The early phase change gene in maize

Recessive mutations of the early phase change (epc) gene in maize affect several aspects of plant development. These mutations were identified initially because of their striking effect on vegetative phase change. In certain genetic backgrounds, epc mutations reduce the duration of the juvenile vegetative phase of development and cause early flowering, but they have little or no effect on the number of adult leaves. Except for a transient delay in leaf production during germination, mutant plants initiate leaves at a normal rate both during and after embryogenesis. Thus, the early flowering phenotype of epc mutations is explained completely by their effect on the expression of the juvenile phase. The observation that epc mutations block the rejuvenation of leaf primordia in excised shoot apices supports the conclusion that epc is required for the expression of juvenile traits. This phenotype suggests that epc functions normally to promote the expression of the juvenile phase of shoot development and to suppress the expression of the adult phase and that floral induction is initiated by the transition to the adult phase. epc mutations are epistatic to the gibberellin-deficient mutation dwarf1 and interact additively with the dominant gain-of-function mutations Teopod1, Teopod2, and Teopod3. Genetic backgrounds that enhance the mutant phenotype of epc demonstrate that, in addition to its role in phase change, epc is required for the maintenance of the shoot apical meristem, leaf initiation, and root initiation.     

The ectopically parting cells 1-2 (epc1-2) mutant exhibits an exaggerated response to abscisic acid

The ECTOPICALLY PARTING CELLS 1 (EPC1) gene encodes a putative retaining glycosyltransferase of the GT64 family, and epc1-1 mutant plants have a severely dwarfed phenotype. A new mutant allele of this gene, epc1-2, has been isolated. Reduced cell adhesion that has previously been reported for the epc1-1 mutant was not observed for either the epc1-1 or epc1-2 mutants grown in our conditions, suggesting that EPC1 does not affect cell adhesion but is involved in some other process affecting plant growth and development. It is shown that the epc1-2 mutant exhibits hypersensitivity to the phytohormone abscisic acid in germination and root elongation assays, however it shows an unaltered response to gibberellin, epi-brassinosteroid, auxin, or ethylene. An EPC1:YFP fusion protein is localized to small motile structures within the cytosol that are similar in size and number to the Golgi apparatus. Analysis of cell wall pectins revealed that levels of beta-(1,4)-galactan in the epc1-2 mutant are reduced by 50%, whilst other pectic polysaccharides (homogalacturonan, arabinan, and rhamnogalacturonan II) are unchanged.     

Mechanisms for recurrent and complex human genomic rearrangements

During the last two decades, the importance of human genome copy number variation (CNV) in disease has become widely recognized. However, much is not understood about underlying mechanisms. We show how, although model organism research guides molecular understanding, important insights are gained from study of the wealth of information available in the clinic. We describe progress in explaining nonallelic homologous recombination (NAHR), a major cause of copy number change occurring when control of allelic recombination fails, highlight the growing importance of replicative mechanisms to explain complex events, and describe progress in understanding extreme chromosome reorganization (chromothripsis). Both nonhomologous end-joining and aberrant replication have significant roles in chromothripsis. As we study CNV, the processes underlying human genome evolution are revealed.     

Point process models of single-neuron discharges

In most neural systems, neurons communicate via sequences of action potentials. Contemporary models assume that the action potentials' times of occurrence rather than their waveforms convey information. The mathematical tool for describing sequences of events occurring in time and/or space is the theory of point processes. Using this theory, we show that neural discharge patterns convey time-varying information intermingled with the neuron's response characteristics. We review the basic techniques for analyzing single-neuron discharge patterns and describe what they reveal about the underlying point process model. By applying information theory and estimation theory to point processes, we describe the fundamental limits on how well information can be represented by and extracted from neural discharges. We illustrate applying these results by considering recordings from the lower auditory pathway.     

The perception of self-agency in chimpanzees (Pan troglodytes)

The ability to distinguish actions and effects caused by oneself from events occurring in the external environment is a fundamental aspect of human cognition. Underlying such distinctions, self-monitoring processes are often assumed, in which predicted events accompanied by one's own volitional action are compared with actual events observed in the external environment. Although many studies have examined the absence or presence of a certain type of self-recognition (i.e. mirror self-recognition) in non-human animals, the underlying cognitive mechanisms remain unclear. Here, we provide, to our knowledge, the first behavioural evidence that chimpanzees can perform self/other distinction for external events on the basis of self-monitoring processes. Three chimpanzees were presented with two cursors on a computer display. One cursor was manipulated by a chimpanzee using a trackball, while the other displayed motion that had been produced previously by the same chimpanzee. Chimpanzees successfully identified which cursor they were able to control. A follow-up experiment revealed that their performance could not be explained by simple associative responses. A further experiment with one chimpanzee showed that the monitoring process occurred in both temporal and spatial dimensions. These findings indicate that chimpanzees and humans share the fundamental cognitive processes underlying the sense of being an independent agent.     

In vivo and in vitro studies of immunoglobulin gene somatic hypermutation

Following antigen encounter, two distinct processes modify immunoglobulin genes. The variable region is diversified by somatic hypermutation while the constant region may be changed by class-switch recombination. Although both genetic events can occur concurrently within germinal centre B cells, there are examples of each occurring independently of the other. Here we compare the contributions of class-switch recombination and somatic hypermutation to the diversification of the serum immunoglobulin repertoire and review evidence that suggests that, despite clear differences, the two processes may share some aspects of their mechanism in common.     

Blood clotting in space

We describe herein a novel in vitro approach that can be used effectively to obtain valuable insights into the role of platelets, various coagulation proteins as well as proteins of the subendothelial extracellular matrix involved in the hemostatic and thrombotic processes occurring under microgravity. At difference with other experimental approaches proposed in the past our device operates in a closed system and under different shear forces, which better mimics flow conditions occurring in vessels. Furthermore our device by allowing real time monitoring of the thrombotic process and its underlying mechanisms can be regarded as a reliable system for the precise assessment of platelet function.     

A signal transduction pathway model prototype I: From agonist to cellular endpoint

The postgenomic era is providing a wealth of information about the genes involved in many cellular processes. However, the ability to apply this information to understanding cellular signal transduction is limited by the lack of tools that quantitatively describe cellular signaling processes. The objective of the current studies is to provide a framework for modeling cellular signaling processes beginning at a plasma membrane receptor and ending with a measurable endpoint in the signaling process. Agonist-induced Ca(2+) mobilization coupled to down stream phosphorylation events was modeled using knowledge of in vitro and in vivo process parameters. The simulation process includes several modules that describe cellular processes involving receptor activation phosphoinositide metabolism, Ca(2+)-release, and activation of a calmodulin-dependent protein kinase. A Virtual Cell-based simulation was formulated using available literature data and compared to new and existing experimental results. The model provides a new approach to facilitate hypothesis-driven investigation and experimental design based upon simulation results. These investigations may be directed at the timing of multiple phosphorylation/dephosphorylation events affecting key enzymatic activities in the signaling pathway.     

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca²⁺ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca²⁺ events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.     

Engineering the melanocortin-4 receptor to control constitutive and ligand-mediated G(S) signaling in vivo

The molecular and functional diversity of G protein-coupled receptors is essential to many physiological processes. However, this diversity presents a significant challenge to understanding the G protein-mediated signaling events that underlie a specific physiological response. To increase our understanding of these processes, we sought to gain control of the timing and specificity of G(s) signaling in vivo. We used naturally occurring human mutations to develop two G(s)-coupled engineered receptors that respond solely to a synthetic ligand (RASSLs). Our G(s)-coupled RASSLs are based on the melanocortin-4 receptor, a centrally expressed receptor that plays an important role in the regulation of body weight. These RASSLs are not activated by the endogenous hormone alpha-melanocyte-stimulating hormone but respond potently to a selective synthetic ligand, tetrahydroisoquinoline. The RASSL variants reported here differ in their intrinsic basal activities, allowing the separation of the effects of basal signaling from ligand-mediated activation of the G(s) pathway in vivo. These RASSLs can be used to activate G(s) signaling in any tissue, but would be particularly useful for analyzing downstream events that mediate body weight regulation in mice. Our study also demonstrates the use of human genetic variation for protein engineering.     

Spontaneous deletions and reciprocal translocations in Saccharomyces cerevisiae: influence of ploidy

Studying spontaneous chromosomal rearrangements throws light on the rules underlying the genome reshaping events occurring in eukaryotic cells, which are part of the evolutionary process. In Saccharomyces cerevisiae, translocation and deletion processes have been frequently described in haploids, but little is known so far about these processes at the diploid level. Here we investigated the nature and the frequency of the chromosomal rearrangements occurring at this ploidy level. Using a positive selection screen based on a particular mutated allele of the URA2 gene, spontaneous diploid revertants were selected and analysed. Surprisingly, the diploid state was found to be correlated with a decrease in chromosome rearrangement frequency, along with an increase in the complexity of the rearrangements occurring in the target gene. The presence of short DNA tandem repeat sequences seems to be a key requirement for deletion and reciprocal translocation processes to occur in diploids. After discussing the differences between the haploid and diploid levels, some mechanisms possibly involved in chromosome shortening and arm exchange are suggested.     

Homology-mediated end-capping as a primary step of sister chromatid fusion in the breakage-fusion-bridge cycles

Breakage-fusion-bridge (BFB) cycle is a series of chromosome breaks and duplications that could lead to the increased copy number of a genomic segment (gene amplification). A critical step of BFB cycles leading to gene amplification is a palindromic fusion of sister chromatids following the rupture of a dicentric chromosome during mitosis. It is currently unknown how sister chromatid fusion is produced from a mitotic break. To delineate the process, we took an integrated genomic, cytogenetic and molecular approach for the recurrent MCL1 amplicon at chromosome 1 in human tumor cells. A newly developed next-generation sequencing-based approach identified a cluster of palindromic fusions within the amplicon at ∼50-kb intervals, indicating a series of breaks and fusions by BFB cycles. The physical location of the amplicon (at the end of a broken chromosome) further indicated BFB cycles as underlying processes. Three palindromic fusions were mediated by the homologies between two nearby inverted Alu repeats, whereas the other two fusions exhibited microhomology-mediated events. Such breakpoint sequences indicate that homology-mediated fold-back capping of broken ends followed by DNA replication is an underlying mechanism of sister chromatid fusion. Our results elucidate nucleotide-level events during BFB cycles and end processing for naturally occurring mitotic breaks.     

Alcohol‐dependent molecular adaptations of the NMDA receptor system

Phenotypes such as motivation to consume alcohol, goal‐directed alcohol seeking and habit formation take part in mechanisms underlying heavy alcohol use. Learning and memory processes greatly contribute to the establishment and maintenance of these behavioral phenotypes. The N‐methyl‐d ‐aspartate receptor (NMDAR) is a driving force of synaptic plasticity, a key cellular hallmark of learning and memory. Here, we describe data in rodents and humans linking signaling molecules that center around the NMDARs, and behaviors associated with the development and/or maintenance of alcohol use disorder (AUD). Specifically, we show that enzymes that participate in the regulation of NMDAR function including Fyn kinase as well as signaling cascades downstream of NMDAR including calcium/calmodulin‐dependent protein kinase II (CamKII), the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR) and the mammalian target of rapamycin complex 1 (mTORC1) play a major role in mechanisms underlying alcohol drinking behaviors. Finally, we emphasize the brain region specificity of alcohol's actions on the above‐mentioned signaling pathways and attempt to bridge the gap between the molecular signaling that drive learning and memory processes and alcohol‐dependent behavioral phenotypes. Finally, we present data to suggest that genes related to NMDAR signaling may be AUD risk factors.     

Involvement of extracellular matrix and integrin-like proteins on conidial adhesion and appressorium differentiation in Magnaporthe oryzae

Conidial adhesion and appressorium formation of Magnaporthe oryzae on the rice surface are important early events in the infection process. As an initiative step to understand the mechanisms underlying these cellular processes at a biochemical level, the effect of a human fibronectin antibody (HFA) and RGD peptides on conidial adhesion and appressorium formation was evaluated. HFA inhibited conidial adhesion and appressorium formation in a dosage-dependent manner. RGD peptides also inhibited these cellular events. Conidial adhesion and appressorium formation inhibited by RGD peptides were restored by chemicals involved in the cyclic AMP-dependent signaling pathway. These results suggest that extracellular matrix proteins might be involved in conidial adhesion and appressorium formation through integrin-like receptor mediation and modulation of cAMP-dependent signaling in the cells.     

Characterization of a naturally-occurring polymorphism in the UHR-1 gene encoding the putative rat prolactin-releasing peptide receptor

The rat orphan receptor UHR-1 and its human orthologue, GPR10, were first isolated in 1995. The ligand for this receptor, prolactin-releasing peptide (PrRP), was identified in 1998 by reverse pharmacology and has subsequently been implicated in a number of physiological processes. As supported by its localization and regulation in the hypothalamus and brainstem, we have shown previously that PrRP is involved in energy homeostasis. Here we describe a naturally occurring polymorphism in the UHR-1 gene that results in an ATG to ATA change at the putative translational initiation site. The presence of the polymorphism abolished the binding of 125I PrRP in rat brain slices but did not affect the ability of PrRP to reduce fast-induced food intake. Together this data suggest that PrRP may be exerting its feeding effects through a receptor other than UHR-1.