Ranpirnase (onconase, ONC) is a new drug, with weak RNase activity and strong cytotoxicity to various tumor cells in vitro and in vivo. This study is to obtain recombination onconase (rONC) with high bioactivity. Based on the codon preference of Pichia pastoris, we designed and synthesized the gene according to cDNA sequences of ONC and the α mating factor's prepeptide. We screened positive clones after transforming the recombination plasmids into P. pastoris X-33, GSS115 and SMD1168. We screened the best combination of seven different vectors and host strains. Moreover, we optimized culture condition in shake flasks and 10 L bioreactor, and purified rONC from the supernatant after inducing it with 0.25% methanol by aqueous two-phase extraction coupling G50 molecular exclusion method. The highest rONC production was 13 mg/L in pPICZα-A/X-33/ONC combination under the condition of pH 5.5 and 23 degrees C in shake flasks for 7 d; and that the highest rONC production was 180 mg/L when the induction is performed in the lower basic salt medium with pH 5.5 in the 10 L bioreactor for 7 d. The yield of rONC is more than 90% at a purity of above 95%. rONC can kill various tumor cells in vitro. The expression and purification of rONC would be useful for further investigation of this new drug.
Antineoplastic Agents ; metabolism ; Bioreactors ; Cell Line, Tumor ; Codon ; DNA, Complementary ; Genetic Vectors ; Humans ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; Ribonucleases ; biosynthesis

Mammalian mitochondrial genome encodes a small set of tRNAs, rRNAs, and mRNAs. The RNA synthesis process has been well characterized. How the RNAs are degraded, however, is poorly understood. It was long assumed that the degradation happens in the matrix where transcription and translation machineries reside. Here we show that contrary to the assumption, mammalian mitochondrial RNA degradation occurs in the mitochondrial intermembrane space (IMS) and the IMS-localized RNASET2 is the enzyme that degrades the RNAs. This provides a new paradigm for understanding mitochondrial RNA metabolism and transport.
Cell Line ; Humans ; Mitochondrial Membranes ; metabolism ; Protein Transport ; RNA ; biosynthesis ; chemistry ; metabolism ; RNA Stability ; RNA, Mitochondrial ; Ribonucleases ; metabolism ; Tumor Suppressor Proteins ; metabolism

MCP-1-induced protein-1 (MCPIP1) is a newly identified protein that is crucial to immune regulation. Mice lacking MCPIP1 gene suffer from severe immune disorders, and most of them cannot survive longer than 12 weeks. Considerable progress has been made in revealing the mechanism underlying the immune regulatory function of MCPIP1. MCPIP1 can act as an RNase to promote the mRNA degradation of some inflammatory cytokines, such as IL-6 and IL-1. Pre-microRNAs are also confirmed to be the substrate of MCPIP1 RNase. The structure of MCPIP1 N-terminal conserved domain shows a PilT N-terminus-like RNase structure, further supporting the notion that MCPIP1 has RNase activity. MCPIP1 can also deubiquitinate TNF receptor-associated factor family proteins, which are known to mediate immune and inflammatory responses. In this review, we summarize recent progress on the immune regulatory role of MCPIP1 and discuss the mechanisms underlying its function.
Amino Acid Sequence ; Animals ; Humans ; Immunity ; Molecular Sequence Data ; Ribonucleases ; metabolism ; Transcription Factors ; chemistry ; metabolism ; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins ; metabolism ; Ubiquitination

The monitoring of airway inflammation has assessed in bronchial asthma directly by sputum examination, and indirectly by measurements in peripheral blood. To investigate the diagnostic value of these two methods, we compared nitric oxide (NO) metabolites, eosinophils, and eosinophil cationic protein (ECP) in sputum and blood in patients with asthma and control subjects. Sputum and serum were obtained from fifteen patients with asthma, and then were examined before anti-asthma treatment, including steroid preparations. ECP was measured by fluoroimmunoassay. NO metabolites were assayed by using modified Griess reaction. Asthmatic patients, compared with control subjects, had significantly higher level of NO metabolites, higher proportion of eosinophils, and higher levels of ECP in sputum. Asthmatic patients, compared with control subjects, however, had significantly higher number of eosinophils, and were at higher levels of ECP in blood. FEV1, FEV1 /FVC was negatively correlated with sputum eosinophils. The area under receiver operating characteristic(ROC) curve showed that eosinophils in sputum are significantly accurate markers than NO metabolites in sputum and blood. These findings suggest that the proportion of eosinophils in sputum have more accurate diagnostic marker of asthmatic airway inflammation than NO metabolites in sputum in differentiating asthmatic patients from control subjects.
Adult ; Area Under Curve ; Asthma/*blood/*metabolism ; Blood Proteins/*metabolism ; Comparative Study ; Eosinophils/*metabolism ; Female ; Fluoroimmunoassay ; Human ; Inflammation ; Male ; Nitrates/metabolism ; Nitric Oxide/blood/*metabolism ; Nitrites/metabolism ; ROC Curve ; Ribonucleases/blood/*metabolism ; Sputum/*metabolism

To prepare virus-like particles containing FluA/B mRNA as RNA standard and control in Influenza RNA detection, the genes coding the coat protein and maturase of E. coli bacteriophage MS2 were amplified and cloned into D-pET32a vector. Then we inserted 6 histidines to MS2 coat protein by QuikChange Site-Directed Mutagenesis Kit to construct the universal expressing vector D-pET32a-CP-His. In addition, the partial gene fragments of FluA and FluB were cloned to the down-stream of expressing vector. The recombinant plasmid D-pET32a-CP-His-FluA/B was transformed to BL21 with induction by IPTG. The virus-like particles were purified by Ni+ chromatography. The virus-like particles can be detected by RT-PCR, but not PCR. They can be conserved stably for at least 3 months at both 4 degrees C and -20 degrees C. His-tagged virus-like particles are more stable and easier to purification. It can be used as RNA standard and control in Influenza virus RNA detection.
Escherichia coli ; genetics ; metabolism ; Influenza A virus ; genetics ; metabolism ; Influenza B virus ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; RNA, Viral ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Ribonucleases ; chemistry ; Virion ; genetics ; metabolism

The traditional light microscopy has limitations for precise growth assays of malaria parasites in culture or for assessment of new compounds for antimalarial activity; the speed and high reproducibility of flow cytometry can overcome these limitations. A flow cytometric method using PicoGreen, a DNA-binding fluorochrome, was developed with optimal precision suitable for performing growth assays of low-parasitemia field isolates. In addition, intra- and inter-person reproducibility of the flow cytometric and the microscopic method were compared in order to quantitatively demonstrate the improved precision. RNase treatment contributed to the precision of the flow cytometric measurements by enhancing the signal-to-noise ratios. Coefficients of variation of the method were smaller than 10% for 0.1% or higher parasitemia samples. The intra- and inter-person coefficients of variation of the flow cytometric method were three to six times smaller than those of the microscopic method. The flow cytometric method developed in this study yielded substantially more precise results than the microscopic method, allowing determination of parasitemia levels of 0.1% or higher, with coefficients of variation smaller than 10%. Thus, the PicoGreen method could be a reliable high sensitivity assay for analysis of low parasitemia samples and might be applied to a high throughput system testing antimalarial drug activity.
*Flow Cytometry ; Fluorescent Dyes/chemistry ; Humans ; *Microscopy ; Organic Chemicals/chemistry ; Parasitemia/*diagnosis ; Plasmodium falciparum/*isolation & purification ; Reproducibility of Results ; Ribonucleases/metabolism ; Signal-To-Noise Ratio

PURPOSE: Growth hormone(GH) produces a variety of effects in adipose tissue via GHRs on the cell membrane. In mouse, alternative splicing of the nascent transcript from the GHR gene produces two major transcripts:GHR mRNA and GHR binding protein(GHBP) mRNA. These two transcripts share the common extracellular ligand-binding domain, but differ in the C-terminal sequence. Since GHR plays an important role in mediating the actions of GH in adipose metabolism, I initiated these studies to examine GHR gene expression during mouse 3T3-Ll preadipocyte-adipocyte differentiation. METHODS: GHR and GHBP transcripts were detected by RNase protection assay (RPA) using the antisense riboprobes complementary either to the specific sequence of the GHR or to the sequence shared by both GHR and GHBP mRNAs. RNA prepared from 3T3-L1 cells at day 0(preadipocytes) and day 7(adipocytes) after treatment with actinomycin D was analyzed by RPA. RESULTS: After stimulation of differentiation, mRNA abundance increased 25-fold and reached a maximal level by day 7 of adipogenesis. The GHR mRNA:GHBP mRNA ratio was 1.3+/-.15 and remained unchanged during differentiation. The decay rate for both mRNAs, estimated by treating the cells with actinomycin D, was approximately 24 hours and showed no significant difference between preadipocytes and adipocytes. CONCLUSION: GHR gene expression is upregulated during preadipocyte-adipocyte differentiation.
3T3-L1 Cells ; Adipocytes ; Adipogenesis ; Adipose Tissue ; Alternative Splicing ; Animals ; Cell Membrane ; Dactinomycin ; Gene Expression ; Growth Hormone* ; Metabolism ; Mice ; Negotiating ; Receptors, Somatotropin* ; Ribonucleases ; RNA ; RNA, Messenger

It is well known that the glomerular basement membrane heparan sulfate proteoglycan(GBM HSPG) synthesized by glomerular epithelial cell(GEC) has an important role in the permeability of glomerular basement membrane and cyclic AMP(cAMP) is involved in regulation of a wide variety of genes maybe including GBM HSPG gene. The direct effect of cAMP on GBM HSPG gene expression and metabolism was not evaluated as yet. Proteinuria represents an impairment of permselectivity function of glomerular basement membrane regulated by GBM HSPG and could be associated with increased glomerular level of cAMP in nephrotic syndrome of diverse causes. RPD-I(rat GBM HSPG core protein domain-I) detected a >9.5kb transcript of GBM HSPG in RNA of rat GEC. Emp1oying a riboprobe synthesized from RPD-I in RNase protection assay, we examined whether cAMP regulated perlecan expression in the GEC. At l, 6, 24 and 48 hrs of incubation, l mM cAMP caused 43%, 32%, 47% and 40% reduction in mRNA expression of perlecan, respectively. Immunoprecipitation showed a corresponding reduction of 51%, 70% and 68% in the synthesis of 35SO4 labeled GBM HSPG by the GEC fol1owing l2, 24 and 48 hrs of incubation with cAMP. Our results show that decrease in GBM HSPG gene expression and synthesis by cAMP may be of relevance to proteinuric states characterized by activation of these mediators.
Animals ; Cyclic AMP* ; Epithelial Cells* ; Gene Expression ; Glomerular Basement Membrane* ; Heparan Sulfate Proteoglycans* ; Heparitin Sulfate* ; Immunoprecipitation ; Metabolism ; Nephrotic Syndrome ; Permeability ; Proteinuria ; Rats* ; Ribonucleases ; RNA ; RNA, Messenger

OBJECTIVETo observe the effect of Pingchuan Mixture (PCM) on plasma eosinophil cation protein (ECP), interleukin-5 in bronchial alveolar lavage fluid (BALF) and inflammatory cell count in experimental guinea pigs with asthma.

METHODSThe eosinophil, neutrophil, lymphocyte count were conducted by conventional method, IL-5 was detected by ELISA and ECP determined by RIA.

RESULTSLevels of eosinophil, neutrophil, lymphocyte, ECP and IL-5 after treatment were significantly lower than those before treatment, the difference between groups treated respectively by PCM, aminophylline, dexamethasone and Dingchuan Zhike Tablet was insignificant.

CONCLUSIONPCM could treat asthma by reducing the inflammatory cell count, ECP and IL-5.

Animals ; Asthma ; chemically induced ; metabolism ; Blood Proteins ; metabolism ; Bronchoalveolar Lavage Fluid ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Eosinophil Granule Proteins ; Eosinophils ; metabolism ; Guinea Pigs ; Interleukin-5 ; metabolism ; Ovalbumin ; Ribonucleases ; metabolism

The brain is particularly vulnerable to oxygen free radicals, and these radicals have been implicated in the pathology of several neurological disorders. In this study, the modulation of TNF-related apoptosis-inducing ligand (TRAIL) expression by oxidative stress was shown in LN215 cells, an astroglioma cell line. Hydrogen peroxide (H2O2) treatment increased TRAIL expression in LN215 cells and H2O2-induced TRAIL augmented apoptosis in Peer cells, a cell line sensitive to TRAIL- mediated cell death. Our findings suggest that the upregulation of TRAIL in astroglial cells may abrogate immune cell effector functions.
*Up-Regulation ; TNF-Related Apoptosis-Inducing Ligand/*biosynthesis ; T-Lymphocytes/*metabolism ; Ribonucleases/metabolism ; Oxidative Stress ; Immunosuppressive Agents/pharmacology ; Hydrogen Peroxide/*pharmacology ; Humans ; *Gene Expression Regulation, Neoplastic ; Cyclosporine/pharmacology ; Cell Line, Tumor ; Astrocytes/*metabolism ; *Apoptosis ; Anoxia ; Allergy and Immunology