Fine filaments observed within the cytoplasm surrounding the leading edge of the septum in telophase cells of Spirogyra (Zygnematales, Chlorophyta)

Electron microscope observation revealed the presence of many fine filaments within the cytoplasm surrounding the leading edge of the septum in telophase cells of Spirogyra verruculosa Jao. These filaments, about 7 nm each in diameter, ran parallel to one another along the leading edge of the septum and, sometimes, they appeared to be gathered into at least two bundles. These filament distribution patterns coincided well with those of the fluorescence of rhodamine-labeled phalloidin in the vicinity of the septum in telophase cells. The present results suggest that the fine filaments observed within the cytoplasm surrounding the leading edge of the septum may be actin filaments.     

Shoots grafted into the upper crowns of tall Japanese cedar (Cryptomeria japonica D. Don) show foliar gas exchange characteristics similar to those of intact shoots

The lower foliar photosynthetic rates seen in shoots in the upper crowns of tall trees than those in seedlings could be caused by extrinsic factors, such as hydraulic conductance, for shoots or by irreversible intrinsic change in the meristems during tree development. To clarify which is most significant, we compared foliar gas exchange characteristics and water relations among scions of Japanese cedar (Cryptomeria japonica D. Don) grafted into the upper crowns of tall trees, rooted cuttings developed from scions of the same clones, and intact shoots in the upper crowns of the tall trees. Grafted shoots had the same water regime as intact shoots, as confirmed by their similar water potentials at the turgor loss point, which were more negative than those of the rooted cuttings. No significant difference was observed between the grafted and intact shoots in their light-saturated photosynthetic rate (P_max), stomatal conductance (g_s), photosynthetic capacity, carboxylation efficiency, ratio of intercellular to ambient CO₂ concentration (C_i/C_a), and carbon isotope composition (¹³C). Compared with the rooted cuttings, the grafted shoots showed significantly lower P_max, g_s, photosynthetic capacity, and carboxylation efficiency (to 49%, 31%, 68%, and 65%, respectively). The C_i/C_a and ¹³C indicated significantly stronger instantaneous and long-term stomatal limitation in the grafted shoots than in the rooted cuttings. These indicate that changes in extrinsic factors can reduce foliar photosynthetic rates in shoots in the upper crowns of tall trees as a result of stronger stomatal limitation and reduced photosynthetic activity, without irreversible intrinsic changes in the meristems.     

Feline bone marrow-derived mesenchymal stromal cells (MSCs) show similar phenotype and functions with regards to neuronal differentiation as human MSCs

Mesenchymal stromal cells (MSCs) show promise for treatment of a variety of neurological and other disorders. Cat has a high degree of linkage with the human genome and has been used as a model for analysis of neurological disorders such as stroke, Alzheimer's disease and motor disorders. The present study was designed to characterize bone marrow-derived MSCs from cats and to investigate the capacity to generate functional peptidergic neurons. MSCs were expanded with cells from the femurs of cats and then characterized by phenotype and function. Phenotypically, feline and human MSCs shared surface markers, and lacked hematopoietic markers, with similar morphology. As compared to a subset of human MSCs, feline MSCs showed no evidence of the major histocompatibility class II. Since the literature suggested Stro-1 as an indicator of pluripotency, we compared early and late passages feline MSCs and found its expression in >90% of the cells. However, the early passage cells showed two distinct populations of Stro-1-expressing cells. At passage 5, the MSCs were more homogeneous with regards to Stro-1 expression. The passage 5 MSCs differentiated to osteogenic and adipogenic cells, and generated neurons with electrophysiological properties. This correlated with the expression of mature neuronal markers with concomitant decrease in stem cell-associated genes. At day 12 induction, the cells were positive for MAP2, Neuronal Nuclei, tubulin βIII, Tau and synaptophysin. This correlated with electrophysiological maturity as presented by excitatory postsynaptic potentials (EPSPs). The findings indicate that the cat may constitute a promising biomedical model for evaluation of novel therapies such as stem cell therapy in such neurological disorders as Alzheimer's disease and stroke.     

Serine phosphorylation of focal adhesion kinase in interphase and mitosis: a possible role in modulating binding to p130(Cas)

Focal adhesion kinase (FAK) is an important regulator of integrin signaling in adherent cells and accordingly its activity is significantly modulated during mitosis when cells detach from the extracellular matrix. During mitosis, FAK becomes heavily phosphorylated on serine residues concomitant with its inactivation and dephosphorylation on tyrosine. Little is known about the regulation of FAK activity by serine phosphorylation. In this report, we characterize two novel sites of serine phosphorylation within the C-terminal domain of FAK. Phosphorylation-specific antibodies directed to these sites and against two previously characterized sites of serine phosphorylation were used to study the regulated phosphorylation of FAK in unsynchronized and mitotic cells. Among the four major phosphorylation sites, designated pS1-pS4, phosphorylation of pS1 (Ser722) is unchanged in unsynchronized and mitotic cells. In contrast, pS3 and pS4 (Ser843 and Ser910) exhibit increased phosphorylation during mitosis. In vitro peptide binding experiments provide evidence that phosphorylation of pS1 (Ser722) may play a role in modulating FAK binding to the SH3 domain of the adapter protein p130(Cas).     

Relationship of nucleolus-associated bodies with the nucleolar organizer tracks in plant interphase nuclei (Pisum sativum)

Nucleolus-associated bodies (NABs) have long been noted in interphase nuclei of a wide variety of plant species. We have recently shown that these bodies consist largely of snRNPs and that they are located on the nucleolar surface in the immediate vicinity of the nucleolar organizer tracks. The present study revealed that, following exposure of roots to KCN, an agent that induces nucleolar segregation, NABs were intimately associated with intranucleolar chromatin. Although immunocytochemical tests with anti-DNA indicated that NABs contained no demonstrable amounts of DNA, our observations nevertheless add further support to the notion that these bodies are somehow related to the nucleolar chromosomes.     

Cell cycle arrest in early mitosis and induction of caspase-dependent apoptosis in U937 cells by diallyltetrasulfide (Al2S4)

Naturally occurring organic sulfur compounds (OSCs), such as linear allylsulfides from Allium species, are attracting attention in cancer research, since several OSCs were shown to act beneficially both in chemoprevention and in chemotherapy, while hardly exerting any harmful side effects. Hence, we investigated the possible role of different OSCs in the treatment of leukemia. Thereby, we found that the compounds tested in this study induced apoptosis in U937 cells, with an efficiency depending on the number of sulfides, and selected the most promising candidate, diallyltetrasulfide (Al2S4), for detailed mechanistic studies. Here we show that Al2S4 induced an accumulation of cells in early mitosis (G2/M phase), followed by the activation of caspase-dependent apoptosis. The compound counteracted different anti-apoptotic Bcl-2 family members (Bcl-xL, phospho-Bad and Bcl-2), promoted activation of Bax and Bak and induced the release of cytochrome c into the cytoplasm. Treatment by Al2S4 let to the identification of early apoptotic events including Bcl-xL degradation, Bak activation and release of cytochrome c followed by late events including Bcl-2 proteolysis, Bax activation, Bad dephosphorylation, caspase activation, nuclear fragmentation and phosphatidylserine exposure. Claudia Cerella and Christiane Scherer, both authors equally contributed to this work.     

Centriole behavior in early mitosis of rat kangaroo cells (PTK2)

Behavior of the centriolar duplexes during early mitosis was investigated. In general, both duplexes are capable of migrating about the cell as a unit with little change in their center to center spacing prior to separation to form the spindle poles. This duplex separation may occur at any point within the mid-prophase-prometaphase period. If it is delayed to prometaphase, transitory monopolar spindles were observed.     

The ontogeny of lipid bodies (spherosomes) in plant cells

Maturing embryos of 16 oil plants, anise suspension culture cells, and Neurospora crassa cells were prepared for electron microscopy at different stages during massive lipid accumulation. Lipid-rich structures of certain species were best preserved by dehydration of fixed tissues in ethanol without propylene oxide, embedding in Spurr's Medium, and polymerization at room temperature. In all cells examined, spherical lipid bodies (spherosomes) showed a moderately osmiophilic, amorphous matrix and displayed a delimiting half-unit membrane when sectioned medially. Associations with the endoplasmic reticulum (ER) were viewed at any stage during lipid body development but with different frequency in the different plant species. Plastids of fat-storing cells exhibited conspicuously undulate outer and inner envelope membranes that formed multiple contact sites with each other and protuberances into both cytoplasm and stroma. Some species, e.g., Linum, have plastids with tubular structures that connect the inner membrane to the thylakoid system; in addition, in the stroma vesicles fusing with or apparently passing through the envelope were observed. The outer envelope membrane may be associated with ER-like cytoplasmic membrane structures. In addition, lipid bodies of various sizes were found in contact with the plastid envelope. The ultrastructural observations are interpreted to match the published biochemical evidence, indicating that both plastids and ER may be involved in the synthesis of storage lipids and lipid body production.     

Medaka (Oryzias latipes) as a sentinel species for aquatic animals: Medaka cells exhibit a similar genotoxic response as North Atlantic right whale cells

Hexavalent chromium (Cr(VI)) is emerging as a major concern for aquatic environments, particularly marine environments. Medaka (Oryzias latipes) has been used as a model species for human and aquatic health, including the marine environment, though few studies have directly compared toxicological responses in medaka to humans or other aquatic species. We used a medaka fin cell line to compare the genotoxic response of medaka to Cr(VI) to the response observed in North Atlantic right whale cells to see if responses in medaka were similar to those of other aquatic species, particularly aquatic mammals. We used the production of chromosomal aberrations as a measure of genotoxicity. We found that in medaka cells, concentrations of 1, 5 and 10 microM sodium chromate damaged 17, 32 and 43% of metaphases, respectively and these same concentrations 1, 2.5, 5 and 10 microM sodium chromate damaged 14, 24 and 49% of metaphases, respectively, in North Atlantic right whale lung cells and 11, 32 and 41% of metaphases, respectively, in North Atlantic right whale testes cells. These data show that genotoxic responses in medaka are comparable to those seen in North Atlantic right whale cells, consistent with the hypothesis that medaka are a useful model for other aquatic species.     

Cell cycle regulation of the murine 8-oxoguanine DNA glycosylase (mOGG1): mOGG1 associates with microtubules during interphase and mitosis

8-Oxoguanine DNA glycosylase (OGG1) is a major DNA repair enzyme in mammalian cells. OGG1 participates in the repair of 8-oxoG, the most abundant known DNA lesion induced by endogenous reactive oxygen species in aerobic organisms. In this study, antibodies directed against purified recombinant human OGG1 (hOGG1) or murine (mOGG1) protein were chemically conjugated to either the photosensitizer Rose Bengal or the fluorescent dye Texas red. These dye-protein conjugates, in combination with binding assays, were used to identify associations between mOGG1 and the cytoskeleton of NIH3T3 fibroblasts. Results from these binding studies showed that mOGG1 associates with the cytoskeleton by specifically binding to the centriole and microtubules radiating from the centrosome at interphase and the spindle assembly at mitosis. Similar results were obtained with hOGG1. Together results reported in this study suggest that OGG1 is a microtubule-associated protein itself or that OGG1 utilizes yet to be identified motor proteins to ride on microtubules as tracks facilitating the movement and redistribution of cytoplasmic OGG1 pools during interphase and mitosis and in response to oxidative DNA damage.     

Activation of c-Jun N-terminal kinase (JNK) during mitosis in retinal progenitor cells

Most studies of c-Jun N-terminal Kinase (JNK) activation in retinal tissue were done in the context of neurodegeneration. In this study, we investigated the behavior of JNK during mitosis of progenitor cells in the retina of newborn rats. Retinal explants from newborn rats were kept in vitro for 3 hours and under distinct treatments. Sections of retinal explants or freshly fixed retinal tissue were used to detect JNK phosphorylation by immunohistochemistry, and were examined through both fluorescence and confocal microscopy. Mitotic cells were identified by chromatin morphology, histone-H3 phosphorylation, and location in the retinal tissue. The subcellular localization of proteins was analyzed by double staining with both a DNA marker and an antibody to each protein. Phosphorylation of JNK was also examined by western blot. The results showed that in the retina of newborn rats (P1), JNK is phosphorylated during mitosis of progenitor cells, mainly during the early stages of mitosis. JNK1 and/or JNK2 were preferentially phosphorylated in mitotic cells. Inhibition of JNK induced cell cycle arrest, specifically in mitosis. Treatment with the JNK inhibitor decreased the number of cells in anaphase, but did not alter the number of cells in either prophase/prometaphase or metaphase. Moreover, cells with aberrant chromatin morphology were found after treatment with the JNK inhibitor. The data show, for the first time, that JNK is activated in mitotic progenitor cells of developing retinal tissue, suggesting a new role of JNK in the control of progenitor cell proliferation in the retina.     

Differentiation of interphase nucleolus organizers in embryonic pig kidney cells (PK cell line)

The prometaphase karyotype of cell line PK contains two heteromorphous pairs of nucleolus organizers that belong to chromosomes 8a and 8, and to 10L and 10s. It was proposed that such heteromorphism may promote chromosome differentiating of interphase nucleolus organizers (INOs) with linear configuration. To test this assumption, we used two-dimensional (2D) preparations of methanol fixed PK cells surface stretched without hypotonic treatment. It was shown that in these preparations the large bulk of interphase PK cells contained 3-4 necklace-like linear structures arranged in nucleolar domains. The observed structures were positive in phase contrast and after DAPI-staining. Complimentary rDNA-FISH revealed that these structures were INOs, the largest iNO in individual cells containing prominent terminal rDNA FISH/DAPI signal. In accordance with the data on prometaphase analysis, the latter INOs belong to chromosomes 8a. As reported by Smetana and coworkers (1999), proteins of the nucleolar fibrillar center reacted preferentially with silver in methanol fixed unwashed smears of human peripheral lymphocytes. It was established that the same specific silver reaction is characteristic most probably of 2D preparations of methanol fixed PK cells. Both silver stained and rDNA-FISH linearized INOs had necklace-like or banded structure with different degrees of resolution. Banded INOs consisted of transverse argyrophilic structures: dense bands and loose interbands. High resolved banded INOs revealed a longitudinal splitting (binemic structure) of interband zone. Necklace-like INOs consisted of argyrophilic beads nearly two-fold more narrow than argyrophilic bands, and uninemic or silver-negative interbead zones. Our findings evidence that necklace-like INOs are typical for G1 and S phase cells, whereas banded INOs are characteristic of G2 cells. Among high resolved linear INOs, we found four reproducible patterns of silver staining, which could be combined it two homologous groups. Because each given pattern is unique for individual PK cells, we concluded that the patterns under study were chromosome specific. Using prometaphase analysis data, we determined chromosome affiliation for each of the four tested patterns of INO silver staining. High resolved INOs, belonging to different chromosomes, were further compared with regard to their average length and the mean of argyrophilic bead number per individual INO, in addition to the length and argyrophilic bead number ratios calculated for different INO pairs of individual cells. Surprisingly, we found that both the ratios, detected for most heteromorphous pair of homologous chromosomes 8a and 8, made only 1.26 +/- 0.02. In comparison, the similar length ratio for nucleolus organizers in chromosomes 8a and 8, calculated for individual prometaphase cells, reached 2.92 +/- 0.30.     

SPontaneous Oscillatory Contraction (SPOC): auto-oscillations observed in striated muscle at partial activation

Striated muscle is well known to exist in either of two states—contraction or relaxation—under the regulation of Ca²⁺ concentration. Described here is a less well-known third, intermediate state induced under conditions of partial activation, known as SPOC (SPontaneous Oscillatory Contraction). This state is characterised by auto-oscillation between rapid-lengthening and slow-shortening phases. Notably, SPOC occurs in skinned muscle fibres and is therefore not the result of fluctuating Ca²⁺ levels, but is rather an intrinsic and fundamental phenomenon of the actomyosin motor. Summarised in this review are the experimental data on SPOC and its fundamental mechanism. SPOC presents a novel technique for studying independent communication and coordination between sarcomeres. In cardiac muscle, this auto-oscillatory property may work in concert with electro-chemical signalling to coordinate the heartbeat. Further, SPOC may represent a new way of demonstrating functional defects of sarcomeres in human heart failure.     

Proteins recognized by antibodies against isolated cytological heterochromatin from rat liver cells change their localization between cell species and between stages of mitosis (interphase vs metaphase)

Heterochromatin in the cell nucleus seems to concentrate various proteins, such as Drosophila heterochromatin protein 1, which maintain the repressed state of gene expression. However, it still remains obscure how protein composition related to chromatin structure is different between heterochromatin and euchromatin in interphase nuclei. We isolated cytological heterochromatin from sonicated interphase nuclei obtained from rat liver cells and prepared antisera against it. The dense heterochromatic bodies seen in the preparation of intact nuclei were duplicated in a relatively pure form during the preparation of heterochromatin. In the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, differences between the fractions of heterochromatin and euchromatin were noted by their protein composition. Isolated heterochromatin was then digested by DNase after partial digestion with trypsin and its dense structure changed to become highly sensitive to DNase. The prepared antibodies reacted with the heterochromatin region of rat liver cell nuclei and isolated cytological heterochromatin; however, they did not react with euchromatin. Using immunohistochemistry, the antibodies bound to each cell nucleus in all tissues observed; some cell types were distinguished by their differential stainability (e.g. staining in the cytoplasm). Staining of the mitotic cells showed that the proteins recognized by the antibodies were localized in the cytoplasm and, in part, on the chromosomes. Based on the results of molecular cloning from rat liver cDNA library using the antibodies as a probe, it seemed that the antibodies mainly recognized two proteins similar to arginase and general vesicular transport factor p115, respectively. The results obtained from these experiments reveal that some proteins located in the heterochromatin of interphase liver cell nuclei seem to play important roles in condensing a portion of the chromatin structure during interphase and suggest that proteins composing heterochromatin might be changed according to cell types or the stage of the cell cycle.     

Acceptors of poly(ADP-ribosylation) in differentiation inducer-treated and untreated Friend erythroleukemia cells

Acceptors of poly(ADP-ribosylation) were identified and compared between inducer-treated and untreated Friend erythroleukemia cells. When permeabilized Friend cells were pulse labeled with 0.6 μM [³²P]NAD for 1 min and labeled proteins analyzed by SDS-polyacrylamide gel electrophoresis, nucleosome core histones were found to be the primary acceptors, with an additional minor radioactive peak at a position corresponding to M_r = 170 000. Friend cells induced to differentiate by DMSO treatment showed a similar distribution of radioactivity, but with a 60% reduction in the overall level of poly(ADP-ribosylation) under identical labeling conditions. When isolated nuclei were pulse labeled with 0.6 μM [³²P]NAD, radioactive peaks were not restricted mainly at the positions of core histones but widely dispersed in the area from 10 to 50 kDa with another peak at 170 kDa. Increase of NAD concentration resulted in the overall shift of peaks to higher molecular weight positions. When pulse-labeled nuclei or permeable cells were chased with 1 mM NAD, radioactive peaks migrated to positions of very high molecular weight (>M_r = 180 000). Remarkable suppression of poly(ADP-ribose) synthesis was observed when DMSO, hexamethylene bisacetamide, butyric acid, or hemin were used as the inducers.     

Recombinant CLIC1 (NCC27) assembles in lipid bilayers via a pH-dependent two-state process to form chloride ion channels with identical characteristics to those observed in Chinese hamster ovary cells expressing CLIC1

CLIC1 (NCC27) is an unusual, largely intracellular, ion channel that exists in both soluble and membrane-associated forms. The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration. Soluble E. coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells. The process of membrane integration of CLIC1 is pH-dependent. Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer. These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics. This has four times the conductance of the SCSK and fast kinetics that characterize the native channel. This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins.     

Condensation of interphase chromatin in nuclei of synchronized chinese hamster ovary (CHO-K1) cells

Reversibly permeabilized cells have been used to visualize interphase chromatin structures in the presence and absence of biotinylated nucleotides. By reversing permeabilization, it was possible to confirm the existence of a flexible chromatin folding pattern through a series of transient geometric forms such as supercoiled, circular forms, chromatin bodies, thin and thick fibers, and elongated chromosomes. Our results show that the incorporation of biotin-11-dUTP interferes with chromatin condensation, leading to the accumulation of decondensed chromatin structures. Chromatin condensation without nucleotide incorporation was also studied in cell populations synchronized by centrifugal elutriation. After reversal of permeabilization, nuclei were isolated and chromatin structures were visualized after DAPI staining by fluorescent microscopy. Decondensed veil-like structures were observed in the early S phase (at an average C-value of 2.21), supercoiled chromatin later in the early S (2, 55 C), fibrous structures in the early mid S phase (2, 76 C), ribboned structures in the mid-S phase (2, 98 C), continuous chromatin strings later in the mid-S phase (3,28), elongated prechromosomes in the late S-phase (3, 72 C), precondensed chromosomes at the end and after the S phase (3, 99 C). Fluorescent microscopy revealed that neither interphase nor metaphase chromosomes are separate entities but form a linear array arranged in a semicircle. Linear arrangement was confirmed by computer image analysis.     

Multitubular bodies in intestinal cells of Amphiuma means/tridactylum (Urodela): ultrastructural characterization

Summary The jejunal absorptive cells of the salamander Amphiuma, when examined using transmission electron microscopy, were found to possess a unique type of intracellular vacuole containing membranous tubules. These vanoles, tentatively named multitubular bodies, were located in the cytoplasm between the nucleus and the brush-border membrane, and were seen with greatest frequency in the summer and fall. The vacuoles containing multitubular bodies had an average diameter of 0.6 m, and the membranous tubules within had an average diameter of 30 nm. The tubules differed morphologically from the vesicles in the multivesicular bodies, and from the primary lysosomes in the polylysosomal vacuoles. The tubules did not exhibit acid phosphatase activity, and were of similar diameter and membrane thickness as the Golgi saccules. In contrast to the multivesicular bodies, the multitubular bodies did not take up exogenous horseradish peroxidase. Early forms of autophagosomes resembling these vacuoles were often seen in the para-Golgi region of the cell. The multitubular bodies may represent a distinct type of autophagosome. Although the exact origin of the tubules as well as their role in cellular activity is unclear, their seasonal appearance within the multitubular bodies of the absorptive cells suggests a unique means of selective down-regulation of Golgi-like organelles.