Reactivation of latent viruses is associated with increased plasma cytokines in astronauts.

Success of long duration space missions will depend upon robust immunity. Decreased immunity has been observed in astronauts during short duration missions, as evident by the reactivation of latent herpes viruses. Seventeen astronauts were studied for reactivation and shedding of latent herpes viruses before, during, and after 9-14 days of 8 spaceflights. Blood, urine, and saliva samples were collected 10 days before the flight (L-10), during the flight (saliva only), 2-3h after landing (R+0), 3 days after landing (R+3), and 120 days after landing (R+120). Values at R+120 were used as baseline levels. No shedding of viruses occurred before flight, but 9 of the 17 (designated "virus shedders") shed at least one or more viruses during and after flight. The remaining 8 astronauts did not shed any of the 3 target viruses (non-virus shedders). Virus-shedders showed elevations in 10 plasma cytokines (IL-1α, IL-6, IL-8, IFNγ, IL-4, IL-10, IL-12, IL-13, eotaxin, and IP-10) at R+0 over baseline values. Only IL-4 and IP-10 were elevated in plasma of non-virus shedders. In virus shedders, plasma IL-4 (a Th2 cytokine) was elevated 21-fold at R+0, whereas IFNγ (a Th1 cytokine) was elevated only 2-fold indicating a Th2 shift. The inflammatory cytokine IL-6 was elevated 33-fold at R+0. In non-shedding astronauts at R+0, only IL-4 and IP-10 levels were elevated over baseline values. Elevated cytokines began returning to normal by R+3, and by R+120 all except IL-4 had returned to baseline values. These data show an association between elevated plasma cytokines and increased viral reactivation in astronauts.

Transport of Fe2+ across lipid bilayers: possible role of free fatty acids.

Fatty acids can form lipid-soluble complexes with Fe2+. Incorporation of fatty acids into phosphatidylcholine/cholesterol liposomes renders them permeable to Fe2+. Of several fatty acids tested, the most effective Fe2+ carriers were linoleic and oleic acids followed, in decreasing order of efficacy, by linolenic, myristic, arachidonic and palmitic acids. The initial Fe2+ transport rate for oleic acid depends on free Fe2+ in the medium which in turn shows a strong pH dependence above pH 7.0. The overall pH dependence of Fe2+ transport for several fatty acids shows an optimum near 6.9. Fe2+ transport catalysed by oleic acid can be inhibited by high NaCl concentrations but not 1 mM Co2+. It is suggested that free fatty acids may act as mediators of Fe2+ transport across biological membranes, particularly isolated intestinal brush-border membrane.

Significance of non-esterified fatty acids in iron uptake by intestinal brush-border membrane vesicles.

Iron uptake from Fe/ascorbate by mouse brush-border membrane vesicles is not greatly inhibited by prior treatment with a variety of protein-modification reagents or heat. Non-esterified fatty acid levels in mouse proximal small intestine brush-border membrane vesicles show a close positive correlation with initial Fe uptake rates. Loading of rabbit duodenal brush-border membrane vesicles with oleic acid increases Fe uptake. Depletion of mouse brush-border membrane vesicle fatty acids by incubation with bovine serum albumin reduces Fe uptake. Iron uptake by vesicles from Fe/ascorbate is enhanced in an O2-free atmosphere. Iron uptake from Fe/ascorbate and Fe3+-nitrilotriacetate (Fe3+-NTA) were closely correlated. Incorporation of oleic acid into phosphatidylcholine/cholesterol (4:1) liposomes leads to greatly increased permeability to Yb3+, Tb3+, Fe2+/Fe3+ and Co2+. Ca2+ and Mg2+ are also transported by oleic acid-containing liposomes, but at much lower rates than transition and lanthanide metal ions. Fe3+ transport by various non-esterified fatty acids was highest with unsaturated acids. The maximal transport rate by saturated fatty acids was noted with chain length C14-16. It is suggested that Fe transport can be mediated by formation of Fe3+ (fatty acid)3 complexes.

Partition and distribution coefficients of solutes and drugs in brush border membrane vesicles.

Partition and distribution coefficients (log P, log D) into rat small intestinal brush border membrane (BBM) were measured for a variety of ionizable and non-ionizable drugs and solutes using a novel technique. The log P values were compared with those determined with model solvents, octanol and propylene glycol dipelargonate (PGDP). Non-ionizable solutes with log P values up to 3.0 showed that octanol was a better model for partition into the BBM than PGDP. With one exception, BBM partition coefficients of greater than 3 were not observed, even for solutes with log P values in model solvents that were greater than 5. Liposomes prepared from BBM lipids, or synthetic lipid mixtures of similar composition to BBM, demonstrated similar trends in partition coefficients to the intact BBM. Two cationic drugs, Atenolol and Xamoterol were investigated for partition into BBM lipid liposomes. An apparent enhancement of log D with respect to octanol was attributed to a "surfactant-like" orientation in the membrane and an interaction of the ionized drug with anionic phospholipid head groups. The anionic drug Proxicromil shows the expected decrease in log D with increasing pH, at low NaCl concentrations. Changes in electrophoretic mobility of liposomes after incorporation of Proxicromil into them were consistent with the negative charge of the ionized drug being at the membrane surface. It was concluded that Proxicromil also associates with membranes in a "surfactant-like" orientation and that increased extraction with increasing NaCl concentrations is a result of ionic strength effects. Partition of solutes into BBM vesicles is more complex than into organic solvents and probably represents an important step in overall intestinal permeation of solutes.

Proteins of the Golgi apparatus. Purification to homogeneity, N-terminal sequence, and unusually large Stokes radius of the membrane-bound form of UDP-galactose:N-acetylglucosamine beta 1-4galactosyltransferase from rat liver.

The Golgi marker enzyme, UDP-galactose:N-acetylglucosamine beta 1-4galactosyltransferase (beta 1-4GalT) was purified 44300-fold in its intact, membrane-bound form from rat liver membranes. The protein was isolated from detergent extracts as a high-M(r) form, having a Stokes radius approximating a globular protein of M(r) 440,000. It is comprised of a single protein component as observed on SDS/polyacrylamide gels, having an M(r) near 51,000, and does not have intermolecular disulfide cross-links. N-terminal sequencing of the enzyme demonstrated that it contains an N-terminal hydrophobic stretch deduced previously from cDNA encoding for the enzyme. Previous studies have indicated that the protein may be translated at either of two AUG sites near the 5' end of the mRNA [Russo, R. N., Shaper, N. L. & Shaper, J. H. (1990) J. Biol. Chem. 265, 3324-3331], giving rise to two polypeptides, one appended with 13 amino acids. In the work described here, evidence was only found for the sequence of the short form, missing a single methionine at the N-terminus. Mild proteolytic treatment cleaved the enzyme, giving rise to low-M(r) forms which were fully catalytically active and which, upon sequencing, were missing a 66-amino-acid stretch from the N-terminus (as compared to the mouse cDNA). Proteolytic treatment was accompanied by conversion of the form having a large Stokes radius to one approximating a globular protein with M(r) near 50,000. The N-terminal stretch appears to contribute to maintenance of the form having a large Stokes radius. This may be the result of interaction with a detergent micelle, dimerization or oligomerization, or interaction with some other large, non-protein molecule, although a detergent exchange still resulted in a form having a large Stokes radius.

Proteome analysis of polyacrylamide gel-separated proteins visualized by reversible negative staining using imidazole-zinc salts.

Identification and characterization of proteins isolated from natural sources by polyacrylamide gel electrophoresis has become a routine technique. However, efficient sample proteolysis and subsequent peptide extraction is still problematic. Here, we present an improved protocol for the rapid detection of polyacrylamide gel-separated proteins, in situ protein modification, proteolytic digestion and peptide extraction for subsequent protein identification and characterization by capillary high-performance liquid chromatography/tandem mass spectrometry. This simple technique employs the rapid imidazole-zinc reverse stain, in-gel S-pyridylethylation and proteolytic digestion of microcrushed polyacrylamide gel pieces with proteases. This technique obviates the need for buffer exchange or gel lyophilisation due to all of the sample manipulation steps being carried out at near neutral pH and thus lends itself readily to automation.

S-pyridylethylation of intact polyacrylamide gels and in situ digestion of electrophoretically separated proteins: a rapid mass spectrometric method for identifying cysteine-containing peptides.

In-gel proteolytic digestion of acrylamide-gel separated proteins is a method widely used for generating peptide fragments for the purpose of identifying proteins by Edman degratation, tandem mass spectrometry, and peptide-mass fingerprinting. However, it is well recognised for disulfide-bonded proteins electrophoresed under reducing conditions that if no precautions are taken to minimise disulfide bond formation during protein digestion or peptide isolation, complex peptide maps can result. Here, we describe an improved method for in-gel protein digestion. It consists of first reducing and S-pyridylethylating Coomassie Brilliant Blue R-250-stained proteins immobilised in the whole gel slab with dithiothreitol and 4-vinylpyridine, excising the individual stained and alkylated proteins, and then digesting them in situ in the gel matrix with trypsin or Achromobacter lyticus protease I. Peptide fragments generated in this manner are extracted from the gel piece and purified to homogeneity by a rapid (< or = 12 min) reversed-phase high performance liquid chromatography (HPLC) procedure, based upon conventional silica supports. Recoveries of peptides are increased by S-pyridylethylation of acrylamide-immobilised proteins prior to in-gel digestion. Further, the levels of gel-related contaminants, which otherwise result in suppression of sample signals during electrosprayionisation mass spectrometry, are greatly reduced by the reduction/alkylation step. Additionally, we demonstrate that S-beta-(4-pyridylethyl)-cysteine containing peptides can be readily identified during reversed-phase HPLC by absorbance at 254 nm, and during electrospray ionisation tandem mass spectrometry by the appearance of a characteristic-pyridylethyl fragment ion of 106 Da. The position of cysteine residues in a sequence can be determined as phenylthiohydantoin S-beta-(4-pyridylethyl)-cysteine during Edman degradation, and by tandem mass spectrometry.

Strategies for internal amino acid sequence analysis of proteins separated by polyacrylamide gel electrophoresis.

An evaluation has been made of various strategies for obtaining internal amino acid sequence data from electrophoretically separated proteins. Electroblotting, in situ proteolysis and extraction, and direct electroelution are compared. Electroblotting of protein or peptides from gels resulted in poor yields (typically, 1-7%). However, higher yields (3-67%) were achieved by in situ enzymatic cleavage followed by acid extraction of the peptides from the gel. Peptides extracted from the gel were separated by reversed-phase high-performance liquid chromatography (RP-HPLC), on short, small-bore columns (100 x 2.1 mm I.D.), to enable recovery of peptides in small volumes (ca. 50 microliters) suitable for microsequence analysis. Capillary zone electrophoresis under acidic conditions (pH 2.5) was used to assess peptide purity before sequence analysis. Cysteine residues were identified in unmodified proteins or peptides by a characteristic phenylthiohydantoin (PTH)-amino acid derivative during sequence analysis. This derivative does not co-chromatograph with any known PTH-amino acid. Direct electrophoretic elution of protein from gels yielded between 45-50% of applied protein. Proteins recovered from gels by electrophoretic elution required further purification by inverse-gradient RP-HPLC [R. J. Simpson, R. L. Moritz, E. C. Nice and B. Grego, Eur. J. Biochem., 165 (1987) 21] to remove sodium dodecylsulphate and acrylamide-related contaminants for sequence analysis.

Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis.

The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.

Peptide mapping and internal sequencing of proteins electroblotted from two-dimensional gels onto polyvinylidene difluoride membranes. A chromatographic procedure for separating proteins from detergents.

Direct sequence analysis of proteins electroblotted from two-dimensional polyacrylamide gels onto immobilizing matrices provides an efficient technique for obtaining N-terminal sequence data for proteins not amenable to purification by reversed-phase high-performance liquid chromatography (RP-HPLC). We present in this paper a procedure for obtaining peptide fragments from electroblotted proteins for internal amino acid sequence analysis. First, Coomassie Blue-stained proteins are extracted from polydivinylidene difluoride membranes, using a detergent mixture of sodium dodecylsulfate and Triton X-100. Proteins are then separated from the detergent mixture by a chromatographic procedure which relies on the ability of proteins to interact with certain reversed-phase sorbents at high organic solvent concentrations. Under these conditions, detergents and Coomassie Blue are not retained and pass through the column. Proteins are recovered by simultaneously: (i) introducing trifluoroacetic acid into the mobile phase and (ii) decreasing the organic solvent concentration. After proteolytic fragmentation, peptides are purified by microbore-column (1-2 mm I.D.) RP-HPLC for microsequence analysis.

Structure of the extracellular domains of the human interleukin-6 receptor alpha -chain.

Dysregulated production of IL-6 and its receptor (IL-6R) are implicated in the pathogenesis of multiple myeloma, autoimmune diseases and prostate cancer. The IL-6R complex comprises two molecules each of IL-6, IL-6R, and the signaling molecule, gp130. Here, we report the x-ray structure (2.4 A) of the IL-6R ectodomains. The N-terminal strand of the Ig-like domain (D(1)) is disulfide-bonded to domain D(2), and domains D(2) and D(3), the cytokine-binding domain, are structurally similar to known cytokine-binding domains. The head-to-tail packing of two closely associated IL-6R molecules observed in the crystal may be representative of the configuration of the physiological dimer of IL-6R and provides new insight into the architecture of the IL-6R complex.

Two-dimensional gel database of human breast carcinoma cell expressed proteins: an update.

Previously, we reported a two-dimensional gel map and database with molecular weight/isoelectric point (Mr/pI) loci for 22 proteins expressed in the breast carcinoma cell line, MDA-MB231 (Rasmussen et al., Electrophoresis 1997, 18, 588-598). Here we update this database with Mr/pI loci for a further nine cytoplasmic proteins and three Triton X-114 solubilised membrane proteins from MDA-MB231 cells. In addition, a novel protein, previously represented only in expressed sequence tag (EST) databases, has been identified as a Triton X-114 soluble protein and assigned an Mr/pI locus. During the course of isolating proteins from the Triton X-114 fraction, we compared recoveries of proteins in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels after isoelectric focusing (IEF) using either immobilised pH gradients or carrier ampholytes. In these experiments, a significantly higher proportion of membrane proteins were visible in SDS-polyacrylamide gels after the use of carrier ampholytes for the first dimension. We also report our mass spectrometric-based procedure for identifying two-dimensional electrophoresis (2-DE) gel-resolved proteins, combining in-gel enzymatic digestion, 0.2 mm internal diameter (ID) capillary column reversed-phase high-performance liquid chromatography (RP-HPLC) peptide mapping and electrospray ionisation--ion trap--mass spectrometry.

Fabrication of stable packed capillary reversed-phase columns for protein structural analysis.

Capillary column (< or = 320-micron ID) liquid chromatography is an essential tool for the separation and concentration of low-picomole amounts of proteins and peptides for mass-spectrometric based structural analysis. We describe a detailed procedure for the fabrication of stable and efficient 50- to 180-micron ID polyimide fused-silica columns. Columns were packed by conventional slurry packing with reversed-phase silica-based supports followed by column bed consolidation with acetonitrile and sonication. PVDF membrane or internal fused-silica particles were employed for column end-frit construction. The ability of these columns to withstand high back pressures (300-400 bar) enabled their use for rapid chromatography (> 3400 cm/hr; i.e., approximately 40 microliters/min for 200-micron ID columns) and the loading of large sample volumes (up to 500 microliters). The accurate low flow rates (0.4-4.0 microliters/min) and precise gradient formation necessary to operate these columns were achieved by a simple modification of conventional HPLC systems [Moritz et al. (1992), J. Chromatogr. 599, 119-130]. Column performance was evaluated for ability to resolve low-fmol amounts of all components of a mixture of PTH-amino acids and to separate peptides for on-line LC/MS analysis of peptide mixtures derived from in situ digestion of 2-DE resolved protein spots.

Electrophoretic analysis of the novel antigen for the gastrointestinal-specific monoclonal antibody, A33.

The murine monoclonal antibody A33 (mAbA33) recognises a human cell membrane-associated antigen selectively expressed in epithelial cells of the lower gastrointestinal tract and > 90% of colonic cancers, but is not detected in a wide range of other normal tissues by immunohistochemical analysis. In phase I/II clinical triasl, mAbA33 has been shown to target advanced colon cancers and the humanized version is currently being evaluated in therapy studies. Although the mAbA33 has been well characterised by immunohistochemical and clinical studies, until recently, the target antigen has remained poorly defined. This was largely attributable to the antigenic determinant recognised by mAbA33 being dependent on the native spatial conformation of the A33 antigen which impeded its identification by conventional two-dimensional electrophoresis (2-DE) and immunoblot analysis. We have developed an immunoblot method, based on nonreducing/non-urea precast 2-DE gels, that has facilitated the purification of the detergent (0.3% Triton X-100) solubilised A33 antigen from the human colon cancer cell lines LIM1215 and SW1222. Under these 2-DE conditions, the A33 antigen electrophoreses with an apparent M(r) approximately 41000 and pI 5.0-6.0. Attempts to isolate the A33 antigen from 2-DE gels for direct structural analysis were unsuccessful, due to its co-electrophoresis with actin and cytokeratin proteins. However, using Western blot and biosensor detection the A33 antigen has been purified chromatographically and N-terminal sequence analysis was possible. Using polyclonal antibodies raised against a synthetic peptide corresponding to the N-terminal region of the A33 antigen we have used Western blot analysis to localise the molecule in our master 2-DE protein database for normal human colon crypts and several colon carcinoma cell lines (URL address: http:(/)/www.ludwig.edu.au). Under reducing 2-DE conditions, the A33 antigen electrophoresis as 6 differentially charged isoforms (pI 4.6-4.8) with a single molecular weight species at M(r) approximately 55000.