Secretion of thiols and disulfide bond formation: retraction.
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Retraction Notice↗
Biomedical subjects
G Fassina
Publications and source records attributed to G Fassina.
TIMP-2 over-expression reduces invasion and angiogenesis and protects B16F10 melanoma cells from apoptosis.
The matrix metalloproteinase (MMP) inhibitor TIMP-2 has a high specificity for gelatinase A/MMP-2. An imbalance between gelatinase A and TIMP-2 in favor of enzymatic activity is linked to the degradation of the extracellular matrix (ECM) associated with several physiologic and pathologic events, including angiogenesis, invasion and metastasis. Since TIMPs are secreted molecules, they have the potential to be used for gene therapy of certain tumors. We transfected B16F10 murine melanoma cells, a highly invasive and metastatic cell line, with an expression vector harboring a cDNA encoding for human TIMP-2. The clones obtained were isolated and examined for TIMP-2 over-expression and changes in tumor cell phenotype. The amount of recombinant TIMP-2 produced correlated with a reduction in invasion. In an in vivo angiogenesis assay, TIMP-2-transfected clones showed reduced levels of blood vessel formation, and in vitro conditioned media from TIMP-2 transfectants showed diminished induction of endothelial cell migration and invasion. TIMP-2 over-expression limited tumor growth in vivo and neoangiogenesis when cells were injected subcutaneously in mice in the presence of Matrigel. However, TIMP-2 overexpressing clones were found to be more resistant to apoptosis than parental and control melanoma cells, while necrosis was increased. Our data confirm the role of TIMP-2 in the down-regulation of metastasis and angiogenesis but indicate a possible involvement in tumor cell survival.
Animals↗
The cAMP analog 8-Cl-cAMP inhibits growth and induces differentiation and apoptosis in retinoblastoma cells.
Retinoblastomas appear to be derived from a multipotential stem cell of the retina, due to alterations of the Rb1 gene. These tumors arise only within a discrete time frame during childhood, prior to terminal differentiation of the retinal precursor cells. Treatment of retinoblastoma cells with certain agents can induce a partial differentiation of cell types resembling those of the mature retina, such as rod and cone photoreceptors, glia, conventional neurons and pigment epithelia. We have tested the effects of 8-Cl-cAMP, a synthetic analog of cAMP which preferentially binds to and activates the RII subunit of protein kinase A on the Y-79 retinoblastoma cell line in vitro. Y-79 cells treated with 8-Cl-cAMP produced short, branching processes and showed a substantial increase in staining for neuron-specific enolase, a marker for conventional neuronal differentiation. In contrast, dibutyryl-cAMP gives a strong increase in the glial marker glial acidic fibrillary protein. Y-79 cell proliferation was strongly inhibited by 8-Cl-cAMP at concentrations as low as 5-25 microM. 8-Cl-cAMP significantly increased the rate of apoptosis of Y-79 cells in a dose-dependent manner. It also modulated expression of the RI regulatory subunit of intracellular cAMP-dependent protein kinase A, which is produced in abnormal quantities by Y-79 cells. A decrease in protein production was observed, with no clear effect on the RI subunit mRNA expression, suggesting that RI regulation occurs post-transcriptionally.
8-Bromo Cyclic Adenosine Monophosphate↗
Cysteine and glutathione secretion in response to protein disulfide bond formation in the ER.
Protein folding in the endoplasmic reticulum (ER) often involves the formation of disulfide bonds. The oxidizing conditions required within this organelle were shown to be maintained through the release of small thiols, mainly cysteine and glutathione. Thiol secretion was stimulated when proteins rich in disulfide bonds were translocated into the ER, and secretion was prevented by the inhibition of protein synthesis. Endogenously generated cysteine and glutathione counteracted thiol-mediated retention in the ER and altered the extracellular redox. The secretion of thiols might link disulfide bond formation in the ER to intra- and intercellular redox signaling.
Animals↗
Astrocytic reaction in long-term resuscitation.
The brain of 34 subjects who died after prolonged resuscitation (more than 20 days) were studied in order to evaluate the possibility of discriminating original cerebral injuries from changes caused by prolonged resuscitation. We focused our attention on the astrocytic reaction and its relationship to the associated lesions. Twelve of the 34 cases showed astrocytosis picture. Of these 5 were anisomorphic and 7 isomorphic. From the analysis of the various morphological findings, taking into account an individual's clinical history, we can conclude that isomorphic astrocytosis is a typical morphological reaction related to resuscitation duration and it is the result of a slow, gradual neuronal degeneration induced by chronic hypoxia. Anisomorphic astrocytosis, on the other hand, can be an indicator of acute and focal cerebral lesion.
Adult↗
Formation of reversible disulfide bonds with the protein matrix of the endoplasmic reticulum correlates with the retention of unassembled Ig light chains.
Exposed thiols act as intracellular retention elements for unassembled secretory molecules. Yet, some free Ig lambda light chains are secreted despite the presence of an unpaired cysteine (Cys214). This is due largely to the presence of a flanking acidic residue: substitution of Asp213 for Gly or Lys increases pre-Golgi retention and degradation of free lambda. Secretion is restored by exogenous reducing agents or by assembly with heavy chains. In the endoplasmic reticulum (ER), lambda chains form covalent complexes with many proteins through Cys214. These complexes are absent from the Golgi. They are more abundant in transfectants expressing the lambdaGly2I3 and lambdaLys213 mutants that are poorly secreted. Radioactive N-ethylmaleimide labels some monomeric lambda chains isolated from the ER, but not from the Golgi or from the medium, indicating that the Cys214 thiol is masked during ER-Golgi transport. Mass spectrometry reveals the presence of a free cysteine residue disulfide-linked to Cys214. We suggest that thiol-mediated retention involves the formation of reversible disulfide bonds with the protein matrix of the ER. The presence of an acidic residue next to the critical cysteine may allow the masking of the thiol and transport to the Golgi.
Base Sequence↗
An expression system for the single-step production of recombinant human amidated calcitonin.
Amidating mouse pituitary cells (AtT-20) have been engineered to secrete human calcitonin (hCT) in the fully active amidated form, without the need of additional enzymatic or chemical modifications. The 141-residue human calcitonin precursor has first been cloned in the eucaryotic expression vector pRc/RSV, and the resulting plasmid pRc/RSV/hCT introduced in AtT-20 cells. After transfection, 122 independent clones resistant to G-418 were selected and screened for calcitonin production using a competitive ELISA specifically designed to detect the amidated form of calcitonin. One of these clones was amplified and showed expression of 17 ng/ml of hCT, with a 70% increase in productivity after cAMP treatment. Calcitonin was partially purified from culture medium by two sequential steps of reverse-phase chromatography and characterized in terms of immunoreactivity and molecular weight by TOF-MALDI mass spectroscopy, which confirmed the intended chemical nature and the presence of the C-terminal amidated residue.
Amino Acid Sequence↗
Kynurenine aminotransferase in hypercholesterolemic rats.
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Animals↗
Effects of N omega-nitro-L-arginine methyl ester on benzodiazepine binding in some limbic areas of hyperlipidaemic rats.
Quantitative autoradiography techniques were used to evaluate the chronic effects of the potent nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester, on the binding pattern of [3H]flunitrazepam (benzodiazepine agonist) in some behaviorally key limbic areas of the genetic hyperlipidaemic Pittsburg Yoshida rat. Administration of this potent synthase inhibitor was capable of supplying higher and moderately higher binding levels in the basolateral amygdala nucleus (+52%) and in the oriens-pyramidalis CA1 hippocampus layer (+38%), respectively. When we tested for the binding changes in the presence of GABA (principal benzodiazepine modulator) we noticed that a physiological concentration (20 microM) of this inhibitory neurotransmitter was sufficient to induce notable changes in other limbic areas. In fact, lower binding values (-65%) were reported for the bed nucleus of stria terminalis whereas moderately higher values (+38%) were obtained for the radiatum-lacunosum molecular CA1 hippocampus layer. From the saturation studies, it was possible to observe that the major receptor variations provoked by the potent synthase inhibitor were not only due to changes in the total number of binding sites because there were variations, as in the case of the basolateral amygdala nucleus, that were instead due to differences in the affinity binding state. These results provide evidences of a GABAergic-nitric oxide synthase inhibitor interaction that might also be involved in the regulation of convulsive, anxiolytic, and aggressive behaviors that are modulated at the benzodiazepine site.
Animals↗
Topological mimicry of cross-reacting enantiomeric peptide antigens.
Rabbit polyclonal antibodies against multimeric peptide antigens were found to cross-react to a significant extent with topologically related variants of the parent antigen, where the chirality of each amino acid residue (inverso derivatives), or the peptide sequence orientation (retro derivatives), was inverted or where both modifications were simultaneously introduced (retro-inverso derivatives). All peptide variants displayed similar recognition properties for antibodies and similar dose-dependent inhibitory effects on the interaction between immobilized parent antigen and corresponding antibodies. Importance of peptide side chain topology on antigenicity was evaluated analyzing the recognition properties of two sequence-simplified parent peptide variants, one lacking of the side chains in the sequence odd position and the other in even position. These two variants, prepared introducing glycine residues alternatively in the parent peptide sequence, were found to cross-react to a significant extent with the original antibody raised against the parent peptide. Analysis of molecular models of peptide enantiomeric variants in the elongated all-trans configuration suggested that the topological equivalence of alternating side chains could lead to the formation of similar recognition surfaces, thus mimicking the parent peptide antigenic structure.
Amino Acid Sequence↗
Invasive phenotype of MCF10A cells overexpressing c-Ha-ras and c-erbB-2 oncogenes.
Infection with erbB-2 (E) of Ha-ras (H) oncogene-transfected cells has been previously shown to cooperatively induce anchorage-independent growth of the MCF10A human mammary epithelial cell line in vitro, but not to induce nude mouse tumorigenicity. Here we show that oncogene-transformed MCF10A are able to halt in the lungs of nude mice, a sign of organ colonization potential. We have therefore studied the transformants for in vitro migratory and invasive properties known to correlate with the metastatic potential of human mammary carcinoma cells in nude mice. MCF10A transfected with Ha-ras, infected with a recombinant retroviral vector containing the human c-erB-2 proto-oncogene (MCF10A-HE cells), show a higher invasive index than either the single transfectant (MCF10A-H) or MCF10A-erB-2(MCF10A-E) cells in the Boyden chamber chemotaxis and chemoinvasion assays. The MCF10A-HE cells also adopted an invasive stellate growth pattern when plated or embedded in Matrigel, in contrast to the spherical colonies formed by the single transformants MCF10A-H, MCF10A-E, and the parental cells. Dot-blot analysis of gelatinase A and TIMP-2 mRNA levels revealed increasing gelatinase A mRNA levels (HE > E > H > MCF10A) and reduced TIMP-2 expression in both single and double transformants. Furthermore, MCF10A-HE cells show more MMP-2 activity than parental MCF10A cells or the single transformants. CD44 analysis revealed differential isoform banding for the MCF10A-HE cells compared to parental cells, MCF10A-H and MCF10A-E, accompanied by increased binding of hyaluronan by the double transformants. Our results indicate that erB-2 and Ha-ras co-expression can induce a more aggressive phenotype in vitro, representative of the malignancy of mammary carcinomas.
Animals↗
Antigenicity of topochemically related peptides.
Antibodies raised in rabbits against multimeric all-L peptides (MAP's) were first made monospecific by affinity chromatography on immobilized antigen columns and then tested for their ability to cross-react with topologically related variants of the parent antigen, where the chirality of each amino-acid residue (inverso derivatives), or the peptide sequence orientation (retro derivatives), was inverted, or where both modifications were simultaneously introduced (retro-inverso derivatives). Retro, inverso, and retro-inverso forms of the parent peptide were prepared, both in the linear as well as in the BSA-conjugated form, and found to cross-react to a significant extent with affinity purified polyclonal antibodies raised against the parent peptide. Peptide variants displayed similar dose-dependent inhibitory effects on the interaction between immobilized parent antigen and affinity purified antibodies. Analysis of molecular models of the peptide variants in the trans-configuration suggested that the topological equivalence of alternating side chains in the series of related peptides may be responsible for the observed cross-recognition, leading to the formation of similar recognition surfaces which could mimic the parent peptide antigenic structure.
Amino Acid Sequence↗
Achalasia and sudden death: a case report.
A 23-year-old pregnant woman was found dead at home. The anamnesis was completely negative, except for a slight dysphagia. The most important autopsy finding was a remarkable previously undiagnosed, megaesophagus. Conjunctival and pleural petechiae were also observed. The most likely etiopathogenetic factors are considered and discussed.
Adult↗
Inhibition of interleukin-2/p55 receptor subunit interaction by complementary peptides.
Complementary peptides to interleukin-2 (IL-2) sequences important for receptor binding were tested for their ability to mimic natural receptors and act as inhibitors of the IL-2/p55 receptor subunit interaction. Peptides hydropathically complementary to IL-2 sequences 15-27 and 40-54 were synthesized in a linear and in a multimeric form and then characterized first by solid-phase binding assays for their ability to interact with IL-2. Binding between the multimeric complementary peptides and biotinylated IL-2 was specific, saturable, and inhibited by linear as well as multimeric complementary peptides. Saturable interactions, characterized by dissociation constants in the micromolar range, occurred also between IL-2 immobilized on microtiter plates and biotinylated linear and multimeric complementary peptides. Peptides corresponding to the IL-2 target sequences were able to interfere with this interaction, as well as full-length IL-2. Peptide recognition was sequence dependent, since scrambling of complementary peptide sequences or IL-2 target peptide sequences abolished binding. Multimeric complementary peptides after immobilization on solid supports proved useful also for affinity purifications of recombinant IL-2 or IL-2 fragments corresponding to the target sites, directly from crude mixtures, in high yield and with high recovery. Complementary peptides to IL-2 sequence 15-27, but not to IL-2 sequence 40-54, in the linear or in the multimeric form, even if with different potency, interfered with the IL-2/p55 receptor subunit interaction in vitro, thus suggesting a possible role of this IL-2 site in receptor recognition.
Amino Acid Sequence↗
Peptide immobilization on calcium alginate beads: applications to antibody purification and assay.
Two different procedures were developed for the non-covalent immobilization of peptide antigens on calcium alginate beads. The antigenic peptide is first synthesized in a multimeric form starting from a polydentate lysine core, and then immobilized on alginate beads (average volume 0.05 ml) by entrapment or simply by non-covalent adsorption. Coupling yields, as monitored by RP-HPLC analysis of the immobilization time course and/or by amino acid analysis of derivatized beads, were close to 1-2 mg of peptide per ml of gel. Immobilization yields were not dependent on the peptide net charge, hydrophobicity or length, but mainly on the extent of peptide multimerization. After immobilization on alginate gel, peptide antigenic properties were fully retained, as clearly demonstrated by the batchwise micropreparative purification of anti-peptide antibodies in good yields and with a high degree of purity, directly from crude sera in a single adsorption-elution step. Derivatized beads were sufficiently stable towards repeated washing-equilibration procedures, allowing very limited peptide leakage from the matrix. Peptide beads were also successfully used for the development of solid-phase immunoassays in test-tubes to characterize the corresponding antibodies, with the immobilization yield and signal-to-noise ratio being greatly enhanced in comparison with other types of conventional supports.
Adsorption↗
Affinity purification of polyclonal antibodies using immobilized multimeric peptides.
The possibility of using multiple antigenic peptides (MAP) not only for the production and characterisation of antibodies but also for their purification by affinity chromatography, has been explored with two different tetrameric MAPs synthesised starting from a tetradentate lysine core. Recognition selectivity and specificity of the multimeric antigens were retained after immobilization on preactivated affinity supports, allowing convenient antibody purification directly from crude sera in a single chromatographic step. Since antibodies raised against MAPs recognise very frequently the N-terminal portion of the peptide antigen, results suggest that only a limited number of peptide chains remains covalently linked to the solid phase, leaving the others uncoupled and free to interact fully with the antibody. Recovery of antibody immunoreactivity from affinity purifications on MAP-columns was much higher than that obtained from columns prepared by immobilizing at the same density the corresponding linear peptide antigen. The purity of thus obtained antibodies is also far superior, as detected by SDS-PAGE analysis. Retention of the multimeric peptide recognition properties for the corresponding antibodies after immobilization on solid supports suggests that production, characterization, and even the affinity purification of anti-peptide antibodies, could be carried out simply and conveniently via the synthesis of a single multimeric antigen, without additional steps.
Amino Acid Sequence↗
Binding of the LXCXE insulin motif to a hexapeptide derived from retinoblastoma protein.
Peptides corresponding to retinoblastoma protein (RB) fragment 649-654 (LFYKKV) were tested for their ability to recognize the LXCXE sequence motif in human papilloma virus type 16E7 protein (HPV-16E7) encompassing E7 residues 21-26 (DLYCYE) and an identical motif in human insulin comprising insulin B-chain residues 16-21 (YLVCGE), respectively. Interaction between these complementary peptide sequences was observed by several approaches, including direct and competitive ELISA as well as affinity chromatography. Moreover, we demonstrated that immobilized RB649-654 displays specific recognition properties towards full-length insulin. Hence, this study provides a first experimental support for the previously anticipated complex formation between insulin and RB.
Amino Acid Sequence↗
Complementary peptides as recognition molecules.
The possibility of designing sequence-directed recognition peptides (complementary peptides) able to non covalently associate target peptides or proteins is one of the most important applications deriving from the Molecular Recognition Theory [MRT]. Complementary peptides can be used widely not only as synthetic ligands for the development of affinity purification strategies to isolate target peptides or proteins from crude sources, but more importantly as peptidyl antagonists to inhibit biologically relevant interactions, or to probe functional sites in proteins and corresponding receptors.
Amino Acid Sequence↗