CytokineDB: a database collecting biological information

Cytokines are subdivided in 12 sub-families and are described as multi-functional molecules that play an important biological activity in host defense system against pathogens, in homeostasis, tissue repair, cell growth and development. CytokineDB is an annotated database that collects biological information regarding the cytokines family in human and will be periodically updated by including new biological information. This database is freely available online and can be accessed at the URL: http://www.cro-m.eu/CytokineDB/     

Human Papillomavirus Type 16 (HPV-16) Virus-Like Particle L1-Specific CD8+ Cytotoxic T Lymphocytes (CTLs) Are Equally Effective as E7-Specific CD8+ CTLs in Killing Autologous HPV-16-Positive Tumor Cells in Cervical Cancer Patients: Implications for L1 Dendritic Cell-Based Therapeutic Vaccines

Papillomavirus-like particles (VLPs) based on L1 capsid protein represent a promising prophylactic vaccine against human papillomavirus (HPV) infections. However, cell-mediated immune responses against this antigen are believed to be of limited therapeutic value in established HPV-infected cervical lesions and, for this reason, have not been intensively investigated in cervical cancer patients. In this study we analyzed and quantified by real-time PCR (RT-PCR) the RNA expression levels of E6, E7, and L1 genes in flash-frozen HPV-16 cervical carcinomas. In addition, the kinetics of expression of E6, E7, and L1 in HPV-16-infected primary cell lines established as long-term cultures in vitro was also evaluated at RNA and protein levels. Finally, in order to evaluate the therapeutic potential of L1-specific CD4⁺ and CD8⁺ T lymphocytes responses in cervical cancer patients, L1 VLP-loaded dendritic cells (DCs) were used to stimulate peripheral blood lymphocytes from cervical cancer patients and such responses were compared to those elicited by the E7 oncoprotein. We show that 22 of 22 (100%) flash-frozen cervical biopsy samples collected from HPV-16-positive cervical cancer patients harbor L1, in addition to E6 and E7 RNA, as detected by RT-PCR. E7 RNA copy number (mean, 176.2) was significantly higher in HPV-16-positive cervical cancers compared to the E6 RNA copy number (mean, 47.3) and the L1 copy number (mean, 58.3) (P < 0.0001 and P < 0.001, respectively). However, no significant differences in expression levels between E6 and L1 were found. Kinetic studies of E6, E7, and L1 RNA and protein expression levels in primary tumors showed a sharp reduction in L1 expression after multiple in vitro passages compared to E6 and E7. Autologous DCs pulsed with HPV-16 VLPs or recombinant full-length E7 elicited strong type 1 L1- and E7-specific responses in CD4⁺ and CD8⁺ T cells from cervical cancer patients. Importantly, L1 VLP-specific CD8⁺ T lymphocytes expressed strong cytolytic activity against autologous tumor cells and were as effective as E7-specific cytotoxic T lymphocytes in lysing naturally HPV-16-infected autologous tumor cells. Taken together, these data demonstrate a consistent expression of L1 in primary cervical tumors and the possibility of inducing effective L1/tumor-specific CD4⁺ and CD8⁺ T-lymphocyte responses in patients harboring HPV-infected cervical cancer. These results may have important implications for the treatment of patients harboring established HPV-infected lesions with L1 VLPs or combined E7/L1 DC-based vaccinations.Human papillomavirus (HPV) infection represents the most important risk factor for the development of cervical cancer. Although more than 100 distinct HPV genotypes have been described, and at least 20 are associated with cervical cancer, HPV type 16 (HPV-16) is by far the most frequently detected in cervical neoplasia regardless of the geographical origin of the patients (4). In the last few years significant advances have been made in the development of candidate prophylactic vaccine against cervical cancer and HPV-related infections. In several large prospective randomized studies, virus-like particles consisting of the HPV-16 and HPV-18 major capsid protein L1 (L1-VLPs) have shown promise in protecting young healthy females against persistent infection with HPV-16 and HPV-18 and their associated cervical intraepithelial neoplasia (reviewed in reference 12). These data strongly suggest that the implementation of large-scale L1-VLP-based prophylactic vaccinations have the potential to dramatically reduce worldwide cervical cancer rates in the years to come.Unfortunately, because HPV infection is endemic in humans and there is a long latency from HPV infection to the development of invasive cervical cancer in women, even if prophylactic L1-based vaccinations are implemented on a worldwide scale today it would take decades to perceive any significant benefit. Consistent with this view, an estimated 5 million cervical cancer deaths will occur in the next 20 years due to existing HPV infections (4, 12). Thus, the current development of therapeutic vaccines for protection against persistent HPV infections, cervical cancer, and its precursor lesions remains an area of great interest.Although the interactions between the host immune system and HPV-infected cells are still not completely understood, several lines of evidence suggest that protection against HPV-related infections by L1-VLP-based vaccines is likely conferred by the generation of high levels of neutralizing antibodies (12, 38). Nevertheless, a potential crucial role of L1-specific T-cell responses and the involvement of T cells in mediating the production of neutralizing antibodies and antiviral effect in infected hosts has been previously hypothesized (8, 24). This point may be particularly noteworthy in patients harboring HPV-infected cervical lesions because several studies have demonstrated the critical importance of both cytotoxic (CD8⁺) and helper (CD4⁺) T cells in achieving clinical responses (1, 5, 16-18, 20, 23). However, limited information is currently available to evaluate whether cell-mediated immune responses to L1-VLP may have any significant therapeutic effect in cervical cancer patients harboring HPV-16 positive tumors. Furthermore, to our knowledge, no direct comparison of the therapeutic efficacy of L1 and E7-specific immune responses against naturally HPV-16-infected cervical cancer have been yet reported in human patients.In the present study we have analyzed and quantified by highly sensitive real-time PCR (RT-PCR) the RNA levels of E6, E7, and L1 in flash-frozen biopsy specimens obtained from HPV-16-infected cervical carcinomas and in short- and long-term primary cultures of HPV-16-positive cervical tumors. In addition, we have studied the kinetics of expression of these genes and proteins during the establishment of HPV-16-positive primary tumors in vitro. Finally, using completely autologous systems of naturally infected HPV-16-positive human tumors, we have carefully studied the phenotype and function of L1-specific CD4⁺ and CD8⁺ T-lymphocyte responses generated by VLP-loaded dendritic cells (DCs) and compared their therapeutic potential to those elicited by DC loaded with the E7 oncoprotein.     

Molecular and cytogenetic characterization of an AT-rich satellite DNA family in <Emphasis Type="Italic">Urvillea chacoensis</Emphasis> Hunz. (Paullinieae,Sapindaceae)

Urvillea chacoensis is a climber with 2n = 22 and some terminal AT-rich heterochromatin blocks that differentiate it from other species of the genus. The AT-rich highly repeated satellite DNA was isolated from U. chacoensis by the digestion of total nuclear DNA with HindIII and XbaI and cloned in Escherichia coli. Satellite DNA structure and chromosomal distribution were investigated. DNA sequencing revealed that the repeat length of satDNA ranges between 721 and 728 bp, the percentage of AT-base pairs was about 72–73% and the studied clones showed an identity of 92.5–95.9%. Although this monomer has a tetranucleosomal size, direct imperfect repetitions of ~180 bp subdividing it in four nucleosomal subregions were observed. The results obtained with FISH indicate that this monomer usually appears distributed in the terminal regions of most chromosomes and is associated to heterochromatin blocks observed after DAPI staining. These observations are discussed in relation to the satellite DNA evolution and compared with other features observed in several plant groups.     

Ancestral Origin of the ATTCT Repeat Expansion in Spinocerebellar Ataxia Type 10 (SCA10)

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurodegenerative disease characterized by cerebellar ataxia and seizures. The disease is caused by a large ATTCT repeat expansion in the ATXN10 gene. The first families reported with SCA10 were of Mexican origin, but the disease was soon after described in Brazilian families of mixed Portuguese and Amerindian ancestry. The origin of the SCA10 expansion and a possible founder effect that would account for its geographical distribution have been the source of speculation over the last years. To unravel the mutational origin and spread of the SCA10 expansion, we performed an extensive haplotype study, using closely linked STR markers and intragenic SNPs, in families from Brazil and Mexico. Our results showed (1) a shared disease haplotype for all Brazilian and one of the Mexican families, and (2) closely-related haplotypes for the additional SCA10 Mexican families; (3) little or null genetic distance in small normal alleles of different repeat sizes, from the same SNP lineage, indicating that they are being originated by a single step mechanism; and (4) a shared haplotype for pure and interrupted expanded alleles, pointing to a gene conversion model for its generation. In conclusion, we show evidence for an ancestral common origin for SCA10 in Latin America, which might have arisen in an ancestral Amerindian population and later have been spread into the mixed populations of Mexico and Brazil.     

Spatial and temporal control of cofilin activity is required for directional sensing during chemotaxis

BACKGROUND: Previous work has led to the hypothesis that cofilin severing, as regulated by PLC, is involved in chemotactic sensing. We have tested this hypothesis by investigating whether activation of endogenous cofilin is spatially and temporally linked to sensing an EGF point source in carcinoma cells. RESULTS: We demonstrate that inhibition of endogenous cofilin activity with either siRNA or overexpression of LIMK suppresses directional sensing in carcinoma cells. LIMK siRNA knockdown, which suppresses cofilin phosphorylation, and microinjection of S3C cofilin, a cofilin mutant that is constitutively active and not phosphorylated by LIMK, also inhibits directional sensing and chemotaxis. These results indicate that phosphorylation of cofilin by LIMK, in addition to cofilin activity, is required for chemotaxis. Cofilin activity concentrates rapidly at the newly formed leading edge facing the gradient, whereas cofilin phosphorylation increases throughout the cell. Quantification of these results indicates that the amplification of asymmetric actin polymerization required for protrusion toward the EGF gradient occurs at the level of cofilin but not at the level of PLC activation by EGFR. CONCLUSIONS: These results indicate that local activation of cofilin by PLC and its global inactivation by LIMK phosphorylation combine to generate the local asymmetry of actin polymerization required for chemotaxis.     

Thyroid hormone regulation of cell migration and oxidative metabolism in polymorphonuclear leukocytes: clinical evidence in thyroidectomized subjects on thyroxine replacement therapy

Migration and superoxide anion (O2-) generation were studied in polymorphonuclear leukocytes (PMNs) from 14 athyreotic patients, previously treated by total thyroidectomy and radioiodine therapy for differentiated thyroid carcinoma, and from age- and sex-matched euthyroid healthy controls. Patients were studied twice: in hypothyroidism (visit 1) and after TSH-suppressive L-T4 replacement therapy (visit 2). Random migration and N-formyl-Met-Leu-Phe (fMLP) 0.1-microM induced chemotaxis were similar in cells from patients at both visit 1 and visit 2 and from healthy controls. On the contrary, resting O2- generation in cells from patients was significantly lower than control values, both at visit 1 and 2. At visit 1, fMLP 0.1 muM-induced O2- generation was significantly lower than control values, while phorbol-myristate acetate (PMA) 100-ng/ml induced O2- generation was similar in cells from patients and from controls. At visit 2 both responses increased, resulting in fMLP-induced O2- generation superimposable to control values and PMA-induced O2- generation significantly higher with respect to both visit 1 and cells from controls. In vitro exposure of PMNs from healthy subjects to L-T4 did not affect O2- generation in resting cells, and significantly increased that induced by fMLP or PMA only at high, supra-physiological concentrations. Neither TSH nor T3 had significant effects at any of the concentrations tested. The present results document the existence of a correlation between thyroid status and oxidative metabolism of human PMNs, which is however unlikely to depend upon a direct action of thyroid hormones on these cells.     

A draft of the human septin interactome

Background

Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins.

Methodology/Principal Findings

Here, we performed yeast two-hybrid screens with human septins 1–10, which include representatives of all four septin groups. Among the interactors detected, we found predominantly other septins, confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments.

Conclusions/Significance

If we take as reference the reported arrangement of the septins 2, 6 and 7 within the heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the “group rule”, i.e. members of the same group (e.g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of the SEPT6 group preferentially interacted with septins of the SEPT2 group (p<0.001), SEPT3 group (p<0.001) and SEPT7 group (p<0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p<0.001) aside from being the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted preferentially with septins of the SEPT7 group (p<0.001). Furthermore, we found non-septin interactors which can be functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis, and apoptosis.     

Hyperdominance in fruit production in the Brazilian Atlantic rain forest: the functional role of plants in sustaining frugivores

The availability of fruits is critical for tropical forests, where the majority of plant species rely upon animal vectors for seed dispersal. However, we do not know how fruit production is temporally distributed over species and families. Two plant families are particularly important in floristic inventories of Atlantic rain forests: Arecaceae, a few species of which are highly abundant; and Myrtaceae, which is abundant and displays outstanding species diversity. In this context, we asked whether hyperdominance occurs in fruit production in the Atlantic rain forest, and whether it occurs in the abundant species of Arecaceae and Myrtaceae. We investigated whether the temporal fruit production patterns differ between Myrtaceae, Arecaceae, and the plant community as a whole. We also applied a functional dispersion index to assess the temporal fruit diversity over a 2‐yr period, with regard to morphological and phenological traits. We found that the phenomenon of hyperdominance occurs in fruit production: five species accounted for more than half of the pulp biomass. Arecaceae fruit biomass peaked at the end of wet season, overlapping with the community peak; whereas Myrtaceae species fruited throughout the year and were an important resource during periods of food scarcity. Myrtaceae filled more of the fruit morphospace over time because their fruits exhibit a large range of morphologies and phenological strategies. Our results demonstrated the importance of combining phenology and fruit morphology in the evaluation of resource availability, which revealed periods of high fruit diversity that could support a range of frugivore sizes and maintain overall ecosystem functionality.     

Determination of Residues Responsible for Substrate and Product Specificity of Solanum habrochaites Short-Chain cis-Prenyltransferases

Prenyl residues confer divergent biological activities such as antipathogenic and antiherbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthones. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate as the prenyl donor, while most of the complementary DNAs encoding these genes have been isolated from the Leguminosae. In this study, we describe the identification of a novel PT gene from lemon (Citrus limon), ClPT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to geranyl diphosphate from citrus species.Prenylation is an important derivatization of plant aromatics, contributing to the chemical diversification of phenolic secondary metabolites in plants due to differences in prenylation positions, prenyl chain lengths, and further modifications of prenyl chains. To date, about 1,000 prenylated aromatic compounds have been isolated as biologically active substances from various plant species, including many medicinal plants.Coumarins (α-benzopyrones) are a large group of plant secondary metabolites. Many biologically active coumarins are prenylated, with the prenyl residue enhancing the biological activities of the aromatic core compound. For example, imperatorin (dimethylallylated xanthotoxol), a strong inhibitor of a Manduca sexta midgut cytochrome P450, has 100-fold greater activity than the nonprenylated coumarin compound, suggesting that prenylation is involved in chemoprevention against biotic stress in plants (Neal and Wu, 1994). Prenylated compounds are also beneficial for human health. For example, geranylation of umbelliferone at the OH position to form auraptene results in a 25-fold enhancement of the inhibition of Epstein Barr virus activity, a test used to screen antitumor compounds (Murakami et al., 1997). Moreover, in tuberculosis, 8-geranyloxypsoralen was reported to decrease the growth rate of Mycobacterium smegmatis (Adams et al., 2006).There are many reports on the detection of prenyltransferase (PT) activities for coumarins in various plant species. For example, umbelliferone-dimethylallyltransferase activities were reported in cultured parsley (Petroselinum crispum) cells, Ruta graveolens, and Ammi majus, and plastidial localization of the enzyme activity is also reported (Ellis and Brown, 1974; Dhillon and Brown, 1976; Tietjen and Matern, 1983; Hamerski and Matern, 1988; Hamerski et al., 1990). In addition, bergaptol 5-O-geranyltransferase activity, which yields bergamottin, a major coumarin derivative, was characterized using the microsomal fraction of lemon (Citrus limon) peel flavedo, the outer part of the lemon fruit (Frérot and Decorzant, 2004; Munakata et al., 2012). In the lemon flavedo, 8-geranyltransferase activity for umbelliferone was also detected (Munakata et al., 2012). To date, only one gene encoding these enzymes has been described; this gene, which encodes a parsley PT (PcPT), was very recently isolated (Karamat et al., 2014).The first flavonoid-specific PT identified was naringenin 8-dimethylallyltransferase (SfN8DT1) from a leguminous medicinal plant, Sophora flavescens (Sasaki et al., 2008). Since then, genes encoding various flavonoid PTs have been identified in Leguminosae (Akashi et al., 2009; Sasaki et al., 2011; Shen et al., 2012). Although other prenylated aromatic compounds, including coumarins, xanthons, phenylpropanoids, and phloroglucinols, have been isolated from many plant species, no gene encoding a PT for those aromatics has been isolated, except for the gene encoding a phloroglucinol-specific enzyme (HlPT1) from hops (Humulus lupulus) and a the recently isolated coumarin dimethylallyltransferase from parsley (Tsurumaru et al., 2010, 2012; Karamat et al., 2014). These isolated plant aromatic PTs show strong preference for dimethylallyl diphosphate (DMAPP) as the prenyl donor substrate, although in nature, many geranylated phenolics and less farnesylated phenolics have been described. This raises questions about the enzymes and reaction mechanisms involved in the synthesis of these phenolic compounds, such as substrate specificity and prenylation sites. Better understanding of these reactions requires the identification of PTs with other enzymatic activities. It is also necessary to identify PTs producing prenylated phenolics in nonleguminosaeous plants. Four different tracks should be explored to identify enzymes that (1) recognize nonflavonoid substrates, e.g. coumarins, phenylpropanoids, and xanthons, (2) are specific for longer chain prenyl diphosphates such as geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), (3) are from nonlegume origins, and (4) catalyze O-prenylation.Citrus species, including lemons, contain large quantities of geranylated coumarins. We therefore isolated a complementary DNA (cDNA) encoding a PT from lemon peel, identifying the novel PT-encoding gene ClPT1. Phylogenetic analysis showed that this enzyme shares homologies with homogentisate PTs involved in vitamin E and plastoquinone biosynthesis but is located in a new clade. We provide evidence showing that this unique enzyme is highly specific for GPP as a prenyl donor and coumarin as a prenyl acceptor. We also show that the gene product is targeted to plastid in plant cells.     

Two non-vesicular ATP release pathways in the mouse erythrocyte membrane

Erythrocytes are exceptionally suited for analysis of non-exocytotic release mechanisms of ATP, because these cells under physiological conditions lack vesicles. Previous studies have indicated, that Pannexin1 (Panx1) provides a key ATP permeation pathway in many cell types, including human and frog erythrocytes. Here we show that erythrocytes of Panx1(-/-) mice lend further support to this conclusion. However, ATP release, although attenuated, was still observed in Panx1(-/-) mouse erythrocytes. In contrast to Panx1(+/+) cells, this release was not correlated with uptake of extracellularly applied dyes, was insensitive to Panx1 channel blockers, and was inhibited by dipyridamole and stimulated by iloprost. Thus, in erythrocytes, two independent pathways mediate the release of ATP. We also show that glyburide is a strong inhibitor of Panx1 channels.