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.     

Formation Of Aldehydes During Ozonation

This study investigated the formation of aldehydes after ozonation of three real and three model waters reconstituted from hydrophobic organic material. The four main aldehyde species formaldehyde, acetaldehyde, glyoxal, and methyl glyoxal were analyzed. Formaldehyde was the dominant species formed as a result of ozonation. The different waters varied greatly with respect to aldehyde production under similar treatment conditions. Studies conducted with model waters allowed for exploring aldehyde formation as a function of various water quality parameters. Overall, the hydrophobic organic extracts appeared to have greater aldehyde formation potential than the hydrophilic organics. Aldehydes were formed in direct proportion to the total organic carbon (TOC) concentration of the water at a 1:1 ozone-to-TOC ratio. Greater aldehyde formation was observed at lower ozonation pH values. Bromide ion and inorganic carbon added to model waters appeared to have no effect on aldehyde production. Finally, Assimilable Organic Carbon (AOC) measurements exhibited a strong correlation between aldehyde production and AOC production.     

Linear blending of curvature profiles

A technique is presented for blending curvature profiles and creating a set of intermediate curves which gradually change their shape from that of one boundary curve to that of a second boundary curve. The use of this technique for calculating a 3D shape and its extension to blending both curvature and torsion profiles are discussed.     

Inhomogeneous complexation of trace metals in water with organic nano-complexants

The complexation of heavy metals, such as Cd²⁺ and Ni²⁺, with organic complexants such as 1-(2-pyridylazo)-2-naphthol (PAN) and 1-(2-thiazolylazo)-2-naphthol (TAN) in water has been investigated. Under such conditions, both the reagents and the products form nano-particulates. These materials are important because their spectrum changes upon exposure to heavy metals and they may be used for design of new optical detectors. The kinetic schemes so far suggested for these complexation reactions are not valid for such experimental conditions, since they assume homogeneous behavior. We provide evidences to the inhomogeneous nature of these reactions. The complexation has been studied using TEM imaging, zeta-potentiometry, time-dependent particulate size analysis and time-dependent spectroscopy. Many of the experimental results are explained in terms of the nature of the nano-particulates of these two complexants. Several processes were identified, including crystal growing of the complexant, its reaction with metal ions in solution and on the surface area, chemical erosion of complexant crystallites and their decomposition, re-crystallization of the formed complexes and long term aggregation of both the complexant and the resulted complex. It was found that the needle-like nano-structures on the surface of the TAN particulates governs its reaction and particulate behavior. The known optimal complexation conditions, such as pH, and delay time are now understood in terms of the zeta-potential minima of the suspensions and in terms of the kinetic parameters. Also the interferences of some ions in the Ni–TAN complexation are now quantified and the kinetic data indicate the best delay time when the interfering effects are minimal.     

Spectral characterization of surfaces using laser multi-photon ionization

Multi-photon ionization (MPI) spectroscopy of solid surfaces under ambient conditions and in nitrogen has been established and exemplified for a variety of materials. This was accomplished using a dedicated experimental setup that monitors the photoelectron yield as a function of the laser wavelength. The MPI spectra resemble the absorption characteristics, however, possess more peaks and are more detailed. This demonstrated the possibility to apply MPI spectroscopy for fast analysis of solids. The dependence of the signals upon the laser flux implies that the ionization mechanism depends on the examined molecule and in many cases it is a two-step process, via a long-living intermediate energy state. The method provides quantification in the pmole range and allows for surface imaging.     

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.     

Aging effects in analysis of polycyclic aromatic hydrocarbon aerosols

Polycyclic aromatic hydrocarbon (PAH) aerosols undergo time-dependent processes such as various aging effects and material loss due to sublimation. Therefore, when their analysis is delayed, the results may not properly represent the true environmental composition as sampled. Moreover, even when fast analysis is performed, one may still be interested in the initial concentration of these aerosols, at the moment of their formation. In both cases, a characterization of the time dependence of aerosol mass losses and other possible modifications is of interest. In this study we monitored these time-dependent processes for three different PAH aerosol particulates (anthracene, perylene, and pyrene). We applied two experimental techniques: Fourier transform spectral imaging microscopy (FT-SIM) and traditional HPLC analysis. The former provided a full fluorescence spectrum at each pixel of the particulate, as well as PAH identification and quantification. The chemical imaging information was also used for morphological characterization of the PAH aerosols, which was applied for developing of a simple mathematical model. The effect of the time-delay upon the actual aerosol mass can thus be estimated. The original (as-formed or as-sampled) aerosol mass can be calculated using the proposed empirical model.     

Treatment of rat fibrosarcoma by radiotherapy plus immune adjuvant

Combined radiotherapy and nonspecific adjuvant C. parvum or Piromen treatment of rat tumors show improvement over radiotherapy alone. The most effective protocol, resulting in complete remission in 6 of 6 rats was obtained with C. parvum given i.p. in three doses 1 day prior to tumor X-irradiation of three doses of 1500 R each given on days 1, 4, and 8. Animals receiving the same dose schedule without adjuvant had only partial regression of their tumor. Without adjuvant, increasing the dose to 6000 R also resulted in tumor regression, but at the expense of marked necrosis to the leg. One mechanism for the observed results may be stimulation of the reticulo-endothelial system to produce macrophages activated against the tumor. It is also possible that C. parvum causes increased rate of clearance of soluble antigens released as a result of radiation destruction of the tumor, as suggested by Proctor et al (3).