The 2.2 A crystal structure of human chymase in complex with succinyl-Ala-Ala-Pro-Phe-chloromethylketone: structural explanation for its dipeptidyl carboxypeptidase specificity

Human chymase (HC) is a chymotrypsin-like serine proteinase expressed by mast cells. The 2.2 A crystal structure of HC complexed to the peptidyl inhibitor, succinyl-Ala-Ala-Pro-Phe-chloromethylketone (CMK), was solved and refined to a crystallographic R-factor of 18.4 %. The HC structure exhibits the typical folding pattern of a chymotrypsin-like serine proteinase, and shows particularly similarity to rat chymase 2 (rat mast cell proteinase II) and human cathepsin G. The peptidyl-CMK inhibitor is covalently bound to the active-site residues Ser195 and His57; the peptidyl moiety juxtaposes the S1 entrance frame segment 214-217 by forming a short antiparallel beta-sheet. HC is a highly efficient angiotensin-converting enzyme. Modeling of the chymase-angiotensin I interaction guided by the geometry of the bound chloromethylketone inhibitor indicates that the extended substrate binding site contains features that may generate the dipeptidyl carboxypeptidase-like activity needed for efficient cleavage and activation of the hormone. The C-terminal carboxylate group of angiotensin I docked into the active-site cleft, with the last two residues extending beyond the active site, is perfectly localized to make a favorable hydrogen bond and salt bridge with the amide nitrogen of the Lys40-Phe41 peptide bond and with the epsilon-ammonium group of the Lys40 side-chain. This amide positioning is unique to the chymase-related proteinases, and only chymases from primates possess a Lys residue at position 40. Thus, the structure conveniently explains the preferred conversion of angiotensin I to angiotensin II by human chymase.     

Production of active recombinant human chymase from a construct containing the enterokinase cleavage site of trypsinogen in place of the native propeptide sequence

Human chymase, a chymotrypsin-like proteinase found in mast cells, was produced in an enzymatically active recombinant form. The protein was expressed in Escherichia coli as part of an insoluble fusion protein which was solubilized and renatured. The structure of the fusion protein was NH2-ubiquitin-enterokinase cleavage site-chymase-COOH. The enterokinase cleavage site of trypsinogen replaced the native propeptide sequence of chymase, allowing for activation by a readily available proteinase (enterokinase) of known specificity. Characterization of refolded-activated recombinant chymase with substrates and inhibitors demonstrated properties identical to that of the native proteinase isolated from skin.     

Mapping surface sequences of the tubulin dimer and taxol-induced microtubules with limited proteolysis

Native tubulin alpha beta dimers and microtubules have been subjected to limited proteolysis with trypsin, chymotrypsin, elastase, clostripain, proteinase lysine-C, thermolysin, protease V8, papain, subtilisin, proteinase K, proteinase aspartic-N, and bromelain. Eighty nicking points have been mapped onto the alpha- and beta-tubulin sequences with the aid of site-directed antibodies, of which 18 sites have been exactly determined by N-terminal sequencing, and the probable position of 6 others deduced from protease specificities. Proteolytic sites cluster into five characteristic zones, including the C termini of both chains. Residues accessible to proteases in the tubulin dimer include alpha-tubulin Lys40-Thr41-Ile42, Glu168-Phe169-Ser170, Ser178-Thr179-Ala180-Val181, Lys280-Ala281, Glu290-Ile291, Ala294-Cys295, Arg339-Ser340 (plus probably Lys60-His61 and Glu183-Pro184) and beta-tubulin Gly93-Gln94, Lys174-Val175, Gly277-Ser278, Tyr281-Arg282-Ala283, Cys354-Asp355 (plus probably Arg121-Lys122, Phe167-Ser168, Tyr183-Asn184, and Glu426-Asp427 or Ala430-Asp431). While the majority of these sites remain accessible at the outer surface of taxol-induced microtubules, alpha-tubulin Lys280-Ala281, Arg339-Ser340 and beta-tubulin Tyr281-Arg282-Ala283 (and probably Arg121-Lys122) become protected from limited proteolysis, suggesting that they are close to or at intermolecular contacts in the assembled structure. The protease nicking points constitute sets of surface constraints for any three-dimensional model structures of tubulin and microtubules. The dimer tryptic site at alpha-tubulin 339-340 jumps approximately 12-22 residues upstream (probably to Lys326-Asp327 or Lys311-Tyr312) in taxol microtubules, suggesting a tertiary structural change. The cleavage of the approximately 10 C-terminal residues of alpha-tubulin by protease V8, papain, and subtilisin is inhibited in taxol microtubules compared to tubulin dimers, while the approximately 20 C-terminal residues of beta-tubulin are similarly accessible to protease V8, subtilisin, proteinase K, proteinase AspN, and bromelain and show enhanced papain cleavage. This is consistent with models in which the alpha-tubulin C-terminal zone is near the interdimer contact zone along the protofilaments, whereas the C terminus of beta is near the interface between both subunits.     

Comparative study of active site structure in bovine and Pacific salmon trypsins

The kinetic investigation was carried out on the inhibition of hydrolysis of N, alpha-benzoyl-D, L-arginine-p-nitroanilide (BApNA) for bovine and salmon trypsin by phenylmethanesulphonyl fluoride (PMSF), N, alpha-tosyl-L-lysine chloromethyl ketone (N-TLCK), N, alpha-tosyl-L-phenylalanine chloromethyl ketone (N-TPCK). Kinetic parameters of inhibition (Ki, k2) by PMSF for salmon and bovine trypsin differ insignificantly. The k2/Ki value of N-TPCK for salmon trypsin is 10 times more than of bovine trypsin. Kinetic parameters of inhibition by N-TLCK had the less difference. The Ki value of this inhibitor for salmon trypsin is 5 times less than that of bovine trypsin and k2 value is 1.7 times less.     

Chymase cleavage of stem cell factor yields a bioactive, soluble product

Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.     

Trypsin-like neutral protease associated with soluble elastin

Isolation of tropoelastin is complicated by the presence of a neutral protease closely associated with tropoelastin that is capable of sequentially degrading tropoelastin to small peptides. Substrate and inhibitor specificities of this neutral protease associated with purified tropoelastin were examined. The enzyme displayed proteolytic activity against casein, and esterase activity was detected when assayed against N-tosyl-L-arginine methyl ester but not against tert-butyl-oxycarbonyl-L-alanine p-nitrophenyl ester. No appreciable elastinolytic activity was detectable against either insoluble sodium dodecyl sulfate treated elastin or maleylated tropoelastin. The enzyme was not inhibited by the chymotrypsin inhibitor toluenesulfonylphenylalanine chloromethyl ketone. The enzyme was inhibited by phenylmethanesulfonyl fluoride and, to various degrees, by metal chelators. Tosyllysyl chloromethyl ketone, epsilon-aminocaproic acid, and Aprotinin (pancreatic trypsin inhibitor--Kunitz type), all inhibitors of trypsin-like enzymes, were very effective inhibitors, as were soybean trypsin inhibitor and human alpha-1-antitrypsin. The data suggest that the tropoelastin-associated enzyme is a neutral serine protease with trypsin-like specificity.     

Detection of staphylococcal enterotoxin in gastric juice

The gastric juice of a patient showing symptoms of staphylococcal food poisoning was examined by a radioimmunoassay for the presence of enterotoxins. Assays gave markedly higher results at 35 degrees than at 5 degrees. The source for this discrepancy was attributed to interference due to trypsin activity on the basis of (1) the demonstration of hydrolysis of p-toluenesulfonyl-L-arginine methyl ester by the specimen, (2) inhibition of this activity by trypsin inhibitor from lima bean, and (3) lowered values produced for enterotoxins in gastric juice when the inhibitor was included in the assay system.     

Human mast cell chymase induces the accumulation of neutrophils, eosinophils and other inflammatory cells in vivo

The roles of chymase in acute allergic responses are not clear, despite the relative abundance of this serine proteinase in the secretory granules of human mast cells. We have isolated chymase to high purity from human skin tissue by heparin-agarose affinity chromatography and Sephacryl S-200 gel filtration procedures, and have investigated the ability of human mast cell chymase to stimulate cell accumulation following injection into laboratory animals. Injection of chymase provoked marked neutrophilia and eosinophilia in the skin of Dunkin Hartley guinea-pigs. Compared with saline injected control animals, there were some 60 fold more neutrophils and 12 fold more eosinophils present at the injection site. Following injection of chymase into the peritoneum of BALB/c mice, there were up to 700 fold more neutrophils. 21 fold more eosinophils, 19 fold more lymphocytes and 7 fold more macrophages recovered than from saline injected controls at 16 h. Doses of chymase as low as 5 ng (1.7 x 10(-13) mole) stimulated an inflammatory infiltrate, and significant neutrophilia was elicited within 3 h. The chymase induced cell accumulation in both the guinea-pig and mouse models was dependent on an intact catalytic site, being reduced by co-injection of proteinase inhibitors or heat inactivation of the enzyme. Co-injection of histamine or heparin significantly reduced the chymase induced neutrophil accumulation, whereas neither histamine nor heparin by themselves had any effect on the accumulation of nucleated cells. No synergistic or antagonist interactions between chymase and tryptase were observed when these two major mast cell proteinases were co-injected into the mouse peritoneum. Our findings suggest that chymase may provide an potent stimulus for inflammatory cell recruitment following mast cell activation.     

Reappraisal of the expression of mast cell proteases of Mongolian gerbils (Meriones unguiculatus)

Mast cell proteases in the tongue and jejunum of Mongolian gerbils (Meriones unguiculatus) were examined by enzyme-histochemical methods. Both trypsin-like (tryptase) and chymotrypsin-like (chymase) protease activities were demonstrated in mast cells in the tongue of fresh cryosections. When frozen sections of the tongue were post-fixed in various fixatives, those fixed in Carnoy's fluid showed strongest enzyme activities. Tryptase and chymase activities in paraffin sections of both tissues were well preserved when tissues were fixed in Carnoy's fluid at 4 degrees C for 15 min. However, enzyme activities in both tissues, especially in the tongue, were drastically reduced by longer fixation time and higher temperature. When Carnoy-fixed (4 degrees C for 15 min) paraffin sections were treated with heparinase I or chondroitinase ABC before enzyme-histochemical stainings for proteases, tryptase activities were lost entirely in the tongue and mostly in the jejunum by heparinase I digestion, and slightly in both organs by chondroitinase ABC digestion. In contrast, chymase activities at both sites were not influenced by these pretreatments. These results show that although mast cells in the tongue as well as in the jejunum of Mongolian gerbils contain both tryptase and chymase activities, their stability to fixations is variable among organs so that tissue fixation conditions are crucial for the preservation. At least some part of the stability of mast cell proteases is dependent on the proteoglycans present in mast cell granules.     

Isolation and some molecular properties of a trypsin-like enzyme from larvae of European corn borer Ostrinia nubilalis Hübner (Lepidoptera: Pyralidae)

A one-step high-yielding procedure is presented for the purification of a trypsin-like proteinase from Ostrinia nubilalis larvae, consisting of benzamidine-sepharose affinity chromatography. The purified enzyme was homogeneous as judged by SDS-PAGE. The enzyme presents a molecular mass of 24 650 Da, a maximum pH activity profile of 9.5, a remarkable thermal stability and an optimum temperature of about 53 degrees C Km values determined using N alpha-benzoyl-DL-arginine-ethylester and N alpha-benzoyl-DL-arginine-p-nitro-anilide were 3.2 x 10(-5) M and 4.1 x 10(-4) M respectively. The proteinase was inhibited by some typical serine proteinase inhibitors such as N alpha-tosyl-L-lysine chloromethyl ketone, soybean trypsin inhibitors, benzamidine and phenylmethylsulfonyl fluoride. In particular, it was competitively inhibited by benzamidine with a Ki of 1.2 x 10(-5) M, whereas it was not affected by cysteine proteinases inhibitors. Comparative analysis of the amino acid composition and N-terminal sequence of O. nubilalis proteinase confirmed that this enzyme is very similar to other serine proteinases from lepidopteran larvae.     

Zyme, a novel and potentially amyloidogenic enzyme cDNA isolated from Alzheimer's disease brain

The deposition of the beta amyloid peptide in neuritic plaques and cerebral blood vessels is a hallmark of Alzheimer's disease (AD) pathology. The major component of the amyloid deposit is a 4.2-kDa polypeptide termed amyloid beta-protein of 39-43 residues, which is derived from processing of a larger amyloid precursor protein (APP). It is hypothesized that a chymotrypsin-like enzyme is involved in the processing of APP. We have discovered a new serine protease from the AD brain by polymerase chain reaction amplification of DNA sequences representing active site homologous regions of chymotrypsin-like enzymes. A cDNA clone was identified as one out of one million that encodes Zyme, a serine protease. Messenger RNA encoding Zyme can be detected in some mammalian species but not in mice, rats, or hamster. Zyme is expressed predominantly in brain, kidney, and salivary gland. Zyme mRNA cannot be detected in fetal brain but is seen in adult brain. The Zyme gene maps to chromosome 19q13.3, a region which shows genetic linkage with late onset familial Alzheimer's disease. When Zyme cDNA is co-expressed with the APP cDNA in 293 (human embryonic kidney) cells, amyloidogenic fragments are detected using C-terminal antibody to APP. These co-transfected cells release an abundance of truncated amyloid beta-protein peptide and shows a reduction of residues 17-42 of Abeta (P3) peptide. Zyme is immunolocalized to perivascular cells in monkey cortex and the AD brain. In addition, Zyme is localized to microglial cells in our AD brain sample. The amyloidogenic potential and localization in brain may indicate a role for this protease in amyloid precursor processing and AD.     

Purification and characterization of lymphocyte chymase I, a granzyme implicated in perforin-mediated lysis

One mechanism of killing by cytotoxic lymphocytes involves the exocytosis of specialized granules. The released granules contain perforin, which assembles into pores in the membranes of cells targeted for death. Serine proteases termed granzymes are present in the cytotoxic granules and include several chymases (with chymotrypsin-like specificity of cleavage). One chymase is selectively reactive with an inhibitor, Biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2, that blocks perforin lysis. We report the purification and characterization of this chymase, lymphocyte chymase I, from rat natural killer cell (RNK)-16 granules. Lymphocyte chymase I is 30 kDa with a pH 7.5 to 9 optimum and primary substrate preference for tryptophan, a preference distinct from rat mast cell chymases. This chymase also reacts with other selective serine protease inhibitors that block perforin pore formation. It elutes by Cu2+-immobilized metal affinity chromatography with other granzymes and has the N-terminal protein sequence conserved among granzymes. Chymase I reduces pore formation when preincubated with perforin at 37 degrees C. In contrast, addition of the chymase without preincubation had little effect on lysis. It should be noted that the perforin preparation contained sufficient residual chymase activity to support lysis. Thus, the reduction of lysis may represent an effect of excess prolytic chymase I or a means to limit perforin lysis of bystander cells. In contrast, other chymases and granzyme K were without effect when added to perforin during similar preincubation. Identification of the natural substrate of chymase I will help resolve how it regulates perforin-mediated pore formation.     

Cleavage of type I procollagen by human mast cell chymase initiates collagen fibril formation and generates a unique carboxyl-terminal propeptide

The ability of human mast cell chymase and tryptase to process procollagen was examined. Purified human intestinal smooth muscle cell procollagen was incubated with human mast cell tryptase or human mast cell chymase. Purified chymase, but not tryptase, exhibited procollagen proteinase activity in the presence of EDTA. Addition of purified porcine heparin over a range of 0.1-100 microg/ml did not affect either the rate or the products of procollagen chymase cleavage. The cleavage site of chymase on the pro-alpha1(I) collagen carboxyl terminus was found to be in the propeptide region at Leu-1248-Ser-1249. Cleavage at this site suggested that the collagen products would form fibrils and confirmed the production of a unique carboxyl-terminal propeptide. Turbidometric fibril formation assay demonstrated de novo formation of chymase-generated collagen fibrils with characteristic lag, growth, and plateau phases. When observed by dark field microscopy, these fibrils were similar to fibrils formed by the action of procollagen proteinases. Thus, mast cell chymase, but not tryptase, exhibits procollagen peptidase-like activity as evidenced by its ability to process procollagen to fibril-forming collagen with concurrent formation of a unique carboxyl-terminal propeptide. These data demonstrate that mast cell chymase has a potential role in the regulation of collagen biosynthesis and in the pathogenesis of fibrosis.     

Potent inactivator of alpha-chymotrypsin: 2,2-dimethyl-3-(N-4-cyanobenzoyl)amino-5-phenyl pentanoic anhydride

We synthesized a novel potent alpha-chymotrypsin inactivator, 2,2-dimethyl-3-(N-4-cyanobenzoyl) amino-5-phenyl pentanoic anhydride, which fulfilled the criteria of a mechanism-based inactivator: first-order kinetics, irreversibility, saturation kinetics and substrate protection. The inactivation rate constant (kinact) and the enzyme-inhibitor dissociation constant (KI) were calculated to be 0.017s-1 and 0.071 microM, respectively (kinact/KI = 242,000 M-1s-1). These kinetic parameters indicate that this compound is one of the most powerful alpha-chymotrypsin inactivators ever reported. The average number of alpha-chymotrypsin turnovers per inactivation (partition ratio) was calculated to be 1, which indicates that it is a stoichiometrically ideal inactivator of alpha-chymotrypsin. We compared the IC50 values of this compound with those of several chymotrypsin-like serine proteinases (bovine alpha-chymotrypsin, recombinant human chymase and human neutrophil cathepsin G) and a metallo proteinase, rabbit angiotensin converting enzyme (ACE). Our compound, 2,2-dimethyl-3-(N-4-cyanobenzoyl) amino-5-phenyl pentanoic anhydride, inhibited bovine alpha-chymotrypsin potently (IC50 = 1.0 (+/- 0.2) x 10(-9) M) as well as other chymotrypsin-like serine proteinase; recombinant human chymase (IC50 = 7.0 (+/- 1.0) x 10(-8) M) and human neutrophil cathepsin G (IC50 = 1.8 (+/- 0.2) x 10(-7) M). However, rabbit ACE was not inhibited by this compound (IC50 > 1 x 10(-4) M).     

Proteolysis of bovine alpha s2-casein by chymosin

Proteolysis of bovine alpha s2-casein by chymosin (E. C. 3.4.23.4) in solution in 100 mM Na phosphate buffer, pH 6.5, at 30 degrees C was studied by reversed-phase (RP)-HPLC and urea-polyacrylamide gel electrophoresis (PAGE). Chymosin hydrolyzed alpha s2-casein in solution to eight peptides detectable by urea-PAGE. Peptides soluble in acetate buffer, pH 4.6, were isolated by RP-HPLC on a C18 column using an acetonitrile/water gradient and identified from their N-terminal amino acid sequence. The chymosin cleavage sites were at the bonds Phe88-Tyr89, Tyr95-Leu96, Gln97-Tyr98, Tyr98-Leu99, Phe163-Leu164, Phe174-Ala175 and Tyr179-Leu180. Chymosin cleavage sites were restricted to the hydrophobic regions of the molecule. The bond-type in alpha s2-casein cleaved by chymosin was in agreement with that found to be susceptible to chymosin in other caseins. The primary site of chymosin action on alpha s2-casein appeared to be at Phe88-Tyr89.     

Two distinct forms of human mast cell chymase--differences in affinity for heparin and in distribution in skin, heart, and other tissues

Chymase, a chymotrypsin-like protease secreted by human mast cells, is generally considered to be a single enzyme. However, by heparin-agarose chromatography of high-salt extracts of human skin, we have consistently resolved three peaks of chymotryptic activity, eluting at 0.4 M NaCl (peak A), 1.0-1.2 M NaCl (peak B) and 1.8-2.0 M NaCl (peak C), with peak B containing 75-90% of the recovered activity. Each peak retained its identity upon rechromatography. The three peaks of activity were similar in substrate specificity and inhibitor profile and distinctly different from other chymotryptic enzymes, including cathepsin G and the stratum corneum chymotryptic enzyme. Examination of different tissues revealed that peak C was virtually absent from synovial tissue, was present as a minor component in skin and heart, but constituted the predominant chymotryptic activity in lung. Peaks B and C from skin tissue were further purified by chromatography on Sephacryl S-200. Both had a molecular mass of 28-29 kDa, yielded the N-terminal sequence reported for chymase, and on western blots reacted with a panel of polyclonal, monoclonal and antipeptide antibodies against chymase. Chymase C required higher concentrations of NaCl to overcome the stimulatory effects of heparin than did chymase B, but had a similar pH profile. Thus, human chymase exists in at least two distinct but similar forms, and the differences in heparin binding and tissue distribution could have important consequences for enzyme function.     

Autoprocessing and peptide substrates for human herpesvirus 6 proteinase

Autoprocessing of the precursor form of human herpesvirus 6 (HHV-6) proteinase at two sites (termed M and R) is required to generate the mature enzyme. Kinetic constants were determined for the hydrolysis of a series of synthetic peptide substrates by mature HHV-6 proteinase, purified to homogeneity. Truncation or replacement of individual residues in peptides mimicking the R-site sequence, indicated that the minimum length for effective hydrolysis by the viral enzyme was P4-P3-P2-Ala*Ser-P2'-P3'-P4' and revealed the importance of the P1 Ala and P4 Tyr residues. Consequently, relevant (P1 or P4) mutations were introduced into the precursor form of the proteinase and the ability of these altered proteins to autoprocess was examined. Introduction of Val in place of the P1 Ala at the M-site essentially abrogated cleavage but mature HHV-6 proteinase was still generated by cleavage at the R-site, indicating that processing of the M-site is not a prerequisite for cleavage of the R-site in the precursor. At the R-site, mutation of the P1 Ala, or of the preceding P4 Tyr residue, prevented processing at the R-site in the precursor so that the mature form of HHV-6 proteinase was not generated. The accumulated data suggest a possible new approach to the design of inhibitors for therapeutic intervention in the life cycle of herpesviruses.     

Proteinases of rat mast cells. Peritoneal but not intestinal mucosal mast cells express mast cell proteinase 5 and carboxypeptidase A

Six basic proteins of 26 to 38 kDa with isoelectric points (pI) > or = 8.5 were abundant in proteins separated by two-dimensional SDS-PAGE from adult rat peritoneal mast cells (MC). One was identified previously as rat mast cell proteinase (RMCP) 1, a chymase of 26 to 28 kDa, pI > 9.0. Microsequence analyses showed that two polypeptides of about 29 and 30 kDa had NH2 terminal amino acid sequences homologous to mouse MC proteinase 5 (MCP-5), whereas the amino terminals of the 33, 35, and 36 kDa proteins were homologous to MC carboxypeptidase A (MC-CPA). Rabbit Abs produced against synthetic peptides of the identified NH2 terminal sequences were used in immunoblot studies. At least three proteins reacted with Abs to MC-CPA, whereas Abs to MCP-5 detected three adjacent polypeptides, rather than just the two identified by using microsequence analysis. Removal of oligosaccharide side chains using peptide:N-glycosidase F reduced the heterogeneity of each set of three polypeptides (MCP-5 and MC-CPA) to a band of each protein of a lower M(r). The serine proteinase inhibitor [3H]diisopropylfluorophosphate ([3H]DFP) bound to a proteinase of 30 to 35 kDa, which is probably MC tryptase (pI < or = 6.0). Immunoblot analysis of proteins from intestinal mucosal mast cells showed RMCP-2, but not RMCP-1, MCP-5, or MC-CPA. This is the first report of MCP-5 in the rat and of clearly distinguishable glycosylated forms of MC CPA. These proteinases appear to be restricted in their distribution to selected MC populations, but little is known about their functions.     

Structure of the has operon promoter and regulation of hyaluronic acid capsule expression in group A Streptococcus

The death of poliovirus-infected cells may occur in two forms: canonical cytopathic effect (CPE) (on productive infections) or apoptosis (when the viral reproduction is hindered by certain drugs or some other restrictive conditions). Morphological manifestations of the CPE and apoptosis, being distinct, share some traits (e.g., chromatin condensation and nuclear deformation). It was shown here that a permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (zVAD.fmk), prevented the development of the poliovirus-induced apoptosis on abortive infection. The apoptotic pathway could be dissected by an inhibitor of chymotrypsin-like serine proteases, N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), which prevented the cleavage of DNA to oligonucleosome-sized pieces and nuclear fragmentation but did not suppress cellular shrinkage, cytoplasmic blebbing, and partial chromatin condensation. These results demonstrate that caspase activation is involved in the execution phase of the viral apoptosis and suggest that a nuclear subset of the apoptotic program is under a separate control, involving a TPCK-sensitive event. Neither zVAD.fmk nor TPCK, at the concentrations affecting the apoptotic response, exerted appreciable influence on the virus growth or cellular pathological changes on productive infection, indicating that the pathways leading to the poliovirus-evoked CPE and apoptosis are different.     

Deficient differentiation of mast cells in the skin of mi/mi mice. Usefulness of in situ hybridization for evaluation of mast cell phenotype

The staining property of skin mast cells changed from Alcian blue+/berberine sulfate- to Alcian blue+/berberine sulfate+ in the skin of normal (+/+) and Wv/Wv mice. In contrast, this change did not occur in the skin of mi/mi mice. Heparin content and histamine content per a mi/mi skin mast cell were estimated to be 34% and 18% those of a +/+ skin mast cell, respectively. The low heparin content of mi/mi skin mast cells seemed to be consistent with the Alcian blue+/berberine sulfate- staining property. Expression of genes encoding mast cell-specific proteolytic enzymes was examined by Northern blotting and in situ hybridization. Messenger RNA of mast cell carboxypeptidase A was expressed most of all by +/+, Wv/Wv, and mi/mi skin mast cells, but mRNA of mouse mast cell protease (MMCP)-6 was expressed by approximately a half of +/+ and Wv/Wv skin mast cells and by only 3% of mi/mi skin mast cells. A significant amount of MMCP-2 mRNA was not expressed in the skin of all +/+, Wv/Wv and mi/mi mice. This shows the presence of at least three phenotypes in skin mast cells of mice: berberine sulfate+/MMCP-6+, berberine sulfate+/MMCP-6-, and berberine sulfate-/MMCP-6-. The in situ hybridization of mRNA of mast cell-specific proteolytic enzymes seemed to be useful to describe abnormalities of mast cell differentiation in the skin of mi/mi mice.