番木瓜proteinase omega基因启动子的克隆及功能初步研究

采用PCR技术从番木瓜基因组中克隆了proteinase omega基因的部分序列及其5′侧翼序列。序列分析表明,克隆到的基因序列与GenBank中的序列同源性为96%,长1039bp的5′端侧翼序列在GenBank数据库中没有同源片段。预测5′端侧翼序列有两处基础启动子区域,转录起始位点(TSS)分别为是A,T。在基础启动子区域都存在TATA-box,上游发现多处CAAT-box,G-box,I-box等顺式作用元件和AT富含区。构建了植物表达载体并用基因枪轰击番木瓜的叶组织,GUS基因瞬间表达结果表明,该长1039bp的5′端侧翼序列具有驱动GUS基因在乳管中表达的功能。该启动子的发现对进一步研究启动子的功能和开发番木瓜作为生物反应器具有重要意义。

Proteinase activated-receptors-associated signaling in the control of gastric cancer

Gastric cancer(GC)is the fourth most common cancer in the world and the second cause of cancer-related death.Gastric carcinogenesis is a multifactorial process,in which environmental and genetic factors interact to activate multiple intracellular signals thus leading to uncontrolled growth and survival of GC cells.One such a pathway is regulated by proteinase activated-receptors(PARs),seven transmembrane-spanning domain G protein-coupled receptors,which comprise four receptors(i.e.,PAR-1,PAR-2,PAR-3,and PAR-4)activated by various proteases.Both PAR-1 and PAR-2 are over-expressed on GC cells and their activation triggers and/or amplifies intracellular pathways,which sustain gastric carcinogenesis.There is also evidence that expression of either PAR-1 or PAR-2 correlates with depth of wall invasion and metastatic dissemination and inversely with the overall survival of patients.Consistently,data emerging from experimental models of GC suggest that both these receptors can be important targets for therapeutic interventions in GC patients.In contrast,PAR-4levels are down-regulated in GC and correlate inversely with the aggressiveness of GC,thus suggesting a negative role of this receptor in the control of GC.In this article we review the available data on the expression and role of PARs in GC and discuss whether manipulation of PAR-driven signals may be useful for interfering with GC cell behavior.

Role of saliva proteinase 3 in dental caries

Salivary analysis can be used to assess the severity of caries. Of the known salivary proteins, a paucity of information exists concerning the role of proteinase 3（PR3）, a serine protease of the chymotrypsin family, in dental caries. Whole, unstimulated saliva was collected from children with varying degrees of active caries and tested using a Human Protease Array Kit and an enzyme-linked immunosorbent assay.A significantly decreased concentration of salivary PR3 was noted with increasing severity of dental caries（P,0.01）; a positive correlation（r50.87; P,0.01; Pearson’s correlation analysis） was also observed between salivary p H and PR3 concentration. In an antibacterial test,a PR3 concentration of 250 ng？m L21 or higher significantly inhibited Streptococcus mutans UA159 growth after 12 h of incubation（P,0.05）. These studies indicate that PR3 is a salivary factor associated with the severity of dental caries, as suggested by the negative relationship between salivary PR3 concentration and the severity of caries as well as the susceptibility of S. mutans to PR3.

Correlation of Inhibitor Proteinase in Varieties and Lines of Cotton(Gossypium hirsutum L.) to Different Geographic Population of Verticillium dathliae Klebahn

Breeding for wilt resistance and its theoretical basis are primarily responsible for increases in cotton yield and fiber quality. Breeding for immunity is the most efficient method in our struggle with infectious diseases.

Molecular Cloning of a Thiol Proteinase Inhibitor Gene and Its Expression in E.coli

A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotide probe,which contained a 309bp open read-ing frame,84bp 5′-end noncoding region and a poly(A)signal AATAAA at the 3′-end fol-lowed by 31Nt poly(A).Then the coding region of OC was amplified and inserted into thedownstream of λP_RP_L promoter for thermal-inducible expression in E.coli.Shifting the cul-ture temperature from 30℃ to 42℃ led to a high level expression of OC,which exhibited adistinct band of 12.0 kDa and accounted for at least 10% of the total soluble proteins fromSDS-PAGE.The papain-inhibitory activity of the expressed OC was further confirmed.

On the Activities of Pancreatic Proteases and Alpha-1 Proteinase Inhibitor in Meat-Type Chicken

The study was aimed at the evaluation of the effects of breed, age, different digestion stimulators, and dietary crude protein (CP) level on the activities of proteolytic enzymes in pancreatic tissue and duodenal chymus (in vivo), serum trypsin and α1-proteinase inhibitor (A1PI) concentrations in meat-type chicks. The study of age dynamics of trypsin and A1PI concentrations was performed on the chicks of hybrid cross “Smena-8”and two parental lines (Plymouth Rock and Cornish) fed standard commercial corn-wheat-SBM diets. Twenty birds per breed were euthanized at 1, 7, 14, 21, 28 and 35 days of age to obtain blood samples and pancreatic homogenate. Experiments on the effects of different digestion promotors (probiotic, acidifier, phytobiotic, enzymatic preparation) and different CP levels (finisher diet, CP 20%, vs. ground corn, CP 8.5%) were performed on 12 hybrid chicks with fistulated duodenum from 14 to 50 days of age. The following conclusions were made: 1) At 1 day of age high proteolytic activity in pancreatic tissue and maximal serum concentrations of trypsin and A1PI were found in both hybrid and parental lines. Since 7 to 35 days of age A1PI concentration was nearly constant, serum trypsin concentration decreased while proteolytic activity in pancreatic tissue exhibited undulate increase;2) Proteolytic activity in pancreatic tissue was higher in hybrids compared to the parental lines from 7 to 35 days of age (p 0.05);3) Supplementation of diet with exogenous enzymes stimulated the digestion due to the increase in protease activity in duodenal chymus by 9.1% compared to unsupplemented control (p 0.05);4) Proteolytic activity in duodenal chymus significantly responded to the substitution of ground corn for the complete diet by 2-fold decrease while serum trypsin concentration responded by 2.5-fold increase (p 0.001). This fact can indicate that physiological functions of digestive proteases are not confined to the digestive processes.

Expression of Candida Albicans Secreted Aspartyl Proteinase in Acute Vaginal Candidiasis

In order to analyze the in vivo expression of Candida albicans secreted aspartyl pro- teinases (SAP) in human vaginal infection, the vaginal secretion from 29 human subjects was col- lected by vaginal swab, and the expression of SAP1–SAP6 was detected by reverse-transcriptase po- lymerase chain reaction using specific primer sets. It was found that Sap2 and Sap5 were the most common genes expressed during infection; Sap3 and Sap4 were detected in all subjects and all 6 SAP genes were simultaneously expressed in some patients with vaginal candidiasis. It was suggested that the SAP family is expressed by Candida albicans during infection in human and that Candida albi- cans infection is associated with the differential expression of individual SAP genes which may be involved in the pathogenesis of vaginal candidiasis.

Cloning and Sequence Analysis of a Cysteine Proteinase Inhibitor Gene from Seedless Litchi

[Objective] This study aimed to clone and analyze the cysteine proteinase inhibitor gene from seedless litchi. [Method] According to the EST se- quence of cysteine proteinase inhibitor in constructed SSH snhtraetive library of seedless litchi abortion, nucleotide sequence of the cysteine proteinase inhibitor gene was obtained by using RACE technology and analyzed by using bioinformatics software. [ Result ] A cysteine protease inhibitor gene was obtained with the sequence of 635 bp containing a 321 bp open reading frame. It was predicted that the erlcoded protein contained 106 amino acids with conserved domain of cysteine proteinase inhibitor and had relatively high homology with the cysteine proteinase inhibitor gene of several species, [ Conclusion] This study laid the foundation for further ex- ploring the physiological functions of this cysteine proteinase inhibitor gene in plants.

Studies on Introduction of Arrowhead Proteinase Inhibitor Gene into N． tobacco Protoplasts

The Arrowhead Proteinase Inhibitor(API)gene was introduced into the protoplasts or mesophyll cells of N.tobacco by PEG-mediated.The transformed protoplasts undergoing dirrerentiation of callus and regeneration of plantlet have been growing into transgenic plants. Restriction endonuclease analysis of products amplificated by PCR indicates the existence of the API gene in the transformed plantlet.The extract of the leaves from the transformed plants shows trypsin inhibitory activity,which indicates the expression of the introduced API gene and the transformed plants can accumulate the inhibitor. However, the variation of the inhlbitory activity of the transformed plants reveals the importance of the integration site of the API gene in the genome.

Cloning of a Potato Proteinase Inhibitor Gene PINII-2x from Diploid Potato(Solanum phurejia L.) and Transgenic Investigation of Its Potential to Confer Insect Resistance in Rice

Both cDNA and a genomic DNA fragment encoding a new potato proteinase inhibitor Ⅱ were isolated from a diploid potato IVP101 （Solanum phurejia L.） and named PINII-2x. Nucleotlde sequencing confirmed that the DNA fragment of PINll-2xwas 580 bp, including a 115-bp intron and two exons. The deduced PINII-2x proteln contained an Intact signal peptide and two active sites. The PINII-2x gene and its deduced PINII-2x protein had 88% and 93% homology with another tetraploid potato proteinase inhibitor Ⅱ, respectively. Northern blotting analysis Indicated that the mRNA of PINII-2x gene was wound induced in potato leaves. Binary vector pNAR301 and pNAR302 were constructed for rice transformation, in which the PINII-2x cDNA was driven, respectively, by rice actin I promoter （Actl） and maize ubiquitin promoter （Ubll）. Via an Agrobacteriummediated method, these two constructs were transferred into japonica rice cv. Xiushui 63. PCR and Southern blotUng analysis for transgenic rice revealed the integration of the PINII-2x gene. Northern blotting analysis also confirmed transcripts of the PINII.2x gene in transgenic rice plants. Insect bloassays using stripe stem borer （Chilo auppressalis Walker） demonstrated that the average weight and body length of larvae In transgenic plants were only nearly 50% and 61% of those of larvae in control plants, respectively. These results Indicate that the PINII-2x gene should be an effective insect-resistance gene and could be valuable for application in crop breeding for insect resistance.

Co-expression of the proteinase inhibitors oryzacystatin I and oryzacystatin II in transgenic potato alters Colorado potato beetle larval development

Colorado potato beetle （CPB; Leptinotarsa decemlineata Say, Coleoptera： Chrysomelidae） has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II （OCI and OCII） have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato （Solanum tuberosum L.） cultivars Desiree, Draga6evka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition ofcysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long-term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae.

Isolation of tomato proteinase inhibitor Ⅱ gene and the function of its intron

The genomic DNA sequence of tomato pro-teinase inhibitor II gene (named tin2i, whose accession number in GenBank is AF007240) was isolated by PCR techniques. The intron sequence (TPI), with a length of 109 bp, owns typical structures of GT/AG dinucieotides at both ends and high content of AT base pairs which accounts for 80.7% of the total nucleotides. As shown by recombination experiment, the TPI sequence could efficiently promote the expression of the reporter gene gusA and this effect was independent of the position and orientation of the intron, thus showing its role as an enhancer. Such experiments as gel retardation assays, GUS histochemical staining and GUS fluorometric assays further demonstrated that TPI sequence maybe has promoter-like activity.

Novel alleles among soybean Bowman-Birk proteinase inhibitor gene families

Trypsin inhibitors have been found in various animals, plants and microorganisms.There were two types of trypsin inhibitors in soybean including Bowman-Birk protease inhibitors(BBI) and Kunitz in-hibitors(KTI).The different BBI genes from wild soybean(G.soja) and cultivated soybean(G.max) formed a multigene family.We constructed a cDNA library of cultivar 'SuiNong 14' seed at the R7 growth stage using the SMART Kit.Seventeen contigs or singletons were highly homologous to soy-bean protease inhibitors.Contigs of 5, 35, 8 and 9 were highly homologous to BBI family members BBI-A1, BBI-A2, BBI-C and BBI-D, respectively.Sequence analyses showed there were novel allelic varia-tions among the 4 BBI members in SuiNong 14.Based on the comparison of soybean seed cDNA li-braries from different developmental stages, it was apparent that the expression of trypsin inhibitors increased during seed development in soybean.Phylogenetic analysis of BBI gene sequences among dicotyledonous and monocotyledonous plants demonstrated that these genes shared a common pro-genitor.

INHIBITORY PROPERTY CHARACTERIZATION AND REACTIVE SITE EXPLORATION OF THE ARROWHEAD PROTEINASE INHIBITOR

Affinity chromatography was used to separate two components A and B of the crystalline arrowhead proteinase inhibitor. Both A and B are double-headed and multifunctional proteinase inhibitors. Inhibitor A is capable of inhibiting equimolarly trypsin and chymo-trypsin simultaneously, and has a weak inhibitory activity toward kallikrein; whereas inhibitor B can inhibit two molecules of trypsin simultaneously, and shows rather higher inhibitory activity toward kallikrein than inhibitor A, but its inhibitory activity toward chymo-trypsin is much weaker than that of inhibitor A. The results of chemical modification and the competitive binding of trypsin and chymotrypsin with inhibitor A showed that the two reactive sites of both inhibitors A and B are Lys and Arg residues. Among them the Lys reactive site is specific for inhibiting mainly trypsin, whereas the active domain composed of the Arg reactive site appears to be multifunctional and capable of inhibiting many different Ser proteinases. Based on the

A new method of research on molecular evolution of proteinase superfamily

The molecular evolutionary tree, also known as a phylogenetic tree, of the serine proteinase superfamily was constructed by means of structural alignment. Three-dimensional structures of proteins were aligned by the SSAP program of Orengo and Taylor to obtain evolutionary distances. The resulting evolutionary tree provides a topology graph that can reflect the evolution of structure and function of homology proteinase. Moreover, study on evolution of the serine proteinase superfamily can lead to better understanding of the relationship and evolutionary difference among proteins of the superfamily, and is of significance to protein engineering, molecular design and protein structure prediction. Structure alignment is one of the useful methods of research on molecular evolution of protein.

NMR studies of the interaction between Bacillis subtilis neutral proteinase and inorganic metal compounds

The interaction between Bacillis subtilis neutral proteinase (B.S.NP) and inorganic metal compounds (CoCI2, NiCI2) was investigated by 1H NMR spectroscopy. It has been shown that the Zn(Ⅱ) ion in the active center of the native enzyme may directly interact with external CoCI2 and NiCI2, producing Co(Ⅱ)- and Ni(Ⅱ)-substituted derivatives, and their 1H NMR spectra were obtained for the first time. From the 1H NMR spectra, the coordinated structure of the active center in the native enzyme was described.

Transgenic Tobacco Plants by Cotransformation With Proteinase Inhibitor Ⅱ and δ-endotoxin Genes

In recent years, great progress has been made in. producing plants resistant to insect attack by genetic engineering of B. t δ-endotoxin gene or plant proteinase inhibitor genes, and how to avoid the emergence of the insect resistance to B. t δ-endotoxin produced by transgenic plants has been paid attention to. To combine one B. t δ-endotoxin gene with an incompatible B. t δ-endotoxin gene or with other genes such as proteinase inhibitor gene might alleviate the insect resistance problem. In this note, we study the

CRYSTALLOGRAPHIC STUDY OF ORTHOR-HOMBIC CRYSTALS OF PROTEINASE INHIBITOR FROM ARROWHEAD (agittaria sagittifolia, Linn)

Ⅰ. INTRODUCTION Proteinases and their inhibitors are widely distributed in nature. They play important roles in the biological regulation and control of a number of physiological processes. More than one hundred inhibitors from different sources have been isolated and some of them investigated biochemically in detail. Among them,

THE PRIMARY STRUCTURE OF ARROWHEAD PROTEINASE INHIBITOR

After the reduction and carboxymethylation of disulfide bonds, arrowhead proteinase inhibitors A and B were cleaved either by proteinases or by cyanogen bromide, the fractionated and purified peptides were then subjected to sequencing by a gas phase automatic sequencer, the primary structures were completed by the alignment of the peptides sequenced with overlapping peptides. Both inhibitors A and B consist of 150 amino acid residues with three pairs of disulfide bonds, share 90% homology in structure, and are markedly different from all other Ser proteinase inhibitors so far known. Hence, the arrowhead inhibitor may belong to a new inhibitor family. Based on their structure characteristics, it was deduced that both their two reactive sites might be located in the positions of Lys-Ser (45-46) and Arg-Tyr-Lys (77-79), respectively. Among 13 mutated residues in inhibitors A and B, the substitution of residue Arg in position 87 of inhibitor B for residue Leu in A might be the main cause of leading to

Successive digestion of tilapia collagen by serine proteinase and proline specific endopeptidase to produce novel angiotensin l-converting enzyme inhibitory peptides

Serine proteinase,purified from the hepatopancreas of Pacific white shrimp(Litopenaeus vannamei), was used to hydrolyze acid solubilized collagen(ASC)isolated from Nile tilapia(Oreochromis sp.)skin to produce angiotensin I-converting enzyme(ACE)inhibitory peptides(ACEIPs).A series of column chromatography assays were used to separate the ACEIPs.A peptide,NPARTCR,was isolated as it exhibited high ACE inhibition potential.Further digestion of this peptide by a proline specific endopeptidase(PSEP),produced a pentapeptide ARTCR with ACE inhibitory activity(IC_(50))of 77.0 pmol/L.Both NPARTCR and ARTCR inhibited ACE in a non-competitive manner.An in vivo study in rats demonstrated that ARTCR has ACE inhibitory activity via lowering systolic blood pressure in spontaneously hypertensive rats(SHRs).These results suggest that processing by-products from shrimp and tilapia are ideal raw materials for the production of serine proteinase and collagen,respectively.Serine proteinase and collagen are both ideal raw materials that can be used to derive ACE inhibitory active peptides against hypertension.