Biomedical subjects
Publications and source records attributed to E Gianazza.
Oxidative stress induces various post-translational modifications (PTM); some are reversible in vivo via enzymatic catalysis. The present paper reviews specific procedures for the detection of oxidative PTM in proteins, most of them including electrophoresis. Main topics are carbonylated and glutathionylated proteins as well as modification of selected amino acids (Cys, Tyr, Met, Trp, Lys).
The plant metabolic response to heavy metal stress is largely unknown. The present investigation was undertaken to examine the influence of different concentrations of potassium dichromate on the Zea mays L. plantlets. A clear effect of chromium on maize plantlets growth and seed germination was observed strating from 100-300 ppm up to 1500 ppm. In this concentration range, chromium uptake was dependent on the concentration in the medium. Metallothioneins, involved in heavy metal binding, were measured by capillary electrophoresis (CE), and showed a dose-response induction. Protein profile analyzed by two-dimensional gel electrophoresis showed differential expression of several proteins. Identification of spots of upregulated proteins was performed by MALDI mass spectrometry. Results showed that proteins induced by heavy metal exposure are principally involved in oxidative stress tolerance or in other stress pathways. Induction of proteins implicated in sugar metabolism was also observed. Identification of factors involved in plant response may lead to a better understanding of the mechanisms involved in cell protection and tolerance. This information could be used to improve agricultural production and environmental quality.
Two-dimensional electrophoresis is usually run on fully reduced samples. Under these conditions even covalently bound oligomers are dissociated and individual polypeptide chains may be fully unfolded by both, urea and SDS, which maximizes the number of resolved components and allows their pI and M(r) to be most accurately evaluated. However, various electrophoretic protocols for protein structure investigation require a combination of steps under varying redox conditions. We review here some of the applications of these procedures. We also present some original data about a few related samples -- serum from four species: Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus -- which we run under fully unreduced and fully reduced conditions as well as with reduction between first and second dimension. We demonstrate that in many cases the unreduced proteins migrate with a better resolution than reduced proteins, mostly in the crowded 'alpha-globulin' area of pI 4.5-6 and M(r) 50-70 kDa.
We have investigated the biological fluids--serum, cerebrospinal fluid, and urine--of three strains of rats; the present data extend our database (also available on-line) and may be of interest for pharmacological and toxicological investigation. Specifically, we have defined reference maps of the major protein components in cerebrospinal fluid and urine. Compartment-specific isoforms were recognized for transferrin and transthyretin. Mass spectrometric data established the cleavage site of the signal peptide and identified the N-terminal blocking group of prostaglandin D synthase from rat cerebrospinal fluid. A previously undescribed member of the family of low molecular mass rat urinary proteins was characterized as containing a sequence similar, but not identical, to the N-terminal region of rat urinary protein-2 (RUP-2), and divergent from RUP-1.
BACKGROUND AND PURPOSE: A high degree of proteinuria has been reported in stroke-prone spontaneously hypertensive rats (SHRSP). We studied the effect of salt loading on the detailed protein pattern of serum and urine in 3 rat strains: Wistar-Kyoto, spontaneously hypertensive rats, and SHRSP, an inbred animal model for a complex form of cerebrovascular disorder resembling the human disease. METHODS: Rats were given a permissive diet and received 1% NaCl in drinking water. The protein pattern in body fluids was assessed over time by 2-dimensional electrophoretic analysis. Brain alterations were monitored by MRI and histology. RESULTS: Several proteins were excreted in urine after weeks of treatment and in advance of stroke: transferrin, hemopexin, albumin, alpha(2)-HS-glycoprotein, kallikrein-binding protein, alpha(1)-antitrypsin, Gc-globulin, and transthyretin. Markers of an inflammatory response, including very high levels of thiostatin, were detected in the serum of SHRSP at least 4 weeks before a stroke occurred. CONCLUSIONS: In SHRSP subjected to salt loading, an atypical inflammatory condition and widespread alterations of vascular permeability developed before the appearance of anomalous features in the brain detected by MRI. Urinary concentrations of each of the excreted serum proteins correlated positively with time before stroke occurred.
BACKGROUND: Chronic myeloid leukemia is caused by a chromosomal translocation that results in an oncogenic fusion protein, Bcr-Abl. Bcr-Abl is a tyrosine kinase whose activity is inhibited by the antineoplastic drug STI571. This drug can cure mice given an injection of human leukemic cells, but treatment ultimately fails in animals that have large tumors when treatment is initiated. We created a mouse model to explore the mechanism of resistance in vivo. METHODS Nude mice were injected with KU812 Bcr-Abl(+) human leukemic cells. After 1 day (no evident tumors), 8 days, or 15 days (tumors >1 g), mice were treated with STI571 (160 mg/kg every 8 hours). Cells recovered from relapsing animals were used for in vitro experiments. Statistical tests were two-sided. RESULTS: Tumors regressed initially in all STI571-treated mice, but all mice treated 15 days after injection of tumor cells eventually relapsed. Relapsed animals did not respond to further STI571 treatment, and their Bcr-Abl kinase activity in vivo was not inhibited by STI571, despite high plasma concentrations of the drug. However, tumor cells from resistant animals were sensitive to STI571 in vitro, suggesting that a molecule in the plasma of relapsed animals may inactivate the drug. The plasma protein alpha1 acid glycoprotein (AGP) bound STI571 at physiologic concentrations in vitro and blocked the ability of STI571 to inhibit Bcr-Abl kinase activity in a dose-dependent manner. Plasma AGP concentrations were strongly associated with tumor load. Erythromycin competed with STI571 for AGP binding. When animals bearing large tumors were treated with STI571 alone or with a combination of STI571 and erythromycin, greater tumor reductions and better long-term tumor-free survival (10 of 12 versus one of 13 at day 180; P:<.001) were observed after the combination treatment. CONCLUSION: AGP in the plasma of relapsed animals binds to STI571, preventing this compound from inhibiting the Bcr/Abl tyrosine kinase. Molecules such as erythromycin that compete with STI571 for binding to AGP may enhance the therapeutic potential of this drug.
Structural properties, and especially the differential stability, of complexes between carbonic anhydrase (CA) and three sulfonamide inhibitors, acetazolamide, dorzolamide and methazolamide, were investigated by spectroscopic and electrophoretic techniques. These included denaturant gradient gel electrophoresis either across a urea or a steady-state transverse sodium dodecyl sulfate (SDS) gradient. Acetazolamide, the smallest and most hydrophilic of the sulfonamides, forms the most stable complex in the presence of urea, whereas dorzolamide, with a bulky and hydrophobic structure, is most stable against the effects of SDS. At pH 7.4, complexes with dorzolamide show minimal changes in mobility across the SDS gradient, as if unaffected by the detergent, both in the presence and in the absence of excess ligand in the gel. When bound to both acetazolamide and methazolamide, on the other hand, CA displays an increase in mobility above 0.05% SDS, lower in the presence than in the absence of excess ligand. The finding of a distinct pattern for the unliganded enzyme, however, suggests the complexes can still retain the ligand, although binding of the surfactant changes their charge density. Under saturating conditions and in the presence of SDS, the surface charge of all complexes is much lower than for unliganded, denatured CA. Circular dichroism (CD) spectra clearly indicate that the increase in secondary structure and the decrease in tertiary structure brought about in CA by the presence of low concentrations of SDS are largely prevented by complexing with the inhibitors. These observations point out peculiar properties of each CA inhibitor, of potential value in the definition of their biological activities and also in the potential development of novel antagonist molecules.
The effect of adjuvant arthritis (AA) on the pattern of rat serum proteins includes the upregulation of haptoglobin, orosomucoid, alpha2-macroglobulin, serine protease inhibitor-3, thiostatin, alpha1-antitrypsin, C-reactive protein, and the downregulation of kallikrein-binding protein, alpha1-inhibitor III, apolipoprotein A-I, alpha2-HS-glycoprotein, albumin, apolipoprotein A-IV, transthyretin and transferrin. Minor changes (+/- 20%) are observed for Gc-globulin, ceruloplasmin, and alpha1-macroglobulin. AA thus grossly resembles the acute inflammatory response elicited by the injection of turpentine, although the changes in the levels of negative acute-phase proteins (APP) are smaller in acute inflammation. Indomethacine and ibuprofen inhibit the effects of arthritis on the synthesis of rat serum proteins in different ways: The former is, on average, three times as effective as the latter. Each drug interferes differently with different proteins. In animals without AA, both nonsteroidal anti-inflammatory drugs (NSAID) mimic the inflammatory pattern to a certain extent, with more effect on the negative than on the positive APPs. Overall, the shifts in serum protein levels parallel changes in inflammatory parameters such as joint swelling and serum interleukin-6 (IL-6) activity. Protein quantitation after two-dimensional electrophoresis (2-DE) reveals some effects of the drugs per se which escape detection by other routine tests.
Human alpha1-microglobulin (alpha1-m; also called protein HC), a glycoprotein belonging to the lipocalin superfamily, was isolated by sequential anion-exchange chromatography and gel filtration from the urine of hemodialized patients and from amniotic fluid collected in the week 16-18 of pregnancy. The carbohydrate chains of the protein purified from the two sources, which are organized in two Asn-linked and one Thr-linked oligosaccharides, were structurally characterized using matrix-assisted laser desorption ionization and electrospray mass spectrometry. The glycans attached to Thr5 are differently truncated NeuHexHexNAc sequences, and O-glycosylation in the amniotic fluid protein is only partial. Asn96 has both diantennary and triantennary structures attached in the case of urinary alpha1-m and only diantennary glycans in the amniotic fluid protein. The main carbohydrate units attached to Asn17 are in both proteins monosialylated and disialylated diantennary glycans. The position of the oligosaccharide chains in a three-dimensional model of the protein, produced using the automated Swiss-Model service, is also discussed.
The activation of LDL receptors was described recently in a human hepatoma cell line (Hep G2) exposed both to alpha + alpha' subunits from 7S soy globulin and to Croksoy(R)70, a commercial isoflavone-poor soy concentrate. To assess the final identity of the peptide(s) putatively responsible for the biochemical effect, experiments were performed in Hep G2 cells, exposed either to synthetic peptides corresponding to specific sequences of 7S soy globulin or to peptides from the in vitro digestion of Croksoy(R)70. Moreover, the ability of the whole 7S globulin, its subunits and whole Croksoy(R)70 to interfere in the apolipoprotein B (apo B) secretion in the medium as well as in sterol biosynthesis was evaluated in the same model. Increased (125)I-LDL uptake and degradation vs. controls were shown after Hep G2 incubation with a synthetic peptide (10(-)(4) mol/L, MW 2271 Da) corresponding to positions 127-150 of the 7S globulin. Cells exposed to Croksoy(R)70 enzyme digestion products showed a more marked up-regulation of LDL receptors vs. controls, compared with vs. Hep G2 cells incubated with undigested Croksoy(R)70. Among soy-derived products, only the 7S globulin inhibited apo B secretion and (14)C-acetate incorporation when tested in Hep G2 cells at a concentration of 1.0 g/L. These findings support the hypothesis that if one or more peptides can reach the liver after intestinal digestion, they may elicit a cholesterol-lowering effect. Moreover, the protein moiety, devoid of isoflavone components, is likely to be responsible for this major biochemical effect of soy protein.
Migration across a stationary pH gradient results in the electrophoretic titration of a protein's dissociable groups. From the resulting curves, some properties of the protein may be derived, including overall amino acid composition and type of mutation between polymorphic variants, as well as range of stability or, for enzymes, of catalytic activity. Analysis with this technique is a stringent purity criterion; other applications allow the study of interacting systems and the planning of chromatographic fractionations based on differences in surface charge.
We describe a site http://users.unimi.it/-ratserum/homeframed.ht ml with clickable maps of serum proteins of control and inflamed rats as well as quantitative data on the expression of such serum proteins under varying physiological and experimental conditions. This information enhances the value of minimally invasive techniques, thus reducing the number of animals to be treated, and eventually sacrificed, in pharmacological/toxicological research projects.
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Self-tolerance, a key feature of the immune system, is still a matter of intense debate. We give here evidence for a peculiar behavior of an antiserum against Mycobacterium tuberculosis chaperonin 10 (m-Cpn10), which could have implications for the mechanism of self-recognition by antibodies against non-self. We show that this antiserum can interact in terms of both inhibition of biological activity and physical association (immunoprecipitation), with the mammalian homologue of m-Cpn10, but only if the bacterial protein is present. Several lines of evidence led us to exclude that the two proteins physically associate to form heterocomplexes: (1) the behavior of the antiserum was not shared by a monoclonal antibody against m-Cpn10; (2) a matrix selective for human Cpn10 (h-Cpn10) did not co-purify m-Cpn10; (3) the distribution pattern in non-denaturing isoelectric focusing of labeled m-Cpn10 was not altered by the presence of the unlabeled h-Cpn10. We conclude therefore that the antiserum against M. tuberculosis Cpn10 also recognizes mammalian Cpn10, with an affinity/avidity regulated by the mycobacterial protein, or by the promotion of hetero-oligomerization. This emergence of self-recognition in the presence of M. tuberculosis Cpn10 could imply a breaking of self-tolerance in situations of infection or vaccination.
In the present investigation, we have identified 56 major spots, or spot rows, corresponding to 22 proteins, in the 2-DE pattern of adult male rats. This was done mainly by applying two complementary techniques, namely immunoblotting and high performance liquid chromatography-mass spectrometry (HPLC-MS) peptide mapping. Glycoproteins were characterized by affinity blotting with six lectins. We have also detailed how rat serum differs from human serum in two main respects: (i) relative abundance of individual proteins, which amounts in some cases to a complete absence in either sample, and (ii) varying molecular parameters for homologous proteins. It was thus possible to establish a first-generation reference map of rat serum proteins, which can be accessed through http://weber.u.washington.edu/ruedilab/aebersold++ +.html. We hope the present database will be a useful reference for the evaluation of changes in serum protein distribution in the course of pharmacological and toxicological studies. The recognition of species-specific proteins appears of special relevance in this respect.
This report complements the database already detailed for serum proteins of healthy adult male rats (P. Haynes et al., Electrophoresis 1998, 19, 1484-1492). The influence on the two-dimensional electrophoresis (2-DE) pattern of certain physiological conditions (sex, age) was studied as well as of changes in thyroid metabolism. We have extended the information about the major components of rat serum by identifying the proteins typical for the response to acute inflammation. Analyzing 27 spots, six proteins not found in control sera could be recognized; migration at overlapping or close positions with five already characterized proteins was observed for some. A compilation of all our rat data can be accessed through: http://weber.u.washington.edu/ruedilab/ aebersold.html.
We describe a new protocol for denaturant gradient gel electrophoresis, namely the migration of proteins across transverse sodium dodecyl sulfate (SDS)-gradients. We show how such gradients may be reproducibly cast, and demonstrate their stability with time once an appropriate SDS reservoir is arranged at the cathode. SDS affects both size and surface charge of the molecules, and influences the secondary and tertiary structure to a variable extent, even in opposite directions. Hence, distinct, sometimes complex, denaturation patterns may be observed for different proteins.
The alpha-estrogen receptor (ER alpha) transcriptional activity can be regulated either by binding to the cognate ligand or by intracellular signaling pathways responsive to a variety of factors acting through cell membrane receptors. Studies carried out in HeLa and COS-1 cells demonstrated that the cross-coupling between estrogen and growth factor receptors is mediated by p21ras and requires phosphorylation of a specific serine residue (Ser 118 in the human ER alpha and Ser 122 in mouse ER alpha) located in the ER alpha N-terminal activation function 1 (AF-1). Likewise, in the SK-N-BE neuroblastoma cell line p21ras is involved in the cross-coupling between insulin and ER alpha receptors. However, in this cell line Ser 122 is not necessary for insulin-dependent activation of unliganded ER alpha. In addition, after insulin activation, the electrophoretic mobility associated to serine hyperphosphorylation of ER alpha in SK-N-BE and in COS-1 cells is different. Our study rules out the possibility of tyrosine phosphorylation in unliganded ER alpha activation by means of transactivation studies of ER alpha tyrosine mutants and analysis of Tyr phosphorylation immunoreactivity. The two cofactors for steroid receptors RIP 140 and SRC-1 do not seem to be specifically involved in the insulin-induced ER alpha transactivation. The present study demonstrates the possibility of an alternative, cell-specific pathway of cross-coupling between intracellular and membrane receptors, which might be of importance for the understanding of the physiological significance of this mode of activation in the nervous system.