Src kinases mediate VEGFR2 transactivation by the osteostatin domain of PTHrP to modulate osteoblastic function

Parathyroid hormone‐related protein (PTHrP) stimulates osteoblastic function through its N‐ and C‐terminal domains. Since the osteogenic action of the latter domain appears to depend at least in part on its interaction with the vascular endothelial growth factor (VEGF) system, we aimed to explore the putative mechanism underlying this interaction in osteoblasts. Using native conditions for protein extraction and immunoblotting, we found that both PTHrP (107–139) and the shorter PTHrP (107–111) peptide (known as osteostatin), at 100 nM, promoted the appearance of a VEGF receptor (VEGFR) 2 protein band of apparent Mr. wt. 230 kDa, which likely represents its activation by dimer formation, in mouse osteoblastic MC3T3‐E1 cells. Moreover, osteostatin (100 nM) maximally increased VEGFR2 phosphorylation at Tyr‐1059 within 5–10 min in both MC3T3‐E1 and rat osteoblastic osteosarcoma UMR‐106 cells. This phosphorylation elicited by osteostatin appears to be VEGF‐independent, but prevented by the VEGFR2 activation inhibitor SU1498 and also by the Src kinase inhibitors SU6656 and PP1. Furthermore, osteostatin induced phosphorylation of Src, extracellular signal‐regulated kinase (ERK) and Akt with a similar time course to that observed for VEGFR2 activation in these osteoblastic cells. This osteostatin‐dependent induction of ERK and Akt activation was abrogated by SU6656. Up‐regulation of VEGF and osteoprotegerin gene expression as well as the pro‐survival effect induced by osteostatin treatment were all prevented by both SU1498 and SU6656 in these osteoblastic cells. Collectively, these findings demonstrate that the osteostatin domain of C‐terminal PTHrP phosphorylates VEGFR2 through Src activation, which represents a mechanism for modulating osteoblastic function. J. Cell. Biochem. 114: 1404–1413, 2013. © 2012 Wiley Periodicals, Inc.     

Depression in Medical School: The Influence of Morningness‐Eveningness

Medical students are at higher risk for depression, affecting not only their lives but also patient care. This article studied a population of medical students engaged in lecture‐based learning regarding the presence of depressive symptoms and its relation to morningness‐eveningness. Depressive symptoms were assessed by the Beck Depressive Inventory scale (BDI>10), and diurnal preference was assessed by the Horne & Östberg Morningness/Eveningness Questionnaire (MEQ). Family history of depression and involvement in regular physical activity were also investigated. A total of 161 students, 77 (47.8%) males, aged 19 to 30 yrs (22.1±2.1) living in a city close to the equator were evaluated. Fifty‐three individuals (32.9%) had depressive symptoms. Depressive individuals showed a trend to be female (p=0.07). Also, female gender showed a non‐significant shift toward morningness. Fifty‐eight (36.0%) subjects participated in regular physical activity. In 57 cases (35.4%), there was a history of depression in the family. Fifteen individuals (9.3%) were definitely evening type, 42 (26.1%) were moderately evening type, 44 (27.3%) were indifferent, 42 (26.1%) were moderately morning type, and 18 (11.2%) were definitely morning type. Family history of depression (OR=0.29, 95% CI=1.37–6.12) and sedentary life (OR=0.28, 95% CI=0.12–0.65) were associated with depressive symptoms. Eveningness was associated with depressive symptoms (OR=0.66, 95% CI=0.50–0.88), and this association remained significant after adjusting for the presence of familial depression and physical activity (OR=0.71, 95% CI=0.52–0.95). In conclusion, depressive symptoms are independently associated with “eveningness” in medical students. These results should be confirmed by future studies involving a larger number of subjects.     

Can we disentangle predator–prey interactions from species distributions at a macro‐scale? A case study with a raptor species

There is a debate on whether an influence of biotic interactions on species distributions can be reflected at macro‐scale levels. Whereas the influence of biotic interactions on spatial arrangements is beginning to be studied at local scales, similar studies at macro‐scale levels are scarce. There is no example disentangling, from other similarities with related species, the influence of predator–prey interactions on species distributions at macro‐scale levels. In this study we aimed to disentangle predator–prey interactions from species distribution data following an experimental approach including a factorial design. As a case of study we selected the short‐toed eagle because of its known specialization on certain prey reptiles. We used presence–absence data at a 100 km2 spatial resolution to extract the explanatory capacity of different environmental predictors (five abiotic and two biotic predictors) on the short‐toed eagle species distribution in peninsular Spain. Abiotic predictors were relevant climatic and topographic variables, and relevant biotic predictors were prey richness and forest density. In addition to the short‐toed eagle, we also obtained the predictor's explanatory capacities for 1) species of the same family Accipitridae (as a reference), 2) for other birds of different families (as controls) and 3) artificial species with randomly selected presences (as null models). We run 650 models to test for similarities of the short‐toed eagle, controls and null models with reference species, assessed by regressions of explanatory capacities. We found higher similarities between the short‐toed eagle and other species of the family Accipitridae than for the other two groups. Once corrected by the family effect, our analyses revealed a signal of predator–prey interaction embedded in species distribution data. This result was corroborated with additional analyses testing for differences in the concordance between the distributions of different bird categories and the distributions of either prey or non‐prey species of the short‐toed eagle. Our analyses were useful to disentangle a signal of predator–prey interactions from species distribution data at a macro‐scale. This study highlights the importance of disentangling specific features from the variation shared with a given taxonomic level.     

Polyamines affect the cellular growth and structure of pro‐embryogenic masses in Araucaria angustifolia embryogenic cultures through the modulation of proton pump activities and endogenous levels of polyamines

Polyamines (PAs) are abundant polycationic compounds involved in many physiological processes in plants, including somatic embryogenesis. This study investigates the role of PAs on cellular growth and structure of pro‐embryogenic masses (PEMs), endogenous PA and proton pump activities in embryogenic suspension cultures of Araucaria angustifolia. The embryogenic suspension cultures were incubated with putrescine (Put), spermidine (Spd), spermine (Spm) and the inhibitor methylglyoxal‐bis(guanylhydrazone) (MGBG), respectively (1 mM). After 24 h and 21 days, the cellular growth and structure of PEMs, endogenous PA contents and proton pump activities were analyzed. The addition of Spm reduced the cellular growth and promoted the development of PEMs in embryogenic cultures, which could be associated with a reduction in the activities of proton pumps, such as H⁺‐ATPase P‐ and V‐types and H⁺‐PPases, and alterations in the endogenous PA contents. Spm significantly affected the physiology of the A. angustifolia somatic embryogenesis suspension, as it potentially affects cellular growth and structure of PEMs through the modulation of proton pump activities. This work demonstrates the involvement of exogenous PAs in the modulation of cellular growth and structure of PEMs, endogenous PA levels and proton pump activities during somatic embryogenesis. To our knowledge, this study is the first to report a relationship between PAs and proton pump activities in these processes. The results obtained in this study offer new perspectives for studies addressing the role of PAs and proton pump on somatic embryogenesis in this species.     

The effect of plant growth-promoting rhizobacteria on the growth of rice (Oryza sativa L.) cropped in southern Brazilian fields

Background and Aims

Several strains of rhizobacteria may be found in the rhizospheric soil, on the root surface or in association with rice plants. These bacteria are able to colonize plant root systems and promote plant growth and crop yield through a variety of mechanisms. The objectives of this study were to isolate, identify, and characterize putative plant growth-promoting rhizobacteria (PGPR) associated with rice cropped in different areas of southern Brazil.

Methods

Bacterial strains were selectively isolated based on their growth on three selective semi-solid nitrogen-free media. Bacteria were identified at the genus level by PCR-RFLP 16S rRNA gene analysis and partial sequencing methodologies. Bacterial isolates were evaluated for their ability to produce indolic compounds and siderophores and to solubilize phosphate. In vitro biological nitrogen fixation and the ability to produce 1-aminocyclopropane-1-carboxylate deaminase were evaluated for each bacterial isolate used in the inoculation experiments.

Results

In total, 336 bacterial strains were isolated representing 31 different bacterial genera. Strains belonging to the genera Agrobacterium, Burkholderia, Enterobacter, and Pseudomonas were the most prominent isolates. Siderophore and indolic compounds producers were widely found among isolates, but 101 isolates were able to solubilize phosphate. Under gnotobiotic conditions, eight isolates were able to stimulate the growth of rice plants. Five of these eight isolates were also field tested in rice plants subjected to different nitrogen fertilization rates.

Conclusions

The results showed that the condition of half-fertilization plus separate inoculation with the isolates AC32 (Herbaspirillum sp.), AG15 (Burkholderia sp.), CA21 (Pseudacidovorax sp.), and UR51 (Azospirillum sp.) achieved rice growth similar to those achieved by full-fertilization without inoculation, thus highlighting the potential of these strains for formulating new bioinoculants for rice crops.     

Contrasting patterns of genetic differentiation in Macaronesian lineages of Ilex (Aquifoliaceae)

Islands offer an interesting framework in which to study the effect of geographical isolation on population genetic differentiation. For plant species with high dispersal abilities, however, oceanic barriers may not represent a factor promoting strong population structure. In this work, we analysed seven nuclear microsatellite loci in Ilex (Aquifoliaceae), a bird‐dispersed plant group, to infer patterns of genetic differentiation among Macaronesian taxa: I. canariensis, I. perado ssp. lopezlilloi, I. perado ssp. platyphylla (Canary Islands) and I. perado ssp. azorica (Azores). In agreement with current taxonomic classification, our results revealed a high genetic differentiation between Ilex lineages (I. canariensis and the I. perado complex), and also supported previous hypotheses that these are the result of independent dispersal events to the islands. In contrast, genetic differentiation between I. perado ssp. azorica and the two subspecies from the Canaries was high, suggesting that taxonomic revision may be necessary. Levels of genetic variation at microsatellite loci in ssp. azorica were, in addition, the lowest reported among Macaronesian bird‐dispersed taxa. Lastly, low genetic differentiation was observed between subspecies occurring on the same island (sspp. platyphylla and lopezlilloi). In summary, our results revealed contrasting patterns between Macaronesian Ilex lineages: I. canariensis displayed moderate population structure across islands, whereas the I. perado complex showed strong differentiation among populations sampled on different islands. Thus, the Macaronesian Ilex taxa show that long‐distance dispersal syndromes (ornithochory) do not always ensure genetic connectivity across large areas in island systems. Plant groups that successfully colonized the islands on multiple occasions may have found barriers to gene flow within certain lineages. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 258–268.     

Spatiotemporal Interactions Among Three Neighboring Groups of Free-Ranging White-Footed Tamarins (Saguinus leucopus) in Colombia

Successful conservation requires an understanding of animal movement patterns and space use. Such data are hard to obtain, however, when difficult terrain, nocturnal habits, or lack of habituation make direct observation impractical. White-footed tamarins (Saguinus leucopus) are small primates endemic to Colombia that are in danger of extinction due to habitat loss, fragmentation, and the illegal pet trade. Here, we report the results of the first study to use radio-tracking to investigate white-footed tamarin ranging behavior. We recorded the movements of three neighboring tamarin groups simultaneously for 3 month using radio-telemetry. Home range sizes (estimated using both minimum convex polygon and fixed kernel contour methods) were substantially larger than reported in previous studies that did not use remote-tracking. Monte Carlo resampling procedures revealed that home range size differed significantly among the three groups but that the mean daily path length did not. As in other tamarin species, the degree of range overlap between neighboring social groups was high, ranging from 27 to 81%. Using a randomization test, we showed that the observed mean distance between groups was significantly lower than expected by chance for two of the three group dyads. This pattern of intergroup “attraction,” in conjunction with substantial range overlap and high population density, implies that the Bellavista Forest, one of the few remaining habitats of Saguinus leucopus, may be saturated, and promoting habitat restoration should be a priority for the conservation of this species.     

Sorting Nexin 1 Loss Results in D5 Dopamine Receptor Dysfunction in Human Renal Proximal Tubule Cells and Hypertension in Mice

The peripheral dopaminergic system plays a crucial role in blood pressure regulation through its actions on renal hemodynamics and epithelial ion transport. The dopamine D5 receptor (D₅R) interacts with sorting nexin 1 (SNX1), a protein involved in receptor retrieval from the trans-Golgi network. In this report, we elucidated the spatial, temporal, and functional significance of this interaction in human renal proximal tubule cells and HEK293 cells stably expressing human D₅R and in mice. Silencing of SNX1 expression via RNAi resulted in the failure of D₅R to internalize and bind GTP, blunting of the agonist-induced increase in cAMP production and decrease in sodium transport, and up-regulation of angiotensin II receptor expression, of which expression was previously shown to be negatively regulated by D₅R. Moreover, siRNA-mediated depletion of renal SNX1 in C57BL/6J and BALB/cJ mice resulted in increased blood pressure and blunted natriuretic response to agonist in salt-loaded BALB/cJ mice. These data demonstrate a crucial role for SNX1 in D₅R trafficking and that SNX1 depletion results in D₅R dysfunction and thus may represent a novel mechanism for the pathogenesis of essential hypertension.     

Eliminating a Set of Four Penicillin Binding Proteins Triggers the Rcs Phosphorelay and Cpx Stress Responses in Escherichia coli

Penicillin binding proteins (PBPs) are responsible for synthesizing and modifying the bacterial cell wall, and in Escherichia coli the loss of several nonessential low-molecular-weight PBPs gives rise to abnormalities in cell shape and division. To determine whether these proteins help connect the flagellar basal body to the peptidoglycan wall, we surveyed a set of PBP mutants and found that motility in an agar migration assay was compromised by the simultaneous absence of four enzymes: PBP4, PBP5, PBP7, and AmpH. A wild-type copy of any one of these restored migration, and complementation depended on the integrity of the PBP active-site serine. However, the migration defect was caused by the absence of flagella instead of improper flagellar assembly. Migration was restored if the flhDC genes were overexpressed or if the rcsB or cpxR genes were deleted. Thus, migration was inhibited because the Rcs and Cpx stress response systems were induced in the absence of these four specific PBPs. Furthermore, in this situation Rcs induction depended on the presence of CpxR. The results imply that small changes in peptidoglycan structure are sufficient to activate these stress responses, suggesting that a specific cell wall fragment may be the signal being sensed. The fact that four PBPs must be inactivated may explain why large perturbations to the envelope are required to induce stress responses.