) occurred in 6 cases. 29 patients had abnormal karyotypes, accounting for 59.2% of the 49 patients subjected cytogenetic examination. The abnormal chromosome was the major cytogenetic abnormality, which occurred more often in secondary MDS and the patients with RAEB or RAEB-T. Among the 49 patients who had received cytogenetic examination, 15 patients transformed into AML with the incidence of 30.61%, but only 3 out of 20 patients in the group of normal chromosome transformed into AML (15%), while 12 out of 29 patients in the group of abnormal karyotypes transformed into AML (41.4%). The median time of following up was 35 months (range 2 - 106 months). The median survival time was 26.8 months and 8 months in the patients with normal karyotype and chromosome aberrations respectively. In conclusion, the incidence of MDS in our country is younger than that in Western countries, the rate of abnormal chromosome in high risk MDS is higher than that in low risk MDS. Meanwhile, those who have the change of chromosome are related to the transformation of MDS into AML and have shorter survival time than those MDS patients with normal karyotypes.
Adult ; Aged ; Aged, 80 and over ; Bone Marrow ; pathology ; Chromosome Aberrations ; Chromosome Disorders ; Female ; Humans ; Karyotyping ; Male ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; pathology ; Prognosis ; Retrospective Studies ; Young Adult

OBJECTIVETo investigate the effect of COX1 and COX2 on angiogenesis and endothelial progenitor cell mobilization in rats with experimental myocardial infarction (MI).

METHODSThe rats were randomly divided into 3 groups: MI group, MI plus rofecoxib group and MI plus valeryl salicylate group. At the 7th day after operation, circulating EPCs, plasma VEGF and HIF-1alpha mRNA of ischemic myocardium were measured. At the 28th day post operation, capillary densities were also measured in ischemic myocardium.

RESULTCompared with the MI group and the MI plus valeryl salicylate group, circulating EPCs, plasma VEGF, HIF-1alpha mRNA and capillary densities of ischemic myocardium were all decreased in MI plus rofecoxib group.

CONCLUSIONThe present study revealed that COX2 play an important role with angiogenesis and endothelial progenitor cell mobilization in rat with experimental MI by modulating expression of VEGF and HIF-1alpha.

Animals ; Cyclooxygenase 2 ; biosynthesis ; Cyclooxygenase Inhibitors ; pharmacology ; Endothelial Cells ; cytology ; Female ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; Myocardial Infarction ; drug therapy ; pathology ; Neovascularization, Physiologic ; drug effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; metabolism ; Vascular Endothelial Growth Factor A ; blood

OBJECTIVETo observe the effect of SSd on reversing the malignant phenotype of HepG2 cells and to investigate its mechanism in order to prove that SSd is a new choice to prevent and treat HCC.

METHODSHepG2 cells were cultured and treated by different concentration (0 mg/L, 2.5 mg/L, 5.0 mg/L, 10.0 mg/L and 20.0 mg/L) of SSd for 24 h, and treated by 10 mg/L of SSd for 0 h, 6 h, 12 h, 24 h, 48 h and 72h respectively. The cell inhibition rates were measured by MTT assay. Then cells were treated by 10 mg/L SSd for 48 hr in experimental group and treated by no SSd as a control, their morphological changes were observed by contrast phase microscope. The concentrations of ALB and AFP in clear supernatant liquid of cells were detected by radio-immunity and chemiluminescence. The cell migration rates were observed by transwell method, the relative expression levels of p27 mRNA were measured by RT-PCR.

RESULTSThe inhibitive effect of 10 mg/L SSd was the most significant among different concentrations ( F = 265.06, P less than 0.01). The shape of HepG2 from experimental group turned into small and round, and their volume ratios of nucleus to plasma decreased. ALB in supernatant liquid of HepG2 was higher ( t = 7.83, P less than 0.05, and its AFP was lower ( t = -10.72, P less than 0.01) as compared to control group. Cells migrated were fewer and p27 mRNA expression of HepG2 was higher in experimental group than that in control group (t = 22.00, P less than 0.05).

CONCLUSIONSSd could reverse the malignant phenotype of HepG2 cells. It was suggested that the up-regulation of p27 mRNA expression play an important role in the differentiation of HepG2 cells treated by SSd.

Carcinoma, Hepatocellular ; pathology ; Hep G2 Cells ; drug effects ; Humans ; Liver Neoplasms ; pathology ; Oleanolic Acid ; analogs & derivatives ; pharmacology ; RNA, Messenger ; genetics ; Saponins ; pharmacology

OBJECTIVETo report a protocol using biotin-labelled PrP protein in cell free conversion assay instead of isotope.

METHODSA hamster PrP protein (HaPrP) was expressed in E. coli and purified with HIS-tag affinity chromatograph. After being labelled with biotin, HaPrP was mixed with PrPSc preparation from scrapie strain 263K.

RESULTSProtease-resistant bands were detected after four-day incubation.

CONCLUSIONThe new conversion model provides a reliable, easily handling, and environment-friendly method for studies of prion and transmissible spongiform encephalopathies.

Animals ; Biotin ; Blotting, Western ; Cell-Free System ; Chromatography, Affinity ; methods ; Cricetinae ; Escherichia coli ; genetics ; Gene Expression Regulation ; Molecular Weight ; Peptide Hydrolases ; analysis ; metabolism ; PrPSc Proteins ; genetics ; metabolism ; Prion Diseases ; genetics ; pathology ; Recombinant Proteins ; genetics ; metabolism ; Staining and Labeling

OBJECTIVETo prepare the PrP specific monoclonal antibodies (mAbs) that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases.

METHODSSeveral BALB/c mice were immunized with recombinant hamster prion protein (HaPrP). Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry.

RESULTSThe mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrPSc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPC from several other mammalian species, including humans and cattles. Immunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPsSc.

CONCLUSIONThe mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.

Animals ; Antibodies, Monoclonal ; immunology ; Blotting, Western ; Brain ; metabolism ; Cell Line, Tumor ; Cricetinae ; Enzyme-Linked Immunosorbent Assay ; Female ; Immunization ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; PrPC Proteins ; genetics ; immunology ; PrPSc Proteins ; genetics ; immunology ; Recombinant Proteins ; immunology