Testing the validity of comparisons between the rheological and the calorimetric glass transition temperatures

Dynamic mechanical techniques are used increasingly in the investigation of vitrification phenomena in biological materials, thus posing the question of whether the rheological T(g) should be compared with the established practice of obtaining T(g) values from differential scanning calorimetry. The nature of the rheological T(g) is discussed and its frequency dependence is established with a view to facilitating comparisons with calorimetric data. Despite claims made in the literature, results on high sugar-kappa-carrageenan mixtures, hydrated gelatin films, and thermoset epoxy resins demonstrate that there is no clear reference point for comparison of the glass transition temperatures derived with the two techniques. Furthermore, the structure-forming ability of kappa-carrageenan and other biopolymers impacts primarily upon the mechanical manifestation of vitrification and contributes to the state of complexity of comparisons between thermal and mechanical data.     

Detection of protease inhibitors by a reverse zymography method, performed in a tris(hydroxymethyl)aminomethane-Tricine buffer system

A new detecting method for protease inhibitors, especially for low-molecular-weight inhibitors, is reported. Inhibitor samples were separated on a protein substrate-SDS-polyacrylamide gel in a Tris-Tricine buffer system that improves the separation and identification of peptides and low-molecular-weight proteins. After electrophoresis, the gel was incubated with the target proteases to hydrolyze the background protein substrate. The inhibitor bands, which were protected from proteolysis by the target proteases, were stained. Standard low-molecular-weight inhibitors, such as pepstatin A for pepsin or matrix metalloproteases inhibitor I for collagenase, as well as larger inhibitors, such as soybean trypsin inhibitor or aprotinin for tryspin and cystatin C for papain, were demonstrated by this method and showed clear blue inhibitor bands in the white background when the gels were treated with the target proteases. Some significant applications of this method are introduced. This method is an ideal system for discovering new protease inhibitors in small natural samples.     

Novel potentiometric immunosensor for hepatitis B surface antigen using a gold nanoparticle-based biomolecular immobilization method

A novel potentiometric immunosensor for the detection of hepatitis B surface antigen has been developed by means of self-assembly to immobilize hepatitis B surface antibody on a platinum disk electrode based on gold nanoparticles, Nafion, and gelatin as matrices in this study. The modification procedure of the immunosensor was further characterized by using cyclic voltammetry and the enzyme-linked immunosorbent assay (ELISA) method. The detection is based on the change in the electric potential before and after the antigen-antibody reaction. In contrast to the commonly applied methods (e.g., the glutaraldehyde crosslinking procedure), this strategy could allow for antibodies immobilized with a higher loading amount and better retained immunoactivity, as demonstrated by the potentiometric measurements. A dynamic concentration range of 4-800 ng ml(-1) and a detection limit of 1.3 ng ml(-1) were observed. Analytical results of several human serum samples obtained using the developing technique are in satisfactory agreement with those given by ELISA. In addition, the technique presents some distinct advantages over the traditional sandwich format in that the analyzing performances are direct, rapid, and simple without multiple separation and labeling steps.     

Enhanced activity of serum and urinary hyaluronidases in streptozotocin-induced diabetic Wistar and GK rats

Using streptozotocin-induced diabetic Wistar and GK rats as models of type 1 and type 2 diabetes, respectively, we investigated the changes in serum and urinary hyaluronidase activity with the pathological progress. The serum hyaluronidase levels of streptozotocin-induced rats started to increase on the third day after injection and thereafter maintained approximately threefold higher levels compared with control rats; those of GK rats were already higher ( approximately twofold) from the beginning of the experiment. The increases of serum hyaluronidase activity in both diabetic rats were similar to those of blood glucose level, indicating that diabetes mellitus was accompanied by enhanced activity of circulating hyaluronidase from the early phase of its development. In zymography, every serum from diabetic and control rats gave two hyaluronidase isomers, a major 73-kDa band (Hyal-1 type) and a minor 132-kDa band, suggesting that the increases in serum hyaluronidase activity were not due to the appearance of novel isomers. The hyaluronidase activity in 24-h urine of streptozotocin-induced rats was 3-, 7-, and 11-fold higher at the 8th, 15th, and 18th week than that of control rats, respectively, and the urinary hyaluronidase activity of GK rats was not significantly different from controls. There was a good correlation between the urinary hyaluronidase activity and the albumin excretion. Thus the increase in urinary hyaluronidase activity may reflect enhanced glomerular permeability in streptozotocin-induced diabetic rats and may be a useful marker for diabetic nephropathy. Relative resistance to SDS-denaturation in zymography of rat serum and urinary hyaluronidases compared with human serum hyaluronidase are also shown.     

Immobilization of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> α-galactosidase in gelatin and its application in removal of flatulence-inducing sugars in soymilk

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean-derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. In this study, α-galactosidase from Aspergillus oryzae was entrapped in gelatin using formaldehyde as the hardener. The immobilization yield was 64.3% under the optimum conditions of immobilization. The immobilized α-galactosidase showed a shift in optimum pH from 4.8 to 5.4 in acetate buffer. The optimum temperature also shifted from 50°C to 57°C compared with soluble enzyme. Immobilized α-galactosidase was used in batch, repeated batch and continuous mode to degrade RO present in soymilk. In the repeated batch, 45% reduction of RO was obtained in the fourth cycle. The performance of immobilized α-galactosidase was tested in a fluidized bed reactor at different flow rates and 86% reduction of RO in soymilk was obtained at 25 ml h⁻¹ flow rate. The study revealed that immobilized α-galactosidase in continuous mode is efficient in reduction of RO present in soymilk.     

Heterogeneity of serum gelatinases MMP‐2 and MMP‐9 isoforms and charge variants

The matrix metalloproteinases (MMPs) gelatinase A (MMP‐2) and gelatinase B (MMP‐9) are mediators of brain injury in multiple sclerosis (MS) and valuable biomarkers of disease activity. We applied bidimensional zymography (2‐DZ) as an extension of classic monodimensional zymography (1‐DZ) to analyse the complete pattern of isoforms and post‐translational modifications of both MMP‐9 and MMP‐2 present in the sera of MS patients. The enzymes were separated on the basis of their isoelectric points (pI) and apparent molecular weights (Mw) and identified both by comparison with standard enzyme preparations and by Western blot analysis. Two MMP‐2 isoforms, and at least three different isoforms and two different states of organization of MMP‐9 (the multimeric MMP‐9 and the N‐GAL‐MMP‐9 complex) were observed. In addition, 2‐DZ revealed for the first time that all MMP‐9 and MMP‐2 isoforms actually exist in the form of charge variants: four or five variants in the N‐GAL complex, more charge variants in the case of MMP‐9; and five to seven charge variants for MMP‐2. Charge variants were also observed in recombinant enzymes and, after concentration, also in sera from healthy individuals. Sialylation (MMP‐9) and phosphorylation (MMP‐2) contributed to molecular heterogeneity. The detection of charge variants of MMP‐9 and MMP‐2 in MS serum samples illustrates the power of 2‐DZ and demonstrates that in previous studies MMP mixtures, rather than single molecules, were analysed. These observations open perspectives for better diagnosis and prognosis of many diseases and need to be critically interpreted when applying other methods for MS and other diseases.     

明胶酶谱实验方法的优化简

目的：优化明胶酶谱实验方法,并检测不同浓度凝血酶刺激人皮肤成纤维细胞分泌的明胶酶B（MMP-9）活性的变化。方法：用含明胶的聚丙烯酰胺凝胶电泳分开上清中各蛋白条带,经2．5％TritonX-100洗脱、明胶酶缓冲液孵育、考马斯亮蓝染色液染色及脱色液脱色后,经光密度扫描记录,应用ImageJ软件进行密度计量分析。结果：凝血酶刺激人皮肤成纤维细胞分泌MMP一9具有浓度依赖性。结论：利用优化的明胶酶谱实验方法可做出高质量的明胶酶谱图片,对检测明胶酶活性有重要意义。     

Correlation between sagging and shear elasticity in pectin, gelatin and polyacrylamide gels

The relationship between the height of gels determined by a sag test and their elastic shear modulus (G′) has been both investigated experimentally and simulated using a finite element analysis for the inhomogeneous deformation of gels due to gravity. It was assumed in the simulations that gels can be modeled as incompressible linear elastic materials. General relationships between the sag of gels and their elastic modulus were obtained from the simulations for slip and no-slip conditions. The relationships were tested experimentally on pectin, gelatin and polyacrylamide gels with a range of concentrations and rigidities. The good agreement between the predictions and the results shows that these gels can be modeled accurately as incompressible elastic materials. A standard 150° SAG pectin gel, which sags 23.5% in the SAG test, has G′ moduli of 429 and 379 Pa under slip and no-slip conditions, respectively.     

Keratinolytic activity of Bacillus subtilis AMR using human hair

Aims:  To determine the ability of a novel Bacillus subtilis AMR isolated from poultry waste to hydrolyse human hair producing peptidases including keratinases and hair keratin peptides.
Methods and Results:  The Bacillus subtilis AMR was identified using biochemical tests and by analysis of 16S rDNA sequence. The isolate was grown in medium containing human hair as the sole source of carbon and nitrogen. The supplementation of hair medium (HM) with 0·01% yeast extract increased the keratinolytic activity 4·2-fold. B. subtilis AMR presented high keratinase production on the 8th day of fermentation in hair medium (HM) supplemented with 0·01% yeast extract (HMY) at pH 8·0. Keratinase yield was not correlated with increase in biomass. Zymography showed keratin-degrading peptidases migrating at c. 54, 80 and 100 kDa and gelatin-degrading bands at c. 80, 70 63, 54 32 and 15 kDa. Keratinases were optimally active at 50°C and pH 9·0 and was fully inhibited by the serine proteinase inhibitor (PMSF). Scanning electron microscopy showed complete degradation of the hair cuticle after exposure to B. subtilis AMR grown in HMY. MALDI-TOF analysis of culture supernatant containing peptides produced during enzymatic hydrolysis of hair by B. subtilis AMR revealed fragments in a range of 800–2600 Da.
Conclusions:  This study showed that B. subtilis AMR was able to hydrolyse human hair producing serine peptidases with keratinase and gelatinase activity as well as hair keratin peptides.
Significance and Impact of the Study:  This is the first report describing the production and partial characterization of keratinases by a B. subtilis strain grown in a medium containing human hair . These data suggest that peptides obtained from enzymatic hair hydrolysis may be useful for future applications on pharmaceutical and cosmetic formulations.     

Enzymatic degradation of formaldehyde-crosslinked gelatin

A general method is proposed for characterizing the enzymatic degradability of formaldehyde-crosslinked gelatin which is simple and uses subtilisin as a readily available, commercial alkaline proteinase. Solubilization of gelatin involved dissolution of species not chemically bonded to the crosslinked network, as well as the soluble fractions resulting to the enzymatic degradation. There was a linear relationship between the complete solubilization time of the gelatin and its exposure time to the formaldehyde crosslinking procedure. © Rapid Science Ltd. 1998