Two new 2-carboxymethyl-3-hexyl-maleic anhydride derivatives, arthrianhydride A (1) and B (2), along with three known compounds 3-5, were isolated from the fermentation broth of a grasshopper-associated fungus Arthrinium sp. NF2410. The structures of new compounds 1 and 2 were determined based on the analysis of the HR-ESI-MS and NMR spectroscopic data. Furthermore, compounds 1 and 2 were evaluated on inhibitory activity against the enzyme SHP2 and both of them showed moderate inhibitory activity against SHP2.
Anhydrides/pharmacology* ; Animals ; Biological Products/pharmacology* ; Enzyme Inhibitors/pharmacology* ; Fungi/chemistry* ; Grasshoppers/microbiology* ; Molecular Structure ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors* ; Secondary Metabolism

The transition from spermatogonia to spermatocytes and the initiation of meiosis are key steps in spermatogenesis and are precisely regulated by a plethora of proteins. However, the underlying molecular mechanism remains largely unknown. Here, we report that Src homology domain tyrosine phosphatase 2 (Shp2; encoded by the protein tyrosine phosphatase, nonreceptor type 11 [Ptpn11] gene) is abundant in spermatogonia but markedly decreases in meiotic spermatocytes. Conditional knockout of Shp2 in spermatogonia in mice using stimulated by retinoic acid gene 8 (Stra8)-cre enhanced spermatogonial differentiation and disturbed the meiotic process. Depletion of Shp2 in spermatogonia caused many meiotic spermatocytes to die; moreover, the surviving spermatocytes reached the leptotene stage early at postnatal day 9 (PN9) and the pachytene stage at PN11-13. In preleptotene spermatocytes, Shp2 deletion disrupted the expression of meiotic genes, such as disrupted meiotic cDNA 1 (Dmc1), DNA repair recombinase rad51 (Rad51), and structural maintenance of chromosome 3 (Smc3), and these deficiencies interrupted spermatocyte meiosis. In GC-1 cells cultured in vitro, Shp2 knockdown suppressed the retinoic acid (RA)-induced phosphorylation of extracellular-regulated protein kinase (Erk) and protein kinase B (Akt/PKB) and the expression of target genes such as synaptonemal complex protein 3 (Sycp3) and Dmc1. Together, these data suggest that Shp2 plays a crucial role in spermatogenesis by governing the transition from spermatogonia to spermatocytes and by mediating meiotic progression through regulating gene transcription, thus providing a potential treatment target for male infertility.
Animals ; Cell Cycle Proteins/genetics* ; Cell Line ; Cell Survival ; Chondroitin Sulfate Proteoglycans/genetics* ; Chromosomal Proteins, Non-Histone/genetics* ; Gene Expression Regulation ; Gene Knockdown Techniques ; Infertility, Male ; Male ; Meiosis/genetics* ; Mice ; Mice, Knockout ; Mice, Transgenic ; Phosphate-Binding Proteins/genetics* ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics* ; Rad51 Recombinase/genetics* ; Real-Time Polymerase Chain Reaction ; Spermatocytes/metabolism* ; Spermatogenesis/genetics* ; Spermatogonia/metabolism*

BACKGROUNDSrc homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is a kind of intracellular protein tyrosine phosphatase. Studies have revealed its roles in various disease, however, whether SHP-2 involves in renal fibrosis remains unclear. The aim of this study was to explore the roles of myeloid cells SHP-2 in renal interstitial fibrosis.

METHODSMyeloid cells SHP-2 gene was conditionally knocked-out (CKO) in mice using loxP-Cre system, and renal interstitial fibrosis was induced by unilateral ureter obstruction (UUO). The total collagen deposition in the renal interstitium was assessed using picrosirius red stain. F4/80 immunostaing was used to evaluate macrophage infiltration in renal tubular interstitium. Quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay were used to analyze the production of cytokines in the kidney. Transferase-mediated dUTP nick-end labeling stain was used to assess the apoptotic renal tubular epithelial cells.

RESULTSSrc homology 2 domain-containing protein tyrosine phosphatase-2 gene CKO in myeloid cells significantly reduced collagen deposition in the renal interstitium after UUO. Macrophage infiltration was evidently decreased in renal tubular interstitium of SHP-2 CKO mice. Meanwhile, the production of pro-inflammatory cytokines was significantly suppressed in SHP-2 CKO mice. However, no significant difference was observed in the number of apoptotic renal tubular epithelial cells between wild-type and SHP-2 CKO mice.

CONCLUSIONSOur observations suggested that SHP-2 in myeloid cells plays a pivotal role in the pathogenesis of renal fibrosis, and that silencing of SHP-2 gene in myeloid cells may protect renal from inflammatory damage and prevent renal fibrosis after renal injury.

Animals ; Enzyme-Linked Immunosorbent Assay ; Female ; Fibrosis ; enzymology ; pathology ; Immunohistochemistry ; Kidney Diseases ; enzymology ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Myeloid Cells ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; genetics ; metabolism ; Ureteral Obstruction ; enzymology ; pathology

The Src homology-2 domain-containing phosphatase SHP-2 encoded by PTPN11 is an essential component in several signaling pathways.Different types of mutation in SHP-2 have been confirmed in several types of leukemia and solid tumors. Elucidation of the events underlying Shp2-evoked transformation may provide new insights into the novel targets for anti-cancer therapy.
Humans ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; chemistry ; metabolism ; physiology

Recent evidence supports a neuroprotective role of Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2) against ischemic brain injury. However, the molecular mechanisms of SHP-2 activation and those governing how SHP-2 exerts its function under oxidative stress conditions are not well understood. Recently we have reported that reactive oxygen species (ROS)-mediated oxidative stress promotes the phosphorylation of endogenous SHP-2 through lipid rafts, and that this phosphorylation strongly occurs in astrocytes, but not in microglia. To investigate the molecules involved in events leading to phosphorylation of SHP-2, raft proteins were analyzed using astrocytes and microglia. Interestingly, caveolin-1 and -2 were detected only in astrocytes but not in microglia, whereas flotillin-1 was expressed in both cell types. To examine whether the H2O2-dependent phosphorylation of SHP-2 is mediated by caveolin-1, we used specific small interfering RNA (siRNA) to downregulate caveolin-1 expression. In the presence of caveolin-1 siRNA, the level of SHP-2 phosphorylation induced by H2O2 was significantly decreased, compared with in the presence of control siRNA. Overexpression of caveolin-1 effectively increased H2O2-induced SHP-2 phosphorylation in microglia. Lastly, H2O2 induced extracellular signal-regulated kinase (ERK) activation in astrocytes through caveolin-1. Our results suggest that caveolin-1 is involved in astrocyte-specific intracellular responses linked to the SHP-2-mediated signaling cascade following ROS-induced oxidative stress.
Animals ; Astrocytes/*metabolism ; Caveolin 1/*genetics/metabolism ; Caveolin 2/genetics ; Cell Line ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Gene Expression ; Humans ; Microglia/metabolism ; Phosphoric Monoester Hydrolases/*metabolism ; Phosphorylation ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/*metabolism ; Rats ; Reactive Oxygen Species/*metabolism

OBJECTIVETo investigate the mutations in protein tyrosine phosphatase, nonreceptor-type 11 (PTPN11) gene in patients with Noonan syndrome (NS).

METHODSThree sporadic patients with NS were studied. Genomic DNAs were extracted from peripheral blood leukocytes. All 15 coding exons and their flanking intronic boundaries of the PTPN11 gene were amplified by polymerase chain reaction and followed by direct sequencing. DNAs from parents were sequenced in the corresponding region when the mutation was detected in their affected child. The identified mutation was screened in 100 healthy individuals for exclusion of polymorphism by restriction endonuclease digestion of the PCR products. Protein conservation analysis was performed among 10 species using an online ClustalW tool.

RESULTSDirect DNA sequence analysis identified a heterozygous 181G to A change in exon 3 of the PTPN11 gene in one patient, which resulted in the substitution of an aspartic acid residue by an asparagine at codon 61. The mutation was absent in his parents and 100 controls, and is located in a highly conserved amino acid site. No mutation in the coding region of PTPN11 gene was observed in the other two patients.

CONCLUSIONThe p.D61N mutation was reported previously in Caucasians and is a de-novo mutation in this patient. Our study further confirmed that the p.D61N is a pathogenic mutation for NS and consistent with the clinical diagnosis. Additional genes may be involved in the other two patients with NS, indicating high genetic heterogeneity of this disease.

Amino Acid Sequence ; Base Sequence ; Case-Control Studies ; Child ; Exons ; Female ; Humans ; Male ; Molecular Sequence Data ; Mutation, Missense ; Noonan Syndrome ; enzymology ; genetics ; Point Mutation ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Young Adult

BACKGROUND AND OBJECTIVEPrecious studies proven that aberrant tyrosine phosphorylation has linked with cancer. The aim of this work is to study the expression and significance of SHP2 in non-small cell lung cancer (NSCLC) through tissue microarray technique and immunohistochemical method.

METHODSEighty NSCLC specimens were constructed into tissue microarray and performed using immunohistochemistry.

RESULTSThe total positive rates of SHP2 were 70.7% (56/80) in NSCLC, 72.5% (29/40) in squamous cell carcinoma and 75% (27/40) in adenocarcinoma, which was not significant difference in sex, age, the size of tumor, histology, clinical stages and differentiation (P > 0.05), the positive rates of SHP2 were significantly higher in the cases with lymphnode metastasis than those without (P < 0.05).

CONCLUSIONThe expression rate of SHP2 is high and closely correlated to lymphnode metastasis in NSCLC, which implies the occurrence and development of lung cancer maybe related to SHP2, and SHP2 maybe a new marker and therapeutic targets for lung cancer.

Adenocarcinoma ; metabolism ; pathology ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Lung Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; pathology ; Male ; Middle Aged ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; metabolism ; Tissue Array Analysis

In order to express and purify the catalytic domain of SHP-1/SHP-2 (named as D1C and D2C respectively) and determine their kinetics, the constructed D1C and D2C plasmids were transformed into Escherichia coli BL21 and the expression was induced with IPTG. The harvested cells were suspended in extraction buffer. After sonication, the solution was applied to HPLC and the results were confirmed by SDS-PAGE. The purified peptides were further subjected to kinetic specificity study using synthetic phosphotyrosine (pY) as substrate by malachite green method and analyzed by Lineweaver-Burk plot calculation. From this study, we found D1C and D2C were expressed successfully in soluble state in Escherichia coli BL21 and purified efficiently with HPLC system. The molecular weight of D1C was 34.6 kD, and its Michaelis constant (K(m)) was 2.04 mmol catalytic constant (K(cat)) was 44.98 s(-1), specific constant (K(cat)/K(m)) was 22.05 L/(mmol x s); the molecular weigh of D2C was 35.3 kD, and its Michaelis constant (K(m)) was 2.47 mmol, catalytic constant (K(cat)) was 27.45 s(-1), specific constant (K(cat)/K(m)) was L/(mmol x s). The enzyme activity of D1C is stronger than that of D2C.
Catalytic Domain ; Chromatography, High Pressure Liquid ; Escherichia coli ; genetics ; metabolism ; Kinetics ; Plasmids ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; genetics ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

OBJECTIVETo study the effect of Panax notoginseng saponins (PNS) on (synaptophysin, syp) and tau gene expression in the brain tissue in senescence accelerated mouse prone 8 (SAMP 8).

METHODSAMP8 were randomly divided into 4 groups: PNS 23.38, 93.50 mg x kg(-1) group, huperzin A 0.038 6 mg x kg(-1) x d(-1) group and blank control group; the drug groups were treated with the designed drugs respectively per day by intragastric administration for 4 consecutive weeks, and double distilled water was given to blank control group. After treatment, the mRNA content of tau and syp were assayed by reverse transcription (RT) and real-time polymerase chain reaction (real-time PCR).

RESULTCompared with blank control group, the syp mRNA contents were increased in PNS groups (P < 0.05 or P < 0.01), and the tau mRNA content were not significant difference in all groups.

CONCLUSIONThis study suggests that PNS can up-regulate syp gene expression at transcriptional level in the brain of SAMP 8.

Aging ; drug effects ; metabolism ; Animals ; Brain ; drug effects ; metabolism ; Gene Expression ; drug effects ; genetics ; Mice ; Panax notoginseng ; chemistry ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; genetics ; metabolism ; Saponins ; pharmacology ; tau Proteins ; genetics ; metabolism

OBJECTIVETo explore the expression of tyrosine phosphatase containing C-src homology SH-2 (SHP-1 and SHP-2) in benign prostate hyperplasia.

METHODSWith En Vision two-step method, the expression of SHP-1 and SHP-2 was detected in 10 cases of normal prostate tissue, 30 cases of BPH, 20 cases of PIN, 20 cases of high differential Pca and 20 cases of low differential Pca.

RESULTThe expression of SHP-2 in normal group was mainly distributed in the cytoplasm of secretive cells and basal cells, and a little part in the nucleu. In BPH it was distributed equally in the plasm and nucleu. In PIN, high differential Pca and low differential Pca, SHP-2 expressed mainly in nucleu. The average dyeing index of SHP-2 in each group is 0.4, 1.7, 2.1, 2.2 and 2.6. SHP-1 positive expression in normal prostate, BPH, PIN and high differential Pca showed differentiating layer staining in the cytoplasm of secretive cells and basal cells, while not in low differential Pca. The average dyeing index of SHP-1 in each group is 1.8, 1.8, 1.5, 1.2 and 0.4.

CONCLUSIONThere are transformation in signal transduction relation with SHP-1 and SHP-2 in the progress of prostate cell proliferation, differentiation and malignant. The abnormal activation and distribution of SHP-2 might induce prostate reconstruction and hyperplasia, even carcinoma.

Adult ; Aged ; Cell Nucleus ; enzymology ; Cytoplasm ; enzymology ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Prostatic Hyperplasia ; enzymology ; pathology ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; metabolism ; Protein Tyrosine Phosphatases ; metabolism ; SH2 Domain-Containing Protein Tyrosine Phosphatases ; metabolism ; src-Family Kinases ; metabolism