Use of multidimensional separation protocols for the purification of trace components in complex biological samples for proteomics analysis.

The routine detection of low abundance components in complex samples for detailed proteomics analysis continues to be a challenge. Whilst the potential of multidimensional chromatographic fractionation for this purpose has been proposed for some years, and was used effectively for the purification to homogeneity of trace components in bulk biological samples for N-terminal sequence analysis, its practical application in the proteomics arena is still limited. This article reviews some of the recent data using these approaches, including the use of microaffinity purification as part of multidimensional protocols for downstream proteomics analysis.

Mutagenesis and selection of PDZ domains that bind new protein targets.

PDZ domains are a recently characterized protein-recognition module. In most cases, PDZ domains bind to the C-terminal end of target proteins and are thought thereby to link these target proteins into functional signaling networks. We report the isolation of artificial PDZ domains selected via a mutagenesis screen in vivo, each recognizing a different C-terminal peptide. We demonstrate that the PDZ domains isolated can bind selectively to their target peptides in vitro and in vivo. Two of the target peptides chosen are the C-terminal ends of two cellular transmembrane proteins with which no known PDZ domains have been reported to interact. By targeting these artificial PDZ domains to the nucleus, interacting target peptides were efficiently transported to the same subcellular localization. One of the isolated PDZ domains was tested and shown to be efficiently directed to the plasma membrane when cotransfected with the full-length transmembrane protein in mammalian cells. Thus, artificial PDZ domains can be engineered and used to target intracellular proteins to different subcellular compartments.

Specific localization, gamma camera imaging, and intracellular trafficking of radiolabelled chimeric anti-G(D3) ganglioside monoclonal antibody KM871 in SK-MEL-28 melanoma xenografts.

The chimeric monoclonal antibody KM871, directed against the G(D3) antigen, is under evaluation for its potential to target melanoma. To facilitate the in vivo evaluation of biodistribution properties and measurement of pharmacokinetics, KM871 was radiolabeled with (125)I via tyrosine residues and with (111)In via the bifunctional metal ion chelator C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid (CHX-A"-DTPA) to lysine residues. Using antigen-positive SK-MEL-28 melanoma cells, immunoreactivities of 42 and 40% cell binding were obtained, respectively, for the two radioconjugates. Binding was enhanced in the presence of added unlabeled antibody. A humanized A33 antibody was similarly labeled with the two isotopes and used as a control. To determine and compare in vivo biodistribution characteristics of KM871 radiolabeled with (111)In or (125)I, mixtures of the radioconjugates were injected i.v. into BALB/c nude mice bearing G(D3)-positive-SK-MEL-28 melanoma xenografts. Gamma camera images were acquired; groups of five mice were sacrificed at various time intervals, and tumors, blood, and tissues were analyzed. (111)In-labeled CHX-A"-DTPA-KM871 showed a maximum tumor uptake of 41.9 +/- 7.0% injected dose/g at 72 h with prolonged retention over a 15-day period. The tumor:blood ratio was 3:1 by 72 h, and higher ratios were observed at later time points. No abnormal accumulation of (111)In-labeled conjugate was found in normal tissues. In contrast, there was little accumulation of (125)I-labeled KM871 in the same tumors. The specificity of antibody localization was confirmed by the low tumor uptake values for radiolabeled control antibody. Gamma camera imaging demonstrated excellent uptake of (111)In-labeled CHX-A"-DTPA-KM871 in the xenografts. Chromatographic analyses of xenograft cytosolic extracts demonstrated tumor internalization and catabolism of radiolabeled KM871 with the formation of small molecular weight metabolites. Laser scanning confocal microscopy demonstrated that the majority of intracellular KM871 is localized to lysosomes. Despite the catabolism of the radioconjugate, a dose-dependent increase in KM871 tumor localization was shown through immunohistochemical examination of xenograft biopsies. This study demonstrates for the first time the in vivo localization of a radiolabeled anti-G(D3) monoclonal antibody to G(D3)-expressing xenografts using gamma camera scanning techniques and tumor cell internalization of KM871 tagged with a green fluorescent dye, Alexa Fluor 488, through confocal microscopy. KM871 has potential for targeting tumors in patients with melanoma.

Kinetics of the autologous red cell agglutination test.

The kinetics of the autologous red cell agglutination test for detecting circulating antibodies to HIV-1 were studied. Two monoclonal anti-red blood cell antibodies (1C3/86 and 10F7MN) were used to construct Fab-peptide conjugates for the test. Both antibodies recognize glycophorin alpha on the surface of erythrocytes by immunoprecipitation or immunoblotting techniques. The number of binding sites, association and dissociation constants of 1C3/86 Fab and 10F7MN Fab' fragments were determined (n = 4.80 X 10(5) sites/erythrocyte, Ka = 0.43 X 10(7) M-1, Kd = 23 X 10(-8) M for 1C3/86, n = 4.66 X 10(5) sites/erythrocyte, Ka = 1.05 X 10(7) M-1, Kd = 9.5 X 10(-8) M for 10F7MN. The binding studies were performed under the same conditions as the autologous red blood cell agglutination test. When 0.9 microgram of anti-glycophorin Fab was added to 10 microliters of blood 0.25 microgram of 1C3/86 Fab was bound whereas 0.29 microgram for 10F7MN Fab' was bound. Antibody binding reached a plateau after 2 min and once bound did not exchange with unbound Fab over the time scale of the test. The binding of the anti-peptide antibody (cross-linking antibody) was also complete within 2 min. Addition of approximately 0.1 microgram of anti-peptide antibody gave half a maximal agglutination score. This is equivalent to 10 micrograms/ml circulating antibody. Under the agglutination test conditions, Fab-peptide conjugate was bound to 14% of available glycophorin molecules. Half maximal agglutination occurred when approximately 1.1% of the bound Fab-peptide conjugates were cross-linked. A maximum agglutination score of four occurred in the presence of 1 microgram of anti-peptide antibody equivalent to 100 micrograms/ml circulating antibody whereas an agglutination score of 1+ was elicited by only 0.32 microgram anti-peptide antibody and involved the cross-linking of approximately 160 glycophorin molecules per red cell.

Simplified conjugation chemistry for coupling peptides to F(ab') fragments: autologous red cell agglutination assay for HIV-1 antibodies.

The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on agglutination of autologous red blood cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579-613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous red cell agglutination test is described. F(ab')2 fragments of the anti-glycophorin antibody were prepared by pepsin digestion and reduced to F(ab') fragments with the use of tri-n-butylphosphine (TBP). This permitted the simultaneous reduction of the F(ab') fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579-613) [Cys-Acm 598, Lys-BrAc 604] containing epsilon-bromoacetyl-lysine at residue 604 to the resultant F(ab') fragment. Conjugation to the F(ab') fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab'). The resultant F(ab')-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous red cell agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.

Aggregation to thrombin and collagen of platelets from a Glanzmann thrombasthenic patient lacking glycoproteins IIb and IIIa.

The aim of this study was to investigate the platelets of a Glanzmann thrombasthenic patient, which in citrated PRP failed to respond to various agonists, but aggregated and secreted to high concentrations of thrombin (0.36, 0.72 and 1 U/ml) and collagen (4, 10 and 20 micrograms/ml) when washed and resuspended in a Tyrode-albumin solution (containing 2 mM Ca2+). Aggregation of the patient platelets was not affected by anti-IIb/IIIa monoclonal antibody (P18) which strongly inhibits thrombin or collagen induced aggregation of normal platelets. Washed platelets of this patient did not aggregate to ADP (10-100 microM) in the presence of added fibrinogen (2 mg/ml) nor bind 125I-labelled fibrinogen (40 to 320 micrograms/ml) when thrombin-stimulated. Different anti-IIb/IIIa monoclonal antibodies (P2, P18) when used in binding or crossed immunoelectrophoretic studies showed a complete absence of the IIb-IIIa glycoprotein complex on the patient platelets. Moreover, glycoproteins IIb or IIIa were absent on silver-stained two-dimensional (non-reduced/reduced) polyacrylamide gel separations of the patient platelets and were not detected by Western blots used in combination with anti-PLA1 (antigen present on IIIa), anti-Leka (antigen present on IIb). This study shows that platelets lacking glycoproteins IIb or IIIa can aggregate in response to high concentrations of collagen or thrombin when resuspended in the presence of physiological concentrations of calcium. Results obtained in this study could indicate the existence of other mechanisms (other than the IIb-IIIa glycoprotein complex) involving glycolipids, heparans, proteoglycans, and/or unknown membrane glycoproteins to mediate platelet aggregation of stimulated thrombasthenic platelets.

Micropreparative ligand fishing with a cuvette-based optical mirror resonance biosensor.

We have previously demonstrated the role of an optical biosensor (BIAcore 2000) as a specific detector to monitor chromatographic fractions during the purification and characterisation of ligands for orphan biomolecules. We have now extended this application to perform micropreparative ligand fishing directly on the sensor surface using an automated cuvette-based optical biosensor (Iasys Auto+) equipped with a high-capacity carboxymethyldextran surface (surface area 16 mm2). Using a F(ab)2' fragment of the A33 monoclonal antibody as bait, we have recovered microgram quantities of essentially homogeneous A33 ligand from the sensor surface in a form suitable for subsequent sensitive and specific down stream analysis (micropreparative HPLC, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting). The design of the cuvette-based system facilitates recovery of desorbed material from the constrained workspace in small volumes at high concentration. The use of on-surface detection allows the surface viability to be continuously monitored and permits direct quantitation of both bound and recovered material.

Kinetic analysis of the interaction between the monoclonal antibody A33 and its colonic epithelial antigen by the use of an optical biosensor. A comparison of immobilisation strategies.

The interaction between the humanised A33 monoclonal antibody and the corresponding F(ab)'2 or Fab' fragments with the colonic epithelial A33 antigen, purified by micropreparative HPLC from membrane extracts of the colonic carcinoma cell line LIM 1215, has been studied with the BIAcore 2000 biosensor using surface plasmon resonance detection. The surface orientation of immobilised antibody and the Fab' fragment onto the biosensor surface was controlled using alternative immobilisation chemistries. This resulted in significantly higher molar binding activities compared with the conventional N-hydroxysuccinimide (NHS)/N-ethyl-N'-dimethylaminopropylcarbodiimide (EDC) chemistry. This increase in signal resulted in a concomitant increase in sensitivity of detection, which facilitates analysis of low levels of A33 antigen. The apparent association rate (ka) and dissociation rate (kd) constants obtained with the different immobilisation chemistries were determined. These analyses showed that the kinetic constants obtained for the IgG were not significantly affected by the method of immobilisation. F(ab)'2 and Fab' fragments immobilised using NHS/EDC chemistry showed significantly lower apparent affinity. By contrast the use of the thiol coupling chemistry with the Fab' fragment gave a five fold increase in observed KA, resulting in a similar affinity to that observed with the intact IgG molecule.

Micro-sequencing strategies for the human A33 antigen, a novel surface glycoprotein of human gastrointestinal epithelium.

Monoclonal antibody (mAb) A33, which recognizes a M(r) approximately 43,000 differentiation antigen (A33) expressed in normal human colonic and small bowel epithelium as well as in 95% of colon cancers, shows specific targeting of colon cancer in humans and is currently being evaluated for clinical use. Here, we describe strategies for the purification and structural analysis of the A33 antigen from the human colorectal carcinoma cell lines LIM1215 and SW1222. Edman degradation of the intact protein and nine peptides, derived by proteolytic digestion of the A33 antigen with Asp-N endoproteinase, thermolysin, trypsin and pepsin followed by micropreparative reversed-phase high-performance liquid chromatography, allowed the unambiguous sequence assignment of 153 amino acid residues; these data reveal one N-glycosylation sequeon in Asp-N endoproteinase peptide D4, and a disulfide linkage between peptides D1 and D4. This amino acid sequence information has facilitated the cloning and subsequent sequencing of a cDNA for the A33 antigen which demonstrates that it is a novel human cell surface molecule of the immunoglobulin superfamily.

Genomic fingerprinting of 80 strains from the WHO multicenter international typing study of listeria monocytogenes via pulsed-field gel electrophoresis (PFGE).

An international multicenter typing study of Listeria monocytogenes was initiated by the World Health Organization (Food Safety Unit, Geneva) in order to evaluate the usefulness of various phenotypic and genotypic typing methods for L. monocytogenes, to select and standardize the most appropriate methods to define common nomenclature of varieties and to select specific reference strains. Pulsed-field gel electrophoresis was used in four laboratories for molecular characterization of a set of 80 'coded' strains distributed to all participating laboratories. The endonucleases ApaI and SmaI, used in all four laboratories, yielded between 21 and 28 restriction endonuclease digestion profiles (REDP). AscI was used, in addition, in laboratory A and displayed 21 REDP. The combination of ApaI, SmaI or AscI REDP established 25 to 33 genomic groups. depending on the laboratory and the number of viable strains. Agreement of typing data among the four laboratories ranged from 79 to 90%. Forty-nine (69%) of the 71 strains viable in all four laboratories were placed into exactly the same genomic groups in all four laboratories. The epidemiological relevance of the strains became known after decoding and it was shown that most of the epidemiologically related strains were correctly identified by the four groups of investigators. i.e., most related strains were placed into the same genomic groups by all four laboratories. Similar results were obtained when 11 duplicate cultures were analyzed-on average 84% of the duplicates were identified. Comparison of REDP obtained by laboratory A with REDP from previously analyzed set of 176 L. monocytogenes strains allowed the prediction of the serovar-groups of the 80 strains. These predictions of serovar-groups were later confirmed by serotyping results obtained by laboratories involved in the WHO multicenter typing study of L. monocytogenes. In general this study reconfirmed that PFGE is a very accurate and reproducible method for fine structure comparison and molecular typing of L. monocytogenes.

Recruitment of adenomatous polyposis coli and beta-catenin to axin-puncta.

The adenomatous polyposis coli (APC) tumour suppressor is a multifunctional protein involved in the regulation of Wnt signalling and cytoskeletal dynamics. Little is known about how APC controls these disparate functions. In this study, we have used APC- and axin-fluorescent fusion proteins to examine the interactions between these proteins and show that the functionally distinct populations of APC are also spatially separate. Axin-RFP forms cytoplasmic punctate structures, similar to endogenous axin puncta. Axin-RFP recruits beta-catenin destruction complex proteins, including APC, beta-catenin, glycogen synthase kinase-3-beta (GSK3-beta) and casein kinase-1-alpha (CK1-alpha). Recruitment into axin-RFP puncta sequesters APC from clusters at cell extensions and this prevents its microtubule-associated functions. The interaction between APC-GFP and axin-RFP within the cytoplasmic puncta is direct and dramatically alters the dynamic properties of APC-GFP. However, recruitment of APC to axin puncta is not absolutely required for beta-catenin degradation. Instead, formation of axin puncta, mediated by the DIX domain, is required for beta-catenin degradation. An axinDeltaDIX mutant did not form puncta, but still mediated recruitment of destruction complex proteins and phosphorylation of beta-catenin. We conclude that there are distinct pools of APC and that the formation of axin puncta, rather than the axin/APC complex, is essential for beta-catenin destruction.

Biosensor-based micro-affinity purification for the proteomic analysis of protein complexes.

A biosensor-based micro-affinity purification method to recover protein binding partners and their complexes for down stream proteomics analysis has been developed using the BIAcore 3000 fitted with a prototype Surface Prep Unit (SPU). The recombinant GST-intracellular domain of E-cadherin or the recombinant GST-beta-catenin binding domain of Adenomatous Polyposis Coli (APC) were immobilized onto the SPU and used to affinity purify binding partners from chromatographically enriched SW480 colon cancer cell lysates. A GST- immobilized surface was used as a control. Samples recovered from the SPU were subjected to SDS-PAGE with sensitive Coomassie staining followed by automated in-gel digestion and LC-MS/MS. The results obtained using the SPU were compared with similar experiments performed using Sepharose beads.

International Phage Typing Center for Listeria: report for 1987.

A total of 3400 Listeria strains were sent for identification and/or phage typing during 1987. These strains mainly originated from Europe. They were isolated from humans, animals and mostly from foodstuffs, thus reflecting the increasing interest concerning the view that listeriosis is a foodborne disease. Phage typing proved to be a useful tool for epidemiological survey. That was especially evidenced during studies of the outbreak of human listeriosis in Switzerland, for which a contaminated cheese was incriminated as the source of contamination.

[Biochemical characterization of species in the genus Listeria]. / Caractérisation biochimique des espèces du genre Listeria.

Seventy Listeria strains, including L. monocytogenes (30 strains), L. ivanovii (9), L. innocua (11), L. welshimeri (6), L. seeligeri (10), L. grayi (2) and L. murrayi (2) were subjected to biochemical characterization. The biochemical tests selected comprised: Api 50CH, Api 20E and Api Zym strips, hydrolysis of DNA and tween 20, 40, 60, 80, egg yolk agar reaction, growth in the presence of 7,5 and 10% NaCl, growth at 42 degrees C and 45 degrees C, and susceptibility to antibiotics and trypaflavin. No further marker in addition to previously described ones, allowing to distinguish L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri and L. seeligeri could be detected. L. grayi and L. murrayi differed from the other Listeria species by gluconate fermentation, lack of phosphoamidase and acid phosphatase and susceptibility to trypaflavin.

[Isolation of Listeria seeligeri and L. welshimeri bacteriophages. Lysotyping of L. monocytogenes, L. ivanovii, L. innocua, L. seeligeri and L. welshimeri]. / Isolement de bactériophages de Listeria seeligeri et L. welshimeri. Lysotypie de L. monocytogenes, L. ivanovii, L. innocua, L. seeligeri et L. welshimeri.

Recent taxonomic reexamination of Listeria monocytogenes strains as defined in the 8th edition of Bergey's Manual of Determinative Bacteriology allowed to distinguish 5 species among these strains: L. monocytogenes, L. innocua, L. ivanovii, L. welshimeri, and L. seeligeri. As till now only the behaviour of the species L. monocytogenes, L. innocua, and L. ivanovii towards bacteriophages was characterized, the present study aimed at the search for phages occurring on these two new species and to determine their lytic activities towards representative strains of the whole genus. Seven different bacteriophages were isolated from the culture filtrates of 45 strains of L. seeligeri, whereas only one phage was obtained from the filtrates of 17 strains of L. welshimeri. No phage was isolated from the filtrates of L. grayi and L. murrayi strains with our methods. The phage preparations proved stable over a period of 6 to 12 months with titers ranging from 7 X 10(8) to 5 X 10(10). By use of a set of lytic phages which contained in addition to the eight new phages from L. welshimeri and L. seeligeri 23 known phages from L. monocytogenes, L. innocua, and L. ivanovii, 511 selected strains, originating from various sources, especially environment (125 strains), and belonging to all species of Listeria, were studied. 345 (68%) of the 511 strains could be assigned to a lysovar by means of the extended set. The eight new phages showed genus- and serogroup specificity but did not present species-specific reactions. The strains of L. grayi and L. murrayi were untypable using these 31 phages. No relationship between lysovar and serovar on the one side and host and geographic source on the other side was apparent.

Instrumental biosensors: new perspectives for the analysis of biomolecular interactions.

The use of instrumental biosensors in basic research to measure biomolecular interactions in real time is increasing exponentially. Applications include protein-protein, protein-peptide, DNA-protein, DNA-DNA, and lipid-protein interactions. Such techniques have been applied to, for example, antibody-antigen, receptor-ligand, signal transduction, and nuclear receptor studies. This review outlines the principles of two of the most commonly used instruments and highlights specific operating parameters that will assist in optimising experimental design, data generation, and analysis.

Pulsed-field gel electrophoresis applied for comparing Listeria monocytogenes strains involved in outbreaks.

Recent food-borne outbreaks of human listeriosis as well as numerous sporadic cases have been mainly caused by Listeria monocytogenes serovar 4b strains. Thus, it was of interest to find out whether a certain clone or a certain few clones were responsible for these cases and especially for outbreaks. We used pulsed-field gel electrophoresis of large chromosomal DNA restriction fragments generated by ApaI, SmaI, or NotI to analyse 75 L. monocytogenes strains isolated during six major and eight smaller recent listeriosis outbreaks. These strains could be divided into 20 different genomic varieties. Thirteen of 14 strains isolated during major epidemics in Switzerland (1983-1987), the United States (California, 1985) and Denmark (1985-1987) demonstrated indistinguishable DNA restriction patterns. In contrast, strains responsible for the outbreaks in Canada (Nova Scotia, 1981), the United States (Massachusetts, 1983), France (Anjou, 1975-1976), New Zealand (1969), and Austria (1986) and some smaller outbreaks in France (1987, 1988, 1989) were each characterized by particular combinations of DNA restriction patterns. Seventy-seven percent of the tested strains could be classified into the previously described ApaI group A (Brosch et al. 1991), demonstrating a very close genomic relatedness. Because 49% of the epidemic strains selected for this study belonged to phagovar 2389/2425/3274/2671/47/108/340 or 2389/47/108/340, fifty-six additional strains of these phagovars, isolated from various origins, were also typed to determine whether differences in DNA restriction profiles between epidemic and randomly selected strains of the same phagovars could be pointed out. Variations in DNA patterns appeared more frequently within randomly selected strains than within epidemic strains.

New families of adhesion molecules play a vital role in platelet functions.

Adhesion molecules play a crucial part in cell-matrix and in cell-cell interactions. These interactions, which are essential to the body's defense processes, involve adhesion molecules belonging to different families: integrins, immunoglobulins and selectins. Integrins are expressed by a large number of tissues, whereas other adhesion molecule families are restricted to a small number of cell types. A recent symposium dealt with the recruitment of circulating platelets at specific sites, their adhesion to extracellular matrix components and their activation by agonists leading to aggregation or attachment to other cells. These events, supporting hemostasis and thrombosis, involve integrins, selectins and other adhesion molecules. This report focuses on newly reported integrins (GPIa, GPIc, GPIIa), selectins (GMP-140) and GPIIIb, previously known as 'minor' surface oriented platelet glycoproteins. Major membrane glycoproteins such as GPIIb-IIIa (an integrin) and GPIb, which also play a vital role in platelet functions, have been extensively reviewed elsewhere.

Virulence Heterogeneity of Listeria monocytogenes Strains From Various Sources (Food, Human, Animal) in Immunocompetent Mice and its Association With Typing Characteristics.

One hundred and twenty-five Listeria monocytogenes strains were screened for their ability to infect immunocompetent white Swiss mice. Mice were infected by intravenous injection of 2.5 × 104 to 7.5 × 104 CFU. Virulence was evaluated by counting viable bacteria in the mouse spleen 2 d after inoculation. Splenic bacterial counts ranged from less than 103 to 4 × 108 CFU per organ; values were between 1 × 106 and 4 × 108 for 88% of the strains. No systematic differences in virulence were noticed among strains of different origins, serovars, phagovars, ribovars, or DNA macrorestriction patterns. All strains isolated from human infections were found to be virulent within this assay. Among the 63 strains isolated from food, two were not virulent (<103 CFU per spleen). Results of this study suggest that L. monocytogenes strains are potential hazards for human health, regardless of their origin and certain strain-specific characteristics, such as serovar, phagovar, ribovar, and DNA macrorestriction patterns.