Mining Microarrays for Metabolic Meaning: Nutritional Regulation of Hypothalamic Gene Expression

DNA microarray analysis has been used to investigate relative changes in the level of gene expression in the CNS, including changes that are associated with disease, injury, psychiatric disorders, drug exposure or withdrawal, and memory formation. We have used oligonucleotide microarrays to identify hypothalamic genes that respond to nutritional manipulation. In addition to commonly used microarray analysis based on criteria such as fold-regulation, we have also found that simply carrying out multiple t tests then sorting by P value constitutes a highly reliable method to detect true regulation, as assessed by real-time polymerase chain reaction (PCR), even for relatively low abundance genes or relatively low magnitude of regulation. Such analyses directly suggested novel mechanisms that mediate effects of nutritional state on neuroendocrine function and are being used to identify regulated gene products that may elucidate the metabolic pathology of obese ob/ob, lean Vgf-/Vgf-, and other models with profound metabolic impairments.     

细胞周期中MicroRNA的调控作用

MicroRNA是近年来发现并热点研究的一类重要的非编码RNA,在干细胞的更新与分化、体细胞性状与数量的维持、甚至肿瘤细胞的恶性增生等生物学过程中都具有重要的调控作用.microRNA通过与靶位点结合而快速有效地降解靶基因mRNA或抑制蛋白的翻译,下调E2F、CDK、cyclin、p21、p27、DNA多聚酶α等关键的细胞周期调控因子的表达,加速或减慢细胞增殖的速度.microRNA对细胞周期的调控还将涉及到微生物感染机体的过程、免疫系统的调控、妊娠期母体的变化、组织的修复、细胞的凋亡与衰老等诸多方面.随着对microRNA调控细胞周期机制的深入研究,microRNA及其靶基因不仅可以作为某些疾病的分子标记物,而且可以用于指导疾病的预防和治疗.     

表达谱基因芯片

表达谱基因芯片具有高通量、缩微、多参数、平行化等优点.在功能基因组学研究中正发挥越来越重要的作用.就其原理、应用、存在问题及解决策略等进行了综述.     

Cell Cycle Regulation

Drosophila researchers met in sunny San Diego for the 49th annual meeting of The Genetics Society of America. It was cold outside and even colder inside. Like last year, ‘Mitosis, Meiosis and Cell Division’ was no longer a session. Instead, we searched out and covered talks and posters in ‘Cell Division and Growth Control’, ‘Gametogenesis’, ‘Cytoskeleton and Cell Biology’ and ‘Genome and Chromosome Structure’. We split up for maximal coverage and re-grouped later for the Workshop on Cell Cycle and Checkpoints. We apologize in advance for the brevity or omission of some reports.     

Global Effects of DDX3 Inhibition on Cell Cycle Regulation Identified by a Combined Phosphoproteomics and Single Cell Tracking Approach

DDX3 is an RNA helicase with oncogenic properties. The small molecule inhibitor RK-33 is designed to fit into the ATP binding cleft of DDX3 and hereby block its activity. RK-33 has shown potent activity in preclinical cancer models. However, the mechanism behind the antineoplastic activity of RK-33 remains largely unknown. In this study we used a dual phosphoproteomic and single cell tracking approach to evaluate the effect of RK-33 on cancer cells. MDA-MB-435 cells were treated for 24?hours with RK-33 or vehicle control. Changes in phosphopeptide abundance were analyzed with quantitative mass spectrometry using isobaric mass tags (Tandem Mass Tags). At the proteome level we mainly observed changes in mitochondrial translation, cell division pathways and proteins related to cell cycle progression. Analysis of the phosphoproteome indicated decreased CDK1 activity after RK-33 treatment. To further evaluate the effect of DDX3 inhibition on cell cycle progression over time, we performed timelapse microscopy of Fluorescent Ubiquitin Cell Cycle Indicators labeled cells after RK-33 or siDDX3 exposure. Single cell tracking indicated that DDX3 inhibition resulted in a global delay in cell cycle progression in interphase and mitosis. In addition, we observed an increase in endoreduplication. Overall, we conclude that DDX3 inhibition affects cells in all phases and causes a global cell cycle progression delay.     

Regulation of podJ Expression during the Caulobacter crescentus Cell Cycle

The polar organelle development gene, podJ, is expressed during the swarmer-to-stalked cell transition of the Caulobacter crescentus cell cycle. Mutants with insertions that inactivate the podJ gene are nonchemotactic, deficient in rosette formation, and resistant to polar bacteriophage, but they divide normally. In contrast, hyperexpression of podJ results in a lethal cell division defect. Nucleotide sequence analysis of the podJ promoter region revealed a binding site for the global response regulator, CtrA. Deletion of this site results in increased overall promoter activity, suggesting that CtrA is a negative regulator of the podJ promoter. Furthermore, synchronization studies have indicated that temporal regulation is not dependent on the presence of the CtrA binding site. Thus, although the level of podJ promoter activity is dependent on the CtrA binding site, the temporal control of podJ promoter expression is dependent on other factors.     

Regulation of lipA Gene Expression by Cell Surface Proteins in Arthrobacter photogonimos

Expression of the light-inducible lipA gene in Arthrobacter photogonimos by photodynamic compounds and visible light was inhibited by washing cells with 1 M KCl. Addition of cell surface extract to KCl-washed cells restored the induction. Washing cells with 1 M MgCl₂ removed a 14-kDa polypeptide and concomitantly caused expression of lipA gene without photodynamic treatment. The purified 14-kDa polypeptide inhibited photodynamic induction of lipA gene. These results indicated that regulation of lipA gene expression occurred at the cell surface and involved positive and negative factors, probably through a signal transduction system. Received: 29 July 1998 / Accepted: 31 August 1998     

Cell Type-Specific Gene Expression Analyses by RNA Sequencing Reveal Local High Nitrate-Triggered Lateral Root Initiation in Shoot-Borne Roots of Maize by Modulating Auxin-Related Cell Cycle Regulation

Plants have evolved a unique plasticity of their root system architecture to flexibly exploit heterogeneously distributed mineral elements from soil. Local high concentrations of nitrate trigger lateral root initiation in adult shoot-borne roots of maize (Zea mays) by increasing the frequency of early divisions of phloem pole pericycle cells. Gene expression profiling revealed that, within 12 h of local high nitrate induction, cell cycle activators (cyclin-dependent kinases and cyclin B) were up-regulated, whereas repressors (Kip-related proteins) were down-regulated in the pericycle of shoot-borne roots. In parallel, a ubiquitin protein ligase S-Phase Kinase-Associated Protein1-cullin-F-box protein^{S-Phase Kinase-Associated Protein 2B}-related proteasome pathway participated in cell cycle control. The division of pericycle cells was preceded by increased levels of free indole-3-acetic acid in the stele, resulting in DR5-red fluorescent protein-marked auxin response maxima at the phloem poles. Moreover, laser-capture microdissection-based gene expression analyses indicated that, at the same time, a significant local high nitrate induction of the monocot-specific PIN-FORMED9 gene in phloem pole cells modulated auxin efflux to pericycle cells. Time-dependent gene expression analysis further indicated that local high nitrate availability resulted in PIN-FORMED9-mediated auxin efflux and subsequent cell cycle activation, which culminated in the initiation of lateral root primordia. This study provides unique insights into how adult maize roots translate information on heterogeneous nutrient availability into targeted root developmental responses.Roots have developed adaptive strategies to reprogram their gene expression and metabolic activity in response to heterogeneous soil environments (Osmont et al., 2007). By this way, local environmental stimuli can be integrated into the developmental program of roots (Forde, 2014; Giehl and von Wirén, 2014). In resource-depleted environments, an important heterogeneously distributed soil factor is nutrient availability, which then directs lateral root growth preferentially into nutrient-rich patches (Zhang and Forde, 1998; Lima et al., 2010; Giehl et al., 2012). Such directed lateral root development depends on regulatory networks that integrate both local and systemic signals to coordinate them with the overall plant nutritional status (Ruffel et al., 2011; Guan et al., 2014). As shown by the impact of the N status-dependent regulatory module CLAVATA3/EMBRYO-SURROUNDING REGION-related peptides-CLAVATA1 leucine-rich repeat receptor-like kinase, economizing the costs for root development is pivotal for a resource-efficient strategy in nutrient acquisition (Araya et al., 2014). In recent years, strategies on yield and efficiency improvement have been developed that are primarily based on the manipulation of root system architecture (Gregory et al., 2013; Lynch, 2014; Meister et al., 2014). A common imperative of these strategies is to develop crops that use water and nutrients more efficiently, allowing the reduction of fertilizer input and potentially hazardous environmental contamination.Maize (Zea mays) plays an eminent role in global food, feed, and fuel production, which is also a consequence of its unique root system (Rogers and Benfey, 2015). The genetic analysis of maize root architecture revealed a complex molecular network coordinating root development during the whole lifecycle (for review, see Hochholdinger et al., 2004a, 2004b). Identification of root type-specific lateral root mutants in maize emphasized the existence of regulatory mechanisms involved in the branching of embryonic roots, which are distinct from those in postembryonic roots (Hochholdinger and Feix, 1998; Woll et al., 2005). Under heterogeneous nutrient supplies, nitrate-rich patches increased only the length of lateral roots in primary and seminal roots, whereas they increased both length and density of lateral roots from shoot-borne roots of adult maize plants (Yu et al., 2014a). Remarkably, modulation of the extensive postembryonic shoot-borne root stock has a great potential to improve grain yield and nutrient use efficiency (Hochholdinger and Tuberosa, 2009).Lateral root branching is critical to secure anchorage and ensure adequate uptake of water and nutrients. In maize, these roots originate from concentric single-file layers of pericycle and endodermis cells (Fahn, 1990; Jansen et al., 2012). Lateral root initiation is the result of auxin-dependent cell cycle progression (Beeckman et al., 2001; Jansen et al., 2013a). Most of the molecular changes during the cell cycle like, for instance, the induction of positive regulators, such as cyclins (CYCs) and cyclin-dependent kinases (CDKs), and the repression of Kip-related proteins (KRPs), thus account for a reactivation of the cell cycle (Beeckman et al., 2001; Himanen et al., 2002, 2004). In eukaryotes, ubiquitin-mediated degradation of cell cycle proteins plays a critical role in the regulation of cell division (Hershko, 2005; Jakoby et al., 2006). Conjugation of ubiquitin to a substrate requires the sequential action of three enzymes: ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin-protein ligase (E3). The E3 enzymes are responsible for the specificity of the pathway, and several classes of E3 enzymes have been implicated in cell cycle regulation, including the S-Phase Kinase-Associated Protein1-cullin-F-box protein (SCF) and Really Interesting New Gene (RING) finger-domain ubiquitin ligases (Del Pozo and Manzano, 2014). The F-box protein S-Phase Kinase-Associated Protein 2B (SKP2B) encodes an F-box ubiquitin ligase, which plays an important role in the cell cycle by regulating the stability of KRP1 and pericycle founder cell division during lateral root initiation (Ren et al., 2008; Manzano et al., 2012).It has been shown that auxin is involved in long-distance signaling to adjust root growth in response to local nutrient availability (Giehl et al., 2012), and it is likely to serve in long-distance signaling for local nutrient responses as well (for review, see Rubio et al., 2009; Krouk et al., 2011; Saini et al., 2013; Forde, 2014). Polar auxin transport is instrumental for the generation of local auxin maxima, which guide these cells to switch their developmental program (Vanneste and Friml, 2009; Lavenus et al., 2013). In Arabidopsis (Arabidopsis thaliana), the PIN-FORMED (PIN) family of auxin efflux carrier proteins controls the directionality of auxin flows to maximum formation at the tip or pericycle cells (Benková et al., 2003; Laskowski et al., 2008; Marhavý et al., 2013). Auxin responses in protoxylem or protophloem cells of the basal meristem coincide with the site of lateral root initiation (De Smet et al., 2007; Jansen et al., 2012). In these defined pericycle cells, the phloem pole pericycle founder cells are primed before auxin accumulation occurs (De Smet et al., 2007; Jansen et al., 2012, 2013a). In contrast to dicots, the larger PIN family in monocots has a more divergent phylogenetic structure (Paponov et al., 2005). It is likely that monocot-specific PIN genes regulate monocot-specific morphogenetic processes, such as the development of a complex root system (Wang et al., 2009; Forestan et al., 2012).The molecular control of lateral root initiation of the root system to heterogeneous nitrate availabilities is not yet understood in maize. In this study, the plasticity of lateral root induction in adult shoot-borne roots of maize in response to local high concentration of nitrate was surveyed in an experimental setup that simulated patchy nitrate distribution. RNA-sequencing (RNA-Seq) experiments and cell type-specific gene expression analyses showed that local nitrate triggers progressive cell cycle control during pericycle cell division. In addition, tissue-specific determination of indole-3-acetic acid (IAA) and its metabolites combined with auxin maxima determination by DR5 supported a role of basipetal auxin transport during lateral root initiation in shoot-borne roots. Thereby, this study provides unique insights in how auxin orchestrates cell cycle control under local nitrate stimulation in the shoot-borne root system of maize.     

Monitoring Gene Expression in Mixed Microbial Communities by Using DNA Microarrays

A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested. Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Ralstonia eutropha JMP134, and the inducing agent 2,4-D. Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D. This strain was diluted into a constructed mixed microbial community derived from a laboratory scale sequencing batch reactor. Induction of two of five 2,4-D catabolic genes (tfdA and tfdC) from populations of JMP134 as low as 10⁵ cells/ml was clearly detected against a background of 10⁸ cells/ml. Induction of two others (tfdB and tfdE) was detected from populations of 10⁶ cells/ml in the same background; however, the last gene, tfdF, showed no significant induction due to high variability. In another experiment, the induction of resin acid degradative genes was statistically detectable in sludge-fed pulp mill effluent exposed to dehydroabietic acid in batch experiments. We conclude that microarrays will be useful tools for the detection of bacterial gene expression in wastewaters and other complex systems.     

Cell Cycle Regulation by Light in Prochlorococcus Strains

The effect of light on the synchronization of cell cycling was investigated in several strains of the oceanic photosynthetic prokaryote Prochlorococcus using flow cytometry. When exposed to a light-dark (L-D) cycle with an irradiance of 25 μmol of quanta · m⁻² s⁻¹, the low-light-adapted strain SS 120 appeared to be better synchronized than the high-light-adapted strain PCC 9511. Submitting L-D-entrained populations to shifts (advances or delays) in the timing of the “light on” signal translated to corresponding shifts in the initiation of the S phase, suggesting that this signal is a key parameter for the synchronization of population cell cycles. Cultures that were shifted from an L-D cycle to continuous irradiance showed persistent diel oscillations of flow-cytometric signals (light scatter and chlorophyll fluorescence) but with significantly reduced amplitudes and a phase shift. Complete darkness arrested most of the cells in the G₁ phase of the cell cycle, indicating that light is required to trigger the initiation of DNA replication and cell division. However, some cells also arrested in the S phase, suggesting that cell cycle controls in Prochlorococcus spp. are not as strict as in marine Synechococcus spp. Shifting Prochlorococcus cells from low to high irradiance translated quasi-instantaneously into an increase of cells in both the S and G₂ phases of the cell cycle and then into faster growth, whereas the inverse shift induced rapid slowing of the population growth rate. These data suggest a close coupling between irradiance levels and cell cycling in Prochlorococcus spp.     

细胞周期和调控因子

近期对细胞周期调控的研究获得了突破性进展.人们深入地研究了周期蛋白家族和pp³⁴蛋白家族在周期调控中的作用及二者之间的相互关系,同时还发现了很多与之相关的调控因子,它们彼此相互作用,形成了极为复杂的级联调控网络.     

Multiple Antibody Targets on Herpes B Glycoproteins B and D Identified by Screening Sera of Infected Rhesus Macaques with Peptide Microarrays

Herpes B virus (or Herpesvirus simiae or Macacine herpesvirus 1) is endemic in many populations of macaques, both in the wild and in captivity. The virus elicits only mild clinical symptoms (if any) in monkeys, but can be transmitted by various routes, most commonly via bites, to humans where it causes viral encephalitis with a high mortality rate. Hence, herpes B constitutes a considerable occupational hazard for animal caretakers, veterinarians and laboratory personnel. Efforts are therefore being made to reduce the risk of zoonotic infection and to improve prognosis after accidental exposure. Among the measures envisaged are serological surveillance of monkey colonies and specific diagnosis of herpes B zoonosis against a background of antibodies recognizing the closely related human herpes simplex virus (HSV). 422 pentadecapeptides covering, in an overlapping fashion, the entire amino acid sequences of herpes B proteins gB and gD were synthesized and immobilized on glass slides. Antibodies present in monkey sera that bind to subsets of the peptide collection were detected by microserological techniques. With 42 different rhesus macaque sera, 114 individual responses to 18 different antibody target regions (ATRs) were recorded, 17 of which had not been described earlier. This finding may pave the way for a peptide-based, herpes B specific serological diagnostic test.     

Regulation of the Cell Cycle by Focal Adhesion Kinase

In this report, we have analyzed the potential role and mechanisms of integrin signaling through FAK in cell cycle regulation by using tetracycline-regulated expression of exogenous FAK and mutants. We have found that overexpression of wild-type FAK accelerated G1 to S phase transition. Conversely, overexpression of a dominant-negative FAK mutant ΔC14 inhibited cell cycle progression at G1 phase and this inhibition required the Y397 in ΔC14. Biochemical analyses indicated that FAK mutant ΔC14 was mislocalized and functioned as a dominant-negative mutant by competing with endogenous FAK in focal contacts for binding signaling molecules such as Src and Fyn, resulting in a decreases of Erk activation in cell adhesion. Consistent with this, we also observed inhibition of BrdU incorporation and Erk activation by FAK Y397F mutant and FRNK, but not FRNKΔC14, in transient transfection assays using primary human foreskin fibroblasts. Finally, we also found that ΔC14 blocked cyclin D1 upregulation and induced p21 expression, while wild-type FAK increased cyclin D1 expression and decreased p21 expression. Taken together, these results have identified FAK and its associated signaling pathways as a mediator of the cell cycle regulation by integrins.