T mutation resulting in p.G1015V from an X linked Alport patient, and that of corresponding cDNA from a control were expressed in E.coli. The recombinant and mutant polypeptide was a fragment of COL4A5 , containing 158 amino acid residues with a glycine to valine substitution mutation in it. The secondary structure of the two recombinant proteins was analyzed using circular dichroism(CD) spectroscopy. Results: CD spectra of the control exhibited a negative peak near 200 nm whereas that of the patient exhibited a negative peak near 220 nm. The magnitude of the negative peak of the patient decreased as compared with that of the control. Furthermore, secondary structure of the control polypeptide was mainly composed of ? sheet and random coil without ? helix, whereas that of the patient presented 12.9% ? helix. Conclusion: Not only local structure of the substitution site but also folding kinetics of the entire ?5 chain may be changed due to Gly→Val substitution in Alport syndrome. We speculate that the abnormally folded polypeptide chain may not be assembled into the triple helix and the network of type Ⅳ collagen, or may be assembled into loosen triple helix then degraded easily, resulting in the pathognomonic ultrastructural changes of the glomerular basement membrane.

Objective To explore the effect of ? - aminobutyric acid B receptor(GABABR)on brain damage induced by recurrent febrile seizures (FS). Methods Rats were randomly divided into four groups: control group (37. 0 ℃ water, n = 8), FS group (45.2 ℃ water,n=8), FS + baclofen group (45.2 ℃ water,77 = 8), FS + phaclofen group (45. 2 ℃ water,n=8). FS in rats were induced for ten times in a bath of warm water, once every 2 days. The intensity, latency and duration of the seizure in rats were recorded. The expression of c - fos gene and Fos protein were examined by in situ hybridization and immunohistochemistry, respectively. Results Compared with those of FS group, the seizure latency gradually prolonged, and the seizure duration was shortened in FS + baclofen group. In FS+ phaclofen group, the seizure latency was shorter and the seizure duration was longer than those of FS group. The seizure intensity was lessened in FS + baclofen group while aggravated in FS + phaclofen group compared with that of FS group. The expression of c - fos gene and Fos protein increased significantly after recurrent FS. Baclofen down regulated the expression of c -fos gene and Fos protein, while phaclofen enhanced the expression of them. Conclusion The study by using the agonist and the inhibitor of GABABR showed that GABABR might play a crucial role in the development of FS- induced brain damage.

Objective To investigate the effect of carbon monoxide(CO) on hydrogen sulfide(H_2S)/cystathionine-?-lyase system(CSE) in vascular smooth muscle cells(VSMC) of spontaneous hypertensive rats(SHR).Methods The SHR VSMC were divided into 2 groups:experimental group(hemin was added to the culture media at a concentration of 10 ?mol/L)and control group (dimethyl sulfoxide was added to the culture media at an equal volume).The content of H_2S in the cultrue media was measured by sulfide electrode method,and the CSEmRNA level was assayed by competitive reversed transcription-polymerase chain reaction(RT-PCR).Results Compared with control group,the content of H_2S in the culture media of hemin-treated SMCs was significantly lower[(16.03? 2.14) ?mol/L vs (13.31?1.74)?mol/L],and the CSEmRNA level in hemin-treated SMCs was decreased markedly(0.17?0.04 vs 0.13?0.03).Conclusion CO can down-regulate the H_2S/CSE system in SMC of SHR.

Objective To investigate the effect of nitric oxide on hydrogen sulfide/ cystathionine-?-lyase(CSE) system in the vascular smooth muscle cells of spontaneously hypertensive rats(SHR).Methods The SHR aortic smooth muscle cells(SMCs) were divided into 2 groups: sodium nitroprusside(SNP) group and control group.The content of hydrogen sulfide in the culture media was measured by sulfide electrode method,and the CSE mRNA level was assayed by competitive reversed transcription-polymerase chain reaction((RT-PCR)).Results Compared with control group,the content of hydrogen sulfide in the culture media of SNP group was significantly higher [(16.16?3.71) ?mol/L vs(22.71?4.84) ?mol/L],and the CSE mRNA level in SMCs in SNP group was lower than that of control group.Conclusion Nitric oxide exerted complicated effect on the hydrogen sulfide/CSE system in the SHR smooth muscle cells.J Appl Clin Pediatr,2006,21(3):140-141

OBJECTIVEAlport syndrome (AS) is a progressive hereditary nephritis presented with hematuria and renal failure, frequently associated with sensorineural deafness and ocular lesions. So far, more than 300 gene mutations in AS have been identified which provides a better way to analyze the association between genotype and phenotype. It is hard to understand all the phenotype according to the gene mutations, because the structure and function changes of the relevant protein, alpha5(IV) chain, encoded by mutated COL4A5 gene are rare to know. This study aimed to detect the proteins structure encoded by COL4A5 gene with different missense mutations and to analyze the effect of gene mutations on the secondary structure of alpha5(IV) chain and structure-phenotype relations.

METHODSTwo X-linked AS patients with different missense mutations (g.3246G > T resulting in p.G1015V and g.3290G > A resulting in p.G1030S, respectively) diagnosed by clinical manifestations, family history and skin or renal biopsy examinations, as well as a control were included in this study. The fragments of cDNA with the two mutations, respectively, and that of corresponding cDNA from the control were expressed in E. coli. The secondary structure of the recombinant polypeptides were analyzed by using circular dichroism (CD) spectroscopy.

RESULTSCD spectra of the control exhibited a negative peak near 200 nm whereas that of the patient 1 exhibited a negative peak near 220 nm. Furthermore, the magnitude of the negative peak of patient 1 decreased from -9000 deg x cm2 x dmol(-1) to -4000 deg x cm2 x dmol(-1) as compared with that of the control. CD spectra of the patient 2 were slightly changed with the negative peak remaining near 220 nm but the magnitude increasing from -9000 deg x cm2 x dmol(-1) to -11000 deg x cm2 x dmol(-1) as compared with that of the control. In addition, the secondary structure of the control polypeptide was mainly composed of beta-sheet and random coil without alpha-helix, whereas that of the patient 1 presented 12.9% alpha-helix. Although the secondary structure of polypeptide of the patient 2 was also mainly composed of beta-sheet and random coil, the composition of beta-sheet reduced and random coil increased.

CONCLUSIONAlthough the glycine substitutions existed in the same domain of alpha5(IV) chain, the patient 1 with the severe AS phenotype and g.3246G > T mutation, and patient 2 with the mild AS phenotype and g.3290G > A mutation were revealed with different secondary structures of alpha5(IV) chain. Moreover, the secondary structure changes of alpha5(IV) chain were consistent with their corresponding phenotype severity.

Collagen Type IV ; genetics ; Humans ; Mutation, Missense ; Nephritis, Hereditary ; genetics ; Phenotype ; Point Mutation ; Protein Structure, Secondary ; genetics ; Spectrum Analysis

Objective To explore the effect of nitric oxide (NO) on ?-aminobutyric acid B receptor (GABA_BR) subunits during recurrent febrile seizures (FS).Methods Twenty-four Sprague-Dawley rats aged 21 days were randomly divided into 4 groups: control group (37.0 ℃ water,n=8), FS group (45.2 ℃ water,n=8), FS + SNP group (45.2 ℃ water,n=8), FS+L-NMMA group (45.2 ℃ water,n=8). FS rats were induced 10 times in a warm-water bath, once every 2 days. The plasma level of NO was detected by the spectrophotometer. The expressions of GABA_BR subunit mRNA and c-fos gene were examined by in situ hybridization. The expressions of GABA_BR subunit and Fos protein were observed by immunohistochemistry. Results The plasma level of NO increased in FS + SNP group while decreased in FS+L-NMMA group compared with that in FS group. The expressions of GABA_BR_2 were down-regulated in FS+SNP group, while GABA_BR_1 hardly changed compared with those in FS group. In FS+L-NMMA group, both the expression of GABA_BR_2 and GABA_BR_1 up regulated compared with those in FS group. The expressions of c-fos gene and Fos protein were significantly enhanced after recurrent FS. SNP elevated the expressions of c-fos gene and Fos protein, while L-NMMA down regulated the expressions of them.Conclusion NO may play a regulatory role through modulating GABA_BR function in the pathogenesis of recurrent FS.

Objective To explore the influence of ?-aminobutyric acid B receptor(GABA_BR)on carbon monoxide (CO)/heme oxygenase(HO-1)system during recurrent febrile seizures (FS).Methods Sprague-Dawley rats aged 21 days were randomly divi- ded into 4 groups:control group and FS group,FS+baclofen group,FS+phaclofen group.FS in rats were induced 10 times in a bath of warm water, once every 2 days.The plasma level of CO was detected by the dual wave lengh spectrophotometer;the expressions of GABA_BR and HO-1 mRNA were examined by insitu hybridization;the expressions of GABA_BR and HO-1 protein were observed by immunohistochemistry.Results The plasma level of CO increased in FS+baclofen group,while decreased in FS+phaclofen group compared with FS group.The expressions of GABA_BR and HO-1 upregulated in FS+baclofen group,while decreased in FS+phaclofen group compared with FS group.There were significant difference (All P

OBJECTIVETo investigate the cell features of 6 Castleman's disease patients and evaluate their prognosis.

METHODSThe resected tumors were investigated by routine histopathology and immunohistochemistry. Reverse-transcript PCR (RT-PCR) and sequencing of RT-PCR products were used to assess the clonal characters of the main tumor cells.

RESULTSHistologically, all of the 6 tumors were classified as the hyaline vascular type. B-cells dominated the follicular germinal centers, with T-cells dispersing inter-follicularly. The results of RT-PCR each obtained a single band of either 128 bp or 122 bp and sequencing showed that there was highly homogeneity within the same length sequences, accompanied by fewer different nucleotide acids.

CONCLUSIONMonoclonal and/or oligoclonal B cells were identified in Castleman's disease. These B cells were originated from germinal center cells.

Adolescent ; Adult ; Antigens, CD20 ; analysis ; B-Lymphocytes ; metabolism ; pathology ; Castleman Disease ; genetics ; metabolism ; pathology ; Clone Cells ; Female ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; genetics ; Humans ; Immunohistochemistry ; Leukocyte Common Antigens ; analysis ; Male ; Prognosis ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUNDThe presence of autoantibodies against multiple epidermal proteins is an important feature in paraneoplastic pemphigus (PNP). Circulating anti-desmoglein 3 autoantibody, the major pathogenic autoantibody in pemphigus vulgaris (PV), has been proved pathogenic in PNP. Because of many clinical differences between PNP and PV, we speculate about the involvement of other autoantibodies in the pathogenesis of PNP. Envoplakin (EPL) and periplakin (PPL) are recognized by most PNP sera. Their linker subdomains are highly homologous and necessary for the association of intermediate filaments.

METHODSWe characterized the autoantibodies against the linker subdomains of EPL and PPL in PNP patients' sera and their associated tumors by enzyme-linked immunosorbent assay (ELISA) and immunofluorence. We also applied the purified autoantibodies against EPL and PPL from PNP sera to cultured human epidermal keratinocytes (HEK), to evaluate the changes of cell-cell adhesion.

RESULTSAutoantibodies against EPL and PPL were detected in most PNP patients by ELISA, and the decrease of these autoantibodies after removal of the tumors was roughly comparable to the improvement of clinical symptoms. Cultured tumor cells from PNP patients secreted these autoantibodies. Specific immunoglobulin receptors for EPL and PPL were found on B lymphocytes in tumors from PNP. Furthermore, purified anti-EPL and anti-PPL autoantibodies from PNP sera were capable of dissociating cultured human epidermal keratinocytes.

CONCLUSIONAutoantibodies against EPL and PPL may also be pathogenic in PNP.

Adolescent ; Adult ; Autoantibodies ; immunology ; pharmacology ; Cell Adhesion ; drug effects ; Cells, Cultured ; Desmoglein 3 ; immunology ; Enzyme-Linked Immunosorbent Assay ; Epidermis ; cytology ; Female ; Humans ; Keratinocytes ; cytology ; drug effects ; Male ; Membrane Proteins ; immunology ; Middle Aged ; Paraneoplastic Syndromes ; immunology ; metabolism ; Pemphigus ; immunology ; metabolism ; Plakins ; immunology ; Protein Precursors ; immunology ; Young Adult

OBJECTIVECongenital nephrotic syndrome (CNS) is defined as heavy proteinuria or nephrotic syndrome occurring before 3 months of age. It is characterized by early onset, resistance to steroid therapy and progressing to end-stage renal disease (ESRD). In recent years, several genes associated with CNS have been identified, such as NPHS1, NPHS2 and WT1. The mutations of these genes have been identified in the patients with CNS in Finland, other European countries, North Africa, North America, and Asia, respectively. However, the investigation of the above genes has not been performed in Chinese CNS patients. In this study, NPHS1 mutations in a Chinese family with CNS were detected and analyzed.

METHODSThere were two CNS patients in the investigated family. The proband, a 45-day-old boy, was born at fullterm and weighed 2700 g at birth. The placenta weighed 450 g. At the age of 10 days, generalized edema, proteinuria, hypoproteinemia, and hypoalbuminemia were found without renal insufficiency. The proband's sister, with the same phenotype and normal renal function, underwent renal biopsy at 5 years of age. Their parents and elder half-sister all had normal phenotypes. Genomic DNA samples were extracted from peripheral bloods of the proband, his family members and 50 unrelated, normal individuals. All 29 exons and exon-intron boundaries of NPHS1 were detected in the proband by polymerase chain reaction (PCR), direct DNA sequencing, and restriction enzyme analysis.

RESULTSThree heterozygous mutations of NPHS1, namely, G928A (D310N), 1893-1900del 8 (CGAAACCG), and G2869C (V957L) were identified in the proband. These mutations involved exons 8, 14, and 21. The same genotype was found in the proband's sister who had the same phenotype, but was not detected in proband's elder half-sister who had normal phenotype. Fifty normal individuals had no these mutations. The proband's mother with normal urinalysis had G928A (D310N) heterozygous mutation, and the father with normal urinalysis had two heterozygous mutations of 1893-1900del 8 (CGAAACCG) and G2869C (V957L). At the same time, three types of single nucleotide polymorphisms (SNPs), E117K (rs3814995), S1105S (rs2071327), and IVS27+45c > t, were confirmed in the proband. Another variant, IVS8+68 a > g had also been found.

CONCLUSIONThis is the first report about NPHS1 mutations in Chinese CNS kindred. These three heterozygous mutations of NPHS1 are novel genetic defects of CNS, which have not been described before.

Asian Continental Ancestry Group ; genetics ; Exons ; Genotype ; Humans ; Infant ; Male ; Membrane Proteins ; genetics ; Mutation ; Nephrotic Syndrome ; congenital ; genetics ; Pedigree ; Phenotype ; Polymerase Chain Reaction ; Sequence Analysis, DNA