PLASMA MEMBRANE GLYCOPROTEINS OF RAT HEPATOCELLULAR CARCINOMA CELLS

Previous investigations have demonstrated qualitative differences in the plasma membrane glycoproteins of normal and malignant rat liver cells. The present investigations were designed to identify and characterize the spectrum of glycoproteins present on the surface of Novikoff and AS-30D hepatocellular carcinoma cells. Three cell-surface radiolabeling techniques were employed to tag specifically the plasma membrane glycoproteins: lactoperoxidase catalyzed iodination, specific for tyrosine residues; galactose oxidase/NaB{('3)H}(,4), specific for galactosyl residues; and NaIO(,4)/NaB{('3)H}(,4), specific for sialic acids. The glycoproteins were resolved by one- and two-dimensional gel electrophoresis and visualized by fluorography or autoradiography. It was found that these glycoproteins are a complex population of molecules. The complexity of this system is reflected not only in the number of individual components that can be detected (> 25), but in the charge heterogeneity of individual glycoproteins due to variable sialic acid content. Certain glycoproteins behaved anamolously on SDS-polyacrylamide gel electrophoresis; the apparent molecular weight decreasing with increasing acrylamide concentrations suggesting a high % carbohydrate. Cell-surface radiolabeling techniques were employed in combination with lectin affinity chromatography, using lectins of different saccharide specificity, to analyze the saccharide determinants present on the spectrum of cell-surface molecules. It was also found that particular glycoproteins differed in their lability to protease or neuraminidase digestion and in their extractability by non-ionic detergents. From these studies, detailed models of the plasma membrane of Novikoff and AS-30D cells were constructed which incorporates information concerning the structure and accessibility of heterosaccharide and peptide moieties, the relationship of the glycolipids, and the interaction of particular glycoproteins with the lipid bilayer. These investigations provide basic information concerning the molecular composition and properties of the plasma membrane of glycoproteins of malignant rat liver cells and lay the groundwork for future comparison to normal hepatocytes. ^

Novel prognostic markers revealed by a proteomic approach separating benign from malignant insulinomas.

The prognosis of pancreatic neuroendocrine tumors is related to size, histology and proliferation rate. However, this stratification needs to be refined further. We conducted a proteome study on insulinomas, a well-defined pancreatic neuroendocrine tumor entity, in order to identify proteins that can be used as biomarkers for malignancy. Based on a long follow-up, insulinomas were divided into those with metastases (malignant) and those without (benign). Microdissected cells from six benign and six malignant insulinomas were subjected to a procedure combining fluorescence dye saturation labeling with high-resolution two-dimensional gel electrophoresis. Differentially expressed proteins were identified using nano liquid chromatography-electrospray ionization/multi-stage mass spectrometry and validated by immunohistochemistry on tissue microarrays containing 62 insulinomas. Sixteen differentially regulated proteins were identified among 3000 protein spots. Immunohistochemical validation revealed that aldehyde dehydrogenase 1A1 and voltage-dependent anion-selective channel protein 1 showed significantly stronger expression in malignant insulinomas than in benign insulinomas, whereas tumor protein D52 (TPD52) binding protein was expressed less strongly in malignant insulinomas than in benign insulinomas. Using multivariate analysis, low TPD52 expression was identified as a strong independent prognostic factor for both recurrence-free and overall disease-related survival.

STUDIES ON THE CLASS I GLYCOPROTEINS OF THE MURINE MAJOR HISTOCOMPATIBILITY COMPLEX (TRANSPLANTATION ANTIGEN, LYMPHOCYTE, CELL SURFACE MOLECULE)

A series of studies were undertaken to analyze and compare various aspects of murine class I glycoproteins. An initial area of investigation characterized the Qa-1 alloantigens using two-dimensional gel electrophoresis. Analysis of the products of the Qa-1('b), Qa-1('c) and Qa-1('d) alleles indicated that these were distinct molecules as determined by their lack of comigration upon comparative two-dimensional gel analysis. The importance of asparagine-linked glycosylation in the cell surface expression of class I molecules was also examined. These studies employed tunicamycin, an inhibitor of N-linked glycosylation. Tunicamycin treatment of activated T lymphocytes diminished the surface expression of Qa-1 to undetectable levels; the levels of other class I molecules exhibited little or no decrease. These results indicated that N-linked glycosylation has a differential importance in the cell surface expression of various class I molecules. The molecular weight diversity of class I molecules was also investigated. Molecular weight determination of both the fully glycosylated and unglycosylated forms of H-2 and Qa/Tla region encoded molecules established that there is a significant variation in the sizes of these forms of various class I molecules. The most significant difference ((TURN)9,000 daltons) exists between the unglycosylated forms of H-2K('b) and Qa-2, suggesting that the structural organization of these two molecules may be very different. A comparative two-dimensional gel analysis of various class I glycoproteins isolated from resting and activated T and B lymphocytes indicated that class I molecules expressed on activated T cells exhibited an isoelectrophoretic pattern that was distinct from the isoelectrophoretic pattern of class I molecules expessed on the other cell populations. This difference was attributed to a lower sialic acid content of the molecules expressed on activated T cells. Analysis of cell homogenates determined that activated T cells contained a higher level of endogenous neuraminidase activity than was detected in the other populations, suggesting that this may be the basis of the lower sialic acid content. The relationship of the Qa-4 and Qa-2 alloantigens was also examined. It was established that upon mitogen activation, the expression of Qa-4 was greatly decreased, whereas Qa-2 expression was not decreased. However, an anti-Qa-2 monoclonal antibody blocked the binding of an anti-Qa-4 monoclonal antibody to resting cells. These studies established that Qa-4 is a determinant restricted to resting cells, which is closely associated on the surface with the Qa-2 molecule. ^

Characterization of humoral immune responses against Treponema pallidum antigens

The spirochete Treponema pallidum subsp. pallidum is the causative agent of syphilis, a sexually transmitted disease with an estimated 12 million new cases per year worldwide. There is no vaccine currently available for the prevention of syphilis. In the present study, the T. pallidum hypothetical protein TP0693 was examined to determine its cellular location, and its potential for use as a vaccine candidate and immunodiagnostic for syphilis. TP0693 was demonstrated to be strongly reactive with sera from rabbits infected experimentally with T. pallidum for >25 days. Results from proteinase K digestion, immunofluorescence and immunoelectron microscopy were consistent with outer surface localization of TP0693. Serum reactivity against TP0693 was detected in only 68% of syphilis patients, which does not support its use as an immunodiagnostic for syphilis. Immunization of rabbits with TP0693 or three other outer membrane candidates did not alter the course of lesion development atter T. pallidum inoculation. We also examined the T. pallidum proteome by two-dimensional gel electrophoresis coupled with mass spectrometry analysis and immunoblotting. This approach resulted in the identification of 95 unique polypeptides, several of which were reactive with sera from infected rabbits and syphilis patients. The analyses described here enabled us to identify antigens potentially useful as vaccine candidates or diagnostic markers, and may provide insight into host-pathogen interactions during T. pallidum infection. ^

Effects of increased CO2 on fish gill and plasma proteome

Ocean acidification and warming are both primarily caused by increased levels of atmospheric CO2, and marine organisms are exposed to these two stressors simultaneously. Although the effects of temperature on fish have been investigated over the last century, the long-term effects of moderate CO2 exposure and the combination of both stressors are almost entirely unknown. A proteomics approach was used to assess the adverse physiological and biochemical changes that may occur from the exposure to these two environmental stressors. We analysed gills and blood plasma of Atlantic halibut (Hippoglossus hippoglossus) exposed to temperatures of 12°C (control) and 18°C (impaired growth) in combination with control (400 µatm) or high-CO2 water (1000 µatm) for 14 weeks. The proteomic analysis was performed using two-dimensional gel electrophoresis (2DE) followed by Nanoflow LC-MS/MS using a LTQ-Orbitrap. The high-CO2 treatment induced the up-regulation of immune system-related proteins, as indicated by the up-regulation of the plasma proteins complement component C3 and fibrinogen beta chain precursor in both temperature treatments. Changes in gill proteome in the high-CO2 (18°C) group were mostly related to increased energy metabolism proteins (ATP synthase, malate dehydrogenase, malate dehydrogenase thermostable, and fructose-1,6-bisphosphate aldolase), possibly coupled to a higher energy demand. Gills from fish exposed to high-CO2 at both temperature treatments showed changes in proteins associated with increased cellular turnover and apoptosis signalling (annexin 5, eukaryotic translation elongation factor 1 gamma, receptor for protein kinase C, and putative ribosomal protein S27). This study indicates that moderate CO2-driven acidification, alone and combined with high temperature, can elicit biochemical changes that may affect fish health.

The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo

Abf2p is a high mobility group (HMG) protein found in yeast mitochondria that is required for the maintenance of wild-type (ρ+) mtDNA in cells grown on fermentable carbon sources, and for efficient recombination of mtDNA markers in crosses. Here, we show by two-dimensional gel electrophoresis that Abf2p promotes or stabilizes Holliday recombination junction intermediates in ρ+ mtDNA in vivo but does not influence the high levels of recombination intermediates readily detected in the mtDNA of petite mutants (ρ−). mtDNA recombination junctions are not observed in ρ+ mtDNA of wild-type cells but are elevated to detectable levels in cells with a null allele of the MGT1 gene (Δmgt1), which codes for a mitochondrial cruciform-cutting endonuclease. The level of recombination intermediates in ρ+ mtDNA of Δmgt1 cells is decreased about 10-fold if those cells contain a null allele of the ABF2 gene. Overproduction of Abf2p by ≥ 10-fold in wild-type ρ+ cells, which leads to mtDNA instability, results in a dramatic increase in mtDNA recombination intermediates. Specific mutations in the two Abf2p HMG boxes required for DNA binding diminishes these responses. We conclude that Abf2p functions in the recombination of ρ+ mtDNA.

A novel amplicon at 8p22–23 results in overexpression of cathepsin B in esophageal adenocarcinoma

Cathepsin B (CTSB) is overexpressed in tumors of the lung, prostate, colon, breast, and stomach. However, evidence of primary genomic alterations in the CTSB gene during tumor initiation or progression has been lacking. We have found a novel amplicon at 8p22–23 that results in CTSB overexpression in esophageal adenocarcinoma. Amplified genomic NotI–HinfI fragments were identified by two-dimensional DNA electrophoresis. Two amplified fragments (D4 and D5) were cloned and yielded unique sequences. Using bacterial artificial chromosome clones containing either D4 or D5, fluorescent in situ hybridization defined a single region of amplification involving chromosome bands 8p22–23. We investigated the candidate cancer-related gene CTSB, and potential coamplified genes from this region including farnesyl-diphosphate farnesyltransferase (FDFT1), arylamine N-acetyltransferase (NAT-1), lipoprotein lipase (LPL), and an uncharacterized expressed sequence tag (D8S503). Southern blot analysis of 66 esophageal adenocarcinomas demonstrated only CTSB and FDFT1 were consistently amplified in eight (12.1%) of the tumors. Neither NAT-1 nor LPL were amplified. Northern blot analysis showed overexpression of CTSB and FDFT1 mRNA in all six of the amplified esophageal adenocarcinomas analyzed. CTSB mRNA overexpression also was present in two of six nonamplified tumors analyzed. However, FDFT1 mRNA overexpression without amplification was not observed. Western blot analysis confirmed CTSB protein overexpression in tumor specimens with CTSB mRNA overexpression compared with either normal controls or tumors without mRNA overexpression. Abundant extracellular expression of CTSB protein was found in 29 of 40 (72.5%) of esophageal adenocarcinoma specimens by using immunohistochemical analysis. The finding of an amplicon at 8p22–23 resulting in CTSB gene amplification and overexpression supports an important role for CTSB in esophageal adenocarcinoma and possibly in other tumors.

Identification of a new mammalian centrin gene, more closely related to Saccharomyces cerevisiae CDC31 gene

Among the numerous centrin isoforms identified by two-dimensional gel electrophoresis in human cells, an acidic and slow-migrating isoform is particularly enriched in a centrosome fraction. We report here that this isoform specifically reacts with antibodies raised against Saccharomyces cerevisiae Cdc31p and is present, as other centrin isoforms, in the distal lumen of centrioles. It is encoded by a new centrin gene, which we propose to name HsCEN3 (Homo sapiens centrin gene 3). This gene is more closely related to the yeast CDC31 gene, and shares less identity with algae centrin than HsCEN1 and HsCEN2. A murine CDC31-related gene was also found that shows 98% identity and 100% similarity with HsCEN3, demonstrating a higher interspecies conservation than the murine centrin gene MmCEN1 (Mus musculus centrin gene 1) with either HsCEN1, or HsCEN2. Finally, immunological data suggest that a CDC31-related gene could exist in amphibians and echinoderms as well. All together, our data suggest the existence of two divergent protein subfamilies in the current centrin family, which might be involved in distinct centrosome-associated functions. The possible implication of this new mammalian centrin gene in centrosome duplication is discussed.

Short DNA Fragments without Sequence Similarity Are Initiation Sites for Replication in the Chromosome of the Yeast Yarrowia lipolytica

We have previously shown that both a centromere (CEN) and a replication origin are necessary for plasmid maintenance in the yeast Yarrowia lipolytica (Vernis et al., 1997). Because of this requirement, only a small number of centromere-proximal replication origins have been isolated from Yarrowia. We used a CEN-based plasmid to obtain noncentromeric origins, and several new fragments, some unique and some repetitive sequences, were isolated. Some of them were analyzed by two-dimensional gel electrophoresis and correspond to actual sites of initiation (ORI) on the chromosome. We observed that a 125-bp fragment is sufficient for a functional ORI on plasmid, and that chromosomal origins moved to ectopic sites on the chromosome continue to act as initiation sites. These Yarrowia origins share an 8-bp motif, which is not essential for origin function on plasmids. The Yarrowia origins do not display any obvious common structural features, like bent DNA or DNA unwinding elements, generally present at or near eukaryotic replication origins. Y. lipolytica origins thus share features of those in the unicellular Saccharomyces cerevisiae and in multicellular eukaryotes: they are discrete and short genetic elements without sequence similarity.

Proteomic analysis of the bacterial cell cycle

A global approach was used to analyze protein synthesis and stability during the cell cycle of the bacterium Caulobacter crescentus. Approximately one-fourth (979) of the estimated C. crescentus gene products were detected by two-dimensional gel electrophoresis, 144 of which showed differential cell cycle expression patterns. Eighty-one of these proteins were identified by mass spectrometry and were assigned to a wide variety of functional groups. Pattern analysis revealed that coexpression groups were functionally clustered. A total of 48 proteins were rapidly degraded in the course of one cell cycle. More than half of these unstable proteins were also found to be synthesized in a cell cycle-dependent manner, establishing a strong correlation between rapid protein turnover and the periodicity of the bacterial cell cycle. This is, to our knowledge, the first evidence for a global role of proteolysis in bacterial cell cycle control.

Molecular Cloning and Expression Analysis of the Mitochondrial Pyruvate Dehydrogenase from Maize1

Four cDNAs, one encoding an α-subunit and three encoding β-subunits of the mitochondrial pyruvate dehydrogenase, were isolated from maize (Zea mays L.) libraries. The deduced amino acid sequences of both α- and β-subunits are approximately 80% identical with Arabidopsis and pea (Pisum sativum L.) homologs. The mature N terminus was determined for the β-subunit by microsequencing the protein purified from etiolated maize shoot mitochondria and was resolved by two-dimensional gel electrophoresis. This single isoelectric species comprised multiple isoforms. Both α- and β-subunits are encoded by multigene families in maize, as determined by Southern-blot analyses. RNA transcripts for both α- and β-subunits were more abundant in roots than in young leaves or etiolated shoots. Pyruvate dehydrogenase activity was also higher in roots (5-fold) compared with etiolated shoots and leaves. Both subunits were present at similar levels in all tissues examined, indicating coordinated gene regulation. The protein levels were highest in heterotrophic organs and in pollen, which contained about 2-fold more protein than any other organ examined. The relative abundance of these proteins in nonphotosynthetic tissues may reflect a high cellular content of mitochondria, a high level of respiratory activity, or an extra plastidial requirement for acetate.

Evidence that the 50-kDa substrate of brefeldin A-dependent ADP-ribosylation binds GTP and is modulated by the G-protein beta gamma subunit complex.

Brefeldin A, a fungal metabolite that inhibits membrane transport, induces the mono(ADP-ribosyl)ation of two cytosolic proteins of 38 and 50 kDa as judged by SDS/PAGE. The 38-kDa substrate has been previously identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We report that the 50-kDa BFA-induced ADP-ribosylated substrate (BARS-50) has native forms of 170 and 130 kDa, as determined by gel filtration of rat brain cytosol, indicating that BARS-50 might exist as a multimeric complex. BARS-50 can bind GTP, as indicated by blot-overlay studies with [alpha-32P]GTP and by photoaffinity labeling with guanosine 5'-[gamma-32P] [beta,gamma-(4-azidoanilido)]triphosphate. Moreover, ADP-ribosylation of BARS-50 was completely inhibited by the beta gamma subunit complex of G proteins, while the ADP-ribosylation of GAPDH was unmodified, indicating that this effect was due to an interaction of the beta gamma complex with BARS-50, rather than with the ADP-ribosylating enzyme. Two-dimensional gel electrophoresis and immunoblot analysis shows that BARS-50 is a group of closely related proteins that appear to be different from all the known GTP-binding proteins.

In vivo estradiol-dependent dephosphorylation of the repressor MDBP-2-H1 correlates with the loss of in vitro preferential binding to methylated DNA.

We have previously shown that estradiol treatment of roosters resulted in a rapid loss of binding activity of the repressor MDBP-2-H1 (a member of the histone H1 family) to methylated DNA that was not due to a decrease in MDBP-2-H1 concentration. Here we demonstrate that MDBP-2-H1 from rooster liver nuclear extracts is a phosphoprotein. Phosphoamino acid analysis reveals that the phosphorylation occurs exclusively on serine residues. Two-dimensional gel electrophoresis and tryptic phosphopeptide analysis show that MDBP-2-H1 is phosphorylated at several sites. Treatment of roosters with estradiol triggers a dephosphorylation of at least two sites in the protein. Phosphatase treatment of purified rooster MDBP-2-H1 combined with gel mobility shift assay indicates that phosphorylation of MDBP-2-H1 is essential for the binding to methylated DNA and that the dephosphorylation can occur on the protein bound to methylated DNA causing its release from DNA. Thus, these results suggest that in vivo modification of the phosphorylation status of MDBP-2-H1 caused by estradiol treatment may be a key step for the down regulation of its binding to methylated DNA.

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

The las and rhl quorum sensing (QS) systems regulate the expression of several genes in response to cell density changes in Pseudomonas aeruginosa. Many of these genes encode surface-associated or secreted virulence factors. Proteins from stationary phase culture supernatants were collected from wild-type and P. aeruginosa PAO1 mutants deficient in one or more of the lasRI, rhIRI and vfr genes and analysed using two-dimensional gel electrophoresis. All mutants released significantly lower amounts of protein than the wild-type. Protein spot patterns from each strain were compared using image analysis and visible spot differences were identified using mass spectrometry. Several previously unknown OS-regulated proteins were characterized, including an aminopeptidase (PA2939), an endoproteinase (PrpL) and a unique 'hypothetical' protein (PA0572), which could not be detected in the culture supernatants of Delta/as mutants, although they were unaffected in Deltarhl mutants. Chitin-binding protein (CbpD) and a hypothetical protein (PA4944) with similarity to host factor I (HF-1) could not be detected when any of the lasRI or rhIRI genes were disrupted. Fourteen proteins were present at significantly greater levels in the culture supernatants of OS mutants, suggesting that QS may also negatively control the expression of some genes. Increased levels of two-partner secretion exoproteins (PA0041 and PA4625) were observed and may be linked to increased stability of their cognate transporters in a CS-defective background. Known QS-regulated extracellular proteins, including elastase (lasB), LasA protease (lasA) and alkaline metalloproteinase (aprA) were also detected.

Measurement of free drug and clinical end-point by high-performance liquid chromatography-mass spectrometry: Applications and implications for pharmacokinetic and pharmacodynamic studies

Free drug measurement and pharmacodymanic markers provide the opportunity for a better understanding of drug efficacy and toxicity. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) is a powerful analytical technique that could facilitate the measurement of free drug and these markers. Currently, there are very few published methods for the determination of free drug concentrations by HPLC-MS. The development of atmospheric pressure ionisation sources, together with on-line microdialysis or on-line equilibrium dialysis and column switching techniques have reduced sample run times and increased assay efficiency. The availability of such methods will aid in drug development and the clinical use of certain drugs, including anti-convulsants, anti-arrhythmics, immunosuppressants, local anaesthetics, anti-fungals and protease inhibitors. The history of free drug measurement and an overview of the current HPLC-MS applications for these drugs are discussed. Immunosuppressant drugs are used as an example for the application of HPLC-MS in the measurement of drug pharmacodynamics. Potential biomarkers of immunosuppression that could be measured by HPLC-MS include purine nucleoside/nucleotides, drug-protein complexes and phosphorylated peptides. At the proteomic level, two-dimensional gel electrophoresis combined with matrix-assisted laser desorption/ionisation time-of-flight (TOF) MS is a powerful tool for identifying proteins involved in the response to inflammatory mediators. (C) 2003 Elsevier Science B.V. All rights reserved.