Putative Step Research Articles

Differential expression of meiosis and homologous recombination-related genes in the life cycle of Trypanosoma cruzi.

Trypanosoma cruzi has a complex life cycle consisting of four morphological and distinct biological stages. Although some authors suggest that T. cruzi primarily follows clonal reproduction, recent genomic and transcriptomic studies indicate an unorthodox capacity for recombination. We aimed to estimate the differential gene expression of 10 meiosis/homologous recombination-related genes during the T. cruzi life cycle, including epimastigotes, under two different types of stress (oxidative stressand pH changes). We performed RT-qPCR tests using novel-designed primers to estimate the differential gene expression (∆Ct and ∆∆Ct) of nine genes (SPO11, HAP2, RAD50, MRN complex, BRCA2, DMC1, MND1, and RPA1) and RAD51, which was previously reported. Our results show basal expression of all genes during the life cycle, indicating their hypothetical role in several cellular processes but with specific signatures of differential gene expression during the life cycle (HAP2, RPA, RAD50, BRCA2, MND1, and DMC1) and oxidative stress (RPA, MRE11, NBS1, BRCA2, MND1, and RAD51). Additionally, we found that the MRN complex has an independent level of expression in T. cruzi, with profiles of MRE11 and NBS1 upregulated in some stages. Recent studies on other trypanosomatids have highlighted the influence of HAP2 and RPA in recombination and hybridization. If T. cruzi uses the same repertoire of genes, our findings could suggest that metacyclogenesis may be the putative step that the parasite uses to undergo recombination. Likewise, our study reveals the differential profiles of genes expressed in response to oxidative and pH stress. Further studies are necessary to confirm our findings and understand the recombination mechanism in T. cruzi.

Brain functional connectivity mirrors genetic pleiotropy in psychiatric conditions.

Pleiotropy occurs when a genetic variant influences more than one trait. This is a key property of the genomic architecture of psychiatric disorders and has been observed for rare and common genomic variants. It is reasonable to hypothesize that the microscale genetic overlap (pleiotropy) across psychiatric conditions and cognitive traits may lead to similar overlaps at the macroscale brain level such as large-scale brain functional networks. We took advantage of brain connectivity, measured by resting-state functional MRI to measure the effects of pleiotropy on large-scale brain networks, a putative step from genes to behaviour. We processed nine resting-state functional MRI datasets including 32 726 individuals and computed connectome-wide profiles of seven neuropsychiatric copy-number-variants, five polygenic scores, neuroticism and fluid intelligence as well as four idiopathic psychiatric conditions. Nine out of 19 pairs of conditions and traits showed significant functional connectivity correlations (rFunctional connectivity), which could be explained by previously published levels of genomic (rGenetic) and transcriptomic (rTranscriptomic) correlations with moderate to high concordance: rGenetic-rFunctional connectivity = 0.71 [0.40-0.87] and rTranscriptomic-rFunctional connectivity = 0.83 [0.52; 0.94]. Extending this analysis to functional connectivity profiles associated with rare and common genetic risk showed that 30 out of 136 pairs of connectivity profiles were correlated above chance. These similarities between genetic risks and psychiatric disorders at the connectivity level were mainly driven by the overconnectivity of the thalamus and the somatomotor networks. Our findings suggest a substantial genetic component for shared connectivity profiles across conditions and traits, opening avenues to delineate general mechanisms-amenable to intervention-across psychiatric conditions and genetic risks.

A Genetic Screen Identifies PRP18a, a Putative Second Step Splicing Factor Important for Alternative Splicing and a Normal Phenotype in Arabidopsis thaliana.

Splicing of pre-mRNA involves two consecutive trans-esterification steps that take place in the spliceosome, a large dynamic ribonucleoprotein complex situated in the nucleus. In addition to core spliceosomal proteins, each catalytic step requires step-specific factors. Although the Arabidopsis thaliana genome encodes around 430 predicted splicing factors, functional information about these proteins is limited. In a forward genetic screen based on an alternatively-spliced GFP reporter gene in Arabidopsis thaliana, we identified a mutant impaired in putative step II factor PRP18a, which has not yet been investigated for its role in pre-mRNA splicing in plants. Step II entails cleavage at the 3′ splice site accompanied by ligation of the 5′ and 3′ exons and intron removal. In the prp18 mutant, splicing of a U2-type intron with non-canonical AT-AC splice sites in GFP pre-mRNA is reduced while splicing of a canonical GT-AG intron is enhanced, resulting in decreased levels of translatable GFP mRNA and GFP protein. These findings suggest that wild-type PRP18a may in some cases promote splicing at weak, non-canonical splice sites. Analysis of genome-wide changes in alternative splicing in the prp18a mutant identified numerous cases of intron retention and a preponderance of altered 3′ splice sites, suggesting an influence of PRP18a on 3′ splice site selection. The prp18a mutant featured short roots on synthetic medium and small siliques, illustrating that wild-type PRP18a function is needed for a normal phenotype. Our study expands knowledge of plant splicing factors and provides foundational information and resources for further functional studies of PRP18 proteins in plants.

Pressure modulates the self-cleavage step of the hairpin ribozyme

The ability of certain RNAs, denoted as ribozymes, to not only store genetic information but also catalyse chemical reactions gave support to the RNA world hypothesis as a putative step in the development of early life on Earth. This, however, might have evolved under extreme environmental conditions, including the deep sea with pressures in the kbar regime. Here we study pressure-induced effects on the self-cleavage of hairpin ribozyme by following structural changes in real-time. Our results suggest that compression of the ribozyme leads to an accelerated transesterification reaction, being the self-cleavage step, although the overall process is retarded in the high-pressure regime. The results reveal that favourable interactions between the reaction site and neighbouring nucleobases are strengthened under pressure, resulting therefore in an accelerated self-cleavage step upon compression. These results suggest that properly engineered ribozymes may also act as piezophilic biocatalysts in addition to their hitherto known properties.

Unusual antibiotic presence in gym trained subjects with food intolerance; a case report.

Great interest is raising in food intolerances due to the lack, in many cases, of a particular sensitizing agent. We investigated the serum level of possible new haptens in 15 heavy meat consumers for sport fitness affected by various kinds of food intolerance and who had ever been administered antibiotics in their life for clinical problems. Forty ml of blood were drawn from each patient and analyzed, by means of an ELISA test, in order to possibly identify the presence of an undue contaminant with hapten properties. Four out of fifteen subjects (26%) showed a serum oxytetracycline amount > 6 ng/g (which is considered the safety limit), 10 of 15 (66%) a serum doxycycline amount > of 6 ng/g and 3 out of 15 (30%) subjects had high serum level of both molecules. Although a direct ratio between body antibiotics remnant storage in the long run and chronic gut dysfunctions and/or food allergy did not reached the evidence yet, the blood traces of these compounds in a food intolerant otherwise healthy population might be considered the preliminary putative step of a sensitizing pathway. Our next goals foresee a deeper insight into the sensitizing trigger from human chronic antibiotic exposure via the zootechnical delivery of poultry food.

Raltegravir flexibility and its impact on recognition by the HIV‐1 IN targets

HIV-1 IN is a pertinent target for the development of AIDS chemotherapy. The first IN-specific inhibitor approved for the treatment of HIV/AIDS, RAL, was designed to block the ST reaction. We characterized the structural and conformational features of RAL and its recognition by putative HIV-1 targets - the unbound IN, the vDNA, and the IN•vDNA complex - mimicking the IN states over the integration process. RAL binding to the targets was studied by performing an extensive sampling of the inhibitor conformational landscape and by using four different docking algorithms: Glide, Autodock, VINA, and SurFlex. The obtained data evidenced that: (i) a large binding pocket delineated by the active site and an extended loop in the unbound IN accommodates RAL in distinct conformational states all lacking specific interactions with the target; (ii) a well-defined cavity formed by the active site, the vDNA, and the shortened loop in the IN•vDNA complex provide a more optimized inhibitor binding site in which RAL chelates Mg(2+) cations; (iii) a specific recognition between RAL and the unpaired cytosine of the processed DNA is governed by a pair of strong H-bonds similar to those observed in DNA base pair G-C. The identified RAL pose at the cleaved vDNA shed light on a putative step of RAL inhibition mechanism. This modeling study indicates that the inhibition process may include as a first step RAL recognition by the processed vDNA bound to a transient intermediate IN state, and thus provides a potentially promising route to the design of IN inhibitors with improved affinity and selectivity.

Gamete fusion site on the egg cell and autonomous establishment of cell polarity in the zygote

Gamete fusion activates the egg in animals and plants, and the gamete fusion site on the zygote might provide a possible cue for zygotic development and/or embryonic patterning. In angiosperms, a zygote generally divides into a two-celled proembryo consisting of an apical and a basal cell with different cell fates. This is a putative step in the formation of the apical–basal axis of the proembryo. We observed the positional relationship between the gamete fusion site and the division plane formed by zygotic cleavage using an in vitro fertilization system with rice gametes. There was no relationship between the gamete fusion site and the division plane leading to the two-celled proembryo. Thus, the gamete fusion site on the rice zygote does not appear to function as a determinant for positioning the zygote division plane, and the zygote apparently possesses autonomous potential to establish cell polarity along the apical–basal axis for its first cleavage.

Correlation of 3-Isobutyl-2-methoxypyrazine to 3-Isobutyl-2-hydroxypyrazine during Maturation of Bell Pepper (Capsicum annuum) and Wine Grapes (Vitis vinifera)

Environmental factors affecting degradation of 3-isobutyl-2-methoxypyrazine (IBMP, "green pepper aroma") in wine grapes (V. vinifera) are widely studied, but the degradation pathway is not defined. We hypothesized that IBMP is demethylated to 3-isobutyl-2-hydroxypyrazine (IBHP) during fruit maturation effectively reversing the final putative step of IBMP biosynthesis. A quantification method for IBHP was developed using solid-phase extraction coupled to one- or two-dimensional gas chromatography-mass spectrometry with a recovery of ca. 80%. IBMP and IBHP in bell peppers (Capsicum annuum) and V. vinifera (cv. 'Cabernet Franc', 'Riesling', 'Pinot noir') were then measured at different maturities. IBMP and IBHP were inversely correlated in both bell peppers (R2=0.958) and Cabernet Franc grapes (R2=0.998) over a range of maturities. In bell peppers, we observed a significant decline in IBMP (125 to 15 ng/mL) and increase in IBHP (undetectable to 42 ng/mL) during ripening. In grapes, all cultivars had comparable IBHP concentrations preveraison (64 to 88 pg/mL) but differed in IBHP concentration by 2 orders of magnitude at the final sampling point (undetectable to 235 pg/mL). Higher preveraison IBMP was correlated with higher final IBHP across the three grape cultivars, with the order Cabernet Franc>Riesling>Pinot noir for both IBMP and IBHP. Acid hydrolysis resulted in a significant increase (33%) in IBHP in Cabernet Franc, indicating that IBHP exists partially in a bound form in grapes.

Positional relationship between the gamete fusion site and the first division plane in the rice zygote.

In angiosperms, a zygote generally divides into a two-celled proembryo consisting of an apical and a basal cell that possess different cell fates. This first division of the zygote is a putative step in the formation of the apical–basal axis of the proembryo. The gamete fusion activates the egg, and the gamete fusion site on the zygote has been reported to provide a possible cue for subsequent zygotic development and/or embryonic patterning in animals and plants. In this study, the gamete fusion site on the rice zygote was labelled by in vitro fertilization of a rice egg cell with a fluorescence-stained sperm cell. The positional relationship between the gamete fusion site and the division plane formed by zygotic cleavage was monitored using a fixed culture of the fusion site-labelled zygote until the two-celled proembryo stage. The results indicate that gamete fusion sites exist on two-celled proembryos with no relation to the position of the first division plane, and that the gamete fusion site on the rice zygote does not function as a determinant for positioning the zygote division plane.

Abstract 463: Hypoxia leads to impaired differentiation and sustained global histone acetylation in immortalized mammary epithelial cells cultured as acini

Abstract We study the effect of hypoxia on mammary epithelial cell differentiation at a non-malignant stage as a putative step in early malignant progression. Due to inadequate vasculature solid tumors often contain regions with low oxygen levels (hypoxia). Hypoxia is associated with increased metastatic potential and worse patient outcome. The hypoxic response is in large driven by the two transcription factors hypoxia inducible factor (HIF) 1 and HIF 2. We have reported that hypoxia leads to a less differentiated phenotype in breast cancer and in neuroblastoma, and in the clinic low tumor cell differentiation is related to poor prognosis in these diseases. To investigate the effect of hypoxia on mammary epithelial cell differentiation we employed the model of acinar morphogenesis. In this model immortalized mammary epithelial cells in three-dimensional (3D) culture on a laminin-rich matrix differentiate and form acini-like structures with evacuated lumen surrounded by polarized cells. We have previously seen that hypoxia (1% oxygen) impairs the process of acinar morphogenesis. The hypoxic cells failed to obtain polarity and markers of differentiation were affected at expression level as well as cellular localization. Addressing the mechanism behind the hypoxia-impaired differentiation, we find that hypoxia impairs the global histone deacetylation that is associated with changes in cell morphology and differentiation of the mammary epithelial cells. Three days post-seeding, during the proliferation stage, both normoxic and hypoxic cells stained positive for acetylated histone 4 (AcH4). When the normoxic cells go through morphological changes and acinar formation the acetylation of histone 4 is reduced whereas it is maintained in the hypoxic cell-clusters 10 days after seeding. Transfer of acini from normoxia to hypoxia at this time-point leads to an increase in AcH4. Thus, the hypoxic condition prevents global deacetylation, which is generally associated with high level of transcription and an undifferentiated proliferative cell state, rendering the cells an open chromatin state and hindering acinar formation. These data indicate that hypoxia promotes loss of differentiation also at a non-malignant stage. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 463.

Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors

RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation.

Translation step size measured in single sarcomeres and single filament pairs.

The quest to identify the size of the molecular contractile stroke has been paramount in the field of muscle contraction. The most common approach has been to employ the optical trap to study the interaction between single myosin molecules and actin filaments, and the results of these studies have been variable. Step size has ranged between ∼4 nm and ∼20 nm. Among the difficulties attendant with such measurements is high noise: peak-to-peak noise is not uncommonly on the order of 30 nm, which is many times the size of the putative step.

Pyridoxal isonicotinoyl hydrazone inhibits iron-induced ascorbate oxidation and ascorbyl radical formation

Previous work from our laboratory demonstrated that pyridoxal isonicotinoyl hydrazone (PIH) has in vitro antioxidant activity against iron plus ascorbate-induced 2-deoxyribose degradation due to its ability to chelate iron; the resulting Fe(III)–PIH 2 complex is supposedly unable to catalyze oxyradical formation. A putative step in the antioxidant action of PIH is the inhibition of Fe(III)-mediated ascorbate oxidation, which yields the Fenton reagent Fe(II) [Biochim. Biophys. Acta 1523 (2000) 154]. In this work, we demonstrate that PIH inhibits Fe(III)–EDTA-mediated ascorbate oxidation (measured at 265 nm) and the formation of ascorbyl radical (in electron paramagnetic resonance (EPR) studies). The efficiency of PIH against ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation was dose dependent and directly proportional to the period of preincubation of PIH with Fe(III)–EDTA. The efficiency of PIH in inhibiting ascorbate oxidation and ascorbyl radical formation was also inversely proportional to the Fe(III)–EDTA concentration in the media. When EDTA was replaced by the weaker iron ligand nitrilotriacetic acid (NTA), PIH was much more effective in preventing ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation. Moreover, the replacement of EDTA with citrate, a physiological chelator with a low affinity for iron, also resulted in PIH having a higher efficiency in inhibiting iron-mediated ascorbate oxidation and 2-deoxyribose degradation. These results demonstrate that PIH removes iron from EDTA (or from either NTA or citrate), forming an iron–PIH complex that cannot induce ascorbate oxidation effectively, thus inhibiting iron-mediated oxyradical formation. These results are of pharmacological relevance because PIH has been considered for experimental chelating therapy in iron-overload diseases.

Ants Induce Domatia in a Rain Forest Tree (Vochysia vismiaefolia)1

ABSTRACTIn Amazonian rain forest trees of Vochysia vismiaefolia (Vochysiaceae), ants were found to induce twig structures that resembled classical ant domatia. This phenomenon is novel for ant‐plants, which commonly develop domatia without the activity of ants. Eight species of ants were recorded inside the domatia of six individual trees, but domatia were most numerous and characteristic when induced and inhabited by an undescribed species of Pseudomyrmex on two trees. To investigate the mechanism of domatium growth, we drilled holes into young twigs: the expansion of the twig diameter surrounding the holes was significantly accelerated, comparable to domatia formation. Domatia induction is discussed as a putative step in the evolution of ant‐plants.

Ants Induce Domatia in a Rain Forest Tree (Vochysia vismiaefolia)1

In Amazonian rain forest trees of Vochysia vismiaefolia (Vochysiaceae), ants were found to induce twig structures that resembled classical ant domatia. This phenomenon is novel for ant-plants, which commonly develop domatia without the activity of ants. Eight species of ants were recorded inside the domatia of six individual trees, but domatia were most numerous and characteristic when induced and inhabited by an undescribed species of Pseudomyrmex on two trees. To investigate the mechanism of domatium growth, we drilled holes into young twigs: the expansion of the twig diameter surrounding the holes was significantly accelerated, comparable to domatia formation. Domatia induction is discussed as a putative step in the evolution of ant-plants.

The Hdj-2/Hsc70 chaperone pair facilitates early steps in CFTR biogenesis.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel constructed from two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBD) and a regulatory (R) domain. The NBDs and R-domain are cytosolic and how they are assembled with the MSDs to achieve the native CFTR structure is not clear. Human DnaJ 2 (Hdj-2) is a co-chaperone of heat shock cognate 70 (Hsc70) which is localized to the cytosolic face of the ER. Whether Hdj-2 directs Hsc70 to facilitate the assembly of cytosolic regions on CFTR was investigated. We report that immature ER forms of CFTR and DeltaF508 CFTR can be isolated in complexes with Hdj-2 and Hsc70. The DeltaF508 mutation is localized in NBD1 and causes the CFTR to misfold. Levels of complex formation between DeltaF508 CFTR and Hdj-2/Hsp70 were approximately 2-fold higher than those with CFTR. The earliest stage at which Hdj-2/Hsc70 could bind CFTR translation intermediates coincided with the expression of NBD1 in the cytosol. Interestingly, complex formation between Hdj-2 and nascent CFTR was greatly reduced after expression of the R-domain. In experiments with purified components, Hdj-2 and Hsc70 acted synergistically to suppress NBD1 aggregation. Collectively, these data suggest that Hdj-2 and Hsc70 facilitate early steps in CFTR assembly. A putative step in the CFTR folding pathway catalyzed by Hdj-2/Hsc70 is the formation of an intramolecular NBD1-R-domain complex. Whether this step is defective in the biogenesis of DeltaF508 CFTR will be discussed.

A putative step in steroid hormone action involves insertion of steroid ligands into DNA facilitated by receptor proteins

The hypothesis is advanced that hormonal ligands in the steroid/thyroid superfamily act through insertion between base pairs in partially unwound DNA. Using published X-ray coordinates of the complex of the glucocorticoid hormone response element (GRE) with the glucocorticoid receptor DNA binding domain, the interface between the protein and the gene was examined. The site 5′-TG-3′-5′-CA-3′ previously shown to accommodate cortisol was found in the first two bases of the GRE half sites, 5′-TGTTCT-3′. These base pairs were sufficiently exposed at the receptor-gene interface to permit access by the steroid. Docking of cortisol into the receptor/DNA complex resulted in a favorable van der Waals energy. Given the general lack of correlation of receptor binding with hormonal activity, we propose that hormone action involves an additional step in which the receptor protein in concert with other transcription factors inserts the hormone into the DNA. This notion provides an explanation for earlier paradoxical observations including structural analogies between base pairs and steroid hormones. The insertion hypothesis suggests that receptor bound ligand facilitates DNA unwinding, stereospecific control of donor/acceptor functional groups on the DNA followed by insertion and release of the ligand between base pairs at 5′-TG-3′-5′-CA-3′.

Protein kinase C is not involved in secretion by permeabilized human neutrophils

The generally accepted sequence of intracellular signal transduction involves: (1) cell surface receptor-ligand interaction; (2) activation of G-proteins; (3) activation of phospholipase C, leading to inositol phosphate (IP 3) and diacylglycerol production; (4) parallel mobilization of intracellular Ca 2+ by IP 3, and; (5) activation of protein kinase C (PKC) by diacylglycerol and Ca 2+, leading to; (6) cellular responses. Human neutrophils appear to utilize this cascade, at least in general, and some, but not all, elements of the intracellular signal cascade known to be operating in intact cells also function in permeabilized cell systems. We have previously shown that permeabilized neutrophils can be induced to secrete lysosomal enzymes in response to elevated levels of Ca 2+ alone and this secretion can be synergistically enhanced by the presence of guanine nucleotides. We now show that Ca 2+, in the presence and absence of guanine nucleotides, can stimulate the production of solubel inositol phosphates. Furthermore, neomyxin, a putative inhibitor of phospholipase C, can block Ca 2+-induced secretion. These data thus suggest a role for phospholipase C activity or its products in the transduction process. The next enzymatic activity ‘downstream’ is PKC. Consequently, we looked at the role Mg-ATP, one of the substrates of PKC, plays in degranulation by permeabilized neutrophils. We found no obligatory role for this nucleotide in the secretory process. We then looked at the activity of oleoyl-acetyl-glycerol (OAG), synthetic diacylglycerol and PKC agonist, on degranulation. We found that OAG was largely additive with Ca 2+. Another PKC agonist, phorbol myristate acetate (PMA), also did not display notable synergy. Finally, inhibitors of PKC activity were not capable of blocking secretion, either in the presence or absence of guanine nucleotides. Thus, while circumstantial evidence seems to point towards a requirement for phospholipase C activation and diacylglycerol production in secretion, we were unable to demonstrate the next putative step in signal transduction, namely activation of PKC.