Thin-layer chromatography and polyacrylamide gel electrophoresis-based assays for sialyltransferases using tetramethylrhodamine-labeled acceptors

Two novel assay systems for the determination of sialyltransferase activity using a tetramethylrhodamine-labeled disaccharide Galbeta1-4GlcNAc (2) as the acceptor are described. The TMR-labeled disaccharide 2 was synthesized by directly coupling Galbeta1-4GlcNAc-O-(CH(2))(6)NH(2) (1) with 5-tetramethylrhodamine N-hydroxysuccinimide ester. The K(m) value for compound 2 obtained with alpha-2,6-sialyltransferase from rat liver (EC 2.4.99.1) was 160 +/- 20 microM. After incubation of compound 2 with sialyltransferase the product and the unreacted acceptor substrate were separated either by thin-layer chromatography (TLC) on C-18 silica gel plates or by polyacrylamide gel electrophoresis (PAGE). The density of the spots on the TLC plates and the fluorescence of the bands on the gel were quantified. The assay conditions were optimized using crude bovine colostrum extract and also alpha-2, 6-sialyltransferase from rat liver. The detection limits for the TLC and PAGE assays were 1 and 0.4 microU of the rat liver enzyme, respectively. Either assay allows the parallel investigation of several samples at a time and is useful for the testing of fractions during enzyme purification.     

Adaptation of a sliding microtome for sectioning of material embedded in epoxy resins]

The employment of a sliding microtome of sectioning plastic embedded material with glass knives is described. Using a new knife holder and a modificated device for fixing plastic blocks succeeded in cutting sections 1--10 micron thick of relatively large pieces of tissue.     

Use of DNA ladders for reproducible protein fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for quantitative proteomics

In proteomics, one-dimensional (1D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is widely used for protein fractionation prior to mass spectrometric analysis to enhance the dynamic range of analysis and to improve the identification of low-abundance proteins. Such protein prefractionation works well for quantitation strategies if the proteins are labeled prior to separation. However, because of the poor reproducibility of cutting gel slices, especially when small amounts of samples are analyzed, its application in label-free and peptide-labeling quantitative proteomics methods has been greatly limited. To overcome this limitation, we developed a new strategy in which a DNA ladder is mixed with the protein sample before PAGE separation. After PAGE separation, the DNA ladder is stained to allow for easy, precise, and reproducible gel cutting. To this end, a novel visible DNA-staining method was developed. This staining method is fast, sensitive, and compatible with mass spectrometry. To evaluate the reproducibility of DNA-ladder-assisted gel cutting for quantitative protein fractionation, we used stable isotope labeling with amino acids in cell culture (SILAC). Our results show that the quantitative error associated with fractionation can be minimized using the DNA-assisted fractionation and multiple replicates of gel cutting. In conclusion, 1D PAGE fractionation in combination with DNA ladders can be used for label-free comparative proteomics without compromising quantitation.     

Antioxidant effects of carnitine supplementation on 14-3-3 protein isoforms in the aged rat hippocampus detected using fully automated two-dimensional chip gel electrophoresis

Abstract

We here described the antioxidant effects of carnitine supplementation on 14-3-3 protein isoforms in the aged rat hippocampus detected using the fully automated two-dimensional chip gel electrophoresis system (Auto2D). This system was easy and convenient to use, and the resolution obtained was more sensitive and higher than that of conventional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). We separated and identified five isoforms of the 14-3-3 protein (beta/alpha, gamma, epsilon, zeta/delta, and eta) using the Auto2D system. We then examined the antioxidant effects of carnitine supplementation on the protein profiles of the cytosolic fraction in the aged rat hippocampus, demonstrating that carnitine supplementation suppressed the oxidation of methionine residues in these isoforms. Since methionine residues are easily oxidized to methionine sulfoxide, the convenient and high-resolution 2-D PAGE system can be available to analyze methionine oxidation avoiding artifactual oxidation. We showed here that the Auto2D system was a very useful tool for studying antioxidant effects through proteomic analysis of protein oxidation.     

A new isoelectric focusing gel for two-dimensional electrophoresis constructed in microporous hollow fiber membranes

We describe the preparation of IEF tube gels inside a nonwetting microporous plastic tubing. The gel in the tube need not be extruded after the first dimension separation. Instead, the porous structure of the tubes is made wettable, and the proteins are electrophoresed "through-the-wall" into the second dimension PAGE gel. Commercial ampholytes and reagents are suitable for the procedure. A useful p/ range of 4.5-9.5 can be obtained when p/ 3-10 ampholyte mixtures are used. Because of the high surface area of the porous material, precautions must be exercised to reduce oxygen inhibition during polymerization and dehydration of the gel during storage and use. A sheath device is described that satisfies these requirements. The plastic tubes can be disposed of by incineration and pose no biohazard.     

江西弯螺属一新种记述(肺螺亚纲,柄眼目,扭轴蜗牛科)

在整理江西地区陆生贝类标本时,经比对鉴定发现1新种,龙潭弯螺Sinoennea longtanensis sp.nov.,隶属肺螺亚纲、柄眼目、扭轴蜗牛科、弯螺属。对新种形态特征、栖息环境作了记述,并与其近似种进行讨论。     

Efficient Detection, Quantification and Enrichment of Subtle Allelic Alterations

Gene targeting (GT) can introduce subtle alterations into a particular locus and represents a powerful tool for genome editing. Engineered zinc finger nucleases (ZFNs) are effective for generating minor allelic alterations. Efficient detection of such minor alterations remains one of the challenges in ZFN-mediated GT experiments. Here, we report the establishment of procedures allowing for efficient detection, quantification and enrichment of such subtle alterations. In a biallelic model, polyacrylamide gel electrophoresis (PAGE) is capable of detecting rare allelic variations in the form of DNA heteroduplexes at a high efficiency of ∼0.4% compared with ∼6.3% by the traditional T7 endonuclease I-digestion and agarose gel electrophoresis. In a multiple allelic model, PAGE could discriminate different alleles bearing addition or deletion of 1–18 bp as distinct bands that were easily quantifiable by densitometry. Furthermore, PAGE enables enrichment for rare alleles. We show for the first time that direct endogenous GT is possible in medaka by ZFN RNA injection, whereas PAGE allows for detection and cloning of ZFN-targeted alleles in adults arising from ZFN-injected medaka embryos. Therefore, PAGE is effective for detection, quantification and enrichment of multiple fine allelic differences and thus offers a versatile tool for screening targeted subtle gene alterations.     

Partial Purification and Characterization of Messenger RNA Coding 14-3-2 Protein from Rat Brain

14-3-2 Protein (neuron-specific enolase) is a neuron-specific protein. Using a reticulocyte lysate cell-free system for translation of 14-3-2 protein mRNA, we have partially purified this mRNA by several procedures, including formamide sucrose density centrifugation, formamide polyacrylamide gel electrophoresis (PAGE) and polyuridylic acid (poly(U))-Sepharose affinity chromatography. Using mRNA obtained by these procedures, we could increase the translation ratio of 14-3-2 protein synthesized/total soluble protein synthesized to 7.31%. The overall purification was 37.8-fold. The size of 14-3-2 protein mRNA appears to be about 19-20S, because translation activity of mRNA obtained by sucrose density gradient centrifugation or formamide PAGE was the most active in this RNA size.     

软枝青冈,福建壳斗科一新种

报道了分布于我国福建西部的壳斗科（Fagaceae）青冈属（Cyclobalanopsis Oerst.）一新种——软枝青冈（Cyclobalanopsis reclinatocaulis M.M.Lin）。该新种与青冈（Cyclobalanopsis glauca（Thunb.）Oerst.）相似，但冬芽淡红褐色；叶薄革质，椭圆形至卵状椭圆形，边缘常具浅锯齿，叶背干后常呈暗灰色；壳斗环带8~10环；小坚果常为倒卵球形或倒卵状椭球形，先端略凹陷，密被短柔毛，柱座底宽3~4 mm，有7~8圆环而与后者相区别。     

TARGET DISCRIMINATION BY AN ECHOLOCATING FINLESS PORPOISE, NEOPHOCAENA PHOCAENOIDES

The ability of a finless porpoise (Neophocaena phocaenoides) to discriminate the material and size of a target by echolocation was investigated. The porpoise was required to choose a standard target of a 15-mm diameter solid steel cylinder from two stimuli, a standard and a comparison target, presented simultaneously. The porpoise could distinguish a standard cylinder from acrylic resin and brass targets, but had difficulties distinguishing it from an aluminum cylinder. In size discrimination, the porpoise could distinguish the standard from 12-, 18-, and 20-mm diameter cylinders, but had difficulties distinguishing it from a 14-mm diameter cylinder. Echo measurements suggest that the porpoise is able to detect: (1) time difference between two echo highlights to within approximately 1 μS, (2) frequency shifts of approximately 7 kHz in a broadband echo having a peak frequency of nearly 140 kHz, (3) time-separation pitch of approximately 30 kHz, and (4) target strength differences of approximately 1 dB.     

Micropuncture pressure in small arteries or veins of perfused rabbit lungs

Until now, direct micropuncture measurements of vascular pressure in lung have been limited to small vessels less than 100 microns on the pleural surface. On the other hand, direct pressure measurements using small catheters (less than 1-mm OD) in pulmonary vessels have been limited to those greater than 1.2 mm. We measured pressure in intermediate-sized microvessels (300-700 microns) using the micropuncture method in isolated perfused rabbit lungs. These microvessels are located 2 or 3 mm beneath the pleura. We exposed them by microsurgery and punctured the relatively thick-walled vessels with specially configured micropipettes. We exposed one pulmonary microvessel in each rabbit lung by microsurgery on the left middle lobe. In 15 rabbit lungs we measured pressure in a total of six small arteries (275- to 470-microns diam) and nine small veins (300- to 700-microns diam) under high zone 3 conditions, near the zone 2/3 boundary. We found approximately 35% of the total pulmonary vascular pressure drop in arteries greater than 275-microns diam and 7% in veins greater than 300-microns diam. In veins greater than 500-microns diam, there was no measurable pressure drop. After the measurements, we froze the lung and confirmed that there was no detectable interstitial or alveolar edema in the cross sections of the punctured site. Our data are compatible with those of other investigators who have used isolated perfused rabbit lungs under similar experimental conditions.     

Preparative isoelectric focusing in ampholine electrofocusing columns versus immobiline polyacrylamide gel for the purification of biologically active leukoregulin

Preparative vertical and rotating horizontal (Rotofor) ampholine column and immobiline flat bed polyacrylamide gel isoelectric focusing were evaluated for the isolation of the biologically active acidic form of leukoregulin, a 50,000-Da glycoprotein lymphokine with tumor growth inhibitory activity. Leukoregulin secreted by normal human lymphocytes was concentrated by 10,000 nominal molecular weight size exclusion ultrafiltration and by DEAE anion exchange chromatography using step elution with 0.02 M Tris-HCl: 0.1 M NaCl, pH 7.4. Preparative isoelectric focusing was carried out in a 110-ml vertical column containing 1% ampholines in a pH 4-6 gradient at 15 W constant power for 16-18 h, in a Rotofor 55-ml horizontal column containing 2% ampholines in a pH 4-6 gradient at 12 W constant power for 4-6 h, or in an immobiline pH 4.5-6.5 gradient within a 5% polyacrylamide 120 X 110 X 5-mm flat bed gel at 3 W constant power for 16-18 h. Recovery of biologically active leukoregulin from the vertical and horizontal ampholine columns was similar. The pH 4.9-5.2 fractions from the Rotofor ampholine column contained 4-7% and the fractions from the immobiline gel contained 4% of the leukoregulin activity applied to the electrofocusing column or gel, respectively. Analytical immobiline isoelectric focusing of the leukoregulin in the pH 4.9-5.2 fractions from the Rotofor column demonstrated that a single silver staining band with a pI of 5.1 can be obtained by this rapid method of preparative isoelectric focusing.     

Preparing Single or Serial Sections of Calcified Bones and Teeth

An apparatus for cutting single or serial sections of calcified bone and teeth consists of a motor-driven shaft on which is mounted one saw (for single section cutting) or a gang (for serial sectioning at one cutting operation). The plastic-embedded specimen is attached to a cylindrical plastic holder which is in turn mounted on the machine and fed into the saw. Prior to cutting the specimen may be oriented in two planes, as well as rotated, with respect to the cutting edge. Single or serial sections made by means of repeated cuts with a single saw, may be 0.3 mm or more thick as determined by the setting of a micrometer screw. For serially sectioning a tooth or bone specimen at one cutting operation, the thickness of the separators between adjacent saws (0.5 mm or more) determines the section thickness. After sectioning, specimens may be ground and polished, with or without reimbedding in fresh plastic.     

Morphology and Ultrastructure of the Intestine in a Plant-Parasitic Nematode,Tylenchorhynchus dubius

An unusual feature of the intestine in Tylenchorhynchus dubius is the presence, within the intestinal cytoplasm, of an extensive system of fibrillar bundles consisting of thin (14 nm diam) filaments and thick (70-90 nm diam), rod-like elements arranged in closely packed arrays. The larger of the fibrillar bundles, for which the term "intestinal fasciculi" is proposed, are evident in whole mounts and apparently correspond to the lateral or sinuous canals described in some other tylenchids. The nature and function of fasciculi are not known, but some possibilities are considered. Fasciculi were found in at least seven other species of Tylenchorhynchus. The intestinal cytoplasm also contains the usual sub cellular organelles and large amounts of reserve materials in the form of particulate glycogen and three types of globules. The surface of the cells bordering the lumen is elaborated into numerous microvilli which have central filaments and often bear regular external projections. Although terminal bars delimit the apical margins between cells, the frequent lack of complete lateral boundaries and extensive length of the fasciculi indicate that the intestinal epithelium is a multinucleate mosaic or syncytium.     

Improved Methods for Molding, Facing and Sectioning Glycol Methacrylate Embedded Tissue Blocks

Improvements in glycol methacrylate embedding, block facing, trimming, and sectioning are described. The improvements are derived from a novel molding system, a multipurpose instrument for rapid block facing, trimming and examination, and a device for removing unwanted sections from the microtome knife while sectioning is in progress. Together, these methods facilitate specimen preparation and result in a significant reduction of the time required to prepare high resolution, very thin sections for light microscopy.     

Identification of coated vesicles in Saccharomyces cerevisiae

Clathrin-coated vesicles were found in yeast, Saccharomyces cerevisiae, and enriched from spheroplasts by a rapid procedure utilizing gel filtration on Sephacryl S-1000. The coated vesicles (62-nm diam) were visualized by negative stain electron microscopy and clathrin triskelions were observed by rotary shadowing. The contour length of a triskelion leg was 490 nm. Coated vesicle fractions contain a prominent band with molecular weight of approximately 185,000 when analyzed by SDS PAGE. The presence of coated vesicles in yeast cells suggests that this organism will be useful for studying the function of clathrin-coated vesicles.     

Candida utilis NAD+ kinase: purification, properties and affinity gel studies

1. NAD+ kinase (ATP:NAD+ 2' phosphotransferase, EC 2.7.1.23) has been purified to apparent enzymic homogeneity on Blue Sepharose CL-6B. 2. The molecular weight of the active species is about 260,000 as determined by PAGE and gel chromatography. Protein staining (PAGE) revealed minor bands with molecular weight values of 40,000, 140,000 and 550,000. Subunit studies (SDS-PAGE) gave evidence of a single band of molecular weight approximately 32,000. 3. On the basis of the release patterns of this enzyme from several affinity gels, an elution diagram is proposed as a device to assess the contribution of any of the several displacing agents that can be used to manipulate the desorption of a (enzyme) ligate from an immobilized ligand.     

Identification of proteins from two-dimensional polyacrylamide gels using a novel acid-labile surfactant

Protein identification by peptide mass mapping usually involves digestion of gel-separated proteins with trypsin, followed by mass measurement of the resulting peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Positive identification requires measurement of enough peptide masses to obtain a definitive match with sequence information recorded in protein or DNA sequence databases. However, competitive binding and ionization of residual surfactant introduced during polyacrylamide gel electrophoresis (PAGE) can inhibit solid-phase extraction and MS analysis of tryptic peptides. We have evaluated a novel, acid-labile surfactant (ALS) as an alternative to sodium dodecylsulfate (SDS) for two-dimensional (2-D) PAGE separation and MALDI-MS mapping of proteins. ALS was substituted for SDS at the same concentration in buffers and gels used for 2-D PAGE. Manual and automated procedures for spot cutting and in-gel digestion were used to process Coomassie stained proteins for MS analysis. Results indicate that substituting ALS for SDS during PAGE can significantly increase the number of peptides detected by MALDI-MS, especially for proteins of relatively low abundance. This effect is attributed to decomposition of ALS under acidic conditions during gel staining, destaining, peptide extraction and MS sample preparation. Automated excision and digestion procedures reduce contamination by keratin and other impurities, further enhancing MS identification of gel separated proteins.