Evaluation of signal transduction pathways mediating the nuclear exclusion of the androgen receptor by melatonin

The intracellular signaling pathways mediating the nuclear exclusion of the androgen receptor (AR) by melatonin were evaluated in PC3 cells stably transfected with the AR. The melatonin-induced nuclear exclusion of the AR by melatonin (100 nM, 3 h) was blocked by LY 83583 (an inhibitor of guanylyl cyclases). 8-Bromo-cGMP (a cell-permeable cGMP analog), mimicked the effect of melatonin, as did ionomycin (a calcium ionophore) and PMA [an activator of protein kinase C (PKC)], and their effects were blocked by GF-109203X (a selective PKC inhibitor). BAPTA (an intracellular calcium chelator) blocked the effects of melatonin and 8-bromo-cGMP but not of PMA. Inhibition or activation of the protein kinase A pathway did not affect basal or melatonin-mediated AR localization. We conclude that the melatonin-mediated rise in cGMP elicits AR nuclear exclusion via a pathway involving increased intracellular calcium and PKC activation. These results define a novel signaling pathway that regulates AR localization and androgen responses in target cells. Received 31 July 2001; received after revision 18 September 2001; accepted 30 October 2001     

MAP kinases in plant signal transduction

Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. Distinct MAPK pathways are regulated by different extracellular stimuli and are implicated in a wide variety of biological processes. In plants there is evidence for MAPKs playing a role in the signaling of abiotic stresses, pathogens and plant hormones. The large number and divergence of plant MAPKs indicates that this ancient mechanism of bioinformatics is extensively used in plants and may provide a new molecular handle on old questions.     

The Janus kinase family of protein tyrosine kinases and their role in signaling

In the early 1990s, the search for protein kinases led to the discovery of a novel family of non-receptor tyrosine kinases, the Janus kinases or JAKs. These proteins were unusual because they contained two kinase homology domains and no other known signaling modules. It soon became clear that these were not ‘just another’ type of kinase. Their ability to complement mutant cells insensitive to interferons and to be activated by a variety of cytokines demonstrated their central signaling function. Now, as we approach the end of the decade, it is evident from biochemical studies to knockout mice that JAKs play non-redundant functions in development, differentiation, and host defense mechanisms. Here, recent progress is reviewed, with particular emphasis on structure-function studies aimed at revealing how this family of tyrosine kinases is regulated.     

Genetic analysis of the kinome and phosphatome in cancer

Protein phosphorylation is a well-characterized biochemical process for reversible regulation of protein activity. Protein kinases and protein phosphatases are the key complementary players in this process, and through their coordinated activity cell homeostasis is tightly controlled. If these enzymes display aberrant activity, cells may undergo unrestrained growth, thus giving rise to complex diseases such as cancer. The technological platform gathered during the Human Genome Project recently allowed the systematic identifi cation of the genetic alterations present in the kinase (the kinome) and the phosphatase (the phosphatome) gene families. These studies suggest that most if not all human tumors carry genetic alterations in at least one phosphatase or kinase gene. Here we integrate the biochemical knowledge on the properties of these molecules with the information collected through their systematic genetic analysis in cancer. We also analyze why the molecular profi ling of the kinome and phosphatome in individual cancers is revolutionizing basic and clinical oncology.Received 13 May 2005; received after revision 30 May 2005; accepted 22 June 2005     

植物抗旱分子机理研究进展

干旱胁迫是影响植物生长发育的主要逆境因子之一,高等植物在长期进化过程中,逐渐演变产生了对干旱胁迫的防御机制,以最大限度减轻干旱胁迫造成的伤害。本文概述了植物耐旱生理及其分子机制。     

Pharmacological attenuation of apoptosis in reoxygenated endothelial cells

BRX-235 (Iroxanadine), a novel drug developed by Biorex (Hungary), was previously characterized as a vasculoprotector against atherosclerosis, an activator of p38 kinase, and an enhancer of stress-responsive heat shock protein (Hsp) expression. The present data demonstrate that BRX-235 may improve survival of vascular endothelial cells (ECs) following ischemia/reperfusion stress. ECs cultured from human umbilical veins were exposed to hypoxia/reoxygenation to mimic ischemia/reperfusion. Caspase activation and apoptosis were monitored in the reoxygenated cells. Addition of BRX-235 (0.1–1 M) to culture medium prior to hypoxia or at start of reoxygenation significantly reduced the caspase-dependent apoptosis. The cytoprotection conferred by the pre-hypoxic drug administration was sensitive to quercetin and seems to be based on enhanced Hsp accumulation in stressed ECs. In the case of post-hypoxic drug administration, the cytoprotection was strongly inhibited by SB202190 and SB203580 and appears to be associated with enhanced p38 kinase activation in reoxygenated ECs.Received 12 May 2004; received after revision 7 September 2004; accepted 24 September 2004     

If phosphatases go up, memory goes down

Previous work has provided conclusive support for a role of various protein kinases in processes underlying learning and memory formation. While these processes are not yet established in full detail, it is interesting to entertain the idea of protein phosphatases being involved in such mechanisms as well. Recent advances in this respect have provided preliminary support of this view. From the pharmacological as well as the transgenic analysis, it appears that especially the calcineurin/inhibitor-1 cascade plays an important role in the transition of intermediate-term into long-term memory formation.     

Molecular aspects of membrane fission in the secretory pathway

Membrane fission is essential in various intracellular dissociative transport steps. The molecular mechanisms by which endocytic vesicles detach from the plasma membrane are being rapidly elucidated. Much less is known about the fission mechanisms operating at Golgi tubular networks; these include the Golgi transport and sorting stations, the trans-Golgi and cis-Golgi networks, where the geometry and physical properties of the membranes differ from those at the cell surface. Here we discuss the lipid and protein machineries that have so far been related to the fission process, with emphasis on those acting in the Golgi complex. Received 10 May 2002; received after revision 20 June 2002; accepted 26 June 2002 RID="*" ID="*"Corresponding author.     

T-cell signal transduction and the role of protein kinase C

The T lymphocyte has a vital part to play in maintaining the host response to bacterial and viral infection and also appears to play a key pathological role in autoimmune diseases such as rheumatoid arthritis. In this review, we summarize the signalling pathways which trigger antigen-driven T-cell proliferation and examine the evidence which suggests that protein kinase C (PKC) is fundamental to this process. Finally, we discuss the therapeutic potential that PKC inhibitors may have in the treatment of autoimmune disease. Received 31 March 1998; received after revision 19 May 1998; accepted 19 May 1998     

The effect of hydrogen peroxide on the cyclin D expression in fibroblasts

Activation of mitogen-activated protein (MAP) kinase is essential for cyclin D1 expression and provides a link between mitogenic signalling and cell cycle progression. Hydrogen peroxide (H₂ O₂ ) activates MAP kinase; however, it is not known whether this leads to cyclin D expression. Sustained expression of cyclin D1 and D2 was observed when Her14 fibroblasts were incu-bated with 3 mM or higher H₂ O₂ concentrations. Similar results were obtained when cells were incubated in the presence of serum (FCS). However, the sustained expres-complex sion of cyclin D1 and D2 upon H₂ O₂ treatment was not due to the MAP kinase pathway, because MAP kinase kinase inhibitors did not inhibit cyclin D expression. Furthermore, cyclin D1 and D2 levels remained constant even after addition of a protein synthesis inhibitor, indicating that the effect of H₂ O₂ was not due to induction of protein synthesis. These results indicate that H₂ O₂ reversibly inhibits the ubiquitin-proteasome dependent degra-dation of cyclin D1 and D2, probably by transiently in-hibiting ubiquitination and/or the proteasome. Received 12 March 2001; received after revision 5 April 2001; accepted 9 April 2001     

Ethanol inhibits insulin expression and actions in the developing brain

Ethanol-induced cerebellar hypoplasia is associated with inhibition of insulin-stimulated survival signaling. The present work explores the mechanisms of impaired insulin signaling in a rat model of fetal alcohol syndrome. Real-time quantitative RT-PCR demonstrated reduced expression of the insulin gene in cerebella of ethanol-exposed pups. Although receptor expression was unaffected, insulin and insulin-like growth factor (IGF-I) receptor tyrosine kinase (RTK) activities were reduced by ethanol exposure, and these abnormalities were associated with increased PTP1b activity. In addition, glucose transporter molecule expression and steady-state levels of ATP were reduced in ethanol-exposed cerebellar tissue. Cultured cerebellar granule neurons from ethanol-exposed pups had reduced expression of genes encoding insulin, IGF-II, and the IGF-I and IGF-II receptors, and impaired insulin- and IGF-I-stimulated glucose uptake and ATP production. The results demonstrate that ethanol inhibits insulin-mediated actions in the developing brain by reducing local insulin production and insulin RTK activation, leading to inhibition of glucose transport and ATP production.Received 30 December 2004; received after revision 1 March 2005; accepted 10 March 2005     

Memory formation and the regulation of gene expression

On a cellular level, formation of memory is based on a selective change in synaptic efficacy that is both fast and, in case of important information, long-lasting. Rapidity of cellular changes is achieved by modifying preexisting synaptic molecules (receptors, ion channels), which instantaneously alters the efficacy of synaptic transmission. Endurance, that is the formation of long-term memory (LTM), is based on transient and perhaps also long-lasting changes in protein synthesis. A number of different methods exist to interfere with the synthesis of specific proteins or proteins in general. Other methods, in turn, help to identify proteins whose synthesis is changed following learning. These mostly molecular methods are briefly described in the present review. Their successful application in a variety of memory paradigms in invertebrates and vertebrates is illustrated. The data support the importance of selective changes in gene expression for LTM. Proteins newly synthesized during memory consolidation are likely to contribute to restructuring processes at the synapse, altering the efficiency of transmission beyond the scope of STM. Increased or, less often, decreased synthesis of proteins appears during specific time windows following learning. Recent evidence supports older data suggesting that two or even more waves of protein synthesis exist during the consolidation period. It is expected that the new molecular methods will help to identify and characterize molecules whose expression changes during LTM formation even in complex vertebrate learning paradigms.     

Regulation of G1 phase progression by growth factors and the extracellular matrix

Cell cycle progression is regulated by both intracellular and extracellular control mechanisms. Intracellular controls ensure that cell cycle progression is stopped in response to irregularities such as DNA damage or faulty spindle assembly, whereas extracellular factors may determine cell fate such as differentiation, proliferation or programmed cell death (apoptosis). When extracellular factors bind to receptors at the outside of the cell, signal transduction cascades are activated inside the cell that eventually lead to cellular responses. We have shown previously that MAP kinase (MAPK), one of the proteins involved in several signal transduction processes, is phosphorylated early after mitosis and translocates to the nucleus around the restriction point. The activation of MAPK is independent of cell attachment, but does require the presence of growth factors. Moreover, it appears that in Chinese hamster ovary cells, a transformed cell line, growth factors must be present early in the G1 phase for a nuclear translocation of MAPK and subsequent DNA replication to occur. When growth factors are withdrawn from the medium immediately after mitosis, MAPK is not phosphorylated, cell cycle progression is stopped and cells appear to enter a quiescent state, which may lead to apoptosis. Furthermore, in addition to this growth-factor-regulated decision point in early G1 phase, another growth-factor-sensitive period can be distinguished at the end of the G1 phase. This period is suggested to correlate with the classical restriction point (R) and may be related to cell differentiation.     

Molecular modeling of mammalian cytochrome P450s

The cytochrome P450s are a superfamily of hemoprotein enzymes responsible for the metabolism of a wide variety of xenobiotic and endogenous compounds. The individual P450s exhibit unique substrate specificity and stereoselectivity profiles which reflect corresponding differences in primary sequence and tertiary structure. In the absence of an experimental structure, models for mammalian P450s have been generated by their homology with bacterial P450s of known structure. The rather low sequence identity between target and template proteins renders P450 modeling a challenging task. However, the substrate recognition properties of several P450s are consistent with recently developed working models. This review summarizes the major concepts and current approaches of molecular modeling of P450s. Received 28 September 1999; received after revision 25 November 1999; accepted 31 December 1999     

Chromatin assembly during S phase: contributions from histone deposition, DNA replication and the cell division cycle

During S phase of the eukaryotic cell division cycle, newly replicated DNA is rapidly assembled into chromatin. Newly synthesised histones form complexes with chromatin assembly factors, mediating their deposition onto nascent DNA and their assembly into nucleosomes. Chromatin assembly factor 1, CAF-1, is a specialised assembly factor that targets these histones to replicating DNA by association with the replication fork associated protein, proliferating cell nuclear antigen, PCNA. Nucleosomes are further organised into ordered arrays along the DNA by the activity of ATP-dependent chromatin assembly and spacing factors such as ATP-utilising chromatin assembly and remodelling factor ACF. An additional level of controlling chromatin assembly pathways has become apparent by the observation of functional requirements for cyclin-dependent protein kinases, casein kinase II and protein phosphatases. In this review, we will discuss replication-associated histone deposition and nucleosome assembly pathways, and we will focus in particular on how nucleosome assembly is linked to DNA replication and how it may be regulated by the cell cycle control machinery.     

Structure and evolution of the genetic code viewed from the perspective of the experimentally expanded amino acid repertoire in vivo

Much effort has been devoted recently to expanding the amino acid repertoire in protein biosynthesis in vivo. From such experimental work it has emerged that some of the non-canonical amino acids are accepted by the cellular translational machinery while others are not, i.e. we have learned that some determinants must exist and that they can even be anticipated. Here, we propose a conceptual framework by which it should be possible to assess deeper levels of the structure of the genetic code, and based on this experiment to understand its evolution and establishment. First, we propose a standardised repertoire of 20 amino acids as a basic set of conserved building blocks in protein biosynthesis in living cells to be the main criteria for genetic code structure and evolutionary considerations. Second, based on such argumentation, we postulate the structure and evolution of the genetic code in the form of three general statements: (i) the nature of the genetic code is deterministic; (ii) the genetic code is conserved and universal; (iii) the genetic code is the oldest known level of complexity in the evolution of living organisms that is accessible to our direct observation and experimental manipulations. Such statements are discussed as our working hypotheses that are experimentally tested by recent findings in the field of expanded amino acid repertoire in vivo. Received 30 June 1999; accepted 9 July 1999