Immunogenic complexes of the soluble surface antigens of Sh. sonnei]

The comparative study of the immunogenic properties of Sh. sonnei (phases I and II) soluble surface antigens obtained by the modified method of aqueous-saline extraction and Sh. sonnei (phase I) antigen obtained by Boivin's method was made with the use of the keratoconjunctival test in guinea pigs. The protective activity of a high molecular fraction obtained by the fractionation of phase I soluble surface antigens in Sepharose 4B was studied. Boivin's antigen, when used for immunization in optimum doses, was found to have pronounced protective properties, whereas phase II soluble surface antigens showed no protective activity. A high molecular fraction obtained from phase I soluble surface antigen was found to be the most immunogenic. Protective activity was largely connected with protein antigen. The question whether protein antigen was an independent protective antigen or whether it constituted a part of a complex which determined the protective activity of a high molecular fraction remained unsolved.     

Isolation of pig platelet membranes and granules distribution and validity of marker enzymes

A method for the subcellular fractionation of pig platelet homogenates by sucrose density gradient centrifugation is described. The procedure is simple, highly reproducible and yields two major particulate fractions and a soluble phase. One particulate fraction consists almost entirely of membrane fragments and is relatively free from granule contamination. The other particulate zone contains the platelet granules and mitochondria. The distribution on the gradients of the enzymes lactate dehydrogenase, succinate dehydrogenase, 5′-nucleotidase, leucyl β-naphthylamidase and cholinesterase has been studied and organelle localisation further substantiated by electron microscopy. The degree of solubilisation of certain marker enzymes during homogenisation has been investigated and the parallel release of these enzymes with the soluble phase marker enzyme lactate dehydrogenase, suggests they have a true biphasic location between the soluble and particulate components of the cell. No significant difference was found in the molar ratios of cholesterol to phospholipid in the subcellular fractions but the content of each lipid was twice as high in the membrane fraction as in the granule fraction.     

Characterization of the fast and slow components of axonal transport in retinal ganglion cells

The constituent proteins of the fast (110–150 mm/day) and slow (1.5–2 mm/day) components of axonal transport in the retinal ganglion cells of the rabbit were investigated. The fast and slow components were labelled by intraocular injection of (³H)- and (¹⁴C)-leucine, respectively. Subcellular fractionation of the optic nerve and tract and subsequent gel electrophoresis of the fractions showed that most of the soluble proteins moved with the slow phase of axonal transport, whereas only some of the soluble proteins were transported with the rapid phase. Extraction of the microsomal fraction with triton X-100 resulted in the solubilization of highly labelled proteins belonging to the rapid phase. These proteins showed a relatively low electrophoretic mobility.     

Identification and characterization of the Sulfolobus solfataricus P2 proteome

Via combined separation approaches, a total of 1399 proteins were identified, representing 47% of the Sulfolobus solfataricus P2 theoretical proteome. This includes 1323 proteins from the soluble fraction, 44 from the insoluble fraction and 32 from the extra-cellular or secreted fraction. We used conventional 2-dimensional gel electrophoresis (2-DE) for the soluble fraction, and shotgun proteomics for all three cell fractions (soluble, insoluble, and secreted). Two gel-based fractionation methods were explored for shotgun proteomics, namely: (i) protein separation utilizing 1-dimensional gel electrophoresis (1-DE) followed by peptide fractionation by iso-electric focusing (IEF), and (ii) protein and peptide fractionation both employing IEF. Results indicate that a 1D-IEF fractionation workflow with three replicate mass spectrometric analyses gave the best overall result for soluble protein identification. A greater than 50% increment in protein identification was achieved with three injections using LC-ESI-MS/MS. Protein and peptide fractionation efficiency; together with the filtration criteria are also discussed.     

Components of fast and slow phases of axoplasmic flow

Abstract— Cat ventral roots removed at times from hours to days after injection of [³H]leucine into the seventh lumbar and first sacral spinal cord segments were homogenized and subjected to sub-cellular fractionation. The soluble fraction was further analysed for protein content by column chromatography with Sephadex. The fast phase of axoplasmic flow carries down labelled free leucine, polypeptide, and soluble protein. The slow phase of axoplasmic flow carries down labelled soluble protein. The sub-cellular components were labelled with the small particulate fraction showing a higher specific activity than the other sub-cellular fractions.     

Antioxidant properties ofErigeron annuus extract and its three phenolic constituents

The antioxidant activity of the extract ofErigeron annuus was assessed by means of two differentin vitro tests: bleaching of the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH test) and the scavenging of authentic peroxynitrite in company with peroxynitrite generation from 3-morpholinosydnonimine (SIN-1). In both tests, the 85% aq. MeOH andn-BuOH soluble fractions of the crude extract showed a significant scavenging effect on peroxynitrite and DPPH radical in comparison to L-ascorbic acid. And bioassay-guided fractionation of then-BuOH soluble fraction led to the isolation of three compounds: Apigenin (1), quercetin-3-O-glucoside (2), and caffeic acid (3). The structures of the isolated compounds were elucidated on the basis of their spectroscopic data and their antioxidant activities were measured by determining their capacity to scavenge peroxynitrite and the DPPH radical.     

Protein microarrays using liquid phase fractionation of cell lysates

We describe an approach in which protein microarrays are produced using a two-dimensional (2-D) liquid phase fractionation of cell lysates. The method involves a pI-based fractionation using chromatofocusing in the first dimension followed by nonporous reversed-phase high-performance liquid chromatography (HPLC) of each pI fraction in the second dimension. This allows fractionation of cellular proteins in the liquid phase that could then be arrayed on nitrocellulose slides and used to study humoral response in cancer. Protein microarrays have been used to identify potential serum biomarkers for prostate cancer. It is shown that specific fractions are immunoreactive against prostate cancer serum but not against serum from healthy individuals. These proteins could serve as sero-diagnostic markers for prostate cancer. Importantly, this method allows for use of post-translationally modified proteins as baits for detection of humoral response. Proteins eliciting an immune response are identified using the molecular mass and peptide sequence data obtained using mass spectrometric analysis of the liquid fractions. The fractionation of proteins in the liquid phase make this method amenable to automation.     

Distribution and subcellular localization of acylpeptide hydrolase and acylase I along the hog gastro-intestinal tract

The distribution of acylase I and acylpeptide hydrolase along the hog small intestine was investigated. No significant changes in their respective specific activity was found when the intestine was cut off and divided into eight segments (taken every 200 cm) so as to specifically study the duodenum, jejunum and ileum. Upon performing subcellular fractionation of hog enterocytes, it was observed that acylpeptide hydrolase is a soluble enzyme, while acylase I is essentially a soluble protein accounting for only 5% of the activity associated with the whole membrane fraction. The membrane-bound acylase I was neither solubilized by phosphatidylinositol-specific phospholipase C from Bacillus cereus nor by detergents which are commonly used to solubilize alkaline phosphatase, a glycosylphosphatidylinositol-anchored protein. When a phase separation was carried out in Triton X-114, all the anchored-membrane proteins of the intestinal membranes were located in the detergent-rich phase, while acylase I was present in the detergent-poor phase. Finally, the immunolabeling of intestinal cells with specific antibodies definitively established the cytoplasmic localization of acylase I. Acylpeptide hydrolase and acylase I therefore both are located in the enterocyte cytoplasm.     

Crystallin distribution patterns in concentric layers from toad eye lenses

Protein distribution patterns across eye lenses from the Asiatic toad Bufo gargarizans were investigated and individual crystallin classes characterised. Special fractionation that follows the growth mode of the lens was used to yield nine fractions corresponding to layers laid down at different chronological (developmental) stages. Proportions of soluble and insoluble crystallins within each fraction were measured by Bradford assay. Water‐soluble proteins in all fractions were separated by size‐exclusion HPLC and constituents of each class further characterised by electrophoresis, RP‐HPLC and MS analysis. In outer lens layers, α‐crystallin is the most abundant soluble protein but is not found in soluble proteins in the lens centre. Water‐soluble β‐crystallins also decrease from their highest level in the outer lens to negligible mounts in the central lens. The proportion of soluble γ‐crystallin increases significantly towards the lens centre where this is the only soluble protein present. Insoluble protein levels increase significantly towards the lens centre. In B. gargarizans lenses, as with other anurans, the predominant water‐soluble protein class is γ‐crystallin. No taxon‐specific crystallins were found. The relationship between the protein distribution patterns and the functional properties of the lens this species is discussed.     

STUDIES ON ISOLATED CELL COMPONENTS : XVI. The Distribution of Acid Phenyl Phosphatase Activities in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge has been used to separate the major components of rat liver brei (soluble phase, ribosomes, microsomes, mitochondria, membranous fragments, and nuclei) during one centrifugation, by using a 1200 ml sucrose gradient varying linearly with radius from 17 to 55 per cent (w/w) with a "cushion" of 66 per cent sucrose at the rotor edge at speeds up to 30,000 RPM. Liver brei was found to contain a family of phosphatases (phenol disodium phosphate substrate, sodium malonate buffers and Turgitol NPX, a non-ionic detergent). Activity maxima at pH 4.1 and 5.9 were observed in untreated brei prepared in 0.25 M sucrose. The addition of the non-ionic detergent Turgitol NPX selectively caused the release of considerable additional activity between these optima. The activity measured at pH 4.1 was primarily associated with the cytoplasmic granules, while the activities at pH 4.8, 5.4 and 5.9 were found in both soluble phase and particulate-mitochondria and membranous fractions. The activities present beyond the region of the gradient occupied by the soluble phase (sample layer) were all bound to particles sedimentable at 105,536 g (average) in the preparative ultracentrifuge. The data suggest that the different activities are not similarly distributed between soluble phase and particulate fractions. When the data are expressed in terms of specific activity, the area in the gradient between the microsomes and mitochondria now appears richest in all the acid phenyl phosphatase activities measured, while the soluble phase and larger particulate fractions appear relatively poor in activity. This part of the gradient is occupied by small, dense granules which may be the so called lysosomes. Pretreatment of the brei with Turgitol NPX prior to fractionation in the zonal ultracentrifuge resulted in the solubilization of acid phenyl phosphatase activities (almost all the activity was in the sample zone of the gradient) and the non-specific destruction of the formed elements of the brei. Essentially all of the activities present in the original brei measured under these conditions were recovered after zonal ultracentrifuge fractionations.     

Pattern of labeling of rabbit cerebral cortex soluble proteins 24 hours after a single electroconvulsive shock

The labeling of proteins in the particulate subcellular fractions and in the cell sap was studied in the cerebral cortex of rabbits 24 hours after a single electroconvulsive shock (ECS). To this end the animals were injected with [³H]valine subarachnoidally and sacrificed 30 minutes later. The incorporation of the labeled aminoacid into proteins was slightly increased in all subcellular fractions but the increase was significant only for soluble proteins.Referring to the type of soluble polypeptides found in the cortex at this post ECS phase, no modification was found in the SDS electrophoresis pattern of steady state proteins. The fractionation pattern of labeled soluble proteins showed in one third (3 out of 9) of the experiments a remarkable stimulation of the synthesis of two polypeptides (25,000 and 54,000 Dalton MW), which were not labeled in the controls.     

Theoretical evaluation of isotopic fractionation factors in oxidation reactions of benzene,phenol and chlorophenols

We have studied theoretically the rate determining steps of reactions of benzene with permanganate, perchlorate, ozone and dioxygen in the gas phase and aqueous solution as well as phenol and dichlorophenol in protonated and unprotonated forms in aqueous solution. Kinetic isotope effects were then calculated for all carbon atoms and based on their values isotopic fractionation factors corresponding to compound specific isotopic analysis have been evaluated. The influence of the oxidant, substituents, environment and protonation on the isotopic fractionation factors has been analyzed.     

Aminoacylase I is not a glycolipid-anchored ectoenzyme in pig kidney.

Subcellular fractionation of pig kidney cortex revealed that aminoacylase I (EC 3.5.1.14, N-acyl-L-amino-acid aminohydrolase) is predominantly a soluble enzyme with only 0.5% of the total activity being recovered in the membrane fraction. The aminoacylase I activity associated with the membrane preparations displayed neither rapid release following incubation with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis nor the distinctive differential pattern of detergent solubilization which was seen with glycosyl-phosphatidylinositol-anchored proteins (renal dipeptidase, alkaline phosphatase). When fractionated by phase separation in Triton X-114, integral membrane proteins of kidney microvillar membranes partitioned predominantly (greater than 90%) into the detergent-rich phase. In contrast, only 3.7% of aminoacylase I activity associated with microvillar membranes partitioned into the detergent-rich phase. Aminoacylase I activity of pig kidney would therefore appear to be a hydrophilic protein in nature and is not, as suggested previously, a G-PI-anchored integral membrane protein.     

Human embryonic stem cells secrete soluble factors that inhibit cancer cell growth

Objectives:  The aim of this study was to determine whether normal human embryonic stem cells (hESC) would secrete factors that arrest growth of human epithelial cancer cell lines.
Materials and methods:  Cell proliferation was examined using the MTT assay then haemocytometer cell counts. Staining with propidium iodide followed by flow cytometry was used to detect cell cycle stages. Heat denaturation and molecular fractionation experiments were also performed.
Results:  We found that hESC conditioned medium (hESC CM) inhibited SKOV-3 and HEY cell proliferation. Similar results were also obtained when we used breast and prostate cancer cell lines, whereas little or no inhibitory effect was observed when human fibroblasts were tested. Moreover, a co-culture model confirmed that inhibition of cancer cell proliferation is mediated by soluble factors produced by hESCs. We also determined that the proportion of cancer cells in G₁ phase was increased by hESC CM treatment, accompanied by decrease in cells in S and G₂/M phases, suggesting that the factors slow progression of cancer cells by cell cycle inhibition. Heat denaturation and molecular fractionation experiments indicated a low molecular weight thermostable factor was responsible for these properties.
Conclusions:  Our findings provide evidence that the human embryonic microenvironment contains soluble factor(s) that are capable of inhibiting growth of cancer cells, and that exposure to such factors may represent a new cancer treatment strategy.     

Two dimensional liquid phase separations of proteins using online fractionation and concentration between chromatographic dimensions

Multi-dimensional liquid chromatography is often presented as an alternative to two-dimensional (2-D) gel electrophoresis for separating complex protein mixtures. The vast majority of analytical-scale 2-D LC systems have employed either off-line fractionation or stepped gradients in the first dimension separation. The latter severely restrict flexibility in setting up the first dimension gradient. We propose a novel two-dimensional LC system that employs online fractionation of proteins into a series of small reversed phase trapping columns. These traps effectively decouple the two separation dimensions and avoid problems associated with off-line fraction collection. Flexibility in determining the gradient programs for the two separations is thus enhanced. The reduced diameter of the trapping columns concentrates analyte between chromatographic dimensions. The apparatus is coupled with online electrospray time-of-flight mass spectrometry to characterize ribosomal proteins of Caulobacter crescentus.     

Identification of the Soluble Starch Synthase Activities of Maize Endosperm

This study identified the complement of soluble starch synthases (SSs) present in developing maize (Zea mays) endosperm. The product of the du1 gene, DU1, was shown to be one of the two major soluble SSs. The C-terminal 450 residues of DU1 comprise eight sequence blocks conserved in 28 known or predicted glucan synthases. This region of DU1 was expressed in Escherichia coli and shown to possess SS activity. DU1-specific antisera detected a soluble endosperm protein of more than 200 kD that was lacking in du1- mutants. These antisera eliminated 20% to 30% of the soluble SS activity from kernel extracts. Antiserum against the isozyme zSSI eliminated approximately 60% of the total soluble SS, and immunodepletion of du1- mutant extracts with this antiserum nearly eliminated SS activity. Two soluble SS activities were identified by electrophoretic fractionation, each of which correlated specifically with zSSI or DU1. Thus, DU1 and zSSI accounted for the great majority of soluble SS activity present in developing endosperm. The relative activity of the two isozymes did not change significantly during the starch biosynthetic period. DU1 and zSSI may be interdependent, because mutant extracts lacking DU1 exhibited a significant stimulation of the remaining SS activity.     

Fractionation of soluble proteins in Escherichia coli using DEAE-, SP-, and phenyl sepharose chromatographies.

In an effort to simplify a complex mixture of soluble proteins from Escherichia coli, methods to fractionate the samples prior to two-dimensional (2D) gel electrophoresis were developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins from an E. coli extract with DEAE-Sepharose resulted in a threefold increase in the number of detectable 2D gel spots. These gel spots were amenable to protein identification by using in-gel trypsin digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and peptide mass fingerprinting. Significantly, the DEAE-Sepharose column fractionation effectively partitioned the soluble proteins from the cell extracts. Similarly, an SP-Sepharose column was used to fractionate the soluble proteins from E. coli and resulted in over a twofold increase in the number of detectable gel spots. Lastly, fractionation of the cell extract with the phenyl Sepharose column resulted in a threefold increase in the number of detectable 2D gel spots. This work describes an easy, inexpensive way to fractionate the soluble proteins in E. coli and a way to better profile the E. coli proteome.     

Modification of glass channel walls for separation of biological particles by gravitational field-flow fractionation

In the gravitational field-flow fractionation of complex samples, various interaction and adsorption phenomena can occur in separation channels that influence fractionation and complicate the explanation of resulting fractograms. To overcome these problems, the glass surface was modified to create charge-free, non-adsorbing hydrophilic media for the mild treatment of hydrophilic biological particles. The modification was carried out in two steps: (1) by a simple lacquering of the glass surface with polystyrene diluted in toluene and (2) subsequent adsorption of a detergent layer on polystyrene. Essential suppression of ionic interactions between soluble low-molecular-mass compounds and the channel wall and decreased adsorption effects were demonstrated in separations of blood samples by gravitational field-flow fractionation.     

Partial purification and properties of phosphatidate phosphatase in Saccharomyces cerevisiae

Using an aqueous dispersion of [32P]phosphatidate as substrate we detected phosphatidate phosphatase (EC 3.1.3.4) activity in a cell-free extract of the yeast, Saccharomyces cerevisiae. The activity was found in both the membrane and the soluble fractions. The enzyme was purified from the soluble fraction about 600-fold. The purification procedure involved (NH4)2SO4 fractionation, poly(ethylene glycol) 6000 fractionation and column chromatography on DEAE-Sepharose, Sephadex G-100 and Blue-Sepharose. The purified enzyme almost absolutely required Mg2+ for activity. The molecular weight of the enzyme was estimated by analytical gel filtration on Sephadex G-100 to be approx. 75000. The enzyme was highly specific for phosphatidate. The apparent Km for phosphatidate was approx. 0.05 mM. The optimum pH was between 7.0 and 8.0.     

Proteins of Vesicular Stomatitis Virus: II. Immunological Comparisons of Viral Antigens

The antigens of the nucleoprotein core and the coat of vesicular stomatitis virus (VSV) particles of the Indiana serotype were prepared and purified by sucrose gradient fractionation. Antibody was prepared separately to each of the two antigen fractions. By immunological procedures, it was shown that soluble antigens of VSV preparations sedimenting at 20S and in the leading edge of the 6S region are antigenically related to VP3, the protein of the virus core, whereas the 6S soluble antigen cross-reacts only with viral coat antibodies. These results confirm previous results obtained by polyacrylamide gel analysis of the antigens. It has further been demonstrated that the 6S antigen is a glycoprotein. Comparing antigens of the New Jersey and Indiana serotype showed that the coat antigens of virus particles and the 6S antigen are immunologically distinct in the two serotypes. In complement-fixation tests, the core antigens and the soluble 20S antigens from one serotype showed a cross-reaction with antiserum prepared against core proteins of the other serotype.