Laura Buonamici

Toxicity and cytotoxicity of nigrin b, a two-chain ribosome-inactivating protein from Sambucus nigra : comparison with ricin

Abstract Nigrin b, a lectin isolated from the bark of elderberry (Sambucus nigra L.), has structure and enzymatic activity similar to that of ricin and other type 2 ribosome-inactivating proteins (RIPs), and yet is much less toxic to cells and animals. In an attempt to explain this difference, we studied (1) the cytotoxicity of both lectins at 18 and 37 °C, and in the presence of substances interfering with intracellular routing, and (2) the binding of nigrin b to, and its uptake and degradation by HeLa cells, in parallel with ricin. As compared with the latter, (1) less nigrin b was bound and more was degraded by cells, with a resulting lower concentration remaining inside the cells, and (2) there is evidence for a different intracellular routing followed by the two lectins. These results may explain at least partly the different cytotoxicity and consequently the lower toxicity to mice of nigrin b compared with ricin.

Interaction of volkensin with HeLa cells: binding, uptake, intracellular localization, degradation and exocytosis

AbstractAmong two-chain ribosome-inactivating proteins (RIPs), volkensin is the most toxic to cells and animals, and is retrogradely axonally transported in the rat central nervous system, being an effective suicide transport agent. Here we studied the binding, endocytosis, intracellular routeing, degradation and exocytosis of this RIP. The interaction of volkensin with HeLa cells was compared to that of nigrin b, as an example of a type 2 RIP with low toxicity, and of ricin, as a reference toxin. Nigrin b and volkensin bound to cells with comparable affinity (approx. 10-10 M) and had a similar number of binding sites (2 × 105/cell), two-log lower than that reported for ricin. The cellular uptake of volkensin was lower than that reported for nigrin b and ricin. Confocal microscopy showed the rapid localization of volkensin in the Golgi stacks with a perinuclear localization similar to that of ricin, while nigrin b was distributed between cytoplasmic dots and the Golgi compartment. Consistently, brefeldin A, which disrupts the Golgi apparatus, protected cells from the inhibition of protein synthesis by volkensin or ricin, whereas it was ineffective in the case of nigrin b. Of the cell-released RIPs, 57% of volkensin and only 5% of ricin were active, whilst exocytosed nigrin b was totally inactive. Despite the low binding to, and uptake by, cells, the high cytotoxicity of volkensin may depend on (i) routeing to the Golgi apparatus, (ii) the low level of degradation, (iii) rapid recycling and (iv) the high percentage of active toxin remaining after exocytosis.

Ribosome-inactivating lectins with polynucleotide:adenosine glycosidase activity

Lectins from Aegopodium podagraria (APA), Bryonia dioica (BDA), Galanthus nivalis (GNA), Iris hybrid (IRA) and Sambucus nigra (SNAl), and a new lectin‐related protein from Sambucus nigra (SNLRP) were studied to ascertain whether they had the properties of ribosome‐inactivating proteins (RIP). IRA and SNLRP inhibited protein synthesis by a cell‐free system and, at much higher concentrations, by cells and had polynucleotide:adenosine glycosidase activity, thus behaving like non‐toxic type 2 (two chain) RIP. APA and SNAl had much less activity, and BDA and GNA did not inhibit protein synthesis.

Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA).

Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, the lowest detectable amount being 0.03 pmol of enzymatic protein. Thus, the test (i) is sensitive enough to determine xanthine oxidase in human serum, being more sensitive than the spectrophotometric method, (ii) it is more convenient for clinical laboratories than other sensitive tests and (iii) it has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases, and it was found that any concentration below 1 mg/L is in the normal range.

Cytogenetic Investigation of Chordomas of the Skull

We report the first cytogenetic investigation of cranial chordoma. Three cranial chordomas were examined, two of which could be further histopathologically classified as chondroid chordomas. In addition, we have included a case of chordoma of a cervical vertebra to compare the cytogenetic abnormalities. Diagnosis was made at histological and immunohistochemical levels. The three cases of cranial chordoma showed a normal karyotype, while one vertebra showed 46,XY,t(6;11)(q12;q23). Chordomas, particularly those containing cartilage, have to be distinguished from chondrosarcomas of the skull base. Such a distinction is normally based on expression of epithelial markers which usually are lacking in chondrosarcoma. Cytogenetic investigation may eventually prove to be useful in the distinction of the two lesions, if chromosome anomalies are consistently absent in chordoma, although some chondrosarcomas may also present a normal karyotype. Such a distinction has clinical implications because chondroid chordomas show better survival, whereas chondrosarcomas show a propensity to infiltrate the surrounding tissues.

Mammary foam cells. Characterization by immunohistochemistry and in situ hybridization.

Abstract Cells showing abundant, finely vacuolized cytoplasm (foam cells) are found frequently in most benign lesions of the breast and in certain malignant breast tumours. The origin of mammary foam cells (FCs) has not been clarified, and we therefore studied the morphological features of mammary FCs in a series of 50 benign lesions. The FCs were subdivided, on the basis of their distribution into FCs lining the glandular lumina, intraluminal FCs, intraepithelial-pagetoid FCs, and stromal FCs. The lesions were tested with a panel of antibodies against macrophage (MAC 387, CD68) and epithelial (epithelial membrane antigen [EMA], gross cystic disease fluid protein 15 [GCDFP15] and cytokeratin) markers. The lesions were examined for the presence of PIP/GCDFP15-specific mRNA by an in situ hybridization technique. Three different types of FCs were identified. Type A FCs are epithelial cells (positivity with EMA and cytokeratin) and show apocrine differentiation (positivity with GCDFP15 antiserum and expression of PIP/GCDFP15 mRNA). Type B FCs are of macrophage origin, as they are positive with the macrophage markers and lack cytokeratin and PIP/GCDFP15 mRNA. Finally, type C FCs show an intermediate profile between an epithelial cell and a macrophage: they are both CD68 and GCDFP15 positive and show a thin peripheral rim of positivity with anti-cytokeratin antibody. They lack PIP/GCDFP15 mRNA. Our results indicate the possibility of a spectrum of phenotypes in mammary FCs, from epithelial-apocrine cells to macrophage-derived phagocytic cells.

Toxicity of ricin and volkensin, two ribosome-inactivating proteins, to microglia, astrocyte, and neuron cultures

Ricin and volkensin, two potent toxins belonging to the family of ribosome‐ inactivating proteins (RIPs), have been largely exploited in recent years in in vivo experiments of neuronal degeneration consequent to suicide transport or immunolesioning. We have determined both the toxicity of, and the inhibition of, protein synthesis by ricin and volkensin in in vitro cultures enriched in microglial cells, astrocytes, or neurons. In microglial cultures, 50% of toxicity (estimated by LDH released from dead cells) after 24 h exposure to RIPs was obtained with volkensin at 2.2×10−12 M concentration and 50% of protein synthesis inhibition at 2×10−14 M concentration. Both values were higher by about one order of magnitude in astrocyte‐enriched cultures. Toxicity of, and inhibition of, protein synthesis by, ricin were lower for both cell types by about 1 order of magnitude as compared to volkensin. Cerebellar granule neurons in culture survived remarkably well to 24 h exposure to ricin or volkensin, although their protein synthesis was effectively inhibited by the two toxins with a potency similar to that found for astrocytes. These results demonstrate that glial cells, in particular microglia, are very sensitive to RIPs toxicity and should, therefore, be a primary target of these toxins when injected in vivo. Thus, the damage observed after in vivo experiments could be partly related to diffusion of toxic substances from early‐affected glial cells. GLIA 20:203‐209, 1997.

Hepatotoxicity of ricin, saporin or a saporin immunotoxin: xanthine oxidase activity in rat liver and blood serum

Male Wistar rats each received an i.p. injection of the ribosome-inactivating proteins ricin or saporin, or a Ber-H2 (anti-CD30)-saporin immunotoxin at a dose corresponding to three times the LD50 calculated for mice. Animals were killed 24, 48 or 72 h after treatment. Histological examination showed hepatic necrosis in all treated animals, although the sinusoidal lining was affected only in ricin-poisoned rats. The activities of xanthine dehydrogenase (D-form) and oxidase (O form) were determined spectrophotometrically in liver and serum samples. In ricin-treated animals the liver enzyme was progressively converted from the D- to the O-form, which accounted for more than 60% of total activity after 48 h of poisoning, whilst no change in the xanthine oxidase activity was found in the serum. In the liver of rats treated with free or Ber-H2-conjugated saporin, the D-form was more than 75%, as in normal animals. In the same animals the serum xanthine oxidase activity was up to three-fold control values. The determination of serum xanthine oxidase may prove helpful in the evaluation of liver damage in patients treated with immunotoxins. It may become a diagnostic tool for the differential diagnosis of liver diseases.

Intracranial germ cell tumour (embryonal carcinoma with teratoma) with complex karyotype including isochromosome 12p

Abstract We report the chromosomal characteristics of a malignant teratoma with embryonal carcinoma component located in the pineal region of a 15-year-old boy. Chromosome analysis showed a near-triploid complex karyotype (62 chromosomes), including two copies of an isochromosome 12p, confirmed by fluorescence in situ hybridization analysis. The present findings indicate that isochromosome 12p formation is probably associated with the development of malignant germ cell tumours.

Simulated ischaemia-reperfusion conditions increase xanthine dehydrogenase and oxidase activities in rat brain slices

Xanthine dehydrogenase and oxidase activities increased by 87% in rat brain slices after 30 min in vitro ischaemia. A further 41% increase was induced by 30 min simulated reperfusion of ischaemic slices. No conversion from the dehydrogenase to the oxidase activity was observed. The increment of enzyme activity was not due to neosynthesis of the enzyme, since it was not affected by the addition of cycloheximide during the ischaemic incubation. The increased oxygen-dependent form of the enzyme could aggravate the ischaemic brain injury by free radicals production, in particular after reperfusion.