Giordana Marcozzi

A reinvestigation on the quaternary structure of ascorbate oxidase from Cucurbita pepo medullosa

SummaryThe optical properties, copper content, catalytic activity and quaternary structure of many preparations of ascorbate oxidase purified with two different methods were examined.Fresh samples appeared identical and were characterized by optical ratios A280/A610 = 25 ± 1 and A330/A610 = 0.8 ± 0.05, by specific activity toward ascorbate of 3.48 ± 0.05 mol g−1 min−1 and by a copper content of 8 ± 0.3 mol/145 000 Mr.The enzyme is composed of two non-covalently linked subunits of slightly different molecular mass (75 000 and 72 000 respectively). These subunits cannot be further resolved by reduction of disulfide bonds. Proteolytic cleavage of the protein chains was observed during purification and storage in the absence of the protease inhibitor 6-amino caproic acid.Ascorbate oxidase exists as a monomer at neutral pH and undergoes reversible association into higher molecular weight species at slightly acid pH values. Association is not accompanied by spectroscopic or catalytic changes.

Differential expression of saporin genes upon wounding, ABA treatment and leaf development

Saporins are type 1 ribosome-inactivating proteins (RIPs: EC 3.2.2.22) produced in various organs of Saponaria officinalis L. Two distinct saporin types, saporin-L and saporin-S isoforms, were respectively purified from the intra- and extra-cellular fractions of soapwort leaves. The saporin-L isoform was lowly identical, differed for toxicity, molecular mass and amino acid composition from saporin-S proteins forming a new monophyletic group. Genes encoding both L- and S-type isoforms were cloned from leaf-specific cDNA library; the encoded products included the N-terminal diversity observed by protein sequencing and showed compatible weights with those from mass spectra. These genes were intron-less belonging to small gene families. Reverse transcription polymerase chain reaction/quantitative reverse transcription polymerase chain reaction experiments evidenced their differential expression during leaf development, wounding and abscisic acid treatment. These results suggest that the saporin-L and -S proteins may play diversified roles during stress responses.

Age- and Gender-Related Differences in Human Lacrimal Fluid Peroxidase Activity

There is evidence to suggest that estrogen influences lacrimal fluid peroxidase activity in women. In this study we investigated changes in peroxidase activity related to ageing and gender. These changes might help to explain the common problem of dry-eye syndrome in menopausal women. Unstimulated tears were collected from 70 healthy subjects of both sexes (age range 24–90 years). Tear samples were collected from 9 to 10 a.m., when peroxidase activity remained stable. In women, lacrimal fluid peroxidase activity decreased significantly during the menopause (p < 0.05 by one-way ANOVA), and thereafter remained unchanged. Conversely, in men, lacrimal fluid peroxidase activity decreased later, declining significantly only towards the age of 80 (p < 0.05). Lacrimal fluid peroxidase activity differs in men and women: the gender-related difference accentuates during ageing, probably owing to changing estrogen levels.

A rapid method for the quantification of artemisinin in Artemisia annua L. plants cultivated for the first time in Burundi

We describe a simple, rapid combined method for extracting the antimalarial compound artemisinin from the leaves of Artemisia annua L. cultivated for the first time in Burundi, and quantitating the active principle by high-performance liquid chromatography–electrospray mass spectrometry.

A simple method for the purification of type 1 RIPs.

We describe a straightforward and simple method for obtaining pure and active preparations of type 1 ribosome inactivating proteins (RIPs). The very high isoelectric point values, characteristic of these proteins, allow this purification in a single chromatographic step.

Antiproliferative effect and apoptotic response in vitro of human melanoma cells to liposomes containing the ribosome-inactivating protein luffin

The present study describes the liposome-mediated delivery of the type 1 ribosome-inactivating protein luffin to human melanoma cells in vitro. Luffin from Luffa cylindrica seeds has been successfully incorporated into lecithin/cholesterol and lecithin/cholesterol/dicetylphosphate negatively charged liposomes. The exposure of melanoma cells to the two types of liposomes resulted in the inhibition of protein synthesis and cell growth; apoptotic cell death was verified by means of TUNEL reaction and quantitation of cytosolic oligonucleosome-bound DNA. The toxicity of encapsulated luffin varied with the lipid composition of the vesicles; the strongest effect was observed with lecithin/cholesterol liposomes. These results identify liposome-incorporated luffin as a possible alternative to immunotoxins for the treatment of human melanoma in situ.

Variations of Lacrimal Fluid Peroxidase Activity in Female and Male Rats

Previous research from this laboratory showed that human lacrimal fluid peroxidase has cyclic variations during the menstrual cycle, correlated with plasma levels of 17β-oestradiol. In the present investigation, variations of enzyme activity and total protein content during the oestrous cycle of young adult female rats are analysed. Effects from circadian rhythm and a gender-related influence are also examined. In female rats, as in women, lacrimal fluid peroxidase activity shows cyclic variations; in fact, it significantly (p < 0.05) changes during the different phases of the oestrous cycle. In contrast, in males such variations do not occur. Thus, we suggest that gender seems to exert a significant influence on the secretion of this specific tear protein, probably by a direct effect of oestrogens.

A Rapid Procedure for the Purification of Human Salivary Peroxidase

An improvement to the purification method for human salivary peroxidase is presented. The enzyme is obtained at a higher degree of purity in two chromatographic steps instead of four, and avoiding lyophilation treatment. Differences in electrophoretic pattern confirm the genetic polymorphism of the peroxidase of human saliva.

Role of reduction potentials in copper abstraction from the trinuclear cluster of blue oxidases

Abstract The crucial point in the selective removal of the type 2 Cu from laccase and ascorbate oxidase by chelating agents was found to be the nature of the reducing agent employed in the reaction. No copper was lost when the reductant was either absent or too strong (ascorbate), namely when the trinuclear Cu cluster was respectively fully oxidized or fully reduced. From both proteins, the type 2 Cu was removed only in the presence of a mild reductant such as [Fe(CN) 6 ] 4− , the reduction potential of which could be varied by addition of [Fe(CN) 6 ] 3− and set at an intermediate value between that of type 3 and type 2 Cu ions. In this way the type 3 Cu ions were reduced with labilization of the oxidized type 2 Cu. Also, on prolonged incubation some type 3 Cu was removed from ascorbate oxidase. The applicability of the procedure to caeruloplasmin and fungal laccase is discussed.

Effect of oral contraceptives on lacrimal fluid peroxidase activity in women.

Lacrimal fluid peroxidase has been supposed to be involved in the protection against oxidative damage to the ocular surface. Our recent findings showed the existence of significant cyclic variations in lacrimal fluid peroxidase activity that were positively correlated with those of 17β-estradiol plasma levels throughout the menstrual cycle of fertile women. In the present study lacrimal fluid peroxidase activity of 8 healthy normocyclic women using low-dose oral contraceptives during the monthly cycle was determined. Data showed that low-dose oral contraceptives caused a decrease in lacrimal fluid peroxidase activity and a lack of its cyclic pattern with respect to the enzyme activity of 8 untreated age-matched women. Moreover, this result suggests that lacrimal fluid peroxidase activity could be regulated by estrogen.