The phylogeny of Patrinieae sensu Graebner (Valerianaceae) revisited: additional evidence from ndhF sequence data

 DNA sequences of both 5′ and 3′ regions of the plastid ndhF gene were generated in order to study the position of Patrinia and Nardostachys, to check the potential paraphyletic nature of Patrinieae, and to evaluate the possible link between the tribe and Linnaeaceae. Parsimony analysis showed very strong support for Patrinia as sister to all members of Valerianaceae (including Nardostachys) and indicated the paraphyletic nature of the tribe Patrinieae. Additionally, trees were constructed from available rbcL data separately and supplemented with ndhF sequences. Topologies of these combined cladograms are in agreement with the ndhF phylogeny, suggesting that the traditionally circumscribed Patrinieae can no longer be recognized but must be considered as part of a basal grade in Valerianaceae. Parsimony analysis based on a morphological data set supported a monophyletic Patrinieae; combination with the molecular data showed a paraphyletic Patrinieae. Furthermore, the possible link between Patrinieae and Linnaeaceae is evaluated. Received July 12, 2001 Accepted February 25, 2002     

Cytokine-induced cell death in immortalized Schwann cells: roles of nitric oxide and cyclic AMP

Tumor necrosis factor-alpha and interferon-gamma are pleiotropic cytokines that regulate Schwann cell responses during injury and inflammatory demyelination. We have previously shown that cyclic AMP (cAMP)-elevating agents decrease the demyelination and Wallerian degeneration in experimental allergic neuritis. In this study, we examined the role of cAMP in cytokine-mediated signaling in a spontaneously immortal Schwann cell clone (iSC). We found that tumor necrosis factor-alpha and interferon-gamma exert synergistic inhibitory action on Schwann cell viability via the production of nitric oxide (NO) and ceramide (cer). Furthermore, we found that: (i) NO synthase inhibitors attenuate the cytokine-induced cer accumulation and cell death indicating that NO acts upstream of cer; and (ii) cytokine-induced cell death is decreased in iSCs pretreated continuously for 48-72 h with forskolin, an activator of adenylate cyclase. Although forskolin modulates the phosphorylation of ERKs and Akt, it decreases the susceptibility of iSC to cytokines via a separate mechanism operating after NO induction and before cer accumulation. We propose that the protective effect of cAMP-elevating agents in experimental allergic neuritis may be mediated in part via modulation of Schwann cell responses to cytokines.     

Geographic isolation and evolution of Mediterranean endemic Cyclamen: insights from chloroplast trnL (UAA) intron sequence variation

 In this study we construct a phylogenetic hypothesis for the relatedness among disjunct subspecies of Cyclamen repandum and its two allopatric congeners, C. creticum and C. balearicum in order to examine the evolutionary divergence of currently isolated populations across the western Mediterranean. The most parsimonious phylogenetic tree obtained from sequencing the cpDNA trnL (UAA) intron suggests a major phylogeographic divide in southern Greece between two clades. The first clade comprises samples of C. repandum subsp. peloponnesiacum (from the Peloponnese) and C. creticum (from Crete). The second comprises samples of C. repandum subsp. repandum (from Croatia, Italy, southern France, Corsica, Sardinia and Sicily), C. repandum subsp. rhodense (from Rhodes and Kos) and C. balearicum (from the Balearic Islands and southern France). These data suggest that C. creticum has evolved in allopatry from C. repandum subsp. peloponnesiacum and that C. balearicum and C. repandum ssp. rhodense have diverged from C. repandum subsp. repandum at its western and eastern distribution limits. At one small site on Corsica, a population of C. repandum may have introgressed with relictual populations of C. balearicum. These divergence patterns illustrate how a phylogenetic perspective can be used to better understand the evolution of endemism in the Mediterranean flora. Received February 19, 2001 Accepted August 22, 2001     

Physiological function as regulation of large transcriptional programs: the cellular response to genotoxic stress

The responses to ionizing radiation and other genotoxic environmental stresses are complex and are regulated by a number of overlapping molecular pathways. One such stress signaling pathway involves p53, which regulates the expression of over 100 genes already identified. It is also becoming increasingly apparent that the pattern of stress gene expression has some cell type specificity. It may be possible to exploit these differences in stress gene responsiveness as molecular markers through the use of a combined informatics and functional genomics approach. The techniques of microarray analysis potentially offer the opportunity to monitor changes in gene expression across the entire set of expressed genes in a cell or organism. As an initial step in the development of a functional genomics approach to stress gene analysis, we have recently demonstrated the utility of cDNA microarray hybridization to measure radiation-stress gene responses and identified a number of previously unknown radiation-regulated genes. The responses of some of these genes to DNA-damaging agents vary widely in cell lines from different tissues of origin and different genetic backgrounds. While this again highlights the importance of a cellular context to genotoxic stress responses, it also raises the prospect of expression-profiling of cell lines, tissues, and tumors. Such profiles may have a predictive value if they can define regions of ‘expression space’ that correlate with important endpoints, such as response to cancer therapy regimens, or identification of exposures to environmental toxins.     

N-terminal Acetylation Stabilizes N-terminal Helicity in Lipid- and Micelle-bound α-Synuclein and Increases Its Affinity for Physiological Membranes

The Parkinson disease protein α-synuclein is N-terminally acetylated, but most in vitro studies have been performed using unacetylated α-synuclein. Binding to lipid membranes is considered key to the still poorly understood function of α-synuclein. We report the effects of N-terminal acetylation on α-synuclein binding to lipid vesicles of different composition and curvature and to micelles composed of the detergents β-octyl-glucoside (BOG) and SDS. In the presence of SDS, N-terminal acetylation results in a slightly increased helicity for the N-terminal ∼10 residues of the protein, likely due to the stabilization of N-terminal fraying through the formation of a helix cap motif. In the presence of BOG, a detergent used in previous isolations of helical oligomeric forms of α-synuclein, the N-terminally acetylated protein adopts a novel conformation in which the N-terminal ∼30 residues bind the detergent micelle in a partly helical conformation, whereas the remainder of the protein remains unbound and disordered. Binding of α-synuclein to lipid vesicles with high negative charge content is essentially unaffected by N-terminal acetylation irrespective of curvature, but binding to vesicles of lower negative charge content is increased, with stronger binding observed for vesicles with higher curvature. Thus, the naturally occurring N-terminally acetylated form of α-synuclein exhibits stabilized helicity at its N terminus and increased affinity for lipid vesicles similar to synaptic vesicles, a binding target of the protein in vivo. Furthermore, the novel BOG-bound state of N-terminally acetylated α-synuclein may serve as a model of partly helical membrane-bound intermediates with a role in α-synuclein function and dysfunction.     

Differential susceptibility to hydrogen sulfide-induced apoptosis between PHLDA1-overexpressing oral cancer cell lines and oral keratinocytes: Role of PHLDA1 as an apoptosis suppressor

Hydrogen sulfide (H₂S) is a novel gasotransmitter that plays multiple biological roles in various body systems. In addition to its endogenous production, H₂S is produced by bacteria colonizing digestive organs, including the oral cavity. H₂S was previously shown to enhance pro-apoptotic effects in cancer cell lines, although the mechanisms involved remain unclear. To properly assess the anti-cancer effects of H₂S, however, investigations of apoptotic effects in normal cells are also necessary. The aims of this study were (1) to compare the susceptibility to H₂S-induced apoptosis between the oral cancer cell line Ca9-22 and oral keratinocytes that were derived from healthy gingiva, and (2) to identify candidate genes involved in the induction of apoptosis by H₂S. The susceptibility to H₂S-induced apoptosis in Ca9-22 cells was significantly higher than that in keratinocytes. H₂S exposure in Ca9-22 cells, but not keratinocytes, enhanced the expression of pleckstrin homology-like domain, family A, member 1 (PHLDA1), which was identified through a differential display method. In addition, PHLDA1 expression increased during actinomycin D-induced apoptosis in Ca9-22 cells. Knockdown of PHLDA1 expression by small interfering RNA in Ca9-22 cells led to expression of active caspase 3, thus indicating apoptosis induction. The tongue cancer cell line SCC-25, which expresses PHLDA1 at a high level, showed similar effects. Our data indicate that H₂S is an anti-cancer compound that may contribute to the low incidence of oral cancer. Furthermore, we demonstrated the role of PHLDA1 as an apoptosis suppressor.     

DNA replication stress: Causes,resolution and disease

DNA replication is a fundamental process of the cell that ensures accurate duplication of the genetic information and subsequent transfer to daughter cells. Various pertubations, originating from endogenous or exogenous sources, can interfere with proper progression and completion of the replication process, thus threatening genome integrity. Coordinated regulation of replication and the DNA damage response is therefore fundamental to counteract these challenges and ensure accurate synthesis of the genetic material under conditions of replication stress. In this review, we summarize the main sources of replication stress and the DNA damage signaling pathways that are activated in order to preserve genome integrity during DNA replication. We also discuss the association of replication stress and DNA damage in human disease and future perspectives in the field.     

Comprehensive DNA microarray expression profiles of tumors in tenascin-C-knockout mice

Tenascin-C (TNC), an extracellular matrix glycoprotein, plays a pivotal role in tumor growth. However, the mechanism whereby TNC affects tumor biology remains unclear. To investigate the exact role of TNC in primary tumor growth, a mouse mammary tumor cell line, GLMT1, was first developed. Subsequently, global gene expression in GLMT1-derived tumors was compared between wild-type (WT) and TNC-knockout (TNKO) mice. Tumors in WT mice were significantly larger than those in TNKO mice. DNA microarray analysis revealed 447 up and 667 downregulated in the tumors inoculated into TNKO mice as compared to tumors in WT mice. Validation by quantitative gene expression analysis showed that Tnc, Cxcl1, Cxcl2, and Cxcr2 were significantly upregulated in WT mice. We hypothesize that TNC stimulates the CXCL1/2-CXCR2 pathway involved in cancer cell proliferation.     

Splenic gene expression profiling in White Leghorn layer inoculated with the Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis (SE) is a foodborne pathogen that can threaten human health through contaminated poultry products. Live poultry, chicken eggs and meat are primary sources of human salmonellosis. To understand the genetic resistance of egg‐type chickens in response to SE inoculation, global gene expression in the spleen of 20‐week‐old White Leghorn was measured using the Agilent 4 × 44 K chicken microarray at 7 and 14 days following SE inoculation (dpi). Results showed that there were 1363 genes significantly differentially expressed between inoculated and non‐inoculated groups at 7 dpi (I7/N7), of which 682 were up‐regulated and 681 were down‐regulated genes. By contrast, 688 differentially expressed genes were observed at 14 dpi (I14/N14), of which 371 were up‐regulated genes and 317 were down‐regulated genes. There were 33 and 28 immune‐related genes significantly differentially expressed in the comparisons of I7/N7 and I14/N14 respectively. Functional annotation revealed that several Gene Ontology (GO) terms related to immunity were significantly enriched between the inoculated and non‐inoculated groups at 14 dpi but not at 7 dpi, despite a similar number of immune‐related genes identified between I7/N7 and I14/N14. The immune response to SE inoculation changes with different time points following SE inoculation. The complicated interaction between the immune system and metabolism contributes to the immune responses to SE inoculation of egg‐type chickens at 14 dpi at the onset of lay. GC, TNFSF8, CD86, CD274, BLB1 and BLB2 play important roles in response to SE inoculation. The results from this study will deepen the current understanding of the genetic response of the egg‐type chicken to SE inoculation at the onset of egg laying.     

Endoplasmic reticulum stress in insulin resistance and diabetes

The endoplasmic reticulum is the main intracellular Ca²⁺ store for Ca²⁺ release during cell signaling. There are different strategies to avoid ER Ca²⁺ depletion. Release channels utilize first Ca²⁺-bound to proteins and this minimizes the reduction of the free luminal [Ca²⁺]. However, if release channels stay open after exhaustion of Ca²⁺-bound to proteins, then the reduction of the free luminal ER [Ca²⁺] (via STIM proteins) activates Ca²⁺ entry at the plasma membrane to restore the ER Ca²⁺ load, which will work provided that SERCA pump is active. Nevertheless, there are several noxious conditions that result in decreased activity of the SERCA pump such as oxidative stress, inflammatory cytokines, and saturated fatty acids, among others. These conditions result in a deficient restoration of the ER [Ca²⁺] and lead to the ER stress response that should facilitate recovery of the ER. However, if the stressful condition persists then ER stress ends up triggering cell death and the ensuing degenerative process leads to diverse pathologies; particularly insulin resistance, diabetes and several of the complications associated with diabetes. This scenario suggests that limiting ER stress should decrease the incidence of diabetes and the mobility and mortality associated with this illness.