A New Member of the TBC1D15 Family from Chiloscyllium plagiosum: Rab GTPase-Activating Protein Based on Rab7 as a Substrate.

APSL (active peptide from shark liver) is a hepatic stimulator cytokine from the liver of Chiloscyllium. It can effectively protect islet cells and improve complications in mice with alloxan-induced diabetes. Here, we demonstrate that the APSL sequence is present in the N-terminus of novel TBC (Tre-2, Bub2 and Cdc16) domain family, member 15 (TBC1D15) from Chiloscyllium plagiosum. This shark TBC1D15 gene, which contains an ORF of 2088 bp, was identified from a cDNA library of regenerating shark liver. Bioinformatic analysis showed that the gene is highly homologous to TBC1D15 genes from other species. Moreover, the N-terminus of shark TBC1D15 contains a motif of unknown function (DUF3548), which encompasses the APSL fragment. Rab-GAP activity analysis showed that shark TBC1D15 is a new member of the TBC1D15 family. These results demonstrated that shark TBC1D15 possesses Rab-GAP activity using Rab7 as a substrate, which is a common property of the TBC1D15 family. The involvement of APSL at the N-terminus of TBC1D15 also demonstrates that this protein might be involved in insulin signaling and may be associated with the development of type 2 diabetes. The current findings pave the way for further functional and clinical studies of these proteins from marine sources.

Anti-diabetic effects of CTB-APSL fusion protein in type 2 diabetic mice.

To determine whether cholera toxin B subunit and active peptide from shark liver (CTB-APSL) fusion protein plays a role in treatment of type 2 diabetic mice, the CTB-APSL gene was cloned and expressed in silkworm (Bombyx mori) baculovirus expression vector system (BEVS), then the fusion protein was orally administrated at a dose of 100 mg/kg for five weeks in diabetic mice. The results demonstrated that the oral administration of CTB-APSL fusion protein can effectively reduce the levels of both fasting blood glucose (FBG) and glycosylated hemoglobin (GHb), promote insulin secretion and improve insulin resistance, significantly improve lipid metabolism, reduce triglycerides (TG), total cholesterol (TC) and low density lipoprotein (LDL) levels and increase high density lipoprotein (HDL) levels, as well as effectively improve the inflammatory response of type 2 diabetic mice through the reduction of the levels of inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Histopathology shows that the fusion protein can significantly repair damaged pancreatic tissue in type 2 diabetic mice, significantly improve hepatic steatosis and hepatic cell cloudy swelling, reduce the content of lipid droplets in type 2 diabetic mice, effectively inhibit renal interstitial inflammatory cells invasion and improve renal tubular epithelial cell nucleus pyknosis, thus providing an experimental basis for the development of a new type of oral therapy for type 2 diabetes.

Immunological features and efficacy of the reconstructed epitope vaccine CtUBE against Helicobacter pylori infection in BALB/c mice model.

Urease is an essential virulence factor and colonization factor for Helicobacter pylori, of which the urease B subunit (UreB) is considered as an excellent vaccine candidate antigen. In previous study, an epitope vaccine with cholera toxin B subunit (CTB) and an epitope (UreB321-339) named CtUBE was constructed and the mice were protected significantly after intragastric vaccination with the CtUBE liposome vaccine. However, the fusion protein CtUBE was expressed as inclusion bodies and was difficultly purified. Besides, the immunogenicity and specificity of the CtUBE vaccine was not investigated in a fairly wide and detailed way. In this study, the fusion peptide CtUBE was reconstructed and expressed as a soluble protein with pectinase signal peptide at the N terminus and the 6-his tag at its C-terminal, and then the immunogenicity, specificity, prophylactic, and therapeutic efficacy of the reconstructed CtUBE (rCtUBE) vaccine were evaluated in BALB/c mice model after purification. The experimental results indicated that mice immunized with rCtUBE could produce comparatively high level of specific antibodies which could respond to natural H. pylori urease, UreB, or the minimal epitope UreB327-334 involved with the active site of urease, and showed effectively inhibitory effect on the enzymatic activity of urease. Besides, oral prophylactic or therapeutic immunization with rCtUBE significantly decreased H. pylori colonization compared with oral immunization with rCTB or PBS, and the protection was correlated with antigen-specific IgG, IgA, and mucosal sIgA antibody responses, and a Th2 cells response. This rCtUBE vaccine may be a promising vaccine candidate for the control of H. pylori infection.

A shark liver gene-derived active peptide expressed in the silkworm, Bombyx mori: preliminary studies for oral administration of the recombinant protein.

Active peptide from shark liver (APSL) is a cytokine from Chiloscyllium plagiosum that can stimulate liver regeneration and protects the pancreas. To study the effect of orally administered recombinant APSL (rAPSL) on an animal model of type 2 diabetes mellitus, the APSL gene was cloned, and APSL was expressed in Bombyx mori N cells (BmN cells), silkworm larvae and silkworm pupae using the silkworm baculovirus expression vector system (BEVS). It was demonstrated that rAPSL was able to significantly reduce the blood glucose level in mice with type 2 diabetes induced by streptozotocin. The analysis of paraffin sections of mouse pancreatic tissues revealed that rAPSL could effectively protect mouse islets from streptozotocin-induced lesions. Compared with the powder prepared from normal silkworm pupae, the powder prepared from pupae expressing rAPSL exhibited greater protective effects, and these results suggest that rAPSL has potential uses as an oral drug for the treatment of diabetes mellitus in the future.

[Synthesis and antitumor activity of novel tetrahydro-beta-carboline derivatives as KSP inhibitors].

Inhibitors of kinesin spindle protein (KSP) are a promising class of anticancer agents that cause mitotic arrest and induce apoptosis of tumor cells. A series of novel tetrahydro-beta-carboline derivatives were synthesized as kinesin spindle protein inhibitor and evaluated as potential antitumor agents. All compounds showed promising KSP inhibitiory activity. Compounds 8 and 9 exhibited better antitumor activity (Lung/A549, Stomach/AGS) than CK0106023 with GI50/IC50 values (1.07/1.62 and 1.46/3.27 micromol x L(-1), 1.09/>10 and 1.22/6.33 micromol x L(-1), respectively).

MT-α-glucan from the fruit body of the maitake medicinal mushroom Grifola frondosa (higher Basidiomyetes) shows protective effects for hypoglycemic pancreatic ß-cells.

The hypoglycemic effect of an α-glucan (designated here as MT-α-glucan) from the fruit body of the Maitake medicinal mushroom, Grifola frondosa, on a murine type 2 diabetes mellitus (T2DM) model was evaluated. Body weight and levels of fasting plasma glucose, glycosylated hemoglobin, triglycerides, cholesterol, free fatty acid, nitric oxide (NO), NO synthase, inducible NO synthase, and hepatic malondialdehyde content decreased significantly when MT-α-glucan was administered to T2DM mice. The content of serum insulin, hepatic glycogen, and reduced glutathione and the activity of superoxide dismutase and glutathione peroxidase increased significantly when MT-α-glucan was administered to T2DM mice. Histopathological changes of the pancreas were ameliorated in the treatment group. These data suggest that MT-α-glucan has a hypoglycemic effect on T2DM mice, which might be related to its protective effect of pancreatic ß-cells exerted by decreasing levels of factors that destroy ß-cells, such as oxidative stress and NO synthesis.

Construction and characterization of a cDNA library from shark regenerated hepatic tissue.

Sharks are a type of fish with a full cartilaginous skeleton and have big livers. To better understand liver regeneration in sharks and screening for the important genes participated in disease-defense, in this study, a cDNA library of regenerated liver tissues of shark, Chiloscyllium plagiosum, was constructed. A total of 2103 expressed sequence tags (ESTs), which represents 997 unique genes, were sequenced. Among these genes, 434 (43.53%) of them showed significant similarity (E-values < 10⁻5) to the sequences in NCBI Nt database, 685 (68.71%) of these unique genes showed significant similarity (E-values < 10⁻5) to the sequences in NCBI Nr database, and 662 (66.40%) of these unique genes showed significant similarity (E-values < 10⁻5) to the Swiss-Prot database. Preliminary analysis of unique genes according to COG database showed that unigenes were further grouped into 21 functional categories including inorganic ion transport and metabolism, energy production and conversion, posttranslational modification, protein turnover and chaperones, general function prediction only, translation, and ribosomal structure and biogenesis. Several possible candidate genes involved in liver regeneration were selected to analyze their expression with relative quantification real-time PCR. This study may contribute to our better understanding of the molecular mechanism of regeneration in shark liver. Furthermore, the EST cataloguing and profiling of shark will be also benefited to the functional genomic research in this marine species.

De novo design, synthesis and biological evaluation of 1,4-dihydroquinolin-4-ones and 1,2,3,4-tetrahydroquinazolin-4-ones as potent kinesin spindle protein (KSP) inhibitors.

Kinesin spindle protein (KSP) inhibitors are a promising class of anticancer agents that cause mitotic arrest in cells from a failure to form functional bipolar mitotic spindles. Here, we report the design, synthesis and biological evaluation of a novel series of 1,4-dihydroquinolin-4-ones and 1,2,3,4-tetrahydroquinazolin-4-ones using de novo design method. The synthesized compound was evaluated and proved to have potent inhibitory activities in the KSP ATPase. Compounds 15j and 15p show potent inhibitory activities in cell proliferation assays. Preferred compound 15j markedly induced G2/M phase cell cycle arrest with characteristic monoastral spindles and subsequent cell death in A549 cells. In vivo evaluation of 15j on the growth of transplantable S180 sarcoma in mice suggested its therapeutic potential for further development.

Design, synthesis and bioevaluation of dihydropyrazolo[3,4-b]pyridine and benzo[4,5]imidazo[1,2-a]pyrimidine compounds as dual KSP and Aurora-A kinase inhibitors for anti-cancer agents.

Four series of dihydropyrazolo[3,4-b]pyridines and benzo[4,5]imidazo[1,2-a]pyrimidines were designed and synthesized as dual KSP and Aurora-A kinase inhibitors for anti-cancer agents by introducing some fragments of Aurora-A kinase inhibitors into our KSP inhibitor CPUYL064. A total of 19 target compounds were evaluated by two related enzyme inhibition assays and a cytotoxicity assay in vitro. The results showed that some target compounds could inhibit both enzymes, and several of them showed significant inhibition activity against HCT116 cell line. Despite showing moderate KSP and Aurora-A kinase inhibition, the lead compounds 6a and 6e displayed significant cytotoxic activity in the micromolar range, especially against the HCT116 cell line and HepG2 cell line. The results may be useful for developing a new class of inhibitors having a dual function, KSP inhibition and Aurora-A kinase inhibition, for the treatment of cancer.

Discovery of tetrahydro-beta-carbolines as inhibitors of the mitotic kinesin KSP.

Inhibitors of kinesin spindle protein (KSP) are a promising class of anticancer agents that cause mitotic arrest in cells from a failure to form functional bipolar mitotic spindles. Here, we report the synthesis and biological evaluation of a novel series of tetrahydro-beta-carboline analogs based on the structure of the known KSP inhibitor HR22C16. Preferred compounds 11b, 12a and 19b were identified as potent inhibitors in a KSP ATPase assay with good anti-proliferative activity in A549 cells.

Study on the antidiabetic mechanism of a shark liver peptide, S-8300, in alloxan-induced diabetes.

OBJECTIVES: The aim was to evaluate the antidiabetic mechanism of S-8300 in alloxan-diabetic mice. METHODS: Diabetes was induced by a single intravenous injection of alloxan (60 mg/kg). The effects of S-8300 on diabetic mice were investigated by observing the change in fasting plasma glucose, detecting Fas mRNA by reverse transcriptase-polymerase chain reaction, Fas protein expression in the pancreas by immunohistochemistry and Western blot, and the DNA fragmentation pattern forming a ladder by electrophoresis. KEY FINDINGS: A significant decrease in fasting plasma glucose was observed, and Fas mRNA and Fas protein expression in the pancreas were attenuated in diabetic mice treated with S-8300. Treatment with S-8300 also attenuated DNA ladder formation. CONCLUSIONS: The results suggest that S-8300 inhibits Fas protein-mediated apoptosis of pancreas cells.

Expression, purification and bioactivities analysis of recombinant active peptide from shark liver.

The Active Peptide from Shark Liver (APSL) was expressed in E. coli BL21 cells. The cDNA encoding APSL protein was obtained from shark regenerated hepatic tissue by RT-PCR, then it was cloned in the pET-28a expression vector. The expressed fusion protein was purified by Ni-IDA affinity chromatography. SDS-PAGE and HPLC analysis showed the purity of the purified fusion protein was more than 98%. The recombinant APSL (rAPSL) was tested for its biological activity both in vitro, by its ability to improve the proliferation of SMMC7721 cells, and in vivo, by its significant protective effects against acute hepatic injury induced by CCl(4) and AAP (acetaminophen) in mice. In addition, the rAPSL could decrease the blood glucose concentration of mice with diabetes mellitus induced by alloxan. Paraffin sections of mouse pancreas tissues showed that rAPSL (3 mg/kg) could effectively protect mouse islets from lesions induced by alloxan, which indicated its potential application in theoretical research and industry.

Enhanced anti-tumor effects achieved in a murine tumor model using combination therapy of recombinant human manganese superoxide dismutase and adriamycin.

Manganese superoxide dismutase (MnSOD) is the only primary antioxidant enzyme in mitochondria that scavenges superoxide radicals. Overexpressing MnSOD in cancer cells by cDNA transfection suppresses tumor formation and reverses malignant growth. In this study, we examined the effect of recombinant human manganese superoxide dismutase (rhMnSOD) alone and in combination with adriamycin (ADR) against solid tumors of sarcoma 180 in Institute of Cancer Research (ICR) mice. Administration of rhMnSOD alone and in combination with ADR significantly inhibited tumor growth in a dose-dependent manner. The use of rhMnSOD in combination with ADR enhanced ADR's anti-tumor potency without increasing toxicity. Histopathological examination provided evidence of the anti-tumor effect. In addition, we found lymphocyte infiltration of the tumors, with an increase in both CD4- and CD8-positive cells in the treated tumors. The expression of CD4 and CD8 was up-regulated with increasing dose of rhMnSOD, and the combination treatment with ADR further enhanced this up-regulation. Collectively, these data indicate that rhMnSOD may exhibit an anti-tumor effect by stimulating the immune system and promoting the recruitment of lymphocytes into the tumor to kill tumor cells. Thus MnSOD may constitute a potential new therapeutic agent to be exploited as an adjuvant in cancer therapy.

Potential use of hirudin in diabetic cataract: a study of galactose mediated human lens epithelial cells injury.

Osmotic stress, together with weakened antioxidant defense mechanisms, is attributed to the changes observed in human diabetic cataract. The use of hirudin, an antithrombic agent, in the pathogenesis of human cataracts has not been studied so far. Since the epithelium is the metabolic unit of the lens, the effect of recombinant hirudin variant III (rHV3) on galactose-induced morphological changes and antioxidant status of human lens epithelial line SRA01/04 in culture was evaluated in this study. The human lens epithelial cells (hLECs) were cultured in D/F(12) medium (normal group), D/F(12) medium + 50 mM D-galactose (control group) or D/F(12) medium + 50 mM D-galactose+rHV3 (test group) for 24 or 72 h. The cells were observed under the light, fluorescence and transmission electron microscope for any morphological changes, while the cell viability was assessed by methylthiazol tetrazolium (MTT) assay. The cells in flasks were harvested for the estimation of various antioxidant parameters. Cell morphology, viability, malondialdeyde, glutathione and antioxidant enzymes were significantly altered in the control group as compared with the normal group. Administration of rHV3 confers significant protection against these changes in the human lens epithelial cells. These results demonstrated that rHV3 could effectively protect galactose-induced hLEC injury and suggested that it could have potential use in diabetic cataracts.

Purification of Escherichia coli L-asparaginase mutants by a native polyacrylamide gel electrophoresis.

The antigenicity of L-asaparaginase (L-ASP) has been problematic for the treatment of leukemia for many years. In order to establish a relationship between the antigenic epitope of L-asparaginase and its antigenicity, several L-asparaginase mutants (mL-ASPs) are constructed and expressed. To effectively purify these enzyme mutants for further investigation, a native preparative polyacrylamide gel electrophoresis is developed. The simplicity and reproducibility of this approach permits the purification of different mutants from the crude enzyme extracts, with a sufficient activity to perform immunological and biological studies. Furthermore, the newly developed method is efficient and cost-effective compared with other methods, such as column chromatography and affinity chromatography. As a result, the enzyme mutants with specific activity of 300 approximately 400 U/mg are obtained by the single-step purification with a high degree of purity.

[Synthesis and biological evaluation of tetrahydro-beta-carline derivatives].

Kinesin spindle protein (KSP/Eg5) is essential for the formation and maintenance of bipolar spindles during mitosis. Inhibition of this protein leads to cell cycle arrest and apoptosis without interfering other microtubule-dependent processes. Therefore, it is a potential target in cancer therapy. Here, a series of tetrahydro-beta-carboline derivatives 5a - k were synthesized as kinesin spindle protein inhibitor. Their structures were confirmed with 1H NMR, ESI-MS and elemental analysis. The synthesized compounds were evaluated for their inhibition of KSP.

[Primary structure determination of hirudin and reteplase fusion protein by LC/ESI-MS/MS spectrometry].

The aim is to determine the primary structure of a new hirudin and reteplase fusion protein (HV12p-rPA) by LC-ESI-MS/MS spectrometry. The molecular weight of the hirudin and reteplase fusion protein (HV12p-rPA) was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The HV12p-rPA was digested with trypsin and chymotrypsin separately and the peptides in the digest mixtures were identified by LC-ESI-MS/MS. The molecular weight of HV12p-rPA was 41,472 Da, which was in accordance with the theoretical value. The peptide fragments of HV12p-rPA digested with trypsin were identified by LC-ESI-MS/MS spectrometry and the results indicated that the fusion protein contained r-PA. Then, the peptides of HV12p-rPA digested with chymotrypsin were identified by the same method. The results indicated that the fusion protein contained HV12p and the linker-containing peptide, DEGGGSY. MASDF and LDWIRDNMRP were identified as the N-terminal and C-terminal containing peptides in the chymotryptic digest mixture of the fusion protein. All of the Xcorr values exceeded 1.5, some of which were above 3.0, showing that the results were correct and credible and a sequence coverage of 85% was achieved. HPLC/MS analysis coupled with uncompleted digestion indicated that all these peptides were arranged with the correct order as expected. Thus, sequence of the fusion protein was confirmed and it was consistent with our design in upstream construction.

Enhanced secretion of adhesive recognition sequence containing hirudin III mutein in E. coli.

It has been previously shown that Escherichia coli L-asparaginase II (L-ASP) signal peptide is capable of being utilized to direct extracellular secretion of hirudin III (HV3) in shake flask. In this study HV3 muteins R33G34D35(S36)-HV3 were generated by introduction of adhesive recognition sequence RGD(S) into the non-functional region of HV3. The resultant recombinants were cultivated on 30 l bioreactor scale using L-ASP signal peptide expression system and the optimized fed-batch cultivation was well established. After cultivation for approximately 11 h the secreted product accumulated up to approximately 1 g l(-1), which means 17-fold increase in productivity compared to initial expression in shake flask. N-terminal analysis, pI measurement, and MALDI mass spectral analysis on mutein R33G34D35S36-HV3 confirmed the authenticity of the product. Compared to wild-type HV3 and R33G34D35HV3, the mutein R33G34D35S36-HV3 exhibits the improved pharmacological activity. Collectively, a novel secretion strategy using L-ASP signal peptide for the rapid, efficient and cost-effective production of HV3 mutein possessing improved pharmacological activity on bioreactor scale has been well established. Using this expression system downstream processing becomes very simple because secreted product is mature, soluble, active, and without N-terminal extension of Met, which is quite critical for most therapeutic protein to reduce the side effect in clinic use. Thus, it provides a promising alternative for extracellular production of other difficult-to-express protein for biopharmaceutical use.

[Application of HPLC-ESI-ITMS in the quality control of carboxyterminal sequence confirmation for insulin and insulin chain B].

Application of HPLC-ESI-ITMS in the quality control of carboxyterminal sequence confirmation for insulin and insulin chain B was studied. The solution of intact insulin or insulin chain B was added to the solution of carboxypeptidase P (CPP) and carboxypeptidase Y (CPY). Fractions of appropriate volume were removed at some appointed time points, acidified with the same amount of 1% formic acid to stop the digestion, and then briefly vortexed for HPLC-ESI-ITMS analysis. Mobile phase A consisted of 0.02% TFA in 98% ultra-pure water and 2% acetonitrile. Mobile phase B consisted of 0.02% TFA in 98% acetonitrile and 2% ultra-pure water. The solution used for post-column fix consisted of propionic acid and isopropyl alcohol (20 : 80, v/v). Chromatographic separation was carried out on a reversed-phase column (Zorbax Prosphere C18, 300A, 5 microm, 2.1 mm ID x 150 mm length). The molecular weights of the multiply charged ions representing consecutive truncated losses of carboxyterminal amino acids were determined by the use of HPLC-ESI-ITMS. The differences between the consecutive truncated peptides are the experimental weights of the carboxyterminal amino acid residues. The carboxyterminal amino acid residue Ala, which released from chain B of intact insulin, was confirmed in the nanomolar concentration range by analyzing the molecular weight of the truncated peptides. Another one carboxyterminal amino acid Ala was confirmed in the nanomolar concentration range of insulin chain B. In the quality control for recombinant DNA product or natural protein, the confirmation of 1 - 3 carboxyterminal amino acid residues is regarded to be up to standard. One amino acid residue of insulin or insulin chain B could be confirmed accurately in the nanomolar concentration range. The results showed that intact insulin could be directly sequenced in the quality control without separating chain B from chain A. There would be no need to separate chain A from chain B to identify carboxyterminal of intact insulin. Furthermore, the method saved us a lot of trouble from the preparation and purification of insulin chain A and chain B.

Expression, purification and identification of recombinant mouse interleukin 21 protein in E. coli.

Interleukin 21 (IL-21) is a novel type I cytokine that is significantly homologous to IL-2, IL-4 and IL-15. Its receptor complex contains gammac chain which is also a component of receptors for IL-2, IL-4, IL-7, IL-9 and IL-15, so there may be overlapping or relevancies in their biological functions. IL-21 is capable of co-stimulating mature T cells, B cells, NK cells, and of stimulating CD16 expression on the surface of NK cells to induce ADCC in innate immune response. It can also strengthen the anti-tumor effect of the cellular immunity, especially via enhancing the activities of NK and antigen specific CTL cells. Thus, IL-21 is a potential useful therapeutic molecule for immunotherapy of malignancies, by eliciting innate and adaptive anti-tumor responses in tumor-bearing hosts. In order to study the biological functions of IL-21, we constructed a mIL-21 prokaryotic expression plasmid and expressed the recombinant mIL-21 protein in E. coli in present study. The recombinant plasmid pET28a/mIL-21 with a carboxyl terminal His-tag was subcloned from the pcDNA3.1/mIL-21 and expressed in E. coli. The induced protein was detected by SDS-PAGE, and identified by Western-blot assay with anti-mIL-21 antibody. The recombinant protein was purified via Ni+ affinity chromatography, and renatured with GSH/GSSG system. Our mouse T cell proliferation experiment showed that the recombinant mIL-21 protein could enhance the mouse T cell proliferation either by itself alone or in the presence of Con A.