Controls on calcium ion fluxes in injured or shocked corn root cells: Importance of proton pumping and cell membrane potential

Passive influx of ⁴⁵Ca²⁺ into non-growing corn root tissue ( Zea mays L.) was increased as a result of actions (cutting, rubbing, chilling, heating, acidifying) or agents (cyanide, uncouplers) known to depolarize the cell membrane, and was decreased by actions (washing) or agents (fusicoccin) known to hyperpolarize it. These responses indicate the presence of Ca²⁺ channels which are voltage controlled. If the injuries were extensive, however, voltage control was lost and hyperpolarization with fusicoccin was expressed by increased ⁴⁵Ca²⁺ influx. Control could be regained by tissue washing, and millimolar levels of external Ca²⁺ would protect against loss of control. Influx of Ca²⁺ was strongly inhibited by La³⁺, but only weakly by verapamil. Intact roots showed greater cold shock sensitivity in maturing cells than in growing cells. We conclude that corn roots normally restrict Ca²⁺ influx by a mechanism linked to hyper-polarization of the plasmalemma.
Calcium ions which enter cold-shocked tissue are partially extruded during the early phase of recovery by a process stimulated by fusicoccin and subject to uncoupling.     

Copper distribution in the normal human brain

Copper concentration was determined in samples from 38 areas of 7 normal human brains. The grey matter contained higher concentrations of copper than the white matter. Identical areas of the grey and white matter of the cerebral cortex showed significant differences between individuals. In the caudate nucleus the highest concentrations of copper were found in the tail followed by the body and the head, respectively. A negative linear regression between age and brain copper levels was demonstrated.     

The population dynamics of three polyphagous owlet moths (Lepidoptera: Noctuidae) and the influence of meteorological factors and ENSO on them

The owlet moths (Lepidoptera: Noctuidae) Anicla infecta (Ochsenheimer 1816), Elaphria agrotina (Guenée 1852) and Spodoptera frugiperda (J.E. Smith 1797) occur in the entire American continent. These polyphagous moths have a preference for grasses, and have different biological habits. In this study, the populations of these three species were evaluated monthly with light traps in the Brazilian Savannah, ranging a span of four crop seasons (from July, 2013 to June, 2017). The population data were analyzed and correlated with the meteorological variables: maximum temperature, minimum temperature, relative humidity and precipitation. A total of 4719 individuals were collected in the following percentages: A. infecta (n = 459; 9.73%), E. agrotina (n = 1809; 38.33%) and S. frugiperda (n = 2451; (51.94%). The abundance of all species went down from the first crop season (2013/2014) to the third (2015/2016). In the fourth crop season (2016/2017), the populations of A. infecta and E. agrotina stabilized, but the abundance of S. frugiperda experienced further decrease. The numbers of individuals of three species declined when precipitation was much above (crop season 2014/2015) and below (crop season 2015/2016) than expected by the climatological normal. There were significant, but different degrees of correlation, between the meteorological factors and the ONI index (Oceanic Niño Index - indicator for monitoring El Niño-Southern Oscillation or “ENSO”) with respect to monthly population variations. The results are discussed in accordance with principles of the Integrated Pest Management (IPM) in mind, given the continental distribution and agricultural importance of the three owlet moth species studied.     

Hepatic Proliferation and Angiogenesis Markers Are Increased after Portal Deprivation in Rats: A Study of Molecular,Histological and Radiological Changes

MethodsWe conducted an experimental study comparing portacaval shunt (PCS), total portal vein ligation (PVL), and sham (S) operated rats. Each group were either sacrificed at 6 weeks (early) or 6 months (late). Arterial liver perfusion was studied in vivo using CT, and histopathological changes were noted. Liver mRNA levels were quantified by RT-QPCR for markers of inflammation (Il10, Tnfa), proliferation (Il6st, Mki67, Hgf, Hnf4a), angiogenesis: (Vegfa, Vegfr 1, 2 and 3; Pgf), oxidative stress (Nos2, and 3, Hif1a), and fibrosis (Tgfb). PCS and PVL were compared to the S group.ResultsPeriportal fibrosis and arterial proliferation was observed in late PCS and PVL groups. CT imaging demonstrated increased arterial liver perfusion in the PCS group. RT-QPCR showed increased inflammatory markers in PCS and PVL early groups. Tnfa and Il10 were increased in PCS and PVL late groups respectively. All proliferative markers increased in the PCS, and Hnf4a in the PVL early groups. Mki67 and Hnf4a were increased in the PCS late group. Nos3 was increased in the early and late PCS groups, and Hif1a was decreased in the PVL groups. Markers of angiogenesis were all increased in the early PCS group, and Vegfr3 and Pgf in the late PCS group. Only Vegfr3 was increased in the PVL groups. Tgf was increased in the PCS groups.ConclusionsPortal deprivation in rats induces a sustained increase in intrahepatic markers of inflammation, angiogenesis, proliferation, and fibrosis.     

A possible mechanism and sequence of events that lead to the Al3+-induced [Ca2+]cyt transients and inhibition of root growth

Fluorescence resonance energy transfer (FRET)-sensitized emission imaging of Arabidopsis thaliana roots expressing the yellow cameleon 3.60 calcium (Ca²⁺) reporter showed that the concentration of calcium in the cytosol ([Ca²⁺]_cyt) increased upon aluminum ion (Al³⁺) treatment in root cells from the transition zone within seconds. The Al³⁺-induced [Ca²⁺]_cyt transients were biphasic and were modified by Ca²⁺ channel blockers and by an antagonist of neuronal glutamate receptors, 2-amino-5-phosphonopentanoate (AP-5), and by the anion channel blocker, 5-nitro-2-(3′-phenylpropyl-amino) benzoate (NPPB). The [Ca²⁺]_cyt transients were not uniquely associated with Al³⁺ toxicity mechanisms since lanthanum (La³⁺) and gadolinium (Gd³⁺) also elicited [Ca²⁺]_cyt transients that were similar to those induced by Al³⁺. Here a testable model that describes a possible mechanism and sequence of events that lead to the Al³⁺-induced [Ca²⁺]_cyt transients and inhibition of root growth is proposed. This model can be applied to study also the signal-response coupling of the trivalent ions La³⁺ and Gd³⁺.Key words: aluminum toxicity, Al³⁺ transport, Ca²⁺ signaling, fluorescence resonance energy transfer (FRET), yellow cameleonAluminum (Al) is a naturally occurring component of soil particles and is the third most abundant element in the earth''s crust.¹ In acidic soils, Al dissolves in the soil solution and different ionic Al species form.^2,3 The most toxic Al species in acidic soils is ionic Al, Al³⁺.⁴ Al³⁺ toxicity stems from its interference with a plethora of cellular processes that control plant growth and development.^3,5–7The interactions between calcium (Ca²⁺) and Al³⁺ are well documented in the literature. One of the toxic effects of Al³⁺ on plant growth and development has been ascribed to the disruption of Ca²⁺ homeostasis by Al³⁺.^8,9 The fact that Al³⁺ inhibits Ca²⁺ uptake by roots,¹⁰ blocks voltage-regulated Ca²⁺ channels,^11,12 and affects the concentration of Ca²⁺ in the cytosol ([Ca²⁺]_cyt)^13–18 support this view. Ca²⁺ alleviates Al³⁺ toxicity^19–22 perhaps by inhibiting Al³⁺ accumulation in the roots and cells.^23,24Rincón-Zachary et al.¹⁸ using fluorescence resonance energy transfer (FRET)-sensitized emission to image Arabidopsis thaliana roots expressing the yellow cameleon 3.60 Ca²⁺ reporter demonstrated increases in the concentration of free Ca²⁺ in the cytosol ([Ca²⁺]_cyt) within seconds of Al³⁺ application. Al³⁺ induced distinct [Ca²⁺]_cyt signatures in cells from the different developmental root regions-meristem, elongation and maturation zones. The [Ca²⁺]_cyt signature in the transition zone, which is the most Al-sensitive root region,²⁵ was biphasic and was modified by treatments that chelate external Ca²⁺ (EGTA), block Ca²⁺ entry through the plasma membrane (verapamil), by an antagonist of neuronal glutamate receptors, 2-amino-5-phosphonopentanoate (AP-5), and by the anion channel blocker, 5-nitro-2-(3′-phenylpropyl-amino) benzoate (NPPB). All of these agents affected the first peak of the Al³⁺-induced [Ca²⁺]_cyt signature by reducing its magnitude or abolishing it. These results support the notion that Al³⁺ interacts with different types of plasma membrane Ca²⁺ channels, causing them to open. Al³⁺-induced [Ca²⁺]_cyt transients were also observed in the Arabidopsis Al-resistant and Al-sensitive mutants alr104 and als3, respectively. In addition, the trivalent ions lanthanum (La³⁺) and gadolinium (Gd³⁺) evoked [Ca²⁺]_cyt signatures in the transition zone of the wild-type Arabidopsis and of the alr104 and als3 roots similar to those elicited by Al³⁺. Hence the authors concluded that the observed [Ca²⁺]_cyt transients were not uniquely associated with Al³⁺ toxicity mechanisms. Al³⁺, La³⁺ and Gd³⁺ appear to elicit the same Ca²⁺ signaling pathway.I would like to propose a testable model that describes the possible sequence of events during Ca²⁺ signaling triggered by trivalent ions using Al³⁺ as a prototype (Fig. 1). (1) Al³⁺ causes Ca²⁺ channels in cells of the root transition zone to open allowing Ca²⁺ influx into the cells. (2) [Ca²⁺]_cyt rises producing the first peak of the biphasic [Ca²⁺]_cyt signature. (3) Increased [Ca²⁺]_cyt activates internal Ca²⁺ channels located in membranes of internal Ca²⁺ stores such as the vacuole, ER, mitochondria or plastids producing the second peak of the [Ca²⁺]_cyt signature. Ca²⁺-induced Ca²⁺ release from internal stores has been described in plant cells.²⁶ (4) Al³⁺ may permeate plasma membrane Ca²⁺ and non-selective cation channels and interact with internal Ca²⁺ channels allowing Ca²⁺ to be released into the cytosol, contributing to the rise in [Ca²⁺]_cyt. In this context, supporting data come from unpublished results (Leblanc J and Rincón-Zachary M) that show Al³⁺ transport across plasma membrane (PM) vesicles isolated from 5 mm wheat (Triticum aestivum) root tips by aqueous two-phase partitioning²⁷ (Fig. 2). In this experiment isolated PM vesicles were loaded with the fluorescent histochemical aluminum indicator morin (2′, 3′, 4′, 5, 7-pentahydroxyflavone) for 30 min at room temperature and then centrifuged at 100,000 xg for 15 min at 4°C and the pellet was washed twice to remove excess morin. The PM vesicles (25 µg protein mL⁻¹) were then incubated in a 2 mL buffer (250 mM sucrose, 50 mM K₂SO⁴, 1 mM DTT, 5 mM MES-Tris [pH 7.0]) containing different concentrations of Al³⁺ for 10 min at room temperature. Al³⁺uptake by the PM vesicles was monitored by fluorometry (excitation at 420 nm; emission at 475 nm). The results show that PM vesicles isolated from the Al-sensitive wheat cultivar Scout 66 root tips are more permeable to Al³⁺ than those isolated from the Al-tolerant cultivar Atlas 66 (Fig. 2A). In this experiment, the relationship between the rate of Al³⁺ uptake and the Al³⁺ concentration in the solution was linear for both Scout 66 (Y = 0.114X + 0.741, R² = 0.99) and Atlas 66 (Y = 0.108X + 0.193, R² = 0.98) PM vesicles. In addition, Leblanc²⁸ showed that compounds known to block Ca²⁺ channels inhibited Al³⁺ uptake by plasma membrane vesicles (Fig. 2B; Leblanc J and Rincón-Zachary M, unpublished data). La³⁺, verapamil and nifedipine were very effective in inhibiting Al³⁺ uptake by plasma membrane vesicles: 5 µM La³⁺ and 1 mM nifedipine caused 67% and 73% inhibition, respectively, and 1 mM verapamil completely abolished the Al³⁺ uptake by the vesicles. Thus, it is feasible that Al³⁺ permeates non-selective cation channels or/and Ca²⁺ channels. (5) Lastly, the overall [Ca²⁺]_cyt elevation could set off mechanisms that inhibit root growth (e.g., callose synthesis and its deposition in the cell wall, disruption of the cytoskeleton organization, formation of reactive oxygen species, etc.). Testing these hypotheses is underway.Open in a separate windowFigure 1A model that describes a possible mechanism and sequence of events that lead to the [Ca²⁺]_cyt transients and inhibition of root growth. (1) Al³⁺ interacts with Ca²⁺ channels in the plasma membrane of root cells in the root transition zone. The Ca²⁺channels open and external Ca²⁺ enters the cytosol. (2) [Ca²⁺]_cyt rises producing the first peak of the biphasic [Ca²⁺]_cyt signature. (3) Increased [Ca²⁺]_cyt activates internal Ca²⁺ channels located in membranes of internal Ca²⁺ stores (e.g., tonoplast, ER, mitochondria or plastids) producing the second peak of the [Ca²⁺]_cyt signature. (4) Al³⁺ permeates the PM through Ca²⁺- and non-selective cation channels. (5) Al³⁺ opens internal Ca²⁺ channels in the tonoplast, ER, mitochondria or plastids and as a result more Ca²⁺ is released into the cytosol. (6) The overall [Ca²⁺]_cyt elevation stimulates mechanisms that inhibit root growth.Open in a separate windowFigure 2Al³⁺ uptake by PM vesicles isolated from 5 mm root tips of both the Al-sensitive cultivar Scout 66 and the Al-tolerant cultivar Atlas 66. (A) Rate of Al³⁺ uptake by PM vesicles incubated in increasing concentrations of Al³⁺. The PM vesicles from the Al sensitive cultivar Scout 66 were more permeable to Al³⁺ than those of the Al-tolerant cultivar Atlas 66. The values are means ± SD. Rates of Al³⁺ uptake are expressed in Fluorescence Intensity Units (FIU) mg⁻¹ protein min⁻¹. (B) Effect of Ca²⁺ channel blockers on the rate of Al³⁺ uptake by PM vesicles s percent of the control. All Ca²⁺ channel blockers tested inhibited the rate Al³⁺ uptake by the PM vesicles in both cultivars. The accumulation of Al³⁺ in the PM vesicles was monitored by measuring the fluorescence emitted by the Al-morin complex as described in the text. Both experiments were repeated three times in triplicate (n = 9). The PM vesicles were pooled from multiple independent membrane isolations in order to obtain enough membrane protein for the assays.     

Production of yam microtubers using a temporary immersion system

Yam clones ‚Pacala Duclos’ and ‚Belep’ in temporary immersion system culture showed favourable results on shoot growth stage and in the development of microtubers in comparison with solid culture media. Cultures in temporary immersion systems in both clones obtained a higher microtuber number per plant, with greater fresh weight and diameter in comparison with solid culture media. Besides, 45 and 47% of microtubers greater than 3.0 gFW for ‚Belep’ and ‚Pacala Duclos’ clones respectively, were obtained. Those tubers may be planted without acclimatization and may be stored for a prolonged period of time.     

Do pollination syndromes cause modularity and predict interactions in a pollination network in tropical high‐altitude grasslands?

The concept of pollination syndromes has been widely questioned, since plant–pollinator interactions have proved to be more generalist than was previously thought. We examined whether the network of a tropical high‐altitude grassland contained groups of plants and pollinators that interact preferentially with each other. A general binary matrix was created. To assess the robustness of myophily, in all analyses we considered: 1) the whole network, 2) the network after the wasps were removed, and 3) the network after the flies were removed. For each network we evaluated whether: 1) the observed interactions were more related to syndromes than expected by chance, compared to an expected matrix; 2) there was a modular structure; 3) the modules found were more related to syndromes than expected by chance, compared to another expected matrix; 4) the syndromes were equally robust. For this analysis, the general matrix was subdivided into smaller matrices that included each pollination syndrome separately. To test the influence of the functional groups of pollinators and the phylogeny of plants, in addition to the general matrix, we also considered the first expected matrix, a quantitative functional group and a plant phylogeny matrix. The pollination syndromes determined the pattern of interactions in the network: 69% of the total interactions resulted from the functional group of pollinators predicted by the plant syndrome. The network showed greater modularity (13 modules) than expected by chance, mostly consisting of the expected functional groups of pollinators and plant syndromes. The modules were associated with pollination syndromes more than was predicted by chance. Most of the variation in interactions was explained by functional groups of pollinators or by plant syndromes. Plant phylogeny did not account for a significant amount of variation in the interactions. Our findings support the concept of pollination syndromes. However, the interactions were not equally predicted by different pollination syndromes, and the accuracy of the prediction was strongest for ornithophily and melittophily.     

CoMFA/CoMSIA 3D-QSAR of pyrimidine inhibitors of Pneumocystis carinii dihydrofolate reductase

Pneumocystis carinii is typically a non-pathogenic fungus found in the respiratory tract of healthy humans. However, it may cause P. carinii pneumonia (PCP) in people with immune deficiency, affecting mainly premature babies, cancer patients and transplant recipients, and people with acquired immunodeficiency syndrome (AIDS). In the latter group, PCP occurs in approximately 80% of patients, a major cause of death. Currently, there are many available therapies to treat PCP patients, including P. carinii dihydrofolate reductase (PcDHFR) inhibitors, such as trimetrexate (TMX), piritrexim (PTX), trimethoprim (TMP), and pyrimethamine (PMT). Nevertheless, the high percentage of adverse side effects and the limited therapeutic success of the current drug therapy justify the search for new drugs rationally planned against PCP. This work focuses on the study of pyrimidine inhibitors of PcDHFR, using both CoMFA and CoMSIA 3D-QSAR methods.     

Genome engineering in Vibrio cholerae: a feasible approach to address biological issues

Although bacteria with multipartite genomes are prevalent, our knowledge of the mechanisms maintaining their genome is very limited, and much remains to be learned about the structural and functional interrelationships of multiple chromosomes. Owing to its bi-chromosomal genome architecture and its importance in public health, Vibrio cholerae, the causative agent of cholera, has become a preferred model to study bacteria with multipartite genomes. However, most in vivo studies in V. cholerae have been hampered by its genome architecture, as it is difficult to give phenotypes to a specific chromosome. This difficulty was surmounted using a unique and powerful strategy based on massive rearrangement of prokaryotic genomes. We developed a site-specific recombination-based engineering tool, which allows targeted, oriented, and reciprocal DNA exchanges. Using this genetic tool, we obtained a panel of V. cholerae mutants with various genome configurations: one with a single chromosome, one with two chromosomes of equal size, and one with both chromosomes controlled by identical origins. We used these synthetic strains to address several biological questions--the specific case of the essentiality of Dam methylation in V. cholerae and the general question concerning bacteria carrying circular chromosomes--by looking at the effect of chromosome size on topological issues. In this article, we show that Dam, RctB, and ParA2/ParB2 are strictly essential for chrII origin maintenance, and we formally demonstrate that the formation of chromosome dimers increases exponentially with chromosome size.