Zone Precipitation Chromatography: Its Use in the Isolation of Different Collagen Types

Zone Precipitation Chromatography is useful tech-nique for the initial isolation of the different colla-gen types in their native configuration. Small quan-tities of collagen mixtures can be rapidly separated into different collagen types with relatively high degree of purity, based upon stained protein patterns on sodium dodecyl sulfate polyacrylamide gel electro-phoresis (SDS-PAGE) slab gels. Tn the commonly used bulk salt preparative method for isolating the different collagens, 50 mg of starting material was needed. Three days were required to complete the procedure. The stained protein patterns on SDS-PAGE slab gels showed about 25% contamination with the bulk purified Type III fraction and 20% contamination with the bulk purified type AB collagen. With Zone Precipitation Chromatography 5 mg of starting material was used and in less than 4 hours the mixture was separated with Types III and AB fractions showing less than 10% contamination from other collagen types. The technique is patterned after the Zone Precinitation method reported by Porath seventeen years ago and utilizes a step-wise sodium chloride gradient to precipitate and redissolve the collagens, eluting from the interbead spaces of a molecular sieve column.     

Can indicator species predict restoration outcomes early in the monitoring process? a case study with peatlands

Success in ecological restoration is rarely assessed rigorously due to insufficient planning for post-restoration monitoring programs, limited funding and, especially, lack of scientifically validated evaluation criteria and protocols. In this article, we propose the use of the Indicator Value Index technique (IndVal), which statistically determines the association of species to one or several particular site types, to obtain indicators of success at the early stages of the recovery process in restoration projects. Peat bogs extracted by the vacuum method, subsequently restored by a moss-transfer technique and regularly monitored for ~10 years were used as a model system to test this approach. We first identified 34 restored sectors of ~10 ha from 4 to 11 years old in twelve eastern-Canadian bogs. These sectors were then classified according to their degree of success in recovering a typical sphagnum moss carpet (restoration goal). Then, we retrospectively reviewed vegetation communities recorded at the third year after restoration to identify indicator species of different categories of restoration success, using the IndVal methodology. By identifying early indicator species, our method provides a tool that guides intervention soon after restoration if a site is not on a desired successional trajectory. Typical bog species, namely the bryophytes S. rubellum and Mylia anomala and the tree Picea mariana, were indicative of successful restoration; while bare peat, lichens and one species of ericaceous shrubs (Empetrum nigrum), which cope better under drier conditions, indicated sites where restoration failed. A surprising finding was that the moss Polytrichum strictum, which is known to facilitate the colonization of sphagnum in disturbed peatlands, is an early indicator of unsuccessful restoration. This finding made us question the nursing role of P. strictum at a cover threshold above ca. 30%, when P. strictum could be outcompeting sphagnum and become dominant. We conclude that the IndVal method is an effective tool to identify early indicators of restoration success when combined with a thoughtful examination of species frequency and cover within each site type.     

Studies on the antioxidant activity and phenolic compounds of enzyme-assisted water extracts from Du-zhong (Eucommia ulmoides Oliv.) leaves

Enzyme-assisted water extracts (EWEDL) and ethanol extracts of Du-zhong leaves (EEDL) were evaluated for their antioxidant activities using the DPPH radical-scavenging assay, Fe²⁺-chelating assay, and inhibition ability of the linoleic acid peroxidation assay. In general, the antioxidant activity of Du-zhong leaf extracts increased with increasing concentration. Based on the two extracting methods with different antioxidative reactions, it was shown that the enzyme-assisted water extracting method was more effective for antioxidant extraction from Du-zhong leaves. By HPLC-MS analysis, the main phenolic compounds (geniposidic acid, epicatechin, and chlorogenic acid) identified in EWEDL and EEDL were similar. EWEDL and EEDL had total phenolic contents of 13.84?±?0.11 and 14.72?±?0.14?mg chlorogenic acid equivalents (CAE) in each gram of extract, respectively. However, there was no positive correlation between total phenolic content and antioxidant activities of EWEDL and EEDL measured by the three different assays.     

News in Brief

Archival formalin-fixed, paraffin-embedded (FFPE) tissue and their associated diagnostic records represent an invaluable source of retrospective proteomic information on diseases for which the clinical outcome and response to treatment are known. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hindered by the adverse effects of formaldehyde fixation and subsequent tissue histology. This review examines recent methodological advances for extracting proteins from FFPE tissue suitable for proteomic analysis. These methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, allow at least a qualitative analysis of the proteome of FFPE archival tissues. The authors also discuss recent advances in the proteomic analysis of FFPE tissue; including liquid-chromatography tandem mass spectrometry, reverse phase protein microarrays and imaging mass spectrometry.     

Continuous countercurrent salting-out extraction of 1,3-propanediol from fermentation broth in a packed column

The possibility of continuous extraction of 1,3-propanediol in a experimental packed column was investigated using a salting-out extraction system of dipotassium phosphate/ethanol. Mass transfer of 1,3-propanediol takes place from the dispersed phase (salt-rich solution) to the continuous phase (ethanol). The influences of flow rate of dispersed phase and size of packing material on partition coefficient and recovery of 1,3-propanediol were investigated and the results were compared with those obtained in spray column and test tube. Furthermore, the influences of various system compositions on hold up of dispersed phase, mass transfer coefficient, and system stability were also studied in the column packed by stainless steel Dixon 3 × 3 mm. It was found that the packed column showed a good extraction efficiency and stability. Besides, 1,3-propanediol recovery of 90.30% was obtained during a 11 h continuous operation when the real fermentation broth was used. At the same time, 94.4% of phosphate could be recovered when 0.2 volume of anhydrous ethanol was added into the raffinate phase at pH 4.0.     

Blood Perfusion in a Murine Fibrosarcoma Tumor Model After Direct Current Electrotherapy a Study with 86Rb Extraction Technique

Electrotherapy with low-level direct current (DC) can induce antitu-mor effects in various tumor models. Applied in combination with certain anticancer drugs, it can significantly increase their effectiveness. It has been suggested that the demonstrated effects of electrotherapy arise from its modification of tumor blood flow. The effect of such treatment on blood perfusion of solid subcutaneous Sa-1 fibrosarcoma tumors in A/J mice was investigated with a ⁸⁶rubidium extraction technique. Following electrotherapy, the relative tissue perfusion of tumors was decreased by more than 50%. Three days after treatment, partial reperfusion of tumors occurred. The dynamics of the perfusion changes induced by electrotherapy are in agreement with tumor growth dynamics following this procedure. The effect of electrotherapy on the blood supply of tumors may be the major mechanism of antitumor action in our model. Electrotherapy could be useful as an adjuvant local procedure to other treatment modalities that require a hypoxic environment for their effectiveness.     

High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker

Sample homogenization is an essential step for genomic DNA extraction, with multiple downstream applications in Molecular Biology. Genotyping hundreds or thousands of samples requires an automation of this homogenization step, and high throughput homogenizer equipment currently costs 7000 euros or more. We present an apparatus for homogenization of individual Drosophila adult flies in 96-well micro-titer dishes, which was built from a small portable paint-shaker (F5 portable paint-shaker, Ushake). Single flies are disrupted in each well that contains extraction buffer and a 4-mm metal ball. Our apparatus can hold up to five 96-well micro-titer plates. Construction of the homogenizer apparatus takes about 3–4 days, and all equipment can be obtained from a home improvement store. The total material cost is approximately 700 euros including the paint-shaker. We tested the performance of our apparatus using the ZR-96 Quick-gDNA? kit (Zymo Research) homogenization buffer and achieved nearly complete tissue homogenization after 15 minutes of shaking. PCR tests did not detect any cross contamination between samples of neighboring wells. We obtained on average 138 ng of genomic DNA per fly, and DNA quality was adequate for standard PCR applications. In principle, our tissue homogenizer can be used for isolation of DNA suitable for library production and high throughput genotyping by Multiplexed Shotgun Genotyping (MSG), as well as RNA isolation from single flies. The sample adapter can also hold and shake other items, such as centrifuge tubes (15–50 mL) or small bottles.     

Fabrication,Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we present the fabrication protocol for a multilayered SAW acoustic counterflow device. The device is fabricated starting from a lithium niobate (LN) substrate onto which two interdigital transducers (IDTs) and appropriate markers are patterned. A polydimethylsiloxane (PDMS) channel cast on an SU8 master mold is finally bonded on the patterned substrate. Following the fabrication procedure, we show the techniques that allow the characterization and operation of the acoustic counterflow device in order to pump fluids through the PDMS channel grid. We finally present the procedure to visualize liquid flow in the channels. The protocol is used to show on-chip fluid pumping under different flow regimes such as laminar flow and more complicated dynamics characterized by vortices and particle accumulation domains.     

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca²⁺-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP₃ and Ca²⁺ that initiate the propagation of the Ca²⁺-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca²⁺-wave propagation are provided by gap junction channels through the direct transfer of IP₃ and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca²⁺-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca²⁺-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca²⁺-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.