Selection of synthetic proteins to modulate the human frataxin function

Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreich's Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigated the possibility of obtaining small proteins exhibiting a high affinity for frataxin. In this study, we applied the ribosome display approach, using human frataxin as the target. We focused on Affi_224, one of the proteins that we were able to select after five rounds of selection. We have studied the interaction between both proteins and discussed some applications of this specific molecular tutor, concerning the modulation of the supercomplex activity. Affi_224 and frataxin showed a K_D value in the nanomolar range, as judged by surface plasmon resonance analysis. Most likely, it binds to the frataxin acidic ridge, as suggested by the analysis of chemical shift perturbations (nuclear magnetic resonance) and computational simulations. Affi_224 was able to increase Cys NFS1 desulfurase activation exerted by the FRDA frataxin variant G130V. Importantly, Affi_224 interacts with frataxin in a human cellular model. Our results suggest quaternary addition may be a new tool to modulate frataxin function in vivo. Nevertheless, more functional experiments under physiological conditions should be carried out to evaluate Affi_224 effectiveness in FRDA cell models.     

Neuroprotection against CCl4 induced brain damage with crocin in Wistar rats

Owing to its lipophilic property, carbon tetrachloride (CCl₄) is rapidly absorbed by both the liver and brain. We investigated the protective effects of crocin against brain damage caused by CCl₄. Fifty rats were divided into five groups of ten: control, corn oil, crocin, CCl₄ and CCl₄ + crocin. CCl₄ administration decreased glutathione (GSH) and total antioxidant status (TAS) levels, and catalase (CAT) activity, while significant increases were observed in malondialdehyde (MDA) and total oxidant status (TOS) levels and superoxide dismutase (SOD) activity. The cerebral cortex nuclear lamina developed a spongy appearance, neuronal degeneration was observed in the hippocampus, and heterochromatic and pyknotic neurons with increased cytoplasmic eosinophilia were observed in the hippocampus after CCl₄ treatment. Because crocin exhibits strong antioxidant properties, crocin treatment increased GSH and TAS levels and CAT activities, and decreased MDA and TOS levels and SOD activity; significant improvements also were observed in histologic architecture. We found that crocin administration nearly eliminated CCl₄ induced brain damage by preventing oxidative stress.     

A partnership project for a new Red List of the Italian Flora

Abstract

The IUCN Red Lists assessment provides an internationally accepted system to verify the extinction risk of species. Working Groups of the Italian Botanical Society have recently discussed the importance of producing a reliable list of species at the national level. This list could be the starting point for future in situ and ex situ plant conservation activities.     

1alpha,25(OH)2D3 and parathyroid hormone (PTH) signaling in rat intestinal cells: activation of cytosolic PLA2

In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to the calciotropic hormones, 1alpha,25,dihydroxy-vitamin D(3) [1alpha,25(OH)(2)D(3)] and PTH. Stimulation of rat enterocytes with either hormone, increased release of arachidonic acid (AA) 3H-AA] one-two fold in a concentration and time-dependent manner. The effect of either hormone on enterocytes was totally reduced by preincubation with the intracellular Ca(2+) chelator BAPTA-AM (5 microM), suggesting that the release of AA following cell exposure to the calciotropic hormones occurs mainly through a Ca(2+)-dependent mechanism involving activation of Ca(2+)-dependent cPLA2. Calciotropic homone stimulation of rat intestinal cells increases cPLA2 phosphorylation (three to four fold). This effect was decreased by PD 98059 (20 microM), a MAP kinase inhibitor, indicating that this action is, in part, mediated through activation of the MAP kinases ERK 1 and ERK2. Enterocytes exposure to 1alpha,25(OH)(2)D(3) (1nM) or PTH (10 nM) also resulted in P-cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase subtrates reside. Collectively, these data suggest that PTH and 1alpha,25(OH)(2)D(3) activate in duodenal cells, a Ca(2+)-dependent cytosolic PLA2 and attendant arachidonic acid release and that this activation requieres prior stimulation of intracellular ERK1/2. 1alpha,25(OH)(2)D(3) and PTH modulation of cPLA2 activity may change membrane fluidity and permeability and thereby affecting intestinal cell membrane function.     

Extracellular fatty acid binding protein (Ex-FABP) modulation by inflammatory agents: "physiological" acute phase response in endochondral bone formation

Ex-FABP, extracellular fatty acid binding protein, is a 21 kDa lipocalin expressed in hypertrophic cartilage, muscle and heart during chick embryo development and in granulocytes. Ex-FABP synthesis was increased in chondrocyte and myoblast cultures by inflammatory agents (LPS; IL6) and repressed by antiinflammatory agents. Expression of Ex-FABP and specific gelatinases is paralleled in hypertrophic cartilage; LPS specifically induced high molecular weight gelatinase ( > 200 kDa). LPS-treated hypertrophic chondrocytes showed increased chemotactic activity for endothelial cells paralleled by increased expression of transferrin. A high amount of Ex-FABP was expressed in adult pathological cartilage both in dyschondroplastic and osteoarthritic chickens. Controls were negative. Ex-FABP could represent a stress protein physiologically expressed in tissues where active remodelling is taking place during development and in tissues characterized by an acute phase response due to pathological conditions. We also suggest that during endochondral bone formation other responses characteristic of a local inflammatory status, such as gelatinase production and angiogenic factor secretion, are "physiologically" activated.     

The use of <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis> mutant cell line for removal of cholesterol from milk

The nonpathogenic ciliate Tetrahymena thermophila converts cholesterol from foodstuffs into provitamin D compounds in high yields. However, prolonged incubation with wild-type strain CU-399 at high densities results in a final deterioration of milk properties, possibly as a result of secreted hydrolases. Here we attempted to solve this problem using MS-1 Tetrahymena strain, a stable mutant with a low rate of hydrolase secretion. Densities of to 2 × 10 ⁶ cells/ml can be incubated for up to 5 h in milk, without any clotting or change in appearance. Moreover, centrifugation of this suspension eliminates most of the cells, and results in an about 75% ± 10 (n = 10) decrease of the initial cholesterol. Sterols are recovered in the cell pellets, which show that Tetrahymena is able to avidly capture them from the medium. Therefore, this mutant strain is optimal for milk cholesterol depletion, avoiding unfavorable sensory alterations.     

A bioreactor model system specifically designed for <Emphasis Type="Italic">Tetrahymena</Emphasis> growth and cholesterol removal from milk

This work describes the configuration and operation of a bioreactor system especially designed for Tetrahymena cultivation and its use for milk improvement, particularly cholesterol elimination by the action of this cell. An advantage of the proposed method is the re-use of the growth medium; thus, the medium is used twice to provide two batches of Tetrahymena biomass without the need of further inoculation. This makes the procedure of producing the cell biomass faster and more economical. Cells are concentrated in the culture vessels by sedimentation at room temperature and then transferred to milk suspensions, where they can further grow for at least one generation with the benefit of reducing steeply cholesterol level. Milk treated according to this process is separated from the biomass by centrifugation. Under these conditions, less than 5% of the cells remain in the milk, and cholesterol elimination amounts to 75 +/- 10% of that initially present. No changes in sensorial properties of the milk, such as clotting or butyric odor, were observed as a result of this treatment. In addition, the bioreactor allows the aseptic recovery of the spent growth medium, which contains diverse enzymes of interest, and the cell pellets, to exploit particular lipids like phosphonolipids, abundant poly-unsaturated fatty acids and co-enzyme Q(8).     

Differential regulation of suppressor of cytokine signaling-3 in the liver and adipose tissue of the sheep fetus in late gestation

It is unknown whether the JAK/STAT/suppressor of cytokine signaling-3 (SOCS-3) intracellular signaling pathway plays a role in tissue growth and metabolism during fetal life. We investigated whether there is a differential profile of SOCS-3 expression in the liver and perirenal adipose tissue during the period of increased fetal growth in late gestation and the impact of fetal growth restriction on SOCS-3 expression in the fetal liver. We also determined whether basal SOCS-3 expression in the fetal liver and perirenal adipose tissue is regulated by endogenous fetal prolactin (PRL). SOCS-3 mRNA abundance was higher in the liver than in the pancreas, spleen, and kidney of the sheep fetus during late gestation. In the liver, SOCS-3 mRNA expression was increased (P < 0.05) between 125 (n = 4) and 145 days (n = 7) gestation and lower (P < 0.05) in growth-restricted compared with normally grown fetal sheep in late gestation. The relative expression of SOCS-3 mRNA in the fetal liver was directly related to the mean plasma PRL concentrations during a 48-h infusion of either a dopaminergic agonist, bromocriptine (n = 7), or saline (n = 5), such that SOCS-3 mRNA expression was lower when plasma PRL concentrations decreased below approximately 20 ng/ml [y = 0.99 - (2.47/x) + (4.96/x(2)); r(2) = 0.91, P < 0.0001, n = 12]. No relationship was shown between the abundance of phospho-STAT5 in the fetal liver and circulating PRL. SOCS-3 expression in perirenal adipose tissue decreased (P < 0001) between 90-91 (n = 6) and 140-145 days (n = 9) gestation and was not related to endogenous PRL concentrations. Thus SOCS-3 is differentially expressed and regulated in key fetal tissues and may play an important and tissue-specific role in the regulation of cellular proliferation and differentiation before birth.     

Cranio-Mandibular Biometrics and Skull Maturation of Macaca sylvanus L., 1758

We report on the biometrics of cranial and dental features in 42 Macaca sylvanus specimens from various museums across Europe. Age classes were determined following dental criteria. Standard biometric landmarks were used to take 17 measurements on the cranium and five on the mandible. The permanent cheek dentition (except the whole C₁/C¹–P₃/P³ complex) was also recorded with four measurements for each molar tooth (three for the P₄/P⁴). Skull measurements show strong sexual dimorphism for characters tied to the mandibular and muzzle/facial portions. The unusual molar dimorphism of M. sylvanus is an unexpected result, because the Cercopithecoid molar pattern is often considered as generally conservative and not very dimorphic. A significant difference between M. sylvanus and other macaques also regards the absolute narrowing of the molar distal loph. This difference may result from a retained plesiomorphic condition, perhaps correlated with the less specialized and tougher diet maintained by M. sylvanus. A multivariate comparison for cranial (both sexual and ontogenetic) difference in M. sylvanus suggests that the full maturation of the male skull shape is delayed longer than what would be expected according to the most restrictive dental criteria adopted here for subadult/adult discrimination. The clustering results, indeed, indicate that significant phenetic differences persist between most of the male crania which are far advanced in their latest dental steps and those of the “fully developed adults”. Outcomes of final growth such as those observed in M. sylvanus suggest that polymorphic maturational patterns can mislead the assessment for a true adult skull shape.