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1.
PLoS One ; 9(6): e100505, 2014.
Article in English | MEDLINE | ID: mdl-24949742

ABSTRACT

OBJECTIVE: Mutations in the type II collagen gene are associated with certain human disorders, collectively termed type II collagenopathies. They include Legg-Calvé-Perthes disease (LCPD) and avascular necrosis of the femoral head (ANFH). These two diseases are skeletal dysplasias, inherited in an autosomal dominant fashion, characterized by groin pain, dislocation of the hip and diminished joint mobility. Coxa vara and elevation of the greater trochanter of the femur comprise the typical phenotype of LCPD, but do not occur in ANFH. Lack of synthesis of type II collagen and structural defects are responsible for the major clinical outcomes, because collagen is the essential matrix protein of all connective tissues. Type II collagen, encoded by the COL2A1 gene, contains N- and C- terminal regions that are cleaved after secretion into the extracellular matrix, and the core area is composed of a triple helical (Gly-X-Y) domain. If the Gly in this specific region is replaced by other amino acids, the structure of type II collagen will be destroyed. METHOD: Forty-five members of a four-generation family were recruited and investigated. Diagnosis was made by independent orthopedic surgeons and radiologists. A mutation of the COL2A1 gene was detected. RESULT: In our research, we identify a heterozygous mutation (c.1888 G>A, p. Gly630Ser) in exon 29 of COL2A1 in the Gly-X-Y domain, in a Chinese family affected by LCPD and ANFH. Our findings provide significant clues to the phenotype-genotype relationships in these syndromes and may be helpful in clinical diagnosis. Furthermore, these results should assist further studies of the mechanisms underlying collagen diseases. CONCLUSION: Our data add new variants to the repertoire of COL2A1 mutation resulting in related collagenopathies.


Subject(s)
Asian People/genetics , Collagen Type II/genetics , Femur Head Necrosis/genetics , Legg-Calve-Perthes Disease/genetics , Mutation , Pedigree , Amino Acid Sequence , Animals , Child , Child, Preschool , Collagen Type II/chemistry , Female , Femur Head Necrosis/diagnostic imaging , Humans , Legg-Calve-Perthes Disease/diagnostic imaging , Male , Mice , Molecular Sequence Data , Phenotype , Radiography , Rats
2.
Zhonghua Nan Ke Xue ; 19(11): 1011-5, 2013 Nov.
Article in Chinese | MEDLINE | ID: mdl-24341097

ABSTRACT

OBJECTIVE: Globozoospermia is mostly associated with homozygous deletion of the DPY19L2 gene. This study aimed to investigate the DPY19L2 gene mutation in a globozoospermia patient. METHODS: We observed the sperm histomorphology of a patient with globozoospermia using Wright-Giemsa's staining and transmission electron microscopy, detected the mutation of the DPY19L2 gene by PCR amplification and DNA sequencing, and compared the findings with the sequences issued in the Genbank. RESULTS: Wright-Giemsa's staining showed that all the spermatozoa were round-headed and lacked the acrosome, with the head nucleus darkly, fully and densely stained. Transmission electron microscopy revealed larger round sperm heads, with an even layer of unit membrane surrounding the nuclei and dispersed cytoplasmic vacuoles but no acrosomal structure. No DPY19L2 gene mutation was found by PCR amplification and DNA sequencing. CONCLUSION: No homozygous mutation of the DPY19L2 gene was found in the globozoospermia patient, and therefore some other disease-causing genes might be involved.


Subject(s)
Gene Deletion , Infertility, Male/genetics , Membrane Proteins/genetics , Acrosome/pathology , Acrosome/ultrastructure , DNA Mutational Analysis , Humans , Male , Microscopy, Electron, Transmission , Spermatozoa/pathology , Spermatozoa/ultrastructure
3.
Zhonghua Nan Ke Xue ; 18(11): 1028-31, 2012 Nov.
Article in Chinese | MEDLINE | ID: mdl-23214256

ABSTRACT

Globozoospermia is a severe teratozoospermia, and the cases with 100% round-headed sperm are rare clinically. Globozoospermia is generally characterized by absence or abnormality of acrosome, accompanied by round-headed sperm with deranged midpiece and tails. The acrosome normally contains the enzymes that enable sperm to fertilize oocytes, while defective sperm cannot independently fertilize oocytes either in vivo or in vitro, and therefore globozoospermia makes males infertile clinically. Recent studies show that the deletion of the DPY19L2 (dpy-19-like 2) gene is a major cause of globozoospermia. This paper updates the relationship between DPY19L2 and globozoospermia to provide some evidence for further studies on the gene diagnosis and molecular mechanisms of globozoospermia.


Subject(s)
Infertility, Male/genetics , Membrane Proteins/genetics , Spermatozoa/abnormalities , Acrosome , Humans , Male , Sequence Homology , Sperm Head
4.
Cell Res ; 14(5): 434-8, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15538975

ABSTRACT

The basal activity of JNK is low in normal growing cells and inactivated JNK targets p53 for ubiquitination. To elucidate if the C-terminal part of JNK is responsible for its binding to p53, the low background tet-off inducible NIH3T3 cell line was selected by luciferase reporter gene and a double stable C-JNK Aa (203-424) cell line was established. After withdrawing tetracycline, the C-JNK fragment expression was induced and cell growth was dramatically inhibited 24 h later. However, the expression of p53 was found to be increased after the induction of C-JNK fragment, evaluated by transfecting p21waf-luciferase reporter genes. Our further studies showed that C-JNK fragment could form complex with p53 both in vivo and in vitro. Induction of C-JNK fragment in vivo can increase p53 stability by inhibiting p53 ubiquitination.


Subject(s)
Cell Proliferation/drug effects , Mitogen-Activated Protein Kinase 9/metabolism , Peptide Fragments/pharmacology , Tumor Suppressor Protein p53/drug effects , 3T3 Cells , Animals , Cell Line , Mice , Mitogen-Activated Protein Kinase 9/chemistry , Time Factors , Tumor Suppressor Protein p53/metabolism
5.
Shi Yan Sheng Wu Xue Bao ; 37(3): 176-82, 2004 Jun.
Article in Chinese | MEDLINE | ID: mdl-15323418

ABSTRACT

Site-directed mutagenesis was used to generate three cysteine mutants of GSTp, C(47/101), C(14/47/101) and C(14/47/101/169). GSTp, C(47/101), C(14/47/101) and C(14/47/101/169) were transfected into 293 cells separately and GST activity was determined by using CDNB as substrate. Data showed that each cysteine mutant inhibited endogenous GST catalyzatic activity and had remarkable dominant negative effect. The expression vectors of wide type GSTp and its cysteine mutants were co-transfected with c-Jun, NF-kappaB, or p21 luciferase reporting vector, into 293 cells separately, luciferase activity showed that C(14/47/101) and C(14/47/101/169) can dramatically activate c-Jun and p21 transcriptional activity. Each cysteine mutant can increase endogenous p21 level, and also increased mortality rate of 293 cells when exposed to H2O2. These results suggest that cysteine residues of GSTp play an important role in protecting cells against oxitative stress.


Subject(s)
Cysteine/physiology , Glutathione S-Transferase pi/genetics , Glutathione S-Transferase pi/metabolism , Oxidative Stress/physiology , Cell Line , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cysteine/genetics , Humans , Mutagenesis, Site-Directed , Oxidative Stress/genetics , Structure-Activity Relationship
6.
Sheng Wu Gong Cheng Xue Bao ; 20(3): 456-60, 2004 May.
Article in Chinese | MEDLINE | ID: mdl-15971624

ABSTRACT

Glutamine is an important conditionally necessary amino acid in human body. The effort is to establish a new and high efficient L-glutamine production system instead of traditional fermentaion. In this paper, high efficiency of L-glutamine production is obtained by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system. Glutamine Synthetase gene (glnA) was amplified from Bacillus subtilis genomic DNA with primers designed according to sequences reported in EMBL data bank, then it was inserted into expression vector PET28b, the sequence of glnA was proved to be the same as that reported in the data bank by DNA sequencing. After transformation of this recombinant plasmid PET28b-glnA into BL-21 (DE3) strain, Lactose and IPTG were used to induce GS expression at 37 degrees C separately. Both of them can induce GS expression efficiently. The induced protein is proved to be soluble and occupies about 80% of the total proteins by SDS-PAGE analysis. The soluble GS was purified by Ni2+ chelating sepharose colum. After purification, the purified enzyme was proved active. Results reveal that the optmum temperature of this enzyme is 60 degrees C and optmum pH is 6.5 in biosynthetic reaction by using glutamate, ammonium choloride and ATP as substrates. After induction, the enzyme activity in crude extract of BL-21/PET28b-glnA is 83 times higher than that of original BL-21 extract. Mn2+ can obviously increase the activity and stability of this enzyme. Experiments show that the transformation efficiency of glutamate to glutamine is more than 95%. Because of the high cost from ATP, a system coupling GS with yeast for ATP regenaration was established. In this system, GS utilizes ATP released by yeast fermentation to synthesize L-glutamine. Yeast was treated by 2% toluence to increase its permeability and a yeast named YC001 with high yield of glutamine by coupling with recombinant GS was obtained. The good efficiency was achieved with the presence of 250 mmol/L glucose and 200 mmol/L phosphate, the transformation efficiency of glutamate to glutamine in this system is more than 80%, the average yield of glutamine is about 22g/L. This provides the basis for future large scale production of L-glutamine.


Subject(s)
Genetic Engineering/methods , Glutamate-Ammonia Ligase/biosynthesis , Glutamine/biosynthesis , Yeasts/metabolism , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Fermentation , Glutamate-Ammonia Ligase/genetics , Glutamic Acid/metabolism , Glutamine/genetics , Yeasts/genetics
7.
Article in English | MEDLINE | ID: mdl-12050808

ABSTRACT

Glutathione S-transferase pi (GSTpi) protects cells from death by altering intracellular oxidative stress. In order to understand the mechanism of GSTpi protection, a cell death model induced by serum depletion as the stress was established. Cotransfection of apoptosis signal-regulating kinase 1 (ASK1) and GSTpi cDNA was performed to elucidate the impact of GSTpi on ASK1 activity, as well as on its downstream signals, MKK7 and JNK, and to elucidate the potential protection of GSTpi on 293 cell death induced by serum depletion. The dominant negative mutant of JNK was used to explore if the blocking of the JNK pathway led to cell death inhibition. It was found that GSTpi had a dose-dependent inhibitory effect on activation induced by serum depletion, and also on inhibition both on MKK7 and JNK. Intracellular expression of GSTpi significantly inhibited serum depletion-induced cell death. Blocking the JNK pathway by transfection of a dominant negative form of JNK (JNK (APF)) brought about significant inhibition of cell death induced by serum depletion with an inhibiting rate as high as 15%. All the results suggest that the mechanism of GSTpi protection on serum depletion-induced cell death works through an ASK1-MKK7-JNK pathway.

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