Respiratory pathways in germinating maize radicles correlated with desiccation tolerance and soluble sugars

We have assessed the activities of the cytochrome and alternative pathways in total respiration and their role in each stage of germination of Zea mays L. radicles. Throughout imbibition, the salicylhydroxamic acid (SHAM) concentration needed to inhibit the cyanide-resistant pathway, without any side effects, decreased from 15 m M in quiescent embryos to 5 m M at 72 h after imbibition. Electrons predominantly flowed through the cytochrome pathway although the alternative pathway was already present at early imbibition. The capacity of the alternative path was about 70% of the control rate of respiration. Its engagement progressively increased from 18% after 10 min of imbibition to 70% at the radicle emergence and then decreased to 50% at 96 h after imbibition, concomitant with the onset of radicle growth. The alternative pathway was, however, not essential for germination. The observed activity of the alternative path correlated with the monosaccharide (glucose + fructose) content, suggesting that the alternative pathway could be acting according to the 'energy overflow model'. On the other hand, up to 24 h after imbibition at 16°C, maize radicles tolerate a severe desiccation, becoming intolerant at 72 h. On reimbibition of tolerant radicles, respiration increased immediately and the alternative pathway was rapidly engaged. At 72 h, no respiration was measured, indicating a total loss of the respiratory systems. The possible correlation between carbohydrate content, loss of desiccation tolerance and activity of the two respiratory pathways is discussed.     

Formation of multilayer rosettes in phytohemagglutinin-stimulated lymphocytes: correlation with chromatin condensation conformation in premature chromosome condensation

We found that the formation of multilayer rosettes by transformed human blood lymphocytes after phytohemagglutinin (PHA) stimulation is correlated with conformational changes of the chromatin as seen by premature chromosome condensation (PCC). The frequency distribution of grades of PCC and multilayer rosette formation suggests that changes in chromatin are a prerequisite for rosette formation. Rosette formation was most pronounced for 24-h and 48-h cultures. Chromatin decondensation and rosette formation showed identical patterns. The possibility that multilayer rosette formation is directly dependent on conformational changes of chromatin is discussed.     

Changing desiccation tolerance of pea embryo protoplasts during germination

Protoplasts were isolated from pea (Pisum sativum L. cv. Alaska) embryonic axes during and after germination to determine whether the loss of desiccation tolerance in the embryos also occurs in the protoplasts. At all times studied, protoplast survival decreased as water content decreased; however, the sensitivity to dehydration was less when the protoplasts were isolated from embryos that were still desiccation-tolerant (12 h and 18 h of imbibition) than when protoplasts were derived from axes that were sensitive (24 h and 36 h of imbibition). The water content at which 50% of the population was killed (WC50) increased throughout germination and early seedling growth for both the intact tissue and the protoplasts derived from them. Prior to radicle emergence, protoplasts were less desiccation-tolerant than the intact axes; however, protoplasts isolated from radicles shortly after emergence had lower WC50s than the intact radicles. A comparison of protoplast survival after isolation and dehydration in either 500 mM sucrose/raffinose or 700 mM sucrose revealed no difference in tolerance except at 24 h of imbibition, when protoplasts treated in the more concentrated solution had improved tolerance of dehydration. Although intact epicotyls are generally more desiccation-tolerant than radicles, protoplasts isolated separately from epicotyls and radicles did not differ in tolerance. Collectively, these data suggest that protoplasts gradually lose desiccation tolerance during germination, as do the orthodox embryos from which they were derived. However, even prior to radicle emergence, protoplasts display a sensitivity to progressive dehydration that is similar to that shown by recalcitrant and ageing embryos.     

Chromatin structure during the prereplicative phases in the life cycle of mammalian cells

The object of this study was to determine the kinetics of chromosome decondensation during the G₁ period of the HeLa cell cycle. HeLa cells synchronized in the G₁ period following the reversal of mitotic block were fused with Colcemid-arrested mitotic HeLa cells at 1.5, 3, 5, and 7 h after the reversal of N₂O block. The resulting prematurely condensed chromosomes (PCC) were classified into six categories depending on the degree of their condensation. The frequency of occurrence of each category was plotted as a function of time after mitosis. The results of this study indicate that the process of chromosome decondensation, initiated during the telophase of mitosis continues throughout the G₁ period without any interruption, thus the chromatin reaches an ultimate state of decondensation by the end of G₁ period, when DNA synthesis is initiated.     

In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins

Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg‐jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø_o (sperm‐specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca²⁺, pH, and this activity could be a serine‐proteinase. Thermal denaturalization of the oocyte extracts (80°C, 10–15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø_o) will be a first condition in the process of sperm chromatin remodeling.     

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in naïve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI     

Experimental Visualization of Chromoneme as a Higher Level of Chromatin Compactization in the Mitotic Chromosome

We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1–0.2 μm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase chromosomes on ultrathin sections. We studied the ultrastructure and behavior of the chromatin of mitotic chromosomes in situ in cultured mouse L-197 cells under the conditions selectively demonstrating the chromoneme structure of the mitotic chromosomes in the presence of Ca²⁺. Loosely packaged dense chromatin bands, ca. 100 nm in diameter, chromonemes, were detected in chromosome arms in a solution containing 3 mM CaCl₂. When transferred in a hypotonic solution containing 10 mM tris-HCl, these chromosomes swelled, lost the chromoneme level of structure, and rapidly transformed in loose aggregates of elementary DNP fibrils, 30 nm in diameter. After this decondensation in the low ionic strength solution, the chromoneme structure of mitotic chromosomes was restored when they were transferred in a Ca₂₊ containing solution. The morphological characteristics of the chromoneme and pattern of its packaging in the chromosome were preserved. However, when the mitotic cells with chromosomes, in which the chromoneme structure was visualized with the help of 3 mM CaCl₂, were treated with a photosensitizer, ethidium bromide, and illuminate with a light with the wavelength 460 nm, chromatic decondensation under the hypotonic solution was not observed. The chromoneme elements in a stabilized chromatin of the mitotic chromosome preserved specific interconnection and the general pattern of their packaging in the chromatid was also preserved. The chromoneme elements in the chromosomes stabilized by light preserved their density and diameter even in a 0.6 M NaCl solution, which normally leads to chromoneme destruction. An even more rigid treatment of the stabilized chromosomes with a 2 M NaCl solution, which normally fully decondenses the chromosomes, made it possible to detect a 3D reticular skeleton devoid of any axial structures. __________ Translated from Ontogenez, Vol. 36, No. 5, 2005, pp. 323–332. Original Russian Text Copyright ? 2005 by Burakov, Tvorogova, Chentsov.     

Mediterranean flour moth Ephestia kuehniella eggs and larvae exposed to a static magnetic field and preference by Trichogramma embryophagum

This study investigated the effect of strong magnetic fields as insecticidal activity on Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) larvae and eggs at different stages of development and their preference by the egg parasitoid, Trichogramma embryophagum Hartig (Hymenoptera: Trichogrammatidae). Eggs ranging in age from 24-h to 48-h and 72-h-old and larvae (1 to 2 days) were exposed to 1.4 Tesla (T) magnetic fields from a DC power supply at 50 Hz for different time periods (3, 6, 12, 24, 48 and 72 h). Twelve hours of exposure at 1.4 T was toxic to 24-h-old eggs of E. kuehniella. The 72-h-old host eggs treated with 1.4 T for 6–72 h were not significantly preferred by T. embryophagum. The magnetic field was toxic to 24-h-old eggs of E. kuehniella exposed to 1.4 T for 12. The treatment of magnetic fields on the 72-h-old host egg with 1.4 T at 6–72 h was not significantly preferred by T. embryophagum. Magnetization of 24-h-old eggs of E. kuehniella for 3 h could be effectively used with T. embryophagum as sterilised host eggs. These eggs were markedly preferred by T. embryophagum. The LT₅₀ and LT₉₉ values of magnetic fields at different egg stages of E. kuehniella, and larvae were measured. A level of 1.4 T at 72 h completely prevented the development of the larvae. There was no significant effect on larval survival at 1.4 T at 48 and 72 h. Increasing magnetic fields exposure times for eggs that were 24-h, 48-h and 72-h-old prevented larval emergence and increased their mortality rate. Consequently, magnetic fields could be used in controlling stored-product pest eggs and larvae of E. kuehniella.     

The disruption in actin-perinuclear theca interactions are related with changes induced by cryopreservation observed on sperm chromatin nuclear decondensation of boar semen

The perinuclear theca (PT) is a cytoskeletal structure that surrounds the mammal sperm nucleus which must be disrupted once the sperm has penetrated the oocyte to permit normal chromatin decondensation and formation of male pronucleus. F-actin is a thermo sensitive protein found in the equatorial segment which is involved in the stability of PT. It has been reported that cryopreservation induces alterations in nuclear decondensation of spermatozoa, which have been interpreted as an over condensation. The aims of the present study were identified the presence of changes in sperm sPT integrity of frozen–thawed boar spermatozoa and its effect in sperm nuclei decondensation; and whether changes in the actin cytoskeleton are involved using an in vitro model to test probably differences in a chemical decondensation (DTT/heparin) between fresh (FS) and frozen–thawed (TS) spermatozoa. Results showed an increase on sPT damage in TS (P < 0.001), and significant changes in sperm chromatin nuclear decondensation (P < 0.05). In same way differences on the swelling degree was found assessed by measures in equatorial region of head sperm (P < 0.05). Evaluation with rodamine-labeled actin (0.2 μM) showed two different patterns with differences in percentages before and after cryopreservation (P < 0.001). F-actin stabilization constrained the equatorial segment of FS while this was not observed in TS. The data showed that the presence of early changes in sPT integrity and changes in the F-actin localization on TS may suggest the participation in F-actin in decondensation process and probably that the disruption of actin-PT interaction during freezing–thawing process could have far-reaching consequences for the subsequent fertility of spermatozoa.     

Dehydration-induced redistribution of amphiphilic molecules between cytoplasm and lipids is associated with desiccation tolerance in seeds

This study establishes a relationship between desiccation tolerance and the transfer of amphiphilic molecules from the cytoplasm into lipids during drying, using electron paramagnetic resonance spectroscopy of amphiphilic spin probes introduced into imbibed radicles of pea (Pisum sativum) and cucumber (Cucumis sativa) seeds. Survival following drying and a membrane integrity assay indicated that desiccation tolerance was present during early imbibition and lost in germinated radicles. In germinated cucumber radicles, desiccation tolerance could be re-induced by an incubation in polyethylene glycol (PEG) before drying. In desiccation-intolerant radicles, partitioning of spin probes into lipids during dehydration occurred at higher water contents compared with tolerant and PEG-induced tolerant radicles. The difference in partitioning behavior between desiccation-tolerant and -intolerant tissues could not be explained by the loss of water. Consequently, using a two-phase model system composed of sunflower or cucumber oil and water, physical properties of the aqueous solvent that may affect the partitioning of amphiphilic spin probes were investigated. A significant relationship was found between the partitioning of spin probes and the viscosity of the aqueous solvent. Moreover, in desiccation-sensitive radicles, the rise in cellular microviscosity during drying commenced at higher water contents compared with tolerant or PEG-induced tolerant radicles, suggesting that the microviscosity of the cytoplasm may control the partitioning behavior in dehydrating seeds.     

Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.     

DNA methylation is not involved in the structural alterations of Ornithine Decarboxylase or Total Chromatin of c-Ha-rasVal 12 Oncogene-Transformed NIH-3T3 Fibroblasts

The ornithine decarboxylase (odc) gene is an early response gene, whose increased expression and relaxed chromatin structure is closely coupled to neoplastic growth. In various tumour cells, the odc gene displays hypomethylation at the sequences CCGG. Hypomethylation of genes is believed to correlate with chromatin decondensation and gene expression. Since a given pattern of DNA methylation may not be preserved in neoplastic cells, we studied the methylation status of odc gene at the CCGG sequences in c-Ha-ras^{Val 12} oncogene-transformed NIH-3T3 fibroblasts during the growth cycle and relative to their normal counterparts. We found that the methylation state of the odc gene and its promoter and mid-coding and 3' regions remain unaltered during the cell cycle. We also found that in ras oncogene-transformed cells, which display a more decondensed nucleosomal organization of chromatin than the normal cells, the CCGG sequences in bulk DNA and at the odc gene were methylated to the same extent as in the nontransformed cells. These data suggest that DNA hypomethylation at the CCGG sequences is not a prerequisite for chromatin decondensation and cell transformation by the c-Ha-ras^{Val 12} oncogene.     

Gene Expression in the Cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120 under Desiccation

The N₂-fixing cyanobacterium Anabaena sp. PCC7120 showed an inherent capacity for desiccation tolerance. A DNA microarray covering almost the entire genome of Anabaena was used to determine the genome-wide gene expression under desiccation. RNA was extracted from cells at intervals starting from early to late desiccation. The pattern of gene expression in DNA fragments was categorized into seven types, which include four types of up-regulated and three types of down-regulated fragments. Validation of the data was carried out by RT-PCR on selected up-regulated DNA fragments and was consistent with the changes in mRNA levels. Our conclusions regarding desiccation tolerance for Anabaena sp. PCC7120 are as follows: (i) Genes for osmoprotectant metabolisms and the K⁺ transporting system are up-regulated from early to mid-desiccation; (ii) genes induced by osmotic, salt, and low-temperature stress are up-regulated under desiccation; (iii) genes for heat shock proteins are up-regulated after mid-desiccation; (iv) genes for photosynthesis and the nitrogen-transporting system are down-regulated during early desiccation; and (v) genes for RNA polymerase and ribosomal protein are down-regulated between the early and the middle phase of desiccation. Profiles of gene expression are discussed in relation to desiccation acclimation.     

Stress-associated factors increase after desiccation of germinated seeds of <Emphasis Type="Italic">Tabebuia impetiginosa</Emphasis> Mart.

Improved re-establishment of desiccation tolerance was studied in germinated seeds of Tabebuia impetiginosa Mart. by exposing to a polyethylene glycol solution prior to desiccation. The effects of different osmotic potentials and drying rates were studied. In addition, temporary temperature stress and exogenous abscisic acid were applied to evaluate their effect on desiccation tolerance of the protruded radicle. An osmotic potential of −1.7 MPa at 5°C followed by slow drying was most effective in the re-establishment of desiccation tolerance in protruded radicles with a length up to 3 mm. An osmotic potential of −1.4 or −2.0 MPa was less effective. Fast drying completely prevented the re-induction of desiccation tolerance. Cold shock or heat shock prior to osmotic treatment as well as abscisic acid added to the osmotic solution improved desiccation tolerance of protruded radicles. Surprisingly, survival of the germinated seed did not depend on re-establishment of desiccation tolerance in the protruded radicle. Even after the protruded radicle became necrotic and died, the production of adventitious roots from the hypocotyls allowed for survival and the development of high quality seedlings. Thus, T. impetiginosa appeared to be well adapted to the seasonally dry biome in which the species thrives via mechanisms that offer protection against desiccation in the young seedling stage.     

Formation of Chromoneme-like Fibrils Induced in Infusorian Somatic Nuclei by Magnesium Ions

A study was made of the effect of Mg²⁺ on higher-order chromatin structure in macronuclei (Ma) of infusoria Paramecium aurelia and Bursaria truncatella. In infusorian Ma, inactive chromatin is commonly packed in chromatin bodies sized 60–200 nm. When isolated chromatin or Ma were treated with Mg²⁺ (about 3 mM), chromatin bodies arranged into fibrils 100–300 nm in diameter, which resembled higher eukaryotic chromonemes. The dynamics of chromoneme-like fibril formation was described. The results testified to the similarity of chromatin bodies to chromomeres of higher eukaryotes. Structurally intact central chromomere cores proved to be essential for the formation of chromoneme-like fibrils. Chromatin organization in infusoria was shown to follow the discrete-level model (nucleosomes–nucleomeres–chromomeres–chromonemes) assumed for higher eukaryotes.     

Sex-specific alterations in chromatin conformation of the brain of aging mouse

Chromatin conformation has been analysed in the brain cortex of adult (24±2 weeks) and old (65±4 weeks) male and female mice. Nuclei purified from different groups of mice were digested with MNase and DNase I for varying time periods (0–90 min), and with endogenous endonucleases for 1 h. MNase and DNase I digestion kinetics showed that the percentage of acid solubility of chromatin was relatively lower in old than adult and in female than male. This was further supported by electrophoretic analysis of nuclease digested DNA fragments. When the nuclei were incubated with only Ca²⁺or mg²⁺, no endonuclease digestion was observed. However, under similar conditions, the liver DNA was cleaved substantially. When divalent cations were added together, they activated endogenous endonucleases and digested the brain chromatin. The activity of Ca²⁺/Mg²⁺-dependent endogenous endonucleases was higher in male than female. Thus the accessibility of chromatin to MNase, DNase I and endogenous endonucleases was higher in male than female, and MNase as well as DNase I were more active in adult than old. Such sex- and age-dependent conformation of chromatin may attribute to differential expression of genes in the mouse brain.     

Inhibition of cation-induced DNA condensation by intercalating dyes

Several intercalating dyes are shown to inhibit the cation-induced condensation of λ-DNA when Co³⁺(NH₃)₆ is the condensing agent. The dyes that have been studied are ethidium, propidium, proflavin, quinacrine, and actinomycin D. Earlier work has shown that intercalating dyes inhibit ψ-DNA condensation. [Lerman, L. S. (1971) Prog. Mol. Subcell. Biol. 2 , 382–391; Cheng, S. & Mohr, S. C. (1975) Biopolymers 14 , 663–674.] Dye-induced decondensation of intramolecularly condensed DNA has been studied by making use of conditions in which Co³⁺(NH₃)₆ produces intramolecular condensation without significant aggregation. Some aggregation is caused, however, during dye-induced decondensation. Dye titration curves of DNA decondensation have been measured by excess light scattering to monitor decondensation and by fluorescence to monitor intercalation. All of the dyes studied act as competing cations in displacing the condensing cation Co³⁺(NH₃)₆ from the DNA. Competition occurs both in and below the transition zone for condensation. The effectiveness of a dye as a competing cation increases with its net positive charge. Before decondensation begins, no intercalated dye can be detected, suggesting that intercalation might be incompatible with the proper helix packing needed for cation-induced DNA condensation. To test this last point, methidium–spermine was synthesized: it contains an intercalating methidium head group combined with a polyamine tail. Methidium–spermine is found to cause λ-DNA condensation, but aggregation accompanies condensation, as has been found earlier for spermine and spermidine. Fluorescence and absorption spectra indicate that the methidium group is intercalated when the DNA is condensed, indicating that intercalation need not be incompatible with DNA condensation. The presence of aggregates among the condensed DNA molecules makes this last conclusion tentative.     

Metabolic dysfunction and unabated respiration precede the loss of membrane integrity during dehydration of germinating radicles

This study shows that dehydration induces imbalanced metabolism before loss of membrane integrity in desiccation-sensitive germinated radicles. Using a photoacoustic detection system, responses of CO(2) emission and fermentation to drying were analyzed non-invasively in desiccation-tolerant and -intolerant radicles of cucumber (Cucumis sativa) and pea (Pisum sativum). Survival after drying and a membrane integrity assay showed that desiccation tolerance was present during early imbibition and lost in germinated radicles. However, tolerance could be re-induced in germinated cucumber radicles by incubation in polyethylene glycol before drying. Tolerant and polyethylene glycol (PEG)-induced tolerant radicles exhibited a much-reduced CO(2) production before dehydration compared with desiccation-sensitive radicles. This difference was maintained during dehydration. In desiccation-sensitive tissues, dehydration induced an increase in the emission of acetaldehyde and ethanol that peaked well before the loss of membrane integrity. Acetaldehyde emission from sensitive radicles was significantly reduced when dehydration occurred in 50% O(2) instead of air. Acetaldehyde/ethanol were not detected in dehydrating tolerant radicles of either species or in polyethylene glycol-induced tolerant cucumber radicles. Thus, a balance between down-regulation of metabolism during drying and O(2) availability appears to be associated with desiccation tolerance. Using Fourier transform infrared spectroscopy, acetaldehyde was found to disturb the phase behavior of phospholipid vesicles, suggesting that the products resulting from imbalanced metabolism in seeds may aggravate membrane damage induced by dehydration.