Zfyve16 (a.k.a. endofin or endosome-associated FYVE-domain protein), a member of the FYVE-domain protein family, is involved in endosomal trafficking and in TGF-β, BMP, and EGFR signaling. The FYVE protein SARA regulates the TGF-β signaling pathway by recruiting Smad2/3 and accelerating their phosphorylation, thereby altering their susceptibility to TGF-β-mediated T cell suppression. Zfyve16 binds to Smad4 and their binding affects the formation of Smad2/3-Smad4 complex in TGF-β signaling. However, the in vivo function of Zfyve16 remains unknown. In this study, we generated a Zfyve16 knockout mouse strain (Zfyve16) and examined its hematopoietic phenotypes and hematopoietic reconstruction ability. The proportion of Tcells in the peripheral blood of Zfyve16 mice increases compared with that in wild-type mice. This finding is consistent with the role of Zfyve16 in facilitating TGF-β signaling. Unpredictably, B cell proliferation is inhibited in Zfyve16 mice. The proliferation potential of Zfyve16 B-lymphoid cells also significantly decreases in vitro. These results suggest that Zfyve16 inhibits the proliferation of T cells, possibly through the TGF-β signaling, but upregulates the proliferation of B-lymphoid cells.
Animals ; B-Lymphocytes ; metabolism ; CD4-Positive T-Lymphocytes ; metabolism ; Cell Movement ; Cell Proliferation ; genetics ; Intracellular Signaling Peptides and Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Mice ; Mice, Knockout ; Serine Endopeptidases ; genetics ; metabolism ; Signal Transduction ; Smad Proteins, Receptor-Regulated ; metabolism ; Transforming Growth Factor beta ; metabolism ; Up-Regulation

OBJECTIVETo observe the expression of serine protease HtrA1 in human periodontal ligament tissue and to explore the effect of HtrA1 on the proliferation of human periodontal ligament cells (hPDLC).

METHODSSix human premolars and three human third molars(patient's ages ranging from 12 to 25, with intact root, without caries and/or periodontitis) were obtained in the Department of Maxillofacial Surgery of Wuhan University Hospital of Stomatology. Reverse transcription-PCR(RT-PCR) and immunohistochemistry analysis were applied to investigate the expression of HtrA1. Primary hPDLC were obtained by tissue-culture method in vitro. The proliferation of hPDLC was determined by methyl thiazolytetrazolium(MTT). Lentivirus-mediated over-expression and reduction of HtrA1 level was performed. An empty vector was used as negative control. On days 1, 3, 5, 7 and 9, the growth of hPDLC was characterized using cell counting kit-8(CCK-8) assay.

RESULTSRT-PCR data indicated that HtrA1 mRNA was expressed in human periodontal ligament tissue. Immunohistochemistry analysis showed HtrA1 was expressed in human periodontal ligament, mainly in the cytoplasm of hPDLC and the extracellular matrix. The MTT result suggested that the growth curve was consistent with the growth characteristics of hPDLC. The stable over-expression and knockdown cell lines was successfully established by lentivirus with more than 90% transfection efficiency. CCK-8 assay showed that HtrA1 over-expression inhibited the proliferation of hPDLC(0.897±0.060, 0.890±0.083, 1.631±0.038, 1.111±0.041, 1.110±0.189), while cell proliferation increased after down-regulation of HtrA1(0.329±0.021, 0.529±0.044, 0.973±0.056, 1.626±0.102, 2.344±0.198)(P<0.05).

CONCLUSIONSHtrA1 is expressed in human periodontal ligament tissue at both mRNA and protein levels, and may play an important role in regulating the proliferation of hPDLC.

Adolescent ; Adult ; Cell Count ; Cell Proliferation ; Cells, Cultured ; Child ; Down-Regulation ; Genetic Vectors ; High-Temperature Requirement A Serine Peptidase 1 ; Humans ; Lentivirus ; physiology ; Periodontal Ligament ; cytology ; metabolism ; RNA, Messenger ; metabolism ; Serine Endopeptidases ; genetics ; metabolism ; Transfection ; Young Adult

Animals ; Cell Differentiation ; Hepatocyte Growth Factor ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; metabolism ; Protein Precursors ; genetics ; metabolism ; Serine Endopeptidases ; genetics ; metabolism

This study was aimed to investigate the expression of RHBDD1 gene in patients with chronic myeloid leukemia (CML) and explore its clinical significance. The relative expression levels of RHBDD1 in bone marrow mononuclear cells of healthy controls and CML patients were detected by using real time PCR. The results showed that the expression level of RHBDD1 in CML patients was significantly higher than that in healthy controls. The expression level of RHBDD1 in CML patients with negative BCR/ABL p210 was remarkably higher than that in patients with positive BCR/ABL p210. In patients ≥ 50 years old RHBDD1 expression was lower than the patients < 50 years old. There were no significant relation of RHBDD1 expression with sex of patients. It is concluded that RHBDD1 gene may be involved in the pathogenesis and progression of CML, particularly reflects in the pathogenesis of the patients with negative BCR/ABL p210.
Bone Marrow Cells ; metabolism ; pathology ; Case-Control Studies ; Female ; Gene Expression ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Serine Endopeptidases ; genetics ; metabolism

PURPOSE: The purpose of this study was to determine the pharmacogenetic effects of complement factor H (CFH) Y402H, LOC387715 and high-temperature requirement factor A1 (HTRA1) genotypes on the treatment of exudative age-related macular degeneration (AMD) by intravitreal bevacizumab injection in a Korean population. METHODS: Seventy-five patients diagnosed with exudative AMD were treated with intravitreal bevacizumab (2.5 mg) monotherapy. All patients received three initial intravitreal bevacizumab injections every four weeks and were then treated "as needed" based on clinical findings, optical coherence tomography and fluorescein angiography during the 12 month follow-up period after the third injection. RESULTS: The difference in visual acuity improvement among the three genotypes of LOC387715 were statistically significant at six months post-treatment (logarithm of the minimum angle of resolution; TT, 0.346; GT, 0.264; GG, 0.188; p = 0.037). Among the LOC387715 genotypes, the number of additional injections was lower in patients who had the risk T allele (GG, 2.143; GT, 2.000; TT, 1.575; p = 0.064). There was no significant difference between visual acuity and central macular thickness change in the CFH Y402H polymorphism group during the 12 month follow-up period. However, the TC group of CFH Y402H required more additional bevacizumab injections than the TT group (TT, 1.517; TC, 3.363; p = 0.020). CONCLUSIONS: This study demonstrated that different LOC387715/HTRA1 genotypes resulted in different bevacizumab treatment responses on exudative AMD. Patients with the risk allele had an improved treatment response and less need for additional injections. However, patients with the CFH Y402H risk allele needed more additional injections of bevacizumab in order to improve visual acuity. This study illustrates how pharmacogenetic factors may help determine treatment modality and dosing. This could ultimately provide basic data for 'personalized medicine' in AMD.
Aged ; Alleles ; Angiogenesis Inhibitors/administration & dosage/therapeutic use ; Antibodies, Monoclonal, Humanized/*administration & dosage/therapeutic use ; DNA/*genetics ; Female ; Follow-Up Studies ; Genotype ; Humans ; Intravitreal Injections ; Macular Degeneration/drug therapy/epidemiology/*genetics ; Male ; Pharmacogenetics/*methods ; *Polymorphism, Genetic ; Retrospective Studies ; Serine Endopeptidases/*genetics/metabolism ; Vascular Endothelial Growth Factor A/antagonists & inhibitors ; Visual Acuity

OBJECTIVESTo investigate the prognostic significance of Omi/HtrA2 expression, and the correlation between Omi/HtrA2 and Hypoxia-inducible factor (HIF)-1α in primary hepatocellular carcinoma cells.

METHODSThe expression of HIF-1α and Omi/HtrA2 in 43 cases of hepatic carcinoma tissues were detected immunohistochemically. Follow-up data were obtained to perform the Kaplan-Meier survival analysis. The change of Omi/HtrA2 expression in HepG2 cell was measured after HIF-1α expression of HepG2 in vitro was regulated by Tet-on expression system.

RESULTSOmi/HtrA2 expression was correlated with lymph node metastasis and recurring within liver during 2 years. Statistical analysis estimation showed the cumulative survival rate of post-hepatectomy for the patients with the positive expression of Omi/HtrA2 was higher than that for other patients with the negative expression of Omi/HtrA2 (χ(2) = 6.13, P = 0.013). In the common paraffin-embedded specimen of hepatocellular carcinoma, most of the samples showing negative or weak positive HIF-1α immunopositivity showed moderate positive or strong positive Omi/HtrA2 immunopositivity, while most of the samples showing moderate positive or strong positive HIF-1α immunopositivity showed negative or weak positive Omi/HtrA2 immunopositivity. The mRNA expression intensity of Omi/HtrA2 was decreasing with the HIF-1α expression increasing, and the difference was statistically significant(F = 106.766, P < 0.01).

CONCLUSIONSOmi/HtrA2 may be an important prognostic marker for primary hepatocellular carcinoma. Omi/HtrA2 expression is reversely correlated with HIF-1α expression in hepatocellular carcinoma. During the apoptotic process Omi/HtrA2 participating in hepatocellular carcinoma cells, HIF-1α is involved in the controlling and regulating of Omi/HtrA2 expression.

Adolescent ; Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Female ; Follow-Up Studies ; Hep G2 Cells ; High-Temperature Requirement A Serine Peptidase 2 ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Liver Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Mitochondrial Proteins ; metabolism ; Neoplasm Recurrence, Local ; metabolism ; pathology ; Prognosis ; Serine Endopeptidases ; metabolism ; Transfection ; Tumor Cells, Cultured ; Young Adult

Objective of this study was to detect the expression of HtrA2 and WT1 mRNA in acute myeloid leukemia (AML) and investigate the relationship of their expression levels with clinical variates and correlation between them. The expression levels of HtrA2 and WT1 were measured by RQ-PCR in bone marrow cells in 104 newly diagnosed AML patients and leukemia cell lines (K562, HL-60, NB4, Kasumi-1, U937), and the relationship between expression level and clinical parameters (age, sex, WBC count, diagnosis and prognosis) was investigated. The results showed that (1) the expression of HtrA2 gene in newly diagnosed AML was lower than that of the normal controls (P < 0.01), while expression of WT1 gene in newly diagnosed AML was higher than that of the normal controls (P < 0.01), the expression levels of HtrA2 and WT1 genes both did not correlate with age, sex and WBC counts of patients. There were no significant difference of HtrA2 gene expression between different NCCN prognosis group, while WT1 gene expression in better-risk group was significantly lower than that in intermediate-risk group (P = 0.003). The HtrA2 expression level rose after treatment in both CR group and non-CR group (P < 0.05), while WT1 expression level significantly decreased after treatment only in CR group (P < 0.01). Negative correlation between HtrA2 and WT1 expression was also observed (r = -0.249, P = 0.011). It is concluded that the low expression of HtrA2 and high expression of WT1 are closely related with occurrence and development of acute leukemia, so up-regulating expression of HtrA2 and interfering expression of WT1 may become the targets for leukemia therapy in the future.
Adolescent ; Adult ; Aged ; Cell Line, Tumor ; Female ; High-Temperature Requirement A Serine Peptidase 2 ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; Middle Aged ; Mitochondrial Proteins ; genetics ; metabolism ; Serine Endopeptidases ; genetics ; metabolism ; WT1 Proteins ; genetics ; metabolism ; Young Adult

Carcinoma ; genetics ; metabolism ; pathology ; surgery ; China ; Humans ; Male ; Oncogene Proteins, Fusion ; genetics ; Prostatic Hyperplasia ; genetics ; metabolism ; pathology ; surgery ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; surgery ; Proto-Oncogene Proteins c-ets ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Serine Endopeptidases ; genetics ; metabolism

OBJECTIVETo express and purify Hap protein of nontypeable Haemophilus influenzae (NTHi) in prokaryotic system, and study its immunogenicity and adhesive activity.

METHODHap protein was expressed in E.coli BL21 with pET32a (+)-Hap and purified by affinity chromatography. The adhesive activity of the recombinant Hap protein was observed in competitive adhesion assay using scanning electron microscope and bacterial counting. BALB/C mice were immunized intranasally with the purified recombinant Hap protein and cholera toxin B subunit (CT-B), and anti-Hap IgA and IgG were detected by enzyme-linked immunosorbent assay.

RESULTSSDS-PAGE analysis showed a single band of the target protein, whose purity reached 85% according to the result of Gel analysis software. The concentration of the protein was 3.2 g/L after ultrafiltration and condensation. Competitive adhesion assay showed that compared with control group, the recombinant Hap protein significantly inhibited the adhesion of NTHi to ECM (P<0.01). Compared with Hap immunization alone, immunization with Hap combined with CT-B resulted in significantly higher titers of anti-Hap IgG and IgA in mice (P<0.05).

CONCLUSIONHighly purified recombinant Hap protein has been obtained in a prokaryotic system and shows good immunogenic and adhesive activities. These results will establish the basis for further study of NTHi vaccine.

Adhesiveness ; Animals ; Bacterial Outer Membrane Proteins ; biosynthesis ; genetics ; immunology ; Cholera Toxin ; immunology ; Escherichia coli ; genetics ; metabolism ; Haemophilus Infections ; prevention & control ; Haemophilus influenzae ; metabolism ; Immunization ; Immunoglobulin A ; blood ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Serine Endopeptidases ; biosynthesis ; genetics ; immunology

OBJECTIVETo explore the expression of serine protease Omi/HtrA2 in gastric carcinoma tissue and its association with clinicopathological features and prognosis.

METHODSOmi/HtrA2 protein expression levels were detected by immunohistochemistry method in resected gastric carcinomas(n=68), adjacent noncancerous tissues(n=15), and normal tissues(n=15), and its association with clinicopathological features and prognosis were analyzed.

RESULTSOmi/HtrA2 expression was positive in 73.5%(50/68) of gastric cancer tissues, which was significantly higher than that in adjacent noncancerous tissues and normal tissues(P<0.05). There were no significant differences in Omi/HtrA2 expression with respect to sex, age, tumor size, and depth of invasion(all P>0.05). Omi/HtrA2 expression level was significantly associated with tumor differentiation, extent of lymph node metastasis, and tumor stage(all P<0.05). Overall 5-year survival rate of patients with gastric carcinoma was 63.3%. Five-year survival rate was higher in Omi/HtrA2 positive cases than Omi/HtrA2 negative cases(72.0% vs. 61.1%), however the difference was not statistically significant.

CONCLUSIONSOmi/HtrA2 expression is more common in gastric carcinoma. Omi/HtrA2 expression is associated with tumor differentiation, extent of lymph node metastasis, and tumor stage.

Adult ; Aged ; Aged, 80 and over ; Female ; High-Temperature Requirement A Serine Peptidase 2 ; Humans ; Male ; Middle Aged ; Mitochondrial Proteins ; genetics ; metabolism ; Neoplasm Staging ; Prognosis ; Serine Endopeptidases ; genetics ; metabolism ; Stomach ; metabolism ; pathology ; Stomach Neoplasms ; metabolism ; pathology ; Young Adult