Early habituation of maize (Zea mays) suspension‐cultured cells to 2,6‐dichlorobenzonitrile is associated with the enhancement of antioxidant status

The cellulose biosynthesis inhibitor 2,6‐dichlorobenzonitrile (DCB) has been widely used to gain insights into cell wall composition and architecture. Studies of changes during early habituation to DCB can provide information on mechanisms that allow tolerance/habituation to DCB. In this context, maize‐cultured cells with a reduced amount of cellulose (～20%) were obtained by stepwise habituation to low DCB concentrations. The results reported here attempt to elucidate the putative role of an antioxidant strategy during incipient habituation. The short‐term exposure to DCB of non‐habituated maize‐cultured cells induced a substantial increase in oxidative damage. Concomitantly, short‐term treated cells presented an increase in class III peroxidase and glutathione S‐transferase activities and total glutathione content. Maize cells habituated to 0.3–1 µM DCB (incipient habituation) were characterized by a reduction in the relative cell growth rate, an enhancement of ascorbate peroxidase and class III peroxidase activities, and a net increment in total glutathione content. Moreover, these cell lines showed increased levels of glutathione S‐transferase activity. Changes in antioxidant/conjugation status enabled 0.3 and 0.5 µM DCB‐habituated cells to control lipid peroxidation levels, but this was not the case of maize cells habituated to 1 μM DCB, which despite showing an increased antioxidant capacity were not capable of reducing the oxidative damage to control levels. The results reported here confirm that exposure and incipient habituation of maize cells to DCB are associated with an enhancement in antioxidant/conjugation activities which could play a role in incipient DCB habituation of maize‐cultured cells.     

Spatio-temporal expression of Prospero is finely tuned to allow the correct development and function of the nervous system in Drosophila melanogaster

Adaptive animal behaviors depend upon the precise development of the nervous system that underlies them. In Drosophila melanogaster, the pan-neural prospero gene (pros), is involved in various aspects of neurogenesis including cell cycle control, axonal outgrowth, neuronal and glial cell differentiation. As these results have been generally obtained with null pros mutants inducing embryonic lethality, the role of pros during later development remains poorly known. Using several pros-Voila (prosV) alleles, that induce multiple developmental and behavioral anomalies in the larva and in adult, we explored the relationship between these phenotypes and the variation of pros expression in 5 different neural regions during pre-imaginal development. We found that the quantity of pros mRNA spliced variants and of Pros protein varied between these alleles in a tissue-specific and developmental way. Moreover, in prosV1 and prosV13 alleles, the respective decrease or increase of pros expression, affected (i) neuronal and glial cell composition, (ii) cell proliferation and death and (iii) axonal-dendritic outgrowth in a stage and cellular context dependant way. The various phenotypic consequences induced during development, related to more or less subtle differences in gene expression, indicate that Pros level needs a precise and specific adjustment in each neural organ to allow its proper function.     

Specific myosin heavy chain mutations suppress troponin I defects in Drosophila muscles

We show that specific mutations in the head of the thick filament molecule myosin heavy chain prevent a degenerative muscle syndrome resulting from the hdp2 mutation in the thin filament protein troponin I. One mutation deletes eight residues from the actin binding loop of myosin, while a second affects a residue at the base of this loop. Two other mutations affect amino acids near the site of nucleotide entry and exit in the motor domain. We document the degree of phenotypic rescue each suppressor permits and show that other point mutations in myosin, as well as null mutations, fail to suppress the hdp2 phenotype. We discuss mechanisms by which the hdp2 phenotypes are suppressed and conclude that the specific residues we identified in myosin are important in regulating thick and thin filament interactions. This in vivo approach to dissecting the contractile cycle defines novel molecular processes that may be difficult to uncover by biochemical and structural analysis. Our study illustrates how expression of genetic defects are dependent upon genetic background, and therefore could have implications for understanding gene interactions in human disease.     

Immunocytochemical characterization of the cell walls of bean cell suspensions during habituation and dehabituation to dichlobenil

The effects of the cellulose inhibitor dichlobenil on the cell wall composition and structure during the habituation/dehabituation process of suspension‐cultured bean cells were assessed. A range of techniques were used including cell wall fractionation, sugar analysis, immunofluorescence and fluorochrome labelling of resin‐embedded sections, and immunodot assays (IDAs) of cell wall fractions. The cell walls from bean cell suspensions with initial levels of habituation to dichlobenil had decreased levels of cellulose, but this effect lessened with increasing numbers of subcultures. All cell walls analysed showed calcofluor‐stained appositions. However, in habituated and dehabituated cells, appositions were not recognized by an anticallose antibody. This finding suggested the accumulation of an extracellular polysaccharide different to callose, probably a 1,4‐β‐glucan in these cell lines. Appositions in habituated cells also contained homogalacturonan (HG) with a high degree of methyl esterification (DE), rhamnogalacturonan (RG) and xyloglucan. Habituated cell walls were also enriched in pectins, particularly HG, with a low DE, and RG. The levels of extensin epitope that colocalized with RG in habituated cells also diminished with the increasing number of subcultures. Habituated cells also liberated less extensin into the medium. In habituated cells, a decrease in the cell wall arabinogalactan protein (AGP) labelling was observed both in cell walls and in the culture medium. The increase in the number of subcultures in 0.3 µM dichlobenil was accompanied by an increment in some pectic epitopes (JIM5 and LM5) and a decrease in other pectic and in protein epitopes (JIM7, PAM1, LM6, LM2 and MAC207), indicating a re‐structuring of cell walls throughout the habituation procedure. Dehabituated cells showed an overall composition similar to that of non‐habituated cells, with exception of an increase in glucose in hemicellulosic fractions tightly bound to cellulose. However, these cells also showed reduced levels of extensin and AGP labelling. These differences could be related to the high tolerance to dichlobenil observed in dehabituated cells.     

Predator occurrence and perceived predation risk determine grouping behavior in guanaco (Lama guanicoe)

Grouping behavior of social ungulates may depend on both predator occurrence and perceived predation risk associated with habitat structure, reproductive state, and density of conspecifics. Over 3 years, we studied grouping behavior of guanaco (Lama guanicoe) families in Chilean Patagonia during the birthing season and determined their response to variation in predator occurrence and perceived predation risk (habitat structure, calf/adult rate, and density of conspecifics). We considered the effect of two predators, puma (Puma concolor) and culpeo fox (Lycalopex culpaeus). We measured two common (family group size and vigilance) and one novel (family group cohesion) behavioral responses of guanaco. Our results show that guanaco family groups adapted their grouping behavior to both predator occurrence and perceived predation risk. Larger family groups were found in open habitats and areas with high puma occurrence, while guanacos stayed in small family groups in areas with high shrub cover or low visibility. Group cohesion increased in areas with higher occurrence of pumas and culpeo foxes, and also increased in smaller family groups and in areas with low guanaco density. Vigilance (number of vigilant adults) was mainly related to group size and visibility, increasing in areas with low visibility, while residual vigilance (vigilance after removing the group‐size effect) did not vary with the explanatory variables examined. Our results suggest that a mix of predator occurrence and perceived predation risk influences guanaco grouping behavior and highlights the importance of evaluating different antipredator responses together and considering all predator species in studies aimed at understanding ungulate behavior.     

Photo‐identification matches of humpback whales (Megaptera novaeangliae) from feeding areas in Russian Far East seas and breeding grounds in the North Pacific

Humpback whales migrate seasonally from high latitude feeding areas to lower latitude breeding areas for mating and calving. In 2004–2006, a North Pacific basin‐wide study called SPLASH was conducted as an international collaboration among various groups of researchers. The Russian Far East consists of multiple high latitude feeding areas and during SPLASH, 102 whales were identified and compared to catalogs from breeding areas. Our goal in this study was to further investigate the migratory destinations of whales from the Russian Far East using a total of 1,459 photographs of whales identified from 2004 to 2014. We compared the latest Russian catalog with the SPLASH catalog from wintering areas (2004–2006) and with two additional regional catalogs from Okinawa (1989–2006) and the northern Philippines (2000–2006). We found a total of 152 matches: 106 with Asian, 35 with Hawaiian, and 11 with Mexican breeding grounds. The match rate was higher in mainland Kamchatka and consisted mostly of whales from the Asian breeding ground. In the Commander Islands, the proportion of whales from Asia was twice that of Hawaii and six times higher than Mexico. The total match rate was low, supporting the hypothesis of some undiscovered humpback whale breeding location in the North Pacific.     

Habituation and dehabituation to dichlobenil: Simply the equivalent of Penélope's weaving and unweaving process?

The habituation of cell cultures to cellulose biosynthesis inhibitors constitutes a valuable method for learning more about the plasticity of plant cell wall composition and structure. The subculture of habituated cells in the absence of an inhibitor (dehabituation) offers complementary information: some habituation-associated modifications revert, whereas others remain, even after longterm (3–5 years) dehabituation processes. However, is dehabituation simply the opposite to the process of habituation, in the same way that the cloth woven by Penélope during the day was unwoven during the night? Principal Component Analysis applied to Fourier Transformed Infrared (FTIR) spectra of cell walls from dichlobenil-habituated and dehabituated bean cell lines has shown that dehabituation follows a different pathway to that of habituation. Principal component loadings show that dehabituated cells have more pectins, but that these display a lower degree of methyl-esterification, than those of habituated ones. Further analysis of cell walls focusing on the first steps of habituation would serve to identify which specific modifications in pectins are responsible to the fine modulation of cell wall architecture observed during the habituation/dehabituation process.Key words: cell-wall, cellulose, dichlobenil, habituation-dehabituation, Fourier transform infrared spectroscopy, principal component analysisThe habituation of cell cultures to the presence of lethal concentrations of cellulose biosynthesis inhibitors illustrates the ability of cells to survive with a modified cell wall and is therefore a valuable experimental technique for gaining an insight into the plasticity of plant cell wall composition and structure. Dichlobenil-habituated cultures usually display some common features: slower growth rates, irregularly shaped cells, a trend to grow in clumps when cultured in suspension and compensation of reduced cell wall cellulose content with other cell wall components.^1–3Most of the cell wall changes induced during the habituation to dichlobenil reverted when cells were dehabituated by culturing them in an inhibitor-free medium.^4–7 However, even in long term (3–5 years) dehabituated cell cultures, some habituation-induced cell wall modifications remain, such as altered extractability of pectins, a decrease in arabinogalactan proteins and hydroxyproline-rich glycoproteins epitopes, and the presence of a soluble β-(1,4)-glucan, although cellulose levels were restored.^5–7 Most remarkably, in addition to these stable changes in cell wall architecture, bean dehabituated cells retained a high capacity to cope with lethal concentrations of dichlobenil, as dehabituated cells were forty times more tolerant to dichlobenil than non-habituated cells.⁵ In an attempt to explain the dichlobenil resistance of dehabituated cells it was found that they had a constitutively increased peroxidase activity, indicating a positive relationship between habituation to dichlobenil and antioxidant capacity.⁷If most of the cell wall modifications induced during the habituation to dichlobenil eventually revert to those of non-habituated cells during the dehabituation process, a question arises: is dehabituation simply the inverse of habituation, in the same way that the cloth woven by Penelope during the day was unwoven during the night, as narrated in Homer''s The Odyssey?Principal Component Analysis applied to Fourier Transformed InfraRed spectra of cell walls has been demonstrated to be a powerful technique for conducting comparative analysis of a wide range of cell wall samples.^3,8 Therefore, a suitable approach to answering this question consists in comparison of cell walls from dichlobenil-habituated and dehabituated bean cell lines using this technique.Clearly, FTIR spectra of cell walls from dehabituated cells with few subcultures in the absence of the herbicide resemble those from cultures habituated to high dichlobenil concentrations.⁵ However, the spectra from cells habituated to low inhibitor concentrations and from cells dehabituated for long periods of time⁷ were more similar to those from non-habituated ones. In fact, when Principal Component Analysis is applied to the entire range, Principal Component 2 (PC2) discriminates between Sh₁₂ (corresponding to cells habituated to high dichlobenil concentration) and the rest of the spectra, which is indicative of the above-mentioned similarity (Fig. 1). Nevertheless, PC1 clearly discriminates between spectra from long-term dehabituated cell walls (located at the positive side) and those from cells habituated to low dichlobenil concentrations (at the negative side). This indicates that progression towards dehabituation follows a different path to that of habituation.Open in a separate windowFigure 1Principal Component Analysis of spectra of cell walls from different calluses. A plot of the first two Principal Components scores is represented based on the FTIR spectra of cell walls from non-habituated cells (Snh, ○), cells habituated to different dichlobenil concentrations (Sh, ▲), and cells previously habituated to 12 µm dichlobenil, with a different number of subcultures in the absence of the herbicide (Sd, ◆). Subindexes indicate dichlobenil concentrations in the growth media of habituated cells (0.3, 0.4 or 12 µm); superindexes indicate number of subcultures in the same media. Arrows indicate the different pathways followed by dichlobenil habituation and dehabituation: black arrows, from non-habituated to habituated cells (habituation), and white arrows, from habituated to non-habituated cells (dehabituation).With the aim of identifying those factors which determine this different pathway, PC1 and PC2 loading factors were analyzed (Fig. 2). This analysis indicated that PC2 (explaining 26.4% of total variance) has a positive correlation with wavenumbers attributed to uronic acids (1,420 and 1,600 cm⁻¹) and galactose (950 cm⁻¹), and a negative correlation with wavenumbers associated with cellulose (1,040, 1,060, 1,175, 1,320 and 1,370 cm⁻¹) and xyloglucan (1,125 cm⁻¹). Thus, Sh₁₂ cell walls (clearly located at the positive side of PC2) are pectin enriched and cellulose/xyloglucan impoverished. As explained above, PC1 discriminates between cell walls from dehabituated cell lines and those from cells habituated to low concentrations of dichlobenil. PC1 (accounting for 42.55% of total variance) has a negative correlation with wavenumbers associated with methylester groups (negative peaks at 1,250 and 1,720 cm⁻¹), and a positive correlation with the so called “fingerprint” region (980–1,200 cm⁻¹). Therefore, cell walls from dehabituated cells (those located at the positive side of PC1) would have lower methyl-esterified pectins when compared with cells habituated to low concentrations of dichlobenil.Open in a separate windowFigure 2Loadings for PC1 and PC2 corresponding to Figure 1. White arrowheads point wavenumbers associated with methyl-esterification; black arrowheads, those associated with cellulose and hemicelluloses, and grey arrowheads indicate wavenumbers associated with uronic acids and galactose.Previous results had revealed that dichlobenil habituated cells experienced a progressive reversion in their cell wall composition when they were subcultured in an inhibitor-free medium, gradually increasing their xyloglucan and cellulose content,^5,6 and that both dichlobenil habituated and dehabituated cells showed changes in the distribution of pectin among cell wall fractions: cell suspensions with a low habituation level had cell walls with a higher amount of pectins, and these were more methyl-esterified.⁶Now, FTIR spectroscopy in association to Principal Component Analysis has shown that, although some of the changes observed in the first steps of habituation and in the last steps of dehabituation are common (i.e., reversion of cellulose content), some other changes affect habituated and dehabituated cells differently, and that these changes involve mainly pectin composition and organization. A more detailed analysis of cell walls focusing on the first steps of habituation will serve to identify which specific modifications are responsible for the differences observed in the pectic component and, consequently, responsible for the fine modulation of cell wall architecture.     

The phenolic profile of maize primary cell wall changes in cellulose-deficient cell cultures

Cultured maize cells habituated to grow in the presence of the cellulose synthesis inhibitor dichlobenil (DCB) have a modified cell wall in which the amounts of cellulose are reduced and the amounts of arabinoxylan increased. This paper examines the contribution of cell wall-esterified hydroxycinnamates to the mechanism of DCB habituation. For this purpose, differences in the phenolic composition of DCB-habituated and non-habituated cell walls, throughout the cell culture cycle and the habituation process were characterized by HPLC. DCB habituation was accompanied by a net enrichment in cell wall phenolics irrespective of the cell culture phase. The amount of monomeric phenolics was 2-fold higher in habituated cell walls. Moreover, habituated cell walls were notably enriched in p-coumaric acid. Dehydrodimers were 5–6-fold enhanced as a result of DCB habituation and the steep increase in 8,5′-diferulic acid in habituated cell walls would suggest that this dehydrodimer plays a role in DCB habituation. In summary, the results obtained indicate that cell wall phenolics increased as a consequence of DCB habituation, and suggest that they would play a role in maintaining the functionality of a cellulose impoverished cell wall.     

Serum insulin-like growth factor levels do not predict functional recovery after hospitalization in nonagenarian patients

AIM: To analyze the insulin-like growth factor 1 (IGF-1) serum levels in nonagenarian patients and to investigate the predictive capacity of this measure to assess the functional recovery of this population following hospitalization. METHODS: We performed a prospective study in 60 consecutive nonagenarian patients admitted for medical or surgical diseases. We assessed IGF-1 serum levels and nutritional status. The functional status was assessed using the Barthel index. Thirty-four patients were reinvestigated 3 months after discharge from hospital. RESULTS: The mean levels of IGF-1 were lower in nonagenarians than in younger patients. No relationship was found between IGF-1 levels and nutritional status. The decline in Barthel index values 3 months after discharge from hospital did not correlate with serum levels of IGF-1 on admission. CONCLUSION: IGF-1 serum levels in nonagenarian patients do not predict functional recovery after hospitalization.