LTCI, a novel chymotrypsin inhibitor of the potato I family from the earthworm Lumbricus terrestris. Purification, cDNA cloning, and expression

A novel chymotrypsin inhibitor of the potato I protease inhibitor family from the earthworm Lumbricus terrestris was purified. The inhibitor, named LTCI, was isolated by methanol extraction, affinity chromatography on immobilized methylchymotrypsin, and ion exchange chromatography followed by RP–HPLC. The 7076 Da inhibitor consists of a single polypeptide chain of 64-amino-acid residues without disulfide bridges. LTCI is the first of the potato I protease inhibitors with Tyr in position P1 of the reactive site. cDNA analysis revealed that LTCI is produced as a 86-amino-acid precursor with a 22-amino-acid secretory signal peptide. RT–PCR analysis demonstrates that LTCI mRNA is expressed in body wall, intestine, and coelomocytes. The recombinant LTCI was produced in Escherichia coli as a fusion protein with intein and chitin binding domain using IMPACT™–CN system.     

A new member of the bacterial ribonuclease inhibitor family from Saccharopolyspora erythraea

We have identified Sti, the gene of a ribonuclease inhibitor from Saccharopolyspora erythraea, by using a T7 phage display system. A specific phage has been isolated from a genome library by a biopanning procedure, using RNase Sa3, a ribonuclease from Streptomyces aureofaciens, as bait. Sti, a protein of 121 amino acid residues, with molecular mass 13059 Da, is a homolog of barstar and other microbial ribonuclease inhibitors. To overexpress its gene in Escherichia coli, we optimized the secondary structure of its mRNA by introducing a series of silent mutations. Soluble protein was isolated and purified to homogeneity. Inhibition constants of complex of Sti and RNase Sa3 or barnase were determined at pH 7 as 5 x 10(-12) or 7 x 10(-7), respectively.     

Isolation and characterization of antistasin. An inhibitor of metastasis and coagulation

The purpose of this study was to purify and characterize the agent responsible for the antimetastatic activity of an extract of the salivary glands (SGE) of the Mexican leech Haementeria officinalis. When administered intravenously in mice on the same day as the intravenous inoculation of T241 sarcoma cells, SGE markedly reduces the number and size of lung tumor colonies. In designing a purification protocol for the antimetastatic agent, we postulated that the antimetastatic agent would also display anticoagulant activity. Thus, we discovered that heparin affinity chromatography followed by anion-exchange chromatography results in a fraction highly enriched in both potent anticoagulant activity and potent antimetastatic activity. Approximately, 200-300 micrograms of purified protein is isolated from 150 mg of SGE. As little as 15 micrograms of this material inhibits tumor cell metastasis to the same extent as 1.0 mg of the unfractionated SGE. When analyzed on sodium dodecyl sulfate gels the active fraction consists mainly of one polypeptide band having an apparent molecular weight of approximately 17,000 under either reducing or nonreducing conditions. The protein has a pI of approximately 9.5 and a molecular weight of approximately 17,000 under nondenaturing conditions. A specific antiserum prepared against the 17,000-dalton protein indicated that this protein is the major anticoagulant and antimetastatic agent of leech salivary gland extract. We have termed this anticoagulant, antimetastatic agent "antistasin." We hypothesize that antistatin inhibits coagulation via factor Xa, and not thrombin, since factor Xa, but not thrombin, is rapidly inactivated upon addition of antistasin. The mechanism of antistasin's antimetastatic activity is currently under investigation.     

Synthesis and structure-activity relationship of new analogues of antistasin.

Intensive investigation connected with the development of new anticoagulant agents for the treatment of cardiovascular diseases was carried out. Direct and specific inhibition of thrombin and Factor Xa-like serine proteases in the coagulation cascade has been the focus of many efforts to design novel anticoagulants over the past decade. This work reports the synthesis and biological activity of new anticoagulant peptide analogues of natural isoforms 2 and 3 of antistasin. In addition they include different tripeptide sequences in their molecules, which are highly active inhibitors of different serine proteases such as plasmin, trypsin, thrombin and Factor Xa.     

A new member of the plasma protease inhibitor gene family.

A 2.1-kb cDNA clone representing a new member of the protease inhibitor family was isolated from a human liver cDNA library. The inhibitor, named human Leuserpin 2 (hLS2), comprises 480 amino acids and contains a leucine residue at its putative reactive center. HLS2 is about 25-28% homologous to three human members of the plasma protease inhibitor family: antithrombin III, alpha 1-antitrypsin and alpha 1-antichymotrypsin. A comparison with published partial amino acid sequences shows that hLS2 is closely related to the thrombin inhibitor heparin cofactor II.     

Characterisation of a highly specific, endogenous inhibitor of cysteine protease from Staphylococcus epidermidis, a new member of the staphostatin family

Staphostatins, a novel family of cysteine protease inhibitors with a unique mechanism of action and distinct protein fold has recently been discovered. In this report we describe the properties of Staphylococcus epidermidis staphostatin A (EcpB), a new member of the family. As for other staphostatins, the recombinant S. epidermidis staphostatin A exerted very narrow inhibitory specificity, limited to cysteine protease from the same species. The closely related proteases from S. aureus cleaved the inhibitor at the reactive site peptide bond and inactivated it. The EcpB homologue, S. aureus staphostatin A (ScpB), was also susceptible to proteolytic cleavage at the same site by non-target cysteine proteases. Conversely, S. aureus staphostatin B (SspC) was resistant to such proteolysis. The difference in the susceptibility of individual inhibitors to proteolytic cleavage at the reactive site suggests subtle variations in the mechanism of interaction with cysteine proteases.     

Purification and characterization of recombinant antistasin: a leech-derived inhibitor of coagulation factor Xa

Antistasin (ATS) is a selective, tight-binding inhibitor of blood coagulation Factor Xa originally isolated from the salivary glands of the Mexican leech Haementeria officinalis. In order to provide sufficient quantities of ATS to further investigate the role of Factor Xa in blood coagulation, a recombinant version of ATS has been produced in an insect baculovirus host-vector system. In this study, we describe the purification and in vitro and in vivo characterization of a single recombinant antistasin (rATS) isoform. The purified protein constitutes a minor isoform relative to the more abundant ATS isoforms present in leech salivary gland extracts. In vitro, rATS inhibits purified human Factor Xa stoichiometrically, prolongs plasma-based clotting assays at nanomolar concentrations, and like native ATS, is cleaved at a single position by Factor Xa during the course of inhibition. An initial evaluation of the in vivo efficacy of rATS was addressed utilizing a rhesus monkey model of mild disseminated intravascular coagulation. rATS was shown to fully suppress thromboplastin-induced fibrinopeptide A generation in a dose-dependent fashion. The availability of rATS should provide a valuable tool for the critical evaluation of the specific role played by Factor Xa in coagulation.     

Tissue inhibitor of metalloproteinase (TIMP-2). A new member of the metalloproteinase inhibitor family

Human melanoma cells secret a 21-kDa protein, termed CSC-21K, which binds with 1:1 molar stoichiometry to the matrix metalloproteinase type IV collagenase proenzyme (70-kDa gelatinase) secreted by the same cells. This binding protein has been purified and its complete primary structure determined by sequencing overlapping peptides which span the entire protein. The amino acid sequence demonstrates that this protein shares significant homology with human TIMP (tissue inhibitor of metalloproteinase), including conservation of the positions of the 12 cysteine residues and 3 of 4 tryptophan residues. The identification of CSC-21K now indicates that a family of TIMP-related proteins exists. Individual members of this family may possess selective affinities for different members of the matrix metalloproteinase family. CSC-21K produced by tumor cells is isolated as a 1:1 molar complex with type IV procollagenase, as demonstrated by amino acid composition analysis. Addition of purified CSC-21K to the activated metalloproteinase results in inhibition of the collagenolytic activity in a stoichiometric fashion. Based on its sequence homology to TIMP and ability to inhibit type IV collagenolysis we propose the name TIMP-2 for this inhibitor.     

Paecilomide,a new acetylcholinesterase inhibitor from Paecilomyces lilacinus

Fungi are some of the most important organisms in the production of bioactive secondary metabolites. This success is related to the advances in biotechnology and also to the possibility of working with techniques such as the “OSMAC” (one strain-many compounds) to achieve different fungal secondary metabolites profiles upon modifying the culturing conditions. Using this approach, the fungal species Paecilomyces lilacinus was cultivated in potato dextrose broth under 14 different fermentative conditions by adding the bacterium Salmonella typhimurium to the growing medium in order to provide biotic stress. S. typhimurium was added alive or after inactivation by autoclave or microwave irradiation in different stages of fungal growth. Extracts were prepared by liquid–liquid extraction using ethyl acetate, a medium polarity solvent in order to avoid extracting culturing media components. Production of fatty acids of relevance for the pharmaceutical and food industries was enhanced by the modified fermentative conditions and they were identified and quantified. The extracts were evaluated for acetylcholinesterase inhibition and the more active extract (91 ± 2.91% inhibition) was prepared in large scale. From this active P. lilacinus extract, a novel pyridone alkaloid, named Paecilomide, was isolated and its structure was elucidated by modern nuclear magnetic resonance techniques and mass spectrometric analyses. Paecilomide (1) was also evaluated for acetylcholinesterase inhibition, presenting 57.5 ± 5.50% of acetylcholinesterase inhibition.     

A novel chitinase from the earthworm,Eisenia andrei

A novel chitinase gene, EaChi, and its expression pattern from the earthworm, E. andrei are demonstrated. Based on a deduced amino acid sequence, in EaChi, two specific domains for GH family 18 are well conserved with two essential amino acid residues for enzyme activity. The phylogenetic analysis shows that earthworm chitinase, EaChi, is evolutionarily close to other lophotrochozoan chitinases. The expression pattern analysis of EaChi indicates that the major expression is localized at intestinal epithelium and epidermis, possibly suggesting that the prime functions of the chitinase activity could be related to not only digestive process but also self-defending immunity as a biochemical barrier to protect the invasion of chitin-containing pathogens, including fungi, nematodes and protozoa.     

A new inhibitor of metalloproteinases from chicken: ChIMP-3. A third member of the TIMP family.

We report cDNA cloning and primary structure of a new metalloproteinase inhibitor (ChIMP-3) produced by chicken embryo fibroblasts. ChIMP-3, formerly called the 21-kDa protein, is one of five ChIMPs (Chicken Inhibitor of MetalloProteinases). In this paper, we report that of the three most abundant ChIMPs, ChIMP-3 and ChIMP-a are extracellular matrix components, whereas ChIMP-2 is found in the media conditioned by the cells. Treatment of ChIMP-3 and ChIMP-a with N-glycosidase-F indicates that ChIMP-a is N-glycosylated whereas ChIMP-3 is not. The deduced amino acid sequence of ChIMP-3 predicts a protein whose properties are consistent with experimental measurements. Analysis of sequence alignments with the two previously described members of the TIMP (tissue inhibitor of metalloproteinases) family, TIMP-1 and TIMP-2, from various species indicates that ChIMP-3 is a related but distinct protein. This conclusion is supported by lack of significant binding with anti-TIMP-1 and anti-TIMP-2 antibodies. Based on these data, its unusual solubility properties, and its exclusive location in the matrix, we propose that ChIMP-3 is a new member of this family of metalloproteinase inhibitors, a TIMP-3.     

广西蕨类植物新记录科——光叶藤蕨科

报道了广西蕨类植物一新记录科——光叶藤蕨科.该科植物以茎圆柱形攀缘;叶二型,通常为奇数羽状;侧生羽片以关节着生于叶轴;不育叶羽片边缘具软骨质硬齿,羽片基部上侧具一腺体;叶脉细密,中肋两侧各具1行窄长网眼,向外伸出分离小脉;能育叶线形,孢子囊群密被羽片下面,无隔丝而与其他蕨类物种相区分.目前该科在广西仅记录光叶藤蕨1种,...     

Cloning a sequence, coding an SKT1 family protease inhibitor from potato

Seven structurally similar clones from potato (Solanum tuberosum L.), cv. Istrinskii genomic DNA were isolated by cloning of the PCR products. It was suggested that five of these clones were the amplified copies of the same gene. Based on comparative and structural analysis of these clones, initial nucleotide structure of the gene was reconstructed. It appeared to be highly homologous (98%) to the already published sequences encoding the proteins belonging to the soybean Kunitz trypsin inhibitor family (SKTI). Comparison of the results with the previously published data on the SKTI-type proteinase inhibitors from potato of cv. Istrinskii suggests that the gene examined encodes both chains of the earlier described PSPI-21-6.3 protein [9].     

Expression and characterization of the N-terminal half of antistasin, an anticoagulant protein derived from the leech Haementeria officinalis

Antistasin, a 15-kDa anticoagulant protein isolated from the salivary glands of the Mexican leech Haementeria officinalis, has been shown to be a potent inhibitor of factor Xa in the blood coagulation cascade. Antistasin possesses a twofold internal homology between the N- and C-terminal halves of the molecule, suggesting a gene duplication event in the evolution of the antistasin gene. This structural feature also suggests that either or both halves of the protein may possess biological activity if expressed as separate domains. Because the N-terminal domain contains a factor Xa P1-reactive site, we chose to express this domain in an insect cell baculovirus expression system. Characterization of this recombinant half antistasin molecule reveals that the N-terminal domain inhibits factor Xa in vitro, with a K(i) of 1.7 nM.     

Raphanusol A,a new growth inhibitor from sakurajima radish seedlings

A new growth inhibitor, raphanusol A, isolated from an acetone extract of light-exposed seedlings of Sakurajima radish, was characterized as 1-,β,4-di-O-(4-hydroxy-3,5-dimethoxycinnamoyl) gentiobiose by chemical methods and spectral data.     

A new family,new genus,and new species allied to the Microsporida

A new and unusual microsporidian species was found in the intestinal epithelium of dipteran larvae of genus Sciara. Merogony is by binary and multiple fission. Sporogonial plasmodia become enclosed in ornate, thick-walled cysts. The cyst contents become cut up by temporary cytoplasmic partitions to make numerous binucleate cells (sporonts) Each divides to produce one normal and one abortive sporoblast. Because this species has a number of unusual features, a new family and a new genus are proposed to contain it.