The solubilization of elastin by Bacillus licheniformis elastase cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate complex has no physical meaning. In this paper we report the optimization of elastolysis conditions and analysis of elastolytic kinetics. Our results indicated that the hydrolyzing temperature and time are very important factors affecting elastolysis rate. The optimized conditions using central composite design were as follows: elastolysis temperature 50 degrees C, elastase concentration 1 x 10(4) U/ml, elastin 80 mg, elastolytic time 4 h. Investigation of the effects of substrate content, elastase concentration and pH was also revealed that low or high elastin content inhibits the elastolysis process. Increasing elastase improves elastin degradation, but high elastase may change the kinetics characterization. Alkaline environment can decrease elastin degradation rate and pH may affect elastolysis by changing elastase reaction pH. To further elucidate the elastolysis process, the logistic model was used to elastolysis kinetics study showing clearly that the logistic model can reasonably explain the elastolysis process, especially under lower elastase concentration. However, there is still need for more investigations with the aid of other methods, such as biochemical and molecular methods.
Bacillus ; enzymology ; Colorimetry ; Elastin ; metabolism ; Hydrogen-Ion Concentration ; Kinetics ; Pancreatic Elastase ; metabolism ; Regression Analysis ; Temperature

In order to provide morphological data and theoretical basis for pig-to-human hepatic xenotransplantation, the difference in morphological parameters and vessel wall structural factors between human and porcine hepatic portal vein was studied. From human subjects and pigs of varying ages, hepatic portal veins were collected, paraffin-embedded and cut into sections. The histological structures were stained with HE, and elastin, collagen and smooth muscles were stained with Weigert, Aniline blue and orange G, respectively. Morphological parameters and relative contents of structural components were determined under microscopy and by computer image analysis system, respectively. The results showed that histological structures of human and porcine hepatic portal vein wall were similar. Caliber, wall thickness, lumen and wall area in pigs increased with age, all in linear correlation to months. Morphological parameters of 6- month-old pigs were similar to those of human. In pigs, collagen content increased gradually with months, elastin content remained relatively stable, smooth muscle content reached the peak at the 3rd month, and collagen/elastin (C/E) rose gradually. The contents of collagen and elastin in porcine hepatic portal vein wall were lower, while the content of smooth muscle was higher than in human, and C/E at the 5th and 6th month was similar to that in human. It is concluded that morphological parameters and contents of structural components of porcine hepatic portal vein vary with age. At the 6 month, its caliber, wall thickness, lumen and wall area are similar to those of human. There are differences in contents of structural components between human and pigs. However, in terms of C/E, mechanic properties of pigs at the 5th and 6th month mimic those of human, hence inosculation is viable in xenotrans-plantation between pigs and human.
Collagen/*analysis ; Elastin/*analysis ; Image Processing, Computer-Assisted ; Liver Transplantation ; Muscle, Smooth, Vascular/cytology ; Portal Vein/*anatomy & histology ; Portal Vein/chemistry ; Swine ; Transplantation, Heterologous

To understand the mechanical properties of aortic artery of atheroselerosis (AS), the aortic artery of rat with AS was studied by mechanical test. Wistar rats were used for establishing the model. The mechanical measurements of opening angle in zero-stress state and the vessel loading test were conducted on the isolated aortic arteries of AS rats. Data on the stress-strain of aortic artery were obtained. Determination of percentage of collagen content was made with the use of electron microscope. The relationship between mechanical measurements and collagen concentration was evaluated. The opening angle in the group of AS was significantly smaller than that in control (87.74 degrees +/-9.67 degrees vs. 196.03 degrees +/- 27.76 degrees, P < 0.001). Significant decrease of material constants (alpha0, alpha1, alpha2, b0, b1, b2) in both long axis and radial axis was observed in AS group(campared with control, P < 0.05-0.001). Close relationship between the mechanical constants and the percentage of elastin and collagen content was observed (r = -0.7523 to -0.8423, P < 0.001). In conclusion, mechanical remodeling in aortic artery of AS might be related with histological remodeling.
Animals ; Aorta, Abdominal ; metabolism ; pathology ; Atherosclerosis ; metabolism ; pathology ; physiopathology ; Biomechanical Phenomena ; Collagen ; analysis ; Elastin ; analysis ; Female ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Stress, Mechanical

BACKGROUND: Fibroblasts produce many components of the extracellular matrix (ECM) and so they contribute to the maintenance of connective tissue integrity. OBJECTIVE: The aim of this study is to evaluate the effect of velvet antler extract (VAE) on the ECM production of dermal fibroblasts cultured in vitro. METHODS: Primary cultured human dermal fibroblasts were treated with VAE, and then the ECM production was determined by RT-PCR, ELISA and Western blot analysis. Furthermore, the change of gene expression according to VAE treatment was evaluated by cDNA microarray. RESULTS: VAE accelerated the growth of fibroblasts in a dose-dependent manner. VAE increased the production of several ECM components, including type 1 collagen, fibronectin and elastin. In line with these results, the phosphorylations of p42/44 ERK and p38 mitogen-activated protein kinase were markedly increased by VAE, suggesting that the enhancement of ECM production may be linked to the activation of intracellular signaling cascades. VAE also significantly increased cell migration on an in vitro scratch wound test. In cDNA microarray, many genes related with connective tissue integrity were identified to be up-regulated by VAE. CONCLUSION: These results suggest that VAE has a potential to stimulate ECM production, and VAE may be applicable for maintaining the skin's texture.
Animals ; Antlers ; Blotting, Western ; Cell Movement ; Collagen Type I ; Connective Tissue ; Elastin ; Enzyme-Linked Immunosorbent Assay ; Extracellular Matrix ; Fibroblasts ; Fibronectins ; Gene Expression ; Humans ; Oligonucleotide Array Sequence Analysis ; Phosphorylation ; Protein Kinases

PURPOSE: Elastin is a major arterial structural protein, and elastin-derived peptides are related to arterial change. We previously reported on a novel assay developed using aortic elastin peptides; however, its clinical implications remain unclear. In this study, we assessed whether anti-elastin antibody titers reflect the risk of coronary artery disease (CAD) or its characteristics. MATERIALS AND METHODS: We included 174 CAD patients and 171 age- and sex-matched controls. Anti-elastin antibody titers were quantified by enzyme-linked immunosorbent assay. Parameters of arterial stiffness, including the augmentation index (AI) and heart-to-femoral pulse wave velocity (hfPWV), were measured non-invasively. The clinical and angiographic characteristics of CAD patients were also evaluated. Associations between anti-elastin levels and vascular characteristics were examined by linear regression analysis. RESULTS: The median blood level of anti-elastin was significantly lower in the CAD group than in the controls [197 arbitrary unit (a.u.) vs. 63 a.u., p<0.001]. Levels of anti-elastin were significantly lower in men and in subjects with hypertension, diabetes mellitus, hyperlipidemia, or high hfPWV. Nevertheless, anti-elastin levels were not dependent on atherothrombotic events or the angiographic severity of CAD. In a multivariate analysis, male sex (beta=-0.38, p<0.001), diabetes mellitus (beta=-0.62, p<0.001), hyperlipidemia (beta=-0.29, p<0.001), and AI (beta=-0.006, p=0.02) were ultimately identified as determinants of anti-elastin levels. CONCLUSION: Lower levels of anti-elastin are related to CAD. The association between antibody titers and CAD is linked to arterial stiffness rather than the advancement of atherosclerosis.
Aged ; Angiography ; Antibodies/*blood ; Atherosclerosis/*blood/immunology ; Coronary Artery Disease/blood/*immunology ; Elastin/*blood/immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Hyperlipidemias ; Hypertension/complications ; Male ; Middle Aged ; Pulse Wave Analysis ; Vascular Stiffness/*immunology/physiology