The Preparation of Silica Gel Thin Layers with a New Apparatus

An apparatus for the preparation of uniform thin layer chromatography gel layers is described. The apparatus employs rubber cushion runners to compensate for differences in plate thickness and a gel applicator which functions independently of plate edge variability. Consistently uniform layers are prepared by careful establishment of the plate-to-applicator distance. Silica gel layers averaged 87% of the applied thickness with a variability of ±2% within a single run and ±8% between independent runs.     

Parallel plate equipment for preparation of uniform gel sheets

A method for the preparation of uniform gel-disks for enzyme and cell immobilisation, as well as for characterisation of gel mechanical stability, is described. The apparatus comprises a stainless steel base unit and glass parallel plates, designed to permit easy and fast production of multiple homogeneous gel sheets of variable thickness.     

Improvements of DNA sequencing gels

We describe three simple modifications of DNA sequencing gels which all result in improved oligonucleotide resolution as visualized by autoradiography. First, it was possible to reduce the thickness of the gel to 0.2 mm by using new gel molding techniques. Second, the gel could be dried without any distortions of its dimensions by prior binding of the gel to the surface of the glass plate. Third, a uniform high temperature was obtained in all parts of the gel during electrophoresis by replacing one of the glass plates with an inexpensive thermostating plate with circulating water. The use of this heating plate resulted in a straight band pattern all over the gel and also in the resolution of such bands which were not resolved in other electrophoresis systems.     

Opening angle and residual strain in a three-layered model of pig oesophagus

Studies of various biological tissues have shown that residual strains are important for tissue function. Since a force balance exists in whole wall thickness specimens cut radially, it is evident that layer separation is an important procedure in the understanding of the meaning of residual stresses and strains. The present study investigated the zero-stress state and residual strain distribution in a three-layer model of the pig oesophagus. The middle part of the oesophagus was obtained from six slaughterhouse pigs. Four 3-mm-wide rings were serially cut from each oesophagus. Two of them were used for separating the wall into mucosa-submucosa, inner and outer muscle layers. The remaining two rings were kept as intact rings. The inner and outer circumferences and wall thickness of different layers in intact and separated rings were measured from the digital images in the no-load state and zero-stress state. The opening angle was measured and the residual strain at the inner and outer surface of different layers and the intact wall were computed. Compared with intact sectors (62.8+/-9.8 degrees ), the opening angles were smaller in the inner muscle sectors (37.2+/-11.4 degrees , P<0.01), whereas the opening angles of mucosa-submucosa (63.9+/-6.8 degrees ) and outer muscle sectors (63.9+/-6.8 degrees ) did not differ (P>0.1). Referenced to the zero-stress state of the intact sectors, the inner and outer residual strains of the intact rings was -0.128+/-0.043 and outer residual strain was 0.308+/-0.032. Referenced to the "true" zero-stress state of separated three-layered sectors, the inner residual strain of intact rings were -0.223+/-0.021 (P<0.01) and 0.071+/-0.022 (P<0.01). Referenced to the "true" zero-stress state, the residual strain distribution of different layers in intact rings was shown that the inner surface residual strain was negative at mucosa-submucosa and inner muscle layers and was positive at outer muscle layer, whereas the outer surface residual strain was negative at the mucosa-submucosa layer and positive at the inner and outer muscle layers. For the separated different layered rings, the inner residual strain was negative and outer residual strain was positive; however, the absolute values did not differ (P>0.1). In conclusion, it is possible to microsurgically separate the oesophagus into three layers, i.e., mucosa-submucosa, inner muscle and outer muscle layers, the residual strain differ between the layers, and the residual strain distribution was more uniform after the layers were separated.     

An automated apparatus for the real-time monitoring of bioluminescence in plants

We developed an automated, high-throughput, bioluminescence-monitoring apparatus that can monitor 1920 individual plant seedlings under uniform light conditions. The apparatus is composed of five units: (i) a plate platform that can hold 20 96-well microplates under uniform light conditions, (ii) a scintillation counter, (iii) a robot that conveys plates between the plate platform and a scintillation counter, (iv) a sequence controller, and (v) an external computer that collects and analyzes bioluminescence data automatically. The apparatus gave reproducible and reliable results for both bioluminescence photon counts and period length of bioluminescence rhythms; neither was affected by the well position in a plate or the plate position on the platform. The apparatus is a powerful tool for both large-scale detailed analysis of gene expression and large-scale screening of mutants.     

Ultrasonic methodology coupled to ATP bioluminescence for the non-invasive detection of fouling in food processing equipment--validation and application to a dairy factory

The use of an ultrasonic apparatus (40 kHz) for the non-destructive, rapid and reproducible removal of biofilm from standard materials (stainless steel and polypropylene) in a dairy factory was investigated. The application of ultrasound with the tested conditions (10 s and 40 kHz) was found not to be detrimental for standard ATP (concentration ranging between 5 x 10(-9) and 10(-5) mol 1(-1)) and for prokaryotic cells, including both rods and coccoid-shaped bacteria (Escherichia coli and Staphylococcus aureus). It allowed the use of the ATP bioluminescence measurement for quantifying the biofilm removal. The repeatability of industrial milk removal was determined on fouled stainless steel and polypropylene sheets. The variability of the results with the sonication method was constant, +/-24% (coefficient of variation) for both surfaces, and was variable with the swabbing method, +/-42% for the stainless steel sheet and +/-74% for the polypropylene sheet. The ultrasonic apparatus removed twice the amount of industrial milk biofilm compared with the swabbing method in the case of the polypropylene sheets. The apparatus was used to validate the industrial cleaning protocols of a milk factory.     

Rapid electrotransfer of proteins from polyacrylamide gel to nitrocellulose membrane using surface-conductive glass as anode

A new type of inexpensive horizontal apparatus for the electrophoretic transfer of proteins from a gel to an immobilization membrane has been developed. In this system, gel and membrane were directly pressed between two flat electrodes. A surface-conductive glass was used as anode and a stainless-steel plate as cathode. Proteins could be transferred from polyacrylamide gel to nitrocellulose sheet, with a yield of at least 90% in 60-90 min, without overheating, using a voltage gradient of 30-40 V/cm. Moderate volumes of separate anodic (with 20% methanol) and cathodic (with 0.1% sodium dodecyl sulfate) buffers were suited for optimal transfer of proteins with relative molecular mass (Mr) in the 14,000-94,000 range.     

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.     

A simplified procedure for preparation of undecalcified human bone sections

A new type of apparatus for sectioning samples of hard, undecalcified bone is described. Slices of fresh or archeological human bone 4-5 mm thick are dehydrated and then embedded in epoxy resin. The apparatus used to prepare sections from the resulting blocks consists of a low-speed rim-type diamond cut-off wheel and a slowly advancing table carrying the specimen held in a rotating mount. Sections may be cut at a thickness of 80 micron +/- 1%. After cleaning in an ultrasonic bath, these can be mounted on slides for quantitative microscopic examination with transmitted light. Grinding and polishing are not necessary. The results obtained are illustrated.     

Separation of biomacromolecules by electrofiltration through gel layers

A straightforward method for concomitant separation and isolation of biomacromolecules from a mixture in solution was developed. Three gel layers that comprise a middle separation layer of 10% polyacrylamide gel were constructed. This gel system was formed in an electroconcentration apparatus above a collection chamber surrounded at the bottom by a dialysis membrane. The mixture is applied over the gel layers where biomacromolecules are caused to migrate by electrophoresis through the gel system, where they are separated into discrete bands and electroeluted into the collection chamber without dismantling the apparatus. The isolated biomacromolecules are removed from the chamber in a highly pure and concentrated form ready for further investigations. Cooling can be applied throughout the whole process, and the setup and conditions of run can be modified according to the characteristics of the biomacromolecules to be purified. The components of a mixture containing the glycoprotein ovalbumin and bovine serum albumin monomer, dimer, and tetramer were successfully isolated as concentrated and highly pure fractions with good recoveries ranging from 70 to 89%. Other proteins were successfully isolated under denaturing conditions in the presence of sodium dodecyl sulfate (SDS) or 6 M urea.     

Determination of layer-specific mechanical properties of human coronary arteries with nonatherosclerotic intimal thickening and related constitutive modeling

At autopsy, 13 nonstenotic human left anterior descending coronary arteries [71.5 +/- 7.3 (mean +/- SD) yr old] were harvested, and related anamnesis was documented. Preconditioned prepared strips (n = 78) of segments from the midregion of the left anterior descending coronary artery from the individual layers in axial and circumferential directions were subjected to cyclic quasi-static uniaxial tension tests, and ultimate tensile stresses and stretches were documented. The ratio of outer diameter to total wall thickness was 0.189 +/- 0.014; ratios of adventitia, media, and intima thickness to total wall thickness were 0.4 +/- 0.03, 0.36 +/- 0.03, and 0.27 +/- 0.02, respectively; axial in situ stretch of 1.044 +/- 0.06 decreased with age. Stress-stretch responses for the individual tissues showed pronounced mechanical heterogeneity. The intima is the stiffest layer over the whole deformation domain, whereas the media in the longitudinal direction is the softest. All specimens exhibited small hysteresis and anisotropic and strong nonlinear behavior in both loading directions. The media and intima showed similar ultimate tensile stresses, which are on average three times smaller than ultimate tensile stresses in the adventitia (1,430 +/- 604 kPa circumferential and 1,300 +/- 692 kPa longitudinal). The ultimate tensile stretches are similar for all tissue layers. A recently proposed constitutive model was extended and used to represent the deformation behavior for each tissue type over the entire loading range. The study showed the need to model nonstenotic human coronary arteries with nonatherosclerotic intimal thickening as a composite structure composed of three solid mechanically relevant layers with different mechanical properties. The intima showed significant thickness, load-bearing capacity, and mechanical strength compared with the media and adventitia.     

Standard specimens for stain calibration and their application to the Papanicolaou stain

Standardized specimens composed of extracts of biologic objects (nucleoprotamine and bovine liver) were developed as tools for the quantitative evaluation of stain performance on biologic substrates. The specimens are mixtures of proteins and nucleic acids and thus mimic the staining characteristics of cytologic smears. The concentration of each mixture and the specimen thickness can be precisely controlled, ensuring the production of a large number of samples with a nearly identical capability for dye binding. The transmitted light spectra of the standardized specimens varied depending on the extract and the preparation conditions. Spectra similar to those reported from the nuclei and cytoplasm of cell types in Papanicolaou-stained cervicovaginal smears were observed. Light transmission was uniform to +/- 5% across each specimen and from specimen to specimen. The specimen thickness was uniform within +/- 2%. Studies with these standardized samples could reveal the much-needed correlations between the chemical and optical characteristics of dyes and dye solutions and the performance of the dyes on biologic substrates.     

Electronic speckle pattern interferometry: a tool for determining diffusion and partition coefficients for proteins in gels

The aim of this study was to demonstrate electronic speckle pattern interferometry (ESPI) as a powerful tool in determining diffusion coefficients and partition coefficients for proteins in gels. ESPI employs a CCD camera instead of a holographic plate as in conventional holographic interferometry. This gives the advantage of being able to choose the reference state freely. If a hologram at the reference state is taken and compared to a hologram during the diffusion process, an interferometric picture can be generated that describes the refraction index gradients and thus the concentration gradients in the gel as well as in the liquid. MATLAB is then used to fit Fick's law to the experimental data to obtain the diffusion coefficients in gel and liquid. The partition coefficient is obtained from the same experiment from the flux condition at the interface between gel and liquid. This makes the comparison between the different diffusants more reliable than when the measurements are performed in separate experiments. The diffusion and partitioning coefficients of lysozyme, BSA, and IgG in 4% agarose gel at pH 5.6 and in 0.1 M NaCl have been determined. In the gel the diffusion coefficients were 11.2 +/- 1.6, 4.8 +/- 0.6, and 3.0 +/- 0.3 m(2)/s for lysozyme, BSA, and IgG, respectively. The partition coefficients were determined to be 0.65 +/- 0.04, 0.44 +/- 0.06, and 0.51 +/- 0.04 for lysozyme, BSA, and IgG, respectively. The current study shows that ESPI is easy to use and gives diffusion coefficients and partition coefficients for proteins with sufficient accuracy from the same experiment.     

A simple method for the release of asparagine-linked oligosaccharides from a glycoprotein purified by SDS-polyacrylamide gel electrophoresis.

A simple method for the release of oligosaccharides from glycoproteins separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) has been developed. Asialo-alpha 1-acid glycoprotein, which was tritiated at the nonreducing terminal D-galactopyranosyl residue by reduction with sodium borotritide after incubation with D-galactose oxidase, was used as a model compound. After electrophoretic separation of the glycoprotein, oligosaccharides were released by the use of a gas-phase hydrazinolysis apparatus. In the first method, the gel was stained with Coomassie Blue and the glycoprotein together with the gel was directly subjected to gas-phase hydrazinolysis after removal of water in a P2O5 desiccator. The recovery of released oligosaccharides was 25.9 +/- 2.4%, based on the amount of the glycoprotein loaded on the gel within the range of 3.5-28.5 micrograms. In the second method, the glycoprotein was electroblotted onto an Immobilon transfer membrane and was visualized by staining with Coomassie Blue. A small piece of the membrane with the corresponding band was cut out, dried in a desiccator and subjected to gas-phase hydrazinolysis. In this case, the recovery of released oligosaccharides was 15.2 +/- 1.0%. These procedures, particularly the first one, should be widely applicable for the isolation of oligosaccharides from glycoproteins separated by SDS-PAGE.     

An apparatus for thin layer vertical polyacrylamide gel electrophoresis with discontinuous buffer and gel system

A vertical polyacrylamide gel slab electrophoresis apparatus with a discontinuous gel and buffer system and a running gel of 1 mm in thickness was devised. Using this apparatus, which employs stacking and sieving effects, sharp bands comparable to those of disc electrophoresis were obtained.Furthermore, a new application using detection with ultraviolet method was introduced for isozyme study.     

Improved coating and fixation methods for scanning electron microscope autoradiography

A simple apparatus for emulsion coating is described. The apparatus is inexpensive and easily assembled in a standard glass shop. Emulsion coating for scanning electron microscope autoradiography with this apparatus consistently yields uniform layers. When used in conjunction with newly described fixation methods, this new approach produces reliable autoradiographs of undamaged specimens.     

Persistent alterations in heart rate variability, baroreflex sensitivity, and anxiety-like behaviors during development of heart failure in the rat

Depressed heart rate variability and mood are associated with increased mortality in patients with congestive heart failure (CHF). Here autonomic indexes were assessed 3 and 7 wk after left coronary artery ligation in telemetered rats, after which anxiety-like behaviors were assessed in an elevated plus maze. Low frequency (LF) and high frequency (HF) heart rate variability were reduced in CHF rats 3 wk after infarction (LF, 1.60 +/- 0.52 vs. 6.97 +/- 0.79 ms(2); and HF, 1.53 +/- 0.39 vs. 6.20 +/- 1.01 ms(2); P < 0.01). The number of sequences of interbeat intervals that correlated with arterial pressure was decreased in CHF rats at 3 and 7 wk (week 3, 26.60 +/- 10.85 vs. 59.75 +/- 11.4 sequences, P < 0.05; and week 7, 20.80 +/- 8.97 vs. 65.38 +/- 5.89 sequences, P < 0.01). Sequence gain was attenuated in CHF rats by 7 wk (1.34 +/- 0.06 vs. 2.70 +/- 0.29 ms/mmHg, P < 0.01). Coherence between interbeat interval and mean arterial blood pressure variability in the LF domain was reduced in CHF rats at 3 (0.12 +/- 0.03 vs. 0.26 +/- 0.05 k(2), P < 0.05) and 7 (0.16 +/- 0.02 vs. 0.31 +/- 0.05 k(2), P < 0.05) wk. CHF rats invariably entered the open arm of the elevated plus maze first and spent more time in the open arms (36.0 +/- 15% vs. 4.6 +/- 1.9%, P < 0.05). CHF rats also showed a tendency to jump head first off the apparatus, whereas controls did not. Together the data indicate that severe autonomic dysfunction is accompanied by escape-seeking behaviors in rats with verified CHF.     

Large-Plate Method for the Assay of Neomycin in Serum

A large-plate method employing radial diffusion from small paper discs for assaying serum levels of neomycin is described. More than 120 discs placed on a loading plate and loaded with 20 muliters of sample could all be brought into contact with the agar plate at one time. The requirement for elaborate statistical design to compensate for time-dependent bias was thus eliminated. The dose-response curve was linear for a range of at least 0.5 to 2.5 mug/ml. The experimental limits for the actual zone width (distance from the edge of the paper disc to the outer edge of the inhibition zone) for 120 repetitions of the assay of neomycin in one and the same serum, carried out simultaneously on one large plate, were about +/- 6% (95% confidence interval). The 95% confidence interval for the distribution of difference between duplicate zones obtained for the assay of neomycin in 34 different sera, also carried out on one plate, was about +/- 10%. The dose-response lines for a standard and for three unknown sera, when carried out together on one plate, were parallel within the variability (+/- SD one) of the zone width. The large-plate method is considered to be more efficient than the use of the smaller petri dishes. The method is suitable for the assay of penicillin in serum and can most probably be used for the assay of a wide variety of substances for which radial diffusion from paper discs into agar is feasible.     

Analysis of PEG 400 and 4000 in urine for gut permeability assessment using solid phase extraction and gel permeation chromatography with refractometric detection

We developed a treatment of urine samples allowing the analysis of two intestinal permeability markers: polyethylene glycol (PEG) 400 (highly diffusible; basal permeability indicator) and PEG 4000 (poorly diffusible; indicator of an abnormal increase of permeability) by a unique gel permeation chromatography (GPC) with refractometric detection. Urinary PEG were extracted using a mixed-bed resin composed of C2 and C18 layers. Permeability mean values determined in 11 human healthy subjects were 24.20 +/- 9.30% and 0.12 +/- 0.08% for, respectively, PEG 400 and 4000. The percentage of the PEG 4000 permeability value to the one of PEG 400 corresponded to an intestinal permeability index (IPI) of 0.52 +/- 0.35 expressing a low diffusion of this poorly permeability marker.     

Computer analysis of double-labeled two-dimensional electrophoresis gels

A computerized process for the automatic analysis of double-label autoradiography after two-dimensional polyacrylamide gel electrophoresis has been developed. Matching fluorographs and autoradiographs produced from gels containing 3H- and 14C-labeled proteins are digitized by a rotating drum densitometer and analyzed by the Man-computer Interactive Data Analysis System III. This system locates corresponding protein spots in the films with edge-detection algorithms, converts spot density readings to isotopic disintegrations by reference to standard curves, and computes a 3H:14C ratio for each spot in the gels. On the average, calculated ratios are accurate to approximately 9% for test strips of polyacrylamide gel containing uniform mixtures of 3H and 14C. Values obtained for two-dimensional gels containing n protein spots with a known 3H:14C ratio of 8.6 +/- 0.1 are as follows: 8.1 +/- 1.4 (n = 268), 8.8 +/- 2.1 (n = 278), 9.1 +/- 1.7 (n = 245), and 8.8 +/- 2.2 (n = 223). The computer process greatly reduces the time required to precisely compare two complex protein mixtures and has sufficient precision to detect a doubling in the biosynthesis of any individual protein.