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

Summary: Abscisic acid (ABA) regulates a diverse array of processes including seed maturation and dormancy, root growth, leaf senescence, and the transition between vegetative and reproductive growth. ABA is also a major stress hormone that regulates the adaptation of plants to environmental stresses including drought, cold and salt. It controls the stomatal aperture to regulate water content and modulates the expression of stress-responsive genes. The most remarkable progress in recent studies of the ABA signaling pathways is the identification of three ABA receptors: the flowering-time control protein FCA, the Mg-chelatase H subunit, and the G protein-coupled receptor GCR2. The first reported ABA receptor was FCA, a nuclear localized RNA-binding protein that physically interacts with the RNA 3'-end processing factors FY and PCFS4 proteins. By a mechanism that remains to be elucidated, FCA-FY-PCFS4 complex prevents the accumulation of FLC transcripts encoding a transcription factor that inhibits flowering. Second intracellular ABA receptor is a plastid CHLH subunit of the magnesium-protoporphyrinIX chelatase that catalyses the insertion of Mg2+ into protoporphyrinIX. CHLH binds ABA independently of protoporphyrinIX and plays a key role in mediating plastid-to-nucleus retrograte signaling, as well functions as a plastid ABA receptor positively controlling major ABA responses. Unlike the above intracellular ABA receptors, the third ABA-binding protein GCR2, associated with plasma membrane, interacts with trimeric G proteins. Binding of ABA to GCR2 causes it to dissociate from G??and initiates of a classical G protein signaling cascade. In fact, it is currently unclear whether or not GCR2 is a canonical G protein coupled receptor or a peripheral membrane protein. Moreover, the recent studies have revealed a connection between protein phosphatases and protein kinases that are pivotal components of the ABA signaling network. Genetic and biochemical studies have demonstrated the key functions of SnRK2 and SnRK3-type kinases in stomatal movement, transcriptional regulation, RNA processing and stress responses.

Summary: Alternative splicing is a process in which more than one isoform of mRNA can be produced from one pre-mRNA. Quite complex regulatory mechanisms decide which isoform is produced in a given case. These mechanisms lead e.g. to a tissue-specific or stadium-dependent splicing, which is possible due to cooperation of regulatory factors. These factors can be considered (though somewhat artificially) at several levels: pre-mRNA sequence, secondary structure of pre-mRNA, trans-acting factors and additional protein and non-protein factors. The pre-mRNA molecule apart from such splicing signals as splicing sites, branch site or polypyrimidine tract, contains cis-acting regulatory elements. These include Intron Splicing Enhancers (ISE), Intron Splicing Silencers (ISS), Exon Splicing Enhancers (ESE) and Exon Splicing Silencers (ESS). In the splicing regulation they are bound by the trans-acting factors, which in turn affect the splice site recognition by the spliceosom. The role of the secondary stucture of pre-mRNA in the regulation of alternative splicing is that it modulates the accessibility of cis-acting elements for trans-acting factors. Firstly, some RNA-binding factors recognize only double-stranded fragments of RNA and changes of the secondary structure modulate their function. Secondly, the secondary structure changes the localization of cis-acting elements in space which is another possibility for splicing regulation. However, trans-acting factors play a central role in alternative splicing. They include among others SR and hnRNP proteins. SR proteins usually bind to enhancers, being splicing activators. Phosphorylation state of SR proteins changes throughout the splicing process and is subject to a complex modulation. hnRNP proteins bind to silencers, playing a role of splicing inhibitors. Their best known member is probably PTB (Polypyrimidine Tract Binding protein), a splicing inhibitor that is engaged mainly in tissue-specific splicing. In fact splicing activators and inhibitors act together in splicing regulation and the final effect depends on the amounts of these factors, their ability to interact with other proteins (including the components of spliceosom) and even proteins engaged in other cellular processes. Transcription is quite tightly coupled with alternative splicing. RNA Pol II plays a central role there, especially its C-Terminal Domain (CTD). CTD is responsible for the nuclear localization of splicing and transcription factors. Multiple factors coupling splicing and transcription are known – they usually phosphorylate or dephosphorylate the CTD domain. The function of RNA Pol II in splicing depends also on the promoter it recognizes. The promoter may decide about the ability of SR proteins to bind to CTD or about the processivity of polymerase (that in some cases affects splicing as well). Such a co-regulation of splicing and transcription is possible due to spatial and temporal coupling of the processes. Splicing is inhibited during mitosis; this effect is achieved mainly through changes in phosphorylation state of some splicing factors. For example SRp38 protein is dephosphorylated at the beginning of mitosis and in this form (as dSRp38) it can affect proper function of SR proteins at an early stage of splicing. Phosphorylation of dSRp38 after mitosis makes a cell able to conduct splicing again. Splicing is modulated by even more cellular processes. For example Nonsense-Mediated mRNA Decay (NMD) is a way of degradation of alternative splicing products that contain Premature Termination Codon (PTC). Some splicing factors use NMD to regulate the level of its own expression, e.g. PTB. The cell is affected by extracellular stimuli, such as growth factors, hormones and factors leading to the depolarisation of a cell membrane. The modulation of alternative splicing is one of the ways a cell can use to respond to these factors. Signal transduction pathways are engaged in this process, changing the phosphorylation state of trans-acting factors (mainly SR proteins). Splicing can also be regulated artificially through the introduction of chemical compounds to the cell. These factors include low molecular weight splicing inhibitors that affect splicing at diferent stages of the process. Such inhibitors seem to be promising in treatment of diseases caused by abnormal splicing.

Summary: Transposable elements (TEs) were discovered in all studying organisms, including animal, fungi and Protozoa species. They are abundant component of plant genomes too. They undergo constant amplification and proliferate throughout genome. Transposable elements are usually classified into two major groups. The class I – retrotransposons, elements that use an RNA intermediate and reverse transcriptase and the class II, DNA transposons that use a DNA intermediate and transposase. First class of TEs includes long terminal repeat (LTR) and non-LTR retrotransposons. Non-LTR retrotransposons are further divided into two major superfamilies – long interspersed nuclear elements (LINE) and short interspersed nuclear elements (SINE). SINE are short nonautonomous retroelements without discernible open reading frames (ORF) and do not encode any proteins. Presumably LINE elements might be a source of the enzymatic machinery required for retroposition of SINEs (some of tRNA-derived SINEs have sequence similarity in their 3'-end regions to the partner LINE found in the same genome). All SINEs share key characteristics, including an internal polymerase III promoter (made of A and B boxes) in their 5' tRNA-related region, a tRNA-unrelated region of variable length, a short strech of T or A at their 3'-end. In contrast to animal all of known plant SINEs originate from tRNA. They are also much less abundant in plant genomes than in animal, they reach 104 copies per haploid genome (in comparison to 104–106 copies in animal genomes). First plant SINE p-SINE1 were identified in intron region of Waxy gene in Oryza sativa genome. Subsequent SINEs elements were found in genomes of another Gramineae family members (Zea mays, Aegilops umbellulata, Triticum aestivum). They have been identified also in dicots species genomes including Brassicaceae (Arabidopsis thaliana, Brassica napus, Brassica oleracea), Solanaceae (Nicotiana tabacum, Capsicum annuum, Lycopersicon esculentum), Fabaceae (Medicago truncatula, Lotus japonicus, Glycine max). Plant SINEs are mainly dispersed randomly in genomes although they are rarely present in heterochromatic, pericentromeric regions, and have a preference for gene-rich regions. Great number and variety of these elements caused that they were grouped into families. High polymorphism of SINEs makes possible to isolate subfamilies within families. This feature makes SINEs very convenient as markers, they can be use in classification cultivars, species and in filogenetic studies.

Key words: SINE elements, non-LTR retrotransposons, plant genome sequences.

Summary: A wide array of research programs have been directed towards a comprehension of roles the intra-S-phase checkpoint which controls either frequency of DNA replication initiation (origin densities) or replication fork movement (rates of elongation). In response to treatment with either hydroxyurea or aphidicolin and after the addition of the DNA-damaging agents, the total rate of DNA replication per cell is reduced. This reduction is due to activation of an intra-S-phase checkpoint-dependent biochemical pathway network. The activated intra-S-phase checkpoint slows down or arrests replication forks, inhibits the premature firing of late origins, starts up the DNA damage response pathways to prevent replication of a damaged DNA, and delays the onset of mitosis until the cells are exposed to replicational stress. This work focuses on ATR, ATM, Chk1 and Chk2 protein kinases that are required for the control of the S phase, illustrates the state of knowledge about the other proteins involved in DNA-replication stress-response, and in addition explains their relationship. Ataxia telangiectasia mutated kinase (ATM) and ataxia telangiectasia and Rad3-related kinase (ATR) are PI-3 Kinase-related Kinase (PIKK) family members. Despite the essential role of ATM and ATR in cell cycle signaling, little is known about their activation. The activated ATM and ATR kinases turn on their downstream target proteins (like Chk2 and Chk1) by phosphorylating specific serine or threonine residues. ATM responds primarily to double strand breaks and phosphorylates Chk2 protein kinase at the amino-terminal domain contains a threonine residue (Thr68). Phosphorylation on Thr68 is a precondition for the successive activation step, which is attributable to autophosphorylation of Chk2 on Thr383 and Thr387. ATR is activated by replicational stress or UV-induced DNA damages and in response phosphorylates Chk1 protein kinase at serine residues (Ser317 and Ser345). Phosphorylation at Ser345 serves to localize Chk1 to the nucleus following checkpoint activation, while phosphorylation at Ser317 was shown to forbid entry into G2 phase and mitosis following stalled DNA replication. It is known, however, that ATM and ATR protein kinases share some phosphorylation targets and their precise roles in the intra-S-phase checkpoint pathway may differ depending on the nature of stress involved. Chk1-mediated Cdc25A-C phosphorylation leading to blocking of Cdk1 and Cdk2 (thus preventing cell cycle progression). Chk1 can stabilize the replisome, possibly by targeting replication proteins (e.g., Cdc6, MCM2-7), and after resolving the replication problems can restart of stalled replication forks. Functional changeability of the ATM/ATR-Chk2/Chk1-Cdc25/Cdk axis underlie the molecular foundation of the intra-S-phase checkpoint. ATR also phosphorylates histone H2AX on serine 139. After DSB-like DNA damage a number of Ser139-phosphorylated-H2AX localizes to sites of DNA damage at subnuclear foci. Although most of them spread throughout the whole area of nucleoplasm, the largest of them, localized at perinucleolar heterochromatin regions. This newly phosphorylated-H2AX forming a platform for the recruitment DNA repair and signaling proteins. This paper also briefly describes abrogating the intra-S-phase checkpoint function will result in overriding the S-M dependency and induction of premature chromosome condensation (PCC). Apart from nume-rous mutations that eliminate particular elements of the intra-S-phase checkpoint pathway, systems which monitor the course of DNA replication can be affected by many types of chemical agents. Caffeine, can override the S-M dependency and induce PCC in cells not prepared to undertake mitotic division, i.e. those which did not complete DNA replication and stay underreplicated. S-phase-blocked cells treated with caffeine start out aberrant mitotic divisions. The full array of aberrations includes: chromosomal breaks and gaps lost and lagging chromatids and chromosomes, chromosome bridges and micronuclei. Thus, drug-induced PCC (due to caffeine action) clearly provided the new insight that DNA replication is tightly coupled with the construction of the higher-ordered structure of the eukaryote chromosome. In the hope of unraveling targets for cytostatic drugs and cellular factors which inhibit or potentiate healing of cancer, a wide array of research programs have been directed towards an understanding of molecular mechanisms that underlie the intra-S-phase signaling pathways. A bulk of research-work is thus focused on methods increasing the effects of radio- and chemotherapy.

Key words: DNA-replication stress, intra-S-phase checkpoint, S-M dependency.

Structural and Functional Reorganisation of Podocytes in Idiopathic Nephrotic Syndrome in Children

Summary: The major reason of idiopathic nephrotic syndrome in children is the increased permeability of glomerular filtration barrier following some immunological and non-immunological disorders. Podocytes – consisting of specific cytoskeletal proteins (cytokeratins, vimentin and actin) as well as molecules stabilizing their intracellular organization (complex podocalixin - ezrin - Na-H exchanger - actin and synaptopodin) and proteins composing inter-podocytal diaphragms (podocin, nephrin), are responsible for the correct process of glomerular filtration. Stated above elements could be impaired during several inflammation processes. Some of the factors, which are crucially important in this disorder, are certain proinflammatory cytokins (including vascular endothelial growth factor, VEGF) and lectins (i.e. galectins-1). What is more, also glomeruli with morphological signs of their immaturity could also affect the correct process of glomerular filtration. In this cases, proteinuria is usually resistant to steroids and serve as the unfavorable risk factor. In this review article the actual opinions about the signification of described proteins in the pathogenesis, clinical course and prognosis of nephrotic syndrome in children were described.

Key words: podocyte, nephritic syndrome, ezrin, podocalyxin, galectin, vascular endothelial growth factor, cytokeratin, vimentin, nephrin.

Summary: hiols exist as low-molecular-weight thiols and protein thiols in cells. Thiol groups in proteins can be present as free thiols, disulfides and mixed disulfides when conjugated with glutathione, cysteine, homocysteine, ?-glutamylcysteine. Blood platelets, enucleate cells derived from megakaryocytes and functioning in haemo-stasis, and reactions in platelets involving thiol groups metabolism play an important role in platelet functions. Different low-molecular-weight thiols (glutathione, cysteine, cysteinyloglycine, homocysteine and its thiolactone) and changes of redox potential of platelets play an essential role in various steps of platelet activation, the exposure receptors on platelet surface, and signal transduction. Moreover, protein disulfide isomerase was found on the platelet surface, where it appears to play an important role in the platelet activation, including the exposure of integrin ??IIb?3 and blood platelet aggregation. This review outlines current knowledge of different low-molecular-weight thiols (particularly glutathione, homocysteine and homocysteine thiolactone) action on blood platelets.

Summary: athogenesis-related proteins belong to group of proteins activated during plant defense response to pathogen attack, insects and abiotic stress factors. Recently, 17 families of PR proteins have been classified based on their aminoacid sequences, biochemical properties and biological activity, but differences in structure and/or function between proteins which belong to one family are very often observed. Expression and accumulation of specific PR proteins is very precisely associated with the type of the pathogen, type of the plant cell, biosynthesis and activation of signaling compounds such as salicylic acid (SA), jasmonic acid (JA) and/or ethylene. For this reason, transcript levels of several PR genes can be used as markers of the specific defense response in plant tissues. It has to be emphasized that many PR proteins occur in plant cells constitutively, although at low concentration. Some of them appear in cells at different developmental stages and show tissue-specific localization, others can be only induced by the specific stress factors. Despite the progress in molecular phytopathology methods, mode of action and function of many PR proteins remain vague. Possibilities to exploit PR proteins as source of natural compounds in plant protection against pathogen attack open the new vistas in plant biotechnology. However, we have to be aware that some of the PR proteins can cause allergic reactions. In this paper, we describe selected families of PR proteins which show antifungal properties and which activity is associated with plant cell response to the necrotrophic fungi attack and additionally, we describe PR proteins that are induced specifically through the activation of JA-dependent signaling pathway.

Keywords: jasmonic acid, necrotrophic fungi, pathogenesis-related (PR) proteins, plant defense response.

The Role of Connective Tissue Growth Factor (CTGF) in Fibroproliferative Processes and Tissues Fibrosis

Summary: Cytokinins are one of the main group of plant hormones. They stimulate anabolic processes and cells growth and divisions. First invented cytokinin was kinetin, N-6-furfuryloadenine. Kinetin is important component of metabolic pathway, which enable cells to release excess free radicals. This is response to oxidative stress. Cytokinin binding proteins (CBP) can be found in plant cells, especially in cytosol, microsomes and thylacoid membranes. Cytokinins combined with CBP cause certain physiological response. Kinetin is a strong inhibitor of proteins and nucleic acids oxidation and glycosylation in vitro. It is a strong antioxidant both in vitro and in vivo. It delays the onset of human cells ageing in cell culture in vitro. Kinetin influences on cell growth and shape, rate of their growth, cytoskeleton structure, macromolecules biosynthesis and lipofuscin content. It acts favorably on skin, enhances barier functions of stratum corneum, decreases TEWL (Transepidermal Water Loss) and improves skin colour. Both kinetin and its ribosides are cytotoxic to melanoma cells. Another cytokinin is N-6-benzyloadenine. It influences mainly on the cell divisions. It can also inhibit some of human kinases, including CDKs (Cyclin Dependent Kinases). It is important especially for cancer cells. Benzyloadenine derivatives stimulate apoptosis in many types of cancer cells. N-9-benzyladenine inhibits phosphodiesterases (PDE) activity. Furthermore, it was shown in fibroblasts culture that N-6-benzyladenine derivatives stimulate cells elongation, decreasing of their movement and increase of their adhesion to medium.