The use of polymer fibers as a substrate for cultured nerve tissue

A study was made of the reaction of cells to administration of polymeric fibers containing amino acids--phenylalanine and leucine--to the culture of nerve tissue. The data obtained have shown that polymeric fibers are some acceptable substrate for the migration of astrocytes and oligodendrocytes and for the growth of nerve cell axons.     

Comparison of substrate specificities of transglutaminases using synthetic peptides as acyl donors

Transglutaminase (TGase) is an enzyme that catalyzes acyl transfer reactions between primary amines and Gln residues in proteins or peptides. Substrate specificities of TGase, Ca2+-independent microbial transglutaminase (MTGase), and Ca2+-dependent tissue type transglutaminase from guinea pig liver (GTGase) and fish, Red sea bream (Pagrus major), liver (FTGase), for acyl donors were investigated using synthetic peptides containing Gln residues and Gln analogues with different lengths of side chain. MTGase dose not recognize the Gln analogues as a substrate and has strict substrate specificities toward L-Gln. Substrate peptides with a variety of sequences around the Gln residue, GXXQXXG (X=G, A, S, L, V, F, Y, R, N, E, L) were synthesized and used as acyl donors. As an acyl acceptor, the fluorescent reagent monodancyl cadaverine was used and the reactions analyzed with RP-HPLC. Substitution of the C-terminal of a Gln residue with a hydrophobic amino acid accelerated the reaction by GTGase and FTGase. N-terminal substitution of Gln residues had similar effects on the reaction by MTGase.     

The use of indole as a spectrophotometric assay substrate for toluene dioxygenase

Abstract Cell extracts of toluene-grown Pseudomonas putida produced a soluble yellow dye during aerobic incubations with indole and NADH. Accumulation of indoxyl in reaction mixtures corresponded with a linear increase in absorbance at 400 nm. The rate of increase in absorbance was shown to be a specific measure of toluene dioxygenase activity. The primary product of toluene oxidation, cis -toluene dihydrodiol, inhibited dioxygenase activity in cell extracts containing no detectable activity of cis -toluene dihydrodiol dehydrogenase.     

Renin activity determination using human plasma as a substrate

A method for human renin activity determination using human plasma as the substrate is described. Angiotensin I is generated by incubating renin with human plasma, which is available along with the commercial radioimmunoassay kit for angiotensin I. The method obviates the need to isolate and purify the substrate, human angiotensinogen, from human plasma. In addition, the assay is highly renin specific, sensitive, and convenient to use for the routine determination of active human renin during its isolation and purification from tissue extracts or from genetically engineered bacterial and nonbacterial expression systems.     

The effects of preservation procedures on amniotic membrane's ability to serve as a substrate for cultivation of endothelial cells

Amniotic membrane (AM) has been used as a scaffold for the ex vivo expansion of different types of cells and a cell delivery matrix in regenerative medicine. Since the preservation procedures can influence the AM properties for experimental and clinical purposes, this study was established to investigate the feasibility of using the AM after different preservation methods to serve as substrates for endothelial cell expansion ex vivo. The effects of cryopreservation and lyophilization were evaluated on mechanical and histological characteristics of the AM, and the results were compared with the fresh AM. The ECM components of the basement membrane were well conserved in all groups. Although lyophilization resulted in more histological changes and lower level of physical variables including thickness, F_max, elongation at break and suture retention than the fresh and cryopreserved AM, endothelial cells grown on the lyophilized AM were better attached to the basement membrane. Cytotoxicity assay by MTT showed that the lyophilized AM is a compatible substrate for endothelial cells cultivation. The findings of this study suggest that the lyophilized AM is a suitable matrix for cultivation of endothelial cells due to this fact that lyophilization led to exposure of basement membrane of the AM.     

Mammalian transglutaminases. Identification of substrates as a key to physiological function and physiopathological relevance

Transglutaminases form a large family of intracellular and extracellular enzymes that catalyse the Ca2+-dependent post-translational modification of proteins. Despite significant advances in our understanding of the biological role of most mammalian transglutaminase isoforms, recent findings suggest new scenarios, most notably for the ubiquitous tissue transglutaminase. It is becoming apparent that some transglutaminases, normally expressed at low levels in many tissue types, are activated and/or overexpressed in a variety of diseases, thereby resulting in enhanced concentrations of cross-linked proteins. As applies to all enzymes that exert their metabolic function by modifying the properties of target proteins, the identification and characterization of the modified proteins will cast light on the functions of transglutaminases and their involvement in human diseases. In this paper we review data on the properties of mammalian transglutaminases, particularly as regards their protein substrates and the relevance of transglutaminase-catalysed reactions in physiological and disease conditions.     

The ability of bacterial DNA methyltransferases to use methylcobalamine as a cofactor in DNA methylation reactions

The ability of bacterial DNA methyltransferases Alu I, Cfr I, Cfr 6, Cfr 10, Eco RI, Eco RII, Msp I, Mva I, Pvu I, Pvu II, and Sau 3A to use methylcobalamine and methylmethionine as cofactors of DNA methylation in vitro has been investigated. These bacterial DNA methyltransferases used methylcobalamine, but not methylmethionine for DNA methylation.     

Characterization of plasma gelsolin as a substrate for matrix metalloproteinases

We previously showed that plasma gelsolin, a major component of the extracellular actin scavenging system, is an matrix metalloproteinase (MMP)-14 substrate. Here we confirmed that plasma gelsolin is cleaved by MMP-14 at the plasma level, and found that it was most efficiently digested by MMP-3 followed by MMP-2, MMP-1, MMP-14, and MMP-9, in that order. Plasma gelsolin (90 kDa) was cut into several fragments of 43-48 kDa by MMP-3. The MMP-3 cleavage sites in plasma gelsolin were determined by labeling the C termini generated by in-gel digestion with 50% H2 18O combined with peptide mass mapping, and sequencing of the N-terminal amino acids. Plasma gelsolin was cleaved at Asn416-Val417, Ser51-Met52, and Ala435-Gln436. Proteolytic cleavage by MMP-3 resulted in considerable loss of its actin filament-depolymerizing activity. This suggests that MMPs weaken the extracellular actin-scavenging system by cleaving plasma gelsolin and may, therefore, be involved in pathological conditions induced by extracellular actin, such as endothelial injury, respiratory distress syndrome, hepatic necrosis, and septic shock.     

The use of deionised date syrup as a substrate for citric acid fermentation

Summary Production of citric acid from date syrup by fermentation has been undertaken usingAspergillus niger as the producer organism. Poor yields were obtained when untreated and ferrocyanide treated date syrup were used. However, acceptable yields, in excess of 60% were obtained when the date syrup was deionised using an ion exchange column at 80°C.     

The use of babassu oil as substrate to produce bioemulsifiers by Candida lipolytica.

Candida lipolytica IA 1055 produced an extracellular emulsifier when using babassu oil as its sole carbon source during batch and fed batch fermentations at 27 degrees C. Emulsification activity was detected after 60 h of growth in all conditions studied. The bioemulsifier was isolated after 144 h of fermentation from the best condition studied. The biopolymer seems to be a polysaccharide-protein-lipid complex.     

The use of canola meal as a substrate for xylanase production by Trichoderma reesei

Summary Tests made utilizing canola meal as a substrate for the production of xylanase indicate that Trichoderma reesei produced this enzyme in similar or better yields from canola meal than from Solka-floc, xylan or glucose. The maximum xylanase activity obtained from canola meal was 210 IU/ml in 9–12 days. The enzyme system produced using canola meal also contained a higher proportion of acetyl-xylan esterase, cellulase, and xylosidase activities. This system was more than or equally efficient as that produced using Solka-floc in hydrolysing canola meal, corn cobs, corn and wheat brans, straw, and larchwood xylan to fermentable sugars. Offprint requests to: Z. Duvnjak     

Isolation and characterization of cDNA clones to mouse macrophage and human endothelial cell tissue transglutaminases.

The deduced amino acid sequences for tissue transglutaminases from human endothelial cells and mouse macrophages have been derived from cloned cDNAs. Northern blot analysis of both tissue transglutaminases shows a message size of approximately 3.6-3.7 kilobases. The molecular weights calculated from the deduced amino acid sequences were 77,253 for human endothelial tissue transglutaminase and 76,699 for mouse macrophage tissue transglutaminase. The deduced amino acid sequence for the human endothelial transglutaminase was confirmed by comparison with the amino acid sequence obtained by cyanogen bromide digestion of the human erythrocyte transglutaminase. The amino acid sequences of both human endothelial and mouse macrophage tissue transglutaminases were compared to other transglutaminases. A very high degree of homology was found between human endothelial, mouse macrophage, and guinea pig liver tissue transglutaminase (greater than 80%). Moreover, human endothelial tissue transglutaminase was compared with human Factor XIIIa and a very high degree of homology (75% identity) was found in the active site region.     

Synthesis of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and its use as a substrate to assess feruloyl esterase activity

A synthetic scheme was developed for the production of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside (FA-Ara) in gram quantities. This molecule accurately models the chemical attachment of ferulic acid to polysaccharides found in cell walls of plants in the Gramineae family. It is therefore a realistic substrate that can be used to monitor feruloyl esterase activity. Ultraviolet spectral analysis indicated that FA-Ara has an absorption maximum distinct from the hydrolytic product, ferulic acid (FA), over a wide range of solution pH values. The log molar extinction coefficient ranges from 4.16 to 4.36 for FA-Ara and 4.16 to 4.33 for FA depending upon the pH of the buffered solution. Consequently a convenient spectrophotometric assay can be utilized to monitor esterase activity. Three different methods were developed for using this model substrate to assess esterase activity, including thin-layer chromatography, a spectrophotometric assay, and the use of high-performance liquid chromatography.     

Monoacylmonoalkylglycerol as a substrate for diacylglycerol hydrolase activity in adipose tissue

The synthesis and use of 1(3)-[3H]oleoyl-2-0-oleylglycerol as a substrate for the assay of diacylglycerol hydrolase activity in adipose tissue is described. Neither the compound nor its reaction product are hydrolyzed by purified adipose tissue monoacylglycerol lipase.     

The collagen substrate specificity of rat uterus collagenase

The collagen substrate specificity of rat uterus collagenase was studied as a function of both collagen type and species of substrate origin. For each collagen examined, values for the basic kinetic parameters, Km and Vmax (kcat), were determined on collagen in solution at 25 degrees C. In all cases, Lineweaver-Burk plots were linear and rat uterus collagenase behaved as a normal Michaelis-Menten enzyme. Collagen types I, II, and III of all species tested were degraded by rat uterus collagenase. Collagen types IV and V were resistant to enzymatic attack. Both enzyme-substrate affinity and catalytic rates were very similar for all susceptible collagens (types I-III). Values for Km ranged from 0.9 to 2.5 X 10(-6) M. Values for kcat varied from 10.7 to 28.1 h-1. The homologous rat type I collagen was no better a substrate than the other animal species type I collagens. The ability of rat uterus collagenase to degrade collagen types I, II, and III with essentially the same catalytic efficiency is unlike the action of human skin fibroblast collagenase or any other interstitial collagenase reported to date. The action of rat uterus collagenase on type I collagen was compared to that of human skin fibroblast collagenase, with regard to their capacity to cleave collagen as solution monomers versus insoluble fibrils. Both enzymes had essentially equal values for kcat on monomeric collagen, yet the specific activity of the rat uterus collagenase was 3- to 6-fold greater on collagen fibrils than the skin fibroblast enzyme. Thus, in spite of their similar activity on collagen monomers in solution, the rat uterus collagenase can degrade collagen aggregated into fibrils considerably more readily than can human skin fibroblast collagenase.