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G Legname

Publications and source records attributed to G Legname.

27 records · Page 2Linked to original sources
PubMed1995

Heterologous expression, purification, activity and conformational studies of different forms of dianthin 30.

Dianthin 30, a ribosome inactivating protein (RIP) from Dianthus caryophyllus, has been expressed in Escherichia coli. Heterologous expression of a deletion mutant dianthin 30 delta 255-270 resulted in the production of a protein identical to carnation mature dianthin 30, including the absence at the carboxy-terminal of a putative 16 amino acid long pro-signal peptide. The production of a form of dianthin 30, which includes the pro-signal, is described as well. Both dianthin 30 delta 255-270 and dianthin 30 expressed in E. coli are mainly localized (90%) in the soluble fraction. Dianthin 30 delta 255-270 and dianthin 30 have been purified to homogeneity and were shown to inhibit protein synthesis in vitro with an IC50 of 8 and of 11 ng/ml, respectively. Secondary structure analysis, carried out by circular dichroism spectroscopy, indicated that the naturally occurring and the recombinant forms of dianthin 30 and dianthin 30 delta 255-270 possess the same secondary structure composition, accounting for an alpha + beta type architecture. RIPs as immunotoxins in clinical trial and as mitotoxins in experimental models have been extremely efficacious. In addition, growing evidence indicates their effective use as antiviral agents, including in HIV-1 infection. These data indicate the ability to produce either chemically linked or recombinant fusion proteins with dianthin 30 and cell-binding ligands for production of new reagents for clinical and experimental use.

Antiviral Agents↗
PubMed1994

Identification and cloning of human chaperonin 10 homologue.

We have identified a heat-shock-inducible 10 kDa protein in the human hepatoma cell line HepG2. The total RNA extracted from the heat-shocked cells was amplified by reverse transcription PCR (polymerase chain reaction) using 21 5' and 18 3' oligonucleotides of rat cpn10 (chaperonin10) cDNA as primers. Sequencing of the above PCR fragment showed a very high homology between human, bovine and rat cpn10 cDNA. The predicted amino acid sequence revealed a 100% identity with the bovine homologue.

Amino Acid Sequence↗
PubMed1993

Expression and activity of pre-dianthin 30 and dianthin 30.

Dianthin 30 is a ribosome inactivating protein (RIP 1) found in different tissues of the carnation (Dianthus caryophyllus). Recently we have isolated and sequenced a cDNA clone from a lambda gt11 expression library [Legname et al. (1991) Biochim. Biophys. Acta 1090, 119-122]. Here we describe specific PCR amplifications of either the full length pre-dianthin 30 or dianthin 30, the mature polypeptide lacking the 23 amino acid signal peptide. In vitro expression of both proteins in reticulocyte lysate generated products of the expected molecular weight. Moreover, the activity of both proteins has been evaluated confirming the characteristics of the natural product. A first attempt to produce recombinant dianthin 30 in Escherichia coli is described.

Animals↗
PubMed1991

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA.

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3' end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A2660, which lies in a sequence that is highly conserved in all species.

Abrin↗
PubMed1991

Nucleotide sequence of cDNA coding for dianthin 30, a ribosome inactivating protein from Dianthus caryophyllus.

Rabbit antibodies raised against dianthin 30, a ribosome inactivating protein from carnation (Dianthus caryophyllus) leaves, were used to identify a full length dianthin precursor cDNA clone from a lambda gt11 expression library. N-terminal amino acid sequencing of purified dianthin 30 and dianthin 32 confirmed that the clone encoded dianthin 30. The cDNA was 1153 basepairs in length and encoded a precursor protein of 293 amino acid residues. The first 23 N-terminal amino acids of the precursor represented the signal sequence. The protein contained a carboxy-terminal region which, by analogy with barley lectin, may contain a vacuolar targeting signal.

Amino Acid Sequence↗
PubMed1990

Motor neuron disease in a patient with a monoclonal IgMk directed against GM1, GD1b, and high-molecular-weight neural-specific glycoproteins.

In a patient with motor neuron disease and benign IgMk monoclonal gammopathy, the M protein reacted with the glycolipids GM1, GD1b, and asialo GM1 and, by immunoblot, with some high-molecular-weight neural-specific glycoproteins. The main reactive bands had an approximate molecular weight of 250 and 400 kd, were most concentrated in the spinal cord, and were also bound by the lectin peanut agglutinin. The presence of the Ga1(beta 1-3)Ga1NAc epitope on these neural-specific glycoproteins may help to explain the selective neurological impairment of the patient.

Gangliosides↗
PubMed1990

Anti-GM1 IgM antibodies in motor neuron disease and neuropathy.

We found anti-GM1 IgM antibodies in 23% of 56 patients with motor neuron disease (MND), in 19% of 69 patients with neuropathy, and in 7% of 107 controls with other neurologic and nonneurologic diseases. Most of these patients had anti-GM1 IgM antibody titers of 1:80 or less; slightly higher antibody titers (up to 1:640) were found in 3 patients, 1 with MND and 2 with neuropathy, and very high titers (1:20,480) in a patient with MND and an IgM kappa M protein that reacted with GM1, GD1b, and asialo GM1. Six other patients with anti-GM1 IgM that also bound to GD1b. Reactivity with GD1b did not correlate with anti-GM1 titers but was only present in patients with MND or neuropathy. Anti-GM1 IgM antibodies may be a normal constituent of the human antibody repertoire but their frequency and, in some cases, their levels are higher in patients with MND and neuropathy. The origin and the pathogenetic role of these antibodies in neural impairment remain to be established.

Antibodies, Antinuclear↗
PubMed1989

Anti-myelin-associated glycoprotein IgM antibody titers in neuropathy associated with macroglobulinemia.

Twenty-seven patients with neuropathy and IgM monoclonal gammopathy were tested for antigen specificity of the M-protein and for anti-myelin-associated glycoprotein (MAG) IgM levels by immunoblot. In 16 patients (59.2%) the M-protein reacted with MAG and with cross-reactive glycoconjugates. Anti-MAG IgM titers in these patients ranged between 1:12,800 and 1:100,000. A fainter IgM reactivity with MAG and related glycoconjugates was detected in 3 additional patients with neuropathy, but also in 8 of 24 patients with IgM M-protein without neuropathy (33.3%). This reactivity was not due to the M-protein and corresponded to antibody titers of 1:400 or less in all but 1 patient with a titer of 1:3,200. Low titers of anti-MAG IgM (1:200 or less) were also detected in 17 of 101 control patients without IgM M-proteins (16.8%), while 1 patient with neuropathy of unknown cause had anti-MAG IgMK titers of 1:25,600. In 1 patient with neuropathy and IgM M-protein that was not anti-MAG, the M-protein bound to other antigens in nerve, while in 6, other possible causes or mechanisms for the neuropathy were found. In this study, high titers of anti-MAG IgM antibodies were always associated with neuropathy. The presence of low levels of anti-MAG IgM in a significant proportion of controls suggests that monoclonal expansion of naturally occurring B-cell clones secreting anti-MAG IgM may be responsible for the high incidence of this antigen specificity of the M-protein.

Adult↗