Advances in Cell biology, vol. 32, 2005, issue 1

Summary: Extracellular nucleotides (eNTP) occur outside the cytoplasm in the extracellular matrix, were discovered almost 100 years ago in animal cells. These studies showed that the nucleotides are not only building a strand of DNA/RNA or constitute reservoir of energy (eg ATP or GTP) for various biochemical processes, but also may play role in cell growth and development. Extracellular nucleotides are also involved in maintaining homeostasis in the whole animal organism by maintaining adequate blood pressure and immune response. Characterization of receptors that recognize extracellular nucleotides (purynoceptors) and enzymes that act in the extracellular matrix caring out the hydrolysis of eNTP, and thus regulating their levels, made possible a better understanding of the role of extracellular nucleotides play in cells. Extracellular nucleotides are synthesized within the cytoplasm of the cell and then secreted into the extracellular matrix. Three different ways of eNTP transferring were identified, including their gradual secretion by exocytosis of secretory vesicles, rapid – by the ion channels or in the association with multidrug resistance transporters. The prevalence of eNTP and the important role they play in animals allowed for assumption that a similar system of regulation of cell growth and development may occur in plants. First reports about the possibility of the presence of extracellular nucleotides in plants were given in the 70's last century. First studies were focused on the effect of exogenously administered ATP on closure of the trap flycatchers, on the modulation of stomatal guard cell aperture and on the cell division. However, evidences that they may serve as important function as in the animal cells appeared recently. Botanists have only just started to explore the mechanisms underlying their actions. This paper presents results of the researches that show the importance of the eNTP in plant response to biotic and abiotic stresses and on cell development and growth. The family of proteins called apyrases are identified in increasing number of plant species. Their mutations cause disruption in Arabidopsis seedling growth, and plants are sterile due to lack of germination of pollen grains. Studies on development of rice root hairs (that also show a tip growth as in a case of germination of pollen grains) revealed a major role of apyrases. Studies carried out on plant cells showed that extracellular nucleotides are signaling molecules inducing cell response – production of reactive oxygen species or changes in calcium ion concentration in the cytoplasm. These mechanisms are the most universal and fundamental in the world of plants. Additionally, they proved to increase the concentration of eATP in wounding tissues or in a response to the attack of fungal pathogens and to osmotic stress. This means that extracellular nucleotides play an important role in plant defense and in the adaptation to new environmental conditions. The publications in recent years also suggest that extracellular nucleotides may be evolutionally very old and accompanied plants for millions of years. Some fungal elicitors induce an uptake of Ca2+ in plant cells, and this could be a mechanism for release of ATP from cells under the attack of fungal pathogens. Similar results were obtained for cells of algae in response to wounding. It is not known how ancient the reactive oxygen species strategy of plant pathogen defense is, but the fossil record indicates that plants had already developed fungal defense mechanisms in the Devonian era a billion years ago. Assembling information about the prevalence of extracellular nucleotides, their participation in the coordination of growth and development of plant cells, early evolutionary origin of eNTP and important functions in response to stress indicated that there are a key signal molecules in the plant world. The paper presents a review of articles supporting this claim.

Key words: extracellular nucleotides (eNTP), apyrases, purinoceptors, signaling, response to stress

Summary: Growth and development of plant occur under great influence of light, that quality and quantity changes are received by the specific photoreceptors. Various photoreceptors perceive the changing light condition and transform them into a molecular signal that results in the appropriate response. The photoreceptors of red/far-red light are phytochromes – the dimeric proteins covalently linked with phytochromobilin that acts as a chromophore. The decoding process starts with the perception of red light (666 nm), which occurs through photoisomerization of a chromophore leading to structural changes in apoprotein. This form of the phytochrome is called Pfr and is considered the biologically active form. This change is reversible, with far-red light (730 nm) illumination restoring Pr form. The phytochromes are synthesized in their inactive Pr form and are localized in the cytoplasm. Upon light excitation they are activated and translocated into the nucleus, where they interact with different proteins and modulate gene expression. PhyB enters the nucleus in response to red light, but phyA is efficiently transported into nucleus in response to far-red light and in response to very law levels of light over a broad range of colours. Nuclear accumulation of phyA is dependent on two proteins FHY1 and FHL which preferentially interact with the light activated form of phyA. Phytochromes, after translocation into the nucleus, interact with nuclear proteins. To date, more than 20 phytochrome-interacting proteins have been reported. Several recent studies have shown that multiple related bHLH (basic helix-loop-helix) class transcription factors play key roles in phytochrome signal transduction. All the bHLH proteins involved in light signaling belong to a single evolutionarily related subclass. These bHLH transcription factors are known as PIF (phytochrome interacting factor) or PIL (phytochrome interacting factor-like). Some PIF/PILs preferentially interact with phyB whereas others interact with equal affinity with both phyB and phyA. Analysis of pif/pil mutants have led to the suggestion that they mainly act as negative rather than positive regulators. Because phytochromes can phosphorylate PIF1/PIL5, PIF3, PIF4, PIF5 and HFR1, it is suggesting that phosphorylated proteins may then by ubiquitinylated by an E3 ubiquitin ligase, leading to degradation by the 26S proteasome. Recent studies demonstrated that several PIF/PIL proteins may interact with DELLA proteins – the key repressors of gibberellic acid signaling. The DELLA proteins physically interact with several members of the PIF/PIL family in such a way that the interaction inhibits the ability of the PIF/PILs to bind to, and regulate their target genes. Members of the PIF/PIL transcription factors subclass are involved in phytochrome-regulated processes such as seed germination, seedling de-etiolation, and response to shade signals. Light, specifically red light, is a crucial external factor that induces seed germination. On the other hand, the plant hormones, gibberellins (GA) and abscisic acid (ABA) are internal cues that play important but antagonistic roles in seed germination. Recent research has identified PIF1/PIL5 as a key negative regulator in phytochrome-mediated seed germination. In the dark, PIF1/PIL5 represses germination through reducing GA responsiveness and regulating GA and ABA levels. Light-activated phytochromes directly interact with PIF1/PIL5 and promote its degradation, negating PIF1/PIL5 repressive effects. To a postgerminative seedling, light is a decisive environmental factor that determines its developmental program. In the dark, a seedling undergoes scotomorphogenesis, however, under light, it adopts genetic program of photomorphogenesis. Light signals from phytochromes and cryptochromes converge on a group of conserved proteins termed COP/DET/FUS, which are central repressors of photomorphogenesis. In darkness, they work in concert to target a number of photomorphogenesis-promoting transcription factors, such as HY5, HYH and LAF1, for degradation. In the dark, PIF/PIL proteins are active and regulate gene expression to promote scotomorphogenic growth. Under light, activated phytochromes in their Pfr form, interact with PIF/PILs and result in the phosphorylation and subsequent degradation of PIF/PILs by the proteasome, resulting in photomorphogenesis. Besides, PIF/PIL and HY5 proteins are signaling integrators that link light signals to the signaling of phytohormones. Phytochromes sense changes in light quality due to shading by competing vegetation, using the ratio of red to far-red light (R/FR). PIF4 and PIF5 have recently been shown to be positive regulators of shade avoidance responses, participating in the regulation of some key players in these responses, such as ATHB2, ATHB4 and PIL1, the proteins positively regulating shade avoidance, and HFR1, a transcription factor with a negative role in shade avoidance. Responses to low R/FR ratio are primarily mediated by phyB. In daylight, phyB exists predominantly in the Pfr form, and following import into the nucleus, Pfr B binds PIF4 and PIF5 proteins, resulting in their degradation via the 25S proteasome. In vegetational shade, a reduction in R/FR ratio results in conversion of phyB to the inactive Pr form. The reduction in phyB Pfr would therefore result in increased abundance of nuclear PIF4 and PIF5 proteins.

Summary: Neuropeptides produced in neuro-endocrine system are involved in many vital processes of insects – the most numerous group of animals. In last ten years many new neuropeptides have been identified in this group of animals. In many cases they have pleiotropic action and play a crucial role as neurotransmitters, neuromodulators and classical hormones. In the present review several families of insect peptidic hormons are characterized, including: AKH/RPCH family, pyrokinins, tachykinins, myosuppressins, peptides of FMRFa/FLRFa family, sulfakinins, peptides involved in development and moulting, allatotropins, allatostatins, diuretic and antidiuretic peptides, peptides of CAP_2b family, periviscerokinins, proctolin and CCAP. Additional aspects discussed in the paper concern applications of insect neuropeptides as safety new generation of insecticides and pharmaceutics used in medicine.

Key words: insects, peptide hormones, neuropeptides, pseudopeptides, peptidomimetics, insecticides

Summary: Eleven linker histone subtypes have been identified to date in mammalian cells. For many years has been thought that members of H1 family are mainly responsible for the formation of higher order structures of chromatin, its stabilization and as DNA accessibility inhibitor. Experimental data from recent years revealed, however, that H1 variants may participate in specific gene regulation, proliferation, senescence, and apoptosis induction. Still little is known which H1 variants take part in above processes and with what efficiacy. It is supposed that particular H1 representatives may performed different functions and regulate diverse gene promotors or the same ones but in a different way. The silencing of gene expression coding some H1 variants allows to show diversity in their expression. Among particular H1 subtypes H1.2 only indicates proapoptotic activity. Obtained results revealed that this H1 variant participates in mitochondrial pathway of apoptosis and induces apoptogenic agents from mitochondria. At the beginning it has been demonstrated that H1.2 translocation from cell nucleus to cytosol is induced by DNA double strand breaks only. However, next data revealed, that other DNA damages, activity of non-genotoxic agents as well as spontaneous apoptosis may trigger the release of H1.2 from nucleus. Pioneer reports about this H1 variant participation in apoptosis suggest, that this protein may be recognized as early apoptosis marker. Viewed from this perspective, H1.2 presence in cancer cell cytosol may indicate that applied therapy has good prognostic value for patient.

Key words: H1 variants, apoptosis, mitochondrial pathway, apoptosis marker

Summary: For many years mitochondria have been implicated in the process of carcinogenesis. At the beginning of 20th century Otto Warburg has started research focused on failure of oxadative metabolism in cancer cells. In his work he described „disruption of respiration” as typical for cancer cells. Warburgs discovery resulted in establishment of many projects focused on the role of mitochondria in cell transformation. Since that time multiple reasearch groups have reported mitochondria DNA mutations in majority types of cancer. Recently re-analyses of raw data has been published and have show multiple methodical errors in previous reports. This paper presents critical analysis and summary of mitochondria polymorphisms and somatic mutations reasearch in oncology. Literature analysis that includes latest methodological guidelines established for mtDNA analysis and evidence based medicine reports proves that cancer patients harbour specific pattern of inherited mtDNA polymorphisms and low number of somatic mutations. It seems that mitochondrial genotype (including haplotype) may be classified as cancer predisposing factor.

Key words: cancer, molecular marker, mitochondria, MtDNA mutation, mtDNA polymorphism, Evidence Based Medicine – EBM

Summary: Calcium is an ubiquitous, crucial second messenger, that plays an essential and versatile role in cellular signaling. It has been shown to act as an intracellular regulator in many aspects of plant growth, development and stress responses. Many distinct signals induce spatial and temporal Ca²⁺ spikes as well as the frequency and amplitude of Ca²⁺ oscillations. Such stimulus-specific elevations in cytosolic Ca²⁺ ions concentration called 'Ca²⁺ signatures' are sensed, interpreted, and transduced to downstream elements by several types of Ca²⁺ sensor proteins such as calmodulins (CaMs), calcineurin B-like proteins (CBLs) or calcium - dependent protein kinases (CDPKs). All of them contain high-affinity Ca²⁺-binding sites, called the 'EF-hand' motif. The calmodulin family is a major class of calcium sensor proteins, which play a crucial role in cellular signaling cascades. It has no catalytic activity of its own but upon binding Ca²⁺ undergoes conformational changes and complex Ca²⁺/CaM activates or modulates numerous target proteins.CaM is one of the most conserved proteins in all eukaryotes and there are some similarities in Ca²⁺/calmodulin-mediated signaling. However, several features of CaM and its downstream effector proteins are unique to plants. While yeats have only a single CaM gene, and animal genomes typically contain only a few CaM genes, plant ones have multiple CaM genes that encode identical CaMs or highly similar isoforms within a single plant species. A particular set of CaM isoforms and CaM-like proteins (CMLs) are present in plant cells. The family of CaM-related proteins (CMLs), exhibiting significant differences with the typical CaM, was identified as encoding proteins that contain CaM-like EF hand structures and share at least 15% homology with CaM in amino acid residues. Diverse CaM isoforms and CMLs interact with downstream targets containing CaM-binding domains. To date, more than 80 different types of CaM-binding proteins have been identified and their physiological functions are implicated in diverse aspects of cellular processes. This review summarizes recent views on the role of CaM and CML proteins in plant development and adaptation to environmental stimuli.

Key words: calcium ions, calmodulin, CaM-like proteins

Summary: In the course of its evolution, plants have developed various mechanisms that act on the local and systemic levels and which role is to adapt plants to the environment they live in. One of these mechanisms is widely recognized plant defense system directed against biotic factors, to which belongs, among the others, pathogen attack. The first line of the defense against pathogen attack is so called structural constitutive resistance determined by plant anatomical structure and chemical constitutive resistance consisting of unfavorable effect of plant secondary metabolites against the pathogens. Epidermis and periderm play a main role in structural constitutive defense. Layers and waxes are present in epidermis and there are trichomes and thorns on its surface, and on the surface of roots – hairy roots. Cell wall itself is also a very important structural barrier. Lack of the continuity of epidermis, caused by wounding or the presence of the natural openings (stomata, spiracles and hydathodes that are the potential places of the pathogen attack), enables pathogens to overcome the first line of defense. Systemic acquired resistance (SAR) and induced systemic resistance (ISR) constitute the second line of plant defense system. Induced systemic resistance has been studied in respect of mechanism of its development and in respect of signaling pathways that transmit the ISR signal to distant plant tissues and organs. The main reason of research, however, is prospect to exploit this phenomenon as the plant protection system against pathogens. Utilization of ISR in agriculture could possibly at least significantly decrease the amount of chemical pesticides. The largest group among plant pathogens is fungal pathogens, mainly necrotrophic fungi including grey fungus – Botrytis cinerea, Fusarium oxysporum, Alternaria species and pathogenic oomycetes – Phytophthora infestans. B. cinerea infection causes grey mold disease, seedling blight and the death of stem in many plant species – in pot plants and crops as well as in conifer and broadleaved trees and shrubs. Fungi of Alternaria genus cause potato or tomato alternariosis, blight of tomato seedlings and infest vegetables and black spot disease in crucifers. Fusarium wilt caused by F. oxysporum is the most destructive disease of potato and tomato, while P. infestans causes potato blight. Necrotrophic fungi can infect living tissues, but their development and reproduction take place only on death tissue. Necrotrophic fungi secrete specific enzymes and mycotoxins during the penetration of host tissues. These substances cause the death of host cells and enable pathogen to feed in the host`s death tissue. Plant diseases caused by described above necrotrophic fungi are very often epidemic, and lead to destruction of entire field causing significant yield losses in agriculture, horticulture and fruit farming. Plant protection against infection of necrotrophic fungi is currently based on keeping the agrotechnical rules regarding soil quality, term of planting, use of fertilizers and chemical protection. At the beginning of 90` last century it has been reported on ISR induction by inoculation of cucumber rhizosphere with bacteria of Pseudomonas genus against Colletotrichum orbiculare, that causes anthracnose in cucumbers. Further research has shown, that this kind of resistance was induced by plant growth-promoting rhizobacteria (PGPR), which colonizing plant roots promote plant growth, rhizoremediation and soil fertilization (Azospirillum, Herbaspirillum, Enterobacter, Acetobacter, Azotobater i Pseudomonas). On the other hand it reduced disease symptoms caused by pathogens and herbivorous insects (Bacillus, Pseudomonas i Serattia). In this paper, we describe molecular mechanisms of ISR induced against necrotrophic fungal pathogens in a light of the current results of research. We provide also examples of ISR induction by nonpathogenic Pseudomonas strains that belong to PGPR. Selected Pseudomonas factors responsible for ISR induction have been divided into direct and indirect factors. Direct factors constitute a competition for iron and antibiotics production and indirect factors bacterial lipopolysaccharide (LPS), Pseudomonas metabolites massetolide A, N-alkylated benzylamine derivative (NABA) and N-acyl-L-homoserine lactone (AHL).

Keywords: induced systemic resistance, necrotrophic fungi, plant growth-promoting rhizobacteria, Pseudomonas

The NF-kB-dependent Cellular Signaling Pathway and its Interference with p53 and HSF1-dependent Pathways

Summary: The NF-kB-dependent signaling pathways are essential components of cellular response to stress. Mammalian family of NF-kB consists of five NF-kB/Rel proteins, which are subunits of the NF-kB transcription factor, and four IkB proteins, which are their specific inhibitors. Activation of NF-kB requires degradation of IkB, which allows nuclear translocation of NF-kB and its binding to cis-acting DNA regulatory elements. NF-kB transcription factors regulate expression of numerous genes, which are involved in cell proliferation, apoptosis, immune response and inflammatory response. The NF-kB-dependent signaling pathways interfere with two other stress-related pathways regulated by p53 and HSF1 transcription factors. All three pathways are essential for both pathogenesis of serious human diseases, including cancer, and for response to therapeutic treatment.

Key words: cellular stress, NF-kB, regulation of gene expression, signal transduction, transcription factors

Summary: Estrogens plays an essential role in the etiology of human breast, ovarian and endometrial carcinoma. Activation of estrogen receptors contributes to the growth and metastasis of these common estrogen-dependent tumors found in women. Recent studies reveal that melatonin, main hormone secreted by pineal gland, has been involved in the regulation of cancer cells growth. The oncostatic properties of this molecule has been considered on different kinds of tumors but especially on hormone – dependent breast cancer. There is general agreement that in vivo melatonin reduces the incidence and the activity of growth rate of tumor and inhibits the proliferation and invasiveness of cancer cells in vitro. The actual anticancer therapeutic strategy treats estrogens as a target and considered possibility of using melatonin as an antiestrogenic drug. The interaction of this indolamine with estrogen-signaling pathways could be explained through several mechanisms. Melatonin may act: a) indirect by down-regulation hypothalamic-pituitary-reproductive axis leading to decrease level of circulating gonadal estrogens or b) behaving as selective estrogen receptor modulator (SERM) and as a selective estrogen enzyme modulator (SEEM). Either indirectly or directly melatonin and estrogen interaction suggest that melatonin may be useful drug in the prevention and treatment hormone-dependent cancers.

Key words: melatonin, melatonin receptors, estrogen, estrogen receptors, aromatase, estrogen-dependent cancers, breast cancer, ovary cancer, endometrial cancer