Dissociation of single cells from lung or kidney tissue with elastase

Summary Experiments were conducted to determine the capacity of various enzyme preparations to dissociate single cells from guinea pig lung tissue. The number of cells separated from tissue progressively increased as the concentration of crude trypsin was increased from 25 to 250 mg per 100 ml. This action could be inhibited by soy bean trypsin inhibitor. Elastase, but not ethylenediaminetetraacetate (disodium salt), crystalline trypsin, nor chymotrypsin, dissociated cells from lung tissues. Crude trypsin (Trypsin 1∶300) was found to contain 3.0 Sachar units of elastase per mg. Elastase was also inhibited by soy bean trypsin inhibitor. Only some collagenase preparations dissociated cells from lung tissue. Impure bacterial proteases dissociated lung cells. Our data suggest that the term “trypsinization” to denote dissociation of cells from tissue with crude preparations of trypsin is misleading and should be discontinued. Partially supported bv Armour-Baldwin Laboratories and the National Institute of Health, Grant, AM 12919.     

Purification and Characterization of α1-Proteinase Inhibitor from Carp (Cyprinus carpio) Serum

α₁-Proteinase inhibitor was purified to homogeneity from carp serum with an increase in specific inhibitory activity of 17-fold and a 3% recovery rate. The inhibitor was estimated to have molecular weight of 55,000 under reducing and nonreducing conditions, indicating its composition of a single polypeptide. The inhibitor immunologically crossreacted faintly with carp muscular serine proteinase inhibitor but had no crossreactivity with serine proteinase inhibitors from other species. Carp serum inhibitor exhibited marked stability over broad pH ranges of 4.0 to 10.0 and temperatures below 55°C. The inhibitor potently inhibited the activities of carp intestinal and fish myofibril-binding proteinases, and its respective inhibitions of trypsin-type and carp muscular proteinases were more severe than those of chymotrypsin-type and white croaker muscular proteinases. Its inhibitions were similar to those of bovine pancreatic trypsin and α-chymotrypsin, and the amount required to completely inactivate 0.2 μg of each of these two proteinases was evaluated as 0.43 to 0.45 μg. This indicates a molar ratio close to 1:1 during combination of the inhibitor with each proteinase. In addition, its ability to form irreversible complexes with the proteinases was observed electrophoretically and immunologically under denaturing and reducing conditions. Received April 3, 1998; accepted June 30, 1998.     

Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens

The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50 900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42° C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 μg/ml) and not inhibited by antipain dihydrochloride (120 μg/ml), aprotinin (4 μg/ml), bestatin (80 μg/ml), chymostatin (50 μg/ml), E-64 (20 μg/ml), leupeptin (4 μg/ml), Pefabloc SC (2000 μg/ml), pepstatin (4 μg/ml), phosphoramidon (660 μg/ml), or phenylmethylsulfonyl fluoride (400 μg/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties. Received: 22 November 1996 / Received revision: 27 February 1997 / Accepted: 7 March 1997     

Immunolocalisation of BPTI-like serine proteinase inhibitory proteins in mast cells, chondrocytes and intervertebral disc fibrochondrocytes of ovine and bovine connective tissues. An immunohistochemical and biochemical study

A polyclonal anti-bovine pancreatic trypsin inhibitor (BPTI) IgY was raised in chickens immunised with aprotinin. The anti-BPTI IgY was subsequently isolated from egg yolks and purified to homogeneity by affinity chromatography on immobilised aprotinin and by Superose 6 size exclusion fast protein liquid chromatography (FPLC). Immunoblotting with the chicken IgY demonstrated its specificity for BPTI; 3.9 ng BPTI could be detected by this technique. There was no crossreactivity against alpha1-proteinase inhibitor (human and sheep), inter-alpha-trypsin inhibitor (human and sheep), secretory leucocyte proteinase inhibitor or a range of serine proteinase inhibitory proteins (SPIs) isolated from plant sources (soybean and lima bean trypsin inhibitor, potato trypsin and chymotrypsin inhibitors) or serum SPIs (antithrombin-III, alpha2-macroglobulin). Immunoblotting using the anti-BPTI IgY identified the 6- to 12- and 58-kDa forms of endogenous ovine cartilage SPIs in cartilage extracts, confirming the interrelationship of the ovine cartilage SPIs with BPTI. BPTI-domain SPIs were immunolocalised within mast cells of ovine and bovine duodenum, lung and pancreas, and in ovine and bovine bronchial cartilage chondrocytes, chondrocytes of the superficial and intermediate zones of articular cartilage and in the fibrochondrocytes/chondrocytes of the nucleus     

Duct,exocrine, and endocrine components of cultured fetal mouse pancreas

Summary Twenty to twenty-two days postcoitum mouse fetal pancreas organ bits were cultured on the dermal surface of irradiated pigskin as a substrate. The medium used for long term culture consisted of Eagle’s Minimum Essential Medium with the addition of 10% bovine serum, 0.02 U/ml insulin, 0.025 μg/ml glucagon, 3.63 μg/ml hydrocortisone, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine. When the medium lacked trypsin inhibitor or atropine but contained the three hormones, the pigskin support began to be destroyed after 2 to 4 wk in culture. Thereafter, the cultured cells could not grow and survive on the digested pigskin. When 10⁻⁶ M atropine was added to the medium, amylase secretion from cultured cells and destruction of pigskin were inhibited completely but pancreas cells could not grow or survive. In contrast, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine permitted cell growth, permitted amylase secretion from the cultured acinar cells, and prevented the destruction of pigskin. Under these conditions pancreas cells migrated or grew or both from the organ bits onto the surface of the pigskin dermis and organoid aggregations formed. Hydrocortisone was needed to permit growth for more than 2 wk. Glucagon and insulin had additive effects. Light and electron microscopic observations indicated the culture of at least five kinds of cells, i.e., duct, acinar, centroacinar, endocrine, and mesenchymal. The majority of cultured cells were duct cells and acinar cells. There were few mesenchymal cells. Mouse pancreas cells were cultured for at least 12 wk by this method. This investigation was supported by PHS Grant CA 30220 awarded by the National Cancer Institute, DHHS, Grant 1203M awarded by the Council for Tobacco Research, Inc., and Grant RD-65 (for equipment) awarded by the American Cancer Society. Nude mice were provided by Dr. Wendall M. Farrow of Life Sciences, Inc., Resource Laboratory N01, CP6-1005 of the National Cancer Institute.     

Trypsin sensitivity of mammalian cells grown in a serum-free medium

Summary Two mammalian cell lines which multiply in vitro in culture medium devoid of serum were investigated for sensitivity to twice crystallized trypsin. The minimum trypsin concentration which showed a concomitant increase in cell numbers and detachment from the surface of the culture vessel in one cell line was 0.01 μg per ml or approximately 10⁻⁸ μg per cell. These effects could be neutralized by normal human or calf serum at a dilution of 1∶500. The second cell line, which normally grows in suspension, was unaffected by these concentrations of trypsin.     

Differential Effects of selenium on the proliferation of human pulmonary adenocarcinoma cells and human embryonic lung diploid cells in vitro

The effect of different concentrations of Na₂SeO₃ on human pulmonary adenocarcinoma cells and human embryonic lung diploid cells in vitro was investigated. For human pulmonary adenocarcinoma cells, mitotic index and cell count decreased with increasing selenium concentrations. At 1 μg/mL sodium selenite, mitotic activity and growth of human lung cancer cells were partially inhibited, and the progression of human lung cancer cell cycle was partially arrested. When human embryonic lung diploid cells were treated with 1 μg/mL sodium selenite for five continuous days, cell counts of the treated group were closely parallel to those of the control group. After treating human embryonic lung diploid cells with 1–5μg/mL sodium selenite for 1–3 d, the mitotic index (MI), labeled index (LI), and average silver grain (SG) number per 20 labeled nuclei were the same as those of the control. In mixed cultures of human embryonic lung diploid cells and human pulmonary adenocarcinoma cells, treated with 3 and 5 μg/mL sodium selenite for 24 h, the lung diploid cells showed a normal fusiform morphology, whereas the lung cancer cells showed heavily vacuolated cytoplasms and distorted nuclei.     

New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro

Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human α₁-antitrypsin in double immunodiffusion. PI-1 corresponding to α₁ - antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200–300 Kd) than that of PI-1, and did not crossreact with antisera against human α₁-antitrypsin, α₂-macroglobulin, α₁-antichymotrypsin, α₂-plasmin inhibitor, inter-α-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including α₁-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors.     

OR-1--a mixture of esters of glyceric acid produced by Penicillium funiculosum and its antitrypsin activity

A mixture of related metabolites (denoted OR-1) was isolated from the fermentation broth ofPenicillium funiculosum together with mitorubrinic acid. Structurally OR-1 is glyceric acid esterified with C₁₄−C₁₈ fatty acids. Steady-state studies revealed that OR-1 behaved like an uncompetitive trypsin inhibitor withK _i 17.6 μmol/L.     

DNA methylation levels in porcine fetal fibroblasts induced by an inhibitor of methylation, 5-azacytidine

Removal of the somatic DNA methylation pattern from donor cells and remodeling of embryonic status have been suggested as integral processes for successful nuclear transfer (NT) reprogramming. This study has investigated the effects of 5-azacytidine (5-azaC), a DNA methylation inhibitor, on global methylation changes in porcine fetal fibroblasts (PFF); this may improve NT attributable to the potential reprogramming of the methyl groups. PFF in 5th passage cultures were treated with 0, 0.5, 1.0, 2.0, and 3.0 μM 5-azaC for 96 h; 5-azaC inhibited the growth at all tested concentrations. At the higher concentrations of 5-azaC used, cells appeared to exhibit morphological changes and to become apoptotic as observed by TUNEL assay. Thus, cells were negatively affected by 5-azaC. Differences in cellular ploidy were also observed at higher concentrations. Analysis showed no considerable changes in the proportion of cells at the G₁-phase of the cell cycle with 5-azaC concentrations. The fractional part of the methylated DNA of these cells was significantly reduced by 5-azaC treatment. Confocal microscopy confirmed the inhibition of methylation levels in PFF with increased concentrations of 5-azaC. Exposure to 5-azaC altered the expression of genes involved in imprinting (IGF2) or pro-apoptosis (BAX), whereas there was a reduction in the expression of the main enzyme responsible for replicating the DNA methylation pattern (DNMT1) and anti-apoptosis (BCL2L1). Therefore, 5-azaC induces a relative reduction in methylation in PFF, and cells treated with 0.5 μM 5-azaC may have enhanced potential for porcine NT.The financial support of BioGreen 21 (grant no. 100052004002000) and KOSEF (grant no. R05-2004-000-10702-0) in Korea is gratefully acknowledged.     

A trypsin inhibitor from <Emphasis Type="Italic">Cassia obtusifolia</Emphasis> seeds: isolation,characterization and activity against <Emphasis Type="Italic">Pieris rapae</Emphasis>

A trypsin inhibitor was isolated from Cassia obtusifolia by ammonium sulfate precipitation, Sepharose 4B-trypsin affinity and Sephadex G-75 chromatography. The inhibitor consisted of a single polypeptide chain with a molecular mass of 19, 812.55 Da. It was stable from pH 2 to 12 for 24 h, whereas it was unstable either above 70°C for 10 min or under reduced conditions. The inhibitor, which inhibited trypsin activity with an apparent Ki of 0.3 μM, had one reactive site involving a lysine residue. The native inhibitor was resistant to pepsin digestion, whereas the heated inhibitor produced 40% degree of susceptibility. The disulfide linkage and lysine residue were important in maintaining its conformation. Partial amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz inhibitor family. Moreover, the inhibitor showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Pieris rapae and could suppress the growth of larvae.     

Cytogenetic study of <Emphasis Type="Italic">Ascaris</Emphasis> trypsin inhibitor in cultured human lymphocytes with metabolic activation

The trypsin inhibitor (ATI) isolated from gastrointestinal nematode Ascaris suum was tested in vitro for induction of chromosome aberrations and sister chromatid exchanges (SCE). Genotoxicity assessment of purified ATI was carried out on metaphase plates received from peripheral blood lymphocyte macroculture (48 h test of structural chromosome aberrations and 72 h test of SCE) with exogenous metabolic activation. ATI was tested in dose of 25, 50 and 100 μg per ml of culture. Kinetics of cell divisions were determined by the replication index (RI). The mitotic index (MI) was expressed as a number of metaphases per 1000 nuclei analysed. Analysis of chromosome aberrations showed that higher doses of ATI (50 and 100 μg/ml) significantly increased the frequency of chromosome aberrations (mainly of chromatid gaps and breaks) compared to the negative control. All concentrations of ATI caused a statistically significant reduction in the MI and RI. In comparison with the negative control, a significant increase in the SCE frequency was observed in all applied doses of ATI. Thus, in the presence of S9 activation, the Ascaris trypsin inhibitor showed potential clastogenic activity and inhibition of the dynamics of lymphocyte divisions.     

A Trypsin Inhibitor from <Emphasis Type="Italic">Sapindus saponaria</Emphasis> L. Seeds: Purification,Characterization, and Activity Towards Pest Insect Digestive Enzyme

The present paper describes the purification, characterization and determination of the partial primary structure of the first trypsin inhibitor isolated from the family Sapindaceae. A highly stable, potent trypsin inhibitor (SSTI) was purified to homogeneity. SDS–PAGE analysis revealed that the protein consists of a two-polypeptide chain with molecular masses of approximately 15 and 3 kDa. The purified inhibitor inhibited bovine trypsin at a 1:1 M ratio. Kinetic analysis revealed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 10⁻⁹ M for trypsin. The partial NH₂- terminal sequence of 36 amino acids in SSTI indicates homology with other members of the trypsin-inhibitor family from different sources. This inhibitor is highly stable in the presence of denaturing agents. SSTI showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Anagasta kuehniella, Corcyra cephalonica, Diatreae saccharalis and Anticarsia gemmatalis.     

Loss of soybean trypsin inhibitor in callus as monitored by inhibition enzyme immunoassay

Summary A monoclonal antibody was produced against Kunitz soybean inhibitor (KSBTI) and used in an inhibition enzyme immunoassay (EIA). The inhibition EIA was as sensitive as competetive EIAs and was easily modified for other protein-antibody interactions. The KSBTI assay described detected KSBTI in complex mixtures from 100 μg/ml to 50 ng/ml and did not react with the Bowman-Birk trypsin inhibitor. The assay was used to examine levels of KSBTI inGlycine max hypocotyl-derived callus tissue. The developing hypocotyls contained 0.21 μg KSBTI per mg of fresh tissue. This level of KSBTI rapidly decreased when placed in culture and was undetectable 6 days later. The decrease in KSBTI correlated with the development of callus.     

Proteolytic sensitivity of the α subunit in luciferases ofPhotobacterium species

Bacterial luciferases isolated from strains of three species ofPhotobacterium (P. leiognathi, P. phosphoreum andP. fischeri) have been found to be considerably more sensitive to trypsin inactivation than luciferase fromBeneckea harveyi. P. leiognathi luciferase has a pseudo-first-order rate constant of inactivation of 0.14 min⁻¹ when exposed to 1 μg/ml (42 nM) trypsin. As judged by sodium dodecyl sulfate electrophoresis of the products of proteolysis, only the α subunit of the αβ heterodimeric luciferases ofPhotobacterium species was attacked by trypsin. After treatment of these enzymes with trypsin, the β subunit of eachPhotobacterium species retained its ability to reform active luciferase when renatured with native complementary α subunit.     

High-performance liquid chromatographic separation of aprotinin-like inhibitors and their determination in very small amounts

A reversed-phase high-performance liquid chromatographic method for the determination and quantitative recovery of fully active aprotinin (the basic pancreatic trypsin inhibitor or Kunitz inhibitor) and aprotinin-like inhibitors in amounts down to 0.5 μg is reported. The method, which allows separation of aprotinin isoinhibitors characterized by small differences in the primary structure with respect to aprotinin itself, appears to be suitable for the quantitation and identification of aprotinin-like inhibitors in human biological fluids, in which they appear to be present at very low levels.     

Purification of recombinant aprotinin from transgenic corn germ fraction using ion exchange and hydrophobic interaction chromatography

Plants have attracted interest as hosts for protein expression because of the promise of a large production capacity and a low production cost. However, recovery costs remain a challenge as illustrated for recovery of recombinant aprotinin, a trypsin inhibitor, with removal of native corn trypsin inhibitor from transgenic corn (Azzoni et al. in Biotechnol Bioeng 80:268–276, 2002). When expression is targeted to corn grain fractions, dry milling can separate germ and endosperm fractions. Hence, only the product-containing fraction needs to be extracted, reducing the cost of extraction and the impurity level of the extract. Selective extraction conditions can reduce impurity levels to the point that low-cost adsorbents can result in relatively high purity levels. In this work, we attempted to achieve comparable purity with these lower cost methods. We replaced whole grain extraction and purification of recombinant aprotinin with sequential trypsin affinity and IMAC steps with an alternative of germ fraction extraction and purification with ion exchange and hydrophobic interaction chromatography (HIC). Using germ extraction at acidic pH supplemented with heat precipitation to remove additional host proteins resulted in a higher specific activity feed to the chromatographic steps. The cation exchange step provided 7.6× purification with 76.4% yield and no sodium dodecyl sulfate–polyacrylamide gel electrophoresis detectable native corn trypsin inhibitor. After the HIC step (2.7× step purification with 44.0% yield), the final product had a specific activity that was 75.3% of that of the affinity-purified aprotinin.     

Effects of micromolar concentrations of Mn, Mo, and Si on α1adrenoceptor-mediated contraction in porcine coronary artery

We studied the effects of trace elements, Mn, Mo, and Si, on vasoconstriction induced by norepinephrine (NE) or electrical field stimulation in isolated porcine right coronary arteries. α₁-Adrenoceptor (AR) antagonist prazosin dose-despondently suppressed vasoconstriction in response to NE or field stimulation indicating an α₁-AR mediated response. Mn, Mo, and Si at 0.3-3 μmol/L dosedespondently inhibited NE mediated contraction (allp < 0.05). In contrast, Mn, Mo, and Si at the same concentrations (0.3-3 μmol/L) enhanced the maximal contractile response to field stimulation in a dose-dependent manner (allp < 0.05), but these elements at 10 μmol/L suppressed the vasoconstrictive response. The results indicate that in porcine right coronary arteries, the α₁-AR-mediated vasoconstriction by NE or electrical field stimulation was affected differently by micromolar concentrations of Mn, Mo, and Si and that the elements might facilitate NE release presynaptically but inhibit the contractile response postsynaptically.     

Enzyme inhibitors from plants. Isolation and characterization of a protease inhibitor from arrow root (Maranta arundinaceae) tuber

A protease inhibitor from arrow root (Maranta arundinaceae) tuber has been isolated in a homogeneous form. The inhibitor has a Mr of 11,000-12,000; it inhibited bovine trypsin, bovine enterokinase, bovine α-chymotrypsin and the proteolytic activity of human and bovine pancreatic preparations. The inhibitor is resistant to pepsin, and elastase. It could withstand heat treatment at 100°C for 60 min and exposure to a wide range of pH (1.0–12.5) for 72 h at 4°C without loss of activity. Arginyl groups are essential for the action of the inhibitor. Preincubation of the inhibitor at pH 3.7 with trypsin or chymotrypsin caused nearly a two-fold increase in inhibitor potency