Restricted Cell Division Research Articles

Regulatory role of Prunus mume DAM6 on lipid body accumulation and phytohormone metabolism in the dormant vegetative meristem.

Bud dormancy is a crucial process in the annual growth cycle of woody perennials. In Rosaceae fruit tree species, DORMANCY-ASSOCIATED MADS-box (DAM) transcription factor genes regulating bud dormancy have been identified, but their molecular roles in meristematic tissues have not been thoroughly characterized. In this study, molecular and physiological analyses of transgenic apple plants overexpressing the Japanese apricot DAM6 gene (PmDAM6) and Japanese apricot cultivars and F1 individuals with contrasting dormancy characteristics revealed the metabolic pathways controlled by PmDAM6. Our transcriptome analysis and transmission electron microscopy examination demonstrated that PmDAM6 promotes the accumulation of lipid bodies and inhibits cell division in the dormant vegetative meristem by down-regulating the expression of lipid catabolism genes (GDSL ESTERASE/LIPASE and OIL BODY LIPASE) and CYCLIN genes, respectively. Our findings also indicate PmDAM6 promotes abscisic acid (ABA) accumulation and decreases cytokinin (CTK) accumulation in vegetative buds by up-regulating the expression of the ABA biosynthesis gene ARABIDOPSIS ALDEHYDE OXIDASE and the CTK catabolism gene CYTOKININ DEHYDROGENASE, while also down-regulating the expression of the CTK biosynthesis genes ISOPENTENYL TRANSFERASE (IPT) and CYP735A. Additionally, PmDAM6 modulates gibberellin (GA) metabolism by up-regulating GA2-OXIDASE expression and down-regulating GA3-OXIDASE expression. Furthermore, PmDAM6 may also indirectly promote lipid accumulation and restrict cell division by limiting the accumulation of CTK and GA in buds. In conclusion, using our valuable genetic platform, we clarified how PmDAM6 modifies diverse cellular processes, including lipid catabolism, phytohormone (ABA, CTK, and GA) biosynthesis and catabolism, and cell division, in the dormant vegetative meristem.

IgG antibodies mediated gold nanoparticles conjugated to methotrexate as targeted chemotherapy for lung cancer

Vincamine, a natural chemical, was used as a reducing agent in the synthesis of IgG antibodies mediated biogenic gold nanoparticles (IgGAuNPs). Eventually, the synthesised IgGAuNPs were bioconjugated with the chemotherapeutic drug methotrexate (MTX-IgGAuNPs). The IgG isotype can target cancer cells through polymorphic Fc gamma receptors (FcγRs) and have therapeutic effects. They can restrict cell division by inhibiting different intracellular signal transduction pathways and activating NK cells and macrophages through antibody-dependent cellular cytotoxicity and macrophage-mediated antibody-dependent phagocytosis, respectively. Further, IgGAuNPs and MTX-IgGAuNPs were characterised by physical techniques. Moreover, 3D conformational changes in the structure of IgG were analysed by fluorescence spectroscopy during and after the synthesis of IgGAuNPs. Furthermore, the IgGAuNPs and MTX-IgGAuNPs were effective against lung cancer (A549 cells), while they were found to be non-toxic against normal cells (NRK cells). The effectiveness of IgGAuNPs and MTX-IgGAuNPs was examined by MTT cytotoxicity assay, DCFDA method for the production of ROS, and release of Cyt-c from the mitochondria for caspase-3-mediated apoptosis. Moreover, the confirmation of internalisation of particles into the nucleus was examined under the DAPI assay, and it was found that particles caused nuclear fragmentation, which was also an indication of apoptosis.

EFFECT OF DROUGHT ON MORPHOPHYSIOLOGICAL TRAITS OF RICE: A REVIEW

Drought is the condition of prolonged paucity in the water supply, whether atmospheric, surface water, or groundwater. This significant abiotic stress is a severe limiting factor of plant growth and rice productivity, adversely affecting the economic rice production globally. Drought stress has a considerable effect on the rice plant’s morphological and physiological traits preventing it from reaching the maximum yield. Morphological alteration in rice includes inhibition of seed germination, reduced tillers, early maturity, reduced biomass while physiological changes involve restricted cell division and elongation, stomatal closure, loss of turgor adjustment, lower photosynthesis, slower growth rate, yield reduction, etc. The stress further brings about a biochemical change by accumulating stress metabolites, increasing Reactive Oxygen Species (ROS) accumulations, increasing antioxidative enzymes and, Abscisic acid (ABA) accumulation. Various mitigation measures are applied to adhere to the effect of drought on rice plants by selecting drought-tolerant cultivars, planting early varieties, sufficient moisture maintenance, conventional breeding, molecular maintenance, and development of varieties with high yielding attributes. Thus, this review aims to assess the complex responses of rice plants to drought in morpho-physiological characters of rice along with its mitigation strategies in adoption to drought stress.

KSHV-encoded vCyclin can modulate HIF1α levels to promote DNA replication in hypoxia.

The cellular adaptive response to hypoxia, mediated by high HIF1α levels includes metabolic reprogramming, restricted DNA replication and cell division. In contrast to healthy cells, the genome of cancer cells, and Kaposi's sarcoma associated herpesvirus (KSHV) infected cells maintains replication in hypoxia. We show that KSHV infection, despite promoting expression of HIF1α in normoxia, can also restrict transcriptional activity, and promoted its degradation in hypoxia. KSHV-encoded vCyclin, expressed in hypoxia, mediated HIF1α cytosolic translocation, and its degradation through a non-canonical lysosomal pathway. Attenuation of HIF1α levels by vCyclin allowed cells to bypass the block to DNA replication and cell proliferation in hypoxia. These results demonstrated that KSHV utilizes a unique strategy to balance HIF1α levels to overcome replication arrest and induction of the oncogenic phenotype, which are dependent on the levels of oxygen in the microenvironment.

ZipN is an essential FtsZ membrane tether and contributes to the septal localization of SepJ in the filamentous cyanobacterium Anabaena

The organismic unit of heterocyst-forming cyanobacteria is a filament of communicating cells connected by septal junctions, proteinaceous structures bridging the cytoplasms of contiguous cells. This distinct bacterial organization is preserved during cell division. In Anabaena, deletion of the zipN gene could not be segregated. We generated strain CSL109 that expresses zipN from a synthetic regulatable promoter. Under conditions of ZipN depletion, cells progressively enlarged, reflecting restricted cell division, and showed drastic morphological alterations including cell detachment from the filaments, to finish lysing. In contrast to the wild-type localization in midcell Z-rings, FtsZ was found in delocalized aggregates in strain CSL109. Consistently, the proportion of membrane-associated to soluble FtsZ in fractionated cell extracts was lower in CSL109. Bacterial two-hybrid analysis showed that ZipN interacts with FtsZ and other cell-division proteins including cytoplasmic Ftn6 and SepF, and polytopic FtsW, FtsX, FtsQ and FtsI. Additionally, ZipN interacted with the septal protein SepJ, and in CSL109 depletion of ZipN was concomitant with a progressive loss of septal specificity of SepJ. Thus, in Anabaena ZipN represents an essential FtsZ membrane tether and an organizer of the divisome, and it contributes to the conformation of septal structures for filament integrity and intercellular communication.

Longitudinal patterning in roots: a GATA2-auxin interaction underlies and maintains the root transition domain.

In Arabidopsis thaliana root meristems the GATA2 transcription factor is a marker for the root transition domain, is auxin regulated, and functions to restrict cell division activity. The growing part of roots is comprised of three discrete regions; the proliferative domain (PD), an elongation zone, and interposed between these two, the transition domain (TD), which is the focus of this investigation. Within the TD, it is hypothesized that cells are reprogrammed, losing the capacity to divide and begin to differentiate. In recently germinated Arabidopsis thaliana seedlings, a TD is not anatomically evident, but subsequently forms in a region of the root in which there has occurred prior expression of both AUX1/PIN2 proteins and of transcripts of the GATA transcription factor family (pGATA2:H2B-YFP or pGATA2:GUS). pGATA2:GUS expression is regulated by auxin and is reduced in seedlings in which either auxin transport or auxin sensitivity is perturbed. Application of cytokinin results in a reduction in both pGATA2:GUS expression and in TD cell number, via a pathway involving ARR1 and ARR12. Overexpression of GATA2 is accompanied by a reduction in cell number in the PD, but has no effect on cell number in the TD, whereas in knockdowns of GATA transcription factors, cell number is reduced in both the PD and TD. We conclude: (1) that GATA2 expression is localized to (a marker for) the TD; (2) that development and maintenance of the TD are associated with an auxin-regulation of GATA2 expression; (3) that GATA transcription factors function to restrict cell division activity.

HwHog1 kinase activity is crucial for survival of Hortaea werneckii in extremely hyperosmolar environments

Although suggested, the involvement of the HOG pathway in adaptation processes in extremely halotolerant fungus Hortaea werneckii has never been specifically demonstrated. Here, we show that the H. werneckii HOG pathway is very robust, and that it includes two functionally redundant MAPK homologues, HwHog1A and HwHog1B, that show osmolyte-type-dependent phosphorylation. Inhibition of HwHog1 kinase activity with the ATP analogue BPTIP restricts H. werneckii colony growth at 3.0M NaCl, KCl and sorbitol, most likely due to restricted cell division. On the other hand, HwHog1-regulated transcription of a selected group of genes (HwSTL1, HwGUT2, HwOPI3, HwGDH1, HwUGP1, HwGPD1) is an osmolyte-specific process that is important for induction of gene transcription with high NaCl, for regulation of specific genes with high sorbitol, and has no role in KCl stressed cells. Survival of H. werneckii at moderate NaCl and KCl concentrations is not dependent on HwHog1 activity or the calcineurin pathway, and thus alternative mechanisms must exist. The HOG pathway described here is vital for the extreme osmotolerance of H. werneckii, and its regulation shows important differences from the homologue pathways characterised in other mesophilic and halotolerant fungi.

Euglena gracilis (Euglenophyceae) produces abscisic acid and cytokinins and responds to their exogenous application singly and in combination with other growth regulators

The main objective of this study was to determine the optimal concentrations of a wide spectrum of exogenous phytohormones for effective stimulation of cell division and production of maximum cell yield in Euglena gracilis Klebs cultured in vitro. Results indicate that two hormones combined exert more effective growth stimulation than a single hormone or three, four or five different hormones combined. Specifically, trans-zeatin at 10−7 M combined with abscisic acid at 10−9 M produced optimal conditions for growth, yielding the maximum cell concentration. High concentrations of exogenous phytohormones were toxic to Euglena. The addition of trans-zeatin, N6-isopentenyladenine, and benzylaminopurine to Euglena cultures resulted in dense, dark green chloroplasts, suggesting that exogenous phytohormones increased the production of chlorophyll. Given the response to exogenous growth regulators, the study identified and quantified the types of endogenous cytokinins (CKs) and abscisic acid (ABA) synthesized in vitro by Euglena gracilis. HPLC-(ESI) MS/MS analysis revealed that the algal cells produced and released into the medium a mixture of CKs and ABA. The main CKs identified in the cell pellets and supernatant samples were from a t-RNA degradation pathway and included: cis-zeatin (cZ) derivatives cZR, cZNT, MeSZ and MeSZR, and to a lesser extent, the free base N6-isopentenyladenine (iP) and its derivatives iPR, iPNT, MeSiP and MeSiPA. A positive response to ABA, and the relatively high levels detected in E. gracilis, suggest that this hormone is important for alleviating stress conditions of in vitro culture that might otherwise restrict cell division.

Adaptation to statins restricts human tumour growth in Nude mice

BackgroundStatins have long been used as anti-hypercholesterolemia drugs, but numerous lines of evidence suggest that they may also bear anti-tumour potential. We have recently demonstrated that it was possible to isolate cancer cells adapted to growth in the continuous presence of lovastatin. These cells grew more slowly than the statin-sensitive cells of origin. In the present study, we compared the ability of both statin-sensitive and statin-resistant cells to give rise to tumours in Nude mice.MethodsHGT-1 human gastric cancer cells and L50 statin-resistant derivatives were injected subcutaneously into Nude mice and tumour growth was recorded. At the end of the experiment, tumours were recovered and marker proteins were analyzed by western blotting, RT-PCR and immunohistochemistry.ResultsL50 tumours grew more slowly, showed a strong decrease in cyclin B1, over-expressed collagen IV, and had reduced laminin 332, VEGF and CD34 levels, which, collectively, may have restricted cell division, cell adhesion and neoangiogenesis.ConclusionsTaken together, these results showed that statin-resistant cells developed into smaller tumours than statin-sensitive cells. This may be reflective of the cancer restricting activity of statins in humans, as suggested from several retrospective studies with subjects undergoing statin therapy for several years.

Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae

In this study Fourier transform infrared micro-spectroscopy (FTIR) was used to determine lipid and carbohydrate content over time in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus subspicatus grown in batch culture in limiting concentrations of nitrogen (N). Both algae exhibited restricted cell division and increased cell size following N-limitation. FTIR spectra of cells in N-limited media showed increasing lipid:amide I and carbohydrate:amide I ratios over time. The use of lipid- and starch-staining dyes confirmed that the observed ratio changes were due to increased lipid and carbohydrate synthesis. These results demonstrate rapid metabolic responses of C. reinhardtii and S. subspicatus to changing nutrient availability, and indicate the efficiency of FTIR as a reliable method for high-throughput determination of lipid induction.

CDC-25.1 stability is regulated by distinct domains to restrict cell division during embryogenesis inC. elegans

Cdc25 phosphatases are key positive cell cycle regulators that coordinate cell divisions with growth and morphogenesis in many organisms. Intriguingly in C. elegans, two cdc-25.1(gf) mutations induce tissue-specific and temporally restricted hyperplasia in the embryonic intestinal lineage, despite stabilization of the mutant CDC-25.1 protein in every blastomere. We investigated the molecular basis underlying the CDC-25.1(gf) stabilization and its associated tissue-specific phenotype. We found that both mutations affect a canonical beta-TrCP phosphodegron motif, while the F-box protein LIN-23, the beta-TrCP orthologue, is required for the timely degradation of CDC-25.1. Accordingly, depletion of lin-23 in wild-type embryos stabilizes CDC-25.1 and triggers intestinal hyperplasia, which is, at least in part, cdc-25.1 dependent. lin-23(RNAi) causes embryonic lethality owing to cell fate transformations that convert blastomeres to an intestinal fate, sensitizing them to increased levels of CDC-25.1. Our characterization of a novel destabilizing cdc-25.1(lf) intragenic suppressor that acts independently of lin-23 indicates that additional cues impinge on different motifs of the CDC-25.1 phosphatase during early embryogenesis to control its stability and turnover, in order to ensure the timely divisions of intestinal cells and coordinate them with the formation of the developing gut.

Dmp53 Activates the Hippo Pathway to Promote Cell Death in Response to DNA Damage

Developmental and environmental signals control a precise program of growth, proliferation, and cell death. This program ensures that animals reach, but do not exceed, their typical size . Understanding how cells sense the limits of tissue size and respond accordingly by exiting the cell cycle or undergoing apoptosis has important implications for both developmental and cancer biology. The Hippo (Hpo) pathway comprises the kinases Hpo and Warts/Lats (Wts), the adaptors Salvador (Sav) and Mob1 as a tumor suppressor (Mats), the cytoskeletal proteins Expanded and Merlin, and the transcriptional cofactor Yorkie (Yki) . This pathway has been shown to restrict cell division and promote apoptosis. The caspase repressor DIAP1 appears to be a primary target of the Hpo pathway in cell-death control. Firstly, Hpo promotes DIAP1 phosphorylation, likely decreasing its stability. Secondly, Wts phosphorylates and inactivates Yki, decreasing DIAP1 transcription. Although we understand some of the events downstream of the Hpo kinase, its mode of activation remains mysterious. Here, we show that Hpo can be activated by Ionizing Radiations (IR) in a Dmp53 (Drosophila melanogaster p53)-dependent manner and that Hpo is required (though not absolutely) for the cell death response elicited by IR or Dmp53 ectopic expression.

Overexpression of Arabidopsis ACK1 alters leaf morphology and retards growth and development

Cyclin dependent kinases (CDKs) play important roles in the plant cell cycle, a highly coordinated process in plant growth and development. To understand the regulatory network involving the CDKs, we have examined the role of ACK1, a gene that has significant homology to known ICKs (inhibitors of CDKs), but occupies a distinct branch of the ICK phylogenetic tree. Overexpression of ACK1 in transgenic Arabidopsis significantly inhibited growth, leading to effects such as serration of leaves, as a result of strong inhibition of cell division in the leaf meristem. ACK1 transgenic plants also differed morphologically from control Arabidopsis plants, and the cells of ACK1 transgenics were more irregular than the corresponding cells of control plants. These results suggest that ACK1 acts as a CDK inhibitor in Arabidopsis, and that the alterations in leaf shape may be the result of restricted cell division.

Amiloride Suppresses Erythropoietin-Induced Proliferation and MAP Kinase, but Potentiates Differentiation of J2E Cells

The J2E erythroid cell line proliferates and differentiates in response to erythropoietin (epo). Here we demonstrate that the diuretic amiloride can suppress normal and hormone-induced cell division in a dose-dependent manner. In the presence of amiloride, cell numbers did not increase, [3H]thymidine incorporation decreased, and fewer cells were observed in the S, G2, and M phases of the cell cycle. In addition, the levels of proliferating cell nuclear antigen, a subunit of DNA polymerase δ, fell. In marked contrast, epoinitiated differentiation was potentiated when J2E cells were cultured with the drug: the number of benzidine-positive cells increased, hemoglobin content per cell rose, and more morphologically mature cells were produced. Immunoblotting with anti-phosphotyrosine antibodies revealed that amiloride reduced the number of phosphorylated proteins in epo-stimulated cells. Moreover, the protein content of p42 and p44 MAP kinases was noticeably downregulated in amiloridetreated cultures. These data indicate that amiloride may interfere with epo-induced signaling cascades within J2E cells which result in restricted cell division and promotion of maturation.

Transcriptional and post-transcriptional regulation of c- myc, c- myb, and p53 during Proliferation and differentiation of murine erythroleukernia cells treated with DFMO and DMSO

The proto-oncogenes myc, myb, and p53 produce nuclear proteins which have been implicated in the regulation of proliferation or differentiation in a number of systems. The expression of these proto-oncogenes was studied in murine erythroleukemia (MEL) cells during (i) normal replication, (ii) DMSO-induced differentiation and (iii), α-difluoromethylornithine (DFMO)-restricted cell division and differentiation. The RNA levels of c- myc, c- myb, and p53 were all elevated during normal cellular proliferation; only c- myc expression declined when the cells stopped dividing although the rate of transcription for the gene was unaltered. In contrast, treatment of the cells with DFMO resulted in gradual cessation of cell replication and a decrease in transcription of c- myc, c- myb and p53. When the MEL cells were induced to differentiate with dimethyl sulfoxide (DMSO), a transient reduction in c- myc and c- myb RNA levels occurred immediately prior to the G 1 arrest with a concomitant decrease in transcriptional activity, while p53 mRNA production was elevated without an increase in transcription. Similar changes of the proto-oncogene levels were observed when the MEL cells were incubated with DFMO and then later induced with DMSO, a protocol which restricts differentiation of the MEL cells. From these experiments we conclude that (i) c- myc, c- myb, and p53 are regulated independently at both the transcriptional and post-transcriptional levels, (ii) DFMO inhibits MEL cell proliferation and expression of several genes, including c- myc, c- myb and p53, and (iii) DFMO suppresses terminal differentiation but is unable to alter proto-oncogene changes associated with the early stages of differentiation.

The role of methionine in the intracellular accumulation and function of folates.

It is suggested that mammalian cells have evolved to respond to methionine deficiency since in such circumstances vital methylation reactions are put at risk, due to decreased levels of S-adenosyl-methionine. Enzymatic changes occurring as a result of decreased methionine, S-adenosylmethionine and S-adenosylhomocysteine, optimize the remethylation of homocysteine to methionine by decreasing homocysteine catabolism and channelling cellular folates into 5-methyltetrahydropteroylglutamate (5-CH3-H4 PteGlu). The latter, in addition to optimising the remethylation cycle, directs the folate cofactors away from purine and pyrimidine biosynthesis and decreases the rate of proliferation of rapidly dividing cells thus reducing competition for methionine incorporation into proteins. Decreased cellular homocysteine, as a result of decreased methionine, would also restrict cell division by decreased conversion of plasma 5-CH3-H4PteGlu into intracellular polyglutamates. Cobalamin deficiency, either nutritional or due to exposure to the Co (I) cobalamin inactivating agent nitrous oxide, prevents the demethylation of 5-CH3-H4PteGlu, which even in the presence of adequate amounts of homocysteine and methionine prevents rapidly proliferating cells from converting enough of the plasma 5-CH3-H4 PteGlu into folylpolyglutamate forms to permit normal DNA biosynthesis and cell replication. This, together with the trapping of the cellular folate cofactors in the 5-CH3-H4PteGlu form, results in megaloblastic changes occurring in tissues such as the marrow. The vital role of the methylation reactions was demonstrated by exposing monkeys to nitrous oxide which inactivated their methionine synthetase. The resultant ataxia and severe demyelination was prevented and diminished by methionine supplementation. When methionine synthetase was similarly inactivated in mice it was shown that while 5-CH3-H4PteGlu enters mammalian cells, it is not converted into a polyglutamyl form and subsequently leaves the cell unmetabolised. In similar experiments in rats methionine was found to have only a small effect in restoring folylpolyglutamate biosynthesis, contrary to previous reports using nutritionally cobalamin deficient animals. It was found that a decrease in the deoxythymidine salvage pathway by methionine, under the experimental conditions used, has led others to the mistaken conclusion that methionine has an 'anti-folate' effect in bone marrow, i.e. that it decreases folate availability for thymidylate synthetase.

Cyclic-AMP-induced differentiation in neuroblastoma is independent of cell division rate.

CLONED, cultured mouse neuroblastoma cells, when grown in conditions which restrict cell division, extend neurites1–6 and increase the activity of neurotransmitter-related enzymes7–9. Treatments that can induce this differentiation include lowered serum concentrations3, prostaglandins4, phosphodiesterase inhibitors5 and analogues of cyclic-3′,5′-AMP (cyclic-AMP)6,10–11. These treatments have been proposed to act by elevating intracellular cyclic-AMP levels, but because they also inhibit cell division it has been difficult to determine whether their effects are due directly to cyclic-AMP or indirectly to inhibition of cell division12. As neurite growth and enzyme elevation occur during the interphase portion of the cell cycle (and are retarded during cell division), cyclic-AMP may simply allow differentiation by stopping the cell cycle in interphase3. The butyryl analogues of cyclic-AMP, in particular, markedly restrict cell division (ref. 10; see also Fig. 1a). Because butyrate, which can dissociate from the dibutyryl analogue of cyclic-AMP, can itself markedly inhibit cell division we reasoned that other analogues may exist which are not inhibitors of cell division but which effectively mimic the action of cyclic-AMP. We report here that 8-bromo-cyclic-3′,5′-AMP (8Br-cyclic-AMP) effects biochemical and morphological changes in the neuroblastoma clone NBD-2 (ref. 9) without concomitant inhibition of cell division.