真鲷仔鱼用微粒子饲料中肽酶与大豆卵磷脂添加效果的研究

本研究以酪蛋白分解物为蛋白源配制三种微粒子饲料MD-S、MD-T和MD-U对真鲷开口仔鱼进行饲养试验。以MD-S的配方为基准,MD-T采用粉状大豆卵磷脂和麸质代替液状大豆卵磷脂;MD-U则另外添加0.1%的肽酶。结果表明,微粒子饲料在水中浸泡15min后,MD-T的溶出率(35.5%)低于MD-S(46.8%)和MD-U(45.8%);实验结束时(20日龄),仔鱼的成活率以生物饵料(轮虫)组为最高(86.3%),其次是MD-T组为20.7%,显著高于(P<0.05)MD-S组(13.3%)和MD-U组(13.6%);生物饵料组的仔鱼全长(6.14±0.49mm)显著大于微粒子饲料组(4.23±0.30mm~4.46±0.30mm),各微粒子饲料组之间仔鱼的全长并不存在显著差异(P>0.05)。在孵化后第12d,微粒子饲料组的仔鱼肠上皮细胞发育良好,但至孵化后第18d,仔鱼肠上皮细胞大部分萎缩、并发生脱落。鱼体的蛋白质、DNA与RNA日间增长率微粒子饲料MD-T组高于MD-S和MD-U组,但都低于生物饵料组。由此可见,微粒子饲料中添加肽酶并无助真鲷仔鱼对其消化吸收;可是,使用粉状大豆卵磷脂与麸质代替液状卵磷脂能增强微粒子饲料的黏合性,可减少其营养成分的溶出率,从而提高微粒子饲料的饲育效果。     

Peptidase inhibitors in the MEROPS database

The MEROPS website (http://merops.sanger.ac.uk) includes information on peptidase inhibitors as well as on peptidases and their substrates. Displays have been put in place to link peptidases and inhibitors together. The classification of protein peptidase inhibitors is continually being revised, and currently inhibitors are grouped into 67 families based on comparisons of protein sequences. These families can be further grouped into 38 clans based on comparisons of tertiary structure. Small molecule inhibitors are important reagents for peptidase characterization and, with the increasing importance of peptidases as drug targets, they are also important to the pharmaceutical industry. Small molecule inhibitors are now included in MEROPS and over 160 summaries have been written.     

The Pseudo Signal Peptide of the Corticotropin-releasing Factor Receptor Type 2a Decreases Receptor Expression and Prevents Gi-mediated Inhibition of Adenylyl Cyclase Activity

The corticotropin-releasing factor receptor type 2a (CRF_2(a)R) belongs to the family of G protein-coupled receptors. The receptor possesses an N-terminal pseudo signal peptide that is unable to mediate targeting of the nascent chain to the endoplasmic reticulum membrane during early receptor biogenesis. The pseudo signal peptide remains uncleaved and consequently forms an additional hydrophobic receptor domain with unknown function that is unique within the large G protein-coupled receptor protein family. Here, we have analyzed the functional significance of this domain in comparison with the conventional signal peptide of the homologous corticotropin-releasing factor receptor type 1 (CRF₁R). We show that the presence of the pseudo signal peptide leads to a very low cell surface receptor expression of the CRF_2(a)R in comparison with the CRF₁R. Moreover, whereas the presence of the pseudo signal peptide did not affect coupling to the G_s protein, G_i-mediated inhibition of adenylyl cyclase activity was abolished. The properties mediated by the pseudo signal peptide were entirely transferable to the CRF₁R in signal peptide exchange experiments. Taken together, our results show that signal peptides do not only influence early protein biogenesis. In the case of the corticotropin-releasing factor receptor subtypes, the use of conventional and pseudo signal peptides have an unexpected influence on signal transduction.     

Cysteine proteases of malaria parasites

A number of cysteine proteases of malaria parasites have been described, and many more putative cysteine proteases are suggested by analysis of the Plasmodium falciparum genome sequence. Studies with protease inhibitors have suggested roles for cysteine proteases in hemoglobin hydrolysis, erythrocyte rupture, and erythrocyte invasion by erythrocytic malaria parasites. The best characterised Plasmodium cysteine proteases are the falcipains, a family of papain-family (clan CA) enzymes. Falcipain-2 and falcipain-3 are hemoglobinases that appear to hydrolyse host erythrocyte hemoglobin in the parasite food vacuole. This function was recently confirmed for falcipain-2, with the demonstration that disruption of the falcipain-2 gene led to a transient block in hemoglobin hydrolysis. A role for falcipain-1 in erythrocyte invasion was recently suggested, but disruption of the falcipain-1 gene did not alter parasite development. Other papain-family proteases predicted by the genome sequence include dipeptidyl peptidases, a calpain homolog, and serine-repeat antigens. The serine-repeat antigens have cysteine protease motifs, but in some the active site Cys is replaced by a Ser. One of these proteins, SERA-5, was recently shown to have serine protease activity. As SERA-5 and some other serine-repeat antigens localise to the parasitophorous vacuole in mature parasites, they may play a role in erythrocyte rupture. The P. falciparum genome sequence also predicts more distantly related (clan CD and CE) cysteine proteases, but biochemical characterisation of these proteins has not been done. New drugs for malaria are greatly needed, and cysteine proteases may provide useful new drug targets. Cysteine protease inhibitors have demonstrated potent antimalarial effects, and the optimisation and testing of falcipain inhibitor antimalarials is underway.     

Generation of Leishmania mutants lacking antibiotic resistance genes using a versatile hit-and-run targeting strategy

The development of a method to create defined mutants of Leishmania parasites lacking foreign genes conferring resistance to antibiotics has both experimental and practical applications. Mutants deficient in specific virulence genes have potential as attenuated live vaccines, but these can only be of clinical relevance if the antibiotic resistance genes used for selection of the mutants are subsequently removed. In addition, the limited number of antibiotic resistance genes that can be used for genetic manipulation of Leishmania means that a system for recycling them for subsequent use would be highly beneficial when multiple genetic modifications are wanted. In the method we report here, a cassette carrying in tandem the hygromycin resistance gene as a positive marker and thymidine kinase gene as a negative marker is first integrated into the locus of interest and then replaced by a null targeting fragment containing no exogenous DNA. The application of this hit-and-run strategy for removal of one allele of the CPB cysteine peptidase gene array of Leishmania infantum is described.     

Proteolytic cleavage of the puromycin-sensitive aminopeptidase generates a substrate binding domain

The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K. The two proteinase K fragments remain associated and retained enzymatic activity. Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies. A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis. The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme.     

Studies on the catabolism of glycated haemoglobin: Glycated globin is not a substrate for the glucosyl-galactosyl-hydroxylysine glucosidase but for a peptidase activity present in rat kidney and spleen

Summary Rat kidney and spleen glucosyl-galactosyl-hydroxylysine glucosidase (EC.3.2.1.107) whose specificity for the hydroxylysine-linked disaccharide units present in collagens depends upon the substrate's free amino group was tested for its glycosidase activity on the ketoamine form of glycated [¹⁴C]Glc-Globin. The most stable preparations of the enzyme from normal and diabetic rat tissues, partially purified by ultracentrifugation and ammonium sulphate fractionation, were used. These glucosidase preparations did not release any significant amount of radioactive neutral hexose. But a radioactive glycopeptide of about 1,400 Da was released from [¹⁴C]Glc-Globin at a pH optimum of 4.0. It appears to be released by a peptidase activity present in the kidney and spleen of normal and diabetic rats.     

Characterization of Nuclear Localization Signal in the N Terminus of Integrin-linked Kinase-associated Phosphatase (ILKAP) and Its Essential Role in the Down-regulation of RSK2 Protein Signaling

Integrin-linked kinase-associated phosphatase (ILKAP) is a serine/threonine (S/T) phosphatase that belongs to the protein phosphatase 2C (PP2C) family. Many previous studies have demonstrated that ILKAP plays key roles in the regulation of cell survival and apoptosis. Researchers have thus far considered ILKAP a cytoplasmic protein that negatively regulates integrin signaling by interacting with and phosphorylating integrin-linked kinase 1 (ILK1). In this study, we found that both endogenous and tagged ILKAP mainly localize to the nucleus and that the nuclear transport of ILKAP is nuclear localization signal (NLS) importin-mediated. The ILKAP protein interacts directly with importin α1, α3, and α5. The NLS in ILKAP is located in the N-terminal region between amino acids 71 and 86, and the NLS-deleted ILKAP protein was distributed in the cytoplasm. In addition, we show that Lys-78 and Arg-79 are critical for the binding of ILKAP to importin α. We also found that nuclear ILKAP interacts with ribosomal protein S6 kinase-2 (RSK2) and induces apoptosis by inhibiting RSK2 activity and down-regulating the expression level of the RSK2 downstream substrate cyclin D1. These results indicate that ILKAP is a nuclear protein that regulates cell survival and apoptosis through the regulation of RSK2 signaling.     

Peptidase Regulation of Gonadotropin-Releasing Hormone Levels During In Vitro Incubations of the Rat Hypothalamus

Abstract: The fate of gonadotropin-releasing hormone (GnRH) was examined by a GnRH radioimmunoassay during in vitro incubations of the rat medial basal hypothalamus (MBH). There was a progressive disappearance of exogenous GnRH during MBH incubations. The GnRH degradation could be explained by the release of peptidases from the MBH into the incubation medium. The cytoplasmic marker lactic dehydrogenase (LDH) was also released into the incubation medium. The peptide antibiotic bacitracin produced a dose-dependent inhibition of GnRH degradation during MBH incubations; 1 mM-bacitracin completely inhibited exogenous GnRH degradation during 4-h incubations. Bacitracin also produced dose-dependent increases in the recovery of endogenous GnRH released from the MBH under basal conditions or when stimulated with the depolarizing agent veratrine. Veratrine also was found to decrease the GnRH peptidase activity significantly but not the LDH activity during MBH incubations. The present results indicate that peptidase activity can be an important regulator of endogenous GnRH released from the hypothalamus during in vitro incubations.     

Lentivirus mediated silencing of Ubiquitin Specific Peptidase 39 inhibits cell proliferation of human hepatocellular carcinoma cells in vitro

Background

Ubiquitin Specific Peptidase 39 (USP39) is a 65 kDa SR-related protein involved in RNA splicing. Previous studies showed that USP39 is related with tumorigenesis of human breast cancer cells.

Results

In the present study, we investigated the functions of USP39 in human hepatocellular carcinoma (HCC) cell line SMMC-7721. We knocked down the expression of USP39 through lentivirus mediated RNA interference. The results of qRT-PCR and western blotting assay showed that both the mRNA and protein levels were suppressed efficiently after USP39 specific shRNA was delivered into SMMC-7721 cells. Cell growth was significantly inhibited as determined by MTT assay. Crystal violet staining indicated that colony numbers and sizes were both reduced after knock-down of USP39. Furthermore, suppression of USP39 arrested cell cycle progression at G₂/M phase in SMMC-7721cells. In addition, Annexin V showed that downregulation of USP39 significantly increased the population of apoptotic cells.

Conclusions

All our results suggest that USP39 is important for HCC cell proliferation and is a potential target for molecular therapy of HCC.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0006-y) contains supplementary material, which is available to authorized users.