Clinical features and molecular genetic analysis of congenital lipoid adrenal hyperplasia caused by steroidogenic acute regulatory protein gene mutation / 中华围产医学杂志

Objective@#To investigate the clinical and molecular genetic features of neonatal congenital lipoid adrenal hyperplasia (CLAH) caused by mutations in steroidogenic acute regulatory protein (StAR) encoding gene.@*Methods@#This study retrospectively analyzed the clinical data of a CLAH neonate admitted to Fujian Provincial Maternity and Children's Hospital, Affiliated Hospital of Fujian Medical University in April 2017. StAR gene was analyzed using high-throughput sequencing and Sanger sequencing. Relevant literature retrieved from databases including China National Knowledge Infrastructure (CNKI), Wanfang and PubMed were reviewed, and the reported cases with relatively complete clinical data and results of serum hormone test and StAR gene mutation analysis were collected.@*Results@#The index patient presented with hyperpigmentation and growth retardation soon after birth. Laboratory tests revealed hyponatremia, hyperkalemia, increased serum adrenocorticotrophic hormone (263.4 pmol/L) and decreased 17-hydroxyprogesterone (0.16 ng/ml), dehydroepiandrosterone (<0.95 μmol/L), androstenedione (<1.0 nmol/L), testosterone (<0.025 ng/ml), progesterone (0.02 ng/ml) and cortisol (1.6 μg/ml). High-throughput sequencing showed that the patient carried a compound heterozygous mutation of p.Thr240fs in exon 6 and p.Gln258X in exon 7, inherited from the father and mother, respectively. Sanger sequencing confirmed the diagnosis of CLAH caused by StAR gene mutation. After steroid replacement therapy, the patient's symptoms resolved and the concentrations of electrolytes returned to normal. The neonate was followed up to two years of age and no abnormality was found in physical or neurological development. Two Chinese and 11 English publications were retrieved and altogether 96 cases of neonatal CLAH, including the index one, were reviewed and 42 of them had detailed clinical data. The most common clinical manifestations were skin pigmentation (85.7%, 36/42). Other manifestations included vomiting (35.7%, 15/42) and growth retardation (14.3%, 6/42). All patients with physical examination records had female external genitalia (100.0%, 35/35). The common laboratory abnormalities included hyponatremia (95.2%, 40/42), hyperkalemia (88.1%, 37/42), elevated serum adrenocorticotrophic hormone (100.0%, 37/37) and decreased 17-hydroxyprogesterone (90.5%, 19/21), cortisol (86.2%, 25/29), testosterone (9/10) and dehydroepiandrosterone (14/14). p.Gln258X was the most common StAR gene mutation in neonates in Eastern Asia, including China. Most cases had a good prognosis after appropriate steroid replacement.@*Conclusions@#CLAH should be considered for neonates with adrenocortical hypofunction, especially with female phenotypes and low 17-hydroxyprogesterone. Karyotyping and StAR gene analysis may be helpful in diagnosis. Timely and appropriate treatment could improve the prognosis.

Clinical features and molecular genetic analysis of congenital lipoid adrenal hyperplasia caused by steroidogenic acute regulatory protein gene mutation / 中华围产医学杂志

Objective To investigate the clinical and molecular genetic features of neonatal congenital lipoid adrenal hyperplasia (CLAH) caused by mutations in steroidogenic acute regulatory protein (StAR) encoding gene.Methods This study retrospectively analyzed the clinical data of a CLAH neonate admitted to Fujian Provincial Matemity and Children's Hospital,Affiliated Hospital of Fujian Medical University in April 2017.StAR gene was analyzed using high-throughput sequencing and Sanger sequencing.Relevant literature retrieved from databases including China National Knowledge Infrastructure (CNKI),Wanfang and PubMed were reviewed,and the reported cases with relatively complete clinical data and results of serum hormone test and StAR gene mutation analysis were collected.Results The index patient presented with hyperpigrnentation and growth retardation soon after birth.Laboratory tests revealed hyponatremia,hyperkalemia,increased serum adrenocorticotrophic hormone (263.4 pmol/L) and decreased 17-hydroxyprogesterone (0.16 ng/ml),dehydroepiandrosterone (＜0.95 μmol/L),androstenedione (＜1.0 nmol/L),testosterone (＜0.025 ng/ml),progesterone (0.02 ng/ml) and cortisol (1.6 μ g/ml).High-throughput sequencing showed that the patient carried a compound heterozygous mutation of p.Thr240fs in exon 6 and p.Gln258X in exon 7,inherited from the father and mother,respectively.Sanger sequencing confirmed the diagnosis of CLAH caused by StAR gene mutation.After steroid replacement therapy,the patient's symptoms resolved and the concentrations of electrolytes returned to normal.The neonate was followed up to two years of age and no abnormality was found in physical or neurological development.Two Chinese and 11 English publications were retrieved and altogether 96 cases of neonatal CLAH,including the index one,were reviewed and 42 of them had detailed clinical data.The most common clinical manifestations were skin pigmentation (85.7％,36/42).Other manifestations included vomiting (35.7％,15/42) and growth retardation (14.3％,6/42).All patients with physical examination records had female external genitalia (100.0％,35/35).The common laboratory abnormalities included hyponatremia (95.2％,40/42),hyperkalemia (88.1％,37/42),elevated serum adrenocorticotrophic hormone (100.0％,37/37) and decreased 17-hydroxyprogesterone (90.5％,19/21),cortisol (86.2％,25/29),testosterone (9/10) and dehydroepiandrosterone (14/14).p.Gln258X was the most common StAR gene mutation in neonates in Eastern Asia,including China.Most cases had a good prognosis after appropriate steroid replacement.Conclusions CLAH should be considered for neonates with adrenocortical hypofunction,especially with female phenotypes and low 17-hydroxyprogesterone.Karyotyping and StAR gene analysis may be helpful in diagnosis.Timely and appropriate treatment could improve the prognosis.

Preparation and identification of monoclonal antibody against phosphoprotein of Borna disease virus / 中国人兽共患病学报

In order to prepare the monoclonal antibodies (MAbs) by the recombinant phosphoprotein (p24) of Borne dis ease virus (BDV) as immunogen,the spleen cells of immunized balb/c mice with the recombinant BDV p24 were fused with the myeloma cells SP2/0.The hybridoma cell lines secreting MAbs against p24 were obtained after selected by indirect ELISA and subcloned for 3 times.The MAbs were prepared by intraperitoneal injection in mice,purified by affinity chromatography,identified by western blot and immunofluorescence assay (IFA) for specificity.The purified MAbs against BDV p24 from ascites belonged to IgG1 showed a purity of 98％ and 93％,and a titer of 1 ∶ 81 000,and specific reaction with the BDV p24 restructured and expressed in Oligodendroglia cells (OL).The MAbs against BDV p24,with high specificity and sensitivity,were prepared successfully,which laid a basis for the study of diagnostic reagents and pathogenic mechanism of BDV.

Preparation and identification of monoclonal antibody against phosphoprotein of Borna disease virus / 中国人兽共患病学报

In order to prepare the monoclonal antibodies (MAbs) by the recombinant phosphoprotein (p24) of Borne dis ease virus (BDV) as immunogen,the spleen cells of immunized balb/c mice with the recombinant BDV p24 were fused with the myeloma cells SP2/0.The hybridoma cell lines secreting MAbs against p24 were obtained after selected by indirect ELISA and subcloned for 3 times.The MAbs were prepared by intraperitoneal injection in mice,purified by affinity chromatography,identified by western blot and immunofluorescence assay (IFA) for specificity.The purified MAbs against BDV p24 from ascites belonged to IgG1 showed a purity of 98％ and 93％,and a titer of 1 ∶ 81 000,and specific reaction with the BDV p24 restructured and expressed in Oligodendroglia cells (OL).The MAbs against BDV p24,with high specificity and sensitivity,were prepared successfully,which laid a basis for the study of diagnostic reagents and pathogenic mechanism of BDV.

Expression of Ezrin and its significance in giant-cell tumor of bone / 国际肿瘤学杂志

Objective To detect the expression of Ezrin in giant-cell tumor of bone,and to investigate its cilincal significance. Methods 60 cases of biopsy which had been confirmed as bone giant-cell tumors in our hospital from January 2008 to December 2013 were set as observation group;tumor tissues from 8 cases of reactive new bone in nonmalignant bone diseases,12 cases of osteoid osteoma and 11 cases of osteoblastoma in the corresponding period were set as control group. Protein and gene levels of Ezrin were tested with Western blotting method and real-time PCR detection,simultaneously proceeded the corresponding analysis combined with the clinical data of patients;60 cases of bone giant-cell tumor patients accepted tumor resection and pros-thesis replacement,2 courses of preoperative chemotherapy;mitochondria morphological changes of tumor tissue and Ezrin protein and genetic changes were observed before and after chemotherapy. Results In the giant-cell tumors of bone,the Ezrin protein mainly located in the cytoplasm,and its expression positive rate was much higher than that in reactive new bone of nonmalignant bone diseases(19. 7% ),osteoid osteoma(21. 2% )and osteoblastoma(20. 7% );the difference was statistically significant(χ2 = 4. 18,P = 0. 024),but no statistical difference in the Ezrin expression among the groups of osteosarcoma,osteoid osteoma and osteblastoma(χ2 =6. 18,P = 0. 087). In the giant-cell tumors of bone tissue after chemotherapy,mitochondria pyknosis and the phenomenon of liquid cavitation was less than that before the treatment,and Ezrin protein expression decreased and gene levels reduced[(23. 99 ± 1. 49)vs(20. 11 ± 1. 11),t = 5. 03,P = 0. 018)]. Conclusion The expression of Ezrin in giant-cell tumor of bone is much higher than other benign bone tumor,and it could be a biological marker for differentiating benign and malignant bone tumor. Early intervention in Ezrin may be helpful for reatment of giant-cell tumor of bone.

Expression of Ezrin and phos-ezrin in skin squamous carcinomas and its clinical significance / 中国医师杂志

Objective To investigate the expressions of Ezrin and phos-ezrin in squamous cell carcinomas (SCC),and to an alyze their clinic significance.Methods Immunohistochemistry was used to detect the expressions of Ezrin and phos-Ezrin in 20 cases of normal skin,31 cases of seborrheic Keratosis (SK),36 cases of basal cell carcinoma (BCC),and 37 cases of SCC.Results (1) The positive rates of Ezrin in normal skin (NS),SK,BCC,and SCC were 20.0％,25.8％,66.7％,and 89.2％,respectively.The positive reates of phos-Ezrin in NS,SK,BCC,and SCC were 10.0 ％,22.6％,77.8％,and 94.6％,respectively.Ezrin and phos ezrin in cancers were higher than that in noncancer tissues (P ＜ 0.01).(2) The expressions of Ezrin,and phos-Ezrin were closely correlated with the cutaneous tumor's type(R1 =0.87,r1 =0.89,P ＜ 0.01),malignant degree (patho-grading of SCC) (R2 =0.80,r2=0.86,P ＜ 0.01),metastasis via lymph node (R3 =0.89,r3 =0.91,P ＜ 0.01).Conclusions Ezrin and phos-Ezrin expressions were closely related to the types of tumor,malignant degrees,and metastasis.The combined-detection of Ezrin and phos-ezrin would provide a novel clue to predicting metastasis and prognosis of cutaneous tumor.

Phosphoproteomic analysis identifies activated MET-axis PI3K/AKT and MAPK/ERK in lapatinib-resistant cancer cell line

Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases, has shown promising results as a growth inhibitor of HER2-positive cancer cells in vitro. However, similar to other EGFR-targeting drugs, acquired resistance to lapatinib by HER2-positive cancer cells remains a major clinical challenge. To elucidate resistance mechanisms to EGFR/HER2-targeting agents, we performed a systematic quantitative comparison of the phosphoproteome of lapatinib-resistant (LR) human gastric cancer cells (SNU216-LR) versus parental cells (SNU216) using a titanium dioxide (TiO2) phosphopeptide enrichment method and analysis with a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer. Biological network analysis of differentially expressed phosphoproteins revealed apparent constitutive activation of the MET-axis phosphatidylinositide 3-kinase (PI3K)/alpha-serine/threonine-protein kinase (AKT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways in SNU216-LR. Inhibition of the PI3K/AKT and MAPK/ERK signaling pathways in SNU216-LR also leads to cell cycle arrest, confirming the biological network analysis. Lapatinib sensitivity was restored when cells were treated with several molecular targeting agents in combination with lapatinib. Thus, by integrating phosphoproteomic data, protein networks and effects of signaling pathway modulation on cell proliferation, we found that SNU216-LR maintains constitutive activation of the PI3K/AKT and MAPK/ERK pathways in a MET-dependent manner. These findings suggest that pathway activation is a key compensatory intracellular phospho-signaling event that may govern gastric cancer cell resistance to drug treatment.

Ezrin-associated signal transduction in tumor invasion and metastasis / 国际肿瘤学杂志

Ezrin is an important membrane-cytoskeleton linker protein.It is highly related with tumor invasion and metastasis and indicates poor prognosis.It is reported that aberrant Ezrin expressed in many carcinomas,such as osteosarcoma and breast carcinoma.However,the complex mechanisms of Ezrin in tumor invasion and metastasis remain unclear.It is involved in several different tumor associated signal pathway based on different tumor types,including adhesion molecule signal transduction,Rho and Akt signal transduction.Therefore,research on the signal transduction of Ezrin has great significance for the understanding of cancer progression and Ezrin is probable to be a new treatment target.

Ezrin and tumor metastasis / 国际肿瘤学杂志

Ezrin is a member of ezrin-radixin-moesin(ERM)family acting as correlationship between plasma membrane and cytoskeleton,which plays an important role in maintaining shape,polarity,and movement as well as signal transduction of cells.The recent studies indicate that Ezrin abnormally overexpresses in majority of tumor cells,which has close relationship with carcinogenesis,invading and metastasis of tumor cells.

Expression of occludin and zona occluden-1 and their morphologic changes in vagina mucosal cells in patients undergoing vaginal construction by using sigmoid colon / 中华妇产科杂志

quamous metaplasia occurred, the expression of occludin and ZO-1 in cells were similar to those in control group, which might play a role in the defense function of neovaginal mucosa.

Expression and activation of insulin receptor substrate-1 in endometrtal carcinoma / 中华妇产科杂志

Objective To investigate the mRNA,protein expression and tyrosine phosphorylation of insulin receptor substrate.1(IRS-1)in endometrial carcinoma.Methods Sixty-three endometrial carcinoma(EC)patients,21 endometrial atypical hyperplasia(AHE)patients and 22 normal control(NE) entered this smdy.Their clinical information were colleeted Fasting serum C-peptide concentration was measured.Expression of IRS-1 in endometrium was examined by RT-PCR and western blol Immunoprecipitation was used to measure the tyrosine phosphorylation of IRS-1.Results C-peptide 0.007].There were no significant differences in IRS-l mRNA and protein expression among the three grouDs.Tyrosine phosphorylation of IRS-1 in EC group[(62±36)%]was higher than that in AHE and NE groups[(53 4-34)%and(35 4-33)%;P=0.048,0.002].IRS-1 activation in AHE group was also higher than nornlal control(P=0.045).IRs-1 activation in endometrioid carcinoma[(69 4-33)%]was higher than that in other histological types[(34±31)%;t=2.300,P=0.025].IRs-l tyrosine phosphorylation was significantly higher in patients with advanced stage,high grade,deep myometrial invasion and pelvic lymph node metastasis.IRS-l activation in endometrium was positively correlated with fasting sertlm C-peptide concentration(r=0.491,P=0.001).Conclusions There is excessive activation of IRS-1 in endometrial carcinoma and atypical hyperplasia.Activation of IRS-I in endometrial carcinoma is related with poor clinical-pathologic fleatures and may be a prognostic predictor for this tumor.Over-activationof IRS-1 may be an intermediate event linking the hyperinsulinemia and endometrial carcinoma.

Relationship between tyrosine phosphorylation and protein expression of insulin receptor substrate-1 and insulin resistance in gestational diabetes mellitus / 中华妇产科杂志

Objective To study the relationship between tyrosine phosphorylation(TP)and protein expression of insulin receptor substrate-1(IRS-1)and insulin resistance in patients with gestational diabetes mellitus(GDM).Methods IRS-1 expression and TP in skeleton muscle tissue were determined by Western blot and immunoprecipitation in women with GDM(GDM group,n=22),normal pregnant women(normal pregnancy group,n=22)and normal nonpregnant women(normal nonpregnant group,n=13).Fasting plasma glucose(FPG)and fasting insulin(FINS)were measured by oxidase assay and immunoradioassay. Results(1)The levels of FPG,FINS,and insulin resistance index were calculated according to homeostasis model assessment [ HOMA-IR;(5.6?0.8)mmol/L,(15.4?5.1)mU/L,and 1.2?0.5 ] in GDM group were significantly higher than those in normal pregnancy group [(4.4+0.5)mmol/L,(10.6 ?3.1)mU/L,and 0.8?0.3;P

The Construction of Human Cytomegalovirus pp65 Prokaryotic Expressing Vector and the Inducement of Specific Immune Response / 中华围产医学杂志

Objective To construct human cytomegalovirus pp65 prokaryotic expressing vector and induce specific T lymphocyte immune response with recombinant pp 65 protein of human cytomegalovirus(HCMV) to observe the effect of CTL on infected HEL cells. Methods The whole cDNA of HCMV pp65 was amplified by PCR and inserted into prokaryotic expressing vector pRSET by gene engineer technique. The product was purified by affinity chromphotography and identified with Western blot after that recombinant plasmid was expressed by the induction of IPTG. With MTT technique, we observed the stimulating and proliferating effect of recombinant HCMV pp65 protein on PBMC in vitro. Cytotoxicity of PBMC on HCMV-infected HEL cells was detected. Results The pp65 prokaryotic expressing vector was successfully constructed and could express in engineering bacteria DE3. High dose of pure recombinant protein was acquired and had been identified. The rhHCMV pp65 protein can activate PBMC and cause the proliferation of it in vitro. The proliferated PBMC have the specific cytotoxicity to HEL cells infected by HCMV. Conclusions The acquired recombinant HCMV pp65 protein could induce specific T cells immune response in vitro to kill the HCMV infected HEL cells. And it is very important for the immune therapy of the HCMV infections.

Dephosphorylation of cell-surface phosphoproteins of goat spermatozoa.

Multiple ecto-phosphoproteins of the goat cauda-epididymal intact spermatozoa have been shown to undergo dephosphorylation in vitro by endogenous phosphoprotein phosphatase(s) located on the sperm outer surface. The major ecto-phosphoproteins that are dephosphorylated have molecular masses of 27, 40, 70, 116 and 205 kDa. The cell surface dephosphorylation reaction is not dependent on bivalent metal ions. Mg2+ (5 mM), Mn2+ (5 mM), orthovanadate (200 μΜ) and cAMP (5 μΜ) have no effect on this surface reaction whereas it is inhibited nearly 50% by Co2+ or Zn2+ (1 mM). Spermidine (5 mM), or Ca2+ (1mM) inhibited to a small extent (approx. 25%) the cell surface dephosphorylation of proteins.