The influence of fixation on the relative amount of cytoplasmic ribosomes in mouse epidermal basal keratinocytes

The nature and significance of so-called dark keratinocytes in the epidermis during chemical carcinogenesis is still a matter of concern and debate. Based on ultrastructural observations it has been suggested that dark cells most often are shrunken cells. Reports on skin carcinogenesis, however, claim that dark cells are a sign of ongoing tumor promotion and represent those stem cells in the epidermis from which the tumors originate. It is therefore important to find out whether these cells are simply injured and shrunken cells, or vital cells of great importance for carcinogenesis. Dark cells are assumed to be rich in ribosomes. There is evidence, however, that the observed number of dark cells is highly dependent on tissue fixation. In the present ultrastructural study, morphometric methods were used to compare the effects of two different fixation procedures on the amount of cytoplasmic ribosomes in dark cells from both untreated and carcinogen-treated hairless mouse epidermis. The results show that the ultrastructural features of both dark and clear cells vary considerably with different fixation procedures. In acetone-treated controls typical dark cells are only observed when the fixative has a lower osmotic activity than the plasma. With iso-osmolal fixation typical dark cells are not observed. After an abortive two-stage carcinogenesis treatment, in which a single application of 9,10-dimethyl-l,2-benzanthracene (DMBA) in acetone was followed by a single application of 12-O-tetradecanoyl-13-acetate (TPA) in acetone, signs of cell injury could be found after both fixation procedures. With DMBA/TPA and hypo-osmolal fixation the number of dark cells seemed to increase, whereas only signs of cell injury with occurrence of some heavily altered “clear cells” dominated the picture with iso-osmolal fixation. Morphometry showed that both the numerical and the volumetric densities of cytoplasmic ribosomes in basal keratinocytes varied most significantly with the fixation procedure used. The cytoplasmic volumes did not vary in a way that could explain these differences. One might therefore assume that the number of ribosomes depends on the fixative. Large swelling artifacts occurred when a fixative with low osmotic activity was used, leading to compression of neighboring cells. Hence, an increased ribosomal density reported previously in dark cells is probably related to such cell volume artifacts and does not reflect an actually increased quantity of ribosomes. With both fixation procedures, a single application of DMBA followed by one of TPA appeared to produce an increased number of ribosomes in basal keratinocytes. When hypo-osmolal fixation was used, however, treatment with DMBA/TPA did not influence the cytoplasmic volume or the numerical density of ribosomes, in dark cells. This might indicate that so-called dark keratinocytes following DMBA/TPA treatment are functionally inactive cells that appear more vulnerable than active cells to compression during hypo-osmolal fixation.     

Energy metabolism in the cyanobacterium Plectonema boryanum. Participation of the thylakoid photosynthetic electron transfer chain in the dark respiration of NADPH and NADH

The oxidation of NADPH and NADH was studied in the light and in the dark using sonically derived membrane vesicles and osmotically shocked spheroplasts. These two types of cell-free membrane preparations mostly differ in that the cell and thylakoid membranes are scrambled in the former type and that they are more or less separated in the latter type of preparations. In the light, using both kinds of preparations, each of NADPH and NADH donates electrons via the plastoquinone-cytochrome $\text{b}\text{c}$ redox complex (Qbc redox complex) to the thylakoid membrane-bound cytochrome c-553 preoxidized by a light flash and to methylviologen via Photosystem I. NADPH donates electrons to the thylakoid membrane via a weakly rotenone-sensitive dehydrogenase to a site that is situated beyond the 3(3′,4′-dichlorophenyl)-1,1-dimethylurea sensitive site and before plastoquinone. Ferredoxin and easily soluble cytoplasmic proteins are presumably not involved in light-mediated NADPH oxidation. Inhibitors of electron transfer at the Qbc redox complex as the dinitrophenylether of 2-iodo-4-nitrothymol, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2-n-heptyl-4-hydroxy-quinone-N-oxide are effective, but antimycin A and KCN are not. The oxidation of NADH showed comparable sensitivity to these inhibitors. However, the oxidation of NADH is antimycin-A-sensitive regardless of the kind of membrane preparation used, indicating that in this case electrons are donated to a different site on the thylakoid membrane. In the dark, NADPH and NADH donate electrons at sites that behave similar to those of light-mediated oxidation, indicating that the initial steps of electron transfer are situated at the thylakoid membranes. However, NADPH oxidation is in some cases not sensitive to inhibitors active at the Qbc redox complex. It is concluded that O₂ reduction takes place at two different sites, one partly developed in vitro, situated near the rotenone-sensitive NADPH dehydrogenase, and another, highly KCN-sensitive one, situated beyond the Qbc redox complex and used in vivo. The terminal oxygen-reducing step of NADPH and NADH oxidation in the dark showed a preparation-dependent sensitivity for KCN, more than 80% inhibition in sonically derived membrane vesicles and less than 30% inhibition in osmotically shocked spheroplasts. From this result we tentatively conclude that the highly KCN-sensitive oxidase is not necessarily located at the thylakoid membrane and could be located at the cytoplasmic membrane.     

Dark modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase in the chloroplast

The properties of the system which reverses light modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase activity in pea chloroplasts were examined. A factor catalyzing dark modulation of these enzymes was found. This factor cochromatographed with thioredoxin in all systems used (Sephacryl S-200, Sephadex G-75, DEAE-cellulose). Inhibition of dithiothreitol-dependent modulation and of dark reversal by antibody against Escherichia coli thioredoxin further suggest that the dark factor is in fact thioredoxin. It appears that the reaction is the reverse of the previously described dithiothreitol-dependent thioredoxin-catalyzed modulation of enzymes. The limiting step in vitro seems to be the oxidation of thioredoxin during the dark period.     

Dark requirement for cell regeneration and colony formation by mesophyll protoplasts ofNicotiana plumbaginifolia Viv.

Summary The protoplasts ofNicotiana plumbaginifolia required darkness for cell regeneration and colony formation. Maximal plating efficiency of the protoplasts could be achieved by keeping the cultures in dark instead of light or dark/light sequence. Only two days of darkness prior to the illumination at 400 or 3,000 lux resulted in appreciable plating efficiency, than those of light from the beginning, but these values could not match the high plating efficiency in total darkness.     

Differences in the response of sunflower (Helianthus annuus) subjected to flooding and drought stress

Wample, R. L. and Thornton, R. K. 1984. Differences in the response of sunflower ( Helianthus annuus ) subjected to flooding and drought stress.
Comparison of drought- and flood-stressed sunflower plants ( Helianthus annuus L. hybrid 894) showed some similarities in response but differences in the mechanisms responsible for the responses to stress. Drought–stressed plants showed typical reductions in leaf water potential with increasing stress accompanied by increased leaf resistance. Photosynthesis declined while photorespiration increased after 48 and 96 h of drought stress. A primary reason for reduced photosynthesis in drought-stressed plants was increased stomatal resistance. No significant 0change in leaf water potential or in leaf resistance in flooded plants was found in this study. However, photosynthesis declined in a manner similar to that in drought-stressed plants and photorespiration showed only a transient increase at 48 h. Dark respiration was significantly higher at 48 and 96 h but the magnitude of the increase cannot account for the reduction in photosynthesis. Since the photosynthetic rate of flooded plants declined while stomata remained open, an effect at a more fundamental level is suggested and is thought to be related to disruption of carbohydrate transport.     

ABA and BAP improve the accumulation of carbohydrates and alter carbon allocation in potato plants at elevated CO2

Elevated CO₂ interactions with other factors affects the plant performance. Regarding the differences between cultivars in response to CO₂ concentrations, identifying the cultivars that better respond to such conditions would maximize their potential benefits. Increasing the ability of plants to benefit more from elevated CO₂ levels alleviates the adverse effects of photoassimilate accumulation on photosynthesis and increases the productivity of plants. Despite its agronomic importance, there is no information about the interactive effects of elevated CO₂ concentration and plant growth regulators (PGRs) on potato (Solanum tuberosum L.) plants. Hence, the physiological response and source-sink relationship of potato plants (cvs. Agria and Fontane) to combined application of CO₂ levels (400 vs. 800 µmol mol⁻¹) and plant growth regulators (PGR) [6-benzylaminopurine (BAP) + Abscisic acid (ABA)] were evaluated under a controlled environment. The results revealed a variation between the potato cultivars in response to a combination of PGRs and CO₂ levels. Cultivars were different in leaf chlorophyll content; Agria had higher chlorophyll a, b, and total chlorophyll content by 23, 43, and 23%, respectively, compared with Fontane. The net photosynthetic rate was doubled at the elevated compared with the ambient CO₂. In Agria, the ratio of leaf intercellular to ambient air CO₂ concentrations [C_i:C_a] was declined in elevated-CO₂-grown plants, which indicated the stomata would become more conservative at higher CO₂ levels. On the other hand, the increased C_i:C_a in Fontane showed a stomatal acclimation to higher CO₂ concentration. The higher leaf dark respiration of the elevated CO₂-grown and BAP + ABA-treated plants was associated with a higher leaf soluble carbohydrates and starch content. Elevated CO₂ and BAP + ABA shifted the dry matter partitioning to the belowground more than the above-media organs. The lower leaf soluble carbohydrate content and greater tuber yield in Fontane might indicate a more efficient photoassimilate translocation than Agria. The results highlighted positive synergic effects of the combined BAP + ABA and elevated CO₂ on tuber yield and productivity of the potato plants.     

scGET: Predicting Cell Fate Transition During Early Embryonic Development by Single-cell Graph Entropy

During early embryonic development, cell fate commitment represents a critical transition or"tipping point"of embryonic differentiation, at which there is a drastic and qualitative shift of the cell populations. In this study, we presented a computational approach, scGET, to explore the gene–gene associations based on single-cell RNA sequencing (scRNA-seq) data for critical transition prediction. Specifically, by transforming the gene expression data to the local network entropy, the single-cell graph entropy (SGE) value quantitatively characterizes the stability and criticality of gene regu-latory networks among cell populations and thus can be employed to detect the critical signal of cell fate or lineage commitment at the single-cell level. Being applied to five scRNA-seq datasets of embryonic differentiation, scGET accurately predicts all the impending cell fate transitions. After identifying the"dark genes"that are non-differentially expressed genes but sensitive to the SGE value, the underlying signaling mechanisms were revealed, suggesting that the synergy of dark genes and their downstream targets may play a key role in various cell development processes. The application in all five datasets demonstrates the effectiveness of scGET in analyzing scRNA-seq data from a network perspective and its potential to track the dynamics of cell differentiation. The source code of scGET is accessible at https://github.com/zhongjiayuna/scGET_Project.     

Could a dysfunction of ferritin be a determinant factor in the aetiology of some neurodegenerative diseases?

Background

The concentration of iron in the brain increases with aging. Furthermore, it has also been observed that patients suffering from neurological diseases (e.g. Parkinson, Alzheimer…) accumulate iron in the brain regions affected by the disease. Nevertheless, it is still not clear whether this accumulation is the initial cause or a secondary consequence of the disease. Free iron excess may be an oxidative stress source causing cell damage if it is not correctly stored in ferritin cores as a ferric iron oxide redox-inert form.

Scope

Both, the composition of ferritin cores and their location at subcellular level have been studied using analytical transmission electron microscopy in brain tissues from progressive supranuclear palsy (PSP) and Alzheimer disease (AD) patients.

Major conclusions

Ferritin has been mainly found in oligodendrocytes and in dystrophic myelinated axons from the neuropili in AD. In relation to the biomineralization of iron inside the ferritin shell, several different crystalline structures have been observed in the study of physiological and pathological ferritin. Two cubic mixed ferric–ferrous iron oxides are the major components of pathological ferritins whereas ferrihydrite, a hexagonal ferric iron oxide, is the major component of physiological ferritin. We hypothesize a dysfunction of ferritin in its ferroxidase activity.

General significance

The different mineralization of iron inside ferritin may be related to oxidative stress in olygodendrocites, which could affect myelination processes with the consequent perturbation of information transference.     

Transcriptional Induction of Two Genes for CCaPs, Novel Cytosolic Proteins, in Arabidopsis thaliana in the Dark

Ca2+-signaling in downstream effectors is supported by many kinds of Ca2+-binding proteins, which function as a signal mediator and a Ca2+-buffering protein. We found in Arabidopsis thaliana a new type of Ca2+-binding protein, CCaP1, which consists of 152 amino acid residues, and binds (45)Ca2+ even in the presence of a high concentration of Mg2+. We found two other proteins with similar motifs, CCaP2 and CCaP3. These three proteins had no organelle localization signal and their green fluorescent protein (GFP) fusions were detected in the cytosol. Real-time PCR and histochemical analysis of promoter-beta-glucuronidase fusions revealed that CCaP1 was predominantly expressed in petioles while CCaP2 was expressed in roots. CCaP3 was hardly expressed. Expression of CCaP1 and CCaP2 was enhanced in darkness and became maximal after 24 h. Immunoblotting revealed petiole-specific accumulation of CCaP1. Expression of CCaP1 and CCaP2 was suppressed by a high concentration of Ca2+ and other metal ions. Deletion of sucrose from the medium markedly increased the mRNA levels of CCaP1 and CCaP2 within 2 h. Gibberellic acid enhanced the expression of CCaP1 and CCaP2 by 5- and 2.5-fold, respectively, after 6 h. CCaP1 and CCaP2 were suppressed in the petiole and the root, respectively, by light and the product of photosynthesis (sucrose) or both. These results suggest that CCaP1 functions as a mediator in response to continuous dark or gibberellic acid.     

Prehistorically modified soils of central Amazonia: a model for sustainable agriculture in the twenty-first century

Terra Preta soils of central Amazonia exhibit approximately three times more soil organic matter, nitrogen and phosphorus and 70 times more charcoal compared to adjacent infertile soils. The Terra Preta soils were generated by pre-Columbian native populations by chance or intentionally adding large amounts of charred residues (charcoal), organic wastes, excrements and bones. In this paper, it is argued that generating new Terra Preta sites ('Terra Preta nova') could be the basis for sustainable agriculture in the twenty-first century to produce food for billions of people, and could lead to attaining three Millennium Development Goals: (i) to combat desertification, (ii) to sequester atmospheric CO2 in the long term, and (iii) to maintain biodiversity hotspots such as tropical rainforests. Therefore, large-scale generation and utilization of Terra Preta soils would decrease the pressure on primary forests that are being extensively cleared for agricultural use with only limited fertility and sustainability and, hence, only providing a limited time for cropping. This would maintain biodiversity while mitigating both land degradation and climate change. However, it should not be overlooked that the infertility of most tropical soils (and associated low population density) is what could have prevented tropical forests undergoing large-scale clearance for agriculture. Increased fertility may increase the populations supported by shifting cultivation, thereby maintaining and increasing pressure on forests.