Sex chromosome analysis in Turner Syndrome by a pentaplex PCR assay.

In this study, we describe a pentaplex PCR to determine the parental origin of the X chromosome and the presence of mosaicism, via amplification of four polymorphic markers located along the X chromosome (DXS10011, DXS6807, HUMARA, DXS101) and the X-Y amelogenin marker, in 41 families having a daughter with Turner Syndrome. Our results confirmed the cytogenetic findings and we found that the parental origin of the single X chromosome to be maternal in 84% of cases.

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.

The DRPLA CAG repeats in an Italian population sample: evaluation of the polymorphism for forensic applications.

The DRPLA CAG repeats polymorphism has been studied in an Italian population sample. PCR amplification, manual PAGE and silver staining were employed. A total of 16 different alleles, spanning the range from 5 to 21 CAG triplettes, was observed. The heterozygosity was 0.81 and no significant deviation from Hardy-Weinberg equilibrium was found 81 meioses from parentage testing were also analyzed and a Mendelian pattern of inheritance was observed in all cases. In addition, we could successfully type DRPLA locus in some forensic specimens, 1 ng of DNA allowing clear definition of alleles. The authors conclude that the DRPLA CAG repeats analysis may be useful for forensic applications.

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.

Excitotoxic increase of xanthine dehydrogenase and xanthine oxidase in the rat olfactory cortex.

Excitotoxic lesions induced by systemic injection of kainic acid, resulted in 2-3-fold increase of xanthine dehydrogenase and xanthine oxidase activities in the rat olfactory cortex 48-72 h after drug administration. A significant increase of the xanthine oxidase/dehydrogenase ratio was also observed at 4 and 48 h post-injection. No similar changes were noticed in the hippocampus. The enhancement of enzyme activity seems to be primarily a consequence of the altered cell composition in damaged area. Free radicals produced by the increased oxygen-dependent form of the enzyme could in turn aggravate the excitotoxic brain injury.

Purification and partial characterization of single-chain ribosome-inactivating proteins from the seeds of Phytolacca dioica L.

Three ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe et al. (1992) Bio/Technology 10, 405-412) were purified from the seeds of Phytolacca dioica. These proteins, called Phytolacca dioica RIPs (PD-S1, PD-S2 and PD-S3 RIPs), are glycoproteins, with M(r) approx. 30,000, inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and depurinate rat liver rRNA in an apparently identical manner as the A-chain of ricin and other RIPs (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Part of the purified rat liver ribosomes appeared resistant to the action of PD-S RIPs. The most abundant protein, PD-S2 RIP, gave a weak or nil cross-reaction with sera against various other RIPs, including a pokeweed antiviral protein from the roots of Phytolacca americana. PD-S2 RIP was linked to a monoclonal antibody (Ber-H2) against the CD30 human lymphocyte antigen and the resulting immunotoxin was selectively toxic to the CD30 + Hodgkin's lymphoma-derived L540 cell line.

Purification and partial characterization of a mitogenic lectin from the latex of Euphorbia marginata.

A lectin was purified from the latex of Euphorbia marginata by affinity chromatography on acid-treated Sepharose 6B and elution with lactose. The lectin is a glycoprotein composed of two identical subunits with M(r) 30,000, approx. The haemagglutinating activity of the lectin is not specific for any human blood group, and is inhibited by galactose and galactose-containing sugars and by gentiobiose. The lectin is strongly mitogenic for human T-lymphocytes and induces the release of interleukin-1 beta and tumor necrosis factor-alpha from cultured mononuclear cells.

Effects of hypoxia and ethanol on xanthine oxidase of isolated rat hepatocytes: conversion from D to O form and leakage from cells.

The combined effects of ethanol and hypoxia on the conversion of xanthine dehydrogenase (D form) to xanthine oxidase (O form) and on the leakage of the enzyme from isolated rat hepatocytes was studied. Time-dependent death of cells occurred during incubation in hypoxic conditions. Ethanol (40 mM) had only a moderate effect on viability in aerobiosis, but accelerated the loss of hypoxic cells, which was 96% after 3 h of incubation. In hypoxic conditions, the xanthine oxidase was gradually converted from D into O form. The conversion was complete in 3 h, and was accelerated by 1 mM xanthine or by ethanol, in a concentration-related manner. Hypoxia brought about a progressive leakage of xanthine oxidase from hepatocytes, which was accelerated by ethanol in a concentration-dependent manner. The enzyme found outside hepatocytes was mostly in its O form. The xanthine oxidase of hepatocytes cytosol was converted from D into O form by human plasma or serum. In all cases the conversion could be completely reverted by treatment of the extract with dithiothreitol.

T lymphocyte killing by a xanthine-oxidase-containing immunotoxin.

We report on the preparation of an immunotoxin consisting of xanthine oxidase, a free-radical-producing enzyme, covalently linked to an anti-CD3 monoclonal antibody. The immunotoxin retained both enzymic and immunological properties and its toxicity to target cells (a) was greater than that of the free enzyme, (b) was proportional to the enzyme concentration, and (c) was reduced either in the absence of hypoxanthine or by an excess of free anti-CD3 monoclonal antibody. The cytotoxicity and selectivity of the hypoxanthine/conjugated xanthine oxidase system were potentiated by the addition of chelated iron and by washing away the unbound immunotoxin prior to the addition of substrate. The same system was not toxic to bone marrow progenitor cells. A possible use of this immunotoxin for the ex vivo purging of organs to be transplanted from T lymphocytes, to avoid the graft-versus-host reaction, is suggested.

Cytotoxicity of, and DNA damage by, active oxygen species produced by xanthine oxidase.

Toxicity to Raji cells of the xanthine oxidase/hypoxanthine system is related to the formation of single-strand DNA breaks. DNA damage was proportional to the concentration of xanthine oxidase and to the time of exposure. It was prevented by the absence of hypoxanthine, or by the presence of allopurinol, or both superoxide dismutase and catalase. The release of 51Cr from damaged cells was detectable 12 h after the inhibition of cloning efficiency and the production of DNA breakage. These data suggest that DNA damage induced by the oxygen products precedes the severe lesion to the cellular membrane.

Purification and properties of new ribosome-inactivating proteins with RNA N-glycosidase activity.

Ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe & Barbieri (1986) FEBS Lett. 195, 1-8) were purified from the seeds of Asparagus officinalis (two proteins, asparin 1 and 2), of Citrullus colocynthis (two proteins, colocin 1 and 2), of Lychnis chalcedonica (lychnin) and of Manihot palmata (mapalmin), from the roots of Phytolacca americana (pokeweed antiviral protein from roots, PAP-R) and from the leaves of Bryonia dioica (bryodin-L). The two latter proteins can be considered as isoforms, respectively, of previously purified PAP, from the leaves of P. americana, and of bryodin-R, from the roots of B. dioica. All proteins have an Mr at approx, 30,000, and an alkaline isoelectric point. Bryodin-L, colocins, lychnin and mapalmin are glycoproteins. All RIPs inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes and alter rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912).

Bone marrow purging by a xanthine oxidase-antibody conjugate.

The selective cytotoxicity of the xanthine oxidase conjugated to an 8A monoclonal antibody recognizing a human plasma cell-associated antigen has been described. The selectivity and the toxicity of the hypoxanthine/conjugated xanthine oxidase system was increased by removing the excess of conjugate and by adding chelated iron. Under these experimental conditions the cytotoxicity of the conjugate exceeded that of free xanthine oxidase by one order of magnitude. The conjugate effectively purged bone marrow from infiltrating neoplastic plasma cells and added target Raji cells, provided blood was removed and bone marrow peroxidases were exhausted. In conditions of purging effectiveness the conjugate had no toxicity to CFU-GM. No toxicity to mice was observed after i.v. injection of xanthine oxidase-antibody conjugate up to 2.9 U/kg body weight. Thus the hypoxanthine/conjugated xanthine oxidase system could be an effective and nontoxic tool for the ex vivo bone marrow purging in multiple myeloma patients for autologous transplantation.

Purification and properties of a new ribosome-inactivating protein with RNA N-glycosidase activity suitable for immunotoxin preparation from the seeds of Momordica cochinchinensis.

A ribosome-inactivating protein similar to those already known (Stirpe and Barbieri (1986) FEBS Lett. 195, 1-8) was purified from the seeds of Momordica cochinchinensis. This protein, for which the name of momorcochin-S is proposed, is a glycoprotein, has an Mr of approx. 30,000, and an alkaline isoelectric point and can be considered as an iso-form of the previously purified momorcochin from the roots of M. cochinchinensis. Momorcochin-S inhibits protein synthesis by a rabbit-reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and alters rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Momorcochin-S was linked to a monoclonal antibody (8A) against human plasma cells, and the resulting immunotoxin was selectively toxic to target cells.

Xanthine oxidase status in ethanol-intoxicated rat liver.

The status of xanthine oxidase in ethanol-induced liver injury has been investigated in the rat, by acute and chronic ethanol treatments. A 38% increase of the enzyme O-form was observed after repeated ethanol administration. Chronic intoxication caused a significant decrease of total xanthine oxidase activity after both prolonged ethanol feeding and life span ethanol ingestion. The intermediate D/O-form of xanthine oxidase (that can act either as an oxidase or as a dehydrogenase, being able to react with O2 as well as with NAD+ as electron acceptor) increased 5.5-fold after prolonged ethanol feeding.

Targeting of a plasma cell line with a conjugate containing xanthine oxidase and the monoclonal antibody 62B1.

We report on the preparation of an antibody-conjugated enzyme consisting of xanthine oxidase, a free-radical-producing enzyme, linked to the 62B1 monoclonal antibody, which recognizes the last steps of differentiation of B cell lineage (plasma cell and hairy cells). The conjugate specifically kills target cells, retaining both enzymic and immunological properties, without any damage to normal myeloid clonogenic efficiency. The model is suitable for ex vivo bone marrow purging in multiple myeloma patients.

Purification and partial characterization of a lectin from the seeds of Trichosanthes kirilowii Maximowicz.

A lectin was purified from the seeds of Trichosanthes kirilowii, belonging to the family Cucurbitaceae, growing in China. The lectin is a glycoprotein of 57 kDa, consists of two subunits with apparent molecular masses of 37 and 25 kDa, is specific for galactose, and is not mitogenic for human lymphocytes.

Selective cytotoxicity of an oxygen-radical-generating enzyme conjugated to a monoclonal antibody.

The monoclonal antibody 8A, which recognizes a human plasma cell-associated antigen, was covalently linked to xanthine oxidase in a conjugate maintaining both immunological and enzymatic properties. A significant degree of target cell lysis was obtained at an enzyme concentration that was ineffective on non-target cells and on myeloid staminal cells (CFU-GM). The cytotoxic activity was abolished by an excess of antibody, by allopurinol and by superoxide dismutase and catalase. A possible use of the conjugate for bone marrow purging in multiple myeloma patients is suggested.

Purification, characterization and biological activity of tulipin, a novel inhibitor of DNA synthesis of plant origin.

A DNA synthesis-inhibiting protein (for which the term tulipin is proposed) was isolated from the bulbs of Tulipa sp. The yield ranged from 3.4 to 4.1 per cent of total protein content of the crude extract. Mr, isoelectric point, neutral and amino sugar and amino acid composition were determined. Inhibition of DNA synthesis varied in intact cells according to the cellular types studied, with a minimum ID 50% (concentration giving 50% inhibition) of 400 ng/ml in neuroblastoma cells. The effect was reversible. No effect was obtained in cell-lysate. RNA and protein synthesis were unaffected. The acute toxicity, evaluated in Swiss mice, gave an LD of 6.1 mg/kg body wt. Results of electron microscopy are also given. A second protein, called tulipin 2, has been isolated and partially characterized.

Bryodin, a ribosome-inactivating protein from the roots of Bryonia dioica L. (white bryony).

Bryodin is a strongly basic (pI greater than or equal to 9.5) glycoprotein (neutral sugar content 6.3%) with Mr 30,000, purified from the roots of Bryonia dioica (white bryony). This protein inhibits protein synthesis by a rabbit reticulocyte lysate with and ID50 (concentration causing 50% inhibition) of 0.12 nM (3.6 ng/ml) and has much less effect on protein synthesis by whole cells, with ID50 values ranging from 46 nM to 2.27 microM (1.4-67 micrograms/ml). Bryodin acts by inactivating ribosomes, with a less-than-equimolar ratio, which suggests a catalytic action. Bryodin decreases the number of local lesions induced by tobacco mosaic virus in the leaves of Nicotiana glutinosa. From all its properties, bryodin can be considered to be a ribosome-inactivating protein, similar to those already known [reviews: Barbieri & Stirpe (1982) Cancer Surveys 1, 489-520; Stirpe & Barbieri (1986) FEBS Lett. 195, 1-8].

Properties of volkensin, a toxic lectin from Adenia volkensii.

Volkensin, a highly toxic protein from the roots of Adenia volkensii (kilyambiti, kinoria), was purified by affinity chromatography on acid-treated Sepharose 6B. The toxin is a glycoprotein (Mr 62,000, neutral sugar content 5.74%) consisting of an A subunit (Mr 29,000) and of a B subunit (Mr 36,000) linked by disulfide and noncovalent bond(s). The amino acid, amino sugar, and neutral sugar composition of the protein were determined. Volkensin is a galactose-specific lectin and is a potent inhibitor of eukaryotic protein synthesis in whole cells as well as in a cell-free system (a rabbit reticulocyte lysate). The inhibitory and the lectin activities are functions of the A and B subunits, respectively. Volkensin can be included amongst the ricin-like toxins and resembles most closely modeccin, the toxin of Adenia digitata.