Embryonic β-globin in the non-Antarctic notothenioid fish Cottoperca gobio (Bovichtidae)

Haemoglobins are sensitive to temperature and their properties mirror the thermal conditions encountered by species during their evolutionary histories. This paper provides data on molecular phylogeny of the haemoglobin chains of Cottoperca gobio, a notothenioid fish of sub-Antarctic latitudes, belonging to the basal family Bovichtidae. Unlike most Antarctic notothenioids, C. gobio has two major haemoglobins sharing the β chain. In the molecular phylogenetic analysis, the β chain is included in the clade of the “embryonic” or minor Antarctic globins. Although, in the majority of notothenioids, “embryonic” (minor) α and β globins are expressed in traces or small amounts in the adult stage, in C. gobio the present analysis supports the occurrence of a complete “switch” to exclusive expression of the embryonic β-globin gene in adult fish. The α and β chains sequences have been used to expand our knowledge of the evolution of notothenioid haemoglobins.The protein sequence data reported in this paper will appear in the UniProt Knowledge base under the accession number: P84652 (β chain), P84653 (α ¹ chain).     

c-Jun N-terminal kinase upregulation as a key event in the proapoptotic interaction between transforming growth factor-beta1 and 4-hydroxynonenal in colon mucosa

Cells of colonic mucosa are sensitive to the Smad-mediated growth-inhibitory effect of transforming growth factor-beta1 (TGF-beta1). Another important cell growth inhibitor is the polyunsaturated lipid peroxidation end product, 4-hydroxynonenal (HNE), which triggers apoptosis through c-Jun N-terminal kinase (JNK) activation. Interestingly, a close association between TGF-beta1 and HNE was found in the progression of human colon cancer, with concentration of both molecules inversely related to the malignancy. We investigated the cross talk between Smads and JNK signal transduction pathways in inducing apoptosis. To this purpose TGF-beta1 and HNE were added singly or in combination to CaCo-2 human colon adenocarcinoma cells. The cotreatment induced a marked enhancement of apoptosis and of JNK and Smad4 activities much more than either individual molecule. Cell preincubation with the JNK inhibitor SP600125 significantly prevented JNK and Smad4 enhancement and, subsequently, the cooperative proapoptotic effect was abolished. The primary role of JNK activity in TGF-beta1/HNE cooperative signaling was fully confirmed in a second set of experiments by using JNKi I, a more selective kinase inhibitor. Hence, in tumor cells becoming resistant to TGF-beta1-mediated growth inhibition, increased induction of the remaining TGF-beta1 pathways by interaction with other antiproliferative molecules, such as HNE, could help in inhibiting tumor growth.     

The P2X7 receptor: a key player in IL-1 processing and release

Human IL-1 family proteins are key mediators of the host response to infections, injury, and immunologic challenges. The mechanism by which IL-1 activates proinflammatory responses in target cells, and the plasma membrane receptors involved, is fairly well known. This has led to the development of innovative drugs that block IL-1 downstream to its synthesis and secretion. On the contrary, the mechanism of IL-1 and other IL-1 family members (e.g., IL-18) maturation and release is incompletely understood. Accruing evidence points to a plasma membrane receptor for extracellular ATP, the P2X(7) receptor, as a key player in both processes. A deeper understanding of the mechanism by which the P2X(7) receptor triggers IL-1 maturation and exteriorization may suggest novel avenues for the treatment of inflammatory diseases and provide a deeper insight in the fundamental mechanism of protease activation and cellular export of proteins lacking a leader sequence.     

Molecular study of genes involved in virulence regulatory pathways in Bacillus anthracis vaccine strain "Carbosap"

This study investigated the genetic bases of attenuation in the Bacillus anthracis vaccine strain "Carbosap" used in Italy against anthrax in cattle and sheep. Twelve genes involved in virulence regulatory pathways underwent sequence analysis in comparison with a B. anthracis virulent strain.     

Molecular characterization of the rpoB gene in Brucella species: new potential molecular markers for genotyping

The rpoB gene encoding the beta subunit of the DNA-dependent RNA polymerase was molecularly characterized by PCR amplification and DNA sequencing in 26 Brucella reference strains by using primers selected according to the B. melitensis 16 M rpoB published sequence. Comparison of the rpoB nucleotide sequence of all Brucella strains analysed revealed specific nucleotide variations associated with different Brucella species and biovars. 17 rpoB alleles were recognized and new Brucella typing is proposed. Our results suggest that the rpoB gene polymorphism can be used to identify all Brucella species and most of the biovars, offering an improvement over conventional typing methods.     

Genetic analysis of candidate genes modifying the age-at-onset in Huntington’s disease

The expansion of a polymorphic CAG repeat in the HD gene encoding huntingtin has been identified as the major cause of Huntington’s disease (HD) and determines 42–73% of the variance in the age-at-onset of the disease. Polymorphisms in huntingtin interacting or associated genes are thought to modify the course of the disease. To identify genetic modifiers influencing the age at disease onset, we searched for polymorphic markers in the GRIK2, TBP, BDNF, HIP1 and ZDHHC17 genes and analysed seven of them by association studies in 980 independent European HD patients. Screening for unknown sequence variations we found besides several silent variations three polymorphisms in the ZDHHC17 gene. These and polymorphisms in the GRIK2, TBP and BDNF genes were analysed with respect to their association with the HD age-at-onset. Although some of the factors have been defined as genetic modifier factors in previous studies, none of the genes encoding GRIK2, TBP, BDNF and ZDHHC17 could be identified as a genetic modifier for HD.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Physicochemical characterization of a peptide deriving from the glycoprotein gp36 of the feline immunodeficiency virus and its lipoylated analogue in micellar systems

P59 is the Trp-rich 20-mer peptide ((767)L-G(786)), partial sequence of the membrane-proximal external region (MPER) of the FIV gp36. It has potent antiviral activity, possibly due to a mechanism that inhibits the fusion of the virus with the cell membranes. In the hypothesis that a lipophilic tail could enhance the adhesion of P59 to the membrane so improving its antiviral activity, we synthesized its lipoylated analogue lipo-P59. Fluorescence, CD and NMR investigations in membrane mimicking environments (such as SDS and DPC micelles) were aimed to assess the potential of the lipo-P59 lipophilic tail to affect the biophysical and conformational behaviour of the peptide. In vitro inhibitory assays using lymphoid cell cultures to check the antiviral activity of peptides were also performed. The data show that the biophysical properties and the conformational preferences of the peptides are not dramatically affected by the hydrophobic tail, suggesting that the lipopeptide is capable of preserving all the biophysical peculiarities. Similarly, antiviral experimental data show that the membrane-anchored lipo-P59 peptide is also effective in inhibiting virus replication. Moreover, the lipophilic tail allows P59 to preserve its antiviral activity even in conditions in which the non lipoylated peptide is devoid of activity. In accordance with the unusual high Trp presence, the peptides confirm the preference to be positioned on the membrane interface. Furthermore, the data point out a peculiarity of interaction of the peptides with SDS as compared with DPC.     

Expression of osteoblast marker genes in rat calvarial osteoblast-like cells, and effects of the endocrine disrupters diphenylolpropane, benzophenone-3, resveratrol and silymarin

Compelling evidence indicates that some endocrine disrupters (EDs), acting as selective estrogen-receptor modulators, interfere with osteoblast differentiation and function. Hence, we investigated whether four EDs [bisphenol-A (BSP), benzophenone-3 (BP3), resveratrol and silymarin] affect differentiation and growth of rat calvarial osteoblast-like (ROB) cells in primary in vitro culture. ROB cells were cultured for up 30 days in a medium supplemented with fetal calf serum (FCS), and conventional RT-PCR detected the expression of collagen-1alpha and osteonectin mRNAs through the entire culture period. Real time-PCR demonstrated that at days 2 and 7 of culture the expressions of collagen-1alpha and osteonectin were very low, and underwent a 192- and a 334-fold increase, respectively, at day 21 of culture. In contrast, osteocalcin expression remained unchanged from days 2 to 21 of culture. EIA showed that ROB cells secreted sizeable amounts of osteocalcin and osteopontin between days 13 and 15 of culture. EDs were added at day 13 of culture at concentrations ranging from 10(-10) to 10(-6) M, being the culture medium deprived of FCS, and their effects were tested 48 h later. None of EDs was found to affect osteocalcin and osteopontin secretion from ROB cells, suggesting that their effects were tested at a relatively earlier stage of culture, when ROB cell differentiation into osteoblats is not fully accomplished, and/or the presence of estrogens contained in FCS is needed for EDs to exert their osteoblast-differentiation modulating action. BSP and BP3, but not resveratrol and silymarin, decreased proliferative activity of cultured ROB cells, a cytotoxic effect conceivably independent of their estrogen-receptor modulating activity.     

Resistance of neuroblastoma GI-ME-N cell line to glutathione depletion involves Nrf2 and heme oxygenase-1

Cancer cell survival is known to be related to the ability to counteract oxidative stress, and glutathione (GSH) depletion has been proposed as a mechanism to sensitize cells to anticancer therapy. However, we observed that GI-ME-N cells, a neuroblastoma cell line without MYCN amplification, are able to survive even if GSH-depleted by l-buthionine-(S,R)-sulfoximine (BSO). Here, we show that in GI-ME-N cells, BSO activates Nrf2 and up-regulates heme oxygenase-1 (HO-1). Silencing of Nrf2 restrained HO-1 induction by BSO. Inhibition of HO-1 and silencing of Nrf2 or HO-1 sensitized GI-ME-N cells to BSO, leading to reactive oxygen/nitrogen species overproduction and decreasing viability. Moreover, targeting the Nrf2/HO-1 axis sensitized GI-ME-N cells to etoposide more than GSH depletion. Therefore, we have provided evidence that in GI-ME-N cells, the Nrf2/HO-1 axis plays a crucial role as a protective factor against cellular stress, and we suggest that the inhibition of Nfr2/HO-1 signaling should be considered as a central target in the clinical battle against neuroblastoma.     

Oldenlandia diffusa extracts exert antiproliferative and apoptotic effects on human breast cancer cells through ERα/Sp1-mediated p53 activation

Breast cancer is the most frequent tumor and a major cause of death among women. Estrogens play a crucial role in breast tumor growth, which is the rationale for the use of hormonal antiestrogen therapies. Unfortunately, not all therapeutic modalities are efficacious and it is imperative to develop new effective antitumoral drugs. Oldenlandia diffusa (OD) is a well-known medicinal plant used to prevent and treat many disorders, especially cancers. The aim of this study was to investigate the effects of OD extracts on breast cancer cell proliferation. We observed that OD extracts strongly inhibited anchorage-dependent and -independent cell growth and induced apoptosis in estrogen receptor alpha (ERα)-positive breast cancer cells, whereas proliferation and apoptotic responses of MCF-10A normal breast epithelial cells were unaffected. Mechanistically, OD extracts enhance the tumor suppressor p53 expression as a result of an increased binding of ERα/Sp1 complex to the p53 promoter region. Finally, we isolated ursolic and oleanolic acids as the bioactive compounds able to upregulate p53 expression and inhibit breast cancer cell growth. These acids were greatly effective in reducing tamoxifen-resistant growth of a derivative MCF-7 breast cancer cell line resistant to the antiestrogen treatment. Our results evidence how OD, and its bioactive compounds, exert antiproliferative and apoptotic effects selectively in ERα-positive breast cancer cells, highlighting the potential use of these herbal extracts as breast cancer preventive and/or therapeutic agents.