Inhibitory effects of a manganese superoxide dismutase isolated from garlic (Allium sativum L.) on in vitro tumoral cell growth

Reactive oxygen species are implicated in cancer development and antioxidants in general and superoxide dismutases and superoxide dismutase mimetic in particular, and they inhibit malignant transformation. We examinate the effects of an isolated manganese superoxide dismutase from a medicinal plant Allium sativum. The protein was prepared by a serial of chromatographic techniques: gel filtration and diethylaminoethyl ions exchanger. The enzyme has a specific activity equal to 55 U/mg. Two tumoral cell lines, porcine endothelial cells and mouse melanoma cells were exposed to garlic superoxide dismutase. The exogenous manganese superoxide dismutase is able to modify the intracellular level of reactive oxygen species by eliminating superoxide anion and producing hydrogen peroxide. The cell viability of the two lines was not significantly affected but the cell multiplication was arrested. This effect obtained in the presence of manganese superoxide dismutase correlates with the activation and modulation of phospho‐extracellular signal‐regulated kinases proteins, implicated in the control of several biological processes including cell proliferation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Relaxin-3: improved synthesis strategy and demonstration of its high-affinity interaction with the relaxin receptor LGR7 both in vitro and in vivo

Relaxin-3 is a member of the human relaxin peptide family, the gene for which, RLN3, is predominantly expressed in the brain. Mapping studies in the rodent indicate a highly developed network of RLN3, RLN1, and relaxin receptor-expressing cells in the brain, suggesting that relaxin peptides have important functional roles in the central nervous system. A regioselective disulfide-bond synthesis protocol was developed and used for the chemical synthesis of human (H3) relaxin-3. The selectively S-protected A and B chains were combined by stepwise formation of each of the three insulin-like disulfides via aeration, thioloysis, and iodolysis. Judicious positioning of the three sets of S-protecting groups was crucial for acquisition of synthetic H3 relaxin in a good overall yield. The activity of the peptide was tested against relaxin family peptide receptors. Although the highest activity was demonstrated on the human relaxin-3 receptor (GPCR135), the peptide also showed high activity on relaxin receptors (LGR7) from various species and variable activity on the INSL3 receptor (LGR8). Recombinant mouse prorelaxin-3 demonstrated similar activity to H3 relaxin, suggesting that the presence of the C peptide did not influence the conformation of the active site. H3 relaxin was also able to activate native LGR7 receptors. It stimulated increased MMP-2 expression in LGR7-expressing rat ventricular fibroblasts in a dose-dependent manner and, following infusion into the lateral ventricle of the brain, stimulated water drinking in rats, activating LGR7 receptors located in the subfornical organ. Thus, H3 relaxin is able to interact with the relaxin receptor LGR7 both in vitro and in vivo.     

Beta defensin-2 is reduced in central but not in distal airways of smoker COPD patients

Background

Altered pulmonary defenses in chronic obstructive pulmonary disease (COPD) may promote distal airways bacterial colonization. The expression/activation of Toll Like receptors (TLR) and beta 2 defensin (HBD2) release by epithelial cells crucially affect pulmonary defence mechanisms.

Methods

The epithelial expression of TLR4 and of HBD2 was assessed in surgical specimens from current smokers COPD (s-COPD; n = 17), ex-smokers COPD (ex-s-COPD; n = 8), smokers without COPD (S; n = 12), and from non-smoker non-COPD subjects (C; n = 13).

Results

In distal airways, s-COPD highly expressed TLR4 and HBD2. In central airways, S and s-COPD showed increased TLR4 expression. Lower HBD2 expression was observed in central airways of s-COPD when compared to S and to ex-s-COPD. s-COPD had a reduced HBD2 gene expression as demonstrated by real-time PCR on micro-dissected bronchial epithelial cells. Furthermore, HBD2 expression positively correlated with FEV1/FVC ratio and inversely correlated with the cigarette smoke exposure. In a bronchial epithelial cell line (16 HBE) IL-1β significantly induced the HBD2 mRNA expression and cigarette smoke extracts significantly counteracted this IL-1 mediated effect reducing both the activation of NFkB pathway and the interaction between NFkB and HBD2 promoter.

Conclusions

This study provides new insights on the possible mechanisms involved in the alteration of innate immunity mechanisms in COPD.     

Changes in gamma-aminobutyric acid release induced by topical administration of drugs affecting its metabolism and receptors: studies in freely moving guinea pigs with epidural cups.

The effect of local application of drugs affecting gamma-aminobutyric acid metabolism and receptors on cortical aminoacid release has been investigated in freely-moving guinea pigs equipped with epidural cups. Topical treatment with gamma-aminobutyric acid reuptake and/or metabolism inhibitors (alone and in combination) produced a slow and progressive increase in cortical aminoacid release. The inhibition of gamma-aminobutyric acid-transaminase with ethanolamino-O-sulphate seemed to be a suitable procedure for enhancing the gamma-aminobutyric acid efflux without interfering with its autoreceptor-mediated negative feedback, tested with the gamma-aminobutyric acid agonist (+/-)baclofen and antagonist phaclofen. A substantial part of the gamma-aminobutyric acid outflowing from the cortex was of neuronal origin since tetrodotoxin halved the basal efflux in the presence of gamma-aminobutyric acid reuptake and/or metabolism inhibitors. These results, considered together, indicate that the epidural cup technique may be a useful approach to study changes in cortical gamma-aminobutyric acid release induced by drugs acting on gabaergic transmission and directly applied on the surface of the cortex.     

Active site structure of bacteriorhodopsin and mechanism of action.

Pressure experiments with freeze-dried bacteriorhodopsin indicate that water is an essential part of the chromophore. This observation is combined with already known information on (a) the pH dependence of proton pumping, (b) the secondary protein-chromophore interaction with lysine-40, and (c) the proton transfer in the initial photochemical step to give a detailed structure of the active site and a mechanism for proton pumping which is consistent with the bacteriorhodopsin polypeptide sequence.     

The amount of heterochromatic proteins in the egg is correlated with sex determination in <Emphasis Type="Italic">Planococcus citri</Emphasis> (Homoptera,Coccoidea)

In the mealybug Planococcus citri, there are no identifiable sex chromosomes. Early in the development of embryos destined to become males, the genome contributed by the sperm undergoes heterochromatization and, following an inverted type of meiosis, will be eliminated. Only two vital sperms are therefore produced, both carrying the same maternally derived genome. A differential distribution observed on the two spermatids during male germline cyst formation of chromatin remodeling factors such as HP1 and methylated K9 histone H3 prompted us to propose an imprinting/sex determination model in which the imprinted sperm is the one to undergo heterochromatization at syngamy. The sex ratio is normally 1:1, but aged females are known to produce almost exclusively male progeny, suggesting that the imprinting pattern of the male gamete in P. citri, though necessary, is apparently not sufficient for sex determination. We report here that egg cells of aged females show larger amounts of HP1 and Su(Var)3–9 than egg cells of young females. These data suggest that a determinant of sex may be the amount of maternally derived heterochromatic proteins.     

Cilomilast counteracts the effects of cigarette smoke in airway epithelial cells

Cigarette smoke extracts (CSE) alter TLR4 expression and activation in bronchial epithelial cells. Cilomilast, a phosphodiesterase-4 inhibitor, inhibits cigarette smoke-induced neutrophilia.This study was aimed to explore whether cilomilast, in a human bronchial epithelial cell line (16-HBE), counteracted CSE effects. In particular, TLR4 expression, IP-10 and IL-8 release, lymphocyte and neutrophil chemotactic activity and ERK and IkBa phosphorylation in CSE and LPS-stimulated 16-HBE were assessed.CSE increased TLR4 expression, reduced IP-10 release and lymphocyte chemotactic activity and increased IL-8 release and neutrophil chemotactic activity. Cilomilast reduced TLR4 expression, IL-8 release and neutrophil chemotactic activity as well as it increased IP-10 release and lymphocyte chemotactic activity. All these cilomilast mediated effects were associated with a reduced ERK1/2 and with an increased IkBa phosphorylation.In conclusion, the present study provides compelling evidences that cilomilast may be considered a possible valid therapeutic option in controlling inflammatory processes present in smokers.     

Endogenous transplacental transmission of Neospora hughesi in naturally infected horses

Over a 2-yr study period, we investigated possible endogenous transplacental transmission of Neospora hughesi in 74 mare and foal pairs following the diagnosis of neuronal neosporosis in a weanling foal. Presuckle and postsuckle serum of each foal, serum and colostrum of each periparturient mare, and serum of each mare and foal pair, collected at 3-mo intervals thereafter, were tested for N. hughesi using an indirect fluorescent antibody test (IFAT). Furthermore, whole blood and colostrum samples and placentae were tested for the presence of N. hughesi by real-time PCR. The mares' seroprevalence at foaling based on IFAT (titer ≥ 160) was 52 and 6% in 2006 and 2007, respectively. Colostral antibodies against N. hughesi were detected in 96 and 11% of the mares in the 2-yr study. With the exception of 3 foals, all remaining foals were born seronegative to N. hughesi. Passive transfer of colostral antibodies to N. hughesi was documented in 15 foals. Three foals born from 2 different mares had presuckle antibodies at a titer ranging from 2,560 to 20,480. All 3 foals were born healthy. Two foals were born to the same dam that also gave birth to the weanling diagnosed with neuronal neosporosis in 2005. The third foal was born to a second mare with no previous foaling history at the farm. Seroconversion was documented in 10 foals and 9 mares over the 2-yr study. All blood and colostrum samples tested PCR negative for N. hughesi. Only 1 placenta collected in 2007 from the mare with the 2 congenitally infected foals tested PCR positive for N. hughesi. In conclusion, N. hughesi persisted in this population via endogenous transplacental infection.     

Immune and inflammatory responses to Leishmania amazonensis isolated from different clinical forms of human leishmaniasis in CBA mice

Leishmania amazonensis causes different diseases depending on the host and parasitic virulence factors. In this study, CBA mice were infected with L. amazonensis isolates from patients with localized (Ba125), diffuse cutaneous (Ba276) or visceral leishmaniasis (Ba109). Mice infected with Ba125 and Ba276 progressed rapidly and lesions displayed an infiltrate rich in parasitized macrophages and were necrotic and ulcerated. Ba109 induced smaller lesions and a mixed inflammatory infiltrate without necrosis or ulceration. Ba109 induced an insidious disease with lower parasite load in CBA mice, similar to human disease. Levels of IFN-γ, IL-4 and IL-10 did not differ among the groups. Because all groups were unable to control the infection, expression of IL-4 associated with low production of IFN-γ in the early phase of infection may account for susceptibility, but others factors may contribute to the differences observed in inflammatory responses and infection progression. Evaluation of some parasitic virulence factors revealed that Ba276 exhibits higher ecto-ADPase and 5'-nucleotidase activities compared to the Ba109 and Ba125 strains. Both Ba276 and Ba125 had higher arginase activity in comparison to Ba109. Finally, these data suggest that the differences in enzyme activities among parasites can account for differences in host inflammatory responses and infection progression.