Biomedical subjects

Fiorenzo Stirpe

Publications and source records attributed to Fiorenzo Stirpe.

23 records · Page 2Linked to original sources

PubMed2002

Ribosome-inactivating and adenine polynucleotide glycosylase activities in Mirabilis jalapa L. tissues.

Several tissues of Mirabilis jalapa L. (Nyctaginaceae) were assayed for inhibition of translation by a rabbit reticulocyte lysate (as a signal of ribosome-inactivating activity) and for adenine DNA glycosylase activity, activities that are both due to the presence of a class of enzymes called ribosome-inactivating proteins (RIPs), currently classified as rRNA N-glycosylases (EC ). These activities were highest in seed; intermediate in flower bud, immature seed, sepal + gynoecium, leaf, and root; and very low in all other tissues. By cation-exchange chromatography, four protein peaks with inhibitory activity on cell-free translation were identified in extracts from seeds, and two proteins were isolated from peaks 1 and 4, all of which have the properties of single-chain type 1 RIP. One is Mirabilis antiviral protein (MAP), so far purified only from roots. The second is a new protein that we propose to call MAP-4. The distribution of MAP and MAP-4 in several tissues was determined with a novel experimental approach based on liquid chromatography/mass spectrometry. The direct enzymatic activity of MAP on several substrates is described here for the first time. MAP depurinated not only rRNA in intact ribosomes, thus inhibiting protein synthesis, but also other polynucleotides such as poly(A), DNA, and tobacco mosaic virus RNA. Autologous DNA was depurinated more extensively than other polynucleotides. Therefore, the enzymatic activity of this protein may be better described as adenine polynucleotide glycosylase activity rather than rRNA N-glycosylase activity. Finally, MAP does not cross-react immunologically with other commonly utilized RIPs.

Adenine↗

PubMed2002

Cloning and expression of cDNA coding for bouganin.

Bouganin is a ribosome-inactivating protein that recently was isolated from Bougainvillea spectabilis Willd. In this work, the cloning and expression of the cDNA encoding for bouganin is described. From the cDNA, the amino-acid sequence was deduced, which correlated with the primary sequence data obtained by amino-acid sequencing on the native protein. Bouganin is synthesized as a pro-peptide consisting of 305 amino acids, the first 26 of which act as a leader signal while the 29 C-terminal amino acids are cleaved during processing of the molecule. The mature protein consists of 250 amino acids. Using the cDNA sequence encoding the mature protein of 250 amino acids, a recombinant protein was expressed, purified and characterized. The recombinant molecule had similar activity in a cell-free protein synthesis assay and had comparable toxicity on living cells as compared to the isolated native bouganin.

Base Sequence↗

PubMed2002

Selective lesions of rabbit extraocular muscles injected with the anti-AChR immunotoxin saporin-mAb 73.

PURPOSE: To evaluate the effects on extraocular muscles of a skeletal muscle-specific immunotoxin, saporin-mAb 73, as an alternative to botulinum toxin to induce a permanent correction of oculo-facial dystonias or some forms of ocular motility disorders. METHODS: An immunotoxin was prepared with a monoclonal antibody (mAb 73) against acetylcholine receptors of skeletal muscle, linked to saporin, a type 1 ribosome-inactivating protein (RIP) from Saponaria officinalis. Sixteen New Zealand white rabbits were treated with a single injection of immunotoxin directly into the medial rectus muscle of one eye. Four different dosages of 2, 5, 20, or 50 ng saporin-mAb 73 were used. The rabbits were sacrificed at two, 7 and 14 days post-injection. The medial rectus muscle and the retractor bulbi muscle of both the injected and the fellow eyes were taken and serial sections were examined by light microscopy in a blinded manner. RESULTS: Saporin-mAb 73, even at the dosage of 2 ng, brought about focal damage in the extraocular muscles of rabbits without histological changes in adjacent muscles. The histological examination revealed necrotic/apoptotic lesions restricted to the sites of inoculation and largely infiltrated by macrophages. No evident inflammatory reaction was detected at any time and neutrophils were substantially absent. At 14 days after injection, necrosis/apoptosis was still evident and the sclerotic reaction was minimal. CONCLUSIONS: The immunotoxin saporin-mAb 73 injections into the extraocular muscles of rabbits caused focal damage to the muscles. There was no significant inflammatory reaction and muscle fiber loss was present even at the lower doses. Although the lesions were followed for only 14 days, our results suggest that saporin-mAb 73 has potential to cause safe focal muscle damage but longer-term follow-up are needed to investigate the persistence of muscle weakness.

Animals↗

PubMed2002

Damage to nuclear DNA induced by Shiga toxin 1 and ricin in human endothelial cells.

Ribosome-inactivating proteins (RIPs) remove a specific adenine from 28S rRNA leading to inactivation of ribosomes and arrest of translation. Great interest as to a possible second physiological substrate for RIPs came from the observation that in vitro RIPs remove adenine from DNA. This paper addresses the problem of nuclear lesions induced by RIPs in human endothelial cells susceptible to the bacterial RIP Shiga toxin 1 and the plant RIP ricin. With both toxins, nuclear DNA damage as evaluated by two independent techniques (alkaline-halo assay and alkaline filter elution) appears early, concomitant with (ricin) or after (Shiga toxin 1) the inhibition of protein synthesis. At this time, the annexin V binding assay, caspase 3 activity, the formation of typical < or = 50 Kb DNA fragments, and changes in morphology associated with apoptosis were negative. Furthermore, a block of translation comparable to that induced by RIPs, but obtained with cycloheximide, did not induce nuclear damage. Such damage is consistent with the enzymatic activity (removal of adenine) of RIPs acting in vitro on RNA-free chromatin and DNA. The results unequivocally indicate that RIPs can damage nuclear DNA in whole cells by means that are not secondary to ribosome inactivation or apoptosis.

Adenine↗

PubMed2002

Xanthine oxidoreductase activity in human liver disease.

OBJECTIVES: The aim of this study was to investigate the level and the form of xanthine oxidoreductase (XOR) in severely diseased human livers, to ascertain whether the modifications of the enzyme activity reported in experimental pathology also occur in human liver disease. METHODS: Total, dehydrogenase, and oxidase activities of XOR were measured in samples of human liver removed for transplantation or partial hepatectomy. Samples included four groups: 1) histologically normal liver tissue, adjacent to metastases from extrahepatic tumors (controls), 2) liver with virus-related cirrhosis; 3) liver with virus-negative cirrhosis, and 4) hepatocellular carcinoma tissue (HCC). RESULTS: The level of total XOR was significantly higher in liver with virus-related cirrhosis, but not in virus-negative cirrhosis, than in controls. In virus-positive cirrhosis, the total XOR activity correlated positively with the level of ALT. The percentage of XOR oxidase activity in cirrhotic liver, regardless of virus infection, correlated positively with aspartate amino-transferase, bilirubin concentration, and partial thromboplastin time, and negatively with prothrombin time. The activity of XOR was significantly lower in HCC than in control tissue or in a nonneoplastic area of the same liver. CONCLUSIONS: Consistent with previous reports in experimental pathology, the level of XOR was increased in cirrhotic liver, in association with viral infection. This increment correlated with ALT, suggesting a relationship between XOR activity and the extent of liver injury caused by viral replication. The percentage of oxidase activity seems to be correlated with tissue damage and consequent liver impairment. The low XOR activity observed in HCC is consistent with reported experimental pathology.

Adult↗