Solid-state NMR studies of collagen-based parchments and gelatin

Historical collagen-based parchments have been studied by solid-state NMR. In addition, new parchment (produced according to traditional methods) and gelatin from bovine skin were also studied. Wideline 1H and MAS 13C measurements were carried out directly on intact parchments. A simple approach is proposed for evaluation of the extent of parchment degradation based on the linewidth changes in the 13C CPMAS spectra relative to new parchment and gelatin. Structural (bound) water content was estimated from wideline 1H NMR lineshape and relaxation time measurements. It was found that the relative water content in parchments correlates linearly with 13C MAS linewidths. Its decrease on parchment degradation indicates that structural water molecules are of primary importance in stabilizing higher order collagen structures. Backbone and side chain dynamics of collagen in parchments were compared to those of gelatin based on the 13C dipolar-dephased experiments. Carbonyl 13C chemical shift anisotropies were measured to deduce the geometry of the collagen backbone motion. Unlike previous studies, we found that the collagen backbone motion is similar to that found in other proteins and occurs primarily via small-angle librations about internal bond directions.     

Inhibition of Tumor Cell Growth in vitro by Damavaricin C Derivatives

Damavaricin C, a degradative derivative of streptovaricin C, has a new active hydroxyl group at the C–19 position of the naphthoquinone moiety where various groups can be substituted by ether linkage. The biological activities of these ethers were compared against animal cells, including normal, virus transformed, and human cancer cells in vitro. Some of derivatives showed preferential lethal activity on virus transformed cells in vitro.     

Screening of Enterobacterial Contamination During Gelatin Production and its Effect on Pharmaceutical Grade Gelatin

Summary Gelatin, an animal protein derived from collagen has many industrial applications; mainly in food and pharmaceutical products. In this study, enterobacterial contamination of gelatin during different stages of its manufacturing was examined. Since gelatin is extracted in the form of liquor by hot water treatment of ossein and undergoes a complex series of processing stages before being finally blended and packaged off as dry gelatin product, contamination at any stage affects the quality of the final product. In total, 142 samples of gelatin were analysed for the presence of enteric bacteria, which were obtained from different stages of gelatin processing. The Enterobacteriaceae-positive samples were processed and these enterobacterial species were isolated and identified as Proteus mirabilis, Serratia marcescens, E. coli, Salmonella typhi, Klebsiella oxytoca, Klebsiella pneumoniae, Shigella flexneri and Serratia liquefaciens. These were tested for gelatinase activity. The eight species producing maximum gelatinolysis were selected and protease zymography was performed using 1% (w/v) gelatin as a substrate in 7.5% (w/v) acrylamide gel and their molecular weights were determined. The effect of these bacteria on the quality of gelatin was assessed chromatographically in terms of change in amino acid content of gelatin broth inoculated by these species, which showed a marked change with length of incubation. Since these enteric bacteria possess pathogenic properties, some of them are gelatinolytic and also have a significant effect on the quality and amino acid content of gelatin, they are of great concern both for the manufacturers as well as for the consumers.     

Immobilization of Kluyveromyces marxianus cells containing inulinase activity in open pore gelatin matrix: 2. Application for high fructose syrup production

Batch and continuous production of high fructose syrup from Jerusalem artichoke tubers has been studied using yeast cells immobilized in open pore gelatin matrix. In a batch reactor, the hydrolysis was 93% ($\text{d}\text{-fructose/}\text{d}\text{-glucose}\text{= 90/10}$) and 42 mg d-fructose per ml was produced from the artichoke tuber extract by immobilized cells in 3 h. The same immobilized cells were recycled and used repeatedly for 10 batch cycles starting with fresh juice at the beginning of each cycle. It was found that immobilized cells were extremely stable and the percent hydrolysis was almost constant for all 10 batch cycles. In a continuous reactor using an immobilized cell concentration of 65.7 g (dry wt) l^?1 of total working bioreactor volume, the percent hydrolysis was found to remain constant at ～100% at dilution rates <1.26 h^?1, but beyond that it decreased. Volumetric productivity attained its maximum value at D = 2.08 h^?1 and was found to be 100 g l^?1 h^?1. This was achieved at a feed sugar conversion of 80%. At 90% conversion and D = 1.66 h^?1, the productivity was found to be 90 g l^?1 h^?1. Continuous operation of the immobilized cell bioreactor at a constant dilution rate of 1.65 h^?1 for 240 h resulted in only 2% loss of original activity.     

Inhibition by zinc of the binding of C1 to antigen-antibody complexes

Zinc sulphate in the range of 10^?4 to 2×10^?5 M prevents the binding of $\text{C}\text{1}$ to antigen antibody complexes, and the initation of the cascade of events in the classical complement pathway leading to cell lysis. Other heavy metals, Co⁺⁺, Cd⁺⁺, Cu⁺⁺, or Mn⁺⁺ were without effect in this concentration range. Zinc was ineffective when added after $\text{C}\text{1}$ was bound and failed to displace $\text{C}\text{1}$ which was already bound to antigen antibody complexes. The ability of zinc to regulate the binding of the zymogen or activated form of $\text{C}\text{1}$ to antigen-antibody complexes represents a new method of controlling the initiation of the classical complement pathway.     

Crystal structure of a ternary complex between human prostate-specific antigen, its substrate acyl intermediate and an activating antibody

Human prostate-specific antigen (PSA or KLK3) is an important marker for the diagnosis and management of prostate cancer. This is an androgen-regulated glycoprotein of the kallikrein-related protease family secreted by prostatic epithelial cells. Its physiological function is to cleave semenogelins in the seminal coagulum and its enzymatic activity is strongly modulated by zinc ions.Here we present the first crystal structure of human PSA in complex with monoclonal antibody (mAb) 8G8F5 that enhances its enzymatic activity. The mAb recognizes an epitope composed of five discontinuous segments including residues from the kallikrein loop and stabilizes PSA in an “open and active conformation” that accelerates catalysis.We also present the crystal structure of PSA in complex with both the mAb 8G8F5 and a fluorogenic substrate Mu-KGISSQY-AFC, derived from semenogelin I. By exploiting the inhibition of PSA by zinc ions, we were able to obtain a substrate acyl intermediate covalently linked to the catalytic serine, at pH 7.3 but not at pH 5.5.Moreover, the inhibition of PSA activity by zinc was found to be modulated by pH variations but not by the antibody binding. The correlation of the different data with the physiological conditions under which PSA can cleave semenogelins is discussed.     

Influence of anaerobic culture acclimation on the degradation kinetics of various substrates

The adaptation of an anaerobic culture (anaerobic sludge) to a specific substrate brings significant changes to its microbial population. These changes can be described by the sludge's ability to treat various substrates such as carbohydrates or proteins or "intermediate" products of anaerobic metabolism such as L-lactic, propionic, and acetic acids. The activity of the sludge with respect to a specific substrate is a critical parameter, because the anaerobic degradability of wastewaters depends strongly on it. This work examines and quantifies the differentiation of two anaerobic sludges of the same origin, following an adaptation period of about 18 months to lactose and gelatin, respectively. The acclimation has a significant effect on the maximum specific utilization rates of various compounds and on their apparent consumption kinetics. It is noticeable, however, that even if the anaerobic cultures were not exposed to a specific substrate for a prolonged period of time (more than a year), they still kept the ability of hydrolyzing or degrading it. In addition, the acclimation has an unquestionable effect on the stoichiometry of the production of volatile fatty acids and L-lactate. Finally, from codigestion experiments it is shown that codigestion of lactose and gelatin appears to have no effect on their hydrolysis kinetics in any of the lactose or gelatin acclimated cultures; specifically, the hydrolysis kinetics remained the same as calculated when lactose or gelatin were the only fed substrates. Similarly, the kinetics of L-lactate and D-glucose biodegradation seemed to be unchanged. On the other hand, codigestion has a significant effect on the production of L-lactic, propionic, and acetic acids, which can be attributed to the increased hydrogen production accompanying gelatin biodegradation.     

Poly(l-lactide) degradation by Kibdelosporangium aridum

A new poly(L-lactide) (PLA)-degrading actinomycete, Kibdelosporangium aridum, degraded more than 97 mg out of 100 mg added high molecular weight PLA film (Mn: 3.4 x 10(5)) within 14 d in liquid culture. L-Lactic acid, the monomeric degradation product of PLA, was totally assimilated by the strain. In solid culture, many distinct grooves formed by the morphology of filamentous microorganisms on the surface of a PLA film were observed by scanning electron microscopy.     

Functional mimetic peptide discovery isolated by phage display interacts selectively to fibronectin domain and inhibits gelatinase

Matrix metalloproteinases (MMPs) play critical roles in a multiple number of autoimmunity diseases progression and metastasis of solid tumor. Gelatinases including MMP-2 and MMP-9 are extremely overexpressed in multiple pathological processes. MMP-9 and MMP-2 breakdown the extracellular matrix component gelatin very efficaciously. Therefore, designing and expansion of MMPs inhibitors can be an engrossing plan for therapeutic intermediacy. Anyway, a wide range of MMPs inhibitors face failure in several clinical trials. Due to sequence and structural conservation across the various MMPs, achieving specific and selective inhibitors is very demanding. In the current study, a phage-displayed peptide library was screened using active human recombinant MMP-9 protein and evaluated by enzyme-linked immunosorbent assay. Here, we isolate novel peptide sequence from phage display peptide libraries that can be a specific gelatinase inhibitor. Interestingly, in silico molecular docking showed strong interactions between the peptide three-dimensional models and some important residues of the MMP-9 and MMP-2 proteins at the fibronectin domain. A consensus peptide sequence was then synthesized (named as RSH-12) to evaluate its inhibitory potency by in vitro assays. Zymography assay was employed to evaluate the effect of RSH-12 on gelatinolysis activity of MMP-2 and MMP-9 secretion from the HT1080 cells using different concentrations of RSH-12 and inhibiting MMP-9- and MMP-2-driven gelatin proteolysis, measured by fluorescein isothiocyanate-gelatin degradation assay and HT1080 cell invasion assay on Matrigel (gelatinous protein mixture). The negative control peptide (CP) with the irrelevant sequence and no MMP inhibition properties and the positive control compound (GM6001) as a potent inhibitor of MMPs were used to assess the selectivity and specificity of gelatinases inhibition by RSH-12. Therefore, RSH-12 decreased the gelatin degradation by specifically preventing gelatin binding to MMP-9 and MMP-2. Selective gelatinase inhibitors may prove the usefulness of the new peptide discovered in tumor targeting and anticancer and anti-inflammation therapies.