In vitro and in vivo assessment of vitamin A encapsulation in a liposome–protein delivery system

Vitamin A (VA) is an essential nutrient needed in small amounts by humans and supports a wide range of biological actions. Retinol, the most common and most biologically active form of VA has also been found to inhibit peroxidation processes in membranes and it has been widely used as an ingredient with pharmaceutical and nutritional applications. VA is a lipophilic molecule, sensitive to air, oxidizing agents, ultraviolet light and low pH levels. For these reasons, it is necessary for VA to be protected against oxidation. Another disadvantage in the application of VA is its low solubility in aqueous media. Both issues (sensitivity and solubility) can be solved by employing encapsulation techniques. Liposomes can efficiently encapsulate lipid-soluble materials, such as VA. The encapsulated materials are protected from environmental and chemical changes. A new liposome/β-lactoglobulin formulation has been developed as a stable delivery system for VA. The aim of this study was the encapsulation of VA into β-lactoglobulin–liposome complexes, recently developed in our laboratory. The in vivo bioavailability characterization of VA was tested after administration in laboratory animals (mice). In this report, we demonstrate that VA could be efficiently entrapped and delivered in a phospholipid–sterol–protein membrane resembling system, a newly synthesized promising carrier. Based on this finding, the phospholipid–sterol–protein membrane resembling system may be one of the promising approaches to enhance VA absorption and to overcome the formulation difficulties associated with lipophilic means. The carrier system described here has huge potential in food fortification applications to treat VA deficiency.     

Neuroblastoma in relation to joint effects of vitamin A and maternal and offspring variants in vitamin A-related genes: A report of the Children’s Oncology Group

BackgroundThere is evidence vitamin A plays a role in neuroblastoma. Not only is 13-cis-retinoic acid used as maintenance therapy for high-risk cases, but prenatal vitamin intake use may decrease neuroblastoma risk. We hypothesized that single nucleotide polymorphisms (SNPs) in vitamin A-related genes are may be associated with neuroblastoma risk and potentially be modified by vitamin A intake.MethodsThe Neuroblastoma Epidemiology in North America (NENA) study recruited 563 case-parent sets through the Children’s Oncology Group’s Childhood Cancer Research Network. We ascertained dietary nutrient intake through questionnaires and genotyped 463 SNPs in vitamin A-related genes from saliva DNA. Offspring and maternal log-additive risk ratios (RR) and stratum-specific RR for gene-environment interaction were estimated with a log-linear model. We avoided false positives due to multiple testing by using the false discovery rate (FDR).ResultsWhen all neuroblastoma cases were considered together, no offspring variants met the significance criteria (FDR Q-value < 0.2). One maternal SNP (rs12442054) was associated with decreased risk of neuroblastoma (RR: 0.61; 95% Confidence Interval (CI): 0.47–0.79, Q = 0.076). When the cases were categorized according to prognostic risk category and age at onset, nine offspring SNPs were significantly associated with intermediate-risk neuroblastoma. Maternal rs6776706 was associated with (RR: 0.49; 95% CI: 0.33–0.72, Q = 0.161) high-risk neuroblastoma and maternal rs11103603 (RR: 0.60; 95% CI: 0.45–0.79, Q = 0.127) was associated with neuroblastoma aged <1 year. For gene-environment interaction, maternal rs729147 was associated with decreased risk of neuroblastoma among mothers with vitamin A consumption above the recommendation.ConclusionsAlthough there is biologic plausibility for the role of vitamin A in neuroblastoma, we found weak evidence of a relationship between vitamin A related genes and neuroblastoma.     

Haptoglobin-α1, -α2, vitamin D-binding protein and apolipoprotein C-III as predictors of etanercept drug response in rheumatoid arthritis

IntroductionThe introduction of tumor necrosis factor-alpha (TNF-α) antagonists has substantially improved patient’s clinical outcome in rheumatoid arthritis (RA). However, nearly 20% to 40% of RA patients do not respond to anti-TNF-α treatment strategies. To identify valid predictors of TNF-α antagonist response in RA, serum proteome profiles from responders (R) and non-responders (NR) to etanercept, a soluble recombinant TNF-α receptor/IgG Fc fusion protein receptor, were compared in a prospective cohort study.MethodsIn this clinical study 50 RA patients with inadequate response to conventional DMARDs were included and treated with etanercept. The primary efficacy endpoint was response according to the European League against Rheumatism (EULAR) improvement criteria. Serum samples collected prior to initiation and after six months of etanercept therapy were cleared of the most abundant major proteins by immunoaffinity chromatography. After separation by two-dimensional differential gel electrophoresis (2D-DIGE) and identification by mass spectrometry (MS) data were validated by Western blot analysis.ResultsAfter six months of etanercept treatment 62% (n = 31) of RA patients achieved response. Haptoglobin-α1 (Hp-α1) and -α2 (Hp-α2) and vitamin D-binding protein (VDBP) were found to be significantly upregulated in responder sera (P ≤0.02) at study entry. In contrast, apolipoprotein C-III (ApoC-III) showed significantly higher levels in non-responders (P = 0.0162). At study end ApoA-II, Hp-α1, Hp-α2 and VDBP were identified to be expressed at significantly higher levels (P <0.05) in responder sera.ConclusionsBy application of clinical proteomics in immunodepleted sera we could identify and validate for the first time Hp-α1, -α2, VDBP and ApoC-III as potential biomarkers for prediction of etanercept drug response in RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0553-1) contains supplementary material, which is available to authorized users.     

Modulation of the immune system by UV radiation: more than just the effects of vitamin D?

Humans obtain most of their vitamin D through the exposure of skin to sunlight. The immunoregulatory properties of vitamin D have been demonstrated in studies showing that vitamin D deficiency is associated with poor immune function and increased disease susceptibility. The benefits of moderate ultraviolet (UV) radiation exposure and the positive latitude gradients observed for some immune-mediated diseases may therefore reflect the activities of UV-induced vitamin D. Alternatively, other mediators that are induced by UV radiation may be more important for UV-mediated immunomodulation. Here, we compare and contrast the effects of UV radiation and vitamin D on immune function in immunopathological diseases, such as psoriasis, multiple sclerosis and asthma, and during infection.     

Identification and characterization of the ATP·Mg-dependent protein phosphatase activator (F A) as a microtubule protein kinase in the brain

The activating factor of ATP·Mg-dependent protein phosphatase (F _A) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates,F _A could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with aK _m value of 0.4 µM, and tau proteins to 4 moles of phosphates per mole of proteins with aK _m value of about 3 µM. When using microtubules as substrates,F _A could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca⁺²/phospholipid-dependent protein kinase, theF _A-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced byF _A. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed thatF _A could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca⁺²/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due toF _A. Taken together, the results provide initial evidence that the ATP·Mg-dependent protein phosphatase activating factor (F _A) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules.     

Elastic deformations of the rotary double motor of single F(o)F(1)-ATP synthases detected in real time by Förster resonance energy transfer

Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, F?rster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary F(O) motor in the Escherichia coli enzyme is connected to a 3-stepped F(1) motor, where ATP is synthesized. To operate the double motor with a mismatch of step sizes smoothly, elastic deformations within the rotor parts have been proposed by W. Junge and coworkers. Here we extend a single-molecule FRET approach to observe both rotary motors simultaneously in individual F(O)F(1)-ATP synthases at work. We labeled this enzyme with two fluorophores specifically, that is, on the ε- and c-subunits of the two rotors. Alternating laser excitation was used to select the FRET-labeled enzymes. FRET changes indicated associated transient twisting within the rotors of single enzyme molecules during ATP hydrolysis and ATP synthesis. Supported by Monte Carlo simulations of the FRET experiments, these studies reveal that the rotor twisting is greater than 36° and is largely suppressed in the presence of the rotation inhibitor DCCD. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Modulation of Paraoxonase 2 (PON2) in Mouse Brain by the Polyphenol Quercetin: A Mechanism of Neuroprotection?

Quercetin is a common flavonoid polyphenol which has been shown to exert neuroprotective actions in vitro and in vivo. Though quercetin has antioxidant properties, it has been suggested that neuroprotection may be ascribed to its ability of inducing the cell’s own defense mechanisms. The present study investigated whether quercetin could increase the levels of paraoxonase 2 (PON2), a mitochondrial enzyme expressed in brain cells, which has been shown to have potent antioxidant properties. PON2 protein, mRNA, and lactonase activity were highest in mouse striatal astrocytes. Quercetin increased PON2 levels, possibly by activating the JNK/AP-1 pathway. The increased PON2 levels induced by quercetin resulted in decreased oxidative stress and ensuing toxicity induced by two oxidants. The neuroprotective effect of quercetin was significantly diminished in cells from PON2 knockout mice. These findings suggest that induction of PON2 by quercetin represents an important mechanism by which this polyphenol may exert its neuroprotective action.     

A pentanucleotide tandem duplication polymorphism in the 3′ untranslated region of the HLA-linked heat-shock protein 70-2 (HSP70-2) gene

A novel two-allele pentanucleotide tandem duplication polymorphism is described within the 3 untranslated region of the HLA-linked HSP70-2 gene, for which a PstI polymorphism is known. All four haplotype combinations were found.     

Role of mitochondria in apoptosis induced by the 2‐5A system and mechanisms involved

The 2‐5A system (2-5OAS/RNaseL) is composed of the 2′,5′oligoadenylate synthetase 1 (2-5OAS1) and 2-5A-dependent RNase (RNaseL), enzymes that play a key role in antiviral defence mechanisms. Activation of the 2-5A system by double stranded RNA (dsRNA) induces degradation of ribosomal RNAs and apoptosis in mammalian cells. To obtain further information into the molecular mechanisms by which RNaseL induces apoptosis, we expressed human RNaseL and 2-5OAS in HeLa cells using recombinant vaccinia viruses as vectors and we analysed in detail different biochemical markers of apoptosis. In this expression virus-cell system the activation of RNaseL, as index of rRNA degradation, is an upstream event of apoptosis induction. RNaseL induces apoptosis in a caspase-dependent manner (caspases 8, 9 and 2). At the beginning of apoptosis RNaseL and 2-5OAS are localized in the mitochondria and cytosol fractions, while at the onset of apoptosis both enzymes are largely in mitochondria. The 2-5A system induces the release of Cytochrome c from mitochondria to cytosol in a caspase dependent manner. The onset of apoptosis elicits the disruption of mitochondrial membrane potential (ΔΦm), as well as the generation of reactive oxygen species (ROS). Moreover, the activation of RNaseL induces morphological alterations in the mitochondria. Apoptosis induced by the 2-5A system involves mitochondrial proteins, such as the human anti-apoptotic protein Bcl-2, which blocks both the apoptosis and the change of ΔΦm induced by the activation of RNaseL. These findings provide new insights into the molecular mechanisms of apoptosis induction by the 2-5A system, demonstrating the importance of mitochondria in 2-5OAS/RNaseL-induced apoptosis.     

Virus-mediated inhibition of the ppp(A2′p)n A system and its prevention by interferon

Encephalomyocarditis virus infection of mouse Ehrlich ascites tumour or L-cells or human HeLa cells inhibits the activation of the ppp(A2′p)_n A-dependent nuclease and prevents the binding of a radioactive analogue of ppp(A2′p)_n A to the ppp(A2′p)_n A binding protein. This loss or inactivation of the ppp(A2′p)_n A-dependent nuclease in response to virus infection is prevented by interferon pretreatment of the cells.     

Gene duplication and gene conversion in class II MHC genes of New Zealand robins (Petroicidae)

In contrast to mammals, the evolution of MHC genes in birds appears to be characterized by high rates of gene duplication and concerted evolution. To further our understanding of the evolution of passerine MHC genes, we have isolated class II B sequences from two species of New Zealand robins, the South Island robin (Petroica australis australis), and the endangered Chatham Island black robin (Petroica traversi). Using an RT-PCR based approach we isolated four transcribed class II B MHC sequences from the black robin, and eight sequences from the South Island robin. RFLP analysis indicated that all class II B loci were contained within a single linkage group. Analysis of 3-untranslated region sequences enabled putative orthologous loci to be identified in the two species, and indicated that multiple rounds of gene duplication have occurred within the MHC of New Zealand robins. The orthologous relationships are not retained within the coding region of the gene, instead the sequences group within species. A number of putative gene conversion events were identified across the length of our sequences that may account for this. Exon 2 sequences are highly diverse and appear to have diverged under balancing selection. It is also possible that gene conversion involving short stretches of sequence within exon 2 adds to this diversity. Our study is the first report of putative orthologous MHC loci in passerines, and provides further evidence for the importance of gene duplication and gene conversion in the evolution of the passerine MHC.Nucleotide sequence data reported in this paper are available in the GenBank database under the accession numbers AY258333–AY258335, AY428561–AY428570, and AY530534–AY530535     

Niche expansion and the niche variation hypothesis: does the degree of individual variation increase in depauperate assemblages?

The niche expansion and niche variation hypotheses predict that release from interspecific competition will promote niche expansion in depauperate assemblages. Niche expansion can occur by different mechanisms, including an increase in within-individual, among-individual, or bimodal variation (sexual dimorphism). Here we explore whether populations with larger niche breadth have a higher degree of diet variation. We also test whether populations from depauperate lizard assemblages differ in dietary resource use with respect to variation within and/or among individuals and sexual dimorphism. We found support for the niche expansion and niche variation hypotheses. Populations in assemblages with low phylogenetic diversity had a higher degree of individual variation, suggesting a tendency for niche expansion. We also found evidence suggesting that the mechanism causing niche expansion is an increase in variation among individuals rather than an increase in within-individual variation or an increase in bimodal variation due to sexual dimorphism.     

Cloning of AAC(3) and AAD(2″) genes fromAcinetobacter: differential expression in the host strain

The mechanism of resistance to gentamicin and tobramycin in a clinical isolate ofAcinetobacter baumannii, in which aminoglycoside-modifying enzymes were not detected, was investigated. For increase of the resistance gene product, DNA prepared from theA. baumannii isolate was cloned into pUC18 and introduced intoEscherichia coli by transformation. Gentamicin-resistant transformants were screened for aminoglycoside-modifying enzymes. This approach identified two genes encoding AAC(3) and AAD(2) activity, respectively. To determine whether both genes are expressed in the hostAcinetobacter strain, we extracted total cellular RNA from this strain, and Northern blots were hybridized with the cloned AAC(3) and AAD(2) structural genes. mRNA transcribed from the AAC(3) gene alone was detected. This shows that cloning a functional resistance gene is not sufficient in itself to investigate mechanisms of resistance in bacterial strains without detectable aminoglycoside-modifying activity. Furthermore, this study suggests a potential limitation of antibiotic resistance gene probes for studying mechanisms of resistance.     

Determination of α2HS-glycoprotein phenotypes by isoelectric focusing and immunoblotting: polymorphic occurrence of HSGA *5 in Okinawa

Summary A print immunofixation is a very useful procedure for the demonstration of ₂HS-glycoprotein (HSGA) following polyacrylamide gel isoelectric focusing. However, this technique has the one disadvantage of requiring a large volume of expensive antiserum. In this paper an alternative detection system is presented which involves the non-electric transfer of HSGA from a focused gel to a nitrocellulose filter and the immunologic detection of HSGA immobilized on nitrocellulose. Using this method the distribution of HSGA polymorphism in the Nepalese and Japanese populations was investigated. All the populations tested were found to lack the common HSGA ^*3 of the Caucasians. A rare HSGA ^*5 in Honshu, a main island of Japan, was observed at polymorphic frequency in Okinawa, Southern Japan.     

IFN-α and TRAIL: A double edge sword in HIV-1 disease?

IFN-α is rapidly upregulated in response to viral infections and it is an essential player in innate immunity against viruses. pDCs are the most potent IFN-α-producing cells and serve as an essential link between innate and adaptive immunity. The fate of pDCs in the course of HIV-1 infection is still a matter of debate, and the question of the detrimental role of chronic production of IFN-α remains open. In particular, IFN-α has been shown to induce the expression of the death ligand TRAIL on pDCs, transforming them into killer pDCs that may contribute to the destruction of CD4(+) T cells, the hallmark of HIV-1-induced disease. In this review, we discuss our current understanding of the protective and pathogenic roles of both IFN-α and TRAIL in HIV-1 disease.     

Immunolocalization of BRCA1 protein in tumor breast tissue: prescreening of BRCA1 mutation in Tunisian patients with hereditary breast cancer?

BRCA1 is a tumor suppressor gene which is inactivated by mutation in familial breast and ovarian cancers. Over 300 different disease causing germ-line mutations have been described; 60% are unique to an individual family. This diversity and the large size of the gene lead us to search for a prescreening method for BRCA1 mutations. Since BRCA1 is a nuclear protein in normal cells, but reported by some authors to be cytoplasmic in breast tumor cells of patients with BRCA1 mutation, we evaluated immunohistochemistry as a prescreening technique to identify BRCA1 mutations in patients with familial presentation of breast cancer. Using a monoclonal antibody against the carboxy-terminal region of BRCA1, we performed immunohistochemistry on 18 tumor samples from patients with hereditary breast cancer. Cytoplasmic staining of BRCA1 was observed in 10 cases. Of the 18 tumors, 12 (66%) showed either BRCA mutation or BRCA1 accumulation or both, indicating that BRCA1 function might be lost in breast tumor cells not only through mutation, but also via abnormal cytoplasmic location. The immunohistochemical test used in this study would not be efficient as a pre-screening method of deleterious mutations, but it appeared useful to investigate tumor physiology.     

Reactivation of protein aggregates by mortalin and Tid1—the human mitochondrial Hsp70 chaperone system

The mitochondrial 70-kDa heat shock protein (mtHsp70), also known in humans as mortalin, is a central component of the mitochondrial protein import motor and plays a key role in the folding of matrix-localized mitochondrial proteins. MtHsp70 is assisted by a member of the 40-kDa heat shock protein co-chaperone family named Tid1 and a nucleotide exchange factor. Whereas, yeast mtHsp70 has been extensively studied in the context of protein import in the mitochondria, and the bacterial 70-kDa heat shock protein was recently shown to act as an ATP-fuelled unfolding enzyme capable of detoxifying stably misfolded polypeptides into harmless natively refolded proteins, little is known about the molecular functions of the human mortalin in protein homeostasis. Here, we developed novel and efficient purification protocols for mortalin and the two spliced versions of Tid1, Tid1-S, and Tid1-L and showed that mortalin can mediate the in vitro ATP-dependent reactivation of stable-preformed heat-denatured model aggregates, with the assistance of Mge1 and either Tid1-L or Tid1-S co-chaperones or yeast Mdj1. Thus, in addition of being a central component of the protein import machinery, human mortalin together with Tid1, may serve as a protein disaggregating machine which, for lack of Hsp100/ClpB disaggregating co-chaperones, may carry alone the scavenging of toxic protein aggregates in stressed, diseased, or aging human mitochondria.