Nocathiacin I, a glycosylated thiopeptide antibiotic, displays excellent antibacterial activities against multidrug resistant bacterial pathogens. Previously, a novel nocathiacin I formulation for intravenous administration has been successfully developed and its aqueous solubility is greatly enhanced for clinical application. The purpose of the present study was to increase the fermentation titer of nocathiacin I and reduce or eliminate analogous impurities by screening the medium ingredients using response surface methodology. After a sysmatic optimization, a water-soluble medium containing quality-controllable components was developed and validated, resulting in an increase in the production of nocathiacin I from 150 to 405.8 mg·L at 150-L scale. Meanwhile, the analogous impurities existed in reported processes were greatly reduced or eliminated. Using optimized medium for fermentation, nocathiacin I with pharmaceutically acceptable quality was easily obtained with a recovery of 67%. In conclusion, the results from the present study offer a practical and efficient fermentation process for the production of nocathiacin I as a therapeutic agent.
Actinobacteria ; growth & development ; metabolism ; Anti-Bacterial Agents ; biosynthesis ; chemistry ; Bioreactors ; Culture Media ; Fermentation ; Intercellular Signaling Peptides and Proteins ; Peptides ; chemistry ; metabolism ; Quality Improvement

OBJECTIVE: In this study, we have detected the expression of STC2 protein in laryngeal cancer and The safe incisal margin by immunohistochemical staining, to evaluate its correlation with clinical features and the prognosis value in laryngeal squamous cell cancer. METHOD: Eighty-one paraffin-embedd specimens of patients who underwent surgery for laryngeal cancer were collected. Clinical data, including date of birth, gender, tumor subsite, tumor stage, RT, and survival status have been recorded. The samples including laryngeal cancer tissues and the safe incisal margin were analyzed for the expression of STC2 protein by immunohistochemical staining. We analyzed the correlations with STC2 expression level and clinical data with software SPSS 18. 0. RESULT: (1)fifty-six cases in 81 cases (69. 1 %) are positive. In 30 cases of the safe incisal margin tissues, STC2 is not expressed, which shows statistically significant differences between the two groups (P<0. 05). (2)Overexpression of STC2 in laryngeal cancer were correlated with the tumor clinical stage and N stage, T stage, tumor site location and histological grade (P<0. 05), but not with genderor age (P> 0. 05). (3)The result showed that the 3-year survival rate is 74. 1%, according to the survival analysis by STC2, STC2 positive group has poorer outcome versus the negative group. (4)The clinical stage displayed the independent value in predicting the tumor prognosis of laryngeal cancer (P<0. 05), not the expression level of STC2. CONCLUSION Overexpression of STC2 in laryngeal cancer patients is correlated with poor outcome, which means STC2 positive group has poorer prognosis than the STC2 negative group.
Carcinoma, Squamous Cell ; metabolism ; mortality ; Glycoproteins ; biosynthesis ; Head and Neck Neoplasms ; metabolism ; mortality ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; Laryngeal Neoplasms ; metabolism ; mortality ; Prognosis ; Squamous Cell Carcinoma of Head and Neck ; Survival Analysis ; Survival Rate

HIV-1 integrase (IN) is a key enzyme for the viral replication. The protein-protein interaction (PPI) between HIV-1 IN and a cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is a validated target for anti-HIV drug discovery. In order to build the platform for screening inhibitor against PPI between IN and LEDGF/p75, the vector containing the LEDGF/p75 protein cDNA was constructed and expressed in Escherichia coli and the function of the LEDGF/p75 protein was assayed. The LGDGF/p75 encoding gene optimized according to the preference codon usage of E. coli, was synthesized and cloned into the expression vector pGEX-4T-1 to form a recombined plasmid, then transformed into host cell E. coli BL21 (DE3). The recombined clones were identified and confirmed by BamH I/Sal I digestion and sequencing, the successfully recombined plasmid in the host cell was induced by IPTG and the condition of the expression was optimized. The expressed protein was purified by the Ni2+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombinant protein. The recombinant LGDGF/p75 was soluble, and expressed highly and stably in E. coli. The protein was proved to enhance HIV-1 IN strand transfer activity in vitro by ELISA. It will be helpful to build the platform of screening inhibitors against PPI between IN and LEDGF/p75.
Cloning, Molecular ; Escherichia coli ; metabolism ; HIV Integrase ; metabolism ; HIV-1 ; physiology ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; Protein Binding ; Virus Replication

This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.
Calcium-Binding Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; metabolism ; Genetic Vectors ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Jagged-1 Protein ; Membrane Proteins ; genetics ; metabolism ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serrate-Jagged Proteins

This study was aimed to investigated the mRNA expression levels of Notch ligands- Delta-like-1 and Jagged-1 in bone marrow mesenchymal stem cells of patients with myelodysplastic syndrome (MDS), and to explore their relation with onset of MDS. Bone marrow mesenchymal stem cells of 38 patients with MDS and 16 normal subjects as control were collected to detect mRNA expression of Delta-like-1 and Jagged-1 by using real-time quantitative polymerase chain reaction. The results showed that the expression levels of Delta-like-1 and Jagged-1 in mesenchymal stem cells of MDS patients were significantly higher than that in normal controls (P < 0.05). According to WHO criteria, the mRNA expression of Delta-like-1 in RA/RAS, RCMD and RAEB groups were significantly higher than that in normal controls (P < 0.05), the mRNA expression of Jagged-1 in RAEB group was also significantly higher than that in normal controls (P < 0.05). The mRNA expression of Delta-like-1 was significantly correlated with the proportion of blasts in the bone marrow of MDS patients (r = 0.502, P < 0.05). The expression levels of Delta-like-1 and Jagged-1 in MDS patients with abnormal karyotypes were significantly higher than those in MDS patients with normal karyotypes (P < 0.05). The mRNA expression of Delta-like-1 in higher risk group according to International Prognostic Scoring System was significantly higher than that in lower risk group (P < 0.05), there was no significant difference in Jagged-1 expression levels between higher risk group and lower risk group (P > 0.05). It is concluded that the changes of Delta-like-1 and Jagged-1 expression level in MSC may play a role in the pathogenesis of myelodysplastic syndrome.
Calcium-Binding Proteins ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; Intracellular Signaling Peptides and Proteins ; genetics ; Jagged-1 Protein ; Membrane Proteins ; genetics ; Mesenchymal Stromal Cells ; metabolism ; Myelodysplastic Syndromes ; genetics ; RNA, Messenger ; biosynthesis ; Serrate-Jagged Proteins

OBJECTIVE: To explore the down-expression mechanism of MYETS1 gene in multiple myeloma cell lines ARH-77 or KM3, and express MYETS1 gene in prokaryotic express system. METHODS: The region of chromosome 13q14.3 in ARH-77 and KM3 was detected by FISH. MYETS1 gene was amplified by RT-PCR and cloned into prokaryotic expression vector pGEX-4T. RESULTS: Positive consequence was acquired in 13q14.3 where MYETS1 located by FISH in ARH- 77 and KM3 cell lines. Bioinformatics indicated highly sequence homology between MYETS1 and LECT1, but excluded the homology of open reading frame between MYETS1 and that of LECT1 by RT-PCR. Myets1 protein was expressed and harvested successfully. CONCLUSION The region of chromosome 13q14.3 ,where MYETS1 gene located, was not defected in ARH-77 and KM3 cell lines. Down-expression of MYETS1 might be regulated by other mechanisms in multiple myeloma cell lines.
Cell Line, Tumor ; Chromosomes, Human, Pair 13 ; genetics ; Gene Deletion ; Genetic Vectors ; genetics ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Membrane Proteins ; biosynthesis ; genetics ; Multiple Myeloma ; genetics ; metabolism ; pathology ; Neoplasm Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

Wnt inhibitor factor-1 (WIF-1) is an extracellular antagonist of Wnts secreted proteins, first characterized as an expressed sequence tag in the human retina. WIF-1 belongs to the secreted Frizzled-related protein (sFRP) class, which can directly bind to Wnt proteins, prevent Wnts from binding to their receptors in vertebrates. Wif1 is expressed in the nervous system of mouse, Xenopus, zebrafish and human. It has been shown that WIF-1 affects the formation of somites in Xenopus embryos and inhibits rod production in retinal histogenesis by binding to Wnt4 in mice. Histological information of Wif1 expression during the development of the central nervous system has been reported in mouse, Xenopus and zebrafish and the strong embryonic expression suggests Wif1 may play an essential role in the spatial and temporal regulation of Wnt signals in development of central nervous system. The Wnt pathway plays a key role in the patterning of the nervous system. However, insights into the function of Wif1 in the development of the central nervous system are rather limited. Selecting suitable stage and target according to the expression pattern may contribute to understanding the function of Wif1.
Adaptor Proteins, Signal Transducing ; biosynthesis ; physiology ; Animals ; Brain ; embryology ; metabolism ; Central Nervous System ; embryology ; metabolism ; Extracellular Matrix Proteins ; biosynthesis ; physiology ; Gene Expression ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; physiology ; Mice ; Repressor Proteins ; biosynthesis ; physiology ; Signal Transduction ; physiology ; Spinal Cord ; embryology ; metabolism ; Xenopus

OBJECTIVETo construct a small interfering RNA (siRNA) expression vector targeting hepatoma-derived growth factor (HDGF) and establish a lung adenocarcinoma cell line stably expressing siRNA-HDGF.

METHODRT-PCR was used to examine HDGF expression in lung adenocarcinoma samples and the matched adjacent lung tissues, and also in lung adenocarcinoma SPC-A-1 cell line. A recombinant lentivirus shRNA-HDGF vector was constructed and transfected into SPC-A-1 cells via Lipofectamine 2000, and the cells with stable expression of HDGF-siRNA was screened by blasticidin selection. The interference effect of siRNA-HDGF was assessed by real-time PCR.

RESULTSCompared to the adjacent lung tissues, lung adenocarcinoma and SPC-A-1 cells showed increased expression of HDGF. The recombinant lentivirus shRNA-HDGF vector was successfully constructed and verified by sequence analysis. siRNA-HDGF recombinants markedly inhibited the expression of HDGF in SPC-A-1 cells.

CONCLUSIONHDGF expression increases in lung adenocarcinoma and SPC-A-1 cell lines. The recombinant siRNA-HDGF lentivirus vector can inhibit the expression of HDGF in SPC-A-1 cells.

Adenocarcinoma ; metabolism ; pathology ; Base Sequence ; Cell Line, Tumor ; Female ; Genetic Vectors ; genetics ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Lentivirus ; genetics ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Male ; Molecular Sequence Data ; RNA Interference ; RNA, Small Interfering ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA

OBJECTIVETo investigate the role of tazarotene induced gene-2 (TIG2) in psoriasis vulgaris.

METHODSTIG2 protein and mRNA expressions in normal tissues, psoriatic lesions and uninvolved skin tissues were detected by immunohistochemistry and in situ hybridization, respectively.

RESULTSTIG2 protein and mRNA were expressed in all the layers of normal and uninvolved epidermis. TIG2 expression was detected in the upper layers of the stratum spinosum of the marginal region of the psoriatic lesions, but not in the central area of the lesions. TIG2 expression was significantly lower in the basal layers of the central area of the paoriasis than that in the normal skin and uninvolved tissues (P < 0.01), and also lower in the marginal regions of the lesions (P < 0.01).The suprabasal layers of the marginal region in the lesion showed significantly lower TIG2 expression than the central area of the lesion (P < 0.01).

CONCLUSIONTIG2 may maintain the normal differentiation of epidermal keratinocytes and implicate in the pathogenesis and development of psoriasis vulgaris.

Adolescent ; Adult ; Chemokines ; Chemotactic Factors ; biosynthesis ; genetics ; Female ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Keratinocytes ; metabolism ; Male ; Middle Aged ; Psoriasis ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo explore the role of exogenous connective tissue growth factor (CTGF) in the collagen III synthesis of human renal tubular epithelial cell line HK2 in vitro.

METHODSCultured HK2 cells were randomly assigned to three groups: placebo-control, low-dose CTGF-treated (2.5 ng/mL) and high-dose CTGF-treated groups (20 ng/mL). Cell morphological changes were observed under an inverted microscope. Collagen III alpha mRNA expression was detected using RT-PCR. Immunohistochemistry staining was used to assess the levels of intracellular collagen III alpha protein.

RESULTSAfter 48 hrs of low- or high- dose CTGF treatment, the appearances of HK2 cells were changed from oval to fusiform. High-dose CTGF treatment increased collagen III alpha mRNA expression (0.4461+/-0.0274 vs 0.2999+/-0.0115; P<0.05) as well as the protein expression of collagen III alpha (0.4075+/-0.0071 vs 0.3503+/-0.0136; P<0.05) compared with the placebo-control group.

CONCLUSIONSCTGF can induce morphological changes of human renal tubular epithelial cells in vitro. High concentration of CTGF may increase the synthesis of collagen III alpha.

Cells, Cultured ; Collagen Type III ; biosynthesis ; genetics ; Connective Tissue Growth Factor ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Immediate-Early Proteins ; pharmacology ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Kidney Tubules ; drug effects ; metabolism ; RNA, Messenger ; analysis