Microbial Volatile Organic Compounds Produced by Bacillus amyloliquefaciens GB03 Ameliorate the Effects of Salt Stress in Mentha piperita Principally Through Acetoin Emission

Journal of Plant Growth Regulation - We investigated the effects of microbial volatile organic compounds (mVOC) emitted by Bacillus amyloliquefaciens GB03 on Mentha piperita growing under different...     

Ecto-5’-Nucleotidase Overexpression Reduces Tumor Growth in a Xenograph Medulloblastoma Model

Background

Ecto-5’-nucleotidase/CD73 (ecto-5’-NT) participates in extracellular ATP catabolism by converting adenosine monophosphate (AMP) into adenosine. This enzyme affects the progression and invasiveness of different tumors. Furthermore, the expression of ecto-5’-NT has also been suggested as a favorable prognostic marker, attributing to this enzyme contradictory functions in cancer. Medulloblastoma (MB) is the most common brain tumor of the cerebellum and affects mainly children.

Materials and Methods

The effects of ecto-5’-NT overexpression on human MB tumor growth were studied in an in vivo model. Balb/c immunodeficient (nude) 6 to 14-week-old mice were used for dorsal subcutaneous xenograph tumor implant. Tumor development was evaluated by pathophysiological analysis. In addition, the expression patterns of adenosine receptors were verified.

Results

The human MB cell line D283, transfected with ecto-5’-NT (D283hCD73), revealed reduced tumor growth compared to the original cell line transfected with an empty vector. D283hCD73 generated tumors with a reduced proliferative index, lower vascularization, the presence of differentiated cells and increased active caspase-3 expression. Prominent A₁ adenosine receptor expression rates were detected in MB cells overexpressing ecto-5’-NT.

Conclusion

This work suggests that ecto-5’-NT promotes reduced tumor growth to reduce cell proliferation and vascularization, promote higher differentiation rates and initiate apoptosis, supposedly by accumulating adenosine, which then acts through A₁ adenosine receptors. Therefore, ecto-5’-NT might be considered an important prognostic marker, being associated with good prognosis and used as a potential target for therapy.     

Overexpression of NTPDase2 in gliomas promotes systemic inflammation and pulmonary injury

Gliomas are the most common and devastating type of primary brain tumor. Many non-neoplastic cells, including immune cells, comprise the tumor microenvironment where they create a milieu that appears to dictate cancer development. ATP and the phosphohydrolytic products ADP and adenosine by activating P2 and P1 receptors may participate in these interactions among malignant and immune cells. Purinergic receptor-mediated cell communication is closely regulated by ectonucleotidases, such as by members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, which hydrolyze extracellular nucleotides. We have shown that gliomas, unlike astrocytes, exhibit low NTPDase activity. Furthermore, ATP induces glioma cell proliferation and the co-administration of apyrase decreases progression of injected cells in vivo. We have previously shown that NTPDase2 reconstitution dramatically increases tumor growth in vivo. Here we evaluated whether NTPDase2 reconstitution to gliomas modulates systemic inflammatory responses. We observed that NTPDase2 overexpression modulated pro-inflammatory cytokine production and platelet reactivity. Additionally, pathological alterations in the lungs were observed in rats bearing these tumors. Our results suggest that disruption of purinergic signaling via ADP accumulation creates an inflammatory state that may promote tumor spread and dictate clinical progression.     

The role of ecto-5′-nucleotidase/CD73 in glioma cell line proliferation

The antioxidant activity of β-carotene and oxygenated carotenoids lutein, canthaxanthin, and astaxanthin was investigated during spontaneous and peroxyl-radical-induced cholesterol oxidation. Cholesterol oxidation, measured as generation of 7-keto-cholesterol (7-KC), was evaluated in a heterogeneous solution with cholesterol, AAPH, and carotenoids solubilized in tetrahydrofuran and in water, and in a homogeneous solution of chlorobenzene, with AIBN as a prooxidant. The formation of 7-KC was dependent on temperature and on cholesterol and prooxidant concentrations. All the carotenoids tested, exhibited significant antioxidant activity by inhibiting spontaneous, AAPH- and AIBN-induced formation of 7-KC, although the overall order of efficacy of these compounds was astaxanthin > canthaxanthin > lutein = β-carotene. The finding that carotenoids exert protective effects on spontaneous and free radical-induced cholesterol oxidation may have important beneficial effects on human health, by limiting the formation of atheroma and by inhibiting cholesterol oxidation in food processing or storage.     

Mecardonia tenella (Plantaginaceae) Attracts Oil-, Perfume-, and Pollen-Gathering Bees in Southern Brazil

Floral characteristics often indicate the pollinators' functional group visiting the plant and the pollination syndromes associated with them. This idea has been challenged in the past decades due to increasing evidence that most plants, including those exhibiting floral syndromes, are visited by large arrays of species that differ in their effectiveness as pollinators. Our study focuses on Mecardonia tenella (Plantaginaceae) from the Araucaria forest of southern Brazil, which exhibits characteristics of the oil flower pollination syndrome. However, it is visited by three types of functional groups of bees: male orchid bees, oil-collecting bees, and pollen-seeking bees. The relative contribution of each functional group to the plant's reproductive success was estimated based on their pollen load, visitation frequency, and morphology. We assessed resources, phenology, and breeding system of M. tenella . Our results indicate that flowers lack nectar, but volatiles, lipids, and pollen are resources that can be gathered by visitors. This combination of floral traits and visitors' assemblage makes M. tenella a challenge to the concept of pollination syndromes. Our findings indicate that the current interactions may not reflect the circumstances under which some floral traits of this plant were selected.     

Musculoskeletal Geometry,Muscle Architecture and Functional Specialisations of the Mouse Hindlimb

Mice are one of the most commonly used laboratory animals, with an extensive array of disease models in existence, including for many neuromuscular diseases. The hindlimb is of particular interest due to several close muscle analogues/homologues to humans and other species. A detailed anatomical study describing the adult morphology is lacking, however. This study describes in detail the musculoskeletal geometry and skeletal muscle architecture of the mouse hindlimb and pelvis, determining the extent to which the muscles are adapted for their function, as inferred from their architecture. Using I₂KI enhanced microCT scanning and digital segmentation, it was possible to identify 39 distinct muscles of the hindlimb and pelvis belonging to nine functional groups. The architecture of each of these muscles was determined through microdissections, revealing strong architectural specialisations between the functional groups. The hip extensors and hip adductors showed significantly stronger adaptations towards high contraction velocities and joint control relative to the distal functional groups, which exhibited larger physiological cross sectional areas and longer tendons, adaptations for high force output and elastic energy savings. These results suggest that a proximo-distal gradient in muscle architecture exists in the mouse hindlimb. Such a gradient has been purported to function in aiding locomotor stability and efficiency. The data presented here will be especially valuable to any research with a focus on the architecture or gross anatomy of the mouse hindlimb and pelvis musculature, but also of use to anyone interested in the functional significance of muscle design in relation to quadrupedal locomotion.     

Cigarette toxicity triggers Leber's hereditary optic neuropathy by affecting mtDNA copy number,oxidative phosphorylation and ROS detoxification pathways

Leber''s hereditary optic neuropathy (LHON), the most frequent mitochondrial disease, is associated with mitochondrial DNA (mtDNA) point mutations affecting Complex I subunits, usually homoplasmic. This blinding disorder is characterized by incomplete penetrance, possibly related to several genetic modifying factors. We recently reported that increased mitochondrial biogenesis in unaffected mutation carriers is a compensatory mechanism, which reduces penetrance. Also, environmental factors such as cigarette smoking have been implicated as disease triggers. To investigate this issue further, we first assessed the relationship between cigarette smoke and mtDNA copy number in blood cells from large cohorts of LHON families, finding that smoking was significantly associated with the lowest mtDNA content in affected individuals. To unwrap the mechanism of tobacco toxicity in LHON, we exposed fibroblasts from affected individuals, unaffected mutation carriers and controls to cigarette smoke condensate (CSC). CSC decreased mtDNA copy number in all cells; moreover, it caused significant reduction of ATP level only in mutated cells including carriers. This implies that the bioenergetic compensation in carriers is hampered by exposure to smoke derivatives. We also observed that in untreated cells the level of carbonylated proteins was highest in affected individuals, whereas the level of several detoxifying enzymes was highest in carriers. Thus, carriers are particularly successful in reactive oxygen species (ROS) scavenging capacity. After CSC exposure, the amount of detoxifying enzymes increased in all cells, but carbonylated proteins increased only in LHON mutant cells, mostly from affected individuals. All considered, it appears that exposure to smoke derivatives has a more deleterious effect in affected individuals, whereas carriers are the most efficient in mitigating ROS rather than recovering bioenergetics. Therefore, the identification of genetic modifiers that modulate LHON penetrance must take into account also the exposure to environmental triggers such as tobacco smoke.Leber''s hereditary optic neuropathy (LHON) is among the most frequent mitochondrial diseases, affecting about 1 in 35 000–60 000 in Europe.^{1, 2} LHON is associated in over 90% of cases with one of three common mitochondrial DNA (mtDNA) point mutations affecting the Complex I subunit genes ND4 (m.11778G>A), ND1 (m.3460G>A) and ND6 (m.14484 T>C), usually occurring in homoplasmic fashion^{3, 4} (100% of mtDNA is mutant). This maternally inherited blinding disorder is caused by selective degeneration of retinal ganglion cells, particularly those originating the small axons composing the papillomacular bundle, which leads to optic atrophy.^{5, 6, 7} Clinically, a subacute loss of central vision develops in weeks/months, mostly affecting young adult men, with a peculiar pattern of fiber depletion⁸ and a relatively predictable natural history of visual function decline.⁹ Exceptions apply to age of onset, with childhood or late cases,^{10, 11} to propensity in recovering vision, more frequent with the m.14484 T>C mutation,¹² and to clinical expression that in a subset of patients may be more widespread.⁴The mtDNA mutations are necessary but not sufficient to cause LHON,¹³ with penetrance being on average about 50% for males and 10% for females. The association of specific mtDNA haplotypes of haplogroup J with the m.14484 T>C and m.11778G>A mutations has been consistently documented in patients of European descent, indicating that mtDNA background modulates to a certain extent disease penetrance.^{14, 15} However, in a prototypical LHON maternal lineage, despite all the individuals carry the homoplasmic mtDNA mutation regardless the haplogroup, only some develop the disease, pointing to further factors that may be genetic and environmental.¹⁶ Thus, male prevalence and incomplete penetrance remain the two investigated and problematic issues in LHON. Both issues have been recently mechanistically related to the efficiency of compensatory mitochondrial biogenesis.^{17, 18} Estrogens ameliorate mitochondrial dysfunction by activating mitochondrial biogenesis, suggesting that females are naturally protected during their fertile period.^{17, 19} Furthermore, by studying different experimental systems (blood cells, skeletal muscle, skin-derived fibroblasts and ocular tissue) we found that the unaffected mutation carriers had a significantly higher mtDNA copy number and mitochondrial mass compared with their affected relatives,¹⁸ indicating that efficiently increasing mitochondrial biogenesis may overcome the pathogenic effect of the mtDNA mutation. Recently, others obtained similar results in different LHON cohorts.²⁰ Notwithstanding, nuclear modifiers remain elusive. In particular, association of LHON with genetic variants was not consistent across different studies.^{18, 21} Similarly inconsistent was the association with chromosome X-linked loci, hypothesized to explain male prevalence.^{22, 23, 24}Several other factors have been implicated in LHON, including exposure to cigarette smoke, alcohol and chemical toxins, head trauma, acute physical illness, psychological stress, antiretroviral and antituberculosis drugs.^{4, 25} These and other environmental factors can have a triggering role in LHON pathogenesis. For example, in vitro exposure to 2,5 exanedione had a toxic effect on LHON cybrid cells, with an increased sensitivity if they harbored a haplogroup J background.²⁶ A major environmental trigger of LHON is cigarette smoke; Sadun et al.²⁷ and Kirkman et al.²⁵ showed that LHON penetrance is significantly increased in smokers, independently of gender and alcohol intake.In the current study, we explored further the effect of cigarette smoking in LHON, showing in white blood cells from patients of large LHON cohorts, and in skin-derived fibroblasts, that cigarette derivatives exert their toxicity by depressing mtDNA copy number and oxidative phosphorylation (OXPHOS). However, unaffected mutation carriers displayed the most efficient capacity for reactive oxygen species (ROS) detoxification, which was not hampered by exposure to cigarette derivatives.     

Alternative Splicing Modulates Autoinhibition and SH3 Accessibility in the Src Kinase Fyn

Src family kinases are central regulators of a large number of signaling pathways. To adapt to the idiosyncrasies of different cell types, these kinases may need a fine-tuning of their intrinsic molecular control mechanisms. Here, we describe on a molecular level how the Fyn kinase uses alternative splicing to adapt to different cellular environments. Using structural analysis, site-directed mutagenesis, and functional analysis, we show how the inclusion of either exon 7A or 7B affects the autoinhibition of Fyn and how this changes the SH3-dependent interaction and tyrosine phosphorylation of Sam68, with functional consequences for the Sam68-regulated survival of epithelial cells. Our results illustrate a novel mechanism of evolution that may contribute to the complexity of Src kinase regulation.The Src family of nonreceptor protein tyrosine kinases comprises nine members, including Src, Blk, Fgr, Fyn, Hck, Lyn, Lck, Yes, and Yrk. These kinases play crucial roles in a variety of cellular processes, such as cell cycle control, cell adhesion, cell motility, cell proliferation, and cell differentiation (41). Extensive studies indicate that the complexity of functional roles of Src kinases derives mainly from their ability to communicate with a large number of upstream receptors and downstream effectors, which vary by cell type (31). Given their critical role, diverse mechanisms of autoregulation have evolved, and their importance is highlighted by the implication of elevated Src expression levels and/or activity in epithelial cancers (for a review, see reference 48). The autoregulatory mechanisms depend on the composition and order of various domains and on posttranslational modification sites in the linker segments that connect the domains (35). From the N to C terminus, Src contains a myristoyl group attached to a unique domain, an Src homology 3 (SH3) domain that typically binds left-handed polyproline type II sequence motifs, an SH2 domain that binds to tyrosine-phosphorylated protein motifs, a protein-tyrosine kinase domain (SH1), and a C-terminal regulatory segment. Early biochemical studies suggested that these domains were critical for keeping Src catalytic activity under control (4, 23, 39, 40). The validation of the autoinhibitory role of these regulatory moieties came from the structures of Src and Hck kinases (36, 37, 43, 46, 47). The structures showed how interdomain interactions, stabilized by the binding of the SH2 domain to the tyrosine-phosphorylated C terminus (pTyr528), lock the molecule in a closed conformation. They further showed the unanticipated finding that residues in the linker region between the SH2 domain and the kinase domain, the SH2-kinase linker, make direct contact with the catalytic domain and adopts a polyproline type II helix conformation that docks onto the SH3 domain. This intramolecular interaction hinders the formation of a salt bridge that is crucial for the kinase activity, thereby eliciting an inhibitory effect. However, these interactions are suboptimal, and other phosphotyrosine- or polyproline-containing sequences can compete favorably with Src''s own sequences for SH2 or SH3 domain binding (3, 25). These binding events lead to the stimulation of Src kinase activity by disrupting the intramolecular constraints imposed on the kinase domain. Once released from the repressed state, the autophosphorylation of tyrosine residue Tyr416 (pTyr416) in the activation loop rapidly occurs, resulting in a conformational change that releases a fully active kinase.Remarkably, recent advances have highlighted the crucial role of linker regions in establishing the structural and functional assembly of multimodular proteins in signal transduction, and Src kinases are influential in our understanding of these mechanisms (13). The nine Src family members are very similar in terms of sequence identity, with, for example, the strong conservation of the SH3 binding surface and the cores of the kinase domain (44). Nevertheless, high sequence variability is noted in the SH2-kinase linker segment, except that the overall hydrophobicity is conserved. The interactions that this linker makes with both the SH3 domain and the back of the kinase domain probably result in a high-specificity binding. Indeed, the activities of chimeras in which the SH3 domain of Src kinases have been swapped show altered regulation (12, 14, 16). Furthermore, in contrast to deletion or point mutations in the SH3 domain, Src mutants in the linker segment or in the linker-interacting surface on the catalytic domain can transform fibroblast, suggesting specific function(s) (14).Src kinases originated by the duplication and diversification of the same ancestral gene with an original 10-intron structure before the separations of Teleostei from Tetrapoda (6). One of the Src-related kinases, Fyn, possesses two kinds of exon 7, exon 7A and exon 7B, essentially encoding for the SH2-kinase linker segment and the N terminus of the SH1 domain. The alternative splicing of exon 7A or 7B yields two major Fyn isoforms, FynB (exon 7A) and FynT (exon 7B) (7). Exon 7A shows a different evolutionary pattern from that of the other parts of the gene, suggesting that it is derived by a recombinatorial event with another gene (33). The newly captured exon, encoding FynB, was coopted by the central nervous system and possibly other tissues, while the ancestral isoform, encoding FynT, is expressed mainly in the hematopoietic system (7, 32). Whether this diversification process generated intrinsic biochemical functional novelty in addition to the differential tissue distribution and related functional divergence currently is unknown. Since the alternatively spliced exon that distinguishes the two isoforms essentially encodes for the SH2-kinase linker segment, it is possible that it confers distinct regulatory features. Thus, this evolutionary divergence in the SH2-linker segment of FynT and FynB, which maintain identical SH2, SH3, and kinase domains, offers the unique opportunity to explore the specific functions that this linker segment may impose on Src kinase function and/or regulation.Here, we have investigated how exons 7A and 7B affect the functional interaction of Fyn with the RNA-binding protein Sam68. Sam68 is known to activate Fyn by binding to its SH3 domain and also to serve as a substrate for phosphorylation by Fyn. We show that FynT and FynB display a distinct capacity to bind and phosphorylate Sam68. This differential interaction with a substrate is functionally relevant, because it allows the specific phosphorylation-mediated regulation of the Sam68-dependent alternative splicing of Bcl-x by FynT and results in the differential regulation of apoptosis in epithelial cells. Swapping experiments identify core residues of the exon 7A- or 7B-encoded SH2-kinase linker segment as both required and sufficient to confer this distinct function. In agreement with structural models, our data show that exon 7B reinforces the autoinhibitory lock that the SH2-linker region imposes onto the kinase domain and on SH3 domain accessibility. These results uncover a novel specific function that the SH2-kinase linker segment can play in Src biology and highlight the importance of alternative splicing for the acquisition of fine-tuning regulatory functions during evolution.