Physical and chemical characteristics of Aloe ferox leaf gel

Aloe ferox leaf gel differs substantially from that of Aloe vera but almost no commercially relevant data is available this species. Leaf dimensions, gel yields and gel compositions were studied, based on samples from several natural populations. Glucose is the only free sugar in aloe gel (0.1 to 0.4 mg ml^− 1 in A. ferox). Monosaccharides released after hydrolysis show potential for gel fingerprinting and allow for a distinction between A. ferox and A. vera. The former yields various combinations of glucose and galactose as main monosaccharides, while the latter yields only mannose. Further variation studies are recommended because A. ferox appears to have three different gel chemotypes. Conductivity shows species-specific ranges — in A. ferox below 3000 μS cm^− 1 in fresh gel and above 3100 μS cm^− 1 in aged gel (corresponding values for A. vera were 1670 and 1990 μS cm^− 1). The level of phenolic (bitter) compounds in A. ferox gel can be reduced by treatment with activated charcoal, resulting in a small loss of total dissolved solids. Alcohol precipitable solids and insolubility are useful variables for quality control of gel powder. The methods and data presented are the first steps towards developing quality criteria for A. ferox leaf gel.     

In vitro models of angiogenesis and vasculogenesis in fibrin gel

In vitro models of endothelial assembly into microvessels are useful for the study of angiogenesis and vasculogenesis. In addition, such models may be used to provide the microvasculature required to sustain engineered tissues. A large range of in vitro models of both angiogenesis and vasculogenesis have utilized fibrin gel as a scaffold. Although fibrin gel is conducive to endothelial assembly, its ultrastructure varies substantially based on the gel formulation and gelation conditions, making it challenging to compare between models. This work reviews existing models of endothelial assembly in fibrin gel and posits that differerences between models are partially caused by microstructural differences in fibrin gel.     

Isolation and properties of chloroperoxidase isozymes

A new purification method for chloroperoxidase from Caldariomyces fumago is described. This method involves dialysis, alumina gel adsorption, DEAE-cellulose chromatography at pH 6, and then at pH 3.85 and crystallization. Two isozymes have been isolated and one has been crystallized. The MWs estimated by gel filtration are 46000 for chloroperoxidase A and 40000 for chloroperoxidase B. The guaiacol peroxidation catalysed by these isozymes is proportional to their chlorination activity.     

Quantitative Determination of the Spatial Distribution of Nitrosomonas europaea and Nitrobacter agilis Cells Immobilized in κ-Carrageenan Gel Beads by a Specific Fluorescent-Antibody Labelling Technique

A novel technique, combining labelling and stereological methods, for the determination of spatial distribution of two microorganisms in a biofilm is presented. Cells of Nitrosomonas europaea (ATCC 19718) and Nitrobacter agilis (ATCC 14123) were homogeneously distributed in a κ-carrageenan gel during immobilization and allowed to grow out to colonies. The gel beads were sliced in thin cross sections after fixation and embedding. A two-step labelling method resulted in green fluorescent colonies of either N. europaea or N. agilis in the respective cross sections. The positions and surface areas of the colonies of each species were determined, and from that a biomass volume distribution for N. europaea and N. agilis in κ-carrageenan gel beads was estimated. This technique will be useful for the validation of biofilm models, which predict such biomass distributions.     

Bakers' yeast protease a purification and enzymatic and molecular properties

It has been previously demonstrated in our laboratory that uridine nucleosidase (EC 3.2.2.3) is inactivated by yeast protease A (EC 3.4.23.8). A complete purification procedure for protease A from bakers' yeast, which lacks the acidic activation step used by other workers, and the major properties of the enzyme are shown. The enzyme is homogeneous as judged by disc gel electrophoresis. Its molecular weight, calculated from both sodium dodecyl sulfate-disc gel electrophoresis and gel filtration experiments, is around 45,000. The protein does not possess quaternary structure. The isoelectric point is 4.1. Carbohydrate content is around 8%. Amino acids analysis and sulfur analysis reveal the presence of 1-SH group and two disulfide bridges. The free-SH group does not seem to be involved in catalysis. Amino terminal analysis shows that isoleucine is at the amino terminal position. The pH optima are 2.4 for the hydrolysis of azocasein and casein, and 3.3 for the hydrolysis of hemoglobin. The K_m value for hemoglobin is 1.7 × 10^?5m. The inhibition exerted by pepstatin on the proteolytic activity of protease A is pH dependent. Among various yest enzyme substrates only uridine nucleosidase is inactivated by protease A.     

Effect of inorganic salts and glucose additives on dose–response,melting point and mass density of genipin gel dosimeters

Genipin gel dosimeters are hydrogels infused with a radiation-sensitive material which yield dosimetric information in three dimensions (3D). The effect of inorganic salts and glucose on the visible absorption dose–response, melting points and mass density of genipin gel dosimeters has been experimentally evaluated using 6-MV LINAC photons. As a result, the addition of glucose with optimum concentration of 10% (w/w) was found to improve the thermal stability of the genipin gel and increase its melting point (T_m) by 6 °C accompanied by a slight decrease of dose–response. Furthermore, glucose helps to adjust the gel mass density to obtain the desired tissue-equivalent properties. A drop of T_m was observed when salts were used as additives. As the salt concentration increased, gel T_m decreased. The mass density and melting point of the genipin gel could be adjusted using different amounts of glucose that improved the genipin gel suitability for 3D dose measurements without introducing additional toxicity to the final gel.     

Studies on the "aerobic" acetyl-coenzyme A synthetase of Saccharomyces cerevisiae: purification, crystallization, and physical properties of the enzyme.

A procedure for the purification of a stable acetyl-coenzyme A synthetase (ACS) from aerobic cells of Saccharomyces cerevisiae is presented. The steps include differential centrifugation, solubilization of the bound enzyme from the crude mitochondrial fraction, ammonium sulfate fractionation, crystallization to constant specific activity from ammonium sulfate solutions followed by Bio-Gel A-1.5 m column chromatography. The resulting enzyme preparation is homogeneous as judged by chromatography on Bio-Gel columns, QAE-Sephadex A-50 anion exchange columns, analytical ultracentrifugal studies, and polyacrylamide gel electrophoresis.Sedimentation velocity runs revealed a single symmetric peak with an s_20,w value of 10.6. The molecular weight of the native enzyme, as determined by gel filtration and analytical ultracentrifugation, is 250,000 ± 500. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the single polypeptide chain is 83,000 ± 500. The purified enzyme is inhibited by palmityl-coenzyme A with a Hill interaction coefficient, n, of 2.88. These studies indicate that the ACS of aerobic S. cerevisiae is composed of three subunits of identical or nearly identical size.     

Activity assay for nisin-like acidic bacteriocins using an optimal pH-conditioned gel matrix

A new zymography for detecting nisin-like acidic bacteriocins was developed using a tricine-sodium dodecyl sulfate (SDS) gel and an acidic gel matrix (pH 4.0). After electrophoresis, proteins in the tricine gel were electrotransferred to an optimal pH-conditioned gel matrix (OP-CGM). The OP-CGM was overlaid with indicator cells (Bacillus cereus) embedded in nutrient broth soft agar (0.8%, w/v). Antibacterial activity shown as a growth inhibition using B. cereus was detected at approximately 3.8 kDa. Because nisin is unstable in buffers at pH values over 6.0, the common electrophoretic systems, SDS-polyacrylamide gel electrophoresis and tricine gel, are not suitable for detection of nisin-like acidic bacteriocins.     

Comparative Electrophoretic Analyses of Soluble Proteins from Heterodera glycines Races 1-4 and Three Other Heterodera Species

Modified polyacrylamide gel and SDS-polyacrylamide gel electrophoretic systems using a low molarity tris-HCl buffer and equal pH of homogenizing buffer and stacking gel provided improved stacking for separation of soluble proteins from Heterodera schachtii, H. trifolii, H. lespedezae, and H. glycines races 1, 2, 3, and 4, compared with previous studies with cyst nematodes, The four Heterodera species were easily distinguished using the polyacrylamide gel system, but H. trifolii and H. lespedezae had similar protein patterns. H. glycines races were not separable by that system. The SDS-polyacrylamide gel system produced different protein patterns for all four Heterodera species although H. trifolii and H. lespedezae differed by only a single band, suggesting that these two may be subspecifically related. A protein band unique to H. glycines races 3 and 4 was not detected in SDS-polyacrylamide gel profiles from races 1 and 2. Molecular weight determinations were 55,000 for distinctive proteins in profiles of H. trifolii and 75,000 for H. glycines races 3 and 4.     

Measuring Dilution of Microbicide Gels with Optical Imaging

We present a novel approach for measuring topical microbicide gel dilution using optical imaging. The approach compares gel thickness measurements from fluorimetry and multiplexed low coherence interferometry in order to calculate dilution of a gel. As a microbicide gel becomes diluted at fixed thickness, its mLCI thickness measurement remains constant, while the fluorimetry signal decreases in intensity. The difference between the two measurements is related to the extent of gel dilution. These two optical modalities are implemented in a single endoscopic instrument that enables simultaneous data collection. A preliminary validation study was performed with in vitro placebo gel measurements taken in a controlled test socket. It was found that change in slope of the regression line between fluorimetry and mLCI based measurements indicates dilution. A dilution calibration curve was then generated by repeating the test socket measurements with serial dilutions of placebo gel with vaginal fluid simulant. This methodology can provide valuable dilution information on candidate microbicide products, which could substantially enhance our understanding of their in vivo functioning.     

Comparison of malate dehydrogenase isoenzymes in someAllium species by isoelectric focusing

Malate dehydrogenase isoenzymes were studied in tenAllium species and in six cultivars ofA. cepa by isoelectric focusing in polyacrylamide gel with Ampholine pH 3.5–10.0. Using this method better resolution was obtained than by polyacrylamide gel electrophoresis. The number of MDH isoenzymes obtained by isoelectric focusing is from five to ten in the range of pH 3.65 to 6.75. MDH isoenzymes can be used for characterization on the level of species and cultivars (inA. cepa), but its use on the level of sections and subgenera is questionable.     

Coenzyme B12-dependent diol dehydrase: purification, subunit heterogeneity, and reversible association.

A purification procedure for diol dehydrase (dl-1,2-propanediol hydro-lyase, EC 4.2.1.28) of Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 has been developed which gives the highest specific activity for this enzyme obtained so far. The purified enzyme is homogeneous by the criteria of ultracentrifugation (s_20,w = 8.9 S) and disc gel electrophoresis in the presence of substrate. The molecular weight of approximately 230,000 was obtained by gel filtration and ultracentrifugal sedimentation equilibrium. The enzyme is composed of components F and S whose molecular weights were determined to be approximately 26,000 and 200,000, respectively, by gel filtration. The incubation of both components F and S with the substrate leads to complete reassociation of the components. Disc gel electrophoresis in the presence of sodium dodecyl sulfate and terminal amino acid analyses indicate that component S consists of at least four nonidentical subunits. The reversible association and heterogeneity of the subunits were also demonstrated with the crude enzyme by immunoelectrophoresis.     

Purification and subunit structure of mouse liver cystathionase

Cystathionase has been purified from mouse liver by ammonium sulfate precipitation, ethanol precipitation, column chromatography on DEAE-cellulose and on hydrox-ylapatite, as well as Sephadex G-200 gel filtration. These procedures yielded a chromatographically homogeneous enzyme which was purified more than 1000-fold relative to whole liver extract. Overall recovery was approximately 4%. The purified enzyme does not contain detectable carbohydrate and migrates as a single protein component on analytical disc gel electrophoresis. A sedimentation coefficient of 8.3 S has been determined for the active enzyme by rate zonal centrifugation in glycerol gradients. This value suggests a molecular weight for the native enzyme of approximately 160,000 g/mol, a value similar to that estimated by gel filtration. Following sodium dodecyl sulfate gel electrophoresis in the presence of reducing agent and at different gel concentrations, a single protein component with a molecular weight of 40,000 g/mol was obtained. Thus, the enzyme appears to consist of four subunits of equal size. The K_m value for cystathionine at pH 8.1, 37 °C, and in the presence of 1 mm dithioerythritol is approximately 1 mm.     

A simplified and efficient system for separating proteins by preparative polyacrylamide electrophoresis. With notes on horse heart myoglobin

A simplified but effective method for preparative polyacrylamide gel electrophoresis is presented, suitable for single runs of ∼15 mg of protein, which allows: (1) a gel casting shaped and cooled so that proteins do not assume curvature as they migrate down the column; (2) direct contact of the bottom of the gel with buffer; (3) use of reduced electrical field since anode and cathode are spaced only 9.5 cm apart; (4) increased stability of the anodal face of the gel, by employing a dilute running buffer and consequently a still lower current (7 to 8 mA at 150 V); (5) obtaining successive effluents in separate Visking sacs, at appropriate time intervals, and removal of proteins in absence of an electrical field; (6) rinsing of the column between collections; (7) advance planning of the actual times of collection, calculated from the rates of migration of proteins of interest relative to the time of passage of (yellow) “myoglobin standard” determined in analytical columns run under the same conditions.     

Creatine amidinohydrolase of Pseudomonas putida: crystallization and some properties.

Creatine amidinohydrolase (EC 3.5.3.3, creatinase) of Pseudomonas putida var. naraensis C-83 was purified by column chromatography on sarcosine-hexamethylenediamine-Sepharose and Sephadex G-200 and then crystallized in the presence of ammonium sulfate. The purified preparation appeared homogeneous on disc gel electrophoresis and ultracentrifugal analysis. It was most active at pH 8 and showed a K_m value of 1.33 mm for creatine. Estimation of the molecular weight by the meniscus depletion method yielded a value of 94,000. A value of 47,000 was obtained, however, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the enzyme is composed of two subunits. Inhibition experiments suggested that a sulfhydryl group is closely related to the creatinase activity.     

Identification of Frankia Strains by Two-Dimensional Polyacrylamide Gel Electrophoresis

Fifteen Frankia strains from five different plant species were analyzed by two-dimensional polyacryl-amide gel electrophoresis to determine their relatedness by comparing the polypeptide patterns obtained. Three major subgroups (A, C, and D) were found in the Alnus-Comptonia-Myrica cross-inoculation group. An isolate from Purshia tridentata had a unique protein pattern and represents a distinct group of frankiae. Members of group A were isolated from root nodules of Alnus incana subsp. rugosa and Alnus viridis subsp. crispa. Group C organisms were from A. incana subsp. rugosa and Comptonia peregrina nodules, and group D organisms were from A. incana subsp. rugosa, A. viridis subsp. cripsa, and Myrica pensylvanica root nodules. Isolates from each gel group were obtained at several widely separated geographical locations. The results indicate that two-dimensional polyacrylamide gel electrophoresis is useful for identifying Frankia isolates.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

Multiple forms of glucocorticosteroid receptors in the neural retina of chick embryo,revealed by polyacrylamide gel electrophoresis

The glucocortiocoid receptors in the cytosol of neural retina of the 15-day chick embryo were analyzed by quantitative polyacrylamide gel electrophoresis. Maintenance of the triamcinolone acetonide (TA)-receptor complexes under conditions of electrophoretic analysis is dependent on temperatures not exceeding ?2 °C and is favored by low ionic strength, but is relatively insensitive to changes in pH between 5 and 10. Polyacrylamide gel electrophoresis in highly crosslinked Resolving Gels (15% crosslinking with N,N′-diallyltartardiamide) at low wattage and under temperature control at ?2 °C, allowed for detection and partial characterization of over 80% of the specific TA-binding activity of the tissue. One form of the glucocorticoid receptor, designated as complex II, was found to have a molecular weight (M_r) of 175,000. In addition, specifically bound TA was found in a multimillion M_r aggregate which was unable to enter gels of any concentration investigated and has been designated TA-complex I. The ratio of complex I/complex II increased with increasing gel concentration, indicating physical or chemical interaction between II and I. A polyacrylamide gel electrophoresis rerun of isolated TA-complex II gave rise to two smaller TA-binding species: Component B, of M_r 108,000 and component A, a relatively fast migrating molecule which could not be characterized under the conditions used. The ratio of $\text{B}\text{A}$ appeared constant and close to 2, suggesting that A and B may be significant structural elements of complex II. Polyacrylamide gel electrophoresis of isolated TA-complex I gave rise to component C of M_r 60,000, but not to components A or B. Components A and B associated to a large M_r complex, designated as I′, which was revealed to an extent directly proportional to gel concentration. Similarly, component C aggregated to I″, as evidenced at elevated gel concentrations. In conclusion, it has been possible to define by gel electrophoresis three of the molecular species (A, B, and C) that comprise the glucocorticoid receptor, and the possible relationships between them.     

Diffusion of insulin-like growth factor-I and ribonuclease through fibrin gels

A fluorescence-based method for simultaneously determining the diffusion coefficients of two proteins is described, and the diffusion coefficient of insulin-like growth factor (IGF-I) and ribonuclease (RNase) in a 0.27% fibrin hydrogel is reported. The method is based on two-color imaging of the relaxation of the protein concentration field with time and comparing the results with a transport model. The gel is confined in a thin (200 μm) capillary and the protein is labeled with a fluorescent dye. The experimentally determined diffusion coefficient of RNase (D = 1.21 × 10⁻⁶ cm²/s) agrees with literature values for dilute gels and bulk aqueous solutions, thus indicating the gel and the dye had a negligible effect on diffusion. The experimental diffusion coefficient of IGF-I (D = 1.59 × 10⁻⁶ cm²/s), in the absence of binding to the fibrin matrix, is consistent with the dimensions of the molecule known from x-ray crystallography and a correlation between D and molecular weight based on 14 other proteins. The experimental method developed here holds promise for determining molecular transport properties of biomolecules under a variety of conditions, for example, when the molecule adsorbs to the gel or is convected through the gel by fluid transport.     

A simple electrophoretic procedure for the determination of the polypeptide composition of the subunits of Fraction 1 protein

A rapid and convenient method is described for resolving the polypeptide composition of Fraction 1 protein. Using crude leaf extracts of a number of Lycopersicon species, Fraction 1 protein was first separated by polyacrylamide gel electrophoresis and the gel slices containing the protein were isoelectrofocused in the presence of 8 m urea. Isoelectric focusing was also applied directly on subunits in gel slices obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptide composition produced is in agreement with previous determinations obtained by more elaborated techniques.