Thyroid hormones induce cell proliferation and survival in ovarian granulosa cells COV434

Numerous evidences indicate that thyroid hormones exert an important role in the regulation of the reproductive system in the adult female. Although a clear demonstration of the thyroid–ovarian interaction is still lacking, it is conceivable that thyroid hormones might have a direct role in ovarian physiology via receptors in granulosa cells. In this study we analyzed if thyroid hormone treatment could affect cell proliferation and survival of COV434 cells. To this aim cell growth experiments and cell cycle analyses by flow cytometry were performed. Secondly the T₃ survival action was tested by TUNEL assay and MD30 cleavage analysis. We showed that T₃, and not T₄, can protect ovarian granulosa cells COV434 from apoptosis, regulating cell cycle and growth in the same cells. The increase in cell growth resulted in an augmented percentage of the cells in the S phase and, in a reduction of the doubling time (18%). Subsequently apoptotic pathway induced by serum deprivation has been evaluated in the cells exposed or not to thyroid hormone treatment. The T₃ treatment was able to remarkably counteract the apoptotic process. Even at the ultrastructural level there was an evident protective effect of T₃ in the cells that, besides the maintenance of the original morphology and, the absence of basophilic cytoplasm, conserved normal junctional areas. Furthermore, the protective T₃ effect evaluated by FACS analysis in the presence of a PI3K inhibitor revealed, as also confirmed by Western Blot on pAkt, that the PI3K pathway is crucial in T₃ survival action. J. Cell. Physiol. 221: 242–253, 2009. © 2009 Wiley‐Liss, Inc     

A label‐free internal standard method for the differential analysis of bioactive lupin proteins using nano HPLC‐Chip coupled with Ion Trap mass spectrometry

Quantitative proteomics based on MS is useful for pointing out the differences in some food proteomes relevant to human nutrition. Stable isotope label‐free (SIF) techniques are suitable for comparing an unlimited number of samples by the use of relatively simple experimental workflows. We have developed an internal standard label‐free method based on the intensities of peptide precursor ions from MS/MS spectra, collected in data dependent runs, for the simultaneous qualitative characterization and relative quantification of storage proteins of Lupinus albus seeds in protein extracts of four lupin cultivars (cv Adam, Arés, Lucky, Multitalia). The use of an innovative microfluidic system, the HPLC‐Chip, coupled with a classical IT mass spectrometer, has allowed a complete qualitative characterization of all proteins. In particular, the homology search mode has permitted to identify single amino acid substitutions in the sequences of vicilins (β‐conglutin precursor and vicilin‐like protein). The MS/MS sequencing of substituted peptides confirms the high heterogeneity of vicilins according to the peculiar characteristics of the vicilin‐encoding gene family. Two suitable bioinformatics parameters were optimized for the differential analyses of the main bioactive proteins: the “normalized protein average of common reproducible peptides” (N‐ACRP) for γ‐conglutin, which is a homogeneous protein, and the “normalized protein mean peptide spectral intensity” (N‐MEAN) for the highly heterogenous class of the vicilins.     

Sex differences in neuroactive steroid levels in the nervous system of diabetic and non-diabetic rats

Neuropathy and encephalopathy represent important complications of diabetes. Recent observations obtained in experimental models have suggested that, in male rats, neuroactive steroids are protective agents and that their levels in peripheral (PNS) and central (CNS) nervous system are strongly affected by the disease.It is interesting to highlight that incidence, progression and severity of diabetic neuropathy and diabetic encephalopathy are different in the two sexes. Consequently, it is important to determine the changes in neuroactive steroid levels in the PNS and the CNS of both males and females. To this aim, we have evaluated the levels of neuroactive steroids such as, pregnenolone, progesterone and its metabolites, testosterone and its metabolites, and dehydroepiandrosterone in different CNS regions (i.e., cerebral cortex, cerebellum and spinal cord) and in the sciatic nerve of control and diabetic (i.e., induced by streptozotocin) male and female rats. Data obtained by liquid chromatography-tandem mass spectrometry indicate that the levels of neuroactive steroids show sex and regional differences in control animals. Streptozotocin-induced diabetes resulted in a strong general decrease in neuroactive steroid levels, in both the PNS and the CNS. In addition, the effects of diabetes on neuroactive steroid levels also show sex and regional differences.These findings may have strong implications for the development of new sex-oriented therapies for the treatment of diabetic neuropathy and diabetic encephalopathy, based on the use of neuroactive steroids.     

Glucoraphanin, the bioprecursor of the widely extolled chemopreventive agent sulforaphane found in broccoli, induces phase-I xenobiotic metabolizing enzymes and increases free radical generation in rat liver

Epidemiological and animal studies linking high fruit and vegetable consumption to lower cancer risk have strengthened the belief that long-term administration of isolated naturally occurring dietary constituents could reduce the risk of cancer. In recent years, metabolites derived from phytoalexins, such as glucoraphanin found in broccoli and other cruciferous vegetables (Brassicaceae), have gained much attention as potential cancer chemopreventive agents. The protective effect of these micronutrients is assumed to be due to the inhibition of Phase-I carcinogen-bioactivating enzymes and/or induction of Phase-II detoxifying enzymes, an assumption that still remains uncertain. The protective effect of glucoraphanin is thought to be due to sulforaphane, an isothiocyanate metabolite produced from glucoraphanin by myrosinase. Here we show, in rat liver, that while glucoraphanin slightly induces Phase-II enzymes, it powerfully boosts Phase-I enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), nitrosamines and olefins. Induction of the cytochrome P450 (CYP) isoforms CYP1A1/2, CYP3A1/2 and CYP2E1 was confirmed by Western immunoblotting. CYP induction was paralleled by an increase in the corresponding mRNA levels. Concomitant with this Phase-I induction, we also found that glucoraphanin generated large amount of various reactive radical species, as determined by electron paramagnetic resonance (EPR) spectrometry coupled to a radical-probe technique. This suggests that long-term uncontrolled administration of glucoraphanin could actually pose a potential health hazard.     

Free and conjugated polyamines during de novo floral and vegetative bud formation in thin cell layers of tobacco

The concentrations of three classes of polyamines, trichloroacetic acid-soluble (free), TCA-soluble conjugated (to small molecules) and TCA-insoluble conjugated (to macromolecules), was examined during de novo floral and vegetative bud formation in thin cell layers of Nicotiana tabacum L. cv. Samsun. Explants (consisting of 5–6 layers of epidermal, subepidermal and parenchyma cells) were excised either from floral pedicels or from stem internodes at the unripe fruit stage and cultured on the same medium. In the former, the first de novo formed flower buds appeared on day 8 of culture, while in the latter the first vegetative domes appeared on day 10. In both cases the number of floral and vegetative buds increased up to day 12 and 15, respectively. Changes in dry weight were determined throughout the culture period. Free and conjugated putrescine titer increased 5–60 times in both types of culture and in the three classes of polyamines examined; spermidine content also increased, while spermine, when present, did not show significant changes. TCA-soluble conjugated polyamines were most abundant, being about 2-fold the TCA-insoluble conjugated ones and 10-fold the free ones. The major increment in putrescine and spermidine content occurred in stem internode explants developing vegetative buds. In pedicel explants the maximum putrescine level was reached before or on day 8 in culture (emergence of the first flower buds with calyx initials), while in stem internode explants the maximum level was reached on day 12, at the emergence of the first vegetative buds with leaf primordia. While spermidine prevailed on day 0, putrescine was the most abundant polyamine during both differentiation processes. The putrescine content rapidly increased immediately after the onset of culture. Thus conjugated polyamines, especially putrescine, and not only the free ones, seem to be involved in both the reproductive and vegetative phases of tobacco growth and development.     

Antimicrobial activity of protoanemonin,a lactone from ranunculaceous plants

Protoanemonin, a component of Ranunculus bulbosus, was tested as an antifungal agent on selected strains of dermatophytes and yeasts. The minimum inhibitory concentrations ranged from 2.0 to 7.5×10^–4 M and the minimum lethal concentrations from 3.8×10^–4 M to >1.0×10^–3 M. The most sensitive dermatophyte tested was Epidermophyton floccosum, and the most sensitive yeast Rhodotorula glutinis. The effects of different culture media and of light on the sensitivity of Rhodotorula glutinis to protoanemonin were also tested. Structural analogies between protoanemonin and other cytotoxic unsaturated lactones, and the reversal by the amino acid cysteine of the antifungal action suggest a possible mechanism of action.     

Kinetic properties of gel-immobilized acid phosphatase in a tubular membrane reactor

Summary Acid phosphatase has been immobilized onto the internal surface of tubular ultrafiltration membranes by two different methods, namely copolymerization/gelation and co-gelation. Rate parameters for p-nitrophenyl phosphate hydrolysis by the enzyme in both gel-immobilization conditions have been determined and compared to the corresponding values obtained in previous work using a flat ultrafiltration membrane. Results indicate that the kinetic properties of the enzyme seems not substantially modified by the membrane geometry; however, for industrial purposes an enzyme reactor equipped with tubular membranes should be preferred.