This article reviews the production, metabolism, and clinical application of procalcitonin (PCT). PCT is a useful indicator to differentiate bacterial infection and virus infection. Also, it can be used to determine the infection severity and prognosis.
Animals ; Bacterial Infections ; immunology ; Calcitonin ; genetics ; metabolism ; Calcitonin Gene-Related Peptide ; Humans ; Protein Precursors ; genetics ; metabolism ; Virus Diseases ; metabolism

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

Tumor necrosis factor (TNF) is a cytokine that is produced by immune cells in response to bacterial and viral stimuli and plays important roles in various inflammatory diseases. TNF is produced as a membrane-bound precursor, which is then cleaved to release soluble mature protein. We expressed murine pro-TNF in Saccharomyces cerevisiae and examined processing and cellular localization of the recombinant protein. Yeast cells were transformed with an expression construct carrying the pro-TNF gene under the control of alcohol dehydrogenase promoter. Immunoblotting analysis of cell homogenate revealed expression of 26 kD pro-TNF in transformed cells. Upon centrifugation, pro-TNF transformed cells fractionated into the membrane/particulate. In a clone that expresses a high level of pro-TNF, mature 17 kD TNF was detected in the culture medium, although the amount was far smaller than that of cell-associated pro-TNF. Flow cytometric analysis of yeast spheroplasts demonstrated the presence of TNF on the cell surface. Our results show that pro-TNF expressed in yeast mainly resides in the cellular membrane with an orientation similar to that of pro-TNF produced in mammalian cells. Our data suggest that the transformed yeast cells can be used for the genetic analysis of pro-TNF processing machinery in immune cells.
Animal ; Cell Line ; Cell Membrane/metabolism ; Flow Cytometry ; Immunoblotting ; Mice ; Plasmids ; Protein Precursors/metabolism* ; Protein Precursors/genetics ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae/genetics ; Transformation, Genetic ; Tumor Necrosis Factor/metabolism* ; Tumor Necrosis Factor/genetics

OBJECTIVETo screen proteins in hepatocytes interacting with HBeAg transactivated protein (HBeAgTP) with yeast-two hybrid technique for investigating the biological functions of HBeAgTP.

METHODSSuppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HBeAg. The HBeAgTP gene was amplified by polymerase chain reaction (PCR) and HBeAgTP bait plasmid was constructed with yeast-two hybrid system 3, and then transformed into yeast AH109. The transformed yeast mated with yeast Y187 containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-gal for selecting two times and screening. After extracting and sequencing of plasmid from blue colonies, the results were analyzed by bioinformatics.

RESULTSHBeAgTP gene was successfully cloned and expressed in yeast cells. Fifteen genes in twenty-four positive colonies were obtained using yeast-two hybrid technique.

CONCLUSIONHBeAgTP conjugated protein genes were successfully cloned, along with the genes involved in transcription and translation of proteins, immunoloregulation, materials and energy metabolism in vivo.

Hepatitis B e Antigens ; genetics ; metabolism ; Hepatitis B virus ; immunology ; Hepatocytes ; immunology ; metabolism ; Humans ; Protein Interaction Mapping ; Protein Precursors ; genetics ; metabolism ; Two-Hybrid System Techniques ; Yeasts ; genetics

Nerve growth factor (NGF) promotes neuronal survival and differentiation and stimulates neurite outgrowth. NGF is synthesized as a precursor-proNGF in vivo. In this paper, a pET-proNGF prokaryocyte expression vector was constructed and transformed into E. coli BL21(DE3)pLysS. The proNGF was expressed in the form of non-active aggregated monomer in E. coli after induction with IPTG. SDS-PAGE revealed the proNGF expression product had a Mr.30.2 kD. Western blotting analysis showed that the protein had good antigenicity. Fusion protein was successfully purified by Ni2+-NTA affinity chromatography and cleaved by Enterokinase and 13.1 mg proNGF was obtained from 100 mL cell culture in a typical experiment. The protein was dialyzed in a redox system containing reduced and oxidized glutathione. RP-HPLC was used to analysis the result of the refolding. The refolded proNGF protein can induce neurite outgrowth of PC12 cells, which indicated that pro-form of NGF we obtained had biological activity.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Nerve Growth Factor ; biosynthesis ; genetics ; Protein Precursors ; biosynthesis ; genetics ; Protein Renaturation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

Chymosin is an important industrial enzyme widely used in cheese manufacture. To improve expression efficiency of recombinant bovine chymosin in Kluyveromyces lactis strain GG799, we designed and synthesized a DNA sequence encoding bovine prochymosin gene (GenBank Accession No. AA30448) by using optimized codons. The synthesized prochymosin gene was amplified by two-step PCR method, and then cloned into the expression vector pKLAC1, resulting in pKLAC1-Prochy. pKLAC1-Prochy was linearized and transformed into K. lactis GG799 by electrotransformation. Positive clones were screened by YEPD plates containing 1% casein. A recombinant strain chyl with highest activities and multi-copy integration which was detected by using specifical integration primers was chosen and fermented in flask. Prochymosin was expressed in K. lactis successfully. SDS-PAGE analysis revealed that the purified recombinant bovine prochymosin had a molecular mass of 41 kDa. After acid treatment, molecular weight of chymosin is about 36 kDa, the same as native bovine chymosin. Activity tests showed that the chymosin activity of the culture supernatant was 99.67 SU/mL after 96 h cultivation. The activities of chymosin were not prominent increased when galactose was used as carbon source instead of glucose, which proved that the fermentation of recombinant strain does not need galactose inducing. The recombinant K. lactis strain obtained in this study could be further used to produce recombinant chymosin for cheese making.
Animals ; Cattle ; Chymosin ; biosynthesis ; genetics ; Enzyme Precursors ; biosynthesis ; genetics ; Gene Expression Regulation, Fungal ; genetics ; Genetic Vectors ; genetics ; Kluyveromyces ; genetics ; growth & development ; metabolism ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics

AIMTo perform screening, related to A-kinase anchoring proteins 4 (AKAP4) and tubulin proteins, in spermatozoa with absent or severely reduced motility in order to detect the status of the fibrous sheath and the axonemal structure.

METHODSAn immunocytochemical study of tubulin, used as a positive control, and AKAP4 was carried out to detect the presence and the distribution of these proteins in different sperm samples. The morphological characteristics of sperm were studied by transmission electron microscope (TEM) and the results were elaborated using a formula reported in previous studies. PCR was carried out on DNA extracted from peripheral blood lymphocytes to analyse partial sequences of the Akap4 and Akap3 genes.

RESULTSImmunolabelling of tubulin and AKAP4 showed different patterns, which led us to divide the patients into groups. In group I, the absence of AKAP4 and tubulin was revealed, although these patients did not show alterations in the Akap4/Akap3 binding site. TEM evaluation highlighted that a high presence of necrosis was associated with total sperm immotility. In group II, a regular AKAP4 and tubulin signal was present, although motility was reduced and TEM analysis revealed the presence of immaturity. In group III, in which a weak AKAP4 label associated with normal tubulin staining and reduced motility was observed, a severe disorganization of the fibrous sheath was highlighted by TEM.

CONCLUSIONWhile the role of AKAP4 in sperm motility is unclear, absent or weak AKAP4-labelling seems to be associated with absent or weak sperm motility.

A Kinase Anchor Proteins ; Humans ; Infertility, Male ; genetics ; physiopathology ; Male ; Microscopy, Electron ; Polymerase Chain Reaction ; Protein Precursors ; genetics ; metabolism ; Semen ; physiology ; Sperm Motility ; Spermatozoa ; physiology ; ultrastructure ; Tubulin ; genetics ; metabolism

Apolipoprotein AI (apo AI), the major protein component of human high-density lipoprotein (HDL), is a single-chain polypeptide of 243 amino acids. Several epidemiological studies have shown that the plasma concentrations of HDL has the role of reverse cholesterol transport (RCT) and inversely correlated with the incidence of coronary artery disease. Because apo AI lacks post-translational modifications, it is convenient to express human apo AI in Escherichia coli expression system. However, there is a poor stability of the mRNA and the apo AI protein in E. coli, it is difficult to express mature apo AI in recombinant bacteria, moreover, even as a fusion protein, apo AI is still sensitive to degradation and can not be cleaved efficiently from the fusion tags. In contrast, proapolipoprotein AI (proapo AI, having an additional polypeptide containing the amino acids Arg-His-Phe-Trp-Gln-Gln at the amino-teminal of the mature protein) proved stable and undegraded in Escherichia coli, and therefore, in this research, an expression system of E. coli including a plasmid of P(R)P(L) tandem promoter was adapted to produce proapo AI. Furthermore, site-directed mutagenesis of the proapo AI cDNA was performed to generate a Clu8Asp mutation in the amino-terminal sequence of proapo AI which created an acid labile Asp-Pro peptide bond between amino acid 8 and 9, and permitted specific chemical cleavage to remove pro-peptide. After inducing with a shift of temperature, yields of recombinant proapo AI achieved about 40% of total cell protein and the recombinant proapo AI expressed proved as a form of inclusion body in cells, so protein need to renature. First of all, the protein was dissolved in buffer with denaturant, and renaturation was carried out on a hydrophobic interaction column (Phenyl Sepharose), ion-exchange chromatography and gel-filtration chromatography were then used to further purify the protein. The purified recombinant apo AI was detected by a set of tests including Western-blotting, Circular dichroism spectra and lipid-binding test, the results shown that recombinant apo AI has similar structural and lipid-binding properties identical to those of native plasma apo AI, which facilitates further research and application.
Apolipoprotein A-I ; biosynthesis ; genetics ; Chromatography, Ion Exchange ; methods ; Escherichia coli ; genetics ; metabolism ; Humans ; Mutagenesis, Site-Directed ; Mutation ; Protein Precursors ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo explore the central mechanism of moxibustion on analgesic effect.

METHODSMale Wistar rats were screened by pain threshold value before making model, and 48 rats whose pain threshold was (250 +/- 25) g were selected. Twelve male Wistar rats were randomly selected as a normal group. For the rest rats the rheumatoid arthritis (RA) model was duplicated by raising in a windy, cold and wet environment combined with injection of Freund's complete adjuvant (FCA), and then they were randomly divided into a model group, a moxibustion group and a moxa volatile oil group, 12 rats in each group. The moxibustion and the moxa volatile oil igroup were treated with moxibustion and moxa volatile oil at "Shenshu"(BL 23) and "Zusanli"(ST 36), respectively, for 15 days. No interventions were added on the model group and the normal group. The pain threshold in Iinjured foot and the expression of hypothalamic POMC mRNA and PDYN mRNA in rats were observed.

RESULTSCompared with the normal group, the pain threshold and the expression of hypothalamic POMC mRNA and PDYN mRNA in the model group were increased (all P < 0.01). Compared with the model group, the pain threshold and the expression of hypothalamic POMC mRNA and PDYN mRNA in the moxibustion group were increased significantly (all P < 0.01), but no statistically significance in the moxa volatile oil group (P > 0.05). Compared with the moxa volatile oil group, the above-mentioned observative indices in moxibustion group were all increased significantly (all P < 0.01).

CONCLUSIONMoxibustion has obvious analgesic effect and its mechanism may be related to the increasing expression of hypothalamic POMC and PDYN mRNA through the warming effect of moxibustion.

Animals ; Arthritis, Rheumatoid ; genetics ; metabolism ; therapy ; Enkephalins ; genetics ; metabolism ; Humans ; Hypothalamus ; metabolism ; Male ; Moxibustion ; Pro-Opiomelanocortin ; genetics ; metabolism ; Protein Precursors ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar

Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that -68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of - 2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells.
Animals ; Cell Line ; Endoplasmic Reticulum/metabolism ; Golgi Apparatus/metabolism ; Kinetics ; Peptides/genetics/*metabolism ; Pituitary Gland, Anterior/*cytology ; Protein Precursors/biosynthesis/genetics/*metabolism ; *Protein Processing, Post-Translational ; Protein Sorting Signals/genetics ; Protein Transport ; Rats ; Recombinant Proteins/biosynthesis/metabolism ; Retroviridae/genetics ; Saccharomyces cerevisiae/genetics/*metabolism ; Saccharomyces cerevisiae Proteins/biosynthesis/genetics/*metabolism ; Secretory Vesicles/metabolism ; Somatostatin/biosynthesis/genetics/metabolism/secretion